Sphingomonas Strains Producing Greatly Increased Yield Of PHB-Deficient Sphingan (Diutan)

Abstract

PHB-deficient Sphingomonas strains having improved sphingan yield are provided. Certain of the Sphingomonas strains are diutan-producing strains that exhibit a dramatic improvement in productivity and yield due to a combination of certain genetic modifications that affect PHB and sphingan synthesis. Moreover, the sphingans produced from such strains have superior characteristics including improved filterability, clarity, and improved rheology-modifying characteristics. The sphingans provided are, thus, highly desirable in a variety of commercial and industrial uses, including personal care items, cement applications, and oilfield applications.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is related to U.S. Ser. No. 11/264,268 entitled "High Viscosity Diutan Gums," filed Nov. 1, 2005, now pending, and U.S. Ser. No. 11/292,366 entitled "Mutant bacterial strains of the genus Sphingomonas deficient in production of polyhydroxybutyrate and a process of clarification of sphingans and compositions thereof," filed Dec. 2, 2005, now pending, which is a divisional of U.S. Ser. No. 09/798,642, filed Mar. 2, 2001, now pending, which claims the benefit of provisional application U.S. Ser. No. 60/186,433, filed Mar. 2, 2000, which are hereby incorporated by reference in their entireties to the extent that they are not inconsistent with the disclosure herein.

[0002] The sequence listing in the file named "68492o705000.txt" having a size of 179,295 bytes that was created Jul. 31, 2009 is hereby incorporated by reference in its entirety.

BACKGROUND

[0003] 1. Field of the Art

[0004] This application generally relates to the construction of PHB-deficient Sphingomonas strains that produce high yields of diutan with improved filterability. In another aspect, this application relates to diutan produced from PHB-deficient Sphingomonas strains that produce high yields of diutan with improved filterability.

[0005] 2. Description of Related Art

[0006] A number of bacteria of the genus Sphingomonas produce polysaccharides called sphingans that have related structures with a generally conserved tetrasaccharide backbone structure and different side chains (ref. no. 1, 6, 7, 8, 10). The sphingans gellan, welan, rhamsan and diutan are produced commercially for use in food, oilfield or personal care applications. The value of sphingan polysaccharides lies in their abilities to modify the rheology of aqueous solutions, i.e., to thicken liquids, suspend solids, stabilize emulsions, or form gels and films.

[0007] Sphingans are structurally related to one another, but are not identical. Common members of the genus Sphingomonas and the sphingans they produce include Sphingomonas elodea ATCC 31461, which produces gellan (S-60); Sphingomonas sp. ATCC 31555, which produces welan (S-130); Sphingomonas sp. ATCC 31961, which produces rhamsan (S-194); Sphingomonas sp. ATCC 53159, which produces diutan (S-657); Sphingomonas sp. ATCC 31554, which produces an as yet unnamed polysaccharide (S-88); Sphingomonas sp. ATCC 31853, which produces an as yet unnamed polysaccharide (S-198); Sphingomonas sp. ATCC 21423, which produces an as yet unnamed polysaccharide (S-7); Sphingomonas sp. ATCC 53272, which produces an as yet unnamed polysaccharide (NW-11); Sphingomonas sp. FERM-BP2015 (previously Alcaligenes latus B-16), which produces alcalan (Biopolymer B-16) and the like. A description of the Sphingomonads and the polysaccharides they produce can be found, for example, in U.S. Pat. Nos. 4,377,636; 4,326,053; 4,326,052 and 4,385,123 (for ATCC 31461 and its S-60 polysaccharide); in U.S. Pat. No. 4,342,866 (for ATCC 31555 and S-130); in U.S. Pat. No. 4,401,760 (for ATCC 31961 and S-194); in U.S. Pat. No. 5,175,278 (for ATCC 53159 and S-657); in U.S. Pat. Nos. 4,331,440 and 4,535,153 (for ATCC 31554 and S-88); in U.S. Pat. No. 4,529,797 (for ATCC 31853 and S-198); in U.S. Pat. No. 3,960,832 (for ATCC 21423 and S-7); in U.S. Pat. No. 4,874,044 (for ATCC 53272 and NW-11); in U.S. Pat. No. 5,175,279 (for FERM BP-2015 and B-16), each of which is incorporated by reference herein in its entirety to the extent that they are not inconsistent with the disclosure herein.

[0008] One particular sphingan, diutan (also known as heteropolysaccharide S-657), is prepared by fermentation of strain Sphingomonas sp. ATCC 53159 (ref. no. 17). Diutan exhibits unique rheological properties in aqueous solutions including high thermal stability, superior suspension properties, and the ability to generate high viscosity at low concentrations. The diutan polysaccharide imparts significant pseudoplasticity to polar solvents such as water, such that diutan can act as a rheological modifier that is capable of particle suspension, friction reduction, emulsion and foam stabilization, filter cake deposition and filtration control. Consequently, diutan has found industrial utility as a rheological modifier in a variety of contexts, including cementitious systems as disclosed in U.S. Pat. No. 6,110,271, which is incorporated herein by reference in its entirety to the extent that they are not inconsistent with the disclosure herein.

[0009] Diutan consists of a repeat unit with a backbone comprised of [.fwdarw.4)-.alpha.-L-rhamnose-(1.fwdarw.3)-.beta.-D-glucose-(1.fwdarw.4)- -.beta.-D-glucuronic acid-(1.fwdarw.4)-.beta.-D-glucose-(1.fwdarw.] and a two-sugar L-rhamnose side-chain attached to the (1.fwdarw.4) linked glucose residues (ref. no. 2, 7). Two O-acetyl groups are attached per repeat unit to the 2' and 6' positions of the (1.fwdarw.3) linked glucose (ref. no. 4).

[0010] Progress has been made in elucidating the genetics and biochemistry underlying biosynthesis of diutan and other sphingans. Genes for biosynthesis of sphingans S-88, S-7, and gellan have been identified (ref. no. 5, 12, 13, 15). Genes for several glycosyl transferases of the backbone structure have been analyzed biochemically (ref. no. 11, 14), as have genes geIC and gelE, potentially involved in chain length determination (ref. no. 9). Several of the genes for synthesis of sugar nucleotide precursors have also been elucidated (ref. no. 12). The genetics and biochemistry of polymerization, secretion and control of polysaccharide molecular length are less defined.

[0011] A cluster of genes involved in biosynthesis of diutan has been identified that includes genes for glycosyl transferases, genes encoding enzymes for synthesis of a precursor molecule dTDP rhamnose, and genes for secretion of the polysaccharide (ref. no. 3). Plasmids, e.g., pS8 and pX6, containing some of the genes in the aforementioned cluster, were shown to increase the yield of diutan by about 10%, and one plasmid in particular (pS8) was found to significantly improve the rheological properties of diutan from the wild-type strain (ref. no. 18).

[0012] Growth conditions typically used for producing diutan and other sphingans also promote production of the internal storage polymer polyhydroxybutyrate ("PHB"), which is generally regarded as an undesirable side-product and is difficult to remove during sphingan preparation. The PHB can form small insoluble particles that interfere with clarity and filterability, limiting the usefulness of the sphingans. For example, the turbidity imparted by PHB particles can limit applicability for household and personal care products in which appearance is critical for consumer acceptance. Moreover, certain oilfield uses require filterability; however, the PHB particles can plug small pores in oil field rock formations, preventing the flow of the sphingan solution and/or the return flow of the crude oil after treating the well. Finally, as PHB synthesis and sphingan synthesis compete for the available carbon source, PHB synthesis can have some adverse effect on sphingan yield.

[0013] Accordingly, attempts have been made to eliminate PHB production in sphingan-producing strains. Ref. no. 26 describes a strain of Sphingomonas elodea (a gellan-producing species) that was isolated following chemical mutagenesis. This strain, called LPG-2, has decreased PHB production, but produces gellan of inconsistent quality and yield.

[0014] A more targeted approach to eliminating PHB production was undertaken by deletion of a gene required for PHB synthesis, the phaC gene (ref. no. 20). Precise deletion of phaC from a diutan producing strain (ATCC 53159) reproducibly resulted in poor growth and severely reduced diutan productivity (strains NPD3 and NPD6). These strains exhibit increased carbohydrate hydrolysis and accumulation of organic acids, suggesting a critical role for phaC in maintaining normal cellular metabolism. Derivatives with less impaired diutan productivity were subsequently isolated. Two independent derivatives, PDD3 and PDD6, have uncharacterized spontaneous mutation(s) and remain PHB-deficient (ATCC deposit nos. PTA-4865 and PTA-4866, respectively). Though recovery of up to 90% of total diutan yield has been reported (ref. no. 20), this yield was only obtained following a greatly increased culture growth time and has not been consistently reproducible. Under standard growth conditions, diutan productivity and yield by these strains is only approximately half of wild-type levels.

SUMMARY

[0015] In view of the foregoing, there is a need to overcome the low sphingan productivity that is characteristic of PHB-deficient strains. The present disclosure addresses this need in the art by providing a genetically modified strain of Sphingomonas which not only lacks PHB production but also provides surprisingly high diutan productivity. Unexpectedly, the plasmids pS8 and pX6--which give only modest improvement in diutan productivity in PHB-producing strain--are now shown to greatly improve diutan productivity in a PHB-deficient strain. The great improvement in diutan productivity was particularly surprising because the plasmids contain genes involved in diutan biosynthesis and are not known to contain any genes that would offset the metabolic deficiency of a PHB-deficient strain. Certain embodiments of these genetically modified strains, described infra, fully overcome the poor yield and low productivity of PHB-deficient strains, while simultaneously attaining the desired filterability and clarity of PHB-deficient sphingans.

[0016] Certain embodiments encompass a mutant strain of the genus Sphingomonas having a genetic modification that reduces, or, preferably, substantially or entirely eliminates the production of PHB. In exemplary embodiments, the genetic modification inactivates the phaA gene, phaB gene, phaC gene, or any combination thereof. In another exemplary embodiment, the genetic modification to impair PHB synthesis is obtained by screening or selection for a PHB-deficient organism. The genetic modification that impairs PHB synthesis can reduce or completely eliminate PHB production, and can optionally be conditional, such as conditional induction, suppression, overexpression, knock-out, etc. of a gene involved in PHB synthesis, a gene that suppresses PHB synthesis, or any combination thereof. Optionally, a mutant strain of the genus Sphingomonas having a genetic modification that reduces, or, preferably, substantially or entirely eliminates the production of PHB also includes at least one additional genetic modification that suppresses the poor growth and/or poor diutan productivity of such strains. In an exemplary embodiment, the additional genetic modification can include at least one of the suppressor mutations contained in strains PDD3, PDD6, or both, or a variant of such suppressor mutation(s).

[0017] Certain embodiments encompass a method of increasing sphingan production in a host organism, such as an organism of the genus Sphingomonas. Exemplary methods of increasing sphingan production include increasing the expression in the host organism of at least one gene involved in sphingan synthesis. Such genes can be involved in sphingan synthesis, secretion, polymerization, synthesis of precursors, control of polysaccharide molecular length, etc. For example, additional copies of at least one gene involved in sphingan production can be introduced on an extrachromosomal element (such as a plasmid) or can be integrated into the host genome, or both. Such genes can be derived from the host strain or can be homologs derived from another species or strain. Homologs can include functional, structural, or sequence homologs of a gene involved in sphingan production or of a gene having an enzymatic activity the same as or similar to a gene involved in sphingan synthesis. In exemplary embodiments, the genes can be obtained by screening or selection for a Sphingomonas strain having increased sphingan production. Exemplary methods of increasing sphingan production also include introduction of genes involved in sphingan production having modified (non-native) sequences, such as modified promoter or enhancer elements, expression-optimized sequences, etc. Additionally, the native chromosomal copy of at least one gene involved in sphingan synthesis can optionally be deleted, or be replaced by any of the foregoing.

[0018] In certain embodiments, an extrachromosomal or integrated sequence element containing at least one gene, such as all of the genes that are contained in the insert in plasmid pS8 and/or pX6, or homolog(s) thereof, can be introduced into a Sphingomonas strain. For example, the at least one gene can include dpsS, dpsG, dpsR, dpsQ, dpsI, dpsK, dpsL, dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, orf7, orf6, orfs, or any combination thereof. In certain exemplary embodiments, the gene(s) include at least one gene encoding a sphingan biosynthetic enzyme, such as a dpsG polymerase. In another exemplary embodiment, such genes encoding a sphingan biosynthetic enzyme can include a dpsG polymerase and a glucose-1-phosphate thymidylyltransferase gene; a dTDP-6-deoxy-D-glucose-3-5-epimerase gene; a dTDP-D-glucose-4,6-dehydratase gene; and a dTDP-6-deoxy-L-mannose-dehydrogenase gene. In another exemplary embodiment, such genes encoding a sphingan biosynthetic enzyme can include a dpsG polymerase and a rhamnosyl transferase IV gene; a beta-1,4-glucuronosyl transferase II gene; a glucosyl isoprenylphosphate transferase I gene; and a glucosyl transferase III gene. In another exemplary embodiment, such a gene encoding a sphingan biosynthetic enzyme can include a dpsG polymerase and one or more of the polysaccharide export genes dpsD, dpsC, and dpsE. In another exemplary embodiment, such a gene encoding a sphingan biosynthetic enzyme can include a rhamnosyl transferase IV gene; a beta-1,4-glucuronosyl transferase II gene; a glucosyl isoprenylphosphate transferase I gene; glucosyl transferase III gene; a glucose-1-phosphate thymidylyltransferase gene; a dTDP-6-deoxy-D-glucose-3-5-epimerase gene; a dTDP-D-glucose-4,6-dehydratase gene; and a dTDP-6-deoxy-L-mannose-dehydrogenase gene. In another exemplary embodiment, such a sphingan biosynthetic enzyme can be selected from the group consisting of a gene encoding a polymerase; lyase; rhamnosyl transferase IV; beta-1,4-glucuronosyl transferase II; glucosyl transferase III; polysaccharide export protein; secretion protein; glucosyl-isoprenylphosphate transferase I; glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase, and any combination thereof. In certain embodiments, any combination of the foregoing genes or homologs thereof can be introduced into a Sphingomonas strain. In one exemplary embodiment, the Sphingomonas strain is a diutan-producing strain, such as ATCC 53159, or a PHB-deficient derivative thereof, such as a phaC deletion strain, such as NPD3, NPD6, PDD3, or PDD6. In another exemplary embodiment, the Sphingomonas strain is derived from Sphingomonas elodea ATCC 31461, Sphingomonas sp. ATCC 31555, Sphingomonas sp. ATCC 31961, Sphingomonas sp. ATCC 53159, Sphingomonas sp. ATCC 31554, Sphingomonas sp. ATCC 31853, Sphingomonas sp. ATCC 21423, Sphingomonas sp. ATCC 53272, Sphingomonas sp. FERM-BP2015, or a PHB-deficient derivative, such as a phaC deletion strain of any of the foregoing, or a phaC deletion strain bearing further mutation(s) that improve growth or sphingan productivity. In an exemplary embodiment, the phaC deletion strain is derived from a gellan-producing strain, such as LPG-2 (ref. no. 26), NPG-1, NPG-2, NPG-3, PDG-1, PDG-3 (ref. no. 20) or a derivative thereof.

[0019] In exemplary embodiments, a gene involved in sphingan synthesis can be derived from a homolog of a gene contained in plasmids pS8 or pX6. Such a homolog can be a Sphingomonas homolog, i.e., derived from an organism of the genus Sphingomonas. Exemplary organisms from which Sphingomonas homologs can be derived include Sphingomonas elodea ATCC 31461, Sphingomonas sp. ATCC 31555, Sphingomonas sp. ATCC 31961, Sphingomonas sp. ATCC 53159, Sphingomonas sp. ATCC 31554, Sphingomonas sp. ATCC 31853, Sphingomonas sp. ATCC 21423, Sphingomonas sp. ATCC 53272, Sphingomonas sp. FERM-BP2015, or any combination thereof. In another exemplary embodiment, a gene involved in sphingan synthesis can encode a polypeptide having at least about 70% sequence identity, such as about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity, to a polypeptide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, or 53. In another exemplary embodiment, a gene involved in sphingan synthesis can be encoded by a polynucleotide having at least about 60% sequence identity, such as about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity, to a polynucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52.

[0020] Certain embodiments of the present compositions include a diutan, particularly a PHB-deficient diutan, exhibiting an improvement (relative to diutan produced from a wild-type strain) in a number of different viscosity measurements. Among these are: i) an intrinsic viscosity of greater than about 150, preferably higher than about 155, more preferably higher than about 160 dL/g; ii) a sea water 3 rpm viscosity greater than about 35, such as greater than about 37, such as greater than about 40, such as greater than about 42, such as greater than about 45, such as greater than about 47, such as greater than about 50 dial reading; iii) a sea water 0.3 rpm viscosity greater than about 35,000, such as greater than about 39,000, such as greater than about 40,000, such as greater than about 42,000, such as greater than about 45,000, such as greater than about 48,000, such as greater than about 50,000, such as greater than about 54,000 centipoise (cP); and a PEG low shear rate viscosity greater than about 3500, such as greater than about 3700, such as greater than about 3900, such as greater than about 4000, such as greater than about 4200, such as greater than about 4500, such as greater than about 4700, such as greater than about 5000, such as greater than about 5200, such as greater than about 5500, such as greater than about 5700, such as greater than about 6000 cP.

[0021] Certain embodiments of the present strains include a mutant strain of the genus Sphingomonas that is able to produce PHB-deficient diutan at a rate of at least about 0.10 g/L/hr, such as at least about 0.11 g/L/hr, such as at least about 0.12 g/L/hr, such as at least about 0.13 g/L/hr, such as at least about 0.14 g/L/hr, such as at least about 0.15 g/L/hr, such as at least about 0.2 g/L/hr, and/or a yield of PHB-deficient diutan of at least about 12 g/L, such as at least about 15 g/L, such as at least about 16 g/L, such as at least about 17 g/L, such as at least about 18 g/L, such as at least about 19 g/L, such as at least about 20 g/L, such as at least about 21 g/L. For example, certain embodiments can include a mutant strain of the genus Sphingomonas able to produce PHB-deficient diutan at a rate of between about 0.15 g/L/hr and about 0.60 g/L/hr, such as between about 0.16 g/L/hr and about 0.5 g/L/hr, such as between about 0.17 g/L/hr and about 0.4 g/L/hr, such as between about 0.18 g/L/hr and about 0.35 g/L/hr, such as between about 0.19 g/L/hr and about 0.3 g/L/hr, such as between about 0.2 g/L/hr and 0.25 g/L/hr, such as between about 0.21 g/L/hr and about 0.22 g/L/hr. Additionally, certain embodiments can include a mutant strain of the genus Sphingomonas able to produce a yield of PHB-deficient diutan between about 12 g/L and about 30 g/L, such as between about 13 g/L and about 25 g/L, such as between about 14 g/L and about 22 g/L, such as between about 19 g/L and about 21 g/L.

[0022] Certain embodiments of the present strains include a mutant strain of the genus Sphingomonas containing a genetic modification that substantially or entirely eliminates the production of PHB and a genetic modification that results in increased production of a sphingan, wherein the mutant strain of the genus Sphingomonas increases the rate of production or yield of PHB-deficient diutan by at least about 50%, such as by at least about 60%, such as by at least about 70%, such as by at least about 80%, such as by at least about 90%, such as by at least about 100%, such as by at least about 110%, such as by at least about 120%, such as by at least about 120%, such as by at least about 130%, such as by at least about 140% relative to a congenic strain containing the genetic modification that substantially or entirely eliminates the production of PHB and lacking the genetic modification that increases the production of a sphingan. For example, the increase in the rate of production or yield of PHB-deficient diutan can be between about 50% and about 200%, such as between about 60% and about 190%, such as between about 70% and about 180%, such as between about 80% and about 170%, such as between about 90% and about 160%, such as between about 100% and about 150%, such as between about 110% and about 140%, such as between about 120% and about 130%.

[0023] In certain embodiments, one or more copies of specific DNA sequences are introduced within certain Sphingomonas strains to provide increased biosynthetic production of high viscosity diutan polysaccharide that is essentially free of PHB. The engineered bacteria containing such genes for increased production produce significantly greater amounts of PHB-deficient diutan polysaccharide compared to non-engineered bacteria and create diutan with the aforementioned resultant high viscosity properties.

[0024] The DNA can be delivered into bacteria of the genus Sphingomonas in multiple copies (via plasmid, other known manner) or increased expression of the genes via a suitable method, e.g., coupling to a stronger promoter. After insertion of the DNA into the target bacteria, the production of diutan can be determined by fermenting the engineered bacteria and comparing the yield in terms of amount produced and quality produced. Increased production and viscosity can both be determined by comparison with other diutan-producing strains.

[0025] Sphingomonas strains, such as the genetically modified strains described herein, can be used to produce sphingans, such as diutan, by fermentation. Generally, a suitable medium for fermentation is an aqueous medium which contains a source of carbon (for example, carbohydrates including glucose, lactose, sucrose, maltose or maltodextrins), a nitrogen source (for example, inorganic ammonium, inorganic nitrate, urea, organic amino acids or proteinaceous materials, such as hydrolyzed yeast, soy flour or casein, distiller's solubles or corn steep liquor), and inorganic salts. A wide variety of fermentation media will support the production of diutan according to the present invention. One of ordinary skill in the art can readily determine an appropriate media formulation.

[0026] Carbohydrates can be included in the fermentation broth in varying amounts--usually between about 1 and 10% by weight (preferably 2-8%) of the fermentation medium. The carbohydrates can be added prior to fermentation or, alternatively, during fermentation. The amount of nitrogen can, for example, range from about 0.01% to about 0.4% by weight of the aqueous medium. A single carbon source or nitrogen source can be used, as well as mixtures of these sources. Among the inorganic salts which are useful in fermenting Sphingomonas bacteria are salts which contain sodium, potassium, ammonium, nitrate, calcium, phosphate, sulfate, chloride, carbonate and similar ions. Trace metals, such as magnesium, manganese, cobalt, iron, zinc, copper, molybdenum, iodide and borate, can also be advantageously included in the broth.

[0027] In certain embodiments of the present method, Sphingomonas strains undergo fermentation. Fermentation can be carried out, for example, at temperatures between about 25 degrees C. and 40 degrees C., preferably between about 27 degrees C. and 35 degrees C. An inoculum can be prepared by standard methods of volume scale-up, including shake flask cultures and small-scale submerged stirred fermentation. The medium for preparing an inoculum can be the same as the production medium or can be any one of several standard media well-known in the art, such as Luria broth or YM medium. More than one seed stage can be used to obtain the desired volume for inoculation. Typical inoculation volumes range from about 0.5% to about 10% of the total final fermentation volume.

[0028] Certain embodiments of the present methods include agitation of the fermentation medium. In some embodiments, an agitator is contained within a fermentation vessel, whereby the contents of the agitation vessel are mixed. The vessel also can have automatic pH and foaming controls. The production medium can be added to the vessel and sterilized in place, e.g., by heating. Alternatively, the media can be sterilized separately before addition. A previously grown seed culture can be added to the cooled medium (typically at the preferred fermentation temperature of about 27 degrees to about 35 degrees C.), and the stirred culture can be fermented for about 48 to about 110 hours, producing a high viscosity broth. The sphingan, such as diutan, can be recovered from the broth by, for example, a standard method of precipitation with an alcohol, generally isopropanol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 shows greatly improved diutan productivity of PHB-deficient strains bearing plasmids pX6 and pS8 relative to PHB-deficient strains without the plasmids.

[0030] FIG. 2 shows greatly improved diutan yield from PHB-deficient strains bearing plasmids pX6 and pS8 relative to PHB-deficient strains without the plasmids.

[0031] FIGS. 3A-3B illustrates poor filterability of two independent PHB-containing diutan preparations (0.04% S657/pS8 diutan in seawater).

[0032] FIGS. 4A-4C illustrates improved filterability of three independent PHB-deficient diutan preparations (0.04% PDD3/pS8 diutan in seawater).

[0033] FIG. 5 presents a map showing the inserts contained in plasmids pS8 and pX6.

[0034] FIG. 6 shows the insert sequence contained in plasmid pS8 (SEQ ID NO: 1).

[0035] FIG. 7 shows the insert sequence contained in plasmid pX6 (SEQ ID NO: 54).

DETAILED DESCRIPTION

[0036] Two PHB-deficient bacterial strains derived from Sphingomonas sp. ATCC 53159 (S657) were previously developed and designated PDD3 and PDD6 (see ref. no. 20). These strains exhibit approximately half of the diutan productivity of the wild-type strain (S657). The plasmid pS8 contains several genes involved in diutan biosynthesis in a multicopy plasmid and has been used to enhance diutan productivity and rheology (ref. no. 18). See also refs. no. 21-23 which describe the use of plasmid mediated gene amplification to increase polysaccharide yield (DNA segments and methods for increasing polysaccharide production).

[0037] As is shown in greater detail below, applicants have now shown that introduction of the plasmids pX6 and pS8--which contain multiple genes involved in diutan biosynthesis, but are not known to contain any genes that would offset the metabolic deficiency of a PHB-deficient strain--into PHB-deficient mutants PDD3 and PDD6 results in an unexpected significantly improved productivity (g/L/hr) and dry weight yield (g/L) of the PDD strains (70% to >100% increase) relative to the PHB-deficient strains without the introduced plasmids. The PHB-deficient strains produced fewer cells and no PHB, thus, more of their dry weight yield is diutan polysaccharide. Due to their increased productivity, these strains can be used for more economical production of PHB-deficient diutan than strains lacking these genetic modifications. Moreover, a clarified diutan produced from such strains exhibits improved filterability and clarity due to the absence of PHB particles relative to PHB-containing diutan. Such PHB-deficient diutan can be particularly desirable in a variety of applications, including household and personal care products, cementitious systems, for enhanced oil recovery, fracturing, well bore clean-up and other `pay zone` applications, or any other application involving particle suspension, friction reduction, emulsion and foam stabilization, filter cake deposition and filtration control, or modification of the rheology of aqueous solutions (such as to thicken liquids, suspend solids, stabilize emulsions, or form gels and films, etc.). Additionally, upon acid hydrolysis, the PHB-deficient diutan leaves little to no residue as compared to PHB-containing diutan. The low acid hydrolysis residue renders the PHB-deficient diutan particularly suitable in oil field applications, such as fracturing, in which a viscosifying fluid is degraded after fracturing the formation, so the return flow of oil is maximized. Unlike the PHB-containing diutan, which contains PHB particles that would plug the pores in the rock formation, PHB-deficient diutan would not plug the pores in the formation, leading to improved oil yield.

[0038] In one exemplary embodiment of the present strains, a plasmid containing the relevant DNA sequence is inserted into a recipient Sphingomonas bacterium and replicates in the recipient cell, typically giving one or several (at least two and usually 4-10) copies of the DNA segment that result in increased production of high viscosity diutan polysaccharide relative to a strain lacking the DNA sequence. Alternatively or in addition to insertion of a plasmid-borne DNA sequence, DNA sequences that integrate into the bacterial chromosome can also be used. The use of conjugation or mobilization to transfer DNA into recipient bacteria is generally effective. Electroporation or chemical transformation of competent cells with purified DNA can also be used. Other vectors or bacteriophages can be used to transfer DNA into the host cell. Maintaining the DNA segments on plasmids (or other well known delivery vectors) in the recipient diutan-producing Sphingomonas is not necessary. It is routine to introduce additional copies of a DNA segment into the bacterial chromosome so that the segments are replicated each generation by the same mechanism that replicates the bacterial DNA. Alternative to or in conjunction with methods that increase the copy number of a DNA sequence, increased gene expression can be achieved by using stronger promoter elements.

[0039] The following terms shall be used throughout the specification in connection with the present invention and have the meaning indicated:

[0040] The term "Sphingomonas" is used throughout the specification to refer to strains of gram-negative bacteria from the genus Sphingomonas.

[0041] The term "inserted" is used throughout the specification to describe the process and outcome of transferring DNA into a Sphingomonas strain. Such isolated DNA can be introduced first into, as one non-limiting possibility, a desired plasmid (such as pLAFR3), by well-known techniques in the art, and then transferred, for example, by conjugation or mobilization into a recipient Sphingomonas bacterium.

[0042] The term "gene amplification" is used to refer to either increased copies of genes, for example, by cloning the target genes on a multicopy plasmid (such as from 4 to 10 copies) or by insertion of multiple copies (such as from 4 to 10) of the genes into the bacterial genome, or alternatively, increased expression of genes by modification of promoter elements to increase gene expression. Both of these methods and others can result in increased amounts of the encoded proteins.

[0043] The term "biosynthesis" is used throughout the specification to describe the biological production or synthesis of a sphingan by Sphingomonas bacteria.

[0044] Cloning of DNA in the present invention relies on general techniques and methods which have become standard in the art. It is noted that any number of methods can be used to clone DNA segments according to the present invention, and the present invention is not limited, for example, to the use of plasmid cloning vectors. For example, DNA fragments can be cloned by insertion into a bacteriophage vector. In certain embodiments of the present methods, cloned DNA sequences are introduced to a Sphingomonas strain via a plasmid or other delivery vector.

[0045] The term "ectopic promoter" is used to refer to a non-native promoter, i.e., a promoter with some sequence difference(s) relative to the native promoter. Such a promoter can be, for example, a strong promoter which drives a measurably increased level of transcription relative to the native promoter. An ectopic promoter can also be a regulated promoter, whereby gene expression is increased or decreased in response to some factor, such as a small molecule, temperature, presence of a gene product, etc. Suitable promoters for a particular use are well known in the art.

[0046] The term "genetic modification" is used throughout the specification to refer to a genetic change. Generally, a genetically modified organism, such as a Sphingomonas strain, is described with reference to a "parent" strain which does not contain the genetic modification. Exemplary genetic modifications include those that increase, decrease, or abolish the expression of a gene. Such changes include modification of chromosomal and extrachromosomal genetic material. Exemplary genetic modifications include introduction of a plasmid, deletion or substitution of a chromosomal sequence. For example, a chromosomal gene can be inactivated by a targeted deletion of part or all of the coding sequence and/or regulatory element (e.g., as described in ref. no. 20), or genetic screen, optionally including mutagenesis (e.g., as described in ref. no. 26). Chromosomal genetic modification can also involve a targeted replacement, e.g., to replace a native gene promoter with an inducible promoter, regulated promoter, strong promoter, etc. Chromosomal gene modification can also involve gene amplification, i.e., introduction of at least one additional copy of at least one gene. Extrachromosomal genetic material can be introduced, for example, on a plasmid, which can be single-copy, multi-copy, or high-copy, as is well known in the art. Genetic modification can be coupled to a selectable marker, such as an antibiotic resistance gene, which helps ensure that the genetic modification is retained.

[0047] The term "essentially free of PHB" is used throughout the specification to refer to a composition, such as a sphingan (e.g., diutan), having a greatly reduced PHB content when compared to a similar composition prepared from a wild-type or PHB-containing strain. Great reduction can be at least a 90% reduction, 95% reduction, 99% reduction, 99.5% reduction, etc. in PHB content (where PHB content is expressed as a fraction of the dry weight of the sphingan composition). Suitable assays for measuring PHB content include the 15% HCl solubility and residue test, HPLC, gas chromatography, and gas chromatography coupled to mass spectrometry (GC-MS). In certain embodiments of the present compositions, a clarified (e.g., cellulase clarified) diutan preparation that is essentially free of PHB can yield less than approximately 1%, such as less than approximately 0.5%, such as less than approximately 0.1%, residue in a 15% HCl solubility and residue test.

[0048] The term "PHB-deficient diutan" is used throughout the specification to refer to a diutan produced from a PHB-deficient strain, such as strain bearing a genetic modification inactivates the phaA gene, phaB gene, phaC gene, or any combination thereof.

[0049] The term "phaC gene" is used throughout the specification to refer to a phaC gene of a Sphingomonas strain. Examples of phaC gene sequences are provided in (ref. no. 20); however, other phaC gene orthologs are also encompassed except where the context indicates otherwise.

[0050] When an amount, concentration, or other value or parameter is given as a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of an upper preferred value and a lower preferred value, regardless of whether ranges are separately disclosed.

[0051] The term "a" or "an" as used herein means "one" or "one or more".

[0052] The term "about" or "approximately" as used herein means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviations, per practice in the art. Where particular values are described in the application and claims, unless otherwise stated, the term "about" means within an acceptable error range for the particular value.

[0053] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about" Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0054] Except wherein indicated otherwise, all measurements and protocols are conducted at standard temperature and pressure, i.e., approximately 20.degree. C. and approximately 1 atmosphere. Except where indicated otherwise, "sea water 3 rpm viscosity," "sea water 0.3 rpm viscosity," and "low shear rate viscosity in the presence of polyethylene glycol" are measured as described in Example 2 (paragraphs [0065]-[0066]), below.

[0055] The invention will now be described in more detail with respect to the following, specific, non-limiting examples.

EXAMPLES

Example 1

Production of Diutan

[0056] This example described an increased yield of PHB-deficient diutan produced from several genetically modified Sphingomonas strains.

[0057] Methods

[0058] The plasmids pS8 and pX6 were transferred into PHB-deficient Sphingomonas strains PDD3 and PDD6 by triparental conjugal mating as described previously (ref. no. 3) and which is well known in the art. Strains PDD3, PDD6, 5657, and S657/pS8 are as described previously (ref. nos. 17, 18, and 20). Strains PDD3/pS8, PDD6/pS8, PDD3/pX6, PDD6/pX6, PDD3, PDD6, 5657, and S657/pS8 were grown in 15 L volumes in 20 L Applikon fermentors with agitation and aeration. For the plasmid containing strains, the antibiotic tetracycline at 5 mg/L was added throughout the fermentation to ensure retention of the plasmid. KOH was added as needed to control pH. Two seed stages were used with 1% to 6% inoculum transfers. The fermentation media contained corn syrup as carbohydrate source, an assimilable nitrogen source, and salts.

[0059] At the end of the fermentation, each broth was treated by introduction of glucoamylase enzyme to hydrolyze any remaining oligosaccharides from the corn syrup. The viscosities of the fermentation broths were measured via a Brookfield.RTM. viscometer run at 60 rpm with a spindle #4. The diutan gums produced were then precipitated from an aliquot of broth with two volumes of isopropyl alcohol. The diutan fibers were collected on a filter and dried. For some strains, multiple replicates were prepared, and the results presented below are the average values across these replicates.

[0060] Results

[0061] The presence of a plasmid containing genes involved in diutan synthesis (pX6 or pS8, see FIG. 5) greatly improved the diutan production by PHB-deficient strains compared to the parent PHB-deficient strains PDD3 and PDD6. Diutan productivity of PHB-deficient strains was greatly improved by between 70% and 142% (FIG. 1 and Table 1B) relative to the parental strains. This increased productivity was much greater than for the wild-type (S657) strain with the introduced pS8 plasmid, which demonstrated increased productivity by only 33%. Three of the four PHB-deficient, plasmid-containing strains had higher productivity than the wild-type (S657) strain, and strain PDD3/pX6 had productivity essentially equal to S657/pS8. Diutan yield of PHB-deficient strains bearing these plasmids was also greatly improved by between 53% and 90% (FIG. 2 and Table 1B) relative to the parental strains. This increase was much greater than for the wild-type (S657) strain with the introduced pS8 plasmid, which only increased diutan yield by 18%.

[0062] Consistent with these results, the PHB-deficient strains also exhibited increases in final broth viscosity due to introduction of the plasmids, indicating greater diutan content. The PHB-deficient, plasmid-containing strains also had lower cell density (measured by (O).sub.am) than the wild-type strain with or without plasmid pS8 (Table 1B), indicating that the unclarified products from these strains are expected to contain a higher proportion of diutan (due to the presence of fewer bacterial cells). Due to the higher purity of the diutan produced from PHB-deficient, plasmid containing strains (both due to lower cell content and absence of PHB), the extent of productivity and yield improvement in these strains compared to wild-type strains is likely to be even greater than these measurements indicate.

