JPH09252775A - Dna segments and method for increasing polysaccharide production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain DNA sequences useful for production in a large amount of sphingans useful as a specific polymer, an application to fibers, a food, a cosmetic material, an additive as a viscosity adjusting agent for a paint, etc., an application to various coating materials, an adhesive, an additive, a specific chemical agent, etc.

SOLUTION: The DNA sequences are isolated from the DNA of a bacterium capable of producing sphingan, e.g. a strain S88 (ATCC No.31554) of the genus Sphingomonas sp., and encode a genetic information useful of indispensable for the bacterium of Sphingomonas sp., etc., as a recipient to bio-synthesize the sphingan. By integrating a multiple number of copies of the DNA sequences into the bacterial of the genus Sphingomonas sp. or the related bacterial, these bacterial can produce a larger amount of sphingan than that by a wild strain and also reviving the sphingan production capability of a mutant not producing the sphingan.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to biosynthesis and production of sphingan polysaccharides of Sphingomonas sp., And DN isolated from the sphingomonas sp.
A sequence, and fragments thereof. Isolated D
Multiple copies of the NA fragment can be inserted into the same or different strains of Sphingomonas or related strains to increase production of polysaccharides, preferably sphingans.
By such an operation, the amount of polysaccharide produced by the bacterium containing the exogenous DNA is significantly larger than that of the bacterium which is not manipulated under the same fermentation conditions. Furthermore, the present invention relates to a method of manipulating strains of Sphingomonas sp. It also describes methods for identifying and isolating DNA sequences that are useful for Sphingomonas spp. To produce sphingan polysaccharides.

[0002]

BACKGROUND OF THE INVENTION Many microorganisms produce exopolysaccharides, or extracellular polysaccharides also known as EPS. Exopolysaccharides include xanthan gum and a group of polysaccharides known as "sphingans.""Sphingans" is the genus Sphingomonas (genus S
Phingomonas ) Gram-negative bacteria.

"Sphingans" are capsular polysaccharides that are similar but not identical in structure and are secreted by Sphingomonas bacteria (Pollock, TJ.1).
993, J.C. Gen. Microbiol. , 139:
1939-1945). Although sphingans have different side groups, either L-rhamnose or L-mannose is present where the backbone is located. L-mannose itself is very rare in nature. Aqueous solutions of polymers have unique and useful rheological properties (Moorho
use, R.M. 1987, "Structure / pro
party relations of of a
family of microbioal poly
saccharides, "p189-206. In
M. Yalpani (ed.), Industria
l polysaccharides: genetic
engineering, structure / pr
operator relation and appli
notes. Elsevier Science
Publishers B.A. V. , Amsterdam
See). It is not yet known how changing the structure of the polymer results in different flow properties.

Xanthomonas campestris ( Xan
thomonas campestris ) is a Gram-negative bacterium that constitutively mass-produces xanthan gum, which is one of exopolysaccharides (Jeanes, et al.
al. , J. Appl. Polymer Sci. ,
5, 519-526, 1961). The biosynthesis of xanthan gum has been studied in considerable detail due to its industrial importance. Recently, a new bacterial exopolysaccharide, gellan, was developed as a gelling agent. Gellan is S-88 (Kan
g and Veeder, US Pat. No. 4,535,1
53), welan (Kang)
and Veeder, U.S. Pat. No. 4,342,866.
No.), NW11 (Robison and St.
ipanovic, see U.S. Pat. No. 4,874,044), rhamsan (Peik et al.
al., U.S. Pat. No. 4,401,760),
S-198 (Peik et al., U.S. Pat. No. 4,5,5).
29, 797), S-657 (Peiket
al., European Patent Application No. 209277A1),
And heteropolysaccharide-7 (Kang and
McNeeely, U.S. Pat. No. 4,342,866), which is one of a group of related polysaccharides.

The above cited references contain several patents relating to sphingan polysaccharide compositions. None of these patents have any relevance to the subject matter of the present invention.

[0006]

[Table 1]

The chemical structures of sphingan polysaccharides are all related to some degree. The main chain of each sphingan is
It consists of related sequences of four sugars: D-glucose, D-glucuronic acid, L-mannose and L-rhamnose. Polysaccharides of the sphingan group can be distinguished from each other by a carbohydrate containing a polymer main axis (main chain) and side chains. An acetyl group or a pyruvyl group may be bound to the constituent sugars of the sphingan.

Various sphingans are used as specialty polymers, in fiber applications, as additives in foods, cosmetics, papers, paints, cements, such as viscosity modifiers, in various coatings, and in petroleum. It is useful as a product adhesive, additive, and special chemical.

[0009]

In the early stage of research leading to the present invention, the first step of biosynthesis of S-88, which is a typical sphingan polysaccharide, was an important subject. This sphingan is Sphingomonas strain S88
Biosynthesized by. Prior to this study, it was known that in some but not all biosynthesis of bacterial polysaccharides other than sphingan, isoprenyl phosphate was used as a carrier. For example, in the case of xanthan gum biosynthesized by X. campestris, the first step of the synthesis is probably UDP-glucose-glucose-because the main chain of xanthan gum contains only glucose.
Phosphoric acid needs to be transferred to a C55-isoprenyl phosphate (IP) carrier. Ielpi et al. ( FEBS Let
t. , 130, 253, 1982 and J. Bacter .
iol. , 175, 2490, 1993) performed a cell-free uptake assay to sequentially add glucose followed by a second glucose, followed by mannose, glucuronic acid, and mannose to an IP carrier in succession to obtain xanthan gum. It was confirmed that the repeating unit was composed. In exactly the same way, Escherichia coli ( Escher
The repeating unit of the subunit of cholenic acid (E. coli , Escherichia coli) is also prepared by first adding glucose-P to carrier I.
It is composed by transferring to P (Johnso
n and Wilson, J. Bacteri
l. , 129, 225, 1977). In contrast, Rhizobium melilot
i ) succinoglycan
an) In the case of polysaccharide synthesis, first galactose-P
Is transferred to IP (Tolmasky, et a
l. , J. Biol. Chem. , 257, 6751,
1982). However, since the isoprenyl carrier does not participate in the synthesis of dextran or levan polysaccharides, the role of the isoprenyl carrier in alginate synthesis is unknown.

Prior to the study of the present invention, it was not known that the role of the carrier was important in view of the complex kinetics of polysaccharide biosynthesis. Furthermore, it was not known what role the isoprenyl phosphate carrier plays throughout Sphingomonas synthesis of sphingan polysaccharides.

At the same time, a special class of X. campestris mutants that are both Bac ^r and Gum ⁻ (bacitracin resistance and xanthan gum negative) are involved in the transfer of glucose-P from UDP-Glc to IP to form Glc-PPI. It was previously demonstrated by a gene complementation assay that it was found in the required gumD gene. (Po
llock, et al. , 1994, J. Bacte
riol. , Vol.176, pp.6229-623.
7, Vanderslice, et al. , "Gen
etic Engineering of Polys
accharidestructure in Xan
thomonas campestris ”, p. 14
5 to 156, in V. Crescenzi, et a
l. , Biomedical and Biotech
logical Advances in Ind
customary Polysaccharides , G
ordonand Break Science P
ublishers, New York and N.
E. Harding and Y. N. Patel, 1
993, Faceb Journal , Vol. 7, N
number7). The latter reference discloses a DNA fragment capable of restoring the synthesis of Sphingan S-60 by a mutant strain having no production ability, but does not mention whether the amount of synthesis is larger than that of the wild-type strain. Furthermore Further, X by the wild type gumD gene of X. campestris, a similar bacteria S88 and NW11 Sphingomonas strain Bac ^r Sps -. ^(Sphingan polysaccharide-negative) mutant bacteria to be able to restore the synthesis of sphingan such been demonstrated in experiments It was Furthermore, it was suggested that the Bac ^r Sps ^- Sphingomonas mutant might inhibit glucose-P translocation to IP.

It is an object of the present invention to provide a DNA segment isolated from Sphingomonas sp. That many microorganisms, and in particular many Sphingomonas strains, use to increase the production of sphingan polysaccharides. .

Another object of the invention is that the production of sphingan polysaccharide is significantly greater than that of the non-engineered strain,
It is to provide a microorganism, and particularly a mass-produced strain obtained from many Sphingomonas strains.

Yet another object of the present invention is to provide a method for producing a strain of a microorganism which is a sphingan polysaccharide mass-producing bacterium, and particularly a strain of the genus Sphingomonas.

Still another object of the present invention is to provide a method for isolating a DNA segment and inserting it into a Sphingomonas strain so that the microorganism thus engineered becomes a sphingan polysaccharide mass-producing bacterium. is there.

These and / or other objects of the invention can be readily understood by the following description of the invention.

[0017]

The DNA sequence of the present invention comprises:
Either the segment or the fragment is isolated from a sphingan-producing bacterium, generally a Sphingomonas strain. The genetic material thus obtained is cloned, and then a large number of copies are incorporated into mutants of Sphingomonas or related strains with or without the ability to produce sphingans. Such DNA sequences have proved useful in restoring sphingan production in mutant strains that do not produce sphingan. Furthermore, unexpectedly, not only are these mutants able to produce sphingans, but their sphingan production is significantly higher than expected for wild-type strains that are naturally capable of producing sphingans. It was found.

Furthermore, when a DNA segment or fragment is isolated from one strain of Sphingomonas and a large number of copies are inserted into the same Sphingomonas mutant strain or a different strain with or without the ability to produce sphingans, It was discovered that the bacteria manipulated in this way became a sphingan mass-producing bacterium,
It was unexpected. This discovery was unexpected, especially in the following points. That is, a DNA segment or fragment is labeled with, for example, Sphingomonas S
When isolated from S. 60 and inserted into a wild-type or non-mucoid mutant of Sphingomonas S88, this operation produces a large-scale production of S-88 sphingan, and S-88 produced is generally S-60 sphingan. Not mixed. This complementarity can be applied fairly widely among various strains of Sphingomonas (inter-strain complementarity), and may be applicable to xanthan gum production of Xanthomonas campestris (intergeneric complementarity).

Furthermore, the inventors of the present invention have isolated and incorporated a DNA segment or fragment from a Sphingomonas strain capable of producing sphingans.
A method for producing a Sphingomonas strain capable of mass production has been discovered. DNA isolated from a sphingan-producing bacterium is first cloned, and then inserted into a sphingomonas-producing sphingomonas strain or a non-mucoid mutant derived from a sphingan-producing strain.

Furthermore, the present invention is obtained by inserting the above-mentioned isolated DNA segment or fragment,
Includes engineered Sphingomonas. These engineered bacteria contain multiple copies of the DNA segment or fragment isolated according to the present invention. The bacterium engineered by the present invention is a sphingan mass-producing bacterium.

According to the invention, the DNA fragment is
It can be isolated and recovered and cloned by a technique easily available to those skilled in the art. The multiple copies of the DNA are then inserted into Sphingomonas bacteria, generally as extrachromosomal or plasmid DNA. After inserting into the target bacterium, the bacterium is engineered to ferment at the same concentration as the non-engineered sphingan-producing bacterium of the same strain, and the sphingan production amount is measured. Those that produce more sphingan than those that have not been manipulated and that have the ability to produce are determined as mass-produced bacteria. Using this method, it is possible to easily determine a DNA sequence that can increase the production of sphingan polysaccharides obtained from Sphingomonas spp.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION The following terms are used throughout the specification of the present invention, and their meanings are defined as follows.

1. The term "sphingan" refers throughout the specification to a group of related but distinct exopolysaccharides secreted by Sphingomonas bacteria (Pollock, J. Gen.
Microbiology , 139: 1939-194.
5, 1993). The structures of sphingans are all of some relevance. The main chain of each sphingan is
D-glucose, D-glucuronic acid, L-mannose,
It consists of the related sequences of the four sugars of L-rhamnose. The sphingan group of polysaccharides can be distinguished from each other by the sequence of the constituent sugars containing the polymer backbone and multiple side chains. The sphingan polysaccharide may also have an acetyl group or a pyruvyl group attached to its constituent sugar (Mikolajczak, et al., App .
L. and Env. Microbiol. , 60: 4
02, 1994). A schematic diagram of the chemical structure of various sphingans produced using the DNA segments and fragments of the present invention and the general method is shown generally in FIG. Gellan (S-60), Welan (S-130), Ramzan (S-194), S-8 of sphingans
The structures of 8, NW-11, S-198 and S-657 are also
Generally shown in FIG.

Typically, each individual member of the sphingan polysaccharide group is generally represented by the following repeating chemical structure:

[0025]

Embedded image

In the formula, Glc is glucose, GlcA is glucuronic acid, Rha is rhamnose, and Man is mannose. X may be either Rha or Man. Z
Is bound to the second Glc residue, α-L-Rha-
It is either (1--6) -α-L-Rha, α-L-Man, or α-L-Rha. W is bound to the No. 1 Glc residue, and W is β-D-Glc- (1--6)
-Α-D-Glc or α-L-Rha, wherein the subscripts v and y are 0, 0.33, 0.5,
It is 0.67 or 1. In the formula, the reducing end of the polymer is
It is located near the X residue of the backbone. The term "backbone" as used herein refers to the remainder of the structure minus the W and Z chains, ie when v and y are equal to zero. The "reducing end" of the polymer is the end of the polymer to which the sugar units are added.

In the sphingan polysaccharide group, some simple substances are acetylated at various positions. However, the polysaccharide can be chemically deacylated by conventional methods to remove the acyl group. For example, gellan has the same carbohydrate backbone as welan (ie, X = Rha) but lacks side chain sugars (ie, v = 0 and y).
= 0), and glucose residue 1 is completely replaced by glycerate. The structure of the gellan subunit is also acylated somewhere, although its position is unknown.

2. The term "Sphingomonas" refers throughout the specification to Gram-negative bacteria of the genus Sphingomonas that produce the above-mentioned exopolysaccharides, ie sphingans. In the present invention, most of the gram-negative bacteria of the genus Sphingomonas are re-inserted into another strain having a sphingan-producing ability as an isolated DNA sequence source (preferably, gram-negative bacteria of the genus Sphingomonas), To produce the sphingan mass-producing bacterium of the present invention,
Alternatively, it can be used as a target bacterium by inserting an exogenous DNA sequence into it to produce a sphingan mass-producing bacterium.

A family of Gram-negative bacteria capable of producing sphingans was first identified in 1993 as being classified into the genus Sphingomonas. (Pollock, J. Ge
n. Microb. , 139, 1939, 1993). However, it has not yet been established exactly which species each strain should be classified into. The species closest to the Sphingomonas strain capable of producing sphingans is Sphingomonas sphingomonas.
s. paucimobilis ). However, it is too early to say that these strains should be classified into the above species until the time when detailed, deterministic taxonomic analysis results are available. It is added that Sphingomonas spp. Of Sphingomonas were originally classified into several different genera.

Among the species of Sphingomonas that have been identified so far, S. porcine mobilis ( S. pau)
cimobilis ), S. paraporus mobilis ( S.
parapaucimobilis ), S. adhaesiva , S. capsulata ( S.
capsulata ) and S. yanoikuyae ( S. ya
noikuyae ) (Yabuuchi,
et al. , Microbiol. Immuno
l. , 34, 99, 1990). In the past, these Sphingomonas species were erroneously mistaken for Pseudomonas (g
It was once classified as enus Pseudomonas ).

3. The terms “donor” and “acceptor” are used to refer to the bacterium from which the DNA sequence was taken and the bacterium into which the DNA sequence was inserted or integrated, respectively.

4. The term "strain" or "sphingomonas strain" is used to refer to a Sphingomonas gram-negative bacterium that produces (depending on chemical structure) a particular sphingan exopolysaccharide. A sphingomonas strain having a sphingan-producing ability will be simply referred to as a sphingan polysaccharide produced by this strain. For example, Sphingomonas strain S88 produces sphingan polysaccharide S-88, Sphingomonas strain S60 produces sphingan polysaccharide S-60 (gellan), and so on. Sphingomonas strain S88 (ATCC No.
31554), S60 (ATCC No. 3146)
1), NW11 (ATCC No. 53272), S1
30 (ATCC No. 31555), S194 (AT
CC No. 31691), S198 (ATCC N
31853), S657 (ATCC No. 531)
59), and S7 (ATCC No. 21423),
It is a representative strain particularly useful in the present invention.

5. The term "mass-producing strain" is used throughout the specification to engineer bacteria which are isolated from bacteria of the same or different strains capable of producing sphingans.
A significant amount of sphingan polysaccharide is produced when fermenting and comparing unengineered bacteria of the same strain or wild-type bacteria containing multiple copies of NA segment or fragment under the same or substantially the same fermentation conditions. Used to refer to bacteria larger than (at least about 5% or more by weight / weight ratio) bacteria.

6. The term "isolated" is used to refer to DNA that has been collected from a microorganism and at least partially purified, ie, which has undergone one or more purification steps. The isolated DNA is preferably prepared in a substantially pure form. That is, DN that is cleaved into fragments or segments by restriction enzymes, made into multiple copies by cloning, inserted into a plasmid vector or other means, or incorporated into bacteria.
Control the pollutant content to the extent that it does not affect the performance of A.

7. The term “DNA” or “chromosomal DNA” refers throughout the specification to DNA isolated from Sphingomonas, generally prior to its isolation from a microorganism, the chromosome or endogenous gene of Sphingomonas. Used to refer to the DNA found in the sex plasmid.

8. The term “sequence” is used throughout the specification to refer to a specific segment of DNA identified by a pattern of cleavage sites by nucleotide units or restriction enzymes, and generally a sphingo-producing sphingo. It is isolated from the DNA of the genus Monas by a restriction enzyme and the DNA sequence thus obtained is inserted into a bacterium to produce a mass-produced bacterium, or further restricted to a small portion of the DNA smaller than the above sequence, or Used to refer to sequences that produce fragments. The term "portion" or "fragment" is generally used to refer to a DNA sequence that is smaller than a DNA segment. A preferred DNA segment for use in the present invention is glycosyltransferase (glucosyl,
Galactosyl, rhamnosyl and glucuronosyl transferases), glycosyl-IP transferases (including glucosyl-IP transferases, galactosyl-IP transferase enzymes, etc.), rhamnose operons (synthesis of rhamnose precursors for insertion into certain rhamnose-containing sphingans), and It is a segment that encodes various proteins including the secretion of bacterial polysaccharides.

9. The terms “inserted”, “insert”, “integrated” or “incorporate” are used throughout the specification to refer to sphingomonas-producing chromosomal DNA or plasmid DNA of a Sphingomonas strain. It refers to the step and result of transferring the separated DNA segment to a Sphingomonas strain that has the ability to produce sphingans and becomes the same or different recipient bacterium. As a result, the transferred D
A mass-produced strain is obtained that contains multiple copies of at least a substantial portion of the NA segment.

As an example, first, the isolated DNA is introduced into a plasmid vector, for example, pRK311 or pSEB24, by a technique well known to those skilled in the art, cloned, and then transferred to Sphingomonas as a recipient by conjugation. . After inserting into the recipient organism Sphingomonas,
The plasmid vector containing the above-mentioned DNA fragment is replicated in the cells of the recipient bacterium, and several copies (at least 2 copies, usually 4 to 20 copies) of the DNA segment necessary for large-scale production of sphingan polysaccharide can be made. In addition to plasmid vectors, bacteriophage vectors,
A transposon vector may be used.

Many plasmid vectors are suitable for inserting isolated DNA segments or fragments into recipient cells. PRK311 and pSEB described above
In addition to 24, the following plasmids are also particularly useful. RK2, which is a broad range host plasmid of incompatibility group P-1, and pRK290 and pRK derived from the plasmid.
293, pRK404, etc. (Ditta, et a
l. , Plasmid , Vol. 13, pp. 149-
153), and other derivatives containing the oriT gene of plasmid RP4 that allow transfer of the plasmid, such as pSUP101 (Simon, et al., Bi .
o / technology , November, 198
3) and plasmids pLAFR1 and pLAF
R3 (Friedman, et al., Gene , 1
8, 289, 1982), and RSF1010 which is a broad host plasmid of the incompatibility group Inc-Q, and pMMB22 and pMMB66 (F
uerste, et al. , Gene , 48, 11
9, 1986).

It is generally effective to use conjugation when inserting a plasmid vector into a recipient bacterium. However, in bacteria belonging to different genera, it is more common to transform competent cells with purified DNA.

In the case of Sphingomonas, electroporation has also been used to introduce DNA fragments and plasmids into bacteria (1992, Monteiro, et al.
l. , J. of App. Bacteriol. , 7
2, 423). Using this method, simply adding the isolated DNA to the bacterium and transferring it across the cell membrane by electroporation will result in two or more cell copies of the isolated DNA segment or fragment becoming a recipient strain of Sphingomonas. Can be incorporated into.

[0042] Monteiro et al., Supra, describe electroporation as a means of introducing DNA into Sphingomonas. The function of electroporation is the same as chemically treating competent cells, eg transforming the cells after treatment with calcium chloride or rubidium salts.
The DNA to be transformed is purified by standard methods, which may or may not be in the form of plasmids. However, in the case of double-stranded closed circular DNA, transformation is usually most efficient. Therefore, in this case, it is not necessary to introduce the DNA into Sphingomonas by the conjugation method, or to clone the segment and then insert it into a plasmid, bacteriophage or transposon vector. However, it is preferred to first introduce the isolated DNA into a plasmid vector and then transfer the plasmid containing the isolated DNA fragment into bacteria.

It is not necessary to continue carrying the DNA segment on a plasmid or other vector such as a bacteriophage or transposon vector in the recipient strain Sphingomonas. It is common practice to further introduce a copy of a DNA segment into bacterial DNA and replicate the segment generation by generation by the same mechanism that replicates bacterial DNA. In the Examples section below, two examples are described that detail a method of further introducing a copy of DNA into bacterial DNA and replicating segments segment by generation by the same mechanism as replicating bacterial DNA.

