Compositions And Methods For Rapid And Dynamic Flux Control Using Synthetic Metabolic Valves

Abstract

This invention relates to metabolically engineered microorganisms, such as bacterial and or fungal strains, and bioprocesses utilizing such strains. These strains enable the dynamic control of metabolic pathways, which can be used to optimize production. Dynamic control over metabolism is accomplished via a combination of methodologies including but not limited to transcriptional silencing and controlled enzyme proteolysis. These microbial strains are utilized in a multi-stage bioprocess encompassing at least two stages, the first stage in which organisms are grown and metabolism can be optimized for microbial growth and at least one other stage in which growth can be slowed or stopped, and dynamic changes can be made to metabolism to improve the production of desired product, such as a chemical or fuel.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 16/849,441, filed Apr. 15, 2020, which is a continuation of U.S. application Ser. No. 15/317,768 which is a .sctn. 371 U.S. National Stage of International Application PCT/US2015/035306, filed Jun. 11, 2015, which claims the benefit of U.S. Provisional Application No. 62/010,574, filed Jun. 11, 2014, the entire content of which are incorporated by reference herein in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0003] This application contains a sequence listing. It has been submitted electronically via EFS-Web as an ASCII text file entitled "OLG Ref 210-44_ST25.txt". The sequence listing is 184,352 bytes in size, and was created on Jun. 11, 2015. It is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0004] This invention relates to metabolically engineered microorganisms, such as bacterial and or fungal strains, and bioprocesses utilizing such strains. These strains enable the dynamic control of metabolic pathways.

BACKGROUND OF THE INVENTION

[0005] Petroleum is the primary feedstock, not only for the fuels we use, but the majority of the chemicals we consume as well. The chemical industry is heavily reliant on this non-renewable resource. Replacement of petroleum with renewable feedstocks ensures longer-term viability and environmental sustainability. Novel fermentation based processes to make chemicals have been a contributing technology, enabling the change to renewable feedstocks (Werpy &Peterson, Top Value Added Chemicals from Biomass. Volume I--Results of Screening for Potential Candidates from Sugars and Synthesis Gas., Yixiang et al. "Green" Chemicals from Renewable Agricultural Biomass--A Mini Review. The Open Agriculture Journal, 2008). These fermentation processes have made rapid advancements in recent years due to technology developments in the fields of fermentation science, synthetic biology, as well as metabolic and enzyme engineering (Jarboe, L. R., et al., Metabolic engineering for production of biorenewable fuels and chemicals: contributions of synthetic biology. J Biomed Biotechnol, 2010, Lee, J. W., et al., Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat Chem Biol, 2012). Despite these substantial advances, most successful examples of rationale directed engineering approaches have also greatly relied on numerous cycles of trial and error. The field of metabolic engineering has historically been limited in predicting the behavior of complex biological systems in-vivo, from simplified models and basic in-vitro biochemical principles. Such rational approaches have required significant a priori knowledge of microbial physiology that in many cases is incomplete. This is particularly true for complex phenotypes that require an intricate balance between the activities of many seemingly unrelated gene products. In many cases it has proven much more difficult than expected to integrate a possibly well characterized production pathway into a living host and balance the complex requirements of both biomass growth and production.

[0006] One solution is the development of platform microbial strains that utilize synthetic metabolic valves (SMVs) that can decouple growth from product formation. These strains enable the dynamic control of metabolic pathways, including those that when altered have negative effects on microorganism growth. Dynamic control over metabolism is accomplished via a combination of methodologies including but not limited to transcriptional silencing and controlled enzyme proteolysis. These microbial strains are utilized in a multi-stage bioprocess encompassing as least two stages, the first stage in which microorganisms are grown and metabolism can be optimized for microbial growth and at least one other stage in which growth can be slowed or stopped, and dynamic changes can be made to metabolism to improve production of desired product, such as a chemical or fuel. The transition of growing cultures between stages and the manipulation of metabolic fluxes can be controlled by artificial chemical inducers or preferably by controlling the level of key limiting nutrients. In addition, genetic modifications may be made to provide metabolic pathways for the biosynthesis of one or more chemical or fuel products. Also, genetic modifications may be made to enable the utilization of a variety of carbon feedstocks including but not limited sugars such as glucose, sucrose, xylose, arabinose, mannose, and lactose, oils, carbon dioxide, carbon monoxide, methane, methanol and formaldehyde.

[0007] This approach allows for simpler models of metabolic fluxes and physiological demands during a production phase, turning a growing cell into a stationary phase biocatalyst. These synthetic metabolic valves can be used to turn off essential genes and redirect carbon, electrons and energy flux to product formation in a multi-stage fermentation process. One or more of the following enables these synthetic valves: 1) transcriptional gene silencing or repression technologies in combination with 2) inducible enzyme degradation and 3) nutrient limitation to induce a stationary or non-dividing cellular state. SMVs are generalizable to any pathway and microbial host. These synthetic metabolic valves allow for novel rapid metabolic engineering strategies useful for the production of renewable chemicals and fuels and any product that can be produced via whole cell catalysis.

[0008] A simplified two-stage bioprocess using synthetic metabolic valves is depicted in FIG. 1, strains are grown in a minimal media with a single limiting nutrient such as inorganic phosphate. During this growth phase cells are not producing any product other than biomass and as a result are not subject to any possible toxic or unwanted side effects of product formation. Biomass growth and yield can be optimized. As the limiting nutrient is depleted, cell growth is stopped. Simultaneously, these strains will be engineered to contain synthetic metabolic valves, which silence genes and enzymes essential for growth and redirect carbon, electrons and energy to any molecule of interest. This process utilizes a novel combination of a two-stage production and concurrent metabolic engineering strategy.

[0009] There is significant precedent in the biotechnology industry for using and scaling two stage processes similar to that described in FIG. 1. Many similar processes are routinely used for the heterologous expression of proteins. In these standard processes cells are grown to a productive or "primed" state for protein synthesis (such as mid-exponential phase in E. coli) and then induced to express a heterologous protein. In many cases, the diversion of cellular amino acids and energy to the heterologous protein has a significant effect on, if not halting, cellular growth. It is not surprising that these types of processes have not been developed for the biological production of small molecules as historically most successful efforts to metabolically engineer the production of small molecules have leveraged the power of anaerobic metabolism to couple product formation with growth.

[0010] Anaerobic growth-coupled product formation enables the use of powerful growth based selections to identify better producers. The faster the cells grow the more product they make. This has allowed for the classical selection of industrial strains for many natural products such as ethanol and isobutanol. However, the requirement for anaerobic production greatly limits the number and variety of different molecules or products that can be made using synthetic biology. Numerous products would require aerobic metabolism to supply the needed energy and cofactors to allow for a thermodynamically feasible metabolic pathway. In these cases a generic and robust aerobic production platform would greatly simplify the optimization and scale up of a diverse number of products. A controlled multi-stage process, enabled by synthetic metabolic valves, supplies such a platform.

[0011] Synthetic metabolic valves enable synthetic biologists and metabolic engineers the ability to decouple the complex metabolic and thermodynamic needs of growth from those of product formation. This decoupling also enables the removal of growth based regulatory mechanisms that may inhibit product formation and allows for the silencing of essential metabolic pathways that may detract from or interfere with production. These essential interfering metabolic pathways could include amino acid biosynthesis or the citric acid cycle as well as the biosynthesis of many secondary metabolites, and those pathways involved in maintaining intracellular redox and energy balances. These pathways have traditionally been off limits to many metabolic engineering strategies, as attempts at manipulation have led to growth defects.

SUMMARY OF THE INVENTION

[0012] According to one embodiment, the invention is directed to methods to construct controllable synthetic metabolic valves. In certain of these embodiments synthetic metabolic valves are used to controllably reduce or eliminate flux through one more metabolic pathways. In further embodiments, flux is reduced or eliminated through one or more metabolic pathways whose enzymes are essential for microbial growth in a given environment. In other embodiments, the invention is related to genetically modified microorganisms that utilize one or more synthetic metabolic valves thereby enabling dynamic control over metabolic pathways. Other embodiments of the invention are directed to multi-stage bioprocesses that utilize genetically modified microorganism that in turn utilize one or more synthetic metabolic valves that enable dynamic flux control. Still in other embodiments of the invention, the transitions between stages in multistage bioprocesses using genetically modified microorganisms are controlled by the addition of chemical inducers or by the control of key nutrient levels. Additional genetic modifications may be added to a microorganism to enable the conversion of carbon feedstocks to chemical or fuel products. In certain embodiments, carbon feedstocks can include, but are not limited to the sugars: glucose, sucrose xylose, arabinose, mannose, lactose, or alternatively carbon dioxide, carbon monoxide, methane, methanol, formaldehyde, or oils. In addition, genetic modifications to produce chemical or fuel products from various carbon feedstocks can include metabolic pathways utilizing, but not limited to, the central metabolites acetyl-CoA, malonyl-CoA, pyruvate, oxaloacetate, erthyrose-4-phosphate, xylulose-5-phosphate, alpha-ketoglutarate and citrate. Products that can be derived from these central metabolites include but are not limited to acetate, alcohols (ethanol, butanol, hexanol, and longer n-alcohols), organic acids (3-hydroxyprpionic acid, lactic acid, itaconic acid), amino acids (alanine, serine, valine), fatty acids and their derivatives (fatty acid methyl esters (FAMEs), fatty aldehydes, alkenes, alkanes) and isoprenoids.

[0013] In various embodiments, the increased production of acetate from acetyl-phosphate may occur via the increased expression of an acetate kinase. A non-limiting example is the acetate kinase from E. coli encoded by the ackA gene. Increased expression of an acetate kinase may optionally be combined with genetic modifications that result decreased activity phosphoacetyltransferase such as that encoded by the pta gene of E. coli.

[0014] In various embodiments, the increased production of ethanol from acetyl-CoA may occur via the increased expression of an oxygen tolerant ethanol dehydrogenase, such as the enzyme from E. coli encoded by the adhE gene with a mutation Glu568Lys as taught by Dellomonaco et al, AEM. August 2010, Vol. 76, No. 15, p 5067. and Holland-Staley et al. JBACs. November 2000, Vol. 182, No. 21, p 6049.

[0015] In various embodiments, the increased production of butyrate from acetyl-CoA may occur via the increased expression of butyrate pathway enzymes including an acetoacetyl-CoA thiolase, crotonase, crotonyl-CoA reductase, butyrate phospho-transferase and butyrate kinase as taught by Fischer et al, Appl Microbiol Biotechnol. 2010, September, Vol. 88, No. 1, p. 265-275. Alternatively, increased butyrate may be accomplished via the increased expression of butyrate pathway enzymes including an acetoacetyl-CoA synthase, crotonase, crotonyl-CoA reductase and butyryl-CoA thioesterase as taught by PCT/US2012/030209.

[0016] In various embodiments, the increased production of n-butanol from acetyl-CoA may occur via the increased expression of n-butanol pathway enzymes including an acetoacetyl-CoA thiolase, crotonase, crotonyl-CoA reductase, butyryl-CoA reductase and butyraldehyde reductase as taught by Atsumi et al, Metabolic Engineering. 2008. November, Vol. 10, No. 6, p. 305).

[0017] In various embodiments, the increased production of fatty acids of chain length greater than 4, from acetyl-CoA may occur via the increased expression of a fatty acid synthesis pathway enzymes including an ketoacetyl-CoA synthase, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, and a acyl-CoA thioesterase as taught by PCT/US2012/030209.

[0018] In various embodiments, the increased production of fatty acid methyl esters from acetyl-CoA may occur via the increased expression of fatty acid methyl ester synthesis pathway enzymes including an ketoacetyl-CoA synthase, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, and a acyl-CoA wax ester synthase as taught by: PCT/US2012/030209 and US 20110146142 A1.

[0019] In various embodiments, the increased production of n-hexanol from acetyl-CoA may occur via the increased expression of a fatty acid synthesis pathway enzymes including an ketoacetyl-CoA thiolases, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, and a acyl-CoA thioesterase as taught by Dekishima et al. J Am Chem Soc. 2011. August. Vol. 133, No. 30, p. 1139.

[0020] In various embodiments, the increased production of n-alcohols of chain length greater than 4, from acetyl-CoA may occur via the increased expression of a fatty acid synthesis pathway enzymes including an ketoacetyl-CoA synthase, 3-hydroxyacyl-CoA dehydratase, an enoyl-CoA reductase, as taught by PCT/US2012/030209 and a fatty acyl-CoA reductase and fatty aldehyde reductase as taught by Yan-Ning Zheng et al. Microbial Cell Factories. 2012. Vol. 11:65.

[0021] In various embodiments, the increased production of n-alkenes can be accomplished by first producing n-alcohols as described elsewhere followed by the chemical dehydration of the n-alcohol to an n-alkene by catalytic methods well known in the art.

[0022] In various embodiments, the increased production of n-alkanes can be accomplished by first producing fatty acids as described elsewhere followed by the chemical decarboxylation of the n-alcohol to an alkane by catalytic methods well known in the art.

[0023] In various embodiments, the increased production of isoprene from acetyl-CoA may occur via the increased expression of pathway enzymes including an acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonte diphosphate decarboxylase, isopentenyl-diphosphate isomerase and isoprene synthase as taught by US 20120276603 A1.

[0024] In various embodiments, the increased production of a product from acetyl-CoA may occur via both the increased expression of an acetyl-CoA carboxylase enzyme which can convert acetyl-CoA into malonyl-CoA and the increased expression of a production pathway comprising multiple pathway enzymes which can convert malonyl-CoA further to a product.

[0025] In various embodiments, the increased production of a product from malonyl-CoA may occur via both the increased activity of an acetyl-CoA carboxylase enzyme which can caused by mutation of one or more fatty acid synthesis enzymes such as is taught by PCT/US2012/030209, PCT/US2011/0222790 and 3. UK Patent GB2473755 and the increased expression of a production pathway comprising multiple pathway enzymes which can convert malonyl-CoA further to a product.

[0026] Within the scope of the invention are genetically modified microorganism, wherein the microorganism is capable of producing an acetyl-CoA derived product at a specific rate selected from the rates of greater than 0.05 g/gDCW-hr, 0.08 g/gDCW-hr, greater than 0.1 g/gDCW-hr, greater than 0.13 g/gDCW-hr, greater than 0.15 g/gDCW-hr, greater than 0.175 g/gDCW-hr, greater than 0.2 g/gDCW-hr, greater than 0.25 g/gDCW-hr, greater than 0.3 g/gDCW-hr, greater than 0.35 g/gDCW-hr, greater than 0.4 g/gDCW-hr, greater than 0.45 g/gDCW-hr, or greater than 0.5 g/gDCW-hr.

[0027] Within the scope of the invention are genetically modified microorganism, wherein the microorganism is capable of producing a product derived from any key metabolic intermediate including but not limited to malonyl-CoA, pyruvate, oxaloacetate, erthyrose-4-phosphate, xylulose-5-phosphate, alpha-ketoglutarate and citrate at a specific rate selected from the rates of greater than 0.05 g/gDCW-hr, 0.08 g/gDCW-hr, greater than 0.1 g/gDCW-hr, greater than 0.13 g/gDCW-hr, greater than 0.15 g/gDCW-hr, greater than 0.175 g/gDCW-hr, greater than 0.2 g/gDCW-hr, greater than 0.25 g/gDCW-hr, greater than 0.3 g/gDCW-hr, greater than 0.35 g/gDCW-hr, greater than 0.4 g/gDCW-hr, greater than 0.45 g/gDCW-hr, or greater than 0.5 g/gDCW-hr.

[0028] In various embodiments, the invention includes a culture system comprising a carbon source in an aqueous medium and a genetically modified microorganism according to any one of claims herein, wherein said genetically modified organism is present in an amount selected from greater than 0.05 gDCW/L, 0.1 gDCW/L, greater than 1 gDCW/L, greater than 5 gDCW/L, greater than 10 gDCW/L, greater than 15 gDCW/L or greater than 20 gDCW/L, such as when the volume of the aqueous medium is selected from greater than 5 mL, greater than 100 mL, greater than 0.5 L, greater than 1 L, greater than 2 L, greater than 10 L, greater than 250 L, greater than 1000 L, greater than 10,000 L, greater than 50,000 L, greater than 100,000 L or greater than 200,000 L, and such as when the volume of the aqueous medium is greater than 250 L and contained within a steel vessel.

[0029] All publications, patents, and patent applications mentioned in this specification 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The novel features of the invention are set forth with particularity in the claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0031] FIG. 1 depicts an overview of a two-phase fermentation processes utilizing a microbe with synthetic metabolic valves. Top Panel: Overview of the fermentation process. Biomass is grown in minimal media with a single limiting macronutrient, such as inorganic phosphate. As the biomass level (black line) or number of cells increases the limiting nutrient (red line) is depleted. When the limiting nutrient is completely consumed, biomass growth is halted. Simultaneously the limitation induces metabolic changes to initiate product biosynthesis through engineered synthetic valves. Lower Panel: Metabolic Changes in the Two Phase Process. In correlation with the system level changes, metabolic changes are induced upon depletion of the limiting nutrient. Specifically, genes encoding metabolic pathways essential for cellular growth "growth genes" are active in the growth phase while genes encoding product biosynthesis "product genes" are silenced. Upon entry into the production phase triggered by nutrient depletion, "growth genes" are silenced and "product genes" are activated.

[0032] FIG. 2 depicts an overview of a synthetic metabolic valve in E. coli using a combination of CRISPR interference gene silencing and controlled protein degradation. Upper Panel: (LEFT) Constructs are made to express small guide RNAs to target a gene of interest in addition to (RIGHT) the controlled induction of a cascade protein complex such as catalytically inactive Cas9 or dCas9 as well as the controlled induction of the chaperone (clpXP enhancing factor) sspB. Expression can be controlled such as by the controlled ptet promoter induced by aTc. The constructs produce dCas9 and sspB proteins in addition to a targeting sgRNA. Bottom Panel: (LEFT) The target gene/protein contains a C-terminal DAS4 tag for binding to sspB. (RIGHT) When expression is induced, dCas9 is targeted to the gene of interest by the targeting sgRNA thereby silencing transcription. Concurrently, the expression of sspB results in the binding of sspB to the DAS4 C-terminal tag of protein that has already been translated. The sspB/DAS4 complex is then targeted for degradation by the clpXP protease.

[0033] FIG. 3 depicts the production of tetrahydroxynapthalene (THN) by redirecting flux from malonyl-CoA. Upper Panel: An overview of redirecting flux from growth to product by controlling fabI (enoyl-coA reductase levels) in E. coli. In E. coli, the primary fate of the intermediate malonyl-CoA is to provide precursors for fatty acid synthesis. The key enzyme controlling the rate of lipid synthesis, acetyl-CoA carboxylase, encoded by the accABCD genes, is strongly inhibited by the fatty acid production intermediates, fatty acyl-ACPs. Removal of fab1 leads to a decrease in acyl-ACP pools and a reduction in inhibition of acetyl-CoA carboxylase allowing malonyl-CoA levels to accumulate and be used for product synthesis. The removal of fab1 limits lipid production and halts growth. Lowe Panel: One potential product from malonyl-CoA is tetrahydroxynapthalene (THN). THN is produced from 5 molecules of malonyl-CoA via the polyketide synthase, THN synthase encoded by the rppA gene of S. coelicolor.

[0034] FIG. 4 depicts increased production of tetrahydroxynapthalene from malonyl-CoA in a two stage process as a result of the controlled inactivation of a temperature sensitive fabI allele. Improved production of THN by redirecting malonyl-CoA flux, using a temperature controlled process to inactivate a temperature sensitive allele of fabI. Strains as listed BWalpdf (BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE), BWalpdf-fab1(ts) (BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE, fab1(F241S), gentR). Plasmids are i) pSMART-HC-Kan-yibD-THNS and ii) pSMART-HC-Kan (control).

[0035] FIG. 5 depicts increased production of tetrahydroxynapthalene from malonyl-CoA in a two stage process as a result of a combination of controlled protein degradation and gene silencing. Improved production of THN by redirecting malonyl-CoA flux, using a synthetic metabolic vlae comprising a combination of CRISPR interference gene silencing and controlled proteolysis as outlined in FIG. 2. THN production at 4 hrs and 20 hrs is compared for two strains. LEFT: Strain BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE, .DELTA.sspB, fabI::DAS4, gentR containing plasmids i) pSMART-HC-Kan-yibD-THNS ii) pdCas9-ptet-sspB and iii) pCDF-control lacking a targeting sgRNA. RIGHT: Strain BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE, .DELTA.sspB, fabI::DAS4, gentR containing plasmids i) pSMART-HC-Kan-yibD-THNS ii) pdCas9-ptet-sspB and iii) pCDF-T2-fabIsgRNA expressing a sgRNA targeting fabI.

[0036] FIG. 6 depicts the low phosphate induction of a GFP reporter with various low phosphate inducible promoters. A comparison of the low phosphate inducible expression for the following gene promoters: amn, phoA, phoB, phoE, phoH, phoU, mipA, pstS, ugpB, waaH and ydfH, is shown. An ultraviolet excitable, green fluorescent protein (GFPuv) reporter gene was used and relative fluorescent units (RFU) are plotted as a function of time. Growth stops and phosphate depletion begins at about 15-20 hrs.

[0037] FIG. 7 depicts the dynamic control over protein levels in E. coli using the CASCADE System and controlled proteolysis. Strain DLF_0025 (enabling low phosphate DAS+4 degradation) has been modified to constitutively express a mCherry protein with a C-terminal DAS+4 degradation tag. In addition the strain has been modified for the low phosphate induction of GFPuv as well as a guide RNA repressing mCherry expression. As cells grow phosphate is depleted, and cells "turn off" mCherry and "turn on" GFPuv. Biomass is plotted as grams cell dry weight per liter, GFPuv and mCherry are plotted as relative fluorescence units (RFU) which are normalized to biomass levels.

[0038] FIG. 8 depicts the production of 3-HP from malonyl-CoA and NADPH at mL scale. Average Maximal 3-HP titers are plotted for several production strains.

[0039] FIG. 9 depicts the production of 3-HP from malonyl-CoA and NADPH at L scale. Biomass and 3-HP titers are plotted as a function of time.

[0040] FIG. 10 depicts the production of alanine from pyruvate and NADPH at mL scale. Biomass and alanine titers are plotted as a function of time.

[0041] FIG. 11 depicts the production of alanine from pyruvate and NADPH at the L scale. Biomass and alanine titers are plotted as a function of time.

[0042] FIG. 12 depicts the production of 2,3-butanediol from pyruvate and NADH at mL scale. Biomass and 2,3-butanediol titers are plotted as a function of time.

[0043] FIG. 13 depicts the production of 2,3-butanediol from pyruvate and NADH at L scale. Biomass and 2,3-butanediol titers are plotted as a function of time.

[0044] FIG. 14 depicts the production of 2,3-butanediol from pyruvate and NADPH at mL scale. Biomass and 2,3-butanediol titers are plotted as a function of time.

[0045] FIG. 15 depicts the production of mevalonic acid from acetyl-CoA and NADPH at L scale. Biomass and mevalonic acid titers are plotted as a function of time.

DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention is related to various production methods and/or genetically modified microorganisms that have utility for fermentative production of various chemical products, to methods of making such chemical products that utilize populations of these microorganisms in vessels, and to systems for chemical production that employ these microorganisms and methods. Among the benefits of the present invention is the increased ability to reduce or eliminate metabolic pathways required for microbial growth that may interfere with production.

Definitions

[0047] As used in the specification and the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "expression vector" includes a single expression vector as well as a plurality of expression vectors, either the same (e.g., the same operon) or different; reference to "microorganism" includes a single microorganism as well as a plurality of microorganisms; and the like.

[0048] As used herein, "reduced enzymatic activity," "reducing enzymatic activity," and the like is meant to indicate that a microorganism cell's, or an isolated enzyme, exhibits a lower level of activity than that measured in a comparable cell of the same species or its native enzyme. That is, enzymatic conversion of the indicated substrate(s) to indicated product(s) under known standard conditions for that enzyme is at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 percent less than the enzymatic activity for the same biochemical conversion by a native (non-modified) enzyme under a standard specified condition. This term also can include elimination of that enzymatic activity. A cell having reduced enzymatic activity of an enzyme can be identified using any method known in the art. For example, enzyme activity assays can be used to identify cells having reduced enzyme activity. See, for example, Enzyme Nomenclature, Academic Press, Inc., New York 2007.

[0049] The term "heterologous DNA," "heterologous nucleic acid sequence," and the like as used herein refers to a nucleic acid sequence wherein at least one of the following is true: (a) the sequence of nucleic acids foreign to (i.e., not naturally found in) a given host microorganism; (b) the sequence may be naturally found in a given host microorganism, but in an unnatural (e.g., greater than expected) amount; or (c) the sequence of nucleic acids comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a heterologous nucleic acid sequence that is recombinantly produced will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid, such as an nonnative promoter driving gene expression.

[0050] The term "synthetic metabolic valve," and the like as used herein refers to either the use of controlled proteolysis, gene silencing or the combination of both proteolysis and gene silencing to alter metabolic fluxes.

[0051] The term "heterologous" is intended to include the term "exogenous" as the latter term is generally used in the art. With reference to the host microorganism's genome prior to the introduction of a heterologous nucleic acid sequence, the nucleic acid sequence that codes for the enzyme is heterologous (whether or not the heterologous nucleic acid sequence is introduced into that genome).

[0052] As used herein, the term "gene disruption," or grammatical equivalents thereof (and including "to disrupt enzymatic function," "disruption of enzymatic function," and the like), is intended to mean a genetic modification to a microorganism that renders the encoded gene product as having a reduced polypeptide activity compared with polypeptide activity in or from a microorganism cell not so modified. The genetic modification can be, for example, deletion of the entire gene, deletion or other modification of a regulatory sequence required for transcription or translation, deletion of a portion of the gene which results in a truncated gene product (e.g., enzyme) or by any of various mutation strategies that reduces activity (including to no detectable activity level) the encoded gene product. A disruption may broadly include a deletion of all or part of the nucleic acid sequence encoding the enzyme, and also includes, but is not limited to other types of genetic modifications, e.g., introduction of stop codons, frame shift mutations, introduction or removal of portions of the gene, and introduction of a degradation signal, those genetic modifications affecting mRNA transcription levels and/or stability, and altering the promoter or repressor upstream of the gene encoding the enzyme.

[0053] Bio-production or Fermentation, as used herein, may be aerobic, microaerobic, or anaerobic.

[0054] When the genetic modification of a gene product, i.e., an enzyme, is referred to herein, including the claims, it is understood that the genetic modification is of a nucleic acid sequence, such as or including the gene, that normally encodes the stated gene product, i.e., the enzyme.

[0055] As used herein, the term "metabolic flux" and the like refers to changes in metabolism that lead to changes in product and/or byproduct formation, including production rates, production titers and production yields from a given substrate.

[0056] Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology.

[0057] Enzymes are listed here within, with reference to a Universal Protein Resource (Uniprot) identification number, which would be well known to one skilled in the art (Uniprot is maintained by and available through the UniProt Consortium).

[0058] Where methods and steps described herein indicate certain events occurring in certain order, those of ordinary skill in the art will recognize that the ordering of certain steps may be modified and that such modifications are in accordance with the variations of the invention. Additionally, certain steps may be performed concurrently in a parallel process when possible, as well as performed sequentially.

[0059] Prophetic examples provided herein are meant to be broadly exemplary and not limiting in any way.

[0060] The meaning of abbreviations is as follows: "C" means Celsius or degrees Celsius, as is clear from its usage, DCW means dry cell weight, "s" means second(s), "min" means minute(s), "h," "hr," or "hrs" means hour(s), "psi" means pounds per square inch, "nm" means nanometers, "d" means day(s), ".mu.L" or "uL" or "ul" means microliter(s), "mL" means milliliter(s), "L" means liter(s), "mm" means millimeter(s), "nm" means nanometers, "mM" means millimolar, ".mu.M" or "uM" means micromolar, "M" means molar, "mmol" means millimole(s), "p mol" or "uMol" means micromole(s)", "g" means gram(s), ".mu.g" or "ug" means microgram(s) and "ng" means nanogram(s), "PCR" means polymerase chain reaction, "OD" means optical density, "OD.sub.600" means the optical density measured at a photon wavelength of 600 nm, "kDa" means kilodaltons, "g" means the gravitation constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "% w/v" means weight/volume percent, "% v/v" means volume/volume percent, "IPTG" means isopropyl-.mu.-D-thiogalactopyranoiside, "aTc" means anhydrotetracycline, "RBS" means ribosome binding site, "rpm" means revolutions per minute, "HPLC" means high performance liquid chromatography, and "GC" means gas chromatography.

[0061] I. Carbon Sources

[0062] Bio-production media, which is used in the present invention with recombinant microorganisms must contain suitable carbon sources or substrates for both growth and production stages. Suitable substrates may include, but are not limited to glucose, sucrose, xylose, mannose, arabinose, oils, carbon dioxide, carbon monoxide, methane, methanol, formaldehyde and glycerol. It is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention as a carbon source(s).

[0063] II. Microorganisms

[0064] Features as described and claimed herein may be provided in a microorganism selected from the listing herein, or another suitable microorganism, that also comprises one or more natural, introduced, or enhanced product bio-production pathways. Thus, in some embodiments the microorganism(s) comprise an endogenous product production pathway (which may, in some such embodiments, be enhanced), whereas in other embodiments the microorganism does not comprise an endogenous product production pathway.

[0065] The examples describe specific modifications and evaluations to certain bacterial and fungal microorganisms. The scope of the invention is not meant to be limited to such species, but to be generally applicable to a wide range of suitable microorganisms.

[0066] More particularly, based on the various criteria described herein, suitable microbial hosts for the bio-production of a chemical product generally may include, but are not limited to the organisms described in the Common Methods Section

[0067] III. Media and Culture Conditions

[0068] In addition to an appropriate carbon source, such as selected from one of the herein-disclosed types, bio-production media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for chemical product bio-production under the present invention.

[0069] Another aspect of the invention regards media and culture conditions that comprise genetically modified microorganisms of the invention and optionally supplements.

[0070] Typically cells are grown at a temperature in the range of about 25.degree. C. to about 40.degree. C. in an appropriate medium, as well as up to 70.degree. C. for thermophilic microorganisms. Suitable growth media are well characterized and known in the art.

[0071] Suitable pH ranges for the bio-production are between pH 2.0 to pH 10.0, where pH 6.0 to pH 8.0 is a typical pH range for the initial condition. However, the actual culture conditions for a particular embodiment are not meant to be limited by these pH ranges.

[0072] Bio-productions may be performed under aerobic, microaerobic or anaerobic conditions with or without agitation.

[0073] IV. Bio-Production Reactors and Systems

[0074] Fermentation systems utilizing methods and/or compositions according to the invention are also within the scope of the invention.

[0075] Any of the recombinant microorganisms as described and/or referred to herein may be introduced into an industrial bio-production system where the microorganisms convert a carbon source into a product in a commercially viable operation. The bio-production system includes the introduction of such a recombinant microorganism into a bioreactor vessel, with a carbon source substrate and bio-production media suitable for growing the recombinant microorganism, and maintaining the bio-production system within a suitable temperature range (and dissolved oxygen concentration range if the reaction is aerobic or microaerobic) for a suitable time to obtain a desired conversion of a portion of the substrate molecules to a selected chemical product. Bio-productions may be performed under aerobic, microaerobic, or anaerobic conditions, with or without agitation. Industrial bio-production systems and their operation are well-known to those skilled in the arts of chemical engineering and bioprocess engineering.

[0076] The following published resources are incorporated by reference herein for their respective teachings to indicate the level of skill in these relevant arts, and as needed to support a disclosure that teaches how to make and use methods of industrial bio-production of chemical product(s) produced under the invention, from sugar sources, and also industrial systems that may be used to achieve such conversion with any of the recombinant microorganisms of the present invention (Biochemical Engineering Fundamentals, 2.sup.nd Ed. J. E. Bailey and D. F. Ollis, McGraw Hill, New York, 1986, entire book for purposes indicated and Chapter 9, pages 533-657 in particular for biological reactor design; Unit Operations of Chemical Engineering, 5.sup.th Ed., W. L. McCabe et al., McGraw Hill, New York 1993, entire book for purposes indicated, and particularly for process and separation technologies analyses; Equilibrium Staged Separations, P. C. Wankat, Prentice Hall, Englewood Cliffs, N.J. USA, 1988, entire book for separation technologies teachings).

[0077] The amount of a product produced in a bio-production media generally can be determined using a number of methods known in the art, for example, high performance liquid chromatography (HPLC), gas chromatography (GC), or GC/Mass Spectroscopy (MS).

[0078] V. Genetic Modifications, Nucleotide Sequences, and Amino Acid Sequences

[0079] Embodiments of the present invention may result from introduction of an expression vector into a host microorganism, wherein the expression vector contains a nucleic acid sequence coding for an enzyme that is, or is not, normally found in a host microorganism.

[0080] The ability to genetically modify a host cell is essential for the production of any genetically modified (recombinant) microorganism. The mode of gene transfer technology may be by electroporation, conjugation, transduction, or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors are tailored to the host organisms based on the nature of antibiotic resistance markers that can function in that host. Also, as disclosed herein, a genetically modified (recombinant) microorganism may comprise modifications other than via plasmid introduction, including modifications to its genomic DNA.

[0081] More generally, nucleic acid constructs can be prepared comprising an isolated polynucleotide encoding a polypeptide having enzyme activity operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a microorganism, such as E. coli, under conditions compatible with the control sequences. The isolated polynucleotide may be manipulated to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well established in the art.

[0082] The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence may contain transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The techniques for modifying and utilizing recombinant DNA promoter sequences are well established in the art.

[0083] For various embodiments of the invention the genetic manipulations may be described to include various genetic manipulations, including those directed to change regulation of, and therefore ultimate activity of, an enzyme or enzymatic activity of an enzyme identified in any of the respective pathways. Such genetic modifications may be directed to transcriptional, translational, and post-translational modifications that result in a change of enzyme activity and/or selectivity under selected and/or identified culture conditions and/or to provision of additional nucleic acid sequences such as to increase copy number and/or mutants of an enzyme related to product production. Specific methodologies and approaches to achieve such genetic modification are well known to one skilled in the art.

[0084] In various embodiments, to function more efficiently, a microorganism may comprise one or more gene deletions. For example, in E. coli, the genes encoding the lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), the clpXP protease specificity enhancing factor (sspB), the ATP-dependent Lon protease (lon), the outer membrane protease (ompT), the arcA transcriptional dual regulator (arcA), and the iclR transcriptional regulator (iclR) may be disrupted, including deleted. Such gene disruptions, including deletions, are not meant to be limiting, and may be implemented in various combinations in various embodiments. Gene deletions may be accomplished by numerous strategies well known in the art, as are methods to incorporate foreign DNA into a host chromosome.