TABLE-US-00001 TABLE 1A Final culture conditions. Final Cell Final Broth Replicates Density Viscosity Strain PHB (n) (OD.sub.600) (cP) S657 (wild-type) + 2 8.22 3000 S657/pS8 + 2 5.84 3775 PDD3 - 1 3.80 3000 PDD3/pX6 - 1 4.14 3650 PDD3/pS8 - 3 3.50 4158 PDD6 - 1 4.15 2500 PDD6/pX6 - 2 3.73 3125 PDD6/pS8 - 3 3.74 3783

TABLE-US-00002 TABLE 1B Diutan productivity and yield. Percent Percent Percent Percent Change Change Change Change Over over Over over S657 PHB- S657 PHB- Productivity (wild- deficient Yield* (wild- deficient Strain PHB (g/L/hr) type) parent (g/L) type) parent S657 (wild- + 0.153 17.5 type) S657/pS8 + 0.203 +33% 20.7 +18% PDD3 - 0.082 -46% 11.4 -35% PDD3/pX6 - 0.197 +140% 20.9 +83% PDD3/pS8 - 0.177 +116% 21.7 +90% PDD6 - 0.067 -56% 9.4 -46% PDD6/pX6 - 0.114 +70% 14.4 +53% PDD6/pS8 - 0.162 +142% 17.3 +84% *Total dry weight of unclarified precipitate (dry weight yield).

Example 2

Diutan Analysis

[0063] The diutan samples produced in the method of Example 1 were analyzed for uses as oilfield additives for oil recovery and for uses requiring good suspension and stabilization (such as for cement additives for water retention and quick set-up).

[0064] Methods

[0065] The oilfield industry relies on a "sea water viscosity" (SWV) test as an indicator of acceptable performance for rheology modifiers in oil recovery. This test indicates whether a rheology modifier can sufficiently increase viscosity in briny conditions of sea water, such as those encountered in seabed oil recovery. Typically, a sea water viscosity test employs synthetic seawater produced by mixing 419.53 grams of sea salt (ASTM D-1141-52) per 9800 grams of deionized water. For a seawater viscosity test, a rheology modifier is dispersed in synthetic seawater by vigorous mixing (e.g., 35 minutes at approximately 11,500 rpm in a Fann Multimixer (Model 9B5, part number N5020)). The sample is cooled to approximately 25.degree. C. before the viscosity is measured. For a 3-rpm viscosity test, the sample is placed on the Fann sample platform (Fann model 35 A; Torsion spring MOC 34/35 F0.2b; Bob Bl; Rotor R1) and the speed is adjusted to 3 rpm by turning the motor to low speed and setting the gearshift in the middle position. The reading is then allowed to stabilize, and the shear stress value is read from the dial and recorded as the SWV 3 rpm dial reading (DR). For the 0.3-rpm reading, a Brookfield viscometer is used (Brookfield LV DY-II or DV-III viscometer, with LV-2C spindle) to measure the viscosity. The speed of the spindle is set to 0.3 rpm, and the spindle is allowed to rotate at least 6 minutes before the viscosity is recorded as the SWV-0.3 rpm reading and expressed in centipoises (cP).

[0066] The LSRV test (a low shear rate viscosity using polyethylene glycol as dispersant as described below) is a general test for viscosity at a low shear rate. Typically, the higher the viscosity the better a sample is at stabilization and suspension. For example, in a cementitious application, a higher viscosity in the LSRV test indicates that a diutan should help suspend particulates in the cement more effectively, giving a more homogeneous cement/concrete, thus, providing better strength and durability. The LSRV test measures the viscosity of a 0.25% solution of biogum in Synthetic Tap Water (STW). STW is prepared by adding 10.0 grams NaCl and 1.47 grams CaCl.sub.2.2H.sub.2O to 10 liters of deionized water. For the viscosity measurement, 0.75 grams of biogum is added to 4.5 grams Polyethylene Glycol 200 (CAS 25322-68-3) in a 400-mL beaker and thoroughly dispersed. Then, 299 grams of STW are added to the beaker and mixed for approximately 4 hours using a low-pitched, propeller-style stirrer at 800.+-.20 rpm. After the 4-hr mixing time, the beaker is placed in a 25.degree. C. water bath and allowed to sit undisturbed for approximately 30 minutes. The viscosity is then measured using a Brookfield LV viscometer equipped with a 2.5+ torque spring (or equivalent instrument, such as Model DVE 2.5+) at 3 rpm using the LV 1 spindle after allowing the spindle to rotate for 3 minutes and expressed in centipoises (cP).

[0067] Results

[0068] The diutan samples produced in Example 1 above were analyzed to determine suitability for use in cement and oilfield applications (Table 2). Utility for stabilization and suspension, such as for cement additives for water retention and quick set-up, was evaluated by low shear rate viscosity (LSRV) testing. Suitability for oil recovery was evaluated using sea water viscosity (SWV) tests at 0.3 rpm and 3 rpm as an indicator of the effectiveness of a gum to increase viscosity in brines.

[0069] In the LSRV test, diutan produced from PHB-deficient strain PDD3 containing either plasmid performed better than or about equal to the wild-type strain bearing pS8, with greater improvement observed for PDD3/pX6 than for PDD3/pS8 (Table 2). In the SWV test at 0.3 rpm, diutan produced from plasmid-containing PHB-deficient strains derived from PDD3 performed better than wild-type strains bearing pS8. In the SWV test at 3 rpm, either PHB-deficient strain bearing pS8 performed essentially equally to the wild-type strains bearing pS8. Together, these results indicate that a PDD3/pS8 diutan is particularly suitable for oilfield applications and cement applications.

TABLE-US-00003 TABLE 2 Rheology of unclarified PHB-deficient diutan. SWV SWV 3 rpm Diutan LSRV 0.3 rpm (dial Strain PHB Productivity n (centipoise) n (centipoise) n reading) S657 + ++ 1 5110 2 37,400 2 40.0 S657/pS8 + +++ 1 6610 2 48,600 2 56.5 PDD3 - + 1 3160 -- nt. -- nt. PDD3/pX6 - +++ 1 6910 1 54,400 1 45.5 PDD3/pS8 - +++ 2 6198 3 51,867 2 57.0 PDD6 - + 1 3020 -- nt. -- nt. PDD6/pX6 - +++ 2 5188 2 41,600 1 43.0 PDD6/pS8 - +++ -- nt. 1 38,400 1 57.0 nt.: Not tested.

Example 3

Low Acid Residue of PHB-Deficient Diutan

[0070] Methods

[0071] The indicated strains were grown in 1000 gallon fermentors and in multiple Applikon.RTM. fermentors to prepare larger samples for testing and analysis. After the fermentations had finaled, the broths were either left untreated or enzyme clarified using one of two methods.

[0072] The first method, clarification with a cellulase, CELLUCLAST.TM. ("Clarified") was as follows: First, the broth temperature was adjusted to 50.degree. C. Next, the pH was adjusted to between 5.0 and 5.4. CELLUCLAST.TM. enzyme (1 g/L) was then added, and the broth was incubated for two hours. Stock solutions of EDTA and Lysozyme in distilled water were then sequentially added to the broth to a final concentration of 0.25 g/L EDTA and 0.05 g/L Lysozyme, and the broth incubated for one hour. The pH was then adjusted to 8.0 to 8.5. Protex 6 L protease was then added to the broth at a final concentration of 0.5 g/L and the broth was incubated for two hours. Finally, the diutan gum was precipitated by addition of three volumes of isopropyl alcohol, dried, and milled.

[0073] The second enzyme clarification ("Treated") was similar to the first method, except the initial pH adjustment and the addition of CELLUCLAST.TM. enzyme were omitted.

[0074] Dried diutan samples were analyzed using the 15% HCl Solubility and Residue Test, as follows: 1.6 grams of a sample is rehydrated in 253 ml Synthetic Tap Water (typically 1 hr mixing at 1000 rpm). The mixing speed was then decreased to 500 RPM, and 147 mL of concentrated HCl (37%) is added to the rehydrated sample and mixed for 10 minutes. The sample container was then sealed and incubated at 150 degrees F. for twenty-four hours. The sample was again mixed, then a 100 gram aliquot was removed. The aliquot was quantitatively transferred to a Gelman filter apparatus containing a 0.5 micron filter. The filter was dried, cooled, and weighed prior to filtration and again after filtration. The weight of residue was reported as a percentage of the dry weight of polymer in the 100 gram aliquot (dry weight is determined by drying a sample of the same starting material).

[0075] Results

[0076] The acid residue test measures the amount of insoluble material that remains in a sample after acid hydrolysis. Low acid residue is preferred for certain uses, for example, an oilfield use in which the diutan is removed by acid hydrolysis and any insoluble residue has the potential to clog pores in the formation. This residue test also provides an indirect indication of the amount of PHB in a diutan preparation because the acid residue of a wild-type diutan is predominantly PHB. For a PHB-deficient diutan, the acid residue indicates an upper bound for the PHB content.

[0077] Results of the acid residue test are provided in Table 3, with residue indicated as a percentage of the starting sample material. Unlike the PHB-containing strains, which contained between 1.8 wt % and 6.8% wt % acid residue, the clarified PHB-deficient strain produced only 0.05 wt % acid residue. These results confirmed that the PHB-deficient strain produced diutan that would not damage an oilfield formation and, moreover, that the PHB-deficient diutan contains less than 0.05% PHB by weight.

TABLE-US-00004 TABLE 3 Low acid residue of clarified PHB-deficient diutan. Weight Percentage Strain PHB Residue S657/pS8 + 1.80% (Treated) S657 + 6.78% (untreated) S657/pS8 + 2.98% (untreated) PDD3/pS8 - 0.05% (Clarified)

Example 4

Confirmation that PHB is Absent from PHB-Deficient Diutan

[0078] Methods

[0079] The analytical method measured the PHB content of diutan preparations and can also be used to measure the PHB content of other polysaccharides. In this method, the diutan is digested with an aqueous hypochlorite solution leaving the PHB intact; the PHB polymer is then hydrolyzed, then esterified to the propyl ester; and finally, the resulting ester is measured by gas chromatography with flame ionization detection. The instrument used was the Hewlett Packard Model 6890 Gas Chromatograph System equipped with a HP model 7673 auto injector, flame ionization detector, and Hewlett Packard HP 5MS column (30 m.times.250 .mu.m.times.0.25 .mu.m nominal id).

[0080] The detailed protocol is as follows. Approximately 35-40 mg of each diutan sample was weighed into a glass centrifuge tube, in duplicate and the weight recorded to the nearest 0.1 mg. Approximately 5 mL of approximately 5% sodium hypochlorite (JT Baker Cat # 4616 or equivalent) was then added to each tube and the tubes vortexed. Samples were then incubated at approximately 37.degree. C. for 12-18 hours, resulting in hypochlorite digestion. Tubes were then centrifuged at approximately 8000 rpm for approximately 40 minutes, and the hypochlorite supernatants were removed with a disposable pipette and discarded. Samples were then washed twice by addition of 5 mL deionized water with centrifugation and supernatant removal as in the previous step. Samples were then evaporated to dryness under reduced pressure using a vacuum oven, optionally with heating to accelerate the drying process. 2.0 mL of internal standard solution (0.513 mg/mL propyl benzoate, Aldrich Cat # 30, 700-9 or equivalent, in 1,2-dichloroethane, Aldrich Cat # 15, 478-4 or equivalent) was then added to each dry sample, followed by 1.0 mL of 20% (vol/vol) HCl (EM Science Cat # HX0603P-1 or equivalent) in n-propanol (Aldrich Cat # 29, 328-8 or equivalent). Samples were then sealed with polytetrafluoroethylene film (Teflon tape or equivalent), capped tightly, and incubated at approximately 100.degree. C. for 3 hours with vortexing approximately every 30 minutes. Samples were then cooled to room temperature. An aqueous extraction was then performed by addition of 2 mL deionized water to each tube, vortexing for 10-20 seconds, allowing the phases to separate, and removal of the aqueous (top) phase. The aqueous extraction was repeated a second time, then the organic (lower) phase was transferred to a GC vial. Calibration standards containing between 0.2 and 10.0 mg/ml sodium 3-hydroxybutyrate (ICN Biomedical Cat # 100964 or equivalent) were also prepared by the same method starting with the step of evaporation to dryness, i.e., the sodium hypochlorite digestion was omitted. Each sample and calibration standard was then analyzed using the Hewlett Packard Model 6890 Gas Chromatograph System.

[0081] The Hewlett Packard Model 6890 Gas Chromatograph System was operated with the following parameters: Sample Inlet: GC; Injection Source: GC ALS; Mass Spectrometer: Disabled; OVEN: Initial temp.: 50 C (On); Maximum temp.: 325 C; Initial time: 2.00 min; Equilibration time: 0.50 min; Ramp #1 Rate 7.00, Final temp. 120 C, Final time 0.00; Ramp #2 Rate 18.00, Final temp., 280 C, Final time 2.00; Ramp #3 Rate 0.0 (Off); Post temp: 0 C; Post time: 0.00 min; Run time: 22.89 min; BACK INLET: Mode: Split; Initial temp: 275 C (On); Pressure: 12.96 psi (On); Split ratio: 10:1; Split flow: 11.0 mL/min; Total flow: 13.1 mL/min; Gas saver: On; Saver flow: 20.0 mL/min; Saver time: 2.00 min; Gas type: Helium; COLUMN 2; Capillary Column; Model Number: HP 19091S-433; HP-5MS 5% Phenyl Methyl Siloxane; Max temperature: 325 C; Nominal length: 30.0 m; Nominal diameter: 250.00 um; Nominal film thickness: 0.25 um; Mode: constant flow; Initial flow: 1.1 mL/min; Nominal init pressure: 12.97 psi; Average velocity: 27 cm/sec; Inlet: Back Inlet; Outlet: Back Detector; Outlet pressure: ambient; BACK DETECTOR (FID); Temperature: 280 C (On); Hydrogen flow: 40.0 mL/min (On); Air flow: 450.0 mL/min (On); Mode: Constant makeup flow; Makeup flow: 15.0 mL/min (On); Makeup Gas Type: Helium; Flame: On; Electrometer: On; Lit offset: 2.0; SIGNAL 1; Data rate: 20 Hz; Type: back detector; Save Data: On; Start Save Time: 4.00 min; Stop Save Time: 22.00 min; Zero: 0.0 (Oft); Range: 0; Fast Peaks: Off; Attenuation: 0; POST RUN: Post Time: 0.00 min; Front Injector: No parameters specified; BACK INJECTOR: Sample Washes: 0; Sample Pumps: 2; Injection Volume: 1.0 microliters; Syringe Size: 10.0 microliters; Nanoliter Adapter: Off; Postlnj Solvent A Washes: 5; Postlnj Solvent B Washes: 5; Viscosity Delay: 0 seconds; Plunger Speed: Fast; Preinjection Dwell: 0.00 minutes; PostInjection Dwell: 0.00 minutes.

[0082] A standard curve was fitted to the calibration standards by linear regression analysis using multilevel calibration with internal standard, resulting in the equation:

y=mx+b

[0083] Where:

y = Area PHB Area Istd = Area ratio ##EQU00001## x = Amount PHB Amount Istd = Amount ratio ##EQU00001.2## [0084] Istd. is the internal standard [0085] m=slope [0086] b=y-intercept

[0087] PHB content of the samples was then calculated using the following equation:

Amount PHB = ( Area PHB / Area Istd ) - b m .times. Amount Istd ##EQU00002##

[0088] Results

[0089] The presence or absence of PHB was confirmed using gas chromatography (GC). Diutan samples from strain S657/pS8 contained an average of 4.0% PHB by weight (Table 4). In contrast, PHB was undetectable in four samples from each of two independent diutan preparations from strain PDD3/pS8 (Table 4). These results indicated that strain PDD3/pS8 produced diutan containing less than approximately 0.05% PHB by weight (the estimated detection limit of the method).

[0090] As discussed above, abolition of PHB production by deletion of the phaC gene resulted in severe metabolic deficiency, poor growth, and greatly impaired diutan productivity. These results provide further confirmation of the unexpected finding that the diutan productivity and yield of a phaC deletion strain can be greatly enhanced by introduction of a plasmid containing genes involved in diutan synthesis, even though PHB production has not been detectably restored.

TABLE-US-00005 TABLE 4 Confirmation of absence of PHB by Gas Chromatography. Sample Weight Calculated Strain (mg) PHB (mg) Wt % PHB S657/pS8 39.8 1.68 4.22 39.8 1.65 4.16 36.0 1.39 3.86 36.0 1.36 3.79 PDD3/pS8 33.1 n.d. n.d. (Preparation #1) 33.1 n.d. n.d. 38.0 n.d. n.d. 38.0 n.d. n.d. PDD3/pS8 38.5 n.d. n.d. (Preparation #2) 38.5 n.d. n.d. 38.0 n.d. n.d. 38.0 n.d. n.d. n.d.: Not detected. The limit of detection was 0.05% by weight.

Example 5

Diutan Filterability for Enhanced Oil Recovery Applications

[0091] Methods

[0092] Diutan fermentation broths were clarified with cellulase and recovered as described in Example 3.

[0093] Filterability studies were performed on 0.04% diutan rehydrated in seawater. The diutan solution was passed through a 47 mm diameter NUCLEPORE.TM. filter (track-etched polycarbonate membranes having stringently controlled pore size, available from Whatman, Inc., Piscataway, N.J.) of the indicated pore size using a flow pressure of 20 psi. The time for each 200 ml of the diutan solution (1 or 2 liters total) to flow through the filter was measured with a graduated cylinder and a stop watch.

[0094] Results

[0095] In this example, the filterability of enzyme-clarified, rehydrated products from the PDD3/pS8 strain were compared to enzyme-clarified, rehydrated products from the 5657/pS8 strain. Enzyme-clarified diutan preparations were filtered through NUCLEPORE.TM. filters of the indicated sizes, and the volume filtered is shown as a function of time (FIGS. 3A-3B and 4A-4C). Clogging of filters is indicated by lines tending towards vertical on the graphs, showing that little additional volume was passing through the filter as time passed. Two preparations containing PHB made from strain S657/pS8 were poorly filterable, clogging filters of 5 microns (FIG. 3A) and 3 microns (FIG. 3B) before one liter could be filtered. In contrast, PHB-deficient diutan preparations made from strain PDD3/pS8 showed improved filterability. Two out of three preparations were filterable at 3 microns (FIG. 4A and FIG. 4C), while the third was filterable at 5 microns (FIG. 4B). Together, these results indicated that the PHB-deficient strains produced diutan with improved filterability.

Example 6

Description of Plasmids pS8 and pX6

[0096] The plasmids pS8 and pX6 are as previously described in U.S. Publication No. 2008/0319186. In brief, these plasmids were obtained by screening an ATCC 53159 genomic sequence library (in cosmid cloning vector pLAFR3) for clones able to restore polysaccharide production in the nonmucoid mutant (GPS2) of S. elodea ATCC 31461 or a nonmucoid mutant of Xanthomonas campestris. Plasmid inserts were end-sequenced and/or shotgun sequenced. A map showing the genes contained in complementing plasmids is shown in FIG. 5. The pS8 insert DNA sequence is provided as SEQ ID NO: 1 (FIG. 6), and the pX6 insert DNA sequence is provided as SEQ ID NO: 54 (FIG. 7). Predicted gene functions were designated based on homology to other genes in public databases. Genes contained in plasmid pS8 and pX6 and their predicted functions are listed in Tables 5 and 6, respectively. Pursuant to the Budapest Treaty for the International Recognition of the Deposit of Microorganisms, strains of E. coli containing plasmids pS8 and pX6 have been deposited with the Patent Depository at the American Type Culture Collection at 10801 University Boulevard, Manassas, Va. 20110, and are available as deposit numbers PTA-10102 (deposit date Jun. 2, 2009) and PTA-10103 (deposit date Jun. 2, 2009), respectively.

[0097] Plasmid pS8 contains the genes dpsS, dpsG, dpsR, dpsQ, dpsl, dpsK, dpsL, dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, and orf7. Plasmid pX6 contains the genes dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, orf7, orf6, and orfs. Based on their homology to known genes, many of the genes contained in these plasmids are predicted to be involved in diutan production. The genes in the genomic region from which plasmids pS8 and pX6 were derived (FIG. 5) include genes that encode the transferases for the four sugars of the diutan backbone and the four genes for dTDP-rhamnose synthesis. Genes for secretion of the polysaccharide, dpsD, dpsC, and dpsE, were identified based on homology to genes for biosynthesis of other polysaccharides. Two genes, atrB and atrD, encode proteins homologous to proteins involved in protein secretion. Two genes, dpsG and dpsR, putatively encode a polymerase and a lyase, respectively. Two genes, dpsM, and dpsN, encode polysaccharide attachment proteins. The insert in plasmid pX6 contained 17 genes including gene dpsB encoding transferase I (which initiates the first step in diutan synthesis), genes for secretion and four genes for dTDP-rhamnose synthesis, but lacks the genes for transferases II, III and IV and the putative genes for polymerase and lyase. Plasmid pS8 contains 20 genes of the dps gene cluster, including genes for all four backbone sugar transferases, the four genes for dTDP-rhamnose synthesis, and genes for secretion of the polysaccharide, including the putative genes for polymerase and lyase, but lacks the genes of unknown function, orfs, orf6, and orf7.

TABLE-US-00006 TABLE 5 Genes contained in plasmid pS8. Start and end coordinates are relative to the pS8 insert sequence contained in SEQ ID NO: 1. Gene SEQ ID NO Start End Name Description DNA Amino Acid 2* 1054 dpsS (partial) homologous to gelS 2 3 2738 1113 dpsG putative polymerase 4 5 4895 2898 dpsR putative lyase 6 7 5093 6031 dpsQ putative rhamnosyl transferase IV 8 9 7082 6111 dpsI unknown 10 11 7121 8167 dpsK beta-1,4-glucuronosyl transferase II 12 13 8164 9030 dpsL glucosyl transferase III 14 15 10467 9079 dpsJ unknown 16 17 11076 12374 dpsF unknown 18 19 12389 13306 dpsD polysaccharide export protein 20 21 13341 14687 dpsC polysaccharide export protein 22 23 14687 15394 dpsE polysaccharide export protein 24 25 15405 16286 dpsM polysaccharide attachment 26 27 16270 16968 dpsN polysaccharide attachment 28 29 18454 17060 atrD secretion protein 30 31 20637 18451 atrB secretion protein 32 33 21229 22641 dpsB glucosyl-isoprenylphosphate transferase I 34 35 22757 23635 rmlA glucose-1-phosphate thymidylyltransferase 36 37 23632 24198 rmlC dTDP-6-deoxy-D-glucose-3-5-epimerase 38 39 24202 25263 rmlB dTDP-D-glucose-4,6-dehydratase 40 41 25263 26129 rmlD dTDP-6-deoxy-L-mannose-dehydrogenase 42 43 26277 26146 orf7 (partial) unknown function 44 45 *First in-frame codon; the start codon is not present.

TABLE-US-00007 TABLE 6 Genes contained in plasmid pX6. Start and end coordinates are relative to the pX6 insert sequence contained in SEQ ID NO: 54. Gene SEQ ID NO Start End Name Description DNA Amino Acid 1 336 dpsL (partial) glucosyl transferase III 46 47 1773 385 dpsJ unknown 16 17 2382 3680 dpsF unknown 18 19 3695 4612 dpsD polysaccharide export protein 20 21 4647 5993 dpsC polysaccharide export protein 22 23 5993 6700 dpsE polysaccharide export protein 24 25 6711 7592 dpsM polysaccharide attachment 26 27 7576 8274 dpsN polysaccharide attachment 28 29 9760 8366 atrD secretion protein 30 31 11943 9757 atrB secretion protein 32 33 12535 13947 dpsB glucosyl-isoprenylphosphate transferase I 34 35 14063 14941 rmlA glucose-1-phosphate thymidylyltransferase 36 37 14938 15504 rmlC dTDP-6-deoxy-D-glucose-3-5-epimerase 38 39 15508 16569 rmlB dTDP-D-glucose-4,6-dehydratase 40 41 16569 17435 rmlD dTDP-6-deoxy-L-mannose-dehydrogenase 42 43 18288 17452 orf7 unknown function 48 49 19433 18618 orf6 unknown function 50 51 19751 20683 orf5 unknown function 52 53

REFERENCES

[0098] The entire disclosure of each patent, publication or other reference cited anywhere herein is hereby incorporated by reference in its entirety to the extent that they are not inconsistent with the disclosure herein.

[0099] Numbered citations in the text above refer to the list below.

[0100] Non-Patent Literature: [0101] 1. Campana, S., J. Ganter, M. Milas, and M. Rinaudo. 1992. On the solution properties of bacterial polysaccharides of the gellan family. Carbohydr. Res. 231: 31-38. [0102] 2. Chowdhury, T. A., B. Lindberg, U. Lindquist, and J. Baird. 1987. Structural studies of an extracellular polysaccharide, S-657, elaborated by Xanthomonas ATCC 53159. Carbohydr. Res. 164: 117-122. [0103] 3. Coleman R. J., N. E. Harding, and Y. N. Patel. 2008. Identification and organization of genes for diutan polysaccharide synthesis from Sphingomonas sp. ATCC 53159. J. Ind. Microbiol. Biotechnol. 35: 263-274. [0104] 4. Diltz, S, and S. G. Zeller. 2001. Location of O-acetyl groups in S-657 using the reductive-cleavage method. Carbohydr. Res. 331: 265-270. [0105] 5. Harding, N. E., Y. N. Patel, and R. J. Coleman. 2004. Organization of Genes Required for Gellan Polysaccharide Biosynthesis in Sphingomonas elodea ATCC 31461 J. Ind. Microbiol. Biotechnol. 31: 70-82. [0106] 6. Kang, K. S., and D. J. Pettitt. 1993. Xanthan, gellan, welan, and rhamsan, p. 341-398. In R. L. Whistler and J. N. BeMiller (ed.), Industrial gums: polysaccharides and their derivatives, 3.sup.rd edition. Academic Press, Inc., New York. [0107] 7. Lee E. J. and R. Chandrasekaran. 1991. X-ray and computer modeling studies on gellan-related polymers: molecular structures of welan, S-657, and rhamsan. Carbohydr. Res. 214: 11-24. [0108] 8. Moorehouse, R. 1987. Structure/property relationships of a family of microbial polysaccharides, p. 187-206. In M. Yalpani (ed.), Industrial polysaccharides: genetic engineering. Structure/property relations and applications. Elsevier Science Publishers BV, Amsterdam. [0109] 9. Moreira, L. M., K. Hoffmann, H. Albano, A. Becker, K. Niehaus and I. Sa-Correia. 2004. The gellan gum biosynthetic genes ge1C and gelE encode two separate polypeptides homologous to the activator and the kinase domains of tyrosine autokinases. J. Mol. Microbiol. Biotechnol. 8: 43-57 [0110] 10. Pollock, T. J. 1993. Gellan-related polysaccharides and the genus Sphingomonas. J. Gen. Microbiology. 139: 1939-1945. [0111] 11. Pollock, T. J., W. VanWorkum, L. Thorne, M. J. Mikolajczak, M. Yamazaki, J. W. Kijne, and R. W. Armentrout. 1998. Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum. J. Bacteriology. 180: 586-593. [0112] 12. Sa-Correia I., A. M. Fialho, P. Videira, L. M. Moreira, A. R. Marques and H. Albano. 2002. Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: Genes, enzymes and exopolysaccharide production engineering. J. Ind. Microbiol. Biotechnol. 29: 170-176. [0113] 13. Thorne, L., M. J. Mikolajczak, R. W. Armentrout, and T. J. Pollock. 2000. Increasing the yield and viscosity of exopolysaccharides secreted by Sphingomonas by augmentation of chromosomal genes with multiple copies of cloned biosynthetic genes. J. Ind. Microbiol. Biotechnol. 25: 49-57. [0114] 14. Videira P., A. Fialho, R. A. Geremia, C. Breton and I. Sa-Correia. 2001. Biochemical characterization of the .beta.-1,4-glucuronosyltransferase Ge1K in the gellan gum-producing strain Sphingomonas paucimobilis ATCC 31461. Biochem. J. 258: 457-464. [0115] 15. Yamazaki, M., L. Thorne, M. Mikolajczak, R. W. Armentrout, and T. J. Pollock. 1996. Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88. J. Bacteriology. 178: 2676-2687.

[0116] Patent Literature: [0117] 16. Harding, N. E., Y. N. Patel, and R. J. Coleman. 2006. Targeted gene deletions for polysaccharide slime formers. U.S. Publication Number 2006/0199201. [0118] 17. Peik, J. A., S. M. Steenbergen, G. T. Veeder. 1992. Heteropolysaccharide S-657. U.S. Pat. No. 5,175,278. [0119] 18. Harding, N. E., Y. N. Patel, R. Coleman, and S. Matzke. 2008. High Viscosity Diutan Gums. U.S. Publication Number 2008/0319186. [0120] 19. Dial, H. D., C. B. Skaggs, and W. G. Rakitsky. 2000. Stable suspension of hydrocolloids. U.S. Pat. No. 6,221,152. [0121] 20. Bower; S., E. Burke, N. E. Harding, Y. N. Patel, J. C. Schneider, D. Meissner, N. A. Morrison, R. Bezanson. 2006. Mutant bacterial strains of the genus sphingomonas deficient in production of polyhydroxybutyrate and a process of clarification of sphingans and compositions thereof. U.S. Publication Number 2006/0121578. [0122] 21. Pollock, T. J., M. Yamazaki, L. Thorne, M. Mikolajczak, and R. W. Armentrout. 1998. DNA segments and methods for increasing polysaccharide production. U.S. Pat. No. 5,854,034. [0123] 22. Pollock, T. J., M. Yamazaki, L. Thorne, M. Mikolajczak, and R. W. Armentrout. 1999. DNA segments and methods for increasing polysaccharide production. U.S. Pat. No. 5,985,623. [0124] 23. Pollock, T. J., M. Yamazaki, L. Thorne, M. Mikolajczak, and R. W. Armentrout. 2001. DNA segments and methods for increasing polysaccharide production. U.S. Pat. No. 6,284,516. [0125] 24. Pollock, T. J. 2004. Production of modified polysaccharide S-7. U.S. Pat. No. 6,709,845. [0126] 25. Bower, S., E. Burke, N. E. Harding, Y. N. Patel, J. C. Schneider, D. Meissner, N. A. Morrison, and R. Bezanson. 2008. Mutant bacterial strains of the genus sphingomonas deficient in production of polyhydroxybutyrate and a process of clarification of sphingans and compositions thereof. U.S. Publication Number 2008/0268527. [0127] 26. Baird, J. K., and J. M. Cleary. 1994. P. elodea mutants are produced which produce gellan gum broth which contains no detectable amount of poly-.beta.-hydroxy-butyrate (PHB). U.S. Pat. No. 5,300,429.

[0128] While the invention has been described by way of examples and preferred embodiments, it is understood that the words which have been used herein are words of description, rather than words of limitation. Changes may be made, within the purview of the appended claims, without departing from the scope and spirit of the invention in its broader aspects. Although the invention has been described herein with reference to particular means, materials, and embodiments, it is understood that the invention is not limited to the particulars disclosed. The invention extends to all equivalent structures, means, and uses which are within the scope of the appended claims.

