Processes And Systems For The Production Of Fermentation Products

Abstract

The present invention relates to processes and systems for the production of fermentation products such as alcohols. The present invention also provides methods for separating feed stream components for improved biomass processing and productivity.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 61/674,607, filed Jul. 23, 2012; U.S. Provisional Application No. 61/699,976, filed Sep. 12, 2012; U.S. Provisional Application No. 61/712,385, filed Oct. 11, 2012; U.S. patent application Ser. No. 13/828,353, filed Mar. 14, 2013; and U.S. patent application Ser. No. 13/836,115, filed Mar. 15, 2013, the entire contents of each are herein incorporated by reference.

[0002] The Sequence Listing associated with this application is filed in electronic form via EFS-Web and hereby incorporated by reference into the specification in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to processes and systems for the production of fermentation products such as alcohols. The present invention also provides processes for separating feed stream components for improved biomass processing productivity.

BACKGROUND OF THE INVENTION

[0004] Alcohols have a variety of industrial and scientific applications such as fuels, reagents, and solvents. For example, butanol is an important industrial chemical with a variety of applications including use as a fuel additive, as a feedstock chemical in the plastics industry, and as a food-grade extractant in the food and flavor industry. Accordingly, there is a high demand for alcohols such as butanol as well as for efficient and environmentally-friendly production methods including, for example, fermentation processes and the use of biomass as feedstock for these processes.

[0005] Production of alcohols by fermentation is one such environmentally friendly production method. Some microorganisms that produce alcohols in high yields also have low toxicity thresholds, such that the alcohol needs to be removed from the fermentor as it is being produced. One method, in situ product removal (ISPR), may be used to remove alcohol from the fermentor as it is produced, thereby allowing the microorganism to produce alcohol at high yields. An example of ISPR that has been described in the art is liquid-liquid extraction (see, e.g., U.S. Patent Application Publication No. 2009/0305370). In order to be technically and economically viable, liquid-liquid extraction requires contact between the extractant and the fermentation broth for efficient mass transfer; phase separation of the extractant from the fermentation broth; efficient recovery and recycle of the extractant; and minimal degradation and/or contamination of the extractant over a long-term operation.

[0006] When the aqueous stream entering the fermentor contains undissolved solids from feedstock, the undissolved solids may interfere with liquid-liquid extraction and the extraction method may not be technically and economically viable, for example, leading to increases in capital and operating costs. The presence of undissolved solids during extractive fermentation may lower the mass transfer coefficient, impede phase separation, result in the accumulation of oil from the undissolved solids in the extractant leading to reduced extraction efficiency over time, increase the loss of extractant because it becomes trapped in solids and ultimately removed as Dried Distillers Grains with Solubles (DDGS), slow the disengagement of extractant drops from the fermentation broth, and/or result in a lower fermentor volume efficiency. Thus, solids removal provides an efficient means to produce and recover an alcohol from a fermentation process.

[0007] In addition to solids removal, removal of oil from feedstock may also provide beneficial effects on the production of alcohols as well as commercial benefits. For example, some oils such as corn oil and soybean oil may be used as feedstock for biodiesel and thus, provide an additional revenue stream for alcohol producers. In addition, removing oil can result in energy savings for the production plant due to more efficient fermentation, less fouling due to the removal of the oil, increased fermentor volume efficiency, and decreased energy requirements, for example, the energy needed to dry distillers grains.

[0008] There is a continuing need to develop more efficient processes and systems for producing alcohols such as ethanol and butanol. The present invention satisfies this need and provides processes and systems for producing alcohols including processes and systems for separating feed stream components prior to the fermentation and controlling the amount of undissolved solids and/or oil in the fermentation process.

SUMMARY OF THE INVENTION

[0009] The present invention relates to processes and systems for separating feed stream components and controlling the amount of undissolved solids and/or oil in a fermentor feed stream in the production of fermentation products. The separated components provide a mechanism for increasing biomass processing productivity, including improving alcohol fermentation co-product profiles. By separating the feed streams into certain components including (1) an aqueous stream comprising fermentable carbon sources, (2) a feed stream comprising oil, and (3) a feed stream comprising undissolved solids, these components may be recombined in a controlled manner, or removed from the system for other uses. This provides a mechanism to develop co-product compositions for the needs of different markets, such as animal feed markets requiring higher protein or higher fat feeds.

[0010] The present invention also relates to processes and systems for removing oil from a fermentor feed stream in the production of fermentation products. In some embodiments, undissolved solids and oil may be removed from a fermentor feed stream.

[0011] The present invention is directed to a method for producing a product alcohol comprising: providing a feedstock slurry comprising fermentable carbon source, undissolved solids, and oil; separating a portion of the undissolved solids and oil from the feedstock slurry whereby an aqueous solution comprising fermentable carbon source, a wet cake comprising solids and an oil stream are formed; and adding the aqueous solution to a fermentation broth comprising microorganisms whereby a product alcohol is produced. In some embodiments, the method further comprises the step of recovering the oil stream. In some embodiments, the method further comprises the step of washing the wet cake to provide an aqueous stream comprising carbohydrate. In some embodiments, the method further comprises the step of adding the aqueous stream to the fermentation broth. In some embodiments, the aqueous solution contains no more than about 5% by weight of undissolved solids. In some embodiments, the oil is corn oil and comprises one or more of triglycerides, fatty acids, diglycerides, monoglycerides, and phospholipids. In some embodiments, the method further comprises the step of combining a portion of the wet cake and a portion of oil to produce a wet cake comprising triglycerides, free fatty acids, diglycerides, monoglycerides, and phospholipids. In some embodiments, the method further comprises the step of combining the aqueous solution with a portion of the wet cake to produce a mixture of the aqueous solution and wet cake and adding the mixture to the fermentation broth. In some embodiments, separating the feedstock slurry is a single step process. In some embodiments, the undissolved solids and oil are separated from feedstock slurry by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, membrane filtration, cross flow filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, one or more control parameters of the separation device is adjusted to improve separation of the feedstock slurry. In some embodiments, the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, weir position, scroll pitch, residence time, and discharge volume. In some embodiments, the product alcohol is selected from ethanol, propanol, butanol, pentanol, hexanol, and fusel alcohols. In some embodiments, the microorganism comprises a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway is a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, or an isobutanol biosynthetic pathway. In some embodiments, real-time measurements are used to monitor separation of the feedstock slurry. In some embodiments, separation is monitored by Fourier transform infrared spectroscopy, near-infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometers, densitometers, tensiometers, droplet size analyzers, particle analyzers, or combinations thereof.

[0012] The present invention is also directed to a method comprising providing a feedstock slurry comprising fermentable carbon source and undissolved solids; separating at least a portion of the undissolved solids from the feedstock slurry whereby an aqueous solution comprising fermentable carbon source and a wet cake comprising solids are generated; contacting the wet cake with a liquid to form a wet cake mixture; and separating at least a portion of undissolved solids from the wet cake mixture whereby a second aqueous solution comprising fermentable carbon source and a second wet cake comprising solids are generated. In some embodiments, the liquid is selected from fresh water, backset, cook water, process water, lutter water, evaporation water, or combinations thereof. In some embodiments, the steps contacting and separating steps may be repeated.

[0013] The present invention is directed to a method comprising: providing a feedstock slurry comprising fermentable carbon source, undissolved solids, and oil; separating at least a portion of the oil and undissolved solids from the feedstock slurry whereby an aqueous solution comprising fermentable carbon source, an oil stream; and a wet cake comprising solids are generated; and contacting the wet cake with a liquid to form a wet cake mixture; and separating at least a portion of undissolved solids and oil from the wet cake mixture whereby a second aqueous solution comprising fermentable carbon source, a second oil stream; and a second wet cake comprising solids are generated. In some embodiments, separating the feedstock slurry is a single step process. In some embodiments, the undissolved solids and oil are separated from feedstock slurry by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, membrane filtration, cross flow filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, one or more control parameters of the separation device is adjusted to improve separation of the feedstock slurry. In some embodiments, the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, weir position, scroll pitch, residence time, and discharge volume. In some embodiments, real-time measurements are used to monitor separation of the feedstock slurry. In some embodiments, separation is monitored by Fourier transform infrared spectroscopy, near-infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometers, densitometers, tensiometers, droplet size analyzers, particle analyzers, or combinations thereof.

[0014] The present invention is directed to a method comprising providing a feedstock slurry comprising fermentable carbon source, undissolved solids, and oil; separating the feedstock slurry whereby (i) a first aqueous solution comprising a fermentable carbon source, (ii) a first wet cake comprising solids, and (iii) a stream comprising oil, solids, and an aqueous stream comprising a fermentable carbon source are formed; and adding the first aqueous solution to a fermentation broth comprising microorganisms whereby a fermentation product is produced. In some embodiments, the method may further comprise separating the stream comprising oil, solids, and aqueous stream comprising a fermentable carbon source whereby (i) a second aqueous solution comprising a fermentable carbon source, (ii) a second wet cake comprising solids, and (iii) an oil stream are formed. In some embodiments, the first and second aqueous solutions may be combined prior to the addition to the fermentation broth. In some embodiments, the second aqueous solution may further comprise oil. In some embodiments, the oil of the second aqueous solution or portion thereof may be treated to generate an extractant. In some embodiments, the oil may be treated chemically or enzymatically. In some embodiments, separation may be performed by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

[0015] The present invention is directed to a method comprising providing a feedstock slurry comprising a fermentable carbon source, undissolved solids, and oil; separating the feedstock slurry whereby (i) a first aqueous solution comprising a fermentable carbon source and solids, (ii) a first wet cake comprising solids, and (iii) a first oil stream are formed; and adding oil to the first aqueous solution whereby an oil layer comprising solids and a second aqueous solution comprising a fermentable carbon source are formed. In some embodiments, the oil layer comprising solids may be separated forming (i) a second oil stream, (ii) a second wet cake comprising solids, and (iii) a third aqueous solution comprising a fermentable carbon source. In some embodiments, the second aqueous solution and the third aqueous solution may be added to a fermentation broth comprising microorganisms whereby a fermentation product is produced. In some embodiments, the second aqueous solution and the third aqueous solution may further comprise oil. In some embodiments, the second aqueous solution and the third aqueous solution may be combined and the oil of the second aqueous solution and the third aqueous solution or portions thereof is treated to generate an extractant. In some embodiments, the oil may be treated chemically or enzymatically. In some embodiments, separation may be performed by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

[0016] The present invention is also directed to a system comprising one or more liquefaction units configured to liquefy a feedstock to create a feedstock slurry, the liquefaction unit comprising: an inlet for receiving the feedstock; and an outlet for discharging a feedstock slurry, wherein the feedstock slurry comprises fermentable carbon source, oil, and undissolved solids; and one or more separation units configured to remove the oil and undissolved solids from the feedstock slurry to create an aqueous solution comprising the fermentable carbon source, an oil stream, and a wet cake comprising the portion of the undissolved solids, the centrifuge comprising: an inlet for receiving the feedstock slurry; a first outlet for discharging the aqueous solution; a second outlet for discharging the wet cake; and a third outlet for discharging the oil. In some embodiments, the system further comprises one or more wash systems configured to recover the fermentable carbon source from the wet cake comprising: one or more mixing units; and one or more separation units. In some embodiments, the separation unit is selected from decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, membrane filtration, cross flow filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, and combinations thereof. In some embodiments, the system further comprises one or more fermentors configured to ferment the aqueous solution to produce product alcohol, the fermentors comprising: an inlet for receiving the aqueous solution and/or wet cake; and an outlet for discharging fermentation broth comprising product alcohol. In some embodiments, the system further comprises on-line measurement devices. In some embodiments, the on-line measurement devices are selected from particle size analyzers, Fourier transform infrared spectroscopes, near-infrared spectroscopes, Raman spectroscopes, high pressure liquid chromatography, viscometers, densitometers, tensiometers, droplet size analyzers, pH meters, dissolved oxygen probes, or combinations thereof.

DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention.

[0018] FIG. 1 schematically illustrates an exemplary process and system of the present invention, in which undissolved solids are removed by separation after liquefaction and before fermentation.

[0019] FIG. 2 schematically illustrates an exemplary alternative process and system of the present invention, in which feedstock is milled.

[0020] FIG. 3 schematically illustrates another exemplary alternative process and system of the present invention, in which undissolved solids and oil are removed by separation.

[0021] FIG. 4 schematically illustrates another exemplary alternative process and system of the present invention, in which the wet cake is subjected to one or more wash cycles.

[0022] FIG. 5 schematically illustrates another exemplary alternative process and system of the present invention, in which undissolved solids and oil are removed by separation and wet cake is subjected to one or more wash cycles.

[0023] FIG. 6 schematically illustrates another exemplary alternative process and system of the present invention, in which the aqueous solution and wet cake are combined and conducted to fermentation.

[0024] FIG. 7 schematically illustrates another exemplary alternative process and system of the present invention, in which the aqueous solution is saccharified prior to fermentation.

[0025] FIG. 8 schematically illustrates another exemplary alternative process and system of the present invention, in which the feedstock slurry is saccharified prior to separation.

[0026] FIGS. 9A-9D schematically illustrate exemplary alternative processes and systems of the present invention, in which additional separation units are utilized to remove undissolved solids and oil.

[0027] FIG. 10 schematically illustrates an exemplary fermentation process utilizing on-line, in-line, at-line, and/or real-time measurements for monitoring fermentation processes.

[0028] FIG. 11 schematically illustrates an exemplary fermentation process of the present invention including downstream processing.

[0029] FIG. 12 illustrates the effect of the presence of undissolved corn mash solids on the overall volumetric mass transfer coefficient, k.sub.La, for the transfer of i-BuOH from an aqueous solution of liquefied corn starch to a dispersion of oleyl alcohol droplets flowing up through a bubble column when a nozzle with an inner diameter of 2.03 mm is used to disperse the oleyl alcohol.

[0030] FIG. 13 illustrates the effect of the presence of undissolved corn mash solids on the overall volumetric mass transfer coefficient, k.sub.La, for the transfer of i-BuOH from an aqueous solution of liquefied corn starch to a dispersion of oleyl alcohol droplets flowing up through a bubble column when a nozzle with an inner diameter of 0.76 mm is used to disperse the oleyl alcohol.

[0031] FIG. 14 illustrates the position of the liquid-liquid interface in the fermentation sample tubes as a function of (gravity) settling time. Phase separation data shown for run times: 5.3, 29.3, 53.3, and 70.3 hr run time. Sample data from extractive-fermentation where solids were removed from the mash feed, and oleyl alcohol was the solvent.

[0032] FIG. 15 illustrates the position of the liquid-liquid interface of the final fermentation broth as a function of (gravity) settling time. Data from extractive-fermentation where solids were removed from the mash feed, and oleyl alcohol was the solvent.

[0033] FIG. 16 illustrates the concentration of glucose in the aqueous phase of the slurries as a function of time for Batch 1 and Batch 2.

[0034] FIG. 17 illustrates concentration of glucose in the aqueous phase of the slurries as a function of time for Batch 3 and Batch 4.

[0035] FIG. 18 illustrates the effect of enzyme loading and +/- a high temperature stage was applied at some time during the liquefaction on starch conversion.

[0036] FIGS. 19A-19E illustrate the effect of three-phase centrifuge conditions on separation of feedstock slurry.

DESCRIPTION OF THE INVENTION

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entireties for all purposes.

[0038] In order to further define this invention, the following terms and definitions are herein provided.

[0039] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0040] Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e., occurrences of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0041] The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

[0042] As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about," the claims include equivalents to the quantities. In one embodiment, the term "about" means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value.

[0043] "Biomass" as used herein refers to a natural product containing hydrolyzable polysaccharides that provide fermentable sugars including any sugars and starch derived from natural resources such as corn, sugar cane (or cane), wheat, cellulosic or lignocellulosic material, and materials comprising cellulose, hemicellulose, lignin, starch, oligosaccharides, disaccharides, and/or monosaccharides, and mixtures thereof. Biomass may also comprise additional components such as protein and/or lipids. Biomass may be derived from a single source or biomass may comprise a mixture derived from more than one source. For example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, and wood and forestry waste (e.g., forest thinnings). Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, rye, wheat straw, spelt, triticale, barley, barley straw, oats, hay, rice, rice straw, switchgrass, potato, sweet potato, cassava, Jerusalem artichoke, sugar cane bagasse, sorghum, sugar cane, sugar beet, fodder beet, soy, palm, coconut, rapeseed, safflower, sunflower, millet, eucalyptus, miscanthus, waste paper, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof. Mash, juice, molasses, or hydrolysate may be formed from biomass by any method known in the art for processing biomass for purposes of fermentation such as milling, treating (e.g., enzymatic, chemical), and/or liquefying. Treated biomass may comprise fermentable sugar and/or water. Cellulosic and/or lignocellulosic biomass may be processed to obtain a hydrolysate containing fermentable sugars by any method known to one skilled in the art. For example, a low ammonia pretreatment is disclosed in U.S. Patent Application Publication No. 2007/0031918A1, the entire contents of which are herein incorporated by reference. Enzymatic saccharification of cellulosic and/or lignocellulosic biomass typically makes use of enzyme mixtures for hydrolysis of cellulose and hemicellulose to produce a hydrolysate containing sugars including glucose, xylose, and arabinose. Saccharification enzymes suitable for cellulosic and/or lignocellulosic biomass are reviewed in Lynd, et al. (Microbiol. Mol. Biol. Rev. 66:506-577, 2002).

[0044] "Fermentable carbon source" or "fermentable carbon substrate" as used herein refers to a carbon source capable of being metabolized by microorganisms. Suitable fermentable carbon sources include, but are not limited to, monosaccharides such as glucose or fructose; disaccharides such as lactose or sucrose; oligosaccharides; polysaccharides such as starch or cellulose; one carbon substrates; and mixtures thereof.

[0045] "Fermentable sugar" as used herein refers to one or more sugars capable of being metabolized by microorganisms for the production of fermentation products such as alcohols.

[0046] "Feedstock" as used herein refers to a feed in a fermentation process. The feed may comprise a fermentable carbon source and may comprise undissolved solids and/or oil. Where applicable, the feed may comprise a fermentable carbon source before or after the fermentable carbon source has been liberated from starch or obtained from the hydrolysis of complex sugars by further processing such as by liquefaction, saccharification, or other process. Feedstock includes or may be derived from biomass. Suitable feedstocks include, but are not limited to, rye, wheat, corn, corn mash, sugar cane, cane mash, barley, cellulosic material, lignocellulosic material, or mixtures thereof. Where reference is made to "feedstock oil," it will be appreciated that the term encompasses the oil produced from a given feedstock.

[0047] "Fermentation broth" as used herein refers to a mixture of water, fermentable carbon sources (e.g., sugars, starch), dissolved solids, optionally microorganisms producing fermentation products, optionally fermentation products (e.g., product alcohols), optionally undissolved solids, and other constituents of the material held in the fermentor in which fermentation product is being made by the metabolism of fermentable carbon sources by the microorganisms to form fermentation product, water, and carbon dioxide (CO.sub.2). From time to time as used herein, the term "fermentation medium" and "fermented mixture" may be used synonymously with "fermentation broth."

[0048] "Fermentor" or "fermentation vessel" as used herein refers to a vessel, unit, or tank in which the fermentation reaction is carried out whereby fermentation product (e.g., product alcohols such as ethanol or butanol) is made from fermentable carbon sources. Fermentor may also refer to a vessel, unit, or tank in which growth of microorganism occurs. In some instances, both microbial growth and fermentation may occur in a fermentor. The term "fermentor" may be used synonymously herein with "fermentation vessel."

[0049] "Saccharification vessel" as used herein refers to a vessel, unit, or tank in which saccharification (i.e., the hydrolysis of oligosaccharides to monosaccharides) is carried out. Where fermentation and saccharification occur simultaneously, the saccharification vessel and the fermentor may be the same vessel.

[0050] "Saccharification enzyme" as used herein refers to one or more enzymes that are capable of hydrolyzing polysaccharides and/or oligosaccharides, for example, alpha-1,4-glucosidic bonds of glycogen, or starch. Saccharification enzymes may include enzymes capable of hydrolyzing cellulosic or lignocellulosic materials as well.

[0051] "Liquefaction vessel" as used herein refers to a vessel, unit, or tank in which liquefaction is carried out. Liquefaction is a process in which starch is hydrolyzed, for example, by an enzymatic process to obtain oligosaccharides. In embodiments where the feedstock is corn, oligosaccharides are hydrolyzed from the corn starch content during liquefaction.

[0052] "Sugar" as used herein refers to oligosaccharides, disaccharides, monosaccharides, and/or mixtures thereof. The term "saccharide" may also include carbohydrates such as starches, dextrans, glycogens, cellulose, pentosans, as well as sugars.

[0053] "Undissolved solids" as used herein refers to non-fermentable portions of feedstock which are not dissolved in the liquid or aqueous phase, for example, germ, fiber, and gluten. The non-fermentable portions of feedstock include the portion of feedstock that remains as solids and can absorb liquid from the fermentation broth. From time to time as used herein, the term "undissolved solids" may be used synonymously with "solids" or "suspended solids."

[0054] "Extractant" as used herein refers to a solvent used to extract a fermentation product. From time to time as used herein, the term "extractant" may be used synonymously with "solvent."

[0055] "In Situ Product Removal" (ISPR) as used herein refers to the selective removal of a product from a biological process such as fermentation to control the product concentration in the biological process as the product is produced.

[0056] "Product alcohol" as used herein refers to any alcohol that may be produced by a microorganism in a fermentation process that utilizes biomass as a fermentable carbon source. Product alcohols include, but are not limited to, C.sub.1 to C.sub.8 alkyl alcohols. In some embodiments, the product alcohols are C.sub.2 to C.sub.8 alkyl alcohols. In other embodiments, the product alcohols are C.sub.2 to C.sub.5 alkyl alcohols. It will be appreciated that C.sub.1 to C.sub.8 alkyl alcohols include, but are not limited to, methanol, ethanol, propanol, butanol, pentanol, hexanol, and isomers thereof. Likewise, C.sub.2 to C.sub.8 alkyl alcohols include, but are not limited to, ethanol, propanol, butanol, pentanol, hexanol, and isomers thereof. The term "alcohol" may also be used herein with reference to a product alcohol.

[0057] "Butanol" as used herein refers to butanol isomers: 1-butanol (1-BuOH), 2-butanol (2-BuOH), tertiary-butanol (tert-BuOH), and/or isobutanol (iBuOH, i-BuOH, or I-BUOH), either individually or as mixtures thereof.

[0058] "Propanol" as used herein refers to the propanol isomers: isopropanol or 1-propanol, either individually or as mixtures thereof.

[0059] "Pentanol" as used herein refers to the pentanol isomers: 1-pentanol, 3-methyl-1-butanol, 2-methyl-1-butanol, 2,2-dimethyl-1-propanol, 3-pentanol, 2-pentanol, 3-methyl-2-butanol, or 2-methyl-2-butanol, either individually or as mixtures thereof.

[0060] "Effective titer" as used herein refers to the total amount of a particular fermentation product (e.g., product alcohol) produced by fermentation. In some embodiments wherein the fermentation product is a product alcohol, effective titer refers to the alcohol equivalent of an alcohol ester produced by alcohol esterification per liter of fermentation medium.

[0061] "Water-immiscible" or "insoluble" as used herein refer to a chemical component such as an extractant or solvent, which is incapable of mixing with an aqueous solution such as a fermentation broth, in such a manner as to form one liquid phase.

[0062] "Aqueous phase" as used herein refers to the aqueous phase of at least a biphasic mixture obtained by contacting a fermentation broth with an extractant, for example, a water-immiscible organic extractant. In an embodiment of a process described herein that includes fermentative extraction, the term "fermentation broth" then may refer to the aqueous phase in biphasic fermentative extraction.

[0063] "Aqueous phase titer" as used herein refers to the concentration of a fermentation (e.g., product alcohol) in the aqueous phase.

[0064] "Organic phase" as used herein refers to the non-aqueous phase of at least a biphasic mixture obtained by contacting a fermentation broth with an extractant, for example, a water-immiscible organic extractant.

[0065] "Portion" as used herein with reference to a process stream refers to any fractional part of the stream which retains the composition of the stream, including the entire stream, as well as any component or components of the stream, including all components of the stream.

[0066] "By-product" or "co-product" as used herein refers to a product produced during the production of another product. In some instances, the term "co-product" may be used synonymously with the term "by-product." Co-products include for example, oil recovered from the feedstock slurry, wet cake, and DDGS. Co-products may also include modification of the oil, wet cake, and DDGS for the purposes of improving value and/or for the manufacture of other products, such as biodiesel from the oil.

[0067] "Distillers co-products" as used herein refers to by-products from a product alcohol production process that can be isolated before or during fermentation. Distillers co-products include non-fermentable products remaining after product alcohol is removed from a fermented mash and solids isolated from a mash. As used herein, distillers co-products may be used in a variety of animal feed and non-animal feed applications. Examples of distillers co-products include, but are not limited to, fatty acids from oil hydrolysis, lipids from evaporation of thin stillage, syrup, distillers grains, distillers grains and solubles, solids from mash before fermentation, and solids from whole stillage after fermentation, biodiesel, and acyl glycerides.

[0068] "Distillers co-products for animal feed" as used herein refers to distillers co-products that are suitable for use in or as animal feed. Examples of distillers co-products for animal feed include, but are not limited to, fatty acids from oil hydrolysis, lipids from evaporation of thin stillage, syrup, distillers grains, distillers grains and solubles, solids from mash before fermentation, and solids from whole stillage after fermentation.

[0069] "Distillers grains" or "DG" as used herein refer to the non-fermentable products remaining after product alcohol is removed from a fermented mash. Distillers grains that are dried are known as "distillers dried grains" or "DDG." Distillers grains that are not dried are known as "wet distillers grains" or "WDG."

[0070] "Distillers grains and solubles" or "DGS" as used herein refer to the non-fermentable products remaining after product alcohol is removed from a fermented mash, that have been blended with solubles. Distillers grains and solubles that are dried are known as "distillers dried grains and solubles" or "DDGS." Distillers grains and solubles that are not dried are known as "wet distillers grains and solubles" or "WDGS."

[0071] "Dried Distillers Grains with Solubles" (DDGS) as used herein refer to a co-product or by-product from a fermentation of a feedstock or biomass (e.g., fermentation of grain or grain mixture that produces a product alcohol). In some embodiments, DDGS may also refer to an animal feed produced from a process of making a product alcohol.

[0072] "Lipid" as used herein refers to any of a heterogeneous group of fats and fat-like substances including fatty acids, neutral fats, waxes, and steroids, which are water-insoluble and soluble in nonpolar solvents. Examples of lipids include monoglycerides, diglycerides, triglycerides, and phospholipids.

[0073] "Lipids from evaporation" as used herein in reference to a process stream refer to a lipid by-product produced by evaporation and centrifugation of thin stillage following fermentation in a product alcohol production process.

[0074] "Syrup" or "condensed distillers solubles" (CDS) as used herein in reference to a process stream refers to a by-product produced by evaporation of thin stillage following fermentation in a product alcohol production process.

[0075] "Process stream" as used herein refers to any by-product or co-product formed by a fermentation product production process. Examples of process streams include, but are not limited to, COFA, lipids from evaporation, syrup, DG, DDG, WDG, DGS, DDGS, and WDGS. Another example of a process stream is solids removed (e.g., by centrifugation) from a mash before fermentation in a fermentation product production process (e.g., the solids removed from a corn mash before fermentation). These solids may be referred to as "wet cake" when they have not been dried, and may be referred to as "dry cake" when they have been dried. Another example of a process stream is solids removed (e.g., by centrifugation) from whole stillage following fermentation in a fermentation product production process. These solids may be referred to as "WS wet cake" when they have not been dried, and may be referred to as "WS dry cake" when they have been dried. From time to time as used herein, the term "process stream" may be used synonymously with "stream."

[0076] The present invention provides processes and methods for producing fermentation products such as product alcohols using fermentation. Other fermentation products that may be produced using the processes and methods described herein include propanediol, butanediol, acetone, acids such as lactic acid, acetic acid, butyric acid, and propionic acid; gases such as hydrogen methane, and carbon dioxide; amino acids; vitamins such as biotin, vitamin B.sub.2 (riboflavin), vitamin B.sub.12 (e.g., cobalamin), ascorbic acid (e.g., vitamin C), vitamin E (e.g., a-tocopherol), and vitamin K (e.g., menaquinone); antibiotics such as erythromycin, penicillin, streptomycin, and tetracycline; and other products such as citric acid, invertase, sorbitol, pectinase, and xylitol.

[0077] As an example of the processes and methods provided herein, a feedstock may be liquefied to create a feedstock slurry which comprises a fermentable carbon source (e.g., soluble sugar) and undissolved solids. In some instances, the terms "feedstock slurry" and "mash" may be used interchangeably. In some embodiments, the feedstock slurry comprises soluble sugar, undissolved solids, and oil. If the feedstock slurry is fed directly to a fermentor, the undissolved solids and/or oil may interfere with efficient removal and recovery of the fermentation product such as a product alcohol. For example, if liquid-liquid extraction is utilized to extract product alcohol from fermentation broth, the presence of undissolved solids may cause system inefficiencies including, but not limited to, decreasing the mass transfer rate of the product alcohol to the extractant by interfering with the contact between the extractant and the fermentation broth; creating an emulsion in the fermentor and thereby interfering with phase separation of the extractant and the fermentation broth; slowing disengagement of the extractant from the fermentation broth; reducing the efficiency of recovering and recycling the extractant because at least a portion of the extractant and product alcohol becomes "trapped" in the solids; shortening the life cycle of the extractant by contamination with oil; and lowering fermentor volume efficiency because there are solids taking up volume in the fermentor. These effects can result in higher capital and operating costs. In addition, the extractant "trapped" in undissolved solids used to generate Distillers Dried Grains with Solubles (DDGS), may detract from DDGS value and qualification for sale as animal feed. Therefore, in order to avoid and/or minimize these problems, at least a portion of the undissolved solids may be removed from the feedstock slurry prior to the addition of the feedstock slurry to the fermentor. Extraction activity and the efficiency of product alcohol production can be increased when extraction is performed on fermentation broth containing an aqueous solution where undissolved solids have been removed relative to extraction performed on fermentation broth containing an aqueous solution where undissolved solids have not been removed.

[0078] The processes and systems of the present invention will be described with reference to the Figures. In some embodiments, as shown, for example, in FIG. 1, the system includes liquefaction 10 configured to liquefy a feedstock to create a feedstock slurry.

[0079] For example, feedstock 12 may be introduced to an inlet in liquefaction 10. Feedstock 12 may be any suitable biomass material that contains a fermentable carbon source such as starch including, but not limited to, barley, oat, rye, sorghum, wheat, triticale, spelt, millet, cane, corn, or combinations thereof. Water may also be introduced to liquefaction 10.

[0080] The process of liquefying feedstock 12 involves hydrolysis of feedstock 12 generating water-soluble sugars. Any known liquefying processes utilized by the industry may be used including, but not limited to, an acid process, an enzyme process, or an acid-enzyme process. Such processes may be used alone or in combination. In some embodiments, the enzyme process may be utilized and an appropriate enzyme 14, for example, alpha-amylase, is introduced to an inlet in liquefaction 10. Examples of alpha-amylases that may be used in the processes and systems of the present invention are described in U.S. Pat. No. 7,541,026; U.S. Patent Application Publication No. 2009/0209026; U.S. Patent Application Publication No. 2009/0238923; U.S. Patent Application Publication No. 2009/0252828; U.S. Patent Application Publication No. 2009/0314286; U.S. Patent Application Publication No. 2010/02278970; U.S. Patent Application Publication No. 2010/0048446; and U.S. Patent Application Publication No. 2010/0021587, the entire contents of each are herein incorporated by reference.

[0081] In some embodiments, enzymes for liquefaction and/or saccharification may be produced by the microorganism (e.g., microorganism 32). Examples of microorganisms producing such enzymes are described in U.S. Pat. No. 7,498,159; U.S. Patent Application Publication No. 2012/0003701; U.S. Patent Application Publication No. 2012/0129229; PCT International Publication No. WO 2010/096562; and PCT International Publication No. WO 2011/153516, the entire contents of each are herein incorporated by reference.

[0082] The process of liquefying feedstock 12 produces feedstock slurry 16 that comprises a fermentable carbon source and undissolved solids. In some embodiments, feedstock slurry 16 may comprise a fermentable carbon source, oil, and undissolved solids. The undissolved solids are non-fermentable portions of feedstock 12. In some embodiments, feedstock 12 may be corn, such as dry milled, unfractionated corn kernels, and the undissolved solids may comprise germ, fiber, and gluten. In some embodiments, feedstock 12 is corn or corn kernels and feedstock slurry 16 is corn mash slurry. Feedstock slurry 16 may be discharged from an outlet of liquefaction 10 and conducted to separation 20.

[0083] In some embodiments, nutrients such as amino acids, nitrogen, minerals, trace elements, and/or vitamins may be added to feedstock slurry 16 or fermentation 30. For example, one or more of the following: biotin, pantothenate, folic acid, niacin, aminobenzoic acid, pyridoxine, riboflavin, thiamine, vitamin A, vitamin C, vitamin D, vitamin E, vitamin K, inositol, potassium (e.g., potassium phosphate), boric acid, calcium, chloride, chromium, copper (e.g., copper sulfate), iodide (e.g., potassium iodide), iron (e.g., ferric chloride), lithium, magnesium (e.g., magnesium sulfate), manganese (e.g., manganese sulfate), molybdenum, calcium chloride, phosphorus, potassium, sodium chloride, vanadium, zinc (e.g., zinc sulfate), yeast extract, soy peptone, and the like may be added to feedstock slurry 16 or fermentation 30. Examples of amino acids include essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine as well as other amine acids such as alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, hydroxylysine, hydroxyproline, ornithine, proline, serine, and tyrosine.

[0084] Separation 20 via an inlet may be configured to remove undissolved solids from feedstock slurry 16. Separation 20 may also be configured to remove oil, or to remove both oil and undissolved solids. Separation 20 may be any device capable of separating solids and liquids. For example, separation 20 may be any conventional centrifuge utilized in the industry, including, for example, a decanter bowl centrifuge, three-phase centrifuge, disk stack centrifuge, filtering centrifuge, or decanter centrifuge. In some embodiments, separation may be accomplished by filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grates or grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or any method or separation device that may be used to separate solids and liquids.

[0085] Feedstock slurry 16, conducted to separation 20, may be separated to form a liquid phase, aqueous stream, or aqueous solution 22 (also known as thin mash) and a solid phase, solid stream, or wet cake 24. Aqueous solution 22 may comprise sugar, for example, in the form of oligosaccharides, and water. In some embodiments, aqueous solution 22 may comprise at least about 10% by weight oligosaccharides, at least about 20% by weight of oligosaccharides, or at least about 30% by weight of oligosaccharides. In some embodiments, aqueous solution 22 may be discharged from an outlet located near the top of separation 20. In some embodiments, aqueous solution 22 may have a viscosity of less than about 20 centipoise (cP). In some embodiments, aqueous solution 22 may comprise less than about 20 g/L of monomeric glucose, less than about 10 g/L of monomeric glucose, or less than about 5 g/L of monomeric glucose. Suitable methodology to determine the amount of monomeric glucose is well known in the art such as high performance liquid chromatography (HPLC).

[0086] Wet cake 24 may be discharged from separation 20. In some embodiments, wet cake 24 may be discharged from an outlet located near the bottom of separation 20. Wet cake 24 may comprise undissolved solids. In some embodiments, wet cake 24 may also comprise a portion of sugar and water. Wet cake 24 may be washed with additional water using separation 20 once aqueous solution 22 has been discharged from separation 20. In some embodiments, wet cake 24 may be washed with additional water using additional separation devices. Washing wet cake 24 will recover the sugar or sugar source (e.g., oligosaccharides) present in the wet cake, and the recovered sugar and water may be recycled to liquefaction 10. After washing, wet cake 24 may be processed to form DDGS using any suitable known process. The formation of DDGS from wet cake 24 has several benefits. For example, since the undissolved solids are not added to the fermentor, the undissolved solids are not subjected to the conditions of the fermentor and therefore, the undissolved solids do not contact the microorganisms present in the fermentor, and fermentation product such as product alcohol or other components such as extractant are not trapped in the undissolved solids. These effects provide benefits to subsequent processing and use of DDGS, for example, as animal feed because the DDGS would not contain microorganism or other components (e.g., product alcohol, extractant) of the fermentation broth.

[0087] In some embodiments, undissolved solids may be separated from feedstock slurry to form two product streams, for example, an aqueous solution of oligosaccharides which contains a lower concentration of solids as compared to the feedstock slurry, and a wet cake which contains a higher concentration of solids as compared to the feedstock slurry. In addition, a third stream containing oil may be generated. As such, a number of product streams may be generated by using different separation techniques or a combination thereof. As an example, feedstock slurry 16 may be separated using a three-phase centrifuge. A three-phase centrifuge allows for three-phase separation yielding two liquid phases (e.g., aqueous stream and oil stream) and a solid stream (e.g., solids or wet cake) (see, e.g., Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany). The two liquid phases may be separated and decanted, for example, from a bowl via two discharge systems to prevent cross-contamination and the solids stream may be removed via a separate discharge system.

[0088] In some embodiments using corn as feedstock 12, a three-phase centrifuge may be used to remove solids and corn oil simultaneously from feedstock slurry 16 (e.g., liquefied corn mash). The solids are the undissolved solids remaining after the starch is hydrolyzed to soluble oligosaccharides during liquefaction, and the corn oil is free oil that is released from the germ during grinding and/or liquefaction. In some embodiments, the three-phase centrifuge may have one feed stream and three outlet streams. The feed stream may consist of liquefied corn mash produced during liquefaction. The mash may consist of an aqueous solution of liquefied starch (e.g., oligosaccharides); undissolved solids which consist of insoluble, non-starch components from the corn; and corn oil which consists of glycerides and free fatty acids. The three outlet streams from the three-phase centrifuge may be a wet cake (i.e., wet cake 24) which contains the undissolved solids from the mash; a heavy centrate stream which contains the liquefied starch from the mash; and a light centrate stream which contains the corn oil from the mash. In some embodiments, the light centrate stream (i.e., oil 26) may be conducted to a storage tank or any vessel that is suitable for oil storage. In some embodiments, the oil may be sold as a co-product, converted to another co-product, or used in processing such as the case in converting corn oil to corn oil fatty acids. In some embodiments, the heavy centrate stream (i.e., aqueous solution 22) may be used for fermentation. In some embodiments, the wet cake may be washed with process recycle water, such as evaporator condensate and/or backset as described herein, to recover the soluble starch in the liquid phase of the cake.

[0089] In some embodiments, wet cake 24 is a composition formed from feedstock slurry 16, and may comprise at least about 50% by weight of the undissolved solids present in the feedstock slurry, at least about 55% by weight of the undissolved solids present in the feedstock slurry, at least about 60% by weight of the undissolved solids present in the feedstock slurry, at least about 65% by weight of the undissolved solids present in the feedstock slurry, at least about 70% by weight of the undissolved solids present in the feedstock slurry, at least about 75% by weight of the undissolved solids present in the feedstock slurry, at least about 80% by weight of the undissolved solids present in the feedstock slurry, at least about 85% by weight of the undissolved solids present in the feedstock slurry, at least about 90% by weight of the undissolved solids present in the feedstock slurry, at least about 95% by weight of the undissolved solids present in the feedstock slurry, or at least about 99% by weight of the undissolved solids present in the feedstock slurry.

[0090] In some embodiments, aqueous solution 22 formed from feedstock slurry 16, and may comprise no more than about 50% by weight of the undissolved solids present in the feedstock slurry, no more than about 45% by weight of the undissolved solids present in the feedstock slurry, no more than about 40% by weight of the undissolved solids present in the feedstock slurry, no more than about 35% by weight of the undissolved solids present in the feedstock slurry, no more than about 30% by weight of the undissolved solids present in the feedstock slurry, no more than about 25% by weight of the undissolved solids present in the feedstock slurry, no more than about 20% by weight of the undissolved solids present in the feedstock slurry, no more than about 15% by weight of the undissolved solids present in the feedstock slurry, no more than about 10% by weight of the undissolved solids present in the feedstock slurry, no more than about 5% by weight of the undissolved solids present in the feedstock slurry, or about 1% by weight of the undissolved solids present in the feedstock slurry.

[0091] Fermentation 30 configured to ferment aqueous solution 22 to produce a fermentation product such as a product alcohol has an inlet for receiving aqueous solution 22. Fermentation 30 may include fermentation broth. In some embodiments, microorganism 32 selected from the group of bacteria, cyanobacteria, filamentous fungi, and yeasts may be introduced to fermentation 30 to be included in the fermentation broth. In some embodiments, microorganism 32 may be bacteria such as Escherichia coli. In some embodiments, microorganism 32 may be Saccharomyces cerevisiae. In some embodiments, microorganism 32 consumes the sugar in aqueous solution 22 and produces butanol. In some embodiments, microorganism 32 may be a recombinant microorganism.

[0092] In some embodiments, microorganism 32 may be engineered to contain a biosynthetic pathway. In some embodiments, the biosynthetic pathway may be a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway may be a 1-butanol biosynthetic pathway, a 2-butanol biosynthetic pathway, or an isobutanol biosynthetic pathway. In some embodiments, the biosynthetic pathway converts pyruvate to a fermentation product. In some embodiments, the biosynthetic pathway converts pyruvate as well as amino acids to a fermentation product. In some embodiments, the biosynthetic pathway comprises at least one heterologous polynucleotide encoding a polypeptide which catalyzes a substrate to product conversion of the biosynthetic pathway. In some embodiments, each substrate to product conversion of the biosynthetic pathway is catalyzed by a polypeptide encoded by a heterologous polynucleotide. Examples of the production of a product alcohol by a microorganism comprising a biosynthetic pathway are disclosed, for example, in U.S. Pat. No. 7,851,188, and U.S. Patent Application Publication Nos. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; and 2009/0305370, the entire contents of each are herein incorporated by reference.

[0093] In some embodiments, microorganism 32 may also be immobilized, such as by adsorption, covalent bonding, crosslinking, entrapment, and encapsulation. Methods for encapsulating cells are known in the art such as in U.S. Patent Application Publication No. 2011/0306116, the entire contents of which are herein incorporated by reference.

[0094] In some embodiments of the processes and systems described herein, in situ product removal (ISPR) may be utilized to remove a fermentation product such as a product alcohol (e.g., butanol) from fermentation broth. In some embodiments, ISPR may be conducted in fermentation 30 as the fermentation product is produced by the microorganism, or external to fermentation 30, using, for example, by liquid-liquid extraction. Methods for producing and recovering product alcohols from a fermentation broth using extractive fermentation are described in U.S. Patent Application Publication No. 2009/0305370; U.S. Patent Application Publication No. 2010/0221802; U.S. Patent Application Publication No. 2011/0097773; U.S. Patent Application Publication No. 2011/0312044; U.S. Patent Application Publication No. 2011/0312043; and U.S. Patent Application Publication No. 2012/0156738; the entire contents of each are herein incorporated by reference.

[0095] In some embodiments, fermentation 30 may have an inlet for receiving extractant 34. In some embodiments, extractant 34 may be added to the fermentation broth external to fermentation 30. In some embodiments, extractant 34 may be added to an external extractor or external extraction loop. Alternative means of additions of extraction 34 to fermentation 30 or external to fermentation 30 are represented by the dotted lines. In some embodiments, extractant 34 may be immiscible organic solvents. In some embodiments, extractant 34 may be water-immiscible organic solvents. In some embodiments, extractant 34 may be an organic extractant selected from the group consisting of saturated, monounsaturated, polyunsaturated compounds, and mixtures thereof. In some embodiments, extractant 34 may be selected from the group consisting of C.sub.12 to C.sub.22 fatty alcohols, C.sub.12 to C.sub.22 fatty acids, esters of C.sub.12 to C.sub.22 fatty acids, C.sub.12 to C.sub.22 fatty aldehydes, C.sub.12 to C.sub.22 fatty amides, and mixtures thereof. In some embodiments, extractant 34 may also be selected from the group consisting of C.sub.4 to C.sub.22 fatty alcohols, C.sub.4 to C.sub.28 fatty acids, esters of C.sub.4 to C.sub.28 fatty acids, C.sub.4 to C.sub.22 fatty aldehydes, and mixtures thereof. In some embodiments, extractant 34 may include a first extractant selected from C.sub.12 to C.sub.22 fatty alcohols, C.sub.12 to C.sub.22 fatty acids, esters of C.sub.12 to C.sub.22 fatty acids, C.sub.12 to C.sub.22 fatty aldehydes, C.sub.12 to C.sub.22 fatty amides, and mixtures thereof; and a second extractant selected from C.sub.7 to C.sub.11 fatty alcohols, C.sub.7 to C.sub.11 fatty acids, esters of C.sub.7 to C.sub.11 fatty acids, C.sub.7 to C.sub.11 fatty aldehydes, and mixtures thereof. In some embodiments, extractant 34 may be carboxylic acids. In some embodiments, extractant 34 may be corn oil fatty acids (COFA) or soybean oil fatty acids (SOFA). In some embodiments, extractant 34 may be an organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol, 1-nonanal, 1-undecanol, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof. For the processes and systems described herein and as illustrated in the figures, extractant may be added to the fermentor or an external extractor.

[0096] In some embodiments, the extractant may be selected based upon certain properties. For example, the extractant may have a high K.sub.d. K.sub.a refers to the partition coefficient of the fermentation product (e.g., product alcohol) between the extractant phase (e.g., organic phase) and aqueous phase. In some embodiments, the extractant may have a high selectivity. For example, selectivity refers to the relative amounts of product alcohol to water taken up by the extractant.

[0097] In some embodiments, the extractant may be biocompatible. In some embodiments, biocompatible refers to a measure of the ability of a microorganism to utilize fermentable carbon sources in the presence of an extractant. In some embodiments, the extractant may be a mixture of biocompatible and non-biocompatible extractants. In some embodiments, a non-biocompatible extractant refers to an extractant that interferes with the ability of a microorganism to utilize fermentable carbon sources. For example, in the presence of a non-biocompatible extractant, the microorganism does not utilize fermentable carbon sources at a rate greater than about 50% of the rate when the extractant is not present. In some embodiments, in the presence of a non-biocompatible extractant, the microorganism does not utilize fermentable carbon sources at a rate greater than about 25% of the rate when the extractant is not present. Examples of mixtures of biocompatible and non-biocompatible extractants include, but are not limited to, oleyl alcohol and nonanol, oleyl alcohol and 1-undecanol, oleyl alcohol and 2-undecanol, oleyl alcohol and 1-nonanal, oleyl alcohol and decanol, and oleyl alcohol and dodecanol. Additional examples of biocompatible and non-biocompatible extractants are described in U.S. Patent Application Publication No. 2011/0097773; the entire contents of which are herein incorporated by reference.

[0098] Extractant 34 contacts the fermentation broth forming stream 36 comprising a biphasic mixture (i.e., aqueous phase and organic phase). In the case that the fermentation product is a product alcohol, product alcohol present in the fermentation broth is transferred to extractant 34 forming extractant rich with product alcohol (e.g., organic phase). In some embodiments, stream 36 may be discharged through an outlet in fermentation 30. Product alcohol may be separated from the extractant in stream 36 using conventional techniques. Feed stream may be added to fermentation 30. Fermentation 30 can be any suitable fermentor known in the art.

[0099] In some embodiments, where extractant 34 is not added to the fermentation broth, stream 36 comprises fermentation broth and product alcohol. Stream 36 or a portion thereof comprising product alcohol and fermentation broth may be discharged from fermentation 30 and further processed for recovery of product alcohol. In some embodiments, fermentation broth may be recycled to fermentation 30.

[0100] In some embodiments, simultaneous saccharification and fermentation (SSF) may occur in fermentation 30. Any known saccharification process utilized by the industry may be used including, but not limited to, an acid process, an enzyme process, or an acid-enzyme process. In some embodiments, enzyme 38 such as glucoamylase, may be introduced to hydrolyze sugars (e.g., oligosaccharides) in feedstock slurry 16 or aqueous solution 22 to form monosaccharides. Examples of glucoamylases that may be used in the processes and systems of the present invention are described in U.S. Pat. No. 7,413,887; U.S. Pat. No. 7,723,079; U.S. Patent Application Publication No. 2009/0275080; U.S. Patent Application Publication No. 2010/0267114; U.S. Patent Application Publication No. 2011/0014681; U.S. Patent Application Publication No. 2011/0020899, the entire contents of each are herein incorporated by reference. In some embodiments, the glucoamylase may be expressed by a recombinant microorganism that also produces the fermentation product (e.g., product alcohol).

[0101] In some embodiments, enzymes such as glucoamylases may be added to liquefaction. The addition of enzymes such as glucoamylases to liquefaction may reduce the viscosity of the feedstock slurry or liquefied mash, and may improve separation efficiency. In some embodiments, any enzyme capable of reducing the viscosity of the feedstock slurry may be used (e.g., Viscozyme.RTM., Sigma-Aldrich, St. Louis, Mo.). Viscosity of the feedstock may be measured by any method known in the art, including the method described in Example 22.

[0102] In some embodiments, stream 35 may be discharged from an outlet in fermentation 30. The discharged stream 35 may include microorganism 32 such as yeast. Microorganism 32 may be separated from the stream 35, for example, by centrifugation (not shown). Microorganism 32 may then be recycled to fermentation 30 which over time can increase the production rate of product alcohol, thereby resulting in an increase in the efficiency of product alcohol production.

[0103] When a portion of stream 35 exits fermentation 30, stream 35 may include no more than about 50% by weight of the undissolved solids present in the feedstock slurry, no more than about 45% by weight of the undissolved solids present in the feedstock slurry, no more than about 40% by weight of the undissolved solids present in the feedstock slurry, no more than about 35% by weight of the undissolved solids present in the feedstock slurry, no more than about 30% by weight of the undissolved solids present in the feedstock slurry, no more than about 25% by weight of the undissolved solids present in the feedstock slurry, no more than about 20% by weight of the undissolved solids present in the feedstock slurry, no more than about 15% by weight of the undissolved solids present in the feedstock slurry, no more than about 10% by weight of the undissolved solids present in the feedstock slurry, no more than about 5% by weight of the undissolved solids present in the feedstock slurry, or no more than about 1% by weight of the undissolved solids present in the feedstock slurry.

[0104] In some embodiments, as shown, for example, in FIG. 2, the process and system of the present invention may include mill 40 configured to dry mill feedstock 12. Feedstock 12 may enter mill 40 through an inlet. Mill 40 can mill or grind feedstock 12. In some embodiments, feedstock 12 may be unfractionated. In some embodiments, feedstock 12 may be unfractionated corn kernels. Mill 40 may be any suitable known mill, for example, a hammer mill. Dry milled feedstock 44 is discharged from mill 40 through an outlet and enters liquefaction 10. The remainder of FIG. 2 is similar to FIG. 1, and therefore will not be described in detail again. In other embodiments, the feedstock may be fractionated and/or wet milled as is known in the industry as an alternative to being unfractionated and/or dry milled.

[0105] Wet milling is a multi-step process that separates biomass into several components such as germ, pericarp fiber, starch, and gluten in order to capture value from each co-product separately. Using corn as a feedstock, this process produces several co-products: starch, gluten feed, gluten meal, and corn oil streams. These streams may be recombined and processed to produce customized products for the feed industry. As an example of a wet milling process, feedstock (e.g., corn) may be conducted to steeping tanks where it is soaked, for example, in a sodium dioxide solution for about 30-50 hours at about 120-130.degree. F. (about 50-55.degree. C.). Nutrients released into the water may be collected and evaporated to produce condensed fermented extractives (or steep liquor). Germ may be removed from the soaked feedstock and further processed to recover oil and germ meal. After removal of the germ, the remaining portion of feedstock may be processed to remove bran and to produce a starch and gluten slurry. The slurry may be further processed to separate the starch and gluten protein which may be dried to form gluten meal. The starch stream may be further processed via fermentation to produce a fermentation product (e.g., product alcohol) or may be utilized by the food, paper, or textile industries. For example, the starch stream may be used to produce sweeteners. The gluten meal and gluten feed stream which both contain protein, fat, and fiber, may be used in feeds for dairy and beef cattle, poultry, swine, livestock, equine, aquaculture, and domestic pets. Gluten feed may also be used as a carrier for added micronutrients. Gluten meal also contains methionine and xanthophylls which may be used a pigment ingredient in, for example, poultry feeds (e.g., pigment provides egg yolks with yellow pigmentation). Condensed fermented extractives which contain protein, growth factors, B vitamins, and minerals may be used as a high energy liquid feed ingredient. Condensed extractives may also be used as a pellet binder. This process provides a purified starch stream; however, it is costly and includes the separation of the biomass into its non-starch components which may not be necessary for fermentation production.

[0106] Fractionation removes fiber and germ, which contains a majority of the lipids (e.g., oil) present in ground whole corn resulting in a fractionated corn that has a higher starch (endosperm) content. In some embodiments, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more of the oil may be removed from the germ by fractionation. In some embodiments, fractionation may reduce the undissolved solids content of the feedstock to at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, or at least about 5% of the feedstock.

[0107] Dry fractionation does not separate the germ from fiber and therefore, it is less expensive than wet milling. The benefits of fractionation may include, for example, improved yield of product, increased volume (i.e., space) in the fermentor, smaller column diameters, lower enzyme loadings and increased efficiency for saccharification, improved oil removal, decreased equipment clogging due to the presence of oil, fewer cleaning shutdowns, increased protein levels in DDGS, reduced drying time for DDGS, and reduced energy consumption. However, fractionation does not remove the entirety of the fiber or germ, and does not result in total elimination of solids. Furthermore, there is some loss of starch in fractionation.

[0108] Dry milling may also be utilized for feedstock processing. Feedstock may be milled, for example, using a hammermill to generate a meal that may then be mixed with water to form a slurry. The slurry may be subjected to liquefaction by the addition of enzymes such as amylases to hydrolyze starch to sugars, forming a mash. The mash may be heated ("cooked") to inactivate the enzyme and then cooled for addition to fermentation. Cooled mash, microorganism, and enzyme such as glucoamylase may be added to fermentation for the production of fermentation product (e.g., product alcohol). Following fermentation, the fermentation broth may be conducted to distillation for recovery of the fermentation product. If the undissolved solids have not been removed, the bottoms stream of the distillation column is whole stillage containing unfermented solids (e.g., distillers grain solids), dissolved materials, and water which may be collected for further processing. For example, the whole stillage may be separated into solids (e.g., wet cake) and thin stillage. Separation may be accomplished by a number of means including, but not limited to, centrifugation, filtration, screen separation, hydrocyclone, or any other means or separation device for separating liquids from solids. Thin stillage may be conducted to evaporation forming condensed distillers solubles (CDS) or syrup. Thin stillage may comprise soluble nutrients, small grain solids (or fine particles), and microorganisms. The solids (e.g., wet cake) may be combined with syrup and then dried to form DDGS. Syrup contains protein, fat, and fiber as well as vitamins and minerals such as phosphorus and potassium; and may be added to animal feeds for its nutritional value and palatability. DDGS contains protein, fat, and fiber; and provides a source of bypass proteins. DDGS may be used in animal feeds for dairy and beef cattle, poultry, swine, livestock, equine, aquaculture, and domestic pets.

[0109] In some embodiments, as shown, for example, in FIG. 3, the processes and systems of the present invention may include discharging oil 26 from an outlet of separation 20. FIG. 3 is similar to FIG. 1, except for oil stream 26 exiting separation 20, and therefore will not be described in detail again.

[0110] Feedstock slurry 16, conducted to separation 20, may be separated into a first liquid phase or aqueous solution 22 containing a fermentable sugar, a second liquid phase containing oil 26, and a solid phase or wet cake 24 containing undissolved solid. Any suitable separation device can be used to discharge aqueous solution 22 (or aqueous stream), wet cake 24 (or solid stream), and oil 26 (or oil stream), for example, a three-phase centrifuge. In some embodiments, feedstock 12 is corn and oil 26 is corn oil (e.g., free corn oil). The term free corn oil as used herein means corn oil that is freed from the corn germ. In some embodiments, oil 26 may be conducted to a storage tank or any vessel that is suitable for oil storage. In some embodiments, a portion of oil from feedstock 12 such as corn oil when the feedstock is corn, remains in wet cake 24.

[0111] In some embodiments, when oil 26 is removed via separation 20 from feedstock 12, the fermentation broth in fermentation 30 includes a reduced amount of corn oil. In some embodiments, the fermentation broth has no more than about 25% by weight of undissolved solids, the fermentation broth has no more than about 15% by weight of undissolved solids, the fermentation broth has no more than about 10% by weight of undissolved solids, the fermentation broth has no more than about 5% by weight of undissolved solids, the fermentation broth has no more than about 1% by weight of undissolved solids, or the fermentation broth has no more than about 0.5% by weight of undissolved solids.

[0112] In some embodiments, the process and system of FIG. 2 may be modified to include discharge of an oil stream from separation 20 as discussed herein in connection to the process and system of FIG. 3.

[0113] As illustrated in FIG. 4, if oil is not discharged separately, it may be removed with wet cake 24. When wet cake 24 is separated via separation 20, in some embodiments, a portion of the oil from feedstock 12, such as corn oil when the feedstock is corn, remains in wet cake 24. Wet cake 24 may be conducted to mix 60 and combined with water or other solvents forming wet cake mixture 65. In some embodiments, water may be fresh water, backset, cook water, process water, lutter water, evaporation water, or any water source available in the fermentation processing facility, or any combination thereof. Wet cake mixture 65 may be conducted to separation 70 producing wash centrate 75 comprising fermentable sugars recovered from wet cake 24, and wet cake 74. Wash centrate 75 may be recycled to liquefaction 10. The remainder of FIG. 4 is similar to FIG. 1, and therefore will not be described in detail again.

[0114] In some embodiments, separation 70 may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

[0115] In some embodiments, wet cake may be subjected to one or more wash cycles or wash systems. For example, wet cake 74 may be further processed by conducting wet cake 74 to a second wash system. In some embodiments, wet cake 74 may be conducted to a second mix 60' forming wet cake mixture 65'. Wet cake mixture 65' may be conducted to a second separation 70' producing wash centrate 75' and wet cake 74'. Wash centrate 75' may be recycled to liquefaction 10 and/or wash centrate 75' may be combined with wash centrate 75 and the combined wash centrates may be recycled to liquefaction 10. Wet cake 74' may be combined with wet cake 74 for further processing as described herein. In some embodiments, separation 70' may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, the wet cake may be subjected to one, two, three, four, five, or more wash cycles or wash systems.

[0116] Wet cake 74 may be combined with solubles and then dried to form DDGS through any suitable known process. The formation of the DDGS from wet cake 74 has several benefits. For example, since the undissolved solids are not added to the fermentor, the undissolved solids are not subjected to the conditions of the fermentor and therefore, the undissolved solids do not contact the microorganisms present in the fermentor, and fermentation product such as product alcohol or other components such as extractant are not trapped in the undissolved solids. These effects provide benefits to subsequent processing and use of DDGS, for example, as animal feed because the DDGS would not contain microorganism or other components (e.g., product alcohol, extractant) of the fermentation broth.

[0117] As shown in FIG. 4, oil is not discharged separately from the wet cake, but rather oil is included as part of the wet cake and is ultimately present in the DDGS. If corn is utilized as feedstock, corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, and phospholipids, which provide a source of metabolizable energy for animals. The presence of oil in the wet cake and ultimately DDGS may provide a desirable animal feed, for example, a high fat content animal feed.

[0118] In some embodiments, oil may be separated from the DDGS and converted to an ISPR extractant for subsequent use in the same or different fermentation processes. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043, U.S. Patent Application Publication No. 2011/0312044, U.S. Patent Application Publication No. 2012/0156738, and PCT International Publication No. WO 2011/159998; the entire contents of each are herein incorporated by reference. Oil may be separated from DDGS using any suitable known process including, for example, a solvent extraction process. In one embodiment of the invention, DDGS are loaded into an extraction vessel and washed with a solvent such as hexane to remove oil. Other solvents that may be utilized include, for example, isobutanol, isohexane, ethanol, petroleum distillates such as petroleum ether, or mixtures thereof. After oil extraction, DDGS may be treated to remove any residual solvent. For example, DDGS may be heated to vaporize any residual solvent using any method known in the art. Following solvent removal, DDGS may be subjected to a drying process to remove any residual water. The processed DDGS may be used as a feed supplement for animals such as dairy and beef cattle, poultry, swine, livestock, equine, aquaculture, and domestic pets.

[0119] After extraction from DDGS, the resulting oil and solvent mixture may be collected for separation of oil from the solvent. In one embodiment, the oil/solvent mixture may be processed by evaporation whereby the solvent is evaporated and may be collected and recycled. The recovered oil may be converted to an ISPR extractant for subsequent use in the same or different fermentation processes.

[0120] Removal of the oil component of the feedstock is advantageous to production because oil present in the fermentor may be hydrolyzed to fatty acids and glycerin. Glycerin can accumulate in water and reduce the amount of water that is available for recycling throughout the fermentation system. Thus, removal of the oil component of feedstock increases the efficiency of production by increasing the amount of water that can be recycled through the system. In addition, removing oil can result in energy savings for the production plant due to more efficient fermentation, less fouling due to the removal of the oil, increased fermentor volume efficiency, and decreased energy requirements, for example, the energy needed to dry distillers grains.

[0121] As illustrated in FIG. 5, oil may be removed at various points during the processes described herein. Feedstock slurry 16 may be separated, for example, using a three-phase centrifuge, into a first liquid phase or aqueous solution 22 (or aqueous stream), a second liquid phase comprising oil 26 (or oil stream), and a solid phase or wet cake 24 (or solid stream). Wet cake 24 may be further processed to recover fermentable sugars and oil. Wet cake 24 may be conducted to mix 60 and combined with water or other solvents forming wet cake mixture 65. In some embodiments, water may be backset, cook water, process water, lutter water, water collected from evaporation, or any water source available in the fermentation processing facility, or any combination thereof. Wet cake mixture 65 may be conducted to separation 70 (e.g., three-phase centrifuge) producing wash centrate 75 comprising fermentable sugars, oil 76, and wet cake 74. Wash centrate 75 may be recycled to liquefaction 10. Oil 76 and oil 26 may be combined and further processed for the manufacture of various consumer products. In some embodiments, the oil (e.g., oil 26, oil 76) may be further processed to generate extractant. For example, the oil may be treated chemically or enzymatically to generate extractant. In some embodiments where the oil is corn oil, the corn oil may be treated chemically or enzymatically to generated fatty acids (e.g., corn oil fatty acids) that may be used as extractant. In some embodiments where the oil is treated enzymatically, the enzymatic reaction may be subjected to a treatment (e.g., heat) post conversion to deactivate the enzyme. In some embodiments, the oil may be enzymatically treated utilizing enzymes such as esterases, lipases, phospholipases, lysophospholipases, or combinations thereof. In some embodiments, the oil may be chemically treated with ammonium hydroxide, anhydrous ammonia, ammonium acetate, hydrogen peroxide, toluene, glacial acetic acid, or combinations thereof. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each are herein incorporated by reference. In some embodiments where the oil is corn oil, the feedstock slurry may comprise at least about 1 wt %, at least about 2 wt %, at least about 3 wt %, at least about 4 wt % corn oil, or at least about 5 wt % corn oil. The remainder of FIG. 5 is similar to FIG. 1, and therefore will not be described in detail again.

[0122] As described herein, wet cake may be subjected to one or more wash cycles or wash systems. In some embodiments, wet cake 74 may be conducted to a second mix 60' forming wet cake mixture 65'. Wet cake mixture 65' may be conducted to a second separation 70' producing wash centrate 75', oil 76', and wet cake 74'. Wash centrate 75' may be recycled to liquefaction 10 and/or wash centrate 75' may be combined with wash centrate 75 and the combined wash centrates may be recycled to liquefaction 10. Wet cake 74' may be combined with wet cake 74 for further processing as described herein. Oil 76, oil 76', and oil 26 may be combined and further processed for the manufacture of various consumer products. In some embodiments, oil (e.g., oil 26, oil 76, oil 76') may be further processed to generate extractant as described herein. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each are herein incorporated by reference.

[0123] As illustrated in FIG. 6, aqueous solution 22 and wet cake 24 may be combined, cooled, and conducted to fermentation 30. Feedstock slurry 16 may be separated, for example, using a three-phase centrifuge, into a first liquid phase or aqueous solution 22, a second liquid phase comprising oil 26, and a solid phase or wet cake 24. In some embodiments, oil 26 (or oil stream) may be conducted to a storage tank or any vessel that is suitable for oil storage. Aqueous solution 22 (or aqueous stream) and wet cake 24 (solid stream) may be conducted to mix 80 and re-slurried forming aqueous solution/wet cake mixture 82. Mixture 82 may be conducted to cooler 90 producing cooled mixture 92 which may be conducted to fermentation 30. In some embodiments, when oil 26 is removed via separation 20 from feedstock 12, mixtures 82 and 92 include a reduced amount of corn oil. The remainder of FIG. 6 is similar to FIG. 1, and therefore will not be described in detail again.

[0124] In some embodiments, as shown, for example, in FIGS. 7 and 8, saccharification may occur in a separate saccharification system 50 which is located between separation 20 and fermentation 30 (FIG. 7) or between liquefaction 10 and separation 20 (FIG. 8). FIGS. 7 and 8 are similar to FIG. 1 except for the inclusion of a separate saccharification 50 and fermentation 30 does not receive enzyme 38. In some embodiments, enzyme 38 may also be added to fermentation 30.

[0125] Any known saccharification processes utilized by the industry may be used including, but not limited to, an acid process, an enzyme process, or an acid-enzyme process. Saccharification 50 may be conducted in any suitable saccharification vessel. In some embodiments, enzyme 38 such as glucoamylase, may be introduced to hydrolyze sugars (e.g., oligosaccharides) in feedstock slurry 16 or aqueous solution 22 to form monosaccharides. For example, in FIG. 7, oligosaccharides present in aqueous solution 22 discharged from separation 20 and conducted to saccharification 50 through an inlet are hydrolyzed to monosaccharides. Aqueous solution 52 containing monosaccharides is discharged from saccharification 50 through an outlet and conducted to fermentation 30. Alternatively, as shown in FIG. 8, oligosaccharides present in feedstock slurry 16 discharged from liquefaction 10 and conducted to saccharification 50 through an inlet are hydrolyzed to monosaccharides. Feedstock slurry 54 containing monosaccharides is discharged from saccharification 50 through an outlet and conducted to separation 20.

[0126] In some embodiments, the system and processes of FIGS. 1-6 may be modified to include a separate saccharification system as discussed herein in connection to the systems and processes of FIGS. 7 and 8.

[0127] In some embodiments, as shown, for example, in FIGS. 9A-9D, the systems and processes of the present invention may include a series of two or more separation devices. FIGS. 9A-9D are similar to FIG. 1, except for the addition of separation systems, and therefore will not be described in detail again.

[0128] Aqueous solution 22 discharged from separation 20 may be conducted to separation 20'. Separation 20' may be identical to separation 20 or may be different to separation 20, and may operate in the same manner. Separation 20' may remove undissolved solids and oil not separated from aqueous solution 22 to generate (i) aqueous solution 22' similar to aqueous solution 22, but containing reduced amounts of undissolved solids and oil in comparison to aqueous solution 22, (ii) wet cake 24' similar to wet cake 24, and (iii) oil 26' similar to oil 26. Aqueous solution 22' may then be introduced to fermentation 30. In some embodiments, there may be one or more additional separation devices following separation 20'.

[0129] In some embodiments, separation 20' may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

[0130] In some embodiments, the systems and processes of FIGS. 1-9 may be modified to include additional separation devices for removing undissolved solids as discussed herein in connection to the systems and processes described herein.

[0131] In some embodiments, stream 35 may be discharged from an outlet in fermentation 30. The absence or minimization of the undissolved solids exiting fermentation 30 via stream 35 has several additional benefits. For example, the need for units and operations in downstream processing may be decreased or eliminated, for example, beer columns or distillation columns, thereby resulting in an increased efficiency for production. Also, some or all of the whole stillage centrifuges may be eliminated as a result of less undissolved solids in the stream exiting the fermentor.

[0132] Referring to FIG. 9B, aqueous solution 22 discharged from separation 20 may be conducted to separation 20'. Separation 20' may be identical to separation 20 or may be different to separation 20. Separation 20' may operate in a manner which could include separation additive 28 such as an extractant or flocculant. Separation additive 28 may aid in the removal of oil or solids. Separation 20' may remove undissolved solids and oil not separated from aqueous solution 22 to generate (i) aqueous solution 22' similar to aqueous solution 22, but containing reduced amounts of undissolved solids and oil in comparison to aqueous solution 22, and (ii) stream 23 which may be similar to a combined stream of oil 26 and wet cake 24, and also contain separation additive 28. Stream 23 may be conducted to separation 20'' and may generate a stream that contains separation additive 28', and wet cake 24'. Separation 20'' may be identical to separation 20 and separation 20' or may be different to separation 20 and separation 20'. Aqueous solution 22' may be introduced to fermentation 30.

[0133] In some embodiments, separation 20, separation 20', and separation 20'' may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

[0134] Referring to FIG. 9C, feedstock slurry 16 may be discharged from liquefaction 10 and conducted to separation 20. Feedstock slurry 16 may be separated to generate streams: (i) aqueous solution 22, (ii) wet cake 24, and (iii) stream 25 comprising oil, solids, and an aqueous stream comprising a fermentable carbon source. In some embodiments, the solids of stream 25 may be light solids. In some embodiments, light solids may be solids that are less dense than water but more dense than oil. In some embodiments, light solids may be coated in oil, resulting in solids that are less dense than water. In some embodiments, solids may have lipophilic and/or hydrophilic properties. In some embodiments, the solids of stream 25 may comprise one or more of the following: germ, fiber, starch, and gluten. In some embodiments, the solids of stream 25 may comprise fine particles. In some embodiments, the solids of stream 25 comprise germ, gluten, and fiber. In some embodiments, aqueous solution 22 comprising a fermentable carbon source may be conducted to fermentation 30 for production of a fermentation product as described herein. In some embodiments, wet cake 24 may be further processed as described herein, for example, processed to form DDGS.

[0135] Stream 25 discharged from separation 20 may be conducted to separation 20'. Separation 20' may be identical to separation 20 or may be different from separation 20. In some embodiments, separation 20 and separation 20' may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, separation may be a single step process.

[0136] Stream 25 may be separated by separation 20' to generate streams: (i) aqueous solution 22', (ii) wet cake 24', and (iii) oil 26. In some embodiments, aqueous solution 22' may be combined with aqueous solution 22, and the combined aqueous solution may be conducted to fermentation 30. In some embodiments, the amount of solids in aqueous solution 22' is reduced compared to the amount of solids in aqueous solution 22. In some embodiments, aqueous solution 22' may comprise oil and the oil in aqueous solution 22' may be further processed to generate an extractant. For example, aqueous solution 22' may be treated chemically or enzymatically to generate an extractant. In some embodiments, aqueous solution 22' may be treated chemically or enzymatically to generate fatty acids (e.g., corn oil fatty acids) that may be used as an extractant. In some embodiments where aqueous solution 22' is treated enzymatically, the enzymatic reaction may be subjected to a treatment (e.g., heat) post conversion to deactivate the enzyme. Converting the oil in aqueous solution 22' which has a relatively small stream volume to fatty acids may reduce the capital cost of generating extractant via enzymatic or chemical conversion. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each are herein incorporated by reference.

[0137] In some embodiments, wet cake 24' may be combined with wet cake 24, and the combined wet cake may be further processed as described herein. In some embodiments, wet cake 24' may comprise oil and this oil-rich wet cake may be combined with wet cake 24 to produce a wet cake with increased fat content (e.g., increased triglyceride content) which would provide a metabolizable energy source for animal feed. In some embodiments, this wet cake with increased fat content may be combined with syrup to generate a high triglyceride, high protein, low carbohydrate DDGS. In some embodiments, wet cake 24' comprising oil may be further processed as described herein, for example, to produce DDGS.

[0138] In some embodiments, oil 26 may be conducted to a storage tank or any vessel that is suitable for oil storage. In some embodiments, oil 26 or a portion thereof may be combined with feedstock slurry 16 (dotted line, FIG. 9C). The addition of oil to the feedstock slurry may improve solids removal via stream 25 by increasing the amount of solids captured.

[0139] In some embodiments, oil 26 may be further processed to generate extractant as described herein. Converting oil 26, which has a relatively small stream volume and would have a reduced flow rate compared to feedstock slurry 16, may reduce the capital cost as well as energy requirements of generating extractant via enzymatic or chemical conversion. In some embodiments, the flow rate of oil 26 may be about 1% to about 10% of the flow rate of feedstock slurry 16. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each are herein incorporated by reference.

[0140] In some embodiments, stream 25 may be generated by adjusting one or more parameters of the separation device. For example, stream 25 may be generated by adjusting the weir (or dip weir) of a centrifuge such as a decanter centrifuge or three-phase centrifuge.

[0141] As described herein, solids may interfere with liquid-liquid extraction and therefore, utilizing an extraction method may not be technically or economically viable. During the extraction process, a rag layer may form at the interface of the aqueous and organic phases, and the rag layer, composed of solids (e.g., light solids), can accumulate and possibly interfere with phase separation. To mitigate the formation of the rag layer, removal of solids via stream 25 prior to fermentation may eliminate the formation of the rag layer and thereby improve downstream processing of the fermentation broth and recovery of fermentation products.

[0142] In another embodiment to mitigate the formation of the rag layer, oil may be added to the aqueous solution as a means to selectively capture solids that form the rag layer. Referring to FIG. 9D, feedstock slurry 16 may be discharged from liquefaction 10 and conducted to separation 20. Feedstock slurry 16 may be separated to generate streams: (i) aqueous solution 22, (ii) wet cake 24, and (iii) oil 26. In some embodiments, wet cake 24 may be further processed as described herein, for example, processed to form DDGS. In some embodiments, oil may be added to aqueous solution 22 and the resulting mixture may be conducted to vessel 80 where the mixture settles or separates forming (i) oil layer comprising solids 86 and (ii) aqueous solution 22'. In some embodiments, aqueous solution 22' may be conducted to fermentation 30 for production of a fermentation product as described herein. In some embodiments, the amount of solids in aqueous solution 22' is reduced compared to the amount of solids in aqueous solution 22. In some embodiments, solids-rich oil layer 86 may be removed (e.g., skimmed from the mixture) and filtered to remove the solids from the oil layer. In some embodiments, solids-rich oil layer 86 may be conducted to separation 20' and may be separated to generate streams: (i) aqueous solution 22'', (ii) wet cake 24', and (iii) oil 26' (a solids-lean oil). In some embodiments, the oil added to aqueous solution 22 may be the oil 26 separated from feedstock slurry 16, oil 26', and/or an external oil source.

[0143] In some embodiments, wet cake 24' may be combined with wet cake 24, and the combined wet cake may be further processed as described herein. In some embodiments, wet cake 24' may comprise oil and this oil-rich wet cake may be combined with wet cake 24 to produce a wet cake with increased fat content and may be further processed as described herein. In some embodiments, wet cake 24' comprising oil may be further processed as described herein.

[0144] In some embodiments, aqueous solution 22'' may be combined with aqueous solution 22', and the combined aqueous solution may be conducted to fermentation 30. The amount of solids in this combined aqueous solution would be reduced compared to aqueous solution 22, and with reduced solids, the formation of the rag layer would be mitigated. In some embodiments, aqueous solution 22' and aqueous solution 22'' may comprise oil and the oil may be further processed to generate an extractant. For example, aqueous solution 22' and aqueous solution 22'' may be combined and may be treated chemically or enzymatically (dotted line in FIG. 9D) to generate an extractant as described herein. Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043 and U.S. Patent Application Publication No. 2011/0312044, the entire contents of each are herein incorporated by reference.

[0145] In some embodiments, separation 20 and separation 20' may be any separation device capable of separating solids and liquids including, for example, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. In some embodiments, separation may be a single step process. In some embodiments, vessel 80 may be a static mixer, mixer-settler, decanter, gravity settler, and combinations thereof. In some embodiments, the process may be maintained at temperatures to minimize contamination (e.g., 70-110.degree. C.).

[0146] In some embodiments, as shown, for example, in FIG. 10, the systems and processes of the present invention may include means for on-line, in-line, at-line, and/or real-time measurements (circles represent measurement devices and dotted lines represent feedback loops). FIG. 10 is similar to FIG. 5, except for the addition of measurement devices for on-line, in-line, at-line, and/or real-time measurements, and therefore will not be described in detail again.

[0147] The processes described herein may be integrated fermentation processes using on-line, in-line, at-line, and/or real-time measurements, for example, of concentrations and other physical properties of the various streams generated during fermentation (e.g., feedstock slurry, aqueous solution, oil stream, wet cake, wet cake mixtures, wash centrate, etc.). These measurements may be used, for example, in feed-back loops to adjust and control the conditions of the fermentation and/or the conditions of the fermentors, liquefaction units, separation units, and mixing units. In some embodiments, the concentration of fermentation products and/or other metabolites and substrates in the fermentation broth may be measured using any suitable measurement device for on-line, in-line, at-line, and/or real-time measurements. In some embodiments, the measurement device may be one or more of the following: Fourier transform infrared spectroscope (FTIR), near-infrared spectroscope (NIR), Raman spectroscope, high pressure liquid chromatography (HPLC), viscometer, densitometer, tensiometer, droplet size analyzer, particle analyzers, pH meter, dissolved oxygen (DO) probe, and the like. In some embodiments, off-gas venting from the fermentor may be analyzed, for example, by an in-line mass spectrometer. Measuring off-gas venting from the fermentor may be used as a means to identify species present in the fermentation reaction. The concentration of fermentation products and other metabolites and substrates may also be measured using the techniques and devices described herein.

[0148] In some embodiments, measured inputs may be sent to a controller and/or control system, and conditions within the fermentor (temperature, pH, nutrients, enzyme and/or substrate concentration), liquefaction units, separation units, and mixing units may be varied to maintain a concentration or concentration profile of the various streams. By utilizing such a control system, process parameters may be maintained in such a way to improve overall plant productivity and economic goals. In some embodiments, real-time control of fermentation may be achieved by variation of concentrations of components (e.g., biomass, sugars, enzymes, nutrients, microorganisms, and the like) in the fermentors, liquefaction units, separation units, and mixing units. In some embodiments, automated systems may be used to adjust separation and mixing conditions, flow rates to and from the separation and mixing operations, solids and oil removal, and sugar and starch recovery.

[0149] During the fermentation processes, it is possible for units of operation to perform at sub-optimal levels over time. It may be necessary to adjust flow rates, mixing rates, equipment settings, and the like for operations such as liquefaction and separation in order to maintain overall plant productivity. The processes and systems described herein may be integrated using on-line, in-line, at-line, and/or real-time measurements for monitoring the concentrations and other physical properties of fermentation streams such as feedstock slurry 16, aqueous solution 22, oil 26, and wet cake 24. These measurements may be used, for example, in feed-back loops to adjust and control the conditions of fermentation, separation of feedstock slurry, and wash cycle performance. By utilizing on-line, in-line, at-line, and/or real-time measurements, immediate feedback and adjustments of process conditions may be made, resulting in an overall improved fermentation process. For example, the amount of fermentable carbon source (e.g., starch, sugars), oil, and solids may be monitored in feedstock slurry 16, aqueous solution 22, and streams 65 and 75 using, for example, FTIR or NIR. By monitoring these parameters, enzyme concentrations and residence time for liquefaction and saccharification may be adjusted to improve preparation of feedstock slurry 16 and separation and mixing conditions may be adjusted to, for example, increase the amount of fermentable carbon source, oil, and/or solids in aqueous solution 22 and streams 65 and 75.

[0150] As another example, washing wet cake 24 allows for recovery of sugars, starch, and oil in the wet cake, minimizing the yield loss of these fermentable carbon sources and oil. By monitoring moisture, sugar, starch, and oil content of streams 24, 65, 74, and 75, the washing performance may be adjusted to improve sugar, starch, and oil recovery. For example, real-time measurement of sugar, starch, and oil content of these streams may be performed by FTIR and NIR, and these measurements allow for immediate feedback and adjustment of mixing (60) and separation (20, 70) conditions. Differential speed, feed rate, bowl speed, scroll differential speed, impeller position, weir position, scroll pitch, residence time, and discharge volume of a separation device may be adjusted to modify the moisture, sugar, starch, and oil content of streams 24, 65, 74, and 75. Mixing (60) conditions such as pump rate or agitator speed may also be adjusted to modify the moisture, sugar, starch, and oil content of streams 24, 65, 74, and 75. In addition, the wash ratio (e.g., ratio between water and wet cake) may also be adjusted to modify the moisture, sugar, starch, and oil content of streams 24, 65, and 74. FTIR measurements and droplet imaging may be used to monitor the water content in oil streams (26, 76). In addition, color and turbidity of oil streams (26, 76) may be monitored to assess the quality of the oil. This real-time measurement would allow for adjustment to separation (20, 70) conditions resulting in a cleaner oil stream (e.g., less water).

[0151] In another embodiment of the processes and systems described herein, moisture content of wet cake 24 may be monitored using real-time measurements. Real-time measurement of moisture content of the wet cake may be performed by NIR, and these measurements allow for immediate feedback and adjustment of separation (20, 70) conditions. By decreasing the water content of the wet cake, less energy is needed to dry the wet cake and therefore, overall energy usage may be improved for the production process. In addition, a lower water content of the wet cake may result in improved starch recovery.

[0152] As another example of process control strategy using real-time measurements, solids in aqueous solution 22 and oil 26, 76 may be measured using particle size analysis such as a process particle analyzer (JM Canty, Inc., Buffalo, N.Y.), focused beam reflectance measurement (FBRM.RTM.), or particle vision and measurement (PVM.RTM.) technologies (Mettler-Toledo, LLC, Columbus Ohio). By monitoring solids in real time, process steps may be adjusted to improve solids removal and thereby, minimize the amount of solids in the aqueous solution and oil streams, maximize the recovery of solids, and improve the overall fermentation process including downstream processing.

[0153] The processes and systems disclosed in FIGS. 1-10 include removing undissolved solids and/or oil from feedstock slurry 16 and as a result, improving the processing productivity and cost effectiveness. The improved productivity can include increased efficiency of fermentation product production and/or increased extraction activity relative to processes and systems that do not remove undissolved solids and/or oil prior to fermentation.

[0154] An exemplary fermentation process of the present invention including downstream processing is described in FIG. 11. Some processes and streams in FIG. 11 have been identified using the same name and numbering as used in FIGS. 1-10 and represent the same or similar processes and streams as described in FIGS. 1-10.

[0155] Feedstock 12 may be processed and undissolved solids and/or oil separated (100) as described herein with reference to FIGS. 1-10. Briefly, feedstock 12 may be liquefied to generate feedstock slurry comprising undissolved solids, fermentable carbon sources, and oil. For example, milled grain and one or more enzymes may be combined to generate a feedstock slurry. This feedstock slurry may be heated (or cooked), liquefied, and/or flashed with flash vapor producing a "cooked" feedstock slurry or mash. In some embodiments, the feedstock slurry may be heated to at least about 100.degree. C. In some embodiments, the feedstock slurry may be heated for about thirty minutes. In some embodiments, the feedstock slurry may be subjected to raw starch hydrolysis (also known as cold cooking or cold hydrolysis). In the raw starch hydrolysis process, the heating (or cooking) step is eliminated, and the elimination of this step reduces energy consumption and steam load (e.g., water consumption). In some embodiments, liquefaction and/or saccharification may be conducted at fermentation temperatures (e.g., about 30.degree. C. to about 55.degree. C.). In some embodiments, liquefaction and/or saccharification may be conducted utilizing raw starch enzymes or low temperature hydrolysis enzymes such as Stargen.TM. (Genencor International, Palo Alto, Calif.) and BPX.TM. (Novozymes, Franklinton, N.C.). In some embodiments, liquefaction and/or saccharification may be conducted at temperatures less than about 50.degree. C.