10. The term "multi-copy" refers throughout the specification to an exogenous DNA sequence, fragment or segment (D above, which incorporates at least 2 copies, preferably at least 4 copies, into a Sphingomonas bacterium.
At least a substantial part of NA). More preferably, DNA to be inserted into Sphingomonas sp.
The copy number of a sequence, fragment or segment actually ranges from about 4 to about 20. In one example, the DNA sequence is incorporated into a single plasmid vector, transferred to Sphingomonas by conjugation, the plasmid is replicated in the recipient strain, and two or more copies of the DNA sequence,
Note that segments or fragments may be obtained.

11. The term “biosynthesis” is used throughout the specification to refer to Sphingomonas sp. To produce or synthesize a sphingan. Sphingan polysaccharides are synthesized per carbohydrate unit by a series of steps controlled by multiple bacterial enzymes.

12. The term “engineered” is used throughout the specification to refer to the recipient strain, Sphingomonas, into which exogenous DNA, preferably multiple copies thereof, has been incorporated. The engineered bacterium of the present invention is a sphingan polysaccharide mass-producing bacterium.

13. The term "encodes genetic information" is used throughout the specification to refer to a DNA sequence containing genetic information in nucleotide units arranged in a specific order. Genetic information of DNA sequence (any length)
Are considered to be "beneficial or essential" for sphingan synthesis by Sphingomonas if the resulting engineered bacterium produces multiple sphingans that incorporate multiple copies. The term "beneficial or indispensable" means that DNA is isolated from a Sphingomonas bacterium, encodes genetic information, and that a large number of copies of this genetic information are incorporated into Sphingomonas bacterium to produce a mass-producing sphingan polysaccharide. Used to refer to the DNA to be converted. Beneficial or essential DNA used in the present invention is, for example, glucosyl IP-transferase, galactosyl IP.
-Genes or operons for the biosynthesis of glycosyltransferases, such as transferases, and biosynthesis of sugar synthons such as rhamnose, mannose, glucose, galactose, and substitution synthons of the above sugars, such as dTDP-L-rhamnose. Gene or operon for and a gene or operon for the biosynthesis of enzymes involved in the polymerization of sugar synthons to produce sphingans, such as polymerases, and secreting polysaccharides from intact cell structures One or more genes or operons for

14. The term "inter-strain complementarity" is used to refer to the isolation of a DNA sequence, segment or fragment from a first Sphingomonas strain and incorporation into a different second Sphingomonas strain. As one of the embodiments of the present invention, it was discovered that, by taking DNA fragments from different Sphingomonas strains and incorporating multiple copies thereof into another Sphingomonas strain, a sphingan polysaccharide mass-producing strain can be produced. It was an unexpected place. The DNA fragments useful in the present invention also exhibit intergeneric complementation (eg, causing Xanthomonas campestris to increase xanthan production).

15. The term “synthon” is used to refer to a sugar or sugar unit that is polymerized during the biosynthesis of a sphingan by a bacterium according to the present invention. Synthons include constituents or units of sphingan polysaccharides, sugar components used in biosynthesis of sphingans, such as glucose, galactose, rhamnose, mannose, and other acetylated and acylated sugars and related precursors. Contains sugar synthons and the like.

16. The word "rhamnose operon"
Rhamnose or rhamnose synthons (dTDP-) used for biosynthesis of sphingan polysaccharide according to the present invention.
L-rhamnose and the like) is used to refer to a DNA sequence encoding a gene or operon involved in biosynthesis of L-rhamnose and the like.

The present invention obtains a DNA sequence from Sphingomonas sp.
The present invention relates to the discovery that when a large number of copies are incorporated into a Sphingomonas bacterium that is a recipient of the same strain or different strains, the recipient Sphingomonas bacterium transforms into a sphingan polysaccharide mass-producing bacterium. Furthermore, by isolating a DNA sequence from a sphingan polysaccharide-producing bacterium and incorporating multiple copies in different strains of Sphingomonas, not only can large-scale production strains be produced from these different strains, In this case, it was also found that contamination with sphingan polysaccharide having the characteristics of the donor bacterium does not occur.

The DNA sequences incorporated into the recipient bacteria include
It encodes genetic information that is beneficial or essential for biosynthesis of sphingan polysaccharide. For example, beneficial or essential genetic information may play or play a variety of roles in bacterial sphingan biosynthesis. Exogenous DNA is
For example, it expresses the synthesis of enzymes and other proteins involved in the process of rate-limiting enzymes, induces the synthesis of enzymes, cofactors, and other biochemical components to increase the production of polysaccharides, It increases production of enzymes such as polymerases that facilitate ligation, binds to one or more repressor genes, promotes secretion of polysaccharides from bacteria, and is usually rate-limiting during polysaccharide biosynthesis. It can bring about a beneficial effect on sphingan biosynthesis, such as suppressing the expression of repressor that inhibits the production of the process.

The DNA sequence is isolated from various strains of Sphingomonas by standard techniques and methods known to those skilled in the art. Generally, the bacteria are cultivated (as described in detail below, by standard fermentation methods using glucose concentrations of less than about 0.5%, preferably about 0.1% to 0.2%), and high concentrations of bacteria. Make a broth containing. The bacterial cells are then centrifuged and resuspended for DNA extraction. DNA is first extracted from bacteria by removing proteins from the mixture and precipitating high molecular weight DNA with ethanol or isopropanol (Birnboim and D).
oly, Nucl. Acids Res. , 7, 151
3, 1979).

After precipitation as described above, the isolated DNA segment or fragment is usually cloned to form a DNA for insertion of Sphingomonas as a recipient. By way of example, the high molecular weight DNA sequences described above are partially digested with a restriction enzyme (eg, SalI enzyme) and electrophoresed by standard methods (Loftus, et al., BioTechni) .
ques , 12, 172, 1992). After electrophoresis, the large DNA fragments (20 kbp and above) are further purified (extraction and precipitation).

Then, the DNA fragment isolated from the bacterium is directly inserted into a cloning vector (generally a plasmid) for cloning the DNA, or alternatively, further treated with a restriction enzyme to obtain a smaller DNA fragment. And insert it into the cloning vector. The DNA cloning of the present invention is performed by general techniques and methods standardized by those skilled in the art. According to the present invention, a number of methods can be used to carry out the cloning of the DNA segment, thus the present invention
For example, the use of a plasmid cloning vector is not limited. For example, the DNA fragment is charon
4A, EMBL3 (Rodriguezand De
nhardt, Vectors, Chapter 2,
pg. 43, 1988, Butterworth Pu
blishers, Boston), or P1 (199)
0, Sternberg, Proc. Natl. Aca
d.Sci. , U.S. S. A. , 87, 103-107)
It can also be inserted into a bacteriophage vector such as and cloned.

As described in detail in Examples 1 and 12 below, first, a DNA fragment to be inserted into a cloning vector is prepared. According to the invention, numerous cloning vectors can be used for the formation of DNA segments or fragments. However, in the present invention, it has been found to be advantageous to use the cloned plasmid vector of the DNA segment or fragment as is and to insert the exogenous DNA into the recipient by conjugation. However, a cloning vector (plasmid or other) different from the vector conjugating to the recipient can also be used, especially if the transformation method is to be used when inserting the DNA into the recipient.

After the insertion into the cloning vector,
The vector containing the isolated DNA is packaged in a bacteriophage and transferred to bacteria (generally E. coli) by the transfection method and replicated in the transfected bacteria. Colonies of bacterial cells containing the DNA clones thus obtained are pooled and either stored or used directly.

The DNA clones are then screened to determine the relative production effect of the DNA fragment on sphingoma production by Sphingomonas. In the screening method, after carrying the DNA on an appropriate vector, the conjugation method (for example, Ditta, e.
t al. , Proc. Natl. Acad. Sc
i. , USA, 77, 7347, 1980, describing tri-parental mating.
g)) was inserted into the recipient strain Sphingomonas and tested for engineered bacteria containing multiple copies of the DNA thus obtained and its sphingan production,
Determine activity.

A DNA segment or fragment determined to have a production-increasing effect on sphingan production is transferred to a recipient strain of Sphingomonas and contains at least two copies of at least a substantial portion of the transferred DNA as described above. Produce mass-produced strains.

A preferred screening method has been developed for use in the present invention. In this method, DNA is inserted into a sphingomonas strain, which is a recipient strain having no production ability, to screen for the presence or absence of a gene useful or essential for sphingan synthesis in this DNA. . That is, a mutant strain having no productivity (eg, Sps ^- Bac ^{r of} strain S88) is derived from a Sphingomonas strain having the ability to produce sphingans, and manipulated so as to contain a large number of copies of DNA to be screened. . Mutants having no production ability, which have been operated in this manner, are grown on a regular agar plate medium containing 1 to 3% glucose. Separately, a sphingomonas mutant having no productivity is grown under the same conditions, and the appearance of colonies of this mutant and the engineered bacteria is compared. After doing so, generally the ability of the DNA to cause Sphingomonas to initiate sphingan synthesis is visually determined.

DNA for improving sphingan production activity
The ability of the segment or fragment is determined by the phenotypic difference between the sphingan-producing bacterium and the non-sphingan-producing mutant that can be easily recognized on the culture dish. For example, since Sphingomonas strains capable of producing sphingans produce mucoids, they often form colonies that can be easily identified by a simple visual inspection (for example, colonies of sphingan-producing strains are rounded up and rounded with a bright ring). Surrounded, whereas non-producing colonies are flat, rough and translucent).

In one case, as will be described in detail in Example 2 below, the phenotypes of the sphingan-producing bacterium and the sphingan-not-producing bacterium in one Sphingomonas strain can be easily or in a short time. In some cases, it cannot be distinguished. In such a case, the screening method is modified so that the activity of the target DNA is judged by the bacteria whose difference between the phenotype of productivity and the phenotype of non-productivity can be easily identified by visual inspection. In this embodiment of the invention, the fact that the DNA fragments useful in the invention show complementation between strains and between genera and that multiple copies of this DNA fragment cause virtually all Sphingomonas strains to produce increased sphingans. It makes use of the fact that you can.

The DNA segments or fragments useful in the present invention also show activity in Xanthomonas campestris. Therefore, DNA that could not be easily screened using one or more Sphingomonas strains
A large number of copies of the fragment were obtained from X. campestris mutants, such as X59 in particular, which are not capable of producing xanthan.
It can be incorporated into m31 to screen xanthan production by visual inspection. X. campestris mutants having no production ability such as X59m31 are formed by selecting survivors after exposure to bacitracin, incorporating duplicate copies of the DNA fragment into these bacitracin-resistant mutants, and culturing in YM agar plates. It can be easily obtained by observing whether the appearance of the colony is mucoid (producing bacterium) or non-mucoid (non-producing bacterium). (Pollock, et al., 1
994 J. Bacteriol. , 176, pp. 6
229-6237 and U.S. Pat. No. 5,338,841.
No.). The DNA from which the screened bacteria increase the production of polysaccharides (sphingan or xanthan) is evidence of complementation between strains or between genera, and not only different Sphingomonas strains but also sphingan polysaccharides by bacteria of different genera (Xanthomonas) The production can be increased.

In general, using the convenient screening method according to the embodiment of the present invention capable of identifying the phenotypic difference between the sphingan-producing strain and the non-producing strain, the cloning and With transfer technology, without undue or unreasonable experimentation,
The DNA segment or fragment used in the present invention can be easily obtained to increase the production of sphingan polysaccharide by Sphingomonas.

Another embodiment of the present invention relates to increased production of sphingan polysaccharide. To produce the sphingan polysaccharide, the engineered bacteria of the present invention are cultivated under suitable fermentation conditions by well known methods. Suitable medium or fermentation broth for culturing the engineered Sphingomonas include, for example, carbon sources such as glucose, lactose, sucrose, carbohydrates generally containing maltose and maltodextrin, such as inorganic ammonium, inorganic nitrates, organic amino acids. Or hydrolysed yeast, proteinaceous material such as soybean flour or casein, distiller's solubles or corn steep liquor provided by distillers.
and an aqueous medium containing a nitrogen source such as inorganic salts and vitamins. In the present invention, sphingans can be produced using a great variety of fermentation media.

The carbohydrate content in the fermentation broth varies, but is usually about 1 to 5% by weight with respect to the fermentation medium.
Carbohydrates are added all at once either before or during fermentation. Nitrogen dose is about 0.
It is in the range of 01% to about 0.4% by weight. As the carbon source and the nitrogen source, only one kind of carbon source or only one kind of nitrogen source may be used, or several kinds of carbon sources or nitrogen sources may be mixed and used.

Examples of inorganic salts used for fermentation of Sphingomonas include sodium, potassium, ammonium, nitrate, calcium, phosphate, sulfate, chloride, carbonate and salts containing ions similar thereto. To be Magnesium, manganese, cobalt, iron, zinc,
Advantageously, trace metals such as copper, molybdenum, iodides and borates are also included. Also, biotin, folate,
Lipoate, niacinamide, vitamins pantothenate, pyridoxine, riboflavin, etc. thiamine and vitamin B _12, and mixtures thereof can also be advantageously used.

The fermentation temperature is about 25 ° C to about 35 ° C,
A temperature range of about 28 ° C. to about 32 ° C. is preferred for optimum productivity. The inoculum is prepared by standard methods, scale-up in volume, including shake flask culture and small scale submerged fermentation. The inoculum preparation medium may be the same as the production medium, or may be Luria Broth medium or YM medium, which is one of several standard mediums well known to those skilled in the art. In seed culture, the carbohydrate concentration can be less than about 1% by weight. There may be more than one seed culture step to obtain the desired inoculum. A typical inoculum is about 0.5% to about 10 based on the total final fermentation amount.
% Range.

The fermentation vessel is typically equipped with a stirrer to stir the contents. The container may be equipped with an automatic pH controller and an automatic foaming controller. The production medium is added to the container and sterilized in situ by heating. Alternatively, the carbohydrate or carbon source may be separately pasteurized prior to addition. The pre-grown seed culture is added to a chilled medium (generally at a fermentation temperature of about 28 ° C to about 32 ° C) and fermented for about 48 to about 96 hours with agitation of the culture, Prepare a high viscosity broth. The sphingan polysaccharide is precipitated from the broth by precipitation with an alcohol, typically isopropanol, by standard methods.

The present application is specifically exemplified by several strains, in particular Sphingomonas strains S88, S60, NW11, S.
198, S7, and S194 (deposit number AT respectively)
CC31554, ATCC31461, ATCC532
72, ATCC31853, ATCC21423, AT
CC31961 as American Type Cul
DNA segments and fragments isolated from (Ture Collection)) are disclosed. By incorporating many copies of these DNA segments or fragments as extrachromosomal (plasmid) DNA into the Sphingomonas strain,
Sphingan S-88, S-60 produced by these strains
And NW-11 have been found to be useful in increasing production.

In the case of Sphingomonas strain S88, the size of the isolated chromosomal DNA segment is 34 kbp.
, Including 23 to 25 genes. As shown in the clone map of S88 in FIG. 1, the 34 kbp region is the combined size of two clones, c3 and c2. This 34 kbp
Of the DNA sequence of each of the genes is divided into a plurality of DNA sequences, and each of them is clΔ3, c2, c3, c4, c5, c6, H1.
5.6, B7.1, B8.6, E5.9, E1.5, E
Labels such as 2.4, E4.5, E6.6, and E12.8 are shown in FIG. Figures 6, 9 and 10 show the isolated S88 chromosomal DNA in more detail.

In the case of Sphingomonas strain S60, multiple DNA sequences including c1, c2 and c3 (see FIG. 2) were isolated from the chromosomal DNA. After cloning the sequence c2, it was placed in the pRK311 vector and inserted into the Sphingomonas strains S88, S60 and NW11 to evaluate the sphingan production activity (see Example 9 for details).

In the case of Sphingomonas strains S198, S7 and S194, DNA segments were also isolated from these strains (see Figures 7 and 8). From these strains, more DNA segments can be isolated by the general methods detailed herein and utilized to increase bacterial sphingan production according to the present invention.

In the case of Sphingomonas strain NW11, D
NA fragments cl, c2, c2.1, c2.2, c
2Hd, c2H1, c2H2, c2H3, c2H10 were isolated (see FIG. 3). After cloning the sequence c2.2, it was placed in the pRK311 vector and inserted into Sphingomonas strains S88, S60 and NW11 to assess sphinggan production activity (see Example 9 for details). ).

The following DNA segments were prepared from Sphingomonas strains S88, S60 and NW11 by the method generally described above. Inserting each of these DNA segments (as a full-length DNA segment in a plasmid vector) into one or more Sphingomonas strains (wild-type sphingan-producing strains or non-mucoid mutants derived from wild-type producing strains) Bacterium of the above turns into a sphingan polysaccharide mass-producing bacterium. These D
Each NA segment or fragment is derived from a DNA segment or fragment isolated from Sphingomonas strains and inserted into a plasmid vector as described above. The characteristics of the DNA segment or fragment are determined by a restriction enzyme cleavage site map (Fig. 1,
2 and 3).

[0076] strain S88 pRK311-S88cl △ 3 pRK311-S88c2 pRK311-S88c3 pRK311-S88c4 pRK311-S88c5 pSEB24-S88H15. 6 pSEB24-S88B8. 6 pSEB24-S88E4. 5 pSEB24-S88E6. 6 strain S60 pRK311-S60c2 strain NW11 pRK311 -NW11c2.2

A DNA clone can be introduced into a wild-type Sphingomonas bacterium having a sphingan-producing ability or a mutant bacterium having no sphingan-producing ability to realize a large-scale production effect. For example, multiple copies of a DNA clone are introduced into each of a wild-type strain capable of producing a sphingan and a non-mucoid mutant strain derived from a strain capable of producing a sphingan. The bacterium manipulated in this manner is a sphingan mass-producing bacterium when compared with a wild-type bacterium having the sphingan-producing ability derived from the same strain or a mutant strain having no sphingan-producing ability.

It was unexpected when the DNA was screened and a large number of copies were introduced into these strains of Sphingomonas. However, the recombinant strains obtained in this manner generally produced sphingan. It was higher than the sphingan production level of wild-type bacteria. This is a general phenomenon, and complementation between strains was observed in the increased production of this sphingan.

In the present invention, the recombinant bacterium obtained by inserting multiple copies of a DNA clone has a higher level of sphingan polysaccharide synthesis activity than the wild type. Also,
DNA segments useful in the present invention include DNA fragments encoding proteins necessary for the binding of the first glucose residue to the isoprenyl phosphate carrier during the initial steps of strain assembly and biosynthesis by the strain. As a result, the Sphingomonas strain has a beneficial or indispensable gene for synthesizing sphingan.

The DNA sequence of the spsB gene (SEQ ID NO: 1) and the deduced amino acid sequence of the SpsB protein (SEQ ID NO: 2) are also disclosed. The SpsB protein (or, depending on the strain of Sphingomonas, especially SgeB, SnwB, S
(Similar proteins such as neB, SssB, and SrhB)
All of the DNA fragments containing the DNA coding for S. cerevisiae can be incorporated into multiple copies to render the Sphingomonas strain an engineered strain, which can increase sphingan production.

Similarly, the same applies to the DNA segment or fragment containing the sgeB gene isolated from S60 Sphingomonas (which encodes the SgeB protein that resembles SpsB) and the snwB gene isolated from NW11 Sphingomonas. sge
The B gene (Fig. 2) is the spsB gene of S88 chromosomal DNA (and the corresponding sneB, sssB, and shrB genes, which are Sphingomonas S198, respectively).
Derived from S7 and S194 strains, SneB, SssB, S
They are similar to each other in that they are believed to encode similar proteins (based on the DNA hybridization of the rhB protein) and (on the basis of DNA hybridization with the fragment corresponding to the spsB gene of S88). The snwB region shown in FIG. 3 is a DNA encoding the SnwB protein.
Corresponds to an array. The snwB gene is the spsB gene of S88 chromosomal DNA and the ssB gene of S60 chromosomal DNA.
Similar to the geB gene. These DNA fragments insert into the plasmid as generally described above to form multiple copies of Sphingomonas to produce sphingans.

The spsB gene of Sphingomonas S88 is believed to encode glucosyl-IP transferase. The deduced amino acid sequence of the SpsB protein has been demonstrated to have considerable homology with putative glycosyl-IP transferases from different genera. This deduced amino acid sequence is commonly glycosyl-IP
Similar to the sequences of other genes believed to encode transferases, this similarity
There is strongest evidence that the gene encodes glucosyl-IP transferase. In fact, there is considerable homology between the carboxyl portion of glucosyl-IP transferase and the carboxyl portion of galactosyl-IP transferase. Although there is no such high homology in the amino-terminal region, S. enterica ( S. e.
N. terica ) is similar to the RfbP protein (19
91, Jiang, et al. , Mol. Microb
iol. , 5, 695). The hydrophobic region of SpsB is 3
Amino acids at positions 5 to 39 (mean hydropathy + 2.
2), the amino acids at positions 68 to 86 (+1.7), 10
Amino acids (+2.3) at positions 5 to 123 and 282
It contains the amino acid at position 303 (+2.9). The location of the latter hydrophobic region is common to these related gene products and is located next to the region of greatest homology.

In the best mode of the present invention, DNA
For segments and fragments, S88, S60, NW
11, S130, S194, S198, S657 and S
DNA sequences encoding glycosyl-IP transferases of various strains of Sphingomonas such as No. 7 are contained. Multiple copies of these DNA segments and fragments can be used in the recipient strain Sphingomonas to achieve the benefit of the recipient producing more sphingan. Gene encoding a glycosyltransferase enzyme or D
In addition to the incorporation of NA fragments, sugar synthons or sugar precursors (ie, sugar moieties that include constituents of sphingan polysaccharides and are used in sphingan biosynthesis, such as glucose, galactose, rhamnose, mannose, and other sugar synthons. Or a precursor) encoding a gene or DNA fragment, or promoting polymerization,
Genes or DNA fragments encoding enzymes or proteins that promote the secretion of polysaccharides from untreated cell structures can be preferably used in the present invention.