Sequence CWU 1

1

54126278DNASphingomonas sp. ATCC53159 1gatcaacggc gccttgctcg gacggcacaa attcgtcctg gtcaatgtgt ccacggtcgc 60ctcttcgata ctgttccagc tgttcccgct tgtcgtcgcc tggatgatcg gcccggacct 120gcgaacgctg ctgatcgccg cgctcgttgg ccgggcggtt ccgatgatcg gcatgctgcc 180cgcgctgtat cgaaaccttt tgcgcggcaa cacgccgcgt tttcacgcca gcgaggcgcg 240cttcctgata ggctatggcg ggtgggcctc gctcacgacc gtggtagcga ccgtgctcat 300gatggcggac cgcttcctga ttggcgcact tcttgggccc gtcgccgtga ccatctacac 360ggcccccctg caactcgcac agcgcgtatc gctgctgccc tccgcactgt ccgccgcgct 420gttcccgcgc ctgcccagcg cgacgccggc ggagcgcatg gcgcttcaga tccgctcgct 480gtcgctgatc atgggcggcc ttaccgggat gatcggcggc ggactattgc tggccgcgcc 540gtttctcgat ctctggatcg gcaagtcgct cggccatgcg ggaacgccgg tcgcgctctt 600cctgttcttc ggcgcatggt ggaatgcgct ggcgatcatt tcgttcagcg gcctgcaggc 660gagcggacgg ccgaaagcga gcgcgatcgt ccagggggca gagctgctac ccgtgttgat 720cgcgctgtat gcagggatcc gatggggcgg cgtgaccggc gccgcagcgg tctttctggg 780acgctccgcc ctggatttcg tcctgttgac ctggcaggca ggcctgctcc gccagacggt 840gaagcaagta tccgtatgcg gcgccgttct caccgtcgcg atgctcgtgg gcgcgaccta 900tcgctattcg gtgccgctct ggtgcgtact cagcgcctgc tgcctggtcg cgctggcagc 960ctgctcctgg tggacattgg cgcgccagga caaggcactg ctgattggac gattgagccg 1020aattctacca aagcagcggc aactcgacct atagcctttc cgcaatgcac cgatggacca 1080caccaacccg ttttaattga cacacacaaa tgctacaccg acaaagacac aggccgagag 1140cgatatagaa gcgctatgcc tagccccagc gtcataaaga tgaacgggtc attgtcacct 1200tgcgacagga ctgaccgcgt atttaaaaga acagccagga aagttgctac ggcgagctca 1260agcgggtagc catctccgct catcttaaga ccacgaaacg cgagcaaaat cattaacgta 1320atcatcgtgc cgtatagcga aacaaaaccc agcaagccgt aatcagccgc tacggacagg 1380aaaccactgt cgatcgatag gaagccttgc tgattacgcc acccgacagc gccagcaccc 1440tctcccgggc catagccgaa gaaagggcgg cgagcgatgg caggcacgcc caagcgaaac 1500tgctcctgcc tgccttgatt gctaagttga gaagcgcctc caccgagaac acggttgtgg 1560acggcaggca cgaacatgac cgccagcgac agcgccacca tcaaggcggg atacgtcaac 1620gtcagcgaaa tgccgacaag cccgcccttt gtggtccgcc accgccgaat tgcccaaata 1680agcaaataca cggtatgcgc caccaatccc cccaccattg ccagtcgaga accgctaaga 1740aatccggacg caactacaag aaaatcgaag aaaatccaaa atgccaatct ccctacgcca 1800cgggaattcg ctatacggtg cagcacgaaa ggaatcgtca aagccgtcaa ctctccccag 1860acaagcggac tgctgaaagt cgtcaaaacg cggtaagtac cccggaaacc gggcgtaagc 1920actacggtaa gaaactgctc atcaacgcgc aggaagctcg gaatcgagta ggcccagagg 1980acgtgcttca cccggaactc cagcacgcca atcgccatca gcacgcccac gcaccaaaac 2040aagcgcgtaa cccaccactc cggggtgcgc gtgtcggtcc cgatcagcca tagcgagatg 2100aatgccatcg gcgtcaccgt cagaacgatg ccaatcaacc gcggaattgt ttgcgaggcc 2160gctggggtcg caatggaggc gacgatctgg accataatga aggcaagcaa tagtcgcgat 2220gggatcggcg ccgcccgcat aatcgccgcc atctcggatc gaaacttttt cgagaccgaa 2280agcgagatca tgagcgtgag caatgcgatc gaaccgatca tccgcctgat cgagatccaa 2340ggcaaaccac caacgctgag cgcaagatag ttcggccaca cgagcgccgc caccatatag 2400gcgaggtata gttttgccag caggcgagta ggcgcctgcc gcgcctcggg tagcgcccag 2460atcactacga gcgccatcag aacgaggggc acggccggga tcgccagcat ctggagcggc 2520agaactgcgg cgagcaggcc gtagactgcg gcaagaaaca tcacgctgac cagcagaacg 2580gtacgccgcg ccgcgatcgt cacgcctgat cgctcggctt tgtagacggg cagtaccggg 2640atcgctggct ttgtcagaaa ccgaaccagt cgcaacctgc gaagccgctg catcgctccg 2700tggaaggccg ggcgacgaaa cgccgaggta gtcgtcatct gcaagtcccc aacaagtccc 2760caagaggcgc tgccgctcgc atgatcgaag ggttcgcgaa aagcaaggtc gatacgccgc 2820actccctgcg atgtgccgcc ggatcgcagg agggcacggg cggcgccggc gcaaggccgc 2880tcaccgcccg cccccgctca ggcgcggtac aggttgtact gatccgccgt agcgctcagt 2940gtcgccgcgc tgcggattgc gcccatcgcc cccgcggtca tcatgtcgac accgatcttg 3000ctgacgagcg cgatctgcga ggacgcggca gtacctatag acagcgtact gcccaccgtg 3060gccaccgtcg caagcggcgt tgccgtgcta gcggcgccgg cacccgccag cagcgcagcg 3120gcctgcgcgg ccgcgccggt gacgaggctg tccttgaccg tcgccgccgc gctggcgctc 3180gacgcggtca ccagcgcctg cacctgggcg gcgctgatcg cgccatcgcg gatctcgatg 3240tcgccgaccg ttccgctgaa tgcggtcgag aacgggctgc cgacatacag cccccaggat 3300tcggcgggcc gggtcgtgcc ggtcatcgtc gctgtgccgc gttgcatgcc gtctacgtac 3360agaatcgcgg ttttccgcgt gctgtcgtag gtcagggcga tcttgtgtgt ggcagcatca 3420agcagcttgg cgccgctcgt caccattgtc tggctgacgc ccgcggcgtt gcgcatggtg 3480aagctcagtt ccccattggc ctgcagcgaa accgaccagc tctggaagat gccaagaatt 3540tgcccggccg tggccgtagc cgagtcccgc ttgaggtcga agctgagcgt gaacgccgac 3600aatgcgtaaa tctgccgcga atagctccgg tttagttcca cccccgtgcc cgtcgagacg 3660tggaaggcgc tgcccacgac cgccgacacg tccaccgcct ttgtcgtctg gccggtattc 3720cagtgcgaaa ggtccacgac gccgctgttg ctgaacgaca gatcgagcag cagcgacgga 3780tttgccgcct tcgcagtcga cagttcggta gtcacctgag cggcagcagc gctcgacacg 3840ggcggctggt acccgacgcc gggaacgatc aaatcgctga gccgcgccgt agccccatcg 3900ttgaggccat agatcttgcg gatcgttgcc gagtcactcg tcagcgtacg attgcctgtc 3960tgcacgatat tgctcgagga gcttgtgacg gtgatcaggt ccgcaacatt gttcttgatc 4020gtcgcgccat tggttttgtc gaggcgaatc caaaatgatg tgccatccac ttgcgatatc 4080acgctattgg attcgatatt gacattaacg ccgttaacaa cgttgatacc gtggtaataa 4140ccattcagat agataagatt gtttttgatg tttacattga catagggaag attaccggcc 4200tcgtcattca tgaaaatccc ttgcgcgcca gagcccgcgc cctgcatgat gacgttattg 4260gagatggtga tgttggtatt gcccttgacc ttgcccgccg tgaagaactg aatggcgtcg 4320ggatgttcgg tgcccacggg aaacaggttc gtgaacgaat ttccgtcgat gacaagattg 4380ttcatctcag tgaagttcgt atgatcgcgc cggttgtcgt ggaagctgct gttctggacc 4440accatgccat cgacgttgta ggcctcaagg cccagaccga agtggtcgat agacgaattc 4500tgcatcgtca ccgacgtgct gttgcgcacg aacaagcccg cccccttcga gagcgaaggg 4560tcaccagtgc cgccgctgaa ccgcacgccg tccaaaacga tgttggccga accctggatc 4620gtattcagtc gattccagtc atcggcgggc ttgtaatcgg tcgcagcgac catgtttttg 4680acggtaacgt tgctactgtt cccgatcacc agcttttgga tattgaccgg gttcgacgag 4740tcgagcgact caattgtcac catgctggta aacgtcttgg tcattacagt gagatctgtg 4800tagaccccgg cggcaagctt gatggtttcg ccacccttcg ccgccgcgat tgcagcattc 4860aactccgtct gattcttgac aatgatatcc ggcatgttga cttaccccgt acgcacgaac 4920ccgggccgat attgaccctt ccattgtcat aaataccaga acagccatga aatttgctcg 4980aagggataca gttaagaact cccttctacg gggccgcatg ccgggcccat gcacgcccga 5040ctttcgccgg caccgtctcg acggcgcaac acagtgcagc tactagggtg cgatgcagat 5100gctcccaacg cccgatgtca gcatactcgt ggtcgctttc aactcgaccg agtatatcga 5160agactgcctg cgcggcatcg ccgaaggagc gggcaagacc ccccacgaag ttctgctgat 5220cgacaatggc gacgggcgaa ccgaagcgct ggtccggcag cggttccacc acgtccgcat 5280cgttcccagt gagggcaata ttggtttcgg ggccggcaat aatcgcctgg cagcgcaggc 5340tgccggcccg ctcctgctgc tcgtcaaccc cgatgccatt ccccagcccg gcgcaatcga 5400tcagttggtc acctttgcca aacagcatcc cgaggcggcg gcatggggcg gccgttccta 5460ctcgcccagc ggcgatctag aacccgcaaa tttcatgtcc ctgccgacgc ccgccgactt 5520tctgacggcg attttcaacg cgcgtgcgct acgcagcggc gggctgcaag aaggcgcgac 5580cacccccgga gcggtcgagg tgttgaatgg cggcttcatg atggtacgca ccgatgtctg 5640gcaggcgatc ggcggttttg acgagagctt ttttctttat tcggaagaga tcgatctctt 5700ccagcgaatc cgcacgttgg ggcacaaggt gctcgtcgac ccctcggtca aagtggtaca 5760caatacgggg agtggtcagt cgatgtccca gaaccgcctg atgtatctca cgaccgggcg 5820catgcactat gcgcgaaagc attttggcgc actcggcacc cttgccaccg ggtgcgcgct 5880ttggctgatc gccgccaaat acacgttggt cggggcggca ctctggcgcc tgtcgccgcg 5940gacgggcacg cgatacaaag agctgagcaa cgggtggcgt gccgtattta gcaatcctgg 6000ccgatggtgg agcggctatc cgcgtcgcta aaagtccagc tccccccccc ctaaaggcgc 6060cgttgggagg cggacgcatc gttgcaacaa cgcgcccgcc tttcagacct tcagttcccc 6120gccggcgttg cgccgctgcc gcgaagctgc ggcggtgcgc tgtagccggc ctgatatttc 6180acggtttccc gcgccttctt caggcggtcg ttgagctgtg cgtcagccgc cttgccgaag 6240cgctcggtac gcagcccgct gagcgcgatc tcgcgcgcct ggtcggccgg caccggcagc 6300accgtggtcg acgtgatgat attcgcggtc agtccctgct gggtcggcag gatgaacatc 6360tcctgtgccg gcagcgacgc gatcttggca gcgatttccg gcggcagcgc agcggtgtcg 6420atctgcgacg gcgcgcgacg gaactggaca ttgtccgccg agagcttggc ggttagctgg 6480tccagcgtct tcagcggcgc gaattgcttg agctttgcgg ccgagctcgg cggagcgaag 6540acgacctgat cgatcgcgta gatcttgcgc tgcgcgaacc gctccggatg cgcggcctga 6600tatttctcga tctcggcatc ggtcggctgg gcgatgccgc cggcgatctt gtcgcgcagc 6660atggcggtga ggatcagctc gtcggcccgg cgctcctgga tcaggaaggc aggcgtcttg 6720tccagcttct gctcgcgggc gaccttggcg aggatcttgc gctcgatgat gcgctgcagc 6780gccagctgct cggccagctt gcgatcggtc cccgggggta cctgggaggc ctgcagttcg 6840gcattcagct cgaagacggt gatttcttcg ccatcgacgc tggcgaccac ctgccccttg 6900tcgagcttgc cgcccttgcc gccacatccg gagacggcca gcgcggccgc agccaccgcc 6960gtaaccaggt acaatttctt catgaagacc tccccgccgg cacggaattg cgcacggcac 7020aaacttctac ttgaacctat tcggacgggc gggcatccgc aatagcgttg gcagtgcagc 7080atggttctaa gcggagccag gcggcaacaa gggggacgag atggcagaag cgaacgcggt 7140agatggaaag gcctccaagc cgctgaaaat gtgccttgca gcgtcgggcg gcggccatct 7200ccggcaaatc ctcgatctgg aatcggtgtg gcgcgaacac gattatttct tcgttactga 7260agataccgcg ctcggccgga gccttgccga aaaacatccc gtcgaactgg tggagcacta 7320tgcgctcggc caggccaagc tgggccatcc cttgcgcatg ctgggcggcg catggcgcaa 7380cctgcgccag agcctttcga tcctgcgccg gcacaagccg gatgtggtga tttccaccgg 7440cgcgggcgca gtctatttca ccgcgctgct cgccaaactg tcgggcgcca agttcgtcca 7500tatcgaaagc ttcgcgcgct tcgaccaccc gtctgccttc ggcaagatgg tgaagggcat 7560cgcgacggtg acgatcgtcc agtcggcggc gctgaaagaa acctggcctg atgccgagct 7620gttcgatccg ttccgcctgc tcgatacacc gcgcccgccc aagcaggcgc taatcttcgc 7680gacggtcggc gccaccctgc ccttcccgcg gctggtgcag gcagtgctcg acctgaagcg 7740cgccggcggg ctgccgggca agctgatcct gcaatatggc gaccaggacc tgcccgatcc 7800cggcatcccc gacgtcgaga tccgccgtac catcccgttc gacgatctgc agctgctgct 7860gcgcgatgcg gatatggtga tatgccacgg cggcaccgga tcgctggtca cggcgctgcg 7920cgccggctgc cgggtcgtcg cctttccgcg ccgccacgat ctgggcgagc attatgacga 7980tcaccaggaa gagatcgccc agaccttcgc cgaccggggc ctgctccagg cggtgcgcga 8040cgagcgccag ctcggcgccg ctgtggaagc ggccaaggca accgagccgc agctggcgac 8100caccgaccac acggccctcg cggcgcggct gcgccagctg ctggcgcagt ggagtgccaa 8160gcgatgagca cgccccggat cagcgtcgtc atcccgcact ataacgatcc gcaatccttg 8220cggctctgcc tggatgcgct ggagcggcag acgatcggtc gcgacgcgtt cgagatcatc 8280gtcggcgaca acaattcgcc ctgtgggctc gcggcggtgg aggcggcggt cgccggacgt 8340gcgcggatcg tgaccattct ggaaaagggg gcgggccccg cgcgcaacgg ggcggcagcc 8400gcagcgcgtg gcgagatcct cgcctttacc gacagtgact gcgtggtgga gcccggctgg 8460ctggcgggcg gcacgaccag ggtcgcgcct ggccgtttca tcggcgggca catgtatgtg 8520cgcaagcccg aagggccgcc gaacggcgcc gaggcgctgg agatggcgct ggcgttcgac 8580aatgaaggct atgtgcggcg cacccagttc acggtcaccg caaacctgtt cgtgatgcgc 8640gccgatttcg aacgggtcgg cggcttccgc gttggcgtgt ccgaggatct ggaatggtgc 8700caccgggcga tcgccagcgg cctcaccatc aactatgcac cggatgcatc ggtgggccac 8760ccgccccggc ccgactggtc ggccctgctg gtgaagacgc ggcgcatcca gcgcgaactc 8820tatctgttca acatcgagcg gccgaagggc aggctgcgct ggctggtccg ttccgtggcg 8880caaccggcga tgatcccaca ggacgtggcc aagatcctgc gcacaccggg taccaagggc 8940gcgcgcctcg ctgcggtcac cacgctggtc cggctgcggc tgtggcgcgg cggcgccggc 9000ttgttgcagt tgctcggccg cgacatctga tcgaccggcg atcggccgac gagcgcgtcg 9060ccggccgatc gcattgcatc agacggtggc cagcgcgtct tccagcgtgc cgctgtcgag 9120ccgcaggcgg ccgatcatca gccacagata gaccggcagc gtatcgtcgg tgaagcggaa 9180gcggcaatcg ccgtcctgcg tttcggattc gaggccgagt tgaccggtga gctcgcccag 9240ctcctgctcg acctgcgccg ccgtgatgtg cgcgcccggc agcagatcca ccacggcttg 9300gccgctgaac cagccatccg ccgagcgcga ggcctcgccc agcgccgcga cgagtggatc 9360gtagcggccg ccgacgaact tgcgcatctc gatcaccgcg cgcggcggca tgcggccctc 9420gatctcaagg atcgcctggt cgagcgcacg acgcagatgc ccggcgtcga ccgtgaggcg 9480gccctggtcc agggcttcca gcgcggaatg gtggcacagc agccgcgcga aatagggcga 9540ccccagcgcg agcaggtgga tcatgtgagt caggtccgga tcgaagcgaa cgcccgaggc 9600ggtttcgccg agcgcgatca tctcctgcac ctccgattcc tccagccggg gcatcggcag 9660gccgatgacg ttgcggcgga tcgacggcgc ataaccgatc agctcctgca ggttcgaggc 9720gacgcccgcg atcaccagct ggacgcgcgc cgaacggtcc gacaggttct tgatcagctc 9780ggcgacctgc tgacggaagg cggaatcgct gacgcgatca tattcgtcga ggatgatcag 9840cacgcgtgtg cccgtgatgt cggcgcacag gtcggccagt tcgccgggcc cgaagctgcc 9900cgtcggcagg cggtcggcca agttgccgcc gctctccgcc tcgccggcgt tgggcgccac 9960gccgcgatgg aacagcagcg gcacgtcttc cagcacggcg cggaagacat cgctgaaatt 10020cgcgttcgca ccgcaggtcg catagctgac gatatagctg gattcgcggg cgacatcggt 10080cagcacgtgg agcagcgagg tcttgccgat gccgcgctcg ccatagagca cgacatggct 10140gcgctggctc tcgatcgagg agattaggcg cgccagcacg ccgaggcgcc cggcgaagct 10200cgaccgatcg gccaccggct gggtgggtgt gaagaaggtc gccagcgcga accgggcgcg 10260cgtgatctcg cggcgctcgt cgcggcggcg atccagcggg cggtccagcg cggaggcacg 10320gaaggttggg aaatccgggc gaccacggcc gctatgggca tcgcgatgcg gcaccactgt 10380cgcagtcagc gggaaatagc cctcttcttc aggttcttct cgacggccga acggccacaa 10440gaatctcagc gcggaaccta cagccactcg aacacctctt aaattcgtgc gccatcggca 10500ccgacggcgc accctggttc gcgccccctg gcgccccctc ctaacgaacc cacgccttgc 10560ctggcctatc ggcgcttgaa gaactcgtac ggtttgatca ccaaggcgat gtacgccagg 10620accagagcga tcgtcaaaat tgcaaagacg tgataattct cattgcccag ataattggcg 10680acggcgcaac cgactgcggg cggcaaatag ctgatcatcg tgtcccggac tgccgaatcg 10740gcttgggacc gttgcaggaa tataacgatc aggccggcaa atatcgcgat ggtgacccaa 10800tcatagggcg tctgcatgca tgtcctttct attcgacacc ggaatcgaac catttccggc 10860gacgctattg cacgcactag cagtgcgcgc ggccgctcgc taggtagcgc cgcaccggat 10920aaaccgacgt taagatggcg cggctcgatc gaaatggagt caaacgggct tgcccggccg 10980accgaagcat ggcgccatgg cgcatgcacc gtattgtgac cacgcaaacc gcgagggtca 11040ttcgatgcgg ttgcttgtac aggaggccat tgataatgaa gccgagaccc gggggaacct 11100ttatgcaagt aaatttcaat cgacaggctc gcaagctcgg tgccggcaat gcgctcgcgc 11160ggggggggcc cgtgcttgcg ctgcttgcga ccgcggcatg gacacaacct gcgctggcgc 11220agcgacaggc atttgagtcc cgcccctccg gtagcgagcg acaggtcgat attcgcgcga 11280cggggtcgct ggaatatgac gacaacgtcg tgctgaacga ccagcggatc acggacggcg 11340cgcgtggcga tgtgatcgca tcgcccgggc tggacgtgac cctagttctg ccccgcgcca 11400ccgggcagct ctacctcacc ggcaatgtcg gatatcgctt ttacaagcga tataccaact 11460ttaaccgcga gcagatctcg ctcaccggcg gcgcagatca gcggttcgcc tcctgcgtcg 11520tgcacgggga agtcggctat cagcgccacc tcaccgacct gtccagcatc ttgatccagg 11580acaccacgcc tgcgctcaac aacaccgaag aggcccggca gtacaccgcg gatatcggct 11640gcggcgcgac ctacggcctg cggcctgccg tttcctacac ccgcaacgaa gtgcgcaaca 11700gccttgccga gcgccgatac gcggactcga ataccaacac ctttaccgca cagcttggcc 11760tgacttcgcc tgccctgggg accgtggcgg tatttgggcg tatgtccgac agcagctatg 11820tccatcgcgt ccttcccggc attaccggcc aggacgggat gaagagctac gcggccggcg 11880tccagctcga gcgctcggtg gccaaccgac tccatttcaa cggctcggtg aattacaccg 11940aggttgaccc aaagctcgca tccaccaaag gattcaaggg cgtaggattt aacgtttccg 12000gcgattatgc tggtgatcag tacagcctcc aattgctggc ttcacgatcg ccccagcctt 12060cacttcttct gttcgtgggt tacgagattg tgacagcggt ttcggcgaat gcgacgcgcc 12120ggctgagcga tcgcattcag atatcgctgc aaggcagccg aacctggcgc gagctcgcgt 12180cttcgcggct gctcaccaac gtgccgattt ccggcaacga caacacctcg acgttgttcg 12240cctccgctac cttccggccg aatcgccggc tgagctttgt gctgggtgcc ggccttcagc 12300ggcgcaccag caacacgcag ctatacagtt acagctccaa acgcatcaat ctctcgacgt 12360cgctttcgct ctgacaaggg ccgtaatcat gcatatcaag aatcgcttcg tgaatatctc 12420gacgttggcc atcgccgccg cgctggccac gccggcggcg gcgcagatcc ccacgcggtc 12480cgtgcccgcg ccggcccgcc cgcggcctgc aacgccgccg gcgcaacagc agaaccaggc 12540gccgtcgacg cccgcagcgg caaccccggc gcagaccgcc gcaaccgttg cccctgcagc 12600aaccgcaccc gcaggttaca aaatcggcgt ggacgacgtg atcgaggccg acgtgctcgg 12660ccagaccgac ttcaagacgc gcgcccgtgt gcaggcggac ggcacggtga ccctgcccta 12720tctgggcgcc gtgcaggtca agggcgagac cgcgacctcg ctcgccgaaa agctggccgg 12780gctgctgcgc gccggcggct attatgccaa gccgatcgtc agcgtcgaaa tcgtcggttt 12840cgtcagcaac tatgtgacgg tgctgggcca ggtgaacagt tccggcctgc agccggtcga 12900ccgcggctat cacgtttccg agatcatcgc ccgtgccggc ggcctgcgcc ccgaagcggc 12960cgatttcgtc gttctcaccc gcgccgatgg ctccagcgcc aagctggact acaagaagct 13020cgcccaaggt ggccccaatg acgatccgat ggtgacgccc ggggacaagg tctttgtccc 13080ggaagtcgag catttctaca tttatggtca aattaacgcg cctggcgtat acgcgattcg 13140atcggacatg acgctccgtc gcgcgctggc ccagggcggt gggcttgccc ccgcaggctc 13200cgtcaagcgt gtgaaggtca cgcgggatgg caatgaactc aagttgaagc tggacgatcc 13260gattctccca ggcgacacga tcgtcatcgg cgaacgattg ttctgatctt ggcaacgatg 13320gcagcggacg aggcccacca gtgaatatca ttcagttctt ccgcattctg tgggtgcgcc 13380gatggatcat cctcccggcg tttctcgttt gcgttaccac tgccaccatt gtggtccagt 13440ttctgcccga acgctacaag gccactacgc gggtggtgct cgacacgttt aagcccgatc 13500ccgtcaccgg acaggtgatg agctcgcagt tcatgcgcgc ctatgtcgag actcagaccc 13560agctgatcga ggactatgcg accgccggtc gcgtggtcga cgaactgggc tgggtgaatg 13620atccggcgaa catctccgcg ttcaacaact cgtccgcggc tgccaccggc gacatccgcc 13680gctggctcgc caagcagatc atcgacaata ccaaggccga tgtgatggag gggagcaaca 13740tcctcgaaat cacctattcg gacagctcgc ccgagcgcgc cgaacgcatc gccaacctga 13800tccgcacctc gttcctcgcc cagtcgctcg ccgccaagcg ccaggccgcg accaagtcgg 13860ccgactggta cgcccagcag gccgaagctg cccgcgattc gctcgctgcg gcggtccagg 13920cccgcaccga tttcgtgaag aagaccggca tcgtgctgac cgaaaccggc gccgacctgg 13980aaacccagaa gctccagcag atcgaggggc agacgacgac cgccaccgcc ccggttgcca 14040tggcccccag cggcatgggc ccggcgcaga tgcagctcgc ccagatcgac cagcagatcc 14100agcaggcagc gaccagccta ggtccgaacc acccaacttt ccaggccttg cagcggcagc 14160gcgaagtgtt cgccaaggca gcggcggcgg aacgcgcgca ggcgaacggc gtatccggtc 14220cggcacgcgg ggccatcgaa agcgcagcca acgcccagcg cgcgcgggtt ctcggcaatc 14280gtcaggatgt cgacaagctt acgcagctgc agcgtgacgt ctcgctgaag caggatcagt 14340acatgaaggc ggcacagcgc gtcgccgatc tgcggctgga agcaagcagc aacgatgtcg 14400gcatgtcgac gctcagcgaa gcatcggcgc cggaaacgcc ctattacccc aaggtgccgc 14460tcatcatcgg tggtgcagcc ggcttcggcc tcgggctcgg tctgctggtc gcgctgctcg 14520tcgagctgct cggccgccgc gtccgcagcc ccgaggatct ggaagttgcg atcgatgcac 14580cggtgctggg cgtgatccag agccgcgcct cgcttgccgc ccgccttcgc cgcgcccaag 14640aaaccctcgg cgaaggtgcc gacacgcacg gagcttcagt aaactgatgg acgcgatgac 14700cagcgaaccg ctgcccgaag gcgatcgtcc gagcgccgtg ccgaccacgc cggatacgat 14760cggcatgctc gaataccagc tcgtcctctc cgatccgacc gggatcgagg cggaagcgat 14820ccgcgcgcta cgcacgcgca tcatgaccca gcacctccgc gagggccggc gcgcgctcgc 14880gatctgcgcc gcctcggcgg gatccggctg cagcttcacc gccgtcaatc tggcgacggc 14940gctggcgcag atcggcgtta agactgcgct ggtcgatgcc aatctgcgcg atcccagcat 15000cggcgcagcc ttcggcctcg ccgccgacaa