[0156] The feedstock slurry may then be subjected to separation to remove undissolved solids, generating wet cake 24, oil 26, and aqueous solution 22 (or centrate) comprising fermentable carbon source, for example, dissolved fermentable sugars. Separation may be accomplished by a number of means including, but not limited to, decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, membrane filtration, cross flow filtration, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof. This separation step may remove at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the undissolved solids from the feedstock slurry. In some embodiments, aqueous solution 22 may comprise at least about 0.5%, at least about 1%, or at least about 2% undissolved solids.

[0157] Wet cake 24 may be re-slurried or washed with water and subjected to separation to remove additional fermentable sugars, generating washed wet cake (e.g., 74, 74' as described in FIGS. 1-10). In some embodiments, about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, or about 15% fermentable sugars may be recovered from the washed wet cake. The wash process may be repeated a number of times, for example, one, two, three, four, five, or more times. The water used to re-slurry or wash the wet cake may be recycled water generated during the fermentation process (e.g., backset, cook water, process water, lutter water, evaporation water). In some embodiments, the wet cake may be re-slurried or washed with beer. The wash centrates (e.g., 75, 75' as described in FIG. 4 and FIG. 5) produced by the wash/separation process may be returned to the mix step to form a slurry with the milled grain or used in the liquefaction process. In some embodiments, the wash centrates may be heated or cooled prior to the mix step.

[0158] Aqueous solution 22 may be further processed as described herein. For example, aqueous solution 22 may be heated with steam or process-to-process heat exchange. A saccharification enzyme may be added to aqueous solution 22 and the dissolved fermentable sugars of aqueous solution 22 may be partially or completely saccharified. The saccharified aqueous solution 22 may be cooled by a number of means such as process-to-process exchange, exchange with cooling water, or exchange with chilled water.

[0159] Aqueous solution 22 and microorganism 32 may be added to fermentation 30 where the fermentable sugars are metabolized by microorganism 32 to produce stream 105 comprising fermentation products (e.g., product alcohol). In some embodiments, microorganism 32 may be a recombinant microorganism capable of producing product alcohol such as 1-butanol, 2-butanol, or isobutanol. In some embodiments, ammonia and recycle streams may also be added to fermentation 30. In some embodiments, the process may include at least one fermentor, at least two fermentors, at least three fermentors, at least four fermentors, at least five fermentors, or more fermentors. In some embodiments, carbon dioxide generated during fermentation may be vented to a scrubber in order to reduce air emissions (e.g., alcohol air emissions) and to increase product yield.

[0160] Stream 105 comprising product alcohol may be conducted to beer column 120 to produce alcohol-rich stream 122 and bottoms stream 125. Alcohol-rich stream 122 may be sent to alcohol recovery 160 for recovery of product alcohol. Product alcohol may be recovered from alcohol-rich stream 122 using methods known in the art including, but not limited to, distillation, adsorption (e.g., by resins), separation by molecular sieves, pervaporation, gas stripping, extraction, and the like. Bottoms stream 125 comprising thin stillage, with most of the solids removed prior to fermentation may be concentrated by evaporation via evaporation 130 to form syrup 135. Syrup 135 may be combined with wet cake (24, 74, 74' as described herein) in mixer 140, and the combined stream 145 of wet cake and syrup may then be dried in a dryer 150 to produce DDGS.

[0161] In some embodiments, stream 105 may be degassed. In some embodiments, stream 105 may be heated before degassing, for example, by process-to-process exchange with hot mash. In some embodiments, vapors may be vented to a condenser and then, to a scrubber. Degassed stream 105 may be heated further, for example, by process-to-process heat exchange with other streams in the distillation and/or alcohol recovery area.

[0162] In another embodiment of FIG. 11, aqueous solution 22, microorganism 32, and extractant may be added to fermentation 30 to produce a biphasic stream. In some embodiments, extractant may be added to fermentation 30 via a recycled loop. In some embodiments, extractant may be added downstream of fermentation 30 or external to fermentation 30. A stream comprising fermentation broth and fermentation product (e.g., product alcohol) may be conducted to an external extractor to produce a stream comprising product alcohol and a bottoms stream. In some embodiments, the stream comprising product alcohol may be conducted to alcohol recovery 160 for recovery of the product alcohol. In some embodiments, the bottoms stream may be conducted to a separation device to separate the bottoms stream into thin stillage and extractant. In some embodiments, the recovered extractant may be recycled for extraction of product alcohol. The thin stillage, with most of the solids removed prior to fermentation, may be concentrated by evaporation 130 to form a syrup. The syrup may be combined with wet cake (24, 74, 74' as described herein) in mixer 140, and the combined stream 145 of wet cake and syrup may then be dried in a dryer 150 to produce DDGS.

[0163] In some embodiments, aqueous solution 22, microorganism 32, and extractant may be added to fermentation 30 to form a single liquid phase stream. In some embodiments, the biphasic stream or single liquid phase stream may be withdrawn batchwise from fermentation 30 or may be withdraw continually from fermentation 30. In some embodiments, extractant may be added downstream of fermentation 30 or external to fermentation 30 to form a single liquid phase stream. In some embodiments, extractant may be added to an external extractor to form a single liquid phase stream.

[0164] An exemplary process for alcohol recovery is described herein, and additional methods for recovering product alcohols from fermentation broth are described in U.S. Patent Application Publication No. 2009/0305370; U.S. Patent Application Publication No. 2010/0221802; U.S. Patent Application Publication No. 2011/0097773; U.S. Patent Application Publication No. 2011/0312044; U.S. Patent Application Publication No. 2011/0312043; U.S. Patent Application Publication No. 2012/0035398; U.S. Patent Application Publication No. 2012/0156738; PCT International Publication No. WO 2011/159998; and PCT International Publication No. WO 2012/030374; the entire contents of each are herein incorporated by reference. For example, vacuum vaporization may be used to recover product alcohol from the fermentation broth. Preheated beer (e.g., aqueous stream 22) and solvent (e.g., extractant) may enter a preflash column which may be a retrofit of a beer column in a conventional dry grind fuel ethanol plant. This column may be operated at sub-atmospheric pressure, driven by water vapor taken from an evaporator train or from the mash cook step. The overheads of the preflash column may be condensed by heat exchange with some combination of cooling water and process-to-process heat exchange including heat exchange with the preflash column feed. The liquid condensate may be directed to an alcohol/water decanter.

[0165] The preflash column bottoms may be advanced to a solvent decanter. The preflash column bottoms may be substantially stripped of product alcohol. The decanter may be a still well, a centrifuge, or a hydrocyclone. Water may be separated from the solvent phase in this decanter, generating a water phase. The water phase including suspended and dissolved solids may be centrifuged to produce a wet cake and thin stillage. The wet cake may be combined with other streams and dried to produce DDGS, it may be dried and sold separate from other streams which produce DDGS, or it may be sold as a wet cake. The water phase may be split to provide a backset which is used in part to re-slurry the wet cake described herein. The split also provides thin stillage which may be pumped to evaporators for further processing.

[0166] The organic phase produced in the solvent decanter may be an ester of an alcohol. The solvent may be hydrolyzed to regenerate reactive solvent and to recover additional alcohol. Alternatively, the organic phase may be filtered and sold as a product. Hydrolysis may be thermal driven, homogeneously catalyzed, or heterogeneously catalyzed. The heat input to this process may be a fired heater, hot oil, electrical heat input, or high pressure steam. Water added to drive the hydrolysis may be from a recycled water stream, fresh water, or steam.

[0167] Cooled hydrolyzed solvent may be pumped into a sub-atmospheric solvent column where it may be substantially stripped of product alcohol with steam. This steam may be water vapor from evaporators, it may be steam from the flash step of the mash process, or it may be steam from a boiler (see, e.g., U.S. Patent Application Publication No. 2009/0171129, the entire contents of which are herein incorporated by reference). A rectifier column from a conventional dry grind ethanol plant may be suitable as a solvent column. The rectifier column may be modified to serve as a solvent column. The bottoms of the solvent column may be cooled, for example, by cooling water or process-to-process heat exchange. The cooled bottoms may be decanted to remove residual water and this water may be recycled to other steps of the process or recycled to liquefaction.

[0168] The solvent column overheads may be cooled by exchange with cooling water or by process-to-process heat exchange, and the condensate may be directed to a vented alcohol/water decanter which may be shared with the preflash column overheads. Other mixed water and product alcohol streams may be added to this decanter including the scrubber bottoms and condensate from the degas step. The vent which comprises carbon dioxide, may be directed to a water scrubber. The aqueous layer of this decanter may also be fed to the solvent column or may be stripped of product alcohol in a small dedicated distillation column. The aqueous layer may be preheated by process-to-process exchange with the preflash column overheads, solvent column overheads, or solvent column bottoms. This dedicated column may be modified from the side stripper of a conventional dry grind fuel ethanol process.

[0169] The organic layer of the alcohol/water decanter may be pumped to an alcohol column. This column may be a super-atmospheric column and may be driven by steam condensation within a reboiler. The feed to the column may be heated by process-to-process heat exchange in order to reduce the energy demand to operate the column. This process-to-process heat exchanger may include a partial condenser of the preflash column, a partial condenser of a solvent column, the product of the hydrolyzer, water vapor from the evaporators, or the alcohol column bottoms. The condensate of the alcohol column vapor may be cooled and may be returned to the alcohol/water decanter. The alcohol column bottoms may be cooled by process-to-process heat exchange including exchange with the alcohol column feed and may be further cooled with cooling water, filtered, and sold as product alcohol.

[0170] Thin stillage generated from the preflash column bottoms as described herein may be directed to a multiple effect evaporator (see e.g., U.S. Patent Application Publication No. 2011/0315541, the entire contents of which are herein incorporated by reference). This evaporator may have two, three, or more stages. The evaporator may have a configuration of four bodies by two effects similar to the conventional design of a fuel ethanol plant, it may have three bodies by three effects, or it may have other configurations. Thin stillage may enter at any of the effects. At least one of the first effect bodies may be heated with vapor from the super-atmospheric alcohol column. The vapor may be taken from the lowest pressure effect to provide heat in the form of water vapor to the sub-atmospheric preflash column and solvent column. Syrup from the evaporators may be added to the distillers grain dryer.

[0171] Carbon dioxide emissions from the fermentor, degasser, alcohol/water decanter, and other sources may be directed to a water scrubber. The water supplied to the top of this scrubber may be fresh water or may be recycled water. The recycled water may be treated (e.g., biologically digested) to remove volatile organic compounds and may be chilled. Scrubber bottoms may be sent to the alcohol/water decanter, to the solvent column, or may be used with other recycled water to re-slurry the wet cake described herein. Condensate from the evaporators may be treated with anaerobic biological digestion or other processes to purify the water before recycling to re-slurry the wet cakes.

[0172] Oil may be separated from the process streams at any of several points. For example, a centrifuge may be operated to produce an oil stream following filtration of cooked mash or the preflash column water phase centrifuge may be operated to produce an oil stream. Intermediate concentration syrup or final syrup may be centrifuged to produce an oil stream.

[0173] In another embodiment, the multi-phase material may leave the bottom of the preflash column and may be processed in a separation system as described herein. The concentrated solids may be redispersed in the aqueous stream and this combined stream may be used to re-pulp and pump the low starch solids that were separated and washed from liquefied mash.

[0174] In another exemplary process for product alcohol recovery, an extractant may be utilized to remove the product alcohol from the fermentation broth during fermentation to maintain the product alcohol in the fermentation broth below a certain concentration. In some embodiments, product alcohol removal may be achieved by esterification with carboxylic acid in the presence of a catalyst to produce alcohol esters. A description of processes and systems for extracting alcohol by formation of alcohol esters may be found in U.S. Patent Application Publication No. 2012/0156738, the entire contents of which are herein incorporated by reference. For example, oil separated from the feedstock slurry may be hydrolyzed by a catalyst such as an esterase (e.g., lipase) converting the triglycerides in the oil to fatty acids such as carboxylic acids. These fatty acids may be used an extractant for the recovery of the product alcohol. In some embodiments, the hydrolysis of the oil may occur in the fermentor by the addition of a catalyst to the fermentor. In some embodiments, the hydrolysis of the oil may occur in a separate vessel, and the fatty acids may be added to the fermentor. For example, the feedstock slurry may be conducted to a vessel or tank, and an esterase such as lipase may be added to the vessel, converting the oil present in the feedstock slurry to fatty acids. The feedstock slurry comprising fatty acids may be conducted to the fermentor.

[0175] The product alcohol produced by fermentation may react with the fatty acids to produce alcohol esters. In some embodiments, these alcohol esters may be extracted from the fermentation broth. For example, the fermentation broth comprising the alcohol esters may be transferred to a separation device such as a three-phase centrifuge to separate the fermentation broth into three streams: undissolved solids (including microorganism), aqueous stream, and organic stream comprising alcohol esters. In some embodiments, the fermentation broth may be separated into two streams: undissolved solids (including microorganism) and a biphasic mixture comprising an aqueous phase and an organic phase. This separation of the fermentation broth may occur continuously during fermentation, for example, by removing a portion of the fermentation broth for separation, or in batch mode, for example, the entire contents of the fermentor may be removed for separation.

[0176] In some embodiments, the biphasic mixture may be separated into an alcohol ester-containing organic phase and aqueous phase and this separation may be achieved using any methods known in the art including, but not limited to, siphoning, aspiration, decantation, centrifugation, gravity settler, membrane-assisted phase splitting, hydrocyclone, and the like. The alcohol ester-containing organic phase may be further processed to recover product alcohol. For example, the alcohol ester-containing organic phase may be transferred to a vessel, where the alcohol esters may be hydrolyzed in the presence of a catalyst to form product alcohol and fatty acids, and this mixture of product alcohol and fatty acids may be processed by distillation to separate the product alcohol and fatty acids.

[0177] In some embodiments, the fatty acids may be recycled to the fermentor or an extractor column. In some embodiments, the aqueous stream and undissolved solids may be recycled to the fermentor.

[0178] In some embodiments, extraction of the product alcohol may occur downstream of the fermentor or external to the fermentor. In some embodiments, the fermentation system may include an external extraction system that includes, for example, a mixing device and a separation system. Fermentation broth may be conducted to the mixing device, and extractant may be added to the mixing device and combined with fermentation broth to produce a biphasic mixture. The biphasic mixture may be introduced to a separation system, in which separation of biphasic mixture produces an alcohol-containing organic phase and an aqueous phase. In some embodiments, the aqueous phase or a portion thereof may be returned to the fermentor. In some embodiments, the alcohol-containing organic phase may be conducted to an extractant column. The biomass processing productivity in these embodiments is substantially improved by the separation of biomass feed stream components after liquefaction but prior to fermentation. In particular, decreasing the amount of undissolved solids and/or oil provides increased efficiency of external alcohol extraction systems.

[0179] In some embodiments, oil may be separated from the feedstock or feedstock slurry and may be stored in an oil storage vessel. For example, oil may be separated from the feedstock or feedstock slurry using any suitable means for separation including three-phase centrifugation or mechanical extraction. To improve the removal of oil from the feedstock or feedstock slurry, oil extraction aids such surfactants, anti-emulsifiers, or flocculants as well as enzymes may be utilized. Examples of oil extraction aids include, but are not limited to, non-polymeric, liquid surfactants; talcum powder; microtalcum powder; enzymes such as Pectinex.RTM. Ultra SP-L, Celluclast.RTM., and Viscozyme.RTM. L (Sigma-Aldrich, St. Louis, Mo.), and NZ 33095 (Novozymes, Franklinton, N.C.); salt (NaOH); and calcium carbonate. Another means to improve oil removal may be pH adjustments such as raising or lowering the pH. Additional benefits for oil removal include increased oil yield, improved oil quality, reduced system deposition, and reduced downtime. In addition, oil removal may also result in cleaner, higher-quality oil.

[0180] The remaining feedstock or feedstock slurry may then be further treated to remove any residual oil. For example, the feedstock or feedstock slurry after oil separation may be conducted to a vessel or tank and a catalyst such as an esterase (e.g., lipase) may be added to the vessel, converting the oil present in the feedstock or feedstock slurry to fatty acids. Removing oil from the feedstock or feedstock slurry may improve enzyme efficiencies as well as reduce the amount of enzyme needed for the processes described herein. The feedstock or feedstock slurry may then be conducted to a fermentor and microorganisms may also be added to the fermentor for the production of product alcohol. In some embodiments, the catalyst may be deactivated, for example, by heating. In some embodiments, deactivation may be conducted in a separate vessel, for example, a deactivation vessel. The deactivated feedstock or feedstock slurry may be conducted to a fermentor and microorganisms may also be added to the fermentor for production of product alcohol. Removing oil from the feedstock or feedstock slurry by converting the oil to fatty acids can result in energy savings for the production plant due to more efficient fermentation, less fouling due to the removal of the oil, and decreased energy requirements, for example, the energy needed to dry distillers grains. Following fermentation, the fermentation broth comprising product alcohol may be conducted to an external vessel, for example, an external extractor or external extraction loop for the recovery of product alcohol. Removal of oil as presented here differs from known techniques in that embodiments of the present invention separate oil from the feedstock slurry such that stable emulsions are less likely to occur by virtue of the feed stream separation process, and the need for the addition of protic solvents to break emulsions formed with recovery entrapped bio-oil after fermentation is less likely (see e.g., U.S. Pat. No. 7,601,858; U.S. Pat. No. 8,192,627). In some embodiments of the present invention, an emulsion may form, but is readily broken by mechanical processing or by other conventional means.

[0181] If extractants are used to recover product alcohol, removing oil prior to the fermentation process can reduce the amount of oil taken up by the extractant and thus extend the effectiveness of the extractant for recovering product alcohol. Oil taken up by the extractant can reduce the K.sub.a as well as the selectivity of the extractant, and in turn can increase the operating costs of the production process. As the extractant may be recycled in the production process, each fermentation cycle exposes the extractant to more oil which is taken up by the extractant and over time can result in a significant decrease in the K.sub.d and selectivity of the extractant. The processes and systems described herein provide a means to maintain the K.sub.d and selectivity of the extractant by removing oil from the feedstock or feedstock slurry and/or converting the oil to fatty acids by the addition of a catalyst.

[0182] The processes and systems described herein can lead to increased extraction activity and/or efficiency in fermentation product (e.g., product alcohol) production as a result of the removal of the undissolved solids. For example, extractive fermentation without the presence of the undissolved solids can lead to increased mass transfer rates of the product alcohol from the fermentation broth to the extractant, better phase separation, and lower hold-up of extractant as a result of increased extractant droplet rise velocities. Also, for example, extractant droplets held up in the fermentation broth during fermentation will disengage from the fermentation broth faster and more completely, thereby resulting in less free extractant in the fermentation broth. In addition, lower hold-up of extractant can decrease the amount of extractant lost in the process. Additional benefits of solids removal include, for example, elimination of agitators in the fermentor and downstream processing equipment such as beer columns and centrifuges resulting in a reduction of capital costs and energy use; increased fermentor volume resulting in increased fermentor productivity; decreased extractant hold-up resulting in increased production efficiency, increased recovery and recycling of extractant, reduced flow rate of extractant which will lower operating costs, and the potential to use continuous fermentation or smaller fermentors. In some embodiments, the volume of the fermentor available for the fermentation may be increased by at least about 5%, at least about 10%, or more.

[0183] Examples of increased extraction efficiency include, for example, stabilization of the partition coefficient, enhanced phase separation, enhanced mass transfer coefficient, operation at a lower titer, increased process stream recyclability, increased fermentation volume efficiency, increased feedstock load feeding, increased product alcohol titer tolerance of the microorganism, water recycling, reduction in energy, increased recycling of extractant, and recycling of the microorganism. For example, because oil in the fermentation broth can be reduced by removing solids from feedstock slurry prior to fermentation, the extractant is exposed to less oil which can combine with the extractant and lower the partition coefficient of the extractant. Therefore, a reduction of oil in the fermentation broth results in a more stable partition coefficient over multiple fermentation cycles. In some embodiments, the partition coefficient may be decreased by less than about 10%, less than about 5%, or less than about 1% over ten or more fermentation cycles. As another example of increased extraction efficiency, a higher mass transfer rate (e.g., in the form of a higher mass transfer coefficient) can result in an increased efficiency of product alcohol production. In some embodiments, the mass transfer coefficient may be increased at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold.

[0184] An increase in phase separation between fermentation broth and extractant reduces the likelihood of emulsion formation resulting in an increased efficiency of product alcohol production. For example, in the absence of an emulsion, phase separation can occur more quickly and can be more complete. In some embodiments, phase separation may occur where previously no appreciable phase separation was observed. In some embodiments, phase separation may occur in 24 hours. In some embodiments, phase separation may occur at least about two times (2.times.) as quickly, at least about five times (5.times.) as quickly, or at least about ten times (10.times.) as quickly as compared to phase separation where solids have not been removed or emulsions have formed.

[0185] As described herein, nutrients such as nitrogen, minerals, trace elements, and/or vitamins may be added to the feedstock slurry or the fermentor. These added nutrients as well as nutrients naturally occurring in the feedstock may be soluble in oil, and thus the presence of oil in the feedstock or feedstock slurry may reduce the concentrations of these nutrients in the fermentation broth. Removing oil from the feedstock or feedstock slurry can minimize the loss of nutrients. In addition, the presence of solids in the fermentation broth may also lead to a reduction in the concentrations of nutrients. Removing the solids and/or oil from the feedstock or feedstock slurry can minimize the loss of nutrients.

[0186] For the processes and systems described herein, the presence of oil in the fermentation process may have an effect on the partition coefficient of the extractant over the course of multiple fermentations. Removing oil from the feedstock or feedstock slurry can reduce the variability of the partition coefficient of the extractant over the course of multiple fermentations, and therefore improve the scalability of the processes and systems described herein. Scalability refers to the ability to modify (e.g., expand or condense) a process or system to accommodate, for example, manufacturing demands (e.g., operating volume) without a penalty in functionality.

[0187] In addition, removing solids from feedstock or feedstock slurry can also have an effect on scalability. For example, if an external extractor is utilized, reduced solids (e.g., reduced total suspended solids, TSS) can results in improved performance and better scalability. Reduced solids enhance the rate of mass transfer of product alcohol between the aqueous phase and organic phase (e.g., fermentation broth and extractant). Solid particles coat the surface of the extractant droplets effectively reducing the area for mass transfer. Solid particles also inhibit phase separation by increasing viscosity and the tendency for emulsification. Since the presence of solids can impede the operation of the external extractor, removing solids from the feedstock or feedstock slurry provides for improved scalability and reliability of an external extractor.

[0188] Therefore, removing solids and/or oil may improve the scalability of the unit operations of processes and systems described herein. For example, removing solids and/or oil may improve unit operations such as, but not limited to, extractor performance, distillation column performance, heat exchanger performance, and/or evaporator performance.

[0189] As an example of improved unit operations, referring to FIG. 11, stream 105 may be conducted to beer column 120 to produce alcohol-rich stream 122 and bottoms stream 125. Bottoms stream 125 comprising thin stillage, with most of the solids removed prior to fermentation may be concentrated via evaporation 130. By removing solids prior to fermentation, less solids may be sent to the evaporators which can result in a lower feed rate to the evaporators. A lower feed rate requires less energy and therefore, lower costs due to lower energy requirements.

[0190] In some embodiments, there may be a need to remove water from the oil recovered from feedstock or feedstock slurry. In some embodiments, water may be removed by a number of methods including gravity separation, coalescing separator, centrifugation (e.g. decanter), adsorption or absorption, distillation, heating, vacuum dehydration, and/or air stripping (e.g., air, nitrogen). Examples of adsorption media include, but are not limited to, activated alumina, bentonite clay, calcium chloride, calcium sulfate, cellulose, magnesium sulfate, molecular sieve, polymers, and/or silica gel. In some embodiments, the adsorption media may be continuously stirred with the oil, or the oil may flow through a packed bed with adsorption media.

[0191] From time to time, it may be necessary to clean and/or sterilize the equipment used in the production of the fermentation products such as product alcohols. Examples of equipment include, but are not limited to, fermentors, liquefaction vessels, saccharification vessels, holding tanks, storage tanks, heat exchangers, pipelines, equipment connections, nozzles, fittings, and valves. Cleaning and sterilization can reduce or eliminate microbial contamination as well as minimize the accumulation of residues (e.g., carbohydrates, sugars) on equipment. Residue build-up on equipment can provide a nutritional source for unwanted microorganisms, leading to the proliferation of these microorganisms. There are a number of methods utilized to clean and/or sterilize fermentation equipment including clean-in-place (CIP) and sterilization-in-place (SIP). CIP and SIP may be performed manually or automated systems are also available. A suitable as well as efficient CIP or SIP can maximize the profitability of the production plant by minimizing the need to shut down operations due to, for example, a microbial contamination.

[0192] In the processes and systems described herein, removal of solids and oil from feedstock or feedstock slurry, for example, prior to fermentation can improve the efficiency of CIP and SIP. The lack of solids in the fermentation equipment would allow for less rinse water, less cleaning solution, and less time to perform CIP and SIP. Thus, removal of solids may improve the efficiency and reduce the costs of CIP and SIP processes. Also, the caustic agents used for CIP (e.g., sodium hydroxide) may react with the triglycerides in oil forming soap (i.e., saponification) which can have an effect on the efficiency of CIP. Therefore, the removal of oil can reduce the formation of soap during CIP and improve the efficiency of CIP.

[0193] During the fermentative process, fouling can have an impact on the productivity and efficiency of the production process. In general, fouling refers to the deposit of extraneous materials or particles, for example, the deposit of materials on the surface of heat exchangers and distillation column reboilers. This deposit of materials on the surface of the heat exchanger can interfere with the transfer of heat and reduce the operational capability of the heat exchanger. For example, the material deposit may interfere with the flow of fluid through the exchanger resulting in an increase in flow resistance. Also, the deposit of material on the surface of heat exchangers or other equipment may require additional cleaning. These additional cleaning requirements may necessitate plant shutdowns which can result in a reduction in plant productivity. In the processes and systems described herein, removal of oil and/or solids from the feedstock or feedstock slurry, for example, prior to fermentation can minimize the deposition of materials and lower the rate of deposition of materials on the surfaces of equipment such as heat exchangers. Thus, solids and oil removal can lower the rate of fouling of the heat exchangers and minimize the effect of fouling on heat transfer and operational capability.

[0194] In another example of an embodiment of the processes and systems of the invention, the material discharged from the fermentor may be processed in a separation system that involves devices such as a centrifuge, settler, hydrocyclone, etc., and combinations thereof to effect the recovery of microorganisms in a concentrated form that may be recycled for reuse in a subsequent fermentations either directly or following re-conditioning. The ability to recycle microorganisms can increase the overall rate of fermentation product production such as product alcohol production, lower the overall titer requirement, and/or lower the aqueous titer requirement, thereby leading to healthier microorganisms and a higher production rate. This separation system may also produce an organic stream that comprises fermentation product (e.g., product alcohol) and other by-products produced from the fermentation, and an aqueous stream containing only trace levels of immiscible organics. This aqueous stream may be used either before or after it is stripped of product alcohol content to wash the solids that were separated from feedstock slurry. This has the advantage of avoiding what might otherwise be a long belt-driven conveying system to transfer these solids from the liquefaction area to the grain drying and syrup blend area. Furthermore, whole stillage produced after product alcohol has been stripped will need to be separated into thin stillage and wet cake fractions either using existing or new separation devices. The thin stillage may form in part the backset that may be combined with cook water for preparing a new batch of fermentable mash. Another advantage of this embodiment is that any residual fermentable sugars that were retained in the solids separated from feedstock slurry would in part be captured and recovered through this backset. Alternatively, microorganisms contained in the solids stream may be redispersed in the aqueous stream and this combined stream distilled of any product alcohol content remaining from fermentation. If the microorganisms are nonviable, the non-viable microorganisms may further be separated for use as a nutrient, for example, in a propagation process.

[0195] In some embodiments of the processes and systems described herein, by-products (or co-products) of the fermentation process may be further processed, for example, undissolved solids may be processed to generate DDGS. Other by-products such as fatty acid esters which may have an inhibitory effect on the microorganisms may be recovered from the fermentation broth and/or by-product streams resulting in an increase in the yield of product alcohol. Recovery of fatty acid esters or other lipids may be accomplished by using a solvent to extract fatty acid esters from the by-product streams. In some embodiments, several by-product streams may be combined and fatty acid esters may be recovered from the combined streams.

[0196] In an embodiment of solvent extraction of lipids (e.g., fatty acid esters), solids may be separated from whole stillage ("separated solids") since this stream would contain a large portion of fatty acid esters. These separated solids may then be fed to an extractor and washed with solvent. In some embodiments, the separated solids may be washed at least two or more times. After washing, the resulting mixture of lipid and solvent, known as miscella, may be collected for separation of the extracted lipid from the solvent. For example, the resulting mixture of lipid and solvent may be conducted to a separator or extractor for further processing. During the extraction process, the solvent not only extracts lipid into solution, but it also collects fine particles ("fines"). These fines are generally undesirable impurities in the miscella and in one embodiment, the miscella may be discharged from the separator or extractor and conducted to a separation device that separates or scrubs the fines from the miscella.

[0197] In order to separate lipid and solvent contained in the miscella, the miscella may be subjected to a distillation step. In this step, the miscella can, for example, be processed through an evaporator which heats the miscella to a temperature that is high enough to cause vaporization of the solvent, but is not sufficiently high to adversely affect or vaporize the extracted lipid. As the solvent evaporates, it may be collected, for example, in a condenser, and recycled for future use. Separation of the solvent from the miscella results in a stock of crude lipid which may be further processed to separate water, fatty acid esters (e.g., fatty acid isobutyl esters), fatty acids, and triglycerides. A solvent-based extraction system for recovering triglycerides is described in U.S. Patent Application Publication No. 2010/0092603, the entire contents of which are herein incorporated by reference.

[0198] After extraction of the lipids, the solids may be conveyed from the extractor and may be conducted to a stripping device (e.g., desolventizer) to remove residual solvent. Recovery of residual solvent can be important to process economics. In some embodiments, the solids may be conveyed to a desolventizer in a vapor tight environment to preserve and collect solvent that may transiently evaporate from the solids. As the solids enter the desolventizer, the solids may be heated to vaporize and remove the residual solvent. In order to heat the solids, the desolventizer may include a mechanism for distributing the solids over one or more trays, and the solids may be heated directly such as through direct contact with heated air or steam, or indirectly such as by heating the tray carrying the solids. In order to facilitate transfer of the solids from one tray to another, the trays carrying the solids may include openings that allow the solids to pass from one tray to the next tray. From the desolventizer, the solids may optionally be conveyed to a mixer where the solids are mixed with other by-products before being conveyed to a dryer. In some embodiments, the solids are conducted to a desolventizer and the solids are contacted by steam. In some embodiments, the flows of steam and solids in the desolventizer may be countercurrent. In some embodiments, vapor exiting the desolventizer may be condensed, optionally mixed with miscella, and then fed to a decanter forming a water-rich phase. This water-rich phase exiting the decanter may be fed to a distillation column where solvent is removed from the water-rich stream. In some embodiments, a solvent-depleted water-rich stream may exit the bottom of the distillation column and may be recycled to the fermentation process, for example, it may be used to process the feedstock. In some embodiments, overhead and bottom products of the distillation column may be recycled to the fermentation process. For example, the lipid-rich bottoms may be added to the feed of a hydrolyzer. The overheads may be, for example, condensed and fed to a decanter forming a solvent-rich stream and a water-rich phase. The solvent-rich stream exiting this decanter may optionally be used as the solvent feed to an extractor, and the water-rich phase exiting this decanter may be fed to a stripping column to strip solvent from water.

[0199] In some embodiments, by-products or co-products may be derived from feedstock slurry used in the fermentation process. As described herein, if corn is used as feedstock, corn oil may be separated from the feedstock slurry prior to fermentation. The benefits of removing corn oil prior to the fermentation process are: recovering more corn oil as compared to corn oil removal at the end of the fermentation process (e.g., from the syrup), recovering higher quality and therefore higher value oil as compared to corn oil removal at end of the fermentation process, generating corn oil as a co-product, and conversion of corn oil to other products.

[0200] For example, as corn oil contains triglycerides, diglycerides, monoglycerides, fatty acids, phytosterols, vitamin E, carotenoids (e.g., .beta.-carotene, .beta.-cryptoxanthin, lutein zeaxanthin), phospholipids, and antioxidants such as tocopherols, it may be added to other co-products at different concentrations or rates, creating the ability to vary the amount of these components in the resulting co-product. In this manner, the fat content of the resulting co-product may be controlled, for example, to yield a lower fat, high protein animal feed that would better suit the needs of dairy cows compared to a high fat product. In another embodiment where a high fat animal feed may be desired, corn oil may be used as a component of animal feed because its high triglyceride content would provide a source of metabolizable energy. In addition, the natural antioxidants in corn oil provide a source of vitamin E as well as reduce the development of rancidity.

[0201] Corn oil separated from feedstock may be further processed to produce refined corn oil or edible oil for consumer use. For example, crude corn oil may be further processed to produce refined corn oil by degumming to remove phosphatides, alkali refining to neutralize free fatty acids, decolorizing for removal of color bodies and trace elements, winterizing to remove waxes, and deodorization (see, e.g., Corn Oil, 5.sup.th Edition, Corn Refiners Association, Washington, D.C., 2006). The refined corn oil may be used, for example, by food manufacturers for the production of food products. The free fatty acids removed by alkali refining may be used as soapstock and waxes recovered from the winterizing step may be utilized in animal feeds.

[0202] Corn oil may be used in the manufacture of resins, plastics, polymers, lubricants, paints, varnishes printing inks, soap, and textiles; and may also be utilized by the pharmaceutical industry as a component of drug formulations. Corn oil may also be used as feedstock for biodiesel or renewable diesel.

[0203] In some embodiments, oils such as corn oil may be used as a feedstock for the generation of extractant for extractive fermentation. For example, oil derived from biomass may be converted into an extractant available for removal of a product alcohol such as butanol from a fermentation broth. The glycerides in the oil may be chemically or enzymatically converted into a reaction product, such as fatty acids, fatty alcohols, fatty amides, fatty acid alkyl esters, fatty acid glycol esters, and hydroxylated triglycerides, or mixtures thereof, which may be used a fermentation product extractant. Using corn oil as an example, corn oil triglycerides may be reacted with a base such as ammonia hydroxide or sodium hydroxide to obtain fatty amides, fatty acids, and glycerol. These fatty amides, fatty acids, or mixtures thereof may be used an extractant. In some embodiments, plant oil such as corn oil may be hydrolyzed by an enzyme such as lipase to form fatty acids (e.g., corn oil fatty acids). Methods for deriving extractants from biomass are described in U.S. Patent Application Publication No. 2011/0312043, U.S. Patent Application Publication No. 2011/0312044, and PCT International Publication No. WO 2011/159998. In some embodiments, extractant may be used, all or in part, as a component of an animal feed or it can be used as feedstock for biodiesel or renewable diesel.

[0204] In some embodiments, corn oil can also be used as feedstock for biodiesel or renewable diesel. In some embodiments, oils or a combination of oils can also be used as feedstock for biodiesel or renewable diesel. Examples of oils include canola, castor, corn, jojoba, karanja, mahua, linseed, soybean, palm, peanut, rapeseed, rice, safflower, and sunflower oils. Biodiesel may be derived from either the transesterification or esterification of plant oils with alcohols such as methanol, ethanol, and butanol. For example, biodiesel may be produced by acid-catalyzed, alkali-catalyzed, or enzyme-catalyzed transesterification or esterification (e.g., transesterification of plant oil-derived triglycerides or esterification of plant oil-derived free fatty acids). Inorganic acids such as sulfuric acid, hydrochloric acid, and phosphoric acid; organic acids such as toluenesulfonic acid and naphthalenesulfonic acid; solid acids such as Amberlyst.RTM. sulfonated polystyrene resins; or zeolites may be used as a catalyst for acid-catalyzed transesterification or esterification. Bases such as potassium hydroxide, potassium methoxide, sodium hydroxide, sodium methoxide, or calcium hydroxide may be used as a catalyst for alkali-catalyzed transesterification or esterification. In some embodiments, biodiesel may be produced by an integrated process, for example, acid-catalyzed esterification of free fatty acids followed by base-catalyzed transesterification of triglycerides.

[0205] Enzymes such as lipases or esterases may be used to catalyze transesterification or esterification reactions. Lipases may be derived from bacteria or fungi, for example, Pseudomonas, Thermomyces, Burkholderia, Candida, and Rhizomucor. In some embodiments, lipases may be derived Pseudomonas fluorescens, Pseudomonas cepacia, Rhizomucor miehei, Burkholderia cepacia, Thermomyces lanuginosa, or Candida antarctica. In some embodiments, the enzyme may be immobilized on a soluble or insoluble support. The immobilization of enzymes may be performed using a variety of techniques including 1) binding of the enzyme to a porous or non-porous carrier support, via covalent support, physical adsorption, electrostatic binding, or affinity binding; 2) crosslinking with bifunctional or multifunctional reagents; 3) entrapment in gel matrices, polymers, emulsions, or some form of membrane; and 4) a combination of any of these methods. In some embodiments, lipase may be immobilized, for example, on acrylic resin, silica, or beads (e.g., polymethacrylate beads). In some embodiments, the lipases may be soluble.

[0206] In some embodiments, biodiesel described herein may comprise one or more of the following fatty acid alkyl esters (FAAE): fatty acid methyl esters (FAME), fatty acid ethyl esters (FAEE), and fatty acid butyl esters (FABE). In some embodiments, biodiesel described herein may comprise one or more of the following: myristate, palmitate, stearate, oleate, linoleate, linolenate, arachidate, and behenate.

[0207] In some embodiments, oil from the fermentation process may be recovered by evaporation forming a non-aqueous stream. This non-aqueous stream may comprise fatty acid esters and fatty acids and this stream may be conducted to a hydrolyzer to recover product alcohol and fatty acids. In some embodiments, this stream may be used as feedstock for biodiesel production.

[0208] In some embodiments, the biodiesel described herein meets the specifications of the American Society for Testing and Materials (ASTM) D6751. In some embodiments, the biodiesel described herein meets the specifications of the European standard EN 14214.

[0209] In some embodiments, reactor configurations for the production of biodiesel include, for example, batch-stirred tank reactors, continuous-stirred tank reactors, packed bed reactors, fluid bed reactors, expanding bed reactors, and recirculation membrane reactors.

[0210] In some embodiments, a composition may comprise at least 2% biodiesel, at least 5% biodiesel, at least 10% biodiesel, at least 20% biodiesel, at least 30% biodiesel, at least 40% biodiesel, at least 50% biodiesel, at least 60% biodiesel, at least 70% biodiesel, at least 80% biodiesel, at least 90% biodiesel, or 100% biodiesel.

[0211] In some embodiments, the biodiesel described herein may be blended with a petroleum-based diesel fuel to form a biodiesel blend. In some embodiments, a biodiesel blend may comprise at least 2% by volume biodiesel, at least 3% by volume biodiesel, at least 4% by volume biodiesel, at least 5% by volume biodiesel, at least 6% by volume biodiesel, at least 7% by volume biodiesel, at least 8% by volume biodiesel, at least 9% by volume biodiesel, at least 10% by volume biodiesel, at least 11% by volume biodiesel, at least 12% by volume biodiesel, at least 13% by volume biodiesel, at least 14% by volume biodiesel, at least 15% by volume biodiesel, at least 16% by volume biodiesel, at least 17% by volume biodiesel, at least 18% by volume biodiesel, at least 19% by volume biodiesel, or at least 20% by volume biodiesel. In some embodiments, a biodiesel blend may comprise up to about 20% by volume biodiesel.

[0212] A by-product of biodiesel production is glycerol. In addition, glycerol may also be a by-product of the generation of extractant from oils and a by-product of the fermentation process. A feedstock for biodiesel may be produced by reacting a fatty acid such as COFA with glycerol. The reaction may be catalyzed by strong inorganic acids such as sulfuric acid or by solid acid catalysts such as Amberlyst.TM. polymeric catalysts and ion exchange resins. High conversions may be obtained by withdrawing water from the reaction mass. The reaction product may contain monoglycerides, diglycerides, and triglycerides in a proportion determined by the ratio of reactants and the extent of reaction. The glyceride mix may be used in lieu of the triglyceride feed normally used to make biodiesel. In some embodiments, the glycerides may be used as a surfactant or as a feedstock for biodiesel.

[0213] In some embodiments, solids may be separated from feedstock slurry and may comprise triglycerides and fatty acids. These solids may be used as an animal feed, either recovered as discharge from centrifugation or after drying. The solids may be particularly suited as feed for ruminants (e.g., dairy cows) because of its high content of available lysine and by-pass or rumen undegradable protein. For example, these solids may be of particular value in a high protein, low fat feed. In some embodiments, these solids may be used as a base, that is, other by-products such as syrup may be added to the solids to form a product that may be used as an animal feed. In some embodiments, different amounts of other by-products may be added to the solids to tailor the properties of the resulting product to meet the needs of a certain animal species (e.g., dairy and beef cattle, poultry, swine, livestock, equine, aquaculture, and domestic pets).

[0214] In some embodiments where a low fat animal feed is desired, oil may be removed from the feedstock prior to fermentation. By removing the corn oil, the DDGS produced would have a low fat, high protein content. If the corn oil is not removed, the oil present in the wet cake can be oxidized by the drying process. This oxidation causes a darkening effect and produces DDGS with a darker color. If the oil is removed from the feedstock prior to fermentation, the DDGS produced would be lighter in color and this lighter color DDGS may be desirable for some animal feed products.

[0215] The composition of solids separated from whole stillage may include, for example, crude protein, fatty acids, and fatty acid esters. In some embodiments, this composition may be used, wet or dry, as an animal feed where, for example, a high protein (e.g., high lysine), low fat, and high fiber content is desired. In some embodiments, fat may be added to this composition, for example, from another by-product stream if a higher fat, low fiber animal feed is desired. In some embodiments, this higher fat, low fiber animal feed may be used for swine or poultry. In some embodiments, a non-aqueous composition of CDS may include, for example, protein, fatty acids, and fatty acid esters as well as other dissolved and suspended solids such as salts and carbohydrates. This CDS composition may be used, for example, as animal feed, either wet or dry, where a high protein, low fat, high mineral salt feed component is desired. In some embodiments, this composition may be used as a component of a dairy cow ration.

[0216] In some embodiments, one or more streams generated by the production of a product alcohol via a fermentation process may be combined to generate a composition comprising at least about 90% fatty acids which may be used as fuel source such as biodiesel.

[0217] The various streams generated by the production of a product alcohol via a fermentation process may be combined in many ways to generate a number of co-products. For example, if crude corn from mash is used to generate fatty acids to be utilized as extractant and lipid is extracted by evaporators, then the remaining streams may be combined and processed to create a co-product composition comprising crude protein, crude fat, triglycerides, fatty acids, and fatty acid esters. In another example, if oil such as corn oil is removed from feedstock slurry, the oil may be added to distillers grains to produce, for example, an animal feed product.

[0218] In some embodiments, compositions of the processes and systems described herein may comprise at least about 20-35 wt % crude protein, at least about 1-20 wt % crude fat, at least about 0-5 wt % triglycerides, at least about 4-10 wt % fatty acids, and at least about 2-6 wt % fatty acid esters. In some embodiments, compositions may comprise about 25 wt % crude protein, about 10 wt % crude fat, about 0.5 wt % triglycerides, about 6 wt % fatty acids, and about 4 wt % fatty acid esters. In some embodiments, compositions may comprise at least about 25-31 wt % crude protein, at least about 6-10 wt % crude fat, at least about 4-8 wt % triglycerides, at least about 0-2 wt % fatty acids, and at least about 1-3 wt % fatty acid esters. In some embodiments, compositions may comprise about 28 wt % crude protein, about 8 wt % crude fat, about 6 wt % triglycerides, about 0.7 wt % fatty acids, and about 1 wt % fatty acid esters. In some embodiments, the fatty acid esters may be fatty acid methyl esters, fatty acid ethyl esters, fatty acid butyl esters, or fatty acid isobutyl ester.

[0219] In some embodiments, solids separated from whole stillage and oil extracted from feedstock slurry may be combined and the resulting composition may comprise crude protein, crude fat, triglycerides, fatty acids, fatty acid esters, lysine, neutral detergent fiber (NDF), and acid detergent fiber (ADF). In some embodiments, compositions may comprise at least about 26-34 wt % crude protein, at least about 15-25 wt % crude fat, at least about 12-20 wt % triglycerides, at least about 1-2 wt % fatty acids, at least about 2-4 wt % fatty acid esters, at least about 1-2 wt % lysine, at least about 11-23 wt % NDF, and at least about 5-11 wt % ADF. In some embodiments, compositions may comprise about 29 wt % crude protein, about 21 wt % crude fat, about 16 wt % triglycerides, about 1 wt % fatty acids, about 3 wt % fatty acid esters, about 1 wt % lysine, about 17 wt % NDF, and about 8 wt % ADF. In some embodiments, the fatty acid esters may be fatty acid methyl esters, fatty acid ethyl esters, fatty acid butyl esters, or fatty acid isobutyl ester. The high fat, triglyceride, and lysine content and the lower fiber content of this composition may be desirable as feed for swine and poultry.

[0220] As described herein, the various streams generated by the production of a product alcohol via a fermentation process may be combined in many ways to generate a composition comprising crude protein, crude fat, triglycerides, fatty acids, and fatty acid esters. For example, a composition comprising at least about 6% crude fat and at least about 28% crude protein may be utilized as an animal feed product for dairy animals. A composition comprising at least about 6% crude fat and at least about 26% crude protein may be utilized as an animal feed product for feedlot cattle whereas a composition comprising at least about 1% crude fat and at least about 27% crude protein may be utilized as an animal feed product for wintering cattle. A composition comprising at least about 13% crude fat and at least about 27% crude protein may be utilized as an animal feed product for poultry. A composition comprising at least about 18% crude fat and at least about 22% crude protein may be utilized as an animal feed product for monogastric animals. The various streams may be combined in such a way as to customize a feed product for a specific animal species (e.g., livestock, ruminant, cattle, dairy animal, swine, goat, sheep, poultry, equine, aquaculture, or domestic pet such as dogs, cats, and rabbits).

[0221] The DDGS generated by the processes of the present invention may be modified to produce a customized high value feed product by the addition of one or more of the following: protein, fat, fiber, ash, lipid, amino acids, vitamins, and minerals. Amino acids include, for example, essential amino acids such as histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine as well as other amine acids such as alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, hydroxylysine, hydroxyproline, ornithine, proline, serine, and tyrosine. Minerals include, for example, calcium, chloride, cobalt, copper, fluoride, iodine, iron, magnesium, manganese, phosphorus, potassium, selenium, sodium, sulfur, and zinc. Vitamins include, for example, vitamins A, C, D, E, K, and B (thiamine, riboflavin, niacin, pantothenic acid, biotin, vitamin B6, vitamin B12 and folate).

[0222] As described herein, the processes and systems of the present invention provide a number of benefits that can result in improved production of a product alcohol such as butanol. For example, an improvement in mass transfer enables operation at a lower aqueous titer resulting in a "healthier" microorganism. A better phase separation can lead to improved fermentor volume efficiency as well as the possibility of processing less reactor contents through beer columns, distillation columns, etc. In addition, there is less solvent loss via solids and there is the possibility of cell recycling. The processes and systems of the present invention may also provide a higher quality of DDGS.

[0223] The processes and systems described herein also provide for the removal of oil prior to fermentation which would then allow the controlled addition of oil to the fermentation. Furthermore, the removal of oil prior to fermentation would allow flexibility in the amount of oil present in DDGS. That is, oil may be added in different amounts to DDGS resulting in the production of DDGS with different fat contents depending on the nutritional needs of a particular animal species.

[0224] The processes and systems described herein may be demonstrated using computational modeling such as Aspen modeling (see, e.g., U.S. Pat. No. 7,666,282). For example, the commercial modeling software Aspen Plus.RTM. (Aspen Technology, Inc., Burlington, Mass.) may be used in conjunction with physical property databases such as DIPPR (Design Institute for Physical Property Research), available from American Institute of Chemical Engineers, Inc. (New York, N.Y.) to develop an Aspen model for an integrated alcohol fermentation, purification, and water management process. This process modeling can perform many fundamental engineering calculations, for example, mass and energy balances, vapor/liquid equilibrium, and reaction rate computations. In order to generate an Aspen model, information input may include, for example, experimental data, water content and composition of feedstock, temperature for mash cooking and flashing, saccharification conditions (e.g., enzyme feed, starch conversion, temperature, pressure), fermentation conditions (e.g., microorganism feed, glucose conversion, temperature, pressure), degassing conditions, solvent columns, preflash columns, condensers, evaporators, centrifuges, etc.

Recombinant Microorganisms

[0225] While not wishing to be bound by theory, it is believed that the processes described herein are useful in conjunction with any alcohol-producing microorganism, particularly recombinant microorganisms which produce alcohol at titers above their tolerance levels.

[0226] Alcohol-producing microorganisms are known in the art. For example, fermentative oxidation of methane by methanotrophic bacteria (e.g., Methylosinus trichosporium) produces methanol, and the yeast strain CEN.PK113-7D (CBS 8340, the Centraal Buro voor Schimmelculture; van Dijken, et al., Enzyme Microb. Techno. 26:706-714, 2000) produces ethanol. Recombinant microorganisms which produce alcohol are also known in the art (e.g., Ohta, et al., Appl. Environ. Microbiol. 57:893-900, 1991; Underwood, et al., Appl. Environ. Microbiol. 68:1071-1081, 2002; Shen and Liao, Metab. Eng. 10:312-320, 2008; Hahnai, et al., Appl. Environ. Microbiol. 73:7814-7818, 2007; U.S. Pat. No. 5,514,583; U.S. Pat. No. 5,712,133; PCT Application Publication No. WO 1995/028476; Feldmann, et al., Appl. Microbiol. Biotechnol. 38: 354-361, 1992; Zhang, et al., Science 267:240-243, 1995; U.S. Patent Application Publication No. 2007/0031918 A1; U.S. Pat. No. 7,223,575; U.S. Pat. No. 7,741,119; U.S. Pat. No. 7,851,188; U.S. Patent Application Publication No. 2009/0203099 A1; U.S. Patent Application Publication No. 2009/0246846 A1; and PCT Application Publication No. WO 2010/075241, which are all herein incorporated by reference).

[0227] In addition, microorganisms may be modified using recombinant technologies to generate recombinant microorganisms capable of producing product alcohols such as ethanol and butanol. Microorganisms that may be recombinantly modified to produce a product alcohol via a biosynthetic pathway include members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula, Issatchenkia, or Saccharomyces. In some embodiments, recombinant microorganisms may be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, Kluyveromyces lactis, Kluyveromyces marxianus and Saccharomyces cerevisiae. In some embodiments, the recombinant microorganism is yeast. In some embodiments, the recombinant microorganism is crabtree-positive yeast selected from Saccharomyces, Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis, Brettanomyces, and some species of Candida. Species of crabtree-positive yeast include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces mikitae, Saccharomyces paradoxus, Zygosaccharomyces rouxii, and Candida glabrata.

[0228] Saccharomyces cerevisiae are known in the art and are available from a variety of sources including, but not limited to, American Type Culture Collection (Rockville, Md.), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. Saccharomyces cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red.RTM. yeast, Ferm Pro.TM. yeast, Bio-Ferm.RTM. XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax.TM. Green yeast, FerMax.TM. Gold yeast, Thermosacc.RTM. yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

[0229] In some embodiments, the microorganism may be immobilized or encapsulated. For example, the microorganism may be immobilized or encapsulated using alginate, calcium alginate, or polyacrylamide gels, or through the induction of biofilm formation onto a variety of high surface area support matrices such as diatomite, celite, diatomaceous earth, silica gels, plastics, or resins. In some embodiments, ISPR may be used in combination with immobilized or encapsulated microorganisms. This combination may improve productivity such as specific volumetric productivity, metabolic rate, product alcohol yields, tolerance to product alcohol. In addition, immobilization and encapsulation may minimize the effects of the process conditions such as shearing on the microorganisms.

[0230] The production of butanol utilizing fermentation, as well as microorganisms which produce butanol, is disclosed, for example, in U.S. Pat. No. 7,851,188, and U.S. Patent Application Publication Nos. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; and 2009/0305370, the entire contents of each are herein incorporated by reference. In some embodiments, the microorganism is engineered to contain a biosynthetic pathway. In some embodiments, the biosynthetic pathway is an engineered butanol biosynthetic pathway. In some embodiments, the biosynthetic pathway converts pyruvate to a fermentation product. In some embodiments, the biosynthetic pathway converts pyruvate as well as amino acids to a fermentation product. In some embodiments, at least one, at least two, at least three, or at least four polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, all polypeptides catalyzing substrate to product conversions of a pathway are encoded by heterologous polynucleotides in the microorganism. In some embodiments, the polypeptide catalyzing the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol are capable of utilizing reduced nicotinamide adenine dinucleotide (NADH) as a cofactor.

Biosynthetic Pathways

[0231] Biosynthetic pathways for the production of isobutanol that may be used include those described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference. In one embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions: [0232] a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0233] b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase; [0234] c) 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase; [0235] d) .alpha.-ketoisovalerate to isobutyraldehyde, which may be catalyzed, for example, by a branched-chain .alpha.-keto acid decarboxylase; and [0236] e) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[0237] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions: [0238] a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0239] b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol-acid reductoisomerase; [0240] c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase; [0241] d) .alpha.-ketoisovalerate to valine, which may be catalyzed, for example, by transaminase or valine dehydrogenase; [0242] e) valine to isobutylamine, which may be catalyzed, for example, by valine decarboxylase; [0243] f) isobutylamine to isobutyraldehyde, which may be catalyzed by, for example, omega transaminase; and [0244] g) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[0245] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions: [0246] a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0247] b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase; [0248] c) 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase; [0249] d) .alpha.-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for example, by branched-chain keto acid dehydrogenase; [0250] e) isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for example, by acylating aldehyde dehydrogenase; and [0251] f) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched-chain alcohol dehydrogenase.

[0252] Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Patent Application Publication No. 2008/0182308, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway comprises the following substrate to product conversions: [0253] a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example, by acetyl-CoA acetyltransferase; [0254] b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which may be catalyzed, for example, by 3-hydroxybutyryl-CoA dehydrogenase; [0255] c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed, for example, by crotonase; [0256] d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by butyryl-CoA dehydrogenase; [0257] e) butyryl-CoA to butyraldehyde, which may be catalyzed, for example, by butyraldehyde dehydrogenase; and [0258] f) butyraldehyde to 1-butanol, which may be catalyzed, for example, by butanol dehydrogenase.

[0259] Biosynthetic pathways for the production of 2-butanol that may be used include those described in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions: [0260] a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0261] b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase; [0262] c) acetoin to 3-amino-2-butanol, which may be catalyzed, for example, acetonin aminase; [0263] d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase; [0264] e) 3-amino-2-butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase; and [0265] f) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol dehydrogenase.

[0266] In another embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions: [0267] a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0268] b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase; [0269] c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase; [0270] d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by dial dehydratase; and [0271] e) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol dehydrogenase.

[0272] Biosynthetic pathways for the production of 2-butanone that may be used include those described in U.S. Patent Application Publication No. 2007/0259410 and U.S. Patent Application Publication No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions: [0273] a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0274] b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase; [0275] c) acetoin to 3-amino-2-butanol, which may be catalyzed, for example, acetonin aminase; [0276] d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase; and [0277] e) 3-amino-2-butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase.

[0278] In another embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions: [0279] a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase; [0280] b) alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase; [0281] c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase; and [0282] d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by diol dehydratase.

[0283] The terms "acetohydroxyacid synthase," "acetolactate synthase," and "acetolactate synthetase" (abbreviated "ALS") are used interchangeably herein to refer to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of pyruvate to acetolactate and CO.sub.2. Example acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These unmodified enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB15618 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO: 3), M73842 (SEQ ID NO: 4)), and Lactococcus lactis (GenBank Nos: AAA25161 (SEQ ID NO: 5), L16975 (SEQ ID NO: 6)).

[0284] The term "ketol-acid reductoisomerase" ("KARI"), "acetohydroxy acid isomeroreductase," and "acetohydroxy acid reductoisomerase" will be used interchangeably and refer to a polypeptide (or polypeptides) having enzyme activity that catalyzes the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. Example KARI enzymes may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP.sub.--418222 (SEQ ID NO: 7), NC.sub.--000913 (SEQ ID NO: 8)), Saccharomyces cerevisiae (GenBank Nos: NP.sub.--013459 (SEQ ID NO: 9), NC.sub.--001144 (SEQ ID NO: 10)), Methanococcus maripaludis (GenBank Nos: CAF30210 (SEQ ID NO: 11), BX957220 (SEQ ID NO: 12)), and Bacillus subtilis (GenBank Nos: CAB14789 (SEQ ID NO: 13), Z99118 (SEQ ID NO: 14)). KARIs include Anaerostipes caccae KARI variants "K9G9" and "K9D3" (SEQ ID NOs: 15 and 16, respectively). Ketol-acid reductoisomerase (KARI) enzymes are described in U.S. Patent Application Publication Nos. 2008/0261230, 2009/0163376, and 2010/0197519, and PCT Application Publication No. WO/2011/041415, which are incorporated herein by reference. Examples of KARIs disclosed therein are those from Lactococcus lactis, Vibrio cholera, Pseudomonas aeruginosa PAO1, and Pseudomonas fluorescens PF5 mutants In some embodiments, the KARI utilizes NADH. In some embodiments, the KARI utilizes reduced nicotinamide adenine dinucleotide phosphate (NADPH).

[0285] The term "acetohydroxy acid dehydratase" and "dihydroxyacid dehydratase" ("DHAD") refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion the conversion of 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate. Example acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. Such enzymes are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: YP.sub.--026248 (SEQ ID NO: 17), NC000913 (SEQ ID NO: 18)), Saccharomyces cerevisiae (GenBank Nos: NP.sub.--012550 (SEQ ID NO: 19), NC 001142 (SEQ ID NO: 20), M. maripaludis (GenBank Nos: CAF29874 (SEQ ID NO: 21), BX957219 (SEQ ID NO: 22)), B. subtilis (GenBank Nos: CAB 14105 (SEQ ID NO: 23), Z99115 (SEQ ID NO: 24)), L. lactis, and N. crassa. U.S. Patent Application Publication No. 2010/0081154, and U.S. Pat. No. 7,851,188, which are incorporated herein by reference, describe dihydroxyacid dehydratases (DHADs), including a DHAD from Streptococcus mutans.

[0286] The term "branched-chain .alpha.-keto acid decarboxylase," ".alpha.-ketoacid decarboxylase," ".alpha.-ketoisovalerate decarboxylase," or "2-ketoisovalerate decarboxylase" ("KIVD") refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of .alpha.-ketoisovalerate to isobutyraldehyde and CO.sub.2. Example branched-chain .alpha.-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166 (SEQ ID NO: 25), AY548760 (SEQ ID NO: 26); CAG34226 (SEQ ID NO: 27), AJ746364 (SEQ ID NO: 28), Salmonella typhimurium (GenBank Nos: NP.sub.--461346 (SEQ ID NO: 29), NC.sub.--003197 (SEQ ID NO: 30)), Clostridium acetobutylicum (GenBank Nos: NP.sub.--149189 (SEQ ID NO: 31), NC.sub.--001988 (SEQ ID NO: 32)), M. caseolyticus (SEQ ID NO: 33), and L. grayi (SEQ ID NO: 34).

[0287] The term "branched-chain alcohol dehydrogenase" ("ADH") refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutyraldehyde to isobutanol. Example branched-chain alcohol dehydrogenases are known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases (specifically, EC 1.1.1.1 or 1.1.1.2). Alcohol dehydrogenases may be NADPH-dependent or NADH-dependent. Such enzymes are available from a number of sources, including, but not limited to, Saccharomyces cerevisiae (GenBank Nos: NP.sub.--010656 (SEQ ID NO: 35), NC.sub.--001136 (SEQ ID NO: 36), NP.sub.--014051 (SEQ ID NO: 37), NC.sub.--001145 (SEQ ID NO: 38)), Escherichia coli (GenBank Nos: NP.sub.--417484 (SEQ ID NO: 39), NC.sub.--000913 (SEQ ID NO: 40)), C. acetobutylicum (GenBank Nos: NP.sub.--349892 (SEQ ID NO: 41), NC.sub.--003030 (SEQ ID NO: 42); NP.sub.--349891 (SEQ ID NO: 43), NC.sub.--003030 (SEQ ID NO: 44)). U.S. Patent Application Publication No. 2009/0269823 describes SadB, an alcohol dehydrogenase (ADH) from Achromobacter xylosoxidans. Alcohol dehydrogenases also include horse liver ADH and Beijerinkia indica ADH (as described by U.S. Patent Application Publication No. 2011/0269199, which is incorporated herein by reference).

[0288] The term "butanol dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutyraldehyde to isobutanol or the conversion of 2-butanone and 2-butanol. Butanol dehydrogenases are a subset of a broad family of alcohol dehydrogenases. Butanol dehydrogenase may be NAD- or NADP-dependent. The NAD-dependent enzymes are known as EC 1.1.1.1 and are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475, AJ491307). The NADP dependent enzymes are known as EC 1.1.1.2 and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169). Additionally, a butanol dehydrogenase is available from Escherichia coli (GenBank Nos: NP 417484, NC.sub.--000913) and a cyclohexanol dehydrogenase is available from Acinetobacter sp. (GenBank Nos: AAG10026, AF282240). The term "butanol dehydrogenase" also refers to an enzyme that catalyzes the conversion of butyraldehyde to 1-butanol, using either NADH or NADPH as cofactor. Butanol dehydrogenases are available from, for example, C. acetobutylicum (GenBank Nos: NP.sub.--149325, NC.sub.--001988; note: this enzyme possesses both aldehyde and alcohol dehydrogenase activity); NP.sub.--349891, NC.sub.--003030; and NP.sub.--349892, NC.sub.--003030) and Escherichia coli (GenBank Nos: NP.sub.--417-484, NC.sub.--000913).

[0289] The term "branched-chain keto acid dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of .alpha.-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), typically using NAD.sup.+ (nicotinamide adenine dinucleotide) as an electron acceptor. Example branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. Such branched-chain keto acid dehydrogenases are comprised of four subunits and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB14336 (SEQ ID NO: 45), Z99116 (SEQ ID NO: 46); CAB14335 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48); CAB14334 (SEQ ID NO: 49), Z99116 (SEQ ID NO: 50); and CAB14337 (SEQ ID NO: 51), Z99116 (SEQ ID NO: 52)) and Pseudomonas putida (GenBank Nos: AAA65614 (SEQ ID NO: 53), M57613 (SEQ ID NO: 54); AAA65615 (SEQ ID NO: 55), M57613 (SEQ ID NO: 56); AAA65617 (SEQ ID NO: 57), M57613 (SEQ ID NO: 58); and AAA65618 (SEQ ID NO: 59), M57613 (SEQ ID NO: 60)).

[0290] The term "acylating aldehyde dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, typically using either NADH or NADPH as an electron donor. Example acylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Such enzymes are available from multiple sources, including, but not limited to, Clostridium beijerinckii (GenBank Nos: AAD31841 (SEQ ID NO: 61), AF157306 (SEQ ID NO: 62)), Clostridium acetobutylicum (GenBank Nos: NP.sub.--149325 (SEQ ID NO: 63), NC.sub.--001988 (SEQ ID NO: 64); NP.sub.--149199 (SEQ ID NO: 65), NC.sub.--001988 (SEQ ID NO: 66)), Pseudomonas putida (GenBank Nos: AAA89106 (SEQ ID NO: 67), U13232 (SEQ ID NO: 68)), and Thermus thermophilus (GenBank Nos: YP.sub.--145486 (SEQ ID NO: 69), NC.sub.--006461 (SEQ ID NO: 70)).

[0291] The term "transaminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of .alpha.-ketoisovalerate to L-valine, using either alanine or glutamate as an amine donor. Example transaminases are known by the EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from a number of sources. Examples of sources for alanine-dependent enzymes include, but are not limited to, Escherichia coli (GenBank Nos: YP.sub.--026231 (SEQ ID NO: 71), NC.sub.--000913 (SEQ ID NO: 72)) and Bacillus licheniformis (GenBank Nos: YP.sub.--093743 (SEQ ID NO: 73), NC.sub.--006322 (SEQ ID NO: 74)). Examples of sources for glutamate-dependent enzymes include, but are not limited to, Escherichia coli (GenBank Nos: YP.sub.--026247 (SEQ ID NO: 75), NC.sub.--000913 (SEQ ID NO: 76)), Saccharomyces cerevisiae (GenBank Nos: NP.sub.--012682 (SEQ ID NO: 77), NC.sub.--001142 (SEQ ID NO: 78)) and Methanobacterium thermoautotrophicum (GenBank Nos: NP.sub.--276546 (SEQ ID NO: 79), NC.sub.--000916 (SEQ ID NO: 80)).

[0292] The term "valine dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of .alpha.-ketoisovalerate to L-valine, typically using NAD(P)H as an electron donor and ammonia as an amine donor. Example valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP.sub.--628270 (SEQ ID NO: 81), NC.sub.--003888 (SEQ ID NO: 82)) and Bacillus subtilis (GenBank Nos: CAB14339 (SEQ ID NO: 83), Z99116 (SEQ ID NO: 84)).

[0293] The term "valine decarboxylase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of L-valine to isobutylamine and CO.sub.2. Example valine decarboxylases are known by the EC number 4.1.1.14. Such enzymes are found in Streptomyces, such as for example, Streptomyces viridifaciens (GenBank Nos: AAN10242 (SEQ ID NO: 85), AY116644 (SEQ ID NO: 86)).

[0294] The term "omega transaminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as an amine donor. Example omega transaminases are known by the EC number 2.6.1.18 and are available from a number of sources, including, but not limited to, Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 87), AY330220 (SEQ ID NO: 88)), Ralstonia eutropha (GenBank Nos: YP.sub.--294474 (SEQ ID NO: 89), NC.sub.--007347 (SEQ ID NO: 90)), Shewanella oneidensis (GenBank Nos: NP.sub.--719046 (SEQ ID NO: 91), NC.sub.--004347 (SEQ ID NO: 92)), and Pseudomonas putida (GenBank Nos: AAN66223 (SEQ ID NO: 93), AE016776 (SEQ ID NO: 94)).

[0295] The term "acetyl-CoA acetyltransferase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (CoA). Example acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP.sub.--416728, NC.sub.--000913; NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NP.sub.--349476.1, NC.sub.--003030; NP.sub.--149242, NC.sub.--001988, Bacillus subtilis (GenBank Nos: NP.sub.--390297, NC.sub.--000964), and Saccharomyces cerevisiae (GenBank Nos: NP.sub.--015297, NC.sub.--001148).

[0296] The term "3-hydroxybutyryl-CoA dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. Example 3-hydroxybutyryl-CoA dehydrogenases may be NADH-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA. Examples may be classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be NADPH-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, Clostridium acetobutylicum (GenBank Nos: NP.sub.--349314, NC.sub.--003030), Bacillus subtilis (GenBank Nos: AAB09614, U29084), Ralstonia eutropha (GenBank Nos: YP.sub.--294481, NC.sub.--007347), and Alcaligenes eutrophus (GenBank Nos: AAA21973, J04987).

[0297] The term "crotonase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H.sub.2O. Example crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and may be classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. Crotonases are available from a number of sources, for example, Escherichia coli (GenBank Nos: NP.sub.--415911, NC.sub.--000913), Clostridium acetobutylicum (GenBank Nos: NP.sub.--349318, NC.sub.--003030), Bacillus subtilis (GenBank Nos: CAB13705, Z99113), and Aeromonas caviae (GenBank Nos: BAA21816, D88825).

[0298] The term "butyryl-CoA dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Example butyryl-CoA dehydrogenases may be NADH-dependent, NADPH-dependent, or flavin-dependent and may be classified as E.C. 1.3.1.44, E.C. 1.3.1.38, and E.C. 1.3.99.2, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, Clostridium acetobutylicum (GenBank Nos: NP.sub.--347102, NC.sub.-- 003030), Euglena gracilis (GenBank Nos: Q5EU90, AY741582), Streptomyces collinus (GenBank Nos: AAA92890, U37135), and Streptomyces coelicolor (GenBank Nos: CAA22721, AL939127).

[0299] The term "butyraldehyde dehydrogenase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as cofactor. Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank Nos: AAD31841, AF157306) and Clostridium acetobutylicum (GenBank Nos: NP.sub.-149325, NC.sub.-001988).

[0300] The term "isobutyryl-CoA mutase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme B.sub.12 as cofactor. Example isobutyryl-CoA mutases are known by the EC number 5.4.99.13. These enzymes are found in a number of Streptomyces, including, but not limited to, Streptomyces cinnamonensis (GenBank Nos: AAC08713 (SEQ ID NO: 95), U67612 (SEQ ID NO: 96); CAB59633 (SEQ ID NO: 97), AJ246005 (SEQ ID NO: 98)), Streptomyces coelicolor (GenBank Nos: CAB70645 (SEQ ID NO: 99), AL939123 (SEQ ID NO: 100); CAB92663 (SEQ ID NO: 101), AL939121 (SEQ ID NO: 102)), and Streptomyces avermitilis (GenBank Nos: NP.sub.--824008 (SEQ ID NO: 103), NC.sub.--003155 (SEQ ID NO: 104); NP.sub.--824637 (SEQ ID NO: 105), NC.sub.--003155 (SEQ ID NO: 106)).

[0301] The term "acetolactate decarboxylase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Example acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (GenBank Nos: AAU43774, AY722056).

[0302] The term "acetoin aminase" or "acetoin transaminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoin to 3-amino-2-butanol. Acetoin aminase may utilize the cofactor pyridoxal 5'-phosphate or NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate as the amino donor. The NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. A suitable example of an NADH-dependent acetoin aminase, also known as amino alcohol dehydrogenase, is described by Ito, et al. (U.S. Pat. No. 6,432,688). An example of a pyridoxal-dependent acetoin aminase is the amine:pyruvate aminotransferase (also called amine:pyruvate transaminase) described by Shin and Kim (J. Org. Chem. 67:2848-2853, 2002).

[0303] The term "acetoin kinase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate) or phosphoenolpyruvate as the phosphate donor in the reaction. Enzymes that catalyze the analogous reaction on the similar substrate dihydroxyacetone, for example, include enzymes known as EC 2.7.1.29 (Garcia-Alles, et al., Biochemistry 43:13037-13046, 2004).

[0304] The term "acetoin phosphate aminase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of phosphoacetoin to 3-amino-2-butanol O-phosphate. Acetoin phosphate aminase may use the cofactor pyridoxal 5'-phosphate, NADH, or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate. The NADH-dependent and NADPH-dependent enzymes may use ammonia as a second substrate. Although there are no reports of enzymes catalyzing this reaction on phosphoacetoin, there is a pyridoxal phosphate-dependent enzyme that is proposed to carry out the analogous reaction on the similar substrate serinol phosphate (Yasuta, et al., Appl. Environ. Microbial. 67:4999-5009, 2001).

[0305] The term "aminobutanol phosphate phospholyase," also called "amino alcohol O-phosphate lyase," refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Amino butanol phosphate phospho-lyase may utilize the cofactor pyridoxal 5'-phosphate. There are reports of enzymes that catalyze the analogous reaction on the similar substrate 1-amino-2-propanol phosphate (Jones, et al., Biochem J. 134:167-182, 1973). U.S. Patent Application Publication No. 2007/0259410 describes an aminobutanol phosphate phospho-lyase from the organism Erwinia carotovora.

[0306] The term "aminobutanol kinase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 3-amino-2-butanol to 3-amino-2-butanol O-phosphate. Amino butanol kinase may utilize ATP as the phosphate donor. Although there are no reports of enzymes catalyzing this reaction on 3-amino-2-butanol, there are reports of enzymes that catalyze the analogous reaction on the similar substrates ethanolamine and 1-amino-2-propanol (Jones, et al., supra). U.S. Patent Application Publication No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.

[0307] The term "butanediol dehydrogenase" also known as "acetoin reductase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanedial dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)-stereochemistry in the alcohol product. (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085, D86412). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP 830481, NC.sub.--004722; AAP07682, AE017000), and Lactococcus lactis (GenBank Nos. AAK04995, AE006323).

[0308] The term "butanediol dehydratase," also known as "dial dehydratase" or "propanediol dehydratase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratase may utilize the cofactor adenosyl cobalamin (also known as coenzyme Bw or vitamin B12; although vitamin B12 may refer also to other forms of cobalamin that are not coenzyme B12). Adenosyl cobalamin-dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: AA08099 (alpha subunit), D45071; BAA08100 (beta subunit), D45071; and BBA08101 (gamma subunit), D45071 (Note all three subunits are required for activity), and Klebsiella pneumonia (GenBank Nos: AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (beta subunit), AF102064, GenBank Nos: AAC98386 (gamma subunit), AF102064). Other suitable dial dehydratases include, but are not limited to, B12-dependent dial dehydratases available from Salmonella typhimurium (GenBank Nos: AAB84102 (large subunit), AF026270; GenBank Nos: AAB84103 (medium subunit), AF026270; GenBank Nos: AAB84104 (small subunit), AF026270); and Lactobacillus collinoides (GenBank Nos: CAC82541 (large subunit), AJ297723; GenBank Nos: CAC82542 (medium subunit); AJ297723; GenBank Nos: CAD01091 (small subunit), AJ297723); and enzymes from Lactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza, et al., J. Agric. Food Chem. 45:3476-3480, 1997), and nucleotide sequences that encode the corresponding enzymes. Methods of dial dehydratase gene isolation are well known in the art (e.g., U.S. Pat. No. 5,686,276).

[0309] The term "pyruvate decarboxylase" refers to a polypeptide (or polypeptides) having enzyme activity that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. Pyruvate dehydrogenases are known by the EC number 4.1.1.1. These enzymes are found in a number of yeast, including Saccharomyces cerevisiae (GenBank Nos: CAA97575 (SEQ ID NO: 107), CAA97705 (SEQ ID NO: 109), CAA97091 (SEQ ID NO: 111)).

[0310] It will be appreciated that microorganisms comprising an isobutanol biosynthetic pathway as provided herein may further comprise one or more additional modifications. U.S. Patent Application Publication No. 2009/0305363 (incorporated by reference) discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity. In some embodiments, the microorganisms may comprise modifications to reduce glycerol-3-phosphate dehydrogenase activity and/or disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or a disruption in at least one gene encoding a regulatory element controlling pyruvate decarboxylase gene expression as described in U.S. Patent Application Publication No. 2009/0305363 (incorporated herein by reference), and/or modifications that provide for increased carbon flux through an Entner-Doudoroff Pathway or reducing equivalents balance as described in U.S. Patent Application Publication No. 2010/0120105 (incorporated herein by reference). Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate-utilizing biosynthetic pathway. Other modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In some embodiments, the polypeptide having acetolactate reductase activity is YMR226C (SEQ ID NOs: 127, 128) of Saccharomyces cerevisiae or a homolog thereof. Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity. In some embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or a homolog thereof. A genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Patent Application Publication No. 2011/0124060, incorporated herein by reference. In some embodiments, the pyruvate decarboxylase that is deleted or down-regulated is selected from the group consisting of: PDC1, PDC5, PDC6, and combinations thereof. In some embodiments, the pyruvate decarboxylase is selected from those enzymes in Table 1. In some embodiments, microorganisms may contain a deletion or down-regulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3-phosphate to glycerate 1,3, bisphosphate. In some embodiments, the enzyme that catalyzes this reaction is glyceraldehyde-3-phosphate dehydrogenase.

TABLE-US-00001 TABLE 1 SEQ ID Numbers of PDC Target Gene coding regions and Proteins SEQ ID NO: SEQ ID NO: Description (Amino Acid) (Nucleic Acid) PDC1 pyruvate 107 108 decarboxylase from Saccharomyces cerevisiae PDC5 pyruvate 109 110 decarboxylase from Saccharomyces cerevisiae PDC6 pyruvate 111 112 decarboxylase Saccharomyces cerevisiae pyruvate decarboxylase 113 114 from Candida glabrata PDC1 pyruvate 115 116 decarboxylase from Pichia stipitis PDC2 pyruvate 117 118 decarboxylase from Pichia stipitis pyruvate decarboxylase 119 120 from Kluyveromyces lactis pyruvate decarboxylase 121 122 from Yarrowia lipolytica pyruvate decarboxylase 123 124 from Schizosaccharomyces pombe pyruvate decarboxylase 125 126 from Zygosaccharomyces rouxii

[0311] In some embodiments, any particular nucleic acid molecule or polypeptide may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence described herein. The term "percent identity" as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Identity and similarity can be readily calculated by known methods, including but not limited to those disclosed in: Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

[0312] Methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment which encompasses several varieties of the algorithm including the Clustal V method of alignment corresponding to the alignment method labeled Clustal V (disclosed by Higgins and Sharp, CABIOS. 5:151-153, 1989; Higgins, et al., Comput. Appl. Biosci. 8:189-191, 1992) and found in the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a percent identity by viewing the sequence distances table in the same program. Additionally the Clustal W method of alignment is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153, 1989; Higgins, et al., Comput. Appl. Biosci. 8:189-191, 1992) and found in the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a percent identity by viewing the sequence distances table in the same program.

[0313] Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, et al. (Sambrook, J., Fritsch, E. F. and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, here in referred to as Maniatis) and by Ausubel, et al. (Ausubel, et al., Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience, 1987). Examples of methods to construct microorganisms that comprise a butanol biosynthetic pathway are disclosed, for example, in U.S. Pat. No. 7,851,188, and U.S. Patent Application Publication Nos. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308; 2008/0274525; 2009/0155870; 2009/0305363; and 2009/0305370, the entire contents of each are herein incorporated by reference.

[0314] Further, while various embodiments of the present invention have been described herein, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the claims and their equivalents.

[0315] All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

EXAMPLES

[0316] The following nonlimiting examples will further illustrate the invention. It should be understood that, while the following examples involve corn as feedstock, other biomass sources can be used for feedstock without departing from the present invention.

[0317] As used herein, the meaning of abbreviations used was as follows: "g" means gram(s), "kg" means kilogram(s), "lbm" means pound mass, "gpm" means gallons per minute, "gal" means gallon(s), "MMGPY" means million gallon per year, "L" means liter(s), "mL" means milliliter(s), ".mu.L" means microliter(s), "mL/L" means milliliter(s) per liter, "mL/min" means milliliter(s) per min, "min" means minute(s), "hr" means hour(s), "DI" means deionized, "uM" means micrometer(s), "nm" means nanometer(s), "w/v" means weight/volume, "wt %" means weight percent, "OD" means optical density, "OD.sub.600" means optical density at a wavelength of 600 nM, "dcw" means dry cell weight, "rpm" means revolutions per minute, ".degree. C." means degree(s) Celsius, ".degree. C./min" means degrees Celsius per minute, "slpm" means standard liter(s) per minute, "ppm" means part per million, "cP" means centipoise, "ID" means inner diameter, and "GC" means gas chromatograph.

Example 1

Effect of Undissolved Solids on the Rate of Mass Transfer

[0318] The following experiment was performed to measure the effect of undissolved solids on the rate of mass transfer of i-BuOH from an aqueous phase that simulates the composition of a fermentation broth derived from corn mash, which is approximately half way through a simultaneous saccharification and fermentation (SSF) fermentation (i.e., about 50% conversion of the oligosaccharides) in order to mimic the average composition of the liquid phase for an SSF batch.

[0319] Approximately 100 kg of liquefied corn mash was prepared in three equivalent batches using a 30 L glass, jacketed resin kettle. The kettle was set up with mechanical agitation, temperature control, and pH control. The protocol used for the three batches was as follows: (a) mixing ground corn with tap water (30 wt % corn on a dry basis), (b) heating the slurry to 55.degree. C. while agitating, (c) adjusting pH of the slurry to 5.8 with either NaOH or H.sub.2SO.sub.4, (d) adding alpha-amylase (0.02 wt % on a dry corn basis), (e) heating the slurry to 85.degree. C., (f) adjusting pH to 5.8, (g) holding the slurry at 85.degree. C. for 2 hr while maintaining pH at 5.8, and (h) cooling the slurry to 25.degree. C.

[0320] Pioneer 3335 hybrid corn, whole kernel yellow corn, was used (Pioneer Hi-Bred International, Johnston, Iowa), and it was ground in a hammer-mill using a 1 mm screen. The moisture content of the ground corn was 12 wt %, and the starch content of the ground corn was 71.4 wt % on a dry corn basis. The alpha-amylase enzyme was Liquozyme.RTM. SC DS (Novozymes, Franklinton, N.C.). The total amounts of the ingredients used for the three batches combined were: 33.9 kg ground corn (12% moisture), 65.4 kg tap water, and 0.006 kg Liquozyme.RTM. SC DS. A total of 0.297 kg of NaOH (17 wt %) was added to control pH. No H.sub.2SO.sub.4 was required. The total amount of liquefied corn mash recovered from the three 30 L batches was 99.4 kg.

[0321] Solids were removed from the mash by centrifugation in a large floor centrifuge which contained six 1 L bottles. Mash (73.4 kg) was centrifuged at 8000 rpm for 20 min at 25.degree. C. yielding 44.4 kg of centrate and 26.9 kg of wet cake. It was determined that the centrate contained <1 wt % suspended solids, and that the wet cake contained approximately 18 wt % suspended solids. This implies that the original liquefied mash contained approximately 7 wt % suspended solids. This is consistent with the corn loading and starch content of the corn used assuming most of the starch was liquefied. If all of the starch was liquefied, the 44.4 kg of centrate recovered directly from the centrifuge would have contained approximately 23 wt % dissolved oligosaccharides (liquefied starch). About 0.6 kg of i-BuOH was added to 35.4 kg of centrate to preserve it. The resulting 36.0 kg of centrate, which contained 1.6 wt % i-BuOH, was used as a stock solution. The centrate mimics the liquid phase composition at the beginning of SSF. Therefore, a portion of it was diluted with an equal amount of H.sub.2O on a mass basis to generate centrate that mimics SSF at about 50% conversion. More i-BuOH was added to bring the final concentration of i-BuOH in the diluted centrate to 3.0 wt % (about 30 g/L).

[0322] The diluted centrate was prepared as follows: 18 kg of the centrate stock solution which contained 1.6 wt % i-BuOH, was mixed with 18 kg tap water and 0.82 kg i-BuOH was added. The resulting 36.8 kg solution of diluted centrate consisted of approximately 11 wt % oligosaccharides and approximately 30 g/L i-BuOH. This solution mimics the liquid phase of a corn mash fermentation (SSF) at approximately 50% conversion of the oligosaccharides and an aqueous titer of 30 g/L i-BuOH.

[0323] Mass transfer tests were conducted using this solution as the aqueous phase to mimic mass transfer performance in a broth derived from liquefied corn mash after most of the undissolved solids are removed. The objective of the mass transfer tests was to measure the effect of undissolved solids on the overall volumetric mass transfer coefficient (k.sub.La) for the transfer of i-BuOH from a simulated broth, derived from liquefied corn mash, to a dispersion of solvent (extractant) droplets rising through the simulated broth. Correlations of k.sub.La with key design of operating parameters can be used to scale up mass transfer operations. Examples of parameters that should be held constant as much as possible in order to generate correlations of k.sub.La from smaller scale data which are useful for scale up are the physical properties of both phases and design parameters that determine droplet size (e.g., nozzle diameter, velocity of the phase to be dispersed through the nozzle).