[0085] In various embodiments, to function more efficiently, a microorganism may comprise one or more synthetic metabolic valves, composed of enzymes targeted for controlled proteolysis, expression silencing or a combination of both controlled proteolysis and expression silencing. For example, one enzyme encoded by one gene or a combination of numerous enzymes encoded by numerous genes in E. coli may be designed as synthetic metabolic valves to alter metabolism and improve product formation. Representative genes in E. coli may include but are not limited to the following: fab1, zwf, gltA, ppc, udhA, lpd, sucD, aceA, pfkA, lon, rpoS, tktA or tktB. It is appreciated that it is well known to one skilled in the art how to identify homologues of these genes and or other genes in additional microbial species.

[0086] For all nucleic acid and amino acid sequences provided herein, it is appreciated that conservatively modified variants of these sequences are included, and are within the scope of the invention in its various embodiments. Functionally equivalent nucleic acid and amino acid sequences (functional variants), which may include conservatively modified variants as well as more extensively varied sequences, which are well within the skill of the person of ordinary skill in the art, and microorganisms comprising these, also are within the scope of various embodiments of the invention, as are methods and systems comprising such sequences and/or microorganisms.

[0087] Accordingly, as described in various sections above, some compositions, methods and systems of the present invention comprise providing a genetically modified microorganism that comprises both a production pathway to make a desired product from a central intermediate in combination with synthetic metabolic valves to redistribute flux.

[0088] Aspects of the invention also regard provision of multiple genetic modifications to improve microorganism overall effectiveness in converting a selected carbon source into a selected product. Particular combinations are shown, such as in the Examples, to increase specific productivity, volumetric productivity, titer and yield substantially over more basic combinations of genetic modifications.

[0089] In addition to the above-described genetic modifications, in various embodiments genetic modifications, including synthetic metabolic valves also are provided to increase the pool and availability of the cofactor NADPH and/or NADH which may be consumed in the production of a product.

[0090] More generally, and depending on the particular metabolic pathways of a microorganism selected for genetic modification, any subgroup of genetic modifications may be made to decrease cellular production of fermentation product(s) other than the desired fermentation product, selected from the group consisting of acetate, acetoin, acetone, acrylic, malate, fatty acid ethyl esters, isoprenoids, glycerol, ethylene glycol, ethylene, propylene, butylene, isobutylene, ethyl acetate, vinyl acetate, other acetates, 1,4-butanediol, 2,3-butanediol, butanol, isobutanol, sec-butanol, butyrate, isobutyrate, 2-OH-isobutryate, 3-OH-butyrate, ethanol, isopropanol, D-lactate, L-lactate, pyruvate, itaconate, levulinate, glucarate, glutarate, caprolactam, adipic acid, propanol, isopropanol, fused alcohols, and 1,2-propanediol, 1,3-propanediol, formate, fumaric acid, propionic acid, succinic acid, valeric acid, maleic acid and poly-hydroxybutyrate. Gene deletions may be made as disclosed generally herein, and other approaches may also be used to achieve a desired decreased cellular production of selected fermentation products other than the desired products.

[0091] VI. Synthetic Metabolic Valves

[0092] In particular the invention describes the construction of synthetic metabolic valves comprising one or more or a combination of the following: controlled gene silencing and controlled proteolysis. It is appreciated that one well skilled in the art is aware of several methodologies for gene silencing and controlled proteolysis. An example of the combination of CRISPR interference based gene silencing and controlled proteolysis is illustrated in FIG. 2.

[0093] VI.A Gene Silencing

[0094] In particular the invention describes the use of controlled gene silencing to help enable the control over metabolic fluxes in controlled multi-stage fermentation processes. There are several methodologies known in the art for controlled gene silencing, including but not limited to mRNA silencing or RNA interference, silencing via transcriptional repressors and CRISPR interference. Methodologies and mechanisms for RNA interference are taught by Agrawal et al. "RNA Interference: Biology, Mechanism, and Applications" Microbiology and Molecular Biology Reviews, December 2003; 67(4) p 657-685. DOI: 10.1128/MMBR.67.657-685.2003. Methodologies and mechanisms for CRISRPR interference are taught by Qi et al. "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression" Cell February 2013; 152(5) p 1173-1183. DOI: 10.1016/j.cell.2013.02.022. In addition, methodologies and mechanisms for CRISRPR interference using the native E. coli CASCADE system are taught by Luo et al. "Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression" NAR. October 2014; DOI: 10.1093. In additional numerous transcriptional repressor systems are well known in the art and can be used to turn off gene expression.

[0095] VI.B Controlled Proteolysis

[0096] In particular the invention describes the use of controlled protein degradation or proteolysis to help enable the control over metabolic fluxes in controlled multi-stage fermentation processes. There are several methodologies known in the art for controlled protein degradation, including but not limited to targeted protein cleavage by a specific protease and controlled targeting of proteins for degradation by specific peptide tags. Systems for the use of the E. coli clpXP protease for controlled protein degradation are taught by McGinness et al, "Engineering controllable protein degradation", Mol Cell. June 2006; 22(5) p 701-707. This methodology relies upon adding a specific C-terminal peptide tag such as a DAS4 (or DAS+4) tag. Proteins with this tag are not degraded by the clpXP protease until the specificity enhancing chaperone sspB is expressed. sspB induces degradation of DAS4 tagged proteins by the clpXP protease. In additional numerous site specific protease systems are well known in the art. Proteins can be engineered to contain a specific target site of a given protease and then cleaved after the controlled expression of the protease. In some embodiments the cleavage can be expected lead to protein inactivation or degradation. For example Schmidt et al, "ClpS is the recognition component for Escherichia coli substrates of the N-end rule degradation pathway" Molecular Microbiology March 2009. 72(2), 506-517. doi:10.1111, teaches that an N-terminal sequence can be added to a protein of interest in enable clpS dependent clpAP degradation. In addition, this sequence can further be masked by an additional N-terminal sequence, which can be controllable cleaved such as by a ULP hydrolase. This allows for controlled N-rule degradation dependent on hydrolase expression. It is therefore possible to tag proteins for controlled proteolysis either at the N-terminus or C-terminus. The preference of using an N-terminal vs. C-terminal tag will largely depend on whether either tag affects protein function prior to the controlled onset of degradation.

[0097] The invention describes the use of controlled protein degradation or proteolysis to help enable the control over metabolic fluxes in controlled multi-stage fermentation processes, in E. coli. There are several methodologies known in the art for controlled protein degradation in other microbial hosts, including a wide range of gram-negative as well as gram-positive bacteria, yeast and even archaea. In particular, systems for controlled proteolysis can be transferred from a native microbial host and used in a non-native host. For example Grilly et al, "A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae" Molecular Systems Biology 3, Article 127. doi:10.1038, teaches the expression and use of the E. coli clpXP protease in the yeast Saccharomyces cerevisiae. Such approaches can be used to transfer the methodology for synthetic metabolic valves to any genetically tractable host.

[0098] VI.C Synthetic Metabolic Valve Control

[0099] In particular the invention describes the use of synthetic metabolic valves to control metabolic fluxes in multi-stage fermentation processes. There are numerous methodologies known in the art to induce expression that can be used at the transition between stages in multi-stage fermentations. These include but are not limited to artificial chemical inducers including: tetracycline, anhydrotetracycline, lactose, IPTG (isopropyl-beta-D-1-thiogalactopyranoside), arabinose, raffinose, tryptophan and numerous others. Systems linking the use of these well known inducers to the control of gene expression silencing and/or controlled proteolysis can be integrated into genetically modified microbial systems to control the transition between growth and production phases in multi-stage fermentation processes.

[0100] In addition, it may be desirable to control the transition between growth and production in multi-stage fermentations by the depletion of one or more limiting nutrients that are consumed during growth. Limiting nutrients can include but are not limited to: phosphate, nitrogen, sulfur and magnesium. Natural gene expression systems that respond to these nutrient limitations can be used to operably link the control of gene expression silencing and/or controlled proteolysis to the transition between growth and production phases in multi-stage fermentation processes.

[0101] VII. Disclosed Embodiments are Non-Limiting

[0102] While various embodiments of the present invention have been shown and described herein, it is emphasized that such embodiments are provided by way of example only. Numerous variations, changes and substitutions may be made without departing from the invention herein in its various embodiments. Specifically, and for whatever reason, for any grouping of compounds, nucleic acid sequences, polypeptides including specific proteins including functional enzymes, metabolic pathway enzymes or intermediates, elements, or other compositions, or concentrations stated or otherwise presented herein in a list, table, or other grouping (such as metabolic pathway enzymes shown in a figure), unless clearly stated otherwise, it is intended that each such grouping provides the basis for and serves to identify various subset embodiments, the subset embodiments in their broadest scope comprising every subset of such grouping by exclusion of one or more members (or subsets) of the respective stated grouping. Moreover, when any range is described herein, unless clearly stated otherwise, that range includes all values therein and all sub-ranges therein.

[0103] Also, and more generally, in accordance with disclosures, discussions, examples and embodiments herein, there may be employed conventional molecular biology, cellular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. (See, e.g., Sambrook and Russell, "Molecular Cloning: A Laboratory Manual," Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture, R. I. Freshney, ed., 1986.) These published resources are incorporated by reference herein for their respective teachings of standard laboratory methods found therein. Such incorporation, at a minimum, is for the specific teaching and/or other purpose that may be noted when citing the reference herein. If a specific teaching and/or other purpose is not so noted, then the published resource is specifically incorporated for the teaching(s) indicated by one or more of the title, abstract, and/or summary of the reference. If no such specifically identified teaching and/or other purpose may be so relevant, then the published resource is incorporated in order to more fully describe the state of the art to which the present invention pertains, and/or to provide such teachings as are generally known to those skilled in the art, as may be applicable. However, it is specifically stated that a citation of a published resource herein shall not be construed as an admission that such is prior art to the present invention. Also, in the event that one or more of the incorporated published resources differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. Subject matter in the Examples is incorporated into this section to the extent not already present.

EXAMPLES

[0104] The examples herein provide some examples, not meant to be limiting. All reagents, unless otherwise indicated, are obtained commercially. Species and other phylogenic identifications are according to the classification known to a person skilled in the art of microbiology, molecular biology and biochemistry.

[0105] The names and city addresses of major suppliers are provided herein.

Example 1: Dynamic Flux Control Using Temperature Sensitive Enzymes to Improve Malonyl-CoA Flux in E. coli

[0106] This example describes the increased production of tetrahydroxynaphtalene (THN) in E. coli from the intermediate malonyl-CoA using the controlled inactivation of fabI via a temperature sensitive allele. Briefly, strain BWapldf (BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE) was further genetically modified so that the fab1 gene was mutated to contain both a temperature sensitive (ts) mutation (F241S) as well as to incorporate gentamicin resistance cassette a the C-terminus of the fab1 gene. This was accomplished using standard recombineering protocols. The strain was further modified to express the tetrahydroxynapthalene (THN) synthase gene (rppA from Streptomyces coelicolor) under phosphate limiting conditions by transformation with the plasmid pSMART-HC-Kan-yibD-THNS (SEQ ID NO:1). Control strains were made with a control empty vector pSMART-HC-Kan (Genbank Accession #AF532107.1), obtained from Lucigen. This high copy plasmid conferring kanamycin resistance was constructed using routine molecular biology methods utilizing the pSMART-HC-Kan kit obtained from Lucigen. The rppA gene under the control of the promoter of low phosphate induced yibD(waaH) gene of E. coli. This strain, as well as controls, were evaluated for THN production using the two-stage protocol as outline in the Common Methods section "Shake Flask Protocol-1". Relative THN production was quantified by measuring the absorbance of the supernatant at 340 nm. FIG. 4 summarizes the results.

Example 2: A Synthetic Metabolic Valve to Improve Malonyl-CoA Flux in E. coli

[0107] This example describes the increased production of tetrahydroxynaphtalene (THN) in E. coli from the intermediate malonyl-CoA using the controlled repression of fab1 using synthetic metabolic valve technology. In this example a combination of CRISPR interference gene silencing technology and controlled protein degradation was used in a two-stage process. Briefly, strain BWapldf (BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE) was further genetically modified so that the fab1 gene was tagged to contain a C-terminal DAS4 tag as well as to incorporate gentamicin resistance cassette a the C-terminus of the fab1 gene. The C-terminal nucleotide sequence encoding the DAS4 tag was integrated as the following sequence: 5'-GCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCT-34 (SEQ ID NO: 48). This was accomplished using standard recombineering protocols. In addition, the strain was further modified so as to delete the sspB gene. This was also performed with standard recombineering methods. In addition, these strains were still further modified to contain three plasmids, the first plasmid expresses the tetrahydroxynapthalene (THN) synthase gene, pSMART-HC-Kan-yibD-THNS (SEQ ID NO:1), as described above. The second plasmid was constructed to express a small guide RNA targeting the fab1 gene from a high copy spectinomycin resistance plasmid derived from pCDF-1b, which was obtained from EMD Millipore Biosciences. The plasmid, pCDF-T2-fabIsgRNA (SEQ ID NO:2), expresses a small guide RNA to use with S. pyogenes dCas9. The specific fab1 T2 targeting sequence is given by 5'-CAGCCGCTCCGGTCGGACCG-3' (SEQ ID NO:47). A control plasmid was also made missing any targeting sequence as described by Qi et al. Cell February 2013; 152(5) p 1173-1183. DOI: 10.1016/j.cell.2013.02.022. The last plasmid, pdCas9-ptet-sspB (SEQ ID NO:3), was derived from the plasmid pdCas9-bacteria, from Qi et al, which was obtained from Addgene (Cambridge, Mass. 02139; Plasmid ID 44249). Briefly, pdCas9-bacteria was linearized and the sspB gene was introduced under the control of an additional ptet promoter at the 3' of the catalytically inactive dcas9 gene. The addition of anhydrotetracycline (aTc) will induce expression of both dCas9 as well as sspB from this Chloramphenicol resistance conferring plasmid. All plasmids were constructed using standard molecular biology methods and sequences confirmed by DNA sequencing. These strains, as well as controls, were evaluated for THN production using the two-stage protocol as outline in the Common Methods section "Shake Flask Protocol-2". Relative THN production was quantified by measuring the absorbance of the supernatant at 340 nm. FIG. 5 summarizes the results.

Example 3: General Example

[0108] Numerous microbial strains, such as any of the strains listed in the Common Methods Section, may be genetically modified to express enzymes for the biosynthesis of a product. In addition these modified microbial strains can be further modified to contain a controllable synthetic metabolic valve for the dynamic reduction in enzyme activity of one or more metabolic pathways including those required for growth. These valves may utilize one or a combination of methods including gene silencing and controlled proteolysis. Further these modified strains may be used in a multistage fermentation process wherein transition between stages is concurrent with controlled activation of these valves. Specifically, any of these microbial strains may also be further engineered to express a heterologous production pathway enabling the product formation.

Example 4: E coli Host Strain Construction

[0109] Briefly, strain BWapldf (BW25113: .DELTA.ldhA, .DELTA.pflB, .DELTA.poxB, .DELTA.ackA-pta, .DELTA.adhE) was further genetically modified for the deletion of the following genes: arcA, iclR and sspB, to construct strain DLF_0002. This was also performed with standard scarless recombineering methods. To construct a strain capable of both crispr based gene silencing using the native CASCADE system in E. coli as well as controlled proteolysis, the cas3 gene of E. coli was first deleted. This gene was replaced with a sequence to enable both constitutive expression of the casABCDE-cas1,2 operon enabling CASCADE based gene silencing, as well as a construct allowing for the low phosphate induction of the sspB chaperone. The DNA sequence integrated was ordered as a single synthetic construct: SEQ ID NO:4, and integrated using standard recombineering methodologies. In the place of the cas3 gene, this construct integrates a transcriptional terminator, followed by the low phosphate inducible E. coli ugpB gene promoter and the sspB gene. The sspB gene is followed by another transcriptional terminator and a subsequent constitutive proB promoter adapted from (Davis, J H., Rubin, A J., and Sauer, R T. NAR. February 2011; 39(3) p 1131-1141. DOI: 10.1093) to drive constant expression of the CASCADE operon. The resulting strain is termed DLF_0025.

[0110] A derivative of E. coli strain DLF_0025 was constructed to utilize a non-PTS dependent glucose uptake system. PTS (phosphotransferase system) based sugar uptake is well known in the art and links the phosphorylation of glucose to the production of pyruvate. Alternative uptake has been previously described in E. coli, (Hernandez-Montalvo, V., et al., Biotechnol Bioeng. September 2003; 83(6) p 687-694.), and relies on the overexpression of the E. coli galP permease and glucokinase (glk gene) along with the deletion of the E. coli ptsG gene. The ptsG gene was deleted and replaced with a constitutively expressed glucokinase construct, this construct was ordered as a single synthetic linear DNA construct (SEQ ID NO:5) and integrated according to standard methodologies. In addition, the galP promoter was also replaced via chromosomal replacement using another single synthetic linear DNA construct (SEQ ID NO:6), the resulting strain was called DLF_0286. In both cases the proC promoter was used to drive constitutive expression (Davis, J H., Rubin, A J., and Sauer, R T. NAR. February 2011; 39(3) p 1131-1141. DOI: 10.1093).

[0111] E. coli strains DLF_0025 and DLF_0286 were further modified for the controlled proteolysis of key enzymes in central metabolism including: 1) enoyl-ACP reductase encoded by the fab1 gene, involved in fatty acid biosynthesis, 2) citrate synthase encoded by the gltA gene, involved in citric acid cycle, 3) soluble transhydrogenase encoded by the udhA gene, involved in NADPH metabolism, 4) glucose-6-phosphate-1-dehydrogenase encoded by the zwf gene, involved in the pentose phosphate pathway and 5) the lipoamide dehydrogenase or E3 component of the pyruvate dehydrogenase complex encoded by lpd gene. C-terminal DAS+4 tags enabling sspB controlled proteolysis were integrated at the 3' end of each of the above genes as the following sequence: 5'-GCGGCCAACGATGAAAACTATTCTGAAAACTATGCGGATGCGTCT-3' (SEQ ID NO:48). This was accomplished by the insertion of single DNA cassettes containing the DAS4 tags, targeting sequences as well as a downstream antibiotic resistance cassette. The fabI-DAS4 tag and lpd-DAS4 tag were followed by a gentamicin resistance cassette, the gltA-DAS4 tag was followed by a zeocin resistance cassette, and the udhA-DAS4 and zwf-DAS4 tags were both followed by a blasticidin resistance cassette. The integrated sequences used for the C-terminal tagging fabI, lpd, gltA, udhA and zwf are SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 SEQ ID NO:10 and SEQ ID NO:11 respectively. Strains with single and combinations of DAS4 tagged enzymes were constructed. Host strain genotypes are listed in Table 1.

TABLE-US-00001 TABLE 1 E. coli Host Strains Strain ID Genotype BW25113 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514 BWapldf F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt DLF_0002 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB DLF_0025 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB DLF_0286 F-, .lamda..sup.-, .DELTA.(araD-araB)567, AlacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-pro, .DELTA.ptsG::proC-glk, proC-galP DLF_0043 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, AarcA, AsspB, Acas3::ugpBp-sspB-proB, gltA- DAS+4:zeoR DLF_0028 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, fabI- DAS+4:gentR DLF_0031 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, lpd- DAS+4:gentR DLF_0038 F-, .lamda..sup.-, .DELTA.(araD-araB)567, AlacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflBxfrt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, fabI- DAS+4:gentR, udhA-DAS+4:bsdR DLF_0040 F-, .lamda..sup.-, .DELTA.(araD-araB)567, AlacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, fabI- DAS+4:gentR zwf-DAS+4:bsdR DLF_0039 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, fabI- DAS+4:gentR, gltA-DAS+4:zeoR DLF_0047 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, fabI- DAS+4:gentR, gltA-DAS+4:zeoR, udhA-DAS+4:bsdR DLF_0167 F-, .lamda..sup.-, .DELTA.(araD-araB)567, AlacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, fabI- DAS+4:gentR gltA-DAS+4:zeoR zwf-DAS+4:bsdR DLF_0041 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, lpd- DAS+4:gentR, gltA-DAS+4:zeoR, DLF_0165 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, lpd- DAS+4:gentR, zwf-DAS+4:bsdR DLF_0042 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, lpd- DAS+4:gentR, udhA-DAS+ 4:bsdR DLF_0049 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, lpd- DAS+4:gentR, gltA-DAS+4:zeoR, udhA-DAS+4:bsdR DLF_0048 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, lpd- DAS+4:gentR, gltA-DAS+4:zeoR, zwf-DAS+4:bsdR DLF_0045 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflBxfrt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, gltA- DAS+4:zeoR, udhA-DAS+4:bsdR DLF_0044 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-proB, gltA- DAS+4:zeoR, zwf-DAS+4:bsdR DLF_0287 F-, .lamda..sup.-, A(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-pro, .DELTA.ptsG::proC-glk, proC-galP, gltA-DAS+4:zeoR DLF_0288 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-pro, .DELTA.ptsG::proC-glk, proC-galP, gltA-DAS+4:zeoR, zwf-DAS+4:bsdR DLF_0289 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-pro, .DELTA.ptsG::proC-glk, proC-galP, gltA-DAS+4:zeoR, udhA-DAS+4:bsdR DLF_0290 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflBxfrt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-pro, .DELTA.ptsG::proC-glk, proC-galP, gltA-DAS+4:zeoR, zwf-DAS+4:bsdR, fabI-DAS+4:gentR DLF_0291 F-, .lamda..sup.-, .DELTA.(araD-araB)567, .DELTA.lacZ4787(::rrnB-3), rph-1, .DELTA.(rhaD- rhaB)568, hsdR514, .DELTA.ldhA::frt, .DELTA.poxB::frt, .DELTA.pflB::frt, .DELTA.ackA-pta::frt, .DELTA.adhE::frt, .DELTA.iclR, .DELTA.arcA, .DELTA.sspB, .DELTA.cas3::ugpBp-sspB-pro, .DELTA.ptsG::proC-glk, proC-galP, gltA-DAS+4:zeoR, udhA-DAS+4:bsdR, fabI-DAS+4:gentR

Example 5: Low Phosphate Gene Expression in E. coli

[0112] In order to evaluate different low phosphate induction schemes to control synthetic metabolic valves, several known low phosphate inducible promoters form E. coli were evaluated with a ultraviolet excitable, green fluorescent protein (GFPuv) reporter gene. These gene promoters included those for the following genes: amn, phioA, phoB, phioE, phioH, phoU, mipA, pstS, ugpB, waaH and ydfH, were evaluated for low phosphate induction. Reporter plasmids linking each promoter to a GFPuv gene reporter were constructed and sequences are as follows: pSMART-amnp-GFPuv (SEQ ID N0:36), pSMART-phoAp-GFPuv (SEQ ID N0:37), pSMART-phoBp-GFPuv (SEQ ID N0:38), pSMART-phoEp-GFPuv (SEQ ID N0:38), pSMART-phoHp-GFPuv (SEQ ID NO:40), pSMART-phoUp-GFPuv (SEQ ID NO:41), pSMART-mipAp-GFPuv (SEQ ID NO:42), pSMART-pstSp-GFPuv (SEQ ID NO:43), pSMART-ugpBp-GFPuv (SEQ ID NO:12), pSMART-waaHp-GFPuv (SEQ ID NO:44), and pSMART-ydtH-p-GFPuv (SEQ ID NO:45). Briefly, plasmids were transformed into E. coli strain BWapldf (Refer to Example 4). Colonies were used to inoculate 4 mL of SM3 media with kanamycin (Refer to Common Methods Section) and incubated overnight at 37 degrees Celsius and a shaking speed of 225 rpm. After overnight growth, cells were normalized to an optical density at 600 nm of 5, and 40 .mu.L of normalized culture was used to inoculate 760 .mu.L of fresh FGM3 (Refer to Common Methods Section) medium with kanamycin in wells of a 48 well FlowerPlate.TM. B which was transferred into a BioLector Microbioreactor both obtained from M2P Labs (Baesweiler, Germany). The BioLector Microbioreactor can continuously measure fluorescence. Cells were incubated in the Microreactor at 37 degrees Celsius and a shaking speed of 1200 rpm for 60 hrs. Growth stopped and phosphate depletion begins at about 15-20 hrs (data not shown for clarity). Fluorescence results for each reporter construct as well as an empty vector control are reported as relative fluorescence units (R.F.U) in FIG. 6. All plasmids were constructed using standard Gibson Assembly methodology (Gibson Assembly Master Mix, obtained from New England Biolabs, Ipswich, Mass., USA), and synthetic linear double stranded DNA provided as Gblocks.TM. (Integrated DNA Technology, Coralville, Iowa, USA). Eton Bioscience (Research Triangle Park, N.C., USA) was used for plasmid DNA sequence confirmations. Standard codon optimization was performed to optimize constructs for expression in E. coli.

Example 6: pCASCADE Plasmid Cloning

[0113] pCASCADE-control (SEQ ID NO:13) was prepared by swapping the tetracycline inducible promoter in pcrRNA plasmid (Luo et al. "Repurposing endogenous type I CRISPR-Cas systems for programmable gene repression" NAR. October 2014; DOI: 10.1093.) with an insulated ugpB promoter. The plasmid was constructed using standard Gibson Assembly methodology (Gibson Assembly Master Mix, obtained from New England Biolabs, Ipswich, Mass., USA), and synthetic linear double stranded DNA provided as Gblocks.TM. (Integrated DNA Technology, Coralville, Iowa, USA). Eton Bioscience (Research Triangle Park, N.C., USA) was used for plasmid DNA sequence confirmations.

[0114] Additional pCASCADE plasmids with single RNA guides were prepared via Q5 site-directed mutagenesis (New England Biolabs, Ipswich, Mass., USA)) following manufacturer's protocol, except that 5% v/v DMSO was added to the Q5 PCR reaction. For example pCASCADE-gltA2 (SEQ ID NO:14) was prepared using pCASCADE-control as template and the following primers: gltA2-FOR 5'-GGGACAGTTATTAGTTCGAGTTCCCCGCGCCA GCGGGGATAAACCGAAAAAAAAACCCC-3' (SEQ ID NO:49) and gltA2-REV 5'-GAATGAATTGGTCAATACGGTTTATCCCCGCTGGCGCGGGGAACTCGAGGTGGT ACCAGATCT-3' (SEQ ID NO:50). Additional pCASCADE plasmids including pCASCADE-fabI (SEQ ID NO:15), pCASCADE-udhA, (SEQ ID NO:16), pCASCADE-zwf (SEQ ID NO:17) and pCASCADE-gltA1 (SEQ ID NO:18) were prepared in a similar manner by exchanging the guide RNA targeting sequence using Q5 mutagenesis.

[0115] Additional pCASCADE plasmids with multiple RNA guides were prepared as follows. For example pCASCADE-gltA2-udhA (SEQ ID NO:19) plasmid was prepared by amplifying gltA2 guide half and udhA guide half from pCASCADE-gltA2 and pCASCADE-udhA respectively using Q5 High-Fidelity 2.times. Master Mix (NEB, MA). The primers used: G2U-FOR1: 5'-CCGGATGAGCATTCATCAGGCGGGCAAG-3' (SEQ ID NO:51), REV1: 5'-CGGTTTATCCCCGCTGGCGCGGGGAACTCGAACTTCATAACTTTTAC-3' (SEQ ID NO:52) and FOR2: 5'-GCGCCAGCGGGGATAAACCGTTACCATTCTGTTG-3' (SEQ ID NO:53) and REV2: 5'-CTTGCCCGCCTGATGAATGCTCATCCGG-3' (SEQ ID NO:54). PCR products were purified by gel-extraction and were then used for Gibson Assembly (NEB, MA). pCASCADE-fabI-udhA (SEQ ID NO:20), pCASCADE-fabI-gltA1 (SEQ ID NO:21), pCASCADE-fabI-gltA2 (SEQ ID NO:22), pCASCADE-fabI-zwf (SEQ ID NO:23), pCASCADE-gltA1-udhA (SEQ ID NO:24), pCASCADE-gltA2-udhA (SEQ ID NO:25), pCASCADE-gltA1-zwf (SEQ ID NO:26), pCASCADE-gltA2-zwf (SEQ ID NO:27), were all prepared in a similar way by amplification of each guide and part of the vector backbone followed by Gibson Assembly. All plasmid sequences were confirmed by DNA sequencing (Eton Bioscience, Research Triangle Park, N.C., USA).

Example 7: Dynamic Control Over Protein Levels in E. coli Using the CASCADE System and Controlled Proteolysis

[0116] All plasmids were constructed using standard Gibson Assembly methodology (Gibson Assembly Master Mix, obtained from New England Biolabs, Ipswich, Mass., USA), and synthetic linear double stranded DNA provided as Gblocks.TM. (Integrated DNA Technology, Coralville, Iowa, USA). Eton Bioscience (Research Triangle Park, N.C., USA) was used for plasmid DNA sequence confirmations. Standard codon optimization was performed to optimize constructs for expression in E. coli. First a plasmid expressing a low phosphate inducible (utilizing the low phosphate inducible waaH gene promoter from E. coli), ultraviolet excitable, green fluorescent protein (GFPuv) was constructed using standard cloning techniques and called pSMART-waaHp-GFPuv (SEQ ID NO:12). Secondly, a compatible vector with the constitutive expression of a red fluorescent protein (mCherry), tagged with a DAS+4 tag enabling controlled proteolysis was constructed pBT1-mCherry-DAS+4 (SEQ ID NO:28). Constitutive expression was achieved using a proD promoter (Davis, J H., Rubin, A J., and Sauer, R T. NAR. February 2011; 39(3) p 1131-1141. DOI: 10.1093). Lastly, another compatible vector enabling the low phosphate expression (utilizing the low phosphate inducible ugpB gene promoter from E. coli) expression of a gene silencing guide RNA targeting the proD promoter was constructed (Refer to Example 6 for methods) and called pCASCADE-proD (SEQ ID NO:29). These plasmids were transformed into several host strains as described in Example 4, including strain DLF_0025 to create several strains. Colonies were used to inoculate 4 mL of SM3 media with kanamycin (Refer to Common Methods Section) and incubated overnight at 37 degrees Celsius and a shaking speed of 225 rpm. After overnight growth, cells were normalized to an optical density at 600 nm of 5, and 40 .mu.L of normalized culture was used to inoculate 760 .mu.L of fresh FGM3 (Refer to Common Methods Section) medium with kanamycin in wells of a 48 well FlowerPlate.TM. B which was transferred into a BioLector Microbioreactor both obtained from M2P Labs (Baesweiler, Germany). The BioLector Microbioreactor can continuously measure fluorescence and biomass levels. Cells were incubated in the Microreactor at 37 degrees Celsius and a shaking speed of 1200 rpm for 60 hrs. Fluorescence results for each reporter construct as well as an empty vector control are reported as relative fluorescence units (R.F.U) normalized to biomass levels are depicted in FIG. 7. All plasmids were constructed using standard Gibson Assembly methodology (Gibson Assembly Master Mix, obtained from New England Biolabs, Ipswich, Mass., USA), and synthetic linear double stranded DNA provided as Gblocks.TM. (Integrated DNA Technology, Coralville, Iowa, USA). Eton Bioscience (Research Triangle Park, N.C., USA) was used for plasmid DNA sequence confirmations. Standard codon optimization was performed to optimize constructs for expression in E. coli.

Example 8: E. coli Pathway Plasmid Cloning

[0117] All production plasmids were constructed using standard Gibson Assembly methodology (Gibson Assembly Master Mix, obtained from New England Biolabs, Ipswich, Mass., USA), and synthetic linear double stranded DNA provided as Gblocks.TM. (Integrated DNA Technology, Coralville, Iowa, USA). Eton Bioscience (Research Triangle Park, N.C., USA) was used for plasmid DNA sequence confirmations. Standard codon optimization was performed to optimize constructs for expression in E. coli.

[0118] A plasmid expressing an NADPH dependent 3-hydroxypropionic acid (3-HP) production pathway was constructed as an operon of two genes. The mcr gene from Chloroflexus auranticus (CaMCR), encoding a malonyl-CoA reductase (Uniprot #A9WIU3), and the ydfG gene from E. coli, encoding an NADPH dependent 3-HP dehydrogenase (Uniprot #P39831) were used. Only the C-terminal end (residues 550-1219) of the mcr enzyme encoding the malonyl-CoA reductase domain was utilized (Liu, C., Wang, Q., Ding., Y and Zhao, Gu., PLOS One. September 2013. DOI: 10.1371). The operon was assembled into the pSMART-HC-Kan vector, resulting in plasmid pSMART-3HP1, (SEQ ID NO:30).

[0119] A plasmid expressing a malonic acid production pathway was constructed from a single gene encoding a triple mutant (E95N/Q384A/F304R) Pseudomonas fulva (strain 12-X) isobutyryl-CoA thioesterase (Uniprot #F6AA82), with altered specificity (Steen, E., Patent Application PCT/US2014/047645). This gene was cloned behind the phosphate dependent waaH gene promoter from E. coli. The gene was then assembled into the pSMART-HC-Kan vector (Lucigen, Middleton Wis.), resulting in plasmid pSMART-F6AA82M, (SEQ ID NO:31).

[0120] A plasmid expressing an NADPH dependent L-alanine production pathway was constructed from a single gene encoding a double mutant (Leu197Arg Asp196Ala) Bacillus subtilis alanine dehydrogenase (AlaDH) (Uniprot #Q08352), with NADPH cofactor specificity (Haas, T., et al. Patent Application PCT/EP2013/057855). This gene was cloned behind the phosphate dependent waaH gene promoter from E. coli. The gene was then assembled into the pSMART-HC-Kan vector (Lucigen, Middleton Wis.), resulting in plasmid pSMART-Ala1, (SEQ ID NO:32). A additional plasmid expressing the same NADPH dependent L-alanine production pathway was constructed using the phosphate dependent ugpB gene promoter from E. coli. The gene was then assembled into the pSMART-HC-Kan vector (Lucigen, Middleton Wis.), resulting in plasmid pSMART-Ala2, (SEQ ID NO:46).

[0121] A plasmid expressing a mevalonate production pathway was constructed from two genes assembled into two transcriptional units. First, the mvaE gene from Enterococcus faecalis encoding a bifunctional acetoacetyl-CoA thiolase, and NADPH dependent HMG-CoA reductase (Uniprot #Q9FD70) was cloned behind an insulated version of the phosphate dependent waaH gene promoter from E. coli. Additionally, the mvaS gene, also from E. faecalis, encoding a hydroxymethylglutaryl-CoA synthase (Uniprot #Q9FD71) was cloned behind an insulated version of the phosphate dependent mipA gene promoter from E. coli. The mvaS expression construct was cloned behind the mvaE construct and both assembled into the pSMART-HC-Kan vector, resulting in plasmid pSMART-Mev1, (SEQ ID NO:33).