The invention is illustrated by the following examples. The description of the examples should not be mistaken as limiting the scope of the invention in any way.

[0085]

EXAMPLES In the following Examples 1-21, the bacterial strains, plasmids and bacteriophages are listed in Table 2. Luria-Bertani medium and YM medium are used as standard medium (Po
Ilock, et al. , 1994, J .; Bacte
riol. , 176: 6229-6237). The dose of antibiotics (Sigma) is as follows. Bacitracin (Bac) is 73 units / mg or 0.01-8 mg / ml, especially in this example. Rifampicin (Rif)
Is 50 μg / ml. Streptomycin (Stm)
Is 50 μg / ml. Kanamycin (Kan) is 25
μg / ml. Chloramphenicol (Cam) is 15
μg / ml. Tetracycline (Tet) is 4-12
μg / ml.

[0086]

[Table 2]

Example 1 Construction of Sphingomonas DNA segment library
From the adult Sphingomonas strain, a clone of a DNA segment essential for the synthesis of sphingans was formed. A complete library of DNA segments was prepared as follows. The strain (S88 in this example) was placed in 25 ml of YM liquid medium and shaken at 30 ° C. overnight to prepare a mucous broth containing a large amount of cells. In YM medium, 3 liters of Bacto yeast extract per 1 liter of water
g, Bacto malt extract 3 g, Bacto peptone (Difc
o) 5 g and D-glucose (Difco) 10 g were contained. Sodium azide was added to 0.01%, and sphinganases (sphinganas) were added.
e) Enzymes (1994, Mikolajczak, et.
al. , Appl. Environ. Microbio
l. , 60, 402) was added at 37 ° C. for 8 hours to digest the sphingan exopolysaccharide to partially reduce the viscosity and increase the amount of cells. Centrifuge the cells, and use them in Birnboim and Doly, Nucl.
Acids Res. , 7, 1513 (1979) to resuspend the DNA for extraction. To the lysate (separate), an equal amount of a mixture of phenol: CHC ₁₃ : isoamyl alcohol (24: 24: 1) was added, and the mixture was gently shaken at 25 ° C for 16 hours, and then 1 volume of CH was added.
Add a mixture of C ₁₃ : isoamyl alcohol (24: 1),
It was gently shaken at 25 ° C. for 3 hours. In this way, the protein was removed from the clear lysate. 1/10 volume of 3 M sodium acetate (pH 5.2) was added, high molecular weight DNA was precipitated with 2 volumes of ethanol, dried, and then TE (10 mM Tris.HCl (pH
8) Resuspended in 0.5 ml of 1 mM EDTA).

Loftus, Foster and R
oss ( BioTechniques, 12, 172,
S88 according to the cosmid cloning strategy of (1992).
The DNA was partially digested with SalI enzyme and
Electrophoresis was performed on 1% low melting point agarose in acetate / EDTA buffer. Fragments larger than 20 kbp were purified by phenol extraction and ethanol precipitation. This S
The S88 DNA digested with alI was treated with Klenow DNA polymerase, dCMP and dTMP were added to the cohesive ends, and the mixture was heated at 70 ° C. for 20 minutes and then precipitated with ethanol. Vector plasmid pRK311 (1985,
Ditta, et al. , Plasmid , 13, 1
49-153) was completely digested with BamHI enzyme,
It was heated at 5 ° C for 15 minutes and purified by phenol extraction and ethanol precipitation. The BrkHI-digested pRK311 DNA was treated with Klenow DNA polymerase, added with dGMP and dAMP, and purified as described above. The molar amounts of vector and insert included in the ligation reaction with T4 DNA ligase were equal. All restriction enzymes, Klenow DNA polymerase, and T4 DNA ligase were obtained from Stratagene and the manufacturer's reaction conditions were used. After packaging in bacteriophage (Gigapack ^™ IIXL from Stratagene), the ligated molecule is transferred to E. coli DH5α ^™ by transfection,
Cells were plated on LB plates containing tetracycline at a concentration of 4-12 μg / ml. Each of the library of 1700 tetracycline resistant colonies and the library of 1400 tetracycline resistant colonies was pooled separately and frozen. Tetracycline resistant colonies (10 out of 10 tested) have SalI restriction enzyme sites inside,
It contained an insert of 25-30 kbp in length.

Similarly, NW11, S60, S19
A library of chromosomal DNA segments was prepared from various other Sphingomonas strains including 8, S194, and S7. In this case, the cells that were the source of the cloned DNA were grown in a medium containing 0.5% w / v or less glucose, and sphinganase treatment was omitted.

Example 2 Single DNA fragment for sphingan S-88 biosynthesis
Whether a gene essential for the synthesis of sphingan S-88 exists by observing whether or not the sphingan-negative mutant bacterium restores sphingan synthesis by screening a fragment of the DNA cloned in the isolated plasmid. It was judged. For some time, the inventors have found that most natural bacitracin-resistant mutants of the Sphingomonas S88 strain capable of growing on YM dishes containing 500-800 μg / ml bacitracin are unable to produce sphingan polysaccharides (Pollock,
et al. , 1994, J. Bacteriol. ,
176, pp. 6229-6237). Based on this finding, a simple screening method for this special class of mutant strains was completed.

The mutant strain S88m260 is the Sps ^-- Ba
It is a representative mutant of the ^cr group. The appearance of the colonies when S88m260 and other Sps ^- Bac ^r mutants were unable to produce exopolysaccharide was flat, and the surface was rough and translucent as compared with the wild type colonies. The wild-type colony was surrounded by a light-reflecting halo, which was narrow when viewed from below against the light. These phenotypic differences indicate that in X. campestris there is a vast array of mucoids in the wild type, while in the corresponding G
The um ^- mutant was less obvious than the difference with a flat appearance. The colony phenotype was confirmed by growing the culture in liquid YM medium, precipitating the exopolysaccharide with isopropyl alcohol, and dry weighing. Some Sps ^- mutants are sensitive to bacitracin,
It was later found that it has the characteristics of a gene that is essential for sphingan synthesis and that should be distinguished from the gene for the bacitracin resistance phenotype.

Plasmid DNA from gene library
And transferred from E. coli to Xanthomonas or Sphingomonas by the three-way mating method (Ditta, et.
al. , Proc. Natl. Acad. Sci. US
A , 77, 7347, 1980). Mob ⁺ Tra ^- recombinant plasmid (pRK311 with S88 insert)
Of the donor bacterium, containing Mob ⁺ Tra ⁺ pRK20
A mixture of helper cells containing 13 plasmids and cells of exopolysaccharide-negative recipients at a ratio of 5: 2: 10 was spotted on a glucose-free non-selective YM plate at 6 to 16 at 30 ° C. Incubated for hours. Then, in the plate, rifampicin (50 μg /
ml) and to select recombinant plasmids, tetracycline (4-12) for pRK311
μg / ml), or clamphenicol (20 μg / ml) for pSEB24. Onto these plates, one platinum loop of the above-mentioned hybrid mixture was developed, and a strain of Xanthomonas that received the plasmid was isolated. Since Sphingomonas is originally resistant to streptomycin, when Sphingomonas is the recipient strain, Streptomycin is used to select Sphingomonas for donor and helper cells.

[0093] strain plasmid was acceptance of the thus-obtained S88 strain, Sps ⁺ (raised round and opaque, when viewed from below by holding the light, surrounded by a bright ring) or Sps - ^(flat , Rough, semi-transparent colonies, no ring), and their complementarity was evaluated by the naked eye. Similarly, Gum ⁺ colonies grown from Xanthomonas that received the plasmid are mucoid in appearance, compared to the dull, non-shiny colonies of Gum ⁻ recipients.

The clone of S88 in the library was cloned into S8
I tried to identify directly in 8 non-mucoid mutants,
No success (because the phenotype of the mucoid could not be clearly demonstrated). Therefore, the library was once transferred to a non-mucoid mutant of the gumD gene of X. campestris and then switched to a method for finding a clone. This allowed us to find the initial clone "S88cl", which will be described in detail below.

S88 gene library from E. coli
Bac in the gumD gene^rGum ^-Is missing
X. campestris strain X59m31 (P
ollock, et al. ,J. Bacteria
l., 176, pp. 6229-6237, 1994;
Thorne et al. ,J. Bacteria
l., 169, 3593, 1987). In this intergeneric cross
Is some Gum⁺Tet ^rColony plays YM
However, this colony was about 10^-3-10^-Four
It was thought to appear at the frequency of. Is it a complementary mutant?
Each of the plasmids was purified and transferred to E. coli again.
Restriction enzyme analysis was performed. Sphingo the purified plasmid
When crossed with Monas S88m260, about 10% contact
The transponders are Sps
⁺It became. One plasmid (pRK311-S88
cl) was collected and used to continue further experiments.
How many more plasmids pRK311-S88cl
B separately isolated from S. sphingomonas strain S88
ac^rSps^-Mutants and Ba of X. campestris
c^rGum^-Complemented with mutant bacteria. Plas for each intergeneric cross
Exopolysaccharide secreted into the medium by the strain that received Sumid
Ride is isolated and then subjected to thin layer chromatography.
Acid hydrolyzate should be present in the polysaccharide of the recipient cell.
It was found that these contain polysaccharide residues of
It was confirmed to be a kind. Plasmid pRK311-S
88cl is sphingan synthetic in Sphingomonas
X. campestris uses xanthan gum synthesis
I revived each. These results show that plasmid pRK3
11-S88cl is Bac^rLoss of polysaccharide-negative mutants
The exopolysaccharide biosynthesis function
That a gene from one genus has been lost in another genus
It has shown that it was able to restore the function. Segment S8
8cl is the rightmost end of S88clΔ3 shown in FIG.
7.5 kbp HindIII fragment added to the end
It is the same as S88clΔ3, except that it is described.
From S88cl, this 7.5 kbp segment was specifically
Deleted to produce its derivative S88clΔ3
Was.

The method for measuring DNA fragments useful for the restoration of gum production (sphingomonas of Sphingomonas spp., Or xanthan gum of X. campestris) described above is reproducible, and in a separate experiment new clones were identified. Isolated. Sphingomonas S88 Sps
^- a segment that complements the Bac ^s mutations bacteria 76 and 78, when screened directly from clone library, three new clones overlap in S88cl partially formed. Each of these three segment clones had a length of about 23 to 27 kb. 3
Of the individual segments, two complemented the mutant S88m260. Maps of restriction enzyme cleavage sites are shown in FIGS. 1 and 6.

The above method is a DNA segment isolated from Sphingomonas strains S60, NW11, S198, S7, S194 and other sphingan-producing strains useful for increasing the production of each of these strains. Used to determine.

Example 3 DNA sequence of spsB gene and estimation of SpsB protein
Amino acid sequence plasmid pSEB24-S88E4.5 :: Tn # 72
Of the 3300 bp fragment subcloned from, the 1950 bp double-stranded nucleotide sequence of the spsB region was obtained. The sequence of the coding chain is shown in the sequence listing (SEQ ID NO: 1). One long open reading frame
ing frame (ORF), which is sps
I named it B. The coding region begins at the ATG at nucleotide 361 and continues to the TGA stop codon at 1771. This ORF encoded 470 amino acids with a putative ribosome binding site ahead of the ORF. The deduced amino acid sequence by the standard three-letter abbreviation is shown in the sequence listing (SEQ ID NO: 2).

Example 4 S88 segment clone and staining of S88 and S60
In order to prove that the DNA clone of the DNA-DNA hybridization plasmid pRK311-S88clΔ3 with chromosomal DNA is derived from the adjacent S88 DNA sequence, the plasmid S88clΔ3D was used.
NA is labeled and Sphingomonas strain S88, mutant S88m260, and wild-type gellan-producing S
Hybridization was performed with a DNA restriction fragment isolated from 60. About 1.5, 2.4, 4.5, 5.
Hybridization in the 9 and 12 kbp EcoRI fragments resulted in wild-type DNA and the mutant S88D.
It is consistent with the DNA clones of both NAs. Eco
Since one of the RI sites begins at the vector's multi-cloning site, the overlapping clones S88c2, S88c3, and S88c4 were EcoRI.
Digested with, the leftmost 6.6 kbp fragment that was proven to be S88clΔ3 was actually 1
It will be 2.8 kbp. The ability to form a hybrid between S88 DNA and S60 DNA, which produces gellan, suggests that similar gene sequences may exist but the gene composition may differ. Since the structures of exopolysaccharides secreted by these two Sphingomonas strains are similar, it is considered that they have similar transferase genes. S88-S
The 60 homologous regions are shown in FIG. A separate DNA homology analysis determined the location of the region of homology between strains NW11 and S88 as shown in FIG.

Example 5 of strains S60, NW11, S198, S7 and S194
Cloning of Sphingan Biosynthesis Gene Cluster DNA fragments were isolated from Sphingomonas S60, NW11, S198, S7 and S194. The method was the same as in the above-mentioned example regarding the strain S88.
Restriction site maps of the DNA fragments of strains S60, NW11, S198, S7 and S194 are shown in FIG.
This is shown in FIGS. 3, 9 and 10. The size of the restriction enzyme fragments generated by digestion of DNA with one or more restriction enzymes was determined as two independently isolated clones, pRK311-S194c1 and pRK311-S19.
4c2 will be mentioned here. This size was determined by comparing the mobility of the fragment by agarose gel electrophoresis to a fragment of known size, namely bacteriophage lambda DNA after digestion with HindIII, and Stratagene's "KbDNALadder". The size of the fragment is consistent with the DNA sequences contained in the above two clones.

C isolated from Sphingomonas S194
Plasmid pRK311-S1 containing 1 segment
The data of the fragment size of 94c1 is shown below.
The size of the fragment of pRK311-S194c1 is as follows. (EcoRI)> 25, 8.3, 5.4
And 1.5 kbp. (EcoRI + HindIII) 1
3.0, 9.7, 8.3, 5.4, 2.8 and 1.5k
bp. (HindIII)> 25 and 2.8 kbp.
(BamHI + HindIII)> 25, 5.8, 3.
9, 2.0 and 0.8 kbp. (BamHI)> 25,
5.8 and 4.7 kbp. (BamHI + EcoRI +
HindIII) 13.0, 8.3, 5.8, 5.4,
3.9, 2.0, 1.5 and 0.8 kbp. (BamH
I + EcoRI) 15.0, 8.3, 5.8, 5.4,
4.7 and 1.5 kbp. pRK311-S194c2
The fragment size of is as follows. (EcoR
I) 14.5, 10.0, 8.3, 6.1, 2.6 and 1.35 kbp. (EcoRI + HindIII) 1
3, 8.3, 5.7, 4.6, 4.0, 2.6, 1.
8, 1.35, 0.75, 0.35. And 0.25 kb
p. (HindIII)> 20, 12.8, 4.6,
2.1, 0.75 and 0.25 kbp. (BamHI +
HindIII)> 20, 3.8, 3.1, 2.8,
2.3, 1.65, 1.6, 1.3, 1.2, 0.9
5, 0.9, 0.8 and 0.25 kbp. (BamH
I)> 20, 5.8, 3.1, 2.8, 2.6, 2.
3, 2.0, 1.6 and 0.25 kbp. (BamHI
+ EcoRI + HindIII) 13, 8.3, 3.
8, 3.1, 2.4, 2.3, 1.6, 1.35, 1.
3, 0.95, 0.9, 0.85, 0.8, 0.45,
0.35 and 0.3 kbp. (BamHI + EcoR
I) 14.5, 8.3, 4.9, 3.1, 2.4, 2.
3, 2.0, 1.6, 1.35, 1.3, 0.85,
0.45 and 0.2 kbp.

Example 6 Constitution- specific replication and conjugal mating functions of plasmid pSEB26 , and a drug resistance gene suitable for Sphingomonas and compatible with mini-Tn10kan, and containing a multicloning site. Carrying the plasmids pSEB24 and pSEB
26 (FIG. 5) was assembled. While plasmid pSEB24 has a broad host range, pSEB26 can replicate in E. coli but not in either Sphingomonas or Xanthomonas. First, the Staphylococcus aureus plasmid pC194 (198
2, Horinouchi and Weisblue
m, J.M. Bacteriol. The Cam ^r on HpaII-Sau3A fragment of 1031bp taken from 150,815), are processed into blunt ends, plasmid pUC13 (1982, Vieira and Me
Ssing, Gene , 259) was ligated to the blunt end of XbaI. Remove the Cam ^r cassette from this plasmid on the BamHI-SalI segment,
After blunting, it was inserted between the unique SspI site in pUC12 and the closest of the two PvuII sites (also blunt-ended). In this way, Am
plasmid pSEB2 which is p ^r and Cam ^r and forms blue colonies when X-Gal and IPTG are added.
3 to construct pSEB24, pSEB23
From ScaI-PvuII fragment of about 2130 bp from pMMB66EH (1986, Fuerst
e, et al. , Gene , 48, 119) Sca.
Ligated with I-PvuII part, and oriV
(Wide range origin of replication from RSF1010) was retained and the Amp ^r gene was regenerated. Plasmid pNH-
Kan / oriT (Hengen and Iyer,
1992, Bio Technologies , 13:57 .
-2700 bp Hind containing oriT sequence
The III-BamHI fragment has a P of 3200 bp.
The pSEB23 plasmid linearized with vuII was ligated blunt ended to form pSEB26. Bam
Ba regenerated by ligation of HI-PvuII
The mHI site was removed by restriction enzyme treatment followed by capping of the sticky ends and religation.

Example 7 Introduction of a copy of the S88 DNA fragment into strain S88
Was specific restriction fragment of the DNA segment shown in Figure 1 isolated from increased production strain S88 polysaccharides S88 by the, after insertion into multicopy plasmid vector by DNA ligation,
The wild-type strain S was obtained by the three-way mating described in Example 2 above.
88 and S88 were transferred to non-mucoid mutant strains. Transfer of the DNA clone to the mutant bacterium restores polysaccharide synthesis. After culturing the bacteria in a liquid medium, the cumulative amount of sphingan polysaccharide produced by the recombinant plasmid-containing strain and the strain without the plasmid gene was measured.
Baffled flask
s) after shaking for 24 hours at 30 ° C. with shaking, then 2
A volume of isopropyl alcohol was added to precipitate the exopolysaccharide. Experiments were carried out by independently culturing each strain 2-3 times. The precipitate was collected on a filter, dried at 80 ° C and weighed. Table 3 shows the average weight of the precipitate and the standard deviation value for each strain. The sphingan S-88 production amount of the recombinant strain to which a copy of the gene clone was added was higher than that of the wild-type strain which had only a normal set of biosynthetic genes.

[0104]

[Table 3]

The restriction enzyme map, the nucleotide sequence of the spsB gene and the DNA surrounding it are standard recombinant DN.
It is clear to a person skilled in the art that the method A is sufficient information to form a myriad of new subfragments in the region of about 32 kb (FIG. 6). In addition, these new fragments were tested by the methods described herein,
It will also be apparent to those skilled in the art that segments that similarly increase sphingan polysaccharide production can be identified. From Table 3, it can be seen that this subfragment is a small fragment (eg pSE
B24-S88E12.8) It can be read that it does not show a sufficient production stimulating effect. However, almost all subfragments allow for increased production. From experiments to date, we have found that pSEB24-S88B8.
6 and pRK311-S88c3 are believed to have the greatest stimulatory effect on sphingan production.

This method of increased production does not require the presence of antibiotics in the culture for continuous selection of recombinant plasmids.

In this method of increasing production, one or more DNA fragments are inserted into a chromosome or a plasmid originally possessed by an endogenous fungus, and preferably a DNA fragment encoding a gene or genes of this chromosomal segment is inserted. To be achieved. Those of ordinary skill in the art will be able to obtain a DNA fragment containing a gene capable of producing a sphingan in an endogenous bacterial chromosome, a plasmid originally possessed by an endogenous fungus, or an exogenous plasmid vector. By inserting, it is easy to introduce the DNA fragment into a bacterium having a sphingan-producing ability. It is worth noting that the test results demonstrated in this application show that this increase in sphingan is not dependent on the use of a particular plasmid vector.

Within the scope of the present invention, any of the DNA fragments encoding the gene segment or genes described above, DNA sequences known to control the expression of genes, and without limiting the present invention For example, fusion with the E. coli lac promoter is included.

Example 8 Exopolysaccharide produced by the recombinant S88 strain was
Identification of Sphingan S-88 To confirm that the exopolysaccharide produced by the engineered strain is identical to the type of recipient strain, identification of monosaccharides in the acid hydrolyzate by thin layer chromatography did.

Extracellular xanthan of X. campestris and Sphingomonas sphingan S-88 were precipitated from 2-3 volumes of isopropyl alcohol, separated from the liquid medium, dried at 80 ° C. and weighed. Polysaccharides,
High-performance liquid chromatography (HPLC) water 5m
Resuspended at a concentration of g / ml. Trifluoroacetic anhydride (88 μl, Sigma) in a 0.6 ml microcentrifuge tube.
a Chemical Co.) was mixed with 75 μl of HPLC grade water (Baker) and 225 μl of polysaccharide (5 mg / ml) and incubated at 95 ° C. for 16 hours. The hydrolyzate was vacuum dried, resuspended in 200 μl HPLC water, placed in a new microcentrifuge tube, dried again and suspended in 45 μl HPLC water. Samples (25 μg / ml) were frozen and stored.
Sugar standard solutions (D-glucose, D-glucuronic acid, D-mannose, L-mannose, L-rhamnose and L-fucose) were resuspended in HPLC water at a concentration of 4 mg / ml. Coated and channeled (chan
nelled) silica gel chromatography plate (Kieselgel 60 CF254, 1020c)
m, E. Merck) over night with 0.3 M NaH ₂ PO.
After soaking in ₄ for 30 minutes at room temperature, then at 95 ℃ 1
Dry for 0 minutes. Spot 1-2 μl of the sample and leave it at room temperature for 2.5-3 hours 40 ml of acetone and 5 m of butanol.
The chromatogram was exposed to a solvent mixture consisting of 1 and 5 ml of deionized water to raise the mixture. Plate is 65 ℃
After drying for 3 minutes, acetone 25 ml, aniline 0.5 m
1, diphenylamine 0.5 g and phosphoric acid 3.75 ml
The sample was dipped in a solution consisting of 3 to dye and then dried at 95 ° C. for 30 minutes. When X. campestris was the recipient of the S88 DNA fragment, glucose, mannose and glucuronic acid were present. Sphingomonas strain S88
Is a recipient of the gumD gene of X. campestris, rhamnose, glucose, mannose and glucuronic acid were present in approximately equal amounts with S-88 exopolysaccharide.