[0324] A 6-inch diameter, 7-foot tall glass, jacketed column was used to measure the k.sub.La for the transfer of i-BuOH from an aqueous solution of oligosaccharides (derived from liquefied corn mash), both with and without suspended mash solids, to a dispersion of oleyl alcohol (OA) droplets rising through the simulated broth. i-BuOH was added to the aqueous phase to give an initial concentration of i-BuOH of approximately 30 g/L. A certain amount of the aqueous phase (typically about 35 kg) which contained approximately 11 wt % oligosaccharides and approximately 30 g/L i-BuOH, was charged to the column, and the column was heated to 30.degree. C. by flowing warm H.sub.2O through the jacket. There was no flow of aqueous phase in or out of the column during the test.

[0325] Fresh oleyl alcohol (80/85% grade, Cognis Corporation, Cincinnati, Ohio) was sparged into the bottom of the column through a single nozzle to create a dispersion of extractant droplets which flowed up through the aqueous phase. After reaching the top of the aqueous phase, the extractant drops formed a separate organic phase which then overflowed from the top of the column and was collected into a receiver. Typically, 3 to 5 gallons of oleyl alcohol flowed through the column for a single test.

[0326] Samples of the aqueous phase were collected from the column at several times throughout the test, and a composite sample of the total "rich" oleyl alcohol collected from the overflow was collected at the end of the test. All samples were analyzed for i-BuOH using a HP-6890 GC (Agilent Technologies, Inc., Santa Clara, Calif.). The concentration profile for i-BuOH in the aqueous phase (i.e., i-BuOH concentration versus time) was used to calculate the k.sub.La at the given set of operating conditions. The final composite sample of the total "rich" oleyl alcohol collected during the test was used to check the mass balance for i-BuOH.

[0327] The nozzle size and nozzle velocity (average velocity of oleyl alcohol through the feed nozzle) were varied to observe the effects on the k.sub.La. A series of tests were done using an aqueous solution of oligosaccharides (diluted centrate obtained from liquefied corn mash) with the mash solids removed. A similar series of tests were done using the same aqueous solution of oligosaccharides after adding the mash solids back to simulate liquefied corn mash (including the undissolved solids) at the middle of SSF. It is noted that under some operating conditions (e.g., higher oleyl alcohol flow rates), poor phase separation was obtained at the top of the column which made it difficult to obtain a representative composite sample of the total "rich" oleyl alcohol collected during the test. It is also noted that under some operating conditions, samples of the aqueous phase contained a significant amount of organic phase. Special sample handling and preparation techniques were employed to obtain a sample of the aqueous phase that was as representative as possible of the aqueous phase in the column at the time the sample was collected.

[0328] It was determined that the aqueous phase in the column was "well mixed" for all practical purposes because the concentration of i-BuOH did not vary much along the length of the column at a given point in time. Assuming the solvent droplet phase is also well mixed, the overall mass transfer of i-BuOH from the aqueous phase to the solvent phase in the column can be approximated by the following equation:

r B = k L a ( C B , broth - C B , solvent K B ) ( 1 ) ##EQU00001## [0329] where, [0330] r.sub.B=total mass of i-BuOH transferred from the aqueous phase to the solvent phase per unit time per unit volume of the aqueous phase, grams i-BuOH/liter aqueous phase/hr or g/L/hr. [0331] k.sub.La=overall volumetric mass transfer coefficient describing the mass transfer of i-BuOH from the aqueous phase to the solvent phase, hr.sup.-1. [0332] C.sub.B,broth=average concentration of i-BuOH in the simulated broth (aqueous) phase over the entire test, grams i-BuOH/Liter aqueous phase or g/L. [0333] C.sub.B,solvent=average concentration of i-BuOH in the solvent phase over the entire test, grams i-BuOH/Liter solvent phase or g/L. [0334] K.sub.B=average equilibrium distribution coefficient for i-BuOH between the solvent and aqueous phase, (grams i-BuOH/Liter solvent phase)/(grams i-BuOH/Liter aqueous phase).

[0335] The parameters r.sub.B, C.sub.B,broth, and C.sub.B,solvent were calculated for each test from the concentration data obtained from the samples of the aqueous and solvent phases. The parameter K.sub.B was independently measured by mixing aqueous centrate from liquefied corn mash, oleyl alcohol, and i-BuOH and vigorously mixing the system until the two liquid phases were at equilibrium. The concentration of i-BuOH was measured in both phases to determine K.sub.B. After r.sub.B, C.sub.B,broth, C.sub.B,solvent, and K.sub.B were determined for a given test, the k.sub.La could be calculated by rearranging Equation (1):

k L a = r B ( C B , broth - C B , solvent K B ) ( 2 ) ##EQU00002##

[0336] Mass transfer tests were conducted with two different size nozzles at nozzle velocities ranging from 5 ft/s to 21 ft/s using the diluted centrate (solids removed) as the aqueous phase. Three tests were done using a nozzle that has an ID of 0.76 mm, and three tests were done using a nozzle that has an ID of 2.03 mm. All tests were conducted at 30.degree. C. in the 6-inch diameter column described above using oleyl alcohol as the solvent. The equilibrium distribution coefficient for i-BuOH between oleyl alcohol and the diluted centrate which was obtained from liquefied corn mash by removing the solids, was measured to be approximately 5. The results of the mass transfer tests using diluted centrate (with the solids removed) are shown in Table 2.

TABLE-US-00002 TABLE 2 41 42 43 44 45 46 Diluted Diluted Diluted Diluted Diluted Diluted Centrate Centrate Centrate Centrate Centrate Centrate from Liq'd from Liq'd from Liq'd from Liq'd from Liq'd from Liq'd Mash, Mash, Mash, Mash, Mash, Mash, Solids Solids Solids Solids Solids Solids Removed Removed Removed Removed Removed Removed MASS TRANSFER TEST CONDITIONS: Aqueous Phase 36.0 35.0 34.3 32.0 28.0 28.6 Volume of Aqueous Phase, L: Solvent Feed Rate, g/min: 33.2 79.5 145.3 237.7 507.7 875 Superficial Liq. Velocity (Us), ft/hr: 0.42 1.01 1.84 3.02 6.45 11.11 Nozzle I.D., mm: 0.76 0.76 0.76 2.03 2.03 2.03 Nozzle Velocity, ft/s 4.7 11.3 20.6 4.7 10.1 17.4 MASS TRANSFER RESULTS: Initial [i-B] in Aq. Phase, g/L: 28.2 27.0 29.1 31.3 38.7 30.1 Final [i-B] in Aq. Phase, g/L: 25.7 14.8 14.7 24.8 11.5 5.4 Rich OA collected, kg: 4.05 7.47 6.03 7.37 12.82 14.0 [i-B] in OA collected, wt %: 2.22 5.72 8.17 2.83 5.93 5.04 Test time, min: 122 94 41.5 31.0 25.3 16.0 Overall i-BuOH M.T. Rate, g/L/hr 1.23 7.81 20.76 12.62 64.52 92.52 kLa, hr{circumflex over ( )}(-1) 0.05 0.70 2.58 0.54 4.29 10.06 (kLa/Us) 0.12 0.69 1.40 0.18 0.67 0.91

[0337] An aqueous phase that simulates a fermentation broth from liquefied corn mash (containing undissolved solids) half way through SSF was synthesized by adding some of the wet cake (which was initially obtained by removing the solids from liquefied corn mash) to diluted centrate. Some water was also added to dilute the liquid phase held up in the wet cake because this liquid has the same composition as the concentrated centrate. Diluted supernate (17.8 kg), 13.0 kg wet cake (contains .about.18 wt % undissolved mash solids), 5.0 kg H.sub.2O, and 0.83 kg i-BuOH were mixed together yielding 36.6 kg of a slurry containing approximately 6.3 wt % undissolved solids and a liquid phase consisting of approximately 13 wt % liquefied starch and approximately 2.4 wt % i-BuOH (balance H.sub.2O). This slurry mimics the composition of a fermentation broth half way through SSF of corn to i-BuOH at approximately 30% corn loadings because the level of undissolved solids and oligosaccharides found in these types of broths is approximately 6-8 wt % and 10-12 wt %, respectively.

[0338] Mass transfer tests were conducted with two different size nozzles at nozzle velocities ranging from 5 ft/s to 22 ft/s using the slurry of diluted centrate and undissolved mash solids as the aqueous phase. Three tests were done using a nozzle that has an ID of 0.76 mm, and three tests were done using a nozzle that has an ID of 2.03 mm. All tests were conducted at 30.degree. C. in the 6-inch diameter column described above using oleyl alcohol as the solvent. The results of the mass transfer tests using the slurry of diluted centrate and undissolved mash solids are shown in Table 3.

TABLE-US-00003 TABLE 3 52 53 54 49 50 51 Diluted Diluted Diluted Diluted Diluted Diluted Centrate Centrate Centrate Centrate Centrate Centrate from Liq'd from Liq'd from Liq'd from Liq'd from Liq'd from Liq'd Mash, Mash, Mash, Mash, Mash, Mash, +6.3 wt % +6.3 wt % +6.3 wt % +6.3 wt % +6.3 wt % +6.3 wt % Solids Solids Solids Solids Solids Solids MASS TRANSFER TEST CONDITIONS: Aqueous Phase 35.5 35.5 32.5 31.5 30 31.6 Volume of Aqueous Phase, L: Solvent Feed Rate, g/min: 40 64 157 249 549 853 Superficial Liq. Velocity (Us), ft/hr: 0.51 0.81 1.99 3.16 6.97 10.83 Nozzle I.D., mm: 0.76 0.76 0.76 2.03 2.03 2.03 Nozzle Velocity, ft/s 5.7 9.1 22.3 4.9 10.9 17.0 MASS TRANSFER RESULTS: Initial [i-B] in Aq. Phase, g/L: 28.1 26.0 26.2 27.6 26.3 36.8 Final [i-B] in Aq. Phase, g/L: 26.3 23.8 14.0 24.6 13.8 16.1 Rich OA collected, kg: 6.02 5.75 10.23 15.05 16.58 13.22 [i-B] in OA collected, wt %: 1.05 1.35 3.86 0.68 2.30 5.00 Test time, min: 150 90 65 60 30 15.5 Overall i-BuOH M.T. Rate, g/L/hr 0.71 1.46 11.2 3.0 25.0 80.0 kLa, hr{circumflex over ( )}(-1) 0.03 0.06 0.83 0.12 1.55 4.45 (kLa/Us) 0.06 0.07 0.42 0.04 0.22 0.41

[0339] FIG. 12 illustrates the effect of the presence of undissolved corn mash solids on the overall volumetric mass transfer coefficient, k.sub.La, for the transfer of i-BuOH from an aqueous solution of liquefied corn starch (i.e., oligosaccharides) to a dispersion of oleyl alcohol droplets flowing up through a bubble column. The oleyl alcohol was fed to the column through a 2.03 mm ID nozzle. It was discovered that the ratio of the k.sub.La for a system where the solids have been removed to the k.sub.La for a system where the solids have not been removed is 2 to 5 depending on the nozzle velocity for a 2.03 mm nozzle.

[0340] FIG. 13 illustrates the effect of the presence of undissolved corn mash solids on the overall volumetric mass transfer coefficient, k.sub.La, for the transfer of i-BuOH from an aqueous solution of liquefied corn starch (i.e., oligosaccharides) to a dispersion of oleyl alcohol droplets flowing up through a bubble column. The oleyl alcohol was fed to the column through a 0.76 mm ID nozzle. It was discovered that the ratio of the k.sub.La for a system where the solids have been removed to the k.sub.La for a system where the solids have not been removed is 2 to 4 depending on the nozzle velocity for a 0.76 mm nozzle.

Example 2

Effect of Removing Undissolved Solids on Phase Separation Between an Aqueous Phase and a Solvent Phase

[0341] This example illustrates improved phase separation between an aqueous solution of oligosaccharides derived from liquefied corn mash from which undissolved solids have been removed and a solvent phase as compared to an aqueous solution of oligosaccharides derived from liquefied corn mash from which no undissolved solids have been removed and the same solvent. Both systems contained i-BuOH. Adequate separation of the solvent phase from the aqueous phase is important for liquid-liquid extraction to be a viable separation method for practicing ISPR.

[0342] Approximately 900 g of liquefied corn mash was prepared in a 1 L glass, jacketed resin kettle. The kettle was set up with mechanical agitation, temperature control, and pH control. The following protocol was used: mixed ground corn with tap water (26 wt % corn on a dry basis), heated the slurry to 55.degree. C. while agitating, adjusted pH to 5.8 with either NaOH or H.sub.2SO.sub.4, added alpha-amylase (0.02 wt % on a dry corn basis), continued heating to 85.degree. C., adjusted pH to 5.8, held at 85.degree. C. for 2 hr while maintaining pH at 5.8, cool to 25.degree. C.

[0343] Pioneer 3335 hybrid corn, whole kernel yellow corn, was used (Pioneer Hi-Bred International, Johnston, Iowa), and it was ground in a hammer-mill using a 1 mm screen. The moisture content of the ground corn was 12 wt %, and the starch content of the ground corn was 71.4 wt % on a dry corn basis. The alpha-amylase enzyme was Liquozyme.RTM. SC DS from Novozymes (Franklinton, N.C.). The total amounts of the ingredients used were: 265.9 g ground corn (12% moisture), 634.3 g tap water, and 0.056 g Liquozyme.RTM. SC DS. The total amount of liquefied corn mash recovered was 883.5 g.

[0344] Part of the liquefied corn mash was used directly, without removing undissolved solids, to prepare the aqueous phase for phase separation tests involving solids. Part of the liquefied corn mash was centrifuged to remove most of the undissolved solids and used to prepare the aqueous phase for phase separation tests involving the absence of solids.

[0345] The solids were removed from the mash by centrifugation in a large floor centrifuge. Mash (583.5 g) was centrifuged at 5000 rpm for 20 min at 35.degree. C. yielding 394.4 g centrate and 189.0 g wet cake. It was determined that the centrate contained approximately 0.5 wt % suspended solids, and that the wet cake contained approximately 20 wt % suspended solids. This implies that the original liquefied mash contained approximately 7 wt % suspended solids. This is consistent with the corn loading and starch content of the corn used assuming most of the starch was liquefied. If all of the starch was liquefied, the centrate recovered directly from the centrifuge would have contained approximately 20 wt % dissolved oligosaccharides (liquefied starch) on a solids-free basis.

[0346] The objective of the phase separation test was to measure the effect of undissolved solids on the degree of phase separation between a solvent phase and an aqueous phase that simulates a broth that is derived from liquefied corn mash. The aqueous liquid phase contained about 20 wt % oligosaccharides, and the organic phase contained oleyl alcohol (OA) in all tests. Furthermore, i-BuOH was added to all tests to give approximately 25 g/L in the aqueous phase when the phases were at equilibrium. Two shake tests were performed. The aqueous phase for the first test (with solids) was prepared by mixing 60.0 g liquefied corn mash with 3.5 g i-BuOH. The aqueous phase for the second test (solids removed) was prepared by mixing 60.0 g centrate which was obtained from the liquefied corn mash by removing the solids, with 3.5 g i-BuOH. Oleyl alcohol (15.0 g, 80/85% grade, Cognis Corporation, Cincinnati, Ohio) was added to each of the shake test bottles. The oleyl alcohol formed a separate liquid phase on top of the aqueous phase in both bottles resulting in a mass ratio of phases: Aq Phase/Solvent Phase to be about 1/4. Both bottles were shaken vigorously for 2 min to intimately contact the aqueous and organic phases and enable the 1-BuOH to approach equilibrium between the two phases. The bottles were allowed to set for 1 hr. Photographs were taken at various times (0, 15, 30, and 60 min) to observe the effect of undissolved solids on phase separation in systems that contain an aqueous phase derived from liquefied corn mash, a solvent phase containing oleyl alcohol, and i-BuOH. Time zero (0) corresponds to the time immediately after the two minute shake period was complete.

[0347] The degree of separation between the organic (solvent) phase and the aqueous phase as a function of time for the system with solids (from liquefied corn mash) and the system where solids were removed (liquid centrate from liquefied corn mash) appeared about the same in both systems at any point in time. The organic phase was a slightly darker and cloudier, and the interface was a little less distinct (thicker "rag" layer around the interface) for the case with solids. However, for an extractive fermentation where the solvent is operated continuously, the composition of the top of the organic phase is of interest for the process downstream of the extractive fermentation wherein the next step is a distillation.

[0348] It may be advantageous to minimize the amount of microorganisms in the top of the organic phase because the microorganisms will be thermally deactivated in the distillation column. It may be advantageous to minimize the amount of undissolved solvents in the top of the organic phase because they could plug the distillation column, foul the reboiler, cause poor phase separation in the solvent/water decanter located at the base of the column, or any combination of the previously mentioned concerns. It may be advantageous to minimize the amount of phase water in the top of the organic phase. Phase water is water that exists as a separate aqueous phase. Additional amounts of aqueous phase will increase the loading and energy requirement in the distillation column. Ten milliliter (10 mL) samples were removed from the top of the organic layers from the "With Solids" and "Solids Removed" bottles, and both samples were centrifuged to reveal and compare the composition of the organic phases in the "With Solids" and "Solids Removed" bottles after 60 min of settling time. The results show that the organic phases at the end of both shake tests contained some undesired phase(s) (both organic phases are cloudy). However, the results also show that the top layer from the phase separation test involving centrate, from which solids were removed, contained essentially no undissolved solids. On the other hand, undissolved solids are clearly seen at the bottom of the 10 mL sample collected from the top of the organic phase of the test involving mash. It was estimated that 3% of the sample collected from the top of the organic layer wash mash solids. If the rich solvent phase exiting the fermentor of an extractive fermentation process contained 3% undissolved solids, one or more of the following problems could occur: loss of significant amount of microorganisms, fouling of solvent column reboiler, plugging of solvent column. The results also show that the top layer from the phase separation test involving centrate contained less phase water. Table 4 shows an estimate of the relative amount of phases that were dispersed throughout the upper "organic" layers in both shake test bottles after 60 min of settling time.

TABLE-US-00004 TABLE 4 Approximate composition of organic (top) layer from shake tests after 60 min Top Layer from Top Layer from "With Solids" "Solids Removed" Shake Test Shake Test Organic (solvent) Phase: 82% 87% Aqueous (water) Phase: 15% 13% Undissolved Solids: 3% 0%

[0349] This example shows that removing most of the undissolved solids from liquefied corn mash results in improved phase separation after the liquid, aqueous phase obtained from the mash is contacted with a solvent, such as oleyl alcohol. This example shows that the upper phase obtained after phase separation will contain significantly less undissolved solids if the solids are removed first before contacting the liquid part of mash with an organic solvent. This demonstrates advantages of minimizing the undissolved solids content of mash in the upper ("organic") layer of the phase separation for an extractive fermentation.

[0350] Samples were also allowed to sit for several days after completion of sample preparation before repeating the phase separation shake test described in this example. The sample with solids consisted of liquefied corn mash, i-BuOH, and oleyl alcohol, and the sample with solids removed consisted of centrate which was produced by removing most of the undissolved solids from liquefied corn mash, i-BuOH, and oleyl alcohol. The liquefied mash contained approximately 7 wt % suspended solids, and the centrate produced from the mash contained approximately 0.5 wt % suspended solids. If all of the starch in the ground corn was liquefied, the liquid phase in the liquefied mash and the centrate produced from the mash would have contained approximately 20 wt % dissolved oligosaccharides (liquefied starch) on a solids-free basis. Both samples contained oleyl alcohol in an amount to give a mass ratio of phases: Solvent Phase/Aq Phase to be about 1/4. Furthermore, i-BuOH was added to all tests to give approximately 25 g/L in the aqueous phase when the phases were at equilibrium.

[0351] The objective of the phase separation test was to measure the effect of undissolved solids on the degree of phase separation after the multi-phase mixtures sat at room temperature for several days to mimic the potential change in properties of the system throughout an extractive fermentation. Two shake tests were performed. Both bottles were shaken vigorously for 2 min to intimately contact the aqueous and organic phases. The bottles were allowed to sit for 1 hr. Photographs were taken at various times (0, 2, 5, 10, 20, and 60 min) to observe the effect of undissolved solids on phase separation in these systems which had aged for several days. Time zero (0) corresponds to the time immediately after the bottles were placed on the bench.

[0352] Phase separation started to occur in the sample where solids were removed after 2 min. It appeared that almost complete phase separation had occurred in the sample where solids had been removed after only 5-10 min based on the fact that the organic phase occupied approximately 25% of the total volume of the two phase mixture. It would be expected that complete separation would be indicated if the organic phase occupied approximately 20% of the total volume, since that corresponds to the initial ratio of phases. No apparent phase separation occurred in the sample where solids were not removed even after 1 hr.

[0353] The composition of the upper phase for both samples was also compared. The composition of the upper phase has implications for the process downstream of the extractive fermentation wherein the next step is a distillation. It is advantageous to minimize the amount of microorganisms in the top of the organic phase because the microorganisms will be thermally deactivated in the distillation column. Another component to minimize in the top of the organic phase is the amount of undissolved solids because the solids could plug the distillation column, foul the reboiler, cause poor phase separation in the solvent/water decanter located at the base of the column, or any combination of the previously mentioned concerns. In addition, another component to minimize in the top of the organic phase is the amount of phase water which is water that exists as a separate aqueous phase, because this additional amount of aqueous phase will increase the loading and energy requirement in the subsequent distillation column.

[0354] Ten milliliter (10 mL) samples were removed from the top of the organic layers from the "With Solids" and "Solids Removed" bottles, and both samples were centrifuged to reveal and compare the composition of the organic phases in the "With Solids" and "Solids Removed" bottles after 60 min of settling time. The composition of the sample collected from the top of the "With Solids" sample confirms that essentially no phase separation occurred in that sample within 60 min. Specifically, the ratio of the solvent phase to total aqueous phase (aqueous liquid+suspended solids) in the sample collected from the top of the "With Solids" shake test bottle is approximately 1/4 w/w, which is the same ratio used to prepare the sample prior to the test. Also, the amount of undissolved solids in the sample collected from the top of the "With Solids" shake test bottle is approximately the same as what is found in liquefied corn mash, which shows that essentially no solids settled in this shake test bottle within 60 min. On the other hand, the top layer from the phase separation test involving centrate ("Solids Removed") from which solids were removed, contained essentially no undissolved solids. The results also show that the top layer from the phase separation test involving centrate contained less phase water. This is indicated by the fact that the ratio of the solvent phase to aqueous phase in that sample bottle is approximately 1/1 w/w, which shows that the organic phase was enriched with solvent (oleyl alcohol) in the test where solids were removed. Table 5 shows an estimate of the relative amount of phases that were dispersed throughout the upper "organic" layers in both shake test bottles after 60 min of settling time.

TABLE-US-00005 TABLE 5 Approximate composition of organic (top) layer from shake tests after 60 min Top Layer from Top Layer from "With Solids" "Solids Removed" Shake Test Shake Test Organic (solvent) Phase: 19% 50% Aqueous (water) Phase: 47% 50% Undissolved Solids: 34% 0%

[0355] This example shows that removing undissolved solids from liquefied corn mash that contains i-BuOH, contacting it with a solvent phase, letting it set for several days, and mixing the phases again results in improved phase separation when compared to a sample where undissolved solids were not removed from the liquefied mash. In fact, this example shows that essentially no phase separation occurs in the sample where undissolved solids were not removed even after 60 min. This example shows that the upper phase obtained after phase separation contains significantly less undissolved solids if the solids are removed first before contacting the liquid part of mash with an organic solvent. This is important because two of the most important species that should be minimized in the upper ("organic") layer of the phase separation for an extractive fermentation are the level of microorganisms and the level of undissolved solids from mash. The previous example showed that removing solids from liquefied corn mash results in improved phase separation shortly after the aqueous phase is contacted with a solvent phase. This would allow extractive fermentation to be viable at earlier times in the fermentation. This example also shows that removing solids from liquefied corn mash results in improved phase separation in aged samples that contain an aqueous phase (oligosaccharide solution with solids removed) that has been contacted with a solvent phase. This would also allow extractive fermentation to be viable at later times in the fermentation.

Example 3

Effect of Removing Undissolved Solids on the Loss of ISPR Extraction Solvent--Disk Stack Centrifuge

[0356] This example demonstrates the potential for reducing solvent losses via DDGS generated by the extractive fermentation process by removing undissolved solids from the corn mash prior to fermentation using a semi-continuous disk-stack centrifuge.

[0357] Approximately 216 kg liquefied corn mash was prepared in a jacketed stainless steel reactor. The reactor was set up with mechanical agitation, temperature control, and pH control. The protocol used was as follows: mixed ground corn with tap water (25 wt % corn on a dry basis), heated the slurry to 55.degree. C. while agitating at 400 rpm, adjusted pH to 5.8 with either NaOH or H.sub.2SO.sub.4, added alpha-amylase (0.02 wt % on a dry corn basis), continued heating to 85.degree. C., adjusted pH to 5.8, held at 85.degree. C. for 30 min while maintaining pH at 5.8, heated to 121.degree. C. using live steam injection, held at 121.degree. C. for 30 min to simulate a jet cooker, cooled to 85.degree. C., adjusted pH to 5.8, added second charge of alpha-amylase (0.02 wt % on a dry corn basis), held at 85.degree. C. for 60 min while maintaining pH at 5.8 to complete liquefaction. The mash was then cooled to 60.degree. C. and transferred to the centrifuge feed tank.

[0358] Pioneer 3335 hybrid corn, whole kernel yellow corn, was used (Pioneer Hi-Bred International, Johnston, Iowa), and it was ground in a hammer-mill using a 1 mm screen. The moisture content of the ground corn was 12 wt %, and the starch content of the ground corn was 71.4 wt % on a dry corn basis. The alpha-amylase enzyme was Liquozyme.RTM. SC DS from Novozymes (Franklinton, N.C.). The amounts of the ingredients used were: 61.8 kg ground corn (12% moisture), 147.3 kg tap water, a solution of 0.0109 kg Liquozyme.RTM. SC DS in 1 kg water for first alpha-amylase charge, another solution of 0.0109 kg Liquozyme.RTM. SC DS in 1 kg water for second alpha-amylase charge (after the cook stage). About 5 kg H.sub.2O was added to the batch via steam condensate during the cook stage. A total of 0.25 kg NaOH (12.5 wt %) and 0.12 kg H.sub.2SO.sub.4 (12.5 wt %) were added throughout the run to control pH. The total amount of liquefied corn mash recovered was 216 kg.

[0359] The composition of the final liquefied corn mash slurry was estimated to be approximately 7 wt % undissolved solids and 93 wt % liquid. The liquid phase contained about 19 wt % (190 g/L) liquefied starch (soluble oligosaccharides). The rheology of the mash is important regarding the ability to separate the slurry into its components. The liquid phase in the mash was determined to be a Newtonian fluid with a viscosity of about 5.5 cP at 30.degree. C. The mash slurry was determined to be a shear-thinning fluid with a bulk viscosity of about 10 to 70 cP at 85.degree. C. depending on shear rate.

[0360] The liquefied mash (209 kg, 190 L) was centrifuged using a disk-stack split-bowl centrifuge (Alfa Laval Inc., Richmond, Va.). The centrifuge operated in semi-batch mode with continuous feed, continuous centrate outlet, and batch discharge of the wet cake. Liquefied corn mash was continuously fed at a rate of 1 L/min, clarified centrate was removed continuously, and wet cake was periodically discharged every 4 min. To determine an appropriate discharge interval for the solids from the disk stack, a sample of the mash to be fed to the disk stack was centrifuged in a high-speed lab centrifuge. Mash (48.5 g) was spun at 11,000 rpm (about 21,000 g for about 10 min at room temperature. Clarified centrate (36.1 g) and 12.4 g pellet (wet cake) were recovered. It was determined that the clarified centrate contained about 0.3 wt % undissolved solids and that the pellet (wet cake) contained about 27 wt % undissolved solids. Based on this data, a discharge interval of 4 min was chosen for operation of the disk stack centrifuge.

[0361] The disk stack centrifuge was operated at 9000 rpm (6100 g) with a liquefied corn mash feed rate of 1 L/min and at about 60.degree. C. Mash (209 kg) was separated into 155 kg clarified centrate and 55 kg wet cake. The split, defined as (amount of centrate)/(amount of mash fed), achieved by the semi-continuous disk stack was similar to the split achieved in the batch centrifuge. The split for the disk stack semi-batch centrifuge operating at 6100 g, 1 L/min feed rate, and 4 min discharge interval was (155 kg/209 kg)=74%, and the split for the lab batch centrifuge operating at 21,000 g for 10 min was (36.1 g/48.5 g)=74%.

[0362] A 45 mL sample of the clarified centrate recovered from the disk stack centrifuge was spun down in a lab centrifuge at 21,000 g for 10 min to estimate the level of suspended solids in the centrate. About 0.15-0.3 g undissolved solids were recovered from the 45 mL of centrate. This corresponds to 0.3-0.7 wt % undissolved solids in the centrate which is about a ten-fold reduction in undissolved solids from mash fed to the centrifuge. It is reasonable to assume that the ISPR extraction solvent losses via DDGS could be reduced by about an order of magnitude if the level of undissolved solids present in extractive fermentation is reduced by an order of magnitude using some solid/liquid separation device or combination of devices to remove suspended solids from the corn mash before fermentation. Minimizing solvent losses via DDGS is an important factor in the economics and DDGS quality for an extractive fermentation process.

Example 4

Effect of Removing Undissolved Solids on the Loss of ISPR Extraction Solvent--Bottle Spin Test

[0363] This example demonstrates the potential for reducing solvent losses via DDGS generated by the extractive fermentation process by removing undissolved solids from the corn mash prior to fermentation using a centrifuge.

[0364] A lab-scale bottle spin test was performed using liquefied corn mash. The test simulates the operating conditions of a typical decanter centrifuge used to remove undissolved solids from whole stillage in a commercial ethanol plant. Decanter centrifuges in commercial ethanol plants typically operate at a relative centrifugal force (RCF) of about 3000 g and a whole stillage residence time of about 30 seconds. These centrifuges typically remove about 90% of the suspended solids in whole stillage which contains about 5% to 6% suspended solids (after the beer column), resulting in thin stillage which contains about 0.5% suspended solids.

[0365] Liquefied corn mash was made according to the protocol described in Example 2. About 10 mL mash was placed in a centrifuge tube. The sample was centrifuged at an RCF of about 3000 g (4400 rpm rotor speed) for a total of 1 min. The sample spent about 30-40 seconds at 3000 g and a total of 20-30 seconds at speeds less than 3000 g due to acceleration and deceleration of the centrifuge. The sample temperature was about 60.degree. C.

[0366] The mash (10 mL) which contained about 7 wt % suspended solids was separated into about 6.25 mL clarified centrate and 3.75 mL wet cake (pellet at the bottom of the centrifuge tube). The split, defined as (amount of centrate)/(amount of original mash charged), achieved by the bottle spin test was about 62%. It was determined that the clarified centrate contained about 0.5 wt % suspended solids which is more than a ten-fold decrease in suspend solids compared to the level of suspended solids in the original mash. It was also determined that the clarified pellet contained about 18 wt % suspended solids.

[0367] Table 6 summarizes the suspended (undissolved) solids mass balance for the bottle spin test at conditions representative of the operation of a decanter centrifuge to convert whole stillage to thin stillage in a commercial ethanol process. All values given in Table 6 are approximate.

TABLE-US-00006 TABLE 6 Volume, mL Suspend Solids, wt % Liquefied Corn Mash charge: 10 7% Clarified Centrate: 6.25 0.5% Wet Cake (pellet): 3.75 18% Performance Summary Split: 62% Centrate Clarity: 0.5 wt % suspended solids Cake (pellet) Dryness: 18 wt % suspended solids % Removal of Suspended Solids: 95% removal from liquefied mash

[0368] It was also determined that the centrate contained about 190 g/L dissolved oligosaccharides (liquefied starch). This is consistent with the assumption that most of the starch in the ground corn was liquefied (i.e., hydrolyzed to soluble oligosaccharides) in the liquefaction process based on the corn loading used (about 26 wt % on a dry corn basis) and the starch content of the corn used to produce the liquefied mash (about 71.4 wt % starch on a dry corn basis). Hydrolyzing most of the starch in the ground corn at a 26% dry corn loading will result in about 7 wt % suspended (undissolved) solids in the liquefied corn mash charged to the centrifuge used for the bottle spin test.

[0369] The fact that the clarified centrate contained only about 0.5 wt % undissolved solids indicates that the conditions used in the bottle spin test resulted in more than a ten-fold reduction in undissolved solids from mash charged. If this same solids removal performance could be achieved by a continuous decanter centrifuge before fermentation, it is reasonable to assume that the ISPR extraction solvent losses in the DDGS could be reduced by about an order of magnitude. Minimizing solvent losses via DDGS is an important factor in the economics and DDGS quality for an extractive fermentation process.

Example 5

Removal of Corn Oil by Removing Undissolved Solids

[0370] This example demonstrates the potential to remove and recover corn oil from corn mash by removing the undissolved solids prior to fermentation. The effectiveness of the extraction solvent may be improved if corn oil is removed via removal of the undissolved solids. In addition, removal of corn oil via removal of the undissolved solids may also minimize any reduction in solvent partition coefficient and potentially resulting an improved extractive fermentation process.

[0371] Approximately 1000 g liquefied corn mash was prepared in a 1 L glass, jacketed resin kettle. The kettle was set up with mechanical agitation, temperature control, and pH control. The following protocol was used: mixed ground corn with tap water (26 wt % corn on a dry basis), heated the slurry to 55.degree. C. while agitating, adjusted pH to 5.8 with either NaOH or H.sub.2SO.sub.4, added alpha-amylase (0.02 wt % on a dry corn basis), continued heating to 85.degree. C., adjusted pH to 5.8, held at 85.degree. C. for 2 hr while maintaining pH at 5.8, cool to 25.degree. C.

[0372] Pioneer 3335 hybrid corn, whole kernel yellow corn, was used (Pioneer Hi-Bred International, Johnston, Iowa), and it was ground in a hammer-mill using a 1 mm screen. The moisture content of the ground corn was about 11.7 wt %, and the starch content of the ground corn was about 71.4 wt % on a dry corn basis. The alpha-amylase enzyme was Liquozyme.RTM. SC DS from Novozymes (Franklinton, N.C.). The total amounts of the ingredients used were: 294.5 g ground corn (11.7% moisture), 705.5 g tap water, and 0.059 g Liquozyme.RTM. SC DS. Water (4.3 g) was added to dilute the enzyme, and a total of 2.3 g of 20% NaOH solution was added to control pH. About 952 g of mash was recovered.

[0373] The liquefied corn mash was centrifuged at 5000 rpm (7260 g) for 30 min at 40.degree. C. to remove the undissolved solids from the aqueous solution of oligosaccharides. Removing the solids by centrifugation also resulted in the removal of free corn oil as a separate organic liquid layer on top of the aqueous phase. Approximately 1.5 g of corn oil was recovered from the organic layer floating on top of the aqueous phase. It was determined by hexane extraction that the ground corn used to produce the liquefied mash contained about 3.5 wt % corn oil on a dry corn basis. This corresponds to about 9 g corn oil fed to the liquefaction process with the ground corn.

[0374] After recovering the corn oil from the liquefied mash, the aqueous solution of oligosaccharides was decanted away from the wet cake. About 617 g liquefied starch solution was recovered leaving about 334 g wet cake. The wet cake contained most of the undissolved solids that were in the liquefied mash. The liquefied starch solution contained about 0.2 wt % undissolved solids. The wet cake contained about 21 wt % undissolved solids. The wet cake was washed with 1000 g tap water to remove the oligosaccharides still in the cake. This was done by mixing the cake with the water to form a slurry. The slurry was then centrifuged under the same conditions used to centrifuge the original mash in order to recover the washed solids. Removing the washed solids by centrifuging the wash slurry also resulted in the removal of some additional free corn oil that must have remained with the original wet cake produced from the liquefied mash. This additional corn oil was observed as a separate, thin, organic liquid layer on top of the aqueous phase of the centrifuged wash mixture. Approximately 1 g of additional corn oil was recovered from the wash process.

[0375] The wet solids were washed two more times using a 1000 g tap water each time to remove essentially all of the liquefied starch. No visible additional corn oil was removed from the 2.sup.nd and 3.sup.rd water washes of the mash solids. The final washed solids were dried in a vacuum oven overnight at 80.degree. C. and about 20 inches Hg vacuum. The amount of corn oil remaining in the dry solids, presumably still in the germ, was determined by hexane extraction. A sample of relatively dry solids (3.60 g, about 2 wt % moisture) contained 0.22 g corn oil. This result corresponds to 0.0624 g corn oil/g dry solids. This was for washed solids which means there are no residual oligosaccharides in the wet solids. After centrifuging the liquefied corn mash to separate the layer of free corn oil and the aqueous solution of oligosaccharides from the wet cake, it was determined that about 334 g wet cake containing about 21 wt % undissolved solids remained. This corresponds to the wet cake comprising about 70.1 g undissolved solids. At 0.0624 g corn oil/g dry solids, the solids in the wet cake should contain about 4.4 g corn oil.

[0376] In summary, approximately 1.5 g free corn oil was recovered by centrifuging the liquefied mash. An additional 1 g free corn oil was recovered by centrifuging the first (water) wash slurry which was generated to wash the original wet cake produced from the mash. Finally, it was determined that the washed solids still contained about 4.4 g corn oil. It was also determined that the corn charged to the liquefaction contained about 9 g corn oil. Therefore, a total of 6.9 g corn oil was recovered from the following process steps: liquefaction, removal of solids from liquefied mash, washing of the solids from the mash, and the final washed solids. Consequently, approximately 76% of the total corn oil in the corn fed to liquefaction was recovered during the liquefaction and solids removal process described here.

Example 6

Extractive Fermentation Using Mash and Centrate as the Sugar Source

[0377] This example describes extractive fermentations performed using corn mash and corn mash centrate as the sugar source. Corn mash centrate was produced by removing undissolved solids from the corn mash prior to fermentation. Four extractive fermentations were conducted side-by side, two with liquefied corn mash as the sugar source (solids not removed) and two with liquefied mash centrate (aqueous solution of oligosaccharides) obtained by removing most of the undissolved solids from liquefied corn mash. Oleyl alcohol (OA) was added to two of the fermentations, one with solids and one with solids removed, to extract the product (i-BuOH) from the broth as it was formed. A mixture of corn oil fatty acids (COFA) was added to the other two of the fermentations, one with solids and one with solids removed, to extract the product from the broth as it was formed. The COFA was made by hydrolyzing corn oil. The purpose of these fermentations was to test the effect of removing solids on phase separation between the solvent and broth and to measure the amount of residual solvent trapped in the undissolved solids recovered from fermentation broths where solids were or were not removed.

Preparation of Liquefied Corn Mash

[0378] Approximately 31 kg liquefied corn mash was prepared in a 30 L jacketed glass resin kettle. The reactor was outfitted with mechanical agitation, temperature control, and pH control. The protocol used was as follows: mix ground corn with tap water (40 wt % corn on a dry basis), heat the slurry to 55.degree. C. while agitating at 250 rpm, adjust pH to 5.8 with either NaOH or H.sub.2SO.sub.4, add a dilute aqueous solution of alpha-amylase (0.16 wt % on a dry corn basis), hold at 55.degree. C. for 60 min, heat to 95.degree. C., adjust pH to 5.8, hold at 95.degree. C. for 120 min while maintaining pH at 5.8 to complete liquefaction. The mash was transferred into sterile centrifuge bottles to prevent contamination.

[0379] The corn used was whole kernel yellow corn (Pioneer Hi-Bred International, Johnston, Iowa), and it was ground in a pilot-scale hammer-mill using a 1 mm screen. The moisture content of the ground corn was about 12 wt %, and the starch content of the ground corn was about 71.4 wt % on a dry corn basis. The alpha-amylase enzyme used was Spezyme.RTM. Fred-L (Genencor.RTM., Palo Alto, Calif.). The amounts of the ingredients used were: 14.1 kg ground corn (12% moisture), 16.9 kg tap water, a solution of alpha-amylase consisting of 19.5 g Spezyme.RTM. Fred-L in 2.0 kg water. The alpha-amylase was sterile filtered. A total of 0.21 kg NaOH (17 wt %) was added throughout the run to control pH.

[0380] It was estimated that the liquefied corn mash contained approximately 28 wt % (about 280 g/L) of liquefied starch based on the corn loading used, starch content of the corn, and assuming all the starch was hydrolyzed during liquefaction. The mash was prepared with a higher concentration of oligosaccharides than was desired in the fermentations to allow for dilution when adding the nutrients, inoculum, glucoamylase, and base to the initial fermentation broth. After dilution by addition of nutrients, inoculum, glucoamylase, and base, the expected total initial soluble sugars in the mash (solids not removed) was about 250 g/L.

[0381] About 13.9 kg liquefied mash was centrifuged using a bottle centrifuge which contained six 1 L bottles. The centrifuge was operated at 5000 rpm (7260 RCF) for 20 min at room temperature. The mash was separated into about 5.5 kg clarified centrate and about 8.4 kg wet cake (the pellet at the bottom of the centrifuge bottles). The split, defined as (amount of centrate)/(amount of mash fed), was about (5.5 kg/13.9 kg)=40%.

[0382] Solids were not removed from the mash charged to the 2010Y034 and 2010Y036 fermentations described below. The centrate charged to fermentations 2010Y033 and 2010Y035 (also described below) was produced by removing by centrifugation most of the suspended solids from mash according to the protocols above.

General Methods for Fermentation

[0383] Seed Flask Growth

[0384] A Saccharomyces cerevisiae strain (with deletions of pdc1, pdc5, and pdc6) that was engineered to produce isobutanol from a carbohydrate source was grown to 0.55-1.1 g/L dcw (OD.sub.600 1.3-2.6--Thermo Helios .alpha. Thermo Fisher Scientific Inc., Waltham, Mass.) in seed flasks from a frozen culture. The Saccharomyces cerevisiae strain is described in U.S. Patent Application Publication No. 2012/0164302, incorporated herein by reference. The culture was grown at 26.degree. C. in an incubator rotating at 300 rpm. The frozen culture was previously stored at -80.degree. C. The composition of the first seed flask medium was: [0385] 3.0 g/L dextrose [0386] 3.0 g/L ethanol, anhydrous [0387] 3.7 g/L ForMedium.TM. Synthetic Complete Amino Acid (Kaiser) Drop-Out: without HIS, without URA (Reference No. DSCK162CK) [0388] 6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920).

[0389] Twelve milliliters (12 mL) from the first seed flask culture was transferred to a 2 L flask and grown at 30.degree. C. in an incubator rotating at 300 rpm. The second seed flask has

[0390] 220 mL of the following medium: [0391] 30.0 g/L dextrose [0392] 5.0 g/L ethanol, anhydrous [0393] 3.7 g/L ForMedium.TM. Synthetic Complete Amino Acid (Kaiser) Drop-Out: without HIS, without URA (Reference No. DSCK162CK) [0394] 6.7 g/L Difco Yeast Nitrogen Base without amino acids (No. 291920) [0395] 0.2M MES Buffer titrated to pH 5.5-6.0.

[0396] The culture was grown to 0.55-1.1 g/L dcw (OD.sub.600 1.3-2.6). An addition of 30 mL of a solution containing 200 g/L peptone and 100 g/L yeast extract was added at this cell concentration. Then an addition of 300 mL of 0.2 uM filter sterilized, 90-95% oleyl alcohol (Cognis Corporation, Cincinnati, Ohio) was added to the flask. The culture continued to grow to >4 g/L dcw (OD.sub.600>10) before being harvested and added to the fermentation.

Fermentation Preparation

[0397] Initial Fermentor Preparation

[0398] A glass jacked, 2 L fermentor (Sartorius AG, Goettingen, Germany) was charged with liquefied mash either with or without solids (centrate). A pH probe (Hamilton Easyferm Plus K8, part number: 238627, Hamilton Bonaduz AG, Bonaduz, Switzerland) was calibrated through the Sartorius DCU-3 Control Tower Calibration menu. The zero was calibrated at pH=7. The span was calibrated at pH=4. The probe was then placed into the fermentor, through the stainless steel head plate. A dissolved oxygen probe (pO.sub.2 probe) was also placed into the fermentor through the head plate. Tubing used for delivering nutrients, seed culture, extracting solvent, and base were attached to the head plate and the ends were foiled. The entire fermentor was placed into an autoclave (Steris Corporation, Mentor, Ohio) and sterilized in a liquid cycle for 30 min.

[0399] The fermentor was removed from the autoclave and placed on a load cell. The jacket water supply and return line was connected to the house water and clean drain, respectively. The condenser cooling water in and water out lines were connected to a 6-L recirculating temperature bath running at 7.degree. C. The vent line that transfers the gas from the fermentor was connected to a transfer line that was connected to a Thermo mass spectrometer (Prima dB, Thermo Fisher Scientific Inc., Waltham, Mass.). The sparger line was connected to the gas supply line. The tubing for adding nutrients, extract solvent, seed culture, and base was plumbed through pumps or clamped closed. The autoclaved material, 0.9% w/v NaCl was drained prior to the addition of liquefied mash.

[0400] Lipase Treatment Post-Liquefaction

[0401] The fermentor temperature was set to 55.degree. C. instead of 30.degree. C. after the liquefaction cycle was complete. The pH was manually controlled at pH=5.8 by making bolus additions of acid or base when needed. A lipase enzyme stock solution was added to the fermentor to a final lipase concentration of 10 ppm. The fermentor was held at 55.degree. C., 300 rpm, and 0.3 slpm N.sub.2 overlay for >6 hr. After the lipase treatment was complete the fermentor temperature was set to 30.degree. C.

Nutrient Addition Prior to Inoculation

[0402] Added 7.0 mL/L (post-inoculation volume) of ethanol (200 proof, anhydrous) just prior to inoculation. Added thiamine to 20 mg/L final concentration just prior to inoculation. Added 100 mg/L nicotinic acid just prior to inoculation.

Fermentor Inoculation

[0403] The fermentor pO.sub.2 probe was calibrated to zero while N.sub.2 was being added to the fermentor. The fermentor pO.sub.2 probe was calibrated to its span with sterile air sparging at 300 rpm. The fermentor was inoculated after the second seed flask was >4 g/L dcw. The shake flask was removed from the incubator/shaker for 5 min allowing a phase separation of the oleyl alcohol phase and the aqueous phase. The aqueous phase (55 mL) was transferred to a sterile, inoculation bottle. The inoculum was pumped into the fermentor through a peristaltic pump.

Oleyl Alcohol or Corn Oil Fatty Acids Addition after Inoculation

[0404] Added 1 L/L (post-inoculation volume) of oleyl alcohol or corn oil fatty acids immediately after inoculation

Fermentor Operating Conditions

[0405] The fermentor was operated at 30.degree. C. for the entire growth and production stages. The pH was allowed to decrease from a pH between 5.7-5.9 to a control set-point of 5.2 without adding any acid. The pH was controlled for the remainder of the growth and production stage at a pH=5.2 with ammonium hydroxide. Sterile air was added to the fermentor, through the sparger, at 0.3 slpm for the remainder of the growth and production stages. The pO.sub.2 was set to be controlled at 3.0% by the Sartorius DCU-3 Control Box PID control loop, using stir control only, with the stirrer minimum being set to 300 rpm and the maximum being set to 2000 rpm. The glucose was supplied through simultaneous saccharification and fermentation of the liquefied corn mash by adding a .alpha.-amylase (glucoamylase). The glucose was kept excess (1-50 g/L) for as long as starch was available for saccharification.

Sample A

[0406] Experiment identifier 2010Y033 included: Seed Flask Growth method, Initial Fermentor Preparation method with corn mash that excludes solids, Lipase Treatment Post-Liquefaction, Nutrient Addition Prior to Inoculation method, Fermentor Inoculation method, Fermentor Operating Conditions method, and all of the Analytical methods. Corn oil fatty acid was added in a single batch between 0.1-1.0 hr after inoculation.

Sample B

[0407] Experiment identifier 2010Y034 included: Seed Flask Growth method, Initial Fermentor Preparation method with corn mash that includes solids, Lipase Treatment Post-Liquefaction, Nutrient Addition Prior to Inoculation method, Fermentor Inoculation method, Fermentor Operating Conditions method, and all of the Analytical methods. Corn oil fatty acid was added in a single batch between 0.1-1.0 hr after inoculation.

Sample C

[0408] Experiment identifier 2010Y035 included: Seed Flask Growth method, Initial Fermentor Preparation method with corn mash that excludes solids, Nutrient Addition Prior to Inoculation method, Fermentor Inoculation method, Fermentor Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation.

Sample D

[0409] Experiment identifier 2010Y036 included: Seed Flask Growth method, Initial Fermentor Preparation method with corn mash that includes solids, Nutrient Addition Prior to Inoculation method, Fermentor Inoculation method, Fermentor Operating Conditions method, and all of the Analytical methods. Oleyl alcohol was added in a single batch between 0.1-1.0 hr after inoculation. Results for Samples A-D are shown in Table 7.

TABLE-US-00007 TABLE 7 Fermentation conditions and results for Samples A-D Post- Liquefaction Glucose g/kg Undissolved Equivalents glucose Effective Experimental Active Solids Extracting Charged consumed isobutanol Sample ID Lipase Removed Solvent g/kg at EOR g/L A 2010Y033 Yes Yes Corn oil 257 257 30.9 fatty acids B 2010Y034 Yes No Corn oil 239 239 17.3 fatty acids C 2010Y035 No Yes Oleyl 263 72 15.7 alcohol D 2010Y036 No No Oleyl 241 101 20 alcohol

Example 7

Effect of Removing Undissolved Solids from the Fermentor Feed on Improvement in Fermentor Volume Efficiency

[0410] This example demonstrates the effect of removing undissolved solids from the mash prior to fermentation on fermentor volume efficiency. Undissolved solids in corn mash occupy at least 5% of the mash volume depending on corn loading and content starch content. Removing solids before fermentation enables at least 5% more sugar to be charged to the fermentor thus increasing batch productivity.

[0411] It was estimated that the liquefied corn mash produced in Example 5 contained approximately 28 wt % (280 g/L) liquefied starch based on the corn loading used (40% dry corn basis), starch content of the corn (71.4% dry corn basis), and assuming all the starch was hydrolyzed to soluble oligosaccharides during liquefaction. The mash was prepared with a higher concentration of oligosaccharides than was desired in the fermentations to allow for dilution when adding the nutrients, inoculum, glucoamylase, and base to the initial fermentation broth. The mash was diluted by approximately 10% after adding these ingredients. Therefore, the expected concentration of liquefied starch in the mash (including solids) at the beginning of fermentations 2010Y034 and 2010Y036 was about 250 g/L. The actual glucose equivalents charged to the 2010Y034 and 2010Y036 fermentations was measured to be 239 g/kg and 241 g/kg, respectively. By comparison, the glucose equivalents charged to the 2010Y033 and 2010Y035 fermentations was measured to be 257 g/kg and 263 g/kg, respectively. Note that the feed to these fermentations was centrate (mash from which most of the solids had been removed). Approximately 1.2 L of the sugar source (mash or centrate) was charged to each fermentation. Therefore, based on this data, approximately 8.3% more sugar was charged to the fermentors which used centrate (2010Y033 and 2010Y035) compared to mash (2010Y034 and 2010Y036). These results demonstrate that removing undissolved solids from corn mash prior to fermentation can result in a significant increase in sugar charged per unit volume. This implies that when solids are present, they occupy valuable fermentor volume. If solids are removed from the feed, more sugar may be added ("fit") to the fermentor due to the absence of undissolved solids. This example demonstrates that fermentor volume efficiency can be significantly improved by removing undissolved solids from the mash prior to fermentation.

Example 8

Effect of Removing Undissolved Solids on Phase Separation Between the Extraction Solvent and the Broth--Extractive Fermentation

[0412] This example demonstrates improved separation between the solvent phase and the broth phase during and after an extractive fermentation process by removing undissolved solids from the corn mash prior to fermentation. Two extractive fermentations were conducted side-by side, one with liquefied corn mash as the sugar source (solids not removed) and one with centrate (aqueous solution of oligosaccharides) which was generated by removing most of the undissolved solids from liquefied corn mash. Oleyl alcohol (OA) was added to both fermentations to extract the product (i-BuOH) from the broth as it was formed. The fermentation broth referred to in this example where solids were not removed from the feed (used corn mash) was 2010Y036 as described in Example 6. The fermentation broth referred to in this example where solids were removed from the feed (used centrate produced from corn mash) was 2010Y035 as described in Example 6. Oleyl alcohol was the extraction solvent used in both fermentations. The rate and degree of phase separation was measured and compared throughout the fermentations as well as for the final fermentation broths. Adequate phase separation in an extractive fermentation process can lead to minimal loss of the microorganism and minimal solvent losses as well lower capital and operating cost of downstream processing.

Phase Separation Between Solvent and Broth Phases During Fermentation

[0413] Approximately 10 mL samples were pulled from each fermentor at 5.3, 29.3, 53.3, and 70.3 hr, and phase separation was compared for the samples from the fermentation where solids were removed (2010Y035) from the samples and where solids were not removed (2010Y036). Phase separation was observed and compared for all samples from all run times by allowing the samples to set for about 2 hr and tracking the position of the liquid-liquid interface. Essentially no phase separation was observed for any of the samples pulled from the fermentation where solids were not removed. Phase separation was observed for all samples from the fermentation where solids were removed from the liquefied corn mash prior to fermentation. Separation started to occur within about 10-15 min of pulling the samples from the run where solids were removed for all fermentation times and continued to improve over a 2 hr period of time. Phase separation started to occur in the sample pulled at 5.3 hr fermentation run time from the centrate fermentation (solids removed) after about 7 min of settling time. Phase separation started to occur in the sample pulled at 53.3 hr from the centrate fermentation (solids removed) after about 17 min of settling time.

[0414] FIG. 13 is a plot of the position of the liquid-liquid interface in the fermentation sample tubes as a function of (gravity) settling time. The data is for the samples pulled from the extractive fermentation where centrate was fed (solids removed from corn mash) as the sugar source and oleyl alcohol was the ISPR extraction solvent (run 2010Y035 in Example 6). The phase separation data in this plot is for samples pulled at 5.3, 29.3, 53.3, and 70.3 hr run time from fermentation 2010Y035. The interface position is reported as a percentage of the total broth height in the sample tube. For example, the interface position in the sample pulled at 5.3 hr run time from the 2010Y035 fermentation (centrate/oleyl alcohol) increased from the bottom of the sample tube (no separation) to 3.5 mL after 120 min of settling time. There was about 10 mL of total broth in that particular sample tube. Therefore, the interface position for that sample was reported as 35% in FIG. 13. Similarly, the interface position in the sample pulled at 53.3 hr run time from the 2010Y035 fermentation (centrate/oleyl alcohol) increased from the bottom of the sample tube (no separation) to about 3.9 mL after 125 min of settling time. There was about 10 mL of total broth in that particular sample tube. Therefore, the interface position for that sample was reported as 39% in FIG. 13.

Phase Separation Between Solvent and Broth Phases after Completing Fermentation

[0415] After 70 hr of run time, the fermentations were stopped, and the two broths from the oleyl alcohol extractive fermentations were transferred to separate 2 L glass graduated cylinders. The separation of the solvent and broth phases were observed and compared. Almost no phase separation was observed after about 3 hr for the broth where solids were not removed prior to fermentation (run 2010Y036). Phase separation was observed for the broth where solids were removed from the liquefied corn mash prior to fermentation (run 2010Y035). Separation started to occur after about 15 min of settling time and continued to improve over a 3 hr period of time. After 15 min, a liquid-liquid interface was established at a level that was about 10% of the total height of the two phase mixture. This indicates that the aqueous phase splits out from the dispersion first and starts to accumulate at the bottom of the mixture. As time proceeded, more aqueous phase accumulated at the bottom of the mixture causing the position of the interface to rise. After about 3 hr of settling time, the interface had increased to a level that was about 40% of the total height of the two phase mixture. This indicates that almost complete phase separation had occurred after about 3 hr of (gravity) settling time for the final two phase broth where solids were removed based on the amounts of centrate and oleyl alcohol initially charged to the fermentation. Approximately equal volumes of initial centrate and solvent were charged to both fermentations. Approximately 1.2 L of liquefied corn mash and approximately 1.1 L of oleyl alcohol were charged to fermentation 2010Y036. Approximately 1.2 L of centrate, which was produced from the same batch of mash, and approximately 1.1 L of oleyl alcohol were charged to fermentation 2010Y035. After accounting for the fact that approximately 100 g/kg of the initial sugar in the aqueous phase was consumed and the fact that about 75% of the i-BuOH produced was in the solvent phase, it would be expected that the relative volumes of the final aqueous and organic phases would be about 1:1 if complete separation occurred. FIG. 14 is a plot of the liquid-liquid interface position as a function of (gravity) settling time for the final two phase broth from the extractive fermentation where solids were removed (2010Y035). The interface position is reported as a percentage of the total broth height in the 2 L graduated cylinder used to observe phase separation of the final broth. The interface position of the final broth from the 2010Y035 fermentation increased from the bottom of the graduated cylinder (no separation) to a level that was about 40% of the total height of the two phase mixture after 175 min of settling time. Therefore, almost complete separation of the two phases in the final broth occurred after 3 hr of settling time. An interface position of approximately 50% would correspond to complete separation.

[0416] A 10 mL sample was pulled from the top of the organic phase of the final broth (which had settled for about 3 hr) from the fermentation where solids had been removed. The sample was spun in a high-speed lab centrifuge to determine the amount of aqueous phase that was present in the organic phase after allowing the broth to settle for 3 hr. The results showed that about 90% of the top layer of the final broth was solvent phase. About 10% of the top layer of the final broth was aqueous phase, including a relatively small amount of undissolved solids. Some solids were located at the bottom of the aqueous phase (more dense than the aqueous phase) and also a small amount of solids accumulated at the liquid-liquid interface.

[0417] A 10 mL sample was also pulled from the bottom phase of the final broth (which had settled for about 3 hr) from the fermentation where solids had been removed. The sample was spun in a high-speed lab centrifuge to determine the amount of organic phase that was present in the aqueous phase after allowing the broth to settle for 3 hr. It was determined that essentially no organic phase was present in the bottom (aqueous) phase of the final broth from the fermentation from which solids had been removed after the broth had settled for 3 hr. These results confirm that almost complete phase separation had occurred for the final broth from the fermentation where solids had been removed. Almost no phase separation was apparent for the final broth from the fermentation where solids had not been removed. This data implies that removing solids from liquefied corn mash before extractive fermentation may enable a significant improvement in phase separation during and after fermentation resulting in less loss of the microorganism, undissolved solids, and water to downstream processing.

[0418] A 10 mL sample was pulled from the top of the final broth from the fermentation from which solids had not been removed after the broth had set for about 3 hr. The sample was spun in a high-speed lab centrifuge to determine the relative amount of solvent and aqueous phases at the top of the final broth. This broth contained all solids from the liquefied corn mash solids. About half of the sample was aqueous phase, and about half was organic phase. The aqueous phase contained significantly more undissolved solids (from the liquefied mash) compared to the sample of the top layer from the broth where solids were removed. The amounts of aqueous and solvent phases in this sample are approximately the same indicating that essentially no phase separation occurred in the final broth where solids were not removed (even after 3 hr of settling time). This data implies that if solids are not removed from liquefied corn mash before an extractive fermentation, little to no phase separation is likely to occur during and after fermentation. This could result in a significant loss of the microorganism, undissolved solids, and water to downstream processing.

Example 9

Effect of Removing Undissolved Solids on the Loss of ISPR Extraction Solvent--Extractive Fermentation

[0419] This example demonstrates the potential for reducing solvent losses with the DDGS out the back end of an extractive fermentation process by removing undissolved solids from the corn mash prior to fermentation. Example 6 described two extractive fermentations conducted side-by side, one with liquefied corn mash as the sugar source (2010Y036--solids not removed) and one with liquefied mash centrate (2010Y035--aqueous solution of oligosaccharides) obtained by removing most of the undissolved solids from liquefied corn mash. Oleyl alcohol (OA) was added to both fermentations to extract the product isobutanol (i-BuOH) from the broth as it was formed. The amount of residual solvent trapped in the undissolved solids recovered from the final fermentation broths was measured and compared.

[0420] After completion of the fermentations 2010Y035 and 2010Y036 described in Example 6, the broths were harvested and used to conduct the phase separation tests. Then the undissolved solids (fines from the corn mash that did not get removed prior to fermentation) were recovered from each broth and analyzed for total extractable oils. The oil recovered from each lot of solids was analyzed for oleyl alcohol and corn oil. The following protocol was followed for both broths: [0421] The broth was centrifuged to separate the organic, aqueous, and solid phases. [0422] The organic and aqueous phases were decanted away from the solids leaving a wet cake at the bottom of the centrifuge bottle. [0423] The wet cake was thoroughly washed with water to remove essentially all of the dissolved solids held up in the cake, such as residual oligosaccharides, glucose, salts, enzymes, etc. [0424] The washed wet cake was dried in a vacuum oven overnight (house-vacuum at 80.degree. C.) to remove essentially all of the water in the cake. [0425] A portion of the dry solids was thoroughly contacted with hexane in a Soxhlet extractor to remove the oil from the solids. [0426] The oil recovered from the solids was analyzed by GC to determine the relative amount of oleyl alcohol and corn oil present in the oil recovered from the solids. [0427] A particle size distribution (PSD) was measured for the solids recovered from both fermentation broths.

[0428] The data for the recovery and hexane extraction of the undissolved solids from both fermentation broths is shown in Table 8. The data shows that approximately the same amount of oil was absorbed by the solids (per unit mass of solids) in both fermentations.

TABLE-US-00008 TABLE 8 Fermentation ID: 2010Y036 2010Y035 Solids removed from liquefied No (mash) Yes (centrate) mash before fermentation Washed wet cake recovered 290.6 g 15.6 g after removing organic phase, aqueous phase, and washing the wet cake with water, g: Dry solids content in washed wet 23.6% 25.8% cake, wt %: Dry solids recovered from 68.1 g 4.02 g washed wet cake, g: Dry solids charged to Soxhlet, g: 20.11 g 3.91 g Dry Content of solids charged to 97.9% 98.1% Soxhlet via moisture analysis, wt %: Total oil recovered from Soxhlet 2.30 g 0.25 g hexane extraction, g: Oil content of solids (dry solids 0.12 g oil/ 0.07 g oil/ basis), g oil per g of dry solids: g dry solids g dry solids Fraction of oil extracted from 76% 74% solids that is OA (approximate value), wt %:

Example 10

Recovery of Soluble Starch from a Wet Cake Generated from the Removal of Solids from Liquefied Corn Mash by Washing the Wet Cake with Water--Two Stage Process

[0429] This example demonstrated the recovery of soluble starch from a wet cake by washing the cake twice with water, where the cake was generated by centrifuging liquefied mash. Liquefied corn mash was fed to a continuous decanter centrifuge to produce a centrate stream (C-1) and a wet cake (WC-1). The centrate was a relatively solids-free, aqueous solution of soluble starch, and the wet cake was concentrated in solids compared to the feed mash. A portion of the wet cake was mixed with hot water to form a slurry (S-1). The slurry was pumped back through the decanter centrifuge to produce a wash water centrate (C-2) and a washed wet cake (WC-2). C-2 was a relatively solids-free, dilute aqueous solution of soluble starch. The concentration of soluble starch in C-2 was less than the concentration of soluble starch in the centrate produced from the separation of mash. The liquid phase held up in WC-2 was more dilute in starch than the liquid in the wet cake produced from the separation of mash. The washed wet cake (WC-2) was mixed with hot water to form a slurry (S-2). The ratio of water charged to the amount of soluble starch in the wet cake charged was the same in both wash steps. The second wash slurry was pumped back through the decanter centrifuge to produce a second wash water centrate (C-3) and a wet cake (WC-3) that had been washed twice. C-3 was a relatively solids-free, dilute aqueous solution of soluble starch. The concentration of soluble starch in C-3 was less than the concentration of soluble starch in the centrate produced in the first wash stage (C-2), and thus the liquid phase held up in WC-3 (second washed wet cake) was more dilute in starch than in WC-2 (first washed wet cake). The total soluble starch in the two wash centrates (C-2 and C-3) is the starch that was recovered and could be recycled back to liquefaction. The soluble starch in the liquid held up in the final washed wet cake is much less that in the wet cake produced in the original separation of the mash.

Production of Liquefied Corn Mash

[0430] Approximately 1000 gallons of liquefied corn mash was produced in a continuous dry-grind liquefaction system consisting of a hammer mill, slurry mixer, slurry tank, and liquefaction tank. Ground corn, water, and alpha-amylase were fed continuously. The reactors were outfitted with mechanical agitation, temperature control, and pH control using either ammonia or sulfuric acid. The reaction conditions were as follows: [0431] Hammer mill screen size: 7/64'' [0432] Feed Rates to Slurry Mixer [0433] Ground Corn: 560 lbm/hr (14.1 wt % moisture) [0434] Process Water: 16.6 lbm/min (200 F) [0435] Alpha-Amylase: 61 g/hr (Genecor: Spezyme.RTM. ALPHA) [0436] Slurry Tank Conditions: [0437] Temperature: 185.degree. F. (85.degree. C.) [0438] pH: 5.8 [0439] Residence Time: 0.5 hr [0440] Dry Corn Loading: 31 wt % [0441] Enzyme Loading: 0.028 wt % (dry corn basis) [0442] Liquefaction Tank Conditions: [0443] Temperature: 185.degree. F. (85.degree. C.) [0444] pH: 5.8 [0445] Residence Time: about 3 hr [0446] No additional enzyme added.

[0447] The production rate of liquefied corn mash was about 3 gpm. The starch content of the ground corn was measured to be about 70 wt % on a dry corn basis. The total solids (TS) of the liquefied mash was about 31 wt %, and the total suspended solids (TSS) was approximately 7 wt %. The liquid phase contained about 23-24 wt % liquefied starch as measured by HPLC (soluble oligosaccharides).

[0448] The liquefied mash was centrifuged in a continuous decanter centrifuge at the following conditions: [0449] Bowl Speed: 5000 rpm (about 3600 g's) [0450] Differential Speed: 15 rpm [0451] Weir Diameter: 185 mm (weir plate removed) [0452] Feed Rate: Varied from 5-20 gpm.

[0453] Approximately 850 gal of centrate and approximately 1400 lbm of wet cake were produced by centrifuging the mash. The total solids in the wet cake were measured to be about 46.3% (suspended+dissolved) by moisture balance. Knowing that the liquid phase contained about 23 wt % soluble starch, it was estimated that the total suspended solids in the wet cake was about 28 wt %. It was estimated that the wet cake contained approximately 12% of the soluble starch that was present in the liquefied mash prior to the centrifuge operation.

Recovery of Soluble Starch from Wet Cake by Washing the Solids with Water--1.sup.st Wash

[0454] About 707 lbm of the wet cake recovered from separation of the liquefied mash was mixed with 165 gal of hot (91.degree. C.) water in a 300 gallon stainless steel vessel. The resulting slurry was mixed for about 30 min. The slurry was continuously fed to a decanter centrifuge to remove the washed solids from the slurry. The centrifuge used to separate the wash slurry was the same one used to remove solids from the liquefied mash above, and it was rinsed with fresh water before feeding the slurry. The centrifuge was operated at the following conditions to remove solids from the wash slurry: [0455] Bowl Speed: 5000 rpm (about 3600 g's) [0456] Differential Speed: 5 rpm [0457] Weir Diameter: 185 mm (weir plate removed) [0458] Feed Rate: 5 gpm.

[0459] Approximately 600 lbm of washed wet cake was produced by the centrifuge, but only 400 lbm were recovered due to loss of material. The total solids in the wet cake were measured to be about 36.7% (suspended+dissolved) by moisture balance. The total soluble starch (sum of glucose, DP2, DP3, and DP4+) in the liquid phase of the slurry and in the wash water centrate (obtained from the slurry) was measured to be about 6.7 wt % and 6.9 wt %, respectively, by HPLC. DP2 refers to a dextrose polymer containing two glucose units (glucose dimer). DP3 refers to a dextrose polymer containing three glucose units (glucose trimer). DP4+ refers to a dextrose polymer containing four or more glucose units (glucose tetramer and higher). This confirmed that a well-mixed dilution wash stage was achieved. Therefore, the concentration of soluble starch in the liquid phase held up in the washed wet cake must have been about 6.8 wt % (by mass balance) for this dilution wash. Based on the total solids and dissolved oligosaccharide data, it was estimated that the total suspended solids in the washed wet cake was about 32 wt %. It was estimated that the washed wet cake contained approximately 2.6% of the soluble starch that was present in the original liquefied mash if all 600 lbm of the cake produced by the centrifuge could have been washed. This represents about a 78% reduction in soluble starch in the washed wet cake compared to the mash wet cake prior to washing. If the wet cake produced from the separation of liquefied mash was not washed, about 12% of the total starch in the mash would be lost as soluble (liquefied) starch. If the wet cake produced from the separation of mash is washed with water at the conditions demonstrated in this example, 2.6% of the total starch from the mash would be lost as soluble (liquefied) starch.

[0460] About 400 lbm of the washed wet cake recovered from the first re-slurry wash of the liquefied mash wet cake was mixed with 110 gal of hot (90.degree. C.) water in a 300 gallon stainless steel vessel. The resulting slurry was mixed for about 30 min. The slurry was continuously fed to a decanter centrifuge to remove the washed solids from the slurry. The centrifuge used to separate the second wash slurry was the same one used in the first wash above, and it was rinsed with fresh water before feeding the second wash slurry. The centrifuge was operated at the following conditions to remove solids from the wash slurry: [0461] Bowl Speed: 5000 rpm (about 3600 g's) [0462] Differential Speed: 5 rpm [0463] Weir Diameter: 185 mm (weir plate removed) [0464] Feed Rate: 4 gpm.

[0465] Approximately 322 lbm of washed wet cake was produced by the centrifuge. The total solids in the wet cake from the second wash were measured to be about 37.4% (suspended+dissolved) by moisture balance. The total soluble starch (sum of glucose, DP2, DP3, and DP4+) in the liquid phase of the slurry and in the wash water centrate (obtained from the slurry) was measured to be about 1.6 wt % and 1.6 wt %, respectively, by HPLC. This confirmed that a well-mixed dilution wash stage was achieved in the second wash. Therefore, the concentration of soluble starch in the liquid phase held up in the washed wet cake must have been about 1.6 wt % (by mass balance) for this dilution wash. Based on the total solids and dissolved oligosaccharide data, it was estimated that the total suspended solids in the washed wet cake was about 36 wt %. It was estimated that the washed wet cake contained approximately 0.5% of the soluble starch that was present in the original liquefied mash if all 600 lbm of the cake produced in the first wash stage could have been washed. This represents an overall reduction in soluble starch in the doubly washed wet cake compared to the mash wet cake prior to washing of about 96%. If the wet cake produced from the separation of liquefied mash was not washed, about 12% of the total starch in the mash would be lost as soluble (liquefied) starch. If the wet cake produced from the separation of mash is washed twice with water at the conditions demonstrated in this example, 0.5% of the total starch from the mash would be lost as soluble (liquefied) starch.

Example 11

Effect of High Temperature Stage During Liquefaction on the Conversion of Starch in Corn Solids to Soluble (Liquefied) Starch

[0466] This example demonstrates that operating liquefaction with a high temperature (or "cook") stage at some time in the middle of the reaction can result in higher conversion of the starch in corn solids to soluble (liquefied) starch. The "cook" stage demonstrated in this example involves raising the liquefaction temperature at some point after liquefaction starts, holding at the higher temperature for some period of time, and then lowering the temperature back to the original value to complete liquefaction.

A. Procedure to Measure Unhydrolyzed Starch Remaining in Solids after Liquefaction

[0467] Liquefied corn mash was prepared in one run according to the protocol in Example 1 (no intermediate high temperature stage). Liquefied corn mash was prepared in another run at the same conditions as in the first run except for the addition of an intermediate high temperature stage. The mash from both runs was worked up according to the following steps. It was centrifuged to separate the aqueous solution of liquefied starch from the undissolved solids. The aqueous solution of liquefied starch was decanted off to recover the wet cake. The wet cake contained most of the undissolved solids from the mash, but the solids were still wet with liquefied starch solution. The wet cake was thoroughly washed with water, and the subsequent slurry was centrifuged to separate the aqueous layer from the undissolved solids. The cake was washed a total of five times with enough water to remove approximately all of the soluble starch that was held up in the original wet cake recovered from liquefaction. Consequently, the liquid phase held up in the final washed wet cake consisted of water containing essentially no soluble starch.

[0468] The final washed wet cake was re-slurried in water, and large excesses of both alpha-amylase and glucoamylase were added. The slurry was mixed for at least 24 hr while controlling temperature and pH to enable the alpha-amylase to convert essentially all the unhydrolyzed starch remaining in the undissolved solids to soluble oligosaccharides. The soluble oligosaccharides generated from the residual starch (which was not hydrolyzed during liquefaction at the conditions of interest) were subsequently converted to glucose by the glucoamylase present. Glucose concentration was tracked with time by HPLC to make sure all the oligosaccharides generated from the residual starch were converted to glucose and that the glucose concentration was no longer increasing with time.

B. Production of Liquefied Corn Mash

[0469] Two batches of liquefied corn mash were prepared (approximately 1 L each) at 85.degree. C. using Liquozyme.RTM. SC DS (alpha-amylase from Novozymes, Franklinton, N.C.). Both batches operated at 85.degree. C. for a little more than 2 hr. However, a "cook" period was added in the middle of the second batch ("Batch 2"). The temperature profile for Batch 2 was about 30 min at 85.degree. C., raising the temperature from 85.degree. C. to 101.degree. C., holding at 101.degree. C. for about 30 min, cooling down to 85.degree. C., and continuing liquefaction for another 120 min. The ground corn used in both batches was the same as in Example 1. A corn loading of 26 wt % (dry corn basis) was used in both batches. The total amount of enzyme used in both runs corresponded to 0.08 wt % (dry corn basis). The pH was controlled at 5.8 during both liquefaction runs. The liquefactions were carried out in a glass, jacketed resin kettle. The kettle was set up with mechanical agitation, temperature control, and pH control.

[0470] The following protocol was followed to prepare liquefied corn mash for Batch 1: [0471] The alpha-amylase was diluted in tap water (0.418 g enzyme in 20.802 g water) [0472] Charged 704.5 g tap water to the kettle [0473] Turned on agitator [0474] Made first charge of ground corn, 198 g [0475] Heated to 55.degree. C. while agitating [0476] Adjusted pH to 5.8 using H.sub.2SO.sub.4 or NaOH [0477] Made first charge of alpha-amylase solution, 7.111 g [0478] Heated to 85.degree. C. [0479] Held at 85.degree. C. for 30 min [0480] Made second charge of alpha-amylase solution, 3.501 g [0481] Made second charge of ground corn, 97.5 g [0482] Continued to run at 85.degree. C. for another 100 min [0483] After the liquefaction was complete, cooled to 60.degree. C. [0484] Dumped reactor and recovered 998.5 g of liquefied mash.

[0485] The following protocol was followed to prepare liquefied corn mash for Batch 2: [0486] The alpha-amylase was diluted in tap water (0.3366 g enzyme in 16.642 g water) [0487] Charged 562.6 g tap water to the kettle [0488] Turned on agitator [0489] Charged ground corn, 237.5 g [0490] Heated to 55.degree. C. while agitating [0491] Adjusted pH to 5.8 using dilute H.sub.2SO.sub.4 or NaOH [0492] Made first charge of alpha-amylase solution, 4.25 g [0493] Heated to 85.degree. C. [0494] Held at 85.degree. C. for 30 min [0495] Heated to 101.degree. C. [0496] Held at 101.degree. C. for 30 min [0497] Lowered temperature of mash back to 85.degree. C. [0498] Adjusted pH to 5.8 using dilute H.sub.2SO.sub.4 or NaOH [0499] Made second charge of alpha-amylase solution, 4.2439 g [0500] Continued to run at 85.degree. C. for another 120 min. [0501] After the liquefaction was complete, cooled to 60.degree. C.

[0502] C. Removal of Undissolved Solids from the Liquefied Mash and Washing of the Wet Cake with Water to Remove Soluble Starch

[0503] Most of the solids were removed from both batches of liquefied mash by centrifuging them in a large floor centrifuge at 5000 rpm for 20 min at room temperature. Centrifugation of 500 g of mash from Batch 1 yielded 334.1 g of centrate and 165.9 g of wet cake. Centrifugation of 872 g of mash from Batch 2 yielded 654.7 g of centrate and 217 g of wet cake. The wet cakes recovered from each batch of liquefied mash were washed five times with tap water to remove essentially all of the soluble starch held up in the cakes. The washes were performed in the same bottle used to centrifuge the original mash to avoid transferring the cake between containers. For each wash stage, the cake was mixed with water, and the resulting wash slurry was centrifuged (5000 rpm for 20 min) at room temperature. This was done for all five wash stages performed on the wet cakes recovered from both batches of mash. Approximately 165 g of water was used in each of the five washes of the wet cake from Batch 1 resulting in a total of 828.7 g of water used to wash the wet cake from Batch 1. Approximately 500 g of water was used in each of the five washes of the wet cake from Batch 2 resulting in a total of 2500 g of water used to wash the wet cake from Batch 2. The total wash centrate recovered from all five water washes of the wet cake from Batch 1 was 893.1 g. The total wash centrate recovered from all five water washes of the wet cake from Batch 2 was 2566.3 g. The final washed wet cake recovered from Batch 1 was 101.5 g, and the final washed wet cake recovered from Batch 2 was 151.0 g. The final washed wet cakes obtained from each batch contained essentially no soluble starch; therefore, the liquid held up in each cake was primarily water. The total solids (TS) of the wet cakes was measured using a moisture balance. The total solids of the wet cake from Batch 1 was 21.63 wt %, and the TS for the wet cake from Batch 2 was 23.66 wt %.

D. Liquefaction/Saccharification of Washed Wet Cake to Determine the Level of Unhydrolyzed Starch Remaining in the Undissolved Solids after Liquefaction

[0504] The level of unhydrolyzed starch remaining in the solids present in both washed wet cakes was measured by re-slurrying the cakes in water and adding excess alpha-amylase and excess glucoamylase. The alpha-amylase converts residual unhydrolyzed starch in the solids to soluble oligosaccharides which dissolve in the aqueous phase of the slurry. The glucoamylase subsequently converts the soluble oligosaccharides generated by the alpha-amylase to glucose. The reactions were run at 55.degree. C. (maximum recommended temperature for the glucoamylase) for at least 24 hr to ensure all of the residual starch in the solids was converted to soluble oligosaccharides and that all the soluble oligosaccharides were converted to glucose. The residual unhydrolyzed starch that was in the solids, which is the starch that did not get hydrolyzed during liquefaction, can be calculated from the amount of glucose generated by this procedure.

[0505] The alpha-amylase and glucoamylase enzymes used in the following protocols were Liquozyme.RTM. SC DS and Spirizyme.RTM. Fuel, respectively (Novozymes, Franklinton, N.C.). The vessel used to treat the washed wet cakes was a 250 mL jacketed glass resin kettle equipped with mechanical agitation, temperature control, and pH control. The amount of Liquozyme.RTM. used corresponds to an enzyme loading of 0.08 wt % on a "dry corn basis." The amount of Spirizyme.RTM. used corresponds to an enzyme loading of 0.2 wt % on a "dry corn basis." This basis is defined as the amount of ground corn required to give the amount of undissolved solids held up in the washed cakes assuming all the starch is hydrolyzed to soluble oligosaccharides. The undissolved solids held up in the washed cakes are considered to be mostly the non-starch, non-fermentable part of the corn. These enzyme loadings are at least four times higher than is required to give complete liquefaction and saccharification at 26% corn loading. The enzymes were used in large excess to ensure complete hydrolysis of the residual starch in the solids and complete conversion of the oligosaccharides to glucose.

[0506] The following protocol was followed to determine the level of unhydrolyzed starch in the solids present in the washed wet cake from Batch 1 mash: [0507] The alpha-amylase was diluted in tap water (0.1297 g enzyme in 10.3607 g water) [0508] The glucoamylase was diluted in tap water (0.3212 g enzyme in 15.6054 g water) [0509] Charged 132 g tap water to the kettle [0510] Turned on agitator [0511] Charged 68 g of the washed wet cake produced from liquefaction Batch 1 (TS=21.63 wt %) [0512] Heated to 55.degree. C. while agitating [0513] Adjusted pH to 5.5 using dilute H.sub.2SO.sub.4 or NaOH [0514] Charged alpha-amylase solution, 3.4992 g [0515] Charged glucoamylase solution, 5.319 g [0516] Run at 55.degree. C. for 24 hr while controlling pH at 5.5 and periodically sample the slurry for glucose.

[0517] The following protocol was followed to determine the level of unhydrolyzed starch in the solids present in the washed wet cake from Batch 2. [0518] The alpha-amylase was diluted in tap water (0.2384 g enzyme in 11.709 g water) [0519] The glucoamylase was diluted in tap water (0.3509 g enzyme in 17.5538 g water) [0520] Charged 154.3 g tap water to the kettle [0521] Turned on agitator [0522] Charged 70.7 g of the washed wet cake produced from liquefaction Batch 1 (TS=23.66 wt %) [0523] Heated to 55.degree. C. while agitating [0524] Adjusted pH to 5.5 using dilute H.sub.2SO.sub.4 or NaOH [0525] Charged alpha-amylase solution, 2.393 g [0526] Charged glucoamylase solution, 5.9701 g [0527] Run at 55.degree. C. for 24 hr while controlling pH at 5.5 and periodically sample the slurry for glucose.

Comparison of Results for the Liquefaction/Saccharification of the Washed Wet Cakes

[0528] As described above, the washed wet cakes from Batches 1 and 2 were re-slurried in water, and large excesses of both alpha-amylase and glucoamylase were added to the slurries in order to hydrolyze any starch remaining in the solids and convert it to glucose. FIG. 15 shows the concentration of glucose in the aqueous phase of the slurries as a function of time.

[0529] The glucose concentration increased with time and leveled out at a maximum value at approximately 24 hr for both reactions. The slight decrease in glucose between 24 and 48 hr could have been from microbial contamination; therefore, the maximum level of glucose reached in each system was used to calculate the level of residual unhydrolyzed starch that was in the solids of the washed wet cake. The maximum level of glucose reached by reacting (in the presence of alpha-amylase and glucoamylase) the washed wet cake obtained from the Batch 1 liquefaction was 4.48 g/L. By comparison, the maximum level of glucose reached by reacting (in the presence of alpha-amylase and glucoamylase) the washed wet cake obtained from the Batch 2 liquefaction was 2.39 g/L.

[0530] The level of residual unhydrolyzed starch that was in the undissolved solids in the liquefied mash (that did not get hydrolyzed during liquefaction) was calculated based on the glucose data obtained from the washed wet cake obtained from the corresponding batch of mash. [0531] Liquefaction Batch 1: The residual unhydrolyzed starch in the solids corresponds to 2.1% of the total starch in the corn fed to liquefaction. This implies that 2.1% of the starch in the corn was not hydrolyzed during Batch 1 liquefaction conditions. No intermediate high temperature ("cook") stage occurred during liquefaction Batch 1. [0532] Liquefaction Batch 2: The residual unhydrolyzed starch in the solids corresponds to 1.1% of the total starch in the corn fed to liquefaction. This implies that 1.1% of the starch in the corn was not hydrolyzed during Batch 2 liquefaction conditions. A high temperature ("cook") stage did occur during liquefaction Batch 2.