[0122] A plasmid expressing an NADH dependent 2,3-butanediol production pathway was constructed as an operon of three genes. The budA, budB and budC genes from Enterobacter cloacae subsp. dissolvens SDM, encoding an .alpha.-acetolactate decarboxylase, an acetolactate synthase and acetoin reductase, respectively, were cloned behind the phosphate dependent waaH gene promoter from E. coli. The operon was assembled into the pSMART-HC-Kan vector, resulting in plasmid pSMART-2,3-BDO1, (SEQ ID NO:34).

[0123] A plasmid expressing an NADPH dependent 2,3-butanediol production pathway was constructed as an operon of three genes. The budA, budB genes from Enterobacter cloacae subsp. dissolvens SDM, encoding an .alpha.-acetolactate decarboxylase, an acetolactate synthase, and a Glu221Ser/Ile222Arg/Ala223Ser triple mutant bdh1 gene from S. cerevisiae, encoding an NADPH dependent acetoin reductase (Ehsani, M., Fernandez, M R., Biosca J A and Dequin, S. Biotechnol Bioeng. 2009 Oct. 1; 104(2):381-9. doi: 10.1002) respectively, were cloned behind the phosphate dependent waaH gene promoter from E. coli. The operon was assembled into the pSMART-HC-Kan vector, resulting in plasmid pSMART-2,3-BDO2 (SEQ ID NO:35).

Example 9: Production of 3-Hydroxypropionic Acid (3-HP) in E. coli, from Malonyl-CoA and NADPH in 96 Well Plates

[0124] Several E. coli strains were constructed utilizing a combination of host strains as described in Example 5, production pathway plasmids as described in Example 8 and CASCADE based gene silencing constructs such as those described in Example 6. Strains were then evaluated for product formation using the standard 96 well plate evaluation protocol "96 Well Plate Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. These strains and the associated production data are given in Table 2.

TABLE-US-00002 TABLE 2 3-HP Production from malonyl-CoA and NADPH in 96 well plates Final 3- Final 3- HP HP pCASCADE Production Titer Std Strain Host Strain plasmid Plasmid (g/L) Deviation 1 DLF_0028 0 0 2 DLF_0043 0 0 3 DLF_0038 0 0 4 DLF_0040 0 0 5 DLF_0049 0 0 6 DLF_0045 0 0 7 DLF_0039 0 0 8 DLF_0167 0 0 9 DLF_0047 0 0 10 DLF_0286 0 0 11 DLF_0286 Empty 0 0 vector 12 DLF_0039 pSMART- 0 0 3HP1 13 DLF_0028 pCASCADE- pSMART- 0 0 fabI 3HP1 14 DLF_0028 pCASCADE- pSMART- 0 0 fabI-zwf 3HP1 15 DLF_0043 pCASCADE- pSMART- 0 0 fabI 3HP1 16 DLF_0025 pCASCADE- pSMART- 0.02 0.03 fabI 3HP1 17 DLF_0045 pCASCADE- pSMART- 0.11 0.06 udhA-gltA2 3HP1 18 DLF_0025 pSMART- 0.16 0.14 3HP1 19 DLF_0043 pCASCADE- pSMART- 0.19 0.06 gltA2 3HP1 20 DLF_0025 pCASCADE- pSMART- 0.36 0.18 fabI-udhA 3HP1 21 DLF_0046 pSMART- 0.41 0.14 3HP1 22 DLF_0039 pCASCADE- pSMART- 0.45 0.29 fabI-gltA2 3HP1 23 DLF_0028 pSMART- 0.55 0.24 3HP1 24 DLF_0025 pCASCADE- pSMART- 0.57 0.14 udhA 3HP1 25 DLF_0046 pCASCADE- pSMART- 0.58 0.09 fabI-udhA 3HP1 26 DLF_0025 pCASCADE- pSMART- 0.66 0.26 fabI-zwf 3HP1 27 DLF_0046 pCASCADE- pSMART- 0.89 0.11 fabI-zwf 3HP1 28 DLF_0047 pCASCADE- pSMART- 1.00 1.74 fabI-gltA1 3HP1 29 DLF_0038 pCASCADE- pSMART- 1.58 0.32 fabI-udhA 3HP1 30 DLF_0039 pCASCADE- pSMART- 1.66 0.34 gltA1 3HP1 31 DLF_0047 pCASCADE- pSMART- 1.82 0.41 fabI 3HP1 32 DLF_0047 pCASCADE- pSMART- 2.05 0.16 fabI-zwf 3HP1 33 DLF_0038 pSMART- 2.09 0.34 3HP1 34 DLF_0047 pCASCADE- pSMART- 2.28 0.39 fabI-udhA 3HP1 35 DLF_0047 pCASCADE- pSMART- 2.33 1.30 udhA 3HP1 36 DLF_0291 pCASCADE- pSMART- 3.17 0.93 gltA2 3HP1 37 DLF_0291 pCASCADE- pSMART- 4.95 2.18 udhA-gltA2 3HP1

Example 10: Production of 3-Hydroxypropionic Acid (3-HP) in E. coli, from Malonyl-CoA and NADPH at mL Scale

[0125] Several E. coli strains were constructed utilizing a combination of host strains as described in Example 5, production pathway plasmids as described in Example 6 and CASCADE based gene silencing constructs such as those described in Example 7. Strains were then evaluated for product formation using the standard mL scale evaluation protocol "Micro24 Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Summary metrics are listed in Table 3 and shown in FIG. 8.

TABLE-US-00003 TABLE 3 3-HP Summary Production metrics for 3-HP produced from malonyl-CoA and NADPH at mL scale. pCASCADE Production Final 3-HP Strain Host Strain plasmid Plasmid Titer (g/L) 18 DLF_0025 pSMART- Below Detection 3HP1 13 DLF_0028 pCASCADE- pSMART- 1.48 .+-. 0.91 fabI 3HP1 38 DLF_0038 pCASCADE- pSMART- 4.19 .+-. 1.39 fabI 3HP1 39 DLF_0038 pCASCADE- pSMART- 5.07 .+-. 1.03 udhA 3HP1 29 DLF_0038 pCASCADE- pSMART- 1.17 .+-. 0.44 fabI-udhA 3HP1 34 DLF_0047 pCASCADE- pSMART- 8.71 .+-. 0.28 fabI-udhA 3HP1

Example 11: Production of 3-Hydroxypropionic Acid (3-HP) in E. coli, from Malonyl-CoA and NADPH L Scale

[0126] E. coli strain 39 from Example 10, was evaluated at 1 L scale using the standard evaluation protocol "1 L Fermentation Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and 3-HP production are shown in FIG. 9.

Example 12: Production of Malonic Acid in E. coli, from Malonyl-CoA in 96 Well Plates

[0127] Several E. coli strains were constructed utilizing a combination of host strains as described in Example 5, production pathway plasmids as described in Example 8 and CASCADE based gene silencing constructs such as those described in Example 6. Strains were then evaluated for product formation using the standard 96 well plate evaluation protocol "96 Well Plate Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. These strains and the associated production data are given in Table 4.

TABLE-US-00004 TABLE 4 Malonic Acid Production from malonyl-CoA in 96 well plates Final Final Malonic Malonic Acid Acid pCASCADE Production Titer Std Strain Host Strain plasmid Plasmid (g/L) Deviation 1 DLF_0028 0 0 2 DLF_0043 0 0 3 DLF_0038 0 0 4 DLF_0040 0 0 5 DLF_0049 0 0 6 DLF_0045 0 0 7 DLF_0039 0 0 8 DLF_0167 0 0 9 DLF_0047 0 0 10 DLF_0286 0 0 11 DLF_0286 Empty 0 0 vector 40 DLF_0025 pCASCADE- Empty 0 0 control vector 41 DLF_0025 pCASCADE- pSMART- 0 0 control F6AA82M 42 DLF_0028 pCASCADE- pSMART- 0.19 0.095 control F6AA82M 43 DLF_0039 pCASCADE- pSMART- 0 0 control F6AA82M 44 DLF_0039 pCASCADE- pSMART- 0 0 gltA1 F6AA82M 45 DLF_0039 pCASCADE- pSMART- 0 0 gltA2 F6AA82M 46 DLF_0039 pCASCADE- pSMART- 0 0 zwf F6AA82M 47 DLF_0290 pCASCADE- pSMART- 0.017 0.029 control F6AA82M 48 DLF_0167 pCASCADE- pSMART- 0.45 0.04 control F6AA82M

Example 13: Production of Alanine in E. coli, from Pyruvate in 96 Well Plates

[0128] Several E. coli strains were constructed utilizing a combination of host strains as described in Example 5, production pathway plasmids as described in Example 8 and CASCADE based gene silencing constructs such as those described in Example 6. Strains were then evaluated for product formation using the standard 96 well plate evaluation protocol "96 Well Plate Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. These strains and the associated production data are given in Table 5.

TABLE-US-00005 TABLE 5 Alanine Production from pyruvate and NADPH in 96 well plates Final Final Alanine Alanine pCASCADE Production Titer Std Strain Host Strain plasmid Plasmid (g/L) Deviation 1 DLF_0028 0 0 2 DLF_0043 0 0 3 DLF_0038 0 0 4 DLF_0040 0 0 5 DLF_0049 0 0 6 DLF_0045 0 0 7 DLF_0039 0 0 8 DLF_0167 0 0 9 DLF_0047 0 0 49 DLF_0042 pSMART- 2.62 0.069 Ala1 50 DLF_0043 pCASCADE- pSMART- 0 0 udhA-gltAl Ala2 51 DLF_0041 pCASCADE- pSMART- 0.23 0.075 udhA-gltAl Ala2 52 DLF_0041 pSMART- 0.71 0.256 Ala1 53 DLF_0049 pCASCADE- pSMART- 1.26 0.737 udhA-gltA2 Ala2 54 DLF_0025 pSMART- 1.39 0.338 Ala1 55 DLF_0049 pSMART- 1.48 0.136 Ala1 56 DLF_0031 pSMART- 1.62 0.245 Ala1 57 DLF_0042 pCASCADE- pSMART- 1.63 0.190 udhA Ala2 58 DLF_0043 pSMART- 1.64 0.104 Ala1 59 DLF_0043 pCASCADE- pSMART- 1.72 0.355 gltA2 Ala2 60 DLF_0049 pCASCADE- pSMART- 2.42 0.105 udhA Ala2 61 DLF_0045 pCASCADE- pSMART- 2.44 0.125 udhA-gltA2 Ala2 62 DLF_0049 pCASCADE- pSMART- 2.74 0.551 gltA2 Ala2 63 DLF_0041 pCASCADE- pSMART- 3.32 1.501 gltA2 Ala2 64 DLF_0045 pSMART- 3.65 0.441 Ala1 65 DLF_0043 pCASCADE- pSMART- 4.03 0.202 udhA-gltA2 Ala2

Example 14: Production of Alanine in E. coli, from Pyruvate at mL Scale

[0129] E. coli strain 49 from Example 13, was evaluated at mL scale using the standard evaluation protocol "Micro24 Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and alanine production are shown in FIG. 10.

Example 15: Production of Alanine in E. coli, from Pyruvate at L Scale

[0130] E. coli strain 60 from Example 13, was evaluated at 1 L scale using the standard evaluation protocol "1 L Fermentation Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and alanine production are shown in FIG. 11.

Example 16: Production of 2,3-Butanediol in E. coli, from Pyruvate and NADH at mL Scale

[0131] An E. coli strain was made by transforming host strain DLF_00165 with both plasmid pSMART-2,3-BDO1 and pCASCADE-zwf (Refer to Examples 4, 6 and 8). This strain was evaluated at mL scale using the standard evaluation protocol "Micro24 Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and alanine production are shown in FIG. 12.

Example 17: Production of 2,3-Butanediol in E. coli, from Pyruvate and NADH at L Scale

[0132] An E. coli strain was made by transforming host strain DLF_00165 with both plasmid pSMART-2,3-BDO1 and pCASCADE-zwf (Refer to Examples 4, 6 and 8). This strain was evaluated at 1 L scale using the standard evaluation protocol "1 L Fermentation Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and alanine production are shown in FIG. 13.

Example 18: Production of 2,3-Butanediol in E. coli, from Pyruvate and NADPH at mL Scale

[0133] An E. coli strain was made by transforming host strain DLF_00049 with both plasmid pSMART-2,3-BDO2 and pCASCADE-udhA (Refer to Examples 4, 6 and 8). This strain was evaluated at mL scale using the standard evaluation protocol "Micro24 Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and alanine production are shown in FIG. 14.

Example 19: Production of Mevalonic Acid in E. coli, from Acetyl-CoA and NADPH at L scale

[0134] An E. coli strain was made by transforming host strain DLF_0004 with plasmid pSMART-Mev1 (Refer to Examples 4 and 8). This strain was evaluated at 1 L scale using the standard evaluation protocol "1 L Fermentation Protocol-1" as described in the Common Methods Section. Products levels were then measured using the analytical methods as described in the Common Methods Section. Biomass growth and alanine production are shown in FIG. 15.

[0135] Common Methods Section

[0136] All methods in this Section are provided for incorporation into the Examples where so referenced.

[0137] Subsection I. Microorganism Species and Strains, Cultures, and Growth Media

[0138] Microbial species, that may be utilized as needed, are as follows:

[0139] Acinetobacter calcoaceticus (DSMZ #1139) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serial dilutions of the resuspended A. calcoaceticus culture are made into BHI and are allowed to grow for aerobically for 48 hours at 37.degree. C. at 250 rpm until saturated.

[0140] Bacillus subtilis is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing B. subtilis culture are made into Luria Broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to grow for aerobically for 24 hours at 37.degree. C. at 250 rpm until saturated.

[0141] Chlorobium limicola (DSMZ #245) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended using Pfennig's Medium I and II (#28 and 29) as described per DSMZ instructions. C. limicola is grown at 25.degree. C. under constant vortexing.

[0142] Citrobacter braakii (DSMZ #30040) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serial dilutions of the resuspended C. braakii culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30.degree. C. at 250 rpm until saturated.

[0143] Clostridium acetobutylicum (DSMZ #792) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Clostridium acetobutylicum medium (#411) as described per DSMZ instructions. C. acetobutylicum is grown anaerobically at 37.degree. C. at 250 rpm until saturated.

[0144] Clostridium aminobutyricum (DSMZ #2634) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Clostridium aminobutyricum medium (#286) as described per DSMZ instructions. C. aminobutyricum is grown anaerobically at 37.degree. C. at 250 rpm until saturated.

[0145] Clostridium kluyveri (DSMZ #555) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of C. kluyveri culture are made into Clostridium kluyveri medium (#286) as described per DSMZ instructions. C. kluyveri is grown anaerobically at 37.degree. C. at 250 rpm until saturated.

[0146] Corynebacterium glutamicum (DSMZ #1412) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of C. glutamicum culture are made into C. glutamicum medium (#1) as described per DSMZ instructions. C. glutamicum is grown aerobically or anaerobically at 37.degree. C. at 250 rpm until saturated.

[0147] Cupriavidus metallidurans (DMSZ #2839) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serial dilutions of the resuspended C. metallidurans culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30.degree. C. at 250 rpm until saturated.

[0148] Cupriavidus necator (DSMZ #428) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Brain Heart Infusion (BHI) Broth (RPI Corp, Mt. Prospect, Ill., USA). Serial dilutions of the resuspended C. necator culture are made into BHI and are allowed to grow for aerobically for 48 hours at 30.degree. C. at 250 rpm until saturated. As noted elsewhere, previous names for this species are Alcaligenes eutrophus and Ralstonia eutrophus.

[0149] Desulfovibrio fructosovorans (DSMZ #3604) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Desulfovibrio fructosovorans medium (#63) as described per DSMZ instructions. D. fructosovorans is grown anaerobically at 37.degree. C. at 250 rpm until saturated.

[0150] Escherichia coli strain BW25113 is obtained from the Yale Genetic Stock Center (New Haven, Conn. 06520) and is obtained as an actively growing culture. Serial dilutions of the actively growing E. coli K12 culture are made into Luria Broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to grow for aerobically for 24 hours at 37.degree. C. at 250 rpm until saturated.

[0151] Escherichia coli strain BWapldf is a generous gift from George Chen from Tsinghua University in China. Serial dilutions of the actively growing E. coli BWapldf is culture are made into Luria Broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to grow for aerobically for 24 hours at 37.degree. C. at 250 rpm until saturated.

[0152] Halobacterium salinarum (DSMZ #1576) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Halobacterium medium (#97) as described per DSMZ instructions. H. salinarum is grown aerobically at 37.degree. C. at 250 rpm until saturated.

[0153] Lactobacillus delbrueckii (#4335) is obtained from WYEAST USA (Odell, Oreg., USA) as an actively growing culture. Serial dilutions of the actively growing L. delbrueckii culture are made into Brain Heart Infusion (BHI) broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to grow for aerobically for 24 hours at 30.degree. C. at 250 rpm until saturated.

[0154] Metallosphaera sedula (DSMZ #5348) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as an actively growing culture. Serial dilutions of M. sedula culture are made into Metallosphaera medium (#485) as described per DSMZ instructions. M. sedula is grown aerobically at 65.degree. C. at 250 rpm until saturated.

[0155] Methylococcus capsulatus Bath (ATCC #33009) is obtained from the American Type Culture Collection (ATCC) (Manassas, Va. 20108 USA) as a vacuum dried culture. Cultures are then resuspended in ATCC.RTM. Medium 1306: Nitrate mineral salts medium (NMS) under a 50% air 50% methane atmosphere (ATCC, Manassas, Va. 20108 USA) and are allowed to grow at 45.degree. C.

[0156] Methylococcus thermophilus IMV 2 Yu T is obtained. Cultures are then resuspended in ATCC.RTM. Medium 1306: Nitrate mineral salts medium (NMS) under a 50% air 50% methane atmosphere (ATCC, Manassas, Va. 20108 USA) and are allowed to grow at 50.degree. C.

[0157] Methylosinus tsporium (ATCC #35069) is obtained from the American Type Culture Collection (ATCC) (Manassas, Va. 20108 USA) as a vacuum dried culture. Cultures are then resuspended in ATCC.RTM. Medium 1306: Nitrate mineral salts medium (NMS) under a 50% air 50% methane atmosphere (ATCC, Manassas, Va. 20108 USA) and are allowed to grow at 30.degree. C.

[0158] Pichia pastoris (Komagataella pastoris) (DSMZ #70382) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in YPD-medium (#393) as described per DSMZ instructions. Pichia pastoris is grown aerobically at 30.degree. C. at 250 rpm until saturated.

[0159] Propionibacterium freudenreichii subsp. shermanii (DSMZ #4902) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in PYG-medium (#104) as described per DSMZ instructions. P. freudenreichii subsp. shermanii is grown anaerobically at 30.degree. C. at 250 rpm until saturated.

[0160] Pseudomonas putida is a gift from the Gill lab (University of Colorado at Boulder) and is obtained as an actively growing culture. Serial dilutions of the actively growing P. putida culture are made into Luria Broth (RPI Corp, Mt. Prospect, Ill., USA) and are allowed to grow for aerobically for 24 hours at 37.degree. C. at 250 rpm until saturated.

[0161] Saccharomyces cerevisiae strains can be obtained from the American Type Culture Collection (ATCC) (Manassas, Va. 20108 USA) as a vacuum dried culture. Cultures are then resuspended in YPD Media and allowed to grow at 30.degree. C.

[0162] Streptococcus mutans (DSMZ #6178) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in Luria Broth (RPI Corp, Mt. Prospect, Ill., USA). S. mutans is grown aerobically at 37.degree. C. at 250 rpm until saturated.

[0163] Yarrowia lipolytica (DSMZ #1345) is obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) as a vacuum dried culture. Cultures are then resuspended in YPD-medium (#393) as described per DSMZ instructions Yarrowia lipolytica is grown aerobically at 37.degree. C. at 250 rpm until saturated.

[0164] Subsection II. Molecular Biology Techniques--DNA Cloning

[0165] In addition to the above or below specific examples, this example is meant to describe a non-limiting approach to genetic modification of a selected microorganism to introduce, remove or alter a nucleic acid sequence of interest. Alternatives and variations are provided within this general example. The methods of this example are conducted to achieve a combination of desired genetic modifications in a selected microorganism species, such as a combination of genetic modifications as described in sections herein, and their functional equivalents, such as in other bacterial and other microorganism species.

[0166] A gene or other nucleic acid sequence segment of interest is identified in a particular species (such as E. coli as described herein) and a nucleic acid sequence comprising that gene or segment is obtained.

[0167] Based on the nucleic acid sequences at the ends of or adjacent the ends of the segment of interest, 5' and 3' nucleic acid primers are prepared. Each primer is designed to have a sufficient overlap section that hybridizes with such ends or adjacent regions. Such primers may include enzyme recognition sites for restriction digest of transposase insertion that could be used for subsequent vector incorporation or genomic insertion. These sites are typically designed to be outward of the hybridizing overlap sections. Numerous contract services are known that prepare primer sequences to order (e.g., Integrated DNA Technologies, Coralville, Iowa USA).

[0168] Once primers are designed and prepared, polymerase chain reaction (PCR) is conducted to specifically amplify the desired segment of interest. This method results in multiple copies of the region of interest separated from the microorganism's genome. The microorganism's DNA, the primers, and a thermophilic polymerase are combined in a buffer solution with potassium and divalent cations (e.g., Mg or Mn) and with sufficient quantities of deoxynucleoside triphosphate molecules. This mixture is exposed to a standard regimen of temperature increases and decreases. However, temperatures, components, concentrations, and cycle times may vary according to the reaction according to length of the sequence to be copied, annealing temperature approximations and other factors known or readily learned through routine experimentation by one skilled in the art.

[0169] In an alternative embodiment the segment of interest may be synthesized, such as by a commercial vendor, and prepared via PCR, rather than obtaining from a microorganism or other natural source of DNA.

[0170] The nucleic acid sequences then are purified and separated, such as on an agarose gel via electrophoresis. Optionally, once the region is purified it can be validated by standard DNA sequencing methodology and may be introduced into a vector. Any of a number of vectors may be used, which generally comprise markers known to those skilled in the art, and standard methodologies are routinely employed for such introduction. Commonly used vector systems are well known in the art. Similarly, the vector then is introduced into any of a number of host cells. Commonly used host cells are E. coli strains. Some of these vectors possess promoters, such as inducible promoters, adjacent the region into which the sequence of interest is inserted (such as into a multiple cloning site). The culturing of such plasmid-laden cells permits plasmid replication and thus replication of the segment of interest, which often corresponds to expression of the segment of interest.

[0171] Various vector systems comprise a selectable marker, such as an expressible gene encoding a protein needed for growth or survival under defined conditions. Common selectable markers contained on backbone vector sequences include genes that encode for one or more proteins required for antibiotic resistance as well as genes required to complement auxotrophic deficiencies or supply critical nutrients not present or available in a particular culture media. Vectors also comprise a replication system suitable for a host cell of interest.

[0172] The plasmids containing the segment of interest can then be isolated by routine methods and are available for introduction into other microorganism host cells of interest. Various methods of introduction are known in the art and can include vector introduction or genomic integration. In various alternative embodiments the DNA segment of interest may be separated from other plasmid DNA if the former will be introduced into a host cell of interest by means other than such plasmid.

[0173] While steps of the general prophetic example involve use of plasmids, other vectors known in the art may be used instead. These include cosmids, viruses (e.g., bacteriophage, animal viruses, plant viruses), and artificial chromosomes (e.g., yeast artificial chromosomes (YAC) and bacteria artificial chromosomes (BAC)).

[0174] Host cells into which the segment of interest is introduced may be evaluated for performance as to a particular enzymatic step, and/or tolerance or bio-production of a chemical compound of interest. Selections of better performing genetically modified host cells may be made, selecting for overall performance, tolerance, or production or accumulation of the chemical of interest.

[0175] It is noted that this procedure may incorporate a nucleic acid sequence for a single gene (or other nucleic acid sequence segment of interest), or multiple genes (under control of separate promoters or a single promoter), and the procedure may be repeated to create the desired heterologous nucleic acid sequences in expression vectors, which are then supplied to a selected microorganism so as to have, for example, a desired complement of enzymatic conversion step functionality for any of the herein-disclosed metabolic pathways. However, it is noted that although many approaches rely on expression via transcription of all or part of the sequence of interest, and then translation of the transcribed mRNA to yield a polypeptide such as an enzyme, certain sequences of interest may exert an effect by means other than such expression.

[0176] The specific laboratory methods used for these approaches are well-known in the art and may be found in various references known to those skilled in the art, such as Sambrook and Russell, Molecular Cloning: A Laboratory Manual, Third Edition 2001 (volumes 1-3), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (hereinafter, Sambrook and Russell, 2001).

[0177] As an alternative to the above, other genetic modifications may also be practiced, such as a deletion of a nucleic acid sequence of the host cell's genome. One non-limiting method to achieve this is by use of Red/ET recombination, known to those of ordinary skill in the art and described in U.S. Pat. Nos. 6,355,412 and 6,509,156, issued to Stewart et al. and incorporated by reference herein for its teachings of this method. Material and kits for such method are available from Gene Bridges (Gene Bridges GmbH, Dresden, Germany), and the method may proceed by following the manufacturer's instructions. Targeted deletion of genomic DNA may be practiced to alter a host cell's metabolism so as to reduce or eliminate production of undesired metabolic products. This may be used in combination with other genetic modifications such as described herein in this general example.

[0178] In addition to the above, longer purified double stranded DNA fragments can now be specified and ordered from a variety of vendors. These DNA pieces can easily be assembled together into plasmid vectors as well as longer synthetic DNA constructs using Gibson Assembly methodologies as taught by Gibson, D. G., et al. "Enzymatic assembly of DNA molecules up to several hundred kilobases" Nature Methods. May 2009. Vol(6) p. 343-345. doi:10.1038.

[0179] In addition to the above, once synthetic genetic parts such as open reading frames, promoters and terminators have been synthesized, it is well known in the art, that these parts can easily be shuffled into numerous different combinations using numerous variant assembly technologies, such as Golden Gate Assembly taught by Engler, C., Kandzia, R., and Marillonnet, S., "A one pot, one step, precision cloning method with high throughput capability". PLoS ONE 2008; 3(11):e3647. doi: 10.1371.

[0180] Subsection III. Molecular Biology Techniques--Chromosomal Modifications in E. coli

[0181] Chromosomal modifications can be made to E. coli using one of many methods including phage transduction and recombineering. It is appreciated that one skilled in the art is well versed in these methods. Of particular use are scarless recombineering methods, which allow for the precise deletion or addition of sequences to the chromosome without any unneeded sequences remaining such as that taught by Li, X., et al. "Positive and negative selection using the tetA-sacB cassette: recombineering and P1 transduction in Escherichia coli". Nucleic Acids Res. December 2013. 41(22) doi: 10.1093.

[0182] Subsection IV. Molecular Biology Techniques--Chromosomal Modifications in Saccharomyces cerevisiae.

[0183] Chromosomal modifications can be made to many yeast strains including Saccharomyces cerevisiae. using methods well known in the art for homologous recombination. It is appreciated that one skilled in the art is well versed in these methods.

[0184] Subsection V: Media for E. coli

[0185] GM25 media: GM25 minimal growth media for E. coli contained per liter: 736 mL sterile distilled, deionized water, 2.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times.GM phosphate salts, 2.0 mL of 2M MgSO.sub.4, 50 mL of 500 g/L glucose, 100 mL of 1 M MOPS buffer, pH 7.4, and 10.0 mL of 100 g/L Yeast Extract. The 100.times. Trace Metal Stock was prepared in 1.0 L of distilled, deionized water with 10.0 mL of concentrated HCl with 5.0 g CaCl.sub.2)*2H.sub.2O, 1.00 g FeCl.sub.3*6H.sub.2O, 0.05 g CoCl.sub.2*6H.sub.2O, 0.3 g CuCl.sub.2*2H.sub.2O, 0.02 g ZnCl.sub.2, 0.02 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.01 g H.sub.3BO.sub.3, and 0.04 g MnCl.sub.2*4H.sub.2O and 0.2 .mu.m sterile-filtered. The 10.times.GM Phosphate Salts were prepared in 1.0 L of distilled, deionized water with 3 g K.sub.2HPO.sub.4, 2 g KH.sub.2PO.sub.4, 30 g (NH.sub.4).sub.2SO.sub.4, and 1.5 g Citric Acid (anhydrous) and autoclaved. The 2M MgSO.sub.4 was prepared in 1.0 L of distilled, deionized water with 240.0 g of anhydrous MgSO.sub.4 and 0.2 .mu.m sterile-filtered. The 500 g/L Glucose solution was prepared in 1.0 L of heated distilled, deionized water and 500 g of anhydrous dextrose and 0.2 .mu.m sterile-filtered. The 1 M 4-Morpholinopropanesulfonic acid (MOPS) buffer was prepared in 700.0 mL of distilled, deionized water with 210.0 g MOPS and 30.0 mL 50% KOH solution. The pH was measured with stirring and final adjustments made to pH 7.4 by slowly adding 50% KOH and Q.S. to a final volume of 1.0 L. The final pH 7.4 solution was 0.2 .mu.m sterile-filtered.

[0186] PM25 media: PM25 minimal production media for E. coli contained per liter: 636 mL sterile distilled, deionized water, 2.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times.PM phosphate-free salts, 2.0 mL of 2M MgSO.sub.4, 50 mL of 500 g/L glucose, 200 mL of 1 M MOPS buffer, pH 7.4, and 10 mL of 1 mg/mL Thiamine. The 100.times. Trace Metal Stock was prepared in 1.0 L of distilled, deionized water with 10.0 mL of concentrated HCl with 5.0 g CaCl.sub.2)*2H.sub.2O, 1.00 g FeCl.sub.3*6H.sub.2O, 0.05 g CoCl.sub.2*6H.sub.2O, 0.3 g CuCl.sub.2*2H.sub.2O, 0.02 g ZnCl.sub.2, 0.02 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.01 g H.sub.3BO.sub.3, and 0.04 g MnCl.sub.2*4H.sub.2O and 0.2 .mu.m sterile-filtered. The 10.times. PM Phosphate-Free Salts were prepared in 1.0 L of distilled, deionized water with 30 g (NH.sub.4).sub.2SO.sub.4 and 1.5 g Citric Acid (anhydrous) and autoclaved. The 2M MgSO.sub.4 was prepared in 1.0 L of distilled, deionized water with 240.0 g of anhydrous MgSO.sub.4 and 0.2 .mu.m sterile-filtered. The 500 g/L Glucose solution was prepared in 1.0 L of heated distilled, deionized water and 500 g of anhydrous dextrose and 0.2 .mu.m sterile-filtered. The 1 M 4-Morpholinopropanesulfonic acid (MOPS) buffer was prepared in 700.0 mL of distilled, deionized water with 210.0 g MOPS and 30.0 mL 50% KOH solution. The pH was measured with stirring and final adjustments made to pH 7.4 by slowly adding 50% KOH and Q.S. to a final volume of 1.0 L. The final pH 7.4 solution was 0.2 .mu.m sterile-filtered.

[0187] SM3 Media: SM3 minimal media for E. coli contained per liter: 596.2 mL sterile distilled, deionized water, 2.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times. Ammonium Citrate 30 Salts, 3.6 mL of Phosphate Buffer, pH=6.8, 2 mL of 40 mM Fe(II) sulfate, 1.0 mL of 2M MgSO.sub.4, 5.0 mL of 10 mM CaSO.sub.4, 90 mL of 500 g/L glucose, 200 mL of 1 M MOPS buffer, pH 7.4, and 0.2 mL of 1 mg/mL Thiamine and 10.0 mL of 100 g/L Yeast Extract. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 30 salts by mixing 30 g of (NH.sub.4).sub.2SO.sub.4 and 1.5 g Citric Acid in water with stirring. Autoclave and store at room temperature. Prepare a 1 M Potassium 3-(N-morpholino)propanesulfonic Acid (MOPS) and adjust to pH 7.4 with KOH (.about.40 mL). Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a 0.1 M potassium phosphate buffer, pH 6.8 by mixing 49.7 mL of 1.0 M K.sub.2HPO.sub.4 and 50.3 mL of 1.0 M KH.sub.2PO4 and adjust to a final volume of 1000 mL with ultrapure water. Filter sterilize (0.2 um) and store at room temperature. Prepare 2 M MgSO.sub.4 and 10 mM CaSO.sub.4 solutions. Filter sterilize (0.2 um) and store at room temperature. Prepare a solution of 100.times. Trace metals in 1000 mL of water containing 10 mL of concentrated H.sub.2SO.sub.4: 0.6 g CoSO.sub.4*7H.sub.2O, 5.0 g CuSO.sub.4*5H.sub.2O, 0.6 g ZnSO.sub.4*7H.sub.2O, 0.2 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.1 g H.sub.3BO.sub.3, and 0.3 g MnSO.sub.4*H.sub.2O. Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a fresh solution of 40 mM ferrous sulfate heptahydrate in water. Filter sterilize (0.2 um) and discard after 1 day. Prepare a 50 g/L solution of thiamine-HCl. Filter sterilize (0.2 um) and store at 4 degrees Celsius. Prepare a 500 g/L solution of glucose by stirring with heat. Cool, filter sterilize (0.2 um), and store at room temperature.

[0188] SM10 Media: SM10 minimal media for E. coli contained per liter: 574.3 mL sterile distilled, deionized water, 4.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times. Ammonium Citrate 90 Salts, 10.0 mL of Phosphate Buffer, pH=6.8, 4 mL of 40 mM Fe(II) sulfate, 1.25 mL of 2M MgSO.sub.4, 6.25 mL of 10 mM CaSO.sub.4, 90 mL of 500 g/L glucose, 200 mL of 1 M MOPS buffer, pH 7.4, and 0.2 mL of 1 mg/mL Thiamine and 10.0 mL of 100 g/L Yeast Extract. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 90 salts by mixing 90 g of (NH.sub.4).sub.2SO.sub.4 and 2.5 g Citric Acid Autoclave and store at room temperature. 0.1 M potassium phosphate buffer, pH 6.8 by mixing 49.7 mL of 1.0 M K.sub.2HPO.sub.4 and 50.3 mL of 1.0 M KH.sub.2PO4 and adjust to a final volume of 1000 mL with ultrapure water. Prepare a 1 M Potassium 3-(N-morpholino)propanesulfonic Acid (MOPS) and adjust to pH 7.4 with KOH (.about.40 mL). Filter sterilize (0.2 um) and store at room temperature in the dark. Filter sterilize (0.2 um) and store at room temperature. Prepare 2 M MgSO.sub.4 and 10 mM CaSO.sub.4 solutions. Filter sterilize (0.2 um) and store at room temperature. Prepare a solution of 100.times. Trace metals in 1000 mL of water containing 10 mL of concentrated H.sub.2SO.sub.4: 0.6 g CoSO.sub.4*7H.sub.2O, 5.0 g CuSO.sub.4*5H.sub.2O, 0.6 g ZnSO.sub.4*7H.sub.2O, 0.2 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.1 g H.sub.3BO.sub.3, and 0.3 g MnSO.sub.4*H.sub.2O. Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a fresh solution of 40 mM ferrous sulfate heptahydrate in water. Filter sterilize (0.2 um) and discard after 1 day. Prepare a 50 g/L solution of thiamine-HCl. Filter sterilize (0.2 um) and store at 4 degrees Celsius. Prepare a 500 g/L solution of glucose by stirring with heat. Cool, filter sterilize (0.2 um), and store at room temperature.