Example 9 Sphingan S-88, S-60, NW-11 and S-
Strains S88, S60 and NW1 for production of 130
Stimulating effect of the DNA fragment obtained from No. 1 by using the DNA fragments obtained from strains S88, S60 and NW11 according to the general method described in Examples 5, 6 and 7 above Increased production of gellan (S60) and NW11. The results are shown in Table 4 below.

[0112]

[Table 4]

As a general method, a number of copies of the DNA fragment isolated using the techniques and methods described in the above Examples, particularly Example 7, were inserted to isolate a particular Sphingomonas strain. DNA fragments are used to increase the production of sphingans in different Sphingomonas strains (different from those produced by the original strain).

In the general method established in this experiment, a DNA fragment containing the genetic material essential for sphingan production of any Sphingomonas strain is inserted into a suitable plasmid vector or other means. In this way, a large number of copies of the plasmid vector having the DNA inserted therein are introduced into a bacterium having the ability to produce sphingan or a non-mucoid mutant strain of the same strain as or different strains of Sphigomonas from which the DNA was isolated. In this case, the Sphingomonas strain into which the DNA has been introduced may be either a sphingan-producing bacterium or a mutant strain of a sphingan-producing bacterium having no sphingan-producing ability. Under the same fermentation conditions,
The sphingan production of the Sphingomonas bacterium so engineered is generally greater than that of the non-mutant, non-mutant sphingan producer.

The following experiments demonstrate that it is routine to incorporate multiple copies of a DNA fragment into Sphingomonas strains and other bacteria.

Example 10 Lactose Consuming Gene on the Chromosome of X. campestris
(Lactose-utilization gen
The lactose obtained from transposon Tn951 flanked by a precloned DNA segment of X. campestris carried on a plasmid that is replication incompetent in X. campestris by standard cloning methods using the insertion restriction enzyme of e) and DNA ligation. A consuming gene was inserted (Thorne, et al., J. Indus .
t. Microbiol. , 3, 321, 1988).
This recombinant plasmid was then transferred by conjugation into X. campestris. In the recipient, the homologous DNA obtained from X. campestris, which flanks the lactose consuming gene, was recombined with the cellular DNA by normal homologous recombination, ligating the lactose consuming gene side-by-side with the bacterial chromosome. As a result, the cloned segment was stably inserted into the bacterial chromosome.
This is more fully explained in the above paper using a diagram. Besides the present invention, there are many examples in which a gene is inserted into bacteria by this method.

In the DNA recombination described above, a plasmid is used as a vector and exogenous DNA is used in advance.
It is worth noting that it does not have to be introduced into the bacteria. D
It is widely known that when a NA segment is carried on a bacteriophage or transposon, it is naturally inserted into a bacterial chromosome by site-specific DNA recombination. Usually bacteriophages are specifically 1-2
Although only inserted in three positions, transposons can usually be inserted in many, virtually any position. Further, it is not necessary to transport the DNA bound to a cloning vector such as a plasmid into the cell. DNA
It is well known that fragments can, upon transformation, enter bacterial cells while retaining the ability to recombine with cellular DNA.

Example 11 At several different arbitrarily selected positions,
The gene encoding syn resistance is Sphingomonas S8.
Site-Specific Insertion into E. coli DNA In this example, inserting a segment of cloned DNA into cellular DNA of Sphingomonas proves to be feasible. First, the S88E12.8 fragment (see FIG. 1) was ligated to the EcoRI site within the multiple cloning site of plasmid vector pSEB26 (FIG. 7).
Like pSEB24 shown in FIG. 5, the plasmid pSEB26 has an ampicillin resistance gene, a chloramphenicol resistance gene, a multicloning site, a Lac segment and oriT (see FIG. 5). However, the plasmid pSEB24 was found to have a broad host range ori
Although it has a V sequence, the plasmid pSEB26 has an origin of replication in a narrow host range.
Different from 22. The origin of replication of pBR322 allows replication of the plasmid in E. coli but not in Sphingomonas. Therefore, the plasmid pS
The only way that the DNA sequence cloned in EB26 will survive in Sphingomonas is the plasmid pSE.
Incorporating the plasmid pSEB26 into chromosomes or bacterial DNA such as endogenous plasmids before B26 disappears from cells or cultures. Next, the pSEB26-S88E12.8 plasmid thus obtained was
Transposon mini-Tn10kan by conventional method
(1991, Keckner, et al., 20.
4, pp. 139-180). Mutagenesis by rearrangement in the non-suppressive host HMS174 is associated with lambda bacteriophage NK1316.
Derived from Tn10 derivative 103 (mini-
Tn10kan / Ptac-ATStransposa
se). As a result, the kanamycin resistance gene was inserted into the plasmid. Also, several separate inserts of the kanamycin resistance gene at different positions within the S88E12.8 segment were isolated. Next, each of the recombinant plasmids thus obtained was transferred separately into Sphingomonas S88 by the three-way mating method to select kanamycin-resistant progeny. In virtually all cases, S88D flanks the kanamycin resistance gene.
The NA sequence was recombined with the recipient cell chromosome and the kanamycin resistance gene was integrated into the bacterial chromosome. By being integrated into the bacterial chromosome, despite the inability of the vector plasmid to replicate in Sphingomonas,
The kanamycin gene could survive.

This example shows that an exogenous DNA sequence (in this case the kanamycin resistance gene) can be introduced into the recipient strain and the transgene can be integrated into the bacterial chromosome. The location of integration is the D flanking the gene.
Determined by NA sequence. In this example, the exogenous gene was integrated at different sites within the E12.8 segment of strain S88. Integration by homologous recombination is a routine genetic manipulation method.

Example 12 Additional Experiment for Sequencing of DNA and its Analysis As shown in FIG. 6, the sequence between 1 bp and 28.804 bp of the BamHI site was determined for both strands of the DNA. The Sanger dideoxynucleotide chain termination method (Sanger, et al., 197).
7, Proc. Nat. Acad. Sci. , 74: 5
463-5467), exonuclease III and S
PBluescriptIIK using I nuclease
It was used to sequence the DNA cloned on S (+). Internal sequencing primers were also used.
In addition, Coral Software (San Di
ego) SuperClone and SuperSee
Program and Kyteand Doolittle
e method (Kyte and Doolittle,
J. Mol. Biol. , 157: 105-132), and the sequence of the transmembrane protein region was analyzed. Also,
The "blastp" program (Altschul, et.
al. , 1990, J .; Mol. Biol. , 21
5: 403-410) to identify homologous protein segments from a vast NCBI data library. Nylon Membrane and Genius ^™ 1 Kit (Boehr
(Inner Mannheim)
DNA hybridization was performed.

Example 13 Claw of gene involved in sphingan S-88 biosynthesis
Training This example was carried out in the same manner a part of Example 2. Since the present inventors have long been, most of the sphingan polysaccharide negative (Sps ⁻ ) mutants of Sphingomonas strain S88 have
It has been found that YM plates containing bacitracin can also grow (Pollock, et al.,
1994, J.I. Bacteriol. , 176: 622
9-6237). As a result of mapping experiments (described later), all of these mutants were classified into genes designated as spsB. The representative SpsB ⁻ mutants (260, 265 and 102w) listed in FIGS. 9 and 10 were also bacitracin resistant (Bac ^r ). Conversely, the bac8 strain
It was initially a representative strain selected as Bac ^r , but was also shown to be Sps ⁻ at the same time. Example 2
As described in Section 1, attempts to form sphingan S-88 in Sps ^- Bac ^r mutants were unsuccessful, and instead, colonies with a flatter, rougher surface and translucent appearance than wild type colonies All of the colonies obtained were surrounded by narrow halos that refracted the light when viewed from underneath. Since no exopolysaccharides that precipitate in alcohol did not appear, it was judged that the Sps ^- mutant strain cannot secrete the sphingans into the liquid YM medium. A small fraction of the Sps ^- Bac ^r mutant carried the second mutation. For example, mutant strain 1
The 02w colonies were white rather than yellow. The mutant strain 134 had a mutation not only in spsB but also in rhsD. Mutants 54 and 302 lacked not only spsB but also spsK. The Sps ^- mutation shown directly above the genetic map in FIG. 9 includes naturally occurring ones and those mutated by exposure to ultraviolet light or ethyl methanesulfonic acid. Other mutations tested in this example are:
All (prefixed with "Y" or "B",
, As shown in Fig.), And the transposon mini-Tn101k.
It was formed by the random insertion of an.

A broad range of cosmids, pRK311 and
Constructing a gene library from Ningomonas strain S88
The pooled clones were then transformed into E. coli by conjugal mating.
To S88m260. However, Sps
⁺10 colonies were screened^Three-10^Fourof
Tet^rAgain in the strain that received the plasmid
Was. Therefore, the library of X.
Campestris Bac^rGum^-(GumD) mutant
(Pollock, et al., 1994,J. Ba
cteriol., 176: 6229-6237, and
And Thorne, et al. , 1987,J. Ba
cteriol., 169: 3593-3600).
Moved. The gumD gene assembles xanthan gum
In the first step, glucose-6-phosphate is converted into UDP-glucan.
Required for transfer from course to isoprenyl-phosphate
(Capage, et al., October
 1987, International Paten
tWO87 / 05938 and Ielpi, et a.
l. , 1993,J. Bacteriol., 175:
2490-2500). X. Campestris gumD
The DNA segment containing the gene allows the sphinc
Gomonas Bac^rSps^-Mutant is Sphingan S-
Since the 88 synthesis could be recovered, the Sphingan S-88
Assembling starts with glucose and sphingomonas
The enzyme from X. Campestris Bac^rGum^-
We thought it might be possible to complement the mutant strain (Pollo
ck, et al. , 1994,J. Bacterio
l., 176: 6229-6237). From intergeneric mating
Is Tet on YM plate^rStrain that received the plasmid
10 ^Three-10^FourAt a frequency of about 1 per X. Campestri
Gum of Su⁺Colonies were observed.

By transformation with E. coli, the complemented X. After purifying the plasmid from the Campestris mutant, it was mated with Sphingomonas S88 mutant 260. About 5 to 25% of the strains that received the plasmid are Sp
It became s ⁺ . The vector alone (pRK311) had a 100- to 1000-fold higher frequency of transposition than the larger recombinant plasmid. Most recombinant plasmids produce large deletions when crossed with Sphingomonas, but pRK311-S88c
Only one restored polymer productivity without causing deletions and used this to continue the experiment. 1, 6, and 9
Also, in FIG. 10, the leftmost portion of S88c1 consisting of 21 kbp is shown as a subclone c1Δ3. The Bac ^r Sps ^- mutant strain of strain S88 and the X. Several Bac ^r Gum ⁻ mutants of Campestris restored polysaccharide synthesis. At each intergeneric cross, the polysaccharide that was secreted by the strain that received the plasmid into the culture medium was isolated, and thin-layer chromatography was performed. It was confirmed that it contained. Although the exopolysaccharide secreted by the Sphingomonas mutant strain 260 having the plasmid pRK311-S88c1 contained glucose, mannose and rhamnose, the X. The one from Campestris mutant m31 contained only glucose and mannose. Furthermore, the recovery of acidic sugars was consistently low due to the hydrolysis conditions, but each polysaccharide contained glucuronic acid.

Screen library for Sps
^- determined segment to complement the Bac ^r mutants 76 and 78, went to expand the region cloned in the left direction in FIGS. 9 and 10. Strains that received the plasmid 10 ⁴ to 10
⁶ were screened and four more clones (S88c2, c
3, c4 and c5) were obtained. Similarly, clone c6 was isolated by complementing Sps ^- mutants 43, 71 and 104. Each of these five cloned segments is 22-28 kb in length. At least one end of each segment is shown in FIG. Furthermore, a group of about 15 Sps ^- mutant strains that are not complementary to either clone c2 or subclone c1Δ3 was identified. Approximately 8 kbp more than c1Δ3 among these mutants
None was complemented by the c1 full-length clone, which spreads to the right for only long. Also, none of them was complemented by c6, which spreads to the left by about 18 kbp longer than c2 in FIG. Thus, the "unlinked" group 1 mutants are essential for sphingan synthesis but are not directly contiguous with the gene clusters shown in Figures 9 and 10, and Suggests the presence of the gene.

Example 14 Mapping of the sps Gene by Functional Complementation By complementation assay using Sps ^- point mutant strains as recipients of conjugation, spsG, spsK, spsF, spsD,
The boundaries of the spsC, spsE, spsB and rhsD genes were determined. The results are summarized in Figure 9. Subcloned small segment or larger DN
The recombinant plasmid carrying the A segment was exposed to an insertion mutation with mini-Tn10kan in E. coli and transferred by crossing strain S88 with a Sps ^- mutant strain. For this, pRK311 and pSEB24 (FIG. 5), which are wide range host vector capable of mating, were used. The strain that received the plasmid that received the drug resistance genetic marker of the carried plasmid was recorded as Sps ⁺ or Sps ⁻ depending on the appearance of the colony. Bac ^r
The Sps ^- S88 mutant was initially positioned as an E4.5 subclone, and then the E4.5 segment was mini-
Exposed to a random insertion mutation of Tn10kan. Position B2
31 and B230 (FIGS. 9 and 10) had no effect on mutant strain 260 restoring sphingan synthesis, but complementation was blocked when it was inserted into B233, B239 and B238. Mutant strain 134, complemented by segment B8.6, had E4.5 or E
It was not complemented by any of 5.9. Mutant 1
In 34, both the spsB gene and the flanking rhsD gene were deleted. B8.6 segment is mi
Exposed to mutations by ni-Tn10kan, B44
1, the pattern of complementarity by inserting into B440, B438, B437 and B435 was analyzed and rhsD
The more precise location of the mutation was determined. Mutants 54 and 302 were also considered to be double mutants lacking the spsK and spsB genes. The spsF mutants (62, 68 and 94 in Figure 9) are localized and segment B1
It was separated from the adjacent spsDCE because it was not complemented by 2.6 and c1Δ3, and clones c3 and c5 restored the synthesis of Sphingan S-88. Consecutive sp following insertion mutation of E6.6 fragment
The results of complementation of the sD, spsC and spsE genes are also shown in FIGS. 9 and 10. From this complementation result, a group containing mutants 76 and 78, and mutants 69, 72, b1
It was suggested that there are two groups, a group containing 04, 3, 9 and 41. Later analysis of the DNA sequence separated the latter group into two separate, contiguous genes, spsC and spsE. spsG mutations (11, 43, 71, 81 and 104 in FIG. 9)
Is the c6 clone and fragment B12.6 of B4.5.
Complemented by subfragments (FIGS. 9 and 1).
0). Due to the insertion mutation of the B12.6 segment, plasmids Y652, Y635, Y63
6, Y653, Y640 and Y641 were produced. Of these, only Y652 and Y641 were able to complement the spsG mutant.

Example 15 Insertion of mini-Tn10kan into chromosome and plasmid
Narrow-domain host Cam ^r plasmid pSEB26 (Figs. 9 and 10) capable of crossing a segment of the phenotypic S88 DNA clone.
Ligated into E. coli and mini-Tn10k
After exposure to the insertion mutation with an, it was transferred to the wild-type (Sps ⁺ ) Sphingomonas strain S88 by conjugation. Since these plasmids are not replicable in Sphingomonas, recombination with the bacterial chromosome is required to maintain the Kan ^r gene. Kan ^r and C
This group was selected only because the recombinant, which is am ^s , does not carry the plasmid sequence. Although the physical structure of these DNA substitutions has not been confirmed, by always using the same plasmid, strain and selection method, a site-specific chromosomal deletion is generated, and restriction mapping and DNA hybridization are performed. We confirmed these double recombination. Colonies of recombinant Kan ^r Cam ^s chromosomes were determined to be Sps ⁺ or Sps ⁻ (inserts are shown in FIG. 9 with the prefix “c”). Mutants showing Sps ^- phenotype (cY776, cY757, cY77
1, cY770, cY676, cB589, cB58
3, cB580, cB579, cB300, cY72
6, cY725, cY676, cY673, cY721
And cY602), it is reasonable to believe that a double recombination has actually occurred. However, in the Sps ⁺ recombinant that was recombined at one site in the homologous region, the entire plasmid was integrated into the chromosome, and therefore, there is a possibility that one gene is defective and one gene remains normal in that chromosome. There is.

In order to eliminate uncertainties in recombination at two sites in the homologous region, after making a large site-specific deletion in the chromosome, the entire DNA segment of S88c2, which was singly inserted with mini-Tn10kan, or , S88c3
A replicative plasmid carrying any of all the DNA segments was introduced to inactivate some genes. The insertion position and the Sps ⁺ or Sps ⁻ phenotype resulting therefrom are shown in FIG. 9 with the prefix “p”. The chromosomal and plasmid mutation strategies were consistent with each other, and both essential ( ^- ) and nonessential ( ⁺ ) regions were observed.

Example 16 DNA Sequence: G + C Content, Rare Codon Usage and Translation Opening
Both strands were measured for the DNA sequence of the starting sequence 28,804 bp (see SEQ ID NO: 3). The average profile (and standard deviation) of the typical Sphingomonas genes in this cluster was calculated by comparing the G + C content distortion, the rare codon frequency, and the “Shine-Dalg
"arno)" sequence or translation initiation sequence.
(Table 5). Each gene was characterized by a high G or C frequency at the position of the third codon. Earlier in this study, the rhsACBD operon codon, and s
2,500 codons of the psB, D, C and E genes were analyzed to identify a group of rarely used codons in the genus Sphingomonas. The ratio of each rare codon present in this group is 0.2% or less, and the group composition is A
GA, AGG, CGA, TGT, GGA, ATA, CT
A, TTA, TTG, AAA, TTT, CCA, CC
Included T, AGT, TCA, TCT, ACA and ACT. In Escherichia coli, translation is usually initiated from the downstream downstream of a sequence complementary to the 3'end of 16S rRNA (Shaine and Dalgarno, 2).
974, Proc. Natl. , Acad. Sci. U
SA, 71: 134). S. Pouch Mobilis DSM10
The "Shin-Dalgarno" homologous sequence complementary to 98 16S rRNA is TAAGGAGGTG (Mo
ore, et al. , 1993, Lett. Appl
i. Microbiol. , 17: 115-118).

If the genes in this cluster match the mean gene profile and are mutated to give rise to the Sps ^- phenotype, then the very name "sps" can be given. However, our protein similarity search failed to yield significant suggestions for the function of the spsG, I and F genes. Furthermore, although there were four open reading frames (ORFs) that satisfied the typical gene profile, no significant similarity could be found for the protein sequences in the computer databank. However, even if these putative genes were mutated, they did not visibly change the polysaccharide synthesis. Therefore, the abbreviation "Urf" was added in the meaning of unidentified frame. There are four Urf sequences (32, 26, 31 and 34), named according to the size of the putative protein in kilodaltons.

[0130]

[Table 5]

Example 17 Glycosyl-IP transferase spsB gene
Most of the Sps ^- mutations isolated after exposure to ultraviolet radiation or chemical mutagens identified as were on the spsB gene. It is believed that this SpsB protein catalyzes the first step in assembling Sphingan S-88, as the deduced amino acids of SpsB are so similar to other gene products that are believed to encode glycosyl-IP transferases. FIG. 11 shows glycosyl-I
Alignment of amino acid sequences predicted to be P transferase and galactosyl-IP transferase. The C-terminal halves of these proteins are quite homologous to each other. Although the N-terminal region does not have this much homology, the SpsB protein has several hydrophobic regions suggested to be transmembrane domains (underlined portion in FIG. 11), so the SpsB protein has S ． Enterica RfbP protein (Jiang,
et al. , 1991, MolecularMicr
biology , 5 , 695-713). The hydrophobic domain of SpsB is amino acids 35-59.
(+2.2 average hydropathy), 68-86 (+1.
7) 105-123 (+2.3) and 282-303
(+2.9) is included. Also, the position of the last hydrophobic segment of the SpsB protein is common to these related gene products and is located in the middle part of the protein, following the region of greatest homology.

The spsB encoding the domain deduced from the DNA sequence was confirmed by complementation assay. E4.
From 5 varying the insertion of mini-Tn10kan in the segment, Bac ^r Sps ^- was observed whether interfering with complementary mutants. mini-Tn10kan
The insertion site and the "+" or "-" as a result of complementation are shown above the spsB gene in FIG. Mi of three parts
For insertion of ni-Tn10kan, Sps ^- mutant S8
It was not possible to restore sphingan synthesis to 8m260 (B233, B239 and B238). On the other hand, when it was inserted into several adjacent sites including B231 and B230, the lost function could be restored.