gcccggcctg gccgattatc tcgcctcggg 15060cgatgtcgac ctcgcctcga tcatccatgc gacccgcctc gaccagctct cgatcatccc 15120ggccgggcat gtcgagcaca gcccgcagga actgctcgcg tccgaacagt tccatgatct 15180ggcgacgcag ctgctgcgcg agttcgacat cacgatcttc gacaccacgg cgtccaacac 15240ctgcgccgac gcgcagcgtg tcgcgcatat cgccggctat gcgatcatcg tggcgcgcaa 15300ggatgcgagc tacatccgcg acgtgaacac gctcagccgc acgctgcgtg cagaccgcac 15360caacgtcatc ggctgcgtac tgaacggcta ttgatttgga ccatatggca gcgaccgcga 15420tgacgcggca gcaggagagg aagggcggtg gctattggct ggccgttgcc ggtcttgccg 15480cgctaaccat cccgaccttc atcaccctgg gtcgcgaggt ttggagtgcg gaaggcggcg 15540tgcagggtcc gatcgtgctc gccacgggcg cctggatgct ggcccgccag tgctcgacga 15600tcgaggcgct acgccgcccc ggcagcgtgc tgctcggcgc gctgttcctg ctggcgacgc 15660ttgccttcta caccgttgga cgggtgttcg acttcatcag tgtcgaaacc ttcggactgg 15720tcgcgaccta tctggtcgtc gcctatctct atttcggtgc cagggtgctc cgtgccgcct 15780ggttcccggt gctgtggctg ttcttcctgg tgccgccgcc cggctgggcc gtcgaccgca 15840tcaccgcacc gctcaaggag ttcgtctcct atgcggcaac gggcctgctt tcctgggtgg 15900attatccgat cctgcgccag ggcgtgacac tgttcgtcgg cccctatcag ctgctcgtcg 15960aagatgcctg ttcgggtctg cgctcgctgt ccagcctggt cgtcgtgacg ctgctctaca 16020tctacatcaa gaacaagccg tcctggcgct acgcggcgtt catcgcagcg ctggtgatcc 16080cggtggcagt ggtgaccaac gtcctgcgga tcatcatcct ggtactgatc acctatcatc 16140tgggcgacga ggcggcgcag agcttcctcc acgtctccac cggcatggtg atgttcgtgg 16200tcgccctgct ttgcatcttc gcgatcgact gggtggtcga gcaacttctt ctcctgcgtc 16260ggaggcatca tgttcaaccg gcgtgacctg ctgatcggcg caggctgctt cgccgccgct 16320ggcgcctcgc tcggcctgaa gccgcaccgg cggatggacc tgctgggcgg caccaagctc 16380gacacgctga tgcccaaggc attcggcgca tggaaggcag aggataccgg ttcgctgatc 16440gcgccggcgc gcgaaggcag cctggaggac aagctctaca accaggtggt cacccgcgcc 16500ttctcccgcg cggacggtgc ccaagtgatg ctgctgatcg cctatggcaa cgcccagacc 16560gatctactgc agctgcaccg gccggaaata tgctacccgt tcttcggctt caccgtggtg 16620gaaagccatg agcagaccat cccggtgacg ccgcaggtga cgatccccgg tcgcgcgctg 16680accgccacca acttcaaccg caccgagcag atcctctact ggacccgcgt cggcgaatat 16740ctgccgcaga acggcaatca gcagatgctc gcgcggctga agagccaggt ccagggctgg 16800atcgtcgacg gtgtgctggt gcgcatctcg acggtgacgc ccgaggcgga agatggcctg 16860agcgccaatc tcgatttcgc gcgcgagctg gtgaagacgc tcgacccgcg cgtgctgcgc 16920ccgctgctcg ggaacgggct cacacggcag ctcggtcacc aggtctgaac cggtgcgccg 16980cacgcggcgc ccccggcaac aaaaaaggag cggcgcgggc cgccgccgct ccctctcctt 17040ctcatgcggc gccctgccct caccgctcgt gcagcgcgtc actccccgtc tcgagcacgg 17100gccccaccag atagctgaac agggttcgct tgccggtgac gatgtccgcg ctcgcgagca 17160tccccggccg cagcggcacc tgtgcgccat gggccagcac atacccgcgc gccagcgcga 17220tccgcgcctt gtagaccggc ggctggttct ccttcatctg caccgcctcg gggctgatgc 17280ccgccaccgt gccgggaatc atgccgtagc gggtataggg aaaggcctgc agcttcacct 17340ttaccggcat gccgatgtgg acgaagccga tgtcgctgtt gtcgaccatc acctcggcct 17400cgagccgggc attgtcggga accaggctga ggagcggctt ggccccttcc accacgccgc 17460cttcggtgtg gacctgcagc tgcgagacgg taccgctcac cggcgcgcgc agttcgcgga 17520acgagctgcg cagattcgcc ttggcgacgt cctcgccgcg ggcacgcacc tcgtcctgcg 17580ccttgaccag atcctgcagc acctgcgccc gcgcctcctc gcgcgtcttg gccgacaggc 17640tggagacgct cagcgactgc tggccgagtt tggcgagcgt agcgcgcgcc gccgtcaggt 17700cctgccgctc ggcgatcagc tggcgacgca tctccacgac gcgcagcttc gagacatagc 17760ccttggcggc catcgtctcg ttcgcggcga tctgctgttc gagcagcggc agcgactgtt 17820cgagcttccg cacctgtgcc tgcgcctcgg ccgcggccga gacggcggca ccgcgatcgg 17880agcggccgcc ggccagcgcc gcctcgatct ggcccagccg ggcgcgggcg aggccgcgat 17940gcgtcgccac ttcgcccggg ctggcggcgg caggcgcgac gaagcggaag cccctgccgt 18000ccagcgcgtc gatgatcgcc tggttgcgtg cggcgtcgag ctgggcgctg agcagcgcca 18060ccttcgcctg tgccgcctcc gccgacgaca cggtcgggtc gagcgtgatc agcacctggc 18120ccttggcgac cttctgcccc tcgcccacca ggatgcggcg gacgatcccc gattcgggcg 18180actggacgat cttggtctcg ccgatcggcg cgatccgccc ctgcgtcggc gcgacgactt 18240cgaccttgcc gatcgccagc caggcggcgg tgatcgccag cccggccagc atcaccttgg 18300cggtaagccg cgcggtgggc gaaaccggcc gctcgatgat ctccagcgcg gcaggcagga 18360aggcggtgtc ataagcgtcg acgcgggcag gcagcacggt atcgcgcatg cgggcgagcg 18420ggccgccgcg gcgcatcgga acaacggcgt tcatgcggca atctccccat agccgccctg 18480gcggcggtgc aggtcggcat agcggccgcc caggcgcaac aattcgtcgt gtcggccgct 18540ctcgacgatg cggccctgtt cgagcgtgat gatccggtcg cagctgcgca ccgcgctcag 18600gcgatgcgcg atcaccacga gcgtgcggcc ggccgagatg gcgcgcaggt tgttctggat 18660cagctcctcg ctctcggcat cgagcgccga ggtcgcttcg tcgaacacca ggatgcgcgg 18720attgccgacg agcgcgcggg cgatggcgag ccgctggcgc tggccgccgg agagattgac 18780gccgcgctcg acgatctcgg tgtcatagcc gcgcggctgg cgcaggatga aatcatgcgc 18840gccggccagc gtcgccgccg cgacgacatt ctcgaacggc atggcggggt tggagagcgc 18900gatgttctcg cggatcgagc ggctgaacag cagattctcc tgcagcacga cgccgatctg 18960gcgacgcagc caggcgggat cgagctgcgc cacgtcgacc tcgtcgacca gcacgcggcc 19020gagattcggc aggttgagcc gctggagcag cttggccagc gtcgacttgc ccgagcccga 19080cgaaccgacg atgccgagcg aggtgcccgc cggaatgtcg agcgtgatgt cgctcagcac 19140cggcggctgg tcctcggcat agcggaagct gacattctcg aagcgaatcg caccgcgcag 19200caccggcagc gtcgccgccg aggccgggcg cggttccacc ggatggttga gcacgtcgcc 19260cagccgctcg accgagatgc gcacctgctg gaaatcctgc cacagctgcg ccatgcggat 19320caccggcccg gacacgcgct gggcgaacat gttgaacgcc accagcgcgc ctacgctcat 19380cgcgccgccg atcaccgcct tggcgccgaa gaacaggatc gccgcgaagc tcagcttcga 19440gatcagctcg atcgcctggc tgccggtgtt ggcggtattg atcagccgct gcgacgcggc 19500ggtatgggcg gcgagctggc gctcccagcg attctgccag tgcggctcga ccgcggtcgc 19560cttgatcgtg tggatgcccg agacgctctc gacgagcagc gcgttgctgg cggagctctt 19620ctcgaacttg tcctccaccc gcgcgcggag cggcccggcg acgctgaacg atacgatcgc 19680ataggcgatc agcgacacga gcacgatgcc cgagagcatc ggcgagtaga acagcatcgc 19740ggcgaggaac acgaaggtga acagcgggtc caccatcacc gtcagcgagg cgctggtaag 19800gaattcgcgg atcgtctcga gctggcggac gcgggtgacg gtgtcgccca cgcggcgctt 19860ctcgaaatag gcgagcggca gcgccagcag gtggtggaac agccgggcac ccagctcgac 19920gtcgatcttc tgcgtcgtct cggtgaacag gcgggtgcgg atccagccga gcgccacttc 19980ccacaccgaa accgccagga aggcgaaggc gagcacgctc agcgtgctca tgctgttgtg 20040gatcagcacc ttgtcgatca cgctctggaa caacagcggc gcggcgaggc cgagcaggtt 20100gagcgcgagg gtgatgccga gcacctcgag gaacagcgtg cgatagcgcc ggaactgcgc 20160ggtgaaccag gagaggccga accgcagcgg ccgtcccgcc accgcgcggg tggtgagcag 20220caccagcgcg ccggaccaga tcgcgtccag cgcgtcccgg tcgacctgtt ccggggcatg 20280gcccgggcgc tggatgatca cgccatgttc ggtcaggccg ccgatcacga accagccttc 20340gggcccgtcg gcgatcgcgg gcagcggctg gcgggcgagt ccgccgcgcg gcacctcgac 20400ggccttggcg cgcacgccct gctggcgctt ggccaggagg atcaggtcgt cggcgcttgc 20460cgcctcggca tggcccagcg cgtggcgcag ctgttcgggc gtgatggcga tgttgtgcgc 20520gccgagcagc agcgacaacg ccaccagtcc ggattcgcgc agctccgcct cgcgctccgc 20580cgccccatgg gccgcgagcg cgctctgcag ggtggcctgc atttcgtcgc gtgtcatttc 20640cggaactctg cctccatggc gatactgaga gcgccatgat gaagaaggct ggtaaagact 20700cacttaatcc tagcttttct ggtatttacc cgtagctgcc gacccgattt gggacaggcc 20760tggcttagca ggtccttaaa ctcgaccgac tataccgcga cgccgaggag ggggaggatt 20820ggcgccgcat cgcgcggcga aacgcgggtg cgtcgcaaca tttcgccgga gtcgatccgt 20880cgcgaatgct gcacccgcga acgcaatgac ggccgccacg caatccggct tgatcccggg 20940cggcggatcg cgataagccg cgccacggtc gccaaaactc gtcgaaataa ccgacaaaac 21000cacggcatat ggctggatat tgcagcgttt gccctgcgtt tccgtcgttc aaccgccctt 21060cgaatcaggc aggcccagcg tgaccatgat tgatcttcct cttggaacgg cacactttgg 21120tcgacacgga gacttccggt cgggcaattg tcccgttata gtgcaatgca acaggccgaa 21180tcggccgctg tcggcgtgca cattccgttg agggagcccg atgaggcaat gaacgctttc 21240gaagcacagc gcgcctttga ggagcaactt cgggcgcatt cccgggttac gccatctgcc 21300gctcccgtgt ggcgtcgctc gacgctgcgg atggtcctct ataccgagtt gctgctgctg 21360gacagtctct cgatcctggc cggattccac gtcgcggcgg gcacgcgcga cggcaactgg 21420ctgtcgctgg cgggcatcaa cgtcggcgtc ttcctgctgc cgatcgctct cggcaccgcg 21480ctcgcaagcg gcacctactc gctgaactgc ctgcgctacc cggtcagcgg cgtgaagagc 21540atcttctcgg cattcttctt ctcgatcttc gtcgtcctgc tcggcagcta cctgctgacg 21600gccgagctgc cgctgtcccg cgtgcagctg gcggagggcg cgatcctctc gctggtcctc 21660ctgatggtgg gccgcctgat gttccgccgc cacgtccgcg cggttaccgg cggcaggctg 21720ctcgacgaac tggtcatcat cgacggcgtc tcgctcgacg tcgcgggcaa tgcggtcgcg 21780ctcgacgcgc ggatcatcaa tctctcgccg aacccgcgcg atccgcaaat gctgcatcgc 21840ctgggcacca ccgtgatcgg gttcgaccgg gtgatcgtcg cctgcaccaa ggagcatcgc 21900gcggtctggg cgctgctgct caagggcatg aacatcaagg gcgagatcct cgtcccccag 21960ttcaatgcgc tgggcgcgat cggcgtggac gcctttgacg ggaaggatac gctggtcgtc 22020tcgcagggcc cgctcaacat gcccaaccgc gcgaagaagc gcgcgctcga tctcgcgatc 22080accgtaccgg ccgtgctcgc gctggcgccg ctgatgatcc tggtggcgat cctgatcaag 22140ctggagagcc cgggcccggt gttgttcgcg caggatcgcg tcggccgcgg caaccggctg 22200ttcaagatca tgaagttccg ctcgatgcgc gtaacgctgt gcgacgcgaa cggcaacgtc 22260tcggccagcc gcgacgacga tcgcatcacc aaggtcggcc gcttcatccg caagaccagc 22320atcgacgaac tgccgcagct gctgaacgtg ctgcgcggcg acatgagcgt cgtcggcccg 22380cggccgcatg cgctgggctc gcgcgccgcc gatcacctgt tctgggaaat cgacgagcgc 22440tactggcacc gccacacgct caagccgggc atgaccggtc tggcccaggt gcgcggtttc 22500cgcggggcga ccgatcgccg cgtcgatctg accaaccggc tccaggcaga catggaatat 22560atcgacggat gggatatctg gcgcgatatc acgatcctgt tcaagacgct gcgggtgatc 22620gtgcattcga acgcattctg atccgcgcac gacgctgggc cgcagcctcg atccgcaaat 22680ggattgacag cggcccggct tccgttttct cgtttgattt tcgttgcggc cggtccgcgc 22740catgggggat tactgaatga agggcatcat ccttgcgggg ggcagcggga cgcgcctgta 22800ccccgcaacg ctatcgatct cgaagcagct gcttcccgtc tatgacaagc cgatgatctt 22860ctatccgctg tcggtgctga tgctcaccgg catccgggac atcctgatta tctccacccc 22920gcgcgacctg ccgatgttcc aggcgctgct gggcgacggc tcggccttcg gcatcaacct 22980cagctatgcc gagcagccct cccccaacgg gctggccgaa gcgttcatca tcggcgcgga 23040tttcgtcggc aacgatccca gcgcgctgat cctgggcgac aacatctatc acggcgaaaa 23100gatgggcgag cgctgccagg cagccgcagc gcaggcagcg cagggcggtg caaacgtctt 23160cgcctatcat gtcgacgacc ccgagcgcta cggcgtggtc gcgttcgacc cggagacggg 23220cgtcgccacc agcgtcgagg aaaagccggc cgagcccaag tccaactggg cgatcaccgg 23280cctgtatttc tacgacaagg acgtggtcga catcgccaag tcgatccagc cctcggcgcg 23340cggcgaactc gagatcaccg acgtcaaccg cgtttacatg gagcgcggcg acctgcacat 23400cacgcgcctc ggccgcggct atgcctggct cgacaccggc acgcatgaca gcctgcacga 23460agccggctcg ttcgttcgca cgctcgagca tcggacgggc gtgaagatcg cctgcccgga 23520ggaaatcgcc ttcgaaagcg gctggctcgg cgccgaagac ctgctcaagc gcgccgccgg 23580cctcggcaag accggctatg ccgcctatct ccgcaaggtt gcgaccgcag catgacccag 23640gtccatcatc acgaactgtc cggcgtcatc gagttcacgc cgcccaaata tggcgaccac 23700cgcggcttct tctccgaagt gttcaagcag tcggtgctcg atgccgaagg cgtcgaggca 23760cgctgggtgc aggacaatca gagcttctcg gcggccccgg gcacgatccg cggcctgcat 23820ctccaggcgc cgcccttcgc ccaggccaag ctggtccgcg tgttgcgcgg cgcgatcttc 23880gacgtcgcgg tcgacatccg tcgcggctcg cccacctatg gcaaatgggt cggcgtcgag 23940ctctcggccg agaagtggaa ccagctgctg gtccccgccg gctatgcgca cggcttcatg 24000acgctcgttc cggattgcga gatcctctac aaggtcagcg ccaaatattc gaaggattcg 24060gagatggcga tccgttggga cgatcccgat ctcgccatcg cctggccgga catcggcgtc 24120gagccggtcc tctccgaaaa ggacgcggtc gccacgccct tcgccgaatt caacaccccc 24180ttcttctatc agggctgagc catgcagcag accttcctcg tcaccggcgg cgccggcttc 24240atcggctcgg cggtggtgcg ccacctcgtc cgccagggcg cgcgcgtcat caatctcgac 24300aagctcacct atgccggcaa cccggcctcg ctgactgcga tcgagaacgc gcccaactat 24360cgcttcgtcc atgccgacat cgccgacacc gcgacgatcc taccgctgct gcgcgaggag 24420caggtcgatg tggtgatgca cctcgccgcc gagagccatg tcgatcgctc gatcgacggc 24480cctggcgagt tcatcgagac caatgtcgtc ggcaccttca agctgctcca gtcggcgctg 24540caatattggc gcgagctgga gggcgagaaa cgcgacgcgt tccgcttcca ccacatctcc 24600accgacgaag tgttcggcga cctgccgttc gacagcggca tcttcaccga agagacgccc 24660tatgatccct cctcgcccta ttcggcgtcg aaggcggcga gcgaccatct ggtgcgcgcc 24720tggggccaca cctatggcct gccggtggtg ctgtcgaact gctcgaacaa ttacgggccg 24780ttccacttcc ccgagaagct gatcccgttg accatcctca acgcgctcga gggcaagccg 24840ctgccggtct acggcaaggg cgagaatatc cgcgactggc tgtatgtcga cgatcacgcc 24900aaggcgctgg cgaccatcgc caccaccggc aaggtcggcc agagctacaa tgtcggcggc 24960cgcaacgagc ggaccaacct gcaggtggtc gagacgatct gcgacctgct cgaccagcgc 25020attccgctgg ccgacggtcg caagcgccgc gaactgatca ccttcgtcac cgatcgcccc 25080ggccatgacc gccgctacgc gatcgacgcg accaagctcg agaccgagct gggctggaag 25140gctgaggaga atttcgacac cggcatcgcc gcgacgatcg actggtatct ggcgaacgag 25200tggtggtggg gcccgatccg ctccggcaaa tatgccggcg agcggctggg gcagaccgcc 25260tgatgcgtat cctcgtcacc gggcatgacg gccaggtcgc ccagtcgctg gccgagcagg 25320cggtgggcca cgagctggtc ttcaccacct accccgaatt cgatctctcc aagccggaga 25380cgatcgaggc cggtgtggcg cgggtgcacc cggacctgat cgtctccgcc gccgcctaca 25440cggcggtcga caaggcggaa agcgaacccg agctggcgat ggcgatcaac ggcgacggtc 25500ccggcgtgct ggcgcgcgcg ggcgcgaaga tcggcgcgcc gatcatccac ctgtcgaccg 25560attatgtgtt cgacggcagt ctcgaccgcc cttggcgcga ggacgatccc accggcccgc 25620tcggcgtcta tggcgcgacc aagctggccg gcgagcaggc ggtgcaggcc tcgggtgcca 25680ccaacgccgt gatccggctg gcctgggtct acagcccgtt cggcaacaat ttcgtcaaga 25740cgatgctccg cctcgccgag acgcgcgacg cgctgaacgt cgtggaggac cagtggggct 25800gccccagttc ggcgctggac atcgcgaccg cgatcctgac ggtggtcggg cactggcagc 25860aggacggcgc gacgagcggc ctctaccatt tcgccggcac cggcgagacc aactgggccg 25920acttcgcatc gacgatcttc gccgagagcg ccaagcgcgg tggcccctcg gccaccgtca 25980ccggcattcc cagctcgggc tatccgactc cggccacgcg cccggccaat tcgcggctgg 26040actgcacccg cttcgcggag accttcggct accgggcgcc tgcctggcag gattcgctga 26100acgtcgtact ggatcgcctg ctcggctgat ccgaaacggg gggcctcagc gccccccgcc 26160atgctcccgt tcgcgcgccg gcaatgcctc tagcaccgcg cgctttccct taggactcag 26220ctcgctccag ccggcgattt ccttgggcga ccgccagcac cccaggcaca gccggatc 2627821053DNASphingomonas sp. ATCC53159 2atcaacggcg ccttgctcgg acggcacaaa ttcgtcctgg tcaatgtgtc cacggtcgcc 60tcttcgatac tgttccagct gttcccgctt gtcgtcgcct ggatgatcgg cccggacctg 120cgaacgctgc tgatcgccgc gctcgttggc cgggcggttc cgatgatcgg catgctgccc 180gcgctgtatc gaaacctttt gcgcggcaac acgccgcgtt ttcacgccag cgaggcgcgc 240ttcctgatag gctatggcgg gtgggcctcg ctcacgaccg tggtagcgac cgtgctcatg 300atggcggacc gcttcctgat tggcgcactt cttgggcccg tcgccgtgac catctacacg 360gcccccctgc aactcgcaca gcgcgtatcg ctgctgccct ccgcactgtc cgccgcgctg 420ttcccgcgcc tgcccagcgc gacgccggcg gagcgcatgg cgcttcagat ccgctcgctg 480tcgctgatca tgggcggcct taccgggatg atcggcggcg gactattgct ggccgcgccg 540tttctcgatc tctggatcgg caagtcgctc ggccatgcgg gaacgccggt cgcgctcttc 600ctgttcttcg gcgcatggtg gaatgcgctg gcgatcattt cgttcagcgg cctgcaggcg 660agcggacggc cgaaagcgag cgcgatcgtc cagggggcag agctgctacc cgtgttgatc 720gcgctgtatg cagggatccg atggggcggc gtgaccggcg ccgcagcggt ctttctggga 780cgctccgccc tggatttcgt cctgttgacc tggcaggcag gcctgctccg ccagacggtg 840aagcaagtat ccgtatgcgg cgccgttctc accgtcgcga tgctcgtggg cgcgacctat 900cgctattcgg tgccgctctg gtgcgtactc agcgcctgct gcctggtcgc gctggcagcc 960tgctcctggt ggacattggc gcgccaggac aaggcactgc tgattggacg attgagccga 1020attctaccaa agcagcggca actcgaccta tag 10533350PRTSphingomonas sp. ATCC53159 3Ile Asn Gly Ala Leu Leu Gly Arg His Lys Phe Val Leu Val Asn Val1 5 10 15Ser Thr Val Ala Ser Ser Ile Leu Phe Gln Leu Phe Pro Leu Val Val 20 25 30Ala Trp Met Ile Gly Pro Asp Leu Arg Thr Leu Leu Ile Ala Ala Leu 35 40 45Val Gly Arg Ala Val Pro Met Ile Gly Met Leu Pro Ala Leu Tyr Arg 50 55 60Asn Leu Leu Arg Gly Asn Thr Pro Arg Phe His Ala Ser Glu Ala Arg65 70 75 80Phe Leu Ile Gly Tyr Gly Gly Trp Ala Ser Leu Thr Thr Val Val Ala 85 90 95Thr Val Leu Met Met Ala Asp Arg Phe Leu Ile Gly Ala Leu Leu Gly 100 105 110Pro Val Ala Val Thr Ile Tyr Thr Ala Pro Leu Gln Leu Ala Gln Arg 115 120 125Val Ser Leu Leu Pro Ser Ala Leu Ser Ala Ala Leu Phe Pro Arg Leu 130 135 140Pro Ser Ala Thr Pro Ala Glu Arg Met Ala Leu Gln Ile Arg Ser Leu145 150 155 160Ser Leu Ile Met Gly Gly Leu Thr Gly Met Ile Gly Gly Gly Leu Leu 165 170 175Leu Ala Ala Pro Phe Leu Asp Leu Trp Ile Gly Lys Ser Leu Gly His 180 185 190Ala Gly Thr Pro Val Ala Leu Phe Leu Phe Phe Gly Ala Trp Trp Asn 195 200 205Ala Leu Ala Ile Ile Ser Phe Ser Gly Leu Gln Ala Ser Gly Arg Pro 210 215 220Lys Ala Ser Ala Ile Val Gln Gly Ala Glu Leu Leu Pro Val Leu Ile225 230 235 240Ala Leu Tyr Ala Gly Ile Arg Trp Gly Gly Val Thr Gly Ala Ala Ala 245 250 255Val Phe Leu Gly Arg Ser Ala Leu Asp Phe Val Leu Leu Thr Trp Gln 260 265 270Ala Gly Leu Leu Arg Gln Thr Val Lys Gln Val Ser Val Cys Gly Ala 275 280 285Val Leu Thr Val Ala Met Leu Val Gly Ala Thr Tyr Arg Tyr Ser Val 290 295 300Pro Leu Trp Cys Val Leu Ser Ala Cys Cys Leu Val Ala Leu Ala Ala305 310 315 320Cys Ser Trp Trp Thr Leu Ala Arg Gln Asp Lys Ala Leu Leu Ile Gly 325 330 335Arg Leu Ser Arg Ile Leu Pro Lys Gln Arg Gln Leu Asp Leu 340 345 35041626DNASphingomonas sp. ATCC53159 4atgacgacta cctcggcgtt tcgtcgcccg gccttccacg gagcgatgca gcggcttcgc 60aggttgcgac tggttcggtt tctgacaaag ccagcgatcc cggtactgcc cgtctacaaa 120gccgagcgat caggcgtgac gatcgcggcg cggcgtaccg ttctgctggt cagcgtgatg 180tttcttgccg cagtctacgg cctgctcgcc gcagttctgc cgctccagat gctggcgatc 240ccggccgtgc ccctcgttct gatggcgctc gtagtgatct gggcgctacc cgaggcgcgg 300caggcgccta ctcgcctgct ggcaaaacta tacctcgcct atatggtggc ggcgctcgtg 360tggccgaact atcttgcgct cagcgttggt ggtttgcctt ggatctcgat caggcggatg 420atcggttcga tcgcattgct cacgctcatg atctcgcttt cggtctcgaa aaagtttcga 480tccgagatgg cggcgattat

gcgggcggcg ccgatcccat cgcgactatt gcttgccttc 540attatggtcc agatcgtcgc ctccattgcg accccagcgg cctcgcaaac aattccgcgg 600ttgattggca tcgttctgac ggtgacgccg atggcattca tctcgctatg gctgatcggg 660accgacacgc gcaccccgga gtggtgggtt acgcgcttgt tttggtgcgt gggcgtgctg 720atggcgattg gcgtgctgga gttccgggtg aagcacgtcc tctgggccta ctcgattccg 780agcttcctgc gcgttgatga gcagtttctt accgtagtgc ttacgcccgg tttccggggt 840acttaccgcg ttttgacgac tttcagcagt ccgcttgtct ggggagagtt gacggctttg 900acgattcctt tcgtgctgca ccgtatagcg aattcccgtg gcgtagggag attggcattt 960tggattttct tcgattttct tgtagttgcg tccggatttc ttagcggttc tcgactggca 1020atggtggggg gattggtggc gcataccgtg tatttgctta tttgggcaat tcggcggtgg 1080cggaccacaa agggcgggct tgtcggcatt tcgctgacgt tgacgtatcc cgccttgatg 1140gtggcgctgt cgctggcggt catgttcgtg cctgccgtcc acaaccgtgt tctcggtgga 1200ggcgcttctc aacttagcaa tcaaggcagg caggagcagt ttcgcttggg cgtgcctgcc 1260atcgctcgcc gccctttctt cggctatggc ccgggagagg gtgctggcgc tgtcgggtgg 1320cgtaatcagc aaggcttcct atcgatcgac agtggtttcc tgtccgtagc ggctgattac 1380ggcttgctgg gttttgtttc gctatacggc acgatgatta cgttaatgat tttgctcgcg 1440tttcgtggtc ttaagatgag cggagatggc tacccgcttg agctcgccgt agcaactttc 1500ctggctgttc ttttaaatac gcggtcagtc ctgtcgcaag gtgacaatga cccgttcatc 1560tttatgacgc tggggctagg catagcgctt ctatatcgct ctcggcctgt gtctttgtcg 1620gtgtag 16265541PRTSphingomonas sp. ATCC53159 5Met Thr Thr Thr Ser Ala Phe Arg Arg Pro Ala Phe His Gly Ala Met1 5 10 15Gln Arg Leu Arg Arg Leu Arg Leu Val Arg Phe Leu Thr Lys Pro Ala 20 25 30Ile Pro Val Leu Pro Val Tyr Lys Ala Glu Arg Ser Gly Val Thr Ile 35 40 45Ala Ala Arg Arg Thr Val Leu Leu Val Ser Val Met Phe Leu Ala Ala 50 55 60Val Tyr Gly Leu Leu Ala Ala Val Leu Pro Leu Gln Met Leu Ala Ile65 70 75 80Pro Ala Val Pro Leu Val Leu Met Ala Leu Val Val Ile Trp Ala Leu 85 90 95Pro Glu Ala Arg Gln Ala Pro Thr Arg Leu Leu Ala Lys Leu Tyr Leu 100 105 110Ala Tyr Met Val Ala Ala Leu Val Trp Pro Asn Tyr Leu Ala Leu Ser 115 120 125Val Gly Gly Leu Pro Trp Ile Ser Ile Arg Arg Met Ile Gly Ser Ile 130 135 140Ala Leu Leu Thr Leu Met Ile Ser Leu Ser Val Ser Lys Lys Phe Arg145 150 155 160Ser Glu Met Ala Ala Ile Met Arg Ala Ala Pro Ile Pro Ser Arg Leu 165 170 175Leu Leu Ala Phe Ile Met Val Gln Ile Val Ala Ser Ile Ala Thr Pro 180 185 190Ala Ala Ser Gln Thr Ile Pro Arg Leu Ile Gly Ile Val Leu Thr Val 195 200 205Thr Pro Met Ala Phe Ile Ser Leu Trp Leu Ile Gly Thr Asp Thr Arg 210 215 220Thr Pro Glu Trp Trp Val Thr Arg Leu Phe Trp Cys Val Gly Val Leu225 230 235 240Met Ala Ile Gly Val Leu Glu Phe Arg Val Lys His Val Leu Trp Ala 245 250 255Tyr Ser Ile Pro Ser Phe Leu Arg Val Asp Glu Gln Phe Leu Thr Val 260 265 270Val Leu Thr Pro Gly Phe Arg Gly Thr Tyr Arg Val Leu Thr Thr Phe 275 280 285Ser Ser Pro Leu Val Trp Gly Glu Leu Thr Ala Leu Thr Ile Pro Phe 290 295 300Val Leu His Arg Ile Ala Asn Ser Arg Gly Val Gly Arg Leu Ala Phe305 310 315 320Trp Ile Phe Phe Asp Phe Leu Val Val Ala Ser Gly Phe Leu Ser Gly 325 330 335Ser Arg Leu Ala Met Val Gly Gly Leu Val Ala His Thr Val Tyr Leu 340 345 350Leu Ile Trp Ala Ile Arg Arg Trp Arg Thr Thr Lys Gly Gly Leu Val 355 360 365Gly Ile Ser Leu Thr Leu Thr Tyr Pro Ala Leu Met Val Ala Leu Ser 370 375 380Leu Ala Val Met Phe Val Pro Ala Val His Asn Arg Val Leu Gly Gly385 390 395 400Gly Ala Ser Gln Leu Ser Asn Gln Gly Arg Gln Glu Gln Phe Arg Leu 405 410 415Gly Val Pro Ala Ile Ala Arg Arg Pro Phe Phe Gly Tyr Gly Pro Gly 420 425 430Glu Gly Ala Gly Ala Val Gly Trp Arg Asn Gln Gln Gly Phe Leu Ser 435 440 445Ile Asp Ser Gly Phe Leu Ser Val Ala Ala Asp Tyr Gly Leu Leu Gly 450 455 460Phe Val Ser Leu Tyr Gly Thr Met Ile Thr Leu Met Ile Leu Leu Ala465 470 475 480Phe Arg Gly Leu Lys Met Ser Gly Asp Gly Tyr Pro Leu Glu Leu Ala 485 490 495Val Ala Thr Phe Leu Ala Val Leu Leu Asn Thr Arg Ser Val Leu Ser 500 505 510Gln Gly Asp Asn Asp Pro Phe Ile Phe Met Thr Leu Gly Leu Gly Ile 515 520 525Ala Leu Leu Tyr Arg Ser Arg Pro Val Ser Leu Ser Val 530 535 54061998DNASphingomonas sp. ATCC53159 6atgccggata tcattgtcaa gaatcagacg gagttgaatg ctgcaatcgc ggcggcgaag 60ggtggcgaaa ccatcaagct tgccgccggg gtctacacag atctcactgt aatgaccaag 120acgtttacca gcatggtgac aattgagtcg ctcgactcgt cgaacccggt caatatccaa 180aagctggtga tcgggaacag tagcaacgtt accgtcaaaa acatggtcgc tgcgaccgat 240tacaagcccg ccgatgactg gaatcgactg aatacgatcc agggttcggc caacatcgtt 300ttggacggcg tgcggttcag cggcggcact ggtgaccctt cgctctcgaa gggggcgggc 360ttgttcgtgc gcaacagcac gtcggtgacg atgcagaatt cgtctatcga ccacttcggt 420ctgggccttg aggcctacaa cgtcgatggc atggtggtcc agaacagcag cttccacgac 480aaccggcgcg atcatacgaa cttcactgag atgaacaatc ttgtcatcga cggaaattcg 540ttcacgaacc tgtttcccgt gggcaccgaa catcccgacg ccattcagtt cttcacggcg 600ggcaaggtca agggcaatac caacatcacc atctccaata acgtcatcat gcagggcgcg 660ggctctggcg cgcaagggat tttcatgaat gacgaggccg gtaatcttcc ctatgtcaat 720gtaaacatca aaaacaatct tatctatctg aatggttatt accacggtat caacgttgtt 780aacggcgtta atgtcaatat cgaatccaat agcgtgatat cgcaagtgga tggcacatca 840ttttggattc gcctcgacaa aaccaatggc gcgacgatca agaacaatgt tgcggacctg 900atcaccgtca caagctcctc gagcaatatc gtgcagacag gcaatcgtac gctgacgagt 960gactcggcaa cgatccgcaa gatctatggc ctcaacgatg gggctacggc gcggctcagc 1020gatttgatcg ttcccggcgt cgggtaccag ccgcccgtgt cgagcgctgc tgccgctcag 1080gtgactaccg aactgtcgac tgcgaaggcg gcaaatccgt cgctgctgct cgatctgtcg 1140ttcagcaaca gcggcgtcgt ggacctttcg cactggaata ccggccagac gacaaaggcg 1200gtggacgtgt cggcggtcgt gggcagcgcc ttccacgtct cgacgggcac gggggtggaa 1260ctaaaccgga gctattcgcg gcagatttac gcattgtcgg cgttcacgct cagcttcgac 1320ctcaagcggg actcggctac ggccacggcc gggcaaattc ttggcatctt ccagagctgg 1380tcggtttcgc tgcaggccaa tggggaactg agcttcacca tgcgcaacgc cgcgggcgtc 1440agccagacaa tggtgacgag cggcgccaag ctgcttgatg ctgccacaca caagatcgcc 1500ctgacctacg acagcacgcg gaaaaccgcg attctgtacg tagacggcat gcaacgcggc 1560acagcgacga tgaccggcac gacccggccc gccgaatcct gggggctgta tgtcggcagc 1620ccgttctcga ccgcattcag cggaacggtc ggcgacatcg agatccgcga tggcgcgatc 1680agcgccgccc aggtgcaggc gctggtgacc gcgtcgagcg ccagcgcggc ggcgacggtc 1740aaggacagcc tcgtcaccgg cgcggccgcg caggccgctg cgctgctggc gggtgccggc 1800gccgctagca cggcaacgcc gcttgcgacg gtggccacgg tgggcagtac gctgtctata 1860ggtactgccg cgtcctcgca gatcgcgctc gtcagcaaga tcggtgtcga catgatgacc 1920gcgggggcga tgggcgcaat ccgcagcgcg gcgacactga gcgctacggc ggatcagtac 1980aacctgtacc gcgcctga 19987665PRTSphingomonas sp. ATCC53159 7Met Pro Asp Ile Ile Val Lys Asn Gln Thr Glu Leu Asn Ala Ala Ile1 5 10 15Ala Ala Ala Lys Gly Gly Glu Thr Ile Lys Leu Ala Ala Gly Val Tyr 20 25 30Thr Asp Leu Thr Val Met Thr Lys Thr Phe Thr Ser Met Val Thr Ile 35 40 45Glu Ser Leu Asp Ser Ser Asn Pro Val Asn Ile Gln Lys Leu Val Ile 50 55 60Gly Asn Ser Ser Asn Val Thr Val Lys Asn Met Val Ala Ala Thr Asp65 70 75 80Tyr Lys Pro Ala Asp Asp Trp Asn Arg Leu Asn Thr Ile Gln Gly Ser 85 90 95Ala Asn Ile Val Leu Asp Gly Val Arg Phe Ser Gly Gly Thr Gly Asp 100 105 110Pro Ser Leu Ser Lys Gly Ala Gly Leu Phe Val Arg Asn Ser Thr Ser 115 120 125Val Thr Met Gln Asn Ser Ser Ile Asp His Phe Gly Leu Gly Leu Glu 130 135 140Ala Tyr Asn Val Asp Gly Met Val Val Gln Asn Ser Ser Phe His Asp145 150 155 160Asn Arg Arg Asp His Thr Asn Phe Thr Glu Met Asn Asn Leu Val Ile 165 170 175Asp Gly Asn Ser Phe Thr Asn Leu Phe Pro Val Gly Thr Glu His Pro 180 185 190Asp Ala Ile Gln Phe Phe Thr Ala Gly Lys Val Lys Gly Asn Thr Asn 195 200 205Ile Thr Ile Ser Asn Asn Val Ile Met Gln Gly Ala Gly Ser Gly Ala 210 215 220Gln Gly Ile Phe Met Asn Asp Glu Ala Gly Asn Leu Pro Tyr Val Asn225 230 235 240Val Asn Ile Lys Asn Asn Leu Ile Tyr Leu Asn Gly Tyr Tyr His Gly 245 250 255Ile Asn Val Val Asn Gly Val Asn Val Asn Ile Glu Ser Asn Ser Val 260 265 270Ile Ser Gln Val Asp Gly Thr Ser Phe Trp Ile Arg Leu Asp Lys Thr 275 280 285Asn Gly Ala Thr Ile Lys Asn Asn Val Ala Asp Leu Ile Thr Val Thr 290 295 300Ser Ser Ser Ser Asn Ile Val Gln Thr Gly Asn Arg Thr Leu Thr Ser305 310 315 320Asp Ser Ala Thr Ile Arg Lys Ile Tyr Gly Leu Asn Asp Gly Ala Thr 325 330 335Ala Arg Leu Ser Asp Leu Ile Val Pro Gly Val Gly Tyr Gln Pro Pro 340 345 350Val Ser Ser Ala Ala Ala Ala Gln Val Thr Thr Glu Leu Ser Thr Ala 355 360 365Lys Ala Ala Asn Pro Ser Leu Leu Leu Asp Leu Ser Phe Ser Asn Ser 370 375 380Gly Val Val Asp Leu Ser His Trp Asn Thr Gly Gln Thr Thr Lys Ala385 390 395 400Val Asp Val Ser Ala Val Val Gly Ser Ala Phe His Val Ser Thr Gly 405 410 415Thr Gly Val Glu Leu Asn Arg Ser Tyr Ser Arg Gln Ile Tyr Ala Leu 420 425 430Ser Ala Phe Thr Leu Ser Phe Asp Leu Lys Arg Asp Ser Ala Thr Ala 435 440 445Thr Ala Gly Gln Ile Leu Gly Ile Phe Gln Ser Trp Ser Val Ser Leu 450 455 460Gln Ala Asn Gly Glu Leu Ser Phe Thr Met Arg Asn Ala Ala Gly Val465 470 475 480Ser Gln Thr Met Val Thr Ser Gly Ala Lys Leu Leu Asp Ala Ala Thr 485 490 495His Lys Ile Ala Leu Thr Tyr Asp Ser Thr Arg Lys Thr Ala Ile Leu 500 505 510Tyr Val Asp Gly Met Gln Arg Gly Thr Ala Thr Met Thr Gly Thr Thr 515 520 525Arg Pro Ala Glu Ser Trp Gly Leu Tyr Val Gly Ser Pro Phe Ser Thr 530 535 540Ala Phe Ser Gly Thr Val Gly Asp Ile Glu Ile Arg Asp Gly Ala Ile545 550 555 560Ser Ala Ala Gln Val Gln Ala Leu Val Thr Ala Ser Ser Ala Ser Ala 565 570 575Ala Ala Thr Val Lys Asp Ser Leu Val Thr Gly Ala Ala Ala Gln Ala 580 585 590Ala Ala Leu Leu Ala Gly Ala Gly Ala Ala Ser Thr Ala Thr Pro Leu 595 600 605Ala Thr Val Ala Thr Val Gly Ser Thr Leu Ser Ile Gly Thr Ala Ala 610 615 620Ser Ser Gln Ile Ala Leu Val Ser Lys Ile Gly Val Asp Met Met Thr625 630 635 640Ala Gly Ala Met Gly Ala Ile Arg Ser Ala Ala Thr Leu Ser Ala Thr 645 650 655Ala Asp Gln Tyr Asn Leu Tyr Arg Ala 660 6658939DNASphingomonas sp. ATCC53159 8atgcagatgc tcccaacgcc cgatgtcagc atactcgtgg tcgctttcaa ctcgaccgag 60tatatcgaag actgcctgcg cggcatcgcc gaaggagcgg gcaagacccc ccacgaagtt 120ctgctgatcg acaatggcga cgggcgaacc gaagcgctgg tccggcagcg gttccaccac 180gtccgcatcg ttcccagtga gggcaatatt ggtttcgggg ccggcaataa tcgcctggca 240gcgcaggctg ccggcccgct cctgctgctc gtcaaccccg atgccattcc ccagcccggc 300gcaatcgatc agttggtcac ctttgccaaa cagcatcccg aggcggcggc atggggcggc 360cgttcctact cgcccagcgg cgatctagaa cccgcaaatt tcatgtccct gccgacgccc 420gccgactttc tgacggcgat tttcaacgcg cgtgcgctac gcagcggcgg gctgcaagaa 480ggcgcgacca cccccggagc ggtcgaggtg ttgaatggcg gcttcatgat ggtacgcacc 540gatgtctggc aggcgatcgg cggttttgac gagagctttt ttctttattc ggaagagatc 600gatctcttcc agcgaatccg cacgttgggg cacaaggtgc tcgtcgaccc ctcggtcaaa 660gtggtacaca atacggggag tggtcagtcg atgtcccaga accgcctgat gtatctcacg 720accgggcgca tgcactatgc gcgaaagcat tttggcgcac tcggcaccct tgccaccggg 780tgcgcgcttt ggctgatcgc cgccaaatac acgttggtcg gggcggcact ctggcgcctg 840tcgccgcgga cgggcacgcg atacaaagag ctgagcaacg ggtggcgtgc cgtatttagc 900aatcctggcc gatggtggag cggctatccg cgtcgctaa 9399312PRTSphingomonas sp. ATCC53159 9Met Gln Met Leu Pro Thr Pro Asp Val Ser Ile Leu Val Val Ala Phe1 5 10 15Asn Ser Thr Glu Tyr Ile Glu Asp Cys Leu Arg Gly Ile Ala Glu Gly 20 25 30Ala Gly Lys Thr Pro His Glu Val Leu Leu Ile Asp Asn Gly Asp Gly 35 40 45Arg Thr Glu Ala Leu Val Arg Gln Arg Phe His His Val Arg Ile Val 50 55 60Pro Ser Glu Gly Asn Ile Gly Phe Gly Ala Gly Asn Asn Arg Leu Ala65 70 75 80Ala Gln Ala Ala Gly Pro Leu Leu Leu Leu Val Asn Pro Asp Ala Ile 85 90 95Pro Gln Pro Gly Ala Ile Asp Gln Leu Val Thr Phe Ala Lys Gln His 100 105 110Pro Glu Ala Ala Ala Trp Gly Gly Arg Ser Tyr Ser Pro Ser Gly Asp 115 120 125Leu Glu Pro Ala Asn Phe Met Ser Leu Pro Thr Pro Ala Asp Phe Leu 130 135 140Thr Ala Ile Phe Asn Ala Arg Ala Leu Arg Ser Gly Gly Leu Gln Glu145 150 155 160Gly Ala Thr Thr Pro Gly Ala Val Glu Val Leu Asn Gly Gly Phe Met 165 170 175Met Val Arg Thr Asp Val Trp Gln Ala Ile Gly Gly Phe Asp Glu Ser 180 185 190Phe Phe Leu Tyr Ser Glu Glu Ile Asp Leu Phe Gln Arg Ile Arg Thr 195 200 205Leu Gly His Lys Val Leu Val Asp Pro Ser Val Lys Val Val His Asn 210 215 220Thr Gly Ser Gly Gln Ser Met Ser Gln Asn Arg Leu Met Tyr Leu Thr225 230 235 240Thr Gly Arg Met His Tyr Ala Arg Lys His Phe Gly Ala Leu Gly Thr 245 250 255Leu Ala Thr Gly Cys Ala Leu Trp Leu Ile Ala Ala Lys Tyr Thr Leu 260 265 270Val Gly Ala Ala Leu Trp Arg Leu Ser Pro Arg Thr Gly Thr Arg Tyr 275 280 285Lys Glu Leu Ser Asn Gly Trp Arg Ala Val Phe Ser Asn Pro Gly Arg 290 295 300Trp Trp Ser Gly Tyr Pro Arg Arg305 31010972DNASphingomonas sp. ATCC53159 10atgctgcact gccaacgcta ttgcggatgc ccgcccgtcc gaataggttc aagtagaagt 60ttgtgccgtg cgcaattccg tgccggcggg gaggtcttca tgaagaaatt gtacctggtt 120acggcggtgg ctgcggccgc gctggccgtc tccggatgtg gcggcaaggg cggcaagctc 180gacaaggggc aggtggtcgc cagcgtcgat ggcgaagaaa tcaccgtctt cgagctgaat 240gccgaactgc aggcctccca ggtacccccg gggaccgatc gcaagctggc cgagcagctg 300gcgctgcagc gcatcatcga gcgcaagatc ctcgccaagg tcgcccgcga gcagaagctg 360gacaagacgc ctgccttcct gatccaggag cgccgggccg acgagctgat cctcaccgcc 420atgctgcgcg acaagatcgc cggcggcatc gcccagccga ccgatgccga gatcgagaaa 480tatcaggccg cgcatccgga gcggttcgcg cagcgcaaga tctacgcgat cgatcaggtc 540gtcttcgctc cgccgagctc ggccgcaaag ctcaagcaat tcgcgccgct gaagacgctg 600gaccagctaa ccgccaagct ctcggcggac aatgtccagt tccgtcgcgc gccgtcgcag 660atcgacaccg ctgcgctgcc gccggaaatc gctgccaaga tcgcgtcgct gccggcacag 720gagatgttca tcctgccgac ccagcaggga ctgaccgcga atatcatcac gtcgaccacg 780gtgctgccgg tgccggccga ccaggcgcgc gagatcgcgc tcagcgggct gcgtaccgag 840cgcttcggca aggcggctga cgcacagctc aacgaccgcc tgaagaaggc gcgggaaacc 900gtgaaatatc aggccggcta cagcgcaccg ccgcagcttc gcggcagcgg cgcaacgccg 960gcggggaact ga 97211323PRTSphingomonas sp. ATCC53159 11Met Leu His Cys Gln Arg Tyr Cys Gly Cys Pro Pro Val Arg Ile Gly1 5 10 15Ser Ser Arg Ser Leu Cys Arg Ala Gln Phe Arg Ala Gly Gly Glu Val 20 25 30Phe Met Lys Lys Leu Tyr Leu Val Thr Ala Val Ala Ala Ala Ala Leu 35 40 45Ala Val Ser Gly Cys Gly Gly Lys Gly Gly Lys Leu Asp Lys Gly Gln 50 55 60Val Val Ala Ser Val Asp Gly Glu Glu