[0189] SM10++ Media: SM10 minimal media for E. coli contained per liter: 549.3 mL sterile distilled, deionized water, 4.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times. Ammonium Citrate 90 Salts, 10.0 mL of Phosphate Buffer, pH=6.8, 4 mL of 40 mM Fe(II) sulfate, 1.25 mL of 2M MgSO.sub.4, 6.25 mL of 10 mM CaSO.sub.4, 90 mL of 500 g/L glucose, 200 mL of 1 M MOPS buffer, pH 7.4, and 0.2 mL of 1 mg/mL Thiamine and 25.0 mL of 100 g/L Yeast Extract and 25.0 mL of 100 g/L Casamino acids. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 90 salts by mixing 90 g of (NH.sub.4).sub.2SO.sub.4 and 2.5 g Citric Acid Autoclave and store at room temperature. 0.1 M potassium phosphate buffer, pH 6.8 by mixing 49.7 mL of 1.0 M K.sub.2HPO.sub.4 and 50.3 mL of 1.0 M KH.sub.2PO4 and adjust to a final volume of 1000 mL with ultrapure water. Filter sterilize (0.2 um) and store at room temperature. Prepare a 1 M Potassium 3-(N-morpholino)propanesulfonic Acid (MOPS) and adjust to pH 7.4 with KOH (.about.40 mL). Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare 2 M MgSO.sub.4 and 10 mM CaSO.sub.4 solutions. Filter sterilize (0.2 um) and store at room temperature. Prepare a solution of 100.times. Trace metals in 1000 mL of water containing 10 mL of concentrated H.sub.2SO.sub.4: 0.6 g CoSO.sub.4*7H.sub.2O, 5.0 g CuSO.sub.4*5H.sub.2O, 0.6 g ZnSO.sub.4*7H.sub.2O, 0.2 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.1 g H.sub.3B03, and 0.3 g MnSO.sub.4*H.sub.2O. Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a fresh solution of 40 mM ferrous sulfate heptahydrate in water. Filter sterilize (0.2 um) and discard after 1 day. Prepare a 50 g/L solution of thiamine-HCl. Filter sterilize (0.2 um) and store at 4 degrees Celsius. Prepare a 500 g/L solution of glucose by stirring with heat. Cool, filter sterilize (0.2 um), and store at room temperature.

[0190] FGM3 Media: FGM3 media for E. coli contained per liter: 636.2 mL sterile distilled, deionized water, 2.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times. Ammonium Citrate 20 Salts, 3.6 mL of Phosphate Buffer, pH=6.8, 2 mL of 40 mM Fe(II) sulfate, 1.0 mL of 2M MgSO.sub.4, 5.0 mL of 10 mM 2M CaSO.sub.4, 50 mL of 500 g/L glucose, 200 mL of 1 M MOPS buffer, pH 7.4, and 0.2 mL of 1 mg/mL Thiamine. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 20 salts by mixing 20 g of (NH.sub.4).sub.2SO.sub.4 and 1.5 g Citric Acid in water with stirring. Autoclave and store at room temperature. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 30 salts by mixing 30 g of (NH.sub.4).sub.2SO.sub.4 and 1.5 g Citric Acid in water with stirring. Autoclave and store at room temperature. 0.1 M potassium phosphate buffer, pH 6.8 by mixing 49.7 mL of 1.0 M K.sub.2HPO.sub.4 and 50.3 mL of 1.0 M KH.sub.2PO.sub.4 and adjust to a final volume of 1000 mL with ultrapure water. Filter sterilize (0.2 um) and store at room temperature. Prepare 2 M MgSO.sub.4 and 10 mM CaSO.sub.4 solutions. Filter sterilize (0.2 um) and store at room temperature. Prepare a solution of 100.times. Trace metals in 1000 mL of water containing 10 mL of concentrated H.sub.2SO.sub.4: 0.6 g CoSO.sub.4*7H.sub.2O, 5.0 g CuSO.sub.4*5H.sub.2O, 0.6 g ZnSO.sub.4*7H.sub.2O, 0.2 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.1 g H.sub.3BO.sub.3, and 0.3 g MnSO.sub.4*H.sub.2O. Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a fresh solution of 40 mM ferrous sulfate heptahydrate in water. Filter sterilize (0.2 um) and discard after 1 day. Prepare a 50 g/L solution of thiamine-HCl. Filter sterilize (0.2 um) and store at 4 degrees Celsius. Prepare a 500 g/L solution of glucose by stirring with heat. Cool, filter sterilize (0.2 um), and store at room temperature.

[0191] FGM10 Media: FGM10 media for E. coli contained per liter: 824.3 mL sterile distilled, deionized water, 4.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times. Ammonium Citrate 90 Salts, 10.0 mL of Phosphate Buffer, pH=6.8, 4 mL of 40 mM Fe(II) sulfate, 1.25 mL of 2M MgSO.sub.4, 6.25 mL of 10 mM 2M CaSO.sub.4, 50 mL of 500 g/L glucose, and 0.2 mL of 1 mg/mL Thiamine. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 90 salts by mixing 90 g of (NH.sub.4).sub.2SO.sub.4 and 2.5 g Citric Acid Autoclave and store at room temperature. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 90 salts by mixing 90 g of (NH.sub.4).sub.2SO.sub.4 and 2.5 g Citric Acid Autoclave and store at room temperature. 0.1 M potassium phosphate buffer, pH 6.8 by mixing 49.7 mL of 1.0 M K.sub.2HPO.sub.4 and 50.3 mL of 1.0 M KH.sub.2PO.sub.4 and adjust to a final volume of 1000 mL with ultrapure water. Filter sterilize (0.2 um) and store at room temperature. Prepare 2 M MgSO.sub.4 and 10 mM CaSO.sub.4 solutions. Filter sterilize (0.2 um) and store at room temperature. Prepare a solution of 100.times. Trace metals in 1000 mL of water containing 10 mL of concentrated H.sub.2SO.sub.4: 0.6 g CoSO.sub.4*7H.sub.2O, 5.0 g CuSO.sub.4*5H.sub.2O, 0.6 g ZnSO.sub.4*7H.sub.2O, 0.2 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.1 g H.sub.3BO.sub.3, and 0.3 g MnSO.sub.4*H.sub.2O. Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a fresh solution of 40 mM ferrous sulfate heptahydrate in water. Filter sterilize (0.2 um) and discard after 1 day. Prepare a 50 g/L solution of thiamine-HCl. Filter sterilize (0.2 um) and store at 4 degrees Celsius. Prepare a 500 g/L solution of glucose by stirring with heat. Cool, filter sterilize (0.2 um), and store at room temperature.

[0192] 96WPM Media: 96WPM media for E. coli contained per liter: 638.8 mL sterile distilled, deionized water, 2.0 mL of 100.times. Trace Metals Stock, 100 mL of 10.times. Ammonium Citrate 30 Salts, 2 mL of 40 mM Fe(II) sulfate, 2.0 mL of 2M MgSO.sub.4, 5.0 mL of 10 mM 2M CaSO.sub.4, 50 mL of 500 g/L glucose, 200 mL of 1 M MOPS buffer, pH 7.4, and 0.2 mL of 1 mg/mL Thiamine and 10.0 mL of 100 g/L Yeast Extract. Prepare 1 liter of 10.times. concentrated Ammonium-Citrate 30 salts by mixing 30 g of (NH.sub.4).sub.2SO.sub.4 and 1.5 g Citric Acid in water with stirring. Autoclave and store at room temperature. Prepare a 1 M Potassium 3-(N-morpholino)propanesulfonic Acid (MOPS) and adjust to pH 7.4 with KOH (.about.40 mL). Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare 2 M MgSO.sub.4 and 10 mM CaSO.sub.4 solutions. Filter sterilize (0.2 um) and store at room temperature. Prepare a solution of 100.times. Trace metals in 1000 mL of water containing 10 mL of concentrated H.sub.2SO.sub.4: 0.6 g CoSO.sub.4*7H.sub.2O, 5.0 g CuSO.sub.4*5H.sub.2O, 0.6 g ZnSO.sub.4*7H.sub.2O, 0.2 g Na.sub.2MoO.sub.4*2H.sub.2O, 0.1 g H.sub.3B03, and 0.3 g MnSO.sub.4*H.sub.2O. Filter sterilize (0.2 um) and store at room temperature in the dark. Prepare a fresh solution of 40 mM ferrous sulfate heptahydrate in water. Filter sterilize (0.2 um) and discard after 1 day. Prepare a 50 g/L solution of thiamine-HCl. Filter sterilize (0.2 um) and store at 4 degrees Celsius. Prepare a 500 g/L solution of glucose by stirring with heat. Cool, filter sterilize (0.2 um), and store at room temperature.

[0193] Antibiotic concentrations: Unless other wise stated standard final concentrations of antibiotic in media are kanamycin (35 ug/mL), ampicillin (100 ug/ml), spectinomycin (100 ug/ml), chloramphenicol (20 ug/ml), anhydrotetracycline (50 ng/ml), gentamicin (10 ug/ml), zeocin (50 ug/ml), blasticidin (50 ug/ml). Low salt medium such as low salt LB medium is used when using blasticidin or zeocin as selective antibiotics.

[0194] Subsection VI: Protocols for Production in E. coli

[0195] Shake Flask Protocol-1

[0196] Bioproduction is demonstrated at a 50-mL scale using GM25 minimal defined media without phosphate. Cultures are started from single colonies by standard practice into 50 mL of GM25 media containing 3.2 mM phosphate plus appropriate antibiotics and grown to stationary phase overnight at 30.degree. C. with rotation at 200 rpm. The optical density (OD.sub.600, 1 cm pathlength) of each stationary phase culture is measured and the entire culture is transferred to 50 mL conical tubes and centrifuged at 4,000 rpm for 15 minutes. A 20 optical density resuspension is generated for each culture by calculating the volume of GM25 media to add to the pellet. Two and a half mL of this resuspension is added to 50 mL of PM25 media plus appropriate antibiotic in triplicate 250-ml non-baffled flasks and incubated at 30.degree. C., 200 rpm. To monitor cell growth and production by these cultures, samples (2 ml) are withdrawn at designated time points for optical density measurements at 600 nm (OD.sub.600, 1 cm pathlength). Samples are centrifuged at 14,000 rpm for 5 minutes and the supernatant retained at -20.degree. C. for analyte measurements. Cultures are shifted to production by changing the temperature of the shaking incubator to 37.degree. C. at 4 hours post-inoculation. A sample is collected at this time point as well as 6-, 8-, and 24-hours post-inoculation for optical density and product measurement.

[0197] Shake Flask Protocol-2

[0198] Bioproduction is demonstrated at a 50-mL scale in GM25 minimal defined media without phosphate. Cultures are started from single colonies by standard practice into 50 mL of GM25 media containing 3.2 mM phosphate plus appropriate antibiotic(s) and grown to stationary phase overnight at 37.degree. C. with rotation at 200 rpm. The optical density (OD.sub.600, 1 cm pathlength) of each stationary phase culture is measured and the entire culture was transferred to 50 mL conical tubes and centrifuged at 4,000 rpm for 15 minutes. A 20 optical density resuspension is generated for each culture by calculating the volume of GM25 media to add to the pellet. Two and a half mL of this resuspension is added to 50 mL of PM25 media plus antibiotics in triplicate 250-ml non-baffled flasks and incubated at 37.degree. C., 200 rpm. To monitor cell growth and production by these cultures, samples (2 ml) are withdrawn at designated time points for optical density measurements at 600 nm (OD.sub.600, 1 cm pathlength). Samples are centrifuged at 14,000 rpm for 5 minutes and the supernatant retained at -20.degree. C. for analyte measurements. Cultures are shifted to production by inducing the cultures using 50 ng/mL of anhydrotetracycline (aTc) at inoculation. A sample was collected at this time point as well as 4 and 20-hours post-inoculation for optical density and product measurement.

[0199] 96 Well Plate Protocol-1

[0200] Bioproduction is demonstrated at .mu.L in minimal medium. Colonies were used to inoculate individual wells in standard 96 well plates, filled with 150 .mu.L of SM10++ medium with the appropriate antibiotics as needed. Plates were covered with sandwich covers (Model #CR1596 obtained from EnzyScreen, Haarlam, The Netherlands). These covers ensure minimal evaporative loss during incubation. To ensure adequate aeration, the inoculated 96 well plates and sandwich covers were clamped into place into a Mini Shaking Incubator (VWR Catalog #12620-942, VWR International LLC., Radnor, Pa., USA.) at a temperature set to 37 degrees Celsius and a shaking speed of 1100 rpm. The plate clamps used were obtained from Enzyscreen (Model #CR1600, EnzyScreen, Haarlam, The Netherlands). Importantly, the shaker used had an orbit of 0.125 inches or 3 mm. This combination of orbit and minimal shaking speed is required to obtain needed mass transfer coefficient and enable adequate culture oxygenation. Cultures were grown for 16 hours.

[0201] After 16 hours of growth, 10 .mu.L samples were taken to measure the optical density at 600 nm (OD(600 nm)). This was done using a plate spectrophotometer. Overnight cell densities at this point often range from 5-15 OD(600 nm). Cells from 100 .mu.L of overnight growth in each well were pelleted by centrifugation, excess media was removed and cells were resuspended in 150 .mu.L of 96WPM, which contains no phosphate. Subsequently cells were once again pelleted and again excess media was removed. Using the overnight measured optical densities, enough fresh 96WPM was added to each well, so upon re-suspension a final OD(600 nm) of 20 was obtained. 7.5 .mu.L of the normalized and washed cultures of OD(600 nm)=20, was used to inoculate 150 .mu.L of fresh 96WPM, plus appropriate antibiotics, in wells of a new standard 96 well plate. Plates were covered with sandwich covers (Model #CR1596 obtained from EnzyScreen, Haarlam, The Netherlands) and clamped into place into a Mini Shaking Incubator (VWR Catalog #12620-942, VWR International LLC., Radnor, Pa., USA.) at a temperature set to 37 degrees Celsius and a shaking speed of 1100 rpm. The plate clamps used were obtained from Enzyscreen (Model #CR1600, EnzyScreen, Haarlam, The Netherlands). Cultures were incubated for 24 hours. After 16-24 hours of production, 100 .mu.L samples from each well were pelleted by centrifugation and the supernatant collected for subsequent analytical analyses.

[0202] Micro24 Protocol-1

[0203] Bioproduction is demonstrated at mL scale in minimal medium. Seeds were prepared as follows. Colonies were used to inoculate 4 mL of SM10 medium, with appropriate antibiotics as needed, into a sterile 14 mL culture tube. Culture tubes were incubated overnight at 37 degrees Celsius in a standard floor model shaking incubator at 225 rpm. After overnight growth, 2.5 mL of these cultures were used to inoculate 50 mL of fresh SM10 medium, plus appropriate antibiotics as needed, in a 250 mL volume disposable and sterile rectangular cell culture flask, such as a Cellstar.TM. Cell Culture Flask (VWR Catalog #82050-856, VWR International LLC., Radnor, Pa., USA.). These seed cultures were incubated at 37 degrees Celsius in a standard floor model shaking incubator at 225 rpm. Samples were taken every few hours to measure the growth by optical density (OD(600 nm)), until they reached at an OD(600 nm) in the range of 4-10. At this point, cells were harvested by centrifugation, excess media removed and resuspended in fresh SM10 media to obtain a final OD(600 nm) of 10. 500 .mu.L of washed and normalized cells was added to 500 .mu.L of 30% sterile glycerol in water, mixed and frozen in cryovial (seed vials) at minus 80 degrees Celsius in a ultralow temperature freezer.

[0204] The Micro-24.TM. Microreactor system (Pall Corporation, Exton, Pa., USA) was used to evaluate strains at the mL scale. Pall 24-well PERC cassettes (Catalogue #MRT-PRC) were used for cell growth and production along with stainless steel check valve caps (Catalogue #MRT-CAP-E24). The experimental protocol was set up with an initial volume of 3 mL of FGM3 medium, with appropriate antibiotics as needed, and an agitation of 1000 rpm. pH control was initially turned off. The temperature was controlled at 37 degrees Celsius, with an environmental temperature of 35 degrees Celsius. Oxygen control was initially turned off with monitoring enabled. Frozen seed vials were thawed on ice and 150 .mu.L was used to inoculate each 3 mL culture in each Micro24 cassette well. Samples were collected at inoculation and at regular intervals. Optical density of samples was measured at 600 nm, glucose using a YSI biochemistry analyzer was measured as described below. In addition, supernatants were collected for subsequent analytical analyses. pH control was turned on for each well at the point at which the culture's optical densities as measured at 600 nm was greater than 1.0. pH control was achieved with pressured ammonium hydroxide gas. In addition, oxygen control was turned on for each well when the dissolved oxygen reached below 60%. Glucose boluses of 10 g/L were added both 24 and 48 hours post inoculation using a sterile 500 g/L stock solution.

[0205] 1 L Fermentation Protocol-1

[0206] Bioproduction is demonstrated at L scale in minimal medium. Seeds were prepared as follows. Colonies were used to inoculate 4 mL of SM10 medium, with appropriate antibiotics as needed, into a sterile 14 mL culture tube. Culture tubes were incubated overnight at 37 degrees Celsius in a standard floor model shaking incubator at 225 rpm. After overnight growth, 2.5 mL of these cultures were used to inoculate 50 mL of fresh SM10 medium, plus appropriate antibiotics as needed, in a 250 mL volume disposable and sterile rectangular cell culture flask, such as a Cellstar.TM. Cell Culture Flask (VWR Catalog #82050-856, VWR International LLC., Radnor, Pa., USA.). These seed cultures were incubated at 37 degrees Celsius in a standard floor model shaking incubator at 225 rpm. Samples were taken every few hours to measure the growth by optical density (OD(600 nm)), until they reached at an OD(600 nm) in the range of 4-10. At this point, cells were harvested by centrifugation, excess media removed and resuspended in fresh SM10 media to obtain a final OD(600 nm) of 10. 3.5 mL of washed and normalized cells was added to 3.5 mL of 30% sterile glycerol in water, mixed and frozen in cryovial (seed vials) at minus 80 degrees Celsius in a ultralow temperature freezer.

[0207] An Infors-HT Multifors (Laurel, Md., USA) parallel bioreactor system was used to perform 1 L fermentations, including three gas connection mass flow controllers configured for air, oxygen and nitrogen gases. Vessels used had a total volume of 1400 mL and a working volume of up to 1 L. Online pH and PO.sub.2 monitoring and control were accomplished with Hamilton probes. Offgas analysis was accomplished with a multiplexed Blue-in-One BlueSens gas analyzer (BlueSens. Northbrook, Ill., USA). Culture densities were continually monitored using Optek 225 mm OD probes, (Optek, Germantown, Wis., USA). The system used was running IrisV6.0 command and control software and integrated with a Seg-flow automated sampling system (Flownamics, Rodeo, Calif. USA), including FISP cell free sampling probes, a Segmod 4800 and FlowFraction 96 well plate fraction collector.

[0208] Tanks were filled with 800 mL of FGM10 Medium, with enough phosphate to target a final E. coli biomass concentration close to 10 g dry cell weight per liter. Antibiotics were added as appropriate. Frozen seed vials were thawed on ice and 7 mL of seed culture was used to inoculate the tanks. After inoculation, tanks were controlled at 37 degrees Celsius and pH 6.8 using a 10M solution of sodium hydroxide solution as a titrant. The following oxygen control scheme was used to maintain a dissolved oxygen set point of 25%. First gas flow rate was increased from a minimum of 0.3 L/min of air to 0.8 L/min of air, subsequently, if more aeration was needed, agitation was increased from a minimum of 300 rpm to a maximum of 1000 rpm. Finally if more oxygen was required to achieve a 25% set point, oxygen supplementation was included using the integrated mass flow controllers. A constant concentrated sterile filtered glucose feed (500 g/L) was added to the tanks at a rate of 2 mL/hr, once agitation reached 800 rpm. Fermentation runs were extended for up to 70 hrs and samples automatically withdrawn every 2-4 hrs. Samples were saved for subsequent analytical analysis.

[0209] Subsection VII: Analytical Methods

[0210] Analytical Methods have been developed for all anticipated metabolites and products.

[0211] Quantification of Organic and Amino Acids

[0212] A reverse phase UPLC-MS/MS method was developed for the simultaneous quantification of organic and amino acids. Chromatographic separation was performed using an Acquity CSH C.sub.18 column (100 mm.times.2.1 i.d., 1.7 .mu.m; Waters Corp., Milford, Mass., USA) at 45 degrees C. The following eluents were used: solvent A: H.sub.2O, 0.2% formic acid and 0.05% ammonium (v/v); solvent B: MeOH, 0.1% formic acid and 0.05% ammonium (v/v). The gradient elution was as follows: 0-0.2 min isocratic 5% B, 0.2-1.0 min linear from 5% to 90% B, 1.0-1.5 min isocratic 90% B, and 1.5-1.8 min linear from 90% to 5% B, with 1.8-3.0 min for initial conditions of 5% B for column equilibration. The flow rate remained constant at 0.4 ml/min. A 5 .mu.l sample injection volume was used. UPLC method development was carried out using standard aqueous stock solutions of analytes. Separations were performed using an Acquity H-Class UPLC integrated with a Xevo.TM. TQD Mass spectrometer (Waters Corp., Milford, Mass. USA). MS/MS parameters including MR transitions were tuned for each analyte and are listed in Table 6 below. Adipic acid at a concentration of 36 mg/L was used as an internal standard for normalization in all samples. Peak integration and further analysis was performed using Mass Lynx v4.1 software. The linear range for all metabolites was 2-50 mg/L. Samples were diluted as needed to be within the accurate linear range.

TABLE-US-00006 TABLE 6 MS/MS parameters Reten- tion Cone Col- Time ESI MRM Volt- lision Analyte (min) Mode Transition(s) age Energy 3-hydroxypropionic 1.04 - 88.94.fwdarw. 59.09 22 8 Acid Alanine 0.63 + 89.95.fwdarw. 44.08 15 9 .alpha.-ketoglutaric 1.97 - 144.80.fwdarw. 56.90 13 11 Acid Citric Acid 1.76 - 190.87.fwdarw. 110.92 25 11 Fumaric Acid 1.91 - 114.72.fwdarw. 70.94 21 7 Glutamic Acid 0.67 - 145.89.fwdarw. 102.02 29 11 Glyoxylic Acid 0.83 - 72.84.fwdarw. 44.98 33 7 Lactic Acid 1.18 - 88.94.fwdarw. 43.08 26 8 Malic Acid 1.06 - 132.80.fwdarw. 70.98 27 13 Malonic Acid 1.45 - 102.85.fwdarw. 59.09 15 9 Mevalonic Acid 1.85 - 146.91.fwdarw. 59.03 23 11 Pyruvic Acid 1.81 - 87.00.fwdarw. 43.05 20 7 Succinic Acid 1.72 - 116.74.fwdarw. 72.96 25 11 Itaconic Acid 1.86 + 130.87.fwdarw.84.98.sup. 20 12 Adipic Acid 2.0 + 144.77.fwdarw.82.96.sup. 32 12

[0213] Quantification of 2,3 Butanediol using Mass Spectrometry

[0214] A rapid UPLC-MS/MS method was developed for the quantification of 2,3 butanediol (2,3-BDO). Chromatographic separation was performed using an Acquity UPLC BEH C.sub.18 column (50 mm.times.2.1 i.d., 1.7 .mu.m; Waters Corp., Milford, Mass., USA) at 45 degrees C. Isopropanol with 0.1% formic acid and 0.05% ammonium (v/v) was used in an isocratic separation. A 5 .mu.l sample injection volume was used. UPLC method development was carried out using standard aqueous stock solutions of analytes. Separations were performed using an Acquity H-Class UPLC integrated with a Xevo.TM. TQD Mass spectrometer (Waters Corp., Milford, Mass. USA). An MRM transition for 2,3-BDO of 90.972.fwdarw.55.074 was used along with a cone voltage of 16V and Collision Energy of 10V, operating in ESI+ mode. Adipic acid at a concentration of 36 mg/L was used as an internal standard for normalization in all samples. The Adipic acid was measured in ESI- mode with an MRM transition of 144.77.fwdarw.82.96, a cone voltage of 32V and collision energy of 12 V. Both 2,3-BDO and adipic acid eluted at 0.38 minutes. Peak integration and further analysis was performed using Mass Lynx v4.1 software.

[0215] Quantification of Diols Using Refractive Index

[0216] A confirmatory HPLC method was developed for the quantification of 2,3 butanediol stereoisomers. Chromatographic separation was performed using a Biorad Aminex HPX-87H column (300.times.7.8 mm, 1.7 .mu.m; Biorad, Hercules, Calif. USA). The isocratic separation was run at room temperature with 5 mM sulfuric acid as the mobile phase. The flow rate remained constant at 0.4 ml/min for 40 minutes after an injection. A 10 .mu.l sample injection volume was used. Method development was carried out using standard aqueous stock solutions of analytes. Separations were performed using an Acquity H-Class UPLC integrated with an ESAT/IN refractive index (RI) detector. (Waters Corp., Milford, Mass. USA). Meso-2,3-butanediol eluted at 24.9 minutes, while (R,R)-2,3-butanediol eluted at 26.3 minutes. Peaks were integrated using Masslynx Software v4.1.

[0217] Quantification of Glucose

[0218] A YSI biochemistry analyzer, model 2950M (YSI Incorporated, Yellow Springs Ohio, USA) was used to routinely measure glucose concentrations as well as ethanol. The instrument was used according to manufacturer's instructions, using all reagents as supplied from YSI.