Example 18 Sphingomonas S88 rhamnose biosynthesis operon The deduced amino acid sequence of the protein encoded by the rhsACBD gene is dTTP and glucose-.
Synthesis of dTDP-L-rhamnose from 4-phosphate in 4 steps, S. Enterica Group B enzymes and X. It is very similar to the campestris enzyme (Figs. 12 to 1).
5). We have found that glucose-1-phosphate, rhs
Thymidylyl transferase encoded by the A gene
se) and the sequential catalytic steps encoded by the rhsB, C and D genes, conventional nomenclature was used. However, the Sphingomonas operon is unique in the following four points. First, the order of genes ACBD
→ is S. Enterica (BDAC →), X. Different from any of Campestris (BACD →). Second, there is almost no intercistronic region. The start codon and the stop codon overlap, or the spacing is narrow, r
hsA-ATGA-rhsC-TGATCCATG-r
It is hsB-TGATG-rhsD. Third,
The average G + C content of rhsACBD is relatively high (66
%), Especially high at the position of the third codon (89%),
Moreover, it is uniform throughout the operon. Fourth, the G + C content is similarly high in the surrounding genes in this cluster, as well as in genes of other species unrelated to Sphingomonas.

Initially, the mutant strain (# 1
34) Only one was isolated, but at the same time it appeared that a second mutation had also occurred in the spsB gene. The Rhs ^- mutation appeared to be potentially lethal. Therefore, it was tested whether specifically manipulating a single mutation within the rhs cluster would block sphingan synthesis. First, an S88 mutant strain was constructed in which the chromosome was largely deleted across the spsD gene from end to end of the rhs gene (ΔTn365 in FIG. 10), and it had lost DNA and had mini-Tn10k at a specific site.
The plasmid in which an was inserted was introduced. Cells remained Sps ⁻ when the insertion position of the plasmid was at the spsD, C, E, B or rhs operon. However, Urf32, 26, 31, 34, atrD or a
When inserted into either trB, the cells became Sps ⁺ without interfering with complementation of the deletion mutation.

Example 19 Sphingomonas S88 Glycosyl Transferers
Ze spsQ, 3 genes spsK and spsL can be seen as encoding the glycosyltransferase. However, the glycospecificity of glycosyltransferases is limited by the limited local homology of the proteins to other glucosyltransferases and rhamnosyltransferases.
It could not be determined by sequence analysis alone. As others have noted, glycosyltransferases are also quite different from a single bacterial-derived enzyme that attaches to the same sugar in the same bond (Glucks).
man, et al. , 1993, J .; Bacteri
ol. , 175: 7045-7055). Estimated spsQ
The gene product is orf11 (Stevenson, et al., 1) adjacent to the gnd of E. coli K-12, which is believed to encode rhamnosyltransferase.
994, J.I. Bacteriol. , 174: 4144
-4156), and in addition to R. Merilochi Exo
O and ExoU glucosyltransferase (Reu
ber and Walker, 1993, Cell ,
74: 269-280). The spsQ gene is essential for Sphingan S-88 synthesis. S88
The mini-Tn10kan was inserted into the spsQ gene in the plasmid having the c2 segment (Z206 in FIG. 9), and the mutated plasmid was cloned into spsGSR.
It was introduced into S88 cells lacking the chromosome of the QI gene. Recipient cells were viable but polysaccharide synthesis was blocked. The putative spsL gene product was similar to the spsQ product by comparison by Dodd matrix analysis, and was also S. Enterica rhamnosyl transferase (Rt
bN) and Yersinia pseudotuber
culosis also had some local similarity to the putative abequosyl transferase (Liu, et al.
l. , 1995. J. Bacteriol. , 177:
4084-4088). A search for spsK-like proteins was performed and only a slight similarity was observed, the overwhelming majority being glycosyltransferases that commonly contain a putative UDP binding site. This includes UD
It is suggested that it is a glucosyl or glucuronosyl transferase because P may be involved. Mutants 54 and 3 without transposon insertion
Similar to 02, the mutant strain (pY882 in FIG. 9) in which the spsK gene was specifically inserted was viable, but could not produce polysaccharide.

Example 20 Secretion of Polysaccharides by Bacteria Since the sequences of essential gene products are similar, a common mechanism for secreting polysaccharides by various bacteria is conceivable.
In this sequence comparison (Table 6), spsD, spsC, s
It has been shown that as many as five sps genes, psE, spsJ, and spsS, may be involved in the secretion of sphingans. R. Table 6 summarizes the sequence relationships of proteins for which considerable information has accumulated, such as the "exo" protein of Meriloti. However, functionally related proteins are a larger population than those shown in this table. Sps consisting of 51, 29 and 22 amino acids
Each of the three different segments of the D protein had 29%, 31% and 36% identity with ExoF, respectively. The start and stop codons overlap (TGATG), and the spsC and spsE genes that are continuous with each other are
It encodes a protein that resembles two different domains within oP. The similar SpsC-ExoP sequence contains a motif (P
X ₂ PX ₄ SPKX _II GXMXG) was included (B
ecker, et al. , 1995, Mol. Mic
robiol. 16: 191-203). 92, 30
And three segments of SpsC of 19 amino acids are 22%, 30% and 42% identical, respectively, to sequences of similar order taken from the N-terminal half of ExoP and consist of 75 and 98 amino acids. The two segments of SpsE are 32% and 2 in the C-terminal half of ExoP.
9% identical. The three segments of SpsS consisting of 37, 20 and 44 amino acids were 38%, 55% and 23% identical to ExoT, respectively. Estimated Sp
The sJ proteins shared a putative nucleotide binding domain with the KpsT, BexA and ABC carriers and had some similarity to each other. The spsR gene is not required for sphingan synthesis, but its gene product was slightly similar to bacterial and fungal polysaccharide lyases. Therefore, the spsR gene may be important for releasing glucuronic acid-containing sphingans from either the cell or the surface of the matrix, or for reusing the polymer as a carbon source.

As shown in FIG. 9 and FIG. 10, spontaneous point mutations and insertion of mini-Tn10kan into the spsD, spsC, spsE and spsS genes were viable, but sphingan S- was added to the culture supernatant. 88 did not accumulate. On the other hand, R. Mutation of a similar gene in Meliroti (Harding, et al., 1993, J. G.
en. Microbiol. , 139: 447-45.
7), and X. Mutations in similar genes in Campestris were lethal. mini-T on the spsJ chromosome
Inserting n10kan results in Sps ⁻ . However, the spsJ harbored in the multicopy plasmid in the mutant strain with a large deletion of the chromosome was
Insertion of Tn10kan resulted in either Sps ⁺ or Sps ⁻ .

[0138]

[Table 6]

References for the role of secretion of each protein ExoF, ExoP, ExoT (Becker et.
al. , 1995, Mol. Microbiol. , 1
6: 191, Horinouchi and Weisb
lum, 1982, J. Am . Bacteriol. , 15
0,815, and Reuber and Walke
r, 1993, Cell , 74: 269) GumB, GumC, GumJ (Glucksman).
n, et al. , 1993, J .; Bacterio
l. , 175: 7033, Becker et a.
l. , 1995, Mol. Microbiol. , 1
6: 191 and Glucksmann, et a.
l. , 1993, J .; Bacteriol. , 175:
7045) KpsD and KpsT (Wunder, et a.
l. , 1994, J .; Bacteriol. , 176:
4025 and Smith, et al. , 199
0, Mol. Microbiol. , 4: 1863) BexD, BexC, and BexA (Kroll,
et al. , 1990. Mol. Microbio
l. , 4: 1853)

Example 21 ABC Transporter of Lytic or Toxic Protein Two contiguous genes, atrB and atrD,
It was located within the ps cluster. These two genes are thought to encode the ABC transporter of a lytic or toxin-like protein, and a transport auxiliary protein. Until now, the hemolysin gene (hlyA) has been re-established as Sphingomonas. Pseudomonas pauc ( Pseudomonas pauc)
immobilis ). Our strain did not detect the definitive hemolysis phenomenon on agar plates containing sheep red blood cells, so the abbreviation "hly" was not used this time. Approximately 48% of the amino acids deduced from the DNA sequence of the atrB gene account for Bordetella pertussis ( Bor).
It was identical to the amino acid of the cyclolysin ABC carrier of detella pertussis . The atrB gene products are the HlyB protein of E. coli and the Pasteurella haemolytica ( Pasteurella ha) that transport hemolysin and leukocyte toxin, respectively.
and very LktB protein of emolytica) was well similar. In addition, the C-terminal half of atrB is R.
It was similar to many other ABC transporters, including the C-terminal half of the Merloti NdvA protein and the two repeating ATP-binding domains in the human multidrug resistance protein Mdr1. The sequence of the atrD gene product is H.
lyD protein and P. It was similar to the sequence of the Haemolytica LktD protein. Unlike related transport genes from other genera, Sphingomonas atrB and atrD
There were no lytic or virulent analogs flanking the gene or present within the sps cluster.

CONCLUSIONS : Mutual genetic complementation of a polysaccharide negative mutant of one genus with DNA of a second genus was first demonstrated between Xanthomonas and Rhizobium (Borthak).
ur, et al. , 1988, Mol. Gen. Ge
net. , 213: 155). In this early study, it was observed that mucus was restored on agar plates. In later years, X. It was reported that complementation occurred between the gumD gene of Campestris and the spsB gene of Sphingomonas strain S88 among the genera (Pollo).
ck, et al. , 1994, J .; Bacterio
l. , 176: 6229), the present inventors have also demonstrated by composition analysis that the recipient bacterium restored exopolysaccharide synthesis by the complementary gene of the donor bacterium.

The experiments of the present application demonstrate that the spsB gene encodes a glucosyl IP-transferase, which is an important step in sphingan biosynthesis. Therefore, D to be incorporated into the recipient according to the present invention
The NA fragment preferably contains a gene encoding a glucosyl IP-transferase enzyme, which may be glucosyl-transferase, galactosyl-transferase, or a related IP-transferase. For example, Sphingan S-88
The first step to assemble the
It is believed most likely to be a transition to. Therefore, it is advantageous to include a gene encoding an enzyme that promotes this biosynthetic reaction in the DNA fragment according to the present invention.

According to the studies conducted by the present inventors, dTD was found in the large sps gene cluster of Sphingomonas S88.
It has been demonstrated that there is a smaller operon (rhsACBD) that encodes the biosynthesis of PL-rhamnose. r
The sequence similarity of the hs operon and other rhamnose operons suggests that Sphingomonas has the same four enzymatic steps in the synthesis of L-rhamnose, and the rhs operon is essential in DNA fragments. It proves that.

Just as many invading pathogens are capsulated, various sphingan exopolysaccharides can also be construed as protective in nature. Further, exopolysaccharides are also considered to play a role of attaching cells to the substrate. Other strains of Sphingomonas sph
Since there is a gene cluster that is organized similarly to the S88 cluster described in detail in the present specification, it can be seen from the results described in the examples of the present specification that a person having ordinary skill in the art can obtain practical use from all Sphingomonas bacteria. Can be used for
A fragment can be obtained.

DNA encoding a genetic information useful or indispensable for biosynthesis of sphingan is isolated from a donor bacterium, Sphingomonas sp.
By inserting into a bacterium of a recipient bacterium, a bacterium having no sphingan-producing ability or a bacterium having only a normal producing ability (a recipient bacterium) can be easily produced as a sphingan gum mass-producing bacterium. Shown by disclosure. Mass-producing bacteria can be easily obtained, and the method can be generally applied to any strain of the genus Sphingomonas as long as it has a sphingan-producing ability.

In some of the embodiments of this invention, the spsB of S88 encoding a glycosyl-IP transferase (the first step in assembling a sphingan carbohydrate).
DNA fragments obtained from a donor bacterium containing a gene or other DNA fragment such as are advantageously used in the present invention. Other embodiments of the invention, particularly sugar synthons or building materials in which rhamnose is important for the formation of the polysaccharide (eg,
In the case of Sphingan, S-88, in the case of Gellan,
S-60, and in the case of Welan S-130), in order to maximize the production of certain sphingan polysaccharides by the mass-producing bacterium of the present invention, the rhamnose operon or gene (S- 88 rhsABC
(For example, D gene) is preferably used. Furthermore, genes or other DNA fragments encoding glucosyltransferases, such as spsQ, spsK and spsL of Sphingomonas strain S88, or genes or other DNA fragments encoding the final polysaccharide formed for secretion (eg, Sphingomonas strain S88, including spsD, C, E, J and S), can be used to advantage to contribute these enzymes to the final polysaccharide production. Furthermore, other DNA encoding all of the above enzymes or functions
Fragments or genes can also be used to advantage, depending on the desired polysaccharide.

Those skilled in the art can easily obtain DNA fragments according to the disclosure of the present specification, and incorporate these fragments into a recipient bacterium to produce a sphingan mass-producing bacterium. Should be noted. These mass-produced bacteria can be easily produced using known simple genetic manipulation methods.

[0148]

[Deposition of Microorganisms] The following six microorganisms are located at 1231 Parcrown Drive, Rockville, Maryland, pursuant to the Budapest Treaty on the International Recognition of the Deposit of Microorganisms in Patent Procedure,
American Type Culture Collection (Ame
rican type Culture Collec
has been internationally deposited. All restrictions on the availability of deposits will be abolished with irrevocable conditions at the same time as the grant of patents for deposits. The third to bottom microorganisms are publicly available from the American Type Culture Collection in Rockville, Maryland.

[0149]

[Table 7]

The examples and embodiments described above are intended to illustrate the invention by way of illustration and should not be considered as limiting the invention in any way. Various modifications and changes can be made to the matters and objects described in the present invention by a person having ordinary skill in the art, but these are all considered in the present invention in advance and are within the technical scope of the present invention. Included within.

[0151]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1950 Sequence type: Nucleic acid Number of strands: Unknown Topology: Unknown Sequence type: Genomic DNA sequence AGCCCGAATG CTGCATCCGC GAAGTGACTT TCGCCAAAGC AGCTATAGGA TGGCCCGGGG 60 CTTGACAGTC GCCGTGGTATCGGTCATACTCATGTGTCGACCCAT ATGC 180 CCCATGCGCC GCTGACTCTG GTAGATTGAC CGTGGCCTCC ATTGCTCATC GTCTCGAAAA 240 AGGACCCTCT GGTCGCCGCG CGGACTTCCG GGAATCGATT TGTCCCGTTA TAGTGCAATG 300 CAACAGGCCG AATCGGCCGC TGTCAGCGTG CACAATCCGT TGAGGGAGCC CGACGAGGCA 360 ATGAACGCTT TTGAAGCACA GCGCGCCTTT GAGGAGCAGC TCCGGGCCCA TGCCCGTTCT 420 GCCCCCAGCG CCGCACCCAT GCTGCGACGT TCCACGATCC GCATGATCCT CTACACCGAA 480 TTGCTGTTGC TCGACAGCAT CGCAATTCTA CTGGGGTTCT ACATCGCGGC CTGCTCGCGC 540 GACGGCAACT GGCTGTCCCT TGCGGGCGTC AATGTCGGCA TCTTCCTCCT GCCGATCACG 600 CTCGGCACCG CGCTCGCCAG CGGCACCTAT TCGCTGAGCT GCCTGCGCTA CCCGGTCAGC 660 GGGGTGAAGA GCATCTTCTC GGCGTTCTTC TTCTCGGTGT TCATCGTGCT GCTGGGCAGC 720 TACCTGCT CA CCGCGGAGCT GCCGCTGTCG CGCCTGCAGC TCGGCGAGGG CGTGCTCCTG 780 GCGCTCAGCC TGGTGACGAT CTGCCGCCTT GGCTTCCGCT GGCACGTTCG TGCGCTGACA 840 CGCGGCACGC TGCTCGACGA GCTGGTGATC GTCGACGGCG TTGCCCTGGA GGTCGCGAGC 900 GGCGCGGTCG CGCTCGATGC GCGCATCATC AACCTCACGC CCAACCCGCG CGATCCGCAG 960 ATGCTGCATC GCCTCGGCAC CACCGTGGTG GGCTTCGACC GGGTCGTCGT CGCCTGCACC 1020 GAGGAGCACC GGGCAGTATG GGCGCTGCTG CTCAAGGGCA TGAACATCAA GGGCGAGATC 1080 CTCGTCCCCC AGTTCAACGC GCTGGGCGCG ATCGGCGTCG ACTCCTATGA GGGCAAGGAC 1140 ACGCTGGTCG TGTCCCAGGG CCCGCTCAAC ATGCCGAACC GCGCAAAGAA GCGGGCGCTC 1200 GATCTGCTCA TCACCGTCCC CGCGCTGGTC GCGCTGGCGC CGCTGATGAT CGTGGTCGCG 1260 ATCCTGATCA AGCTGGAGAG CCCCGGCCCC GTCTTCTTCG CACAGGACCG CGTCGGCCGC 1320 GGCAACCGAC TGTTCAAGAT CCTCAAGTTC CGCTCGATGC GCGTTGCGCT CTGCGATGCG 1380 AACGGCAACG TCTCGGCCAG CCGCGATGAC GATCGCATCA CCAAGGTAGG CCGGATCATC 1440 CGCAAGACCA GCATCGACGA GCTGCCGCAG CTGCTCAACG TGCTGCGCGG CGACATGAGC 1500 GTCGTCGGCC CGCGCCCGCA CGCACTCGGG TCGCGCGCCG CCAACCATCT CTTCTGGGAA 1560 ATCGACGAGC GCTACTG GCA CCGCCACACG CTCAAGCCGG GCATGACGGG CCTCGCGCAG 1620 ATCCGCGGCT TCCGCGGCGC GACCGATCGC CGCGTCGATC TCACCAATCG CCTGCAGGCG 1680 GACATGGAGT ATATCGACGG CTGGGACATC TGGCGGGACG TCACCATCCT GTTCAAGACG 1740 CTGCGCGTGA TCGTGCACTC CAACGCCTTC TGATCGCGGA GGGGAGCAAC GCGAGCACCG 1800 CTTGGTGCAA GAGCATTGAC ATCCGCCCTG CTTCTGCATT TGTCATTTTA TCATTGTCGT 1860 TGCGGGCCCG CCCGCGCCAT GGGGGATTTT GAATGAAGGG TATCATCCTT GCGGGGGGCA 1920 GCGGCACGCG CCTCTACCCC GCAACGCTGT 1950

SEQ ID NO: 2 Sequence length: 470 Sequence type: Amino acid Number of chains: Unknown Topology: Unknown Sequence type: Protein Sequence Met Asn Ala Phe Glu Ala Gln Arg Ala Phe Glu Glu Gln Leu Arg Ala 1 5 10 15 His Ala Arg Ser Ala Pro Ser Ala Ala Pro Met Leu Arg Arg Ser Thr 20 25 30 Ile Arg Met Ile Leu Tyr Thr Glu Leu Leu Leu Leu Asp Ser Ile Ala 35 40 45 Ile Leu Leu Gly Phe Tyr Ile Ala Ala Cys Ser Arg Asp Gly Asn Trp 50 55 60 Leu Ser Leu Ala Gly Val Asn Val Gly Ile Phe Leu Leu Pro Ile Thr 65 70 75 80 Leu Gly Thr Ala Leu Ala Ser Gly Thr Tyr Ser Leu Ser Cys Leu Arg 85 90 95 Tyr Pro Val Ser Gly Val Lys Ser Ile Phe Ser Ala Phe Phe Phe Ser 100 105 110 Val Phe Ile Val Leu Leu Gly Ser Tyr Leu Leu Thr Ala Glu Leu Pro 115 120 125 Leu Ser Arg Leu Gln Leu Gly Glu Gly Val Leu Leu Ala Leu Ser Leu 130 135 140 Val Thr Ile Cys Arg Leu Gly Phe Arg Trp His Val Arg Ala Leu Thr 145 150 155 160 Arg Gly Thr Leu Leu Asp Glu Leu Val Ile Val Asp Gly Val Ala Leu 165 170 175 G lu Val Ala Ser Gly Ala Val Ala Leu Asp Ala Arg Ile Ile Asn Leu 180 185 190 Thr Pro Asn Pro Arg Asp Pro Gln Met Leu His Arg Leu Gly Thr Thr 195 200 205 Val Val Gly Phe Asp Arg Val Val Val Ala Cys Thr Glu Glu His Arg 210 215 220 Ala Val Trp Ala Leu Leu Leu Lys Gly Met Asn Ile Lys Gly Glu Ile 225 230 235 240 Leu Val Pro Gln Phe Asn Ala Leu Gly Ala Ile Gly Val Asp Ser Tyr 245 250 255 Glu Gly Lys Asp Thr Leu Val Val Ser Gln Gly Pro Leu Asn Met Pro 260 265 270 Asn Arg Ala Lys Lys Arg Ala Leu Asp Leu Leu Ile Thr Val Pro Ala 275 280 285 Leu Val Ala Leu Ala Pro Leu Met Ile Val Val Ala Ile Leu Ile Lys 290 295 300 Leu Glu Ser Pro Gly Pro Val Phe Phe Ala Gln Asp Arg Val Gly Arg 305 310 315 320 Gly Asn Arg Leu Phe Lys Ile Leu Lys Phe Arg Ser Met Arg Val Ala 325 330 335 Leu Cys Asp Ala Asn Gly Asn Val Ser Ala Ser Arg Asp Asp Asp Arg 340 345 350 Ile Thr Lys Val Gly Arg Ile Ile Arg Lys Thr Ser Ile Asp Glu Leu 355 360 365 Pro Gln Leu Leu Asn Val Leu Arg Gly Asp Met Ser Val Val Gly Pro 370 375 380 Arg P ro His Ala Leu Gly Ser Arg Ala Ala Asn His Leu Phe Trp Glu 385 390 395 400 Ile Asp Glu Arg Tyr Trp His Arg His Thr Leu Lys Pro Gly Met Thr 405 410 415 Gly Leu Ala Gln Ile Arg Gly Phe Arg Gly Ala Thr Asp Arg Arg Val 420 425 430 Asp Leu Thr Asn Arg Leu Gln Ala Asp Met Glu Tyr Ile Asp Gly Trp 435 440 445 Asp Ile Trp Arg Asp Val Thr Ile Leu Phe Lys Thr Leu Arg Val Ile 450 455 460 Val His Ser Asn Ala Phe 465 470