[0533] This example demonstrates that the addition of a high temperature "cook" stage at some time during the liquefaction could result in higher starch conversion. This will result in less residual unhydrolyzed starch remaining in the undissolved solids in the liquefied corn mash and will lead to less starch loss in a process where undissolved solids are removed from the mash prior to liquefaction.

Example 12

Effect of High Temperature Stage During Liquefaction on the Conversion of Starch in Corn Solids to Soluble (Liquefied) Starch

[0534] Two batches of liquefied corn mash (Batch 3 and Batch 4) were prepared at 85.degree. C. using Liquozyme.RTM. SC DS (alpha-amylase from Novozymes, Franklinton, N.C.). Both batches operated at 85.degree. C. for a little more than 2 hr. However, a "cook" period was added in the middle of Batch 4. The temperature profile for Batch 4 was about 30 min at 85.degree. C., raising the temperature from 85.degree. C. to 121.degree. C., holding at 121.degree. C. for about 30 min, cooling down to 85.degree. C., and continuing liquefaction for another 90 min. The ground corn used in both batches was the same as in Example 1. A corn loading of 26 wt % (dry corn basis) was used in both batches. The total amount of enzyme used in both runs corresponded to 0.04 wt % (dry corn basis). The pH was controlled at 5.8 during both liquefaction runs. The liquefaction for Batch 3 was carried out in a 1 L glass, jacketed resin kettle, and the liquefaction for Batch 4 was carried out in a 200L stainless steel fermentor. Both reactors were outfitted with mechanical agitation, temperature control, and pH control.

[0535] The experimental conditions for this example were similar to those described for Example 9 with the following differences:

[0536] For the Production of Liquefied Corn Mash for Batch 3: 0.211 g of alpha-amylase was diluted in 10.403 g tap water. The first charge of alpha-amylase solution was 3.556 g. The second charge of alpha-amylase solution was 1.755 g and the reaction was allowed to continue to run at 85.degree. C. for another 90 min.

[0537] For the Production of Liquefied Corn Mash for Batch 4: 22 g of alpha-amylase was diluted in 2 kg tap water, 147.9 kg of tap water was charged to the fermentor, and 61.8 kg of ground corn was charged. The first charge of alpha-amylase solution was 1.0 kg, the reaction was heated to 85.degree. C. and held at 85.degree. C. for 30 min, then the reaction was heated to 121.degree. C. and held at 121.degree. C. for 30 min. The second charge of alpha-amylase solution was 1 kg and the reaction was allowed to continue to run at 85.degree. C. for another 90 min.

Removal of Undissolved Solids from the Liquefied Mash and Washing of the Wet Cake with Water to Remove Soluble Starch

[0538] Most of the solids were removed from both batches of liquefied mash by centrifuging them in a large floor centrifuge at 5000 rpm for 15 min at room temperature. Centrifugation of 500.1 g of mash from Batch 3 yielded 337.2 g of centrate and 162.9 g of wet cake. Centrifugation of 509.7 g of mash from Batch 4 yielded 346.3 g of centrate and 163.4 g of wet cake. The wet cakes recovered from each batch of liquefied mash were washed five times with tap water to remove essentially all of the soluble starch held up in the cakes. The washes were performed in the same bottle used to centrifuge the original mash to avoid transferring the cake between containers. For each wash stage, the cake was mixed with water, and the resulting wash slurry was centrifuged (5000 rpm for 15 min) at room temperature. This was done for all five wash stages performed on the wet cakes recovered from both batches of mash. Approximately 164 g of water was used in each of the five washes of the wet cake from Batch 3 resulting in a total of 819.8 g of water used to wash the wet cake from Batch 3. Approximately 400 g of water was used in each of the five washes of the wet cake from Batch 4 resulting in a total of 2000 g of water used to wash the wet cake from Batch 4. The total wash centrate recovered from all five water washes of the wet cake from Batch 3 was 879.5 g. The total wash centrate recovered from all five water washes of the wet cake from Batch 4 was 2048.8 g. The final washed wet cake recovered from Batch 3 was 103.2 g, and the final washed wet cake recovered from Batch 4 was 114.6 g. The final washed wet cakes obtained from each batch contained essentially no soluble starch; therefore, the liquid held up in each cake was primarily water. The total solids (TS) of the wet cakes were measured using a moisture balance. The total solids of the wet cake from Batch 3 was 21.88 wt %, and the TS for the wet cake from Batch 4 was 18.1 wt %.

[0539] The experimental conditions for this example were similar to those described for Example 9 with the following differences:

[0540] For the Liquefaction/Saccharification of Washed Wet Cake to Determine the Level of

[0541] Unhydrolyzed Starch Remaining in the Undissolved Solids after Liquefaction for Batch 3: 68 g of the washed wet cake produced from liquefaction of Batch 3 was charged (TS=21.88 wt %). 3.4984 g of alpha-amylase solution and 5.3042 g of glucoamylase was charged. The reaction was ran at 55.degree. C. for 47 hr while controlling pH at 5.5 and periodically sampling the slurry for glucose.

[0542] For the Liquefaction/Saccharification of Washed Wet Cake to Determine the Level of Unhydrolyzed Starch Remaining in the Undissolved Solids after Liquefaction for Batch 4: 0.1663 g of alpha-amylase was diluted in 13.8139 g tap water, and 0.213 g of glucoamylase was diluted in 20.8002 g tap water. 117.8 g of tap water was charged to the kettle. 82.24 g of the washed wet cake produced from liquefaction of Batch 4 was charged (TS=18.1 wt %). 3.4952 g of alpha-amylase solution and 10.510 g of glucoamylase was charged. The reaction was ran at 55.degree. C. for 50 hr while controlling pH at 5.5 and periodically sampling the slurry for glucose.

Comparison of Results for the Liquefaction/Saccharification of the Washed Wet Cakes

[0543] As described above, the washed wet cakes from Batches 3 and 4 were re-slurried in water, and large excesses of both alpha-amylase and glucoamylase were added to the slurries in order to hydrolyze any starch remaining in the solids and convert it to glucose. FIG. 16 shows the concentration of glucose in the aqueous phase of the slurries as a function of time.

[0544] The glucose concentration increased with time and leveled out at a maximum value at approximately 26 hr for the washed wet cake from Batch 3. For the Batch 4 washed wet cake, the glucose concentration continued to increase slightly between 24 hr and 47 hr. It is assumed that the glucose concentration measured at 47 hr for the Batch 4 wet cake is approximately equal to the maximum value. The maximum level of glucose reached by reacting (in the presence of alpha-amylase and glucoamylase) the washed wet cake obtained from the Batch 3 liquefaction was 8.33 g/L. By comparison, the maximum level of glucose reached by reacting (in the presence of alpha-amylase and glucoamylase) the washed wet cake obtained from the Batch 4 liquefaction was 4.92 g/L.

[0545] The level of residual unhydrolyzed starch that was in the undissolved solids in the liquefied mash (that did not get hydrolyzed during liquefaction) was calculated based on the glucose data obtained from "hydrolyzing" the washed wet cake (in the presence of excess alpha-amylase and glucoamylase) obtained from the corresponding batch of mash. [0546] Liquefaction Batch 3: The residual unhydrolyzed starch in the solids corresponds to 3.8% of the total starch in the corn fed to liquefaction. This implies that 3.8% of the starch in the corn was not hydrolyzed during Batch 3 liquefaction conditions. No intermediate high temperature ("cook") stage occurred during liquefaction Batch 3. [0547] Liquefaction Batch 4: The residual unhydrolyzed starch in the solids corresponds to 2.2% of the total starch in the corn fed to liquefaction. This implies that 2.2% of the starch in the corn was not hydrolyzed during Batch 4 liquefaction conditions. A high temperature ("cook") stage did occur during liquefaction Batch 4.

[0548] This example demonstrates that the addition of a high temperature "cook" stage at some time during the liquefaction could result in higher starch conversion. This will result in less residual unhydrolyzed starch remaining in the undissolved solids in the liquefied corn mash and will lead to less starch loss in a process where undissolved solids are removed from the mash prior to liquefaction.

Summary and Comparison of Examples 11 and 12

[0549] Liquefaction conditions can influence the conversion of starch in the corn solids to soluble (liquefied) starch. Possible liquefaction conditions that could affect the conversion of starch in the ground corn to soluble starch are temperature, enzyme (alpha-amylase) loading, and +/- a high temperature ("cook") stage occurs at some time during liquefaction. Examples 11 and 12 demonstrated that implementing a high temperature ("cook") stage at some time during liquefaction can result in higher conversion of starch in the corn solids to soluble (liquefied) starch. The high temperature stage in the liquefactions described in Examples 11 and 12 involved raising the liquefaction temperature at some point after liquefaction starts, holding at the higher temperature for some period of time, and then lowering the temperature back to the original value to complete liquefaction.

[0550] The liquefaction reactions compared in Example 11 were run at a different enzyme loading than the reactions compared in Example 12. These examples demonstrate the effect of two key liquefaction conditions on starch conversion: (1) enzyme loading, and (2)+/-a high temperature stage is applied at some time during liquefaction.

[0551] The conditions used to prepare the four batches of liquefied corn mash described in Examples 11 and 12 are summarized below and in Table 9.

[0552] Conditions common for all batches: [0553] Liquefaction temperature--85.degree. C. [0554] Total time at liquefaction temperature--approximately 2 hr [0555] Screen size used to grind corn--1 mm [0556] pH--5.8 [0557] Dry corn loading--26% [0558] Alpha-amylase--Liquozyme.RTM. SC DS (Novozymes, Franklinton, N.C.).

TABLE-US-00009 [0558] TABLE 9 Batch 1 Batch 2 Batch 3 Batch 4 Described in Example: 11 11 12 12 High Temperature Stage No Yes No Yes Implemented Temperature of High NA 101.degree. C. NA 121.degree. C. Temperature Stage, C.: Total Enzyme Loading, wt % 0.08% 0.08% 0.04% 0.04% (dry corn basis): Residual Unhydrolyzed 2.1% 1.1% 3.8% 2.3% Starch in Undissolved Solids after Liquefaction (as a percentage of total starch in corn feed):

[0559] The temperature profile for Batches 2 and 4 was (all values are approximate): 85.degree. C. for 30 min, High Temperature Stage for 30 min, 85.degree. C. for 90 min. Half the enzyme was added before the initial 85.degree. C. period, and half was added after the high temperature stage for the final 85.degree. C. period.

[0560] FIG. 17 illustrates the effect of enzyme loading and +/- a high temperature stage was applied at some time during the liquefaction on starch conversion. The level of residual unhydrolyzed starch in the solids is the starch that was not hydrolyzed during the liquefaction conditions of interest. FIG. 17 shows that the level of unhydrolyzed starch in the solids was reduced by almost half by applying a high temperature ("cook") stage at some point during the liquefaction. This was demonstrated at two different enzyme loadings. The data in FIG. 17 also shows that doubling the enzyme loading resulted in almost half the level of unhydrolyzed starch remaining in the solids whether a high temperature stage was applied during liquefaction or not. These examples demonstrate that operating liquefaction with a higher enzyme (alpha-amylase) loading and/or the addition of a high temperature ("cook") stage at some time during the reaction could result in a significant reduction in residual unhydrolyzed starch in the undissolved solids present in the liquefied corn mash and can reduce the loss of starch in a process where undissolved solids are removed from the mash prior to liquefaction. Any residual starch in the solids after liquefaction will not have the opportunity to hydrolyze during fermentation in a process where solids are removed prior to fermentation.

Example 13

Screen Separation of Starch and Nonsolubles Following 85.degree. C. Enzyme Digestion

[0561] Mash (301 grams) prepared per the method described in Example 1 were maintained at pH 5.8 using drops of NaOH solution when adjustment was necessary, treated with a vendor-specified dose of approximately 0.064 grams of Liquozyme.RTM. alpha-amylase enzyme (Novozyme, Franklinton, N.C.) and held at 85.degree. C. for five hours. The product was refrigerated.

[0562] Refrigerated product was warmed to approximately 50.degree. C. and 48 g was poured onto a filter assembly containing a 100 mesh screen and connected to a house vacuum source at between -15 in Hg and -20 in Hg. The screen dish had an exposed screen surface area of 44 cm.sup.2 and was sealed inside a plastic filter housing provided by Nalgene.RTM. (Thermo Fisher Scientific, Rochester, N.Y.). The slurry was filtered to form a wet cake on the screen and a yellow cloudy filtrate of 40.4 g in the receiver bottle. The wet cake was immediately washed in place with water and then discontinued while the vacuum source continued to pull any free moisture through the final washed cake. Filtration was ended when dripping ceased from the underside of the filter. An additional 28.5 g of wash filtrate were collected over 3 stages where the final stage of filtrate revealed the least color and turbidity. The final wet cake mass of 7.6 g was air dried to 2.1 g over 24 hours at room temperature. The 2.1 g were determined to contain 7.73% water after drying with a heat lamp. The vacuum filtration of this experiment produced a wet cake containing 25% total dry solids.

[0563] A sample of filtrate was combined with oleyl alcohol at room temperature, vigorously mixed and allowed to settle. The interface was restored in approximately 15 min but a hazy rag layer remained.

[0564] Lugol's solution (starch indicator) consisting of 1 g of >99.99% (trace metals basis) iodine, 2 g of ReagentPlus.RTM. grade (>99%) potassium iodide (both from Sigma-Aldrich, St. Louis, Mo.), and 17 g of house deionized water in the amount of one drop was added to samples of the filtrate, dried cake solids re-slurried in water and a control sample of water. The filtrate turned dark blue or purple, the solids slurry turned very dark blue and the water became light amber in color. Any color darker than amber indicates presence of oligosaccharides greater than 12 units long.

[0565] This experiment illustrated that most suspended solids could be separated from starch solution prepared as described above at a moderate rate on a 100 mesh screen and that starch remains with the filter cake solids. This is an indication of incomplete washing of the cake where a portion of hydrolyzed starch is left behind.

[0566] This experiment was repeated with 156 grams of mash on a 63 mm diameter 100 mesh screen. The maximum temperature was 102.degree. C., the enzyme was Spezyme.RTM. and the slurry was held above 85.degree. C. for three hours. The screening rate was measured and determined to be 0.004 or less gallons per minute per square foot of screen area.

Example 14

Screen Separation of Starch and Nonsolubles Following 115.degree. C. Enzyme Digestion

[0567] House deionized water (200 g) were charged into an open Parr Model 4635 1 liter pressure vessel (Moline, Ill.) and heated to a temperature of 85.degree. C. The water was agitated with a magnetic stir bar. Dry ground corn (90 g) prepared as described in Example 1 were added spoon-wise. The pH was raised from 5.2 to near 6.0 with stock aqueous ammonia solution. Approximately 0.064 grams of Liquozyme.RTM. solution were added with a small calibrated pipette. The lid of the pressure vessel was sealed and the vessel was pressurized to 50 psig with house nitrogen. The agitated mixture was heated to 110.degree. C. within 6 min and held between 106 to 116.degree. C. for a total of 20 min. The heating was reduced, the pressure was relieved, and the vessel was opened. An additional 0.064 g of Liquozyme.RTM. was added and the temperature was held at 63-75.degree. C. for an additional 142 min.

[0568] A small amount of the slurry was taken from the Parr vessel and gravity screened through a stack of 100, 140, and 170 mesh screens. Solids were retained only on the 100 mesh screen.

[0569] A portion, about 40%, of the slurry was transferred while hot onto the top of a dual screen assembly of 100 and 200 mesh dishes of 75 millimeter diameter. Some gravity filtration took place. Vacuum, between -15 and -20 inches of mercury, was pulled on the filtrate receiver and steady filtration was established. The filtrate was yellow and cloudy but with a stable dispersion. The cake surface was exposed within 5 min. The cake was washed with a spray of deionized water for 2-3 min and repeated with a change of receiver until the turbidity of the filtrate was constant--a total of five sprayings. The screens were examined with the conclusion that all solids were on the 100 mesh screen and none were on the 200 mesh. The wet cake was 5 mm thick. The wet cake mass was determined to be 18.9 g and the combined filtrate masses were 192 g.

[0570] The remaining mass of slurry was transferred to the filter assembly with a 100 mesh screen in place at 65.degree. C. and filtered over 5-10 min. The cake was washed with a spray of deionized water for 3-4 min and repeated with a change of receiver until the turbidity of the filtrate was constant--a total of eight sprayings. Vacuum was continued until no more drops were observed falling from the underside of the filter. The wet cake was 8 mm thick and 75 mm in diameter with a mass of 36.6 g. The combined filtrates weighed 261 g.

[0571] Three vials were tested for starch per the method described above. One vial contained water and the other two contained samples of wet cake slurried in deionized water. All vials turned yellow-amber in color. This was interpreted to mean that the filter cake was washed free of oligosaccharides of starch. These solids were later analyzed rigorously using prolonged liquefaction and subsequent saccharification to confirm that on a glucose basis, the wet cake contained no more than 0.2% of the total starch that was in the original corn.

[0572] A sample of filtrate was combined with oleyl alcohol in a vial, vigorously mixed and allowed to settle. A clear oil layer was quickly attained and the interface was well defined with little rag layer. This example illustrated that in a process in which corn mash is heated to hydrothermal conditions of .about.110.degree. C. for 20 min of cooking and further liquefied for more than two hours at 85.degree. C. before being filtered and washed, the total filtrate contains essentially all starch supplied in the grain. Furthermore, no significant interference is observed between the oleyl alcohol and the impurities contained in the filtrate.

[0573] This experiment was repeated with 247 grams of mash on a 75 mm diameter 80 mesh screen. The maximum cook temperature was 115.degree. C., the enzyme was Liquozyme.RTM. and the slurry was held at or above 85.degree. C. for three hours. The screening rate was measured and determined to be more than 0.1 gallons per minute per square foot of screen area.

Example 15

[0574] This example illustrated the removal of solids from stillage and extraction by desolventizer to recover fatty acids, esters, and triglycerides from the solids. During fermentation, solids are separated from whole stillage and fed to a desolventizer where they are contacted with 1.1 tons/hr of steam. The flow rates for the whole stillage wet cake (extractor feed), solvent, the extractor miscella, and extractor discharge solids are as shown in Table 10. Table values are short tons/hr.

TABLE-US-00010 TABLE 10 Solids from Extractor whole discharge stillage Solvent Miscella solids Fatty acids 0.099 0 0.0982 0.001 Undissolved solids 17.857 0 0.0009 17.856 Fatty acid butyl esters 2.866 0 2.837 0.0287 Hexane 0 11.02 10.467 0.555 Triglyceride 0.992 0 0.982 0.0099 Water 29.762 0 29.464 0.297

[0575] Solids exiting the desolventizer are fed to a dryer. The vapor exiting the desolventizer contains 0.55 tons/hr of hexane and 1.102 tons/hr of water. This stream is condensed and fed to a decanter. The water-rich phase exiting the decanter contains about 360 ppm of hexane. This stream is fed to a distillation column where the hexane is removed from the water-rich stream. The hexane enriched stream exiting the top of the distillation column is condensed and fed to the decanter. The organic-rich stream exiting the decanter is fed to a distillation column. Steam (11.02 tons/hr) is fed to the bottom of the distillation column. The composition of the overhead and bottom products for this column are shown in Table 11. Table values are tons/hr.

TABLE-US-00011 TABLE 11 Bottoms Overheads Fatty acids 0.0981 0 Fatty acid butyl esters 2.8232 0 Hexane 0.0011 11.12 Triglyceride 0.9812 0 Water 0 11.02

Example 16

By-Product Recovery

[0576] This example illustrates the recovery of by-products from mash. Corn oil separated from mash under the conditions described in Example 6 with the exception that a three-phase centrifuge (Flottweg Tricanter.RTM. Z23-4 bowl diameter, 230 mm, length to diameter ratio 4:1) was used with these conditions: [0577] Bowl Speed: 5000 rpm [0578] Differential Speed: 10 rpm [0579] Feed Rate: 3 gpm [0580] Phase Separator Disk: 138 mm [0581] Impeller Setting: 144 mm.

[0582] The corn oil separate had 81% triglycerides, 6% free fatty acids, 4% diglyceride, and 5% total of phospholipids and monoglycerides as determined by gas chromatography and thin layer chromatography (see, e.g., U.S. Patent Application Publication No. 2012/0164302).

[0583] The solids separated from mash under the conditions described above had a moisture content of 58% as determined by weight loss upon drying and had 1.2% triglycerides and 0.27% free fatty acids as determined by gas chromatography (see, e.g., U.S. Patent Application Publication No. 2012/0164302).

[0584] The composition of solids separated from whole stillage, oil extracted between evaporator stages, by-product extractant and Condensed Distillers Solubles (CDS) in Table 14 were calculated assuming the composition of whole stillage shown in Table 12 and the assumptions in Table 13 (separation at three-phase centrifuge). The values of Table 11 were obtained from an Aspen Plus.RTM. model (Aspen Technology, Inc., Burlington, Mass.). This model assumes that corn oil is not extracted from mash. It is estimated that the protein content on a dry basis of cells, dissolved solids, and suspended solids is approximately 50%, 22%, and 35.5%, respectively. The composition of by-product extractant is estimated to be 70.7% fatty acid and 29.3% fatty acid isobutyl ester on a dry basis.

TABLE-US-00012 TABLE 12 Component Mass % Water 57.386% Cells 0.502% Fatty acids 6.737% Isobutyl esters of fatty acids 30.817% Triglyceride 0.035% Suspended solids 0.416% Dissolved solids 4.107%

TABLE-US-00013 TABLE 13 Hydrolyzer Thin feed stillage Solids Organics 99.175% 0.75% 0.08% Water and dissolved solids 1% .sup. 96% 3% Suspended solids and cells 1% 2% .sup. 97%

TABLE-US-00014 TABLE 14 Stream C. protein triglyceride FFA FABE Whole stillage wet cake 40% trace 0.5% 2.2% Oil at evaporator 0% 0.08% 16.1% 73.8% CDS 22% trace % 0.37% 1.71%

Example 17

Removal of Corn Oil from Liquefied Corn Mash

[0585] This example describes the use of a three-phase centrifuge to remove corn oil from liquefied corn mash. Whole corn kernels typically contain about 3-6 wt % corn oil, most of which resides in the germ. Corn oil is released from the germ during dry milling and liquefaction. Consequently, corn mash contains free corn oil.

[0586] Liquefied corn mash was generated using a standard continuous liquefaction process as used, for example, in a dry-grind corn-to-ethanol process. The ground corn contained 4.16 wt % corn oil (dry corn basis) and had a moisture content of 14.7 wt %. Ground corn and water were fed to a slurry tank at 10.2 lbm/min and 17.0 lbm/min, respectively, to give a dry corn loading of 32 wt %. Alpha-amylase was fed to the slurry tank at a rate that corresponded to an enzyme loading of about 0.025 wt % on a dry corn basis. The slurry and liquefaction tanks were both run at 85.degree. C. and a pH of 5.8. The total residence time at 85.degree. C. was about 2 hr. Mash was produced at a rate of about 3 gpm and contained about 1.3 wt % corn oil on a wet basis. A portion of this oil existed as free oil and a portion was in the undissolved solids. This corresponds to a total corn oil content of the mash to be roughly 2.0 lbm of corn oil/bushel of corn. The total solids (TS) in the mash was 32 wt % and the total suspended solids (TSS) was 7.7 wt %.

[0587] The liquefied corn mash was fed to a three-phase centrifuge (Model Z23-4/441, Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany) at a rate of about 3 gpm. The feed temperature was about 80.degree. C. The mash was separated into three streams: (1) corn oil, (2) aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of the Tricanter.RTM. were as follows: [0588] Bowl Speed: 5000 rpm [0589] G-force: approximately 4000 g's [0590] Differential speed: 10 rpm [0591] Impeller setting: approximately 145 [0592] Phase separator disk: approximately 138 mm.

[0593] Table 15 summarizes data (flow rate, density, solids content, and corn oil content) measured for the feed stream and the three exit streams from the Tricanter.RTM..

TABLE-US-00015 TABLE 15 Aque- Feed ous Wet Corn Mash Centrate Cake Oil Flow Rate, lbm/min: 27.2 19.5 7.6 0.14 Density, g/ml: 1.1008 ~1.09 0.875 Total Solids, wt %: 32.0 28.7 39.1 ~0 Total Suspended Solids, wt %: 7.7 4.3 16.6 ~0 Corn Oil Content (wet basis), 1.3 0.38 1.95 99.4 * wt %: Corn Oil Content, lbm/bushel: 2.0 0.4 0.8 0.8 % of Corn Oil in feed: NA 20 41 39 * Balance is water

[0594] The corn oil removed from the mash by the Tricanter.RTM. accounted for 39% of the total corn oil in the mash feed. The corn oil removal rate was equal to about 0.8 lbm/bushel of corn. The corn oil separated and recovered from the liquefied corn mash contained about 85 wt % glycerides.

[0595] In a process where about 0.8 lb corn oil/bushel of corn is removed, the mash flow rate would decrease by 3.9 gallons per minute:

37 bu corn min * 0.8 lb oil bu corn / 7.65 lb oil gal = 3.9 gallons oil / minute ##EQU00003##

[0596] In a production plant where the total mash flow to fermentation is about 700 gpm, the oil that was removed would make about 0.55% of the total mash flow. Assuming that the production plant proportionally raises throughput to take advantage of the extra volume, the yearly production would increase by 0.55%, which means that a 56 MMGPY plant would produce an additional 310,000 gallons of ethanol.

Example 18

Removal of Corn Oil from Liquefied Corn Mash--Feed Rate Adjustment

[0597] In this example, liquefied corn mash was fed to a three-phase centrifuge at a feed rate of 1 gpm. Liquefied corn mash was generated using a standard continuous liquefaction process as used, for example, in a dry-grind corn-to-ethanol process. The ground corn contained 4.16 wt % corn oil (dry corn basis) and had a moisture content of 14.7 wt %. Ground corn and water were fed to a slurry tank at 8.2 lbm/min and 19.0 lbm/min, respectively, to give a dry corn loading of approximately 26 wt %. Alpha-amylase was fed to the slurry tank at a rate of 50 g/hr, which corresponded to an enzyme loading of about 0.026 wt % on a dry corn basis. The slurry and liquefaction tanks were both run at 85.degree. C. and a pH of 5.8. No jet cooker was used. The total residence time at 85.degree. C. was about 2 hr. Mash was produced at a rate of about 3 gpm and inventoried into a 1500 gal tank for centrifuge testing. The mash contained about 1.1 wt % corn oil on a wet basis. A portion of this oil existed as free oil and a portion was in the undissolved solids. This corresponds to a total corn oil content of the mash to be roughly 2.0 lbm of corn oil/bushel of corn. The TS in the mash was 25.6 wt % and the TSS was 5.3 wt %.

[0598] The liquefied corn mash was fed from a feed tank to a three-phase centrifuge (Model Z23-3, Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany) at a rate of about 1 gpm. The feed temperature was about 80.degree. C. The mash was separated into three streams: (1) corn oil, (2) aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of the Tricanter.RTM. were as follows: [0599] Bowl Speed: 5000 rpm [0600] G-force: approximately 4000 g's [0601] Differential speed: 12 rpm [0602] Impeller setting: approximately 156 [0603] Phase separator disk: approximately 140 mm.

[0604] Table 16 summarizes data (flow rate, density, solids content, and corn oil content) measured for the feed stream and the three exit streams from the Tricanter.RTM.. The quality of the corn oil mass balance was 102% and the quality of the total solids mass balance was 105%.

TABLE-US-00016 TABLE 16 Aque- Feed ous Wet Corn Mash Centrate Cake Oil Flow Rate, lbm/min: 9.2 6.2 3.0 0.016 Density, g/ml: ~1.10 ~1.09 ~0.9 Total Solids, wt %: 25.6 21.6 37.4 ~0 Total Suspended Solids, wt %: 5.3 1.1 13.8 ~0 Corn Oil Content (wet basis), 1.1 0.28 2.2 >99 * wt %: Corn Oil Content, lbm/bushel: 2.0 0.36 1.34 0.34 % of the Corn Oil in the feed: NA 18 67 17 * Balance is water

[0605] The corn oil removed from the mash by the Tricanter.RTM. accounted for 17% of the total corn oil in the mash feed. This corn oil removal rate was equal to about 0.34 lbm/bushel of corn. The corn oil separated and recovered from the liquefied corn mash contained about 81.4 wt % glycerides and 8.3 wt % free fatty acids.

Example 19

Removal of Corn Oil from Liquefied Corn Mash--Feed Rate Adjustment

[0606] In this example, liquefied corn mash was fed to a three-phase centrifuge at a feed rate of 10.1 gpm. Liquefied corn mash was generated using a standard continuous liquefaction process as used, for example, in a dry-grind corn-to-ethanol process. The ground corn contained 4.16 wt % corn oil (dry corn basis) and had a moisture content of 14.7 wt %. Ground corn and water were fed to a slurry tank at 8.2 lbm/min and 19.0 lbm/min, respectively, to give a dry corn loading of approximately 26 wt %. Alpha-amylase was fed to the slurry tank at a rate of 50 g/hr, which corresponded to an enzyme loading of about 0.026 wt % on a dry corn basis. The slurry and liquefaction tanks were both run at 85.degree. C. and a pH of 5.8. No jet cooker was used. The total residence time at 85.degree. C. was about 2 hr. Mash was produced at a rate of about 3 gpm and inventoried into a 1500 gal tank for centrifuge testing. The mash contained about 1.1 wt % corn oil on a wet basis. A portion of this oil existed as free oil and a portion was in the undissolved solids. This corresponds to a total corn oil content of the mash to be roughly 2.0 lbm of corn oil/bushel of corn. The TS in the mash was 26.2 wt % and the TSS was 6.7 wt %.

[0607] The liquefied corn mash was fed from the feed tank to a three-phase centrifuge (Model Z23-4/441, Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany) at a rate of about 10.1 gpm. The feed temperature was about 80.degree. C. The mash was separated into three streams: (1) corn oil, (2) aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of the Tricanter.RTM. were as follows: [0608] Bowl Speed: 5000 rpm [0609] G-force: approximately 4000 g's [0610] Differential speed: 20 rpm [0611] Impeller setting: approximately 148 [0612] Phase separator disk: approximately 138 mm.

[0613] Table 17 summarizes data (flow rate, density, solids content, and corn oil content) measured for the feed stream and the three exit streams from the Tricanter.RTM.. The quality of the corn oil mass balance was 95%.

TABLE-US-00017 TABLE 17 Aque- Feed ous Wet Corn Mash Centrate Cake Oil Flow Rate, lbm/min: 92.2 73.1 18.9 0.177 Density, g/ml: ~1.10 ~1.09 ~0.9 Total Solids, wt %: 26.2 23.3 36.9 ~0 Total Suspended Solids, wt %: 6.7 1.9 25.2 ~0 Corn Oil Content (wet basis), 1.1 0.71 1.4 >99 * wt %: Corn Oil Content, lbm/bushel: 2.0 1.02 0.52 0.36 % of the Corn Oil in the feed: NA 51 26 18 * Balance is water

[0614] The corn oil removed from the mash by the Tricanter.RTM. accounted for 18% of the total corn oil in the mash feed. This corn oil removal rate was equal to about 0.36 lbm/bushel of corn. The corn oil separated and recovered from the liquefied corn mash contained about 81.4 wt % glycerides and 8.3 wt % free fatty acids.

Example 20

Effect of Liquefied Corn Mash pH on the Recovery of Corn Oil from Mash

[0615] Liquefied corn mash was generated using a standard continuous liquefaction process as typically used in a dry-grind corn-to-ethanol process. The ground corn contained 4.6 wt % corn oil (dry corn basis) and had a moisture content of 12.5 wt %. Ground corn and water were fed to the slurry tank at rates produce corn mash at 3 gpm with a dry corn loading of 25.9 wt %. The slurry tank was operated at 85.degree. C. with a 30 min residence time. The slurry was then heated to 105.degree. C. using live steam in a jet cooker and held at that temperature for about 30 min. After exiting the hold tube, the slurry was fed into a liquefaction tank which was operated at 85.degree. C. with a 90 min residence time. Alpha-amylase (Spezyme.RTM. ALPHA, Genencor.RTM., Palo Alto, Calif.) was continuously fed to the process at a rate that corresponded to an overall enzyme loading of 0.04 wt % enzyme on a dry corn basis. Forty percent (40%) of the total enzyme was added to the slurry tank, and 60% was added to the liquefaction tank. The slurry and liquefaction tanks were both run at a pH of 5.8. Mash was produced at a rate of about 3 gpm and inventoried into a 1500 gal tank for centrifuge testing. The liquefied corn mash contained about 1.12 wt % corn oil on a wet basis. This corresponds to a total corn oil content of the mash to be roughly 2.2 lbm of corn oil/bushel of corn. Some of this oil existed as free oil; some still was in the undissolved solids. The ratio of glycerides to free fatty acids in the corn oil in the mash was about 7.6 to 1. The total solids (TS) in the mash were 25.9 wt %, and the total suspended solids (TSS) were 4.7 wt %. The DE (dextrose equivalent) and the pH of the final mash was 15.9 and 5.75, respectively. The density of the mash was 1.08 g/mL.

[0616] The liquefied mash was separated using a three-phase centrifuge (Model Z23-4/441, Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany) at three different feed flow rates: 1.24 gal/min, 5.1 gal/min and 10 gal/min. The feed temperature was about 80.degree. C. The mash was separated into three streams: (1) corn oil, (2) aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The bowl speed was held constant at about 5000 rpm (approximately 4000 g's). Table 18 compares the corn oil recovery as a function of mash feed rate to the Tricanter.RTM. for a mash pH of 5.8. The data shown in Table 18 shows that there is an effect of feed rate to the Tricanter.RTM. on the recovery rate of corn oil at pH=5.8.

TABLE-US-00018 TABLE 18 Mash Feed Differential Impeller Corn Oil Corm Oil Corn Oil Rate, Speed, Setting, in Mash, Recovered, Recovery Test gpm rpm mm g/min g/min % A 1.2 5.2 144 63.3 8.3 13 B 5.1 10.5 146 248.4 73.2 29 C 10 9.8 149 487.1 100.3 21

[0617] Corn oil recovery is based on the total oil contained in the mash (both free oil and oil in the solids). The mash fed to the Tricanter.RTM. contained 1.1-1.2 wt % corn oil (includes free oil and oil in the solids).

[0618] The data in Table 18 shows that there is an effect of mash feed rate on corn oil recovery rate (at the conditions tested). Table 19 summarizes the amount of oil phase in the aqueous centrate, aqueous phase in the oil centrate, and solids in the oil centrate for the three conditions tested.

TABLE-US-00019 TABLE 19 Mash Corn Oil in Aqueous Density Feed Corn Oil Aqueous Phase in Solids in of Corn Rate, Recovery Centrate, Corn Oil, Corn Oil, Oil, Test gpm % vol %* vol %* vol %* g/mL A 1.2 13 0 0 1.8 0.892 B 5.1 29 0 0 1.7 0.892 C 10 21 0 3 1.5 0.906 *Measured using a LuMiSizer .RTM. (L.U.M GmbH, Berlin, Germany)

[0619] The data in Table 19 shows that the corn oil recovered was fairly clean since it contained very little aqueous phase and very little solids. The corn oil separated and recovered from the liquefied corn mash contained about 85.1 wt % glycerides and 8.0 wt % free fatty acids. The balance was solids, aqueous phase, and other extractables (e.g. phospholipids, sterols, etc.,).

[0620] The pH of the remaining liquefied corn mash in the Tricanter.RTM. feed tank was lowered to about 3. The acidic mash was separated using a three-phase centrifuge (Model Z23-4/441, Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany) at two different feed flow rates: 1.3 gal/min and 5 gal/min. The feed temperature was about 80.degree. C. The mash was separated into three streams: (1) corn oil, (2) aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The bowl speed was held constant at about 5000 rpm (approximately 4000 g's). Table 20 compares the corn oil recovery as a function of mash feed rate to the Tricanter.RTM. for a mash pH of 3.0.

TABLE-US-00020 TABLE 20 Mash Feed Differential Impeller Corn Oil Corm Oil Corn Oil Rate, Speed, Setting, in Mash, Recovered, Recovery Test gpm rpm mm g/min g/min % D 1.3 5.2 144.5 47.3 20.8 44 E 5.0 10.3 146 181.9 72.8 40

[0621] Mash was produced at pH=5.8, and the pH of the final mash was then lowered to 3 before feeding the centrifuge. Corn oil recovery is based on the total oil contained in the mash (both free oil and oil in the solids). The mash fed to the Tricanter.RTM. contained about 0.9 wt % corn oil (includes free oil and oil in the solids). Total Solids of mash fed to Tricanter.RTM. were 27.1 wt %, and Total Suspended Solids of mash were 5.5 wt %.

[0622] Table 21 summarizes the amount of oil phase in the aqueous centrate, aqueous phase in the oil centrate, and solids in the oil centrate for the two conditions tested. The data in Table 21 shows that the corn oil recovered was fairly clean since it contained very little aqueous phase and very little solids.

TABLE-US-00021 TABLE 21 Mash Corn Oil in Aqueous Density Feed Corn Oil Aqueous Phase in Solids in of Corn Rate, Recovery Centrate, Corn Oil, Corn Oil, Oil, Test gpm % vol %* vol %* vol %* g/mL D 1.3 44 0 0.2 0 0.895 E 5.0 40 0 0 0 0.895 *Measured using a LuMiSizer .RTM. (L.U.M GmbH, Berlin, Germany)

[0623] Comparing the results of Test A (Table 18) to Test D (Table 20) and comparing the results of Test B (Table 19) to Test E (Table 21) show an effect of mash pH on the corn oil recovery using a Tricanter.RTM.. The data suggests that reducing the pH of the mash before separating it with a Tricanter.RTM. results in higher corn oil recovery. These comparisons are shown in Table 22.

TABLE-US-00022 TABLE 22 pH of Mash fed to Tricanter .RTM. Mash Feed Rate gpm pH = 5.8 pH = 3.0 1.3 13% 44% 5.1 29% 40%

[0624] Percentages shown in Table 22 are corn oil recoveries based on the total oil contained in the mash (both free oil and oil in the solids). The composition of corn oil in the mash fed to the Tricanter.RTM. ranged from 0.9% to 1.2 wt % corn oil (includes free oil and oil in the solids) for all the tests described in this example. The Tricanter.RTM. was operated at 5000 rpm (.about.4000 G's), and the differential speed and impeller setting were 5-10 rpm and 145 mm, respectively.

Example 21

Recovery of Corn Oil and Solids from Corn Mash

[0625] Liquefied corn mash was generated using a standard continuous liquefaction process as used in a dry-grind corn-to-ethanol process with 30-31 wt % on a dry corn basis. Recycle water consisting of cook water and backset was used, which elevated the total solids (TS) to approximately 33 wt %. Alpha-amylase (Spezyme.RTM. RSL, Genencor.RTM., Palo Alto, Calif.) was added to the slurry tank (85.degree. C., pH approximately 5.8, 30 min residence time) at a rate that corresponded to approximately 0.02 wt % dry corn base enzyme load. A jet cooker was used to elevate the temperature to 105-110.degree. C. with 18 min cook time. The liquefaction tank was run at 85.degree. C. with a pH of approximately 5.8. Spezyme.RTM. RSL (Genencor.RTM., Palo Alto, Calif.) was also added to the liquefaction tank at a rate that corresponded to approximately 0.005 wt % dry corn base enzyme load, and the total residence time in the liquefaction tank was about 90 min. A side stream of mash was collected from the liquefaction tank and diverted to a small dilution tank, where process condensate was added to achieve the desired dilution. The original mash contained about 1.55 wt % corn oil on a wet basis. A portion of this oil existed as free oil and a portion was in the undissolved solids. This corresponds to a total corn oil content of the original mash to be roughly 3.0 lbm of corn oil/bushel of corn. The TS in the original mash was 33.2 wt % and the total suspended solids (TSS) was 6.5 wt %. The dilution with process condensate lowered the TS to approximately 27 wt %, the TSS to approximately 5.5 wt %, and the oil content to approximately 1.3 wt % (wet basis).

[0626] The liquefied corn mash was fed from the feed tank to a three-phase centrifuge (Model Z23-4/441, Flottweg Tricanter.RTM., Flottweg AG, Vilsibiburg, Germany) at a rate between 9 and 11 gpm. The feed temperature was about 85.degree. C. The mash was separated into three streams: (1) corn oil, (2) aqueous solution of oligosaccharides (liquefied starch), and (3) wet cake. The operating conditions of the three-phase centrifuge were as follows: [0627] Bowl Speed: 5000 rpm [0628] G-force: approximately 3200 g [0629] Differential speed: 25 rpm [0630] Impeller setting: see table [0631] Phase separator disk: approximately 138 mm

[0632] Table 23 summarizes three-phase centrifuge conditions and properties following separation. Streams at both corn loads 33 wt % and 26 wt % were separated into a very clean corn oil stream and wet cakes at 38-41 wt % total solids. The suspended solids concentration in the heavy phase was strongly affected by the corn load. The 33 wt % sample generated a centrate TSS of approximately 3.5-4 wt % while the 26 wt % TS generate a lower TSS centrate at approximately 1.7-2 wt %.

TABLE-US-00023 TABLE 23 Feed Properties TS (wt %) 33 33 26 26 Feed rate (gpm) 9 11.2 9 11.3 Centrifuge Conditions Bowl speed (rpm) 5000 5000 (4400-5400) 5000 5000 Differential speed (rpm) 25 (25-50) 25 (25-50) 25 15 (15-25) Impeller Speed (mm) 155 (145-158) 155 (155-160) 155 153 (153-155) Light Centrate Properties Water content (ppm) Very low Very low Very low Very low TSS (wt %) Very low Very low Very low Very low Flow rate (mL/min) 230 (150-330) 300 (195-360) 280 (170-280) 364 (364-459) Recovery (on total basis) (%) 43 (30-60) 43 (30-54) 53 (30-53) 54 (54-68) Heavy Centrate Properties TSS (wt %) 3.5 (3.5-4) 4.3 (3.6-4.7) 1.7 (1.7-3.8) 2 (2-3.2) Wet Cake Properties TS (wt %) 41 (36-42) 39 (37-39) 38.7 (38.5-38.7) 39 (30-40)

[0633] Results are also shown in FIGS. 19A to 19E. FIG. 19A shows that at low flow rates of approximately 4 gpm, the centrate TSS was about 3.3%, and the centrate TSS increased to about 4.2-4.7% with a flow rate of 11.5 gpm.

[0634] FIG. 19B shows the suspended solids recovery as a function of flow rate. At low flow rates of approximately 4 gpm, approximately 60% of the suspended solids were recovered in the wet cake. By increasing the flow rate to about 11.5 gpm, the recovery rate decreased to about 40-50%.

[0635] FIG. 19C shows the wet cake total solids as a function of flow rate. At low flow rates of approximately 4 gpm, wet cake total solids were about 41%. By increasing the flow rate to about 11.5 gpm, the wet cake total solids decreased to about 39%.

[0636] FIG. 19D shows the impact of differential rpm on the total wet cake solids. The wet cake solids decreased with increased differential rpm.

[0637] FIG. 19E shows the effect of feed rate on corn oil recovery. At low flow rates, oil recovery was about 48%. When the flow rate was increased to approx. 11.5 gpm, oil recovery decreases to about 35%. It appears that less oil is separated from the feed stream with higher flow rate.

Example 22

Rheological Characteristics of Corn Mash

[0638] The viscosity of corn mash is measured using an AR-G2 rotational rheometer (TA Instruments, New Castle, Del.) configured with vane geometry. A slurry of ground corn and water is prepared, mixed, and heated in a resin kettle to 55.degree. C. The pH is adjusted to 5.8, and enzyme (e.g., alpha-amylase and/or glucoamylase) is added to the slurry. The slurry is heated to 65.degree. C. at a rate of 2.degree. C./min. A sample is removed and transferred to the rheometer equipped with a narrow gap concentric cylinder geometry that is preheated to 65.degree. C. A temperature ramp is then performed raising the temperature from 65.degree. C. to 85.degree. C. at a rate of 2.degree. C./min. The temperature ramp is conducted at a fixed shear rate (e.g., 75-200 s.sup.-1). Viscosity is measured as a function of time.

[0639] While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. It will be apparent to persons skilled in the relevant art that various changes in form and detail can be made therein without departing from the spirit and scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0640] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Sequence CWU 1

1

1281570PRTBacillus subtilis 1Met Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg Gly 1 5 10 15 Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His Val 20 25 30 Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu Gln 35 40 45 Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala Ala 50 55 60 Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val Val 65 70 75 80 Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu Leu 85 90 95 Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn Val 100 105 110 Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn Ala 115 120 125 Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp Val 130 135 140 Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser Ala 145 150 155 160 Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val Asn 165 170 175 Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys Leu 180 185 190 Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile Gln 195 200 205 Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg Pro 210 215 220 Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu Pro 225 230 235 240 Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu Glu 245 250 255 Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly Asp 260 265 270 Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp Pro 275 280 285 Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr Ile 290 295 300 Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln Pro 305 310 315 320 Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile Glu 325 330 335 His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile Leu 340 345 350 Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala Asp 355 360 365 Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu Arg 370 375 380 Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 400 Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr Leu 405 410 415 Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala 420 425 430 Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val Ser 435 440 445 Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala Val 450 455 460 Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr Tyr 465 470 475 480 Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser Ala 485 490 495 Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe Gly 500 505 510 Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val Leu 515 520 525 Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro Val 530 535 540 Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys Glu 545 550 555 560 Phe Gly Glu Leu Met Lys Thr Lys Ala Leu 565 570 21716DNABacillus subtilis 2atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680gaattcgggg aactcatgaa aacgaaagct ctctag 17163559PRTKlebsiella pneumoniae 3Met Asp Lys Gln Tyr Pro Val Arg Gln Trp Ala His Gly Ala Asp Leu 1 5 10 15 Val Val Ser Gln Leu Glu Ala Gln Gly Val Arg Gln Val Phe Gly Ile 20 25 30 Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Ser Leu Leu Asp Ser Ser 35 40 45 Ile Arg Ile Ile Pro Val Arg His Glu Ala Asn Ala Ala Phe Met Ala 50 55 60 Ala Ala Val Gly Arg Ile Thr Gly Lys Ala Gly Val Ala Leu Val Thr 65 70 75 80 Ser Gly Pro Gly Cys Ser Asn Leu Ile Thr Gly Met Ala Thr Ala Asn 85 90 95 Ser Glu Gly Asp Pro Val Val Ala Leu Gly Gly Ala Val Lys Arg Ala 100 105 110 Asp Lys Ala Lys Gln Val His Gln Ser Met Asp Thr Val Ala Met Phe 115 120 125 Ser Pro Val Thr Lys Tyr Ala Ile Glu Val Thr Ala Pro Asp Ala Leu 130 135 140 Ala Glu Val Val Ser Asn Ala Phe Arg Ala Ala Glu Gln Gly Arg Pro 145 150 155 160 Gly Ser Ala Phe Val Ser Leu Pro Gln Asp Val Val Asp Gly Pro Val 165 170 175 Ser Gly Lys Val Leu Pro Ala Ser Gly Ala Pro Gln Met Gly Ala Ala 180 185 190 Pro Asp Asp Ala Ile Asp Gln Val Ala Lys Leu Ile Ala Gln Ala Lys 195 200 205 Asn Pro Ile Phe Leu Leu Gly Leu Met Ala Ser Gln Pro Glu Asn Ser 210 215 220 Lys Ala Leu Arg Arg Leu Leu Glu Thr Ser His Ile Pro Val Thr Ser 225 230 235 240 Thr Tyr Gln Ala Ala Gly Ala Val Asn Gln Asp Asn Phe Ser Arg Phe 245 250 255 Ala Gly Arg Val Gly Leu Phe Asn Asn Gln Ala Gly Asp Arg Leu Leu 260 265 270 Gln Leu Ala Asp Leu Val Ile Cys Ile Gly Tyr Ser Pro Val Glu Tyr 275 280 285 Glu Pro Ala Met Trp Asn Ser Gly Asn Ala Thr Leu Val His Ile Asp 290 295 300 Val Leu Pro Ala Tyr Glu Glu Arg Asn Tyr Thr Pro Asp Val Glu Leu 305 310 315 320 Val Gly Asp Ile Ala Gly Thr Leu Asn Lys Leu Ala Gln Asn Ile Asp 325 330 335 His Arg Leu Val Leu Ser Pro Gln Ala Ala Glu Ile Leu Arg Asp Arg 340 345 350 Gln His Gln Arg Glu Leu Leu Asp Arg Arg Gly Ala Gln Leu Asn Gln 355 360 365 Phe Ala Leu His Pro Leu Arg Ile Val Arg Ala Met Gln Asp Ile Val 370 375 380 Asn Ser Asp Val Thr Leu Thr Val Asp Met Gly Ser Phe His Ile Trp 385 390 395 400 Ile Ala Arg Tyr Leu Tyr Thr Phe Arg Ala Arg Gln Val Met Ile Ser 405 410 415 Asn Gly Gln Gln Thr Met Gly Val Ala Leu Pro Trp Ala Ile Gly Ala 420 425 430 Trp Leu Val Asn Pro Glu Arg Lys Val Val Ser Val Ser Gly Asp Gly 435 440 445 Gly Phe Leu Gln Ser Ser Met Glu Leu Glu Thr Ala Val Arg Leu Lys 450 455 460 Ala Asn Val Leu His Leu Ile Trp Val Asp Asn Gly Tyr Asn Met Val 465 470 475 480 Ala Ile Gln Glu Glu Lys Lys Tyr Gln Arg Leu Ser Gly Val Glu Phe 485 490 495 Gly Pro Met Asp Phe Lys Ala Tyr Ala Glu Ser Phe Gly Ala Lys Gly 500 505 510 Phe Ala Val Glu Ser Ala Glu Ala Leu Glu Pro Thr Leu Arg Ala Ala 515 520 525 Met Asp Val Asp Gly Pro Ala Val Val Ala Ile Pro Val Asp Tyr Arg 530 535 540 Asp Asn Pro Leu Leu Met Gly Gln Leu His Leu Ser Gln Ile Leu 545 550 555 42055DNAKlebsiella pneumoniae 4tcgaccacgg ggtgctgacc ttcggcgaaa ttcacaagct gatgatcgac ctgcccgccg 60acagcgcgtt cctgcaggct aatctgcatc ccgataatct cgatgccgcc atccgttccg 120tagaaagtta agggggtcac atggacaaac agtatccggt acgccagtgg gcgcacggcg 180ccgatctcgt cgtcagtcag ctggaagctc agggagtacg ccaggtgttc ggcatccccg 240gcgccaaaat cgacaaggtc tttgattcac tgctggattc ctccattcgc attattccgg 300tacgccacga agccaacgcc gcatttatgg ccgccgccgt cggacgcatt accggcaaag 360cgggcgtggc gctggtcacc tccggtccgg gctgttccaa cctgatcacc ggcatggcca 420ccgcgaacag cgaaggcgac ccggtggtgg ccctgggcgg cgcggtaaaa cgcgccgata 480aagcgaagca ggtccaccag agtatggata cggtggcgat gttcagcccg gtcaccaaat 540acgccatcga ggtgacggcg ccggatgcgc tggcggaagt ggtctccaac gccttccgcg 600ccgccgagca gggccggccg ggcagcgcgt tcgttagcct gccgcaggat gtggtcgatg 660gcccggtcag cggcaaagtg ctgccggcca gcggggcccc gcagatgggc gccgcgccgg 720atgatgccat cgaccaggtg gcgaagctta tcgcccaggc gaagaacccg atcttcctgc 780tcggcctgat ggccagccag ccggaaaaca gcaaggcgct gcgccgtttg ctggagacca 840gccatattcc agtcaccagc acctatcagg ccgccggagc ggtgaatcag gataacttct 900ctcgcttcgc cggccgggtt gggctgttta acaaccaggc cggggaccgt ctgctgcagc 960tcgccgacct ggtgatctgc atcggctaca gcccggtgga atacgaaccg gcgatgtgga 1020acagcggcaa cgcgacgctg gtgcacatcg acgtgctgcc cgcctatgaa gagcgcaact 1080acaccccgga tgtcgagctg gtgggcgata tcgccggcac tctcaacaag ctggcgcaaa 1140atatcgatca tcggctggtg ctctccccgc aggcggcgga gatcctccgc gaccgccagc 1200accagcgcga gctgctggac cgccgcggcg cgcagctcaa ccagtttgcc ctgcatcccc 1260tgcgcatcgt tcgcgccatg caggatatcg tcaacagcga cgtcacgttg accgtggaca 1320tgggcagctt ccatatctgg attgcccgct acctgtacac gttccgcgcc cgtcaggtga 1380tgatctccaa cggccagcag accatgggcg tcgccctgcc ctgggctatc ggcgcctggc 1440tggtcaatcc tgagcgcaaa gtggtctccg tctccggcga cggcggcttc ctgcagtcga 1500gcatggagct ggagaccgcc gtccgcctga aagccaacgt gctgcatctt atctgggtcg 1560ataacggcta caacatggtc gctatccagg aagagaaaaa atatcagcgc ctgtccggcg 1620tcgagtttgg gccgatggat tttaaagcct atgccgaatc cttcggcgcg aaagggtttg 1680ccgtggaaag cgccgaggcg ctggagccga ccctgcgcgc ggcgatggac gtcgacggcc 1740cggcggtagt ggccatcccg gtggattatc gcgataaccc gctgctgatg ggccagctgc 1800atctgagtca gattctgtaa gtcatcacaa taaggaaaga aaaatgaaaa aagtcgcact 1860tgttaccggc gccggccagg ggattggtaa agctatcgcc cttcgtctgg tgaaggatgg 1920atttgccgtg gccattgccg attataacga cgccaccgcc aaagcggtcg cctccgaaat 1980caaccaggcc ggcggccgcg ccatggcggt gaaagtggat gtttctgacc gcgaccaggt 2040atttgccgcc gtcga 20555554PRTLactococcus lactis 5Met Ser Glu Lys Gln Phe Gly Ala Asn Leu Val Val Asp Ser Leu Ile 1 5 10 15 Asn His Lys Val Lys Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile Asp 20 25 30 Arg Val Phe Asp Leu Leu Glu Asn Glu Glu Gly Pro Gln Met Val Val 35 40 45 Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala Gln Ala Val Gly Arg 50 55 60 Leu Thr Gly Glu Pro Gly Val Val Val Val Thr Ser Gly Pro Gly Val 65 70 75 80 Ser Asn Leu Ala Thr Pro Leu Leu Thr Ala Thr Ser Glu Gly Asp Ala 85 90 95 Ile Leu Ala Ile Gly Gly Gln Val Lys Arg Ser Asp Arg Leu Lys Arg 100 105 110 Ala His Gln Ser Met Asp Asn Ala Gly Met Met Gln Ser Ala Thr Lys 115 120 125 Tyr Ser Ala Glu Val Leu Asp Pro Asn Thr Leu Ser Glu Ser Ile Ala 130 135 140 Asn Ala Tyr Arg Ile Ala Lys Ser Gly His Pro Gly Ala Thr Phe Leu 145 150 155 160 Ser Ile Pro Gln Asp Val Thr Asp Ala Glu Val Ser Ile Lys Ala Ile 165 170 175 Gln Pro Leu Ser Asp Pro Lys Met Gly Asn Ala Ser Ile Asp Asp Ile 180 185 190 Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val Ile Leu 195 200 205 Val Gly Ala Gly Ala Ser Asp Ala Lys Val Ala Ser Ser Leu Arg Asn 210 215 220 Leu Leu Thr His Val Asn Ile Pro Val Val Glu Thr Phe Gln Gly Ala 225 230 235 240 Gly Val Ile Ser His Asp Leu Glu His Thr Phe Tyr Gly Arg Ile Gly 245 250 255 Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg Ser Asp Leu 260 265 270 Val Ile Ala Val Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Arg Asn Trp 275 280 285 Asn Ala Glu Ile Asp Ser Arg Ile Ile Val Ile Asp Asn Ala Ile Ala 290 295 300 Glu Ile Asp Thr Tyr Tyr Gln Pro Glu Arg Glu Leu Ile Gly Asp Ile 305 310 315 320 Ala Ala Thr Leu Asp Asn Leu Leu Pro Ala Val Arg Gly Tyr Lys Ile 325 330 335 Pro Lys Gly Thr Lys Asp Tyr Leu Asp Gly Leu His Glu Val Ala Glu 340 345 350 Gln His Glu Phe Asp Thr Glu Asn Thr Glu Glu Gly Arg Met His Pro 355 360 365 Leu Asp Leu Val Ser Thr Phe Gln Glu Ile Val Lys Asp Asp Glu Thr 370 375 380 Val Thr Val Asp Val Gly Ser Leu Tyr Ile Trp Met Ala Arg His Phe 385 390 395 400 Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr 405 410 415 Leu Gly Val Ala Leu Pro Trp Ala Ile Thr Ala Ala Leu Leu Arg Pro 420 425 430 Gly Lys Lys Val Tyr Ser His Ser Gly Asp Gly Gly Phe Leu Phe Thr 435 440 445 Gly Gln Glu Leu Glu Thr Ala Val Arg Leu Asn Leu Pro Ile Val Gln 450 455 460 Ile Ile Trp Asn Asp Gly His Tyr Asp Met Val Lys Phe Gln Glu Glu 465 470 475 480 Met Lys Tyr Gly Arg Ser Ala Ala Val Asp Phe Gly Tyr Val Asp Tyr 485 490 495 Val Lys Tyr Ala Glu Ala Met Arg Ala Lys Gly Tyr Arg Ala His Ser 500 505 510 Lys Glu Glu Leu Ala Glu Ile Leu Lys Ser Ile Pro Asp Thr Thr Gly 515 520 525 Pro Val Val Ile Asp Val Pro Leu Asp Tyr Ser Asp Asn Ile Lys Leu 530 535 540 Ala Glu Lys Leu Leu Pro Glu Glu Phe Tyr 545 550 63220DNALactococcus lactis 6tagatccgga aacaactgat tacctgagtt aacttagcag

aaattgcaga agataacggt 60aatttggatg aagcattaaa ttacctttat caaattccgg tgaatgatga aaattatatt 120gctgctttaa tcaaaattgc tgacttatat caatttgaag ttgattttga aacagcaatt 180tctaagttag aagaagcaag agaattatcg gattctcctc tgattacttt tgctttggct 240gagtcctact ttgaacaagg tgattattca gctgccatta ccgaatatgc aaaactttca 300gaacgaaaaa ttttacatga aacaaaaatt tctatttatc aaagaattgg tgactcttat 360gcccaattag gtaattttga gaatgccata tcatttcttg aaaaatcact tgaatttgat 420gaaaaaccgg aaaccttgta taaaattgct cttctttatg gagaaactca taatgaaaca 480agagccattg ctaatttcaa acggttagaa aaaatggatg ttgaattttt gaactatgaa 540ttagcctatg cccaaaccct agaagctaat caagaattta aagctgcact agaaatggca 600aagaaaggga tgaaaaaaaa tcctaatgcc gttcctctct tacacttcgc ttcaaaaatt 660tgtttcaaac ttaaggacaa agctgcagca gaacgttatc tcgtggatgc tttaaattta 720ccagaattac atgacgaaac agtctttttg cttgctaatt tatacttcaa cgaagaagat 780tttgaagctg tcattaatct tgaagagctt ttagaagatg aacatttatt agctaaatgg 840ctttttgcag gagcacataa agctttggaa aatgattctg aagcggctgc tttgtatgaa 900gaactcattc aaaccaatct gtcagagaat ccagagtttt tagaagacta tattgatttt 960cttaaagaaa ttggtcaaat ttctaaaaca gaaccaatta ttgaacaata tttggaactt 1020gttccagatg atgaaaatat gagaaattta ctgacagact taaaaaataa ttactgacaa 1080agctgtcagt aattattttt attgtaagct agaaaattca aaaacttgcg tcaaaataat 1140tgtaaaaggt tctattatct gataaaatga ttgtgaagta atccaagaga ttatgaaata 1200tgaattagaa caaatagagg taaaataaaa aatgtctgag aaacaatttg gggcgaactt 1260ggttgtcgat agtttgatta accataaagt gaagtatgta tttgggattc caggagcaaa 1320aattgaccgg gtttttgatt tattagaaaa tgaagaaggc cctcaaatgg tcgtgactcg 1380tcatgagcaa ggagctgctt tcatggctca agctgtcggt cgtttaactg gcgaacctgg 1440tgtagtagtt gttacgagtg ggcctggtgt atcaaacctt gcgactccgc ttttgaccgc 1500gacatcagaa ggtgatgcta ttttggctat cggtggacaa gttaaacgaa gtgaccgtct 1560taaacgtgcg caccaatcaa tggataatgc tggaatgatg caatcagcaa caaaatattc 1620agcagaagtt cttgacccta atacactttc tgaatcaatt gccaacgctt atcgtattgc 1680aaaatcagga catccaggtg caactttctt atcaatcccc caagatgtaa cggatgccga 1740agtatcaatc aaagccattc aaccactttc agaccctaaa atggggaatg cctctattga 1800tgacattaat tatttagcac aagcaattaa aaatgctgta ttgccagtaa ttttggttgg 1860agctggtgct tcagatgcta aagtcgcttc atccttgcgt aatctattga ctcatgttaa 1920tattcctgtc gttgaaacat tccaaggtgc aggggttatt tcacatgatt tagaacatac 1980tttttatgga cgtatcggtc ttttccgcaa tcaaccaggc gatatgcttc tgaaacgttc 2040tgaccttgtt attgctgttg gttatgaccc aattgaatat gaagctcgta actggaatgc 2100agaaattgat agtcgaatta tcgttattga taatgccatt gctgaaattg atacttacta 2160ccaaccagag cgtgaattaa ttggtgatat cgcagcaaca ttggataatc ttttaccagc 2220tgttcgtggc tacaaaattc caaaaggaac aaaagattat ctcgatggcc ttcatgaagt 2280tgctgagcaa cacgaatttg atactgaaaa tactgaagaa ggtagaatgc accctcttga 2340tttggtcagc actttccaag aaatcgtcaa ggatgatgaa acagtaaccg ttgacgtagg 2400ttcactctac atttggatgg cacgtcattt caaatcatac gaaccacgtc atctcctctt 2460ctcaaacgga atgcaaacac tcggagttgc acttccttgg gcaattacag ccgcattgtt 2520gcgcccaggt aaaaaagttt attcacactc tggtgatgga ggcttccttt tcacagggca 2580agaattggaa acagctgtac gtttgaatct tccaatcgtt caaattatct ggaatgacgg 2640ccattatgat atggttaaat tccaagaaga aatgaaatat ggtcgttcag cagccgttga 2700ttttggctat gttgattacg taaaatatgc tgaagcaatg agagcaaaag gttaccgtgc 2760acacagcaaa gaagaacttg ctgaaattct caaatcaatc ccagatacta ctggaccggt 2820ggtaattgac gttcctttgg actattctga taacattaaa ttagcagaaa aattattgcc 2880tgaagagttt tattgattac aatcaagcaa tttgtggcat aacaaaataa aagaagaagg 2940ccttgaacac ctaagcgttc agggcctttt tttgtgaaat aaattagatg aaatttacaa 3000tgagttttgt gaaactagct tctagtttgt gaaaaattgc ctataattgc cgaataaaaa 3060tacccattta ccactccaag aggatgcttc aaattagcta aatacccgtt ttagaggatg 3120cgtaaaaaca acaaaagagg atgagtatag aacgataaaa cttttttatg ataggttgag 3180agaattgaat ataaaatata ataagtagaa ggcagcaatt 32207491PRTEscherichia coli 7Met Ala Asn Tyr Phe Asn Thr Leu Asn Leu Arg Gln Gln Leu Ala Gln 1 5 10 15 Leu Gly Lys Cys Arg Phe Met Gly Arg Asp Glu Phe Ala Asp Gly Ala 20 25 30 Ser Tyr Leu Gln Gly Lys Lys Val Val Ile Val Gly Cys Gly Ala Gln 35 40 45 Gly Leu Asn Gln Gly Leu Asn Met Arg Asp Ser Gly Leu Asp Ile Ser 50 55 60 Tyr Ala Leu Arg Lys Glu Ala Ile Ala Glu Lys Arg Ala Ser Trp Arg 65 70 75 80 Lys Ala Thr Glu Asn Gly Phe Lys Val Gly Thr Tyr Glu Glu Leu Ile 85 90 95 Pro Gln Ala Asp Leu Val Ile Asn Leu Thr Pro Asp Lys Gln His Ser 100 105 110 Asp Val Val Arg Thr Val Gln Pro Leu Met Lys Asp Gly Ala Ala Leu 115 120 125 Gly Tyr Ser His Gly Phe Asn Ile Val Glu Val Gly Glu Gln Ile Arg 130 135 140 Lys Asp Ile Thr Val Val Met Val Ala Pro Lys Cys Pro Gly Thr Glu 145 150 155 160 Val Arg Glu Glu Tyr Lys Arg Gly Phe Gly Val Pro Thr Leu Ile Ala 165 170 175 Val His Pro Glu Asn Asp Pro Lys Gly Glu Gly Met Ala Ile Ala Lys 180 185 190 Ala Trp Ala Ala Ala Thr Gly Gly His Arg Ala Gly Val Leu Glu Ser 195 200 205 Ser Phe Val Ala Glu Val Lys Ser Asp Leu Met Gly Glu Gln Thr Ile 210 215 220 Leu Cys Gly Met Leu Gln Ala Gly Ser Leu Leu Cys Phe Asp Lys Leu 225 230 235 240 Val Glu Glu Gly Thr Asp Pro Ala Tyr Ala Glu Lys Leu Ile Gln Phe 245 250 255 Gly Trp Glu Thr Ile Thr Glu Ala Leu Lys Gln Gly Gly Ile Thr Leu 260 265 270 Met Met Asp Arg Leu Ser Asn Pro Ala Lys Leu Arg Ala Tyr Ala Leu 275 280 285 Ser Glu Gln Leu Lys Glu Ile Met Ala Pro Leu Phe Gln Lys His Met 290 295 300 Asp Asp Ile Ile Ser Gly Glu Phe Ser Ser Gly Met Met Ala Asp Trp 305 310 315 320 Ala Asn Asp Asp Lys Lys Leu Leu Thr Trp Arg Glu Glu Thr Gly Lys 325 330 335 Thr Ala Phe Glu Thr Ala Pro Gln Tyr Glu Gly Lys Ile Gly Glu Gln 340 345 350 Glu Tyr Phe Asp Lys Gly Val Leu Met Ile Ala Met Val Lys Ala Gly 355 360 365 Val Glu Leu Ala Phe Glu Thr Met Val Asp Ser Gly Ile Ile Glu Glu 370 375 380 Ser Ala Tyr Tyr Glu Ser Leu His Glu Leu Pro Leu Ile Ala Asn Thr 385 390 395 400 Ile Ala Arg Lys Arg Leu Tyr Glu Met Asn Val Val Ile Ser Asp Thr 405 410 415 Ala Glu Tyr Gly Asn Tyr Leu Phe Ser Tyr Ala Cys Val Pro Leu Leu 420 425 430 Lys Pro Phe Met Ala Glu Leu Gln Pro Gly Asp Leu Gly Lys Ala Ile 435 440 445 Pro Glu Gly Ala Val Asp Asn Gly Gln Leu Arg Asp Val Asn Glu Ala 450 455 460 Ile Arg Ser His Ala Ile Glu Gln Val Gly Lys Lys Leu Arg Gly Tyr 465 470 475 480 Met Thr Asp Met Lys Arg Ile Ala Val Ala Gly 485 490 81476DNAEscherichia coli 8atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440atgacagata tgaaacgtat tgctgttgcg ggttaa 14769395PRTSaccharomyces cerevisiae 9Met Leu Arg Thr Gln Ala Ala Arg Leu Ile Cys Asn Ser Arg Val Ile 1 5 10 15 Thr Ala Lys Arg Thr Phe Ala Leu Ala Thr Arg Ala Ala Ala Tyr Ser 20 25 30 Arg Pro Ala Ala Arg Phe Val Lys Pro Met Ile Thr Thr Arg Gly Leu 35 40 45 Lys Gln Ile Asn Phe Gly Gly Thr Val Glu Thr Val Tyr Glu Arg Ala 50 55 60 Asp Trp Pro Arg Glu Lys Leu Leu Asp Tyr Phe Lys Asn Asp Thr Phe 65 70 75 80 Ala Leu Ile Gly Tyr Gly Ser Gln Gly Tyr Gly Gln Gly Leu Asn Leu 85 90 95 Arg Asp Asn Gly Leu Asn Val Ile Ile Gly Val Arg Lys Asp Gly Ala 100 105 110 Ser Trp Lys Ala Ala Ile Glu Asp Gly Trp Val Pro Gly Lys Asn Leu 115 120 125 Phe Thr Val Glu Asp Ala Ile Lys Arg Gly Ser Tyr Val Met Asn Leu 130 135 140 Leu Ser Asp Ala Ala Gln Ser Glu Thr Trp Pro Ala Ile Lys Pro Leu 145 150 155 160 Leu Thr Lys Gly Lys Thr Leu Tyr Phe Ser His Gly Phe Ser Pro Val 165 170 175 Phe Lys Asp Leu Thr His Val Glu Pro Pro Lys Asp Leu Asp Val Ile 180 185 190 Leu Val Ala Pro Lys Gly Ser Gly Arg Thr Val Arg Ser Leu Phe Lys 195 200 205 Glu Gly Arg Gly Ile Asn Ser Ser Tyr Ala Val Trp Asn Asp Val Thr 210 215 220 Gly Lys Ala His Glu Lys Ala Gln Ala Leu Ala Val Ala Ile Gly Ser 225 230 235 240 Gly Tyr Val Tyr Gln Thr Thr Phe Glu Arg Glu Val Asn Ser Asp Leu 245 250 255 Tyr Gly Glu Arg Gly Cys Leu Met Gly Gly Ile His Gly Met Phe Leu 260 265 270 Ala Gln Tyr Asp Val Leu Arg Glu Asn Gly His Ser Pro Ser Glu Ala 275 280 285 Phe Asn Glu Thr Val Glu Glu Ala Thr Gln Ser Leu Tyr Pro Leu Ile 290 295 300 Gly Lys Tyr Gly Met Asp Tyr Met Tyr Asp Ala Cys Ser Thr Thr Ala 305 310 315 320 Arg Arg Gly Ala Leu Asp Trp Tyr Pro Ile Phe Lys Asn Ala Leu Lys 325 330 335 Pro Val Phe Gln Asp Leu Tyr Glu Ser Thr Lys Asn Gly Thr Glu Thr 340 345 350 Lys Arg Ser Leu Glu Phe Asn Ser Gln Pro Asp Tyr Arg Glu Lys Leu 355 360 365 Glu Lys Glu Leu Asp Thr Ile Arg Asn Met Glu Ile Trp Lys Val Gly 370 375 380 Lys Glu Val Arg Lys Leu Arg Pro Glu Asn Gln 385 390 395 101188DNASaccharomyces cerevisiae 10atgttgagaa ctcaagccgc cagattgatc tgcaactccc gtgtcatcac tgctaagaga 60acctttgctt tggccacccg tgctgctgct tacagcagac cagctgcccg tttcgttaag 120ccaatgatca ctacccgtgg tttgaagcaa atcaacttcg gtggtactgt tgaaaccgtc 180tacgaaagag ctgactggcc aagagaaaag ttgttggact acttcaagaa cgacactttt 240gctttgatcg gttacggttc ccaaggttac ggtcaaggtt tgaacttgag agacaacggt 300ttgaacgtta tcattggtgt ccgtaaagat ggtgcttctt ggaaggctgc catcgaagac 360ggttgggttc caggcaagaa cttgttcact gttgaagatg ctatcaagag aggtagttac 420gttatgaact tgttgtccga tgccgctcaa tcagaaacct ggcctgctat caagccattg 480ttgaccaagg gtaagacttt gtacttctcc cacggtttct ccccagtctt caaggacttg 540actcacgttg aaccaccaaa ggacttagat gttatcttgg ttgctccaaa gggttccggt 600agaactgtca gatctttgtt caaggaaggt cgtggtatta actcttctta cgccgtctgg 660aacgatgtca ccggtaaggc tcacgaaaag gcccaagctt tggccgttgc cattggttcc 720ggttacgttt accaaaccac tttcgaaaga gaagtcaact ctgacttgta cggtgaaaga 780ggttgtttaa tgggtggtat ccacggtatg ttcttggctc aatacgacgt cttgagagaa 840aacggtcact ccccatctga agctttcaac gaaaccgtcg aagaagctac ccaatctcta 900tacccattga tcggtaagta cggtatggat tacatgtacg atgcttgttc caccaccgcc 960agaagaggtg ctttggactg gtacccaatc ttcaagaatg ctttgaagcc tgttttccaa 1020gacttgtacg aatctaccaa gaacggtacc gaaaccaaga gatctttgga attcaactct 1080caacctgact acagagaaaa gctagaaaag gaattagaca ccatcagaaa catggaaatc 1140tggaaggttg gtaaggaagt cagaaagttg agaccagaaa accaataa 118811330PRTMethanococcus maripaludis 11Met Lys Val Phe Tyr Asp Ser Asp Phe Lys Leu Asp Ala Leu Lys Glu 1 5 10 15 Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser Gln Gly Arg Ala Gln Ser 20 25 30 Leu Asn Met Lys Asp Ser Gly Leu Asn Val Val Val Gly Leu Arg Lys 35 40 45 Asn Gly Ala Ser Trp Asn Asn Ala Lys Ala Asp Gly His Asn Val Met 50 55 60 Thr Ile Glu Glu Ala Ala Glu Lys Ala Asp Ile Ile His Ile Leu Ile 65 70 75 80 Pro Asp Glu Leu Gln Ala Glu Val Tyr Glu Ser Gln Ile Lys Pro Tyr 85 90 95 Leu Lys Glu Gly Lys Thr Leu Ser Phe Ser His Gly Phe Asn Ile His 100 105 110 Tyr Gly Phe Ile Val Pro Pro Lys Gly Val Asn Val Val Leu Val Ala 115 120 125 Pro Lys Ser Pro Gly Lys Met Val Arg Arg Thr Tyr Glu Glu Gly Phe 130 135 140 Gly Val Pro Gly Leu Ile Cys Ile Glu Ile Asp Ala Thr Asn Asn Ala 145 150 155 160 Phe Asp Ile Val Ser Ala Met Ala Lys Gly Ile Gly Leu Ser Arg Ala 165 170 175 Gly Val Ile Gln Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Val Thr Glu Leu Ile Lys Ala 195 200 205 Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr Ala Pro Glu Met Ala Tyr 210 215 220 Phe Glu Thr Cys His Glu Leu Lys Leu Ile Val Asp Leu Ile Tyr Gln 225 230 235 240 Lys Gly Phe Lys Asn Met Trp Asn Asp Val Ser Asn Thr Ala Glu Tyr 245 250 255 Gly Gly Leu Thr Arg Arg Ser Arg Ile Val Thr Ala Asp Ser Lys Ala 260 265 270 Ala Met Lys Glu Ile Leu Arg Glu Ile Gln Asp Gly Arg Phe Thr Lys 275 280 285 Glu Phe Leu Leu Glu Lys Gln Val Ser Tyr Ala His Leu Lys Ser Met 290 295 300 Arg Arg Leu Glu Gly Asp Leu Gln Ile Glu Glu Val Gly Ala Lys Leu 305 310 315 320 Arg Lys Met Cys Gly Leu Glu Lys Glu Glu 325 330 12993DNAMethanococcus maripaludis 12atgaaggtat tctatgactc agattttaaa ttagatgctt taaaagaaaa aacaattgca 60gtaatcggtt atggaagtca aggtagggca cagtccttaa acatgaaaga cagcggatta 120aacgttgttg ttggtttaag aaaaaacggt gcttcatgga acaacgctaa agcagacggt 180cacaatgtaa tgaccattga agaagctgct gaaaaagcgg acatcatcca catcttaata 240cctgatgaat tacaggcaga agtttatgaa agccagataa aaccatacct aaaagaagga 300aaaacactaa gcttttcaca tggttttaac atccactatg gattcattgt tccaccaaaa 360ggagttaacg tggttttagt tgctccaaaa tcacctggaa aaatggttag aagaacatac 420gaagaaggtt tcggtgttcc aggtttaatc tgtattgaaa ttgatgcaac aaacaacgca 480tttgatattg tttcagcaat ggcaaaagga atcggtttat caagagctgg agttatccag 540acaactttca aagaagaaac agaaactgac cttttcggtg aacaagctgt tttatgcggt 600ggagttaccg aattaatcaa ggcaggattt gaaacactcg ttgaagcagg atacgcacca 660gaaatggcat actttgaaac ctgccacgaa ttgaaattaa tcgttgactt aatctaccaa 720aaaggattca aaaacatgtg gaacgatgta agtaacactg cagaatacgg cggacttaca 780agaagaagca gaatcgttac agctgattca aaagctgcaa tgaaagaaat cttaagagaa 840atccaagatg gaagattcac aaaagaattc cttctcgaaa aacaggtaag ctatgctcat 900ttaaaatcaa tgagaagact cgaaggagac ttacaaatcg aagaagtcgg cgcaaaatta 960agaaaaatgt gcggtcttga aaaagaagaa taa 99313342PRTBacillus subtilis 13Met