Sequence CWU 1

1

5413111DNAArtificial SequencePlasmid pSMART-HC-Kan-yibD-THNS 1gtgcgtaatt gtgctgatct cttatatagc tgctctcatt atctctctac cctgaagtga 60ctctctcacc tgtaaaaata atatctcaca ggcttaatag tttcttaata caaagcctgt 120aaaacgtcag gataacttct gtgtaggagg ataatctatg gcaactctct gccgtccgtc 180cgtgagtgtg ccggagcatg ttatcacgat ggaagaaacc cttgaactgg cccgtcgtcg 240tcatacggat catccacagc tgcccctggc gctgcgctta attgaaaaca ccggtgttcg 300cacgcgtcat attgttcaac cgatcgagga taccctggag catccagggt ttgaagatcg 360caataaagta tacgagcgcg aggccaaatc gcgtgtgccg gcggtaatcc aacgcgccct 420ggacgacgcg gagcttctgg cgacggacat tgacgttatt atctatgtct catgcacggg 480ttttatgatg cctagtctta ctgcttggtt aatcaacgaa atgggcttcg acagcacgac 540ccgccaaatt cctatcgcac agcttggctg tgcggccggt ggtgccgcga ttaaccgcgc 600tcacgatttt tgcacggcat atcctgaagc aaatgcgctg atcgttgcct gcgaattctg 660cagcctgtgt tatcagccca cagatctcgg tgtaggttct ctcctgtgca acggtctgtt 720cggtgatgga attgctgcgg ctgtggtgcg cggacgtggt ggtacggggg ttcgcttgga 780gcgtaacggc agctacttaa ttccaaaaac cgaagattgg atcatgtatg atgtgaaagc 840aaccggtttc cacttcttac tggataagcg cgtcccggcc accatggaac ccttggcgcc 900ggctctgaaa gaactcgcgg gcgagcatgg ttgggacgcc agtgatctgg atttttatat 960tgttcacgcc ggtggtccgc gtattttaga cgacttgagt actttccttg aggtggatcc 1020gcatgcgttt cgtttttccc gtgctaccct gaccgagtat ggtaacattg cgtcagcagt 1080cgtgctggat gcgttacgcc gcttgttcga tgaaggcggt gtggaggaag gtgcgcgcgg 1140tctgctggcg gggttcgggc caggtattac agccgaaatg tcactgggct gctggcaaac 1200cgcgtagtaa ccggcttatc ggtcagtttc acctgattta cgtaaaaacc cgcttcggcg 1260ggtttttgct tttggagggg cagaaagatg aatgactgtc cacgacgcta tacccaaaag 1320aaagacgaat tctctagata tcgctcaata ctgaccattt aaatcatacc tgacctccat 1380agcagaaagt caaaagcctc cgaccggagg cttttgactt gatcggcacg taagaggttc 1440caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt 1500ttcaggagct aaggaagcta aaatgagcca tattcaacgg gaaacgtctt gctcgaggcc 1560gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt 1620cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt 1680tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcaggctaaa 1740ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga 1800tgcatggtta ctcaccactg cgatcccagg gaaaacagca ttccaggtat tagaagaata 1860tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc 1920gattcctgtt tgtaattgtc cttttaacgg cgatcgcgta tttcgtctcg ctcaggcgca 1980atcacgaatg aataacggtt tggttggtgc gagtgatttt gatgacgagc gtaatggctg 2040gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt 2100cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg 2160ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg 2220gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat 2280tgataatcct gatatgaata aattgcagtt tcacttgatg ctcgatgagt ttttctaatg 2340agggcccaaa tgtaatcacc tggctcacct tcgggtgggc ctttctgcgt tgctggcgtt 2400tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgatgctcaa gtcagaggtg 2460gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 2520ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 2580cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 2640caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 2700ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 2760taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 2820taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 2880ctcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2940ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3000attttctacc gaagaaaggc ccacccgtga aggtgagcca gtgagttgat tgcagtccag 3060ttacgctgga gtctgaggct cgtcctgaat gatatcaagc ttgaattcgt t 311122386DNAArtificial SequencePlasmid pCDF-T2-fabIsgRNA 2gcactgaaat ctagagcggt tcagtagaaa agatcaaagg atcttcttga gatccttttt 60ttctgcgcgt aatcttttgc cctgtaaacg aaaaaaccac ctggggaggt ggtttgatcg 120aaggttaagt cagttgggga actgcttaac cgtggtaact ggctttcgca gagcacagca 180accaaatctg tccttccagt gtagccggac tttggcgcac acttcaagag caaccgcgtg 240tttagctaaa caaatcctct gcgaactccc agttaccaat ggctgctgcc agtggcgttt 300taccgtgctt ttccgggttg gactcaagtg aacagttacc ggataaggcg cagcagtcgg 360gctgaacggg gagttcttgc ttacagccca gcttggagcg aacgacctac accgagccga 420gataccagtg tgtgagctat gagaaagcgc cacacttccc gtaagggaga aaggcggaac 480aggtatccgg taaacggcag ggtcggaaca ggagagcgca agagggagcg acccgccgga 540aacggtgggg atctttaagt cctgtcgggt ttcgcccgta ctgtcagatt catggttgag 600cctcacggct cccacagatg caccggaaaa gcgtctgttt atgtgaactc tggcaggagg 660gcggagccta tggaaaaacg ccaccggcgc ggccctgctg ttttgcctca catgttagtc 720ccctgcttat ccacggaatc tgtgggtaac tttgtatgtg tccgcagcgc ccgccgcagt 780ctcacgcccg gagcgtagcg accgagtgag ctagctattt gtttattttt ctaaatacat 840tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 900aggaagagta tgagggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt 960ggcgtcatcg agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca 1020gtggatggcg gcctgaagcc acacagtgat attgatttgc tggttacggt gaccgtaagg 1080cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct 1140ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt 1200ccgtggcgtt atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt 1260cttgcaggta tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa 1320gcaagagaac atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt 1380cctgaacagg atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc 1440gactgggctg gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca 1500gtaaccggca aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg 1560gcccagtatc agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat 1620cgcttggcct cgcgcgcaga tcagttggaa gaatttgtcc actacgtgaa aggcgagatc 1680accaaggtag tcggcaaata atgtctaaca attcgttcaa cactataggg cgaattgaag 1740gaaggccgtc aaggccgcat tgaggctcgt cctgaatgat atcaagcttg aattcgttga 1800attctaaaga tctttgacag ctagctcagt cctaggtata atactagtca gcctgctccg 1860gtcggaccgt tttagagcta gaaatagcaa gttaaaataa ggctagtccg ttatcaactt 1920gaaaaagtgg caccgagtcg gtgctttttt tgaagcttgg gcccgaacaa aaactcatct 1980cagaagagga tctgaatagc gccgtcgacc atcatcatca tcatcattga gtttaaacgg 2040tctccagctt ggctgttttg gcggatgaga gaagattttc agcctgatac agattaaatc 2100agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg cggtggtccc 2160acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta gtgtggggtc 2220tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag 2280actgggcctt tcgttttatc tgttgtttgt cggtgaactg gatccttact cgagtctaga 2340ctgcagctgg gcctcatggg ccttcctttc actgcccgct ttccag 238637413DNAArtificial SequencePlasmid pdCas9-ptet-sspB 3gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat cattaattcc taatttttgt tgacactcta 660tcgttgatag agttatttta ccactcccta tcagtgatag agaaaagaat tcaaaagatc 720taaagaggag aaaggatcta tggataagaa atactcaata ggcttagcta tcggcacaaa 780tagcgtcgga tgggcggtga tcactgatga atataaggtt ccgtctaaaa agttcaaggt 840tctgggaaat acagaccgcc acagtatcaa aaaaaatctt ataggggctc ttttatttga 900cagtggagag acagcggaag cgactcgtct caaacggaca gctcgtagaa ggtatacacg 960tcggaagaat cgtatttgtt atctacagga gattttttca aatgagatgg cgaaagtaga 1020tgatagtttc tttcatcgac ttgaagagtc ttttttggtg gaagaagaca agaagcatga 1080acgtcatcct atttttggaa atatagtaga tgaagttgct tatcatgaga aatatccaac 1140tatctatcat ctgcgaaaaa aattggtaga ttctactgat aaagcggatt tgcgcttaat 1200ctatttggcc ttagcgcata tgattaagtt tcgtggtcat tttttgattg agggagattt 1260aaatcctgat aatagtgatg tggacaaact atttatccag ttggtacaaa cctacaatca 1320attatttgaa gaaaacccta ttaacgcaag tggagtagat gctaaagcga ttctttctgc 1380acgattgagt aaatcaagac gattagaaaa tctcattgct cagctccccg gtgagaagaa 1440aaatggctta tttgggaatc tcattgcttt gtcattgggt ttgaccccta attttaaatc 1500aaattttgat ttggcagaag atgctaaatt acagctttca aaagatactt acgatgatga 1560tttagataat ttattggcgc aaattggaga tcaatatgct gatttgtttt tggcagctaa 1620gaatttatca gatgctattt tactttcaga tatcctaaga gtaaatactg aaataactaa 1680ggctccccta tcagcttcaa tgattaaacg ctacgatgaa catcatcaag acttgactct 1740tttaaaagct ttagttcgac aacaacttcc agaaaagtat aaagaaatct tttttgatca 1800atcaaaaaac ggatatgcag gttatattga tgggggagct agccaagaag aattttataa 1860atttatcaaa ccaattttag aaaaaatgga tggtactgag gaattattgg tgaaactaaa 1920tcgtgaagat ttgctgcgca agcaacggac ctttgacaac ggctctattc cccatcaaat 1980tcacttgggt gagctgcatg ctattttgag aagacaagaa gacttttatc catttttaaa 2040agacaatcgt gagaagattg aaaaaatctt gacttttcga attccttatt atgttggtcc 2100attggcgcgt ggcaatagtc gttttgcatg gatgactcgg aagtctgaag aaacaattac 2160cccatggaat tttgaagaag ttgtcgataa aggtgcttca gctcaatcat ttattgaacg 2220catgacaaac tttgataaaa atcttccaaa tgaaaaagta ctaccaaaac atagtttgct 2280ttatgagtat tttacggttt ataacgaatt gacaaaggtc aaatatgtta ctgaaggaat 2340gcgaaaacca gcatttcttt caggtgaaca gaagaaagcc attgttgatt tactcttcaa 2400aacaaatcga aaagtaaccg ttaagcaatt aaaagaagat tatttcaaaa aaatagaatg 2460ttttgatagt gttgaaattt caggagttga agatagattt aatgcttcat taggtaccta 2520ccatgatttg ctaaaaatta ttaaagataa agattttttg gataatgaag aaaatgaaga 2580tatcttagag gatattgttt taacattgac cttatttgaa gatagggaga tgattgagga 2640aagacttaaa acatatgctc acctctttga tgataaggtg atgaaacagc ttaaacgtcg 2700ccgttatact ggttggggac gtttgtctcg aaaattgatt aatggtatta gggataagca 2760atctggcaaa acaatattag attttttgaa atcagatggt tttgccaatc gcaattttat 2820gcagctgatc catgatgata gtttgacatt taaagaagac attcaaaaag cacaagtgtc 2880tggacaaggc gatagtttac atgaacatat tgcaaattta gctggtagcc ctgctattaa 2940aaaaggtatt ttacagactg taaaagttgt tgatgaattg gtcaaagtaa tggggcggca 3000taagccagaa aatatcgtta ttgaaatggc acgtgaaaat cagacaactc aaaagggcca 3060gaaaaattcg cgagagcgta tgaaacgaat cgaagaaggt atcaaagaat taggaagtca 3120gattcttaaa gagcatcctg ttgaaaatac tcaattgcaa aatgaaaagc tctatctcta 3180ttatctccaa aatggaagag acatgtatgt ggaccaagaa ttagatatta atcgtttaag 3240tgattatgat gtcgatgcca ttgttccaca aagtttcctt aaagacgatt caatagacaa 3300taaggtctta acgcgttctg ataaaaatcg tggtaaatcg gataacgttc caagtgaaga 3360agtagtcaaa aagatgaaaa actattggag acaacttcta aacgccaagt taatcactca 3420acgtaagttt gataatttaa cgaaagctga acgtggaggt ttgagtgaac ttgataaagc 3480tggttttatc aaacgccaat tggttgaaac tcgccaaatc actaagcatg tggcacaaat 3540tttggatagt cgcatgaata ctaaatacga tgaaaatgat aaacttattc gagaggttaa 3600agtgattacc ttaaaatcta aattagtttc tgacttccga aaagatttcc aattctataa 3660agtacgtgag attaacaatt accatcatgc ccatgatgcg tatctaaatg ccgtcgttgg 3720aactgctttg attaagaaat atccaaaact tgaatcggag tttgtctatg gtgattataa 3780agtttatgat gttcgtaaaa tgattgctaa gtctgagcaa gaaataggca aagcaaccgc 3840aaaatatttc ttttactcta atatcatgaa cttcttcaaa acagaaatta cacttgcaaa 3900tggagagatt cgcaaacgcc ctctaatcga aactaatggg gaaactggag aaattgtctg 3960ggataaaggg cgagattttg ccacagtgcg caaagtattg tccatgcccc aagtcaatat 4020tgtcaagaaa acagaagtac agacaggcgg attctccaag gagtcaattt taccaaaaag 4080aaattcggac aagcttattg ctcgtaaaaa agactgggat ccaaaaaaat atggtggttt 4140tgatagtcca acggtagctt attcagtcct agtggttgct aaggtggaaa aagggaaatc 4200gaagaagtta aaatccgtta aagagttact agggatcaca attatggaaa gaagttcctt 4260tgaaaaaaat ccgattgact ttttagaagc taaaggatat aaggaagtta aaaaagactt 4320aatcattaaa ctacctaaat atagtctttt tgagttagaa aacggtcgta aacggatgct 4380ggctagtgcc ggagaattac aaaaaggaaa tgagctggct ctgccaagca aatatgtgaa 4440ttttttatat ttagctagtc attatgaaaa gttgaagggt agtccagaag ataacgaaca 4500aaaacaattg tttgtggagc agcataagca ttatttagat gagattattg agcaaatcag 4560tgaattttct aagcgtgtta ttttagcaga tgccaattta gataaagttc ttagtgcata 4620taacaaacat agagacaaac caatacgtga acaagcagaa aatattattc atttatttac 4680gttgacgaat cttggagctc ccgctgcttt taaatatttt gatacaacaa ttgatcgtaa 4740acgatatacg tctacaaaag aagttttaga tgccactctt atccatcaat ccatcactgg 4800tctttatgaa acacgcattg atttgagtca gctaggaggt gactaactcg agccggctta 4860tcggtcagtt tcacctgatt tacgtaaaaa cccgcttcgg cgggtttttg cttttggagg 4920ggcagaaaga tgaatgactg tccacgacgc tatacccaaa agaaatccct atcagtgata 4980gagattgaca tccctatcag tgatagagat actgagcaca tcagcaggac gcactgacca 5040agaggagaaa ggatctatgg atttgtcaca gctaacacca cgtcgtccct atctgctgcg 5100tgcattctat gagtggttgc tggataacca gctcacgccg cacctggtgg tggatgtgac 5160gctccctggc gtgcaggttc ctatggaata tgcgcgtgac gggcaaatcg tactcaacat 5220tgcgccgcgt gctgtcggca atctggaact ggcgaatgat gaggtgcgct ttaacgcgcg 5280ctttggtggc attccgcgtc aggtttctgt gccgctggct gccgtgctgg ctatctacgc 5340ccgtgaaaat ggcgcaggca cgatgtttga gcctgaagct gcctacgatg aagataccag 5400catcatgaat gatgaagagg catcggcaga caacgaaacc gttatgtcgg ttattgatgg 5460cgacaagcca gatcacgatg atgacactca tcctgacgat gaacctccgc agccaccacg 5520cggtggtcga ccggcattac gcgttgtgaa gtaactcgag taaggatctc caggcatcaa 5580ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg tttgtcggtg 5640aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg cgtttatacc 5700tagggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc 5760gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta 5820acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa 5880gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata 5940ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac 6000cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg 6060ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct 6120gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac 6180tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa 6240ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc 6300aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt 6360tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag 6420ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc 6480cccatacgat ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg 6540caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac 6600aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg 6660ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg 6720aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac 6780gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca 6840gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt 6900ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg 6960gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg 7020gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg 7080agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt 7140ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg 7200agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt 7260ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa 7320ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac 7380gtgccgatca acgtctcatt ttcgccagat atc 74134974DNAArtificial SequenceSynthetic Construct Delta-cas3::ugpBp-sspB-proB 4caagacatgt gtatatcact gtaattcgat atttatgagc agcatcgaaa aatagcccgc 60tgatatcatc gataatacta aaaaaacagg gaggctatta ccaggcatca aataaaacga 120aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 180tactagagtc acactggctc accttcgggt gggcctttct gcgtttatat ctttctgaca 240ccttactatc ttacaaatgt aacaaaaaag ttatttttct gtaattcgag catgtcatgt 300taccccgcga gcataaaacg cgtgtgtagg aggataatct atggatttgt cacagctaac 360accacgtcgt ccctatctgc tgcgtgcatt ctatgagtgg ttgctggata accagctcac 420gccgcacctg gtggtggatg tgacgctccc tggcgtgcag gttcctatgg aatatgcgcg 480tgacgggcaa atcgtactca acattgcgcc gcgtgctgtc ggcaatctgg aactggcgaa 540tgatgaggtg cgctttaacg cgcgctttgg tggcattccg cgtcaggttt ctgtgccgct 600ggctgccgtg ctggctatct acgcccgtga aaatggcgca ggcacgatgt ttgagcctga 660agctgcctac gatgaagata ccagcatcat gaatgatgaa gaggcatcgg cagacaacga 720aaccgttatg tcggttattg atggcgacaa gccagatcac gatgatgaca ctcatcctga 780cgatgaacct ccgcagccac cacgcggtgg tcgaccggca ttacgcgttg tgaagtaatt 840gacggctagc tcagtcctag gtacagtgct agccatatga aggagaacaa atgaatttgc 900ttattgataa ctggatccct gtacgcccgc gaaacggggg gaaagtccaa atcataaatc 960tgcaatcgct atac 97451500DNAArtificial SequenceLinear DNA Construct Delta-ptsG::proC-glk 5ggctgtgttg aaaggtgttg ccgttgaaga actggcgcag gtaaccaccg ataacttcgc 60ccgtctgttt cacatcgacg cttcccgcct tcaatccatc cgttgaatga gtttttttaa 120agctcgtaat taatacttcg ctcttcatgc cgccgcaaac cccgcccctg acagggcggg 180gtttcgccgc acgtctccat cgcttgccca agttgtgaag cacagctaac accacgtcgt 240ccctatctgc tgccctaggt ctatgagtgg ttgctggata actttacggg catgcataag 300gctcgtatga tatattcagg gagaccacaa cggtttccct ctacaaataa ttttgtttaa 360ctttcgtaga agagcacttc cacacttctg gaaaaaggag atataccatg accaagtatg 420ccctggtcgg tgacgtaggt ggtaccaatg cacgtctcgc tctctgtgat atcgcaagcg 480gggaaatttc tcaggccaaa acatattccg ggttggatta ccccagctta gaagccgtga 540ttcgtgtcta tttagaagaa cataaagtag aagtcaaaga cggttgtatt gctattgcgt 600gccccatcac tggggattgg gtagcaatga ccaaccatac ctgggcgttt tctattgccg 660agatgaaaaa aaatctgggt ttctcacacc tggagatcat caacgatttt accgcggtga 720gcatggcgat cccaatgtta aaaaaggaac acttaattca

gttcggcggg gcagaacctg 780tggagggcaa gccgatcgcg gtttatggtg caggcacagg cttaggtgtc gcgcacttgg 840tacatgttga caagcgctgg gtgagtttgc cgggcgaagg cggccacgtg gattttgccc 900ccaattctga agaggaggcg attattctgg aaatcttgcg tgcagaaatc ggtcatgtgt 960ctgccgaacg tgtgctgagt ggtccaggtc tggtgaatct gtaccgcgct attgtcaaag 1020cggataaccg cctgccagaa aaccttaaac cgaaagatat caccgaacgt gccttggccg 1080actcctgtac cgattgccgc cgcgcactta gtctgttttg cgttatcatg ggtcgttttg 1140gcggcaacct cgcgctgaac ctggggacct ttggcggtgt ttttattgcg ggaggtattg 1200ttccacgctt tttagaattt ttcaaagcca gtggctttcg cgcggccttc gaagacaagg 1260gacgttttaa agaatacgta catgatatcc cagtctattt aattgttcac gataacccag 1320gactgttagg ctctggtgcc catctgcgtc agacattggg ccatattctg taatccgtaa 1380gacgttgggg agactaaggc agccagatgg ctgccttttt tacaggtgtt attcagaatt 1440gatacgtgcc ggtaatgctg aaattacgcg gtgtgccgta gacgatagaa ccttccacgt 150061332DNAArtificial SequenceLinear DNA Construct proC-galP 6tatgtcgcga aaaacatcgt tgctgctggc ctggccgatc gttgtgaaat tcaggtttcc 60tacgcaatcg gcgtggctga accgacctcc atcatggtag aaactttcgg tactgagaaa 120gtgccttctg aacaactgac cctgctggta cgtgagttct tcgacctgcg cccatacggt 180ctgattcaga tgctggatct gctgcacccg atctacaaag aaaccgcagc atacggtcac 240tttggtcgtg aacatttccc gtgggaaaaa accgacaaag cgcagctgct gcgcgatgct 300gccggtctga agtaatcttt cttcacctgc gttcaaagga cttcgctctt catgccgccg 360caaaccccgc ccctgacagg gcggggtttc gccgcacgtc tccatcgctt gcccaagttg 420tgaagcacag ctaacaccac gtcgtcccta tctgctgccc taggtctatg agtggttgct 480ggataacttt acgggcatgc ataaggctcg tatgatatat tcagggagac cacaacggtt 540tccctctaca aataattttg tttaactttc gtagaagagc acttccacac ttctggaaaa 600aggagatata ccatgccaga tgccaaaaag caaggccgtt ctaacaaggc aatgacattc 660ttcgtgtgct tccttgcggc gcttgccggc ctcttgttcg gcttggacat cggcgtcatt 720gccggtgctt taccatttat cgctgacgaa ttccagatca cctcgcacac gcaagaatgg 780gtcgtaagct ccatgatgtt cggtgcggca gtcggtgcgg tgggcagcgg ctggctctcc 840tttaaactcg ggcgcaaaaa gagcctgatg atcggcgcaa ttttgtttgt tgccggttcg 900ctgttctctg cggctgcgcc aaacgttgaa gtactgattc tttcccgcgt tctactgggg 960ctggcggtgg gtgtggcctc ttataccgca ccgctgtacc tctctgaaat tgcgccggaa 1020aaaattcgtg gcagtatgat ctcgatgtat cagttgatga tcactatcgg gatcctcggt 1080gcttatcttt ctgataccgc cttcagctac accggtgcat ggcgctggat gctgggtgtg 1140attatcatcc cggcaatttt gctgctgatt ggtgtcttct tcctgccaga cagcccacgt 1200tggtttgccg ccaaacgccg ttttgttgat gccgaacgcg tgctgctacg cctgcgtgac 1260accagcgcgg aagcgaaacg cgaactggat gaaatccgtg aaagtttgca ggttaaacag 1320agtggctggg cg 13327970DNAArtificial SequencefabI-DAS+4gentR 7ctattgaaga tgtgggtaac tctgcggcat tcctgtgctc cgatctctct gccggtatct 60ccggtgaagt ggtccacgtt gacggcggtt tcagcattgc tgcaatgaac gaactcgaac 120tgaaagcggc caacgatgaa aactattctg aaaactatgc ggatgcgtct taataggaag 180ttcctattct ctagaaagta taggaacttc cgaatccatg tgggagttta ttcttgacac 240agatatttat gatataataa ctgagtaagc ttaacataag gaggaaaaac atatgttacg 300cagcagcaac gatgttacgc agcagggcag tcgccctaaa acaaagttag gtggctcaag 360tatgggcatc attcgcacat gtaggctcgg ccctgaccaa gtcaaatcca tgcgggctgc 420tcttgatctt ttcggtcgtg agttcggaga cgtagccacc tactcccaac atcagccgga 480ctccgattac ctcgggaact tgctccgtag taagacattc atcgcgcttg ctgccttcga 540ccaagaagcg gttgttggcg ctctcgcggc ttacgttctg cccaagtttg agcagccgcg 600tagtgagatc tatatctatg atctcgcagt ctccggcgag caccggaggc agggcattgc 660caccgcgctc atcaatctcc tcaagcatga ggccaacgcg cttggtgctt atgtgatcta 720cgtgcaagca gattacggtg acgatcccgc agtggctctc tatacaaagt tgggcatacg 780ggaagaagtg atgcactttg atatcgaccc aagtaccgcc acctaagaag ttcctattct 840ctagaaagta taggaacttc cgttctgttg gtaaagatgg gcggcgttct gccgcccgtt 900atctctgtta tacctttctg atatttgtta tcgccgatcc gtctttctcc ccttcccgcc 960ttgcgtcagg 97081026DNAArtificial Sequencelpd-DAS+4gentR 8ggtactaacg gcggcgagct gctgggtgaa atcggcctgg caatcgaaat gggttgtgat 60gctgaagaca tcgcactgac catccacgcg cacccgactc tgcacgagtc tgtgggcctg 120gcggcagaag tgttcgaagg tagcattacc gacctgccga acccgaaagc gaagaagaag 180gcggccaacg atgaaaacta ttctgaaaac tatgcggatg cgtcttaata gcgaatccat 240gtgggagttt attcttgaca cagatattta tgatataata actgagtaag cttaacataa 300ggaggaaaaa catatgttac gcagcagcaa cgatgttacg cagcagggca gtcgccctaa 360aacaaagtta ggtggctcaa gtatgggcat cattcgcaca tgtaggctcg gccctgacca 420agtcaaatcc atgcgggctg ctcttgatct tttcggtcgt gagttcggag acgtagccac 480ctactcccaa catcagccgg actccgatta cctcgggaac ttgctccgta gtaagacatt 540catcgcgctt gctgccttcg accaagaagc ggttgttggc gctctcgcgg cttacgttct 600gcccaagttt gagcagccgc gtagtgagat ctatatctat gatctcgcag tctccggcga 660gcaccggagg cagggcattg ccaccgcgct catcaatctc ctcaagcatg aggccaacgc 720gcttggtgct tatgtgatct acgtgcaagc agattacggt gacgatcccg cagtggctct 780ctatacaaag ttgggcatac gggaagaagt gatgcacttt gatatcgacc caagtaccgc 840cacctaattt ttcgtttgcc ggaacatccg gcaattaaaa aagcggctaa ccacgccgct 900ttttttacgt ctgcaattta cctttccagt cttcttgctc cacgttcaga gagacgttcg 960catactgctg accgttgctc gttattcagc ctgacagtat ggttactgtc gtttagacgt 1020tgtggg 10269869DNAArtificial SequencegltA-DAS+4zeoR 9gtattccgtc ttccatgttc accgtcattt tcgcaatggc acgtaccgtt ggctggatcg 60cccactggag cgaaatgcac agtgacggta tgaagattgc ccgtccgcgt cagctgtata 120caggatatga aaaacgcgac tttaaaagcg atatcaagcg tgcggccaac gatgaaaact 180attctgaaaa ctatgcggat gcgtcttaat agttgacaat taatcatcgg catagtatat 240cggcatagta taatacgact cactatagga gggccatcat ggccaagttg accagtgccg 300ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga gttctggacc gaccggctcg 360ggttctcccg ggacttcgtg gaggacgact tcgccggtgt ggtccgggac gacgtgaccc 420tgttcatcag cgcggtccag gaccaggtgg tgccggacaa caccctggcc tgggtgtggg 480tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt cgtgtccacg aacttccggg 540acgcctccgg gccggccatg accgagatcg gcgagcagcc gtgggggcgg gagttcgccc 600tgcgcgaccc ggccggcaac tgcgtgcact ttgtggcaga ggagcaggac tgaggataag 660taatggttga ttgctaagtt gtaaatattt taacccgccg ttcatatggc gggttgattt 720ttatatgcct aaacacaaaa aattgtaaaa ataaaatcca ttaacagacc tatatagata 780tttaaaaaga atagaacagc tcaaattatc agcaacccaa tactttcaat taaaaacttc 840atggtagtcg catttataac cctatgaaa 86910852DNAArtificial SequenceudhA-DAS+4bsdR 10tctgggtatt cactgctttg gcgagcgcgc tgccgaaatt attcatatcg gtcaggcgat 60tatggaacag aaaggtggcg gcaacactat tgagtacttc gtcaacacca cctttaacta 120cccgacgatg gcggaagcct atcgggtagc tgcgttaaac ggtttaaacc gcctgtttgc 180ggccaacgat gaaaactatt ctgaaaacta tgcggatgcg tcttaatagt tgacaattaa 240tcatcggcat agtatatcgg catagtataa tacgactcac tataggaggg ccatcatgaa 300gaccttcaac atctctcagc aggatctgga gctggtggag gtcgccactg agaagatcac 360catgctctat gaggacaaca agcaccatgt cggggcggcc atcaggacca agactgggga 420gatcatctct gctgtccaca ttgaggccta cattggcagg gtcactgtct gtgctgaagc 480cattgccatt gggtctgctg tgagcaacgg gcagaaggac tttgacacca ttgtggctgt 540caggcacccc tactctgatg aggtggacag atccatcagg gtggtcagcc cctgtggcat 600gtgcagagag ctcatctctg actatgctcc tgactgcttt gtgctcattg agatgaatgg 660caagctggtc aaaaccacca ttgaggaact catccccctc aagtacacca ggaactaaag 720taaaacttta tcgaaatggc catccattct tgcgcggatg gcctctgcca gctgctcata 780gcggctgcgc agcggtgagc caggacgata aaccaggcca atagtgcggc gtggttccgg 840cttaatgcac gg 85211898DNAArtificial Sequencezwf-DAS+4bsdR 11gaagtggaag aagcctggaa atgggtagac tccattactg aggcgtgggc gatggacaat 60gatgcgccga aaccgtatca ggccggaacc tggggacccg ttgcctcggt ggcgatgatt 120acccgtgatg gtcgttcctg gaatgagttt gaggcggcca acgatgaaaa ctattctgaa 180aactatgcgg atgcgtctta atagttgaca attaatcatc ggcatagtat atcggcatag 240tataatacga ctcactatag gagggccatc atgaagacct tcaacatctc tcagcaggat 300ctggagctgg tggaggtcgc cactgagaag atcaccatgc tctatgagga caacaagcac 360catgtcgggg cggccatcag gaccaagact ggggagatca tctctgctgt ccacattgag 420gcctacattg gcagggtcac tgtctgtgct gaagccattg ccattgggtc tgctgtgagc 480aacgggcaga aggactttga caccattgtg gctgtcaggc acccctactc tgatgaggtg 540gacagatcca tcagggtggt cagcccctgt ggcatgtgca gagagctcat ctctgactat 600gctcctgact gctttgtgct cattgagatg aatggcaagc tggtcaaaac caccattgag 660gaactcatcc ccctcaagta caccaggaac taaagtaata tctgcgctta tcctttatgg 720ttattttacc ggtaacatga tcttgcgcag attgtagaac aatttttaca ctttcaggcc 780tcgtgcggat tcacccacga ggcttttttt attacactga ctgaaacgtt tttgccctat 840gagctccggt tacaggcgtt tcagtcataa atcctctgaa tgaaacgcgt tgtgaatc 898123037DNAArtificial SequencePlasmid pSMART-waaHp-GFPuv 12tgcccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct 60gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt 120tctgcgttta tacacagcta acaccacgtc gtccctatct gctgccctag gtctatgagt 180ggttgctgga taacgtgcgt aattgtgctg atctcttata tagctgctct cattatctct 240ctaccctgaa gtgactctct cacctgtaaa aataatatct cacaggctta atagtttctt 300aatacaaagc ctgtaaaacg tcaggataac ttctatattc agggagacca caacggtttc 360cctctacaaa taattttgtt taactttcgt gtgtaggagg ataatctatg gctagcaaag 420gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt gatgttaatg 480ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc tacatacgga aagcttaccc 540ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt gtcactactt 600tctcttatgg tgttcaatgc ttttcccgtt atccggatca tatgaaacgg catgactttt 660tcaagagtgc catgcccgaa ggttatgtac aggaacgcac tatatctttc aaagatgacg 720ggaactacaa gacgcgtgct gaagtcaagt ttgaaggtga tacccttgtt aatcgtatcg 780agttaaaagg tattgatttt aaagaagatg gaaacattct cggacacaaa ctcgagtaca 840actataactc acacaatgta tacatcacgg cagacaaaca aaagaatgga atcaaagcta 900acttcaaaat tcgccacaac attgaagatg gatccgttca actagcagac cattatcaac 960aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac ctgtcgacac 1020aatctgccct ttcgaaagat cccaacgaaa agcgtgacca catggtcctt cttgagtttg 1080taactgctgc tgggattaca catggcatgg atgagctcta caaataatga ggatccccgg 1140cttatcggtc agtttcacct gatttacgta aaaacccgct tcggcgggtt tttgcttttg 1200gaggggcaga aagatgaatg actgtccacg acgctatacc caaaagaaag acgaattctc 1260tagatatcgc tcaatactga ccatttaaat catacctgac ctccatagca gaaagtcaaa 1320agcctccgac cggaggcttt tgacttgatc ggcacgtaag aggttccaac tttcaccata 1380atgaaataag atcactaccg ggcgtatttt ttgagttatc gagattttca ggagctaagg 1440aagctaaaat gagccatatt caacgggaaa cgtcttgctc gaggccgcga ttaaattcca 1500acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg caatcaggtg 1560cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg aaacatggca 1620aaggtagcgt tgccaatgat gttacagatg agatggtcag gctaaactgg ctgacggaat 1680ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca tggttactca 1740ccactgcgat cccagggaaa acagcattcc aggtattaga agaatatcct gattcaggtg 1800aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt cctgtttgta 1860attgtccttt taacggcgat cgcgtatttc gtctcgctca ggcgcaatca cgaatgaata 1920acggtttggt tggtgcgagt gattttgatg acgagcgtaa tggctggcct gttgaacaag 1980tctggaaaga aatgcataag cttttgccat tctcaccgga ttcagtcgtc actcatggtg 2040atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt attgatgttg 2100gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac tgcctcggtg 2160agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat aatcctgata 2220tgaataaatt gcagtttcac ttgatgctcg atgagttttt ctaatgaggg cccaaatgta 2280atcacctggc tcaccttcgg gtgggccttt ctgcgttgct ggcgtttttc cataggctcc 2340gcccccctga cgagcatcac aaaaatcgat gctcaagtca gaggtggcga aacccgacag 2400gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 2460ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 2520atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 2580tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 2640ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 2700gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 2760ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttacctcg gaaaaagagt 2820tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 2880gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgattt tctaccgaag 2940aaaggcccac ccgtgaaggt gagccagtga gttgattgca gtccagttac gctggagtct 3000gaggctcgtc ctgaatgata tcaagcttga attcgtt 3037132780DNAArtificial SequencepCASCADE-Control Plasmid 13gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgaaaa aaaaaccccg cccctgacag ggcggggttt tttttcctag ggatatattc 1080cgcttcctcg ctcactgact cgctacgctc ggtcgttcga ctgcggcgag cggaaatggc 1140ttacgaacgg ggcggagatt tcctggaaga tgccaggaag atacttaaca gggaagtgag 1200agggccgcgg caaagccgtt tttccatagg ctccgccccc ctgacaagca tcacgaaatc 1260tgacgctcaa atcagtggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 1320cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg 1380ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg 1440ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg 1500taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac 1560tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa 1620ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag 1680ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga 1740ttacgcgcag accaaaacga tctcaagaag atcatcttat taatcagata aaatatttct 1800agatttcagt gcaatttatc tcttcaaatg tagcacctga agtcagcccc atacgatata 1860agttgttact agtgcttgga ttctcaccaa taaaaaacgc ccggcggcaa ccgagcgttc 1920tgaacaaatc cagatggagt tctgaggtca ttactggatc tatcaacagg agtccaagcg 1980agctcgatat caaattacgc cccgccctgc cactcatcgc agtactgttg taattcatta 2040agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat cgccagcggc 2100atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg ggcgaagaag 2160ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg attggctgag 2220acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc accgtaacac 2280gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta ttcactccag 2340agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg aacactatcc 2400catatcacca gctcaccgtc tttcattgcc atacgaaatt ccggatgagc attcatcagg 2460cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt tacggtcttt 2520aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc aactgactga 2580aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt atatccagtg 2640atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc aaaaaatacg 2700cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg ccgatcaacg 2760tctcattttc gccagatatc 2780142843DNAArtificial SequencepCASCADE-gltA2 Plasmid 14gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgtatt gaccaattca ttcgggacag ttattagttc gagttccccg cgccagcggg 1080gataaaccga aaaaaaaacc ccgcccctga cagggcgggg ttttttttcc tagggatata 1140ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat 1200ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt 1260gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa 1320atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1380ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt 1440ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt 1500tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc 1560cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc 1620cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg 1680aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg 1740tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag 1800agattacgcg cagaccaaaa cgatctcaag

aagatcatct tattaatcag ataaaatatt 1860tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat 1920ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg 1980ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa 2040gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca 2100ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc 2160ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag 2220aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 2280gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 2340cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 2400cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 2460tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc 2520aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc 2580tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac 2640tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca 2700gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat 2760acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca 2820acgtctcatt ttcgccagat atc 2843152841DNAArtificial SequencepCASCADE-fabI Plasmid 15gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgttga ttataataac cgtttatctg ttcgtatcga gttccccgcg ccagcgggga 1080taaaccgaaa aaaaaacccc gcccctgaca gggcggggtt ttttttccta gggatatatt 1140ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg 1200cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga 1260gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat 1320ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1380ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 1440gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 1500gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 1560gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 1620ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 1680aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 1740gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 1800attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 1860tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 1920aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt 1980ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc 2040gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 2100aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 2160catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa 2220gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 2280gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 2340cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 2400gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 2460ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag 2520gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 2580taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 2640aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 2700gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 2760gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 2820gtctcatttt cgccagatat c 2841162841DNAArtificial SequencepCASCADE-udhA Plasmid 16gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgttac cattctgttg cttttatgta taagaatcga gttccccgcg ccagcgggga 1080taaaccgaaa aaaaaacccc gcccctgaca gggcggggtt ttttttccta gggatatatt 1140ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg 1200cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga 1260gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat 1320ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1380ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 1440gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 1500gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 1560gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 1620ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 1680aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 1740gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 1800attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 1860tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 1920aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt 1980ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc 2040gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 2100aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 2160catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa 2220gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 2280gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 2340cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 2400gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 2460ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag 2520gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 2580taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 2640aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 2700gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 2760gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 2820gtctcatttt cgccagatat c 2841172841DNAArtificial SequencepCASCADE-zwf Plasmid 17gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgctcg taaaagcagt acagtgcacc gtaagatcga gttccccgcg ccagcgggga 1080taaaccgaaa aaaaaacccc gcccctgaca gggcggggtt ttttttccta gggatatatt 1140ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg 1200cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga 1260gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat 1320ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1380ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 1440gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 1500gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 1560gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 1620ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 1680aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 1740gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 1800attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 1860tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 1920aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt 1980ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc 2040gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 2100aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 2160catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa 2220gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 2280gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 2340cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 2400gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 2460ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag 2520gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 2580taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 2640aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 2700gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 2760gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 2820gtctcatttt cgccagatat c 2841182842DNAArtificial SequencepCASCADE-gltA1 Plasmid 18gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgaaaa gcatataatg cgtaaaagtt atgaagttcg agttccccgc gccagcgggg 1080ataaaccgaa aaaaaaaccc cgcccctgac agggcggggt tttttttcct agggatatat 1140tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg 1200gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg 1260agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa 1320tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1380cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc 1440cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt 1500cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc 1560ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc 1620actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga 1680aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt 1740agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga 1800gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt 1860ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata 1920taagttgtta ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt 1980tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag 2040cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat 2100taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg 2160gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga 2220agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg 2280agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac 2340acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc 2400agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat 2460cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca 2520ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct 2580ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact 2640gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag 2700tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata 2760cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa 2820cgtctcattt tcgccagata tc 2842192903DNAArtificial SequencepCASCADE-gltA2-udhA Plasmid 19gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaataaat gtaggctgct ctacacctag cttctgggcg 540agtttacggg ttgttaaacc ttcgattccg acctcattaa gcagctctaa tgcgctgtta 600atcactttac ttttatctaa tctagacatc atccaggcat caaataaaac gaaaggctca 660gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg gtgaacgctc tctactagag 720tcacactggc tcaccttcgg gtgggccttt ctgcgtttat acacagctaa caccacgtcg 780tccctatctg ctgccctagg tctatgagtg gttgctggat aactctttct gacaccttac 840tatcttacaa atgtaacaaa aaagttattt ttctgtaatt cgagcatgtc atgttacccc 900gcgagcataa aacgcgtata ttcagggaga ccacaacggt ttccctctac aaataatttt 960gtttaacttt gaattcaaaa gatctggtac cacctcgagt tccccgcgcc agcggggata 1020aaccgtattg accaattcat tcgggacagt tattagttcg agttccccgc gccagcgggg 1080ataaaccgtt accattctgt tgcttttatg tataagaatc gagttccccg cgccagcggg 1140gataaaccga aaaaaaaacc ccgcccctga cagggcgggg ttttttttcc tagggatata 1200ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat 1260ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt 1320gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa 1380atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1440ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt 1500ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt 1560tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc 1620cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc 1680cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg 1740aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg 1800tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag 1860agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt 1920tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat 1980ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg 2040ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa 2100gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca 2160ttaagcattc tgccgacatg gaagccatca