[0153]

[Sequence Listing] SEQ ID NO: 3 Sequence length: 28804 Sequence type: Nucleic acid Number of chains: Unknown Topology: Unknown Sequence type: Genomic DNA sequence GGATCCACTG GCCGGGAATT GCCGAGAATC CTCCGATGAA GCGCTCGTCG GGTACCAGCG 60 TGCCCCGGGG CGCATCGCTT TGCGCCGGTG CATCGCGCCGGCGTCCCCGC120C ATCCCCGGGC CTGCCTTTAC GCCATGCCCG GCAGCCGAGC 180 TGCCGGGCGC CGAGCATGCG AGCGGCGTAA CCGATAGGGC GAGGCCCCCG CCCAGAAGGG 240 TGCGACGTGT GGTATCGATC ATGCGGCGCG CTCCAAACCG TGCGCGCCGT GACTACAACC 300 AAAAATGCTG CGCTGCGAGC GGGATCAGGC GCCCCGTGCC TGCTTCGAGC GGTACAGCAG 360 CGCGAACGTC AGCCCCACCA GCATGAAGAA GACTTGGTCG TTGTCGGTCT GCGACAGCAC 420 GAGCCTGGTA TTGAGCAGCA CGACCATCGT CGTCGCGACC GCCAGATGCA GCGGATAGCC 480 TTGGGAGGGG TCCGTCAACC CGGCGCGGAT CAACAGCCCG GCACCCAGCA CCATCGTACC 540 GTAGAATGCG ATGAAGCCGA GCACCCCGTA ATCGACGGCC GTCGAAAGGA AGCCGGAGTC 600 GATCGACAGG AACCCGCTCT GGGAACGCCA TCCGACGACC TCCGCGGACT GGAACGGCCC 660 GTAGCCGAAT ACCGGGCGCA TCGCGAGCTT GGGCAAGCCC ATGCGGATCT GCTC GTGGCG 720 CCCGTCGTTG CTCGCCTGGG TCGCGCCGCC GCCAAGAACG CGATTGTGTA CCGCAGGCAC 780 TACCATGATC ATCACCGCGA GAACCACGGC GAAGGCCGGA TACATCATCG TCGTGGAAAT 840 CCCGACGAGC CCGCCACGCT CCTTGATCCA GCGCCGCAGG CCCCAGAGCA ACAGATAGGT 900 GGCATGCGCC ACGACCATGC CGACCATGCT CAGGCGCGCG CCGCTCCAAT AGGCGGACAA 960 TACCATGGCG AGATCGAACA GGATCGTGAG TGCCAGCGCC GACACCGACC GGCTGTTCAC 1020 CATCAGGTGG ATCGCGAAGG GAATCGTCAT CGCCACGAGT TCGCCCCACA CCAGCGGGTT 1080 CCCGAACACG TTCATCACGC GATACGTGCC GCGCACCTGC GAGGTGAGAT GCAGGATGAC 1140 GCTCGGATCG TTGATCTGCA GCCAGCTGGG AATGTGGCCG ACCCACAGAA CGTGCTCGGC 1200 CCGGAACTCG AAGAAGCCGA TCACCATCAG CACGGACACG CAGCCCAGCA TGTTCCGCAC 1260 CCACCATTCG GGTGTGCGCG TGTTCGATCC CAGGCACCAC AGCGTCGCGA AGAAGAACGG 1320 CGTGACCGTC AGCGAGATAT TCACCAGGCG CCCGATCGAA ACGGATGGCT GGCTGGAAAT 1380 GAGCGACGCG ATGATCTGGA TGATCAGGAA GCCCAGCATG AAGCGGGCAA GCCAGGGCGA 1440 CGCCGACAGC GTCACCGCCA TGTCGCGCCG AAACTTCGGC GAAATCGAAT AGCACACCAG 1500 CAGAAGAAGC GTCGTCAGCA CGCCGAACAG GCGGCGGAAG GAGATCCAGG GCAGGCCCGC 1560 CACCGACAGC GACAGATAGT TCGGCCACAC GATCGCGAGG ATCATGAACA GGACGTAGCA 1620 GCGCAGCAGC AACTTGGTGG GCGCCTTGTC GGCCTCCGGG AGCGCCCAGA TGACGAACAG 1680 CGCGAGGATC GCCAGCGGCG CGGCGGCCCC GAGGAGCATG CTGGGCGGCA GGATCGCCGA 1740 AAGCAGCCCG TAGACCATCG ACACGAACAC GATCACGGCG AGCCCGATGA AGCGCCGCCC 1800 GAGCGTGACG AGACCAGAGC GTTGCGGGTG ATAGAGCGGG AGCACCGCTC TGGCGGGGAA 1860 GAACACGATG TCGCGCGCCC GGCGCAGGGG CTGCACCACC CGCGCCAAGC CGCCGCTCCC 1920 CCGAACTCGC GCCGATGTCG CCATGACCAA CCCCTTAGAT AATCGGTATG CCGATCAGCC 1980 GCACCGCGAC CATCGACACG AAGCGCAGGA AGACCGACGG CACCGCGATC GCAATCGCCG 2040 CGCCTAGTGC ACCATAGGGC GGAATCAGGA CCAGCGCGAG TATTGCGGCA AGGATAACCG 2100 ACGACATGGT CAGCACCACG GCCAGACGCT CGCGATTGGC CATGACGAGG ACGCCGCCGC 2160 TCGACGCGAA GACCATCCCG AACACCTGCC CAAGCACCAG CACCTGCATC GCGGCGGCGC 2220 CCGCGGTGAA CTGTTTGCCG AACAGGCCCA TGATCCAATG CGGAGCGACC AGCACCGCCA 2280 GGGCGATGGG CGAGGCGGCG ACCAGCAGCG CGAGAATGGT GATCCGGATG ATGCGGGCGA 2340 TCCGCTTGAC GTCGCCCTGT TCGTAGGAGG CGGCAAAGAC CGGATGCAGG ATCGTCTCGG 2400 AGGTG GCCGA CAGCAACTTG AGCGAGGATG CGATCTGATA GCCCACCCGG AACAGACCGG 2460 CTTCGGCGGG GCCGTGCGTC GCGGCAAGGA TCACGGTGGC AAACCAGTCG ACGAAGAAGT 2520 TGTTGACGTT GGTGATCAGC ACCATGAAGC CGGGGCGAAG CATCGGCCGG TCCAACGGCT 2580 CGGCCGGCGC CCAATCACGC GTCATGCGGC GGACGATGAT CGTCGCGGCA AACATCGTCA 2640 CCAGCCAGCC GACCAGGTAC AGCACCGACG GCAGCAGCGG ATTATGGGCA ACGCCGATCA 2700 GCAGCGCGCC GGCCAGCATC GCCCCACCCA GGAAGGTGCC GAGCGGCCCA TCGACCATCT 2760 GCGACTTGCC GATATCCCCC ATGCCGCGCA GCGTCGTCGA AGCGAGACGG CAATAGGCGC 2820 TGACCGGAAT GAGAAACCCC ATGATCAGAA GGTCCGGCGC CATGGCGGGG CTGCCCAGCA 2880 GGTTGGTGGC AATCTGTTGG TGAAACAGCA GGATCATCAC CATCAGGACC AGGCCACCAC 2940 CCACCGCGAC CCGCGTGGCA TGCCGCACTG CGGTACGCGC CACACCCGTC CGATTTTGCG 3000 ACACGCAGAC GGCCACGGTG CGCACCAGGA TGGTATCGAG GCCGATCAGC GACAGAATGA 3060 CCAGCATCTG CGCAGTCGTG AGCGCCGTAC CGAAGGCACC GACGCCGGCG GGGCCAAAGG 3120 CGCGGGCGAC CAGCCAGGTG AAAGCGAAAC TGGTGACGGC GCCGAAGCCC TTGACGCCGA 3180 AGCCGACCAC CATCTGCCCC CGCAGCCCCC GCAGGTGCAA CTTGCTACGT GTCACGTTGA 3240 ATGCTTGCCC CACAGGAGAT CCCGTCTGTG CCTTATGGCA GGGCCCTCCC GGGGGCAAGC 3300 CTGAGGACGT CATCAGACGT GATAGAAGTC CTGCACCAAC TTCTTGGTGG CGAACAGGCT 3360 ATTCGCCACG GACAGGCTGC CCGTCGCCGA GACGGCCGCA GTGCCGGCCG CATTCATGGC 3420 GATCGCCTGG GCGAGCGACA CTTGCGCGAC GGACGCCGTC GATGCCGATC CCCCCAGCGT 3480 CAGCGTGCCG GTGGTCGCCG CCGGCAGCGC CGTCGACGTG ACCGGGGTGC CGAGAATGGT 3540 TACGGCGCTG GCGGCCAAGC TGCTGGTGAG GCTGGGCTTC ACGGTGGTGG TCGGCTGGCT 3600 GGCGGCGGTC GCCGCGGCAT TCAGCGCAAG GATCTGGGAC GCACTGAGGG CAGCGTCGCG 3660 CATCTCGATC TCGCCCACGC TGCCGCTGAA GACAGCGTTG AACGGGCTGC CGATGTACAG 3720 TCCGGCATAT TCGACCGCCC GCGTGCTGCC GACGATCGTT CCCGATCCCT TCACCACGCC 3780 ATCGACATAG ATGATCGCCT TGCCCTTCGC GCTGTCATAG GTCAGCGCGA TCTTGTGGGT 3840 GGCCGTGTCG GTCATCTTGG CGCCGCTCGT CGCGACGGTA TAGCTCTGCC CGGCGGCATT 3900 CTTGACGGTG AAGACCAGTT CGCCGTCCGC CCGGAGCGAG ATTCCCCAGC TCTGGTTGAC 3960 GCCCATGATC TGGCCGACCG CGCCCGTCGC GGTGGCACGC TTCATGTCGA AGTTGAGCGT 4020 GAAGGCGGGC AGCGCGAAGA GTTGACGTGA ATTGTCCCGC GTAAGCTCGA AGCCGGTGCC 4080 GGTCTTCACC TGGAAC ATGC CGTTGCTGAT GGCGGTGAGA TCCAGCGCCT TCGTGGTCTC 4140 GTCCGTGCTC CAGCGCGTCT GGTCCACGAT TCCGGTCGCA GTGAACTGCA GATCCAGCAG 4200 CAGGTTGGCG CCGGTCGAGG TCTGTGCTGC CGCCTGCTCC TTGGCGACCT GCGCGGCAAA 4260 CGCGCTGCCT GCAGGCGGCT GATACCCGAC ACCACTGACG ATCAGGTTCG CCAGTTGCGC 4320 CTTCGATCCG GCCATGAGAT CGCCGATCTT GCGAAGAGTG ACCGCGTCCG TTGCAAGCAC 4380 GGCGTTGTTC GATTGAGTAA TGCCGCTCGA CGTTGCGGTG ATGACAACCT GGTCCACGAC 4440 ATTGTTGGTG ACCTTGCCGC CGGTCACGCC GTCCAGGCGG ATCCAATCGG CGATCGCATC 4500 CATCTTCGAG ATGATGGTAT TGGAGTCCAC GGTGACGTTC TTGCCCAGAA CGACATTGAT 4560 GCCGTGCGTG AAACCATTCT GGTACACGAG ATTGTTTTTG ATCGTGATGT TTTCGTAGGG 4620 AATGCTGGAT TCATTGCCCA TGAATACGCC CTGGAAGGCC AGGCCGTCCC CCTGCATCAT 4680 CACGTTATTG GTGATCGTGA TGTTCGTGTT GCCCTTGGTC TTGCCGTTCG TCATGAACTG 4740 GATGGCGTCG GGATGCTCAC CATTCACCGG ATAGAGGTTG GTGAACATGT TGTTGTCGAT 4800 GACGACGTTC GACGCTTCGG CGAAATTGGT GTGATCGCGG CGATTGTCGT GGAAGTTGTT 4860 GCCCTGCAGG GTGACACCGT CGACGGTGAG GACGTTCATC CCCAGGGCGA AATGATCGAC 4920 CGAGGAATTC TTGATCGTCA C CCCCTTGCT TTCTCGCAGC AGAAGCCCCC AGCCCATCGA 4980 CTTCGTCACA TCGCCCGTAC CCCCGCTCAG GGTCACGCCG TCGATCACGA CATTGCTGGA 5040 GCCGATGATC CGGTTCGCGT AATTATAGTC CTGTGCCGGC TGGAAGTTTT GTGCGGCCGT 5100 GACGTTCTTC ACCACCAGGT TGCTGCTGTT GATGATCTGC AGGGTCGTCA CATTCACCGG 5160 CTTGCTCGCA TCGAGCGAGG TGATCGTGAC GGGCGTGGTG AAGGTCGTGG TGTGCACGGT 5220 GATGGACGTA TAGGTCCCCG CCGCAAGCTT GATCGTCTCG CCCCCTTTCG CAGCCTTGAT 5280 GGCGGCGTCC AGTTCGCTCT GATTCCTCAC GATGATGTCC GGCATGTACT CTACCCTCGT 5340 TACGCGTCGA CCCCAATCGA CCTGCGATCC CTCGGACCGT CTTGTACCTG CCAAGCCCTG 5400 AAACGGTGGC TAAGAGGCAG GGTTAATGCC CTGTTTTTCA AGCCGATAAC TGGCAGCCCT 5460 CAAGGCACTG CCAGCGTGCG GGCAACACTC TCGACGCCGC AGTGCAGCAC GGGTAAGAAC 5520 GAGGCATGGA AGCCTCGCCC ACACCCGACG TCAGCATCCT GGTGGTTGCC TACCACTCGG 5580 CTCCGTTCAT CGGACAATGC ATCCGGGGCA TCGCCGCGGC GGCACAAGGC ACAGCCCACG 5640 AAATCCTGCT GATCGACAAT GGCGGCGGCG ACACCGAGGC GGTGGTTCGT GCCGAGTTCC 5700 CGCACGTGCG GATCGTGCCG AGCGAGGGCA ATATCGGCTT CGGGGCGGGG AATAACCGGT 5760 GTGCGGCCCA TGCCCGCGCG CCGCGGC TGC TGCTCGTCAA CCCCGACGCC ATTCCCCGCC 5820 CCGGCGCGAT CGACCTGCTG GTCGCCTTCG CCAAGGCGCA CCCGGACGCG GCAGCCTGGG 5880 GCGGGCGTTC CTATTTTCCG AACGGCCAGC TGGACCATGC CAACTTCCTC CCGCTGCCCA 5940 CGGTGCGCGA TTTCGTCGTG TCGATCTTCA GCAGCAGCCC GATGCGGCGC GGCGGCCTTC 6000 CTGCCGACGC CACCGCGCCC GGGCCGGTCG AGGTGCTCAA CGGCGGCTTC ATGATGGTCG 6060 ATGCCCGCGT GTGGCGGGAG ATCGACGGCT TCGACGAAGG CTTCTTCCTC TATTCGGAGG 6120 AAATCGATCT GTTCCAGCGG ATCCGCGCGC GGGGCTATTC CGTGCTGGTC GATCCGGCTG 6180 TGGGCGTGGT GCACGACACC GGTGGCGGGC ATTCGCTCTC GCCCACTCGC GTGCTGTTTC 6240 TCACCACCGG CCGCATGCAT TATGCCCGCA AGCATTTCGG CCACGTCGGT GCCGTCGTGA 6300 CGGGCTGGGC ACTGTGGGCC AATGCCGCCA AATATGTCGT TATCGGCGGC CTGCTCGGGC 6360 GCCTCTCACC CCGCCGCGCG GCGCGCTGGA ACGCGCTGCG CGATGCCTGG AGCATCGTGT 6420 TCGGCCAGCC GCGGCGCTGG TGGCACGGCT GGCGCGACCA CGTTCGTACT TGAGGATAGC 6480 GCCGCGCCAG ACGGCCCGAA ATGGCAACCC GACGCAAGGC GGAAGGCTTG CCGACGGCAA 6540 GCCCCCCGAC TTGTCGCTCA CTGCGCGGCG TTGGGCGCCG GAGCAGGGGC CGCAGCAGGC 6600 GCGGCGGCAG CGCCGCCCTG CAGTTGCGGC GG CGGGCTGT AGCCCGGCTG ATATTTCACC 6660 GACTCGCGCG CCTTCTTCAG ACGATCGTTC AGCTGCGCGT CCGCCGCCTT GCTGAACCGC 6720 TCGGTGCGCA GCGTATTGAG CGCGAGTTCG CGCGCCTGAT CGCCCGCCAG CGGCTGGATC 6780 GTCGTGCCGG TGATGACATT GGCGGTGACG CCCTGCTGCG TCGGCAGGAT GAACAGCTCC 6840 TGCGCCGGCA GCGCCGCAAT CTTGGCGGCG ATCTCCGGCG GCAACGCGGC GGTGTCCAGC 6900 TGGGTCGGCG CGCGGCGGAA CTGCACGCCG TCGGCGGTCA GCTTGGCGGC AAGCTGGTCC 6960 AACGTCTTGA GCGGCGCGAA TTCCTTGAAC TTCGCCGCCG AGCCGGGCGG CGGGAAGACG 7020 ATCTGTTCGA TGCTGTAGAT CTTGCGCTGC GCGAAGCGAT CGGGATGCGC CGCTTCATAT 7080 TGCGCGATCT CGGCATCGGT CGGCTGGGCG ATGCCGCCGG CAATCTTGTC GCGCAGCAGC 7140 GTGGTGAGGA TCAACTCGTC GGCGCGGCGC TGCTGGATCA GGAAGACGGG GGTCTTGTCC 7200 AGCTTCTGCT CGCGGGCGTA CTTCGCGAGA ATCTTGCGCT CGATGATGCG CTGCAGCGCC 7260 ATCTGCTCGG CAAGCTTGCG GTCGGTCCCC TGCGGCACCT GCGTGGCCTG CACTTCGGCA 7320 TTCAGTTCGA AGATGGTGAT CTCGTCGCCG TCCACGCTGG CGACGACCTG CCCCTTATCG 7380 AGCTTGCCTT CCTTGCTGCC ACATCCGGAG ACGGCCAGCG CGGCCGCAGC CACCGCCGTT 7440 ACCAGGTACA ATTTCTTCAT GAAGACCTCC CAGCCGGC AC GGAATTGCGC ACGGCACAAA 7500 CTTCTACTTG AACCTATTCG GGCGGGCGGG CATCCGCAAT AGCGTTGGCA GTGCAGCATG 7560 CCTCCCGGCG GGAGGCAGGC GGGATCAATG GGGGACGGCA TGGCAGAAGC GACGGTGACC 7620 GAAGCGAAGG CGGGCAAACC GCTGAAAATG TGTCTCGCAG CTTCCGGCGG CGGCCATCTG 7680 CGGCAGATCC TCGATCTGGA ATCGGTCTGG AAGGAACATG ACTATTTCTT CGTGACCGAA 7740 GACACCGCGC TGGGCCGCAG CCTTGCCGAA AAACACTCGG TCGCGCTTGT CGATCACTAT 7800 GCCCTCGGCC AGGCCAAGCT CGGCCACCCG CTGCGCATGC TGGGAGGCGC CTGGCGGAAC 7860 CTGCGGCAGA GCCTGTCGAT CATCCGCAAG CACAAGCCCG ATGTGGTGAT CTCCACCGGT 7920 GCGGGCGCGG TCTATTTCAC GGCGCTGCTC GCCAAGCTCT CGGGCGCAAA GTTCGTCCAC 7980 ATCGAAAGCT TCGCCCGGTT CGATCATCCT TCCGCCTTCG GCAAGATGGT CAAGGGCATC 8040 GCGACCGTGA CCATCGTCCA GTCCGCCGCG CTCAAGCAGA CCTGGCCGGA TGCGGAGCTG 8100 TTCGATCCCT TCCGCCTGCT CGACACCCCC CGCCCTCCCA AGCAGGCACT CACCTTCGCC 8160 ACCGTCGGTG CCACCCTGCC CTTTCCGCGG CTCGTGCAGG CCGTGCTCGA TCTCAAGCGG 8220 GCCGGCGGGC TGCCGGGCAA GCTGGTGCTG CAATATGGCG ACCAGGACCT GGCCGACCCC 8280 GGCATCCCCG ACGTGGAGAT CCGCCGGACC ATTCCCTTCG ACG ACCTCCA GCTGCTGCTG 8340 CGCGACGCGG ACATGGTGAT CTGCCACGGC GGCACCGGAT CGCTGGTCAC CGCGCTGCGC 8400 GCCGGCTGCC GCGTCGTCGC CTTCCCGCGC CGCCACGATC TGGGCGAGCA TTATGACGAT 8460 CACCAGGAAG AGATCGCGCA GACCTTCGCC GATCGCGGCC TGCTCCACGC CGTGCGCGAC 8520 GAGCGCGAAC TGGGCGCGGC AGTGGAGGCC GCCAAGGCGA CCGAGCCGCA GCTCGCCACC 8580 ACCGATCACA CGGCGCTCGC CGGCCGCCTG CGCGAGTTGC TGGCACAGTG GAGTGCCAAG 8640 CGATGAGCGC GCCGCGGATC AGCGTCGTCA TCCCGCACTA CAATGATCCG GACTCGCTGC 8700 GACAATGTCT CGATGCACTG CAGCATCAGA CGATCGGGCG AGAGGCCTTC GAGATCATCG 8760 TCGGAGACAA CAACTCCCCC TGCGGCCTGG CGGCAGTGGA AGCCGCCGTA GCCGGGCGCG 8820 CGCGGATCGT CACGATCCTG GAGAAGGGCG CCGGACCGGC GCGGAACGGC GCCGCGGCGG 8880 AAGCGCAGGG CGAGATTCTC GCCTTCACCG ACAGCGACTG CGTCGTCGAG CCCGGCTGGC 8940 TGGCCGGGGG CGTCGCCCAT GTCGCCCCGG GCCGCTTCGT CGGCGGCCAC ATGTATGTGC 9000 TCAAGCCGGA AGGGCGACTG ACCGGCGCGG AAGCACTCGA GATGGCGCTG GCCTTCGACA 9060 ATGAAGGCTA TGTTCGCCGT GCGAAGTTCA CCGTCACTGC CAATCTGTTC GTCATGCGGG 9120 CCGATTTCGA GCGCGTCGGC GGATTTCGTA CCGGAGTCTC GGAAGATCT G GAATGGTGCC 9180 ACCGCGCCAT CGCCACGGGT CTCGCGATCG ACTACGCCCC CGAGGCCTCG GTAGGCCACC 9240 CGCCCCGGCC GGACTGGGCA ACGCTACTGG TCAAGACGCG GCGCATCCAG CGCGAGCTGT 9300 TCCTGTTCAA TATCGAGCGC CCGCGCGGCC GGCTGCGCTG GCTTGCGCGC TCGACGCTGC 9360 AGCCTGCGCT GATTCCGGCG GATACCGCCA AGATCCTGCG CACGCCCGGC ACCCGCGGGT 9420 CCCGTATAGC TGCCGTCGGC ACGCTTGTCC GCCTGCGCTT CTGGCGCGCT GGCGCCGGCC 9480 TCCTGCAACT GCTCGGCAGA CCAATCTGAT GAAGGCGGGG CGGCCATGGT GCGGCGCCCC 9540 GTCTCCTGTC CTCACACCGC CGCGAGCGCC TCTTCCAGCG TCCCGCTGTC GATCCGCAGG 9600 CGTCCCACCA TCAGCCAGAG ATAGACGGGC AGCGAATCGT CGTTGAAGCG GAAGCGGCGC 9660 TCCCCGTCCT GCGCATCGCT CTCCAGGCCG AGCTGGCGGC TCAGCGCGTC GAGTTCCTGC 9720 TCGACCTGCG CCGCAGTGAT CGTGCTCCCC GGCAGCAGCT CGACGACTGC CTGGCCGGTG 9780 AACCAACCAT CGGTCGAACG CGACGCCTCG CCCAGCGCGG CGACCAGCGG ATCGTAGCGA 9840 CCGCCGACGA ACTTGCGCAT CTCCAGCACG GCGCGCGGCG ACATCCGGCC TTCTATTTCC 9900 AGGATGGCCT GGTCGAGCGC GCGGCGCAGA TGGCCCAGAT CGACGGTCAG CCGCCCCTGG 9960 TCGAGCGCCT CGAGCGCCGC ATGGTGGCAC AGCAGCCGCG CGAAATAGGG CGAC CCCAGC 10020 GCCAGCAGGT GGATGATCCG GGTGAGGTTC GGATCGAAGC GCAGGCCCGA GGCGGTCTCG 10080 CCGAGCGCGA TCATCTCCTG TACCTCGGTT TCCTCGAGCC GCGGCATCGG CAGGCCGATG 10140 ATGTTGCGGC GGATCGAGGG TACGTAGCCG ACGAGTTCCT GCAGGTTCGA CGAGACGCCG 10200 GCGATCACCA GCTGTACGCG CGCGGAGCGG TCCGAGAGGT TCTTGATCAG TTCGGCGACC 10260 TGCTGGCGGA ACCGGGTATC CGTCACGCGG TCATATTCGT CGAGGATGAT CAGAACGCGG 10320 GTGCCGGTGA TGTCGGCGCA CAGATCGGCG AGTTCGCCCG AATCGAACGA TCCGGTCGGC 10380 AGGCGATCGG CGAGGCTTCC GCCCGATTCC GCCTCGCCCG CATTGGGCGA GACGCCGCGA 10440 TGGAACAGCA GCGGCACATC CTCTAGCACC GCGCGGAACA GGTCGGCGAA GTTGGCATTG 10500 GCGCCGCAGG TCGCGTAGCT GACGATGTAG CTGGATTCAC GCGCCACGTC GGTCAGCACA 10560 TGGAGCAGCG AGGTCTTGCC GATGCCGCGC TCGCCATAGA GCACGACATG GCTGCGCTGG 10620 CTCTCGATCG CCGAGATCAG CCGCGCCAGC ACCTCGAGGC GACCGGCAAA GCTCGAGCGG 10680 TCCGCCACCG GCTGGGTGGG CGTGAAGAAG GTGGCGAGCG CAAACCGCGC GCGGGTGATC 10740 TCGCGACGCT CTTCCCGGCG CCGGTCGAGC GGGCGATCGA GCGCGGAAGC GCGAAAGGTC 10800 GGAAAGTCGG GTCGCCCGCG GCCCGCATGC GCGTCGCGAT GGGGAAC GAC GGTGGCGGCC 10860 AGCGGGAAAT ATCCGTCCTC CTCCGGTACG TCCCGACGCC CAAAGGGCCA CAAGAACTTC 10920 AGCGCGGATC CTACAGCCAC TCGAACACCT CTTAATTTCG GACGCCGCCA CGCTCGGCAG 10980 CGAACCCCTG GTTCGCGCCT TCTGGCGCCT CCCCCAAACG ATCCGGCCCC GCCTGTATCA 11040 GCGGCGCTTG AAAAACTCGT ACGGTTTGAT CACGAACGCA ATGTACGCCA GCACCAATAC 11100 AATCGTGAGG ATTGCGAAAA CATGATAGTT TTCGTTCCCG AGATAATTGG CGACGGCACA 11160 TCCGACCGCG GGAGGCAAAT AGCTGATCAT CGTGTCGCGC ACTACCGAAT CCGCCTGGGA 11220 TCGTTGCAAG AAGATCACGA TCAGGCCGGC GAATATCGCG ATGGTCACCC AATCATAGGG 11280 CGTCTGCATG CATGTCCTTT CTTTTCGGCG CCGGAATCGA AGGACTTCCG ACGTCGCCCG 11340 AACCGCACTA GCAGCGGACG GTGCAACTCG CTAGATACCG CGGTGCAGGA TAAAAGCTCG 11400 TTAAAACGCG ACCCTAGGAA TAGCGCGGTA GCGCCGGCAT GCGAGAGGTC GGGCATGCGG 11460 AAGGCCGAAG CGGCCGGGAC AGCACCGGAT GGGAGGATAT TCCCGTAGTG GGAGTGGCGA 11520 GGCCATGGCA TCCTCAGATC CGGTTGCTTG TACTGGAGGC CATTGATAAT GAAGCCAGGA 11580 CCCGGGGGAA CATTCGTGCC AGTAAAAGAC GTTCAGCAAG CGGTAGAAGT GCGCCTCGGC 11640 GATCGTGTCT CGCGATCGTG CCGCGTGCTC GCGCTGCTTG CGACGGCAAC GGCGATCCAG 11700 CCCGCGCTCG CGCAGCGACA GGCGTTCACG CCACGCCCGA GCGGCAGCGA GCGCCAGATC 11760 AGCGTGCATG CAACGGGACA GCTCGAGTAC AACGACAATG TCGTGCTCAA CGACCCGCGC 11820 ATCACCAGCG GCGCGCGCGG CGACGTGATC GCCTCCCCCT CCCTCGATCT GAGCATTGTC 11880 CTGCCGCGCG CGACCGGACA GCTCTATCTC GCGGGCACGG TGGGCTATCG CTTCTATCGT 11940 CGCTACACGA ACTTCAATCG CGAGAATATC TCGCTCACCG GCGGCGGCGA CCAGCGGATC 12000 GCGTCCTGCG TGGTGCATGG CGAAGTCGGC TATCAGCGCC ACCTGACGGA CCTGTCCAGC 12060 GTCCTCGTCC AGGATACTGC GCCCGCGCTC AACAACACGG AAGAAGCGCG CGCCTATTCC 12120 GCGGACATCG GCTGCGGGTC