Ile Thr Val Phe Glu Leu Asn65 70 75 80Ala Glu Leu Gln Ala Ser Gln Val Pro Pro Gly Thr Asp Arg Lys Leu 85 90 95Ala Glu Gln Leu Ala Leu Gln Arg Ile Ile Glu Arg Lys Ile Leu Ala 100 105 110Lys Val Ala Arg Glu Gln Lys Leu Asp Lys Thr Pro Ala Phe Leu Ile 115 120 125Gln Glu Arg Arg Ala Asp Glu Leu Ile Leu Thr Ala Met Leu Arg Asp 130 135 140Lys Ile Ala Gly Gly Ile Ala Gln Pro Thr Asp Ala Glu Ile Glu Lys145 150 155 160Tyr Gln Ala Ala His Pro Glu Arg Phe Ala Gln Arg Lys Ile Tyr Ala 165 170 175Ile Asp Gln Val Val Phe Ala Pro Pro Ser Ser Ala Ala Lys Leu Lys 180 185 190Gln Phe Ala Pro Leu Lys Thr Leu Asp Gln Leu Thr Ala Lys Leu Ser 195 200 205Ala Asp Asn Val Gln Phe Arg Arg Ala Pro Ser Gln Ile Asp Thr Ala 210 215 220Ala Leu Pro Pro Glu Ile Ala Ala Lys Ile Ala Ser Leu Pro Ala Gln225 230 235 240Glu Met Phe Ile Leu Pro Thr Gln Gln Gly Leu Thr Ala Asn Ile Ile 245 250 255Thr Ser Thr Thr Val Leu Pro Val Pro Ala Asp Gln Ala Arg Glu Ile 260 265 270Ala Leu Ser Gly Leu Arg Thr Glu Arg Phe Gly Lys Ala Ala Asp Ala 275 280 285Gln Leu Asn Asp Arg Leu Lys Lys Ala Arg Glu Thr Val Lys Tyr Gln 290 295 300Ala Gly Tyr Ser Ala Pro Pro Gln Leu Arg Gly Ser Gly Ala Thr Pro305 310 315 320Ala Gly Asn121047DNASphingomonas sp. ATCC53159 12atggcagaag cgaacgcggt agatggaaag gcctccaagc cgctgaaaat gtgccttgca 60gcgtcgggcg gcggccatct ccggcaaatc ctcgatctgg aatcggtgtg gcgcgaacac 120gattatttct tcgttactga agataccgcg ctcggccgga gccttgccga aaaacatccc 180gtcgaactgg tggagcacta tgcgctcggc caggccaagc tgggccatcc cttgcgcatg 240ctgggcggcg catggcgcaa cctgcgccag agcctttcga tcctgcgccg gcacaagccg 300gatgtggtga tttccaccgg cgcgggcgca gtctatttca ccgcgctgct cgccaaactg 360tcgggcgcca agttcgtcca tatcgaaagc ttcgcgcgct tcgaccaccc gtctgccttc 420ggcaagatgg tgaagggcat cgcgacggtg acgatcgtcc agtcggcggc gctgaaagaa 480acctggcctg atgccgagct gttcgatccg ttccgcctgc tcgatacacc gcgcccgccc 540aagcaggcgc taatcttcgc gacggtcggc gccaccctgc ccttcccgcg gctggtgcag 600gcagtgctcg acctgaagcg cgccggcggg ctgccgggca agctgatcct gcaatatggc 660gaccaggacc tgcccgatcc cggcatcccc gacgtcgaga tccgccgtac catcccgttc 720gacgatctgc agctgctgct gcgcgatgcg gatatggtga tatgccacgg cggcaccgga 780tcgctggtca cggcgctgcg cgccggctgc cgggtcgtcg cctttccgcg ccgccacgat 840ctgggcgagc attatgacga tcaccaggaa gagatcgccc agaccttcgc cgaccggggc 900ctgctccagg cggtgcgcga cgagcgccag ctcggcgccg ctgtggaagc ggccaaggca 960accgagccgc agctggcgac caccgaccac acggccctcg cggcgcggct gcgccagctg 1020ctggcgcagt ggagtgccaa gcgatga 104713348PRTSphingomonas sp. ATCC53159 13Met Ala Glu Ala Asn Ala Val Asp Gly Lys Ala Ser Lys Pro Leu Lys1 5 10 15Met Cys Leu Ala Ala Ser Gly Gly Gly His Leu Arg Gln Ile Leu Asp 20 25 30Leu Glu Ser Val Trp Arg Glu His Asp Tyr Phe Phe Val Thr Glu Asp 35 40 45Thr Ala Leu Gly Arg Ser Leu Ala Glu Lys His Pro Val Glu Leu Val 50 55 60Glu His Tyr Ala Leu Gly Gln Ala Lys Leu Gly His Pro Leu Arg Met65 70 75 80Leu Gly Gly Ala Trp Arg Asn Leu Arg Gln Ser Leu Ser Ile Leu Arg 85 90 95Arg His Lys Pro Asp Val Val Ile Ser Thr Gly Ala Gly Ala Val Tyr 100 105 110Phe Thr Ala Leu Leu Ala Lys Leu Ser Gly Ala Lys Phe Val His Ile 115 120 125Glu Ser Phe Ala Arg Phe Asp His Pro Ser Ala Phe Gly Lys Met Val 130 135 140Lys Gly Ile Ala Thr Val Thr Ile Val Gln Ser Ala Ala Leu Lys Glu145 150 155 160Thr Trp Pro Asp Ala Glu Leu Phe Asp Pro Phe Arg Leu Leu Asp Thr 165 170 175Pro Arg Pro Pro Lys Gln Ala Leu Ile Phe Ala Thr Val Gly Ala Thr 180 185 190Leu Pro Phe Pro Arg Leu Val Gln Ala Val Leu Asp Leu Lys Arg Ala 195 200 205Gly Gly Leu Pro Gly Lys Leu Ile Leu Gln Tyr Gly Asp Gln Asp Leu 210 215 220Pro Asp Pro Gly Ile Pro Asp Val Glu Ile Arg Arg Thr Ile Pro Phe225 230 235 240Asp Asp Leu Gln Leu Leu Leu Arg Asp Ala Asp Met Val Ile Cys His 245 250 255Gly Gly Thr Gly Ser Leu Val Thr Ala Leu Arg Ala Gly Cys Arg Val 260 265 270Val Ala Phe Pro Arg Arg His Asp Leu Gly Glu His Tyr Asp Asp His 275 280 285Gln Glu Glu Ile Ala Gln Thr Phe Ala Asp Arg Gly Leu Leu Gln Ala 290 295 300Val Arg Asp Glu Arg Gln Leu Gly Ala Ala Val Glu Ala Ala Lys Ala305 310 315 320Thr Glu Pro Gln Leu Ala Thr Thr Asp His Thr Ala Leu Ala Ala Arg 325 330 335Leu Arg Gln Leu Leu Ala Gln Trp Ser Ala Lys Arg 340 34514867DNASphingomonas sp. ATCC53159 14atgagcacgc cccggatcag cgtcgtcatc ccgcactata acgatccgca atccttgcgg 60ctctgcctgg atgcgctgga gcggcagacg atcggtcgcg acgcgttcga gatcatcgtc 120ggcgacaaca attcgccctg tgggctcgcg gcggtggagg cggcggtcgc cggacgtgcg 180cggatcgtga ccattctgga aaagggggcg ggccccgcgc gcaacggggc ggcagccgca 240gcgcgtggcg agatcctcgc ctttaccgac agtgactgcg tggtggagcc cggctggctg 300gcgggcggca cgaccagggt cgcgcctggc cgtttcatcg gcgggcacat gtatgtgcgc 360aagcccgaag ggccgccgaa cggcgccgag gcgctggaga tggcgctggc gttcgacaat 420gaaggctatg tgcggcgcac ccagttcacg gtcaccgcaa acctgttcgt gatgcgcgcc 480gatttcgaac gggtcggcgg cttccgcgtt ggcgtgtccg aggatctgga atggtgccac 540cgggcgatcg ccagcggcct caccatcaac tatgcaccgg atgcatcggt gggccacccg 600ccccggcccg actggtcggc cctgctggtg aagacgcggc gcatccagcg cgaactctat 660ctgttcaaca tcgagcggcc gaagggcagg ctgcgctggc tggtccgttc cgtggcgcaa 720ccggcgatga tcccacagga cgtggccaag atcctgcgca caccgggtac caagggcgcg 780cgcctcgctg cggtcaccac gctggtccgg ctgcggctgt ggcgcggcgg cgccggcttg 840ttgcagttgc tcggccgcga catctga 86715288PRTSphingomonas sp. ATCC53159 15Met Ser Thr Pro Arg Ile Ser Val Val Ile Pro His Tyr Asn Asp Pro1 5 10 15Gln Ser Leu Arg Leu Cys Leu Asp Ala Leu Glu Arg Gln Thr Ile Gly 20 25 30Arg Asp Ala Phe Glu Ile Ile Val Gly Asp Asn Asn Ser Pro Cys Gly 35 40 45Leu Ala Ala Val Glu Ala Ala Val Ala Gly Arg Ala Arg Ile Val Thr 50 55 60Ile Leu Glu Lys Gly Ala Gly Pro Ala Arg Asn Gly Ala Ala Ala Ala65 70 75 80Ala Arg Gly Glu Ile Leu Ala Phe Thr Asp Ser Asp Cys Val Val Glu 85 90 95Pro Gly Trp Leu Ala Gly Gly Thr Thr Arg Val Ala Pro Gly Arg Phe 100 105 110Ile Gly Gly His Met Tyr Val Arg Lys Pro Glu Gly Pro Pro Asn Gly 115 120 125Ala Glu Ala Leu Glu Met Ala Leu Ala Phe Asp Asn Glu Gly Tyr Val 130 135 140Arg Arg Thr Gln Phe Thr Val Thr Ala Asn Leu Phe Val Met Arg Ala145 150 155 160Asp Phe Glu Arg Val Gly Gly Phe Arg Val Gly Val Ser Glu Asp Leu 165 170 175Glu Trp Cys His Arg Ala Ile Ala Ser Gly Leu Thr Ile Asn Tyr Ala 180 185 190Pro Asp Ala Ser Val Gly His Pro Pro Arg Pro Asp Trp Ser Ala Leu 195 200 205Leu Val Lys Thr Arg Arg Ile Gln Arg Glu Leu Tyr Leu Phe Asn Ile 210 215 220Glu Arg Pro Lys Gly Arg Leu Arg Trp Leu Val Arg Ser Val Ala Gln225 230 235 240Pro Ala Met Ile Pro Gln Asp Val Ala Lys Ile Leu Arg Thr Pro Gly 245 250 255Thr Lys Gly Ala Arg Leu Ala Ala Val Thr Thr Leu Val Arg Leu Arg 260 265 270Leu Trp Arg Gly Gly Ala Gly Leu Leu Gln Leu Leu Gly Arg Asp Ile 275 280 285161389DNASphingomonas sp. ATCC53159 16gtggctgtag gttccgcgct gagattcttg tggccgttcg gccgtcgaga agaacctgaa 60gaagagggct atttcccgct gactgcgaca gtggtgccgc atcgcgatgc ccatagcggc 120cgtggtcgcc cggatttccc aaccttccgt gcctccgcgc tggaccgccc gctggatcgc 180cgccgcgacg agcgccgcga gatcacgcgc gcccggttcg cgctggcgac cttcttcaca 240cccacccagc cggtggccga tcggtcgagc ttcgccgggc gcctcggcgt gctggcgcgc 300ctaatctcct cgatcgagag ccagcgcagc catgtcgtgc tctatggcga gcgcggcatc 360ggcaagacct cgctgctcca cgtgctgacc gatgtcgccc gcgaatccag ctatatcgtc 420agctatgcga cctgcggtgc gaacgcgaat ttcagcgatg tcttccgcgc cgtgctggaa 480gacgtgccgc tgctgttcca tcgcggcgtg gcgcccaacg ccggcgaggc ggagagcggc 540ggcaacttgg ccgaccgcct gccgacgggc agcttcgggc ccggcgaact ggccgacctg 600tgcgccgaca tcacgggcac acgcgtgctg atcatcctcg acgaatatga tcgcgtcagc 660gattccgcct tccgtcagca ggtcgccgag ctgatcaaga acctgtcgga ccgttcggcg 720cgcgtccagc tggtgatcgc gggcgtcgcc tcgaacctgc aggagctgat cggttatgcg 780ccgtcgatcc gccgcaacgt catcggcctg ccgatgcccc ggctggagga atcggaggtg 840caggagatga tcgcgctcgg cgaaaccgcc tcgggcgttc gcttcgatcc ggacctgact 900cacatgatcc acctgctcgc gctggggtcg ccctatttcg cgcggctgct gtgccaccat 960tccgcgctgg aagccctgga ccagggccgc ctcacggtcg acgccgggca tctgcgtcgt 1020gcgctcgacc aggcgatcct tgagatcgag ggccgcatgc cgccgcgcgc ggtgatcgag 1080atgcgcaagt tcgtcggcgg ccgctacgat ccactcgtcg cggcgctggg cgaggcctcg 1140cgctcggcgg atggctggtt cagcggccaa gccgtggtgg atctgctgcc gggcgcgcac 1200atcacggcgg cgcaggtcga gcaggagctg ggcgagctca ccggtcaact cggcctcgaa 1260tccgaaacgc aggacggcga ttgccgcttc cgcttcaccg acgatacgct gccggtctat 1320ctgtggctga tgatcggccg cctgcggctc gacagcggca cgctggaaga cgcgctggcc 1380accgtctga 138917462PRTSphingomonas sp. ATCC53159 17Val Ala Val Gly Ser Ala Leu Arg Phe Leu Trp Pro Phe Gly Arg Arg1 5 10 15Glu Glu Pro Glu Glu Glu Gly Tyr Phe Pro Leu Thr Ala Thr Val Val 20 25 30Pro His Arg Asp Ala His Ser Gly Arg Gly Arg Pro Asp Phe Pro Thr 35 40 45Phe Arg Ala Ser Ala Leu Asp Arg Pro Leu Asp Arg Arg Arg Asp Glu 50 55 60Arg Arg Glu Ile Thr Arg Ala Arg Phe Ala Leu Ala Thr Phe Phe Thr65 70 75 80Pro Thr Gln Pro Val Ala Asp Arg Ser Ser Phe Ala Gly Arg Leu Gly 85 90 95Val Leu Ala Arg Leu Ile Ser Ser Ile Glu Ser Gln Arg Ser His Val 100 105 110Val Leu Tyr Gly Glu Arg Gly Ile Gly Lys Thr Ser Leu Leu His Val 115 120 125Leu Thr Asp Val Ala Arg Glu Ser Ser Tyr Ile Val Ser Tyr Ala Thr 130 135 140Cys Gly Ala Asn Ala Asn Phe Ser Asp Val Phe Arg Ala Val Leu Glu145 150 155 160Asp Val Pro Leu Leu Phe His Arg Gly Val Ala Pro Asn Ala Gly Glu 165 170 175Ala Glu Ser Gly Gly Asn Leu Ala Asp Arg Leu Pro Thr Gly Ser Phe 180 185 190Gly Pro Gly Glu Leu Ala Asp Leu Cys Ala Asp Ile Thr Gly Thr Arg 195 200 205Val Leu Ile Ile Leu Asp Glu Tyr Asp Arg Val Ser Asp Ser Ala Phe 210 215 220Arg Gln Gln Val Ala Glu Leu Ile Lys Asn Leu Ser Asp Arg Ser Ala225 230 235 240Arg Val Gln Leu Val Ile Ala Gly Val Ala Ser Asn Leu Gln Glu Leu 245 250 255Ile Gly Tyr Ala Pro Ser Ile Arg Arg Asn Val Ile Gly Leu Pro Met 260 265 270Pro Arg Leu Glu Glu Ser Glu Val Gln Glu Met Ile Ala Leu Gly Glu 275 280 285Thr Ala Ser Gly Val Arg Phe Asp Pro Asp Leu Thr His Met Ile His 290 295 300Leu Leu Ala Leu Gly Ser Pro Tyr Phe Ala Arg Leu Leu Cys His His305 310 315 320Ser Ala Leu Glu Ala Leu Asp Gln Gly Arg Leu Thr Val Asp Ala Gly 325 330 335His Leu Arg Arg Ala Leu Asp Gln Ala Ile Leu Glu Ile Glu Gly Arg 340 345 350Met Pro Pro Arg Ala Val Ile Glu Met Arg Lys Phe Val Gly Gly Arg 355 360 365Tyr Asp Pro Leu Val Ala Ala Leu Gly Glu Ala Ser Arg Ser Ala Asp 370 375 380Gly Trp Phe Ser Gly Gln Ala Val Val Asp Leu Leu Pro Gly Ala His385 390 395 400Ile Thr Ala Ala Gln Val Glu Gln Glu Leu Gly Glu Leu Thr Gly Gln 405 410 415Leu Gly Leu Glu Ser Glu Thr Gln Asp Gly Asp Cys Arg Phe Arg Phe 420 425 430Thr Asp Asp Thr Leu Pro Val Tyr Leu Trp Leu Met Ile Gly Arg Leu 435 440 445Arg Leu Asp Ser Gly Thr Leu Glu Asp Ala Leu Ala Thr Val 450 455 460181299DNASphingomonas sp. ATCC53159 18atgaagccga gacccggggg aacctttatg caagtaaatt tcaatcgaca ggctcgcaag 60ctcggtgccg gcaatgcgct cgcgcggggg gggcccgtgc ttgcgctgct tgcgaccgcg 120gcatggacac aacctgcgct ggcgcagcga caggcatttg agtcccgccc ctccggtagc 180gagcgacagg tcgatattcg cgcgacgggg tcgctggaat atgacgacaa cgtcgtgctg 240aacgaccagc ggatcacgga cggcgcgcgt ggcgatgtga tcgcatcgcc cgggctggac 300gtgaccctag ttctgccccg cgccaccggg cagctctacc tcaccggcaa tgtcggatat 360cgcttttaca agcgatatac caactttaac cgcgagcaga tctcgctcac cggcggcgca 420gatcagcggt tcgcctcctg cgtcgtgcac ggggaagtcg gctatcagcg ccacctcacc 480gacctgtcca gcatcttgat ccaggacacc acgcctgcgc tcaacaacac cgaagaggcc 540cggcagtaca ccgcggatat cggctgcggc gcgacctacg gcctgcggcc tgccgtttcc 600tacacccgca acgaagtgcg caacagcctt gccgagcgcc gatacgcgga ctcgaatacc 660aacaccttta ccgcacagct tggcctgact tcgcctgccc tggggaccgt ggcggtattt 720gggcgtatgt ccgacagcag ctatgtccat cgcgtccttc ccggcattac cggccaggac 780gggatgaaga gctacgcggc cggcgtccag ctcgagcgct cggtggccaa ccgactccat 840ttcaacggct cggtgaatta caccgaggtt gacccaaagc tcgcatccac caaaggattc 900aagggcgtag gatttaacgt ttccggcgat tatgctggtg atcagtacag cctccaattg 960ctggcttcac gatcgcccca gccttcactt cttctgttcg tgggttacga gattgtgaca 1020gcggtttcgg cgaatgcgac gcgccggctg agcgatcgca ttcagatatc gctgcaaggc 1080agccgaacct ggcgcgagct cgcgtcttcg cggctgctca ccaacgtgcc gatttccggc 1140aacgacaaca cctcgacgtt gttcgcctcc gctaccttcc ggccgaatcg ccggctgagc 1200tttgtgctgg gtgccggcct tcagcggcgc accagcaaca cgcagctata cagttacagc 1260tccaaacgca tcaatctctc gacgtcgctt tcgctctga 129919432PRTSphingomonas sp. ATCC53159 19Met Lys Pro Arg Pro Gly Gly Thr Phe Met Gln Val Asn Phe Asn Arg1 5 10 15Gln Ala Arg Lys Leu Gly Ala Gly Asn Ala Leu Ala Arg Gly Gly Pro 20 25 30Val Leu Ala Leu Leu Ala Thr Ala Ala Trp Thr Gln Pro Ala Leu Ala 35 40 45Gln Arg Gln Ala Phe Glu Ser Arg Pro Ser Gly Ser Glu Arg Gln Val 50 55 60Asp Ile Arg Ala Thr Gly Ser Leu Glu Tyr Asp Asp Asn Val Val Leu65 70 75 80Asn Asp Gln Arg Ile Thr Asp Gly Ala Arg Gly Asp Val Ile Ala Ser 85 90 95Pro Gly Leu Asp Val Thr Leu Val Leu Pro Arg Ala Thr Gly Gln Leu 100 105 110Tyr Leu Thr Gly Asn Val Gly Tyr Arg Phe Tyr Lys Arg Tyr Thr Asn 115 120 125Phe Asn Arg Glu Gln Ile Ser Leu Thr Gly Gly Ala Asp Gln Arg Phe 130 135 140Ala Ser Cys Val Val His Gly Glu Val Gly Tyr Gln Arg His Leu Thr145 150 155 160Asp Leu Ser Ser Ile Leu Ile Gln Asp Thr Thr Pro Ala Leu Asn Asn 165 170 175Thr Glu Glu Ala Arg Gln Tyr Thr Ala Asp Ile Gly Cys Gly Ala Thr 180 185 190Tyr Gly Leu Arg Pro Ala Val Ser Tyr Thr Arg Asn Glu Val Arg Asn 195 200 205Ser Leu Ala Glu Arg Arg Tyr Ala Asp Ser Asn Thr Asn Thr Phe Thr 210 215 220Ala Gln Leu Gly Leu Thr Ser Pro Ala Leu Gly Thr Val Ala Val Phe225 230 235 240Gly Arg Met Ser Asp Ser Ser Tyr Val His Arg Val Leu Pro Gly Ile 245 250 255Thr Gly Gln Asp Gly Met Lys Ser Tyr Ala Ala Gly Val Gln Leu Glu 260 265 270Arg Ser Val Ala Asn Arg Leu His Phe Asn Gly Ser Val Asn Tyr Thr 275 280 285Glu Val Asp Pro Lys Leu Ala Ser Thr Lys Gly Phe Lys Gly Val Gly 290 295

300Phe Asn Val Ser Gly Asp Tyr Ala Gly Asp Gln Tyr Ser Leu Gln Leu305 310 315 320Leu Ala Ser Arg Ser Pro Gln Pro Ser Leu Leu Leu Phe Val Gly Tyr 325 330 335Glu Ile Val Thr Ala Val Ser Ala Asn Ala Thr Arg Arg Leu Ser Asp 340 345 350Arg Ile Gln Ile Ser Leu Gln Gly Ser Arg Thr Trp Arg Glu Leu Ala 355 360 365Ser Ser Arg Leu Leu Thr Asn Val Pro Ile Ser Gly Asn Asp Asn Thr 370 375 380Ser Thr Leu Phe Ala Ser Ala Thr Phe Arg Pro Asn Arg Arg Leu Ser385 390 395 400Phe Val Leu Gly Ala Gly Leu Gln Arg Arg Thr Ser Asn Thr Gln Leu 405 410 415Tyr Ser Tyr Ser Ser Lys Arg Ile Asn Leu Ser Thr Ser Leu Ser Leu 420 425 43020918DNASphingomonas sp. ATCC53159 20atgcatatca agaatcgctt cgtgaatatc tcgacgttgg ccatcgccgc cgcgctggcc 60acgccggcgg cggcgcagat ccccacgcgg tccgtgcccg cgccggcccg cccgcggcct 120gcaacgccgc cggcgcaaca gcagaaccag gcgccgtcga cgcccgcagc ggcaaccccg 180gcgcagaccg ccgcaaccgt tgcccctgca gcaaccgcac ccgcaggtta caaaatcggc 240gtggacgacg tgatcgaggc cgacgtgctc ggccagaccg acttcaagac gcgcgcccgt 300gtgcaggcgg acggcacggt gaccctgccc tatctgggcg ccgtgcaggt caagggcgag 360accgcgacct cgctcgccga aaagctggcc gggctgctgc gcgccggcgg ctattatgcc 420aagccgatcg tcagcgtcga aatcgtcggt ttcgtcagca actatgtgac ggtgctgggc 480caggtgaaca gttccggcct gcagccggtc gaccgcggct atcacgtttc cgagatcatc 540gcccgtgccg gcggcctgcg ccccgaagcg gccgatttcg tcgttctcac ccgcgccgat 600ggctccagcg ccaagctgga ctacaagaag ctcgcccaag gtggccccaa tgacgatccg 660atggtgacgc ccggggacaa ggtctttgtc ccggaagtcg agcatttcta catttatggt 720caaattaacg cgcctggcgt atacgcgatt cgatcggaca tgacgctccg tcgcgcgctg 780gcccagggcg gtgggcttgc ccccgcaggc tccgtcaagc gtgtgaaggt cacgcgggat 840ggcaatgaac tcaagttgaa gctggacgat ccgattctcc caggcgacac gatcgtcatc 900ggcgaacgat tgttctga 91821305PRTSphingomonas sp. ATCC53159 21Met His Ile Lys Asn Arg Phe Val Asn Ile Ser Thr Leu Ala Ile Ala1 5 10 15Ala Ala Leu Ala Thr Pro Ala Ala Ala Gln Ile Pro Thr Arg Ser Val 20 25 30Pro Ala Pro Ala Arg Pro Arg Pro Ala Thr Pro Pro Ala Gln Gln Gln 35 40 45Asn Gln Ala Pro Ser Thr Pro Ala Ala Ala Thr Pro Ala Gln Thr Ala 50 55 60Ala Thr Val Ala Pro Ala Ala Thr Ala Pro Ala Gly Tyr Lys Ile Gly65 70 75 80Val Asp Asp Val Ile Glu Ala Asp Val Leu Gly Gln Thr Asp Phe Lys 85 90 95Thr Arg Ala Arg Val Gln Ala Asp Gly Thr Val Thr Leu Pro Tyr Leu 100 105 110Gly Ala Val Gln Val Lys Gly Glu Thr Ala Thr Ser Leu Ala Glu Lys 115 120 125Leu Ala Gly Leu Leu Arg Ala Gly Gly Tyr Tyr Ala Lys Pro Ile Val 130 135 140Ser Val Glu Ile Val Gly Phe Val Ser Asn Tyr Val Thr Val Leu Gly145 150 155 160Gln Val Asn Ser Ser Gly Leu Gln Pro Val Asp Arg Gly Tyr His Val 165 170 175Ser Glu Ile Ile Ala Arg Ala Gly Gly Leu Arg Pro Glu Ala Ala Asp 180 185 190Phe Val Val Leu Thr Arg Ala Asp Gly Ser Ser Ala Lys Leu Asp Tyr 195 200 205Lys Lys Leu Ala Gln Gly Gly Pro Asn Asp Asp Pro Met Val Thr Pro 210 215 220Gly Asp Lys Val Phe Val Pro Glu Val Glu His Phe Tyr Ile Tyr Gly225 230 235 240Gln Ile Asn Ala Pro Gly Val Tyr Ala Ile Arg Ser Asp Met Thr Leu 245 250 255Arg Arg Ala Leu Ala Gln Gly Gly Gly Leu Ala Pro Ala Gly Ser Val 260 265 270Lys Arg Val Lys Val Thr Arg Asp Gly Asn Glu Leu Lys Leu Lys Leu 275 280 285Asp Asp Pro Ile Leu Pro Gly Asp Thr Ile Val Ile Gly Glu Arg Leu 290 295 300Phe305221347DNASphingomonas sp. ATCC53159 22gtgaatatca ttcagttctt ccgcattctg tgggtgcgcc gatggatcat cctcccggcg 60tttctcgttt gcgttaccac tgccaccatt gtggtccagt ttctgcccga acgctacaag 120gccactacgc gggtggtgct cgacacgttt aagcccgatc ccgtcaccgg acaggtgatg 180agctcgcagt tcatgcgcgc ctatgtcgag actcagaccc agctgatcga ggactatgcg 240accgccggtc gcgtggtcga cgaactgggc tgggtgaatg atccggcgaa catctccgcg 300ttcaacaact cgtccgcggc tgccaccggc gacatccgcc gctggctcgc caagcagatc 360atcgacaata ccaaggccga tgtgatggag gggagcaaca tcctcgaaat cacctattcg 420gacagctcgc ccgagcgcgc cgaacgcatc gccaacctga tccgcacctc gttcctcgcc 480cagtcgctcg ccgccaagcg ccaggccgcg accaagtcgg ccgactggta cgcccagcag 540gccgaagctg cccgcgattc gctcgctgcg gcggtccagg cccgcaccga tttcgtgaag 600aagaccggca tcgtgctgac cgaaaccggc gccgacctgg aaacccagaa gctccagcag 660atcgaggggc agacgacgac cgccaccgcc ccggttgcca tggcccccag cggcatgggc 720ccggcgcaga tgcagctcgc ccagatcgac cagcagatcc agcaggcagc gaccagccta 780ggtccgaacc acccaacttt ccaggccttg cagcggcagc gcgaagtgtt cgccaaggca 840gcggcggcgg aacgcgcgca ggcgaacggc gtatccggtc cggcacgcgg ggccatcgaa 900agcgcagcca acgcccagcg cgcgcgggtt ctcggcaatc gtcaggatgt cgacaagctt 960acgcagctgc agcgtgacgt ctcgctgaag caggatcagt acatgaaggc ggcacagcgc 1020gtcgccgatc tgcggctgga agcaagcagc aacgatgtcg gcatgtcgac gctcagcgaa 1080gcatcggcgc cggaaacgcc ctattacccc aaggtgccgc tcatcatcgg tggtgcagcc 1140ggcttcggcc tcgggctcgg tctgctggtc gcgctgctcg tcgagctgct cggccgccgc 1200gtccgcagcc ccgaggatct ggaagttgcg atcgatgcac cggtgctggg cgtgatccag 1260agccgcgcct cgcttgccgc ccgccttcgc cgcgcccaag aaaccctcgg cgaaggtgcc 1320gacacgcacg gagcttcagt aaactga 134723448PRTSphingomonas sp. ATCC53159 23Val Asn Ile Ile Gln Phe Phe Arg Ile Leu Trp Val Arg Arg Trp Ile1 5 10 15Ile Leu Pro Ala Phe Leu Val Cys Val Thr Thr Ala Thr Ile Val Val 20 25 30Gln Phe Leu Pro Glu Arg Tyr Lys Ala Thr Thr Arg Val Val Leu Asp 35 40 45Thr Phe Lys Pro Asp Pro Val Thr Gly Gln Val Met Ser Ser Gln Phe 50 55 60Met Arg Ala Tyr Val Glu Thr Gln Thr Gln Leu Ile Glu Asp Tyr Ala65 70 75 80Thr Ala Gly Arg Val Val Asp Glu Leu Gly Trp Val Asn Asp Pro Ala 85 90 95Asn Ile Ser Ala Phe Asn Asn Ser Ser Ala Ala Ala Thr Gly Asp Ile 100 105 110Arg Arg Trp Leu Ala Lys Gln Ile Ile Asp Asn Thr Lys Ala Asp Val 115 120 125Met Glu Gly Ser Asn Ile Leu Glu Ile Thr Tyr Ser Asp Ser Ser Pro 130 135 140Glu Arg Ala Glu Arg Ile Ala Asn Leu Ile Arg Thr Ser Phe Leu Ala145 150 155 160Gln Ser Leu Ala Ala Lys Arg Gln Ala Ala Thr Lys Ser Ala Asp Trp 165 170 175Tyr Ala Gln Gln Ala Glu Ala Ala Arg Asp Ser Leu Ala Ala Ala Val 180 185 190Gln Ala Arg Thr Asp Phe Val Lys Lys Thr Gly Ile Val Leu Thr Glu 195 200 205Thr Gly Ala Asp Leu Glu Thr Gln Lys Leu Gln Gln Ile Glu Gly Gln 210 215 220Thr Thr Thr Ala Thr Ala Pro Val Ala Met Ala Pro Ser Gly Met Gly225 230 235 240Pro Ala Gln Met Gln Leu Ala Gln Ile Asp Gln Gln Ile Gln Gln Ala 245 250 255Ala Thr Ser Leu Gly Pro Asn His Pro Thr Phe Gln Ala Leu Gln Arg 260 265 270Gln Arg Glu Val Phe Ala Lys Ala Ala Ala Ala Glu Arg Ala Gln Ala 275 280 285Asn Gly Val Ser Gly Pro Ala Arg Gly Ala Ile Glu Ser Ala Ala Asn 290 295 300Ala Gln Arg Ala Arg Val Leu Gly Asn Arg Gln Asp Val Asp Lys Leu305 310 315 320Thr Gln Leu Gln Arg Asp Val Ser Leu Lys Gln Asp Gln Tyr Met Lys 325 330 335Ala Ala Gln Arg Val Ala Asp Leu Arg Leu Glu Ala Ser Ser Asn Asp 340 345 350Val Gly Met Ser Thr Leu Ser Glu Ala Ser Ala Pro Glu Thr Pro Tyr 355 360 365Tyr Pro Lys Val Pro Leu Ile Ile Gly Gly Ala Ala Gly Phe Gly Leu 370 375 380Gly Leu Gly Leu Leu Val Ala Leu Leu Val Glu Leu Leu Gly Arg Arg385 390 395 400Val Arg Ser Pro Glu Asp Leu Glu Val Ala Ile Asp Ala Pro Val Leu 405 410 415Gly Val Ile Gln Ser Arg Ala Ser Leu Ala Ala Arg Leu Arg Arg Ala 420 425 430Gln Glu Thr Leu Gly Glu Gly Ala Asp Thr His Gly Ala Ser Val Asn 435 440 44524708DNASphingomonas sp. ATCC53159 24atggacgcga tgaccagcga accgctgccc gaaggcgatc gtccgagcgc cgtgccgacc 60acgccggata cgatcggcat gctcgaatac cagctcgtcc tctccgatcc gaccgggatc 120gaggcggaag cgatccgcgc gctacgcacg cgcatcatga cccagcacct ccgcgagggc 180cggcgcgcgc tcgcgatctg cgccgcctcg gcgggatccg gctgcagctt caccgccgtc 240aatctggcga cggcgctggc gcagatcggc gttaagactg cgctggtcga tgccaatctg 300cgcgatccca gcatcggcgc agccttcggc ctcgccgccg acaagcccgg cctggccgat 360tatctcgcct cgggcgatgt cgacctcgcc tcgatcatcc atgcgacccg cctcgaccag 420ctctcgatca tcccggccgg gcatgtcgag cacagcccgc aggaactgct cgcgtccgaa 480cagttccatg atctggcgac gcagctgctg cgcgagttcg acatcacgat cttcgacacc 540acggcgtcca acacctgcgc cgacgcgcag cgtgtcgcgc atatcgccgg ctatgcgatc 600atcgtggcgc gcaaggatgc gagctacatc cgcgacgtga acacgctcag ccgcacgctg 660cgtgcagacc gcaccaacgt catcggctgc gtactgaacg gctattga 70825235PRTSphingomonas sp. ATCC53159 25Met Asp Ala Met Thr Ser Glu Pro Leu Pro Glu Gly Asp Arg Pro Ser1 5 10 15Ala Val Pro Thr Thr Pro Asp Thr Ile Gly Met Leu Glu Tyr Gln Leu 20 25 30Val Leu Ser Asp Pro Thr Gly Ile Glu Ala Glu Ala Ile Arg Ala Leu 35 40 45Arg Thr Arg Ile Met Thr Gln His Leu Arg Glu Gly Arg Arg Ala Leu 50 55 60Ala Ile Cys Ala Ala Ser Ala Gly Ser Gly Cys Ser Phe Thr Ala Val65 70 75 80Asn Leu Ala Thr Ala Leu Ala Gln Ile Gly Val Lys Thr Ala Leu Val 85 90 95Asp Ala Asn Leu Arg Asp Pro Ser Ile Gly Ala Ala Phe Gly Leu Ala 100 105 110Ala Asp Lys Pro Gly Leu Ala Asp Tyr Leu Ala Ser Gly Asp Val Asp 115 120 125Leu Ala Ser Ile Ile His Ala Thr Arg Leu Asp Gln Leu Ser Ile Ile 130 135 140Pro Ala Gly His Val Glu His Ser Pro Gln Glu Leu Leu Ala Ser Glu145 150 155 160Gln Phe His Asp Leu Ala Thr Gln Leu Leu Arg Glu Phe Asp Ile Thr 165 170 175Ile Phe Asp Thr Thr Ala Ser Asn Thr Cys Ala Asp Ala Gln Arg Val 180 185 190Ala His Ile Ala Gly Tyr Ala Ile Ile Val Ala Arg Lys Asp Ala Ser 195 200 205Tyr Ile Arg Asp Val Asn Thr Leu Ser Arg Thr Leu Arg Ala Asp Arg 210 215 220Thr Asn Val Ile Gly Cys Val Leu Asn Gly Tyr225 230 23526882DNASphingomonas sp. ATCC53159 26atggcagcga ccgcgatgac gcggcagcag gagaggaagg gcggtggcta ttggctggcc 60gttgccggtc ttgccgcgct aaccatcccg accttcatca ccctgggtcg cgaggtttgg 120agtgcggaag gcggcgtgca gggtccgatc gtgctcgcca cgggcgcctg gatgctggcc 180cgccagtgct cgacgatcga ggcgctacgc cgccccggca gcgtgctgct cggcgcgctg 240ttcctgctgg cgacgcttgc cttctacacc gttggacggg tgttcgactt catcagtgtc 300gaaaccttcg gactggtcgc gacctatctg gtcgtcgcct atctctattt cggtgccagg 360gtgctccgtg ccgcctggtt cccggtgctg tggctgttct tcctggtgcc gccgcccggc 420tgggccgtcg accgcatcac cgcaccgctc aaggagttcg tctcctatgc ggcaacgggc 480ctgctttcct gggtggatta tccgatcctg cgccagggcg tgacactgtt cgtcggcccc 540tatcagctgc tcgtcgaaga tgcctgttcg ggtctgcgct cgctgtccag cctggtcgtc 600gtgacgctgc tctacatcta catcaagaac aagccgtcct ggcgctacgc ggcgttcatc 660gcagcgctgg tgatcccggt ggcagtggtg accaacgtcc tgcggatcat catcctggta 720ctgatcacct atcatctggg cgacgaggcg gcgcagagct tcctccacgt ctccaccggc 780atggtgatgt tcgtggtcgc cctgctttgc atcttcgcga tcgactgggt ggtcgagcaa 840cttcttctcc tgcgtcggag gcatcatgtt caaccggcgt ga 88227293PRTSphingomonas sp. ATCC53159 27Met Ala Ala Thr Ala Met Thr Arg Gln Gln Glu Arg Lys Gly Gly Gly1 5 10 15Tyr Trp Leu Ala Val Ala Gly Leu Ala Ala Leu Thr Ile Pro Thr Phe 20 25 30Ile Thr Leu Gly Arg Glu Val Trp Ser Ala Glu Gly Gly Val Gln Gly 35 40 45Pro Ile Val Leu Ala Thr Gly Ala Trp Met Leu Ala Arg Gln Cys Ser 50 55 60Thr Ile Glu Ala Leu Arg Arg Pro Gly Ser Val Leu Leu Gly Ala Leu65 70 75 80Phe Leu Leu Ala Thr Leu Ala Phe Tyr Thr Val Gly Arg Val Phe Asp 85 90 95Phe Ile Ser Val Glu Thr Phe Gly Leu Val Ala Thr Tyr Leu Val Val 100 105 110Ala Tyr Leu Tyr Phe Gly Ala Arg Val Leu Arg Ala Ala Trp Phe Pro 115 120 125Val Leu Trp Leu Phe Phe Leu Val Pro Pro Pro Gly Trp Ala Val Asp 130 135 140Arg Ile Thr Ala Pro Leu Lys Glu Phe Val Ser Tyr Ala Ala Thr Gly145 150 155 160Leu Leu Ser Trp Val Asp Tyr Pro Ile Leu Arg Gln Gly Val Thr Leu 165 170 175Phe Val Gly Pro Tyr Gln Leu Leu Val Glu Asp Ala Cys Ser Gly Leu 180 185 190Arg Ser Leu Ser Ser Leu Val Val Val Thr Leu Leu Tyr Ile Tyr Ile 195 200 205Lys Asn Lys Pro Ser Trp Arg Tyr Ala Ala Phe Ile Ala Ala Leu Val 210 215 220Ile Pro Val Ala Val Val Thr Asn Val Leu Arg Ile Ile Ile Leu Val225 230 235 240Leu Ile Thr Tyr His Leu Gly Asp Glu Ala Ala Gln Ser Phe Leu His 245 250 255Val Ser Thr Gly Met Val Met Phe Val Val Ala Leu Leu Cys Ile Phe 260 265 270Ala Ile Asp Trp Val Val Glu Gln Leu Leu Leu Leu Arg Arg Arg His 275 280 285His Val Gln Pro Ala 29028699DNASphingomonas sp. ATCC53159 28atgttcaacc ggcgtgacct gctgatcggc gcaggctgct tcgccgccgc tggcgcctcg 60ctcggcctga agccgcaccg gcggatggac ctgctgggcg gcaccaagct cgacacgctg 120atgcccaagg cattcggcgc atggaaggca gaggataccg gttcgctgat cgcgccggcg 180cgcgaaggca gcctggagga caagctctac aaccaggtgg tcacccgcgc cttctcccgc 240gcggacggtg cccaagtgat gctgctgatc gcctatggca acgcccagac cgatctactg 300cagctgcacc ggccggaaat atgctacccg ttcttcggct tcaccgtggt ggaaagccat 360gagcagacca tcccggtgac gccgcaggtg acgatccccg gtcgcgcgct gaccgccacc 420aacttcaacc gcaccgagca gatcctctac tggacccgcg tcggcgaata tctgccgcag 480aacggcaatc agcagatgct cgcgcggctg aagagccagg tccagggctg gatcgtcgac 540ggtgtgctgg tgcgcatctc gacggtgacg cccgaggcgg aagatggcct gagcgccaat 600ctcgatttcg cgcgcgagct ggtgaagacg ctcgacccgc gcgtgctgcg cccgctgctc 660gggaacgggc tcacacggca gctcggtcac caggtctga 69929232PRTSphingomonas sp. ATCC53159 29Met Phe Asn Arg Arg Asp Leu Leu Ile Gly Ala Gly Cys Phe Ala Ala1 5 10 15Ala Gly Ala Ser Leu Gly Leu Lys Pro His Arg Arg Met Asp Leu Leu 20 25 30Gly Gly Thr Lys Leu Asp Thr Leu Met Pro Lys Ala Phe Gly Ala Trp 35 40 45Lys Ala Glu Asp Thr Gly Ser Leu Ile Ala Pro Ala Arg Glu Gly Ser 50 55 60Leu Glu Asp Lys Leu Tyr Asn Gln Val Val Thr Arg Ala Phe Ser Arg65 70 75 80Ala Asp Gly Ala Gln Val Met Leu Leu Ile Ala Tyr Gly Asn Ala Gln 85 90 95Thr Asp Leu Leu Gln Leu His Arg Pro Glu Ile Cys Tyr Pro Phe Phe 100 105 110Gly Phe Thr Val Val Glu Ser His Glu Gln Thr Ile Pro Val Thr Pro 115 120 125Gln Val Thr Ile Pro Gly Arg Ala Leu Thr Ala Thr Asn Phe Asn Arg 130 135 140Thr Glu Gln Ile Leu Tyr Trp Thr Arg Val Gly Glu Tyr Leu Pro Gln145 150 155 160Asn Gly Asn Gln Gln Met Leu Ala Arg Leu Lys Ser Gln Val Gln Gly 165 170 175Trp Ile Val Asp Gly Val Leu Val Arg Ile Ser Thr Val Thr Pro Glu 180 185 190Ala Glu Asp Gly Leu Ser Ala Asn Leu Asp Phe Ala Arg Glu Leu Val 195 200 205Lys Thr Leu Asp Pro Arg Val Leu Arg Pro Leu Leu Gly Asn Gly Leu 210 215 220Thr Arg Gln Leu Gly His Gln Val225 230301395DNASphingomonas sp. ATCC53159 30atgaacgccg ttgttccgat gcgccgcggc ggcccgctcg cccgcatgcg cgataccgtg 60ctgcctgccc gcgtcgacgc