caaacggcat gatgaacctg aatcgccagc 2220ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag 2280aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 2340gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 2400cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 2460cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 2520tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc 2580aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc 2640tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac 2700tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca 2760gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat 2820acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca 2880acgtctcatt ttcgccagat atc 2903202902DNAArtificial SequencepCASCADE-fabI-udhA Plasmid 20gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgttga ttataataac cgtttatctg ttcgtatcga gttccccgcg ccagcgggga 1080taaaccgtta ccattctgtt gcttttatgt ataagaatcg agttccccgc gccagcgggg 1140ataaaccgaa aaaaaaaccc cgcccctgac agggcggggt tttttttcct agggatatat 1200tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg 1260gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg 1320agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa 1380tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1440cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc 1500cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt 1560cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc 1620ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc 1680actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga 1740aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt 1800agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga 1860gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt 1920ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata 1980taagttgtta ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt 2040tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag 2100cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat 2160taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg 2220gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga 2280agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg 2340agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac 2400acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc 2460agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat 2520cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca 2580ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct 2640ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact 2700gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag 2760tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata 2820cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa 2880cgtctcattt tcgccagata tc 2902212903DNAArtificial SequencepCASCADE-fabI-gltA1 Plasmid 21gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgttga ttataataac cgtttatctg ttcgtatcga gttccccgcg ccagcgggga 1080taaaccgaaa agcatataat gcgtaaaagt tatgaagttc gagttccccg cgccagcggg 1140gataaaccga aaaaaaaacc ccgcccctga cagggcgggg ttttttttcc tagggatata 1200ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat 1260ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt 1320gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa 1380atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1440ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt 1500ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt 1560tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc 1620cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc 1680cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg 1740aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg 1800tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag 1860agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt 1920tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat 1980ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg 2040ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa 2100gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca 2160ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc 2220ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag 2280aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 2340gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 2400cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 2460cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 2520tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc 2580aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc 2640tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac 2700tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca 2760gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat 2820acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca 2880acgtctcatt ttcgccagat atc 2903222904DNAArtificial SequencepCASCADE-fabI-gltA2 Plasmid 22gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgttga ttataataac cgtttatctg ttcgtatcga gttccccgcg ccagcgggga 1080taaaccgtat tgaccaattc attcgggaca gttattagtt cgagttcccc gcgccagcgg 1140ggataaaccg aaaaaaaaac cccgcccctg acagggcggg gttttttttc ctagggatat 1200attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa 1260tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag 1320tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga 1380aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1440tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat 1500tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag 1560ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 1620ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 1680ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 1740gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 1800gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 1860gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat 1920ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga 1980tataagttgt tactagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc 2040gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca 2100agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc 2160attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag 2220cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa 2280gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc 2340tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta 2400acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact 2460ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact 2520atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat 2580caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt 2640ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga 2700ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc 2760agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa 2820tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 2880aacgtctcat tttcgccaga tatc 2904232902DNAArtificial SequencepCASCADE-fabI-zwf Plasmid 23gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgttga ttataataac cgtttatctg ttcgtatcga gttccccgcg ccagcgggga 1080taaaccgctc gtaaaagcag tacagtgcac cgtaagatcg agttccccgc gccagcgggg 1140ataaaccgaa aaaaaaaccc cgcccctgac agggcggggt tttttttcct agggatatat 1200tccgcttcct cgctcactga ctcgctacgc tcggtcgttc gactgcggcg agcggaaatg 1260gcttacgaac ggggcggaga tttcctggaa gatgccagga agatacttaa cagggaagtg 1320agagggccgc ggcaaagccg tttttccata ggctccgccc ccctgacaag catcacgaaa 1380tctgacgctc aaatcagtgg tggcgaaacc cgacaggact ataaagatac caggcgtttc 1440cccctggcgg ctccctcgtg cgctctcctg ttcctgcctt tcggtttacc ggtgtcattc 1500cgctgttatg gccgcgtttg tctcattcca cgcctgacac tcagttccgg gtaggcagtt 1560cgctccaagc tggactgtat gcacgaaccc cccgttcagt ccgaccgctg cgccttatcc 1620ggtaactatc gtcttgagtc caacccggaa agacatgcaa aagcaccact ggcagcagcc 1680actggtaatt gatttagagg agttagtctt gaagtcatgc gccggttaag gctaaactga 1740aaggacaagt tttggtgact gcgctcctcc aagccagtta cctcggttca aagagttggt 1800agctcagaga accttcgaaa aaccgccctg caaggcggtt ttttcgtttt cagagcaaga 1860gattacgcgc agaccaaaac gatctcaaga agatcatctt attaatcaga taaaatattt 1920ctagatttca gtgcaattta tctcttcaaa tgtagcacct gaagtcagcc ccatacgata 1980taagttgtta ctagtgcttg gattctcacc aataaaaaac gcccggcggc aaccgagcgt 2040tctgaacaaa tccagatgga gttctgaggt cattactgga tctatcaaca ggagtccaag 2100cgagctcgat atcaaattac gccccgccct gccactcatc gcagtactgt tgtaattcat 2160taagcattct gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg 2220gcatcagcac cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga 2280agttgtccat attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg 2340agacgaaaaa catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac 2400acgccacatc ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc 2460agagcgatga aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat 2520cccatatcac cagctcaccg tctttcattg ccatacgaaa ttccggatga gcattcatca 2580ggcgggcaag aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct 2640ttaaaaaggc cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact 2700gaaatgcctc aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag 2760tgattttttt ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata 2820cgcccggtag tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa 2880cgtctcattt tcgccagata tc 2902242903DNAArtificial SequencepCASCADE-gltA1-udhA Plasmid 24gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgaaaa gcatataatg cgtaaaagtt atgaagttcg agttccccgc gccagcgggg 1080ataaaccgtt accattctgt tgcttttatg tataagaatc gagttccccg cgccagcggg 1140gataaaccga aaaaaaaacc ccgcccctga cagggcgggg ttttttttcc tagggatata 1200ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat 1260ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt 1320gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa 1380atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1440ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt 1500ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt 1560tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc 1620cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc 1680cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg 1740aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg 1800tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag 1860agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt 1920tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat 1980ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg 2040ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa 2100gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca 2160ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc 2220ggcatcagca ccttgtcgcc ttgcgtataa

tatttgccca tggtgaaaac gggggcgaag 2280aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 2340gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 2400cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 2460cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 2520tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc 2580aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc 2640tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac 2700tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca 2760gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat 2820acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca 2880acgtctcatt ttcgccagat atc 2903252904DNAArtificial SequencepCASCADE-gltA2-udhA Plasmid 25gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgtatt gaccaattca ttcgggacag ttattagttc gagttccccg cgccagcggg 1080gataaaccgt taccattctg ttgcttttat gtataagaat cgagttcccc gcgccagcgg 1140ggataaaccg aaaaaaaaac cccgcccctg acagggcggg gttttttttc ctagggatat 1200attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa 1260tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag 1320tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga 1380aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1440tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat 1500tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag 1560ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 1620ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 1680ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 1740gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 1800gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 1860gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat 1920ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga 1980tataagttgt tactagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc 2040gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca 2100agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc 2160attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag 2220cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa 2280gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc 2340tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta 2400acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact 2460ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact 2520atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat 2580caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt 2640ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga 2700ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc 2760agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa 2820tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 2880aacgtctcat tttcgccaga tatc 2904262903DNAArtificial SequencepCASCADE-gltA1-zwf Plasmid 26gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgaaaa gcatataatg cgtaaaagtt atgaagttcg agttccccgc gccagcgggg 1080ataaaccgct cgtaaaagca gtacagtgca ccgtaagatc gagttccccg cgccagcggg 1140gataaaccga aaaaaaaacc ccgcccctga cagggcgggg ttttttttcc tagggatata 1200ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt cgactgcggc gagcggaaat 1260ggcttacgaa cggggcggag atttcctgga agatgccagg aagatactta acagggaagt 1320gagagggccg cggcaaagcc gtttttccat aggctccgcc cccctgacaa gcatcacgaa 1380atctgacgct caaatcagtg gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1440ccccctggcg gctccctcgt gcgctctcct gttcctgcct ttcggtttac cggtgtcatt 1500ccgctgttat ggccgcgttt gtctcattcc acgcctgaca ctcagttccg ggtaggcagt 1560tcgctccaag ctggactgta tgcacgaacc ccccgttcag tccgaccgct gcgccttatc 1620cggtaactat cgtcttgagt ccaacccgga aagacatgca aaagcaccac tggcagcagc 1680cactggtaat tgatttagag gagttagtct tgaagtcatg cgccggttaa ggctaaactg 1740aaaggacaag ttttggtgac tgcgctcctc caagccagtt acctcggttc aaagagttgg 1800tagctcagag aaccttcgaa aaaccgccct gcaaggcggt tttttcgttt tcagagcaag 1860agattacgcg cagaccaaaa cgatctcaag aagatcatct tattaatcag ataaaatatt 1920tctagatttc agtgcaattt atctcttcaa atgtagcacc tgaagtcagc cccatacgat 1980ataagttgtt actagtgctt ggattctcac caataaaaaa cgcccggcgg caaccgagcg 2040ttctgaacaa atccagatgg agttctgagg tcattactgg atctatcaac aggagtccaa 2100gcgagctcga tatcaaatta cgccccgccc tgccactcat cgcagtactg ttgtaattca 2160ttaagcattc tgccgacatg gaagccatca caaacggcat gatgaacctg aatcgccagc 2220ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca tggtgaaaac gggggcgaag 2280aagttgtcca tattggccac gtttaaatca aaactggtga aactcaccca gggattggct 2340gagacgaaaa acatattctc aataaaccct ttagggaaat aggccaggtt ttcaccgtaa 2400cacgccacat cttgcgaata tatgtgtaga aactgccgga aatcgtcgtg gtattcactc 2460cagagcgatg aaaacgtttc agtttgctca tggaaaacgg tgtaacaagg gtgaacacta 2520tcccatatca ccagctcacc gtctttcatt gccatacgaa attccggatg agcattcatc 2580aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt gcttattttt ctttacggtc 2640tttaaaaagg ccgtaatatc cagctgaacg gtctggttat aggtacattg agcaactgac 2700tgaaatgcct caaaatgttc tttacgatgc cattgggata tatcaacggt ggtatatcca 2760gtgatttttt tctccatttt agcttcctta gctcctgaaa atctcgataa ctcaaaaaat 2820acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca 2880acgtctcatt ttcgccagat atc 2903272904DNAArtificial SequencepCASCADE-gltA2-zwf Plasmid 27gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgtatt gaccaattca ttcgggacag ttattagttc gagttccccg cgccagcggg 1080gataaaccgc tcgtaaaagc agtacagtgc accgtaagat cgagttcccc gcgccagcgg 1140ggataaaccg aaaaaaaaac cccgcccctg acagggcggg gttttttttc ctagggatat 1200attccgcttc ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa 1260tggcttacga acggggcgga gatttcctgg aagatgccag gaagatactt aacagggaag 1320tgagagggcc gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga 1380aatctgacgc tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1440tccccctggc ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat 1500tccgctgtta tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag 1560ttcgctccaa gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat 1620ccggtaacta tcgtcttgag tccaacccgg aaagacatgc aaaagcacca ctggcagcag 1680ccactggtaa ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact 1740gaaaggacaa gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg 1800gtagctcaga gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa 1860gagattacgc gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat 1920ttctagattt cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga 1980tataagttgt tactagtgct tggattctca ccaataaaaa acgcccggcg gcaaccgagc 2040gttctgaaca aatccagatg gagttctgag gtcattactg gatctatcaa caggagtcca 2100agcgagctcg atatcaaatt acgccccgcc ctgccactca tcgcagtact gttgtaattc 2160attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag 2220cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa 2280gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc 2340tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta 2400acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact 2460ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact 2520atcccatatc accagctcac cgtctttcat tgccatacga aattccggat gagcattcat 2580caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt 2640ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga 2700ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc 2760agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa 2820tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc 2880aacgtctcat tttcgccaga tatc 2904283505DNAArtificial SequencepBT1-mCherry-DAS+4 Vector 28cgcaaaaaac cccgcttcgg cggggttttt tcgcacgtct ccatcgcttg cccaagttgt 60gaagcacagc taacaccacg tcgtccctat ctgctgccct aggtctatga gtggttgctg 120gataacttta cgggcatgca taaggctcgt ataatatatt cagggagacc acaacggttt 180ccctctacaa ataattttgt ttaactttga tcgcatggtt gctactagag aaagaggaga 240aatactagat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc 300gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg 360gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc 420ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg 480tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt 540gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc 600tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg 660gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg 720aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact 780acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct 840acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac 900agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgaactg tacaaggcgg 960ccaacgatga aaactattct gaaaactatg cggatgcgtc ttaataagga cgagcctcag 1020actccagcgt aactggactg aaaacaaact aaagcgccct tgtggcgctt tagttttgtt 1080ccgcggccac cggctggctc gcttcgctcg gcccgtggac aaccctgctg gacaagctga 1140tggacaggct gcgcctgccc acgagcttga ccacagggat tgcccaccgg ctacccagcc 1200ttcgaccaca tacccaccgg ctccaactgc gcggcctgcg gccttgcccc atcaattttt 1260ttaattttct ctggggaaaa gcctccggcc tgcggcctgc gcgcttcgct tgccggttgg 1320acaccaagtg gaaggcgggt caaggctcgc gcagcgaccg cgcagcggct tggccttgac 1380gcgcctggaa cgacccaagc ctatgcgagt gggggcagtc gaaggcgaag cccgcccgcc 1440tgccccccga gcctcacggc ggcgagtgcg ggggttccaa gggggcagcg ccaccttggg 1500caaggccgaa ggccgcgcag tcgatcaaca agccccggag gggccacttt ttgccggagg 1560gggagccgcg ccgaaggcgt gggggaaccc cgcaggggtg cccttctttg ggcaccaaag 1620aactagatat agggcgaaat gcgaaagact taaaaatcaa caacttaaaa aaggggggta 1680cgcaacagct cattgcggca ccccccgcaa tagctcattg cgtaggttaa agaaaatctg 1740taattgactg ccacttttac gcaacgcata attgttgtcg cgctgccgaa aagttgcagc 1800tgattgcgca tggtgccgca accgtgcggc accctaccgc atggagataa gcatggccac 1860gcagtccaga gaaatcggca ttcaagccaa gaacaagccc ggtcactggg tgcaaacgga 1920acgcaaagcg catgaggcgt gggccgggct tattgcgagg aaacccacgg cggcaatgct 1980gctgcatcac ctcgtggcgc agatgggcca ccagaacgcc gtggtggtca gccagaagac 2040actttccaag ctcatcggac gttctttgcg gacggtccaa tacgcagtca aggacttggt 2100ggccgagcgc tggatctccg tcgtgaagct caacggcccc ggcaccgtgt cggcctacgt 2160ggtcaatgac cgcgtggcgt ggggccagcc ccgcgaccag ttgcgcctgt cggtgttcag 2220tgccgccgtg gtggttgatc acgacgacca ggacgaatcg ctgttggggc atggcgacct 2280gcgccgcatc ccgaccctgt atccgggcga gcagcaacta ccgaccggcc ccggcgagga 2340gccgcccagc cagcccggca ttccgggcat ggaaccagac ctgccagcct tgaccgaaac 2400ggaggaatgg gaacggcgcg ggcagcagcg cctgccgatg cccgatgagc cgtgttttct 2460ggacgatggc gagccgttgg agccgccgac acgggtcacg ctgccgcgcc ggtagtacgt 2520aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt 2580atcgagattt tcaggagcta aggaagctaa aatgagtatt caacatttcc gtgtcgccct 2640tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa 2700agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa 2760cagcggtaag atccttgaga gtttacgccc cgaagaacgt tttccaatga tgagcacttt 2820taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg 2880tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca 2940tctcacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa 3000cactgcggcc aacttacttc tggcaacgat cggaggaccg aaggagctaa ccgctttttt 3060gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3120cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa 3180actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga 3240ggcggataaa gttgcaggat cacttctgcg ctcggccctc ccggctggct ggtttattgc 3300tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 3360tggtaagccc tcccgcatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 3420acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aatgaggatc 3480cccctcaagt caaaagcctc cggtc 3505292841DNAArtificial SequencepCASCADE-proD Plasmid 29gacgtcttaa gacccacttt cacatttaag ttgtttttct aatccgcata tgatcaattc 60aaggccgaat aagaaggctg gctctgcacc ttggtgatca aataattcga tagcttgtcg 120taataatggc ggcatactat cagtagtagg tgtttccctt tcttctttag cgacttgatg 180ctcttgatct tccaatacgc aacctaaagt aaaatgcccc acagcgctga gtgcatataa 240tgcattctct agtgaaaaac cttgttggca taaaaaggct aattgatttt cgagagtttc 300atactgtttt tctgtaggcc gtgtacctaa atgtactttt gctccatcgc gatgacttag 360taaagcacat ctaaaacttt tagcgttatt acgtaaaaaa tcttgccagc tttccccttc 420taaagggcaa aagtgagtat ggtgcctatc taacatctca atggctaagg cgtcgagcaa 480agcccgctta ttttttacat gccaatacaa tgtaggctgc tctacaccta gcttctgggc 540gagtttacgg gttgttaaac cttcgattcc gacctcatta agcagctcta atgcgctgtt 600aatcacttta cttttatcta atctagacat catccaggca tcaaataaaa cgaaaggctc 660agtcgaaaga ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctctactaga 720gtcacactgg ctcaccttcg ggtgggcctt tctgcgttta tacacagcta acaccacgtc 780gtccctatct gctgccctag gtctatgagt ggttgctgga taactctttc tgacacctta 840ctatcttaca aatgtaacaa aaaagttatt tttctgtaat tcgagcatgt catgttaccc 900cgcgagcata aaacgcgtat attcagggag accacaacgg tttccctcta caaataattt 960tgtttaactt tgaattcaaa agatctggta ccacctcgag ttccccgcgc cagcggggat 1020aaaccgagtg gttgctggat aactttacgg gcatgctcga gttccccgcg ccagcgggga 1080taaaccgaaa aaaaaacccc gcccctgaca gggcggggtt ttttttccta gggatatatt 1140ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg 1200cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga 1260gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat 1320ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 1380ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 1440gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 1500gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 1560gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 1620ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 1680aggacaagtt ttggtgactg cgctcctcca

agccagttac ctcggttcaa agagttggta 1740gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 1800attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 1860tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 1920aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt 1980ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc 2040gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 2100aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 2160catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa 2220gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 2280gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 2340cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 2400gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 2460ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag 2520gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 2580taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 2640aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 2700gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 2760gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 2820gtctcatttt cgccagatat c 2841304866DNAArtificial SequencepSMART-3HP1 Plasmid 30gtgcgtaatt gtgctgatct cttatatagc tgctctcatt atctctctac cctgaagtga 60ctctctcacc tgtaaaaata atatctcaca ggcttaatag tttcttaata caaagcctgt 120aaaacgtcag gataacttct tttctggaaa aaggagatat accatggcga cgacgggggc 180acgtagcgct agtgttggtt gggccgagag cctgatcggt ctgcatttgg gaaaagtggc 240cttaatcacc ggaggctcag ccggcatcgg cgggcagatc ggccgccttt tagcgctgtc 300tggcgcgcgt gttatgctgg ccgctcgcga ccgtcacaaa ctcgaacaaa tgcaggccat 360gattcaatcc gaactggcgg aagttggtta taccgatgtc gaagaccgcg tgcacatcgc 420gccggggtgt gacgtttcct ctgaagcgca gctggcagat ctggttgaac gcactctgtc 480agcattcggt accgtggatt atctgatcaa taacgcgggt attgcgggtg tcgaagagat 540ggttatcgac atgccggtgg aaggctggcg tcatacgtta tttgccaacc ttatctcaaa 600ttatagcctg atgcgcaaac tggcccctct gatgaagaaa caggggagtg gctatatctt 660gaacgtctcg tcgtactttg gtggcgaaaa agatgcggct atcccatacc caaatcgtgc 720cgattatgcg gtttcaaaag ccggtcaacg tgcaatggct gaagtgttcg cccgtttcct 780cggcccggag atccagatta acgctatcgc cccaggcccg gtggaaggtg accgcctccg 840cggcacgggc gaacgtcccg gcttgtttgc gcgccgcgcg cgtttgattt tagaaaataa 900gcgtttaaac gagctgcatg ctgcccttat tgcggctgcg cgtacagatg agcgctccat 960gcacgaactg gtggaattac tgctgccgaa tgatgtagcg gcactcgagc agaatcccgc 1020agccccaacg gcgttgcgcg aactcgcgcg ccgttttcgc agcgaaggcg acccggccgc 1080gtcaagctcc agtgctctgc tcaaccgcag cattgcggcg aagttactgg cccgcctgca 1140caacggcggc tatgttctgc cggcggatat cttcgccaac ctgccgaacc ctccagaccc 1200gttcttcacg cgcgcgcaga ttgatcgcga agcccgtaaa gtgcgcgacg ggattatggg 1260aatgctgtat ctgcagcgca tgcctaccga gtttgacgta gcaatggcta ccgtttacta 1320tctggcggat cgcaatgtga gtggagagac cttccatccg agtggggggc tgcgttacga 1380gcgcacacct accggcggtg agttatttgg cctgccgtct cccgagcgcc tggcggagtt 1440agttggaagc accgtatatt tgatcggtga acacttaacc gaacatctga acttgctcgc 1500acgtgcgtat cttgaacgtt acggtgcgcg tcaggttgtt atgatcgtgg aaacggagac 1560aggcgcggaa accatgcgcc gcttacttca cgaccatgtc gaagcaggtc gccttatgac 1620cattgtggcg ggtgaccaaa tcgaagccgc catcgaccag gcgattacgc gctacggccg 1680tcctggtccg gttgtgtgca cccccttccg cccccttccg accgtcccgt tagttggccg 1740caaggactcc gattggagca ccgtactgag tgaagccgaa tttgccgaac tgtgtgaaca 1800tcaactgaca catcattttc gcgtagcgcg caaaatcgca ctttcggatg gtgcctcact 1860ggctctggtt acccccgaaa ccacagcgac aagtaccact gaacagttcg ccctggccaa 1920ctttattaag acaaccctgc acgcttttac ggccactatc ggagttgaaa gtgagcgcac 1980ggcgcagcgt atcctgatta atcaggtaga cctcacccgt cgtgctcgtg cggaagaacc 2040acgcgatccg catgaacgtc agcaggaact ggaacgtttc atcgaggcgg tactgctggt 2100tacggcccca ttaccgccgg aagcagatac ccgctacgct ggccgcatcc accgtgggcg 2160tgccattact gtctagtaag ctcttcttct ggaaaaagga gatataccat gatcgtttta 2220gtaactggag caacggcagg ttttggtgaa tgcattactc gtcgttttat tcaacaaggg 2280cataaagtta tcgccactgg ccgtcgccag gaacggttgc aggagttaaa agacgaactg 2340ggagataatc tgtatatcgc ccaactggac gttcgcaacc gcgccgctat tgaagagatg 2400ctggcatcgc ttcctgccga gtggtgcaat attgatatcc tggtaaataa tgccggcctg 2460gcgttgggca tggagcctgc gcataaagcc agcgttgaag actgggaaac gatgattgat 2520accaacaaca aaggcctggt atatatgacg cgcgccgtct taccgggtat ggttgaacgt 2580aatcatggtc atattattaa cattggctca acggcaggta gctggccgta tgccggtggt 2640aacgtttacg gtgcgacgaa agcgtttgtt cgtcagttta gcctgaatct gcgtacggat 2700ctgcatggta cggcggtgcg cgtcaccgac atcgaaccgg gtctggtggg tggtaccgag 2760ttttccaatg tccgctttaa aggcgatgac ggtaaagcag aaaaaaccta tcaaaatacc 2820gttgcattga cgccagaaga tgtcagcgaa gccgtctggt gggtgtcaac gctgcctgct 2880cacgtcaata tcaataccct ggaaatgatg ccggttaccc aaagctatgc cggactgaat 2940gtccaccgtc agtaatagga tcgtcccggc ttatcggtca gtttcacctg atttacgtaa 3000aaacccgctt cggcgggttt ttgcttttgg aggggcagaa agatgaatga ctgtccacga 3060cgctataccc aaaagaaaga cgaattctct agatatcgct caatactgac catttaaatc 3120atacctgacc tccatagcag aaagtcaaaa gcctccgacc ggaggctttt gacttgatcg 3180gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt 3240tgagttatcg agattttcag gagctaagga agctaaaatg agccatattc aacgggaaac 3300gtcttgctcg aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg 3360ggctcgcgat aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga 3420tgcgccagag ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga 3480gatggtcagg ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat 3540ccgtactcct gatgatgcat ggttactcac cactgcgatc ccagggaaaa cagcattcca 3600ggtattagaa gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct 3660gcgccggttg cattcgattc ctgtttgtaa ttgtcctttt aacggcgatc gcgtatttcg 3720tctcgctcag gcgcaatcac gaatgaataa cggtttggtt ggtgcgagtg attttgatga 3780cgagcgtaat ggctggcctg ttgaacaagt ctggaaagaa atgcataagc ttttgccatt 3840ctcaccggat tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga 3900ggggaaatta ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga 3960tcttgccatc ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt 4020tcaaaaatat ggtattgata atcctgatat gaataaattg cagtttcact tgatgctcga 4080tgagtttttc taatgagggc ccaaatgtaa tcacctggct caccttcggg tgggcctttc 4140tgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgatg 4200ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg 4260aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt 4320tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 4380gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 4440cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact 4500ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt 4560cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct 4620gctgaagcca gttacctcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 4680gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 4740caagaagatc ctttgatttt ctaccgaaga aaggcccacc cgtgaaggtg agccagtgag 4800ttgattgcag tccagttacg ctggagtctg aggctcgtcc tgaatgatat caagcttgaa 4860ttcgtt 4866313353DNAArtificial SequencePlasmid pSMART-F6AA82M 31ccatggttga atgactccta taacgaagtt cacagctaac accacgtcgt ccctatctgc 60tgccctaggt ctatgagtgg ttgctggata acgtgcgtaa ttgtgctgat ctcttatata 120gctgctctca ttatctctct accctgaagt gactctctca cctgtaaaaa taatatctca 180caggcttaat agtttcttaa tacaaagcct gtaaaacgtc aggataactt ctatattcag 240ggagaccaca acggtttccc tctacaaata attttgttta actttcgaca tggcaaaatc 300cccccctcgc gacttgctct tcagctttct ggaaaaagga gatataccat gaatgttacg 360tttgaagaac gtgcgagtct gcacggttac cgtatcggca ttgcaagctt ggatgccccg 420gcttccttaa acgccttgag cctgcctatg atcgatgcgc tccaagatcg tttgcgcgct 480tgggcggaag atgccgatat cgcttgcgtt ctgttacgtg gtaatggcag caaggcgttt 540tgcgctggtg gcgatgtagt tcaattggcc aaaaaatgct tagcaagccc aggtgaagcc 600ccggaactgg ccgagcgttt tttcgcccgt agctatcgct tggatcatta tttgcacacc 660taccccaaac cgttgatctg ttgggcccat ggtcacgtgc tgggtggtgg aatgggactt 720ttacagggcg ccggcatccg tattgtgaca ccatcgtctc gcttagctat gccggaaatt 780tctatcgggc tgttccctga cgtgggtggc tcccatttcc tgagtcgcct cccgggaaaa 840ctggggttgt ttttcggtct taccgcgtct ccccttaacg cacgcgacgc gctggactta 900aatctggctg accgtttcct gcttgacacg cagcaggatg cgctgatcga tggtctgatt 960cagttaaatt ggcgcgagca acctgatctg cagctgcact ctcttctgaa agctctggaa 1020cagcaggctc gtagtgagct gccggccgct cagtggttgc ctcgtcgtga acgccttgat 1080gccctcctgg accaagccac gttaccattg tcctggcagg cgctggcgtc gctcgaaaat 1140gatgaggatg ctctgttagc taaggcagct aaaacgatgc tgggcggtag cccgctcacc 1200ggccatctcg tgtggggtca aattcgtcgt gcacgccacc tgtccttggc gcaggtgttt 1260cagatggaat acggtatgtc attgaactgc tgccgccatc cggagttcgc ggaaggcgtc 1320cgcgcccgtt tgattgacaa ggatcacgcc ccccattggc actggccgga cgttaaccag 1380gttccggaac aggtaattgc agcgcatttc gcgccattgg atgatcaccc tttagccgat 1440ctggcatagt aaccggctta tcggtcagtt tcacctgatt tacgtaaaaa cccgcttcgg 1500cgggtttttg cttttggagg ggcagaaaga tgaatgactg tccacgacgc tatacccaaa 1560agaaagacga attctctaga tatcgctcaa tactgaccat ttaaatcata cctgacctcc 1620atagcagaaa gtcaaaagcc tccgaccgga ggcttttgac ttgatcggca cgtaagaggt 1680tccaactttc accataatga aataagatca ctaccgggcg tattttttga gttatcgaga 1740ttttcaggag ctaaggaagc taaaatgagc catattcaac gggaaacgtc ttgctcgagg 1800ccgcgattaa attccaacat ggatgctgat ttatatgggt ataaatgggc tcgcgataat 1860gtcgggcaat caggtgcgac aatctatcga ttgtatggga agcccgatgc gccagagttg 1920tttctgaaac atggcaaagg tagcgttgcc aatgatgtta cagatgagat ggtcaggcta 1980aactggctga cggaatttat gcctcttccg accatcaagc attttatccg tactcctgat 2040gatgcatggt tactcaccac tgcgatccca gggaaaacag cattccaggt attagaagaa 2100tatcctgatt caggtgaaaa tattgttgat gcgctggcag tgttcctgcg ccggttgcat 2160tcgattcctg tttgtaattg tccttttaac ggcgatcgcg tatttcgtct cgctcaggcg 2220caatcacgaa tgaataacgg tttggttggt gcgagtgatt ttgatgacga gcgtaatggc 2280tggcctgttg aacaagtctg gaaagaaatg cataagcttt tgccattctc accggattca 2340gtcgtcactc atggtgattt ctcacttgat aaccttattt ttgacgaggg gaaattaata 2400ggttgtattg atgttggacg agtcggaatc gcagaccgat accaggatct tgccatccta 2460tggaactgcc tcggtgagtt ttctccttca ttacagaaac ggctttttca aaaatatggt 2520attgataatc ctgatatgaa taaattgcag tttcacttga tgctcgatga gtttttctaa 2580tgagggccca aatgtaatca cctggctcac cttcgggtgg gcctttctgc gttgctggcg 2640tttttccata ggctccgccc ccctgacgag catcacaaaa atcgatgctc aagtcagagg 2700tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 2760cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 2820agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 2880tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 2940aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 3000ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 3060cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 3120acctcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg 3180gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt 3240tgattttcta ccgaagaaag gcccacccgt gaaggtgagc cagtgagttg attgcagtcc 3300agttacgctg gagtctgagg ctcgtcctga atgatatcaa gcttgaattc gtt 3353323424DNAArtificial SequencePlasmid pSMART-Ala1 32ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 60gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct 120gcgtttatac acagctaaca ccacgtcgtc cctatctgct gccctaggtc tatgagtggt 180tgctggataa ctctttctga caccttacta tcttacaaat gtaacaaaaa agttattttt 240ctgtaattcg agcatgtcat gttaccccgc gagcataaaa cgcgtatatt cagggagacc 300acaacggttt ccctctacaa ataattttgt ttaactttgg aaaaaggaga tataccatga 360tcattggggt gccgaaggag atcaaaaata atgagaaccg cgtcgcgttg accccgggag 420gtgtcagcca gctgatctct aatggccatc gtgtcttagt tgaaacaggc gctggcctgg 480gttctggctt cgaaaacgag gcctacgaat ctgcaggtgc ggaaattatt gctgatccaa 540aacaggtctg ggatgcagag atggtcatga aagtgaaaga accgctcccg gaagaatatg 600tctattttcg taaaggtctg gtgctgttta catatctgca tctggcagct gaaccggagc 660tcgcacaagc ccttaaagat aaaggtgtca cggccatcgc atacgaaact gtcagcgaag 720ggcgcacgct gccattactg accccgatgt cagaagtggc aggccgtatg gctgcgcaga 780tcggcgcaca gtttcttgaa aaaccaaagg gcgggaaggg tattctctta gcaggagtgc 840cgggcgtcag tcgtgggaaa gtaactatta ttggtggcgg cgtggtagga acaaatgctg 900ccaaaatggc cgtcggtttg ggggccgacg taacaatcat tgcgcgtaat gccgatcgcc 960ttcgtcaatt agacgatatc tttggccacc aaatcaaaac cctgatttcg aacccagtca 1020atatcgcgga tgcggtggcg gaagctgatt tgttgatctg cgccgtgtta attccgggag 1080cgaaagcacc tacattggtg acggaagaaa tggtgaaaca aatgaaaccg ggttcagtca 1140ttgttgatgt ggctattgat cagggtggca tcgtggaaac ggtggaccat attaccactc 1200acgaccagcc gacgtatgaa aaacatggtg tcgtacacta tgcggtggcg aatatgcctg 1260gtgcggtccc acgtacgagt acaatcgcac tgacaaatgt caccgtgccg tatgcgttgc 1320aaatcgcgaa caaaggtgcc gtgaaagcgc tggccgacaa tacggcgtta cgtgccggtc 1380tgaacaccgc taacggtcac gtgacatatg aagcggtcgc gcgtgatttg gggtacgaat 1440atgtaccggc ggaaaaagcc ttacaagacg aatcgagtgt cgctggtgca tagtaagctc 1500ttctaatacg actcactata gggccggctt atcggtcagt ttcacctgat ttacgtaaaa 1560acccgcttcg gcgggttttt gcttttggag gggcagaaag atgaatgact gtccacgacg 1620ctatacccaa aagaaagacg aattctctag atatcgctca atactgacca tttaaatcat 1680acctgacctc catagcagaa agtcaaaagc ctccgaccgg aggcttttga cttgatcggc 1740acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg 1800agttatcgag attttcagga gctaaggaag ctaaaatgag ccatattcaa cgggaaacgt 1860cttgctcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 1920ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 1980cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 2040tggtcaggct aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 2100gtactcctga tgatgcatgg ttactcacca ctgcgatccc agggaaaaca gcattccagg 2160tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 2220gccggttgca ttcgattcct gtttgtaatt gtccttttaa cggcgatcgc gtatttcgtc 2280tcgctcaggc gcaatcacga atgaataacg gtttggttgg tgcgagtgat tttgatgacg 2340agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 2400caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 2460ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 2520ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 2580aaaaatatgg tattgataat cctgatatga ataaattgca gtttcacttg atgctcgatg 2640agtttttcta atgagggccc aaatgtaatc acctggctca ccttcgggtg ggcctttctg 2700cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgatgct 2760caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 2820gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 2880tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 2940aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3000ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3060cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3120tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 3180tgaagccagt tacctcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 3240tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3300agaagatcct ttgattttct accgaagaaa ggcccacccg tgaaggtgag ccagtgagtt 3360gattgcagtc cagttacgct ggagtctgag gctcgtcctg aatgatatca agcttgaatt 3420cgtt 3424336275DNAArtificial SequencePlasmid pSMART-Mev1 33tgcccaggca tcaaataaaa cgaaaggctc agtcgaaaga ctgggccttt cgttttatct 60gttgtttgtc ggtgaacgct ctctactaga gtcacactgg ctcaccttcg ggtgggcctt 120tctgcgttta tacacagcta acaccacgtc gtccctatct gctgccctag gtctatgagt 180ggttgctgga taacgtgcgt aattgtgctg atctcttata tagctgctct cattatctct 240ctaccctgaa gtgactctct cacctgtaaa aataatatct cacaggctta atagtttctt 300aatacaaagc ctgtaaaacg tcaggataac ttctatattc agggagacca caacggtttc 360cctctacaaa taattttgtt taactttcgt ggaaaaagga gatataccat gaagacggta 420gttattatcg acgcactgcg tacccccatt ggaaaataca aaggaagtct gagccaggta 480agcgccgtcg acctgggcac acatgtgacc acgcagttgt tgaagcgtca cagcactatc 540agcgaggaaa ttgatcaggt catttttggt aatgttctgc aggcgggcaa tgggcagaac 600cctgcacgtc agattgcaat caactcaggt ttaagccatg aaattccagc gatgacggtc 660aatgaggtct gtggcagtgg gatgaaagcg gtaatcctgg ccaaacagtt aatccagctg 720ggtgaggcgg aggtacttat cgcaggtggt attgaaaaca tgtcacaggc cccgaaactg 780caacgcttta actacgaaac agaaagctac gatgcgcctt tttcgtccat gatgtatgat 840ggtcttaccg acgcattcag tggtcaggcg atgggtctga cggccgagaa tgttgctgaa 900aaataccacg ttacccgtga ggaacaagac caattctctg tccatagcca actcaaagcg 960gcacaggctc aggcagaagg catttttgcc gatgagattg caccactgga agtttccggc 1020accctggtgg aaaaggacga gggcattcgt ccgaatagca gtgttgaaaa actcggtact 1080ttgaaaaccg tattcaaaga ggacggcacg gtgactgccg gtaatgcctc aactatcaac 1140gacggtgcct cggcactgat tattgcgtct caagaatacg cggaagcgca cggcttgccg 1200tatctcgcga ttatccgcga ttcagtggag gtcggcatcg atcccgcgta catgggcatt 1260tcgccgatca aagcaattca gaagcttctg gcacgcaacc agttgacgac cgaagagatt 1320gatttatacg aaatcaatga agcgttcgcg gcgacctcga ttgtggttca gcgtgaactt 1380gccctcccgg aagaaaaggt caacatctat ggcggaggca tcagtttggg ccatgccatc 1440ggagcgaccg gtgcccgtct gctcaccagc ttatcatatc agttgaacca gaaagaaaaa 1500aagtacggcg ttgcatctct gtgtattggc ggaggtctgg gcctcgccat gttgttagaa 1560cgtccgcagc aaaaaaaaaa ctcccgcttt tatcagatgt cgccggagga acgtctggcg 1620agcttgttga acgaagggca gatctctgcc gacactaaaa aggaattcga aaacacggca 1680ctgagcagtc agattgcgaa ccatatgatt gaaaatcaga tcagcgagac cgaggtgccc 1740atgggcgtgg gccttcatct cacggtggac gaaacggatt atctggtacc aatggccaca 1800gaagaaccgt cggtaatcgc cgcgttgtca aatggcgcga aaatcgcgca agggttcaaa 1860acggtcaacc agcagcgtct catgcgcggc cagatcgtgt tctatgatgt agcagatgca 1920gagagtctga