Val Lys Val Tyr Tyr Asn Gly Asp Ile Lys Glu Asn Val Leu Ala 1 5 10 15 Gly Lys Thr Val Ala Val Ile Gly Tyr Gly Ser Gln Gly His Ala His 20 25 30 Ala Leu Asn Leu Lys Glu Ser Gly Val Asp Val Ile Val Gly Val Arg 35 40 45 Gln Gly Lys Ser Phe Thr Gln Ala Gln Glu Asp Gly His Lys Val Phe 50 55 60 Ser Val Lys Glu Ala Ala Ala Gln Ala Glu Ile Ile Met Val Leu Leu 65 70 75 80 Pro Asp Glu Gln Gln Gln Lys Val Tyr Glu Ala Glu Ile Lys Asp Glu 85 90 95 Leu Thr Ala Gly Lys Ser Leu Val Phe Ala His Gly Phe Asn Val His 100 105 110 Phe His Gln Ile Val Pro Pro Ala Asp Val Asp Val Phe Leu Val Ala 115 120 125 Pro Lys Gly Pro Gly His Leu Val Arg Arg Thr Tyr Glu Gln Gly Ala 130 135 140 Gly Val Pro Ala Leu Phe Ala Ile Tyr Gln Asp Val Thr Gly Glu Ala 145 150 155 160 Arg Asp Lys Ala Leu Ala Tyr Ala Lys Gly Ile Gly Gly Ala Arg Ala 165 170 175 Gly Val Leu Glu Thr Thr Phe Lys Glu Glu Thr Glu Thr Asp Leu Phe 180 185 190 Gly Glu Gln Ala Val Leu Cys Gly Gly Leu Ser Ala Leu Val Lys Ala 195 200 205 Gly Phe Glu Thr Leu Thr Glu Ala Gly Tyr Gln Pro Glu Leu Ala Tyr 210 215 220 Phe Glu Cys Leu His Glu Leu Lys Leu Ile Val Asp Leu Met Tyr Glu 225 230 235 240 Glu Gly Leu Ala Gly Met Arg Tyr Ser Ile Ser Asp Thr Ala Gln Trp 245 250 255 Gly Asp Phe Val Ser Gly Pro Arg Val Val Asp Ala Lys Val Lys Glu 260 265 270 Ser Met Lys Glu Val Leu Lys Asp Ile Gln Asn Gly Thr Phe Ala Lys 275 280 285 Glu Trp Ile Val Glu Asn Gln Val Asn Arg Pro Arg Phe Asn Ala Ile 290 295 300 Asn Ala Ser Glu Asn Glu His Gln Ile Glu Val Val Gly Arg Lys Leu 305 310 315 320 Arg Glu Met Met Pro Phe Val Lys Gln Gly Lys Lys Lys Glu Ala Val 325 330 335 Val Ser Val Ala Gln Asn 340 141476DNABacillus subtilis 14atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440atgacagata tgaaacgtat tgctgttgcg ggttaa 147615343PRTAnaerostipes caccae 15Met Glu Glu Cys Lys Met Ala Lys Ile Tyr Tyr Gln Glu Asp Cys Asn 1 5 10 15 Leu Ser Leu Leu Asp Gly Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser 20 25 30 Gln Gly His Ala His Ala Leu Asn Ala Lys Glu Ser Gly Cys Asn Val 35 40 45 Ile Ile Gly Leu Tyr Glu Gly Ala Lys Glu Trp Lys Arg Ala Glu Glu 50 55 60 Gln Gly Phe Glu Val Tyr Thr Ala Ala Glu Ala Ala Lys Lys Ala Asp 65 70 75 80 Ile Ile Met Ile Leu Ile Asn Asp Glu Lys Gln Ala Thr Met Tyr Lys 85 90 95 Asn Asp Ile Glu Pro Asn Leu Glu Ala Gly Asn Met Leu Met Phe Ala 100 105 110 His Gly Phe Asn Ile His Phe Gly Cys Ile Val Pro Pro Lys Asp Val 115 120 125 Asp Val Thr Met Ile Ala Pro Lys Gly Pro Gly His Thr Val Arg Ser 130 135 140 Glu Tyr Glu Glu Gly Lys Gly Val Pro Cys Leu Val Ala Val Glu Gln 145 150 155 160 Asp Ala Thr Gly Lys Ala Leu Asp Met Ala Leu Ala Tyr Ala Leu Ala 165 170 175 Ile Gly Gly Ala Arg Ala Gly Val Leu Glu Thr Thr Phe Arg Thr Glu 180 185 190 Thr Glu Thr Asp Leu Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Val 195 200 205 Cys Ala Leu Met Gln Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr 210 215 220 Asp Pro Arg Asn Ala Tyr Phe Glu Cys Ile His Glu Met Lys Leu Ile 225 230 235 240 Val Asp Leu Ile Tyr Gln Ser Gly Phe Ser Gly Met Arg Tyr Ser Ile 245 250 255 Ser Asn Thr Ala Glu Tyr Gly Asp Tyr Ile Thr Gly Pro Lys Ile Ile 260 265 270 Thr Glu Asp Thr Lys Lys Ala Met Lys Lys Ile Leu Ser Asp Ile Gln 275 280 285 Asp Gly Thr Phe Ala Lys Asp Phe Leu Val Asp Met Ser Asp Ala Gly 290 295 300 Ser Gln Val His Phe Lys Ala Met Arg Lys Leu Ala Ser Glu His Pro 305 310 315 320 Ala Glu Val Val Gly Glu Glu Ile Arg Ser Leu Tyr Ser Trp Ser Asp 325 330 335 Glu Asp Lys Leu Ile Asn Asn 340 16343PRTAnaerostipes caccae 16Met Glu Glu Cys Lys Met Ala Lys Ile Tyr Tyr Gln Glu Asp Cys Asn 1 5 10 15 Leu Ser Leu Leu Asp Gly Lys Thr Ile Ala Val Ile Gly Tyr Gly Ser 20 25 30 Gln Gly His Ala His Ala Leu Asn Ala Lys Glu Ser Gly Cys Asn Val 35 40 45 Ile Ile Gly Leu Tyr Glu Gly Ala Lys Asp Trp Lys Arg Ala Glu Glu 50 55 60 Gln Gly Phe Glu Val Tyr Thr Ala Ala Glu Ala Ala Lys Lys Ala Asp 65 70 75 80 Ile Ile Met Ile Leu Ile Asn Asp Glu Lys Gln Ala Thr Met Tyr Lys 85 90 95 Asn Asp Ile Glu Pro Asn Leu Glu Ala Gly Asn Met Leu Met Phe Ala 100 105 110 His Gly Phe Asn Ile His Phe Gly Cys Ile Val Pro Pro Lys Asp Val 115 120 125 Asp Val Thr Met Ile Ala Pro Lys Gly Pro Gly His Thr Val Arg Ser 130 135 140 Glu Tyr Glu Glu Gly Lys Gly Val Pro Cys Leu Val Ala Val Glu Gln 145 150 155 160 Asp Ala Thr Gly Lys Ala Leu Asp Met Ala Leu Ala Tyr Ala Leu Ala 165 170 175 Ile Gly Gly Ala Arg Ala Gly Val Leu Glu Thr Thr Phe Arg Thr Glu 180 185 190 Thr Glu Thr Asp Leu Phe Gly Glu Gln Ala Val Leu Cys Gly Gly Val 195 200 205 Cys Ala Leu Met Gln Ala Gly Phe Glu Thr Leu Val Glu Ala Gly Tyr 210 215 220 Asp Pro Arg Asn Ala Tyr Phe Glu Cys Ile His Glu Met Lys Leu Ile 225 230 235 240 Val Asp Leu Ile Tyr Gln Ser Gly Phe Ser Gly Met Arg Tyr Ser Ile 245 250 255 Ser Asn Thr Ala Glu Tyr Gly Asp Tyr Ile Thr Gly Pro Lys Ile Ile 260 265 270 Thr Glu Asp Thr Lys Lys Ala Met Lys Lys Ile Leu Ser Asp Ile Gln 275 280 285 Asp Gly Thr Phe Ala Lys Asp Phe Leu Val Asp Met Ser Asp Ala Gly 290 295 300 Ser Gln Val His Phe Lys Ala Met Arg Lys Leu Ala Ser Glu His Pro 305 310 315 320 Ala Glu Val Val Gly Glu Glu Ile Arg Ser Leu Tyr Ser Trp Ser Asp 325 330 335 Glu Asp Lys Leu Ile Asn Asn 340 17616PRTEscherichia coli 17Met Pro Lys Tyr Arg Ser Ala Thr Thr Thr His Gly Arg Asn Met Ala 1 5 10 15 Gly Ala Arg Ala Leu Trp Arg Ala Thr Gly Met Thr Asp Ala Asp Phe 20 25 30 Gly Lys Pro Ile Ile Ala Val Val Asn Ser Phe Thr Gln Phe Val Pro 35 40 45 Gly His Val His Leu Arg Asp Leu Gly Lys Leu Val Ala Glu Gln Ile 50 55 60 Glu Ala Ala Gly Gly Val Ala Lys Glu Phe Asn Thr Ile Ala Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Gly Gly Met Leu Tyr Ser Leu Pro Ser 85 90 95 Arg Glu Leu Ile Ala Asp Ser Val Glu Tyr Met Val Asn Ala His Cys 100 105 110 Ala Asp Ala Met Val Cys Ile Ser Asn Cys Asp Lys Ile Thr Pro Gly 115 120 125 Met Leu Met Ala Ser Leu Arg Leu Asn Ile Pro Val Ile Phe Val Ser 130 135 140 Gly Gly Pro Met Glu Ala Gly Lys Thr Lys Leu Ser Asp Gln Ile Ile 145 150 155 160 Lys Leu Asp Leu Val Asp Ala Met Ile Gln Gly Ala Asp Pro Lys Val 165 170 175 Ser Asp Ser Gln Ser Asp Gln Val Glu Arg Ser Ala Cys Pro Thr Cys 180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Thr 195 200 205 Glu Ala Leu Gly Leu Ser Gln Pro Gly Asn Gly Ser Leu Leu Ala Thr 210 215 220 His Ala Asp Arg Lys Gln Leu Phe Leu Asn Ala Gly Lys Arg Ile Val 225 230 235 240 Glu Leu Thr Lys Arg Tyr Tyr Glu Gln Asn Asp Glu Ser Ala Leu Pro 245 250 255 Arg Asn Ile Ala Ser Lys Ala Ala Phe Glu Asn Ala Met Thr Leu Asp 260 265 270 Ile Ala Met Gly Gly Ser Thr Asn Thr Val Leu His Leu Leu Ala Ala 275 280 285 Ala Gln Glu Ala Glu Ile Asp Phe Thr Met Ser Asp Ile Asp Lys Leu 290 295 300 Ser Arg Lys Val Pro Gln Leu Cys Lys Val Ala Pro Ser Thr Gln Lys 305 310 315 320 Tyr His Met Glu Asp Val His Arg Ala Gly Gly Val Ile Gly Ile Leu 325 330 335 Gly Glu Leu Asp Arg Ala Gly Leu Leu Asn Arg Asp Val Lys Asn Val 340 345 350 Leu Gly Leu Thr Leu Pro Gln Thr Leu Glu Gln Tyr Asp Val Met Leu 355 360 365 Thr Gln Asp Asp Ala Val Lys Asn Met Phe Arg Ala Gly Pro Ala Gly 370 375 380 Ile Arg Thr Thr Gln Ala Phe Ser Gln Asp Cys Arg Trp Asp Thr Leu 385 390 395 400 Asp Asp Asp Arg Ala Asn Gly Cys Ile Arg Ser Leu Glu His Ala Tyr 405 410 415 Ser Lys Asp Gly Gly Leu Ala Val Leu Tyr Gly Asn Phe Ala Glu Asn 420 425 430 Gly Cys Ile Val Lys Thr Ala Gly Val Asp Asp Ser Ile Leu Lys Phe 435 440 445 Thr Gly Pro Ala Lys Val Tyr Glu Ser Gln Asp Asp Ala Val Glu Ala 450 455 460 Ile Leu Gly Gly Lys Val Val Ala Gly Asp Val Val Val Ile Arg Tyr 465 470 475 480 Glu Gly Pro Lys Gly Gly Pro Gly Met Gln Glu Met Leu Tyr Pro Thr 485 490 495 Ser Phe Leu Lys Ser Met Gly Leu Gly Lys Ala Cys Ala Leu Ile Thr 500 505 510 Asp Gly Arg Phe Ser Gly Gly Thr Ser Gly Leu Ser Ile Gly His Val 515 520 525 Ser Pro Glu Ala Ala Ser Gly Gly Ser Ile Gly Leu Ile Glu Asp Gly 530 535 540 Asp Leu Ile Ala Ile Asp Ile Pro Asn Arg Gly Ile Gln Leu Gln Val 545 550 555 560 Ser Asp Ala Glu Leu Ala Ala Arg Arg Glu Ala Gln Asp Ala Arg Gly 565 570 575 Asp Lys Ala Trp Thr Pro Lys Asn Arg Glu Arg Gln Val Ser Phe Ala 580 585 590 Leu Arg Ala Tyr Ala Ser Leu Ala Thr Ser Ala Asp Lys Gly Ala Val 595 600 605 Arg Asp Lys Ser Lys Leu Gly Gly 610 615 181851DNAEscherichia coli 18atgcctaagt accgttccgc caccaccact catggtcgta atatggcggg tgctcgtgcg 60ctgtggcgcg ccaccggaat gaccgacgcc gatttcggta agccgattat cgcggttgtg 120aactcgttca cccaatttgt accgggtcac gtccatctgc gcgatctcgg taaactggtc 180gccgaacaaa ttgaagcggc tggcggcgtt gccaaagagt tcaacaccat tgcggtggat 240gatgggattg ccatgggcca cggggggatg ctttattcac tgccatctcg cgaactgatc 300gctgattccg ttgagtatat ggtcaacgcc cactgcgccg acgccatggt ctgcatctct 360aactgcgaca aaatcacccc ggggatgctg atggcttccc tgcgcctgaa tattccggtg 420atctttgttt ccggcggccc gatggaggcc gggaaaacca aactttccga tcagatcatc 480aagctcgatc tggttgatgc gatgatccag ggcgcagacc cgaaagtatc tgactcccag 540agcgatcagg ttgaacgttc cgcgtgtccg acctgcggtt cctgctccgg gatgtttacc 600gctaactcaa tgaactgcct gaccgaagcg ctgggcctgt cgcagccggg caacggctcg 660ctgctggcaa cccacgccga ccgtaagcag ctgttcctta atgctggtaa acgcattgtt 720gaattgacca aacgttatta cgagcaaaac gacgaaagtg cactgccgcg taatatcgcc 780agtaaggcgg cgtttgaaaa cgccatgacg ctggatatcg cgatgggtgg atcgactaac 840accgtacttc acctgctggc ggcggcgcag gaagcggaaa tcgacttcac catgagtgat 900atcgataagc tttcccgcaa ggttccacag ctgtgtaaag ttgcgccgag cacccagaaa 960taccatatgg aagatgttca ccgtgctggt ggtgttatcg gtattctcgg cgaactggat 1020cgcgcggggt tactgaaccg tgatgtgaaa aacgtacttg gcctgacgtt gccgcaaacg 1080ctggaacaat acgacgttat gctgacccag gatgacgcgg taaaaaatat gttccgcgca 1140ggtcctgcag gcattcgtac cacacaggca ttctcgcaag attgccgttg ggatacgctg 1200gacgacgatc gcgccaatgg ctgtatccgc tcgctggaac acgcctacag caaagacggc 1260ggcctggcgg tgctctacgg taactttgcg gaaaacggct gcatcgtgaa aacggcaggc 1320gtcgatgaca gcatcctcaa attcaccggc ccggcgaaag tgtacgaaag ccaggacgat 1380gcggtagaag cgattctcgg cggtaaagtt gtcgccggag atgtggtagt aattcgctat 1440gaaggcccga aaggcggtcc ggggatgcag gaaatgctct acccaaccag cttcctgaaa 1500tcaatgggtc tcggcaaagc ctgtgcgctg atcaccgacg gtcgtttctc tggtggcacc 1560tctggtcttt ccatcggcca cgtctcaccg gaagcggcaa gcggcggcag cattggcctg 1620attgaagatg gtgacctgat cgctatcgac atcccgaacc gtggcattca gttacaggta 1680agcgatgccg aactggcggc gcgtcgtgaa gcgcaggacg ctcgaggtga caaagcctgg 1740acgccgaaaa atcgtgaacg tcaggtctcc tttgccctgc gtgcttatgc cagcctggca 1800accagcgccg acaaaggcgc ggtgcgcgat aaatcgaaac tggggggtta a 185119585PRTSaccharomyces cerevisiae 19Met Gly Leu Leu Thr Lys Val Ala Thr Ser Arg Gln Phe Ser Thr Thr 1 5 10 15 Arg Cys Val Ala Lys Lys Leu Asn Lys Tyr Ser Tyr Ile Ile Thr Glu 20 25 30 Pro Lys Gly Gln Gly Ala Ser Gln Ala Met Leu Tyr Ala Thr Gly Phe 35 40 45 Lys Lys Glu Asp Phe Lys Lys Pro Gln Val Gly Val Gly Ser Cys Trp 50 55 60 Trp Ser Gly Asn Pro Cys Asn Met His Leu Leu Asp Leu Asn Asn Arg 65 70 75 80 Cys Ser Gln Ser Ile Glu Lys Ala Gly Leu Lys Ala Met Gln Phe Asn 85 90 95 Thr Ile Gly Val Ser Asp Gly Ile Ser Met Gly Thr Lys Gly Met Arg 100 105 110 Tyr Ser Leu Gln Ser Arg Glu Ile Ile Ala Asp Ser Phe Glu Thr Ile 115

120 125 Met Met Ala Gln His Tyr Asp Ala Asn Ile Ala Ile Pro Ser Cys Asp 130 135 140 Lys Asn Met Pro Gly Val Met Met Ala Met Gly Arg His Asn Arg Pro 145 150 155 160 Ser Ile Met Val Tyr Gly Gly Thr Ile Leu Pro Gly His Pro Thr Cys 165 170 175 Gly Ser Ser Lys Ile Ser Lys Asn Ile Asp Ile Val Ser Ala Phe Gln 180 185 190 Ser Tyr Gly Glu Tyr Ile Ser Lys Gln Phe Thr Glu Glu Glu Arg Glu 195 200 205 Asp Val Val Glu His Ala Cys Pro Gly Pro Gly Ser Cys Gly Gly Met 210 215 220 Tyr Thr Ala Asn Thr Met Ala Ser Ala Ala Glu Val Leu Gly Leu Thr 225 230 235 240 Ile Pro Asn Ser Ser Ser Phe Pro Ala Val Ser Lys Glu Lys Leu Ala 245 250 255 Glu Cys Asp Asn Ile Gly Glu Tyr Ile Lys Lys Thr Met Glu Leu Gly 260 265 270 Ile Leu Pro Arg Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn Ala Ile 275 280 285 Thr Tyr Val Val Ala Thr Gly Gly Ser Thr Asn Ala Val Leu His Leu 290 295 300 Val Ala Val Ala His Ser Ala Gly Val Lys Leu Ser Pro Asp Asp Phe 305 310 315 320 Gln Arg Ile Ser Asp Thr Thr Pro Leu Ile Gly Asp Phe Lys Pro Ser 325 330 335 Gly Lys Tyr Val Met Ala Asp Leu Ile Asn Val Gly Gly Thr Gln Ser 340 345 350 Val Ile Lys Tyr Leu Tyr Glu Asn Asn Met Leu His Gly Asn Thr Met 355 360 365 Thr Val Thr Gly Asp Thr Leu Ala Glu Arg Ala Lys Lys Ala Pro Ser 370 375 380 Leu Pro Glu Gly Gln Glu Ile Ile Lys Pro Leu Ser His Pro Ile Lys 385 390 395 400 Ala Asn Gly His Leu Gln Ile Leu Tyr Gly Ser Leu Ala Pro Gly Gly 405 410 415 Ala Val Gly Lys Ile Thr Gly Lys Glu Gly Thr Tyr Phe Lys Gly Arg 420 425 430 Ala Arg Val Phe Glu Glu Glu Gly Ala Phe Ile Glu Ala Leu Glu Arg 435 440 445 Gly Glu Ile Lys Lys Gly Glu Lys Thr Val Val Val Ile Arg Tyr Glu 450 455 460 Gly Pro Arg Gly Ala Pro Gly Met Pro Glu Met Leu Lys Pro Ser Ser 465 470 475 480 Ala Leu Met Gly Tyr Gly Leu Gly Lys Asp Val Ala Leu Leu Thr Asp 485 490 495 Gly Arg Phe Ser Gly Gly Ser His Gly Phe Leu Ile Gly His Ile Val 500 505 510 Pro Glu Ala Ala Glu Gly Gly Pro Ile Gly Leu Val Arg Asp Gly Asp 515 520 525 Glu Ile Ile Ile Asp Ala Asp Asn Asn Lys Ile Asp Leu Leu Val Ser 530 535 540 Asp Lys Glu Met Ala Gln Arg Lys Gln Ser Trp Val Ala Pro Pro Pro 545 550 555 560 Arg Tyr Thr Arg Gly Thr Leu Ser Lys Tyr Ala Lys Leu Val Ser Asn 565 570 575 Ala Ser Asn Gly Cys Val Leu Asp Ala 580 585 201131DNASaccharomyces cerevisiae 20atgaccttgg cacccctaga cgcctccaaa gttaagataa ctaccacaca acatgcatct 60aagccaaaac cgaacagtga gttagtgttt ggcaagagct tcacggacca catgttaact 120gcggaatgga cagctgaaaa agggtggggt accccagaga ttaaacctta tcaaaatctg 180tctttagacc cttccgcggt ggttttccat tatgcttttg agctattcga agggatgaag 240gcttacagaa cggtggacaa caaaattaca atgtttcgtc cagatatgaa tatgaagcgc 300atgaataagt ctgctcagag aatctgtttg ccaacgttcg acccagaaga gttgattacc 360ctaattggga aactgatcca gcaagataag tgcttagttc ctgaaggaaa aggttactct 420ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 480agagccttgc tatatgtcat ttgctgccct gtgggtcctt attacaaaac tggatttaag 540gcggtcagac tggaagccac tgattatgcc acaagagctt ggccaggagg ctgtggtgac 600aagaaactag gtgcaaacta cgccccctgc gtcctgccac aattgcaagc tgcttcaagg 660ggttaccaac aaaatttatg gctatttggt ccaaataaca acattactga agtcggcacc 720atgaatgctt ttttcgtgtt taaagatagt aaaacgggca agaaggaact agttactgct 780ccactagacg gtaccatttt ggaaggtgtt actagggatt ccattttaaa tcttgctaaa 840gaaagactcg aaccaagtga atggaccatt agtgaacgct acttcactat aggcgaagtt 900actgagagat ccaagaacgg tgaactactt gaagcctttg gttctggtac tgctgcgatt 960gtttctccca ttaaggaaat cggctggaaa ggcgaacaaa ttaatattcc gttgttgccc 1020ggcgaacaaa ccggtccatt ggccaaagaa gttgcacaat ggattaatgg aatccaatat 1080ggcgagactg agcatggcaa ttggtcaagg gttgttactg atttgaactg a 113121550PRTMethanococcus maripaludis 21Met Ile Ser Asp Asn Val Lys Lys Gly Val Ile Arg Thr Pro Asn Arg 1 5 10 15 Ala Leu Leu Lys Ala Cys Gly Tyr Thr Asp Glu Asp Met Glu Lys Pro 20 25 30 Phe Ile Gly Ile Val Asn Ser Phe Thr Glu Val Val Pro Gly His Ile 35 40 45 His Leu Arg Thr Leu Ser Glu Ala Ala Lys His Gly Val Tyr Ala Asn 50 55 60 Gly Gly Thr Pro Phe Glu Phe Asn Thr Ile Gly Ile Cys Asp Gly Ile 65 70 75 80 Ala Met Gly His Glu Gly Met Lys Tyr Ser Leu Pro Ser Arg Glu Ile 85 90 95 Ile Ala Asp Ala Val Glu Ser Met Ala Arg Ala His Gly Phe Asp Gly 100 105 110 Leu Val Leu Ile Pro Thr Cys Asp Lys Ile Val Pro Gly Met Ile Met 115 120 125 Gly Ala Leu Arg Leu Asn Ile Pro Phe Ile Val Val Thr Gly Gly Pro 130 135 140 Met Leu Pro Gly Glu Phe Gln Gly Lys Lys Tyr Glu Leu Ile Ser Leu 145 150 155 160 Phe Glu Gly Val Gly Glu Tyr Gln Val Gly Lys Ile Thr Glu Glu Glu 165 170 175 Leu Lys Cys Ile Glu Asp Cys Ala Cys Ser Gly Ala Gly Ser Cys Ala 180 185 190 Gly Leu Tyr Thr Ala Asn Ser Met Ala Cys Leu Thr Glu Ala Leu Gly 195 200 205 Leu Ser Leu Pro Met Cys Ala Thr Thr His Ala Val Asp Ala Gln Lys 210 215 220 Val Arg Leu Ala Lys Lys Ser Gly Ser Lys Ile Val Asp Met Val Lys 225 230 235 240 Glu Asp Leu Lys Pro Thr Asp Ile Leu Thr Lys Glu Ala Phe Glu Asn 245 250 255 Ala Ile Leu Val Asp Leu Ala Leu Gly Gly Ser Thr Asn Thr Thr Leu 260 265 270 His Ile Pro Ala Ile Ala Asn Glu Ile Glu Asn Lys Phe Ile Thr Leu 275 280 285 Asp Asp Phe Asp Arg Leu Ser Asp Glu Val Pro His Ile Ala Ser Ile 290 295 300 Lys Pro Gly Gly Glu His Tyr Met Ile Asp Leu His Asn Ala Gly Gly 305 310 315 320 Ile Pro Ala Val Leu Asn Val Leu Lys Glu Lys Ile Arg Asp Thr Lys 325 330 335 Thr Val Asp Gly Arg Ser Ile Leu Glu Ile Ala Glu Ser Val Lys Tyr 340 345 350 Ile Asn Tyr Asp Val Ile Arg Lys Val Glu Ala Pro Val His Glu Thr 355 360 365 Ala Gly Leu Arg Val Leu Lys Gly Asn Leu Ala Pro Asn Gly Cys Val 370 375 380 Val Lys Ile Gly Ala Val His Pro Lys Met Tyr Lys His Asp Gly Pro 385 390 395 400 Ala Lys Val Tyr Asn Ser Glu Asp Glu Ala Ile Ser Ala Ile Leu Gly 405 410 415 Gly Lys Ile Val Glu Gly Asp Val Ile Val Ile Arg Tyr Glu Gly Pro 420 425 430 Ser Gly Gly Pro Gly Met Arg Glu Met Leu Ser Pro Thr Ser Ala Ile 435 440 445 Cys Gly Met Gly Leu Asp Asp Ser Val Ala Leu Ile Thr Asp Gly Arg 450 455 460 Phe Ser Gly Gly Ser Arg Gly Pro Cys Ile Gly His Val Ser Pro Glu 465 470 475 480 Ala Ala Ala Gly Gly Val Ile Ala Ala Ile Glu Asn Gly Asp Ile Ile 485 490 495 Lys Ile Asp Met Ile Glu Lys Glu Ile Asn Val Asp Leu Asp Glu Ser 500 505 510 Val Ile Lys Glu Arg Leu Ser Lys Leu Gly Glu Phe Glu Pro Lys Ile 515 520 525 Lys Lys Gly Tyr Leu Ser Arg Tyr Ser Lys Leu Val Ser Ser Ala Asp 530 535 540 Glu Gly Ala Val Leu Lys 545 550 221653DNAMethanococcus maripaludis 22atgataagtg ataacgtcaa aaagggagtt ataagaactc caaaccgagc tcttttaaag 60gcttgcggat atacagacga agacatggaa aaaccattta ttggaattgt aaacagcttt 120acagaagttg ttcccggcca cattcactta agaacattat cagaagcggc taaacatggt 180gtttatgcaa acggtggaac accatttgaa tttaatacca ttggaatttg cgacggtatt 240gcaatgggcc acgaaggtat gaaatactct ttaccttcaa gagaaattat tgcagacgct 300gttgaatcaa tggcaagagc acatggattt gatggtcttg ttttaattcc tacgtgtgat 360aaaatcgttc ctggaatgat aatgggtgct ttaagactaa acattccatt tattgtagtt 420actggaggac caatgcttcc cggagaattc caaggtaaaa aatacgaact tatcagcctt 480tttgaaggtg tcggagaata ccaagttgga aaaattactg aagaagagtt aaagtgcatt 540gaagactgtg catgttcagg tgctggaagt tgtgcagggc tttacactgc aaacagtatg 600gcctgcctta cagaagcttt gggactctct cttccaatgt gtgcaacaac gcatgcagtt 660gatgcccaaa aagttaggct tgctaaaaaa agtggctcaa aaattgttga tatggtaaaa 720gaagacctaa aaccaacaga catattaaca aaagaagctt ttgaaaatgc tattttagtt 780gaccttgcac ttggtggatc aacaaacaca acattacaca ttcctgcaat tgcaaatgaa 840attgaaaata aattcataac tctcgatgac tttgacaggt taagcgatga agttccacac 900attgcatcaa tcaaaccagg tggagaacac tacatgattg atttacacaa tgctggaggt 960attcctgcgg tattgaacgt tttaaaagaa aaaattagag atacaaaaac agttgatgga 1020agaagcattt tggaaatcgc agaatctgtt aaatacataa attacgacgt tataagaaaa 1080gtggaagctc cggttcacga aactgctggt ttaagggttt taaagggaaa tcttgctcca 1140aacggttgcg ttgtaaaaat cggtgcagta catccgaaaa tgtacaaaca cgatggacct 1200gcaaaagttt acaattccga agatgaagca atttctgcga tacttggcgg aaaaattgta 1260gaaggggacg ttatagtaat cagatacgaa ggaccatcag gaggccctgg aatgagagaa 1320atgctctccc caacttcagc aatctgtgga atgggtcttg atgacagcgt tgcattgatt 1380actgatggaa gattcagtgg tggaagtagg ggcccatgta tcggacacgt ttctccagaa 1440gctgcagctg gcggagtaat tgctgcaatt gaaaacgggg atatcatcaa aatcgacatg 1500attgaaaaag aaataaatgt tgatttagat gaatcagtca ttaaagaaag actctcaaaa 1560ctgggagaat ttgagcctaa aatcaaaaaa ggctatttat caagatactc aaaacttgtc 1620tcatctgctg acgaaggggc agttttaaaa taa 165323558PRTBacillus subtilis 23Met Ala Glu Leu Arg Ser Asn Met Ile Thr Gln Gly Ile Asp Arg Ala 1 5 10 15 Pro His Arg Ser Leu Leu Arg Ala Ala Gly Val Lys Glu Glu Asp Phe 20 25 30 Gly Lys Pro Phe Ile Ala Val Cys Asn Ser Tyr Ile Asp Ile Val Pro 35 40 45 Gly His Val His Leu Gln Glu Phe Gly Lys Ile Val Lys Glu Ala Ile 50 55 60 Arg Glu Ala Gly Gly Val Pro Phe Glu Phe Asn Thr Ile Gly Val Asp 65 70 75 80 Asp Gly Ile Ala Met Gly His Ile Gly Met Arg Tyr Ser Leu Pro Ser 85 90 95 Arg Glu Ile Ile Ala Asp Ser Val Glu Thr Val Val Ser Ala His Trp 100 105 110 Phe Asp Gly Met Val Cys Ile Pro Asn Cys Asp Lys Ile Thr Pro Gly 115 120 125 Met Leu Met Ala Ala Met Arg Ile Asn Ile Pro Thr Ile Phe Val Ser 130 135 140 Gly Gly Pro Met Ala Ala Gly Arg Thr Ser Asp Gly Arg Lys Ile Ser 145 150 155 160 Leu Ser Ser Val Phe Glu Gly Val Gly Ala Tyr Gln Ala Gly Lys Ile 165 170 175 Asn Glu Asn Glu Leu Gln Glu Leu Glu Gln Phe Gly Cys Pro Thr Cys 180 185 190 Gly Ser Cys Ser Gly Met Phe Thr Ala Asn Ser Met Asn Cys Leu Ser 195 200 205 Glu Ala Leu Gly Leu Ala Leu Pro Gly Asn Gly Thr Ile Leu Ala Thr 210 215 220 Ser Pro Glu Arg Lys Glu Phe Val Arg Lys Ser Ala Ala Gln Leu Met 225 230 235 240 Glu Thr Ile Arg Lys Asp Ile Lys Pro Arg Asp Ile Val Thr Val Lys 245 250 255 Ala Ile Asp Asn Ala Phe Ala Leu Asp Met Ala Leu Gly Gly Ser Thr 260 265 270 Asn Thr Val Leu His Thr Leu Ala Leu Ala Asn Glu Ala Gly Val Glu 275 280 285 Tyr Ser Leu Glu Arg Ile Asn Glu Val Ala Glu Arg Val Pro His Leu 290 295 300 Ala Lys Leu Ala Pro Ala Ser Asp Val Phe Ile Glu Asp Leu His Glu 305 310 315 320 Ala Gly Gly Val Ser Ala Ala Leu Asn Glu Leu Ser Lys Lys Glu Gly 325 330 335 Ala Leu His Leu Asp Ala Leu Thr Val Thr Gly Lys Thr Leu Gly Glu 340 345 350 Thr Ile Ala Gly His Glu Val Lys Asp Tyr Asp Val Ile His Pro Leu 355 360 365 Asp Gln Pro Phe Thr Glu Lys Gly Gly Leu Ala Val Leu Phe Gly Asn 370 375 380 Leu Ala Pro Asp Gly Ala Ile Ile Lys Thr Gly Gly Val Gln Asn Gly 385 390 395 400 Ile Thr Arg His Glu Gly Pro Ala Val Val Phe Asp Ser Gln Asp Glu 405 410 415 Ala Leu Asp Gly Ile Ile Asn Arg Lys Val Lys Glu Gly Asp Val Val 420 425 430 Ile Ile Arg Tyr Glu Gly Pro Lys Gly Gly Pro Gly Met Pro Glu Met 435 440 445 Leu Ala Pro Thr Ser Gln Ile Val Gly Met Gly Leu Gly Pro Lys Val 450 455 460 Ala Leu Ile Thr Asp Gly Arg Phe Ser Gly Ala Ser Arg Gly Leu Ser 465 470 475 480 Ile Gly His Val Ser Pro Glu Ala Ala Glu Gly Gly Pro Leu Ala Phe 485 490 495 Val Glu Asn Gly Asp His Ile Ile Val Asp Ile Glu Lys Arg Ile Leu 500 505 510 Asp Val Gln Val Pro Glu Glu Glu Trp Glu Lys Arg Lys Ala Asn Trp 515 520 525 Lys Gly Phe Glu Pro Lys Val Lys Thr Gly Tyr Leu Ala Arg Tyr Ser 530 535 540 Lys Leu Val Thr Ser Ala Asn Thr Gly Gly Ile Met Lys Ile 545 550 555 241677DNABacillus subtilis 24atggcagaat tacgcagtaa tatgatcaca caaggaatcg atagagctcc gcaccgcagt 60ttgcttcgtg cagcaggggt aaaagaagag gatttcggca agccgtttat tgcggtgtgt 120aattcataca ttgatatcgt tcccggtcat gttcacttgc aggagtttgg gaaaatcgta 180aaagaagcaa tcagagaagc agggggcgtt ccgtttgaat ttaataccat tggggtagat 240gatggcatcg caatggggca tatcggtatg agatattcgc tgccaagccg tgaaattatc 300gcagactctg tggaaacggt tgtatccgca cactggtttg acggaatggt ctgtattccg 360aactgcgaca aaatcacacc gggaatgctt atggcggcaa tgcgcatcaa cattccgacg 420atttttgtca gcggcggacc gatggcggca ggaagaacaa gttacgggcg aaaaatctcc 480ctttcctcag tattcgaagg ggtaggcgcc taccaagcag ggaaaatcaa cgaaaacgag 540cttcaagaac tagagcagtt cggatgccca acgtgcgggt cttgctcagg catgtttacg 600gcgaactcaa tgaactgtct gtcagaagca cttggtcttg ctttgccggg taatggaacc 660attctggcaa catctccgga acgcaaagag tttgtgagaa aatcggctgc gcaattaatg 720gaaacgattc gcaaagatat caaaccgcgt gatattgtta cagtaaaagc gattgataac 780gcgtttgcac tcgatatggc gctcggaggt tctacaaata ccgttcttca tacccttgcc 840cttgcaaacg aagccggcgt tgaatactct ttagaacgca ttaacgaagt cgctgagcgc 900gtgccgcact tggctaagct ggcgcctgca tcggatgtgt ttattgaaga tcttcacgaa 960gcgggcggcg tttcagcggc tctgaatgag ctttcgaaga aagaaggagc gcttcattta 1020gatgcgctga ctgttacagg aaaaactctt ggagaaacca ttgccggaca tgaagtaaag 1080gattatgacg tcattcaccc gctggatcaa ccattcactg aaaagggagg ccttgctgtt 1140ttattcggta atctagctcc ggacggcgct atcattaaaa caggcggcgt acagaatggg 1200attacaagac acgaagggcc ggctgtcgta ttcgattctc aggacgaggc gcttgacggc 1260attatcaacc gaaaagtaaa agaaggcgac gttgtcatca tcagatacga agggccaaaa 1320ggcggacctg gcatgccgga aatgctggcg ccaacatccc aaatcgttgg aatgggactc 1380gggccaaaag tggcattgat tacggacgga cgtttttccg gagcctcccg tggcctctca 1440atcggccacg tatcacctga ggccgctgag ggcgggccgc ttgcctttgt tgaaaacgga 1500gaccatatta tcgttgatat tgaaaaacgc atcttggatg tacaagtgcc agaagaagag 1560tgggaaaaac gaaaagcgaa ctggaaaggt tttgaaccga aagtgaaaac cggctacctg 1620gcacgttatt ctaaacttgt gacaagtgcc aacaccggcg gtattatgaa aatctag 167725547PRTLactococcus lactis 25Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe

Leu 20 25 30 Asp Gln Ile Ile Ser Arg Glu Asp Met Lys Trp Ile Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Ile 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Asp Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Tyr 130 135 140 Glu Ile Asp Arg Val Leu Ser Gln Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ala Leu Ser Leu Glu Lys Glu Ser Ser Thr Thr Asn Thr Thr Glu Gln 180 185 190 Val Ile Leu Ser Lys Ile Glu Glu Ser Leu Lys Asn Ala Gln Lys Pro 195 200 205 Val Val Ile Ala Gly His Glu Val Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Val Ser Glu Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ala Val Asp Glu Ser Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Lys Leu Ser Glu Ile Ser Leu Lys Asn Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asp Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Ile Ile Phe Asn Lys Val Val Glu Asp Phe Asp Phe 305 310 315 320 Arg Ala Val Val Ser Ser Leu Ser Glu Leu Lys Gly Ile Glu Tyr Glu 325 330 335 Gly Gln Tyr Ile Asp Lys Gln Tyr Glu Glu Phe Ile Pro Ser Ser Ala 340 345 350 Pro Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Ser Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Thr Ile Phe Leu Lys Ser Asn Ser Arg Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ser Ile Arg Glu Lys Leu Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Thr Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Thr Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Val Asn Arg Met Tyr Trp Ile Glu Leu Val Leu Glu Lys 515 520 525 Glu Asp Ala Pro Lys Leu Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys 545 261828DNALactococcus lactis 26tttaaataag tcaatatcgt tgacttattt agaagaaaga gttattcttt aaatgtcaag 60ttagttgact aaattaaata taaaatatgg aggaatgtga tgtatacagt aggagattac 120ctgttagacc gattacacga gttgggaatt gaagaaattt ttggagttcc tggtgactat 180aacttacaat ttttagatca aattatttca cgcgaagata tgaaatggat tggaaatgct 240aatgaattaa atgcttctta tatggctgat ggttatgctc gtactaaaaa agctgccgca 300tttctcacca catttggagt cggcgaattg agtgcgatca atggactggc aggaagttat 360gccgaaaatt taccagtagt agaaattgtt ggttcaccaa cttcaaaagt acaaaatgac 420ggaaaatttg tccatcatac actagcagat ggtgatttta aacactttat gaagatgcat 480gaacctgtta cagcagcgcg gactttactg acagcagaaa atgccacata tgaaattgac 540cgagtacttt ctcaattact aaaagaaaga aaaccagtct atattaactt accagtcgat 600gttgctgcag caaaagcaga gaagcctgca ttatctttag aaaaagaaag ctctacaaca 660aatacaactg aacaagtgat tttgagtaag attgaagaaa gtttgaaaaa tgcccaaaaa 720ccagtagtga ttgcaggaca cgaagtaatt agttttggtt tagaaaaaac ggtaactcag 780tttgtttcag aaacaaaact accgattacg acactaaatt ttggtaaaag tgctgttgat 840gaatctttgc cctcattttt aggaatatat aacgggaaac tttcagaaat cagtcttaaa 900aattttgtgg agtccgcaga ctttatccta atgcttggag tgaagcttac ggactcctca 960acaggtgcat tcacacatca tttagatgaa aataaaatga tttcactaaa catagatgaa 1020ggaataattt tcaataaagt ggtagaagat tttgatttta gagcagtggt ttcttcttta 1080tcagaattaa aaggaataga atatgaagga caatatattg ataagcaata tgaagaattt 1140attccatcaa gtgctccctt atcacaagac cgtctatggc aggcagttga aagtttgact 1200caaagcaatg aaacaatcgt tgctgaacaa ggaacctcat tttttggagc ttcaacaatt 1260ttcttaaaat caaatagtcg ttttattgga caacctttat ggggttctat tggatatact 1320tttccagcgg ctttaggaag ccaaattgcg gataaagaga gcagacacct tttatttatt 1380ggtgatggtt cacttcaact taccgtacaa gaattaggac tatcaatcag agaaaaactc 1440aatccaattt gttttatcat aaataatgat ggttatacag ttgaaagaga aatccacgga 1500cctactcaaa gttataacga cattccaatg tggaattact cgaaattacc agaaacattt 1560ggagcaacag aagatcgtgt agtatcaaaa attgttagaa cagagaatga atttgtgtct 1620gtcatgaaag aagcccaagc agatgtcaat agaatgtatt ggatagaact agttttggaa 1680aaagaagatg cgccaaaatt actgaaaaaa atgggtaaat tatttgctga gcaaaataaa 1740tagatatcaa cggatgatga aaagtaaaat agacaaagtc caataatttt ataaaaagta 1800aaaacattag gattttccta atgttttt 182827548PRTLactococcus lactis 27Met Tyr Thr Val Gly Asp Tyr Leu Leu Asp Arg Leu His Glu Leu Gly 1 5 10 15 Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu 20 25 30 Asp Gln Ile Ile Ser His Lys Asp Met Lys Trp Val Gly Asn Ala Asn 35 40 45 Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys 50 55 60 Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val 65 70 75 80 Asn Gly Leu Ala Gly Ser Tyr Ala Glu Asn Leu Pro Val Val Glu Ile 85 90 95 Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His 100 105 110 His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu 115 120 125 Pro Val Thr Ala Ala Arg Thr Leu Leu Thr Ala Glu Asn Ala Thr Val 130 135 140 Glu Ile Asp Arg Val Leu Ser Ala Leu Leu Lys Glu Arg Lys Pro Val 145 150 155 160 Tyr Ile Asn Leu Pro Val Asp Val Ala Ala Ala Lys Ala Glu Lys Pro 165 170 175 Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln 180 185 190 Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro 195 200 205 Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr 210 215 220 Val Thr Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn 225 230 235 240 Phe Gly Lys Ser Ser Val Asp Glu Ala Leu Pro Ser Phe Leu Gly Ile 245 250 255 Tyr Asn Gly Thr Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser 260 265 270 Ala Asp Phe Ile Leu Met Leu Gly Val Lys Leu Thr Asp Ser Ser Thr 275 280 285 Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn 290 295 300 Ile Asp Glu Gly Lys Ile Phe Asn Glu Arg Ile Gln Asn Phe Asp Phe 305 310 315 320 Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys 325 330 335 Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala 340 345 350 Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln 355 360 365 Ser Asn Glu Thr Ile Val Ala Glu Gln Gly Thr Ser Phe Phe Gly Ala 370 375 380 Ser Ser Ile Phe Leu Lys Ser Lys Ser His Phe Ile Gly Gln Pro Leu 385 390 395 400 Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile 405 410 415 Ala Asp Lys Glu Ser Arg His Leu Leu Phe Ile Gly Asp Gly Ser Leu 420 425 430 Gln Leu Thr Val Gln Glu Leu Gly Leu Ala Ile Arg Glu Lys Ile Asn 435 440 445 Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu 450 455 460 Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr 465 470 475 480 Ser Lys Leu Pro Glu Ser Phe Gly Ala Thr Glu Asp Arg Val Val Ser 485 490 495 Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala 500 505 510 Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys 515 520 525 Glu Gly Ala Pro Lys Val Leu Lys Lys Met Gly Lys Leu Phe Ala Glu 530 535 540 Gln Asn Lys Ser 545 281954DNALactococcus lactis 28ctagagtttt ctttagtcat aattcactcc ttttattagt ctattatact tgataattca 60aataagtcaa tatcgttgac ttatttaaag aaaagcgtta ttctataaat gtcaagttga 120ttgaccaata tataataaaa tatggaggaa tgcgatgtat acagtaggag attacctatt 180agaccgatta cacgagttag gaattgaaga aatttttgga gtccctggag actataactt 240acaattttta gatcaaatta tttcccacaa ggatatgaaa tgggtcggaa atgctaatga 300attaaatgct tcatatatgg ctgatggcta tgctcgtact aaaaaagctg ccgcatttct 360tacaaccttt ggagtaggtg aattgagtgc agttaatgga ttagcaggaa gttacgccga 420aaatttacca gtagtagaaa tagtgggatc acctacatca aaagttcaaa atgaaggaaa 480atttgttcat catacgctgg ctgacggtga ttttaaacac tttatgaaaa tgcacgaacc 540tgttacagca gctcgaactt tactgacagc agaaaatgca accgttgaaa ttgaccgagt 600actttctgca ctattaaaag aaagaaaacc tgtctatatc aacttaccag ttgatgttgc 660tgctgcaaaa gcagagaaac cctcactccc tttgaaaaag gaaaactcaa cttcaaatac 720aagtgaccaa gaaattttga acaaaattca agaaagcttg aaaaatgcca aaaaaccaat 780cgtgattaca ggacatgaaa taattagttt tggcttagaa aaaacagtca ctcaatttat 840ttcaaagaca aaactaccta ttacgacatt aaactttggt aaaagttcag ttgatgaagc 900cctcccttca tttttaggaa tctataatgg tacactctca gagcctaatc ttaaagaatt 960cgtggaatca gccgacttca tcttgatgct tggagttaaa ctcacagact cttcaacagg 1020agccttcact catcatttaa atgaaaataa aatgatttca ctgaatatag atgaaggaaa 1080aatatttaac gaaagaatcc aaaattttga ttttgaatcc ctcatctcct ctctcttaga 1140cctaagcgaa atagaataca aaggaaaata tatcgataaa aagcaagaag actttgttcc 1200atcaaatgcg cttttatcac aagaccgcct atggcaagca gttgaaaacc taactcaaag 1260caatgaaaca atcgttgctg aacaagggac atcattcttt ggcgcttcat caattttctt 1320aaaatcaaag agtcatttta ttggtcaacc cttatgggga tcaattggat atacattccc 1380agcagcatta ggaagccaaa ttgcagataa agaaagcaga caccttttat ttattggtga 1440tggttcactt caacttacag tgcaagaatt aggattagca atcagagaaa aaattaatcc 1500aatttgcttt attatcaata atgatggtta tacagtcgaa agagaaattc atggaccaaa 1560tcaaagctac aatgatattc caatgtggaa ttactcaaaa ttaccagaat cgtttggagc 1620aacagaagat cgagtagtct caaaaatcgt tagaactgaa aatgaatttg tgtctgtcat 1680gaaagaagct caagcagatc caaatagaat gtactggatt gagttaattt tggcaaaaga 1740aggtgcacca aaagtactga aaaaaatggg caaactattt gctgaacaaa ataaatcata 1800atttataaat agtaaaaaac attaggaaat acctaatgtt tttttgttga ctaaatcaat 1860ccctctttat atagaaaacc ttagtttctc aaagacaact taattaagcc tgccaaattg 1920gaactcgcaa aatgtaatct atcctctgct ccta 195429550PRTSalmonella typhimurium 29Met Gln Asn Pro Tyr Thr Val Ala Asp Tyr Leu Leu Asp Arg Leu Ala 1 5 10 15 Gly Cys Gly Ile Gly His Leu Phe Gly Val Pro Gly Asp Tyr Asn Leu 20 25 30 Gln Phe Leu Asp His Val Ile Asp His Pro Thr Leu Arg Trp Val Gly 35 40 45 Cys Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg 50 55 60 Met Ser Gly Ala Gly Ala Leu Leu Thr Thr Phe Gly Val Gly Glu Leu 65 70 75 80 Ser Ala Ile Asn Gly Ile Ala Gly Ser Tyr Ala Glu Tyr Val Pro Val 85 90 95 Leu His Ile Val Gly Ala Pro Cys Ser Ala Ala Gln Gln Arg Gly Glu 100 105 110 Leu Met His His Thr Leu Gly Asp Gly Asp Phe Arg His Phe Tyr Arg 115 120 125 Met Ser Gln Ala Ile Ser Ala Ala Ser Ala Ile Leu Asp Glu Gln Asn 130 135 140 Ala Cys Phe Glu Ile Asp Arg Val Leu Gly Glu Met Leu Ala Ala Arg 145 150 155 160 Arg Pro Gly Tyr Ile Met Leu Pro Ala Asp Val Ala Lys Lys Thr Ala 165 170 175 Ile Pro Pro Thr Gln Ala Leu Ala Leu Pro Val His Glu Ala Gln Ser 180 185 190 Gly Val Glu Thr Ala Phe Arg Tyr His Ala Arg Gln Cys Leu Met Asn 195 200 205 Ser Arg Arg Ile Ala Leu Leu Ala Asp Phe Leu Ala Gly Arg Phe Gly 210 215 220 Leu Arg Pro Leu Leu Gln Arg Trp Met Ala Glu Thr Pro Ile Ala His 225 230 235 240 Ala Thr Leu Leu Met Gly Lys Gly Leu Phe Asp Glu Gln His Pro Asn 245 250 255 Phe Val Gly Thr Tyr Ser Ala Gly Ala Ser Ser Lys Glu Val Arg Gln 260 265 270 Ala Ile Glu Asp Ala Asp Arg Val Ile Cys Val Gly Thr Arg Phe Val 275 280 285 Asp Thr Leu Thr Ala Gly Phe Thr Gln Gln Leu Pro Ala Glu Arg Thr 290 295 300 Leu Glu Ile Gln Pro Tyr Ala Ser Arg Ile Gly Glu Thr Trp Phe Asn 305 310 315 320 Leu Pro Met Ala Gln Ala Val Ser Thr Leu Arg Glu Leu Cys Leu Glu 325 330 335 Cys Ala Phe Ala Pro Pro Pro Thr Arg Ser Ala Gly Gln Pro Val Arg 340 345 350 Ile Asp Lys Gly Glu Leu Thr Gln Glu Ser Phe Trp Gln Thr Leu Gln 355 360 365 Gln Tyr Leu Lys Pro Gly Asp Ile Ile Leu Val Asp Gln Gly Thr Ala 370 375 380 Ala Phe Gly Ala Ala Ala Leu Ser Leu Pro Asp Gly Ala Glu Val Val 385 390 395 400 Leu Gln Pro Leu Trp Gly Ser Ile Gly Tyr Ser Leu Pro Ala Ala Phe 405 410 415 Gly Ala Gln Thr Ala Cys Pro Asp Arg Arg Val Ile Leu Ile Ile Gly 420 425 430 Asp Gly Ala Ala Gln Leu Thr Ile Gln Glu Met Gly Ser Met Leu Arg 435 440 445 Asp Gly Gln Ala Pro Val Ile Leu Leu Leu Asn Asn Asp Gly Tyr Thr 450 455 460 Val Glu Arg Ala Ile His Gly Ala Ala Gln Arg Tyr Asn Asp Ile Ala 465 470 475 480 Ser Trp Asn Trp Thr Gln Ile Pro Pro Ala Leu Asn Ala Ala Gln Gln 485 490 495 Ala Glu Cys Trp Arg Val Thr Gln Ala Ile Gln Leu Ala Glu Val Leu 500 505 510 Glu Arg Leu Ala Arg Pro Gln Arg Leu Ser Phe Ile Glu Val Met Leu 515 520 525 Pro Lys Ala Asp Leu Pro Glu Leu Leu Arg Thr Val Thr Arg Ala Leu 530 535 540 Glu Ala Arg Asn Gly Gly 545 550 301653DNASalmonella typhimurium 30ttatcccccg ttgcgggctt ccagcgcccg ggtcacggta cgcagtaatt ccggcagatc 60ggcttttggc aacatcactt caataaatga cagacgttgt gggcgcgcca accgttcgag 120gacctctgcc agttggatag cctgcgtcac ccgccagcac tccgcctgtt gcgccgcgtt 180tagcgccggt ggtatctgcg tccagttcca gctcgcgatg tcgttatacc gctgggccgc 240gccgtgaatg gcgcgctcta cggtatagcc gtcattgttg agcagcagga tgaccggcgc 300ctgcccgtcg cgtaacatcg agcccatctc ctgaatcgtg agctgcgccg cgccatcgcc 360gataatcaga atcacccgcc gatcgggaca ggcggtttgc gcgccaaacg cggcgggcaa 420ggaatagccg atagaccccc acagcggctg taacacaact tccgcgccgt caggaagcga 480cagcgcggca

gcgccaaaag ctgctgtccc ctggtcgaca aggataatat ctccgggttt 540gagatactgc tgtaaggttt gccagaagct ttcctgggtc agttctcctt tatcaatccg 600cactggctgt ccggcggaac gcgtcggcgg cggcgcaaaa gcgcattcca ggcacagttc 660gcgcagcgta gacaccgcct gcgccatcgg gaggttgaac caggtttcgc cgatgcgcga 720cgcgtaaggc tgaatctcca gcgtgcgttc cgccggtaat tgttgggtaa atccggccgt 780aagggtatcg acaaaacggg tgccgacgca gataacccta tcggcgtcct ctatggcctg 840acgcacttct ttgctgctgg cgccagcgct ataggtgcca acgaagttcg ggtgctgttc 900atcaaaaagc cccttcccca tcagtagtgt cgcatgagcg atgggcgttt ccgccatcca 960gcgctgcaac agtggtcgta aaccaaaacg cccggcaaga aagtcggcca atagcgcaat 1020gcgccgactg ttcatcaggc actgacgggc gtgataacga aaggccgtct ccacgccgct 1080ttgcgcttca tgcacgggca acgccagcgc ctgcgtaggt gggatggccg tttttttcgc 1140cacatcggcg ggcaacatga tgtatcctgg cctgcgtgcg gcaagcattt cacccaacac 1200gcggtcaatc tcgaaacagg cgttctgttc atctaatatt gcgctggcag cggatatcgc 1260ctgactcatg cgataaaaat gacgaaaatc gccgtcaccg agggtatggt gcatcaattc 1320gccacgctgc tgcgcagcgc tacagggcgc gccgacgata tgcaagaccg ggacatattc 1380cgcgtaactg cccgcgatac cgttaatagc gctaagttct cccacgccaa aggtggtgag 1440tagcgctcca gcgcccgaca tgcgcgcata gccgtccgcg gcataagcgg cgttcagctc 1500attggcgcat cccacccaac gcagggtcgg gtggtcaatc acatggtcaa gaaactgcaa 1560gttataatcg cccggtacgc caaaaagatg gccaatgccg catcctgcca gtctgtccag 1620caaatagtcg gccacggtat aggggttttg cat 165331554PRTClostridium acetobutylicum 31Met Lys Ser Glu Tyr Thr Ile Gly Arg Tyr Leu Leu Asp Arg Leu Ser 1 5 10 15 Glu Leu Gly Ile Arg His Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu 20 25 30 Ser Phe Leu Asp Tyr Ile Met Glu Tyr Lys Gly Ile Asp Trp Val Gly 35 40 45 Asn Cys Asn Glu Leu Asn Ala Gly Tyr Ala Ala Asp Gly Tyr Ala Arg 50 55 60 Ile Asn Gly Ile Gly Ala Ile Leu Thr Thr Phe Gly Val Gly Glu Leu 65 70 75 80 Ser Ala Ile Asn Ala Ile Ala Gly Ala Tyr Ala Glu Gln Val Pro Val 85 90 95 Val Lys Ile Thr Gly Ile Pro Thr Ala Lys Val Arg Asp Asn Gly Leu 100 105 110 Tyr Val His His Thr Leu Gly Asp Gly Arg Phe Asp His Phe Phe Glu 115 120 125 Met Phe Arg Glu Val Thr Val Ala Glu Ala Leu Leu Ser Glu Glu Asn 130 135 140 Ala Ala Gln Glu Ile Asp Arg Val Leu Ile Ser Cys Trp Arg Gln Lys 145 150 155 160 Arg Pro Val Leu Ile Asn Leu Pro Ile Asp Val Tyr Asp Lys Pro Ile 165 170 175 Asn Lys Pro Leu Lys Pro Leu Leu Asp Tyr Thr Ile Ser Ser Asn Lys 180 185 190 Glu Ala Ala Cys Glu Phe Val Thr Glu Ile Val Pro Ile Ile Asn Arg 195 200 205 Ala Lys Lys Pro Val Ile Leu Ala Asp Tyr Gly Val Tyr Arg Tyr Gln 210 215 220 Val Gln His Val Leu Lys Asn Leu Ala Glu Lys Thr Gly Phe Pro Val 225 230 235 240 Ala Thr Leu Ser Met Gly Lys Gly Val Phe Asn Glu Ala His Pro Gln 245 250 255 Phe Ile Gly Val Tyr Asn Gly Asp Val Ser Ser Pro Tyr Leu Arg Gln 260 265 270 Arg Val Asp Glu Ala Asp Cys Ile Ile Ser Val Gly Val Lys Leu Thr 275 280 285 Asp Ser Thr Thr Gly Gly Phe Ser His Gly Phe Ser Lys Arg Asn Val 290 295 300 Ile His Ile Asp Pro Phe Ser Ile Lys Ala Lys Gly Lys Lys Tyr Ala 305 310 315 320 Pro Ile Thr Met Lys Asp Ala Leu Thr Glu Leu Thr Ser Lys Ile Glu 325 330 335 His Arg Asn Phe Glu Asp Leu Asp Ile Lys Pro Tyr Lys Ser Asp Asn 340 345 350 Gln Lys Tyr Phe Ala Lys Glu Lys Pro Ile Thr Gln Lys Arg Phe Phe 355 360 365 Glu Arg Ile Ala His Phe Ile Lys Glu Lys Asp Val Leu Leu Ala Glu 370 375 380 Gln Gly Thr Cys Phe Phe Gly Ala Ser Thr Ile Gln Leu Pro Lys Asp 385 390 395 400 Ala Thr Phe Ile Gly Gln Pro Leu Trp Gly Ser Ile Gly Tyr Thr Leu 405 410 415 Pro Ala Leu Leu Gly Ser Gln Leu Ala Asp Gln Lys Arg Arg Asn Ile 420 425 430 Leu Leu Ile Gly Asp Gly Ala Phe Gln Met Thr Ala Gln Glu Ile Ser 435 440 445 Thr Met Leu Arg Leu Gln Ile Lys Pro Ile Ile Phe Leu Ile Asn Asn 450 455 460 Asp Gly Tyr Thr Ile Glu Arg Ala Ile His Gly Arg Glu Gln Val Tyr 465 470 475 480 Asn Asn Ile Gln Met Trp Arg Tyr His Asn Val Pro Lys Val Leu Gly 485 490 495 Pro Lys Glu Cys Ser Leu Thr Phe Lys Val Gln Ser Glu Thr Glu Leu 500 505 510 Glu Lys Ala Leu Leu Val Ala Asp Lys Asp Cys Glu His Leu Ile Phe 515 520 525 Ile Glu Val Val Met Asp Arg Tyr Asp Lys Pro Glu Pro Leu Glu Arg 530 535 540 Leu Ser Lys Arg Phe Ala Asn Gln Asn Asn 545 550 321665DNAClostridium acetobutylicum 32ttgaagagtg aatacacaat tggaagatat ttgttagacc gtttatcaga gttgggtatt 60cggcatatct ttggtgtacc tggagattac aatctatcct ttttagacta tataatggag 120tacaaaggga tagattgggt tggaaattgc aatgaattga atgctgggta tgctgctgat 180ggatatgcaa gaataaatgg aattggagcc atacttacaa catttggtgt tggagaatta 240agtgccatta acgcaattgc tggggcatac gctgagcaag ttccagttgt taaaattaca 300ggtatcccca cagcaaaagt tagggacaat ggattatatg tacaccacac attaggtgac 360ggaaggtttg atcacttttt tgaaatgttt agagaagtaa cagttgctga ggcattacta 420agcgaagaaa atgcagcaca agaaattgat cgtgttctta tttcatgctg gagacaaaaa 480cgtcctgttc ttataaattt accgattgat gtatatgata aaccaattaa caaaccatta 540aagccattac tcgattatac tatttcaagt aacaaagagg ctgcatgtga atttgttaca 600gaaatagtac ctataataaa tagggcaaaa aagcctgtta ttcttgcaga ttatggagta 660tatcgttacc aagttcaaca tgtgcttaaa aacttggccg aaaaaaccgg atttcctgtg 720gctacactaa gtatgggaaa aggtgttttc aatgaagcac accctcaatt tattggtgtt 780tataatggtg atgtaagttc tccttattta aggcagcgag ttgatgaagc agactgcatt 840attagcgttg gtgtaaaatt gacggattca accacagggg gattttctca tggattttct 900aaaaggaatg taattcacat tgatcctttt tcaataaagg caaaaggtaa aaaatatgca 960cctattacga tgaaagatgc tttaacagaa ttaacaagta aaattgagca tagaaacttt 1020gaggatttag atataaagcc ttacaaatca gataatcaaa agtattttgc aaaagagaag 1080ccaattacac aaaaacgttt ttttgagcgt attgctcact ttataaaaga aaaagatgta 1140ttattagcag aacagggtac atgctttttt ggtgcgtcaa ccatacaact acccaaagat 1200gcaactttta ttggtcaacc tttatgggga tctattggat acacacttcc tgctttatta 1260ggttcacaat tagctgatca aaaaaggcgt aatattcttt taattgggga tggtgcattt 1320caaatgacag cacaagaaat ttcaacaatg cttcgtttac aaatcaaacc tattattttt 1380ttaattaata acgatggtta tacaattgaa cgtgctattc atggtagaga acaagtatat 1440aacaatattc aaatgtggcg atatcataat gttccaaagg ttttaggtcc taaagaatgc 1500agcttaacct ttaaagtaca aagtgaaact gaacttgaaa aggctctttt agtggcagat 1560aaggattgtg aacatttgat ttttatagaa gttgttatgg atcgttatga taaacccgag 1620cctttagaac gtctttcgaa acgttttgca aatcaaaata attag 1665331641DNAClostridium acetobutylicum 33atgaaacaac gtatcgggca atacttgatc gatgccctac acgttaatgg tgtcgataag 60atctttggag tcccaggtga tttcacttta gcctttttgg acgatatcat aagacatgac 120aacgtggaat gggtgggaaa tactaatgag ttgaacgccg cttacgccgc tgatggttac 180gctagagtta atggattagc cgctgtatct accacttttg gggttggcga gttatctgct 240gtgaatggta ttgctggaag ttacgcagag cgtgttcctg taatcaaaat ctcaggcggt 300ccttcatcag ttgctcaaca agagggtaga tatgtccacc attcattggg tgaaggaatc 360tttgattcat attcaaagat gtacgctcac ataaccgcaa caactacaat cttatccgtt 420gacaacgcag tcgacgaaat tgatagagtt attcattgtg ctttgaagga aaagaggcca 480gtgcatattc atttgcctat tgacgtagcc ttaactgaga ttgaaatccc tcatgcacca 540aaagtttaca cacacgaatc ccagaacgtc gatgcttaca ttcaagctgt tgagaaaaag 600ttaatgtctg caaaacaacc agtaatcata gcaggtcatg aaatcaattc attcaagttg 660cacgaacaac tggaacagtt tgtcaatcag acaaacatcc ctgttgcaca actttccttg 720ggtaagtctg ctttcaatga agagaatgaa cattaccttg gtatctacga tggcaaaatc 780gcaaaggaaa atgtgagaga gtacgtcgac aatgctgatg tcatattgaa cataggtgcc 840aaactgactg attctgctac agctggattt tcctacaagt tcgatacaaa caacataatc 900tacattaacc ataatgactt caaagctgaa gatgtgattt ctgataatgt ttcactgatt 960gatcttgtga atggcctgaa ttctattgac tatagaaatg aaacacacta cccatcttat 1020caaagatctg atatgaaata cgaattgaat gacgcaccac ttacacaatc taactatttc 1080aaaatgatga acgcttttct agaaaaagat gacatcctac tagctgaaca aggtacatcc 1140tttttcggcg catatgactt atccctatac aagggaaatc agtttatcgg tcagccttta 1200tgggggtcaa tagggtatac ttttccatct ttactaggaa gtcaactagc agacatgcat 1260aggagaaaca ttttgcttat aggcgatggt agtttacaac ttactgttca agccctaagt 1320acaatgatta gaaaggatat caaaccaatc attttcgtta tcaataacga cggttacacc 1380gtcgaaagac ttatccacgg catggaagag ccatacaatg atatccaaat gtggaactac 1440aagcaattgc cagaagtatt tggtggaaaa gatactgtaa aagttcatga tgctaaaacc 1500tccaacgaac tgaaaactgt aatggattct gttaaagcag acaaagatca catgcatttc 1560attgaagtgc atatggcagt agaggacgcc ccaaagaagt tgattgatat agctaaagcc 1620tttagtgatg ctaacaagta a 1641341647DNAListeria grayi 34atgtacaccg tcggccaata cttagtagac cgcttagaag agatcggcat cgataaggtt 60tttggtgtcc cgggtgacta caacctgacc tttttggact acatccagaa ccacgaaggt 120ctgagctggc aaggtaatac gaatgaactg aatgccgcgt acgcagctga tggctatgct 180cgtgaacgcg gtgttagcgc tttggtcacg accttcggcg ttggtgagct gtccgcaatc 240aatggcaccg caggtagctt cgcggagcaa gttccggtga ttcatatcgt gggcagcccg 300accatgaatg ttcagagcaa caagaaactg gttcatcaca gcctgggtat gggcaacttt 360cacaacttca gcgagatggc gaaagaagtc accgccgcaa ccacgatgct gacggaagag 420aatgcggcgt cggagattga tcgtgttctg gaaaccgccc tgctggagaa acgcccagtg 480tacatcaatc tgccgatcga cattgctcac aaggcgatcg tcaagccggc gaaagccctg 540caaaccgaga agagctctgg cgagcgtgag gcacaactgg cggagatcat tctgagccat 600ctggagaagg ctgcacagcc gattgtgatt gcgggtcacg agatcgcgcg cttccagatc 660cgtgagcgtt tcgagaattg gattaatcaa acgaaactgc cggtgaccaa tctggcctac 720ggcaagggta gcttcaacga agaaaacgag catttcattg gtacctatta tcctgcattt 780agcgataaga acgtgctgga ctacgtggat aactccgact ttgtcctgca ctttggtggt 840aaaatcattg ataacagcac ctccagcttc tcccaaggct tcaaaaccga gaacaccctg 900actgcggcga acgatatcat tatgctgccg gacggtagca cgtattctgg tattagcctg 960aatggcctgc tggccgagct ggaaaaactg aatttcacgt ttgccgacac cgcagcaaag 1020caggcggagt tggcggtgtt tgagccgcag gctgaaaccc cgttgaaaca ggaccgtttt 1080caccaggcgg tgatgaattt tctgcaagct gacgatgtcc tggttacgga acagggcacc 1140tcttcttttg gcttgatgct ggcgcctctg aaaaagggta tgaacttgat ctcgcaaacg 1200ctgtggggta gcattggtta cacgttgccg gcgatgattg gtagccaaat tgcggcaccg 1260gagcgtcgtc atatcctgag cattggtgat ggtagctttc agctgactgc gcaggaaatg 1320agcaccattt tccgtgagaa actgacccca gtcatcttca tcattaacaa tgatggctat 1380accgttgagc gtgcgatcca tggcgaagat gaaagctata acgacattcc gacgtggaac 1440ttgcaactgg tggcggaaac cttcggtggt gacgccgaaa ccgtcgacac tcacaatgtg 1500ttcacggaga ctgatttcgc caacaccctg gcggcaattg acgcgacgcc gcagaaagca 1560cacgttgtgg aagttcacat ggaacaaatg gatatgccgg agagcctgcg ccagatcggt 1620ctggcactgt ccaagcagaa tagctaa 164735312PRTSaccharomyces cerevisiae 35Met Pro Ala Thr Leu Lys Asn Ser Ser Ala Thr Leu Lys Leu Asn Thr 1 5 10 15 Gly Ala Ser Ile Pro Val Leu Gly Phe Gly Thr Trp Arg Ser Val Asp 20 25 30 Asn Asn Gly Tyr His Ser Val Ile Ala Ala Leu Lys Ala Gly Tyr Arg 35 40 45 His Ile Asp Ala Ala Ala Ile Tyr Leu Asn Glu Glu Glu Val Gly Arg 50 55 60 Ala Ile Lys Asp Ser Gly Val Pro Arg Glu Glu Ile Phe Ile Thr Thr 65 70 75 80 Lys Leu Trp Gly Thr Glu Gln Arg Asp Pro Glu Ala Ala Leu Asn Lys 85 90 95 Ser Leu Lys Arg Leu Gly Leu Asp Tyr Val Asp Leu Tyr Leu Met His 100 105 110 Trp Pro Val Pro Leu Lys Thr Asp Arg Val Thr Asp Gly Asn Val Leu 115 120 125 Cys Ile Pro Thr Leu Glu Asp Gly Thr Val Asp Ile Asp Thr Lys Glu 130 135 140 Trp Asn Phe Ile Lys Thr Trp Glu Leu Met Gln Glu Leu Pro Lys Thr 145 150 155 160 Gly Lys Thr Lys Ala Val Gly Val Ser Asn Phe Ser Ile Asn Asn Ile 165 170 175 Lys Glu Leu Leu Glu Ser Pro Asn Asn Lys Val Val Pro Ala Thr Asn 180 185 190 Gln Ile Glu Ile His Pro Leu Leu Pro Gln Asp Glu Leu Ile Ala Phe 195 200 205 Cys Lys Glu Lys Gly Ile Val Val Glu Ala Tyr Ser Pro Phe Gly Ser 210 215 220 Ala Asn Ala Pro Leu Leu Lys Glu Gln Ala Ile Ile Asp Met Ala Lys 225 230 235 240 Lys His Gly Val Glu Pro Ala Gln Leu Ile Ile Ser Trp Ser Ile Gln 245 250 255 Arg Gly Tyr Val Val Leu Ala Lys Ser Val Asn Pro Glu Arg Ile Val 260 265 270 Ser Asn Phe Lys Ile Phe Thr Leu Pro Glu Asp Asp Phe Lys Thr Ile 275 280 285 Ser Asn Leu Ser Lys Val His Gly Thr Lys Arg Val Val Asp Met Lys 290 295 300 Trp Gly Ser Phe Pro Ile Phe Gln 305 310 36939DNASaccharomyces cerevisiae 36atgcctgcta cgttaaagaa ttcttctgct acattaaaac taaatactgg tgcctccatt 60ccagtgttgg gtttcggcac ttggcgttcc gttgacaata acggttacca ttctgtaatt 120gcagctttga aagctggata cagacacatt gatgctgcgg ctatctattt gaatgaagaa 180gaagttggca gggctattaa agattccgga gtccctcgtg aggaaatttt tattactact 240aagctttggg gtacggaaca acgtgatccg gaagctgctc taaacaagtc tttgaaaaga 300ctaggcttgg attatgttga cctatatctg atgcattggc cagtgccttt gaaaaccgac 360agagttactg atggtaacgt tctgtgcatt ccaacattag aagatggcac tgttgacatc 420gatactaagg aatggaattt tatcaagacg tgggagttga tgcaagagtt gccaaagacg 480ggcaaaacta aagccgttgg tgtctctaat ttttctatta acaacattaa agaattatta 540gaatctccaa ataacaaggt ggtaccagct actaatcaaa ttgaaattca tccattgcta 600ccacaagacg aattgattgc cttttgtaag gaaaagggta ttgttgttga agcctactca 660ccatttggga gtgctaatgc tcctttacta aaagagcaag caattattga tatggctaaa 720aagcacggcg ttgagccagc acagcttatt atcagttgga gtattcaaag aggctacgtt 780gttctggcca aatcggttaa tcctgaaaga attgtatcca attttaagat tttcactctg 840cctgaggatg atttcaagac tattagtaac ctatccaaag tgcatggtac aaagagagtc 900gttgatatga agtggggatc cttcccaatt ttccaatga 93937360PRTSaccharomyces cerevisiae 37Met Ser Tyr Pro Glu Lys Phe Glu Gly Ile Ala Ile Gln Ser His Glu 1 5 10 15 Asp Trp Lys Asn Pro Lys Lys Thr Lys Tyr Asp Pro Lys Pro Phe Tyr 20 25 30 Asp His Asp Ile Asp Ile Lys Ile Glu Ala Cys Gly Val Cys Gly Ser 35 40 45 Asp Ile His Cys Ala Ala Gly His Trp Gly Asn Met Lys Met Pro Leu 50 55 60 Val Val Gly His Glu Ile Val Gly Lys Val Val Lys Leu Gly Pro Lys 65 70 75 80 Ser Asn Ser Gly Leu Lys Val Gly Gln Arg Val Gly Val Gly Ala Gln 85 90 95 Val Phe Ser Cys Leu Glu Cys Asp Arg Cys Lys Asn Asp Asn Glu Pro 100 105 110 Tyr Cys Thr Lys Phe Val Thr Thr Tyr Ser Gln Pro Tyr Glu Asp Gly 115 120 125 Tyr Val Ser Gln Gly Gly Tyr Ala Asn Tyr Val Arg Val His Glu His 130 135 140 Phe Val Val Pro Ile Pro Glu Asn Ile Pro Ser His Leu Ala Ala Pro 145 150 155 160 Leu Leu Cys Gly Gly Leu Thr Val Tyr Ser Pro Leu Val Arg Asn Gly 165 170 175 Cys Gly Pro Gly Lys Lys Val Gly Ile Val Gly Leu Gly Gly Ile Gly 180 185 190 Ser Met Gly Thr Leu Ile Ser Lys Ala Met Gly Ala Glu Thr Tyr Val 195 200 205 Ile Ser Arg Ser Ser Arg Lys Arg Glu Asp Ala Met Lys Met Gly Ala 210 215 220 Asp His Tyr Ile Ala Thr Leu Glu Glu Gly Asp Trp Gly Glu Lys Tyr 225 230 235 240 Phe Asp Thr Phe Asp Leu Ile Val Val Cys Ala Ser Ser Leu Thr Asp 245 250 255 Ile Asp Phe Asn Ile Met Pro Lys Ala Met Lys Val Gly Gly Arg Ile 260 265 270 Val Ser Ile Ser Ile Pro Glu Gln His Glu Met Leu Ser Leu Lys Pro 275 280 285 Tyr Gly Leu Lys Ala Val Ser Ile Ser Tyr Ser Ala Leu Gly Ser Ile 290 295 300

Lys Glu Leu Asn Gln Leu Leu Lys Leu Val Ser Glu Lys Asp Ile Lys 305 310 315 320 Ile Trp Val Glu Thr Leu Pro Val Gly Glu Ala Gly Val His Glu Ala 325 330 335 Phe Glu Arg Met Glu Lys Gly Asp Val Arg Tyr Arg Phe Thr Leu Val 340 345 350 Gly Tyr Asp Lys Glu Phe Ser Asp 355 360 381083DNASaccharomyces cerevisiae 38ctagtctgaa aattctttgt cgtagccgac taaggtaaat ctatatctaa cgtcaccctt 60ttccatcctt tcgaaggctt catggacgcc ggcttcacca acaggtaatg tttccaccca 120aattttgata tctttttcag agactaattt caagagttgg ttcaattctt tgatggaacc 180taaagcactg taagaaatgg agacagcctt taagccatat ggctttagcg ataacatttc 240gtgttgttct ggtatagaga ttgagacaat tctaccacca accttcatag cctttggcat 300aatgttgaag tcaatgtcgg taagggagga agcacagact acaatcaggt cgaaggtgtc 360aaagtacttt tcaccccaat caccttcttc taatgtagca atgtagtgat cggcgcccat 420cttcattgca tcttctcttt ttctcgaaga acgagaaata acatacgtct ctgcccccat 480ggctttggaa atcaatgtac ccatactgcc gataccacca agaccaacta taccaacttt 540tttacctgga ccgcaaccgt tacgaaccaa tggagagtac acagtcaaac caccacataa 600tagtggagca gccaaatgtg atggaatatt ctctgggata ggcaccacaa aatgttcatg 660aactctgacg tagtttgcat agccaccctg cgacacatag ccgtcttcat aaggctgact 720gtatgtggta acaaacttgg tgcagtatgg ttcattatca ttcttacaac ggtcacattc 780caagcatgaa aagacttgag cacctacacc aacacgttga ccgactttca acccactgtt 840tgacttgggc cctagcttga caactttacc aacgatttca tgaccaacga ctagcggcat 900cttcatattg ccccaatgac cagctgcaca atgaatatca ctaccgcaga caccacatgc 960ttcgatctta atgtcaatgt catgatcgta aaatggtttt gggtcatact ttgtcttctt 1020tgggtttttc caatcttcgt gtgattgaat agcgatacct tcaaatttct caggataaga 1080cat 108339387PRTEscherichia coli 39Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys 1 5 10 15 Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val 20 25 30 Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp 35 40 45 Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly 50 55 60 Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu 65 70 75 80 Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser 85 90 95 Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu 100 105 110 Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys 115 120 125 Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser 130 135 140 Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys 145 150 155 160 Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp 165 170 175 Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val 180 185 190 Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val 195 200 205 Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu 210 215 220 Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val 225 230 235 240 Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile 245 250 255 Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu 260 265 270 Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val 275 280 285 Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu 290 295 300 Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp 305 310 315 320 Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln 325 330 335 Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser 340 345 350 Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu 355 360 365 Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu 370 375 380 Ala Ala Arg 385 40387PRTEscherichia coli 40Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys 1 5 10 15 Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val 20 25 30 Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp 35 40 45 Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly 50 55 60 Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu 65 70 75 80 Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser 85 90 95 Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu 100 105 110 Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys 115 120 125 Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser 130 135 140 Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys 145 150 155 160 Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp 165 170 175 Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val 180 185 190 Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val 195 200 205 Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu 210 215 220 Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val 225 230 235 240 Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile 245 250 255 Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu 260 265 270 Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val 275 280 285 Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu 290 295 300 Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp 305 310 315 320 Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln 325 330 335 Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser 340 345 350 Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu 355 360 365 Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu 370 375 380 Ala Ala Arg 385 41389PRTClostridium acetobutylicum 41Met Leu Ser Phe Asp Tyr Ser Ile Pro Thr Lys Val Phe Phe Gly Lys 1 5 10 15 Gly Lys Ile Asp Val Ile Gly Glu Glu Ile Lys Lys Tyr Gly Ser Arg 20 25 30 Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr 35 40 45 Asp Arg Ala Thr Ala Ile Leu Lys Glu Asn Asn Ile Ala Phe Tyr Glu 50 55 60 Leu Ser Gly Val Glu Pro Asn Pro Arg Ile Thr Thr Val Lys Lys Gly 65 70 75 80 Ile Glu Ile Cys Arg Glu Asn Asn Val Asp Leu Val Leu Ala Ile Gly 85 90 95 Gly Gly Ser Ala Ile Asp Cys Ser Lys Val Ile Ala Ala Gly Val Tyr 100 105 110 Tyr Asp Gly Asp Thr Trp Asp Met Val Lys Asp Pro Ser Lys Ile Thr 115 120 125 Lys Val Leu Pro Ile Ala Ser Ile Leu Thr Leu Ser Ala Thr Gly Ser 130 135 140 Glu Met Asp Gln Ile Ala Val Ile Ser Asn Met Glu Thr Asn Glu Lys 145 150 155 160 Leu Gly Val Gly His Asp Asp Met Arg Pro Lys Phe Ser Val Leu Asp 165 170 175 Pro Thr Tyr Thr Phe Thr Val Pro Lys Asn Gln Thr Ala Ala Gly Thr 180 185 190 Ala Asp Ile Met Ser His Thr Phe Glu Ser Tyr Phe Ser Gly Val Glu 195 200 205 Gly Ala Tyr Val Gln Asp Gly Ile Ala Glu Ala Ile Leu Arg Thr Cys 210 215 220 Ile Lys Tyr Gly Lys Ile Ala Met Glu Lys Thr Asp Asp Tyr Glu Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu 245 250 255 Ser Leu Gly Lys Asp Arg Lys Trp Ser Cys His Pro Met Glu His Glu 260 265 270 Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu 275 280 285 Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asp Asp Thr Leu His Lys 290 295 300 Phe Val Ser Tyr Gly Ile Asn Val Trp Gly Ile Asp Lys Asn Lys Asp 305 310 315 320 Asn Tyr Glu Ile Ala Arg Glu Ala Ile Lys Asn Thr Arg Glu Tyr Phe 325 330 335 Asn Ser Leu Gly Ile Pro Ser Lys Leu Arg Glu Val Gly Ile Gly Lys 340 345 350 Asp Lys Leu Glu Leu Met Ala Lys Gln Ala Val Arg Asn Ser Gly Gly 355 360 365 Thr Ile Gly Ser Leu Arg Pro Ile Asn Ala Glu Asp Val Leu Glu Ile 370 375 380 Phe Lys Lys Ser Tyr 385 421170DNAClostridium acetobutylicum 42ttaataagat tttttaaata tctcaagaac atcctctgca tttattggtc ttaaacttcc 60tattgttcct ccagaatttc taacagcttg ctttgccatt agttctagtt tatcttttcc 120tattccaact tctctaagct ttgaaggaat acccaatgaa ttaaagtatt ctctcgtatt 180tttaatagcc tctcgtgcta tttcatagtt atctttgttc ttgtctattc cccaaacatt 240tattccataa gaaacaaatt tatgaagtgt atcgtcattt agaatatatt ccatccaatt 300aggtgttaaa attgcaagtc ctacaccatg tgttatatca taatatgcac ttaactcgtg 360ttccatagga tgacaactcc attttctatc cttaccaagt gataatagac catttatagc 420taaacttgaa gcccacatca aattagctct agcctcgtaa tcatcagtct tctccattgc 480tatttttcca tactttatac atgttcttaa gattgcttct gctataccgt cctgcacata 540agcaccttca acaccactaa agtaagattc aaaggtgtga ctcataatgt cagctgttcc 600cgctgctgtt tgatttttag gtactgtaaa agtatatgta ggatctaaca ctgaaaattt 660aggtctcata tcatcatgtc ctactccaag cttttcatta gtctccatat ttgaaattac 720tgcaatttga tccatttcag accctgttgc tgaaagagta agtatacttg caattggaag 780aactttagtt attttagatg gatctttaac catgtcccat gtatcgccat cataataaac 840tccagctgca attaccttag aacagtctat tgcacttcct ccccctattg ctaatactaa 900atccacatta ttttctctac atatttctat gccttttttt actgttgtta tcctaggatt 960tggctctact cctgaaagtt catagaaagc tatattgttt tcttttaata tagctgttgc 1020tctatcatat ataccgttcc tttttatact tcctccgcca taaactataa gcactcttga 1080gccatatttc ttaatttctt ctccaattac gtctattttt ccttttccaa aaaaaacttt 1140agttggtatt gaataatcaa aacttagcat 117043390PRTClostridium acetobutylicum 43Met Val Asp Phe Glu Tyr Ser Ile Pro Thr Arg Ile Phe Phe Gly Lys 1 5 10 15 Asp Lys Ile Asn Val Leu Gly Arg Glu Leu Lys Lys Tyr Gly Ser Lys 20 25 30 Val Leu Ile Val Tyr Gly Gly Gly Ser Ile Lys Arg Asn Gly Ile Tyr 35 40 45 Asp Lys Ala Val Ser Ile Leu Glu Lys Asn Ser Ile Lys Phe Tyr Glu 50 55 60 Leu Ala Gly Val Glu Pro Asn Pro Arg Val Thr Thr Val Glu Lys Gly 65 70 75 80 Val Lys Ile Cys Arg Glu Asn Gly Val Glu Val Val Leu Ala Ile Gly 85 90 95 Gly Gly Ser Ala Ile Asp Cys Ala Lys Val Ile Ala Ala Ala Cys Glu 100 105 110 Tyr Asp Gly Asn Pro Trp Asp Ile Val Leu Asp Gly Ser Lys Ile Lys 115 120 125 Arg Val Leu Pro Ile Ala Ser Ile Leu Thr Ile Ala Ala Thr Gly Ser 130 135 140 Glu Met Asp Thr Trp Ala Val Ile Asn Asn Met Asp Thr Asn Glu Lys 145 150 155 160 Leu Ile Ala Ala His Pro Asp Met Ala Pro Lys Phe Ser Ile Leu Asp 165 170 175 Pro Thr Tyr Thr Tyr Thr Val Pro Thr Asn Gln Thr Ala Ala Gly Thr 180 185 190 Ala Asp Ile Met Ser His Ile Phe Glu Val Tyr Phe Ser Asn Thr Lys 195 200 205 Thr Ala Tyr Leu Gln Asp Arg Met Ala Glu Ala Leu Leu Arg Thr Cys 210 215 220 Ile Lys Tyr Gly Gly Ile Ala Leu Glu Lys Pro Asp Asp Tyr Glu Ala 225 230 235 240 Arg Ala Asn Leu Met Trp Ala Ser Ser Leu Ala Ile Asn Gly Leu Leu 245 250 255 Thr Tyr Gly Lys Asp Thr Asn Trp Ser Val His Leu Met Glu His Glu 260 265 270 Leu Ser Ala Tyr Tyr Asp Ile Thr His Gly Val Gly Leu Ala Ile Leu 275 280 285 Thr Pro Asn Trp Met Glu Tyr Ile Leu Asn Asn Asp Thr Val Tyr Lys 290 295 300 Phe Val Glu Tyr Gly Val Asn Val Trp Gly Ile Asp Lys Glu Lys Asn 305 310 315 320 His Tyr Asp Ile Ala His Gln Ala Ile Gln Lys Thr Arg Asp Tyr Phe 325 330 335 Val Asn Val Leu Gly Leu Pro Ser Arg Leu Arg Asp Val Gly Ile Glu 340 345 350 Glu Glu Lys Leu Asp Ile Met Ala Lys Glu Ser Val Lys Leu Thr Gly 355 360 365 Gly Thr Ile Gly Asn Leu Arg Pro Val Asn Ala Ser Glu Val Leu Gln 370 375 380 Ile Phe Lys Lys Ser Val 385 390 441173DNAClostridium acetobutylicum 44gtggttgatt tcgaatattc aataccaact agaatttttt tcggtaaaga taagataaat 60gtacttggaa gagagcttaa aaaatatggt tctaaagtgc ttatagttta tggtggagga 120agtataaaga gaaatggaat atatgataaa gctgtaagta tacttgaaaa aaacagtatt 180aaattttatg aacttgcagg agtagagcca aatccaagag taactacagt tgaaaaagga 240gttaaaatat gtagagaaaa tggagttgaa gtagtactag ctataggtgg aggaagtgca 300atagattgcg caaaggttat agcagcagca tgtgaatatg atggaaatcc atgggatatt 360gtgttagatg gctcaaaaat aaaaagggtg cttcctatag ctagtatatt aaccattgct 420gcaacaggat cagaaatgga tacgtgggca gtaataaata atatggatac aaacgaaaaa 480ctaattgcgg cacatccaga tatggctcct aagttttcta tattagatcc aacgtatacg 540tataccgtac ctaccaatca aacagcagca ggaacagctg atattatgag tcatatattt 600gaggtgtatt ttagtaatac aaaaacagca tatttgcagg atagaatggc agaagcgtta 660ttaagaactt gtattaaata tggaggaata gctcttgaga agccggatga ttatgaggca 720agagccaatc taatgtgggc ttcaagtctt gcgataaatg gacttttaac atatggtaaa 780gacactaatt ggagtgtaca cttaatggaa catgaattaa gtgcttatta cgacataaca 840cacggcgtag ggcttgcaat tttaacacct aattggatgg agtatatttt aaataatgat 900acagtgtaca agtttgttga atatggtgta aatgtttggg gaatagacaa agaaaaaaat 960cactatgaca tagcacatca agcaatacaa aaaacaagag attactttgt aaatgtacta 1020ggtttaccat ctagactgag agatgttgga attgaagaag aaaaattgga cataatggca 1080aaggaatcag taaagcttac aggaggaacc ataggaaacc taagaccagt aaacgcctcc 1140gaagtcctac aaatattcaa aaaatctgtg taa 117345330PRTBacillus subtilis 45Met Ser Thr Asn Arg His Gln Ala Leu Gly Leu Thr Asp Gln Glu Ala 1 5 10 15 Val Asp Met Tyr Arg Thr Met Leu Leu Ala Arg Lys Ile Asp Glu Arg 20 25 30 Met Trp Leu Leu Asn Arg Ser Gly Lys Ile Pro Phe Val Ile Ser Cys 35 40 45 Gln Gly Gln Glu Ala Ala Gln Val Gly Ala Ala Phe Ala Leu Asp Arg 50 55 60 Glu Met Asp Tyr Val Leu Pro Tyr Tyr Arg Asp Met Gly Val Val Leu 65 70 75 80 Ala Phe Gly Met Thr Ala Lys Asp Leu Met Met Ser Gly Phe Ala Lys 85 90 95 Ala Ala Asp Pro Asn Ser Gly Gly Arg Gln Met Pro Gly His Phe Gly 100 105 110 Gln Lys Lys Asn Arg Ile Val Thr Gly