ttatgacacc gccttcctgc ctgccgcgct ggagatcatc 120gagcggccgg tttcgcccac cgcgcggctt accgccaagg tgatgctggc cgggctggcg 180atcaccgccg cctggctggc gatcggcaag gtcgaagtcg tcgcgccgac gcaggggcgg 240atcgcgccga tcggcgagac caagatcgtc cagtcgcccg aatcggggat cgtccgccgc 300atcctggtgg gcgaggggca gaaggtcgcc aagggccagg tgctgatcac gctcgacccg 360accgtgtcgt cggcggaggc ggcacaggcg aaggtggcgc tgctcagcgc ccagctcgac 420gccgcacgca accaggcgat catcgacgcg ctggacggca ggggcttccg cttcgtcgcg 480cctgccgccg ccagcccggg cgaagtggcg acgcatcgcg gcctcgcccg cgcccggctg 540ggccagatcg aggcggcgct ggccggcggc cgctccgatc gcggtgccgc cgtctcggcc 600gcggccgagg cgcaggcaca ggtgcggaag ctcgaacagt cgctgccgct gctcgaacag 660cagatcgccg cgaacgagac gatggccgcc aagggctatg tctcgaagct gcgcgtcgtg 720gagatgcgtc gccagctgat cgccgagcgg caggacctga cggcggcgcg cgctacgctc 780gccaaactcg gccagcagtc gctgagcgtc tccagcctgt cggccaagac gcgcgaggag 840gcgcgggcgc aggtgctgca ggatctggtc aaggcgcagg acgaggtgcg tgcccgcggc 900gaggacgtcg ccaaggcgaa tctgcgcagc tcgttccgcg aactgcgcgc gccggtgagc 960ggtaccgtct cgcagctgca ggtccacacc gaaggcggcg tggtggaagg ggccaagccg 1020ctcctcagcc tggttcccga caatgcccgg ctcgaggccg aggtgatggt cgacaacagc 1080gacatcggct tcgtccacat cggcatgccg gtaaaggtga agctgcaggc ctttccctat 1140acccgctacg gcatgattcc cggcacggtg gcgggcatca gccccgaggc ggtgcagatg 1200aaggagaacc agccgccggt ctacaaggcg cggatcgcgc tggcgcgcgg gtatgtgctg 1260gcccatggcg cacaggtgcc gctgcggccg gggatgctcg cgagcgcgga catcgtcacc 1320ggcaagcgaa ccctgttcag ctatctggtg gggcccgtgc tcgagacggg gagtgacgcg 1380ctgcacgagc ggtga 139531464PRTSphingomonas sp. ATCC53159 31Met Asn Ala Val Val Pro Met Arg Arg Gly Gly Pro Leu Ala Arg Met1 5 10 15Arg Asp Thr Val Leu Pro Ala Arg Val Asp Ala Tyr Asp Thr Ala Phe 20 25 30Leu Pro Ala Ala Leu Glu Ile Ile Glu Arg Pro Val Ser Pro Thr Ala 35 40 45Arg Leu Thr Ala Lys Val Met Leu Ala Gly Leu Ala Ile Thr Ala Ala 50 55 60Trp Leu Ala Ile Gly Lys Val Glu Val Val Ala Pro Thr Gln Gly Arg65 70 75 80Ile Ala Pro Ile Gly Glu Thr Lys Ile Val Gln Ser Pro Glu Ser Gly 85 90 95Ile Val Arg Arg Ile Leu Val Gly Glu Gly Gln Lys Val Ala Lys Gly 100 105 110Gln Val Leu Ile Thr Leu Asp Pro Thr Val Ser Ser Ala Glu Ala Ala 115 120 125Gln Ala Lys Val Ala Leu Leu Ser Ala Gln Leu Asp Ala Ala Arg Asn 130 135 140Gln Ala Ile Ile Asp Ala Leu Asp Gly Arg Gly Phe Arg Phe Val Ala145 150 155 160Pro Ala Ala Ala Ser Pro Gly Glu Val Ala Thr His Arg Gly Leu Ala 165 170 175Arg Ala Arg Leu Gly Gln Ile Glu Ala Ala Leu Ala Gly Gly Arg Ser 180 185 190Asp Arg Gly Ala Ala Val Ser Ala Ala Ala Glu Ala Gln Ala Gln Val 195 200 205Arg Lys Leu Glu Gln Ser Leu Pro Leu Leu Glu Gln Gln Ile Ala Ala 210 215 220Asn Glu Thr Met Ala Ala Lys Gly Tyr Val Ser Lys Leu Arg Val Val225 230 235 240Glu Met Arg Arg Gln Leu Ile Ala Glu Arg Gln Asp Leu Thr Ala Ala 245 250 255Arg Ala Thr Leu Ala Lys Leu Gly Gln Gln Ser Leu Ser Val Ser Ser 260 265 270Leu Ser Ala Lys Thr Arg Glu Glu Ala Arg Ala Gln Val Leu Gln Asp 275 280 285Leu Val Lys Ala Gln Asp Glu Val Arg Ala Arg Gly Glu Asp Val Ala 290 295 300Lys Ala Asn Leu Arg Ser Ser Phe Arg Glu Leu Arg Ala Pro Val Ser305 310 315 320Gly Thr Val Ser Gln Leu Gln Val His Thr Glu Gly Gly Val Val Glu 325 330 335Gly Ala Lys Pro Leu Leu Ser Leu Val Pro Asp Asn Ala Arg Leu Glu 340 345 350Ala Glu Val Met Val Asp Asn Ser Asp Ile Gly Phe Val His Ile Gly 355 360 365Met Pro Val Lys Val Lys Leu Gln Ala Phe Pro Tyr Thr Arg Tyr Gly 370 375 380Met Ile Pro Gly Thr Val Ala Gly Ile Ser Pro Glu Ala Val Gln Met385 390 395 400Lys Glu Asn Gln Pro Pro Val Tyr Lys Ala Arg Ile Ala Leu Ala Arg 405 410 415Gly Tyr Val Leu Ala His Gly Ala Gln Val Pro Leu Arg Pro Gly Met 420 425 430Leu Ala Ser Ala Asp Ile Val Thr Gly Lys Arg Thr Leu Phe Ser Tyr 435 440 445Leu Val Gly Pro Val Leu Glu Thr Gly Ser Asp Ala Leu His Glu Arg 450 455 460322187DNASphingomonas sp. ATCC53159 32atgacacgcg acgaaatgca ggccaccctg cagagcgcgc tcgcggccca tggggcggcg 60gagcgcgagg cggagctgcg cgaatccgga ctggtggcgt tgtcgctgct gctcggcgcg 120cacaacatcg ccatcacgcc cgaacagctg cgccacgcgc tgggccatgc cgaggcggca 180agcgccgacg acctgatcct cctggccaag cgccagcagg gcgtgcgcgc caaggccgtc 240gaggtgccgc gcggcggact cgcccgccag ccgctgcccg cgatcgccga cgggcccgaa 300ggctggttcg tgatcggcgg cctgaccgaa catggcgtga tcatccagcg cccgggccat 360gccccggaac aggtcgaccg ggacgcgctg gacgcgatct ggtccggcgc gctggtgctg 420ctcaccaccc gcgcggtggc gggacggccg ctgcggttcg gcctctcctg gttcaccgcg 480cagttccggc gctatcgcac gctgttcctc gaggtgctcg gcatcaccct cgcgctcaac 540ctgctcggcc tcgccgcgcc gctgttgttc cagagcgtga tcgacaaggt gctgatccac 600aacagcatga gcacgctgag cgtgctcgcc ttcgccttcc tggcggtttc ggtgtgggaa 660gtggcgctcg gctggatccg cacccgcctg ttcaccgaga cgacgcagaa gatcgacgtc 720gagctgggtg cccggctgtt ccaccacctg ctggcgctgc cgctcgccta tttcgagaag 780cgccgcgtgg gcgacaccgt cacccgcgtc cgccagctcg agacgatccg cgaattcctt 840accagcgcct cgctgacggt gatggtggac ccgctgttca ccttcgtgtt cctcgccgcg 900atgctgttct actcgccgat gctctcgggc atcgtgctcg tgtcgctgat cgcctatgcg 960atcgtatcgt tcagcgtcgc cgggccgctc cgcgcgcggg tggaggacaa gttcgagaag 1020agctccgcca gcaacgcgct gctcgtcgag agcgtctcgg gcatccacac gatcaaggcg 1080accgcggtcg agccgcactg gcagaatcgc tgggagcgcc agctcgccgc ccataccgcc 1140gcgtcgcagc ggctgatcaa taccgccaac accggcagcc aggcgatcga gctgatctcg 1200aagctgagct tcgcggcgat cctgttcttc ggcgccaagg cggtgatcgg cggcgcgatg 1260agcgtaggcg cgctggtggc gttcaacatg ttcgcccagc gcgtgtccgg gccggtgatc 1320cgcatggcgc agctgtggca ggatttccag caggtgcgca tctcggtcga gcggctgggc 1380gacgtgctca accatccggt ggaaccgcgc ccggcctcgg cggcgacgct gccggtgctg 1440cgcggtgcga ttcgcttcga gaatgtcagc ttccgctatg ccgaggacca gccgccggtg 1500ctgagcgaca tcacgctcga cattccggcg ggcacctcgc tcggcatcgt cggttcgtcg 1560ggctcgggca agtcgacgct ggccaagctg ctccagcggc tcaacctgcc gaatctcggc 1620cgcgtgctgg tcgacgaggt cgacgtggcg cagctcgatc ccgcctggct gcgtcgccag 1680atcggcgtcg tgctgcagga gaatctgctg ttcagccgct cgatccgcga gaacatcgcg 1740ctctccaacc ccgccatgcc gttcgagaat gtcgtcgcgg cggcgacgct ggccggcgcg 1800catgatttca tcctgcgcca gccgcgcggc tatgacaccg agatcgtcga gcgcggcgtc 1860aatctctccg gcggccagcg ccagcggctc gccatcgccc gcgcgctcgt cggcaatccg 1920cgcatcctgg tgttcgacga agcgacctcg gcgctcgatg ccgagagcga ggagctgatc 1980cagaacaacc tgcgcgccat ctcggccggc cgcacgctcg tggtgatcgc gcatcgcctg 2040agcgcggtgc gcagctgcga ccggatcatc acgctcgaac agggccgcat cgtcgagagc 2100ggccgacacg acgaattgtt gcgcctgggc ggccgctatg ccgacctgca ccgccgccag 2160ggcggctatg gggagattgc cgcatga 218733728PRTSphingomonas sp. ATCC53159 33Met Thr Arg Asp Glu Met Gln Ala Thr Leu Gln Ser Ala Leu Ala Ala1 5 10 15His Gly Ala Ala Glu Arg Glu Ala Glu Leu Arg Glu Ser Gly Leu Val 20 25 30Ala Leu Ser Leu Leu Leu Gly Ala His Asn Ile Ala Ile Thr Pro Glu 35 40 45Gln Leu Arg His Ala Leu Gly His Ala Glu Ala Ala Ser Ala Asp Asp 50 55 60Leu Ile Leu Leu Ala Lys Arg Gln Gln Gly Val Arg Ala Lys Ala Val65 70 75 80Glu Val Pro Arg Gly Gly Leu Ala Arg Gln Pro Leu Pro Ala Ile Ala 85 90 95Asp Gly Pro Glu Gly Trp Phe Val Ile Gly Gly Leu Thr Glu His Gly 100 105 110Val Ile Ile Gln Arg Pro Gly His Ala Pro Glu Gln Val Asp Arg Asp 115 120 125Ala Leu Asp Ala Ile Trp Ser Gly Ala Leu Val Leu Leu Thr Thr Arg 130 135 140Ala Val Ala Gly Arg Pro Leu Arg Phe Gly Leu Ser Trp Phe Thr Ala145 150 155 160Gln Phe Arg Arg Tyr Arg Thr Leu Phe Leu Glu Val Leu Gly Ile Thr 165 170 175Leu Ala Leu Asn Leu Leu Gly Leu Ala Ala Pro Leu Leu Phe Gln Ser 180 185 190Val Ile Asp Lys Val Leu Ile His Asn Ser Met Ser Thr Leu Ser Val 195 200 205Leu Ala Phe Ala Phe Leu Ala Val Ser Val Trp Glu Val Ala Leu Gly 210 215 220Trp Ile Arg Thr Arg Leu Phe Thr Glu Thr Thr Gln Lys Ile Asp Val225 230 235 240Glu Leu Gly Ala Arg Leu Phe His His Leu Leu Ala Leu Pro Leu Ala 245 250 255Tyr Phe Glu Lys Arg Arg Val Gly Asp Thr Val Thr Arg Val Arg Gln 260 265 270Leu Glu Thr Ile Arg Glu Phe Leu Thr Ser Ala Ser Leu Thr Val Met 275 280 285Val Asp Pro Leu Phe Thr Phe Val Phe Leu Ala Ala Met Leu Phe Tyr 290 295 300Ser Pro Met Leu Ser Gly Ile Val Leu Val Ser Leu Ile Ala Tyr Ala305 310 315 320Ile Val Ser Phe Ser Val Ala Gly Pro Leu Arg Ala Arg Val Glu Asp 325 330 335Lys Phe Glu Lys Ser Ser Ala Ser Asn Ala Leu Leu Val Glu Ser Val 340 345 350Ser Gly Ile His Thr Ile Lys Ala Thr Ala Val Glu Pro His Trp Gln 355 360 365Asn Arg Trp Glu Arg Gln Leu Ala Ala His Thr Ala Ala Ser Gln Arg 370 375 380Leu Ile Asn Thr Ala Asn Thr Gly Ser Gln Ala Ile Glu Leu Ile Ser385 390 395 400Lys Leu Ser Phe Ala Ala Ile Leu Phe Phe Gly Ala Lys Ala Val Ile 405 410 415Gly Gly Ala Met Ser Val Gly Ala Leu Val Ala Phe Asn Met Phe Ala 420 425 430Gln Arg Val Ser Gly Pro Val Ile Arg Met Ala Gln Leu Trp Gln Asp 435 440 445Phe Gln Gln Val Arg Ile Ser Val Glu Arg Leu Gly Asp Val Leu Asn 450 455 460His Pro Val Glu Pro Arg Pro Ala Ser Ala Ala Thr Leu Pro Val Leu465 470 475 480Arg Gly Ala Ile Arg Phe Glu Asn Val Ser Phe Arg Tyr Ala Glu Asp 485 490 495Gln Pro Pro Val Leu Ser Asp Ile Thr Leu Asp Ile Pro Ala Gly Thr 500 505 510Ser Leu Gly Ile Val Gly Ser Ser Gly Ser Gly Lys Ser Thr Leu Ala 515 520 525Lys Leu Leu Gln Arg Leu Asn Leu Pro Asn Leu Gly Arg Val Leu Val 530 535 540Asp Glu Val Asp Val Ala Gln Leu Asp Pro Ala Trp Leu Arg Arg Gln545 550 555 560Ile Gly Val Val Leu Gln Glu Asn Leu Leu Phe Ser Arg Ser Ile Arg 565 570 575Glu Asn Ile Ala Leu Ser Asn Pro Ala Met Pro Phe Glu Asn Val Val 580 585 590Ala Ala Ala Thr Leu Ala Gly Ala His Asp Phe Ile Leu Arg Gln Pro 595 600 605Arg Gly Tyr Asp Thr Glu Ile Val Glu Arg Gly Val Asn Leu Ser Gly 610 615 620Gly Gln Arg Gln Arg Leu Ala Ile Ala Arg Ala Leu Val Gly Asn Pro625 630 635 640Arg Ile Leu Val Phe Asp Glu Ala Thr Ser Ala Leu Asp Ala Glu Ser 645 650 655Glu Glu Leu Ile Gln Asn Asn Leu Arg Ala Ile Ser Ala Gly Arg Thr 660 665 670Leu Val Val Ile Ala His Arg Leu Ser Ala Val Arg Ser Cys Asp Arg 675 680 685Ile Ile Thr Leu Glu Gln Gly Arg Ile Val Glu Ser Gly Arg His Asp 690 695 700Glu Leu Leu Arg Leu Gly Gly Arg Tyr Ala Asp Leu His Arg Arg Gln705 710 715 720Gly Gly Tyr Gly Glu Ile Ala Ala 725341413DNASphingomonas sp. ATCC53159 34atgaacgctt tcgaagcaca gcgcgccttt gaggagcaac ttcgggcgca ttcccgggtt 60acgccatctg ccgctcccgt gtggcgtcgc tcgacgctgc ggatggtcct ctataccgag 120ttgctgctgc tggacagtct ctcgatcctg gccggattcc acgtcgcggc gggcacgcgc 180gacggcaact ggctgtcgct ggcgggcatc aacgtcggcg tcttcctgct gccgatcgct 240ctcggcaccg cgctcgcaag cggcacctac tcgctgaact gcctgcgcta cccggtcagc 300ggcgtgaaga gcatcttctc ggcattcttc ttctcgatct tcgtcgtcct gctcggcagc 360tacctgctga cggccgagct gccgctgtcc cgcgtgcagc tggcggaggg cgcgatcctc 420tcgctggtcc tcctgatggt gggccgcctg atgttccgcc gccacgtccg cgcggttacc 480ggcggcaggc tgctcgacga actggtcatc atcgacggcg tctcgctcga cgtcgcgggc 540aatgcggtcg cgctcgacgc gcggatcatc aatctctcgc cgaacccgcg cgatccgcaa 600atgctgcatc gcctgggcac caccgtgatc gggttcgacc gggtgatcgt cgcctgcacc 660aaggagcatc gcgcggtctg ggcgctgctg ctcaagggca tgaacatcaa gggcgagatc 720ctcgtccccc agttcaatgc gctgggcgcg atcggcgtgg acgcctttga cgggaaggat 780acgctggtcg tctcgcaggg cccgctcaac atgcccaacc gcgcgaagaa gcgcgcgctc 840gatctcgcga tcaccgtacc ggccgtgctc gcgctggcgc cgctgatgat cctggtggcg 900atcctgatca agctggagag cccgggcccg gtgttgttcg cgcaggatcg cgtcggccgc 960ggcaaccggc tgttcaagat catgaagttc cgctcgatgc gcgtaacgct gtgcgacgcg 1020aacggcaacg tctcggccag ccgcgacgac gatcgcatca ccaaggtcgg ccgcttcatc 1080cgcaagacca gcatcgacga actgccgcag ctgctgaacg tgctgcgcgg cgacatgagc 1140gtcgtcggcc cgcggccgca tgcgctgggc tcgcgcgccg ccgatcacct gttctgggaa 1200atcgacgagc gctactggca ccgccacacg ctcaagccgg gcatgaccgg tctggcccag 1260gtgcgcggtt tccgcggggc gaccgatcgc cgcgtcgatc tgaccaaccg gctccaggca 1320gacatggaat atatcgacgg atgggatatc tggcgcgata tcacgatcct gttcaagacg 1380ctgcgggtga tcgtgcattc gaacgcattc tga 141335470PRTSphingomonas sp. ATCC53159 35Met Asn Ala Phe Glu Ala Gln Arg Ala Phe Glu Glu Gln Leu Arg Ala1 5 10 15His Ser Arg Val Thr Pro Ser Ala Ala Pro Val Trp Arg Arg Ser Thr 20 25 30Leu Arg Met Val Leu Tyr Thr Glu Leu Leu Leu Leu Asp Ser Leu Ser 35 40 45Ile Leu Ala Gly Phe His Val Ala Ala Gly Thr Arg Asp Gly Asn Trp 50 55 60Leu Ser Leu Ala Gly Ile Asn Val Gly Val Phe Leu Leu Pro Ile Ala65 70 75 80Leu Gly Thr Ala Leu Ala Ser Gly Thr Tyr Ser Leu Asn Cys Leu Arg 85 90 95Tyr Pro Val Ser Gly Val Lys Ser Ile Phe Ser Ala Phe Phe Phe Ser 100 105 110Ile Phe Val Val Leu Leu Gly Ser Tyr Leu Leu Thr Ala Glu Leu Pro 115 120 125Leu Ser Arg Val Gln Leu Ala Glu Gly Ala Ile Leu Ser Leu Val Leu 130 135 140Leu Met Val Gly Arg Leu Met Phe Arg Arg His Val Arg Ala Val Thr145 150 155 160Gly Gly Arg Leu Leu Asp Glu Leu Val Ile Ile Asp Gly Val Ser Leu 165 170 175Asp Val Ala Gly Asn Ala Val Ala Leu Asp Ala Arg Ile Ile Asn Leu 180 185 190Ser Pro Asn Pro Arg Asp Pro Gln Met Leu His Arg Leu Gly Thr Thr 195 200 205Val Ile Gly Phe Asp Arg Val Ile Val Ala Cys Thr Lys Glu His Arg 210 215 220Ala Val Trp Ala Leu Leu Leu Lys Gly Met Asn Ile Lys Gly Glu Ile225 230 235 240Leu Val Pro Gln Phe Asn Ala Leu Gly Ala Ile Gly Val Asp Ala Phe 245 250 255Asp Gly Lys Asp Thr Leu Val Val Ser Gln Gly Pro Leu Asn Met Pro 260 265 270Asn Arg Ala Lys Lys Arg Ala Leu Asp Leu Ala Ile Thr Val Pro Ala 275 280 285Val Leu Ala Leu Ala Pro Leu Met Ile Leu Val Ala Ile Leu Ile Lys 290 295 300Leu Glu Ser Pro Gly Pro Val Leu Phe Ala Gln Asp Arg Val Gly Arg305 310 315 320Gly Asn Arg Leu Phe Lys Ile Met Lys Phe Arg Ser Met Arg Val Thr 325 330 335Leu Cys Asp Ala Asn Gly Asn Val Ser Ala Ser Arg Asp Asp Asp Arg 340 345 350Ile Thr Lys Val Gly Arg Phe Ile Arg Lys Thr Ser Ile Asp Glu Leu 355 360 365Pro Gln Leu Leu Asn Val Leu Arg Gly Asp Met Ser Val Val Gly Pro 370 375 380Arg Pro His Ala Leu Gly Ser Arg Ala Ala Asp His Leu Phe Trp Glu385 390 395 400Ile Asp Glu Arg Tyr Trp His Arg His Thr Leu Lys Pro Gly Met Thr 405 410 415Gly Leu Ala Gln Val Arg Gly Phe Arg Gly Ala Thr Asp Arg Arg Val 420 425 430Asp Leu Thr Asn Arg