ttgacgagtt acaggttcgt gagacggaga tttttcagca agccgagctg 1980tcgtacccga gcattgttaa acgtggcggt ggccttcgtg acttgcagta tcgcgccttc 2040gacgaatcgt tcgtgagtgt cgactttctg gtagacgtga aggacgccat gggggccaat 2100atcgttaatg ccatgctgga aggggttgca gagctgtttc gtgagtggtt cgccgaacaa 2160aaaatcctgt ttagcatctt aagcaattac gcaacggaaa gcgtcgtgac catgaaaacc 2220gcgatccctg ttagccgcct ttcaaagggc agtaacggtc gtgaaatcgc tgaaaaaatt 2280gttctcgcgt cccgctatgc atcgttggat ccttatcgcg cggtgacaca caacaaaggc 2340attatgaatg gtatcgaagc ggtcgttctg gcgaccggca acgatactcg cgccgtgagc 2400gcgtcctgcc atgcttttgc tgtgaaagag ggccgttatc agggcttgac gtcctggacc 2460ctggacggtg aacagctgat cggcgaaatc tcggtgcccc tcgccctggc cactgtgggc 2520ggcgccacaa aagtgttgcc aaaaagccaa gcggcggcgg atctgctggc cgtaactgat 2580gctaaggaac tgagtcgcgt ggttgccgca gtgggcctgg cccaaaacct ggcagcactg 2640cgcgcgctgg tttctgaagg catccagaaa ggtcatatgg ccctgcaagc gcgctctctg 2700gccatgaccg taggggcgac cggcaaggaa gtcgaagcgg tagctcaaca gttaaaacgc 2760cagaaaacta tgaatcagga tcgtgcgctg gccatcctca atgacctgcg caaacagtaa 2820tagtcgcgcc gaaaaccccg cttcggcggg gttttgccgc acgtctccat cgcttgccca 2880agttgtgaag cacagctaac accacgtcgt ccctatctgc tgccctaggt ctatgagtgg 2940ttgctggata accatccata aattttgcat aattaatgta aagaccaggc tcgccagtaa 3000cgctaaattc atttggctgt aagcgcggtg tcatccgcgt caggaaaatt aaacagttac 3060tttaaaaaat gaaaacgtaa aaaggttggg tttcgatgta ttgacgggta aactttgtcg 3120cccgctaaac atttgtttat attcagggag accacaacgg tttccctcta caaataattt 3180tgtttaactt tgctggaaaa aggagatata ccatgaccat tgggattgat aaaatctcgt 3240ttttcgtgcc tccttattat atcgacatga cggccctggc cgaggctcgc aatgtggatc 3300ccggcaaatt tcacatcggt atcggccagg accaaatggc ggtgaatccc atctcgcagg 3360acattgtcac cttcgccgca aacgcagcag aagctatctt gactaaagaa gataaagagg 3420ccatcgacat ggtgatcgtg ggtacggaaa gctctattga cgaaagtaaa gccgcggcgg 3480tggtattaca ccgcctgatg ggtatccagc cgtttgcgcg ctcctttgaa atcaaagagg 3540cctgctacgg cgcaacggct ggactgcaac tcgcgaagaa ccatgttgca ttacatccgg 3600ataaaaaagt cctggttgtc gcggcggaca tcgcgaaata cggcctgaac tccggcgggg 3660aaccaacgca gggtgccggc gcagtggcga tgcttgtcgc aagcgagcct cgtatcctgg 3720ctttaaagga ggacaacgtg atgctgacac aggatattta cgatttttgg cgtcccaccg 3780gtcatccata tccgatggtt gatggtcctc tgtccaatga aacttatatt cagagcttcg 3840cgcaagtttg ggatgaacat aagaaacgta ccggtctgga ttttgcggat tacgacgctc 3900tggcttttca cattccatac acgaaaatgg gcaaaaaagc cctcttagct aaaatctcag 3960accagaccga ggcagaacag gaacgcattt tagcgcgtta cgaagagtca attatctaca 4020gccgccgtgt aggtaattta tatacggggt cgctttatct gggattgatt tccttactcg 4080aaaacgccac aaccctgacg gcgggtaacc aaatcggttt attctcttac ggtagcggtg 4140ccgttgccga attcttcacg ggtgagctgg ttgccggtta ccagaaccac ttacagaaag 4200aaacccacct cgccctgctg gacaaccgta ctgaactcag catcgcagaa tatgaggcca 4260tgttcgccga aacactcgac acggatatcg atcaaacctt agaggatgaa ctcaaatatt 4320ccatttcagc gattaataac accgtccgct cctatcgcaa ttagtaagat cgtcccggct 4380tatcggtcag tttcacctga tttacgtaaa aacccgcttc ggcgggtttt tgcttttgga 4440ggggcagaaa gatgaatgac tgtccacgac gctataccca aaagaaagac gaattctcta 4500gatatcgctc aatactgacc atttaaatca tacctgacct ccatagcaga aagtcaaaag 4560cctccgaccg gaggcttttg acttgatcgg cacgtaagag gttccaactt tcaccataat 4620gaaataagat cactaccggg cgtatttttt gagttatcga gattttcagg agctaaggaa 4680gctaaaatga gccatattca acgggaaacg tcttgctcga ggccgcgatt aaattccaac 4740atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg 4800acaatctatc gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa 4860ggtagcgttg ccaatgatgt tacagatgag atggtcaggc taaactggct gacggaattt 4920atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc 4980actgcgatcc cagggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa 5040aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat 5100tgtcctttta acggcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac 5160ggtttggttg gtgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc 5220tggaaagaaa tgcataagct tttgccattc tcaccggatt cagtcgtcac tcatggtgat 5280ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga 5340cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag 5400ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg 5460aataaattgc agtttcactt gatgctcgat gagtttttct aatgagggcc caaatgtaat 5520cacctggctc accttcgggt gggcctttct gcgttgctgg cgtttttcca taggctccgc 5580ccccctgacg agcatcacaa aaatcgatgc tcaagtcaga ggtggcgaaa cccgacagga 5640ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 5700ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 5760agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 5820cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 5880aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 5940gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 6000agaagaacag tatttggtat ctgcgctctg ctgaagccag ttacctcgga aaaagagttg 6060gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 6120agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgattttc taccgaagaa 6180aggcccaccc gtgaaggtga gccagtgagt tgattgcagt ccagttacgc tggagtctga 6240ggctcgtcct gaatgatatc aagcttgaat tcgtt 6275345364DNAArtificial SequencePlasmid pSMART-2,3-BDO1 34gtgcgtaatt gtgctgatct cttatatagc tgctctcatt atctctctac cctgaagtga 60ctctctcacc tgtaaaaata atatctcaca ggcttaatag tttcttaata caaagcctgt 120aaaacgtcag gataacttct tggaaaaagg agatatacca tgatgcacag cagcgcatgt 180gattgtgaag cgagtttgtg cgagacactc cgtggttttt ccgcaaaaca cccggattcc 240gtaatctacc agacatcact gatgtccgcc cttctgtcag gcgtatatga aggggacacg 300actattgcgg atcttctggc ccacggcgat tttggcctgg gtacgttcaa tgaactcgac 360ggcgaaatga tcgcgttttc ttcgcaagtt tatcagctcc gtgcggatgg gagcgcccgc 420gccgcgaagc cagaacaaaa aacaccgttt gcagtaatga catggttcca accgcagtat 480cgtaaaactt tcgatgcccc ggtgagtcgt cagcagatcc acgatgtaat cgatcaacag 540attccttcag acaacctgtt ttgcgcgctg cgtattgacg ggaatttccg tcatgctcac 600acacgtaccg ttccgcgcca gaccccaccc tatcgcgcga tgaccgatgt gctggatgat 660caaccggtct ttcgttttaa ccagcgcgaa ggagttctgg tgggttttcg taccccgcaa 720catatgcagg gtattaacgt ggcgggctac catgagcatt tcattacaga tgatcgccaa 780ggcggtggtc acctgttgga ttaccagctg gaatctggcg tcctgacttt cggcgagatt 840cacaaactga tgattgacct gccggcggat tctgcattcc tgcaggcaaa tttgcacccc 900agcaaccttg atgccgccat ccgctccgtc gagaactaat aggctcttca cttctggaaa 960aaggagatat accatgaatt ccgaaaaaca atcgcgtcag tgggcacatg gtgctgatat 1020ggttgtgggc cagctggagg cgcagggggt taaacaggtc tttggtattc cgggtgctaa 1080gatcgacaaa gtgtttgatt ctttactgga tagctcaatc gagattatcc cggtgcgtca 1140tgaagcaaac gcagcgttca tggccgcggc agttggtcgc cttacgggta aagctggcgt 1200agccctggtc acaagcggcc ccgggtgctc gaatctcatt accggcattg caaccgcaaa 1260ttctgaggga gatcctgtag tggcactggg gggcgcggta aaacgtgctg ataaagcgaa 1320attagttcac cagagtatgg acaccgtcgc gatgttctct ccagtaacca aatatgcggt 1380tgaagtttct tccccagatg caattgcaga ggtagtatca aacgcttttc gtgccgcgga 1440acatggccgc ccaggtgggg cgttcgtttc gctgccgcag gatattgtag accaaccggc 1500gacaggcgcc atcctgcctg catctggccc ggcactgatg ggcccagcgc cagagtcggc 1560gattaacgat gtggcaaaac ttatcgacaa cgccaaaaac cctgtgattc tgttgggctt 1620aatggcatca cagccggcta attcggctgc attgcgtaag ctgctggaga agagtcgcat 1680cccggtgact tccacctacc aagccgccgg agctgtgaac caagaacatt tcacccgctt 1740cgccggtcgt gttggccttt tcaataacca agcgggagac cgtctgctgc atttggccga 1800tctcattatc tgtattggat actctccagt cgagtatgaa ccgagcatgt ggaactcggg 1860tgacgcaacc ctcgttcata ttgacgtgct gccagcttat gaagaacgca actatgtacc 1920cgatatcgag ttggtaggcg acattgcggc gacactgaac ctgctcgctt cccgcattga 1980tcataaactg gagctctcgc agcgtgcctc cgagatctta gtcgatcgcc aacaccagcg 2040cgatctgctg gatcgccgtg gcgcaagctt aaatcaattt gcgctgcatc cattacgtat 2100cgtccgtgcc atgcaggaca tcgtaaacaa tgacgtaacg ctgaccgtgg acatgggctc 2160atttcatatt tggatcgcac gctatctcta ttcatttcgc gcacgtcagg tcatgattag 2220taatgggcaa caaactatgg gcgtggctct gccttgggct atcggtgcgt ggctggtgaa 2280ccccggccgc aaagtggtga gcgttagcgg tgacggagga tttctgcaga gtagcatgga 2340gttagaaacc gctgtccgcc tgaacgctaa tgtgttacac atcatttggg tggataatgg 2400ttataatatg gttgcaatcc aggaggagaa aaagtatcag cgtttaagcg gtgtggcgtt 2460tggaccggta gatttcaaag cctacgccga tgcattcggc gcccgtggct tcgcggtcga 2520aagcgcggat gccttagaga gcaccttacg tgcggcaatg gatgtgaatg gtccggccgt 2580cgtggcgatt ccggtggatt attcggataa tccgctgctg atgggacaac tgcacctttc 2640gcagatcctg tagtaagctc ttctggaaaa aggagatata ccatgcagaa ggtggcgctc 2700gttaccggat ctggccaagg cattggcaaa gcgattgcgc ttcgtctggt caaagacgga 2760ttcgccgttg caattgctga ttacaacgac gaaacggcgc gtgctgtcgc cgatgaaatc 2820atccgtaatg gtggcaacgc tgtcgcagtg aaagtggacg tctctgatcg cgaccaagta 2880tttgcagcgg tcgagaaagc acgtaccgct ctgggcggtt tcaacgttat cgtgaacaac 2940gcgggcattg cgccgtcgac gcctatcgaa agcatcaccc cggagattgt agataaggtg 3000tacaacatca acgtaaaagg agtaatctgg ggtatgcaag ctgccatcga tgcgttccgc 3060aaagaggggc acggcggtaa aatcattaac gcgtgttcgc aggctggtca tactggtaac 3120ccggaactgg cggtttatag cagcagcaaa ttcgccgtgc gtggcctgac ccagaccgct 3180gcacgcgatc tggcgccgct ggggatcacc gtcaatgcat attgtccggg tatcgtaaaa 3240accccgatgt gggcggaaat tgatcgccag gtatcagagg ccgctggcaa accgctgggc 3300tatggcacgg aaacgtttgc caagcgcatc acgttaggcc gtctgtcgga accggaggat 3360gttgcagcat gcgtctctta cctggcgggc ccggattctg attatatgac gggtcagtcc 3420ctgctgattg atggtggcat ggtctttaac tagtaagatc gtcccggctt atcggtcagt 3480ttcacctgat ttacgtaaaa acccgcttcg gcgggttttt gcttttggag gggcagaaag 3540atgaatgact gtccacgacg ctatacccaa aagaaagacg aattctctag atatcgctca 3600atactgacca tttaaatcat acctgacctc catagcagaa agtcaaaagc ctccgaccgg 3660aggcttttga cttgatcggc acgtaagagg ttccaacttt caccataatg aaataagatc 3720actaccgggc gtattttttg agttatcgag attttcagga gctaaggaag ctaaaatgag 3780ccatattcaa cgggaaacgt cttgctcgag gccgcgatta aattccaaca tggatgctga 3840tttatatggg tataaatggg ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg 3900attgtatggg aagcccgatg cgccagagtt gtttctgaaa catggcaaag gtagcgttgc 3960caatgatgtt acagatgaga tggtcaggct aaactggctg acggaattta tgcctcttcc 4020gaccatcaag cattttatcc gtactcctga tgatgcatgg ttactcacca ctgcgatccc 4080agggaaaaca gcattccagg tattagaaga atatcctgat tcaggtgaaa atattgttga 4140tgcgctggca gtgttcctgc gccggttgca ttcgattcct gtttgtaatt gtccttttaa 4200cggcgatcgc gtatttcgtc tcgctcaggc gcaatcacga atgaataacg gtttggttgg 4260tgcgagtgat tttgatgacg agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat 4320gcataagctt ttgccattct caccggattc agtcgtcact catggtgatt tctcacttga 4380taaccttatt tttgacgagg ggaaattaat aggttgtatt gatgttggac gagtcggaat 4440cgcagaccga taccaggatc ttgccatcct atggaactgc ctcggtgagt tttctccttc 4500attacagaaa cggctttttc aaaaatatgg tattgataat cctgatatga ataaattgca 4560gtttcacttg atgctcgatg agtttttcta atgagggccc aaatgtaatc acctggctca 4620ccttcgggtg ggcctttctg cgttgctggc gtttttccat aggctccgcc cccctgacga 4680gcatcacaaa aatcgatgct caagtcagag gtggcgaaac ccgacaggac tataaagata 4740ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 4800cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 4860taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 4920cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4980acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 5040aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt 5100atttggtatc tgcgctctgc tgaagccagt tacctcggaa aaagagttgg tagctcttga 5160tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 5220cgcagaaaaa aaggatctca agaagatcct ttgattttct accgaagaaa ggcccacccg 5280tgaaggtgag ccagtgagtt gattgcagtc cagttacgct ggagtctgag gctcgtcctg 5340aatgatatca agcttgaatt cgtt 5364355748DNAArtificial SequencePlasmid pSMART-2,3-BDO2 35gtgcgtaatt gtgctgatct cttatatagc tgctctcatt atctctctac cctgaagtga 60ctctctcacc tgtaaaaata atatctcaca ggcttaatag tttcttaata caaagcctgt 120aaaacgtcag gataacttct tggaaaaagg agatatacca tgatgcacag cagcgcatgt 180gattgtgaag cgagtttgtg cgagacactc cgtggttttt ccgcaaaaca cccggattcc 240gtaatctacc agacatcact gatgtccgcc cttctgtcag gcgtatatga aggggacacg 300actattgcgg atcttctggc ccacggcgat tttggcctgg gtacgttcaa tgaactcgac 360ggcgaaatga tcgcgttttc ttcgcaagtt tatcagctcc gtgcggatgg gagcgcccgc 420gccgcgaagc cagaacaaaa aacaccgttt gcagtaatga catggttcca accgcagtat 480cgtaaaactt tcgatgcccc ggtgagtcgt cagcagatcc acgatgtaat cgatcaacag 540attccttcag acaacctgtt ttgcgcgctg cgtattgacg ggaatttccg tcatgctcac 600acacgtaccg ttccgcgcca gaccccaccc tatcgcgcga tgaccgatgt gctggatgat 660caaccggtct ttcgttttaa ccagcgcgaa ggagttctgg tgggttttcg taccccgcaa 720catatgcagg gtattaacgt ggcgggctac catgagcatt tcattacaga tgatcgccaa 780ggcggtggtc acctgttgga ttaccagctg gaatctggcg tcctgacttt cggcgagatt 840cacaaactga tgattgacct gccggcggat tctgcattcc tgcaggcaaa tttgcacccc 900agcaaccttg atgccgccat ccgctccgtc gagaactaat aggctcttca cttctggaaa 960aaggagatat accatgaatt ccgaaaaaca atcgcgtcag tgggcacatg gtgctgatat 1020ggttgtgggc cagctggagg cgcagggggt taaacaggtc tttggtattc cgggtgctaa 1080gatcgacaaa gtgtttgatt ctttactgga tagctcaatc gagattatcc cggtgcgtca 1140tgaagcaaac gcagcgttca tggccgcggc agttggtcgc cttacgggta aagctggcgt 1200agccctggtc acaagcggcc ccgggtgctc gaatctcatt accggcattg caaccgcaaa 1260ttctgaggga gatcctgtag tggcactggg gggcgcggta aaacgtgctg ataaagcgaa 1320attagttcac cagagtatgg acaccgtcgc gatgttctct ccagtaacca aatatgcggt 1380tgaagtttct tccccagatg caattgcaga ggtagtatca aacgcttttc gtgccgcgga 1440acatggccgc ccaggtgggg cgttcgtttc gctgccgcag gatattgtag accaaccggc 1500gacaggcgcc atcctgcctg catctggccc ggcactgatg ggcccagcgc cagagtcggc 1560gattaacgat gtggcaaaac ttatcgacaa cgccaaaaac cctgtgattc tgttgggctt 1620aatggcatca cagccggcta attcggctgc attgcgtaag ctgctggaga agagtcgcat 1680cccggtgact tccacctacc aagccgccgg agctgtgaac caagaacatt tcacccgctt 1740cgccggtcgt gttggccttt tcaataacca agcgggagac cgtctgctgc atttggccga 1800tctcattatc tgtattggat actctccagt cgagtatgaa ccgagcatgt ggaactcggg 1860tgacgcaacc ctcgttcata ttgacgtgct gccagcttat gaagaacgca actatgtacc 1920cgatatcgag ttggtaggcg acattgcggc gacactgaac ctgctcgctt cccgcattga 1980tcataaactg gagctctcgc agcgtgcctc cgagatctta gtcgatcgcc aacaccagcg 2040cgatctgctg gatcgccgtg gcgcaagctt aaatcaattt gcgctgcatc cattacgtat 2100cgtccgtgcc atgcaggaca tcgtaaacaa tgacgtaacg ctgaccgtgg acatgggctc 2160atttcatatt tggatcgcac gctatctcta ttcatttcgc gcacgtcagg tcatgattag 2220taatgggcaa caaactatgg gcgtggctct gccttgggct atcggtgcgt ggctggtgaa 2280ccccggccgc aaagtggtga gcgttagcgg tgacggagga tttctgcaga gtagcatgga 2340gttagaaacc gctgtccgcc tgaacgctaa tgtgttacac atcatttggg tggataatgg 2400ttataatatg gttgcaatcc aggaggagaa aaagtatcag cgtttaagcg gtgtggcgtt 2460tggaccggta gatttcaaag cctacgccga tgcattcggc gcccgtggct tcgcggtcga 2520aagcgcggat gccttagaga gcaccttacg tgcggcaatg gatgtgaatg gtccggccgt 2580cgtggcgatt ccggtggatt attcggataa tccgctgctg atgggacaac tgcacctttc 2640gcagatcctg tagtaagctc ttctggaaaa aggagatata ccatgcgtgc gttggcatat 2700ttcaaaaaag gagacatcca ctttaccaac gatattccgc gtccggagat ccagacggat 2760gatgaagtga ttattgatgt gagctggtgt gggatttgcg gttcggattt gcatgaatat 2820ctggatggtc caatttttat gccgaaggat ggcgaatgtc acaaactgag taacgcggcg 2880ctgcccctgg caatgggaca cgagatgtcg ggaattgtca gtaaagtcgg cccgaaagtg 2940accaaggtca aagtgggcga tcatgttgtt gttgatgctg catcgtcctg tgccgatctc 3000cattgctggc cccacagcaa attctataac tctaaacctt gtgacgcgtg tcaacgcgga 3060tcggagaacc tgtgcacgca tgccggtttt gtcgggcttg gggttatctc tggcggtttt 3120gcggaacaag tggtggtatc tcaacatcac attattcccg tgccgaagga aatccctctg 3180gacgtagcag cactggtgga accgctctcg gtaacctggc acgcagtaaa aatttcgggc 3240tttaagaaag gctcgagtgc actggtgttg ggggccggcc caatcggtct gtgtacgatt 3300ctggtgctga aaggtatggg tgcgagcaaa atcgtagtta gttcgcgttc cgagcgtcgc 3360atcgaaatgg caaaaaaact cggcgtcgaa gtgtttaatc catcgaaaca cggccataag 3420agtattgaaa ttctgcgtgg tctgaccaaa tcacatgacg gtttcgatta tagctatgac 3480tgcagtggaa ttcaggttac cttcgaaacc agccttaaag cccttacttt taaaggcacc 3540gccaccaata tcgctgtttg gggtcccaaa cccgtacctt tccagcctat ggatgtgaca 3600cttcaggaaa aagttatgac gggatccatc ggctacgtgg tggaggactt cgaagaagtg 3660gtccgtgcca ttcacaacgg agatatcgcg atggaagatt gtaagcagct gattaccggc 3720aaacagcgca ttgaggatgg gtgggaaaaa ggcttccagg aattaatgga ccacaaagag 3780tctaatgtaa aaattctgct gaccccgaat aatcatggag aaatgaaata gtaatagtaa 3840gatcgtcccg gcttatcggt cagtttcacc tgatttacgt aaaaacccgc ttcggcgggt 3900ttttgctttt ggaggggcag aaagatgaat gactgtccac gacgctatac ccaaaagaaa 3960gacgaattct ctagatatcg ctcaatactg accatttaaa tcatacctga cctccatagc 4020agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa gaggttccaa 4080ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc 4140aggagctaag gaagctaaaa tgagccatat tcaacgggaa acgtcttgct cgaggccgcg 4200attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg 4260gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct 4320gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca ggctaaactg 4380gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc 4440atggttactc accactgcga tcccagggaa aacagcattc caggtattag aagaatatcc 4500tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat 4560tcctgtttgt aattgtcctt ttaacggcga tcgcgtattt cgtctcgctc aggcgcaatc 4620acgaatgaat aacggtttgg ttggtgcgag tgattttgat gacgagcgta atggctggcc 4680tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt 4740cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg 4800tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa 4860ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga 4920taatcctgat atgaataaat tgcagtttca cttgatgctc gatgagtttt tctaatgagg 4980gcccaaatgt aatcacctgg ctcaccttcg ggtgggcctt tctgcgttgc tggcgttttt 5040ccataggctc cgcccccctg acgagcatca caaaaatcga tgctcaagtc agaggtggcg 5100aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 5160tcctgttccg

accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 5220ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5280gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5340tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5400caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5460ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctc 5520ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 5580tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatt 5640ttctaccgaa gaaaggccca cccgtgaagg tgagccagtg agttgattgc agtccagtta 5700cgctggagtc tgaggctcgt cctgaatgat atcaagcttg aattcgtt 5748362818DNAArtificial SequencePlasmid pSMART-amnp-GFPuv 36tgaggctcgt cctgaatgat atcaagcttg aattcgttag acagtcaacg cgcttgatag 60cctggcgaag atcatccgat cttcgcctta cacttttgtt tcacatttct gtgacatact 120atcggatgtg cggtaattgt atgtgtagga ggataatcta tggctagcaa aggagaagaa 180cttttcacat ggctagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg 240aattagatgg tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg 300ctacatacgg aaagcttacc cttaaattta tttgcactac tggaaaacta cctgttccat 360ggccaacact tgtcactact ttctcttatg gtgttcaatg cttttcccgt tatccggatc 420atatgaaacg gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca 480ctatatcttt caaagatgac gggaactaca agacgcgtgc tgaagtcaag tttgaaggtg 540atacccttgt taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc 600tcggacacaa actcgagtac aactataact cacacaatgt atacatcacg gcagacaaac 660aaaagaatgg aatcaaagct aacttcaaaa ttcgccacaa cattgaagat ggatccgttc 720aactagcaga ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag 780acaaccatta cctgtcgaca caatctgccc tttcgaaaga tcccaacgaa aagcgtgacc 840acatggtcct tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagctct 900acaaataatg aggatccccg gcttatcggt cagtttcacc tgatttacgt aaaaacccgc 960ttcggcgggt ttttgctttt ggaggggcag aaagatgaat gactgtccac gacgctatac 1020ccaaaagaaa gacgaattct ctagatatcg ctcaatactg accatttaaa tcatacctga 1080cctccatagc agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa 1140gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat 1200cgagattttc aggagctaag gaagctaaaa tgagccatat tcaacgggaa acgtcttgct 1260cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg 1320ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag 1380agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca 1440ggctaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc 1500ctgatgatgc atggttactc accactgcga tcccagggaa aacagcattc caggtattag 1560aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt 1620tgcattcgat tcctgtttgt aattgtcctt ttaacggcga tcgcgtattt cgtctcgctc 1680aggcgcaatc acgaatgaat aacggtttgg ttggtgcgag tgattttgat gacgagcgta 1740atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg 1800attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat 1860taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca 1920tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat 1980atggtattga taatcctgat atgaataaat tgcagtttca cttgatgctc gatgagtttt 2040tctaatgagg gcccaaatgt aatcacctgg ctcaccttcg ggtgggcctt tctgcgttgc 2100tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga tgctcaagtc 2160agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 2220tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 2280cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 2340ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 2400ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 2460ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 2520ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 2580cagttacctc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2640cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 2700tcctttgatt ttctaccgaa gaaaggccca cccgtgaagg tgagccagtg agttgattgc 2760agtccagtta cgctggagtc tgaggctcgt cctgaatgat atcaagcttg aattcgtt 2818372839DNAArtificial SequencePlasmid pSMART-phoAp-GFPuv 37tgaggctcgt cctgaatgat atcaagcttg aattcgttcg attacgtaaa gaagttattg 60aagcatcctc gtcagtaaaa agttaatctt ttcaacagct gtcataaagt tgtcacggcc 120gagacttata gtcgctttgt ttttattttt taatgtattt gtagtgtagg aggataatct 180atggctagca aaggagaaga acttttcaca tggctagcaa aggagaagaa cttttcactg 240gagttgtccc aattcttgtt gaattagatg gtgatgttaa tgggcacaaa ttttctgtca 300gtggagaggg tgaaggtgat gctacatacg gaaagcttac ccttaaattt atttgcacta 360ctggaaaact acctgttcca tggccaacac ttgtcactac tttctcttat ggtgttcaat 420gcttttcccg ttatccggat catatgaaac ggcatgactt tttcaagagt gccatgcccg 480aaggttatgt acaggaacgc actatatctt tcaaagatga cgggaactac aagacgcgtg 540ctgaagtcaa gtttgaaggt gatacccttg ttaatcgtat cgagttaaaa ggtattgatt 600ttaaagaaga tggaaacatt ctcggacaca aactcgagta caactataac tcacacaatg 660tatacatcac ggcagacaaa caaaagaatg gaatcaaagc taacttcaaa attcgccaca 720acattgaaga tggatccgtt caactagcag accattatca acaaaatact ccaattggcg 780atggccctgt ccttttacca gacaaccatt acctgtcgac acaatctgcc ctttcgaaag 840atcccaacga aaagcgtgac cacatggtcc ttcttgagtt tgtaactgct gctgggatta 900cacatggcat ggatgagctc tacaaataat gaggatcccc ggcttatcgg tcagtttcac 960ctgatttacg taaaaacccg cttcggcggg tttttgcttt tggaggggca gaaagatgaa 1020tgactgtcca cgacgctata cccaaaagaa agacgaattc tctagatatc gctcaatact 1080gaccatttaa atcatacctg acctccatag cagaaagtca aaagcctccg accggaggct 1140tttgacttga tcggcacgta agaggttcca actttcacca taatgaaata agatcactac 1200cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa atgagccata 1260ttcaacggga aacgtcttgc tcgaggccgc gattaaattc caacatggat gctgatttat 1320atgggtataa atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt 1380atgggaagcc cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg 1440atgttacaga tgagatggtc aggctaaact ggctgacgga atttatgcct cttccgacca 1500tcaagcattt tatccgtact cctgatgatg catggttact caccactgcg atcccaggga 1560aaacagcatt ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc 1620tggcagtgtt cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacggcg 1680atcgcgtatt tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttggtgcga 1740gtgattttga tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata 1800agcttttgcc attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc 1860ttatttttga cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag 1920accgatacca ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac 1980agaaacggct ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc 2040acttgatgct cgatgagttt ttctaatgag ggcccaaatg taatcacctg gctcaccttc 2100gggtgggcct ttctgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 2160acaaaaatcg atgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 2220cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 2280acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2340atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 2400agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 2460acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 2520gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 2580gtatctgcgc tctgctgaag ccagttacct cggaaaaaga gttggtagct cttgatccgg 2640caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2700aaaaaaagga tctcaagaag atcctttgat tttctaccga agaaaggccc acccgtgaag 2760gtgagccagt gagttgattg cagtccagtt acgctggagt ctgaggctcg tcctgaatga 2820tatcaagctt gaattcgtt 2839382819DNAArtificial SequencePlasmid pSMART-phoBp-GFPuv 38tgaggctcgt cctgaatgat atcaagcttg aattcgttgc cacggaaatc aataacctga 60agatatgtgc gacgagcttt tcataaatct gtcataaatc tgacgcataa tgacgtcgca 120ttaatgatcg caacctattt attgtgtagg aggataatct atggctagca aaggagaaga 180acttttcaca tggctagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt 240gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat 300gctacatacg gaaagcttac ccttaaattt atttgcacta ctggaaaact acctgttcca 360tggccaacac ttgtcactac tttctcttat ggtgttcaat gcttttcccg ttatccggat 420catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc 480actatatctt tcaaagatga cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt 540gatacccttg ttaatcgtat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt 600ctcggacaca aactcgagta caactataac tcacacaatg tatacatcac ggcagacaaa 660caaaagaatg gaatcaaagc taacttcaaa attcgccaca acattgaaga tggatccgtt 720caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca 780gacaaccatt acctgtcgac acaatctgcc ctttcgaaag atcccaacga aaagcgtgac 840cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc 900tacaaataat gaggatcccc ggcttatcgg tcagtttcac ctgatttacg taaaaacccg 960cttcggcggg tttttgcttt tggaggggca gaaagatgaa tgactgtcca cgacgctata 1020cccaaaagaa agacgaattc tctagatatc gctcaatact gaccatttaa atcatacctg 1080acctccatag cagaaagtca aaagcctccg accggaggct tttgacttga tcggcacgta 1140agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta 1200tcgagatttt caggagctaa ggaagctaaa atgagccata ttcaacggga aacgtcttgc 1260tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc 1320gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca 1380gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc 1440aggctaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact 1500cctgatgatg catggttact caccactgcg atcccaggga aaacagcatt ccaggtatta 1560gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg 1620ttgcattcga ttcctgtttg taattgtcct tttaacggcg atcgcgtatt tcgtctcgct 1680caggcgcaat cacgaatgaa taacggtttg gttggtgcga gtgattttga tgacgagcgt 1740aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg 1800gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa 1860ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc 1920atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa 1980tatggtattg ataatcctga tatgaataaa ttgcagtttc acttgatgct cgatgagttt 2040ttctaatgag ggcccaaatg taatcacctg gctcaccttc gggtgggcct ttctgcgttg 2100ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg atgctcaagt 2160cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 2220ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 2280tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 2340gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2400tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2460gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2520tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 2580ccagttacct cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2640gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2700atcctttgat tttctaccga agaaaggccc acccgtgaag gtgagccagt gagttgattg 2760cagtccagtt acgctggagt ctgaggctcg tcctgaatga tatcaagctt gaattcgtt 2819392880DNAArtificial SequencePlasmid pSMART-phoEp-GFPuv 39tgaggctcgt cctgaatgat atcaagcttg aattcgttag catggcgttt tgttgcgcgg 60gatcagcaag cctagcggca gttgtttacg cttttattac agatttaata aattaccaca 120ttttaagaat attattaatc tgtaatatat ctttaacaat ctcaggttaa aaactttcct 180gttttcaacg ggactctccc gctggtgtag gaggataatc tatggctagc aaaggagaag 240aacttttcac atggctagca aaggagaaga acttttcact ggagttgtcc caattcttgt 300tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 360tgctacatac ggaaagctta cccttaaatt tatttgcact actggaaaac tacctgttcc 420atggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttccc gttatccgga 480tcatatgaaa cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaacg 540cactatatct ttcaaagatg acgggaacta caagacgcgt gctgaagtca agtttgaagg 600tgataccctt gttaatcgta tcgagttaaa aggtattgat tttaaagaag atggaaacat 660tctcggacac aaactcgagt acaactataa ctcacacaat gtatacatca cggcagacaa 720acaaaagaat ggaatcaaag ctaacttcaa aattcgccac aacattgaag atggatccgt 780tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc 840agacaaccat tacctgtcga cacaatctgc cctttcgaaa gatcccaacg aaaagcgtga 900ccacatggtc cttcttgagt ttgtaactgc tgctgggatt acacatggca tggatgagct 960ctacaaataa tgaggatccc cggcttatcg gtcagtttca cctgatttac gtaaaaaccc 1020gcttcggcgg gtttttgctt ttggaggggc agaaagatga atgactgtcc acgacgctat 1080acccaaaaga aagacgaatt ctctagatat cgctcaatac tgaccattta aatcatacct 1140gacctccata gcagaaagtc aaaagcctcc gaccggaggc ttttgacttg atcggcacgt 1200aagaggttcc aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt 1260atcgagattt tcaggagcta aggaagctaa aatgagccat attcaacggg aaacgtcttg 1320ctcgaggccg cgattaaatt ccaacatgga tgctgattta tatgggtata aatgggctcg 1380cgataatgtc gggcaatcag gtgcgacaat ctatcgattg tatgggaagc ccgatgcgcc 1440agagttgttt ctgaaacatg gcaaaggtag cgttgccaat gatgttacag atgagatggt 1500caggctaaac tggctgacgg aatttatgcc tcttccgacc atcaagcatt ttatccgtac 1560tcctgatgat gcatggttac tcaccactgc gatcccaggg aaaacagcat tccaggtatt 1620agaagaatat cctgattcag gtgaaaatat tgttgatgcg ctggcagtgt tcctgcgccg 1680gttgcattcg attcctgttt gtaattgtcc ttttaacggc gatcgcgtat ttcgtctcgc 1740tcaggcgcaa tcacgaatga ataacggttt ggttggtgcg agtgattttg atgacgagcg 1800taatggctgg cctgttgaac aagtctggaa agaaatgcat aagcttttgc cattctcacc 1860ggattcagtc gtcactcatg gtgatttctc acttgataac cttatttttg acgaggggaa 1920attaataggt tgtattgatg ttggacgagt cggaatcgca gaccgatacc aggatcttgc 1980catcctatgg aactgcctcg gtgagttttc tccttcatta cagaaacggc tttttcaaaa 2040atatggtatt gataatcctg atatgaataa attgcagttt cacttgatgc tcgatgagtt 2100tttctaatga gggcccaaat gtaatcacct ggctcacctt cgggtgggcc tttctgcgtt 2160gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gatgctcaag 2220tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 2280cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 2340ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 2400cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 2460atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 2520agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 2580gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 2640gccagttacc tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 2700agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 2760gatcctttga ttttctaccg aagaaaggcc cacccgtgaa ggtgagccag tgagttgatt 2820gcagtccagt tacgctggag tctgaggctc gtcctgaatg atatcaagct tgaattcgtt 2880402879DNAArtificial SequencePlasmid pSMART-phoHp-GFPuv 40tgaggctcgt cctgaatgat atcaagcttg aattcgttaa tcctgctgaa agcacacagc 60ttttttcatc actgtcatca ctctgtcatc tttccagtag aaactaatgt cactgaaatg 120gtgttttata gttaaatata agtaaatata ttgttgcaat aaatgcgaga tctgttgtac 180ttattaagta gcagcggaag ttcgtgtagg aggataatct atggctagca aaggagaaga 240acttttcaca tggctagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt 300gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat 360gctacatacg gaaagcttac ccttaaattt atttgcacta ctggaaaact acctgttcca 420tggccaacac ttgtcactac tttctcttat ggtgttcaat gcttttcccg ttatccggat 480catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc 540actatatctt tcaaagatga cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt 600gatacccttg ttaatcgtat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt 660ctcggacaca aactcgagta caactataac tcacacaatg tatacatcac ggcagacaaa 720caaaagaatg gaatcaaagc taacttcaaa attcgccaca acattgaaga tggatccgtt 780caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca 840gacaaccatt acctgtcgac acaatctgcc ctttcgaaag atcccaacga aaagcgtgac 900cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc 960tacaaataat gaggatcccc ggcttatcgg tcagtttcac ctgatttacg taaaaacccg 1020cttcggcggg tttttgcttt tggaggggca gaaagatgaa tgactgtcca cgacgctata 1080cccaaaagaa agacgaattc tctagatatc gctcaatact gaccatttaa atcatacctg 1140acctccatag cagaaagtca aaagcctccg accggaggct tttgacttga tcggcacgta 1200agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta 1260tcgagatttt caggagctaa ggaagctaaa atgagccata ttcaacggga aacgtcttgc 1320tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc 1380gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca 1440gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc 1500aggctaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact 1560cctgatgatg catggttact caccactgcg atcccaggga aaacagcatt ccaggtatta 1620gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg 1680ttgcattcga ttcctgtttg taattgtcct tttaacggcg atcgcgtatt tcgtctcgct 1740caggcgcaat cacgaatgaa taacggtttg gttggtgcga gtgattttga tgacgagcgt 1800aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg 1860gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa 1920ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc 1980atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa 2040tatggtattg ataatcctga tatgaataaa ttgcagtttc acttgatgct cgatgagttt 2100ttctaatgag ggcccaaatg taatcacctg gctcaccttc gggtgggcct ttctgcgttg 2160ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg atgctcaagt 2220cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 2280ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 2340tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 2400gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2460tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2520gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2580tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 2640ccagttacct cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2700gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2760atcctttgat