CGCCTACGGC CTGCGCCCTG CACTTGCCTA TTCGCGCAAC 12180 GAGGTTCGCA ACAGCCTCGC CCAGCGCAAG TTCGCCGATT CCGACACCAA CACGGTCACT 12240 GCCCAGTTGG GCCTGACGTC GCCGGCGCTG GGCACCGTGT CGGTGTTTGG ACGCATGTCC 12300 GACAGCAGCT ACATCCATCG CACGGTACCG GGGGTCAGTG GCCGCGACGG CATGAAGAGC 12360 TATGCGGCCG GCGTCCAGCT CGAGCGGGCG GTCTCCAGCC GGCTGAATTT CCGCGGCTCC 12420 GTCAATTATT CGGAGGTCGA CCCCAAGCTC GCCTCGACGC CGGGCTTCAG CGGGATCGGA 12480 TTCGATCTGT CGGCGGTATA TTCGGGCGAT CA ATATGGCG TGCAGCTCCT TGCGTCGCGC 12540 AACCCGCAGC CCTCCACGCT GCTGTTCGTA GGCTATGAAA TTGTGACGAC CGTGTCGGCA 12600 ACGGCAACCC GTAAGCTGAG CGATCGGACC CAACTCTCGC TACAGGCCAC CAAGACCTGG 12660 CGCGAGCTTG CCTCTTCGCG GTTGTTCACT CTTGCGCCGA CGACGGGCAA CGACAACACG 12720 CTGACGCTGT TCGGCACCGT GAACTTCCGA CCCAATCCTC GGCTGAACTT CTCGCTGGGT 12780 GCGGGCTATA ACAAGCGCAC CAGCAATATT GGGCTGTATC AATACCGCTC CAAACGTATC 12840 AATCTCACGA CGTCGCTGTC GCTCTGACAA GGGCCGTATT CATGCATGAC AAACACCGTT 12900 TCGTGATCCT TTCGGCGCTC ACCGGAATTG CCGTACTCGC CGCGCCCGCG GCAGCGCAGA 12960 TTCCCACCCG GTCCGTTCCG ACGCCGGCGC GGGCGCGCCC GGCGACCCCG CCAGCGGCCC 13020 CGCAGCAGCA GACGACGGCA GTGCCGACAA CGGCAGCCAC CGCCACCCCG CCGGCTGCGG 13080 GTGCGGCGCC GGCCGGCTAC AAGATCGGCG TCGACGACGT GATCGAGGCG GACGTTCTGG 13140 GCCAGTCGGA CTTCAAGACC CGCGCGCGCG TGCAAGCGGA CGGTACCGTC ACCCTTCCCT 13200 ATCTCGGCGC CGTGCAGGTA CGGGGCGAGA CCGCCGTCAC GCTGGCCGAG AAGCTCGCCG 13260 GCCTGCTGCG CGCGGGTGGC TATTACGCGA AGCCGATCGT CAGCGTCGAA GTCGTCAGCT 13320 TCGTCAGCAA CTATGTGACG GTGCT GGGCC AGGTGACCAC GGCCGGCCTG CAGCCGGTGG 13380 ATCGCGGCTA TCACGTCTCG GAGATCATCG CGCGCGCCGG CGGCCTTCGC GCCGATGCGG 13440 CCGATTTCGT GGTGCTCACC CGCGCCGACG GCACCAGTGC CAAGCTGAAC TACAAGCAGC 13500 TGGCCCAGGG CGGCCCGGAG CAGGATCCGG TGGTCACGCC TGGCGACAAG CTGTTCGTGC 13560 CGGAAGTCGA GCACTTCTAC ATTTATGGCC AAGTTAACGC GCCTGGGGTA TACGCGATTC 13620 GAACGGACAT GACGCTCCGT CGCGCGCTGG CACAAGGCGG CGGCCTTACC CCCGCCGGCT 13680 CGTCGAAGCG AGTGAAGGTC TCGCGCGACG GCCAGGAAAT CAAGTTGAAG ATGGACGATC 13740 CGATCAAGCC TGGCGACACG ATCGTCATCG GCGAGCGGTT GTTCTGATCT AGGCAATGTT 13800 GACAGCGGAC GAGGCCCACC AGTGAATATC ATTCAGTTCT TCCGCATTCT CTGGGTGCGC 13860 CGGTGGATCA TCCTCCCGGC GTTTCTCGTC TGCGTCACCA CCGCGGCGCT GGTGGTCCAG 13920 TTCCTGCCCG AACGCTACCG CGCGACCACG CGGCTGGTGC TCGACACCTT CAAGCCCGAT 13980 CCCGTCACCG GCCAGGTGAT GAACTCGCAG TTCATGCGCG CCTATGTCCA GACGCAGACC 14040 GAGCTGATCG AGGACTATGC GACCTCCGGC CGCGTGGTCG ACGAACTGGG CTGGGCCAAC 14100 GATCCTGCCA ACATCGCTGC CTTCAACGCC TCGTCCTCGG CGGCGACCGG CGACATTCGC 14160 CGCTGGCTCG CAAAGCAG AT CTCGGACAAC ACCAAGGCGG ATGTGATCGA GGGCAGCAAC 14220 ATCCTCGAAA TCTCCTACTC GGACAGCTCG CCCGAGCGTG CCGAGCGTAT CGCCAACCTG 14280 ATCCGCACCG CATTCCTCGC CCAGTCGCTC GCCGCCAAGC GCCAGGCGGC GGCGAAGTCG 14340 GCCGACTGGT ACACCCAGCA AGCGGAAGCG GCACGCCAGT CGCTGCTCGC GGCGGTGCAG 14400 GCGCGCACCG ACTTCGTGAA GAAGTCCGGC ATCGTGCTGA CCGAGACCGG TTCGGATCTC 14460 GATACGCAGA AGCTCGCACA GCTCCAGGGC GCGAGCGCGA TACCGTCGGC ACCGGTCGTC 14520 GCGGCCGCCA GCGGCATGGG CCCGGCGCAG CTCCAGCTTG CCCAGATCGA CCAGCAGATC 14580 CAGCAGGCGG CCACCAATCT CGGCCCGAAC CACCCGGCCT TCCAGGCCCT GCAGCGCCAG 14640 CGCGAGGTGC TCGCCCGCGC AGCGGCGGCG GAACGCAGCC AGGCAAGCGC CAGCGGCCCC 14700 GGCCGCGGCG CGCTGGAAAG CGAAGCCAAT GCCCAGCGCG CCCGCGTGCT CGGCAACCGC 14760 CAGGATGTCG ACAAGGTCAT GCAGCTCCAG CGGGACGTCA CGCTGAAGCA GGACCAGTAT 14820 ATGAAGGCGG CCCAGCGCGT CGCCGATCTG CGCCTGGAAG CAAGCAGCAA CGACACGGGC 14880 ATGAGCACGC TGAGCGAAGC CAGCGCGCCG GAAACGCCCT ATTACCCCAA GGTGCCGATG 14940 ATCATCGGCG GCGCGGCCGG CTTCGGCCTC GGCCTCGGCG TGCTGGTCGC GCTGCTCGTC 15000 GAACTGCTCG GTCGCCGCGT GCGCAGCGCC GAGGATCTCG AAGTGGCGGT CGATGCGCCG 15060 GTGCTGGGCG TGATCCAGAG CCGTGCCTCG CTCGCCGCAC GCCTGCGCCG CGCCCAAGAA 15120 ACCCTCGGCG ACCGCGCCGA AACGCACGGA GCTTCAGTAA ACTGATGGAC GCGATGACCA 15180 GCGAACCGCT GCCCGAAGGC GAGCGCCCGA GCGCCGTTCC GACGACGCCC GACACCACCG 15240 GCGTCCTGGA ATATCAGCTC GTCCTGTCCG ACCCGAACGG CATCGAAGCG GAAGCCATTC 15300 GCGCGCTGCG CACCCGCATC ATGGCGCAGC ACCTGCGCGA GGGCCGCCGC GCCCTGGCGA 15360 TCTGCGGCGC CTCGGCCGGC GTCGGCTGCA GCTTCACCGC CGCCAACCTC GCGACGGCGC 15420 TGGCGCAGAT CGGCATCAAG ACCGCGCTGG TCGATGCCAA TCTGCGCGAC CCGAGCATCG 15480 GCAGCGCCTT CAACATCGCC GCCGACAAGC CGGGCCTCGC CGACTATCTC GCCTCGGGCG 15540 ATATCGACCT CGCCTCGATC ATCCACCCGA CCAAGCTGGA CCAGCTGTCG GTGATCCATG 15600 CCGGGCATGT CGAGCACAGC CCGCAGGAAC TGCTGTCCTC CGAGCAGTTC CACGACCTCG 15660 TGACGCAGCT GCTGCGCGAG TTCGACATCA CGATCTTCGA CACCACGGCC GCGAACACCT 15720 GCGCCGATGC GCAGCGCGTC GCACATGTCG CCGGCTATGC GATCATCGTG GGGCGGAAGG 15780 ATTCGAGCTA CATCCGCGAC GTCAACACGC TCACCCGCAC GCTGCGGTCG GACCGCACCA 15840 ACG TCATCGG CTGCGTCCTG AACGGCTATT GAATTGGATT CCATGACCGC GACTGCGCTG 15900 GAGCGGCAGC AAGGACGGCG ACAGGGGGGC TATTGGCTCG CGGTCGCCGG CCTTGCGGCA 15960 CTCGCCATTC CCACTTTCGT CACGCTCGGC CGCGAAACCT GGAGCGCCGA AGGTGGCGTG 16020 CAGGGGCCGA TCGTGCTGGC GACCGGCGCC TGGATGCTGG CGCGGCAACG CGACAGCCTC 16080 GTGGCGCTCC GGCGCCCCGG CAATCTGGCG CTGGGCGCAT TGTGCCTGTT GCTGGCGCTG 16140 GGCATCTACA CCGTCGGTCG CGTGTTCGAC TTCATCAGCA TCGAGACGTT CGGGCTGGTC 16200 GCGACCTTCG TGGCGGCTGC GTTCCTCTAT TTCGGCGGCC GGGCGCTGCG CGCTGCGTGG 16260 TTCCCGACCT TGTGGCTGTT CTTCCTCGTG CCGCCGCCGG GCTGGATCGT CGATCGCGTC 16320 ACCGCGCCGC TCAAGGAGTT CGTCTCCTAT GCCGCCACCG GCTTCCTGTC CTGGCTGGAC 16380 TATCCGATCC TGCGCCAGGG CGTGACGCTG TTCGTCGGCC CCTATCAGCT GCTGGTCGAG 16440 GATGCCTGTT CGGGGCTGCG CTCGCTCTCC AGCCTCGTCG TCGTCACGCT GCTGTACATC 16500 TACATCAAGA ACAAGCCGTC CTGGCGCTAC GCGCTGTTCA TCGCCGCGCT GGTGATCCCG 16560 GTCGCGGTGA TCACCAACAT CCTGCGCATC GTCATCCTCG TGCTGATCAC CTATCATATG 16620 GGCGACGAGG CCGCGCAGAG CTTCCTCCAC GTCTCCACCG GCATGGTGAT GTTCGTGGTC 1 6680 GCGCTGCTCT GCATCTTCGC CATCGACTGG GTGGTCGAAC AGCTCTTCAC ACGGCGCCGG 16740 AGGCCCCATG TTCAACCGGC GTGACCTGCT GATCGGCGCG GGCTGCTTCG CCGCCGCCGG 16800 CGCCTCGCTC GGCCTCAAGC CGCACCGTCG CATGGACCTG CTCGGTGCGA CCAAGCTCGA 16860 TGCGCTGATG CCCAAGGCAT TTGGCGGCTG GAAGGCCGAG GATACCGGTG CGCTGATCGC 16920 CCCCGCGCGC GAAGGCAGCC TGGAAGACAA GCTGTACAAC CAGGTGGTCG CCCGTGCCTT 16980 TTCGCGCGCC GACGGCACCC AGGTGATGCT GCTGATCGCC TATGGCAACG CCCAGACGGA 17040 TCTGCTGCAG CTCCACCGAC CGGAAGTCTG CTACCCGTTC TTCGGCTTCA CCGTGGTCGA 17100 GAGCCACGAG CAGATCATCC CGGTGACGCC GCAGGTGACG ATTCCCGGAC GGGCGCTGAC 17160 CGCGACCAAC TTCAACCGCA CCGAGCAGAT CCTCTACTGG ACCCGCGTGG GCGAATATCT 17220 GCCGCAGAAC GGCAACGAGC AGCTGTTCGC CCGCCTCAAG AGCCAGCTCC AGGGCTGGAT 17280 CGTCGACGGG GTGCTGGTCC GCATCTCGAC TGTGACGGCG GAAGCCAAGG ACGGCCTCAA 17340 CGCCAATCTC GATTTCGCGC GCGAGCTGGT GAAGACGCTC GATCCGCGCG TGCTGCGCCC 17400 GTTGCTCGGC ACGCAGGTAA CGCGCGACCT GGCGCCGCGC GCCTGAACGA AAAAGGGGCG 17460 GCGCAGACCG CCGCCCCTCC CTCTCCTTCT CGTCGCGTAC CCGCGCTCAG CGCT CGTGCA 17520 GCGCGTCGCT GCCGGTTTCG AGCATCGGGC CGACGAGATA GCTCAGCAAT GTCCGCTTGC 17580 CGGTGACGAT GTCGGCACTG GCGATCATGC CCGGCCGCAG CGGCACGTGC CCGCCATTGG 17640 CGATGACATA GCCGCGGTCC AGTGCGATCC GCGCCTTGTA GACCGGCGGC TGGCCCTCCT 17700 TCACCTGCAC CGCCTCGGGC GCGATGCCCA CCACCGTGCC GGGGATCATG CCATAGCGGG 17760 TGTGCGGGAA CGCCTGCAGC TTCACCTTTA CCGGCATGCC GGTGCGCACG AAGCCGATAT 17820 CGCTGTTGTC CACCATCACC TCGGCCTCGA GCCGGGCATT GTCCGGCACC AGCGACAGCA 17880 GCGGCTTGGC GCCCTCCACC ACGCCGCCTT CGGTGTGGAC CTGCAGCTGC GAGACCGTGC 17940 CGCTGACCGG CGCGCGCAGT TCGCGGAACG AACTGCGCAG ATTCGCCTTG GCGACTTCCT 18000 CGCTGCGCGC CCGCACGTCG TCCTGCGCCT TCACCAGATC CTGCAACACC TGCGCGCGCG 18060 CCTCCTCGCG CGTCCTGATC GACATGCTGC TGGCACTGCG CGACTGCTGA CCAAGCTTGG 18120 CCACCGTCGC CCGCGCCGCG GTGAGGTCCT GCCGTTCGGA AATGAGCTGG CGGCGCATCT 18180 CGACCACGCG CAGCTTCGAG ACATAGCCCT TGGCGGCCAT CGCCTCGTTC GCGGCGATCT 18240 GCTGCTCGAG CAGCGGCAGC GATTGTTCCA GCTTGCGAAC CTGCGCCTGT GCCTCGGCCG 18300 AGGCGGAAGC GGCGGCACCG CTGTCCGATC GGCCGCCGGC AAGCATC GCC TCGATCTGGC 18360 CGAGCCGCGC GCGTGCGAGG CCGCGATGCG TCTCGACCTC CGCGGCGCCT GCGGCGGCGG 18420 GCGCGGCGAA GCGGAAGCCC TTTCCGTCCA GCGCGTCGAT GATCGCCTGG TTGCGCGCGG 18480 CATCGAGCTG GGCGCTGAGC AGCGCCACGC GCGCCTGCGC GGCTTCGGCT GCCGACATGG 18540 TGGGATCGAG CGTGATCAGC ACCTGGCCCT TCTGAACCTT CTGCCCCTCG CCCACCAGAA 18600 TGCGCCGGAC GATACCGCTT TCGGGGGACT GCACGATCTT GGTCTCGCCG ATCGGGGCGA 18660 TGCGGCCCTG CGTCGGCGCC ACCACTTCCA CGCGGCCGAT TGCCAGCCAG GCGGTGGTGA 18720 TCGCCAGCCC CGCCACCATC ACCCGGCCGG TGAGGCGCGC GGTGGGCGAC ACCGGACGTT 18780 CGATGATCTC GAGCGCGGCC GGCAGGAATT CGGTATCATA GGCATCGGCG CGAGCGGGCA 18840 GCACGGTGCC GCGCATGCGG GCGATCGGGC CGCCGCGGCC GATCGGAACA ACGGCGTTCA 18900 TGCGGCAATC TCCCCATATC CGCTTTGGCG GCGGTGCAGG TCGGCATAGC GGCCGCCCAA 18960 GCGTAGCAGT TCGTCATGCC GGCCGCTCTC GACGATGCGG CCCTGCTCCA GCGTGATGAT 19020 CCGATCGCAG GCGCGTACCG CGGACAGGCG GTGGGCGATG ATCACCAGCG TGCGGCCCGC 19080 CGAGATGGCG CGCAGATTGT TCTGGATCAG CTCCTCGCTC TCGGCATCCA GCGCGGAGGT 19140 CGCCTCGTCG AACACCAGGA TGCGCGGATT GCCGACCAGC GCGCGGGCGA TAGCGAGCCG 19200 CTGGCGCTGG CCGCCCGACA GGTTGACGCC GCGCTCGACG ATCTCGGTGT CATAGCCGCG 19260 CGGCTGACGC AGGATGAAGT CATGCGCACC CGCCAGCGTC GCCGCCGCCA CGACATGCTC 19320 GAACGGCATC GCCGGGTTGG ACAGCGCAAT GTTCTCGCGG ATCGAGCGGC TGAACAGCAG 19380 ATTTTCCTGC AGCACGACGC CGATCTGCCG GCGCAGCCAG GCGGGATCGA GCTGGGCCAC 19440 ATCCACCTCG TCGACCAGCA CGCGGCCCAG ATCGGGGGTG TTGAGGCGCT GCAGCAGCTT 19500 GGCCAGCGTC GACTTGCCCG ACCCCGAGGA GCCGACGATG CCGAGCGACG TGCCGGCGGG 19560 GATGTCGAGC GTGATGTCGC TCAGCACCGG CGGCTGGTCC TCGGCATAGC GGAAGGTCAC 19620 GTTTTCGAAG CGGATCGCGC CGCGCAGCAC CGGCAGCGTC GCGGCGGAGG CCGGCCGCGG 19680 CTCCACCGGA TGGTTGAGCA CGTCGCCGAG GCGCTCGATC GCGATGCGGA CCTGCTGGAA 19740 GTCCTGCCAC AGCTGGGCCA TGCGGATCAC GGGGCCGGAA ACGCGCTGGG CGAACATGTT 19800 GAACGCCACG AGCGCGCCGA CGCTCATCGC GCCACCGATC ACGGCCTTGG CGCCGAAGAA 19860 CAGGATCGCC GCGAAGCTCA GCTTGGAGAT CAGCTCGATC GCCTGGCTGC CGGTGTTGGC 19920 GACGTTGATC AGCCGCTGCG ACGAGGCGGT ATAGGCGGCG AGCTGACGTT CCCAGCGATT 19980 CTGCCAGTGC GGTTCGACTG CGGTCGCCTT GA TGGTGTGG ATGCCGGAGA CGCTCTCGAC 20040 GAGCAGCGCG TTGCTGGCGG AGCTCTTCTC GAACTTGTCC TCGACACGCG TGCGCAGCGG 20100 GCCCGCGACG CCGAACGAGA CCATCGCATA GGCGACCAGC GACACGATCA CGACGCCGAA 20160 CAGCATCGGC GAGTAGAACA GCATCGCGCC GAGGAACACG ACCGTGAACA GCGGATCGAC 20220 CATCACCGTC AGCGACGCAT TGGTGAGGAA TTCCCGGATG GTCTCGAGCT GGCGGACCCG 20280 GGTGACGGTG TCGCCCACCC GCCGCTTTTC GAAATAGCCG AGCGGCAGCG CCAGCAGATG 20340 GTGGAACAGC CGCGCGCCCA GCTCGACGTC GATCTTCTGC GTCGTCTCGG TGAACAGGCG 20400 CGTGCGGATC CAGCCCAGCG CCACCTCCCA GACCGACACG GCCAGGAAGG CGAAGGCGAG 20460 CACGCTCAGC GTGCTCATGC TGTTGTGGAC CAGCACCTTG TCGATCACGC TCTGGAAGAG 20520 CAGCGGCGCC GCGAGGCCGA GCAGGTTGAG CGCCAGGGTG ATGCCCAGCA CCTCGAGAAA 20580 CAGCCTGCGA TACCGCTGGA ACTGTGCGGC GAACCAGGAG AAACCGAATC GCAGCGCCTG 20640 GCCGGCCACG GCGCGCGTCG TCAGCAGCAC GAGCGTGCCG GACCACAGCG CATCCAGCCC 20700 CTCGCGGTCG ACCTGTTCGG GGGCGTGGCC GGGACGCTGG ATGATCACGC CATGCTCGGT 20760 CAGGCCACCG ATCACGAACC AGCCCTCCGG GCCGTCGGCG ATGGCCGGCA GCGGCTGGCG 20820 GGCCAGACCG CCGCGCGGCA CGTCC ACCGC CTTGGCGCGC ACGCCCTGCT GGCGCTTGGC 20880 GAGCAGGATC AGGTCGTCGA CGCTGGCACC CTCGGCATGG CCCAGCATGT GCCGCAGCTG 20940 TTCGGGGGTG ACGGCGATGT TGTGGACGCC GAGCAGCAGC GACAGCGCCA CAAGCCCGGA 21000 TTCGCGCAAT TCGCCCTCGC GCTCGGCGGC AGCCTGGGCG GCGAACGCGC CCTGGAGCTG 21060 TGCCTGCATC TCGTCGCGTG TCATTCCGGT ACTCTGCCTC CATGGCGCTA CTGATCGCAG 21120 CCATGATGAA CGAGCTCGGT AAAGACTCGC TTAAGCCAGA TTTTTCTGTG GTTTATACCT 21180 ATTGCCGGGG ATGCCGGACC GGACCGGATC GGCAGACGGC AGCCTGCGTT AGTCGGGCCT 21240 TAAAGCGTTG CCGCTAGCAC AAGGACAAGA ATTTTATCGG AGAGGGTCGG GAACCATGCC 21300 CACGCATGAA GGTTGCAGCG CAGCAATATC GACGGATCGC CTCGGAGCCC GAATGCTGCA 21360 TCCGCGAAGT GACTTTCGCC AAAGCAGCTA TAGGATGGCC CGGGGCTTGA TTGCCGCCGT 21420 GCGATCAGCA TAAGCGATCC ATGGTCGCCA AAATCTGTCA TCCTTGGTAA CAATCATGCA 21480 GCCGCTAAGG AAGATGTGCA CGTCTGACGA TGCTTTCTTC CGCACCCCAT GCGCCGCTGA 21540 CTCTGGTAGA TTGACCGTGG CCTCCATTGC TCATCGTCTC GAAAAAGGAC CCTCTGGTCG 21600 CCGCGCGGAC TTCCGGGAAT CGATTTGTCC CGTTATAGTG CAATGCAACA GGCCGAATCG 21660 GCCGCTGTCA GCGTGCAC AA TCCGTTGAGG GAGCCCGACG AGGCAATGAA CGCTTTTGAA 21720 GCACAGCGCG CCTTTGAGGA GCAGCTCCGG GCCCATGCCC GTTCTGCCCC CAGCGCCGCA 21780 CCCATGCTGC GACGTTCCAC GATCCGCATG ATCCTCTACA CCGAATTGCT GTTGCTCGAC 21840 AGCATCGCAA TTCTACTGGG GTTCTACATC GCGGCCTGCT CGCGCGACGG CAACTGGCTG 21900 TCCCTTGCGG GCGTCAATGT CGGCATCTTC CTCCTGCCGA TCACGCTCGG CACCGCGCTC 21960 GCCAGCGGCA CCTATTCGCT GAGCTGCCTG CGCTACCCGG TCAGCGGGGT GAAGAGCATC 22020 TTCTCGGCGT TCTTCTTCTC GGTGTTCATC GTGCTGCTGG GCAGCTACCT GCTCACCGCG 22080 GAGCTGCCGC TGTCGCGCCT GCAGCTCGGC GAGGGCGTGC TCCTGGCGCT CAGCCTGGTG 22140 ACGATCTGCC GCCTTGGCTT CCGCTGGCAC GTTCGTGCGC TGACACGCGG CACGCTGCTC 22200 GACGAGCTGG TGATCGTCGA CGGCGTTGCC CTGGAGGTCG CGAGCGGCGC GGTCGCGCTC 22260 GATGCGCGCA TCATCAACCT CACGCCCAAC CCGCGCGATC CGCAGATGCT GCATCGCCTC 22320 GGCACCACCG TGGTGGGCTT CGACCGGGTC GTCGTCGCCT GCACCGAGGA GCACCGGGCA 22380 GTATGGGCGC TGCTGCTCAA GGGCATGAAC ATCAAGGGCG AGATCCTCGT CCCCCAGTTC 22440 AACGCGCTGG GCGCGATCGG CGTCGACTCC TATGAGGGCA AGGACACGCT GGTCGTGTCC 22500 CAGGGCCCGC TCAACATGCC GAACCGCGCA AAGAAGCGGG CGCTCGATCT GCTCATCACC 22560 GTCCCCGCGC TGGTCGCGCT GGCGCCGCTG ATGATCGTGG TCGCGATCCT GATCAAGCTG 22620 GAGAGCCCCG GCCCCGTCTT CTTCGCACAG GACCGCGTCG GCCGCGGCAA CCGACTGTTC 22680 AAGATCCTCA AGTTCCGCTC GATGCGCGTT GCGCTCTGCG ATGCGAACGG CAACGTCTCG 22740 GCCAGCCGCG ATGACGATCG CATCACCAAG GTAGGCCGGA TCATCCGCAA GACCAGCATC 22800 GACGAGCTGC CGCAGCTGCT CAACGTGCTG CGCGGCGACA TGAGCGTCGT CGGCCCGCGC 22860 CCGCACGCAC TCGGGTCGCG CGCCGCCAAC CATCTCTTCT GGGAAATCGA CGAGCGCTAC 22920 TGGCACCGCC ACACGCTCAA GCCGGGCATG ACGGGCCTCG CGCAGATCCG CGGCTTCCGC 22980 GGCGCGACCG ATCGCCGCGT CGATCTCACC AATCGCCTGC AGGCGGACAT GGAGTATATC 23040 GACGGCTGGG ACATCTGGCG GGACGTCACC ATCCTGTTCA AGACGCTGCG CGTGATCGTG 23100 CACTCCAACG CCTTCTGATC GCGGAGGGGA GCAACGCGAG CACCGCTTGG TGCAAGAGCA 23160 TTGACATCCG CCCTGCTTCT GCATTTGTCA TTTTATCATT GTCGTTGCGG GCCCGCCCGC 23220 GCCATGGGGG ATTTTGAATG AAGGGTATCA TCCTTGCGGG GGGCAGCGGC ACGCGCCTCT 23280 ACCCCGCAAC GCTGTCGATC TCGAAGCAGC TGCTTCCCGT CTATGACAAG CCGATGATCT 23340 TCT ACCCCCT GTCGGTGCTG ATGCTCACGG GTATCCGGGA CATCCTGATC ATCTCCACCC 23400 CGCGCGACCT GCCGATGTTC CAGGCGCTGC TCGGCGACGG TTCGGCATTC GGCATCAACC 23460 TGAGCTATGC CGAACAGCCT TCGCCCAACG GCCTTGCGGA AGCCTTCATC ATCGGCGCCG 23520 ATTTCGTCGG CAACGATCCC AGCGCGCTGA TCCTCGGCGA CAACATCTAT CACGGTGAAA 23580 AGATGGGCGA GCGCTGCCAG GCAGCTGCGG CCCAGGCATC GCAGGGCGGC GCGAACGTGT 23640 TCGCCTATCA TGTCGACGAT CCCGAGCGCT ACGGCGTGGT CGCGTTCGAT CCGGAGACGG 23700 GCGTCGCTAC CAGCGTCGAG GAAAAGCCGG CCAACCCCAA GTCCAATTGG GCGATCACCG 23760 GGCTTTATTT CTACGACAAG GACGTGGTCG ACATCGCCAA GTCGATCCAG CCCTCGGCGC 23820 GCGGCGAACT CGAGATCACC GACGTCAACC GCATCTACAT GGAGCGCGGC GACCTCCACA 23880 TCACCCGGCT CGGTCGCGGC TATGCCTGGC TCGACACCGG CACGCATGAC AGCCTGCACG 23940 AGGCCGGCTC GTTCGTCCGC ACGCTGGAGC ACCGCACCGG CGTGAAGATC GCCTGCCCGG 24000 AGGAAATCGC CTTCGAGAGC GGCTGGCTGG GCGCCGACGA TCTGCTCAAG CGCGCCGCCG 24060 GCCTCGGCAA GACGGGGTAT GCCGCCTATC TGCGCAAGCT GGTAGCCGCG GCATGACCCA 24120 GGTGCATCAC CACGCGCTAT CGGGCGTCAT CGAGTTCACC CCGCCCAAGT ACGGCGATCA 2 4180 CCGCGGCTTC TTCTCCGAGG TGTTCAAGCA GTCCACGCTC GACGCCGAAG GCGTCGAGGC 24240