Ser Ser Pro Val Thr Thr Gln 115 120 125 Val Pro His Ala Val Gly Ile Ala Leu Ala Gly Arg Met Glu Lys Lys 130 135 140 Asp Ile Ala Ala Phe Val Thr Phe Gly Glu Gly Ser Ser Asn Gln Gly 145 150 155 160 Asp Phe His Glu Gly Ala Asn Phe Ala Ala Val His Lys Leu Pro Val 165 170 175 Ile Phe Met Cys Glu Asn Asn Lys Tyr Ala Ile Ser Val Pro Tyr Asp 180 185 190 Lys Gln Val Ala Cys Glu Asn Ile Ser Asp Arg Ala Ile Gly Tyr Gly 195 200 205 Met Pro Gly Val Thr Val Asn Gly Asn Asp Pro Leu Glu Val Tyr Gln 210 215 220 Ala Val Lys Glu Ala Arg Glu Arg Ala Arg Arg Gly Glu Gly Pro Thr 225 230 235 240 Leu Ile Glu Thr Ile Ser Tyr Arg Leu Thr Pro His Ser Ser Asp Asp 245 250 255 Asp Asp Ser Ser Tyr Arg Gly Arg Glu Glu Val Glu Glu Ala Lys Lys 260 265 270 Ser Asp Pro Leu Leu Thr Tyr Gln Ala Tyr Leu Lys Glu Thr Gly Leu 275 280 285 Leu Ser Asp Glu Ile Glu Gln Thr Met Leu Asp Glu Ile Met Ala Ile 290 295 300 Val Asn Glu Ala Thr Asp Glu Ala Glu Asn Ala Pro Tyr Ala Ala Pro 305 310 315 320 Glu Ser Ala Leu Asp Tyr Val Tyr Ala Lys 325 330 46993DNABacillus subtilis 46atgagtacaa accgacatca agcactaggg ctgactgatc aggaagccgt tgatatgtat 60agaaccatgc tgttagcaag aaaaatcgat gaaagaatgt ggctgttaaa ccgttctggc 120aaaattccat ttgtaatctc ttgtcaagga caggaagcag cacaggtagg agcggctttc 180gcacttgacc gtgaaatgga ttatgtattg ccgtactaca gagacatggg tgtcgtgctc 240gcgtttggca tgacagcaaa ggacttaatg atgtccgggt ttgcaaaagc agcagatccg 300aactcaggag gccgccagat gccgggacat ttcggacaaa agaaaaaccg cattgtgacg 360ggatcatctc cggttacaac gcaagtgccg cacgcagtcg gtattgcgct tgcgggacgt 420atggagaaaa aggatatcgc agcctttgtt acattcgggg aagggtcttc aaaccaaggc 480gatttccatg aaggggcaaa ctttgccgct gtccataagc tgccggttat tttcatgtgt 540gaaaacaaca aatacgcaat ctcagtgcct tacgataagc aagtcgcatg tgagaacatt 600tccgaccgtg ccataggcta tgggatgcct ggcgtaactg tgaatggaaa tgatccgctg 660gaagtttatc aagcggttaa agaagcacgc gaaagggcac gcagaggaga aggcccgaca 720ttaattgaaa cgatttctta ccgccttaca ccacattcca gtgatgacga tgacagcagc 780tacagaggcc gtgaagaagt agaggaagcg aaaaaaagtg atcccctgct tacttatcaa 840gcttacttaa aggaaacagg cctgctgtcc gatgagatag aacaaaccat gctggatgaa 900attatggcaa tcgtaaatga agcgacggat gaagcggaga acgccccata tgcagctcct 960gagtcagcgc ttgattatgt ttatgcgaag tag 99347327PRTBacillus subtilis 47Met Ser Val Met Ser Tyr Ile Asp Ala Ile Asn Leu Ala Met Lys Glu 1 5 10 15 Glu Met Glu Arg Asp Ser Arg Val Phe Val Leu Gly Glu Asp Val Gly 20 25 30 Arg Lys Gly Gly Val Phe Lys Ala Thr Ala Gly Leu Tyr Glu Gln Phe 35 40 45 Gly Glu Glu Arg Val Met Asp Thr Pro Leu Ala Glu Ser Ala Ile Ala 50 55 60 Gly Val Gly Ile Gly Ala Ala Met Tyr Gly Met Arg Pro Ile Ala Glu 65 70 75 80 Met Gln Phe Ala Asp Phe Ile Met Pro Ala Val Asn Gln Ile Ile Ser 85 90 95 Glu Ala Ala Lys Ile Arg Tyr Arg Ser Asn Asn Asp Trp Ser Cys Pro 100 105 110 Ile Val Val Arg Ala Pro Tyr Gly Gly Gly Val His Gly Ala Leu Tyr 115 120 125 His Ser Gln Ser Val Glu Ala Ile Phe Ala Asn Gln Pro Gly Leu Lys 130 135 140 Ile Val Met Pro Ser Thr Pro Tyr Asp Ala Lys Gly Leu Leu Lys Ala 145 150 155 160 Ala Val Arg Asp Glu Asp Pro Val Leu Phe Phe Glu His Lys Arg Ala 165 170 175 Tyr Arg Leu Ile Lys Gly Glu Val Pro Ala Asp Asp Tyr Val Leu Pro 180 185 190 Ile Gly Lys Ala Asp Val Lys Arg Glu Gly Asp Asp Ile Thr Val Ile 195 200 205 Thr Tyr Gly Leu Cys Val His Phe Ala Leu Gln Ala Ala Glu Arg Leu 210 215 220 Glu Lys Asp Gly Ile Ser Ala His Val Val Asp Leu Arg Thr Val Tyr 225 230 235 240 Pro Leu Asp Lys Glu Ala Ile Ile Glu Ala Ala Ser Lys Thr Gly Lys 245 250 255 Val Leu Leu Val Thr Glu Asp Thr Lys Glu Gly Ser Ile Met Ser Glu 260 265 270 Val Ala Ala Ile Ile Ser Glu His Cys Leu Phe Asp Leu Asp Ala Pro 275 280 285 Ile Lys Arg Leu Ala Gly Pro Asp Ile Pro Ala Met Pro Tyr Ala Pro 290 295 300 Thr Met Glu Lys Tyr Phe Met Val Asn Pro Asp Lys Val Glu Ala Ala 305 310 315 320 Met Arg Glu Leu Ala Glu Phe 325 48984DNABacillus subtilis 48atgtcagtaa tgtcatatat tgatgcaatc aatttggcga tgaaagaaga aatggaacga 60gattctcgcg ttttcgtcct tggggaagat gtaggaagaa aaggcggtgt gtttaaagcg 120acagcgggac tctatgaaca atttggggaa gagcgcgtta tggatacgcc gcttgctgaa 180tctgcaatcg caggagtcgg tatcggagcg gcaatgtacg gaatgagacc gattgctgaa 240atgcagtttg ctgatttcat tatgccggca gtcaaccaaa ttatttctga agcggctaaa 300atccgctacc gcagcaacaa tgactggagc tgtccgattg tcgtcagagc gccatacggc 360ggaggcgtgc acggagccct gtatcattct caatcagtcg aagcaatttt cgccaaccag 420cccggactga aaattgtcat gccatcaaca ccatatgacg cgaaagggct cttaaaagcc 480gcagttcgtg acgaagaccc cgtgctgttt tttgagcaca agcgggcata ccgtctgata 540aagggcgagg ttccggctga tgattatgtc ctgccaatcg gcaaggcgga cgtaaaaagg 600gaaggcgacg acatcacagt gatcacatac ggcctgtgtg tccacttcgc cttacaagct 660gcagaacgtc tcgaaaaaga tggcatttca gcgcatgtgg tggatttaag aacagtttac 720ccgcttgata aagaagccat catcgaagct gcgtccaaaa ctggaaaggt tcttttggtc 780acagaagata caaaagaagg cagcatcatg agcgaagtag ccgcaattat atccgagcat 840tgtctgttcg acttagacgc gccgatcaaa cggcttgcag gtcctgatat tccggctatg 900ccttatgcgc cgacaatgga aaaatacttt atggtcaacc ctgataaagt ggaagcggcg 960atgagagaat tagcggagtt ttaa 98449424PRTBacillus subtilis 49Met Ala Ile Glu Gln Met Thr Met Pro Gln Leu Gly Glu Ser Val Thr 1 5 10 15 Glu Gly Thr Ile Ser Lys Trp Leu Val Ala Pro Gly Asp Lys Val Asn 20 25 30 Lys Tyr Asp Pro Ile Ala Glu Val Met Thr Asp Lys Val Asn Ala Glu 35 40 45 Val Pro Ser Ser Phe Thr Gly Thr Ile Thr Glu Leu Val Gly Glu Glu 50 55 60 Gly Gln Thr Leu Gln Val Gly Glu Met Ile Cys Lys Ile Glu Thr Glu 65 70 75 80 Gly Ala Asn Pro Ala Glu Gln Lys Gln Glu Gln Pro Ala Ala Ser Glu 85 90 95 Ala Ala Glu Asn Pro Val Ala Lys Ser Ala Gly Ala Ala Asp Gln Pro 100 105 110 Asn Lys Lys Arg Tyr Ser Pro Ala Val Leu Arg Leu Ala Gly Glu His 115 120 125 Gly Ile Asp Leu Asp Gln Val Thr Gly Thr Gly Ala Gly Gly Arg Ile 130 135 140 Thr Arg Lys Asp Ile Gln Arg Leu Ile Glu Thr Gly Gly Val Gln Glu 145 150 155 160 Gln Asn Pro Glu Glu Leu Lys Thr Ala Ala Pro Ala Pro Lys Ser Ala 165 170 175 Ser Lys Pro Glu Pro Lys Glu Glu Thr Ser Tyr Pro Ala Ser Ala Ala 180 185 190 Gly Asp Lys Glu Ile Pro Val Thr Gly Val Arg Lys Ala Ile Ala Ser 195 200 205 Asn Met Lys Arg Ser Lys Thr Glu Ile Pro His Ala Trp Thr Met Met 210 215 220 Glu Val Asp Val Thr Asn Met Val Ala Tyr Arg Asn Ser Ile Lys Asp 225 230 235 240 Ser Phe Lys Lys Thr Glu Gly Phe Asn Leu Thr Phe Phe Ala Phe Phe 245 250 255 Val Lys Ala Val Ala Gln Ala Leu Lys Glu Phe Pro Gln Met Asn Ser 260 265 270 Met Trp Ala Gly Asp Lys Ile Ile Gln Lys Lys Asp Ile Asn Ile Ser 275 280 285 Ile Ala Val Ala Thr Glu Asp Ser Leu Phe Val Pro Val Ile Lys Asn 290 295 300 Ala Asp Glu Lys Thr Ile Lys Gly Ile Ala Lys Asp Ile Thr Gly Leu 305 310 315 320 Ala Lys Lys Val Arg Asp Gly Lys Leu Thr Ala Asp Asp Met Gln Gly 325 330 335 Gly Thr Phe Thr Val Asn Asn Thr Gly Ser Phe Gly Ser Val Gln Ser 340 345 350 Met Gly Ile Ile Asn Tyr Pro Gln Ala Ala Ile Leu Gln Val Glu Ser 355 360 365 Ile Val Lys Arg Pro Val Val Met Asp Asn Gly Met Ile Ala Val Arg 370 375 380 Asp Met Val Asn Leu Cys Leu Ser Leu Asp His Arg Val Leu Asp Gly 385 390 395 400 Leu Val Cys Gly Arg Phe Leu Gly Arg Val Lys Gln Ile Leu Glu Ser 405 410 415 Ile Asp Glu Lys Thr Ser Val Tyr 420 501275DNABacillus subtilis 50atggcaattg aacaaatgac gatgccgcag cttggagaaa gcgtaacaga ggggacgatc 60agcaaatggc ttgtcgcccc cggtgataaa gtgaacaaat acgatccgat cgcggaagtc 120atgacagata aggtaaatgc agaggttccg tcttctttta ctggtacgat aacagagctt 180gtgggagaag aaggccaaac cctgcaagtc ggagaaatga tttgcaaaat tgaaacagaa 240ggcgcgaatc cggctgaaca aaaacaagaa cagccagcag catcagaagc cgctgagaac 300cctgttgcaa aaagtgctgg agcagccgat cagcccaata aaaagcgcta ctcgccagct 360gttctccgtt tggccggaga gcacggcatt gacctcgatc aagtgacagg aactggtgcc 420ggcgggcgca tcacacgaaa agatattcag cgcttaattg aaacaggcgg cgtgcaagaa 480cagaatcctg aggagctgaa aacagcagct cctgcaccga agtctgcatc aaaacctgag 540ccaaaagaag agacgtcata tcctgcgtct gcagccggtg ataaagaaat ccctgtcaca 600ggtgtaagaa aagcaattgc ttccaatatg aagcgaagca aaacagaaat tccgcatgct 660tggacgatga tggaagtcga cgtcacaaat atggttgcat atcgcaacag tataaaagat 720tcttttaaga agacagaagg ctttaattta acgttcttcg ccttttttgt aaaagcggtc 780gctcaggcgt taaaagaatt cccgcaaatg aatagcatgt gggcggggga caaaattatt 840cagaaaaagg atatcaatat ttcaattgca gttgccacag aggattcttt atttgttccg 900gtgattaaaa acgctgatga aaaaacaatt aaaggcattg cgaaagacat taccggccta 960gctaaaaaag taagagacgg aaaactcact gcagatgaca tgcagggagg cacgtttacc 1020gtcaacaaca caggttcgtt cgggtctgtt cagtcgatgg gcattatcaa ctaccctcag 1080gctgcgattc ttcaagtaga atccatcgtc aaacgcccgg ttgtcatgga caatggcatg 1140attgctgtca gagacatggt taatctgtgc ctgtcattag atcacagagt gcttgacggt 1200ctcgtgtgcg gacgattcct cggacgagtg aaacaaattt tagaatcgat tgacgagaag 1260acatctgttt actaa 127551474PRTBacillus subtilis 51Met Ala Thr Glu Tyr Asp Val Val Ile Leu Gly Gly Gly Thr Gly Gly 1 5 10 15 Tyr Val Ala Ala Ile Arg Ala Ala Gln Leu Gly Leu Lys Thr Ala Val 20 25 30 Val Glu Lys Glu Lys Leu Gly Gly Thr Cys Leu His Lys Gly Cys Ile 35 40 45 Pro Ser Lys Ala Leu Leu Arg Ser Ala Glu Val Tyr Arg Thr Ala Arg 50 55 60 Glu Ala Asp Gln Phe Gly Val Glu Thr Ala Gly Val Ser Leu Asn Phe 65 70 75 80 Glu Lys Val Gln Gln Arg Lys Gln Ala Val Val Asp Lys Leu Ala Ala 85 90 95 Gly Val Asn His Leu Met Lys Lys Gly Lys Ile Asp Val Tyr Thr Gly 100 105 110 Tyr Gly Arg Ile Leu Gly Pro Ser Ile Phe Ser Pro Leu Pro Gly Thr 115 120 125 Ile Ser Val Glu Arg Gly Asn Gly Glu Glu Asn Asp Met Leu Ile Pro 130 135 140 Lys Gln Val Ile Ile Ala Thr Gly Ser Arg Pro Arg Met Leu Pro Gly 145 150 155 160 Leu Glu Val Asp Gly Lys Ser Val Leu Thr Ser Asp Glu Ala Leu Gln 165 170 175 Met Glu Glu Leu Pro Gln Ser Ile Ile Ile Val Gly Gly Gly Val Ile 180 185 190 Gly Ile Glu Trp Ala Ser Met Leu His Asp Phe Gly Val Lys Val Thr 195 200 205 Val Ile Glu Tyr Ala Asp Arg Ile Leu Pro Thr Glu Asp Leu Glu Ile 210 215 220 Ser Lys Glu Met Glu Ser Leu Leu Lys Lys Lys Gly Ile Gln Phe Ile 225 230 235 240 Thr Gly Ala Lys Val Leu Pro Asp Thr Met Thr Lys Thr Ser Asp Asp 245 250 255 Ile Ser Ile Gln Ala Glu Lys Asp Gly Glu Thr Val Thr Tyr Ser Ala 260 265 270 Glu Lys Met Leu Val Ser Ile Gly Arg Gln Ala Asn Ile Glu Gly Ile 275 280 285 Gly Leu Glu Asn Thr Asp Ile Val Thr Glu Asn Gly Met Ile Ser Val 290 295 300 Asn Glu Ser Cys Gln Thr Lys Glu Ser His Ile Tyr Ala Ile Gly Asp 305 310 315 320 Val Ile Gly Gly Leu Gln Leu Ala His Val Ala Ser His Glu Gly Ile 325 330 335 Ile Ala Val Glu His Phe Ala Gly Leu Asn Pro His Pro Leu Asp Pro 340 345 350 Thr Leu Val Pro Lys Cys Ile Tyr Ser Ser Pro Glu Ala Ala Ser Val 355 360 365 Gly Leu Thr Glu Asp Glu Ala Lys Ala Asn Gly His Asn Val Lys Ile 370 375 380 Gly Lys Phe Pro Phe Met Ala Ile Gly Lys Ala Leu Val Tyr Gly Glu 385 390 395 400 Ser Asp Gly Phe Val Lys Ile Val Ala Asp Arg Asp Thr Asp Asp Ile 405 410 415 Leu Gly Val His Met Ile Gly Pro His Val Thr Asp Met Ile Ser Glu 420 425 430 Ala Gly Leu Ala Lys Val Leu Asp Ala Thr Pro Trp Glu Val Gly Gln 435 440 445 Thr Ile His Pro His Pro Thr Leu Ser Glu Ala Ile Gly Glu Ala Ala 450 455 460 Leu Ala Ala Asp Gly Lys Ala Ile His Phe 465 470 521425DNABacillus subtilis 52atggcaactg agtatgacgt agtcattctg ggcggcggta ccggcggtta tgttgcggcc 60atcagagccg ctcagctcgg cttaaaaaca gccgttgtgg aaaaggaaaa actcggggga 120acatgtctgc ataaaggctg tatcccgagt aaagcgctgc ttagaagcgc agaggtatac 180cggacagctc gtgaagccga tcaattcgga gtggaaacgg ctggcgtgtc cctcaacttt 240gaaaaagtgc agcagcgtaa gcaagccgtt gttgataagc ttgcagcggg tgtaaatcat 300ttaatgaaaa aaggaaaaat tgacgtgtac accggatatg gacgtatcct tggaccgtca 360atcttctctc cgctgccggg aacaatttct gttgagcggg gaaatggcga agaaaatgac 420atgctgatcc cgaaacaagt gatcattgca acaggatcaa gaccgagaat gcttccgggt 480cttgaagtgg acggtaagtc tgtactgact tcagatgagg cgctccaaat ggaggagctg 540ccacagtcaa tcatcattgt cggcggaggg gttatcggta tcgaatgggc gtctatgctt 600catgattttg gcgttaaggt aacggttatt gaatacgcgg atcgcatatt gccgactgaa 660gatctagaga tttcaaaaga aatggaaagt cttcttaaga aaaaaggcat ccagttcata 720acaggggcaa aagtgctgcc tgacacaatg acaaaaacat cagacgatat cagcatacaa 780gcggaaaaag acggagaaac cgttacctat tctgctgaga aaatgcttgt ttccatcggc 840agacaggcaa atatcgaagg catcggccta gagaacaccg atattgttac tgaaaatggc 900atgatttcag tcaatgaaag ctgccaaacg aaggaatctc atatttatgc aatcggagac 960gtaatcggtg gcctgcagtt agctcacgtt gcttcacatg agggaattat tgctgttgag 1020cattttgcag gtctcaatcc gcatccgctt gatccgacgc ttgtgccgaa gtgcatttac 1080tcaagccctg aagctgccag tgtcggctta accgaagacg aagcaaaggc gaacgggcat 1140aatgtcaaaa tcggcaagtt cccatttatg gcgattggaa aagcgcttgt atacggtgaa 1200agcgacggtt ttgtcaaaat cgtggctgac cgagatacag atgatattct cggcgttcat 1260atgattggcc cgcatgtcac cgacatgatt tctgaagcgg gtcttgccaa agtgctggac 1320gcaacaccgt gggaggtcgg gcaaacgatt cacccgcatc caacgctttc tgaagcaatt 1380ggagaagctg cgcttgccgc agatggcaaa gccattcatt tttaa 142553410PRTPseudomonas putida 53Met Asn Glu Tyr Ala Pro Leu Arg Leu His Val Pro Glu Pro Thr Gly 1 5 10 15 Arg Pro Gly Cys Gln Thr Asp Phe Ser Tyr Leu Arg Leu Asn Asp Ala 20 25 30 Gly Gln Ala Arg Lys Pro Pro Val Asp Val Asp Ala Ala Asp Thr Ala 35 40 45 Asp Leu Ser Tyr Ser Leu Val Arg Val Leu Asp Glu Gln Gly Asp Ala 50 55 60 Gln Gly Pro Trp Ala Glu Asp Ile Asp Pro Gln Ile Leu Arg Gln Gly 65 70 75 80 Met Arg Ala Met Leu Lys Thr Arg Ile Phe Asp Ser Arg Met Val Val 85 90 95 Ala Gln Arg Gln Lys Lys Met Ser Phe Tyr

Met Gln Ser Leu Gly Glu 100 105 110 Glu Ala Ile Gly Ser Gly Gln Ala Leu Ala Leu Asn Arg Thr Asp Met 115 120 125 Cys Phe Pro Thr Tyr Arg Gln Gln Ser Ile Leu Met Ala Arg Asp Val 130 135 140 Ser Leu Val Glu Met Ile Cys Gln Leu Leu Ser Asn Glu Arg Asp Pro 145 150 155 160 Leu Lys Gly Arg Gln Leu Pro Ile Met Tyr Ser Val Arg Glu Ala Gly 165 170 175 Phe Phe Thr Ile Ser Gly Asn Leu Ala Thr Gln Phe Val Gln Ala Val 180 185 190 Gly Trp Ala Met Ala Ser Ala Ile Lys Gly Asp Thr Lys Ile Ala Ser 195 200 205 Ala Trp Ile Gly Asp Gly Ala Thr Ala Glu Ser Asp Phe His Thr Ala 210 215 220 Leu Thr Phe Ala His Val Tyr Arg Ala Pro Val Ile Leu Asn Val Val 225 230 235 240 Asn Asn Gln Trp Ala Ile Ser Thr Phe Gln Ala Ile Ala Gly Gly Glu 245 250 255 Ser Thr Thr Phe Ala Gly Arg Gly Val Gly Cys Gly Ile Ala Ser Leu 260 265 270 Arg Val Asp Gly Asn Asp Phe Val Ala Val Tyr Ala Ala Ser Arg Trp 275 280 285 Ala Ala Glu Arg Ala Arg Arg Gly Leu Gly Pro Ser Leu Ile Glu Trp 290 295 300 Val Thr Tyr Arg Ala Gly Pro His Ser Thr Ser Asp Asp Pro Ser Lys 305 310 315 320 Tyr Arg Pro Ala Asp Asp Trp Ser His Phe Pro Leu Gly Asp Pro Ile 325 330 335 Ala Arg Leu Lys Gln His Leu Ile Lys Ile Gly His Trp Ser Glu Glu 340 345 350 Glu His Gln Ala Thr Thr Ala Glu Phe Glu Ala Ala Val Ile Ala Ala 355 360 365 Gln Lys Glu Ala Glu Gln Tyr Gly Thr Leu Ala Asn Gly His Ile Pro 370 375 380 Ser Ala Ala Ser Met Phe Glu Asp Val Tyr Lys Glu Met Pro Asp His 385 390 395 400 Leu Arg Arg Gln Arg Gln Glu Leu Gly Val 405 410 546643DNAPseudomonas putida 54gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 664355352PRTPseudomonas putida 55Met Asn Asp His Asn Asn Ser Ile Asn Pro Glu Thr Ala Met Ala Thr 1 5 10 15 Thr Thr Met Thr Met Ile Gln Ala Leu Arg Ser Ala Met Asp Val Met 20 25 30 Leu Glu Arg Asp Asp Asn Val Val Val Tyr Gly Gln Asp Val Gly Tyr 35 40 45 Phe Gly Gly Val Phe Arg Cys Thr Glu Gly Leu Gln Thr Lys Tyr Gly 50 55 60 Lys Ser Arg Val Phe Asp Ala Pro Ile Ser Glu Ser Gly Ile Val Gly 65 70 75 80 Thr Ala Val Gly Met Gly Ala Tyr Gly Leu Arg Pro Val Val Glu Ile 85 90 95 Gln Phe Ala Asp Tyr Phe Tyr Pro Ala Ser Asp Gln Ile Val Ser Glu 100 105 110 Met Ala Arg Leu Arg Tyr Arg Ser Ala Gly Glu Phe Ile Ala Pro Leu 115 120 125 Thr Leu Arg Met Pro Cys Gly Gly Gly Ile Tyr Gly Gly Gln Thr His 130 135 140 Ser Gln Ser Pro Glu Ala Met Phe Thr Gln Val Cys Gly Leu Arg Thr 145 150 155 160 Val Met Pro Ser Asn Pro Tyr Asp Ala Lys Gly Leu Leu Ile Ala Ser 165 170 175 Ile Glu Cys Asp Asp Pro Val Ile Phe Leu Glu Pro Lys Arg Leu Tyr 180 185 190 Asn Gly Pro Phe Asp Gly His His Asp Arg Pro Val Thr Pro Trp Ser 195 200 205 Lys His Pro His Ser Ala Val Pro Asp Gly Tyr Tyr Thr Val Pro Leu 210 215 220 Asp Lys Ala Ala Ile Thr Arg Pro Gly Asn Asp Val Ser Val Leu Thr 225 230 235 240 Tyr Gly Thr Thr Val Tyr Val Ala Gln Val Ala Ala Glu Glu Ser Gly 245 250 255 Val Asp Ala Glu Val Ile Asp Leu Arg Ser Leu Trp Pro Leu Asp Leu 260 265 270 Asp Thr Ile Val Glu Ser Val Lys Lys Thr Gly Arg Cys Val Val Val 275 280 285 His Glu Ala Thr Arg Thr Cys Gly Phe Gly Ala Glu Leu Val Ser Leu 290 295 300 Val Gln Glu His Cys Phe His His Leu Glu Ala Pro Ile Glu Arg Val 305 310 315 320 Thr Gly Trp Asp Thr Pro Tyr Pro His Ala Gln Glu Trp Ala Tyr Phe 325 330 335 Pro Gly Pro Ser Arg Val Gly Ala Ala Leu Lys Lys Val Met Glu Val 340 345 350 566643DNAPseudomonas putida 56gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag

gtgggcgaca tcatcgccga 4020ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 664357423PRTPseudomonas putida 57Met Gly Thr His Val Ile Lys Met Pro Asp Ile Gly Glu Gly Ile Ala 1 5 10 15 Gln Val Glu Leu Val Glu Trp Phe Val Lys Val Gly Asp Ile Ile Ala 20 25 30 Glu Asp Gln Val Val Ala Asp Val Met Thr Asp Lys Ala Thr Val Glu 35 40 45 Ile Pro Ser Pro Val Ser Gly Lys Val Leu Ala Leu Gly Gly Gln Pro 50 55 60 Gly Glu Val Met Ala Val Gly Ser Glu Leu Ile Arg Ile Glu Val Glu 65 70 75 80 Gly Ser Gly Asn His Val Asp Val Pro Gln Ala Lys Pro Ala Glu Val 85 90 95 Pro Ala Ala Pro Val Ala Ala Lys Pro Glu Pro Gln Lys Asp Val Lys 100 105 110 Pro Ala Ala Tyr Gln Ala Ser Ala Ser His Glu Ala Ala Pro Ile Val 115 120 125 Pro Arg Gln Pro Gly Asp Lys Pro Leu Ala Ser Pro Ala Val Arg Lys 130 135 140 Arg Ala Leu Asp Ala Gly Ile Glu Leu Arg Tyr Val His Gly Ser Gly 145 150 155 160 Pro Ala Gly Arg Ile Leu His Glu Asp Leu Asp Ala Phe Met Ser Lys 165 170 175 Pro Gln Ser Ala Ala Gly Gln Thr Pro Asn Gly Tyr Ala Arg Arg Thr 180 185 190 Asp Ser Glu Gln Val Pro Val Ile Gly Leu Arg Arg Lys Ile Ala Gln 195 200 205 Arg Met Gln Asp Ala Lys Arg Arg Val Ala His Phe Ser Tyr Val Glu 210 215 220 Glu Ile Asp Val Thr Ala Leu Glu Ala Leu Arg Gln Gln Leu Asn Ser 225 230 235 240 Lys His Gly Asp Ser Arg Gly Lys Leu Thr Leu Leu Pro Phe Leu Val 245 250 255 Arg Ala Leu Val Val Ala Leu Arg Asp Phe Pro Gln Ile Asn Ala Thr 260 265 270 Tyr Asp Asp Glu Ala Gln Ile Ile Thr Arg His Gly Ala Val His Val 275 280 285 Gly Ile Ala Thr Gln Gly Asp Asn Gly Leu Met Val Pro Val Leu Arg 290 295 300 His Ala Glu Ala Gly Ser Leu Trp Ala Asn Ala Gly Glu Ile Ser Arg 305 310 315 320 Leu Ala Asn Ala Ala Arg Asn Asn Lys Ala Ser Arg Glu Glu Leu Ser 325 330 335 Gly Ser Thr Ile Thr Leu Thr Ser Leu Gly Ala Leu Gly Gly Ile Val 340 345 350 Ser Thr Pro Val Val Asn Thr Pro Glu Val Ala Ile Val Gly Val Asn 355 360 365 Arg Met Val Glu Arg Pro Val Val Ile Asp Gly Gln Ile Val Val Arg 370 375 380 Lys Met Met Asn Leu Ser Ser Ser Phe Asp His Arg Val Val Asp Gly 385 390 395 400 Met Asp Ala Ala Leu Phe Ile Gln Ala Val Arg Gly Leu Leu Glu Gln 405 410 415 Pro Ala Cys Leu Phe Val Glu 420 586643DNAPseudomonas putida 58gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 664359459PRTPseudomonas putida 59Met Gln Gln Thr Ile Gln Thr Thr Leu Leu Ile Ile Gly Gly Gly Pro 1 5 10 15 Gly Gly Tyr Val Ala Ala Ile Arg Ala Gly Gln Leu Gly Ile Pro Thr 20 25 30 Val Leu Val Glu Gly Gln Ala Leu Gly Gly Thr Cys Leu Asn Ile Gly 35 40 45 Cys Ile Pro Ser Lys Ala Leu Ile His Val Ala Glu Gln Phe His Gln 50 55 60 Ala Ser Arg Phe Thr Glu Pro Ser Pro Leu Gly Ile Ser Val Ala Ser 65 70 75 80 Pro Arg Leu Asp Ile Gly Gln Ser Val Ala Trp Lys Asp Gly Ile Val 85 90 95 Asp Arg Leu Thr Thr Gly Val Ala Ala Leu Leu Lys Lys His Gly Val 100 105 110 Lys Val Val His Gly Trp Ala Lys Val Leu Asp Gly Lys Gln Val Glu 115 120 125 Val Asp Gly Gln Arg Ile Gln Cys Glu His Leu Leu Leu Ala Thr Gly 130 135 140 Ser Ser Ser Val Glu Leu Pro Met Leu Pro Leu Gly Gly Pro Val Ile 145 150 155 160 Ser Ser Thr Glu Ala Leu Ala Pro Lys Ala Leu Pro Gln His Leu Val 165 170 175 Val Val Gly Gly Gly Tyr Ile Gly Leu Glu Leu Gly Ile Ala Tyr Arg 180 185 190 Lys Leu Gly Ala Gln Val Ser Val Val Glu Ala Arg Glu Arg Ile Leu 195 200 205 Pro Thr Tyr Asp Ser Glu Leu Thr Ala Pro Val Ala Glu Ser Leu Lys 210 215 220 Lys Leu Gly Ile Ala Leu His Leu Gly His Ser Val Glu Gly Tyr Glu 225 230 235 240 Asn Gly Cys Leu Leu Ala Asn Asp Gly Lys Gly Gly Gln Leu Arg Leu 245 250 255 Glu Ala Asp Arg Val Leu Val Ala Val Gly Arg Arg Pro Arg Thr Lys 260 265 270 Gly Phe Asn Leu Glu Cys Leu Asp Leu Lys Met Asn Gly Ala Ala Ile 275 280 285 Ala Ile Asp Glu Arg Cys Gln Thr Ser Met His Asn Val Trp Ala Ile 290 295 300 Gly Asp Val Ala Gly Glu Pro Met Leu Ala His Arg Ala Met Ala Gln 305 310 315 320 Gly Glu Met Val Ala Glu Ile Ile Ala Gly Lys Ala Arg Arg Phe Glu 325 330 335 Pro Ala Ala Ile Ala Ala Val Cys Phe Thr Asp Pro Glu Val Val Val 340 345 350 Val Gly Lys Thr Pro Glu Gln Ala Ser Gln Gln Gly Leu Asp Cys Ile 355 360 365 Val Ala Gln Phe Pro Phe Ala Ala Asn Gly Arg Ala Met Ser Leu Glu 370 375 380 Ser Lys Ser Gly Phe Val Arg Val Val Ala Arg Arg Asp Asn His Leu 385 390 395 400 Ile Leu Gly Trp Gln Ala Val Gly Val Ala Val Ser Glu Leu Ser Thr 405 410 415 Ala Phe Ala Gln Ser Leu Glu Met Gly Ala Cys Leu Glu Asp Val Ala 420 425 430 Gly Thr Ile His Ala His Pro Thr Leu Gly Glu Ala Val Gln Glu Ala 435

440 445 Ala Leu Arg Ala Leu Gly His Ala Leu His Ile 450 455 606643DNAPseudomonas putida 60gcatgcctgc aggccgccga tgaaatggtg gaaggtatcg gtaggctggc cctgctcatc 60gctgaacacg ttacgcccgc tgccggtatc gaccaggctc tggtgaatat gcatggaact 120gccaggcgtg cgcgccagcg gtttggccat gcacaccacg gtcagcccgt gcttgagtgc 180cacttccttg agcaggtgtt tgaacaggaa ggtctggtcg gccagcagca gcgggtcgcc 240atgtagcaag ttgatctcga actggctgac gcccatttcg tgcatgaagg tgtcgcgcgg 300caggccgagc gcggccatgc actggtacac ctcattgaag aacgggcgca ggccgttgtt 360ggaactgaca ctgaacgccg aatggcccag ctcgcggcgg ccgtcggtgc ccagcggtgg 420ctggaacggc tgctgcgggt cactgttggg ggcaaacacg aagaactcaa gctcggtcgc 480cactaccggt gccagaccca acgctgcgta gcgggcgatc acggccttca gctggccccg 540ggtggacagt gccgagggcc ggccatccag ttcattggca tcgcagatgg ccagggcgcg 600accgtcatcg ctccagggca agcgatgaac ctggctgggt tccgctacca acgccaggtc 660gccgtcgtcg cagccgtaga atttcgccgg cgggtagccg cccatgatgc attgcagcag 720caccccacgg gccatctgca ggcggcggcc ttcgagaaag ccttcggcgg tcatcacctt 780gccgcgtggg acgccgttga ggtcgggggt gacgcattcg atttcatcga tgccctggag 840ctgagcgatg ctcatgacgc ttgtccttgt tgttgtaggc tgacaacaac ataggctggg 900ggtgtttaaa atatcaagca gcctctcgaa cgcctggggc ctcttctatt cgcgcaaggt 960catgccattg gccggcaacg gcaaggctgt cttgtagcgc acctgtttca aggcaaaact 1020cgagcggata ttcgccacac ccggcaaccg ggtcaggtaa tcgagaaacc gctccagcgc 1080ctggatactc ggcagcagta cccgcaacag gtagtccggg tcgcccgtca tcaggtagca 1140ctccatcacc tcgggccgtt cggcaatttc ttcctcgaag cggtgcagcg actgctctac 1200ctgtttttcc aggctgacat ggatgaacac attcacatcc agccccaacg cctcgggcga 1260caacaaggtc acctgctggc ggatcacccc cagttcttcc atggcccgca cccggttgaa 1320acagggcgtg ggcgacaggt tgaccgagcg tgccagctcg gcgttggtga tgcgggcgtt 1380ttcctgcagg ctgttgagaa tgccgatatc ggtacgatcg agtttgcgca tgagacaaaa 1440tcaccggttt tttgtgttta tgcggaatgt ttatctgccc cgctcggcaa aggcaatcaa 1500cttgagagaa aaattctcct gccggaccac taagatgtag gggacgctga cttaccagtc 1560acaagccggt actcagcggc ggccgcttca gagctcacaa aaacaaatac ccgagcgagc 1620gtaaaaagca tgaacgagta cgcccccctg cgtttgcatg tgcccgagcc caccggccgg 1680ccaggctgcc agaccgattt ttcctacctg cgcctgaacg atgcaggtca agcccgtaaa 1740ccccctgtcg atgtcgacgc tgccgacacc gccgacctgt cctacagcct ggtccgcgtg 1800ctcgacgagc aaggcgacgc ccaaggcccg tgggctgaag acatcgaccc gcagatcctg 1860cgccaaggca tgcgcgccat gctcaagacg cggatcttcg acagccgcat ggtggttgcc 1920cagcgccaga agaagatgtc cttctacatg cagagcctgg gcgaagaagc catcggcagc 1980ggccaggcgc tggcgcttaa ccgcaccgac atgtgcttcc ccacctaccg tcagcaaagc 2040atcctgatgg cccgcgacgt gtcgctggtg gagatgatct gccagttgct gtccaacgaa 2100cgcgaccccc tcaagggccg ccagctgccg atcatgtact cggtacgcga ggccggcttc 2160ttcaccatca gcggcaacct ggcgacccag ttcgtgcagg cggtcggctg ggccatggcc 2220tcggcgatca agggcgatac caagattgcc tcggcctgga tcggcgacgg cgccactgcc 2280gaatcggact tccacaccgc cctcaccttt gcccacgttt accgcgcccc ggtgatcctc 2340aacgtggtca acaaccagtg ggccatctca accttccagg ccatcgccgg tggcgagtcg 2400accaccttcg ccggccgtgg cgtgggctgc ggcatcgctt cgctgcgggt ggacggcaac 2460gacttcgtcg ccgtttacgc cgcttcgcgc tgggctgccg aacgtgcccg ccgtggtttg 2520ggcccgagcc tgatcgagtg ggtcacctac cgtgccggcc cgcactcgac ctcggacgac 2580ccgtccaagt accgccctgc cgatgactgg agccacttcc cgctgggtga cccgatcgcc 2640cgcctgaagc agcacctgat caagatcggc cactggtccg aagaagaaca ccaggccacc 2700acggccgagt tcgaagcggc cgtgattgct gcgcaaaaag aagccgagca gtacggcacc 2760ctggccaacg gtcacatccc gagcgccgcc tcgatgttcg aggacgtgta caaggagatg 2820cccgaccacc tgcgccgcca acgccaggaa ctgggggttt gagatgaacg accacaacaa 2880cagcatcaac ccggaaaccg ccatggccac cactaccatg accatgatcc aggccctgcg 2940ctcggccatg gatgtcatgc ttgagcgcga cgacaatgtg gtggtgtacg gccaggacgt 3000cggctacttc ggcggcgtgt tccgctgcac cgaaggcctg cagaccaagt acggcaagtc 3060ccgcgtgttc gacgcgccca tctctgaaag cggcatcgtc ggcaccgccg tgggcatggg 3120tgcctacggc ctgcgcccgg tggtggaaat ccagttcgct gactacttct acccggcctc 3180cgaccagatc gtttctgaaa tggcccgcct gcgctaccgt tcggccggcg agttcatcgc 3240cccgctgacc ctgcgtatgc cctgcggtgg cggtatctat ggcggccaga cacacagcca 3300gagcccggaa gcgatgttca ctcaggtgtg cggcctgcgc accgtaatgc catccaaccc 3360gtacgacgcc aaaggcctgc tgattgcctc gatcgaatgc gacgacccgg tgatcttcct 3420ggagcccaag cgcctgtaca acggcccgtt cgacggccac catgaccgcc cggttacgcc 3480gtggtcgaaa cacccgcaca gcgccgtgcc cgatggctac tacaccgtgc cactggacaa 3540ggccgccatc acccgccccg gcaatgacgt gagcgtgctc acctatggca ccaccgtgta 3600cgtggcccag gtggccgccg aagaaagtgg cgtggatgcc gaagtgatcg acctgcgcag 3660cctgtggccg ctagacctgg acaccatcgt cgagtcggtg aaaaagaccg gccgttgcgt 3720ggtagtacac gaggccaccc gtacttgtgg ctttggcgca gaactggtgt cgctggtgca 3780ggagcactgc ttccaccacc tggaggcgcc gatcgagcgc gtcaccggtt gggacacccc 3840ctaccctcac gcgcaggaat gggcttactt cccagggcct tcgcgggtag gtgcggcatt 3900gaaaaaggtc atggaggtct gaatgggcac gcacgtcatc aagatgccgg acattggcga 3960aggcatcgcg caggtcgaat tggtggaatg gttcgtcaag gtgggcgaca tcatcgccga 4020ggaccaagtg gtagccgacg tcatgaccga caaggccacc gtggaaatcc cgtcgccggt 4080cagcggcaag gtgctggccc tgggtggcca gccaggtgaa gtgatggcgg tcggcagtga 4140gctgatccgc atcgaagtgg aaggcagcgg caaccatgtg gatgtgccgc aagccaagcc 4200ggccgaagtg cctgcggcac cggtagccgc taaacctgaa ccacagaaag acgttaaacc 4260ggcggcgtac caggcgtcag ccagccacga ggcagcgccc atcgtgccgc gccagccggg 4320cgacaagccg ctggcctcgc cggcggtgcg caaacgcgcc ctcgatgccg gcatcgaatt 4380gcgttatgtg cacggcagcg gcccggccgg gcgcatcctg cacgaagacc tcgacgcgtt 4440catgagcaaa ccgcaaagcg ctgccgggca aacccccaat ggctatgcca ggcgcaccga 4500cagcgagcag gtgccggtga tcggcctgcg ccgcaagatc gcccagcgca tgcaggacgc 4560caagcgccgg gtcgcgcact tcagctatgt ggaagaaatc gacgtcaccg ccctggaagc 4620cctgcgccag cagctcaaca gcaagcacgg cgacagccgc ggcaagctga cactgctgcc 4680gttcctggtg cgcgccctgg tcgtggcact gcgtgacttc ccgcagataa acgccaccta 4740cgatgacgaa gcgcagatca tcacccgcca tggcgcggtg catgtgggca tcgccaccca 4800aggtgacaac ggcctgatgg tacccgtgct gcgccacgcc gaagcgggca gcctgtgggc 4860caatgccggt gagatttcac gcctggccaa cgctgcgcgc aacaacaagg ccagccgcga 4920agagctgtcc ggttcgacca ttaccctgac cagcctcggc gccctgggcg gcatcgtcag 4980cacgccggtg gtcaacaccc cggaagtggc gatcgtcggt gtcaaccgca tggttgagcg 5040gcccgtggtg atcgacggcc agatcgtcgt gcgcaagatg atgaacctgt ccagctcgtt 5100cgaccaccgc gtggtcgatg gcatggacgc cgccctgttc atccaggccg tgcgtggcct 5160gctcgaacaa cccgcctgcc tgttcgtgga gtgagcatgc aacagactat ccagacaacc 5220ctgttgatca tcggcggcgg ccctggcggc tatgtggcgg ccatccgcgc cgggcaactg 5280ggcatcccta ccgtgctggt ggaaggccag gcgctgggcg gtacctgcct gaacatcggc 5340tgcattccgt ccaaggcgct gatccatgtg gccgagcagt tccaccaggc ctcgcgcttt 5400accgaaccct cgccgctggg catcagcgtg gcttcgccac gcctggacat cggccagagc 5460gtggcctgga aagacggcat cgtcgatcgc ctgaccactg gtgtcgccgc cctgctgaaa 5520aagcacgggg tgaaggtggt gcacggctgg gccaaggtgc ttgatggcaa gcaggtcgag 5580gtggatggcc agcgcatcca gtgcgagcac ctgttgctgg ccacgggctc cagcagtgtc 5640gaactgccga tgctgccgtt gggtgggccg gtgatttcct cgaccgaggc cctggcaccg 5700aaagccctgc cgcaacacct ggtggtggtg ggcggtggct acatcggcct ggagctgggt 5760atcgcctacc gcaagctcgg cgcgcaggtc agcgtggtgg aagcgcgcga gcgcatcctg 5820ccgacttacg acagcgaact gaccgccccg gtggccgagt cgctgaaaaa gctgggtatc 5880gccctgcacc ttggccacag cgtcgaaggt tacgaaaatg gctgcctgct ggccaacgat 5940ggcaagggcg gacaactgcg cctggaagcc gaccgggtgc tggtggccgt gggccgccgc 6000ccacgcacca agggcttcaa cctggaatgc ctggacctga agatgaatgg tgccgcgatt 6060gccatcgacg agcgctgcca gaccagcatg cacaacgtct gggccatcgg cgacgtggcc 6120ggcgaaccga tgctggcgca ccgggccatg gcccagggcg agatggtggc cgagatcatc 6180gccggcaagg cacgccgctt cgaacccgct gcgatagccg ccgtgtgctt caccgacccg 6240gaagtggtcg tggtcggcaa gacgccggaa caggccagtc agcaaggcct ggactgcatc 6300gtcgcgcagt tcccgttcgc cgccaacggc cgggccatga gcctggagtc gaaaagcggt 6360ttcgtgcgcg tggtcgcgcg gcgtgacaac cacctgatcc tgggctggca agcggttggc 6420gtggcggttt ccgagctgtc cacggcgttt gcccagtcgc tggagatggg tgcctgcctg 6480gaggatgtgg ccggtaccat ccatgcccac ccgaccctgg gtgaagcggt acaggaagcg 6540gcactgcgtg ccctgggcca cgccctgcat atctgacact gaagcggccg aggccgattt 6600ggcccgccgc gccgagaggc gctgcgggtc ttttttatac ctg 664361468PRTClostridium beijerinckii 61Met Asn Lys Asp Thr Leu Ile Pro Thr Thr Lys Asp Leu Lys Leu Lys 1 5 10 15 Thr Asn Val Glu Asn Ile Asn Leu Lys Asn Tyr Lys Asp Asn Ser Ser 20 25 30 Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Asn Ser Ala Val 35 40 45 His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu 50 55 60 Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Val 65 70 75 80 Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp 85 90 95 Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu 100 105 110 Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val 115 120 125 Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn 130 135 140 Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly 145 150 155 160 Asn Ala Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala 165 170 175 Phe Ala Ile Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro 180 185 190 Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Glu Ser Leu Asp 195 200 205 Ala Ile Ile Lys His Pro Leu Ile Lys Leu Leu Cys Gly Thr Gly Gly 210 215 220 Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly 225 230 235 240 Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile 245 250 255 Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn 260 265 270 Leu Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala 275 280 285 Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn 290 295 300 Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn 305 310 315 320 Glu Thr Gln Glu Tyr Phe Ile Asn Lys Lys Trp Val Gly Lys Asp Ala 325 330 335 Lys Leu Phe Ser Asp Glu Ile Asp Val Glu Ser Pro Ser Asn Ile Lys 340 345 350 Cys Ile Val Cys Glu Val Asn Ala Asn His Pro Phe Val Met Thr Glu 355 360 365 Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu 370 375 380 Ala Val Lys Tyr Thr Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala 385 390 395 400 Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu 405 410 415 Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val 420 425 430 Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr 435 440 445 Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys 450 455 460 Val Leu Ala Gly 465 626558DNAClostridium beijerinckii 62aagcttaaaa tatccatagg ctattgttaa taagactata gcgcttaata ctctaagcgc 60accatctaaa aaattataca taggaattgg ataaactcca atcttctcca taatactttt 120cataggtgaa attgatatta taattaaggc tgcataaagc aaactcatat ctccaataaa 180tgtcactatc ataggtaatt tccttttcat gttcacatac tcccccgtat tctataataa 240tttacaataa acttccatca caaataaatt ataacatata ttgtaaatat agttttatta 300ttcgcatatt tatagataaa caatatataa cttagactta tgcaataacc taccgaaagt 360aaaaacattg ttatttcaca ggactatgaa aatttcgctg aaagtactaa attgcaggtt 420gcaccactaa tgcttgctcc caatttcatt gtgacaagca ttagttgaac aacctacaat 480taagagcctt taacagctca tttccaatgc ctgctacata aaaatgtttc tactttctaa 540tatggtattt acttcaaagt gtaaatcaaa ttcttaagtt gtttatctat atagggttcc 600atattgataa caatacatat ctactgtttc aatttcatga ataccagcga tattgaaatt 660ttgtaagttt gaatatcatt gtgaaaccct atatatcaaa tacaatctca aaattataca 720aaaagatccc aacttcacaa tatgaatttg agatcttcta tcttaacttc ttcaatattt 780ttaagattat ttatactgcg tgcaaatctt ctataagtat caattatcag ctatgtacat 840tgatagtgga cttgtaatct gaacagctta tatatttaca cttttaagtt ctcttccact 900aacccctgct ttgaaacttc tcataaacaa atcgaaagta cctttaatta gctgtgcatt 960ctcaaactca gaatttaact taagtacttc aattgccgct tcgatagtat agagctctcc 1020ttctgaagca ccttttctta aagtatattc tgatttatta attggattta atgaaattct 1080tggaagcttc tttaagtagt cgctctttct tagtatcttc tctgcttctt tccatgtgcc 1140atctaagata ataaatgctg gaattttttc tgaatcttta catttagact ttctttctag 1200aggttcatca tcatccatag gaaataatac acgtatttca taatcatcgc tattaatata 1260ttcaattaat ttttcaggag tctttactct ctcccaaaga attaactcag ttgattctgg 1320attcaccaat ttcaataatc tagcggtatt tgaaggccta ctaaattctc tttctgttga 1380taatatcaat atctttgctt ttgtctctat tttaggcaca atatcgcaga tacaatttat 1440tattggcaac ccacatttat tgcagctctc atataactta gtaatttgct taactttaaa 1500ttcagactcc attttacctc cattattagt tggttagtgt gtcatatctt cttgctatta 1560ctaactgatt ataacatatg tattcaatat atcactccta gttttcaaag cactggcaat 1620acgaattaca aattaatttc tggatttatg tcagtatttc attaataaaa ggtcggactt 1680ttaagatact tgttttagct attgatcata tttattaaag actatgcatt taatgtataa 1740ttataatgaa tattatcaat aatatttatt ttatattaca atcttacagt ctttattcta 1800aatttcactc aaataccaaa cgagctttat tcataaacaa tatataacaa taattccaaa 1860ataatacgat attttatctg taacagccat ataaaaaaaa tatcatatag tcttgtcatt 1920tgataacgtt ttgtcttcct tatatttact ttttcggttt aataggttga ttctgtaaat 1980tttagtgata acatatattt gatgacatta aaaatttaat atttcatata aatttttaat 2040gtctattaat ttttaaatca caaggaggaa tagttcatga ataaagacac actaatacct 2100acaactaaag atttaaaatt aaaaacaaat gttgaaaaca ttaatttaaa gaactacaag 2160gataattctt catgtttcgg agtattcgaa aatgttgaaa atgctataaa cagcgctgta 2220cacgcgcaaa agatattatc ccttcattat acaaaagaac aaagagaaaa aatcataact 2280gagataagaa aggccgcatt agaaaataaa gaggttttag ctaccatgat tctggaagaa 2340acacatatgg gaaggtatga agataaaata ttaaagcatg aattagtagc taaatatact 2400cctggtacag aagatttaac tactactgct tggtcaggtg ataatggtct tacagttgta 2460gaaatgtctc catatggcgt tataggtgca ataactcctt ctacgaatcc aactgaaact 2520gtaatatgta atagcatcgg catgatagct gctggaaatg ctgtagtatt taacggacac 2580ccaggcgcta aaaaatgtgt tgcttttgct attgaaatga taaataaagc aattatttca 2640tgtggcggtc ctgagaattt agtaacaact ataaaaaatc caactatgga atccctagat 2700gcaattatta agcatccttt aataaaactt ctttgcggaa ctggaggtcc aggaatggta 2760aaaaccctct taaattctgg caagaaagct ataggtgctg gtgctggaaa tccaccagtt 2820attgtagatg ataccgctga tatagaaaag gctggtaaga gtatcattga aggctgttct 2880tttgataata atttaccttg tattgcagaa aaagaagtat ttgtttttga gaatgttgca 2940gatgatttaa tatctaacat gctaaaaaat aatgctgtaa ttataaatga agatcaagta 3000tcaaaattaa tagatttagt attacaaaaa aataatgaaa ctcaagaata ctttataaac 3060aaaaaatggg taggaaaaga tgcaaaatta ttctcagatg aaatagatgt tgagtctcct 3120tcaaatatta aatgcatagt ctgcgaagta aatgcaaatc atccatttgt catgacagaa 3180ctcatgatgc caatattacc aattgtaaga gttaaagata tagatgaagc tgttaaatat 3240acaaagatag cagaacaaaa tagaaaacat agtgcctata tttattctaa aaatatagac 3300aacctaaata gatttgaaag agaaattgat actactattt ttgtaaagaa tgctaaatct 3360tttgctggtg ttggttatga agctgaagga tttacaactt tcactattgc tggatctact 3420ggtgaaggca taacctctgc aagaaatttt acaagacaaa gaagatgtgt acttgccggc 3480taacttcttg ctaaatttat acatttattc acataacttt aatatgcaat gttcccacaa 3540aatattaaaa actatttaga agggagatat taaatgaata aattagtaaa attaacagat 3600ttaaagcgca ttttcaaaga tggtatgaca attatggttg ggggtttttt agattgtgga 3660actcctgaaa atattataga tatgctagtt gatttaaata taaaaaatct gactattata 3720agcaatgata cagcttttcc taataaagga ataggaaaac ttattgtaaa tggtcaagtt 3780tctaaagtaa ttgcttcaca tattggaact aatcctgaaa ctgggaaaaa aatgagctct 3840ggtgaactta aagttgagct ttctccacaa ggaacactga tcgaaagaat tcgtgcagct 3900ggatctggac tcggaggtgt attaactcca accggacttg ggactatcgt tgaagaaggt 3960aagaaaaaag ttactatcgg tggcaaagaa tatctattag aacttccttt atccgctgat 4020gtttcattaa taaaaggtag cattgtagat gaatttggaa ataccttcta tagagctgct 4080actaaaaatt tcaatccata tatggcaatg gctgcaaaaa cagttatagt tgaagcagaa 4140aatttagtta aatgtgaaga tttaaaaaga gatgccataa tgactcctgg cgtattagta 4200gattatatcg ttaaggaggc ggcttaattg attgtagata aagttttagc aaaagagata 4260attgccaaaa gagttgcaaa agaactaaaa aaaggccaac tcgtaaacct tggaatagga 4320cttccaactt tagtagctaa ttatgtgcca aaagaaatga acattacttt cgaatcagaa 4380aatggcatgg ttggcatggc acaaatggcc tcatcaggtg aaaatgaccc agatataata 4440aatgctggtg gggaatatgt aacattatta cctcaaggtg cattttttga tagttcaacg 4500tcttttgcac taataagagg aggacatgtt gatgttgctg ttcttggtgc tctagaagtt 4560gatgaagaag gtaatttagc taactggatt gttccaaata aaattgtccc aggtatggga 4620ggcgccatgg atttggcaat aggcgcaaaa aaaataatag tggcaatgca acatacagga 4680aaaggtaaac ctaaaatcgt aaaaaaatgt actctcccac ttactgctaa ggctcaggta 4740gatttaattg ttacagaact ttgtgtaatt gatgtaacaa atgatggttt acttttcaga 4800gaaattcata aagatacaac tattgatgaa ataaaatttt taacagatgc agatttaatt 4860attcccgaca acttaaaaat tatggatatc taaatcattc tattttaaat atataacttt 4920aaaaatctta tgtattaaaa actaagaaaa gaggttgatt attttatgtt agaaagtgaa 4980gtatctaaac aaattacaac tccacttgct gctccagcgt ttcctagagg accatataga 5040tttcacaata gagaatatct aaacattatt tatcgaactg atttagatgc tcttcgaaaa 5100atagtaccag agccacttga attagatgga

gcatatgtta ggtttgagat gatggctatg 5160cctgatacaa ccggactagg ctcatatact gagtgtggtc aagccattcc agtaaaatat 5220aatgaggtta aaggtgacta cttgcatatg atgtacctag ataatgaacc tgctattgct 5280gttggaagag aaagcagtgc ttatcccaaa aagttcggct atccaaagct atttgttgat 5340tcagacgccc tagttggcgc ccttaagtat ggtgcattac cggtagttac tgcgacgatg 5400ggatataagc atgagcccct agatcttaaa gaagcctata ctcaaattgc aagacccaat 5460ttcatgctaa aaatcattca aggttatgat ggtaagccaa gaatttgtga actcatctgt 5520gcagaaaata ctgatataac tatccacggt gcttggactg gaagtgcacg cctacaatta 5580tttagccatg cactagctcc tcttgctgat ttacctgtat tagagatcgt atcagcatct 5640catatcctaa cagatttaac tcttggaaca cctaaggttg tacatgatta tctttcagta 5700aaataaaagc aatatagaat aaccactaca aaagtagtgg ttattctata ttttaaatca 5760aactgtaaaa cttaagtttt atagtaccta ataatatttt actaccagca ttagattagt 5820taaaatacaa agtttgtggt aaaagtattt tagattgcat aatagccttc tatactttta 5880acaatataac caattgctca ccatctgctt agaatatgct tctttaagct ctaaaataca 5940tataaaaaag taggaatttc ttattaaaat tcctacttat attatatata aatttaatcg 6000ttaggtttta ttcgcattgt tcctctttaa tttatctctt ataacatttt attataattg 6060ttcatataat taattcaata tactattata tattttcaag cattaataat tattcagcat 6120ctgtcattac atatgcttcc atactttgac ttcttattaa atcatagcta atccatccat 6180agccattgat tccccagtct ttaccccatg aatttattat ttttacagct tttttactat 6240catcataacc aactacgcaa actgcatgac cacctctatt ttctccatca atctggtcat 6300aaattggatt atcagaattt aaattatcaa aatctggata tactgatatt ccaataacta 6360ctggatttcc agctgctatt tgtgccttta ttgcattata gtcaccatct ggaagttgac 6420tccaactttt tgctttatat ttggctgcat tagccttttg ttcatctgta ggtgtaacct 6480cccaactata ttcactacca tcataaggca tatcagataa tgtagtacaa ccttgttctt 6540ctaataattt aaatgcat 655863862PRTClostridium acetobutylicum 63Met Lys Val Thr Thr Val Lys Glu Leu Asp Glu Lys Leu Lys Val Ile 1 5 10 15 Lys Glu Ala Gln Lys Lys Phe Ser Cys Tyr Ser Gln Glu Met Val Asp 20 25 30 Glu Ile Phe Arg Asn Ala Ala Met Ala Ala Ile Asp Ala Arg Ile Glu 35 40 45 Leu Ala Lys Ala Ala Val Leu Glu Thr Gly Met Gly Leu Val Glu Asp 50 55 60 Lys Val Ile Lys Asn His Phe Ala Gly Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asp Glu Lys Thr Cys Gly Ile Ile Glu Arg Asn Glu Pro Tyr Gly 85 90 95 Ile Thr Lys Ile Ala Glu Pro Ile Gly Val Val Ala Ala Ile Ile Pro 100 105 110 Val Thr Asn Pro Thr Ser Thr Thr Ile Phe Lys Ser Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Gly Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys 130 135 140 Ser Thr Ile Leu Ala Ala Lys Thr Ile Leu Asp Ala Ala Val Lys Ser 145 150 155 160 Gly Ala Pro Glu Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu 165 170 175 Leu Thr Gln Tyr Leu Met Gln Lys Ala Asp Ile Thr Leu Ala Thr Gly 180 185 190 Gly Pro Ser Leu Val Lys Ser Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205 Gly Val Gly Pro Gly Asn Thr Pro Val Ile Ile Asp Glu Ser Ala His 210 215 220 Ile Lys Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Val Ile Val Leu Lys Ser Ile 245 250 255 Tyr Asn Lys Val Lys Asp Glu Phe Gln Glu Arg Gly Ala Tyr Ile Ile 260 265 270 Lys Lys Asn Glu Leu Asp Lys Val Arg Glu Val Ile Phe Lys Asp Gly 275 280 285 Ser Val Asn Pro Lys Ile Val Gly Gln Ser Ala Tyr Thr Ile Ala Ala 290 295 300 Met Ala Gly Ile Lys Val Pro Lys Thr Thr Arg Ile Leu Ile Gly Glu 305 310 315 320 Val Thr Ser Leu Gly Glu Glu Glu Pro Phe Ala His Glu Lys Leu Ser 325 330 335 Pro Val Leu Ala Met Tyr Glu Ala Asp Asn Phe Asp Asp Ala Leu Lys 340 345 350 Lys Ala Val Thr Leu Ile Asn Leu Gly Gly Leu Gly His Thr Ser Gly 355 360 365 Ile Tyr Ala Asp Glu Ile Lys Ala Arg Asp Lys Ile Asp Arg Phe Ser 370 375 380 Ser Ala Met Lys Thr Val Arg Thr Phe Val Asn Ile Pro Thr Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Arg Ile Pro Pro Ser Phe Thr 405 410 415 Leu Gly Cys Gly Phe Trp Gly Gly Asn Ser Val Ser Glu Asn Val Gly 420 425 430 Pro Lys His Leu Leu Asn Ile Lys Thr Val Ala Glu Arg Arg Glu Asn 435 440 445 Met Leu Trp Phe Arg Val Pro His Lys Val Tyr Phe Lys Phe Gly Cys 450 455 460 Leu Gln Phe Ala Leu Lys Asp Leu Lys Asp Leu Lys Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Ser Asp Pro Tyr Asn Leu Asn Tyr Val Asp Ser 485 490 495 Ile Ile Lys Ile Leu Glu His Leu Asp Ile Asp Phe Lys Val Phe Asn 500 505 510 Lys Val Gly Arg Glu Ala Asp Leu Lys Thr Ile Lys Lys Ala Thr Glu 515 520 525 Glu Met Ser Ser Phe Met Pro Asp Thr Ile Ile Ala Leu Gly Gly Thr 530 535 540 Pro Glu Met Ser Ser Ala Lys Leu Met Trp Val Leu Tyr Glu His Pro 545 550 555 560 Glu Val Lys Phe Glu Asp Leu Ala Ile Lys Phe Met Asp Ile Arg Lys 565 570 575 Arg Ile Tyr Thr Phe Pro Lys Leu Gly Lys Lys Ala Met Leu Val Ala 580 585 590 Ile Thr Thr Ser Ala Gly Ser Gly Ser Glu Val Thr Pro Phe Ala Leu 595 600 605 Val Thr Asp Asn Asn Thr Gly Asn Lys Tyr Met Leu Ala Asp Tyr Glu 610 615 620 Met Thr Pro Asn Met Ala Ile Val Asp Ala Glu Leu Met Met Lys Met 625 630 635 640 Pro Lys Gly Leu Thr Ala Tyr Ser Gly Ile Asp Ala Leu Val Asn Ser 645 650 655 Ile Glu Ala Tyr Thr Ser Val Tyr Ala Ser Glu Tyr Thr Asn Gly Leu 660 665 670 Ala Leu Glu Ala Ile Arg Leu Ile Phe Lys Tyr Leu Pro Glu Ala Tyr 675 680 685 Lys Asn Gly Arg Thr Asn Glu Lys Ala Arg Glu Lys Met Ala His Ala 690 695 700 Ser Thr Met Ala Gly Met Ala Ser Ala Asn Ala Phe Leu Gly Leu Cys 705 710 715 720 His Ser Met Ala Ile Lys Leu Ser Ser Glu His Asn Ile Pro Ser Gly 725 730 735 Ile Ala Asn Ala Leu Leu Ile Glu Glu Val Ile Lys Phe Asn Ala Val 740 745 750 Asp Asn Pro Val Lys Gln Ala Pro Cys Pro Gln Tyr Lys Tyr Pro Asn 755 760 765 Thr Ile Phe Arg Tyr Ala Arg Ile Ala Asp Tyr Ile Lys Leu Gly Gly 770 775 780 Asn Thr Asp Glu Glu Lys Val Asp Leu Leu Ile Asn Lys Ile His Glu 785 790 795 800 Leu Lys Lys Ala Leu Asn Ile Pro Thr Ser Ile Lys Asp Ala Gly Val 805 810 815 Leu Glu Glu Asn Phe Tyr Ser Ser Leu Asp Arg Ile Ser Glu Leu Ala 820 825 830 Leu Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Phe Pro Leu Thr Ser 835 840 845 Glu Ile Lys Glu Met Tyr Ile Asn Cys Phe Lys Lys Gln Pro 850 855 860 641665DNAClostridium acetobutylicum 64ttgaagagtg aatacacaat tggaagatat ttgttagacc gtttatcaga gttgggtatt 60cggcatatct ttggtgtacc tggagattac aatctatcct ttttagacta tataatggag 120tacaaaggga tagattgggt tggaaattgc aatgaattga atgctgggta tgctgctgat 180ggatatgcaa gaataaatgg aattggagcc atacttacaa catttggtgt tggagaatta 240agtgccatta acgcaattgc tggggcatac gctgagcaag ttccagttgt taaaattaca 300ggtatcccca cagcaaaagt tagggacaat ggattatatg tacaccacac attaggtgac 360ggaaggtttg atcacttttt tgaaatgttt agagaagtaa cagttgctga ggcattacta 420agcgaagaaa atgcagcaca agaaattgat cgtgttctta tttcatgctg gagacaaaaa 480cgtcctgttc ttataaattt accgattgat gtatatgata aaccaattaa caaaccatta 540aagccattac tcgattatac tatttcaagt aacaaagagg ctgcatgtga atttgttaca 600gaaatagtac ctataataaa tagggcaaaa aagcctgtta ttcttgcaga ttatggagta 660tatcgttacc aagttcaaca tgtgcttaaa aacttggccg aaaaaaccgg atttcctgtg 720gctacactaa gtatgggaaa aggtgttttc aatgaagcac accctcaatt tattggtgtt 780tataatggtg atgtaagttc tccttattta aggcagcgag ttgatgaagc agactgcatt 840attagcgttg gtgtaaaatt gacggattca accacagggg gattttctca tggattttct 900aaaaggaatg taattcacat tgatcctttt tcaataaagg caaaaggtaa aaaatatgca 960cctattacga tgaaagatgc tttaacagaa ttaacaagta aaattgagca tagaaacttt 1020gaggatttag atataaagcc ttacaaatca gataatcaaa agtattttgc aaaagagaag 1080ccaattacac aaaaacgttt ttttgagcgt attgctcact ttataaaaga aaaagatgta 1140ttattagcag aacagggtac atgctttttt ggtgcgtcaa ccatacaact acccaaagat 1200gcaactttta ttggtcaacc tttatgggga tctattggat acacacttcc tgctttatta 1260ggttcacaat tagctgatca aaaaaggcgt aatattcttt taattgggga tggtgcattt 1320caaatgacag cacaagaaat ttcaacaatg cttcgtttac aaatcaaacc tattattttt 1380ttaattaata acgatggtta tacaattgaa cgtgctattc atggtagaga acaagtatat 1440aacaatattc aaatgtggcg atatcataat gttccaaagg ttttaggtcc taaagaatgc 1500agcttaacct ttaaagtaca aagtgaaact gaacttgaaa aggctctttt agtggcagat 1560aaggattgtg aacatttgat ttttatagaa gttgttatgg atcgttatga taaacccgag 1620cctttagaac gtctttcgaa acgttttgca aatcaaaata attag 166565858PRTClostridium acetobutylicum 65Met Lys Val Thr Asn Gln Lys Glu Leu Lys Gln Lys Leu Asn Glu Leu 1 5 10 15 Arg Glu Ala Gln Lys Lys Phe Ala Thr Tyr Thr Gln Glu Gln Val Asp 20 25 30 Lys Ile Phe Lys Gln Cys Ala Ile Ala Ala Ala Lys Glu Arg Ile Asn 35 40 45 Leu Ala Lys Leu Ala Val Glu Glu Thr Gly Ile Gly Leu Val Glu Asp 50 55 60 Lys Ile Ile Lys Asn His Phe Ala Ala Glu Tyr Ile Tyr Asn Lys Tyr 65 70 75 80 Lys Asn Glu Lys Thr Cys Gly Ile Ile Asp His Asp Asp Ser Leu Gly 85 90 95 Ile Thr Lys Val Ala Glu Pro Ile Gly Ile Val Ala Ala Ile Val Pro 100 105 110 Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu 115 120 125 Lys Thr Arg Asn Ala Ile Phe Phe Ser Pro His Pro Arg Ala Lys Lys 130 135 140 Ser Thr Ile Ala Ala Ala Lys Leu Ile Leu Asp Ala Ala Val Lys Ala 145 150 155 160 Gly Ala Pro Lys Asn Ile Ile Gly Trp Ile Asp Glu Pro Ser Ile Glu 165 170 175 Leu Ser Gln Asp Leu Met Ser Glu Ala Asp Ile Ile Leu Ala Thr Gly 180 185 190 Gly Pro Ser Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro Ala Ile 195 200 205 Gly Val Gly Ala Gly Asn Thr Pro Ala Ile Ile Asp Glu Ser Ala Asp 210 215 220 Ile Asp Met Ala Val Ser Ser Ile Ile Leu Ser Lys Thr Tyr Asp Asn 225 230 235 240 Gly Val Ile Cys Ala Ser Glu Gln Ser Ile Leu Val Met Asn Ser Ile 245 250 255 Tyr Glu Lys Val Lys Glu Glu Phe Val Lys Arg Gly Ser Tyr Ile Leu 260 265 270 Asn Gln Asn Glu Ile Ala Lys Ile Lys Glu Thr Met Phe Lys Asn Gly 275 280 285 Ala Ile Asn Ala Asp Ile Val Gly Lys Ser Ala Tyr Ile Ile Ala Lys 290 295 300 Met Ala Gly Ile Glu Val Pro Gln Thr Thr Lys Ile Leu Ile Gly Glu 305 310 315 320 Val Gln Ser Val Glu Lys Ser Glu Leu Phe Ser His Glu Lys Leu Ser 325 330 335 Pro Val Leu Ala Met Tyr Lys Val Lys Asp Phe Asp Glu Ala Leu Lys 340 345 350 Lys Ala Gln Arg Leu Ile Glu Leu Gly Gly Ser Gly His Thr Ser Ser 355 360 365 Leu Tyr Ile Asp Ser Gln Asn Asn Lys Asp Lys Val Lys Glu Phe Gly 370 375 380 Leu Ala Met Lys Thr Ser Arg Thr Phe Ile Asn Met Pro Ser Ser Gln 385 390 395 400 Gly Ala Ser Gly Asp Leu Tyr Asn Phe Ala Ile Ala Pro Ser Phe Thr 405 410 415 Leu Gly Cys Gly Thr Trp Gly Gly Asn Ser Val Ser Gln Asn Val Glu 420 425 430 Pro Lys His Leu Leu Asn Ile Lys Ser Val Ala Glu Arg Arg Glu Asn 435 440 445 Met Leu Trp Phe Lys Val Pro Gln Lys Ile Tyr Phe Lys Tyr Gly Cys 450 455 460 Leu Arg Phe Ala Leu Lys Glu Leu Lys Asp Met Asn Lys Lys Arg Ala 465 470 475 480 Phe Ile Val Thr Asp Lys Asp Leu Phe Lys Leu Gly Tyr Val Asn Lys 485 490 495 Ile Thr Lys Val Leu Asp Glu Ile Asp Ile Lys Tyr Ser Ile Phe Thr 500 505 510 Asp Ile Lys Ser Asp Pro Thr Ile Asp Ser Val Lys Lys Gly Ala Lys 515 520 525 Glu Met Leu Asn Phe Glu Pro Asp Thr Ile Ile Ser Ile Gly Gly Gly 530 535 540 Ser Pro Met Asp Ala Ala Lys Val Met His Leu Leu Tyr Glu Tyr Pro 545 550 555 560 Glu Ala Glu Ile Glu Asn Leu Ala Ile Asn Phe Met Asp Ile Arg Lys 565 570 575 Arg Ile Cys Asn Phe Pro Lys Leu Gly Thr Lys Ala Ile Ser Val Ala 580 585 590 Ile Pro Thr Thr Ala Gly Thr Gly Ser Glu Ala Thr Pro Phe Ala Val 595 600 605 Ile Thr Asn Asp Glu Thr Gly Met Lys Tyr Pro Leu Thr Ser Tyr Glu 610 615 620 Leu Thr Pro Asn Met Ala Ile Ile Asp Thr Glu Leu Met Leu Asn Met 625 630 635 640 Pro Arg Lys Leu Thr Ala Ala Thr Gly Ile Asp Ala Leu Val His Ala 645 650 655 Ile Glu Ala Tyr Val Ser Val Met Ala Thr Asp Tyr Thr Asp Glu Leu 660 665 670 Ala Leu Arg Ala Ile Lys Met Ile Phe Lys Tyr Leu Pro Arg Ala Tyr 675 680 685 Lys Asn Gly Thr Asn Asp Ile Glu Ala Arg Glu Lys Met Ala His Ala 690 695 700 Ser Asn Ile Ala Gly Met Ala Phe Ala Asn Ala Phe Leu Gly Val Cys 705 710 715 720 His Ser Met Ala His Lys Leu Gly Ala Met His His Val Pro His Gly 725 730 735 Ile Ala Cys Ala Val Leu Ile Glu Glu Val Ile Lys Tyr Asn Ala Thr 740 745 750 Asp Cys Pro Thr Lys Gln Thr Ala Phe Pro Gln Tyr Lys Ser Pro Asn 755 760 765 Ala Lys Arg Lys Tyr Ala Glu Ile Ala Glu Tyr Leu Asn Leu Lys Gly 770 775 780 Thr Ser Asp Thr Glu Lys Val Thr Ala Leu Ile Glu Ala Ile Ser Lys 785 790 795 800 Leu Lys Ile Asp Leu Ser Ile Pro Gln Asn Ile Ser Ala Ala Gly Ile 805 810 815 Asn Lys Lys Asp Phe Tyr Asn Thr Leu Asp Lys Met Ser Glu Leu Ala 820 825 830 Phe Asp Asp Gln Cys Thr Thr Ala Asn Pro Arg Tyr Pro Leu Ile Ser 835 840 845 Glu Leu Lys Asp Ile Tyr Ile Lys Ser Phe 850 855 662589DNAClostridium acetobutylicum 66atgaaagtca caacagtaaa ggaattagat gaaaaactca aggtaattaa agaagctcaa 60aaaaaattct cttgttactc gcaagaaatg gttgatgaaa tctttagaaa tgcagcaatg 120gcagcaatcg acgcaaggat agagctagca aaagcagctg ttttggaaac cggtatgggc 180ttagttgaag acaaggttat aaaaaatcat tttgcaggcg aatacatcta taacaaatat 240aaggatgaaa aaacctgcgg tataattgaa cgaaatgaac cctacggaat tacaaaaata 300gcagaaccta taggagttgt agctgctata atccctgtaa caaaccccac atcaacaaca 360atatttaaat ccttaatatc ccttaaaact agaaatggaa ttttcttttc gcctcaccca 420agggcaaaaa aatccacaat actagcagct aaaacaatac ttgatgcagc cgttaagagt 480ggtgccccgg aaaatataat aggttggata gatgaacctt caattgaact aactcaatat 540ttaatgcaaa