Leu Gln Ala Asp Met Glu Tyr Ile Asp Gly Trp 435 440 445Asp Ile Trp Arg Asp Ile Thr Ile Leu Phe Lys Thr Leu Arg Val Ile 450 455 460Val His Ser Asn Ala Phe465 47036879DNASphingomonas sp. ATCC53159 36atgaagggca tcatccttgc ggggggcagc gggacgcgcc tgtaccccgc aacgctatcg 60atctcgaagc agctgcttcc cgtctatgac aagccgatga tcttctatcc gctgtcggtg 120ctgatgctca ccggcatccg ggacatcctg attatctcca ccccgcgcga cctgccgatg 180ttccaggcgc tgctgggcga cggctcggcc ttcggcatca acctcagcta tgccgagcag 240ccctccccca acgggctggc cgaagcgttc atcatcggcg cggatttcgt cggcaacgat 300cccagcgcgc tgatcctggg cgacaacatc tatcacggcg aaaagatggg cgagcgctgc 360caggcagccg cagcgcaggc agcgcagggc ggtgcaaacg tcttcgccta tcatgtcgac 420gaccccgagc gctacggcgt ggtcgcgttc gacccggaga cgggcgtcgc caccagcgtc 480gaggaaaagc cggccgagcc caagtccaac tgggcgatca ccggcctgta tttctacgac 540aaggacgtgg tcgacatcgc caagtcgatc cagccctcgg cgcgcggcga actcgagatc 600accgacgtca accgcgttta catggagcgc ggcgacctgc acatcacgcg cctcggccgc 660ggctatgcct ggctcgacac cggcacgcat gacagcctgc acgaagccgg ctcgttcgtt 720cgcacgctcg agcatcggac gggcgtgaag atcgcctgcc cggaggaaat cgccttcgaa 780agcggctggc tcggcgccga agacctgctc aagcgcgccg ccggcctcgg caagaccggc 840tatgccgcct atctccgcaa ggttgcgacc gcagcatga 87937292PRTSphingomonas sp. ATCC53159 37Met Lys Gly Ile Ile Leu Ala Gly Gly Ser Gly Thr Arg Leu Tyr Pro1 5 10 15Ala Thr Leu Ser Ile Ser Lys Gln Leu Leu Pro Val Tyr Asp Lys Pro 20 25 30Met Ile Phe Tyr Pro Leu Ser Val Leu Met Leu Thr Gly Ile Arg Asp 35 40 45Ile Leu Ile Ile Ser Thr Pro Arg Asp Leu Pro Met Phe Gln Ala Leu 50 55 60Leu Gly Asp Gly Ser Ala Phe Gly Ile Asn Leu Ser Tyr Ala Glu Gln65 70 75 80Pro Ser Pro Asn Gly Leu Ala Glu Ala Phe Ile Ile Gly Ala Asp Phe 85 90 95Val Gly Asn Asp Pro Ser Ala Leu Ile Leu Gly Asp Asn Ile Tyr His 100 105 110Gly Glu Lys Met Gly Glu Arg Cys Gln Ala Ala Ala Ala Gln Ala Ala 115 120 125Gln Gly Gly Ala Asn Val Phe Ala Tyr His Val Asp Asp Pro Glu Arg 130 135 140Tyr Gly Val Val Ala Phe Asp Pro Glu Thr Gly Val Ala Thr Ser Val145 150 155 160Glu Glu Lys Pro Ala Glu Pro Lys Ser Asn Trp Ala Ile Thr Gly Leu 165 170 175Tyr Phe Tyr Asp Lys Asp Val Val Asp Ile Ala Lys Ser Ile Gln Pro 180 185 190Ser Ala Arg Gly Glu Leu Glu Ile Thr Asp Val Asn Arg Val Tyr Met 195 200 205Glu Arg Gly Asp Leu His Ile Thr Arg Leu Gly Arg Gly Tyr Ala Trp 210 215 220Leu Asp Thr Gly Thr His Asp Ser Leu His Glu Ala Gly Ser Phe Val225 230 235 240Arg Thr Leu Glu His Arg Thr Gly Val Lys Ile Ala Cys Pro Glu Glu 245 250 255Ile Ala Phe Glu Ser Gly Trp Leu Gly Ala Glu Asp Leu Leu Lys Arg 260 265 270Ala Ala Gly Leu Gly Lys Thr Gly Tyr Ala Ala Tyr Leu Arg Lys Val 275 280 285Ala Thr Ala Ala 29038567DNASphingomonas sp. ATCC53159 38atgacccagg tccatcatca cgaactgtcc ggcgtcatcg agttcacgcc gcccaaatat 60ggcgaccacc gcggcttctt ctccgaagtg ttcaagcagt cggtgctcga tgccgaaggc 120gtcgaggcac gctgggtgca ggacaatcag agcttctcgg cggccccggg cacgatccgc 180ggcctgcatc tccaggcgcc gcccttcgcc caggccaagc tggtccgcgt gttgcgcggc 240gcgatcttcg acgtcgcggt cgacatccgt cgcggctcgc ccacctatgg caaatgggtc 300ggcgtcgagc tctcggccga gaagtggaac cagctgctgg tccccgccgg ctatgcgcac 360ggcttcatga cgctcgttcc ggattgcgag atcctctaca aggtcagcgc caaatattcg 420aaggattcgg agatggcgat ccgttgggac gatcccgatc tcgccatcgc ctggccggac 480atcggcgtcg agccggtcct ctccgaaaag gacgcggtcg ccacgccctt cgccgaattc 540aacaccccct tcttctatca gggctga 56739188PRTSphingomonas sp. ATCC53159 39Met Thr Gln Val His His His Glu Leu Ser Gly Val Ile Glu Phe Thr1 5 10 15Pro Pro Lys Tyr Gly Asp His Arg Gly Phe Phe Ser Glu Val Phe Lys 20 25 30Gln Ser Val Leu Asp Ala Glu Gly Val Glu Ala Arg Trp Val Gln Asp 35 40 45Asn Gln Ser Phe Ser Ala Ala Pro Gly Thr Ile Arg Gly Leu His Leu 50 55 60Gln Ala Pro Pro Phe Ala Gln Ala Lys Leu Val Arg Val Leu Arg Gly65 70 75 80Ala Ile Phe Asp Val Ala Val Asp Ile Arg Arg Gly Ser Pro Thr Tyr 85 90 95Gly Lys Trp Val Gly Val Glu Leu Ser Ala Glu Lys Trp Asn Gln Leu 100 105 110Leu Val Pro Ala Gly Tyr Ala His Gly Phe Met Thr Leu Val Pro Asp 115 120 125Cys Glu Ile Leu Tyr Lys Val Ser Ala Lys Tyr Ser Lys Asp Ser Glu 130 135 140Met Ala Ile Arg Trp Asp Asp Pro Asp Leu Ala Ile Ala Trp Pro Asp145 150 155 160Ile Gly Val Glu Pro Val Leu Ser Glu Lys Asp Ala Val Ala Thr Pro 165 170 175Phe Ala Glu Phe Asn Thr Pro Phe Phe Tyr Gln Gly 180 185401062DNASphingomonas sp. ATCC53159 40atgcagcaga ccttcctcgt caccggcggc gccggcttca tcggctcggc ggtggtgcgc 60cacctcgtcc gccagggcgc gcgcgtcatc aatctcgaca agctcaccta tgccggcaac 120ccggcctcgc tgactgcgat cgagaacgcg cccaactatc gcttcgtcca tgccgacatc 180gccgacaccg cgacgatcct accgctgctg cgcgaggagc aggtcgatgt ggtgatgcac 240ctcgccgccg agagccatgt cgatcgctcg atcgacggcc ctggcgagtt catcgagacc 300aatgtcgtcg gcaccttcaa gctgctccag tcggcgctgc aatattggcg cgagctggag 360ggcgagaaac gcgacgcgtt ccgcttccac cacatctcca ccgacgaagt gttcggcgac 420ctgccgttcg acagcggcat cttcaccgaa gagacgccct atgatccctc ctcgccctat 480tcggcgtcga aggcggcgag cgaccatctg gtgcgcgcct ggggccacac ctatggcctg 540ccggtggtgc tgtcgaactg ctcgaacaat tacgggccgt tccacttccc cgagaagctg 600atcccgttga ccatcctcaa cgcgctcgag ggcaagccgc tgccggtcta cggcaagggc 660gagaatatcc gcgactggct gtatgtcgac gatcacgcca aggcgctggc gaccatcgcc 720accaccggca aggtcggcca gagctacaat gtcggcggcc gcaacgagcg gaccaacctg 780caggtggtcg agacgatctg cgacctgctc gaccagcgca ttccgctggc cgacggtcgc 840aagcgccgcg aactgatcac cttcgtcacc gatcgccccg gccatgaccg ccgctacgcg 900atcgacgcga ccaagctcga gaccgagctg ggctggaagg ctgaggagaa tttcgacacc 960ggcatcgccg cgacgatcga ctggtatctg gcgaacgagt ggtggtgggg cccgatccgc 1020tccggcaaat atgccggcga gcggctgggg cagaccgcct ga 106241353PRTSphingomonas sp. ATCC53159 41Met Gln Gln Thr Phe Leu Val Thr Gly Gly Ala Gly Phe Ile Gly Ser1 5 10 15Ala Val Val Arg His Leu Val Arg Gln Gly Ala Arg Val Ile Asn Leu 20 25 30Asp Lys Leu Thr Tyr Ala Gly Asn Pro Ala Ser Leu Thr Ala Ile Glu 35 40 45Asn Ala Pro Asn Tyr Arg Phe Val His Ala Asp Ile Ala Asp Thr Ala 50 55 60Thr Ile Leu Pro Leu Leu Arg Glu Glu Gln Val Asp Val Val Met His65 70 75 80Leu Ala Ala Glu Ser His Val Asp Arg Ser Ile Asp Gly Pro Gly Glu 85 90 95Phe Ile Glu Thr Asn Val Val Gly Thr Phe Lys Leu Leu Gln Ser Ala 100 105 110Leu Gln Tyr Trp Arg Glu Leu Glu Gly Glu Lys Arg Asp Ala Phe Arg 115 120 125Phe His His Ile Ser Thr Asp Glu Val Phe Gly Asp Leu Pro Phe Asp 130 135 140Ser Gly Ile Phe Thr Glu Glu Thr Pro Tyr Asp Pro Ser Ser Pro Tyr145 150 155 160Ser Ala Ser Lys Ala Ala Ser Asp His Leu Val Arg Ala Trp Gly His 165 170 175Thr Tyr Gly Leu Pro Val Val Leu Ser Asn Cys Ser Asn Asn Tyr Gly 180 185 190Pro Phe His Phe Pro Glu Lys Leu Ile Pro Leu Thr Ile Leu Asn Ala 195 200 205Leu Glu Gly Lys Pro Leu Pro Val Tyr Gly Lys Gly Glu Asn Ile Arg 210 215 220Asp Trp Leu Tyr Val Asp Asp His Ala Lys Ala Leu Ala Thr Ile Ala225 230 235 240Thr Thr Gly Lys Val Gly Gln Ser Tyr Asn Val Gly Gly Arg Asn Glu 245 250 255Arg Thr Asn Leu Gln Val Val Glu Thr Ile Cys Asp Leu Leu Asp Gln 260 265 270Arg Ile Pro Leu Ala Asp Gly Arg Lys Arg Arg Glu Leu Ile Thr Phe 275 280 285Val Thr Asp Arg Pro Gly His Asp Arg Arg Tyr Ala Ile Asp Ala Thr 290 295 300Lys Leu Glu Thr Glu Leu Gly Trp Lys Ala Glu Glu Asn Phe Asp Thr305 310 315 320Gly Ile Ala Ala Thr Ile Asp Trp Tyr Leu Ala Asn Glu Trp Trp Trp 325 330 335Gly Pro Ile Arg Ser Gly Lys Tyr Ala Gly Glu Arg Leu Gly Gln Thr 340 345 350Ala42867DNASphingomonas sp. ATCC53159 42atgcgtatcc tcgtcaccgg gcatgacggc caggtcgccc agtcgctggc cgagcaggcg 60gtgggccacg agctggtctt caccacctac cccgaattcg atctctccaa gccggagacg 120atcgaggccg gtgtggcgcg ggtgcacccg gacctgatcg tctccgccgc cgcctacacg 180gcggtcgaca aggcggaaag cgaacccgag ctggcgatgg cgatcaacgg cgacggtccc 240ggcgtgctgg cgcgcgcggg cgcgaagatc ggcgcgccga tcatccacct gtcgaccgat 300tatgtgttcg acggcagtct cgaccgccct tggcgcgagg acgatcccac cggcccgctc 360ggcgtctatg gcgcgaccaa gctggccggc gagcaggcgg tgcaggcctc gggtgccacc 420aacgccgtga tccggctggc ctgggtctac agcccgttcg gcaacaattt cgtcaagacg 480atgctccgcc tcgccgagac gcgcgacgcg ctgaacgtcg tggaggacca gtggggctgc 540cccagttcgg cgctggacat cgcgaccgcg atcctgacgg tggtcgggca ctggcagcag 600gacggcgcga cgagcggcct ctaccatttc gccggcaccg gcgagaccaa ctgggccgac 660ttcgcatcga cgatcttcgc cgagagcgcc aagcgcggtg gcccctcggc caccgtcacc 720ggcattccca gctcgggcta tccgactccg gccacgcgcc cggccaattc gcggctggac 780tgcacccgct tcgcggagac cttcggctac cgggcgcctg cctggcagga ttcgctgaac 840gtcgtactgg atcgcctgct cggctga 86743288PRTSphingomonas sp. ATCC53159 43Met Arg Ile Leu Val Thr Gly His Asp Gly Gln Val Ala Gln Ser Leu1 5 10 15Ala Glu Gln Ala Val Gly His Glu Leu Val Phe Thr Thr Tyr Pro Glu 20 25 30Phe Asp Leu Ser Lys Pro Glu Thr Ile Glu Ala Gly Val Ala Arg Val 35 40 45His Pro Asp Leu Ile Val Ser Ala Ala Ala Tyr Thr Ala Val Asp Lys 50 55 60Ala Glu Ser Glu Pro Glu Leu Ala Met Ala Ile Asn Gly Asp Gly Pro65 70 75 80Gly Val Leu Ala Arg Ala Gly Ala Lys Ile Gly Ala Pro Ile Ile His 85 90 95Leu Ser Thr Asp Tyr Val Phe Asp Gly Ser Leu Asp Arg Pro Trp Arg 100 105 110Glu Asp Asp Pro Thr Gly Pro Leu Gly Val Tyr Gly Ala Thr Lys Leu 115 120 125Ala Gly Glu Gln Ala Val Gln Ala Ser Gly Ala Thr Asn Ala Val Ile 130 135 140Arg Leu Ala Trp Val Tyr Ser Pro Phe Gly Asn Asn Phe Val Lys Thr145 150 155 160Met Leu Arg Leu Ala Glu Thr Arg Asp Ala Leu Asn Val Val Glu Asp 165 170 175Gln Trp Gly Cys Pro Ser Ser Ala Leu Asp Ile Ala Thr Ala Ile Leu 180 185 190Thr Val Val Gly His Trp Gln Gln Asp Gly Ala Thr Ser Gly Leu Tyr 195 200 205His Phe Ala Gly Thr Gly Glu Thr Asn Trp Ala Asp Phe Ala Ser Thr 210 215 220Ile Phe Ala Glu Ser Ala Lys Arg Gly Gly Pro Ser Ala Thr Val Thr225 230 235 240Gly Ile Pro Ser Ser Gly Tyr Pro Thr Pro Ala Thr Arg Pro Ala Asn 245 250 255Ser Arg Leu Asp Cys Thr Arg Phe Ala Glu Thr Phe Gly Tyr Arg Ala 260 265 270Pro Ala Trp Gln Asp Ser Leu Asn Val Val Leu Asp Arg Leu Leu Gly 275 280 28544132DNASphingomonas sp. ATCC53159 44atccggctgt gcctggggtg ctggcggtcg cccaaggaaa tcgccggctg gagcgagctg 60agtcctaagg gaaagcgcgc ggtgctagag gcattgccgg cgcgcgaacg ggagcatggc 120ggggggcgct ga 1324543PRTSphingomonas sp. ATCC53159 45Ile Arg Leu Cys Leu Gly Cys Trp Arg Ser Pro Lys Glu Ile Ala Gly1 5 10 15Trp Ser Glu Leu Ser Pro Lys Gly Lys Arg Ala Val Leu Glu Ala Leu 20 25 30Pro Ala Arg Glu Arg Glu His Gly Gly Gly Arg 35 4046336DNASphingomonas sp. ATCC53159 46gctgcaggtc gacggatcgc cagcggcctc accatcaact atgcaccgga tgcatcggtg 60ggccacccgc cccggcccga ctggtcggcc ctgctggtga agacgcggcg catccagcgc 120gaactctatc tgttcaacat cgagcggccg aagggcaggc tgcgctggct ggtccgttcc 180gtggcgcaac cggcgatgat cccacaggac gtggccaaga tcctgcgcac accgggtacc 240aagggcgcgc gcctcgctgc ggtcaccacg ctggtccggc tgcggctgtg gcgcggcggc 300gccggcttgt tgcagttgct cggccgcgac atctga 33647111PRTSphingomonas sp. ATCC53159 47Ala Ala Gly Arg Arg Ile Ala Ser Gly Leu Thr Ile Asn Tyr Ala Pro1 5 10 15Asp Ala Ser Val Gly His Pro Pro Arg Pro Asp Trp Ser Ala Leu Leu 20 25 30Val Lys Thr Arg Arg Ile Gln Arg Glu Leu Tyr Leu Phe Asn Ile Glu 35 40 45Arg Pro Lys Gly Arg Leu Arg Trp Leu Val Arg Ser Val Ala Gln Pro 50 55 60Ala Met Ile Pro Gln Asp Val Ala Lys Ile Leu Arg Thr Pro Gly Thr65 70 75 80Lys Gly Ala Arg Leu Ala Ala Val Thr Thr Leu Val Arg Leu Arg Leu 85 90 95Trp Arg Gly Gly Ala Gly Leu Leu Gln Leu Leu Gly Arg Asp Ile 100 105 11048837DNASphingomonas sp. ATCC53159 48gtgacgacct cggacgagga gcagtttccg ccgctcgtcc gcctgatgat gatgcagttt 60cagtcgggca tcttctgcaa cttcctcgac cggctgagcg gccacaagca cctgctgccg 120gatcccaacc attatggctg cggcctgcac tcgaccggct cgggcgggcg gctgatgctc 180cacatcgatg cctcgcgcca ccccaacaag aagctcagcc agcagatcaa ctgcatctat 240tactgcacgc cagactggca ggaggaatgg ggcggcgacc tggagctgtg ggacgaggat 300gcgaccaggt gcgtttccag catcacgccc aagttcaatc gcctcgcgat cttccgcgtc 360tcgggcaagt cgtggcacgg ccagcccttc ccgctgcaga cgccgccgaa catccgccgc 420aactcgctcg cactctacta ctacagcgca gaggaggata ccgagggtcg cggctattcg 480aacttcgtgc gttggaaggg ccgtctcggc gcacgacaag cgcaccgcgc tgcaccgggt 540gaagggcctg atccgcgact atgcgccgac cccggtgatc aacggcctcg ccaagttcgc 600ccgcaagacg gggctgaact tcaagcgctg atggggctgt tctcgcgctt tgccgccgca 660ccgccggaat cgccctgtcg caaggtctgc cgcctcgaca tggagatccg gctgtgcctg 720gggtgctggc ggtcgcccaa ggaaatcgcc ggctggagcg agctgagtcc taagggaaag 780cgcgcggtgc tagaggcatt gccggcgcgc gaacgggagc atggcggggg gcgctga 83749278PRTSphingomonas sp. ATCC53159 49Val Thr Thr Ser Asp Glu Glu Gln Phe Pro Pro Leu Val Arg Leu Met1 5 10 15Met Met Gln Phe Gln Ser Gly Ile Phe Cys Asn Phe Leu Asp Arg Leu 20 25 30Ser Gly His Lys His Leu Leu Pro Asp Pro Asn His Tyr Gly Cys Gly 35 40 45Leu His Ser Thr Gly Ser Gly Gly Arg Leu Met Leu His Ile Asp Ala 50 55 60Ser Arg His Pro Asn Lys Lys Leu Ser Gln Gln Ile Asn Cys Ile Tyr65 70 75 80Tyr Cys Thr Pro Asp Trp Gln Glu Glu Trp Gly Gly Asp Leu Glu Leu 85 90 95Trp Asp Glu Asp Ala Thr Arg Cys Val Ser Ser Ile Thr Pro Lys Phe 100 105 110Asn Arg Leu Ala Ile Phe Arg Val Ser Gly Lys Ser Trp His Gly Gln 115 120 125Pro Phe Pro Leu Gln Thr Pro Pro Asn Ile Arg Arg Asn Ser Leu Ala 130 135 140Leu Tyr Tyr Tyr Ser Ala Glu Glu Asp Thr Glu Gly Arg Gly Tyr Ser145 150 155 160Asn Phe Val Arg Trp Lys Gly Arg Leu Gly Ala Arg Gln Ala His Arg 165 170 175Ala Ala Pro Gly Glu Gly Pro Asp Pro Arg Leu Cys Ala Asp Pro Gly 180 185 190Asp Gln Arg Pro Arg Gln Val Arg Pro Gln Asp Gly Ala Glu Leu Gln 195 200 205Ala Leu Met Gly Leu Phe Ser Arg Phe Ala Ala Ala Pro Pro Glu Ser 210 215 220Pro Cys Arg Lys Val Cys Arg Leu Asp Met Glu Ile Arg Leu Cys Leu225 230 235 240Gly Cys Trp Arg Ser Pro Lys Glu Ile Ala Gly Trp Ser Glu Leu Ser 245 250 255Pro Lys Gly Lys Arg Ala Val Leu Glu Ala Leu Pro Ala Arg Glu Arg 260 265 270Glu His Gly Gly Gly Arg 27550816DNASphingomonas sp. ATCC53159 50atgctccacg tgatcctgac tcgcttcaac atcgccagcc cgggacgcga ggtggcgatt 60cgcaactcgc cgggctggct ggaacgccgt

ttcggcctgt tcgagcagtt ctgcctgccg 120agcatcgcgg gccagaccga gcgcaacttc cactggctga tctatttcga caaggatacg 180ccggttgaat tccgcgagcg gatcgagcgc gatcgccaga tcttcaattt taccccacgc 240tatgtggcga tgttcgacaa ggcgatgatc gccgaggacg tgcgggcact cgcgacggcg 300ggcgagacgc tgatcgtcac cacgcggctg gacaatgatg atgcggtgtc gagcgatttc 360gtcgcgcggg tgcaggacgc cgccaaggaa gcgccggcgc agaccgtgct gaacttcccc 420cacggcatcg cgatgcgggg cgggcaactc tacaccgcca gcgatcacag cagcccgttc 480acctcgctgg tcgagaaaga cgtggccggg atcgagacga tctgggccaa gccgcaccac 540gagctgggcg agaagtggac gatccgccag gtgccgagca agccgctatg gctgcaggtg 600gtgcacggcg agaatgtaac caaccggatc aagggcaagc tggtttcgga catcgacatc 660atcaatatgt tcaagatccg cagcgatgtc gccgcacggc cggtggcggc cggcgcgatt 720ctgtgggacc atgcggtgcg cacgccgatc cggcgcttcc gcgaattcgg tatccgcctg 780gtcaagccga tcgtggttcg gataagggat cgctaa 81651271PRTSphingomonas sp. ATCC53159 51Met Leu His Val Ile Leu Thr Arg Phe Asn Ile Ala Ser Pro Gly Arg1 5 10 15Glu Val Ala Ile Arg Asn Ser Pro Gly Trp Leu Glu Arg Arg Phe Gly 20 25 30Leu Phe Glu Gln Phe Cys Leu Pro Ser Ile Ala Gly Gln Thr Glu Arg 35 40 45Asn Phe His Trp Leu Ile Tyr Phe Asp Lys Asp Thr Pro Val Glu Phe 50 55 60Arg Glu Arg Ile Glu Arg Asp Arg Gln Ile Phe Asn Phe Thr Pro Arg65 70 75 80Tyr Val Ala Met Phe Asp Lys Ala Met Ile Ala Glu Asp Val Arg Ala 85 90 95Leu Ala Thr Ala Gly Glu Thr Leu Ile Val Thr Thr Arg Leu Asp Asn 100 105 110Asp Asp Ala Val Ser Ser Asp Phe Val Ala Arg Val Gln Asp Ala Ala 115 120 125Lys Glu Ala Pro Ala Gln Thr Val Leu Asn Phe Pro His Gly Ile Ala 130 135 140Met Arg Gly Gly Gln Leu Tyr Thr Ala Ser Asp His Ser Ser Pro Phe145 150 155 160Thr Ser Leu Val Glu Lys Asp Val Ala Gly Ile Glu Thr Ile Trp Ala 165 170 175Lys Pro His His Glu Leu Gly Glu Lys Trp Thr Ile Arg Gln Val Pro 180 185 190Ser Lys Pro Leu Trp Leu Gln Val Val His Gly Glu Asn Val Thr Asn 195 200 205Arg Ile Lys Gly Lys Leu Val Ser Asp Ile Asp Ile Ile Asn Met Phe 210 215 220Lys Ile Arg Ser Asp Val Ala Ala Arg Pro Val Ala Ala Gly Ala Ile225 230 235 240Leu Trp Asp His Ala Val Arg Thr Pro Ile Arg Arg Phe Arg Glu Phe 245 250 255Gly Ile Arg Leu Val Lys Pro Ile Val Val Arg Ile Arg Asp Arg 260 265 27052933DNASphingomonas sp. ATCC53159 52atggcttgcc cctacccgag cccggtgtcg ccccctcgtc ccgacagcat cgccaccggc 60ctggcgcttc gcctgttcgc gatcgcctgc atgtcgacca tgtcggcgct catcaagatg 120tccgaactgc gcggcgcctc gctgatcgag acgatgtttc accgccagct ctgggcggtg 180cccttggtca ccctgtgggt cacgctgggg ccgggcctca agtcgctcag gaccgcgcgg 240ttcggcgcgc atgtctggcg caccgcggtg ggacttaccg gcatgatctt caccttcggc 300gcggtgatcc tgctgccgct cgccgaagcg cagaccttcc agttcaccgt ccccatcttc 360gcgacgctgc tcggcgcgct gatcctaggc gaaccgaccg gctggcaccg ctggagcgcg 420gtgatcctcg ggttcgtcgg cgtgcttatc gtcgtccagc cggggcacga ggcgatcccg 480gtgttcggtg cgttcgtggg cctgatggcg gcgctgttcg tcgccatcgt cgcgatcacg 540ctccgccaga tcgggaagac cgaaagcgcc ggcaccacgg tgttctggtt ctcgctgttg 600tcggtgccgg tgctgggcgc aatctatgcc ttccactaca agccccatga tgccgagacc 660tgggccatcc tgatcgccac gggcctggtc ggcggcgtcg gccagctcgc gctgaccggg 720gcgatgcgct tcgctcccgt gtcggcagtg gtgccgatgg actattcggg gctgctctgg 780gcgacgctct atggctggct gctgttcggc gtgctgccga ccttttccac ctggctcggc 840gcgccggtga tcatcgccag cggcctgtac atcgtctatc gcgagcagaa gctggcgcgc 900ggccaggcta gctacgccga aacgccacta tga 93353310PRTSphingomonas sp. ATCC53159 53Met Ala Cys Pro Tyr Pro Ser Pro Val Ser Pro Pro Arg Pro Asp Ser1 5 10 15Ile Ala Thr Gly Leu Ala Leu Arg Leu Phe Ala Ile Ala Cys Met Ser 20 25 30Thr Met Ser Ala Leu Ile Lys Met Ser Glu Leu Arg Gly Ala Ser Leu 35 40 45Ile Glu Thr Met Phe His Arg Gln Leu Trp Ala Val Pro Leu Val Thr 50 55 60Leu Trp Val Thr Leu Gly Pro Gly Leu Lys Ser Leu Arg Thr Ala Arg65 70 75 80Phe Gly Ala His Val Trp Arg Thr Ala Val Gly Leu Thr Gly Met Ile 85 90 95Phe Thr Phe Gly Ala Val Ile Leu Leu Pro Leu Ala Glu Ala Gln Thr 100 105 110Phe Gln Phe Thr Val Pro Ile Phe Ala Thr Leu Leu Gly Ala Leu Ile 115 120 125Leu Gly Glu Pro Thr Gly Trp His Arg Trp Ser Ala Val Ile Leu Gly 130 135 140Phe Val Gly Val Leu Ile Val Val Gln Pro Gly His Glu Ala Ile Pro145 150 155 160Val Phe Gly Ala Phe Val Gly Leu Met Ala Ala Leu Phe Val Ala Ile 165 170 175Val Ala Ile Thr Leu Arg Gln Ile Gly Lys Thr Glu Ser Ala Gly Thr 180 185 190Thr Val Phe Trp Phe Ser Leu Leu Ser Val Pro Val Leu Gly Ala Ile 195 200 205Tyr Ala Phe His Tyr Lys Pro His Asp Ala Glu Thr Trp Ala Ile Leu 210 215 220Ile Ala Thr Gly Leu Val Gly Gly Val Gly Gln Leu Ala Leu Thr Gly225 230 235 240Ala Met Arg Phe Ala Pro Val Ser Ala Val Val Pro Met Asp Tyr Ser 245 250 255Gly Leu Leu Trp Ala Thr Leu Tyr Gly Trp Leu Leu Phe Gly Val Leu 260 265 270Pro Thr Phe Ser Thr Trp Leu Gly Ala Pro Val Ile Ile Ala Ser Gly 275 280 285Leu Tyr Ile Val Tyr Arg Glu Gln Lys Leu Ala Arg Gly Gln Ala Ser 290 295 300Tyr Ala Glu Thr Pro Leu305 3105420779DNASphingomonas sp. ATCC53159 54gctgcaggtc gacggatcgc cagcggcctc accatcaact atgcaccgga tgcatcggtg 60ggccacccgc cccggcccga ctggtcggcc ctgctggtga agacgcggcg catccagcgc 120gaactctatc tgttcaacat cgagcggccg aagggcaggc tgcgctggct ggtccgttcc 180gtggcgcaac cggcgatgat cccacaggac gtggccaaga tcctgcgcac accgggtacc 240aagggcgcgc gcctcgctgc ggtcaccacg ctggtccggc tgcggctgtg gcgcggcggc 300gccggcttgt tgcagttgct cggccgcgac atctgatcga ccggcgatcg gccgacgagc 360gcgtcgccgg ccgatcgcat tgcatcagac ggtggccagc gcgtcttcca gcgtgccgct 420gtcgagccgc aggcggccga tcatcagcca cagatagacc ggcagcgtat cgtcggtgaa 480gcggaagcgg caatcgccgt cctgcgtttc ggattcgagg ccgagttgac cggtgagctc 540gcccagctcc tgctcgacct gcgccgccgt gatgtgcgcg cccggcagca gatccaccac 600ggcttggccg ctgaaccagc catccgccga gcgcgaggcc tcgcccagcg ccgcgacgag 660tggatcgtag cggccgccga cgaacttgcg catctcgatc accgcgcgcg gcggcatgcg 720gccctcgatc tcaaggatcg cctggtcgag cgcacgacgc agatgcccgg cgtcgaccgt 780gaggcggccc tggtccaggg cttccagcgc ggaatggtgg cacagcagcc gcgcgaaata 840gggcgacccc agcgcgagca ggtggatcat gtgagtcagg tccggatcga agcgaacgcc 900cgaggcggtt tcgccgagcg cgatcatctc ctgcacctcc gattcctcca gccggggcat 960cggcaggccg atgacgttgc ggcggatcga cggcgcataa ccgatcagct cctgcaggtt 1020cgaggcgacg cccgcgatca ccagctggac gcgcgccgaa cggtccgaca ggttcttgat 1080cagctcggcg acctgctgac ggaaggcgga atcgctgacg cgatcatatt cgtcgaggat 1140gatcagcacg cgtgtgcccg tgatgtcggc gcacaggtcg gccagttcgc cgggcccgaa 1200gctgcccgtc ggcaggcggt cggccaagtt gccgccgctc tccgcctcgc cggcgttggg 1260cgccacgccg cgatggaaca gcagcggcac gtcttccagc acggcgcgga agacatcgct 1320gaaattcgcg ttcgcaccgc aggtcgcata gctgacgata tagctggatt cgcgggcgac 1380atcggtcagc acgtggagca gcgaggtctt gccgatgccg cgctcgccat agagcacgac 1440atggctgcgc tggctctcga tcgaggagat taggcgcgcc agcacgccga ggcgcccggc 1500gaagctcgac cgatcggcca ccggctgggt gggtgtgaag aaggtcgcca gcgcgaaccg 1560ggcgcgcgtg atctcgcggc gctcgtcgcg gcggcgatcc agcgggcggt ccagcgcgga 1620ggcacggaag gttgggaaat ccgggcgacc acggccgcta tgggcatcgc gatgcggcac 1680cactgtcgca gtcagcggga aatagccctc ttcttcaggt tcttctcgac ggccgaacgg 1740ccacaagaat ctcagcgcgg aacctacagc cactcgaaca cctcttaaat tcgtgcgcca 1800tcggcaccga cggcgcaccc tggttcgcgc cccctggcgc cccctcctaa cgaacccacg 1860ccttgcctgg cctatcggcg cttgaagaac tcgtacggtt tgatcaccaa ggcgatgtac 1920gccaggacca gagcgatcgt caaaattgca aagacgtgat aattctcatt gcccagataa 1980ttggcgacgg cgcaaccgac tgcgggcggc aaatagctga tcatcgtgtc ccggactgcc 2040gaatcggctt gggaccgttg caggaatata acgatcaggc cggcaaatat cgcgatggtg 2100acccaatcat agggcgtctg catgcatgtc ctttctattc gacaccggaa tcgaaccatt 2160tccggcgacg ctattgcacg cactagcagt gcgcgcggcc gctcgctagg tagcgccgca 2220ccggataaac cgacgttaag atggcgcggc tcgatcgaaa tggagtcaaa cgggcttgcc 2280cggccgaccg aagcatggcg ccatggcgca tgcaccgtat tgtgaccacg caaaccgcga 2340gggtcattcg atgcggttgc ttgtacagga ggccattgat aatgaagccg agacccgggg 2400gaacctttat gcaagtaaat ttcaatcgac aggctcgcaa gctcggtgcc ggcaatgcgc 2460tcgcgcgggg ggggcccgtg cttgcgctgc ttgcgaccgc ggcatggaca caacctgcgc 2520tggcgcagcg acaggcattt gagtcccgcc cctccggtag cgagcgacag gtcgatattc 2580gcgcgacggg gtcgctggaa tatgacgaca acgtcgtgct gaacgaccag cggatcacgg 2640acggcgcgcg tggcgatgtg atcgcatcgc ccgggctgga cgtgacccta gttctgcccc 2700gcgccaccgg gcagctctac ctcaccggca atgtcggata tcgcttttac aagcgatata 2760ccaactttaa ccgcgagcag atctcgctca ccggcggcgc agatcagcgg ttcgcctcct 2820gcgtcgtgca cggggaagtc ggctatcagc gccacctcac cgacctgtcc agcatcttga 2880tccaggacac cacgcctgcg ctcaacaaca ccgaagaggc ccggcagtac accgcggata 2940tcggctgcgg cgcgacctac ggcctgcggc ctgccgtttc ctacacccgc aacgaagtgc 3000gcaacagcct tgccgagcgc cgatacgcgg actcgaatac caacaccttt accgcacagc 3060ttggcctgac ttcgcctgcc ctggggaccg tggcggtatt tgggcgtatg tccgacagca 3120gctatgtcca tcgcgtcctt cccggcatta ccggccagga cgggatgaag agctacgcgg 3180ccggcgtcca gctcgagcgc tcggtggcca accgactcca tttcaacggc tcggtgaatt 3240acaccgaggt tgacccaaag ctcgcatcca ccaaaggatt caagggcgta ggatttaacg 3300tttccggcga ttatgctggt gatcagtaca gcctccaatt gctggcttca cgatcgcccc 3360agccttcact tcttctgttc gtgggttacg agattgtgac agcggtttcg gcgaatgcga 3420cgcgccggct gagcgatcgc attcagatat cgctgcaagg cagccgaacc tggcgcgagc 3480tcgcgtcttc gcggctgctc accaacgtgc cgatttccgg caacgacaac acctcgacgt 3540tgttcgcctc cgctaccttc cggccgaatc gccggctgag ctttgtgctg ggtgccggcc 3600ttcagcggcg caccagcaac acgcagctat acagttacag ctccaaacgc atcaatctct 3660cgacgtcgct ttcgctctga caagggccgt aatcatgcat atcaagaatc gcttcgtgaa 3720tatctcgacg ttggccatcg ccgccgcgct ggccacgccg gcggcggcgc agatccccac 3780gcggtccgtg cccgcgccgg cccgcccgcg gcctgcaacg ccgccggcgc aacagcagaa 3840ccaggcgccg tcgacgcccg cagcggcaac cccggcgcag accgccgcaa ccgttgcccc 3900tgcagcaacc gcacccgcag gttacaaaat cggcgtggac gacgtgatcg aggccgacgt 3960gctcggccag accgacttca agacgcgcgc ccgtgtgcag gcggacggca cggtgaccct 4020gccctatctg ggcgccgtgc aggtcaaggg cgagaccgcg acctcgctcg ccgaaaagct 4080ggccgggctg ctgcgcgccg gcggctatta tgccaagccg atcgtcagcg tcgaaatcgt 4140cggtttcgtc agcaactatg tgacggtgct gggccaggtg aacagttccg gcctgcagcc 4200ggtcgaccgc ggctatcacg tttccgagat catcgcccgt gccggcggcc tgcgccccga 4260agcggccgat ttcgtcgttc tcacccgcgc cgatggctcc agcgccaagc tggactacaa 4320gaagctcgcc caaggtggcc ccaatgacga tccgatggtg acgcccgggg acaaggtctt 4380tgtcccggaa gtcgagcatt tctacattta tggtcaaatt aacgcgcctg gcgtatacgc 4440gattcgatcg gacatgacgc tccgtcgcgc gctggcccag ggcggtgggc ttgcccccgc 4500aggctccgtc aagcgtgtga aggtcacgcg ggatggcaat gaactcaagt tgaagctgga 4560cgatccgatt ctcccaggcg acacgatcgt catcggcgaa cgattgttct gatcttggca 4620acgatggcag cggacgaggc ccaccagtga atatcattca gttcttccgc attctgtggg 4680tgcgccgatg gatcatcctc ccggcgtttc tcgtttgcgt taccactgcc accattgtgg 4740tccagtttct gcccgaacgc tacaaggcca ctacgcgggt ggtgctcgac acgtttaagc 4800ccgatcccgt caccggacag gtgatgagct cgcagttcat gcgcgcctat gtcgagactc 4860agacccagct gatcgaggac tatgcgaccg ccggtcgcgt ggtcgacgaa ctgggctggg 4920tgaatgatcc ggcgaacatc tccgcgttca acaactcgtc cgcggctgcc accggcgaca 4980tccgccgctg gctcgccaag cagatcatcg acaataccaa ggccgatgtg atggagggga 5040gcaacatcct cgaaatcacc tattcggaca gctcgcccga gcgcgccgaa cgcatcgcca 5100acctgatccg cacctcgttc ctcgcccagt cgctcgccgc caagcgccag gccgcgacca 5160agtcggccga ctggtacgcc cagcaggccg aagctgcccg cgattcgctc gctgcggcgg 5220tccaggcccg caccgatttc gtgaagaaga ccggcatcgt gctgaccgaa accggcgccg 5280acctggaaac ccagaagctc cagcagatcg aggggcagac gacgaccgcc accgccccgg 5340ttgccatggc ccccagcggc atgggcccgg cgcagatgca gctcgcccag atcgaccagc 5400agatccagca ggcagcgacc agcctaggtc cgaaccaccc aactttccag gccttgcagc 5460ggcagcgcga agtgttcgcc aaggcagcgg cggcggaacg cgcgcaggcg aacggcgtat 5520ccggtccggc acgcggggcc atcgaaagcg cagccaacgc ccagcgcgcg cgggttctcg 5580gcaatcgtca ggatgtcgac aagcttacgc agctgcagcg tgacgtctcg ctgaagcagg 5640atcagtacat gaaggcggca cagcgcgtcg ccgatctgcg gctggaagca agcagcaacg 5700atgtcggcat gtcgacgctc agcgaagcat cggcgccgga aacgccctat taccccaagg 5760tgccgctcat catcggtggt gcagccggct tcggcctcgg gctcggtctg ctggtcgcgc 5820tgctcgtcga gctgctcggc cgccgcgtcc gcagccccga ggatctggaa gttgcgatcg 5880atgcaccggt gctgggcgtg atccagagcc gcgcctcgct tgccgcccgc cttcgccgcg 5940cccaagaaac cctcggcgaa ggtgccgaca cgcacggagc ttcagtaaac tgatggacgc 6000gatgaccagc gaaccgctgc ccgaaggcga tcgtccgagc gccgtgccga ccacgccgga 6060tacgatcggc atgctcgaat accagctcgt cctctccgat ccgaccggga tcgaggcgga 6120agcgatccgc gcgctacgca cgcgcatcat gacccagcac ctccgcgagg gccggcgcgc 6180gctcgcgatc tgcgccgcct cggcgggatc cggctgcagc ttcaccgccg tcaatctggc 6240gacggcgctg gcgcagatcg gcgttaagac tgcgctggtc gatgccaatc tgcgcgatcc 6300cagcatcggc gcagccttcg gcctcgccgc cgacaagccc ggcctggccg attatctcgc 6360ctcgggcgat gtcgacctcg cctcgatcat ccatgcgacc cgcctcgacc agctctcgat 6420catcccggcc gggcatgtcg agcacagccc gcaggaactg ctcgcgtccg aacagttcca 6480tgatctggcg acgcagctgc tgcgcgagtt cgacatcacg atcttcgaca ccacggcgtc 6540caacacctgc gccgacgcgc agcgtgtcgc gcatatcgcc ggctatgcga tcatcgtggc 6600gcgcaaggat gcgagctaca tccgcgacgt gaacacgctc agccgcacgc tgcgtgcaga 6660ccgcaccaac gtcatcggct gcgtactgaa cggctattga tttggaccat atggcagcga 6720ccgcgatgac gcggcagcag gagaggaagg gcggtggcta ttggctggcc gttgccggtc 6780ttgccgcgct aaccatcccg accttcatca ccctgggtcg cgaggtttgg agtgcggaag 6840gcggcgtgca gggtccgatc gtgctcgcca cgggcgcctg gatgctggcc cgccagtgct 6900cgacgatcga ggcgctacgc cgccccggca gcgtgctgct cggcgcgctg ttcctgctgg 6960cgacgcttgc cttctacacc gttggacggg tgttcgactt catcagtgtc gaaaccttcg 7020gactggtcgc gacctatctg gtcgtcgcct atctctattt cggtgccagg gtgctccgtg 7080ccgcctggtt cccggtgctg tggctgttct tcctggtgcc gccgcccggc tgggccgtcg 7140accgcatcac cgcaccgctc aaggagttcg tctcctatgc ggcaacgggc ctgctttcct 7200gggtggatta tccgatcctg cgccagggcg tgacactgtt cgtcggcccc tatcagctgc 7260tcgtcgaaga tgcctgttcg ggtctgcgct cgctgtccag cctggtcgtc gtgacgctgc 7320tctacatcta catcaagaac aagccgtcct ggcgctacgc ggcgttcatc gcagcgctgg 7380tgatcccggt ggcagtggtg accaacgtcc tgcggatcat catcctggta ctgatcacct 7440atcatctggg cgacgaggcg gcgcagagct tcctccacgt ctccaccggc atggtgatgt 7500tcgtggtcgc cctgctttgc atcttcgcga tcgactgggt ggtcgagcaa cttcttctcc 7560tgcgtcggag gcatcatgtt caaccggcgt gacctgctga tcggcgcagg ctgcttcgcc 7620gccgctggcg cctcgctcgg cctgaagccg caccggcgga tggacctgct gggcggcacc 7680aagctcgaca cgctgatgcc caaggcattc ggcgcatgga aggcagagga taccggttcg 7740ctgatcgcgc cggcgcgcga aggcagcctg gaggacaagc tctacaacca ggtggtcacc 7800cgcgccttct cccgcgcgga cggtgcccaa gtgatgctgc tgatcgccta tggcaacgcc 7860cagaccgatc tactgcagct gcaccggccg gaaatatgct acccgttctt cggcttcacc 7920gtggtggaaa gccatgagca gaccatcccg gtgacgccgc aggtgacgat ccccggtcgc 7980gcgctgaccg ccaccaactt caaccgcacc gagcagatcc tctactggac ccgcgtcggc 8040gaatatctgc cgcagaacgg caatcagcag atgctcgcgc ggctgaagag ccaggtccag 8100ggctggatcg tcgacggtgt gctggtgcgc atctcgacgg tgacgcccga ggcggaagat 8160ggcctgagcg ccaatctcga tttcgcgcgc gagctggtga agacgctcga cccgcgcgtg 8220ctgcgcccgc tgctcgggaa cgggctcaca cggcagctcg gtcaccaggt ctgaaccggt 8280gcgccgcacg cggcgccccc ggcaacaaaa aaggagcggc gcgggccgcc gccgctccct 8340ctccttctca tgcggcgccc tgccctcacc gctcgtgcag cgcgtcactc cccgtctcga 8400gcacgggccc caccagatag ctgaacaggg ttcgcttgcc ggtgacgatg tccgcgctcg 8460cgagcatccc cggccgcagc ggcacctgtg cgccatgggc cagcacatac ccgcgcgcca 8520gcgcgatccg cgccttgtag accggcggct ggttctcctt catctgcacc gcctcggggc 8580tgatgcccgc caccgtgccg ggaatcatgc cgtagcgggt atagggaaag gcctgcagct 8640tcacctttac cggcatgccg atgtggacga agccgatgtc gctgttgtcg accatcacct 8700cggcctcgag ccgggcattg tcgggaacca ggctgaggag cggcttggcc ccttccacca 8760cgccgccttc ggtgtggacc tgcagctgcg agacggtacc gctcaccggc gcgcgcagtt 8820cgcggaacga gctgcgcaga ttcgccttgg cgacgtcctc gccgcgggca cgcacctcgt 8880cctgcgcctt gaccagatcc tgcagcacct gcgcccgcgc ctcctcgcgc gtcttggccg 8940acaggctgga gacgctcagc gactgctggc cgagtttggc gagcgtagcg cgcgccgccg 9000tcaggtcctg ccgctcggcg atcagctggc gacgcatctc cacgacgcgc agcttcgaga 9060catagccctt ggcggccatc gtctcgttcg cggcgatctg ctgttcgagc agcggcagcg 9120actgttcgag cttccgcacc tgtgcctgcg cctcggccgc ggccgagacg gcggcaccgc 9180gatcggagcg gccgccggcc agcgccgcct cgatctggcc cagccgggcg cgggcgaggc 9240cgcgatgcgt cgccacttcg cccgggctgg cggcggcagg cgcgacgaag cggaagcccc 9300tgccgtccag cgcgtcgatg atcgcctggt tgcgtgcggc gtcgagctgg gcgctgagca 9360gcgccacctt cgcctgtgcc gcctccgccg acgacacggt cgggtcgagc gtgatcagca 9420cctggccctt ggcgaccttc tgcccctcgc ccaccaggat gcggcggacg atccccgatt 9480cgggcgactg gacgatcttg gtctcgccga tcggcgcgat ccgcccctgc gtcggcgcga 9540cgacttcgac cttgccgatc gccagccagg cggcggtgat cgccagcccg gccagcatca 9600ccttggcggt aagccgcgcg gtgggcgaaa ccggccgctc gatgatctcc agcgcggcag 9660gcaggaaggc ggtgtcataa