tttctaccga agaaaggccc acccgtgaag gtgagccagt gagttgattg 2820cagtccagtt acgctggagt ctgaggctcg tcctgaatga tatcaagctt gaattcgtt 2879412850DNAArtificial SequencePlasmid pSMART-phoUp-GFPuv 41tgaggctcgt cctgaatgat atcaagcttg aattcgttac cgaactgaag caggattaca 60ccgtggtgat cgtcacccac aacatgcagc aggctgcgcg ttgttccgac cacacggcgt 120ttatgtacct gggcgaattg attgagttca gcaacacgga cgatctgttc accagtgtag 180gaggataatc tatggctagc aaaggagaag aacttttcac atggctagca aaggagaaga 240acttttcact ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa 300attttctgtc agtggagagg gtgaaggtga tgctacatac ggaaagctta cccttaaatt 360tatttgcact actggaaaac tacctgttcc atggccaaca cttgtcacta ctttctctta 420tggtgttcaa tgcttttccc gttatccgga tcatatgaaa cggcatgact ttttcaagag 480tgccatgccc gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaacta 540caagacgcgt gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa 600aggtattgat tttaaagaag atggaaacat tctcggacac aaactcgagt acaactataa 660ctcacacaat gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa 720aattcgccac aacattgaag atggatccgt tcaactagca gaccattatc aacaaaatac 780tccaattggc gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgc 840cctttcgaaa gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc 900tgctgggatt acacatggca tggatgagct ctacaaataa tgaggatccc cggcttatcg 960gtcagtttca cctgatttac gtaaaaaccc gcttcggcgg gtttttgctt ttggaggggc 1020agaaagatga atgactgtcc acgacgctat acccaaaaga aagacgaatt ctctagatat 1080cgctcaatac tgaccattta aatcatacct gacctccata gcagaaagtc aaaagcctcc 1140gaccggaggc ttttgacttg atcggcacgt aagaggttcc aactttcacc ataatgaaat 1200aagatcacta ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa 1260aatgagccat attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga 1320tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat 1380ctatcgattg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag 1440cgttgccaat gatgttacag atgagatggt caggctaaac tggctgacgg aatttatgcc 1500tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc 1560gatcccaggg aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat 1620tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc 1680ttttaacggc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt 1740ggttggtgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa 1800agaaatgcat aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc 1860acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt 1920cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc 1980tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa 2040attgcagttt cacttgatgc tcgatgagtt tttctaatga gggcccaaat gtaatcacct 2100ggctcacctt cgggtgggcc tttctgcgtt gctggcgttt ttccataggc tccgcccccc 2160tgacgagcat cacaaaaatc gatgctcaag tcagaggtgg cgaaacccga caggactata 2220aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 2280gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 2340acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 2400accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 2460ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 2520gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 2580aacagtattt ggtatctgcg ctctgctgaa gccagttacc tcggaaaaag agttggtagc 2640tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 2700attacgcgca gaaaaaaagg atctcaagaa gatcctttga ttttctaccg aagaaaggcc 2760cacccgtgaa ggtgagccag tgagttgatt gcagtccagt tacgctggag tctgaggctc 2820gtcctgaatg atatcaagct tgaattcgtt 2850422900DNAArtificial SequencePlasmid pSMART-mipAp-GFPuv 42tgaggctcgt cctgaatgat atcaagcttg aattcgttca tccataaatt ttgcataatt 60aatgtaaaga ccaggctcgc cagtaacgct aaattcattt ggctgtaagc gcggtgtcat 120ccgcgtcagg aaaattaaac agttacttta aaaaatgaaa acgtaaaaag gttgggtttc 180gatgtattga cgggtaaact ttgtcgcccg ctaaacattt gtttgtgtag gaggataatc 240tatggctagc aaaggagaag aacttttcac atggctagca aaggagaaga acttttcact 300ggagttgtcc caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc 360agtggagagg gtgaaggtga tgctacatac ggaaagctta cccttaaatt tatttgcact 420actggaaaac tacctgttcc atggccaaca cttgtcacta ctttctctta tggtgttcaa 480tgcttttccc gttatccgga tcatatgaaa cggcatgact ttttcaagag tgccatgccc 540gaaggttatg tacaggaacg cactatatct ttcaaagatg acgggaacta caagacgcgt 600gctgaagtca agtttgaagg tgataccctt gttaatcgta tcgagttaaa aggtattgat 660tttaaagaag atggaaacat tctcggacac aaactcgagt acaactataa ctcacacaat 720gtatacatca cggcagacaa acaaaagaat ggaatcaaag ctaacttcaa aattcgccac 780aacattgaag atggatccgt tcaactagca gaccattatc aacaaaatac tccaattggc 840gatggccctg tccttttacc agacaaccat tacctgtcga cacaatctgc cctttcgaaa 900gatcccaacg aaaagcgtga ccacatggtc cttcttgagt ttgtaactgc tgctgggatt 960acacatggca tggatgagct ctacaaataa tgaggatccc cggcttatcg gtcagtttca 1020cctgatttac gtaaaaaccc gcttcggcgg gtttttgctt ttggaggggc agaaagatga 1080atgactgtcc acgacgctat acccaaaaga aagacgaatt ctctagatat cgctcaatac 1140tgaccattta aatcatacct gacctccata gcagaaagtc aaaagcctcc gaccggaggc 1200ttttgacttg atcggcacgt aagaggttcc aactttcacc ataatgaaat aagatcacta 1260ccgggcgtat tttttgagtt atcgagattt tcaggagcta aggaagctaa aatgagccat 1320attcaacggg aaacgtcttg ctcgaggccg cgattaaatt ccaacatgga tgctgattta 1380tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgattg 1440tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat 1500gatgttacag atgagatggt caggctaaac tggctgacgg aatttatgcc tcttccgacc 1560atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatcccaggg 1620aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg 1680ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacggc 1740gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttggtgcg 1800agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat 1860aagcttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac 1920cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca 1980gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta 2040cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt 2100cacttgatgc tcgatgagtt tttctaatga gggcccaaat gtaatcacct ggctcacctt 2160cgggtgggcc tttctgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 2220cacaaaaatc gatgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 2280gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 2340tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 2400tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 2460cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 2520gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 2580ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 2640ggtatctgcg ctctgctgaa gccagttacc tcggaaaaag agttggtagc tcttgatccg 2700gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 2760gaaaaaaagg atctcaagaa gatcctttga ttttctaccg aagaaaggcc cacccgtgaa 2820ggtgagccag tgagttgatt gcagtccagt tacgctggag tctgaggctc gtcctgaatg 2880atatcaagct tgaattcgtt 2900432816DNAArtificial SequencePlasmid pSMART-pstSp-GFPuv 43tgaggctcgt cctgaatgat atcaagcttg aattcgttaa gactttatct ctctgtcata 60aaactgtcat attccttaca tataactgtc acctgtttgt cctattttgc ttctcgtagc 120caacaaacaa tgctttatga gtgtaggagg ataatctatg gctagcaaag gagaagaact 180tttcacatgg ctagcaaagg agaagaactt ttcactggag ttgtcccaat tcttgttgaa 240ttagatggtg atgttaatgg gcacaaattt tctgtcagtg gagagggtga aggtgatgct 300acatacggaa agcttaccct taaatttatt tgcactactg gaaaactacc tgttccatgg 360ccaacacttg tcactacttt ctcttatggt gttcaatgct tttcccgtta tccggatcat 420atgaaacggc atgacttttt caagagtgcc atgcccgaag gttatgtaca ggaacgcact 480atatctttca aagatgacgg gaactacaag acgcgtgctg aagtcaagtt tgaaggtgat 540acccttgtta atcgtatcga gttaaaaggt attgatttta aagaagatgg aaacattctc 600ggacacaaac tcgagtacaa ctataactca cacaatgtat acatcacggc agacaaacaa 660aagaatggaa tcaaagctaa cttcaaaatt cgccacaaca ttgaagatgg atccgttcaa 720ctagcagacc attatcaaca aaatactcca attggcgatg gccctgtcct tttaccagac 780aaccattacc tgtcgacaca atctgccctt tcgaaagatc ccaacgaaaa gcgtgaccac 840atggtccttc ttgagtttgt aactgctgct gggattacac atggcatgga tgagctctac 900aaataatgag gatccccggc ttatcggtca gtttcacctg atttacgtaa aaacccgctt 960cggcgggttt ttgcttttgg aggggcagaa agatgaatga ctgtccacga cgctataccc 1020aaaagaaaga cgaattctct agatatcgct caatactgac catttaaatc atacctgacc 1080tccatagcag aaagtcaaaa gcctccgacc ggaggctttt gacttgatcg gcacgtaaga 1140ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg 1200agattttcag gagctaagga agctaaaatg agccatattc aacgggaaac gtcttgctcg 1260aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat 1320aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag 1380ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcagg 1440ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct 1500gatgatgcat ggttactcac cactgcgatc ccagggaaaa cagcattcca ggtattagaa 1560gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg 1620cattcgattc ctgtttgtaa ttgtcctttt aacggcgatc gcgtatttcg tctcgctcag 1680gcgcaatcac gaatgaataa cggtttggtt ggtgcgagtg attttgatga cgagcgtaat 1740ggctggcctg ttgaacaagt ctggaaagaa atgcataagc ttttgccatt ctcaccggat 1800tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta 1860ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc 1920ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat 1980ggtattgata atcctgatat gaataaattg cagtttcact tgatgctcga tgagtttttc 2040taatgagggc ccaaatgtaa tcacctggct caccttcggg tgggcctttc tgcgttgctg 2100gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgatg ctcaagtcag 2160aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 2220gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 2280ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 2340cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 2400ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 2460actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 2520tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca 2580gttacctcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 2640gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 2700ctttgatttt ctaccgaaga aaggcccacc cgtgaaggtg agccagtgag ttgattgcag 2760tccagttacg ctggagtctg aggctcgtcc tgaatgatat caagcttgaa ttcgtt 2816442808DNAArtificial SequencePlasmid pSMART-ugpBp-GFPuv 44tgaggctcgt cctgaatgat atcaagcttg aattcgtttc tttctgacac cttactatct 60tacaaatgta acaaaaaagt tatttttctg taattcgagc atgtcatgtt accccgcgag 120cataaaacgc gtgtgtagga ggataatcta tggctagcaa aggagaagaa cttttcacat 180ggctagcaaa ggagaagaac ttttcactgg agttgtccca attcttgttg aattagatgg 240tgatgttaat gggcacaaat tttctgtcag tggagagggt gaaggtgatg ctacatacgg 300aaagcttacc cttaaattta tttgcactac tggaaaacta cctgttccat ggccaacact 360tgtcactact ttctcttatg gtgttcaatg cttttcccgt tatccggatc atatgaaacg 420gcatgacttt ttcaagagtg ccatgcccga aggttatgta caggaacgca ctatatcttt 480caaagatgac gggaactaca agacgcgtgc tgaagtcaag tttgaaggtg atacccttgt 540taatcgtatc gagttaaaag gtattgattt taaagaagat ggaaacattc tcggacacaa 600actcgagtac aactataact cacacaatgt atacatcacg gcagacaaac aaaagaatgg 660aatcaaagct aacttcaaaa ttcgccacaa cattgaagat ggatccgttc aactagcaga 720ccattatcaa caaaatactc caattggcga tggccctgtc cttttaccag acaaccatta 780cctgtcgaca caatctgccc tttcgaaaga tcccaacgaa aagcgtgacc acatggtcct 840tcttgagttt gtaactgctg ctgggattac acatggcatg gatgagctct acaaataatg 900aggatccccg gcttatcggt cagtttcacc tgatttacgt aaaaacccgc ttcggcgggt 960ttttgctttt ggaggggcag aaagatgaat gactgtccac gacgctatac ccaaaagaaa 1020gacgaattct ctagatatcg ctcaatactg accatttaaa tcatacctga cctccatagc 1080agaaagtcaa aagcctccga ccggaggctt ttgacttgat cggcacgtaa gaggttccaa 1140ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat cgagattttc 1200aggagctaag gaagctaaaa tgagccatat tcaacgggaa acgtcttgct cgaggccgcg 1260attaaattcc aacatggatg ctgatttata tgggtataaa tgggctcgcg ataatgtcgg 1320gcaatcaggt gcgacaatct atcgattgta tgggaagccc gatgcgccag agttgtttct 1380gaaacatggc aaaggtagcg ttgccaatga tgttacagat gagatggtca ggctaaactg 1440gctgacggaa tttatgcctc ttccgaccat caagcatttt atccgtactc ctgatgatgc 1500atggttactc accactgcga tcccagggaa aacagcattc caggtattag aagaatatcc 1560tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat 1620tcctgtttgt aattgtcctt ttaacggcga tcgcgtattt cgtctcgctc aggcgcaatc 1680acgaatgaat aacggtttgg ttggtgcgag tgattttgat gacgagcgta atggctggcc 1740tgttgaacaa gtctggaaag aaatgcataa gcttttgcca ttctcaccgg attcagtcgt 1800cactcatggt gatttctcac ttgataacct tatttttgac gaggggaaat taataggttg 1860tattgatgtt ggacgagtcg gaatcgcaga ccgataccag gatcttgcca tcctatggaa 1920ctgcctcggt gagttttctc cttcattaca gaaacggctt tttcaaaaat atggtattga 1980taatcctgat atgaataaat tgcagtttca cttgatgctc gatgagtttt tctaatgagg 2040gcccaaatgt aatcacctgg ctcaccttcg ggtgggcctt tctgcgttgc tggcgttttt 2100ccataggctc cgcccccctg acgagcatca caaaaatcga tgctcaagtc agaggtggcg 2160aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 2220tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 2280ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 2340gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 2400tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 2460caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 2520ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctc 2580ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 2640tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatt 2700ttctaccgaa gaaaggccca cccgtgaagg tgagccagtg agttgattgc agtccagtta 2760cgctggagtc tgaggctcgt cctgaatgat atcaagcttg aattcgtt 2808452819DNAArtificial SequencePlasmid pSMART-ydfHp-GFPuv 45tgaggctcgt cctgaatgat atcaagcttg aattcgttgc tatgccggac tgaatgtcca 60ccgtcagtaa tttttatacc cggcgtaact gccgggttat tgcttgtcac aaaaaagtgg 120tagactcatg cagttaactc actgtgtagg aggataatct atggctagca aaggagaaga 180acttttcaca tggctagcaa aggagaagaa cttttcactg gagttgtccc aattcttgtt 240gaattagatg gtgatgttaa tgggcacaaa ttttctgtca gtggagaggg tgaaggtgat 300gctacatacg gaaagcttac ccttaaattt atttgcacta ctggaaaact acctgttcca 360tggccaacac ttgtcactac tttctcttat ggtgttcaat gcttttcccg ttatccggat 420catatgaaac ggcatgactt tttcaagagt gccatgcccg aaggttatgt acaggaacgc 480actatatctt tcaaagatga cgggaactac aagacgcgtg ctgaagtcaa gtttgaaggt 540gatacccttg ttaatcgtat cgagttaaaa ggtattgatt ttaaagaaga tggaaacatt 600ctcggacaca aactcgagta caactataac tcacacaatg tatacatcac ggcagacaaa 660caaaagaatg gaatcaaagc taacttcaaa attcgccaca acattgaaga tggatccgtt 720caactagcag accattatca acaaaatact ccaattggcg atggccctgt ccttttacca 780gacaaccatt acctgtcgac acaatctgcc ctttcgaaag atcccaacga aaagcgtgac 840cacatggtcc ttcttgagtt tgtaactgct gctgggatta cacatggcat ggatgagctc 900tacaaataat gaggatcccc ggcttatcgg tcagtttcac ctgatttacg taaaaacccg 960cttcggcggg tttttgcttt tggaggggca gaaagatgaa tgactgtcca cgacgctata 1020cccaaaagaa agacgaattc tctagatatc gctcaatact gaccatttaa atcatacctg 1080acctccatag cagaaagtca aaagcctccg accggaggct tttgacttga tcggcacgta 1140agaggttcca actttcacca taatgaaata agatcactac cgggcgtatt ttttgagtta 1200tcgagatttt caggagctaa ggaagctaaa atgagccata ttcaacggga aacgtcttgc 1260tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc 1320gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc cgatgcgcca 1380gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc 1440aggctaaact ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact 1500cctgatgatg catggttact caccactgcg atcccaggga aaacagcatt ccaggtatta 1560gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg 1620ttgcattcga ttcctgtttg taattgtcct tttaacggcg atcgcgtatt tcgtctcgct 1680caggcgcaat cacgaatgaa taacggtttg gttggtgcga gtgattttga tgacgagcgt 1740aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc attctcaccg 1800gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa 1860ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc 1920atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa 1980tatggtattg ataatcctga tatgaataaa ttgcagtttc acttgatgct cgatgagttt 2040ttctaatgag ggcccaaatg taatcacctg gctcaccttc gggtgggcct ttctgcgttg 2100ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg atgctcaagt 2160cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 2220ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 2280tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 2340gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 2400tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 2460gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 2520tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 2580ccagttacct cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 2640gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 2700atcctttgat tttctaccga agaaaggccc acccgtgaag gtgagccagt gagttgattg 2760cagtccagtt acgctggagt ctgaggctcg tcctgaatga tatcaagctt gaattcgtt 2819463424DNAArtificial SequencePlasmid pSMART-Ala2 46ccaggcatca aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt 60gtttgtcggt gaacgctctc tactagagtc acactggctc accttcgggt gggcctttct 120gcgtttatac acagctaaca ccacgtcgtc cctatctgct gccctaggtc tatgagtggt 180tgctggataa ctctttctga caccttacta tcttacaaat gtaacaaaaa agttattttt 240ctgtaattcg agcatgtcat gttaccccgc gagcataaaa cgcgtatatt cagggagacc 300acaacggttt

ccctctacaa ataattttgt ttaactttgg aaaaaggaga tataccatga 360tcattggggt gccgaaggag atcaaaaata atgagaaccg cgtcgcgttg accccgggag 420gtgtcagcca gctgatctct aatggccatc gtgtcttagt tgaaacaggc gctggcctgg 480gttctggctt cgaaaacgag gcctacgaat ctgcaggtgc ggaaattatt gctgatccaa 540aacaggtctg ggatgcagag atggtcatga aagtgaaaga accgctcccg gaagaatatg 600tctattttcg taaaggtctg gtgctgttta catatctgca tctggcagct gaaccggagc 660tcgcacaagc ccttaaagat aaaggtgtca cggccatcgc atacgaaact gtcagcgaag 720ggcgcacgct gccattactg accccgatgt cagaagtggc aggccgtatg gctgcgcaga 780tcggcgcaca gtttcttgaa aaaccaaagg gcgggaaggg tattctctta gcaggagtgc 840cgggcgtcag tcgtgggaaa gtaactatta ttggtggcgg cgtggtagga acaaatgctg 900ccaaaatggc cgtcggtttg ggggccgacg taacaatcat tgcgcgtaat gccgatcgcc 960ttcgtcaatt agacgatatc tttggccacc aaatcaaaac cctgatttcg aacccagtca 1020atatcgcgga tgcggtggcg gaagctgatt tgttgatctg cgccgtgtta attccgggag 1080cgaaagcacc tacattggtg acggaagaaa tggtgaaaca aatgaaaccg ggttcagtca 1140ttgttgatgt ggctattgat cagggtggca tcgtggaaac ggtggaccat attaccactc 1200acgaccagcc gacgtatgaa aaacatggtg tcgtacacta tgcggtggcg aatatgcctg 1260gtgcggtccc acgtacgagt acaatcgcac tgacaaatgt caccgtgccg tatgcgttgc 1320aaatcgcgaa caaaggtgcc gtgaaagcgc tggccgacaa tacggcgtta cgtgccggtc 1380tgaacaccgc taacggtcac gtgacatatg aagcggtcgc gcgtgatttg gggtacgaat 1440atgtaccggc ggaaaaagcc ttacaagacg aatcgagtgt cgctggtgca tagtaagctc 1500ttctaatacg actcactata gggccggctt atcggtcagt ttcacctgat ttacgtaaaa 1560acccgcttcg gcgggttttt gcttttggag gggcagaaag atgaatgact gtccacgacg 1620ctatacccaa aagaaagacg aattctctag atatcgctca atactgacca tttaaatcat 1680acctgacctc catagcagaa agtcaaaagc ctccgaccgg aggcttttga cttgatcggc 1740acgtaagagg ttccaacttt caccataatg aaataagatc actaccgggc gtattttttg 1800agttatcgag attttcagga gctaaggaag ctaaaatgag ccatattcaa cgggaaacgt 1860cttgctcgag gccgcgatta aattccaaca tggatgctga tttatatggg tataaatggg 1920ctcgcgataa tgtcgggcaa tcaggtgcga caatctatcg attgtatggg aagcccgatg 1980cgccagagtt gtttctgaaa catggcaaag gtagcgttgc caatgatgtt acagatgaga 2040tggtcaggct aaactggctg acggaattta tgcctcttcc gaccatcaag cattttatcc 2100gtactcctga tgatgcatgg ttactcacca ctgcgatccc agggaaaaca gcattccagg 2160tattagaaga atatcctgat tcaggtgaaa atattgttga tgcgctggca gtgttcctgc 2220gccggttgca ttcgattcct gtttgtaatt gtccttttaa cggcgatcgc gtatttcgtc 2280tcgctcaggc gcaatcacga atgaataacg gtttggttgg tgcgagtgat tttgatgacg 2340agcgtaatgg ctggcctgtt gaacaagtct ggaaagaaat gcataagctt ttgccattct 2400caccggattc agtcgtcact catggtgatt tctcacttga taaccttatt tttgacgagg 2460ggaaattaat aggttgtatt gatgttggac gagtcggaat cgcagaccga taccaggatc 2520ttgccatcct atggaactgc ctcggtgagt tttctccttc attacagaaa cggctttttc 2580aaaaatatgg tattgataat cctgatatga ataaattgca gtttcacttg atgctcgatg 2640agtttttcta atgagggccc aaatgtaatc acctggctca ccttcgggtg ggcctttctg 2700cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgatgct 2760caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa 2820gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc 2880tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt 2940aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg 3000ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg 3060cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct 3120tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc 3180tgaagccagt tacctcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 3240tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 3300agaagatcct ttgattttct accgaagaaa ggcccacccg tgaaggtgag ccagtgagtt 3360gattgcagtc cagttacgct ggagtctgag gctcgtcctg aatgatatca agcttgaatt 3420cgtt 34244721DNAArtificial SequencefabI T2 targeting sequence 47cagcctgctc cggtcggacc g 214845DNAArtificial SequenceC-terminal DAS+4 tag 48gcggccaacg atgaaaacta ttctgaaaac tatgcggatg cgtct 454959DNAArtificial SequencePrimer gltA2-FOR 49gggacagtta ttagttcgag ttccccgcgc cagcggggat aaaccgaaaa aaaaacccc 595063DNAArtificial SequencePrimer gltA2-REV 50gaatgaattg gtcaatacgg tttatccccg ctggcgcggg gaactcgagg tggtaccaga 60tct 635128DNAArtificial SequencePrimer G2U-FOR1 51ccggatgagc attcatcagg cgggcaag 285247DNAArtificial SequencePrimer G2U-REV1 52cggtttatcc ccgctggcgc ggggaactcg aacttcataa cttttac 475334DNAArtificial SequencePrimer G2U-FOR2 53gcgccagcgg ggataaaccg ttaccattct gttg 345428DNAArtificial SequencePrimer G2U-REV2 54cttgcccgcc tgatgaatgc tcatccgg 28

Claims

1.-77. (canceled)

78. A genetically modified microorganism comprising: a production pathway comprising at least one enzyme for the production of a product, and at least one synthetic metabolic valve comprising controlled transcriptional gene silencing of a gene encoding an enzyme that is one of: enoyl-ACP reductase (fabI), citrate synthase (gltA), soluble transhydrogenase (udhA), glucose-6-phosphate-l-dehydrogenase (zwf), lipoamide dehydrogenase (lpd), or combinations thereof; wherein transcriptional gene silencing comprises expression of small guide RNAs specific for the gene; wherein depletion of the limiting nutrient from a growth media in which the genetically modified microorganism is growing will inducing a stationary or non-dividing cellular state; wherein the synthetic metabolic valve of the microorganism may be conditionally activated.

79. The genetically modified microorganism of claim 78, wherein transcriptional gene silencing further comprises expression of a gene encoding a dCas9 protein.

80. The genetically modified microorganism of claim 78, wherein a single plasmid expresses small guide RNAs for two or more genes.

81. The genetically modified microorganism of claim 78, wherein the synthetic metabolic valve directed to controlled transcriptional gene silencing further comprises conditional expression of a proteolysis chaperone protein.

82. The genetically modified microorganism of claim 81, wherein the chaperone protein is a sspB protein.

83. The genetically modified microorganism of claim 78 wherein the genetically modified microorganism is characterized by disruption or deletion of a gene naturally occurring in the genetically modified microorganism, the naturally occurring gene one of a gene encoding lactate dehydrogenase (ldhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), the methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), ATP-dependent Lon protease (ion), outer membrane protease (ompT), arcA transcriptional dual regulator (arcA), iclR transcriptional regulator (icR), cas3, sspB or combinations thereof.

84. The genetically modified microorganism of claim 81 further comprising a synthetic metabolic valve comprising controlled proteolysis of an enzyme that is one of: enoyl-ACP reductase (fabI), citrate synthase (gltA), soluble transhydrogenase (udhA), glucose-6-phosphate-l-dehydrogenase (zwf), lipoamide dehydrogenase (lpd), or combinations thereof.

85. A bioprocess for production of a product from the genetically modified microorganism of claim 78, the bioprocess comprising: in a first stage, growing the genetically modified microorganism in a medium and in a second stage, reducing the genetically modified microorganism growth of the first stage and expressing a heterologous enzyme of the product production pathway, wherein, a transition from the first stage to the second stage is at least partially controlled by depletion of a level of a limiting nutrient from the media and, as the limiting nutrient is depleted from the media growth of the genetically modified microorganism is stopped, and the transition comprising activation of the synthetic metabolic valve.

86. The bioprocess of claim 85, wherein the product is one of an alcohol, a diol, a polyol, an organic acid, an amino acid, a fatty acid, a fatty acid derivative, an ester, an alkane, or an alkene.

87. The bioprocess of claim 85, wherein the limiting nutrient comprises inorganic phosphate.

88. The bioprocess of claim 85, wherein the genetically modified microorganism is characterized by disruption or deletion of a gene naturally occurring in the genetically modified microorganism, the naturally occurring gene one of a gene encoding lactate dehydrogenase (idhA), phosphate acetyltransferase (pta), pyruvate oxidase (poxB), pyruvate-formate lyase (pflB), the methylglyoxal synthase (mgsA), acetate kinase (ackA), alcohol dehydrogenase (adhE), ATP-dependent Lon protease (Ion), outer membrane protease (ompT), arcA transcriptional dual regulator (arcA), iclR transcriptional regulator (icR), or combinations thereof.

89. The bioprocess of claim 85, further comprising a second synthetic metabolic valve controlling at least one enzyme essential for growth of the genetically modified microorganism that is one of: sucD, aceA, pfkA, Ion, rpoS, tktA or tktB.