aagcagatat aacccttgca actggtggtc cctcactagt taaatctgct 600tattcttccg gaaaaccagc aataggtgtt ggtccgggta acaccccagt aataattgat 660gaatctgctc atataaaaat ggcagtaagt tcaattatat tatccaaaac ctatgataat 720ggtgttatat gtgcttctga acaatctgta atagtcttaa aatccatata taacaaggta 780aaagatgagt tccaagaaag aggagcttat ataataaaga aaaacgaatt ggataaagtc 840cgtgaagtga tttttaaaga tggatccgta aaccctaaaa tagtcggaca gtcagcttat 900actatagcag ctatggctgg cataaaagta cctaaaacca caagaatatt aataggagaa 960gttacctcct taggtgaaga agaacctttt gcccacgaaa aactatctcc tgttttggct 1020atgtatgagg ctgacaattt tgatgatgct ttaaaaaaag cagtaactct aataaactta 1080ggaggcctcg gccatacctc aggaatatat gcagatgaaa taaaagcacg agataaaata 1140gatagattta gtagtgccat gaaaaccgta agaacctttg taaatatccc aacctcacaa 1200ggtgcaagtg gagatctata taattttaga ataccacctt ctttcacgct tggctgcgga 1260ttttggggag gaaattctgt ttccgagaat gttggtccaa aacatctttt gaatattaaa 1320accgtagctg aaaggagaga aaacatgctt tggtttagag ttccacataa agtatatttt 1380aagttcggtt gtcttcaatt tgctttaaaa gatttaaaag atctaaagaa aaaaagagcc 1440tttatagtta ctgatagtga cccctataat ttaaactatg ttgattcaat aataaaaata 1500cttgagcacc tagatattga ttttaaagta tttaataagg ttggaagaga agctgatctt 1560aaaaccataa aaaaagcaac tgaagaaatg tcctccttta tgccagacac tataatagct 1620ttaggtggta cccctgaaat gagctctgca aagctaatgt gggtactata tgaacatcca 1680gaagtaaaat ttgaagatct tgcaataaaa tttatggaca taagaaagag aatatatact 1740ttcccaaaac tcggtaaaaa ggctatgtta gttgcaatta caacttctgc tggttccggt 1800tctgaggtta ctccttttgc tttagtaact gacaataaca ctggaaataa gtacatgtta 1860gcagattatg aaatgacacc aaatatggca attgtagatg cagaacttat gatgaaaatg 1920ccaaagggat taaccgctta ttcaggtata gatgcactag taaatagtat agaagcatac 1980acatccgtat atgcttcaga atacacaaac ggactagcac tagaggcaat acgattaata 2040tttaaatatt tgcctgaggc ttacaaaaac ggaagaacca atgaaaaagc aagagagaaa 2100atggctcacg cttcaactat ggcaggtatg gcatccgcta atgcatttct aggtctatgt 2160cattccatgg caataaaatt aagttcagaa cacaatattc ctagtggcat tgccaatgca 2220ttactaatag aagaagtaat aaaatttaac gcagttgata atcctgtaaa acaagcccct 2280tgcccacaat ataagtatcc aaacaccata tttagatatg ctcgaattgc agattatata 2340aagcttggag gaaatactga tgaggaaaag gtagatctct taattaacaa aatacatgaa 2400ctaaaaaaag ctttaaatat accaacttca ataaaggatg caggtgtttt ggaggaaaac 2460ttctattcct cccttgatag aatatctgaa cttgcactag atgatcaatg cacaggcgct 2520aatcctagat ttcctcttac aagtgagata aaagaaatgt atataaattg ttttaaaaaa 2580caaccttaa 258967307PRTPseudomonas putida 67Met Ser Lys Lys Leu Lys Ala Ala Ile Ile Gly Pro Gly Asn Ile Gly 1 5 10 15 Thr Asp Leu Val Met Lys Met Leu Arg Ser Glu Trp Ile Glu Pro Val 20 25 30 Trp Met Val Gly Ile Asp Pro Asn Ser Asp Gly Leu Lys Arg Ala Arg 35 40 45 Asp Phe Gly Met Lys Thr Thr Ala Glu Gly Val Asp Gly Leu Leu Pro 50 55 60 His Val Leu Asp Asp Asp Ile Arg Ile Ala Phe Asp Ala Thr Ser Ala 65 70 75 80 Tyr Val His Ala Glu Asn Ser Arg Lys Leu Asn Ala Leu Gly Val Leu 85 90 95 Met Val Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val Pro Pro 100 105 110 Val Asn Leu Lys Gln His Val Gly Arg Leu Glu Met Asn Val Asn Met 115 120 125 Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val Ser 130 135 140 Arg Val Gln Pro Val Ala Tyr Ala Glu Ile Val Ala Thr Val Ser Ser 145 150 155 160 Arg Ser Val Gly Pro Gly Thr Arg Lys Asn Ile Asp Glu Phe Thr Arg 165 170 175 Thr Thr Ala Gly Ala Ile Glu Gln Val Gly Gly Ala Arg Glu Gly Lys 180 185 190 Ala Ile Ile Val Ile Asn Pro Ala Glu Pro Pro Leu Met Met Arg Asp 195 200 205 Thr Ile His Cys Leu Thr Asp Ser Glu Pro Asp Gln Ala Ala Ile Thr 210 215 220 Ala Ser Val His Ala Met Ile Ala Glu Val Gln Lys Tyr Val Pro Gly 225 230 235 240 Tyr Arg Leu Lys Asn Gly Pro Val Phe Asp Gly Asn Arg Val Ser Ile 245 250 255 Phe Met Glu Val Glu Gly Leu Gly Asp Tyr Leu Pro Lys Tyr Ala Gly 260 265 270 Asn Leu Asp Ile Met Thr Ala Ala Ala Leu Arg Thr Gly Glu Met Phe 275 280 285 Ala Glu Glu Ile Ala Ala Gly Thr Ile Gln Leu Pro Arg Arg Asp Ile 290 295 300 Ala Leu Ala 305 682180DNAPseudomonas putida 68ggtacccctg gagccggtca aggccggcga cttcatgcgc gtcgagatcg gcggcatcgg 60cagcgcctcc gtgcgcttca cctgatcgaa cagaggacaa acccatgagc aagaaactca 120aggcggccat cataggcccc ggcaatatcg gtaccgatct ggtgatgaag atgctccgtt 180ccgagtggat tgagccggtg tggatggtcg gcatcgaccc caactccgac ggcctcaaac 240gcgcccgcga tttcggcatg aagaccacag ccgaaggcgt cgacggcctg ctcccgcacg 300tgctggacga cgacatccgc atcgccttcg acgccacctc ggcctatgtg catgccgaga 360atagccgcaa gctcaacgcg cttggcgtgc tgatggtcga cctgaccccg gcggccatcg 420gcccctactg cgtgccgccg gtcaacctca agcagcatgt cggccgcctg gaaatgaacg 480tcaacatggt cacctgcggc ggccaggcca ccatccccat ggtcgccgcg gtgtcccgcg 540tgcagccggt ggcctacgcc gagatcgtcg ccaccgtctc ctcgcgctcg gtcggcccgg 600gcacgcgcaa gaacatcgac gagttcaccc gcaccaccgc cggcgccatc gagcaggtcg 660gcggcgccag ggaaggcaag gcgatcatcg tcatcaaccc ggccgagccg ccgctgatga 720tgcgcgacac catccactgc ctgaccgaca gcgagccgga ccaggctgcg atcaccgctt 780cggttcacgc gatgatcgcc gaggtgcaga aatacgtgcc cggctaccgc ctgaagaacg 840gcccggtgtt cgacggcaac cgcgtgtcga tcttcatgga agtcgaaggc ctgggcgact 900acctgcccaa gtacgccggc aacctcgaca tcatgaccgc cgccgcgctg cgtaccggcg 960agatgttcgc cgaggaaatc gccgccggca ccattcaact gccgcgtcgc gacatcgcgc 1020tggcttgagg agtagcacca tgaatttgca cggcaagagc gtcatcctgc acgacatgag 1080cctgcgcgac ggcatgcacg ccaagcgcca ccagatcagc ctggagcaga tggtcgcggt 1140cgccaccggc ctcgatcaag ccggtatgcc gctgatcgag atcacccacg gcgacggcct 1200cggcggtcgt tcgatcaact acggcttccc ggcccacagt gacgaggagt acctgcgcgc 1260ggtgatcccg cagctcaagc aggccaaagt ctcggcgctg ctgctgcccg gcatcggcac 1320cgtcgaccac ctgaagatgg ccctggactg cggcgtctcg actattcgcg tggccaccca 1380ctgtaccgag gcggatgtct ccgagcagca catcggcatg gcgcgcaagc tgggggtcga 1440caccgtcggc ttcctgatga tggcgcacat gatcagcgcc gagaaagtcc tggagcaggc 1500caagctgatg gaaagctatg gtgccaactg catctactgc accgactcgg ccggctacat 1560gctgcctgat gaagtcagcg agaaaatcgg cctcctgcgc gccgagctga acccggccac 1620cgaagtcggc ttccacggcc accacaacat gggcatggct atcgccaact cgctggccgc 1680catcgaagcc ggtgccgcgc gcatcgacgg ctcggtcgcc ggcctcggcg ccggtgccgg 1740caacaccccg ctggaagtgt tcgtcgcagt gtgcaaacgc atgggcgtgg agaccggcat 1800cgacctgtac aagatcatgg acgtggccga ggacctggtg gtgccgatga tggatcagcc 1860gatccgcgtc gaccgcgacg ccctgaccct gggctacgcc ggggtgtaca gctcgttcct 1920gctgttcgcc cagcgcgccg agaagaaata tggcgtgtcg gcccgcgaca tcctggtcga 1980actgggccgg cgcggcaccg tcggtggcca ggaagacatg atcgaagacc tcgccctgga 2040catggcccgg gcccgtcagc agcagaaggt gagcgcatga accgtaccct gacccgcgaa 2100caggtgctgg ccctggccga gcacatcgaa aacgccgagc tgaatgtcca cgacatcggc 2160aaggtgacca acgattttcc 218069307PRTThermus thermophilus 69Met Ser Glu Arg Val Lys Val Ala Ile Leu Gly Ser Gly Asn Ile Gly 1 5 10 15 Thr Asp Leu Met Tyr Lys Leu Leu Lys Asn Pro Gly His Met Glu Leu 20 25 30 Val Ala Val Val Gly Ile Asp Pro Lys Ser Glu Gly Leu Ala Arg Ala 35 40 45 Arg Ala Leu Gly Leu Glu Ala Ser His Glu Gly Ile Ala Tyr Ile Leu 50 55 60 Glu Arg Pro Glu Ile Lys Ile Val Phe Asp Ala Thr Ser Ala Lys Ala 65 70 75 80 His Val Arg His Ala Lys Leu Leu Arg Glu Ala Gly Lys Ile Ala Ile 85 90 95 Asp Leu Thr Pro Ala Ala Arg Gly Pro Tyr Val Val Pro Pro Val Asn 100 105 110 Leu Lys Glu His Leu Asp Lys Asp Asn Val Asn Leu Ile Thr Cys Gly 115 120 125 Gly Gln Ala Thr Ile Pro Leu Val Tyr Ala Val His Arg Val Ala Pro 130 135 140 Val Leu Tyr Ala Glu Met Val Ser Thr Val Ala Ser Arg Ser Ala Gly 145 150 155 160 Pro Gly Thr Arg Gln Asn Ile Asp Glu Phe Thr Phe Thr Thr Ala Arg 165 170 175 Gly Leu Glu Ala Ile Gly Gly Ala Lys Lys Gly Lys Ala Ile Ile Ile 180 185 190 Leu Asn Pro Ala Glu Pro Pro Ile Leu Met Thr Asn Thr Val Arg Cys 195 200 205 Ile Pro Glu Asp Glu Gly Phe Asp Arg Glu Ala Val Val Ala Ser Val 210 215 220 Arg Ala Met Glu Arg Glu Val Gln Ala Tyr Val Pro Gly Tyr Arg Leu 225 230 235 240 Lys Ala Asp Pro Val Phe Glu Arg Leu Pro Thr Pro Trp Gly Glu Arg 245 250 255 Thr Val Val Ser Met Leu Leu Glu Val Glu Gly Ala Gly Asp Tyr Leu 260 265 270 Pro Lys Tyr Ala Gly Asn Leu Asp Ile Met Thr Ala Ser Ala Arg Arg 275 280 285 Val Gly Glu Val Phe Ala Gln His Leu Leu Gly Lys Pro Val Glu Glu 290 295 300 Val Val Ala 305 70924DNAThermus thermophilus 70atgtccgaaa gggttaaggt agccatcctg ggctccggca acatcgggac ggacctgatg 60tacaagctcc tgaagaaccc gggccacatg gagcttgtgg cggtggtggg gatagacccc 120aagtccgagg gcctggcccg ggcgcgggcc ttagggttag aggcgagcca cgaagggatc 180gcctacatcc tggagaggcc ggagatcaag atcgtctttg acgccaccag cgccaaggcc 240cacgtgcgcc acgccaagct cctgagggag gcggggaaga tcgccataga cctcacgccg 300gcggcccggg gcccttacgt ggtgcccccg gtgaacctga aggaacacct ggacaaggac 360aacgtgaacc tcatcacctg cggggggcag gccaccatcc ccctggtcta cgcggtgcac 420cgggtggccc ccgtgctcta cgcggagatg gtctccacgg tggcctcccg ctccgcgggc 480cccggcaccc ggcagaacat cgacgagttc accttcacca ccgcccgggg cctggaggcc 540atcggggggg ccaagaaggg gaaggccatc atcatcctga acccggcgga accccccatc 600ctcatgacca acaccgtgcg ctgcatcccc gaggacgagg gctttgaccg ggaggccgtg 660gtggcgagcg tccgggccat ggagcgggag gtccaggcct acgtgcccgg ctaccgcctg 720aaggcggacc cggtgtttga gaggcttccc accccctggg gggagcgcac cgtggtctcc 780atgctcctgg aggtggaggg ggcgggggac tatttgccca aatacgccgg caacctggac 840atcatgacgg cttctgcccg gagggtgggg gaggtcttcg cccagcacct cctggggaag 900cccgtggagg aggtggtggc gtga 92471417PRTEscherichia coli 71Met Thr Phe Ser Leu Phe Gly Asp Lys Phe Thr Arg His Ser Gly Ile 1 5 10 15 Thr Leu Leu Met Glu Asp Leu Asn Asp Gly Leu Arg Thr Pro Gly Ala 20 25 30 Ile Met Leu Gly Gly Gly Asn Pro Ala Gln Ile Pro Glu Met Gln Asp 35 40 45 Tyr Phe Gln Thr Leu Leu Thr Asp Met Leu Glu Ser Gly Lys Ala Thr 50 55 60 Asp Ala Leu Cys Asn Tyr Asp Gly Pro Gln Gly Lys Thr Glu Leu Leu 65 70 75 80 Thr Leu Leu Ala Gly Met Leu Arg Glu Lys Leu Gly Trp Asp Ile Glu 85 90 95 Pro Gln Asn Ile Ala Leu Thr Asn Gly Ser Gln Ser Ala Phe Phe Tyr 100 105 110 Leu Phe Asn Leu Phe Ala Gly Arg Arg Ala Asp Gly Arg Val Lys Lys 115 120 125 Val Leu Phe Pro Leu Ala Pro Glu Tyr Ile Gly Tyr Ala Asp Ala Gly 130 135 140 Leu Glu Glu Asp Leu Phe Val Ser Ala Arg Pro Asn Ile Glu Leu Leu 145 150 155 160 Pro Glu Gly Gln Phe Lys Tyr His Val Asp Phe Glu His Leu His Ile 165 170 175 Gly Glu Glu Thr Gly Met Ile Cys Val Ser Arg Pro Thr Asn Pro Thr 180 185 190 Gly Asn Val Ile Thr Asp Glu Glu Leu Leu Lys Leu Asp Ala Leu Ala 195 200 205 Asn Gln His Gly Ile Pro Leu Val Ile Asp Asn Ala Tyr Gly Val Pro 210 215 220 Phe Pro Gly Ile Ile Phe Ser Glu Ala Arg Pro Leu Trp Asn Pro Asn 225 230 235 240 Ile Val Leu Cys Met Ser Leu Ser Lys Leu Gly Leu Pro Gly Ser Arg 245 250 255 Cys Gly Ile Ile Ile Ala Asn Glu Lys Ile Ile Thr Ala Ile Thr Asn 260 265 270 Met Asn Gly Ile Ile Ser Leu Ala Pro Gly Gly Ile Gly Pro Ala Met 275 280 285 Met Cys Glu Met Ile Lys Arg Asn Asp Leu Leu Arg Leu Ser Glu Thr 290 295 300 Val Ile Lys Pro Phe Tyr Tyr Gln Arg Val Gln Glu Thr Ile Ala Ile 305 310 315 320 Ile Arg Arg Tyr Leu Pro Glu Asn Arg Cys Leu Ile His Lys Pro Glu 325 330 335 Gly Ala Ile Phe Leu Trp Leu Trp Phe Lys Asp Leu Pro Ile Thr Thr 340 345 350 Lys Gln Leu Tyr Gln Arg Leu Lys Ala Arg Gly Val Leu Met Val Pro 355 360 365 Gly His Asn Phe Phe Pro Gly Leu Asp Lys Pro Trp Pro His Thr His 370 375 380 Gln Cys Met Arg Met Asn Tyr Val Pro Glu Pro Glu Lys Ile Glu Ala 385 390 395 400 Gly Val Lys Ile Leu Ala Glu Glu Ile Glu Arg Ala Trp Ala Glu Ser 405 410 415 His 72417PRTEscherichia coli 72Met Thr Phe Ser Leu Phe Gly Asp Lys Phe Thr Arg His Ser Gly Ile 1 5 10 15 Thr Leu Leu Met Glu Asp Leu Asn Asp Gly Leu Arg Thr Pro Gly Ala 20 25 30 Ile Met Leu Gly Gly Gly Asn Pro Ala Gln Ile Pro Glu Met Gln Asp 35 40 45 Tyr Phe Gln Thr Leu Leu Thr Asp Met Leu Glu Ser Gly Lys Ala Thr 50 55 60 Asp Ala Leu Cys Asn Tyr Asp Gly Pro Gln Gly Lys Thr Glu Leu Leu 65 70 75 80 Thr Leu Leu Ala Gly Met Leu Arg Glu Lys Leu Gly Trp Asp Ile Glu 85 90 95 Pro Gln Asn Ile Ala Leu Thr Asn Gly Ser Gln Ser Ala Phe Phe Tyr 100 105 110 Leu Phe Asn Leu Phe Ala Gly Arg Arg Ala Asp Gly Arg Val Lys Lys 115 120 125 Val Leu Phe Pro Leu Ala Pro Glu Tyr Ile Gly Tyr Ala Asp Ala Gly 130 135 140 Leu Glu Glu Asp Leu Phe Val Ser Ala Arg Pro Asn Ile Glu Leu Leu 145 150 155 160 Pro Glu Gly Gln Phe Lys Tyr His Val Asp Phe Glu His Leu His Ile 165 170 175 Gly Glu Glu Thr Gly Met Ile Cys Val Ser Arg Pro Thr Asn Pro Thr 180 185 190 Gly Asn Val Ile Thr Asp Glu Glu Leu Leu Lys Leu Asp Ala Leu Ala 195 200 205 Asn Gln His Gly Ile Pro Leu Val Ile Asp Asn Ala Tyr Gly Val Pro 210 215 220 Phe Pro Gly Ile Ile Phe Ser Glu Ala Arg Pro Leu Trp Asn Pro Asn 225 230 235 240 Ile Val Leu Cys Met Ser Leu Ser Lys Leu Gly Leu Pro Gly Ser Arg 245 250 255 Cys Gly Ile Ile Ile Ala Asn Glu Lys Ile Ile Thr Ala Ile Thr Asn 260 265 270 Met Asn Gly Ile Ile Ser Leu Ala Pro Gly Gly Ile Gly Pro Ala Met 275 280 285 Met Cys Glu Met Ile Lys Arg Asn Asp Leu Leu Arg Leu Ser Glu Thr 290 295 300 Val Ile Lys Pro Phe Tyr Tyr Gln Arg Val Gln Glu Thr Ile Ala Ile 305 310 315 320 Ile Arg Arg Tyr Leu Pro Glu Asn Arg Cys Leu Ile His Lys Pro Glu 325 330 335 Gly Ala Ile Phe Leu Trp Leu Trp Phe Lys Asp Leu Pro Ile Thr Thr 340 345 350 Lys Gln Leu Tyr Gln Arg Leu Lys Ala Arg Gly Val Leu Met Val Pro 355 360 365 Gly His Asn Phe Phe Pro Gly Leu Asp Lys Pro Trp Pro His Thr His 370 375 380 Gln Cys Met Arg Met Asn Tyr Val Pro Glu Pro Glu Lys Ile Glu Ala 385 390 395 400 Gly Val Lys Ile Leu Ala Glu Glu Ile Glu Arg Ala Trp Ala Glu Ser 405 410 415 His 73425PRTBacillus licheniformis 73Met Lys Pro Pro Leu Ser Lys Ile Gly Glu Lys Met Ile Glu Lys Thr 1 5 10 15 Gly Val Arg Ala Val Met Ser Asp Ile Gln Glu Val Leu Ala Gly

Gly 20 25 30 Glu Arg Ser Tyr Ile Asn Leu Ser Ala Gly Asn Pro Met Ile Leu Pro 35 40 45 Gly Val Ser Ala Met Trp Lys Ser Ala Leu Ala Asp Leu Leu Asp Asp 50 55 60 Asp Arg Phe Ser Ser Val Ile Gly Gln Tyr Gly Ser Ser Tyr Gly Thr 65 70 75 80 Asp Glu Leu Ile Ala Ser Val Val Arg Phe Phe Ser Glu Arg Tyr Ser 85 90 95 Ala Gly Ile Arg Lys Glu Asn Val Leu Ile Thr Ala Gly Ser Gln Gln 100 105 110 Leu Phe Phe Leu Ala Ile Asn Ser Phe Cys Gly Met Gly Ser Gly Ser 115 120 125 Val Met Lys Lys Ala Leu Ile Pro Met Leu Pro Asp Tyr Ser Gly Tyr 130 135 140 Ser Gly Ala Ala Leu Glu Arg Glu Met Ile Glu Gly Ile Pro Pro Leu 145 150 155 160 Ile Ser Lys Leu Asp Asp His Thr Phe Arg Tyr Glu Leu Asp Arg Lys 165 170 175 Gly Phe Leu Glu Arg Met Arg Ile Gly Ala Val Leu Leu Ser Arg Pro 180 185 190 Asn Asn Pro Cys Gly Asn Ile Leu Pro Lys Glu Asp Val Ala Phe Ile 195 200 205 Ser Asp Ala Cys Arg Glu Ala Asn Val Pro Leu Phe Ile Asp Ser Ala 210 215 220 Tyr Ala Pro Pro Phe Pro Ala Ile His Phe Ile Asp Met Glu Pro Ile 225 230 235 240 Phe Asn Glu Gln Ile Ile His Cys Met Ser Leu Ser Lys Ala Gly Leu 245 250 255 Pro Gly Glu Arg Ile Gly Ile Ala Ile Gly Pro Ser Arg Tyr Ile Gln 260 265 270 Ala Met Glu Ala Phe Gln Ser Asn Ala Ala Ile His Ser Ser Arg Leu 275 280 285 Gly Gln Tyr Met Ala Ala Ser Val Leu Asn Asp Gly Arg Leu Ala Asp 290 295 300 Val Ser Leu Asn Glu Val Arg Pro Tyr Tyr Arg Asn Lys Phe Met Leu 305 310 315 320 Leu Lys Glu Thr Leu Leu Cys Lys Met Pro Glu Asp Ile Lys Trp Tyr 325 330 335 Leu His Gln Gly Glu Gly Ser Leu Phe Gly Trp Leu Trp Phe Glu Asp 340 345 350 Leu Pro Val Thr Asp Ala Ala Leu Tyr Glu Tyr Met Lys Ala Asp Gly 355 360 365 Val Ile Ile Val Pro Gly Ser Ser Phe Phe His Arg Gln Ser Arg Arg 370 375 380 Leu Ala His Ser His Gln Cys Ile Arg Ile Ser Leu Thr Ala Ala Asp 385 390 395 400 Glu Asp Ile Ile Arg Gly Ile Asp Val Leu Ala Lys Ile Ala Lys Gly 405 410 415 Val Tyr Glu Lys Gln Val Glu Tyr Leu 420 425 741278DNABacillus licheniformis 74ttataagtat tcaacctgtt tctcatatac acccttcgca attttagcta aaacatcgat 60tccccttata atatcttcat ccgccgcggt taggctgatt cgtatacact ggtgtgaatg 120cgccaggcgc cgggattgac ggtgaaagaa agatgatccg ggaacgataa tgactccatc 180cgctttcata tactcataca gcgctgcatc ggtcaccggc aggtcttcaa accacagcca 240tccgaaaagc gatccttccc cttgatgcag ataccatttg atgtcttcag gcatcttgca 300taaaagcgtt tccttgagca gcatgaattt attgcggtaa tatggcctga cttcattcag 360cgacacgtcg gcgaggcgcc cgtcattcaa tactgatgca gccatatact gccccagcct 420tgaagaatgg atcgccgcat tcgactgaaa agcttccatt gcctgaatat accgggacgg 480cccgatggcg attccgatcc tttcgccagg caggccggct tttgaaaggc tcatacagtg 540aatgatctgc tcgttgaaaa tcggttccat gtcgataaag tgaatcgccg gaaaaggcgg 600agcatatgcg gaatcaatga acagcggaac attcgcttct cggcatgcgt ctgaaatgaa 660tgctacatct tctttaggca agatgtttcc gcaaggattg ttcgggcgcg atagcaagac 720agcaccgatg cgcatcctct ctaaaaaccc cttacggtcg agctcatatc gaaacgtatg 780atcatccaat ttcgatatga gcggagggat cccctcaatc atctcccgct ccagtgccgc 840cccgctgtat cccgaatagt caggcagcat cgggatcaag gcttttttca tcacagatcc 900gcttcccatt ccgcaaaacg aattgatcgc cagaaaaaac agctgctggc ttccggctgt 960aatcaacacg ttctcttttc gaatgccggc gctataccgc tctgaaaaga agcggacaac 1020acttgcaatc agttcatcgg ttccatagct cgatccgtat tggccgatca ccgaagaaaa 1080cctgtcatcg tcaaggagat cggcaagagc cgacttccac atggctgaca cgccgggcaa 1140aatcatcgga ttgcccgcac ttaaattaat gtatgaccgt tcaccgccgg ccaggacttc 1200ctgaatatcg ctcatcacag ccctgacccc tgttttctca atcattttct ctccgatttt 1260gcttaatggc ggcttcac 127875309PRTEscherichia coli 75Met Thr Thr Lys Lys Ala Asp Tyr Ile Trp Phe Asn Gly Glu Met Val 1 5 10 15 Arg Trp Glu Asp Ala Lys Val His Val Met Ser His Ala Leu His Tyr 20 25 30 Gly Thr Ser Val Phe Glu Gly Ile Arg Cys Tyr Asp Ser His Lys Gly 35 40 45 Pro Val Val Phe Arg His Arg Glu His Met Gln Arg Leu His Asp Ser 50 55 60 Ala Lys Ile Tyr Arg Phe Pro Val Ser Gln Ser Ile Asp Glu Leu Met 65 70 75 80 Glu Ala Cys Arg Asp Val Ile Arg Lys Asn Asn Leu Thr Ser Ala Tyr 85 90 95 Ile Arg Pro Leu Ile Phe Val Gly Asp Val Gly Met Gly Val Asn Pro 100 105 110 Pro Ala Gly Tyr Ser Thr Asp Val Ile Ile Ala Ala Phe Pro Trp Gly 115 120 125 Ala Tyr Leu Gly Ala Glu Ala Leu Glu Gln Gly Ile Asp Ala Met Val 130 135 140 Ser Ser Trp Asn Arg Ala Ala Pro Asn Thr Ile Pro Thr Ala Ala Lys 145 150 155 160 Ala Gly Gly Asn Tyr Leu Ser Ser Leu Leu Val Gly Ser Glu Ala Arg 165 170 175 Arg His Gly Tyr Gln Glu Gly Ile Ala Leu Asp Val Asn Gly Tyr Ile 180 185 190 Ser Glu Gly Ala Gly Glu Asn Leu Phe Glu Val Lys Asp Gly Val Leu 195 200 205 Phe Thr Pro Pro Phe Thr Ser Ser Ala Leu Pro Gly Ile Thr Arg Asp 210 215 220 Ala Ile Ile Lys Leu Ala Lys Glu Leu Gly Ile Glu Val Arg Glu Gln 225 230 235 240 Val Leu Ser Arg Glu Ser Leu Tyr Leu Ala Asp Glu Val Phe Met Ser 245 250 255 Gly Thr Ala Ala Glu Ile Thr Pro Val Arg Ser Val Asp Gly Ile Gln 260 265 270 Val Gly Glu Gly Arg Cys Gly Pro Val Thr Lys Arg Ile Gln Gln Ala 275 280 285 Phe Phe Gly Leu Phe Thr Gly Glu Thr Glu Asp Lys Trp Gly Trp Leu 290 295 300 Asp Gln Val Asn Gln 305 761476DNAEscherichia coli 76atggctaact acttcaatac actgaatctg cgccagcagc tggcacagct gggcaaatgt 60cgctttatgg gccgcgatga attcgccgat ggcgcgagct accttcaggg taaaaaagta 120gtcatcgtcg gctgtggcgc acagggtctg aaccagggcc tgaacatgcg tgattctggt 180ctcgatatct cctacgctct gcgtaaagaa gcgattgccg agaagcgcgc gtcctggcgt 240aaagcgaccg aaaatggttt taaagtgggt acttacgaag aactgatccc acaggcggat 300ctggtgatta acctgacgcc ggacaagcag cactctgatg tagtgcgcac cgtacagcca 360ctgatgaaag acggcgcggc gctgggctac tcgcacggtt tcaacatcgt cgaagtgggc 420gagcagatcc gtaaagatat caccgtagtg atggttgcgc cgaaatgccc aggcaccgaa 480gtgcgtgaag agtacaaacg tgggttcggc gtaccgacgc tgattgccgt tcacccggaa 540aacgatccga aaggcgaagg catggcgatt gccaaagcct gggcggctgc aaccggtggt 600caccgtgcgg gtgtgctgga atcgtccttc gttgcggaag tgaaatctga cctgatgggc 660gagcaaacca tcctgtgcgg tatgttgcag gctggctctc tgctgtgctt cgacaagctg 720gtggaagaag gtaccgatcc agcatacgca gaaaaactga ttcagttcgg ttgggaaacc 780atcaccgaag cactgaaaca gggcggcatc accctgatga tggaccgtct ctctaacccg 840gcgaaactgc gtgcttatgc gctttctgaa cagctgaaag agatcatggc acccctgttc 900cagaaacata tggacgacat catctccggc gaattctctt ccggtatgat ggcggactgg 960gccaacgatg ataagaaact gctgacctgg cgtgaagaga ccggcaaaac cgcgtttgaa 1020accgcgccgc agtatgaagg caaaatcggc gagcaggagt acttcgataa aggcgtactg 1080atgattgcga tggtgaaagc gggcgttgaa ctggcgttcg aaaccatggt cgattccggc 1140atcattgaag agtctgcata ttatgaatca ctgcacgagc tgccgctgat tgccaacacc 1200atcgcccgta agcgtctgta cgaaatgaac gtggttatct ctgataccgc tgagtacggt 1260aactatctgt tctcttacgc ttgtgtgccg ttgctgaaac cgtttatggc agagctgcaa 1320ccgggcgacc tgggtaaagc tattccggaa ggcgcggtag ataacgggca actgcgtgat 1380gtgaacgaag cgattcgcag ccatgcgatt gagcaggtag gtaagaaact gcgcggctat 1440atgacagata tgaaacgtat tgctgttgcg ggttaa 147677376PRTSaccharomyces cerevisiae 77Met Thr Leu Ala Pro Leu Asp Ala Ser Lys Val Lys Ile Thr Thr Thr 1 5 10 15 Gln His Ala Ser Lys Pro Lys Pro Asn Ser Glu Leu Val Phe Gly Lys 20 25 30 Ser Phe Thr Asp His Met Leu Thr Ala Glu Trp Thr Ala Glu Lys Gly 35 40 45 Trp Gly Thr Pro Glu Ile Lys Pro Tyr Gln Asn Leu Ser Leu Asp Pro 50 55 60 Ser Ala Val Val Phe His Tyr Ala Phe Glu Leu Phe Glu Gly Met Lys 65 70 75 80 Ala Tyr Arg Thr Val Asp Asn Lys Ile Thr Met Phe Arg Pro Asp Met 85 90 95 Asn Met Lys Arg Met Asn Lys Ser Ala Gln Arg Ile Cys Leu Pro Thr 100 105 110 Phe Asp Pro Glu Glu Leu Ile Thr Leu Ile Gly Lys Leu Ile Gln Gln 115 120 125 Asp Lys Cys Leu Val Pro Glu Gly Lys Gly Tyr Ser Leu Tyr Ile Arg 130 135 140 Pro Thr Leu Ile Gly Thr Thr Ala Gly Leu Gly Val Ser Thr Pro Asp 145 150 155 160 Arg Ala Leu Leu Tyr Val Ile Cys Cys Pro Val Gly Pro Tyr Tyr Lys 165 170 175 Thr Gly Phe Lys Ala Val Arg Leu Glu Ala Thr Asp Tyr Ala Thr Arg 180 185 190 Ala Trp Pro Gly Gly Cys Gly Asp Lys Lys Leu Gly Ala Asn Tyr Ala 195 200 205 Pro Cys Val Leu Pro Gln Leu Gln Ala Ala Ser Arg Gly Tyr Gln Gln 210 215 220 Asn Leu Trp Leu Phe Gly Pro Asn Asn Asn Ile Thr Glu Val Gly Thr 225 230 235 240 Met Asn Ala Phe Phe Val Phe Lys Asp Ser Lys Thr Gly Lys Lys Glu 245 250 255 Leu Val Thr Ala Pro Leu Asp Gly Thr Ile Leu Glu Gly Val Thr Arg 260 265 270 Asp Ser Ile Leu Asn Leu Ala Lys Glu Arg Leu Glu Pro Ser Glu Trp 275 280 285 Thr Ile Ser Glu Arg Tyr Phe Thr Ile Gly Glu Val Thr Glu Arg Ser 290 295 300 Lys Asn Gly Glu Leu Leu Glu Ala Phe Gly Ser Gly Thr Ala Ala Ile 305 310 315 320 Val Ser Pro Ile Lys Glu Ile Gly Trp Lys Gly Glu Gln Ile Asn Ile 325 330 335 Pro Leu Leu Pro Gly Glu Gln Thr Gly Pro Leu Ala Lys Glu Val Ala 340 345 350 Gln Trp Ile Asn Gly Ile Gln Tyr Gly Glu Thr Glu His Gly Asn Trp 355 360 365 Ser Arg Val Val Thr Asp Leu Asn 370 375 78376PRTSaccharomyces cerevisiae 78Met Thr Leu Ala Pro Leu Asp Ala Ser Lys Val Lys Ile Thr Thr Thr 1 5 10 15 Gln His Ala Ser Lys Pro Lys Pro Asn Ser Glu Leu Val Phe Gly Lys 20 25 30 Ser Phe Thr Asp His Met Leu Thr Ala Glu Trp Thr Ala Glu Lys Gly 35 40 45 Trp Gly Thr Pro Glu Ile Lys Pro Tyr Gln Asn Leu Ser Leu Asp Pro 50 55 60 Ser Ala Val Val Phe His Tyr Ala Phe Glu Leu Phe Glu Gly Met Lys 65 70 75 80 Ala Tyr Arg Thr Val Asp Asn Lys Ile Thr Met Phe Arg Pro Asp Met 85 90 95 Asn Met Lys Arg Met Asn Lys Ser Ala Gln Arg Ile Cys Leu Pro Thr 100 105 110 Phe Asp Pro Glu Glu Leu Ile Thr Leu Ile Gly Lys Leu Ile Gln Gln 115 120 125 Asp Lys Cys Leu Val Pro Glu Gly Lys Gly Tyr Ser Leu Tyr Ile Arg 130 135 140 Pro Thr Leu Ile Gly Thr Thr Ala Gly Leu Gly Val Ser Thr Pro Asp 145 150 155 160 Arg Ala Leu Leu Tyr Val Ile Cys Cys Pro Val Gly Pro Tyr Tyr Lys 165 170 175 Thr Gly Phe Lys Ala Val Arg Leu Glu Ala Thr Asp Tyr Ala Thr Arg 180 185 190 Ala Trp Pro Gly Gly Cys Gly Asp Lys Lys Leu Gly Ala Asn Tyr Ala 195 200 205 Pro Cys Val Leu Pro Gln Leu Gln Ala Ala Ser Arg Gly Tyr Gln Gln 210 215 220 Asn Leu Trp Leu Phe Gly Pro Asn Asn Asn Ile Thr Glu Val Gly Thr 225 230 235 240 Met Asn Ala Phe Phe Val Phe Lys Asp Ser Lys Thr Gly Lys Lys Glu 245 250 255 Leu Val Thr Ala Pro Leu Asp Gly Thr Ile Leu Glu Gly Val Thr Arg 260 265 270 Asp Ser Ile Leu Asn Leu Ala Lys Glu Arg Leu Glu Pro Ser Glu Trp 275 280 285 Thr Ile Ser Glu Arg Tyr Phe Thr Ile Gly Glu Val Thr Glu Arg Ser 290 295 300 Lys Asn Gly Glu Leu Leu Glu Ala Phe Gly Ser Gly Thr Ala Ala Ile 305 310 315 320 Val Ser Pro Ile Lys Glu Ile Gly Trp Lys Gly Glu Gln Ile Asn Ile 325 330 335 Pro Leu Leu Pro Gly Glu Gln Thr Gly Pro Leu Ala Lys Glu Val Ala 340 345 350 Gln Trp Ile Asn Gly Ile Gln Tyr Gly Glu Thr Glu His Gly Asn Trp 355 360 365 Ser Arg Val Val Thr Asp Leu Asn 370 375 79330PRTMethanobacterium thermoautotrophicum 79Met Arg Leu Trp Arg Ala Leu Tyr Arg Pro Pro Thr Ile Thr Tyr Pro 1 5 10 15 Ser Lys Ser Pro Glu Val Ile Ile Met Ser Cys Glu Ala Ser Gly Lys 20 25 30 Ile Trp Leu Asn Gly Glu Met Val Glu Trp Glu Glu Ala Thr Val His 35 40 45 Val Leu Ser His Val Val His Tyr Gly Ser Ser Val Phe Glu Gly Ile 50 55 60 Arg Cys Tyr Arg Asn Ser Lys Gly Ser Ala Ile Phe Arg Leu Arg Glu 65 70 75 80 His Val Lys Arg Leu Phe Asp Ser Ala Lys Ile Tyr Arg Met Asp Ile 85 90 95 Pro Tyr Thr Gln Glu Gln Ile Cys Asp Ala Ile Val Glu Thr Val Arg 100 105 110 Glu Asn Gly Leu Glu Glu Cys Tyr Ile Arg Pro Val Val Phe Arg Gly 115 120 125 Tyr Gly Glu Met Gly Val His Pro Val Asn Cys Pro Val Asp Val Ala 130 135 140 Val Ala Ala Trp Glu Trp Gly Ala Tyr Leu Gly Ala Glu Ala Leu Glu 145 150 155 160 Val Gly Val Asp Ala Gly Val Ser Thr Trp Arg Arg Met Ala Pro Asn 165 170 175 Thr Met Pro Asn Met Ala Lys Ala Gly Gly Asn Tyr Leu Asn Ser Gln 180 185 190 Leu Ala Lys Met Glu Ala Val Arg His Gly Tyr Asp Glu Ala Ile Met 195 200 205 Leu Asp Tyr His Gly Tyr Ile Ser Glu Gly Ser Gly Glu Asn Ile Phe 210 215 220 Leu Val Ser Glu Gly Glu Ile Tyr Thr Pro Pro Val Ser Ser Ser Leu 225 230 235 240 Leu Arg Gly Ile Thr Arg Asp Ser Val Ile Lys Ile Ala Arg Thr Glu 245 250 255 Gly Val Thr Val His Glu Glu Pro Ile Thr Arg Glu Met Leu Tyr Ile 260 265 270 Ala Asp Glu Ala Phe Phe Thr Gly Thr Ala Ala Glu Ile Thr Pro Ile 275 280 285 Arg Ser Val Asp Gly Ile Glu Ile Gly Ala Gly Arg Arg Gly Pro Val 290 295 300 Thr Lys Leu Leu Gln Asp Glu Phe Phe Arg Ile Ile Arg Ala Glu Thr 305 310 315 320 Glu Asp Ser Phe Gly Trp Leu Thr Tyr Ile 325 330 80993DNAMethanobacterium thermoautotrophicum 80tcagatgtag gtgagccatc cgaagctgtc ctctgtctct gccctgatta tcctgaagaa 60ctcatcctgc agcagctttg taacgggacc ccttcgcccg gcacctatct ctataccatc 120aactgatctg atgggtgtta tctctgcggc tgtacctgtg aagaaggcct catctgcgat 180gtagagcatc tccctggtta tgggttcctc atgcacggta acaccctcgg tcctggctat 240ctttattacg gagtcccttg ttatccccct cagaagggat gatgaaacag ggggggtgta 300aatttcaccc

tcactgacga ggaatatgtt ctccccgcta ccctcactta tgtagccatg 360gtagtccagc attatggcct catcatagcc gtgtctcaca gcctccatct tggcaagctg 420tgagttgagg tagttaccgc cggcctttgc catgttgggc attgtgtttg gtgccatcct 480ccgccaggtt gaaacaccag catcgacacc aacctcaagg gcctctgcac ccagataggc 540cccccattcc caggcagcca cagcgacgtc cactgggcag ttcaccgggt gaacacccat 600ctcaccgtat cccctgaata ccacgggtct tatatagcac tcctcaagtc cgttctccct 660gacggtctca actatggcat cacatatctg ctcctgggtg tagggtatgt ccatccggta 720tatctttgca gaatcaaaaa ggcgtttaac atgctcccgc aaacggaaga tggctgaccc 780cttactgttc ctgtagcacc ttattccctc aaagacagat gatccataat gcacaacatg 840tgagagtacg tggacggtgg cttcttccca ttcaaccatt tcaccgttta accatatctt 900tccactggct tcgcatgaca tgataataac ctcaggtgat ttactaggat aggttatggt 960tggaggccta tataatgctc tccataaccg caa 99381364PRTStreptomyces coelicolor 81Met Thr Asp Val Asn Gly Ala Pro Ala Asp Val Leu His Thr Leu Phe 1 5 10 15 His Ser Asp Gln Gly Gly His Glu Gln Val Val Leu Cys Gln Asp Arg 20 25 30 Ala Ser Gly Leu Lys Ala Val Ile Ala Leu His Ser Thr Ala Leu Gly 35 40 45 Pro Ala Leu Gly Gly Thr Arg Phe Tyr Pro Tyr Ala Ser Glu Ala Glu 50 55 60 Ala Val Ala Asp Ala Leu Asn Leu Ala Arg Gly Met Ser Tyr Lys Asn 65 70 75 80 Ala Met Ala Gly Leu Asp His Gly Gly Gly Lys Ala Val Ile Ile Gly 85 90 95 Asp Pro Glu Gln Ile Lys Ser Glu Glu Leu Leu Leu Ala Tyr Gly Arg 100 105 110 Phe Val Ala Ser Leu Gly Gly Arg Tyr Val Thr Ala Cys Asp Val Gly 115 120 125 Thr Tyr Val Ala Asp Met Asp Val Val Ala Arg Glu Cys Arg Trp Thr 130 135 140 Thr Gly Arg Ser Pro Glu Asn Gly Gly Ala Gly Asp Ser Ser Val Leu 145 150 155 160 Thr Ser Phe Gly Val Tyr Gln Gly Met Arg Ala Ala Ala Gln His Leu 165 170 175 Trp Gly Asp Pro Thr Leu Arg Asp Arg Thr Val Gly Ile Ala Gly Val 180 185 190 Gly Lys Val Gly His His Leu Val Glu His Leu Leu Ala Glu Gly Ala 195 200 205 His Val Val Val Thr Asp Val Arg Lys Asp Val Val Arg Gly Ile Thr 210 215 220 Glu Arg His Pro Ser Val Val Ala Val Ala Asp Thr Asp Ala Leu Ile 225 230 235 240 Arg Val Glu Asn Leu Asp Ile Tyr Ala Pro Cys Ala Leu Gly Gly Ala 245 250 255 Leu Asn Asp Asp Thr Val Pro Val Leu Thr Ala Lys Val Val Cys Gly 260 265 270 Ala Ala Asn Asn Gln Leu Ala His Pro Gly Val Glu Lys Asp Leu Ala 275 280 285 Asp Arg Gly Ile Leu Tyr Ala Pro Asp Tyr Val Val Asn Ala Gly Gly 290 295 300 Val Ile Gln Val Ala Asp Glu Leu His Gly Phe Asp Phe Asp Arg Cys 305 310 315 320 Lys Ala Lys Ala Ser Lys Ile Tyr Asp Thr Thr Leu Ala Ile Phe Ala 325 330 335 Arg Ala Lys Glu Asp Gly Ile Pro Pro Ala Ala Ala Ala Asp Arg Ile 340 345 350 Ala Glu Gln Arg Met Ala Glu Ala Arg Pro Arg Pro 355 360 821095DNAStreptomyces coelicolor 82tcacggccgg ggacgggcct ccgccatccg ctgctcggcg atccggtcgg ccgccgcggc 60cggcggaata ccgtcctcct tcgcacgtgc gaatatggcc agcgtggtgt cgtagatctt 120cgaggccttc gccttgcacc ggtcgaagtc gaacccgtgc agctcgtcgg cgacctggat 180gacaccgccg gcgttcacca catagtccgg cgcgtagagg atcccgcggt cggcgaggtc 240cttctcgacg cccgggtggg cgagctggtt gttggccgcg ccgcacacca ccttggcggt 300cagcaccggc acggtgtcgt cgttcagcgc gccgccgagc gcgcagggcg cgtagatgtc 360caggttctcc acccggatca gcgcgtcggt gtcggcgacg gcgaccaccg acgggtgccg 420ctccgtgatc ccgcgcacca cgtccttgcg cacgtccgtg acgacgacgt gggcgccctc 480ggcgagcagg tgctcgacca ggtggtggcc gaccttgccg acgcccgcga tgccgacggt 540gcggtcgcgc agcgtcgggt cgccccacag gtgctgggcg gcggcccgca tgccctggta 600gacgccgaag gaggtgagca cggaggagtc gcccgcgccg ccgttctccg gggaacgccc 660ggtcgtccag cggcactcgc gggccacgac gtccatgtcg gcgacgtagg tgccgacgtc 720gcacgcggtg acgtagcggc cgcccagcga ggcgacgaac cggccgtagg cgaggagcag 780ctcctcgctc ttgatctgct ccggatcgcc gatgatcacg gccttgccgc caccgtggtc 840cagaccggcc atggcgttct tgtacgacat cccgcgggcg aggttcagcg cgtcggcgac 900ggcctccgcc tcgctcgcgt acgggtagaa gcgggtaccg ccgagcgccg ggcccagggc 960ggtggagtgg agggcgatca cggccttgag gccgctggca cggtcctggc agagcacgac 1020ttgctcatgt cccccctgat ccgagtggaa cagggtgtgc agtacatcag caggtgcgcc 1080gtttacgtcg gtcac 109583364PRTBacillus subtilis 83Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser 195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg 355 360 84364PRTBacillus subtilis 84Met Glu Leu Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val 1 5 10 15 Phe Cys Gln Asp Glu Gln Ser Gly Leu Lys Ala Ile Ile Ala Ile His 20 25 30 Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Thr Arg Met Trp Thr Tyr 35 40 45 Glu Asn Glu Glu Ala Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly 50 55 60 Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys 65 70 75 80 Thr Val Ile Ile Gly Asp Pro Arg Lys Asp Lys Asn Glu Glu Met Phe 85 90 95 Arg Ala Phe Gly Arg Tyr Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr 100 105 110 Ala Glu Asp Val Gly Thr Thr Val Glu Asp Met Asp Ile Ile His Asp 115 120 125 Glu Thr Asp Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly 130 135 140 Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala 145 150 155 160 Ala Ala Lys Ala Ala Phe Gly Thr Asp Ser Leu Glu Gly Lys Thr Ile 165 170 175 Ala Val Gln Gly Val Gly Asn Val Ala Tyr Asn Leu Cys Arg His Leu 180 185 190 His Glu Glu Gly Ala Asn Leu Ile Val Thr Asp Ile Asn Lys Gln Ser 195 200 205 Val Gln Arg Ala Val Glu Asp Phe Gly Ala Arg Ala Val Asp Pro Asp 210 215 220 Asp Ile Tyr Ser Gln Asp Cys Asp Ile Tyr Ala Pro Cys Ala Leu Gly 225 230 235 240 Ala Thr Ile Asn Asp Asp Thr Ile Lys Gln Leu Lys Ala Lys Val Ile 245 250 255 Ala Gly Ala Ala Asn Asn Gln Leu Lys Glu Thr Arg His Gly Asp Gln 260 265 270 Ile His Glu Met Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala 275 280 285 Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Ala Glu 290 295 300 Arg Ala Leu Lys Lys Val Glu Gly Ile Tyr Gly Asn Ile Glu Arg Val 305 310 315 320 Leu Glu Ile Ser Gln Arg Asp Gly Ile Pro Ala Tyr Leu Ala Ala Asp 325 330 335 Arg Leu Ala Glu Glu Arg Ile Glu Arg Met Arg Arg Ser Arg Ser Gln 340 345 350 Phe Leu Gln Asn Gly His Ser Val Leu Ser Arg Arg 355 360 85594PRTStreptomyces viridifaciens 85Met Ser Thr Ser Ser Ala Ser Ser Gly Pro Asp Leu Pro Phe Gly Pro 1 5 10 15 Glu Asp Thr Pro Trp Gln Lys Ala Phe Ser Arg Leu Arg Ala Val Asp 20 25 30 Gly Val Pro Arg Val Thr Ala Pro Ser Ser Asp Pro Arg Glu Val Tyr 35 40 45 Met Asp Ile Pro Glu Ile Pro Phe Ser Lys Val Gln Ile Pro Pro Asp 50 55 60 Gly Met Asp Glu Gln Gln Tyr Ala Glu Ala Glu Ser Leu Phe Arg Arg 65 70 75 80 Tyr Val Asp Ala Gln Thr Arg Asn Phe Ala Gly Tyr Gln Val Thr Ser 85 90 95 Asp Leu Asp Tyr Gln His Leu Ser His Tyr Leu Asn Arg His Leu Asn 100 105 110 Asn Val Gly Asp Pro Tyr Glu Ser Ser Ser Tyr Thr Leu Asn Ser Lys 115 120 125 Val Leu Glu Arg Ala Val Leu Asp Tyr Phe Ala Ser Leu Trp Asn Ala 130 135 140 Lys Trp Pro His Asp Ala Ser Asp Pro Glu Thr Tyr Trp Gly Tyr Val 145 150 155 160 Leu Thr Met Gly Ser Ser Glu Gly Asn Leu Tyr Gly Leu Trp Asn Ala 165 170 175 Arg Asp Tyr Leu Ser Gly Lys Leu Leu Arg Arg Gln His Arg Glu Ala 180 185 190 Gly Gly Asp Lys Ala Ser Val Val Tyr Thr Gln Ala Leu Arg His Glu 195 200 205 Gly Gln Ser Pro His Ala Tyr Glu Pro Val Ala Phe Phe Ser Gln Asp 210 215 220 Thr His Tyr Ser Leu Thr Lys Ala Val Arg Val Leu Gly Ile Asp Thr 225 230 235 240 Phe His Ser Ile Gly Ser Ser Arg Tyr Pro Asp Glu Asn Pro Leu Gly 245 250 255 Pro Gly Thr Pro Trp Pro Thr Glu Val Pro Ser Val Asp Gly Ala Ile 260 265 270 Asp Val Asp Lys Leu Ala Ser Leu Val Arg Phe Phe Ala Ser Lys Gly 275 280 285 Tyr Pro Ile Leu Val Ser Leu Asn Tyr Gly Ser Thr Phe Lys Gly Ala 290 295 300 Tyr Asp Asp Val Pro Ala Val Ala Gln Ala Val Arg Asp Ile Cys Thr 305 310 315 320 Glu Tyr Gly Leu Asp Arg Arg Arg Val Tyr His Asp Arg Ser Lys Asp 325 330 335 Ser Asp Phe Asp Glu Arg Ser Gly Phe Trp Ile His Ile Asp Ala Ala 340 345 350 Leu Gly Ala Gly Tyr Ala Pro Tyr Leu Gln Met Ala Arg Asp Ala Gly 355 360 365 Met Val Glu Glu Ala Pro Pro Val Phe Asp Phe Arg Leu Pro Glu Val 370 375 380 His Ser Leu Thr Met Ser Gly His Lys Trp Met Gly Thr Pro Trp Ala 385 390 395 400 Cys Gly Val Tyr Met Thr Arg Thr Gly Leu Gln Met Thr Pro Pro Lys 405 410 415 Ser Ser Glu Tyr Ile Gly Ala Ala Asp Thr Thr Phe Ala Gly Ser Arg 420 425 430 Asn Gly Phe Ser Ser Leu Leu Leu Trp Asp Tyr Leu Ser Arg His Ser 435 440 445 Tyr Asp Asp Leu Val Arg Leu Ala Ala Asp Cys Asp Arg Leu Ala Gly 450 455 460 Tyr Ala His Asp Arg Leu Leu Thr Leu Gln Asp Lys Leu Gly Met Asp 465 470 475 480 Leu Trp Val Ala Arg Ser Pro Gln Ser Leu Thr Val Arg Phe Arg Gln 485 490 495 Pro Cys Ala Asp Ile Val Arg Lys Tyr Ser Leu Ser Cys Glu Thr Val 500 505 510 Tyr Glu Asp Asn Glu Gln Arg Thr Tyr Val His Leu Tyr Ala Val Pro 515 520 525 His Leu Thr Arg Glu Leu Val Asp Glu Leu Val Arg Asp Leu Arg Gln 530 535 540 Pro Gly Ala Phe Thr Asn Ala Gly Ala Leu Glu Gly Glu Ala Trp Ala 545 550 555 560 Gly Val Ile Asp Ala Leu Gly Arg Pro Asp Pro Asp Gly Thr Tyr Ala 565 570 575 Gly Ala Leu Ser Ala Pro Ala Ser Gly Pro Arg Ser Glu Asp Gly Gly 580 585 590 Gly Ser 861785DNAStreptomyces viridifaciens 86gtgtcaactt cctccgcttc ttccgggccg gacctcccct tcgggcccga ggacacgcca 60tggcagaagg ccttcagcag gctgcgggcg gtggatggcg tgccgcgcgt caccgcgccg 120tccagtgatc cgcgtgaggt ctacatggac atcccggaga tccccttctc caaggtccag 180atccccccgg acggaatgga cgagcagcag tacgcagagg ccgagagcct cttccgccgc 240tacgtagacg cccagacccg caacttcgcg ggataccagg tcaccagcga cctcgactac 300cagcacctca gtcactatct caaccggcat ctgaacaacg tcggcgatcc ctatgagtcc 360agctcctaca cgctgaactc caaggtcctt gagcgagccg ttctcgacta cttcgcctcc 420ctgtggaacg ccaagtggcc ccatgacgca agcgatccgg aaacgtactg gggttacgtg 480ctgaccatgg gctccagcga aggcaacctg tacgggttgt ggaacgcacg ggactatctg 540tcgggcaagc tgctgcggcg ccagcaccgg gaggccggcg gcgacaaggc ctcggtcgtc 600tacacgcaag cgctgcgaca cgaagggcag agtccgcatg cctacgagcc ggtggcgttc 660ttctcgcagg acacgcacta ctcgctcacg aaggccgtgc gggttctggg catcgacacc 720ttccacagca tcggcagcag tcggtatccg gacgagaacc cgctgggccc cggcactccg 780tggccgaccg aagtgccctc ggttgacggt gccatcgatg tcgacaaact cgcctcgttg 840gtccgcttct tcgccagcaa gggctacccg atactggtca gcctcaacta cgggtcaacg 900ttcaagggcg cctacgacga cgtcccggcc gtggcacagg ccgtgcggga catctgcacg 960gaatacggtc tggatcggcg gcgggtatac cacgaccgca gtaaggacag tgacttcgac 1020gagcgcagcg gcttctggat ccacatcgat gccgccctgg gggcgggcta cgctccctac 1080ctgcagatgg cccgggatgc cggcatggtc gaggaggcgc cgcccgtttt cgacttccgg 1140ctcccggagg tgcactcgct gaccatgagc ggccacaagt ggatgggaac accgtgggca 1200tgcggtgtct acatgacacg gaccgggctg cagatgaccc cgccgaagtc gtccgagtac 1260atcggggcgg ccgacaccac cttcgcgggc tcccgcaacg gcttctcgtc actgctgctg 1320tgggactacc tgtcccggca ttcgtatgac gatctggtgc gcctggccgc cgactgcgac 1380cggctggccg gctacgccca cgaccggttg ctgaccttgc aggacaaact cggcatggat 1440ctgtgggtcg cccgcagccc gcagtccctc acggtgcgct tccgtcagcc atgtgcagac 1500atcgtccgca agtactcgct gtcgtgtgag acggtctacg aagacaacga gcaacggacc 1560tacgtacatc tctacgccgt tccccacctc actcgggaac tcgtggatga gctcgtgcgc 1620gatctgcgcc agcccggagc cttcaccaac gctggtgcac tggaggggga ggcctgggcc 1680ggggtgatcg atgccctcgg ccgcccggac cccgacggaa cctatgccgg cgccttgagc 1740gctccggctt ccggcccccg ctccgaggac ggcggcggga gctga 178587440PRTAlcaligenes denitrificans 87Met Ser Ala Ala Lys Leu Pro Asp Leu Ser His Leu Trp Met Pro Phe 1 5 10 15 Thr Ala Asn Arg Gln Phe Lys Ala Asn Pro Arg Leu Leu Ala Ser Ala 20

25 30 Lys Gly Met Tyr Tyr Thr Ser Phe Asp Gly Arg Gln Ile Leu Asp Gly 35 40 45 Thr Ala Gly Leu Trp Cys Val Asn Ala Gly His Cys Arg Glu Glu Ile 50 55 60 Val Ser Ala Ile Ala Ser Gln Ala Gly Val Met Asp Tyr Ala Pro Gly 65 70 75 80 Phe Gln Leu Gly His Pro Leu Ala Phe Glu Ala Ala Thr Ala Val Ala 85 90 95 Gly Leu Met Pro Gln Gly Leu Asp Arg Val Phe Phe Thr Asn Ser Gly 100 105 110 Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr His Arg 115 120 125 Ala Arg Gly Glu Ala Gln Arg Thr Arg Leu Ile Gly Arg Glu Arg Gly 130 135 140 Tyr His Gly Val Gly Phe Gly Gly Ile Ser Val Gly Gly Ile Ser Pro 145 150 155 160 Asn Arg Lys Thr Phe Ser Gly Ala Leu Leu Pro Ala Val Asp His Leu 165 170 175 Pro His Thr His Ser Leu Glu His Asn Ala Phe Thr Arg Gly Gln Pro 180 185 190 Glu Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Ile Ala Leu 195 200 205 His Asp Ala Ser Thr Ile Ala Ala Val Ile Val Glu Pro Met Ala Gly 210 215 220 Ser Thr Gly Val Leu Val Pro Pro Lys Gly Tyr Leu Glu Lys Leu Arg 225 230 235 240 Glu Ile Thr Ala Arg His Gly Ile Leu Leu Ile Phe Asp Glu Val Ile 245 250 255 Thr Ala Tyr Gly Arg Leu Gly Glu Ala Thr Ala Ala Ala Tyr Phe Gly 260 265 270 Val Thr Pro Asp Leu Ile Thr Met Ala Lys Gly Val Ser Asn Ala Ala 275 280 285 Val Pro Ala Gly Ala Val Ala Val Arg Arg Glu Val His Asp Ala Ile 290 295 300 Val Asn Gly Pro Gln Gly Gly Ile Glu Phe Phe His Gly Tyr Thr Tyr 305 310 315 320 Ser Ala His Pro Leu Ala Ala Ala Ala Val Leu Ala Thr Leu Asp Ile 325 330 335 Tyr Arg Arg Glu Asp Leu Phe Ala Arg Ala Arg Lys Leu Ser Ala Ala 340 345 350 Phe Glu Glu Ala Ala His Ser Leu Lys Gly Ala Pro His Val Ile Asp 355 360 365 Val Arg Asn Ile Gly Leu Val Ala Gly Ile Glu Leu Ser Pro Arg Glu 370 375 380 Gly Ala Pro Gly Ala Arg Ala Ala Glu Ala Phe Gln Lys Cys Phe Asp 385 390 395 400 Thr Gly Leu Met Val Arg Tyr Thr Gly Asp Ile Leu Ala Val Ser Pro 405 410 415 Pro Leu Ile Val Asp Glu Asn Gln Ile Gly Gln Ile Phe Glu Gly Ile 420 425 430 Gly Lys Val Leu Lys Glu Val Ala 435 440 881947DNAAlcaligenes denitrificans 88ttcgatggcg cgctgcacgg cggccaccag ctgctccacc aggggtgggc gcctgcccgc 60gcgcgcggtc gggctggaaa tcgatcatgg atgaatctat acagttgtca tgattgcaac 120tatacagtta gcccgttttg cggcaattgt atattttcat tcgctcgtgg acgtccgaga 180atcggtttga tcgcgccgcc cgcccctttc cgcgcagcgg cgtttctttt cctccggagt 240ctccccatga gcgctgccaa actgcccgac ctgtcccacc tctggatgcc ctttaccgcc 300aaccggcagt tcaaggcgaa cccccgcctg ctggcctcgg ccaagggcat gtactacacg 360tctttcgacg gccgccagat cctggacggc acggccggcc tgtggtgcgt gaacgccggc 420cactgccgcg aagaaatcgt ctccgccatc gccagccagg ccggcgtcat ggactacgcg 480ccggggttcc agctcggcca cccgctggcc ttcgaggccg ccaccgccgt ggccggcctg 540atgccgcagg gcctggaccg cgtgttcttc accaattcgg gctccgaatc ggtggacacc 600gcgctgaaga tcgccctggc ctaccaccgc gcgcgcggcg aggcgcagcg cacccgcctc 660atcgggcgcg agcgcggcta ccacggcgtg ggcttcggcg gcatttccgt gggcggcatc 720tcgcccaacc gcaagacctt ctccggcgcg ctgctgccgg ccgtggacca cctgccgcac 780acccacagcc tggaacacaa cgccttcacg cgcggccagc ccgagtgggg cgcgcacctg 840gccgacgagt tggaacgcat catcgccctg cacgacgcct ccaccatcgc ggccgtgatc 900gtcgagccca tggccggctc caccggcgtg ctcgtcccgc ccaagggcta tctcgaaaaa 960ctgcgcgaaa tcaccgcccg ccacggcatt ctgctgatct tcgacgaagt catcaccgcg 1020tacggccgcc tgggcgaggc caccgccgcg gcctatttcg gcgtaacgcc cgacctcatc 1080accatggcca agggcgtgag caacgccgcc gttccggccg gcgccgtcgc ggtgcgccgc 1140gaagtgcatg acgccatcgt caacggaccg caaggcggca tcgagttctt ccacggctac 1200acctactcgg cccacccgct ggccgccgcc gccgtgctcg ccacgctgga catctaccgc 1260cgcgaagacc tgttcgcccg cgcccgcaag ctgtcggccg cgttcgagga agccgcccac 1320agcctcaagg gcgcgccgca cgtcatcgac gtgcgcaaca tcggcctggt ggccggcatc 1380gagctgtcgc cgcgcgaagg cgccccgggc gcgcgcgccg ccgaagcctt ccagaaatgc 1440ttcgacaccg gcctcatggt gcgctacacg ggcgacatcc tcgcggtgtc gcctccgctc 1500atcgtcgacg aaaaccagat cggccagatc ttcgagggca tcggcaaggt gctcaaggaa 1560gtggcttagg gtgaacacgc cctgagccgg ccccggcagg aaacgcgccg ccgcgcggcg 1620gcgcgtccat cgaactcccg catcgagctt ttgcattcat gaagaaaatc acgcatttca 1680tcaacggcca gccccacgaa ggccgcagca accgctacac cgagggcttc aacccggcca 1740cgggcgagtc gtctcctcga tctgcctggg cggggccgaa gaagtggacc tggccgtggc 1800ggccgcccgc gcggcctttc ccgcctggtc cgaaacgccg gcgctcaagc gcgcgcgcgt 1860gctgttcaac ttcaaggcgc tgctggacaa gcaccaggac gagctggccg cgctcatcac 1920gcgcgagcac ggcaaggtgt tttccga 194789443PRTRalstonia eutropha 89Met Asp Ala Ala Lys Thr Val Ile Pro Asp Leu Asp Ala Leu Trp Met 1 5 10 15 Pro Phe Thr Ala Asn Arg Gln Tyr Lys Ala Ala Pro Arg Leu Leu Ala 20 25 30 Ser Ala Ser Gly Met Tyr Tyr Thr Thr His Asp Gly Arg Gln Ile Leu 35 40 45 Asp Gly Cys Ala Gly Leu Trp Cys Val Ala Ala Gly His Cys Arg Lys 50 55 60 Glu Ile Ala Glu Ala Val Ala Arg Gln Ala Ala Thr Leu Asp Tyr Ala 65 70 75 80 Pro Pro Phe Gln Met Gly His Pro Leu Ser Phe Glu Ala Ala Thr Lys 85 90 95 Val Ala Ala Ile Met Pro Gln Gly Leu Asp Arg Ile Phe Phe Thr Asn 100 105 110 Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Ile Ala Leu Ala Tyr 115 120 125 His Arg Ala Arg Gly Glu Gly Gln Arg Thr Arg Phe Ile Gly Arg Glu 130 135 140 Arg Gly Tyr His Gly Val Gly Phe Gly Gly Met Ala Val Gly Gly Ile 145 150 155 160 Gly Pro Asn Arg Lys Ala Phe Ser Ala Asn Leu Met Pro Gly Thr Asp 165 170 175 His Leu Pro Ala Thr Leu Asn Ile Ala Glu Ala Ala Phe Ser Lys Gly 180 185 190 Gln Pro Thr Trp Gly Ala His Leu Ala Asp Glu Leu Glu Arg Ile Val 195 200 205 Ala Leu His Asp Pro Ser Thr Ile Ala Ala Val Ile Val Glu Pro Leu 210 215 220 Ala Gly Ser Ala Gly Val Leu Val Pro Pro Val Gly Tyr Leu Asp Lys 225 230 235 240 Leu Arg Glu Ile Thr Thr Lys His Gly Ile Leu Leu Ile Phe Asp Glu 245 250 255 Val Ile Thr Ala Phe Gly Arg Leu Gly Thr Ala Thr Ala Ala Glu Arg 260 265 270 Phe Lys Val Thr Pro Asp Leu Ile Thr Met Ala Lys Ala Ile Asn Asn 275 280 285 Ala Ala Val Pro Met Gly Ala Val Ala Val Arg Arg Glu Val His Asp 290 295 300 Thr Val Val Asn Ser Ala Ala Pro Gly Ala Ile Glu Leu Ala His Gly 305 310 315 320 Tyr Thr Tyr Ser Gly His Pro Leu Ala Ala Ala Ala Ala Ile Ala Thr 325 330 335 Leu Asp Leu Tyr Gln Arg Glu Asn Leu Phe Gly Arg Ala Ala Glu Leu 340 345 350 Ser Pro Val Phe Glu Ala Ala Val His Ser Val Arg Ser Ala Pro His 355 360 365 Val Lys Asp Ile Arg Asn Leu Gly Met Val Ala Gly Ile Glu Leu Glu 370 375 380 Pro Arg Pro Gly Gln Pro Gly Ala Arg Ala Tyr Glu Ala Phe Leu Lys 385 390 395 400 Cys Leu Glu Arg Gly Val Leu Val Arg Tyr Thr Gly Asp Ile Leu Ala 405 410 415 Phe Ser Pro Pro Leu Ile Ile Ser Glu Ala Gln Ile Ala Glu Leu Phe 420 425 430 Asp Thr Val Lys Gln Ala Leu Gln Glu Val Gln 435 440 901341DNARalstonia eutropha 90atggccgact cacccaacaa cctcgctcac gaacatcctt cacttgaaca ctattggatg 60ccttttaccg ccaatcgcca attcaaagcg agccctcgtt tactcgccca agctgaaggt 120atgtattaca cagatatcaa tggcaacaag gtattagact ctacagcggg cttatggtgt 180tgtaatgctg gccatggtcg ccgtgagatc agtgaagccg tcagcaaaca aattcggcag 240atggattacg ctccctcctt ccaaatgggc catcccatcg cttttgaact ggccgaacgt 300ttaaccgaac tcagcccaga aggactcaac aaagtattct ttaccaactc aggctctgag 360tcggttgata ccgcgctaaa aatggctctt tgctaccata gagccaatgg ccaagcgtca 420cgcacccgct ttattggccg tgaaatgggt taccatggcg taggatttgg tgggatctcg 480gtgggtggtt taagcaataa ccgtaaagcc ttcagcggcc agctattgca aggcgtggat 540cacctgcccc acaccttaga cattcaacat gccgccttta gtcgtggctt accgagcctc 600ggtgctgaaa aagctgaggt attagaacaa ttagtcacac tccatggcgc cgaaaatatt 660gccgccgtta ttgttgaacc catgtcaggt tctgcagggg taattttacc acctcaaggc 720tacttaaaac gcttacgtga aatcactaaa aaacacggca tcttattgat tttcgatgaa 780gtcattaccg catttggccg tgtaggtgca gcattcgcca gccaacgttg gggcgttatt 840ccagacataa tcaccacggc taaagccatt aataatggcg ccatccccat gggcgcagtg 900tttgtacagg attatatcca cgatacttgc atgcaagggc caaccgaact gattgaattt 960ttccacggtt atacctattc gggccaccca gtcgccgcag cagcagcact cgccacgctc 1020tccatctacc aaaacgagca actgtttgag cgcagttttg agcttgagcg gtatttcgaa 1080gaagccgttc atagcctcaa agggttaccg aatgtgattg atattcgcaa caccggatta 1140gtcgcgggtt tccagctagc accgaatagc caaggtgttg gtaaacgcgg atacagcgtg 1200ttcgagcatt gtttccatca aggcacactc gtgcgggcaa cgggcgatat tatcgccatg 1260tccccaccac tcattgttga gaaacatcag attgaccaaa tggtaaatag ccttagcgat 1320gcaattcacg ccgttggatg a 134191446PRTShewanella oneidensis 91Met Ala Asp Ser Pro Asn Asn Leu Ala His Glu His Pro Ser Leu Glu 1 5 10 15 His Tyr Trp Met Pro Phe Thr Ala Asn Arg Gln Phe Lys Ala Ser Pro 20 25 30 Arg Leu Leu Ala Gln Ala Glu Gly Met Tyr Tyr Thr Asp Ile Asn Gly 35 40 45 Asn Lys Val Leu Asp Ser Thr Ala Gly Leu Trp Cys Cys Asn Ala Gly 50 55 60 His Gly Arg Arg Glu Ile Ser Glu Ala Val Ser Lys Gln Ile Arg Gln 65 70 75 80 Met Asp Tyr Ala Pro Ser Phe Gln Met Gly His Pro Ile Ala Phe Glu 85 90 95 Leu Ala Glu Arg Leu Thr Glu Leu Ser Pro Glu Gly Leu Asn Lys Val 100 105 110 Phe Phe Thr Asn Ser Gly Ser Glu Ser Val Asp Thr Ala Leu Lys Met 115 120 125 Ala Leu Cys Tyr His Arg Ala Asn Gly Gln Ala Ser Arg Thr Arg Phe 130 135 140 Ile Gly Arg Glu Met Gly Tyr His Gly Val Gly Phe Gly Gly Ile Ser 145 150 155 160 Val Gly Gly Leu Ser Asn Asn Arg Lys Ala Phe Ser Gly Gln Leu Leu 165 170 175 Gln Gly Val Asp His Leu Pro His Thr Leu Asp Ile Gln His Ala Ala 180 185 190 Phe Ser Arg Gly Leu Pro Ser Leu Gly Ala Glu Lys Ala Glu Val Leu 195 200 205 Glu Gln Leu Val Thr Leu His Gly Ala Glu Asn Ile Ala Ala Val Ile 210 215 220 Val Glu Pro Met Ser Gly Ser Ala Gly Val Ile Leu Pro Pro Gln Gly 225 230 235 240 Tyr Leu Lys Arg Leu Arg Glu Ile Thr Lys Lys His Gly Ile Leu Leu 245 250 255 Ile Phe Asp Glu Val Ile Thr Ala Phe Gly Arg Val Gly Ala Ala Phe 260 265 270 Ala Ser Gln Arg Trp Gly Val Ile Pro Asp Ile Ile Thr Thr Ala Lys 275 280 285 Ala Ile Asn Asn Gly Ala Ile Pro Met Gly Ala Val Phe Val Gln Asp 290 295 300 Tyr Ile His Asp Thr Cys Met Gln Gly Pro Thr Glu Leu Ile Glu Phe 305 310 315 320 Phe His Gly Tyr Thr Tyr Ser Gly His Pro Val Ala Ala Ala Ala Ala 325 330 335 Leu Ala Thr Leu Ser Ile Tyr Gln Asn Glu Gln Leu Phe Glu Arg Ser 340 345 350 Phe Glu Leu Glu Arg Tyr Phe Glu Glu Ala Val His Ser Leu Lys Gly 355 360 365 Leu Pro Asn Val Ile Asp Ile Arg Asn Thr Gly Leu Val Ala Gly Phe 370 375 380 Gln Leu Ala Pro Asn Ser Gln Gly Val Gly Lys Arg Gly Tyr Ser Val 385 390 395 400 Phe Glu His Cys Phe His Gln Gly Thr Leu Val Arg Ala Thr Gly Asp 405 410 415 Ile Ile Ala Met Ser Pro Pro Leu Ile Val Glu Lys His Gln Ile Asp 420 425 430 Gln Met Val Asn Ser Leu Ser Asp Ala Ile His Ala Val Gly 435 440 445 921341DNAShewanella oneidensis 92atggccgact cacccaacaa cctcgctcac gaacatcctt cacttgaaca ctattggatg 60ccttttaccg ccaatcgcca attcaaagcg agccctcgtt tactcgccca agctgaaggt 120atgtattaca cagatatcaa tggcaacaag gtattagact ctacagcggg cttatggtgt 180tgtaatgctg gccatggtcg ccgtgagatc agtgaagccg tcagcaaaca aattcggcag 240atggattacg ctccctcctt ccaaatgggc catcccatcg cttttgaact ggccgaacgt 300ttaaccgaac tcagcccaga aggactcaac aaagtattct ttaccaactc aggctctgag 360tcggttgata ccgcgctaaa aatggctctt tgctaccata gagccaatgg ccaagcgtca 420cgcacccgct ttattggccg tgaaatgggt taccatggcg taggatttgg tgggatctcg 480gtgggtggtt taagcaataa ccgtaaagcc ttcagcggcc agctattgca aggcgtggat 540cacctgcccc acaccttaga cattcaacat gccgccttta gtcgtggctt accgagcctc 600ggtgctgaaa aagctgaggt attagaacaa ttagtcacac tccatggcgc cgaaaatatt 660gccgccgtta ttgttgaacc catgtcaggt tctgcagggg taattttacc acctcaaggc 720tacttaaaac gcttacgtga aatcactaaa aaacacggca tcttattgat tttcgatgaa 780gtcattaccg catttggccg tgtaggtgca gcattcgcca gccaacgttg gggcgttatt 840ccagacataa tcaccacggc taaagccatt aataatggcg ccatccccat gggcgcagtg 900tttgtacagg attatatcca cgatacttgc atgcaagggc caaccgaact gattgaattt 960ttccacggtt atacctattc gggccaccca gtcgccgcag cagcagcact cgccacgctc 1020tccatctacc aaaacgagca actgtttgag cgcagttttg agcttgagcg gtatttcgaa 1080gaagccgttc atagcctcaa agggttaccg aatgtgattg atattcgcaa caccggatta 1140gtcgcgggtt tccagctagc accgaatagc caaggtgttg gtaaacgcgg atacagcgtg 1200ttcgagcatt gtttccatca aggcacactc gtgcgggcaa cgggcgatat tatcgccatg 1260tccccaccac tcattgttga gaaacatcag attgaccaaa tggtaaatag ccttagcgat 1320gcaattcacg ccgttggatg a 134193448PRTPseudomonas putida 93Met Asn Met Pro Glu Thr Gly Pro Ala Gly Ile Ala Ser Gln Leu Lys 1 5 10 15 Leu Asp Ala His Trp Met Pro Tyr Thr Ala Asn Arg Asn Phe Gln Arg 20 25 30 Asp Pro Arg Leu Ile Val Ala Ala Glu Gly Asn Tyr Leu Val Asp Asp 35 40 45 His Gly Arg Lys Ile Phe Asp Ala Leu Ser Gly Leu Trp Thr Cys Gly 50 55 60 Ala Gly His Thr Arg Lys Glu Ile Ala Asp Ala Val Thr Arg Gln Leu 65 70 75 80 Ser Thr Leu Asp Tyr Ser Pro Ala Phe Gln Phe Gly His Pro Leu Ser 85 90 95 Phe Gln Leu Ala Glu Lys Ile Ala Glu Leu Val Pro Gly Asn Leu Asn 100 105 110 His Val Phe Tyr Thr Asn Ser Gly Ser Glu Cys Ala Asp Thr Ala Leu 115 120 125 Lys Met Val Arg Ala Tyr Trp Arg Leu Lys Gly Gln Ala Thr Lys Thr 130 135 140 Lys Ile Ile Gly Arg Ala Arg Gly Tyr His Gly Val Asn Ile Ala Gly 145 150 155 160 Thr Ser Leu Gly Gly Val Asn Gly Asn Arg Lys Met Phe Gly Gln Leu 165 170 175 Leu Asp Val Asp His Leu Pro His Thr Val Leu Pro Val Asn Ala Phe 180 185 190 Ser Lys Gly Leu Pro Glu Glu Gly Gly Ile Ala Leu Ala Asp Glu Met 195 200 205 Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala Val Ile 210 215 220 Val Glu Pro Leu Ala Gly Ser Ala Gly Val Leu Pro Pro Pro Lys Gly 225 230 235 240 Tyr Leu Lys Arg Leu Arg Glu Ile Cys Thr Gln His Asn Ile Leu Leu 245 250

255 Ile Phe Asp Glu Val Ile Thr Gly Phe Gly Arg Met Gly Ala Met Thr 260 265 270 Gly Ser Glu Ala Phe Gly Val Thr Pro Asp Leu Met Cys Ile Ala Lys 275 280 285 Gln Val Thr Asn Gly Ala Ile Pro Met Gly Ala Val Ile Ala Ser Ser 290 295 300 Glu Ile Tyr Gln Thr Phe Met Asn Gln Pro Thr Pro Glu Tyr Ala Val 305 310 315 320 Glu Phe Pro His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala Cys Ala 325 330 335 Ala Gly Leu Ala Ala Leu Asp Leu Leu Gln Lys Glu Asn Leu Val Gln 340 345 350 Ser Ala Ala Glu Leu Ala Pro His Phe Glu Lys Leu Leu His Gly Val 355 360 365 Lys Gly Thr Lys Asn Ile Val Asp Ile Arg Asn Tyr Gly Leu Ala Gly 370 375 380 Ala Ile Gln Ile Ala Ala Arg Asp Gly Asp Ala Ile Val Arg Pro Tyr 385 390 395 400 Glu Ala Ala Met Lys Leu Trp Lys Ala Gly Phe Tyr Val Arg Phe Gly 405 410 415 Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Thr Lys Pro Gln Glu 420 425 430 Leu Asp Arg Leu Phe Asp Ala Val Gly Glu Thr Leu Asn Leu Ile Asp 435 440 445 94930DNAPseudomonas putida 94atgaccacga agaaagctga ttacatttgg ttcaatgggg agatggttcg ctgggaagac 60gcgaaggtgc atgtgatgtc gcacgcgctg cactatggca cttcggtttt tgaaggcatc 120cgttgctacg actcgcacaa aggaccggtt gtattccgcc atcgtgagca tatgcagcgt 180ctgcatgact ccgccaaaat ctatcgcttc ccggtttcgc agagcattga tgagctgatg 240gaagcttgtc gtgacgtgat ccgcaaaaac aatctcacca gcgcctatat ccgtccgctg 300atcttcgtcg gtgatgttgg catgggagta aacccgccag cgggatactc aaccgacgtg 360attatcgctg ctttcccgtg gggagcgtat ctgggcgcag aagcgctgga gcaggggatc 420gatgcgatgg tttcctcctg gaaccgcgca gcaccaaaca ccatcccgac ggcggcaaaa 480gccggtggta actacctctc ttccctgctg gtgggtagcg aagcgcgccg ccacggttat 540caggaaggta tcgcgctgga tgtgaacggt tatatctctg aaggcgcagg cgaaaacctg 600tttgaagtga aagatggtgt gctgttcacc ccaccgttca cctcctccgc gctgccgggt 660attacccgtg atgccatcat caaactggcg aaagagctgg gaattgaagt acgtgagcag 720gtgctgtcgc gcgaatccct gtacctggcg gatgaagtgt ttatgtccgg tacggcggca 780gaaatcacgc cagtgcgcag cgtagacggt attcaggttg gcgaaggccg ttgtggcccg 840gttaccaaac gcattcagca agccttcttc ggcctcttca ctggcgaaac cgaagataaa 900tggggctggt tagatcaagt taatcaataa 93095566PRTStreptomyces cinnamonensis 95Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Lys Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly 20 25 30 Asp Pro Val Asp Pro Val Tyr Gly Pro Arg Pro Gly Asp Thr Tyr Asp 35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly 50 55 60 Leu Tyr Ala Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile 85 90 95 Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr 100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly 115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe 130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln 165 170 175 Gly Val Asp Pro Ala Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe 180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His 195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Arg Asp Ile Pro 210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala 260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu 275 280 285 Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg 290 295 300 Asp Glu Tyr Gly Ala Lys Thr Glu Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr 340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser 355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu 370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr 385 390 395 400 Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe 405 410 415 Glu Gln Ile Arg Glu Arg Gly Arg Arg Ala Cys Pro Asp Gly Gln His 420 425 430 Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly 435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Arg Ser 450 455 460 Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Cys Leu Glu Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu 485 490 495 Arg Glu Gln Val Arg Glu Leu Ala Gly Arg Lys Gly Arg Arg Asp Asp 500 505 510 Ala Arg Val Arg Ala Ser Leu Asp Ala Met Leu Ala Ala Ala Arg Asp 515 520 525 Gly Ser Asn Met Ile Ala Pro Met Leu Glu Ala Val Arg Ala Glu Ala 530 535 540 Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr 545 550 555 560 Val Glu Pro Pro Gly Phe 565 964362DNAStreptomyces cinnamonensis 96tgaggcgctg gatcgcctcg gagagcagct ggtaacggtc cgcgtggtac tcggccgggg 60tgcagccgtc cacgatgtgc gggatcgcgt cgggctcgag gatcaccagg gcgggggcgt 120cgccgatcgc gtcggcgaac gtgtccaccc agctccggta ggcctccgca ctggccgcgc 180cgcccgcgga gtgctgaccg cagtcgcggt gcgggatgtt gtacgcgacg agtacggcgg 240tgcggtcctc cttgaccgcg ccccgcgtcg ccttcgcgac gtcgggcgcc ggatcgtccc 300cggccggcca cacggccatg gcccgttcgg agatgcgcct gagcgtctcg gcgtcctcgg 360cgcggccctg ttcctcccac tgcctgacct ggcgcgcggc ggggctgtcg gggtcgaccc 420agaaggtgcc ggcggggggc ccggcgctcg cggtggcggg cttgcgcacg gccgcctcct 480ccttcgtgcc gtcggacccc gggtctgagg aggagcagcc tgccgggagc ccgagggcgg 540cgagggccgc gagtgccgtg aacgtgcgga gcagccggtg catccagccc ccttgggcga 600tggtgacagt gacggtcagt cagcccggca atcgttacat aaaggactat tcaagctctt 660gtgccacacc gcctccggtg ccgagcgcga acccggcggg caccagagcc ccgccgcggc 720cgcggagccg tacgtacgac cgaattgcga gacggggctg accaccatat gaccggcggg 780taaggtcgat gccgtgccga agccgctcag cctccccttc gatcccatcg cccgcgccga 840cgagctctgg aagcagcgct ggggatcggt cccggccatg ggcgcgatca cctcgatcat 900gcgggcgcac cagatcctgc tcgccgaggt cgacgcggtc gtcaagccgt acggactgac 960cttcgcgcgc tacgaggcgc tggtgctcct caccttctcg caggccggcg agttgccgat 1020gtcgaagatc ggcgagcggc tcatggtgca cccgacctcg gtcacgaaca ccgtggaccg 1080cctggtgaag tccggcctgg tcgacaagcg cccgaacccc aacgacggcc gcggcacgct 1140cgcctccatc acggagaagg gccgcgaggt cgtcgaggcg gccacccgcg agctgatggc 1200gatggacttc gggctcgggg tgtacgacgc ggaggagtgc ggggagatct tcgcgatgct 1260gcggcccctg cgggtggcgg cgcgcgattt cgaggagcag tagggcccgc ccggtgagaa 1320gtgggatcgg gtcgtcccgg tacgggcggg ggcggcgaag atcgcgtgaa aagggcggtt 1380acgctcgtag ccatgaaacg cagcgtgctg acccgctacc gggtgatggc ctacgtcacc 1440gccgtcatgc tcctcatcct gtgcgcctgc atggtggcca agtacggctt cgacaagggc 1500gagggtctga ccctcgtcgt gtcgcaggtg cacggcgtgc tctacatcat ctacctgatc 1560ttcgccttcg acctgggctc caaggcgaag tggccgttcg gcaagctgct ctgggtgctg 1620gtctcgggca cgatcccgac cgccgccttc ttcgtcgagc gcaaggtcgc ccgtgacgtc 1680gagccgctga tcgccgacgg ctccccggtc accgcgaagg cgtaacccgc accgccacgg 1740acaggtccgt ggcggttggc catcgacttt tactaggacg tcctagtaaa ttcgatggta 1800tggacgctga cgcgatcgag gaaggccgcc gacgctggca ggcccgttac gacaaggccc 1860gcaagcgcga cgcggacttc accacgctct ccggggaccc cgtcgacccc gtctacggcc 1920cccggcccgg ggacacgtac gacgggttcg agcggatcgg ctggccgggg gagtacccct 1980tcacccgcgg gctctacgcc accgggtacc gcggccgcac ctggaccatc cgccagttcg 2040ccggcttcgg caacgccgag cagacgaacg agcgctacaa gatgatcctg gccaacggcg 2100gcggcggcct ctccgtcgcc ttcgacatgc cgaccctcat gggccgcgac tccgacgacc 2160cgcgctcgct cggcgaggtc ggccactgcg gtgtcgccat cgactccgcc gccgacatgg 2220aggtcctctt caaggacatc ccgctcggcg acgtcacgac gtccatgacc atcagcgggc 2280ccgccgtgcc cgtcttctgc atgtacctcg tcgcggccga gcgccagggc gtcgacccgg 2340ccgtcctcaa cggcacgctg cagaccgaca tcttcaagga gtacatcgcc cagaaggagt 2400ggctcttcca gcccgagccg cacctgcgcc tcatcggcga cctgatggag cactgcgcgc 2460gcgacatccc cgcgtacaag ccgctctcgg tctccggcta ccacatccgc gaggccgggg 2520cgacggccgc gcaggagctc gcgtacaccc tcgcggacgg cttcgggtac gtggaactgg 2580gcctctcgcg cggcctggac gtggacgtct tcgcgcccgg cctctccttc ttcttcgacg 2640cgcacgtcga cttcttcgag gagatcgcga agttccgcgc cgcacgccgc atctgggcgc 2700gctggctccg ggacgagtac ggagcgaaga ccgagaaggc acagtggctg cgcttccaca 2760cgcagaccgc gggggtctcg ctcacggccc agcagccgta caacaacgtg gtgcggacgg 2820cggtggaggc cctcgccgcg gtgctcggcg gcacgaactc cctgcacacc aacgctctcg 2880acgagaccct tgccctcccc agcgagcagg ccgcggagat cgcgctgcgc acccagcagg 2940tgctgatgga ggagaccggc gtcgccaacg tcgcggaccc gctgggcggc tcctggtaca 3000tcgagcagct caccgaccgc atcgaggccg acgccgagaa gatcttcgag cagatcaggg 3060agcgggggcg gcgggcctgc cccgacgggc agcacccgat cgggccgatc acctccggca 3120tcctgcgcgg catcgaggac ggctggttca ccggcgagat cgccgagtcc gccttccagt 3180accagcggtc cctggagaag ggcgacaagc gggtcgtcgg cgtcaactgc ctcgaaggct 3240ccgtcaccgg cgacctggag atcctgcgcg tcagccacga ggtcgagcgc gagcaggtgc 3300gggagcttgc ggggcgcaag gggcggcgtg acgatgcgcg ggtgcgggcc tcgctcgacg 3360cgatgctcgc cgctgcgcgg gacgggtcga acatgattgc ccccatgctg gaggcggtgc 3420gggccgaggc gaccctcggg gagatctgcg gggtgcttcg cgatgagtgg ggggtctacg 3480tggagccgcc cgggttctga gggcgcgctc cctttgcctg cgggtctgct gtggctggtc 3540gcgcagttcc ccgcacccct gaaagacccc ggcgctttcc cttcctggct cgcctcgtcg 3600ctgtctgcgg ggccgtgggg gctggtcgcg cagttccccg cgcccctgcc cgcacctgcg 3660ccccgccgcc tgcatgccgc ccccaccctg acgggggcgt tcggggccca ccctgacggg 3720tgcggtcggg gcgtgccggg gtcttttagg ggcgcgggga actgcgcgag caacccccac 3780ccacccgcag gtgcacgcgg agcggcggac gccccgcaga cgggggcaaa acgggcggag 3840tgcccccgcc cgccgggcgg cgcgaattcg taggtttaag gggcaggggt cagggcaggc 3900gccgagccgg tcaaccgccc ccgtcccagg agaccccgtg acctcgaccg gccacgcccg 3960caccgccgcc atcgccatcg gagccgccac cgccaccgtc ctcggcgcgc tgctggtcgg 4020cggctccggc gaggtgagtg cgagcccgcc gcccgagccc aaggtccagg acgacttcga 4080ctccctcggc cccgaggtgc gcgccgcgaa gctctccgac gggcggacgg cccactactc 4140ggacacgggc gacaaggacg gcaagccggc cctgttcatc ggcggcaccg gcacgagcgc 4200ccgcgcctcc cacatgaccg acttcttccg ctcgacgcgc gaggacctgg gcctgcgcct 4260catctccgtg gagcgcaacg gcttcggcga caccgcgttc gacgagaagc tgggcaccgc 4320cgacttcgcg aaggacgccc tcgaagtcct cgaccggctc gg 436297136PRTStreptomyces cinnamonensis 97Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg 20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu 35 40 45 Gln Val Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu 50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Arg Val Leu Glu 65 70 75 80 Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala 100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Thr Ser Ile Val Glu Trp Val 115 120 125 Arg Gly Asn Val Arg Gln Ala Val 130 135 981643DNAStreptomyces cinnamonensis 98gtcgacctcc cgtttggcgc acggaaggga ggctctgtcc cccgtgtgcc ctagggggag 60tcgtggtcga ggagtcggct gtgcgatggc gatcccggcc accgccctgc ggtgactccg 120tgcccccgtt gcatcgccga tgcgcggtgt caccacgccg tgcggctgcc ggcgcggtgg 180cccggcgtct cgttgcggct cccctcgcgc ctggtccgga tgcggagcgt gaacccctgg 240gttacggacg ggcgcgcagc gaacgtgtcc cacgtgtgat ttccccctcg ctctccaccg 300cgaaactgcc gcttgcgcga tgctggggat aacgttcgtt cacttccccg gccggtgcgg 360tgcggggtat ctgtgccggg acagactttg tcggtacgga tatcggtaca tggaggcagt 420gatgggtgtg gcagccgggc cgatccgcgt ggtggtcgcc aagccggggc tcgacgggca 480cgatcgcggg gccaaggtga tcgcgcgggc gttgcgtgac gcgggtatgg aggtcatcta 540caccgggctg caccagacgc ccgagcaggt ggtggacacc gcgatccagg aggacgccga 600cgcgatcggc ctctccatcc tctccggagc gcacaacacg ctgttcgcgc gcgtgttgga 660gctcttgaag gagcgggacg cggaggacat caaggtgttt ggtggcggca tcatcccgga 720ggcggacatc gcgccgctga aggagaaggg cgtcgcggag atcttcacgc ccggggccac 780caccacgtcg atcgtggagt gggttcgggg gaacgtgcga caggccgtct gaggcattcc 840ccgtcgcccg tctgccgtgg tcggcgtcat atcggcggac atcgtctcgg tggacgtcat 900ggcggcgggg ggagttcgtc gcgtatcgcc gcgcggaggc gcagggtggt gaccaggcgc 960tggaacgctt ccgaccagta gctgcccgcg ccgggtgacg cgtcctccgc ttcgtcgggg 1020accgcggtga gcgcttccag gcggaccgcc tcggccgggt ccagacagcg ttccgccagg 1080cccatcactc cgctgaagct ccatgggtaa ctgcccgcgt cgcgcgcgat gttcagggcg 1140tccaccacgg cccggccgag agggccggcc cagggcaccg cgcagacgcc gagcagttgg 1200aacgcctccg acaggccgtg tgccgctatg aaccccgcca cccagtccgc gcgctcggcg 1260gcaggcatgg aggcgagcag tttggcccgc tcggcgaggg acacggcgcc aggccccgcc 1320gcgtcgggtg aggcgggggc gccgagcagc gctctggacc aggcgacgtc acgctggcgt 1380acggccgcgc ggcaccatgc ggcgtgcagt tcgccccgcc agtcgtcggc caccgggagc 1440gccacgatct ccgccggggt gcggttgccg agccggggcg gccaggtggc gagcggggcc 1500gattccacga gctggccgag ccaccaggag cgctcgcccc ggccggtggg gggcttcggg 1560acgacgccgt cccgctccat gcccgcgtcg cactcgtgcg gcgcctcgac ggtgagggtc 1620ggcgtgctcg atgtgtggtc gac 164399566PRTStreptomyces coelicolor 99Met Asp Ala His Ala Ile Glu Glu Gly Arg Leu Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Ala Ala Arg Lys Arg Asp Ala Asp Phe Thr Thr Leu Ser Gly 20 25 30 Asp Pro Val Glu Pro Val Tyr Gly Pro Arg Pro Gly Asp Glu Tyr Glu 35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly 50 55 60 Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Met Ile 85 90 95 Leu Arg Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr 100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Pro Arg Ser Leu Gly Glu Val Gly 115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe 130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln 165 170 175 Gly Val Asp Ala Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe 180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His 195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu Tyr Cys Ala Ala Gly Ile Pro 210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala 260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Leu Asp Phe Phe Glu Glu 275 280 285 Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Met Arg 290 295 300