gcgtcgacgc gggcaggcag cacggtatcg cgcatgcggg 9720cgagcgggcc gccgcggcgc atcggaacaa cggcgttcat gcggcaatct ccccatagcc 9780gccctggcgg cggtgcaggt cggcatagcg gccgcccagg cgcaacaatt cgtcgtgtcg 9840gccgctctcg acgatgcggc cctgttcgag cgtgatgatc cggtcgcagc tgcgcaccgc 9900gctcaggcga tgcgcgatca ccacgagcgt gcggccggcc gagatggcgc gcaggttgtt 9960ctggatcagc tcctcgctct cggcatcgag cgccgaggtc gcttcgtcga acaccaggat 10020gcgcggattg ccgacgagcg cgcgggcgat ggcgagccgc tggcgctggc cgccggagag 10080attgacgccg cgctcgacga tctcggtgtc atagccgcgc ggctggcgca ggatgaaatc 10140atgcgcgccg gccagcgtcg ccgccgcgac gacattctcg aacggcatgg cggggttgga 10200gagcgcgatg ttctcgcgga tcgagcggct gaacagcaga ttctcctgca gcacgacgcc 10260gatctggcga cgcagccagg cgggatcgag ctgcgccacg tcgacctcgt cgaccagcac 10320gcggccgaga ttcggcaggt tgagccgctg gagcagcttg gccagcgtcg acttgcccga 10380gcccgacgaa ccgacgatgc cgagcgaggt gcccgccgga atgtcgagcg tgatgtcgct 10440cagcaccggc ggctggtcct cggcatagcg gaagctgaca ttctcgaagc gaatcgcacc 10500gcgcagcacc ggcagcgtcg ccgccgaggc cgggcgcggt tccaccggat ggttgagcac 10560gtcgcccagc cgctcgaccg agatgcgcac ctgctggaaa tcctgccaca gctgcgccat 10620gcggatcacc ggcccggaca cgcgctgggc gaacatgttg aacgccacca gcgcgcctac 10680gctcatcgcg ccgccgatca ccgccttggc gccgaagaac aggatcgccg cgaagctcag 10740cttcgagatc agctcgatcg cctggctgcc ggtgttggcg gtattgatca gccgctgcga 10800cgcggcggta tgggcggcga gctggcgctc ccagcgattc tgccagtgcg gctcgaccgc 10860ggtcgccttg atcgtgtgga tgcccgagac gctctcgacg agcagcgcgt tgctggcgga 10920gctcttctcg aacttgtcct ccacccgcgc gcggagcggc ccggcgacgc tgaacgatac 10980gatcgcatag gcgatcagcg acacgagcac gatgcccgag agcatcggcg agtagaacag 11040catcgcggcg aggaacacga aggtgaacag cgggtccacc atcaccgtca gcgaggcgct 11100ggtaaggaat tcgcggatcg tctcgagctg gcggacgcgg gtgacggtgt cgcccacgcg 11160gcgcttctcg aaataggcga gcggcagcgc cagcaggtgg tggaacagcc gggcacccag 11220ctcgacgtcg atcttctgcg tcgtctcggt gaacaggcgg gtgcggatcc agccgagcgc 11280cacttcccac accgaaaccg ccaggaaggc gaaggcgagc acgctcagcg tgctcatgct 11340gttgtggatc agcaccttgt cgatcacgct ctggaacaac agcggcgcgg cgaggccgag 11400caggttgagc gcgagggtga tgccgagcac ctcgaggaac agcgtgcgat agcgccggaa 11460ctgcgcggtg aaccaggaga ggccgaaccg cagcggccgt cccgccaccg cgcgggtggt 11520gagcagcacc agcgcgccgg accagatcgc gtccagcgcg tcccggtcga cctgttccgg 11580ggcatggccc gggcgctgga tgatcacgcc atgttcggtc aggccgccga tcacgaacca 11640gccttcgggc ccgtcggcga tcgcgggcag cggctggcgg gcgagtccgc cgcgcggcac 11700ctcgacggcc ttggcgcgca cgccctgctg gcgcttggcc aggaggatca ggtcgtcggc 11760gcttgccgcc tcggcatggc ccagcgcgtg gcgcagctgt tcgggcgtga tggcgatgtt 11820gtgcgcgccg agcagcagcg acaacgccac cagtccggat tcgcgcagct ccgcctcgcg 11880ctccgccgcc ccatgggccg cgagcgcgct ctgcagggtg gcctgcattt cgtcgcgtgt 11940catttccgga actctgcctc catggcgata ctgagagcgc catgatgaag aaggctggta 12000aagactcact taatcctagc ttttctggta tttacccgta gctgccgacc cgatttggga 12060caggcctggc ttagcaggtc cttaaactcg accgactata ccgcgacgcc gaggaggggg 12120aggattggcg ccgcatcgcg cggcgaaacg cgggtgcgtc gcaacatttc gccggagtcg 12180atccgtcgcg aatgctgcac ccgcgaacgc aatgacggcc gccacgcaat ccggcttgat 12240cccgggcggc ggatcgcgat aagccgcgcc acggtcgcca aaactcgtcg aaataaccga 12300caaaaccacg gcatatggct ggatattgca gcgtttgccc tgcgtttccg tcgttcaacc 12360gcccttcgaa tcaggcaggc ccagcgtgac catgattgat cttcctcttg gaacggcaca 12420ctttggtcga cacggagact tccggtcggg caattgtccc gttatagtgc aatgcaacag 12480gccgaatcgg ccgctgtcgg cgtgcacatt ccgttgaggg agcccgatga ggcaatgaac 12540gctttcgaag cacagcgcgc ctttgaggag caacttcggg cgcattcccg ggttacgcca 12600tctgccgctc ccgtgtggcg tcgctcgacg ctgcggatgg tcctctatac cgagttgctg 12660ctgctggaca gtctctcgat cctggccgga ttccacgtcg cggcgggcac gcgcgacggc 12720aactggctgt cgctggcggg catcaacgtc ggcgtcttcc tgctgccgat cgctctcggc 12780accgcgctcg caagcggcac ctactcgctg aactgcctgc gctacccggt cagcggcgtg 12840aagagcatct tctcggcatt cttcttctcg atcttcgtcg tcctgctcgg cagctacctg 12900ctgacggccg agctgccgct gtcccgcgtg cagctggcgg agggcgcgat cctctcgctg 12960gtcctcctga tggtgggccg cctgatgttc cgccgccacg tccgcgcggt taccggcggc 13020aggctgctcg acgaactggt catcatcgac ggcgtctcgc tcgacgtcgc gggcaatgcg 13080gtcgcgctcg acgcgcggat catcaatctc tcgccgaacc cgcgcgatcc gcaaatgctg 13140catcgcctgg gcaccaccgt gatcgggttc gaccgggtga tcgtcgcctg caccaaggag 13200catcgcgcgg tctgggcgct gctgctcaag ggcatgaaca tcaagggcga gatcctcgtc 13260ccccagttca atgcgctggg cgcgatcggc gtggacgcct ttgacgggaa ggatacgctg 13320gtcgtctcgc agggcccgct caacatgccc aaccgcgcga agaagcgcgc gctcgatctc 13380gcgatcaccg taccggccgt gctcgcgctg gcgccgctga tgatcctggt ggcgatcctg 13440atcaagctgg agagcccggg cccggtgttg ttcgcgcagg atcgcgtcgg ccgcggcaac 13500cggctgttca agatcatgaa gttccgctcg atgcgcgtaa cgctgtgcga cgcgaacggc 13560aacgtctcgg ccagccgcga cgacgatcgc atcaccaagg tcggccgctt catccgcaag 13620accagcatcg acgaactgcc gcagctgctg aacgtgctgc gcggcgacat gagcgtcgtc 13680ggcccgcggc cgcatgcgct gggctcgcgc gccgccgatc acctgttctg ggaaatcgac 13740gagcgctact ggcaccgcca cacgctcaag ccgggcatga ccggtctggc ccaggtgcgc 13800ggtttccgcg gggcgaccga tcgccgcgtc gatctgacca accggctcca ggcagacatg 13860gaatatatcg acggatggga tatctggcgc gatatcacga tcctgttcaa gacgctgcgg 13920gtgatcgtgc attcgaacgc attctgatcc gcgcacgacg ctgggccgca gcctcgatcc 13980gcaaatggat tgacagcggc ccggcttccg ttttctcgtt tgattttcgt tgcggccggt 14040ccgcgccatg ggggattact gaatgaaggg catcatcctt gcggggggca gcgggacgcg 14100cctgtacccc gcaacgctat cgatctcgaa gcagctgctt cccgtctatg acaagccgat 14160gatcttctat ccgctgtcgg tgctgatgct caccggcatc cgggacatcc tgattatctc 14220caccccgcgc gacctgccga tgttccaggc gctgctgggc gacggctcgg ccttcggcat 14280caacctcagc tatgccgagc agccctcccc caacgggctg gccgaagcgt tcatcatcgg 14340cgcggatttc gtcggcaacg atcccagcgc gctgatcctg ggcgacaaca tctatcacgg 14400cgaaaagatg ggcgagcgct gccaggcagc cgcagcgcag gcagcgcagg gcggtgcaaa 14460cgtcttcgcc tatcatgtcg acgaccccga gcgctacggc gtggtcgcgt tcgacccgga 14520gacgggcgtc gccaccagcg tcgaggaaaa gccggccgag cccaagtcca actgggcgat 14580caccggcctg tatttctacg acaaggacgt ggtcgacatc gccaagtcga tccagccctc 14640ggcgcgcggc gaactcgaga tcaccgacgt caaccgcgtt tacatggagc gcggcgacct 14700gcacatcacg cgcctcggcc gcggctatgc ctggctcgac accggcacgc atgacagcct 14760gcacgaagcc ggctcgttcg ttcgcacgct cgagcatcgg acgggcgtga agatcgcctg 14820cccggaggaa atcgccttcg aaagcggctg gctcggcgcc gaagacctgc tcaagcgcgc 14880cgccggcctc ggcaagaccg gctatgccgc ctatctccgc aaggttgcga ccgcagcatg 14940acccaggtcc atcatcacga actgtccggc gtcatcgagt tcacgccgcc caaatatggc 15000gaccaccgcg gcttcttctc cgaagtgttc aagcagtcgg tgctcgatgc cgaaggcgtc 15060gaggcacgct gggtgcagga caatcagagc ttctcggcgg ccccgggcac gatccgcggc 15120ctgcatctcc aggcgccgcc cttcgcccag gccaagctgg tccgcgtgtt gcgcggcgcg 15180atcttcgacg tcgcggtcga catccgtcgc ggctcgccca cctatggcaa atgggtcggc 15240gtcgagctct cggccgagaa gtggaaccag ctgctggtcc ccgccggcta tgcgcacggc 15300ttcatgacgc tcgttccgga ttgcgagatc ctctacaagg tcagcgccaa atattcgaag 15360gattcggaga tggcgatccg ttgggacgat cccgatctcg ccatcgcctg gccggacatc 15420ggcgtcgagc cggtcctctc cgaaaaggac gcggtcgcca cgcccttcgc cgaattcaac 15480acccccttct tctatcaggg ctgagccatg cagcagacct tcctcgtcac cggcggcgcc 15540ggcttcatcg gctcggcggt ggtgcgccac ctcgtccgcc agggcgcgcg cgtcatcaat 15600ctcgacaagc tcacctatgc cggcaacccg gcctcgctga ctgcgatcga gaacgcgccc 15660aactatcgct tcgtccatgc cgacatcgcc gacaccgcga cgatcctacc gctgctgcgc 15720gaggagcagg tcgatgtggt gatgcacctc gccgccgaga gccatgtcga tcgctcgatc 15780gacggccctg gcgagttcat cgagaccaat gtcgtcggca ccttcaagct gctccagtcg 15840gcgctgcaat attggcgcga gctggagggc gagaaacgcg acgcgttccg cttccaccac 15900atctccaccg acgaagtgtt cggcgacctg ccgttcgaca gcggcatctt caccgaagag 15960acgccctatg atccctcctc gccctattcg gcgtcgaagg cggcgagcga ccatctggtg 16020cgcgcctggg gccacaccta tggcctgccg gtggtgctgt cgaactgctc gaacaattac 16080gggccgttcc acttccccga gaagctgatc ccgttgacca tcctcaacgc gctcgagggc 16140aagccgctgc cggtctacgg caagggcgag aatatccgcg actggctgta tgtcgacgat 16200cacgccaagg cgctggcgac catcgccacc accggcaagg tcggccagag ctacaatgtc 16260ggcggccgca acgagcggac caacctgcag gtggtcgaga cgatctgcga cctgctcgac 16320cagcgcattc cgctggccga cggtcgcaag cgccgcgaac tgatcacctt cgtcaccgat 16380cgccccggcc atgaccgccg ctacgcgatc gacgcgacca agctcgagac cgagctgggc 16440tggaaggctg aggagaattt cgacaccggc atcgccgcga cgatcgactg gtatctggcg 16500aacgagtggt ggtggggccc gatccgctcc ggcaaatatg ccggcgagcg gctggggcag 16560accgcctgat gcgtatcctc gtcaccgggc atgacggcca ggtcgcccag tcgctggccg 16620agcaggcggt gggccacgag ctggtcttca ccacctaccc cgaattcgat ctctccaagc 16680cggagacgat cgaggccggt gtggcgcggg tgcacccgga cctgatcgtc tccgccgccg 16740cctacacggc ggtcgacaag gcggaaagcg aacccgagct ggcgatggcg atcaacggcg 16800acggtcccgg cgtgctggcg cgcgcgggcg cgaagatcgg cgcgccgatc atccacctgt 16860cgaccgatta tgtgttcgac ggcagtctcg accgcccttg gcgcgaggac gatcccaccg 16920gcccgctcgg cgtctatggc gcgaccaagc tggccggcga gcaggcggtg caggcctcgg 16980gtgccaccaa cgccgtgatc cggctggcct gggtctacag cccgttcggc aacaatttcg 17040tcaagacgat gctccgcctc gccgagacgc gcgacgcgct gaacgtcgtg gaggaccagt 17100ggggctgccc cagttcggcg ctggacatcg cgaccgcgat cctgacggtg gtcgggcact 17160ggcagcagga cggcgcgacg agcggcctct accatttcgc cggcaccggc gagaccaact 17220gggccgactt cgcatcgacg atcttcgccg agagcgccaa gcgcggtggc ccctcggcca 17280ccgtcaccgg cattcccagc tcgggctatc cgactccggc cacgcgcccg gccaattcgc 17340ggctggactg cacccgcttc gcggagacct tcggctaccg ggcgcctgcc tggcaggatt 17400cgctgaacgt cgtactggat cgcctgctcg gctgatccga aacggggggc ctcagcgccc 17460cccgccatgc tcccgttcgc gcgccggcaa tgcctctagc accgcgcgct ttcccttagg 17520actcagctcg ctccagccgg cgatttcctt gggcgaccgc cagcacccca ggcacagccg 17580gatctccatg tcgaggcggc agaccttgcg acagggcgat tccggcggtg cggcggcaaa 17640gcgcgagaac agccccatca gcgcttgaag ttcagccccg tcttgcgggc gaacttggcg 17700aggccgttga tcaccggggt cggcgcatag tcgcggatca ggcccttcac ccggtgcagc 17760gcggtgcgct tgtcgtgcgc cgagacggcc cttccaacgc acgaagttcg aatagccgcg 17820accctcggta tcctcctctg cgctgtagta gtagagtgcg agcgagttgc ggcggatgtt 17880cggcggcgtc tgcagcggga agggctggcc gtgccacgac ttgcccgaga cgcggaagat 17940cgcgaggcga ttgaacttgg gcgtgatgct ggaaacgcac ctggtcgcat cctcgtccca 18000cagctccagg tcgccgcccc attcctcctg ccagtctggc gtgcagtaat agatgcagtt 18060gatctgctgg ctgagcttct tgttggggtg gcgcgaggca tcgatgtgga gcatcagccg 18120cccgcccgag ccggtcgagt gcaggccgca gccataatgg ttgggatccg gcagcaggtg 18180cttgtggccg ctcagccggt cgaggaagtt gcagaagatg cccgactgaa actgcatcat 18240catcaggcgg acgagcggcg gaaactgctc ctcgtccgag gtcgtcacct ttcgatcttg 18300cgatcaccgc tgtgcgcgct atcgcccggc ccttccaggc gccagttgac gtcgtccagc 18360ttcgggaagg catccccgag ccgccgcgcc acgtcgtcgg gcaggaaatt gtcgatcgcg 18420acatgctcat agggctcggc gttcaggaag cgatcatgat attcgtccgc gagcgcatat 18480agcttctcgc gcgtgaagaa gaagaagtcc gaagtatctg caccgaccga catgcaatcc 18540cccccgaaga aacggacgca gcgatcataa acgattcacc gcaatcgcgt aacccgtctt 18600gcacagcacc gtaacactta gcgatccctt atccgaacca cgatcggctt gaccaggcgg 18660ataccgaatt cgcggaagcg ccggatcggc gtgcgcaccg catggtccca cagaatcgcg 18720ccggccgcca ccggccgtgc ggcgacatcg ctgcggatct tgaacatatt gatgatgtcg 18780atgtccgaaa ccagcttgcc cttgatccgg ttggttacat tctcgccgtg caccacctgc 18840agccatagcg gcttgctcgg cacctggcgg atcgtccact tctcgcccag ctcgtggtgc 18900ggcttggccc agatcgtctc gatcccggcc acgtctttct cgaccagcga ggtgaacggg 18960ctgctgtgat cgctggcggt gtagagttgc ccgccccgca tcgcgatgcc gtgggggaag 19020ttcagcacgg tctgcgccgg cgcttccttg gcggcgtcct gcacccgcgc gacgaaatcg 19080ctcgacaccg catcatcatt gtccagccgc gtggtgacga tcagcgtctc gcccgccgtc 19140gcgagtgccc gcacgtcctc ggcgatcatc gccttgtcga acatcgccac atagcgtggg 19200gtaaaattga agatctggcg atcgcgctcg atccgctcgc ggaattcaac cggcgtatcc 19260ttgtcgaaat agatcagcca gtggaagttg cgctcggtct ggcccgcgat gctcggcagg 19320cagaactgct cgaacaggcc gaaacggcgt tccagccagc ccggcgagtt gcgaatcgcc 19380acctcgcgtc ccgggctggc gatgttgaag cgagtcagga tcacgtggag catggggttg 19440atcagccctt gtttgcggaa ggaatggcgc ggggcacggc gaccgggcat gccaggaacc 19500gggagcggcg cttcgcgaca tggcggagct tcgccctgaa tggcacgcgc tgcacggctg 19560ctagccccct ttattgccgt tcacctgctt cggttaaggg atattccgga gcccggcaac 19620cggcgattgc tgcgctgcgc aatgaacggc gccgccgcgt ggtggccaag ggcgcgccaa 19680tccacacctg ccgggccggc gatcgcgcgc ccaaagcgcc gccaacgcat tcgcaaggct 19740tgcgaaataa atggcttgcc cctacccgag cccggtgtcg ccccctcgtc ccgacagcat 19800cgccaccggc ctggcgcttc gcctgttcgc gatcgcctgc atgtcgacca tgtcggcgct 19860catcaagatg tccgaactgc gcggcgcctc gctgatcgag acgatgtttc accgccagct 19920ctgggcggtg cccttggtca ccctgtgggt cacgctgggg ccgggcctca agtcgctcag 19980gaccgcgcgg ttcggcgcgc atgtctggcg caccgcggtg ggacttaccg gcatgatctt 20040caccttcggc gcggtgatcc tgctgccgct cgccgaagcg cagaccttcc agttcaccgt 20100ccccatcttc gcgacgctgc tcggcgcgct gatcctaggc gaaccgaccg gctggcaccg 20160ctggagcgcg gtgatcctcg ggttcgtcgg cgtgcttatc gtcgtccagc cggggcacga 20220ggcgatcccg gtgttcggtg cgttcgtggg cctgatggcg gcgctgttcg tcgccatcgt 20280cgcgatcacg ctccgccaga tcgggaagac cgaaagcgcc ggcaccacgg tgttctggtt 20340ctcgctgttg tcggtgccgg tgctgggcgc aatctatgcc ttccactaca agccccatga 20400tgccgagacc tgggccatcc tgatcgccac gggcctggtc ggcggcgtcg gccagctcgc 20460gctgaccggg gcgatgcgct tcgctcccgt gtcggcagtg gtgccgatgg actattcggg 20520gctgctctgg gcgacgctct atggctggct gctgttcggc gtgctgccga ccttttccac 20580ctggctcggc gcgccggtga tcatcgccag cggcctgtac atcgtctatc gcgagcagaa 20640gctggcgcgc ggccaggcta gctacgccga aacgccacta tgaggttgtt ggcgggcatc 20700gccacccgcc gctcgaacac cagcccctgc gcttccgccg ccgccacgac atcgcccagc 20760aaccgcaggc cccaggcgg 20779

Claims

1. A mutant strain of the genus Sphingomonas, containing: at least one genetic modification that substantially or entirely eliminates the production of polyhydroxybutyrate (PHB); at least one genetic modification that results in increased production of a sphingan, comprising a genetic modification that increases the expression of at least one gene involved in sphingan synthesis, wherein said at least one gene involved in sphingan synthesis is selected from the group consisting of the genes contained in the insert in plasmids pS8 (SEQ ID NO: 1), pX6 (SEQ ID NO: 54), the plasmid contained in strain ATCC PTA-10102, the plasmid contained in strain ATCC PTA-10103, and Sphingomonas homologs thereof; whereby the mutant strain of the genus Sphingomonas can produce an increased production of a sphingan that is essentially free of PHB relative to a congenic strain containing the at least one genetic modification that substantially or entirely eliminates the production of PHB and lacking the at least one genetic modification that results in increased production of a sphingan.

2. The mutant strain of the genus Sphingomonas of claim 1, wherein the sphingan is selected from the group consisting of diutan, S-7, gellan, S-88, welan, rhamsan, S-198, NW-11, and alcalan.

3. The mutant strain of the genus Sphingomonas of claim 2, wherein the sphingan is diutan.

4. The mutant strain of the genus Sphingomonas of claim 1, wherein said at least one gene involved in sphingan synthesis is selected from the group consisting of Sphingomonas sp. ATCC 53159 genes dpsS, dpsG, dpsR, dpsQ, dpsl, dpsK, dpsL, dpsJ, dpsF, dpsD, dpsC, dpsE, dpsM, dpsN, atrD, atrB, dpsB, rmlA, rmlC, rmlB, rmlD, orf7, orf6, orfs, and Sphingomonas homologs thereof.

5. The mutant strain of the genus Sphingomonas of claim 1, wherein said Sphingomonas homolog of at least one gene involved in sphingan synthesis is derived from a Sphingomonas species selected from the group consisting of Sphingomonas elodea ATCC 31461, Sphingomonas sp. ATCC 31555, Sphingomonas sp. ATCC 31961, Sphingomonas sp. ATCC 53159, Sphingomonas sp. ATCC 31554, Sphingomonas sp. ATCC 31853, Sphingomonas sp. ATCC 21423, Sphingomonas sp. ATCC 53272, and Sphingomonas sp. FERM-BP2015.

6. The mutant strain of the genus Sphingomonas of claim 1, wherein the at least one genetic modification that results in increased production of a sphingan is selected from the group consisting of: (i) an operable linkage of at least one gene involved in sphingan synthesis to an ectopic promoter; (ii) an increased number of copies per bacterial chromosome of at least one gene involved in sphingan synthesis; and (iii) any combination thereof, wherein each of said at least one gene involved in sphingan synthesis are contained in a bacterial chromosome or extrachromosomal element.

7. The mutant strain of the genus Sphingomonas of claim 1, wherein the at least one genetic modification that substantially or entirely eliminates the production of PHB is a mutation that constitutively or conditionally inactivates or deletes a gene selected from the group consisting the phaA gene, the phaB gene, and the phaC gene or a combination thereof.

8. The mutant strain of the genus Sphingomonas of claim 1, wherein the at least one genetic modification that substantially or entirely eliminates the production of PHB is an insertion or deletion that inactivates the phaC gene.

9. The mutant strain of the genus Sphingomonas of claim 3, wherein the mutant strain of the genus Sphingomonas is able to produce diutan at a rate of at least about 0.15 g/L/hr or a yield of diutan of at least about 12 g/L.

10. The mutant strain of the genus Sphingomonas of claim 3, wherein the mutant strain of the genus Sphingomonas is able to produce diutan at a rate of at least about 0.2 g/L/hr or a yield of diutan of at least about 15 g/L.

11. The mutant strain of the genus Sphingomonas of claim 3, wherein the mutant strain of the genus Sphingomonas increases the rate of production or yield of diutan by at least about 50% relative to a congenic strain containing the at least one genetic modification that substantially or entirely eliminates the production of PHB and lacking the at least one genetic modification that results in increased production of a sphingan.

12. The mutant strain of the genus Sphingomonas of claim 3, wherein a clarified diutan produced from the mutant strain of the genus Sphingomonas yields less than 0.5% residue in a 15% HCl solubility and residue test, or less than 0.1 wt % PHB when measured using gas chromatography.

13. The mutant strain of the genus Sphingomonas of claim 3, wherein a clarified diutan produced from the mutant strain of the genus Sphingomonas and rehydrated as one liter of 0.04% diutan in seawater can pass through a Nuclepore filter in less than five minutes at a flow pressure of approximately 20 psi; wherein the Nuclepore filter is approximately 47 mm in diameter and has a pore size of approximately 3 microns.

14. The mutant strain of the genus Sphingomonas of claim 3, wherein diutan produced from the mutant strain of the genus Sphingomonas is essentially free from PHB, and wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 40 dial reading, a sea water 0.3 rpm viscosity of at least about 37,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 3,500 cp.

15. The mutant strain of the genus Sphingomonas of claim 3 that is a strain of Sphingomonas sp. ATCC 53159 selected from the group consisting of strains PDD3/pS8, PDD3/pX6, PDD6/pS8, and PDD6/pX6.

16. A composition comprising diutan, wherein the composition is essentially free from polyhydroxybutyrate (PHB), and wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 32 dial reading, a sea water 0.3 rpm viscosity of at least about 24,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 3,000 cp.

17. The composition of claim 16 wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 40 dial reading, a sea water 0.3 rpm viscosity of at least about 37,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 3,500 cp.

18. The composition of claim 16 wherein the diutan exhibits a sea water 3 rpm viscosity of at least about 45 dial reading, a sea water 0.3 rpm viscosity of at least about 40,000 cp, or a low shear rate viscosity in the presence of polyethylene glycol dispersant of at least about 5,500 cp.

19. A method of making a sphingan that is essentially free from polyhydroxybutyrate (PHB), comprising: growing a mutant strain of the genus Sphingomonas under conditions that facilitate the production of sphingan; and optionally, isolating the sphingan from the resulting culture, wherein said mutant strain of the genus Sphingomonas contains: at least one genetic modification that results in increased production of a sphingan; and at least one genetic modification that substantially or entirely eliminates the production of PHB.

20. The method of claim 19, wherein the sphingan is diutan.