Asp Val Tyr Gly Ala Arg Thr Asp Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr 340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser 355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu 370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Phe 385 390 395 400 Ile Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe 405 410 415 Glu Gln Ile Lys Glu Arg Gly Leu Arg Ala His Pro Asp Gly Gln His 420 425 430 Pro Val Gly Pro Ile Thr Ser Gly Leu Leu Arg Gly Ile Glu Asp Gly 435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Arg Tyr Gln Gln Ser 450 455 460 Leu Glu Lys Asp Asp Lys Lys Val Val Gly Val Asn Val His Thr Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu 485 490 495 Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Asp Ala Arg Asp Asp 500 505 510 Ala Ala Val Arg Gly Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Ser 515 520 525 Gly Gly Asn Met Ile Gly Pro Met Leu Asp Ala Val Arg Ala Glu Ala 530 535 540 Thr Leu Gly Glu Ile Cys Gly Val Leu Arg Asp Glu Trp Gly Val Tyr 545 550 555 560 Thr Glu Pro Ala Gly Phe 565 1001701DNAStreptomyces coelicolor 100atggacgctc atgccataga ggagggccgc cttcgctggc aggcccggta cgacgcggcg 60cgcaagcgcg acgcggactt caccacgctc tccggagacc ccgtggagcc ggtgtacggg 120ccccgccccg gggacgagta cgagggcttc gagcggatcg gctggccggg cgagtacccc 180ttcacccgcg gcctgtatcc gaccgggtac cgggggcgta cgtggaccat ccggcagttc 240gccgggttcg gcaacgccga gcagaccaac gagcgctaca agatgatcct ccgcaacggc 300ggcggcgggc tctcggtcgc cttcgacatg ccgaccctga tgggccgcga ctccgacgac 360ccgcgctcgc tgggcgaggt cgggcactgc ggggtggcca tcgactcggc cgccgacatg 420gaagtgctgt tcaaggacat cccgctcggg gacgtgacga cctccatgac gatcagcggg 480cccgccgtgc ccgtgttctg catgtacctc gtcgccgccg agcgccaggg cgtcgacgca 540tccgtgctca acggcacgct gcagaccgac atcttcaagg agtacatcgc ccagaaggag 600tggctcttcc agcccgagcc ccacctccgg ctcatcggcg acctcatgga gtactgcgcg 660gccggcatcc ccgcctacaa gccgctctcc gtctccggct accacatccg cgaggcgggc 720gcgacggccg cgcaggagct ggcgtacacg ctcgccgacg gcttcggata cgtggagctg 780ggcctcagcc gcgggctcga cgtggacgtc ttcgcgcccg gcctctcctt cttcttcgac 840gcgcacctcg acttcttcga ggagatcgcc aagttccgcg cggcccgcag gatctgggcc 900cgctggatgc gcgacgtgta cggcgcgcgg accgacaagg cccagtggct gcggttccac 960acccagaccg ccggagtctc gctcaccgcg cagcagccgt acaacaacgt cgtacgcacc 1020gcggtggagg cgctggcggc cgtgctcggc ggcaccaact ccctgcacac caacgcgctc 1080gacgagaccc tcgccctgcc cagcgagcag gccgccgaga tcgccctgcg cacccagcag 1140gtgctgatgg aggagaccgg cgtcgccaac gtcgccgacc cgctgggcgg ttcctggttc 1200atcgagcagc tgaccgaccg catcgaggcc gacgccgaga agatcttcga gcagatcaag 1260gagcgggggc tgcgcgccca ccccgacggg cagcaccccg tcggaccgat cacctccggc 1320ctgctgcgcg gcatcgagga cggctggttc accggcgaga tcgccgagtc cgccttccgc 1380taccagcagt ccttggagaa ggacgacaag aaggtggtcg gcgtcaacgt ccacaccggc 1440tccgtcaccg gcgacctgga gatcctgcgg gtcagccacg aggtcgagcg cgagcaggtg 1500cgggtcctgg gcgagcgcaa ggacgcccgg gacgacgccg ccgtgcgcgg cgccctggac 1560gccatgctgg ccgcggcccg ctccggcggc aacatgatcg ggccgatgct ggacgcggtg 1620cgcgcggagg cgacgctggg cgagatctgc ggtgtgctgc gcgacgagtg gggggtgtac 1680acggaaccgg cggggttctg a 1701101138PRTStreptomyces coelicolor 101Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg 20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu 35 40 45 Gln Ile Val Asp Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu 50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Glu 65 70 75 80 Leu Leu Arg Glu Arg Asp Ala Ala Asp Ile Leu Val Phe Gly Gly Gly 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala 100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Asp Trp Val 115 120 125 Arg Ala Asn Val Arg Glu Pro Ala Gly Ala 130 135 102417DNAStreptomyces coelicolor 102atgggtgtgg cagccggtcc gatccgcgtg gtggtggcca agccggggct cgacggccac 60gatcgcgggg ccaaggtgat cgcgagggcc ctgcgtgacg ccggtatgga ggtgatctac 120accgggctcc accagacgcc cgagcagatc gtcgacaccg cgatccagga ggacgccgac 180gcgatcgggc tgtccatcct ctccggtgcg cacaacacgc tcttcgccgc cgtgatcgag 240ctgctccggg agcgggacgc cgcggacatc ctggtcttcg gcggcgggat catccccgag 300gcggacatcg ccccgctgaa ggagaagggc gtcgcggaga tcttcacgcc cggcgccacc 360acggcgtcca tcgtggactg ggtccgggcg aacgtgcggg agcccgcggg agcatag 417103566PRTStreptomyces avermitilis 103Met Asp Ala Asp Ala Ile Glu Glu Gly Arg Arg Arg Trp Gln Ala Arg 1 5 10 15 Tyr Asp Ala Ser Arg Lys Arg Glu Ala Asp Phe Thr Thr Leu Ser Gly 20 25 30 Asp Pro Val Glu Pro Ala Tyr Gly Pro Arg Pro Gly Asp Ala Tyr Glu 35 40 45 Gly Phe Glu Arg Ile Gly Trp Pro Gly Glu Tyr Pro Phe Thr Arg Gly 50 55 60 Leu Tyr Pro Thr Gly Tyr Arg Gly Arg Thr Trp Thr Ile Arg Gln Phe 65 70 75 80 Ala Gly Phe Gly Asn Ala Glu Gln Thr Asn Glu Arg Tyr Lys Lys Ile 85 90 95 Leu Ala Asn Gly Gly Gly Gly Leu Ser Val Ala Phe Asp Met Pro Thr 100 105 110 Leu Met Gly Arg Asp Ser Asp Asp Arg Arg Ala Leu Gly Glu Val Gly 115 120 125 His Cys Gly Val Ala Ile Asp Ser Ala Ala Asp Met Glu Val Leu Phe 130 135 140 Lys Asp Ile Pro Leu Gly Asp Val Thr Thr Ser Met Thr Ile Ser Gly 145 150 155 160 Pro Ala Val Pro Val Phe Cys Met Tyr Leu Val Ala Ala Glu Arg Gln 165 170 175 Gly Val Asp Pro Ser Val Leu Asn Gly Thr Leu Gln Thr Asp Ile Phe 180 185 190 Lys Glu Tyr Ile Ala Gln Lys Glu Trp Leu Phe Gln Pro Glu Pro His 195 200 205 Leu Arg Leu Ile Gly Asp Leu Met Glu His Cys Ala Ser Lys Ile Pro 210 215 220 Ala Tyr Lys Pro Leu Ser Val Ser Gly Tyr His Ile Arg Glu Ala Gly 225 230 235 240 Ala Thr Ala Ala Gln Glu Leu Ala Tyr Thr Leu Ala Asp Gly Phe Gly 245 250 255 Tyr Val Glu Leu Gly Leu Ser Arg Gly Leu Asp Val Asp Val Phe Ala 260 265 270 Pro Gly Leu Ser Phe Phe Phe Asp Ala His Val Asp Phe Phe Glu Glu 275 280 285 Ile Ala Lys Phe Arg Ala Ala Arg Arg Ile Trp Ala Arg Trp Leu Arg 290 295 300 Asp Val Tyr Gly Ala Lys Ser Glu Lys Ala Gln Trp Leu Arg Phe His 305 310 315 320 Thr Gln Thr Ala Gly Val Ser Leu Thr Ala Gln Gln Pro Tyr Asn Asn 325 330 335 Val Val Arg Thr Ala Val Glu Ala Leu Ala Ala Val Leu Gly Gly Thr 340 345 350 Asn Ser Leu His Thr Asn Ala Leu Asp Glu Thr Leu Ala Leu Pro Ser 355 360 365 Glu Gln Ala Ala Glu Ile Ala Leu Arg Thr Gln Gln Val Leu Met Glu 370 375 380 Glu Thr Gly Val Ala Asn Val Ala Asp Pro Leu Gly Gly Ser Trp Tyr 385 390 395 400 Val Glu Gln Leu Thr Asp Arg Ile Glu Ala Asp Ala Glu Lys Ile Phe 405 410 415 Glu Gln Ile Arg Glu Arg Gly Leu Arg Ala His Pro Asp Gly Arg His 420 425 430 Pro Ile Gly Pro Ile Thr Ser Gly Ile Leu Arg Gly Ile Glu Asp Gly 435 440 445 Trp Phe Thr Gly Glu Ile Ala Glu Ser Ala Phe Gln Tyr Gln Gln Ala 450 455 460 Leu Glu Lys Gly Asp Lys Arg Val Val Gly Val Asn Val His His Gly 465 470 475 480 Ser Val Thr Gly Asp Leu Glu Ile Leu Arg Val Ser His Glu Val Glu 485 490 495 Arg Glu Gln Val Arg Val Leu Gly Glu Arg Lys Ser Gly Arg Asp Asp 500 505 510 Thr Ala Val Thr Ala Ala Leu Asp Ala Met Leu Ala Ala Ala Arg Asp 515 520 525 Gly Ser Asn Met Ile Ala Pro Met Leu Asp Ala Val Arg Ala Glu Ala 530 535 540 Thr Leu Gly Glu Ile Cys Asp Val Leu Arg Glu Glu Trp Gly Val Tyr 545 550 555 560 Thr Glu Pro Ala Gly Phe 565 1041701DNAStreptomyces avermitilis 104tcagaaaccg gcgggctccg tgtagacccc ccactcctcc cggaggacat cgcagatctc 60gcccagcgtg gcctccgcgc ggaccgcgtc cagcatcggg gcgatcatgt tcgacccgtc 120gcgcgcggcg gcgagcatcg cgtccagggc cgcggttacg gccgtgtcgt cgcgccccga 180cttccgctcg cccagcaccc gcacctgctc gcgctccacc tcgtggctga cgcgcaggat 240ctccaggtcg cccgtcacgg acccgtggtg gacgttgacg ccgacgaccc gcttgtcgcc 300cttctccagc gcctgctggt actggaaggc cgactcggcg atctccccgg tgaaccagcc 360gtcctcgatg ccgcgcagga tgccggaggt gatgggcccg atcgggtgcc gcccgtccgg 420gtgggcccgc agcccgcgct ccctgatctg ttcgaagatc ttctcggcgt cggcctcgat 480ccggtcggtc agctgctcca cgtaccagga accgcccagc ggatcggcca cgttggcgac 540gcccgtctcc tccatcagca cctgctgggt gcgcagggcg atctcggccg cctgctcgga 600cggcagggcg agggtctcgt cgagggcgtt ggtgtgcagc gagttcgtcc cgccgagcac 660cgcggcgagg gcctccacgg ccgtccgtac gacgttgttg tacggctgct gcgcggtgag 720cgagacgccc gcggtctggg tgtggaagcg cagccactgc gccttctccg acttcgcccc 780gtacacgtcc cgcagccagc gcgcccagat gcgccgcgcc gcacggaact tggcgatctc 840ctcgaagaag tcgacgtgcg cgtcgaagaa gaaggagagc ccgggcgcga acacgtccac 900gtccaggccg cggctcagcc ccagctccac gtatccgaaa ccgtcggcga gggtgtacgc 960cagctcctgg gcggccgtgg caccggcctc ccggatgtgg tacccggaga cggacagcgg 1020cttgtacgcg gggatcttcg aggcgcagtg ctccatcagg tcgccgatga gccgcagatg 1080gggctcgggc tggaagagcc actccttctg cgcgatgtac tccttgaaga tgtcggtctg 1140gagggtgccg ttgaggacgg aggggtcgac gccctgccgc tcggccgcga ccaggtacat 1200gcagaagacg ggcacggcgg gcccgctgat cgtcatcgac gtcgtcacgt cacccagcgg 1260gatgtccttg aacaggacct ccatgtcggc cgccgagtcg atcgcgaccc cgcagtgccc 1320gacctcgccg agcgcgcggc ggtcgtcgga gtcgcgcccc atgagcgtcg gcatgtcgaa 1380ggccacggac agcccaccgc cgccgttggc gaggatcttc ttgtagcgct cgttggtctg 1440ctcggcgttg ccgaacccgg cgaactgccg gatggtccag gtccggcccc ggtagccggt 1500cggatacaga ccgcgcgtga aggggtactc acccggccag ccgatccgct cgaaaccctc 1560gtacgcgtcc ccgggccggg gcccgtacgc cggctccacg ggatcgccgg agagcgtggt 1620gaaatcggcc tcgcgcttgc gtgaggcgtc gtagcgggcc tgccagcgtc ggcggccttc 1680ctcgatggcg tcagcgtcca t 1701105138PRTStreptomyces avermitilis 105Met Gly Val Ala Ala Gly Pro Ile Arg Val Val Val Ala Lys Pro Gly 1 5 10 15 Leu Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Arg Ala Leu Arg 20 25 30 Asp Ala Gly Met Glu Val Ile Tyr Thr Gly Leu His Gln Thr Pro Glu 35 40 45 Gln Ile Val Gly Thr Ala Ile Gln Glu Asp Ala Asp Ala Ile Gly Leu 50 55 60 Ser Ile Leu Ser Gly Ala His Asn Thr Leu Phe Ala Ala Val Ile Asp 65 70 75 80 Leu Leu Lys Glu Arg Asp Ala Glu Asp Ile Lys Val Phe Gly Gly Gly 85 90 95 Ile Ile Pro Glu Ala Asp Ile Ala Pro Leu Lys Glu Lys Gly Val Ala 100 105 110 Glu Ile Phe Thr Pro Gly Ala Thr Thr Ala Ser Ile Val Glu Trp Val 115 120 125 Arg Ala Asn Val Arg Gln Pro Ala Gly Ala 130 135 1061701DNAStreptomyces avermitilis 106tcagaaaccg gcgggctccg tgtagacccc ccactcctcc cggaggacat cgcagatctc 60gcccagcgtg gcctccgcgc ggaccgcgtc cagcatcggg gcgatcatgt tcgacccgtc 120gcgcgcggcg gcgagcatcg cgtccagggc cgcggttacg gccgtgtcgt cgcgccccga 180cttccgctcg cccagcaccc gcacctgctc gcgctccacc tcgtggctga cgcgcaggat 240ctccaggtcg cccgtcacgg acccgtggtg gacgttgacg ccgacgaccc gcttgtcgcc 300cttctccagc gcctgctggt actggaaggc cgactcggcg atctccccgg tgaaccagcc 360gtcctcgatg ccgcgcagga tgccggaggt gatgggcccg atcgggtgcc gcccgtccgg 420gtgggcccgc agcccgcgct ccctgatctg ttcgaagatc ttctcggcgt cggcctcgat 480ccggtcggtc agctgctcca cgtaccagga accgcccagc ggatcggcca cgttggcgac 540gcccgtctcc tccatcagca cctgctgggt gcgcagggcg atctcggccg cctgctcgga 600cggcagggcg agggtctcgt cgagggcgtt ggtgtgcagc gagttcgtcc cgccgagcac 660cgcggcgagg gcctccacgg ccgtccgtac gacgttgttg tacggctgct gcgcggtgag 720cgagacgccc gcggtctggg tgtggaagcg cagccactgc gccttctccg acttcgcccc 780gtacacgtcc cgcagccagc gcgcccagat gcgccgcgcc gcacggaact tggcgatctc 840ctcgaagaag tcgacgtgcg cgtcgaagaa gaaggagagc ccgggcgcga acacgtccac 900gtccaggccg cggctcagcc ccagctccac gtatccgaaa ccgtcggcga gggtgtacgc 960cagctcctgg gcggccgtgg caccggcctc ccggatgtgg tacccggaga cggacagcgg 1020cttgtacgcg gggatcttcg aggcgcagtg ctccatcagg tcgccgatga gccgcagatg 1080gggctcgggc tggaagagcc actccttctg cgcgatgtac tccttgaaga tgtcggtctg 1140gagggtgccg ttgaggacgg aggggtcgac gccctgccgc tcggccgcga ccaggtacat 1200gcagaagacg ggcacggcgg gcccgctgat cgtcatcgac gtcgtcacgt cacccagcgg 1260gatgtccttg aacaggacct ccatgtcggc cgccgagtcg atcgcgaccc cgcagtgccc 1320gacctcgccg agcgcgcggc ggtcgtcgga gtcgcgcccc atgagcgtcg gcatgtcgaa 1380ggccacggac agcccaccgc cgccgttggc gaggatcttc ttgtagcgct cgttggtctg 1440ctcggcgttg ccgaacccgg cgaactgccg gatggtccag gtccggcccc ggtagccggt 1500cggatacaga ccgcgcgtga aggggtactc acccggccag ccgatccgct cgaaaccctc 1560gtacgcgtcc ccgggccggg gcccgtacgc cggctccacg ggatcgccgg agagcgtggt 1620gaaatcggcc tcgcgcttgc gtgaggcgtc gtagcgggcc tgccagcgtc ggcggccttc 1680ctcgatggcg tcagcgtcca t 1701107139PRTSaccharomyces cerevisiae 107Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile 130 135 1081689DNASaccharomyces cerevisiae 108atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300gtcccatcca tctctgctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960ttcccaggtg tccaaatgaa attcgttttg

caaaagttgt tgaccactat tgctgacgcc 1020gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140ggtgatgttg tcattgctga aaccggtacc tccgctttcg gtatcaacca aaccactttc 1200ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500actttcggtg ctaaggacta tgaaacccac agagtcgcta ccaccggtga atgggacaag 1560ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga aatcatgttg 1620ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680gctaagcaa 1689109563PRTSaccharomyces cerevisiae 109Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Ser Gln 1 5 10 15 Val Asn Cys Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Leu Tyr Glu Val Lys Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ser Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Asn 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Thr Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val 165 170 175 Pro Ala Lys Leu Leu Glu Thr Pro Ile Asp Leu Ser Leu Lys Pro Asn 180 185 190 Asp Ala Glu Ala Glu Ala Glu Val Val Arg Thr Val Val Glu Leu Ile 195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Ala Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Met Asp Leu Thr Gln Phe 225 230 235 240 Pro Val Tyr Val Thr Pro Met Gly Lys Gly Ala Ile Asp Glu Gln His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Arg Pro Glu Val 260 265 270 Lys Lys Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Ile Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser Asp His Ile Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Ala Leu Gln Lys Leu Leu Asp Ala 325 330 335 Ile Pro Glu Val Val Lys Asp Tyr Lys Pro Val Ala Val Pro Ala Arg 340 345 350 Val Pro Ile Thr Lys Ser Thr Pro Ala Asn Thr Pro Met Lys Gln Glu 355 360 365 Trp Met Trp Asn His Leu Gly Asn Phe Leu Arg Glu Gly Asp Ile Val 370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Thr Asp Val Tyr Ala Ile Val Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Val Gly Ala Leu Leu Gly Ala Thr Met Ala Ala Glu Glu Leu 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Ile Phe Val Leu Asn Asn Asn Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro His Ala Glu Tyr Asn Glu Ile Gln Gly Trp Asp His Leu 485 490 495 Ala Leu Leu Pro Thr Phe Gly Ala Arg Asn Tyr Glu Thr His Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Glu Lys Leu Thr Gln Asp Lys Asp Phe Gln 515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Val Met Leu Pro Val Phe Asp 530 535 540 Ala Pro Gln Asn Leu Val Lys Gln Ala Gln Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln 1101689DNASaccharomyces cerevisiae 110atgtctgaaa taaccttagg taaatattta tttgaaagat tgagccaagt caactgtaac 60accgtcttcg gtttgccagg tgactttaac ttgtctcttt tggataagct ttatgaagtc 120aaaggtatga gatgggctgg taacgctaac gaattgaacg ctgcctatgc tgctgatggt 180tacgctcgta tcaagggtat gtcctgtatt attaccacct tcggtgttgg tgaattgtct 240gctttgaatg gtattgccgg ttcttacgct gaacatgtcg gtgttttgca cgttgttggt 300gttccatcca tctcttctca agctaagcaa ttgttgttgc atcatacctt gggtaacggt 360gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc catgatcact 420gatattgcta acgctccagc tgaaattgac agatgtatca gaaccaccta cactacccaa 480agaccagtct acttgggttt gccagctaac ttggttgact tgaacgtccc agccaagtta 540ttggaaactc caattgactt gtctttgaag ccaaacgacg ctgaagctga agctgaagtt 600gttagaactg ttgttgaatt gatcaaggat gctaagaacc cagttatctt ggctgatgct 660tgtgcttcta gacatgatgt caaggctgaa actaagaagt tgatggactt gactcaattc 720ccagtttacg tcaccccaat gggtaagggt gctattgacg aacaacaccc aagatacggt 780ggtgtttacg ttggtacctt gtctagacca gaagttaaga aggctgtaga atctgctgat 840ttgatattgt ctatcggtgc tttgttgtct gatttcaata ccggttcttt ctcttactcc 900tacaagacca aaaatatcgt tgaattccac tctgaccaca tcaagatcag aaacgccacc 960ttcccaggtg ttcaaatgaa atttgccttg caaaaattgt tggatgctat tccagaagtc 1020gtcaaggact acaaacctgt tgctgtccca gctagagttc caattaccaa gtctactcca 1080gctaacactc caatgaagca agaatggatg tggaaccatt tgggtaactt cttgagagaa 1140ggtgatattg ttattgctga aaccggtact tccgccttcg gtattaacca aactactttc 1200ccaacagatg tatacgctat cgtccaagtc ttgtggggtt ccattggttt cacagtcggc 1260gctctattgg gtgctactat ggccgctgaa gaacttgatc caaagaagag agttatttta 1320ttcattggtg acggttctct acaattgact gttcaagaaa tctctaccat gattagatgg 1380ggtttgaagc catacatttt tgtcttgaat aacaacggtt acaccattga aaaattgatt 1440cacggtcctc atgccgaata taatgaaatt caaggttggg accacttggc cttattgcca 1500acttttggtg ctagaaacta cgaaacccac agagttgcta ccactggtga atgggaaaag 1560ttgactcaag acaaggactt ccaagacaac tctaagatta gaatgattga agttatgttg 1620ccagtctttg atgctccaca aaacttggtt aaacaagctc aattgactgc cgctactaac 1680gctaaacaa 1689111533PRTSaccharomyces cerevisiae 111Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asn Val Asn Thr Ile Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Asp Gly Leu Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Leu Ser Val Leu Val Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ala Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ser Met Ile Thr Asp Ile Ala Thr 130 135 140 Ala Pro Ser Glu Ile Asp Arg Leu Ile Arg Thr Thr Phe Ile Thr Gln 145 150 155 160 Arg Pro Ser Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Lys Val 165 170 175 Pro Gly Ser Leu Leu Glu Lys Pro Ile Asp Leu Ser Leu Lys Pro Asn 180 185 190 Asp Pro Glu Ala Glu Lys Glu Val Ile Asp Thr Val Leu Glu Leu Ile 195 200 205 Gln Asn Ser Lys Asn Pro Val Ile Leu Ser Asp Ala Cys Ala Ser Arg 210 215 220 His Asn Val Lys Lys Glu Thr Gln Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Leu Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Gln Asp Val 260 265 270 Lys Gln Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Val Val Glu Phe His Ser Asp Tyr Val Lys Val Lys Asn Ala Thr 305 310 315 320 Phe Leu Gly Val Gln Met Lys Phe Ala Leu Gln Asn Leu Leu Lys Val 325 330 335 Ile Pro Asp Val Val Lys Gly Tyr Lys Ser Val Pro Val Pro Thr Lys 340 345 350 Thr Pro Ala Asn Lys Gly Val Pro Ala Ser Thr Pro Leu Lys Gln Glu 355 360 365 Trp Leu Trp Asn Glu Leu Ser Lys Phe Leu Gln Glu Gly Asp Val Ile 370 375 380 Ile Ser Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Ile Phe 385 390 395 400 Pro Lys Asp Ala Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Asn Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro His Ala Glu Tyr Asn Glu Ile Gln Thr Trp Asp His Leu 485 490 495 Ala Leu Leu Pro Ala Phe Gly Ala Lys Lys Tyr Glu Asn His Lys Ile 500 505 510 Ala Thr Thr Gly Glu Trp Asp Ala Leu Thr Thr Asp Ser Glu Phe Gln 515 520 525 Lys Asn Ser Val Ile 530 1121599DNASaccharomyces cerevisiae 112atgtctgaaa ttactcttgg aaaatactta tttgaaagat tgaagcaagt taatgttaac 60accatttttg ggctaccagg cgacttcaac ttgtccctat tggacaagat ttacgaggta 120gatggattga gatgggctgg taatgcaaat gagctgaacg ccgcctatgc cgccgatggt 180tacgcacgca tcaagggttt atctgtgctg gtaactactt ttggcgtagg tgaattatcc 240gccttgaatg gtattgcagg atcgtatgca gaacacgtcg gtgtactgca tgttgttggt 300gtcccctcta tctccgctca ggctaagcaa ttgttgttgc atcatacctt gggtaacggt 360gattttaccg tttttcacag aatgtccgcc aatatctcag aaactacatc aatgattaca 420gacattgcta cagccccttc agaaatcgat aggttgatca ggacaacatt tataacacaa 480aggcctagct acttggggtt gccagcgaat ttggtagatc taaaggttcc tggttctctt 540ttggaaaaac cgattgatct atcattaaaa cctaacgatc ccgaagctga aaaggaagtt 600attgataccg tactagaatt gatccagaat tcgaaaaacc ctgttatact atcggatgcc 660tgtgcttcta ggcacaacgt taaaaaagaa acccagaagt taattgattt gacgcaattc 720ccagcttttg tgacacctct aggtaaaggg tcaatagatg aacagcatcc cagatatggc 780ggtgtttatg tgggaacgct gtccaaacaa gacgtgaaac aggccgttga gtcggctgat 840ttgatccttt cggtcggtgc tttgctctct gattttaaca caggttcgtt ttcctactcc 900tacaagacta aaaatgtagt ggagtttcat tccgattacg taaaggtgaa gaacgctacg 960ttcctcggtg tacaaatgaa atttgcacta caaaacttac tgaaggttat tcccgatgtt 1020gttaagggct acaagagcgt tcccgtacca accaaaactc ccgcaaacaa aggtgtacct 1080gctagcacgc ccttgaaaca agagtggttg tggaacgaat tgtccaaatt cttgcaagaa 1140ggtgatgtta tcatttccga gaccggcacg tctgccttcg gtatcaatca aactatcttt 1200cctaaggacg cctacggtat ctcgcaggtg ttgtgggggt ccatcggttt tacaacagga 1260gcaactttag gtgctgcctt tgccgctgag gagattgacc ccaacaagag agtcatctta 1320ttcataggtg acgggtcttt gcagttaacc gtccaagaaa tctccaccat gatcagatgg 1380gggttaaagc cgtatctttt tgtccttaac aacgacggct acactatcga aaagctgatt 1440catgggcctc acgcagagta caacgaaatc cagacctggg atcacctcgc cctgttgccc 1500gcatttggtg cgaaaaagta cgaaaatcac aagatcgcca ctacgggtga gtgggatgcc 1560ttaaccactg attcagagtt ccagaaaaac tcggtgatc 1599113564PRTCandida glabrata 113Met Ser Glu Ile Thr Leu Gly Arg Tyr Leu Phe Glu Arg Leu Asn Gln 1 5 10 15 Val Asp Val Lys Thr Ile Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Ile Ser Ser Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Val Thr Asp Ile Ala Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Ile Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Lys Val 165 170 175 Pro Ala Lys Leu Leu Glu Thr Pro Ile Asp Leu Ser Leu Lys Pro Asn 180 185 190 Asp Pro Glu Ala Glu Thr Glu Val Val Asp Thr Val Leu Glu Leu Ile 195 200 205 Lys Ala Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Ala Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Ala Thr Gln Phe 225 230 235 240 Pro Ser Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His 245 250 255 Pro Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Arg Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His Ser Asp Tyr Ile Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Ala Leu Gln Lys Leu Leu Asn Ala 325 330 335 Val Pro Glu Ala Ile Lys Gly Tyr Lys Pro Val Pro Val Pro Ala Arg 340 345 350 Val Pro Glu Asn Lys Ser Cys Asp Pro Ala Thr Pro Leu Lys Gln Glu 355 360 365 Trp Met Trp Asn Gln Val Ser Lys Phe Leu Gln Glu Gly Asp Val Val 370 375 380 Ile Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Pro Phe 385 390 395 400 Pro Asn Asn Ala Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Cys Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile 465 470 475 480 His Gly Glu Lys Ala Gly Tyr Asn Asp Ile Gln Asn Trp Asp His Leu 485 490 495 Ala Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Asn His Arg Val 500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Glu Phe Asn 515 520 525 Lys Asn Ser Lys Ile Arg Met Ile Glu Val Met Leu Pro Val Met Asp 530 535 540 Ala Pro Thr Ser Leu Ile Glu Gln Ala Lys Leu Thr Ala Ser Ile Asn 545 550 555 560 Ala Lys Gln

Glu 1141692DNACandida glabrata 114atgtctgaga ttactttggg tagatacttg ttcgagagat tgaaccaagt cgacgttaag 60accatcttcg gtttgccagg tgacttcaac ttgtccctat tggacaagat ctacgaagtt 120gaaggtatga gatgggctgg taacgctaac gaattgaacg ctgcttacgc tgctgacggt 180tacgctagaa tcaagggtat gtcctgtatc atcaccacct tcggtgtcgg tgaattgtct 240gccttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgtcttgca cgtcgtcggt 300gtcccatcca tctcctctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360gacttcactg tcttccacag aatgtccgct aacatctctg agaccaccgc tatggtcact 420gacatcgcta ccgctccagc tgagatcgac agatgtatca gaaccaccta catcacccaa 480agaccagtct acttgggtct accagctaac ttggtcgacc taaaggtccc agccaagctt 540ttggaaaccc caattgactt gtccttgaag ccaaacgacc cagaagccga aactgaagtc 600gttgacaccg tcttggaatt gatcaaggct gctaagaacc cagttatctt ggctgatgct 660tgtgcttcca gacacgacgt caaggctgaa accaagaagt tgattgacgc cactcaattc 720ccatccttcg ttaccccaat gggtaagggt tccatcgacg aacaacaccc aagattcggt 780ggtgtctacg tcggtacctt gtccagacca gaagttaagg aagctgttga atccgctgac 840ttgatcttgt ctgtcggtgc tttgttgtcc gatttcaaca ctggttcttt ctcttactct 900tacaagacca agaacatcgt cgaattccac tctgactaca tcaagatcag aaacgctacc 960ttcccaggtg tccaaatgaa gttcgctttg caaaagttgt tgaacgccgt cccagaagct 1020atcaagggtt acaagccagt ccctgtccca gctagagtcc cagaaaacaa gtcctgtgac 1080ccagctaccc cattgaagca agaatggatg tggaaccaag tttccaagtt cttgcaagaa 1140ggtgatgttg ttatcactga aaccggtacc tccgcttttg gtatcaacca aaccccattc 1200ccaaacaacg cttacggtat ctcccaagtt ctatggggtt ccatcggttt caccaccggt 1260gcttgtttgg gtgccgcttt cgctgctgaa gaaatcgacc caaagaagag agttatcttg 1320ttcattggtg acggttcttt gcaattgact gtccaagaaa tctccaccat gatcagatgg 1380ggcttgaagc catacttgtt cgtcttgaac aacgacggtt acaccatcga aagattgatt 1440cacggtgaaa aggctggtta caacgacatc caaaactggg accacttggc tctattgcca 1500accttcggtg ctaaggacta cgaaaaccac agagtcgcca ccaccggtga atgggacaag 1560ttgacccaag acaaggaatt caacaagaac tccaagatca gaatgatcga agttatgttg 1620ccagttatgg acgctccaac ttccttgatt gaacaagcta agttgaccgc ttccatcaac 1680gctaagcaag aa 1692115596PRTPichia stipitis 115Met Ala Glu Val Ser Leu Gly Arg Tyr Leu Phe Glu Arg Leu Tyr Gln 1 5 10 15 Leu Gln Val Gln Thr Ile Phe Gly Val Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Asp Ala His Gly Lys Asn Ser 35 40 45 Phe Arg Trp Ala Gly Asn Ala Asn Glu Leu Asn Ala Ser Tyr Ala Ala 50 55 60 Asp Gly Tyr Ser Arg Val Lys Arg Leu Gly Cys Leu Val Thr Thr Phe 65 70 75 80 Gly Val Gly Glu Leu Ser Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala 85 90 95 Glu His Val Gly Leu Leu His Val Val Gly Val Pro Ser Ile Ser Ser 100 105 110 Gln Ala Lys Gln Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe 115 120 125 Thr Val Phe His Arg Met Ser Asn Asn Ile Ser Gln Thr Thr Ala Phe 130 135 140 Ile Ser Asp Ile Asn Ser Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg 145 150 155 160 Glu Ala Tyr Val Lys Gln Arg Pro Val Tyr Ile Gly Leu Pro Ala Asn 165 170 175 Leu Val Asp Leu Asn Val Pro Ala Ser Leu Leu Glu Ser Pro Ile Asn 180 185 190 Leu Ser Leu Glu Lys Asn Asp Pro Glu Ala Gln Asp Glu Val Ile Asp 195 200 205 Ser Val Leu Asp Leu Ile Lys Lys Ser Ser Asn Pro Ile Ile Leu Val 210 215 220 Asp Ala Cys Ala Ser Arg His Asp Cys Lys Ala Glu Val Thr Gln Leu 225 230 235 240 Ile Glu Gln Thr Gln Phe Pro Val Phe Val Thr Pro Met Gly Lys Gly 245 250 255 Thr Val Asp Glu Gly Gly Val Asp Gly Glu Leu Leu Glu Asp Asp Pro 260 265 270 His Leu Ile Ala Lys Val Ala Ala Arg Leu Ser Ala Gly Lys Asn Ala 275 280 285 Ala Ser Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu 290 295 300 Val Lys Asp Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala 305 310 315 320 Leu Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Arg Thr 325 330 335 Lys Asn Ile Val Glu Phe His Ser Asp Tyr Thr Lys Ile Arg Gln Ala 340 345 350 Thr Phe Pro Gly Val Gln Met Lys Glu Ala Leu Gln Glu Leu Asn Lys 355 360 365 Lys Val Ser Ser Ala Ala Ser His Tyr Glu Val Lys Pro Val Pro Lys 370 375 380 Ile Lys Leu Ala Asn Thr Pro Ala Thr Arg Glu Val Lys Leu Thr Gln 385 390 395 400 Glu Trp Leu Trp Thr Arg Val Ser Ser Trp Phe Arg Glu Gly Asp Ile 405 410 415 Ile Ile Thr Glu Thr Gly Thr Ser Ser Phe Gly Ile Val Gln Ser Arg 420 425 430 Phe Pro Asn Asn Thr Ile Gly Ile Ser Gln Val Leu Trp Gly Ser Ile 435 440 445 Gly Phe Ser Val Gly Ala Thr Leu Gly Ala Ala Met Ala Ala Gln Glu 450 455 460 Leu Asp Pro Asn Lys Arg Thr Ile Leu Phe Val Gly Asp Gly Ser Leu 465 470 475 480 Gln Leu Thr Val Gln Glu Ile Ser Thr Ile Ile Arg Trp Gly Thr Thr 485 490 495 Pro Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu 500 505 510 Ile His Gly Val Asn Ala Ser Tyr Asn Asp Ile Gln Pro Trp Gln Asn 515 520 525 Leu Glu Ile Leu Pro Thr Phe Ser Ala Lys Asn Tyr Asp Ala Val Arg 530 535 540 Ile Ser Asn Ile Gly Glu Ala Glu Asp Ile Leu Lys Asp Lys Glu Phe 545 550 555 560 Gly Lys Asn Ser Lys Ile Arg Leu Ile Glu Val Met Leu Pro Arg Leu 565 570 575 Asp Ala Pro Ser Asn Leu Ala Lys Gln Ala Ala Ile Thr Ala Ala Thr 580 585 590 Asn Ala Glu Ala 595 1161788DNAPichia stipitis 116atggctgaag tctcattagg aagatatctc ttcgagagat tgtaccaatt gcaagtgcag 60accatcttcg gtgtccctgg tgatttcaac ttgtcgcttt tggacaagat ctacgaagtg 120gaagatgccc atggcaagaa ttcgtttaga tgggctggta atgccaacga attgaatgca 180tcgtacgctg ctgacggtta ctcgagagtc aagcgtttag ggtgtttggt cactaccttt 240ggtgtcggtg aattgtctgc tttgaatggt attgccggtt cttatgccga acatgttggt 300ttgcttcatg tcgtaggtgt tccatcgatt tcctcgcaag ctaagcaatt gttacttcac 360cacactttgg gtaatggtga tttcactgtt ttccatagaa tgtccaacaa catttctcag 420accacagcct ttatctccga tatcaactcg gctccagctg aaattgatag atgtatcaga 480gaggcctacg tcaaacaaag accagtttat atcgggttac cagctaactt agttgatttg 540aatgttccgg cctctttgct tgagtctcca atcaacttgt cgttggaaaa gaacgaccca 600gaggctcaag atgaagtcat tgactctgtc ttagacttga tcaaaaagtc gctgaaccca 660atcatcttgg tcgatgcctg tgcctcgaga catgactgta aggctgaagt tactcagttg 720attgaacaaa cccaattccc agtatttgtc actccaatgg gtaaaggtac cgttgatgag 780ggtggtgtag acggagaatt gttagaagat gatcctcatt tgattgccaa ggtcgctgct 840aggttgtctg ctggcaagaa cgctgcctct agattcggag gtgtttatgt cggaaccttg 900tcgaagcccg aagtcaagga cgctgtagag agtgcagatt tgattttgtc tgtcggtgcc 960cttttgtctg atttcaacac tggttcattt tcctactcct acagaaccaa gaacatcgtc 1020gaattccatt ctgattacac taagattaga caagccactt tcccaggtgt gcagatgaag 1080gaagccttgc aagaattgaa caagaaagtt tcatctgctg ctagtcacta tgaagtcaag 1140cctgtgccca agatcaagtt ggccaataca ccagccacca gagaagtcaa gttaactcag 1200gaatggttgt ggaccagagt gtcttcgtgg ttcagagaag gtgatattat tatcaccgaa 1260accggtacat cctccttcgg tatagttcaa tccagattcc caaacaacac catcggtatc 1320tcccaagtat tgtggggttc tattggtttc tctgttggtg ccactttggg tgctgccatg 1380gctgcccaag aactcgaccc taacaagaga accatcttgt ttgttggaga tggttctttg 1440caattgaccg ttcaggaaat ctccaccata atcagatggg gtaccacacc ttaccttttc 1500gtgttgaaca atgacggtta caccatcgag cgtttgatcc acggtgtaaa tgcctcatat 1560aatgacatcc aaccatggca aaacttggaa atcttgccta ctttctcggc caagaactac 1620gacgctgtga gaatctccaa catcggagaa gcagaagata tcttgaaaga caaggaattc 1680ggaaagaact ccaagattag attgatagaa gtcatgttac caagattgga tgcaccatct 1740aaccttgcca aacaagctgc cattacagct gccaccaacg ccgaagct 1788117569PRTPichia stipitis 117Met Val Ser Thr Tyr Pro Glu Ser Glu Val Thr Leu Gly Arg Tyr Leu 1 5 10 15 Phe Glu Arg Leu His Gln Leu Lys Val Asp Thr Ile Phe Gly Leu Pro 20 25 30 Gly Asp Phe Asn Leu Ser Leu Leu Asp Lys Val Tyr Glu Val Pro Asp 35 40 45 Met Arg Trp Ala Gly Asn Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala 50 55 60 Asp Gly Tyr Ser Arg Ile Lys Gly Leu Ser Cys Leu Val Thr Thr Phe 65 70 75 80 Gly Val Gly Glu Leu Ser Ala Leu Asn Gly Val Gly Gly Ala Tyr Ala 85 90 95 Glu His Val Gly Leu Leu His Val Val Gly Val Pro Ser Ile Ser Ser 100 105 110 Gln Ala Lys Gln Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe 115 120 125 Thr Val Phe His Arg Met Ser Asn Ser Ile Ser Gln Thr Thr Ala Phe 130 135 140 Leu Ser Asp Ile Ser Ile Ala Pro Gly Gln Ile Asp Arg Cys Ile Arg 145 150 155 160 Glu Ala Tyr Val His Gln Arg Pro Val Tyr Val Gly Leu Pro Ala Asn 165 170 175 Met Val Asp Leu Lys Val Pro Ser Ser Leu Leu Glu Thr Pro Ile Asp 180 185 190 Leu Lys Leu Lys Gln Asn Asp Pro Glu Ala Gln Glu Val Val Glu Thr 195 200 205 Val Leu Lys Leu Val Ser Gln Ala Thr Asn Pro Ile Ile Leu Val Asp 210 215 220 Ala Cys Ala Leu Arg His Asn Cys Lys Glu Glu Val Lys Gln Leu Val 225 230 235 240 Asp Ala Thr Asn Phe Gln Val Phe Thr Thr Pro Met Gly Lys Ser Gly 245 250 255 Ile Ser Glu Ser His Pro Arg Leu Gly Gly Val Tyr Val Gly Thr Met 260 265 270 Ser Ser Pro Gln Val Lys Lys Ala Val Glu Asn Ala Asp Leu Ile Leu 275 280 285 Ser Val Gly Ser Leu Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr 290 295 300 Ser Tyr Lys Thr Lys Asn Val Val Glu Phe His Ser Asp Tyr Met Lys 305 310 315 320 Ile Arg Gln Ala Thr Phe Pro Gly Val Gln Met Lys Glu Ala Leu Gln 325 330 335 Gln Leu Ile Lys Arg Val Ser Ser Tyr Ile Asn Pro Ser Tyr Ile Pro 340 345 350 Thr Arg Val Pro Lys Arg Lys Gln Pro Leu Lys Ala Pro Ser Glu Ala 355 360 365 Pro Leu Thr Gln Glu Tyr Leu Trp Ser Lys Val Ser Gly Trp Phe Arg 370 375 380 Glu Gly Asp Ile Ile Val Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile 385 390 395 400 Ile Gln Ser His Phe Pro Ser Asn Thr Ile Gly Ile Ser Gln Val Leu 405 410 415 Trp Gly Ser Ile Gly Phe Thr Val Gly Ala Thr Val Gly Ala Ala Met 420 425 430 Ala Ala Gln Glu Ile Asp Pro Ser Arg Arg Val Ile Leu Phe Val Gly 435 440 445 Asp Gly Ser Leu Gln Leu Thr Val Gln Glu Ile Ser Thr Leu Cys Lys 450 455 460 Trp Asp Cys Asn Asn Thr Tyr Leu Tyr Val Leu Asn Asn Asp Gly Tyr 465 470 475 480 Thr Ile Glu Arg Leu Ile His Gly Lys Ser Ala Ser Tyr Asn Asp Ile 485 490 495 Gln Pro Trp Asn His Leu Ser Leu Leu Arg Leu Phe Asn Ala Lys Lys 500 505 510 Tyr Gln Asn Val Arg Val Ser Thr Ala Gly Glu Leu Asp Ser Leu Phe 515 520 525 Ser Asp Lys Lys Phe Ala Ser Pro Asp Arg Ile Arg Met Ile Glu Val 530 535 540 Met Leu Ser Arg Leu Asp Ala Pro Ala Asn Leu Val Ala Gln Ala Lys 545 550 555 560 Leu Ser Glu Arg Val Asn Leu Glu Asn 565 1181707DNAPichia stipitis 118atggtatcaa cctacccaga atcagaggtt actctaggaa ggtacctctt tgagcgactc 60caccaattga aagtggacac cattttcggc ttgccgggtg acttcaacct ttccttattg 120gacaaagtgt atgaagttcc ggatatgagg tgggctggaa atgccaacga attgaatgct 180gcctatgctg ccgatggtta ctccagaata aagggattgt cttgcttggt cacaactttt 240ggtgttggtg aattgtctgc tttaaacgga gttggtggtg cctatgctga acacgtagga 300cttctacatg tcgttggagt tccatccata tcgtcacagg ctaaacagtt gttgctccac 360cataccttgg gtaatggtga cttcactgtt tttcacagaa tgtccaatag catttctcaa 420actacagcat ttctctcaga tatctctatt gcaccaggtc aaatagatag atgcatcaga 480gaagcatatg ttcatcagag accagtttat gttggtttac cggcaaatat ggttgatctc 540aaggttcctt ctagtctctt agaaactcca attgatttga aattgaaaca aaatgatcct 600gaagctcaag aagttgttga aacagtcctg aagttggtgt cccaagctac aaaccccatt 660atcttggtag acgcttgtgc cctcagacac aattgcaaag aggaagtcaa acaattggtt 720gatgccacta attttcaagt ctttacaact ccaatgggta aatctggtat ctccgaatct 780catccaagat tgggcggtgt ctatgtcggg acaatgtcga gtcctcaagt caaaaaagcc 840gttgaaaatg ccgatcttat actatctgtt ggttcgttgt tatcggactt caatacaggt 900tcattttcat actcctacaa gacgaagaat gttgttgaat tccactctga ctatatgaaa 960atcagacagg ccaccttccc aggagttcaa atgaaagaag ccttgcaaca gttgataaaa 1020agggtctctt cttacatcaa tccaagctac attcctactc gagttcctaa aaggaaacag 1080ccattgaaag ctccatcaga agctcctttg acccaagaat atttgtggtc taaagtatcc 1140ggctggttta gagagggtga tattatcgta accgaaactg gtacatctgc tttcggaatt 1200attcaatccc attttcccag caacactatc ggtatatccc aagtcttgtg gggctcaatt 1260ggtttcacag taggtgcaac agttggtgct gccatggcag cccaggaaat cgaccctagc 1320aggagagtaa ttttgttcgt cggtgatggt tcattgcagt tgacggttca ggaaatctct 1380acgttgtgta aatgggattg taacaatact tatctttacg tgttgaacaa tgatggttac 1440actatagaaa ggttgatcca cggcaaaagt gccagctaca acgatataca gccttggaac 1500catttatcct tgcttcgctt attcaatgct aagaaatacc aaaatgtcag agtatcgact 1560gctggagaat tggactcttt gttctctgat aagaaatttg cttctccaga taggataaga 1620atgattgagg tgatgttatc gagattggat gcaccagcaa atcttgttgc tcaagcaaag 1680ttgtctgaac gggtaaacct tgaaaat 1707119563PRTKluyveromyces lactis 119Met Ser Glu Ile Thr Leu Gly Arg Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Glu Val Gln Thr Ile Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Asn Ile Tyr Glu Val Pro Gly Met Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Leu 50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Val Val Gly Val Pro Ser Val Ser Ser Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ser Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Asn Thr 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Ser Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Thr Val 165 170 175 Pro Ala Ser Leu Leu Asp Thr Pro Ile Asp Leu Ser Leu Lys Pro Asn 180 185 190 Asp Pro Glu Ala Glu Glu Glu Val Ile Glu Asn Val Leu Gln Leu Ile 195 200 205 Lys Glu Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Ala Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Lys His 245 250 255 Pro Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Ser Pro Ala Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Val Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Ile Val Glu Phe His

Ser Asp Tyr Thr Lys Ile Arg Ser Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Ala Leu Gln Lys Leu Leu Thr Lys 325 330 335 Val Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Pro Val Pro Ser Glu 340 345 350 Pro Glu His Asn Glu Ala Val Ala Asp Ser Thr Pro Leu Lys Gln Glu 355 360 365 Trp Val Trp Thr Gln Val Gly Glu Phe Leu Arg Glu Gly Asp Val Val 370 375 380 Ile Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr His Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile 465 470 475 480 His Gly Glu Thr Ala Gln Tyr Asn Cys Ile Gln Asn Trp Gln His Leu 485 490 495 Glu Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Ala Val Arg Val 500 505 510 Ser Thr Thr Gly Glu Trp Asn Lys Leu Thr Thr Asp Glu Lys Phe Gln 515 520 525 Asp Asn Thr Arg Ile Arg Leu Ile Glu Val Met Leu Pro Thr Met Asp 530 535 540 Ala Pro Ser Asn Leu Val Lys Gln Ala Gln Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Asn 1201689DNAKluyveromyces lactis 120atgtctgaaa ttacattagg tcgttacttg ttcgaaagat taaagcaagt cgaagttcaa 60accatctttg gtctaccagg tgatttcaac ttgtccctat tggacaatat ctacgaagtc 120ccaggtatga gatgggctgg taatgccaac gaattgaacg ctgcttacgc tgctgatggt 180tacgccagat taaagggtat gtcctgtatc atcaccacct tcggtgtcgg tgaattgtct 240gctttgaacg gtattgccgg ttcttacgct gaacacgttg gtgtcttgca cgttgtcggt 300gttccatccg tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360gacttcactg ttttccacag aatgtcctcc aacatttctg aaaccactgc tatgatcacc 420gatatcaaca ctgccccagc tgaaatcgac agatgtatca gaaccactta cgtttcccaa 480agaccagtct acttgggttt gccagctaac ttggtcgact tgactgtccc agcttctttg 540ttggacactc caattgattt gagcttgaag ccaaatgacc cagaagccga agaagaagtc 600atcgaaaacg tcttgcaact gatcaaggaa gctaagaacc cagttatctt ggctgatgct 660tgttgttcca gacacgatgc caaggctgag accaagaagt tgatcgactt gactcaattc 720ccagccttcg ttaccccaat gggtaagggt tccattgacg aaaagcaccc aagattcggt 780ggtgtctacg tcggtaccct atcttctcca gctgtcaagg aagccgttga atctgctgac 840ttggttctat cggtcggtgc tctattgtcc gatttcaaca ctggttcttt ctcttactct 900tacaagacca agaacattgt cgaattccac tctgactaca ccaagatcag aagcgctacc 960ttcccaggtg tccaaatgaa gttcgcttta caaaaattgt tgactaaggt tgccgatgct 1020gctaagggtt acaagccagt tccagttcca tctgaaccag aacacaacga agctgtcgct 1080gactccactc cattgaagca agaatgggtc tggactcaag tcggtgaatt cttgagagaa 1140ggtgatgttg ttatcactga aaccggtacc tctgccttcg gtatcaacca aactcatttc 1200ccaaacaaca catacggtat ctctcaagtt ttatggggtt ccattggttt caccactggt 1260gctaccttgg gtgctgcctt cgctgccgaa gaaattgatc caaagaagag agttatctta 1320ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380ggcttgaagc catacttgtt cgtattgaac aacgacggtt acaccattga aagattgatt 1440cacggtgaaa ccgctcaata caactgtatc caaaactggc aacacttgga attattgcca 1500actttcggtg ccaaggacta cgaagctgtc agagtttcca ccactggtga atggaacaag 1560ttgaccactg acgaaaagtt ccaagacaac accagaatca gattgatcga agttatgttg 1620ccaactatgg atgctccatc taacttggtt aagcaagctc aattgactgc tgctaccaac 1680gctaagaac 1689121571PRTYarrowia lipolytica 121Met Ser Asp Ser Glu Pro Gln Met Val Asp Leu Gly Asp Tyr Leu Phe 1 5 10 15 Ala Arg Phe Lys Gln Leu Gly Val Asp Ser Val Phe Gly Val Pro Gly 20 25 30 Asp Phe Asn Leu Thr Leu Leu Asp His Val Tyr Asn Val Asp Met Arg 35 40 45 Trp Val Gly Asn Thr Asn Glu Leu Asn Ala Gly Tyr Ser Ala Asp Gly 50 55 60 Tyr Ser Arg Val Lys Arg Leu Ala Cys Leu Val Thr Thr Phe Gly Val 65 70 75 80 Gly Glu Leu Ser Ala Val Ala Ala Val Ala Gly Ser Tyr Ala Glu His 85 90 95 Val Gly Val Val His Val Val Gly Val Pro Ser Thr Ser Ala Glu Asn 100 105 110 Lys His Leu Leu Leu His His Thr Leu Gly Asn Gly Asp Phe Arg Val 115 120 125 Phe Ala Gln Met Ser Lys Leu Ile Ser Glu Tyr Thr His His Ile Glu 130 135 140 Asp Pro Ser Glu Ala Ala Asp Val Ile Asp Thr Ala Ile Arg Ile Ala 145 150 155 160 Tyr Thr His Gln Arg Pro Val Tyr Ile Ala Val Pro Ser Asn Phe Ser 165 170 175 Glu Val Asp Ile Ala Asp Gln Ala Arg Leu Asp Thr Pro Leu Asp Leu 180 185 190 Ser Leu Gln Pro Asn Asp Pro Glu Ser Gln Tyr Glu Val Ile Glu Glu 195 200 205 Ile Cys Ser Arg Ile Lys Ala Ala Lys Lys Pro Val Ile Leu Val Asp 210 215 220 Ala Cys Ala Ser Arg Tyr Arg Cys Val Asp Glu Thr Lys Glu Leu Ala 225 230 235 240 Lys Ile Thr Asn Phe Ala Tyr Phe Val Thr Pro Met Gly Lys Gly Ser 245 250 255 Val Asp Glu Asp Thr Asp Arg Tyr Gly Gly Thr Tyr Val Gly Ser Leu 260 265 270 Thr Ala Pro Ala Thr Ala Glu Val Val Glu Thr Ala Asp Leu Ile Ile 275 280 285 Ser Val Gly Ala Leu Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr 290 295 300 Ser Tyr Ser Thr Lys Asn Val Val Glu Leu His Ser Asp His Val Lys 305 310 315 320 Ile Lys Ser Ala Thr Tyr Asn Asn Val Gly Met Lys Met Leu Phe Pro 325 330 335 Pro Leu Leu Glu Ala Val Lys Lys Leu Val Ala Glu Thr Pro Asp Phe 340 345 350 Ala Ser Lys Ala Leu Ala Val Pro Asp Thr Thr Pro Lys Ile Pro Glu 355 360 365 Val Pro Asp Asp His Ile Thr Thr Gln Ala Trp Leu Trp Gln Arg Leu 370 375 380 Ser Tyr Phe Leu Arg Pro Thr Asp Ile Val Val Thr Glu Thr Gly Thr 385 390 395 400 Ser Ser Phe Gly Ile Ile Gln Thr Lys Phe Pro His Asn Val Arg Gly 405 410 415 Ile Ser Gln Val Leu Trp Gly Ser Ile Gly Tyr Ser Val Gly Ala Ala 420 425 430 Cys Gly Ala Ser Ile Ala Ala Gln Glu Ile Asp Pro Gln Gln Arg Val 435 440 445 Ile Leu Phe Val Gly Asp Gly Ser Leu Gln Leu Thr Val Thr Glu Ile 450 455 460 Ser Cys Met Ile Arg Asn Asn Val Lys Pro Tyr Ile Phe Val Leu Asn 465 470 475 480 Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile His Gly Glu Asn Ala Ser 485 490 495 Tyr Asn Asp Val His Met Trp Lys Tyr Ser Lys Ile Leu Asp Thr Phe 500 505 510 Asn Ala Lys Ala His Glu Ser Ile Val Val Asn Thr Lys Gly Glu Met 515 520 525 Asp Ala Leu Phe Asp Asn Glu Glu Phe Ala Lys Pro Asp Lys Ile Arg 530 535 540 Leu Ile Glu Val Met Cys Asp Lys Met Asp Ala Pro Ala Ser Leu Ile 545 550 555 560 Lys Gln Ala Glu Leu Ser Ala Lys Thr Asn Val 565 570 1221713DNAYarrowia lipolytica 122atgagcgact ccgaacccca aatggtcgac ctgggcgact atctctttgc ccgattcaag 60cagctaggcg tggactccgt ctttggagtg cccggcgact tcaacctcac cctgttggac 120cacgtgtaca atgtcgacat gcggtgggtt gggaacacaa acgagctgaa tgccggctac 180tcggccgacg gctactcccg ggtcaagcgg ctggcatgtc ttgtcaccac ctttggcgtg 240ggagagctgt ctgccgtggc tgctgtggca ggctcgtacg ccgagcatgt gggcgtggtg 300catgttgtgg gcgttcccag cacctctgct gagaacaagc atctgctgct gcaccacaca 360ctcggtaacg gcgacttccg ggtctttgcc cagatgtcca aactcatctc cgagtacacc 420caccatattg aggaccccag cgaggctgcc gacgtaatcg acaccgccat ccgaatcgcc 480tacacccacc agcggcccgt ttacattgct gtgccctcca acttctccga ggtcgatatt 540gccgaccagg ctagactgga tacccccctg gacctttcgc tgcagcccaa cgaccccgag 600agccagtacg aggtgattga ggagatttgc tcgcgtatca aggccgccaa gaagcccgtg 660attctcgtcg acgcctgcgc ttcgcgatac agatgtgtgg acgagaccaa ggagctggcc 720aagatcacca actttgccta ctttgtcact cccatgggta agggttctgt ggacgaggat 780actgaccggt acggaggaac atacgtcgga tcgctgactg ctcctgctac tgccgaggtg 840gttgagacag ctgatctcat catctccgta ggagctcttc tgtcggactt caacaccggt 900tccttctcgt actcctactc caccaaaaac gtggtggaat tgcattcgga ccacgtcaaa 960atcaagtccg ccacctacaa caacgtcggc atgaaaatgc tgttcccgcc cctgctcgaa 1020gccgtcaaga aactggttgc cgagacccct gactttgcat ccaaggctct ggctgttccc 1080gacaccactc ccaagatccc cgaggtaccc gatgatcaca ttacgaccca ggcatggctg 1140tggcagcgtc tcagttactt tctgaggccc accgacatcg tggtcaccga gaccggaacc 1200tcgtcctttg gaatcatcca gaccaagttc ccccacaacg tccgaggtat ctcgcaggtg 1260ctgtggggct ctattggata ctcggtggga gcagcctgtg gagcctccat tgctgcacag 1320gagattgacc cccagcagcg agtgattctg tttgtgggcg acggctctct tcagctgacg 1380gtgaccgaga tctcgtgcat gatccgcaac aacgtcaagc cgtacatttt tgtgctcaac 1440aacgacggct acaccatcga gaggctcatt cacggcgaaa acgcctcgta caacgatgtg 1500cacatgtgga agtactccaa gattctcgac acgttcaacg ccaaggccca cgagtcgatt 1560gtggtcaaca ccaagggcga gatggacgct ctgttcgaca acgaagagtt tgccaagccc 1620gacaagatcc ggctcattga ggtcatgtgc gacaagatgg acgcgcctgc ctcgttgatc 1680aagcaggctg agctctctgc caagaccaac gtt 1713123571PRTSchizosaccharomyces pombe 123Met Ser Gly Asp Ile Leu Val Gly Glu Tyr Leu Phe Lys Arg Leu Glu 1 5 10 15 Gln Leu Gly Val Lys Ser Ile Leu Gly Val Pro Gly Asp Phe Asn Leu 20 25 30 Ala Leu Leu Asp Leu Ile Glu Lys Val Gly Asp Glu Lys Phe Arg Trp 35 40 45 Val Gly Asn Thr Asn Glu Leu Asn Gly Ala Tyr Ala Ala Asp Gly Tyr 50 55 60 Ala Arg Val Asn Gly Leu Ser Ala Ile Val Thr Thr Phe Gly Val Gly 65 70 75 80 Glu Leu Ser Ala Ile Asn Gly Val Ala Gly Ser Tyr Ala Glu His Val 85 90 95 Pro Val Val His Ile Val Gly Met Pro Ser Thr Lys Val Gln Asp Thr 100 105 110 Gly Ala Leu Leu His His Thr Leu Gly Asp Gly Asp Phe Arg Thr Phe 115 120 125 Met Asp Met Phe Lys Lys Val Ser Ala Tyr Ser Ile Met Ile Asp Asn 130 135 140 Gly Asn Asp Ala Ala Glu Lys Ile Asp Glu Ala Leu Ser Ile Cys Tyr 145 150 155 160 Lys Lys Ala Arg Pro Val Tyr Ile Gly Ile Pro Ser Asp Ala Gly Tyr 165 170 175 Phe Lys Ala Ser Ser Ser Asn Leu Gly Lys Arg Leu Lys Leu Glu Glu 180 185 190 Asp Thr Asn Asp Pro Ala Val Glu Gln Glu Val Ile Asn His Ile Ser 195 200 205 Glu Met Val Val Asn Ala Lys Lys Pro Val Ile Leu Ile Asp Ala Cys 210 215 220 Ala Val Arg His Arg Val Val Pro Glu Val His Glu Leu Ile Lys Leu 225 230 235 240 Thr His Phe Pro Thr Tyr Val Thr Pro Met Gly Lys Ser Ala Ile Asp 245 250 255 Glu Thr Ser Gln Phe Phe Asp Gly Val Tyr Val Gly Ser Ile Ser Asp 260 265 270 Pro Glu Val Lys Asp Arg Ile Glu Ser Thr Asp Leu Leu Leu Ser Ile 275 280 285 Gly Ala Leu Lys Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr His Leu 290 295 300 Ser Gln Lys Asn Ala Val Glu Phe His Ser Asp His Met Arg Ile Arg 305 310 315 320 Tyr Ala Leu Tyr Pro Asn Val Ala Met Lys Tyr Ile Leu Arg Lys Leu 325 330 335 Leu Lys Val Leu Asp Ala Ser Met Cys His Ser Lys Ala Ala Pro Thr 340 345 350 Ile Gly Tyr Asn Ile Lys Pro Lys His Ala Glu Gly Tyr Ser Ser Asn 355 360 365 Glu Ile Thr His Cys Trp Phe Trp Pro Lys Phe Ser Glu Phe Leu Lys 370 375 380 Pro Arg Asp Val Leu Ile Thr Glu Thr Gly Thr Ala Asn Phe Gly Val 385 390 395 400 Leu Asp Cys Arg Phe Pro Lys Asp Val Thr Ala Ile Ser Gln Val Leu 405 410 415 Trp Gly Ser Ile Gly Tyr Ser Val Gly Ala Met Phe Gly Ala Val Leu 420 425 430 Ala Val His Asp Ser Lys Glu Pro Asp Arg Arg Thr Ile Leu Val Val 435 440 445 Gly Asp Gly Ser Leu Gln Leu Thr Ile Thr Glu Ile Ser Thr Cys Ile 450 455 460 Arg His Asn Leu Lys Pro Ile Ile Phe Ile Ile Asn Asn Asp Gly Tyr 465 470 475 480 Thr Ile Glu Arg Leu Ile His Gly Leu His Ala Ser Tyr Asn Glu Ile 485 490 495 Asn Thr Lys Trp Gly Tyr Gln Gln Ile Pro Lys Phe Phe Gly Ala Ala 500 505 510 Glu Asn His Phe Arg Thr Tyr Cys Val Lys Thr Pro Thr Asp Val Glu 515 520 525 Lys Leu Phe Ser Asp Lys Glu Phe Ala Asn Ala Asp Val Ile Gln Val 530 535 540 Val Glu Leu Val Met Pro Met Leu Asp Ala Pro Arg Val Leu Val Glu 545 550 555 560 Gln Ala Lys Leu Thr Ser Lys Ile Asn Lys Gln 565 570 1241713DNASchizosaccharomyces pombe 124atgagtgggg atattttagt cggtgaatat ctattcaaaa ggcttgaaca attaggggtc 60aagtccattc ttggtgttcc aggagatttc aatttagctc tacttgactt aattgagaaa 120gttggagatg agaaatttcg ttgggttggc aataccaatg agttgaatgg tgcttatgcc 180gctgatggtt atgctcgtgt taatggtctt tcagccattg ttacaacgtt cggcgtggga 240gagctttccg ctattaatgg agtggcaggt tcttatgcgg agcatgtccc agtagttcat 300attgttggaa tgccttccac aaaggtgcaa gatactggag ctttgcttca tcatacttta 360ggagatggag actttcgcac tttcatggat atgtttaaga aagtttctgc ctacagtata 420atgatcgata acggaaacga tgcagctgaa aagatcgatg aagccttgtc gatttgttat 480aaaaaggcta ggcctgttta cattggtatt ccttctgatg ctggctactt caaagcatct 540tcatcaaatc ttgggaaaag actaaagctc gaggaggata ctaacgatcc agcagttgag 600caagaagtca tcaatcatat ctcggaaatg gttgtcaatg caaagaaacc agtgatttta 660attgacgctt gtgctgtaag acatcgtgtc gttccagaag tacatgagct gattaaattg 720acccatttcc ctacatatgt aactcccatg ggtaaatctg caattgacga aacttcgcaa 780ttttttgacg gcgtttatgt tggttcaatt tcagatcctg aagttaaaga cagaattgaa 840tccactgatc tgttgctatc catcggtgct ctcaaatcag actttaacac gggttccttc 900tcttaccacc tcagccaaaa gaatgccgtt gagtttcatt cagaccacat gcgcattcga 960tatgctcttt atccaaatgt agccatgaag tatattcttc gcaaactgtt gaaagtactt 1020gatgcttcta tgtgtcattc caaggctgct cctaccattg gctacaacat caagcctaag 1080catgcggaag gatattcttc caacgagatt actcattgct ggttttggcc taaatttagt 1140gaatttttga agccccgaga tgttttgatc accgagactg gaactgcaaa ctttggtgtc 1200cttgattgca ggtttccaaa ggatgtaaca gccatttccc aggtattatg gggatctatt 1260ggatactccg ttggtgcaat gtttggtgct gttttggccg tccacgattc taaagagccc 1320gatcgtcgta ccattcttgt agtaggtgat ggatccttac aactgacgat tacagagatt 1380tcaacctgca ttcgccataa cctcaaacca attattttca taattaacaa cgacggttac 1440accattgagc gtttaattca tggtttgcat gctagctata acgaaattaa cactaaatgg 1500ggctaccaac agattcccaa gtttttcgga gctgctgaaa accacttccg cacttactgt 1560gttaaaactc ctactgacgt tgaaaagttg tttagcgaca aggagtttgc aaatgcagat 1620gtcattcaag tagttgagct tgtaatgcct atgttggatg cacctcgtgt cctagttgag 1680caagccaagt tgacgtctaa gatcaataag caa 1713125563PRTZygosaccharomyces rouxii 125Met Ser Glu Ile Thr Leu Gly Arg Tyr Leu Phe Glu Arg Leu Lys Gln 1 5 10 15 Val Asp Thr Asn Thr Ile Phe Gly Val Pro Gly Asp Phe Asn Leu Ser 20 25 30 Leu Leu Asp Lys Val Tyr Glu Val Gln Gly Leu Arg Trp Ala Gly Asn 35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Val 50 55 60 Lys Gly Leu Ala Ala Leu Ile Thr Thr Phe Gly Val Gly Glu Leu Ser 65 70 75 80 Ala Leu Asn Gly Ile

Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu 85 90 95 His Ile Val Gly Val Pro Ser Val Ser Ser Gln Ala Lys Gln Leu Leu 100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met 115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Leu Thr Asp Ile Thr Ala 130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Val Ala Tyr Val Asn Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Gln Lys Val 165 170 175 Pro Ala Ser Leu Leu Asn Thr Pro Ile Asp Leu Ser Leu Lys Glu Asn 180 185 190 Asp Pro Glu Ala Glu Thr Glu Val Val Asp Thr Val Leu Glu Leu Ile 195 200 205 Lys Glu Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg 210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ser Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln Asn 245 250 255 Pro Arg Phe Gly Gly Val Tyr Val Gly Thr Leu Ser Ser Pro Glu Val 260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Val Leu Ser Val Gly Ala Leu 275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys 290 295 300 Asn Val Val Glu Phe His Ser Asp His Ile Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Lys Lys Leu Leu Gln Ala 325 330 335 Val Pro Glu Ala Val Lys Asn Tyr Lys Pro Gly Pro Val Pro Ala Pro 340 345 350 Pro Ser Pro Asn Ala Glu Val Ala Asp Ser Thr Thr Leu Lys Gln Glu 355 360 365 Trp Leu Trp Arg Gln Val Gly Ser Phe Leu Arg Glu Gly Asp Val Val 370 375 380 Ile Thr Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr His Phe 385 390 395 400 Pro Asn Gln Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly 405 410 415 Tyr Thr Thr Gly Ser Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile 420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln 435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro 450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Arg Leu Ile 465 470 475 480 His Gly Glu Thr Ala Glu Tyr Asn Cys Ile Gln Pro Trp Lys His Leu 485 490 495 Glu Leu Leu Asn Thr Phe Gly Ala Lys Asp Tyr Glu Asn His Arg Val 500 505 510 Ser Thr Val Gly Glu Trp Asn Lys Leu Thr Gln Asp Pro Lys Phe Asn 515 520 525 Glu Asn Ser Arg Ile Arg Met Ile Glu Val Met Leu Glu Val Met Asp 530 535 540 Ala Pro Ser Ser Leu Val Ala Gln Ala Gln Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln 1261689DNAZygosaccharomyces rouxii 126atgtctgaaa ttactctagg tcgttacttg ttcgaaagat taaagcaagt tgacactaac 60accatcttcg gtgttccagg tgacttcaac ttgtccttgt tggacaaggt ctacgaagtg 120caaggtctaa gatgggctgg taacgctaac gaattgaacg ctgcctacgc tgctgacggt 180tacgccagag ttaagggttt ggctgctttg atcaccacct tcggtgtcgg tgaattgtct 240gctttgaacg gtattgcagg ttcttacgct gaacacgttg gtgttttgca cattgttggt 300gttccatctg tctcttctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360gacttcactg ttttccacag aatgtccgcc aacatctctg aaaccaccgc tatgttgacc 420gacatcactg ctgctccagc tgaaattgac cgttgcatca gagttgctta cgtcaaccaa 480agaccagtct acttgggtct accagctaac ttggttgacc aaaaggtccc agcttctttg 540ttgaacactc caattgatct atctctaaag gagaacgacc cagaagctga aaccgaagtt 600gttgacaccg ttttggaatt gatcaaggaa gctaagaacc cagttatctt ggctgatgct 660tgctgctcca gacacgacgt caaggctgaa accaagaagt tgatcgactt gactcaattc 720ccatctttcg ttactcctat gggtaagggt tccatcgacg aacaaaaccc aagattcggt 780ggtgtctacg tcggtactct atccagccca gaagttaagg aagctgttga atctgctgac 840ttggttctat ctgtcggtgc tctattgtcc gatttcaaca ctggttcttt ctcttactct 900tacaagacca agaacgttgt tgaattccac tctgaccaca tcaagatcag aaacgctacc 960ttcccaggtg ttcaaatgaa attcgttttg aagaaactat tgcaagctgt cccagaagct 1020gtcaagaact acaagccagg tccagtccca gctccgccat ctccaaacgc tgaagttgct 1080gactctacca ccttgaagca agaatggtta tggagacaag tcggtagctt cttgagagaa 1140ggtgatgttg ttattaccga aactggtacc tctgctttcg gtatcaacca aactcacttc 1200cctaaccaaa cttacggtat ctctcaagtc ttgtggggtt ctattggtta caccactggt 1260tccactttgg gtgctgcctt cgctgctgaa gaaattgacc ctaagaagag agttatcttg 1320ttcattggtg acggttctct acaattgacc gttcaagaaa tctccaccat gatcagatgg 1380ggtctaaagc catacttgtt cgttttgaac aacgatggtt acaccattga aagattgatt 1440cacggtgaaa ccgctgaata caactgtatc caaccatgga agcacttgga attgttgaac 1500accttcggtg ccaaggacta cgaaaaccac agagtctcca ctgtcggtga atggaacaag 1560ttgactcaag atccaaaatt caacgaaaac tctagaatta gaatgatcga agttatgctt 1620gaagtcatgg acgctccatc ttctttggtc gctcaagctc aattgaccgc tgctactaac 1680gctaagcaa 1689127267PRTSaccharomyces cerevisiae 127Met Ser Gln Gly Arg Lys Ala Ala Glu Arg Leu Ala Lys Lys Thr Val 1 5 10 15 Leu Ile Thr Gly Ala Ser Ala Gly Ile Gly Lys Ala Thr Ala Leu Glu 20 25 30 Tyr Leu Glu Ala Ser Asn Gly Asp Met Lys Leu Ile Leu Ala Ala Arg 35 40 45 Arg Leu Glu Lys Leu Glu Glu Leu Lys Lys Thr Ile Asp Gln Glu Phe 50 55 60 Pro Asn Ala Lys Val His Val Ala Gln Leu Asp Ile Thr Gln Ala Glu 65 70 75 80 Lys Ile Lys Pro Phe Ile Glu Asn Leu Pro Gln Glu Phe Lys Asp Ile 85 90 95 Asp Ile Leu Val Asn Asn Ala Gly Lys Ala Leu Gly Ser Asp Arg Val 100 105 110 Gly Gln Ile Ala Thr Glu Asp Ile Gln Asp Val Phe Asp Thr Asn Val 115 120 125 Thr Ala Leu Ile Asn Ile Thr Gln Ala Val Leu Pro Ile Phe Gln Ala 130 135 140 Lys Asn Ser Gly Asp Ile Val Asn Leu Gly Ser Ile Ala Gly Arg Asp 145 150 155 160 Ala Tyr Pro Thr Gly Ser Ile Tyr Cys Ala Ser Lys Phe Ala Val Gly 165 170 175 Ala Phe Thr Asp Ser Leu Arg Lys Glu Leu Ile Asn Thr Lys Ile Arg 180 185 190 Val Ile Leu Ile Ala Pro Gly Leu Val Glu Thr Glu Phe Ser Leu Val 195 200 205 Arg Tyr Arg Gly Asn Glu Glu Gln Ala Lys Asn Val Tyr Lys Asp Thr 210 215 220 Thr Pro Leu Met Ala Asp Asp Val Ala Asp Leu Ile Val Tyr Ala Thr 225 230 235 240 Ser Arg Lys Gln Asn Thr Val Ile Ala Asp Thr Leu Ile Phe Pro Thr 245 250 255 Asn Gln Ala Ser Pro His His Ile Phe Arg Gly 260 265 128804DNASaccharomyces cerevisiae 128atgtcccaag gtagaaaagc tgcagaaaga ttggctaaga agactgtcct cattacaggt 60gcatctgctg gtattggtaa ggcgaccgca ttagagtact tggaggcatc caatggtgat 120atgaaactga tcttggctgc tagaagatta gaaaagctcg aggaattgaa gaagaccatt 180gatcaagagt ttccaaacgc aaaagttcat gtggcccagc tggatatcac tcaagcagaa 240aaaatcaagc ccttcattga aaacttgcca caagagttca aggatattga cattctggtg 300aacaatgccg gaaaggctct tggcagtgac cgtgtgggcc agatcgcaac ggaggatatc 360caggacgtgt ttgacaccaa cgtcacggct ttaatcaata tcacacaagc tgtactgccc 420atattccaag ccaagaattc aggagatatt gtaaatttgg gttcaatcgc tggcagagac 480gcatacccaa caggttctat ctattgtgcc tctaagtttg ccgtgggggc gttcactgat 540agtttgagaa aggagctcat caacactaaa attagagtca ttctaattgc accagggcta 600gtcgagactg aattttcact agttagatac agaggtaacg aggaacaagc caagaatgtt 660tacaaggata ctaccccatt gatggctgat gacgtggctg atctgatcgt ctatgcaact 720tccagaaaac aaaatactgt aattgcagac actttaatct ttccaacaaa ccaagcgtca 780cctcatcata tcttccgtgg ataa 804

Claims

1-27. (canceled)

28. A method comprising providing a feedstock slurry comprising fermentable carbon source, undissolved solids, and oil; separating the feedstock slurry whereby (i) a first aqueous solution comprising a fermentable carbon source, (ii) a first wet cake comprising solids, and (iii) a stream comprising oil, solids, and an aqueous stream comprising a fermentable carbon source are formed; and adding the first aqueous solution to a fermentation broth comprising microorganisms whereby a fermentation product is produced.

29. The method of claim 28, further comprising separating the stream comprising oil, solids, and aqueous stream comprising a fermentable carbon source whereby (i) a second aqueous solution comprising a fermentable carbon source, (ii) a second wet cake comprising solids, and (iii) an oil stream are formed.

30. The method of claim 29, wherein the first and second aqueous solutions are combined prior to the addition to the fermentation broth.

31. The method of claim 29, wherein the second aqueous solution further comprises oil.

32. The method of claim 31, wherein the oil of the second aqueous solution or portion thereof is treated to generate an extractant.

33. The method of claim 32, wherein the oil is treated chemically or enzymatically.

34. The method of claim 28, wherein separation is by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

35. A method comprising providing a feedstock slurry comprising a fermentable carbon source, undissolved solids, and oil; separating the feedstock slurry whereby (i) a first aqueous solution comprising a fermentable carbon source and solids, (ii) a first wet cake comprising solids, and (iii) a first oil stream are formed; and adding oil to the first aqueous solution whereby an oil layer comprising solids and a second aqueous solution comprising a fermentable carbon source are formed.

36. The method of claim 35, wherein the oil layer comprising solids is separated forming (i) a second oil stream, (ii) a second wet cake comprising solids, and (iii) a third aqueous solution comprising a fermentable carbon source.

37. The method of claim 36, wherein the second aqueous solution and the third aqueous solution are added to a fermentation broth comprising microorganisms whereby a fermentation product is produced.

38. The method of claim 35, wherein the second aqueous solution and the third aqueous solution further comprise oil.

39. The method of claim 38, wherein the second aqueous solution and the third aqueous solution are combined and the oil of the second aqueous solution and the third aqueous solution or portions thereof is treated to generate an extractant.

40. The method of claim 39, wherein the oil is treated chemically or enzymatically.

41. The method of claim 35, wherein separation is by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

42-47. (canceled)

48. The method of claim 29, wherein separation is by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof.

49. The method of claim 36, wherein separation is by decanter bowl centrifugation, three-phase centrifugation, disk stack centrifugation, filtering centrifugation, decanter centrifugation, filtration, vacuum filtration, beltfilter, pressure filtration, filtration using a screen, screen separation, grating, porous grating, flotation, hydrocyclone, filter press, screwpress, gravity settler, vortex separator, or combination thereof

50. The method of claim 28, wherein one or more control parameters of the separation device is adjusted to improve separation of the feedstock slurry.

51. The method of claim 50, wherein the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, weir position, scroll pitch, residence time, and discharge volume.

52. The method of claim 28, wherein real-time measurements are used to monitor separation of the feedstock slurry.

53. The method of claim 52, wherein separation is monitored by Fourier transform infrared spectroscopy, near-infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometers, densitometers, tensiometers, droplet size analyzers, particle analyzers, or combinations thereof.

54. The method of claim 35, wherein one or more control parameters of the separation device is adjusted to improve separation of the feedstock slurry.

55. The method of claim 54, wherein the one or more control parameters are selected from differential speed, bowl speed, flow rate, impeller position, weir position, scroll pitch, residence time, and discharge volume.

56. The method of claim 35, wherein real-time measurements are used to monitor separation of the feedstock slurry.

57. The method of claim 56, wherein separation is monitored by Fourier transform infrared spectroscopy, near-infrared spectroscopy, Raman spectroscopy, high pressure liquid chromatography, viscometers, densitometers, tensiometers, droplet size analyzers, particle analyzers, or combinations thereof.