[0154] GCGGTGGGTG CAGGACAATC AGAGCTTCTC GGCCGCACCG GGCACGATCC GCGGACTGCA 24300 CCTGCAGGCG CCGCCCTTCG CCCAGGCCAA GCTGGTGCGC GTGCTGCGCG GCGCGATCTA 24360 CGACGTCGCG GTCGACATTC GCCGCGGCTC GCCCACATAC GGCCAGTGGG TCGGCGTCGA 24420 GCTTTCGGCG GACAAGTGGA ACCAGCTGCT GGTGCCGGCC GGCTATGCGC ATGGCTTCAT 24480 GACGCTCGTC CCGGATTGCG AGATCCTCTA CAAGGTCAGC GCCAAATATT CGAAGGAATC 24540 GGAGATGGCG ATCCGCTGGG ATGATCCCGA TCTCGCCATC ACCTGGCCGG ACATCGGCGT 24600 CGAGCCGGTG CTCTCCGAAA AGGACGCGGT CGCTACCCCG TTCGCCGAAT TCAACACCCC 24660 CTTCTTCTAT CAGGGCTGAT CCATGCAGCA GACCTTCCTC GTTACCGGCG GCGCCGGCTT 24720 CATCGGCTCG GCAGTGGTAC GCCACCTCGT TCGCCAGGGC GCGCGCGTCA TCAATCTCGA 24780 CAAGCTCACC TATGCGGGCA ACCCGGCCTC GCTGACCGCG ATCGAGAACG CCCCCAACTA 24840 CCGCTTCGTC CACGCCGATA TCGCCGACAC CGCGACGATC CTGCCGCTGC TGCGCGAAGA 24900 GCAGGTCGAC GTGGTGATGC ACCTCGCCGC CGAGAGCCAT GTCGATCGCT CGATCGACGG 24960 CCCGGGCGAG TTCATCGAGA CCAACGTCGT CGGCACCTTC AAGCTGCTCC AGGCGGCGCT 25020 GCAATATTGG CGCGAGCTGG AAGGGGAGAA GCGCGAGGCT T TCCGCTTCC ACCACATTTC 25080 CACCGACGAG GTGTTCGGCG ACCTGCCGTT CGACAGCGGC ATCTTCACCG AAGAGACGCC 25140 CTATGATCCC TCCTCGCCCT ATTCGGCGTC GAAGGCGGCC AGCGACCATC TGGTCCGCGC 25200 CTGGGGTCAC ACCTATGGCC TGCCCGTGGT GCTGTCGAAC TGCTCGAACA ATTACGGGCC 25260 GTTCCACTTC CCCGAGAAGC TGATCCCGCT GACCATCCTC AACGCGCTGG AAGGCAAGCC 25320 CCTGCCCGTC TACGGCAAGG GCGAGAATAT CCGCGACTGG CTGTACGTCG ACGATCACGC 25380 CAAGGCGCTG GCGACGATCG CCACGACCGG CAAGGTCGGC CAGAGCTACA ATGTCGGCGG 25440 CCGCAACGAG CGCACCAACC TGCAGGTCGT CGAGACGATC TGCGACCTGC TCGATCAGCG 25500 CATTCCGCTG AAGGATGGCA AGAAGCGCCG CGAGCTGATC ACCTTCGTCA CCGATCGCCC 25560 CGGCCATGAC CGCCGCTACG CGATCGACGC GACCAAGCTC GAGACCGAAC TGGGCTGGAA 25620 GGCCGAGGAG AATTTCGACA CCGGCATCGC CGCGACGATC GACTGGTATC TCGAGAATGA 25680 ATGGTGGTGG GGTCCGATCC GCTCCGGCAA ATATGCCGGC GAGCGGTTGG GGCAGACCGC 25740 CTGATGCGCA TCCTCGTCAC CGGGCATGAC GGCCAGGTCG CCCAGGCGCT GGGCGAACAG 25800 GCGGAGGGCC ATGAGCTGAT CTTCACCAGC TATCCCGAGT TCGATCTCTC CAAGCCGGAG 25860 ACGATCGAGG CGGCGGTGGC GAAGATCCAG CCCG AGCTGA TCGTGTCGGC GGCTGCGTAT 25920 ACGGCGGTCG ACAAGTCCGA GAGCGAGCCC GAGCTCGCCA TGGCGATCAA CGGCGACGGC 25980 CCCGGCGTAC TGGCGCGCGC GGGCGCGAAG ATCGGCGCGC CGATCATCCA TCTGTCGACC 26040 GACTATGTGT TCGACGGCAG CCTGGACCGC CCGTGGCGCG AAGACGACCC CACCGGTCCG 26100 CTCGGCGTCT ATGGCGCCAC CAAGCTGGCC GGCGAGCAAG CGGTGCAGGC CTCGGGCGCG 26160 ACCAACGCGG TGATCCGGCT CGCCTGGGTC TACAGCCCGT TCGGCAACAA CTTCGTCAAG 26220 ACGATGCTGC GCCTCGCCGA GACGCGGGAC ACGCTGAACG TGGTCGAGGA CCAGCAGGGC 26280 TGCCCGAGCT CGGCGCTGGA CATCGCCACG GCGATCCTCA AGGTCGTCGG CCACTGGCAG 26340 CAGAACGGCG CCACCAGCGG CCTGTATCAC TTCACCGGAT CGGGCGAGAC CAACTGGGCC 26400 GACTTCGCGC GCGCGATCTT CGCGGAAAGC GCCAAGCACG GCGGTCCGAC CGCCGAGGTG 26460 ACCGGCATTC CGACCTCCGG CTACCCCACC CCGGCGAAGC GCCCGGCCAA TTCGCGGCTC 26520 AATTGCGACA AGTTCGCCGA AACCTTCGGC TATCGTGCAC CCGCCTGGCA GGACTCGGTG 26580 GCGGAAGTGG TAGGCCGCCT CCTGGCATAA AATGCCCGGC CCGACCCTGT GCGCGGCGGG 26640 GTGGCTGCGC ACTCCGGTCG GGTTTCATCG ACATCGCCGG CTGCGGGGAG CATCACCGAT 26700 GCTCCCCGAT CAGCGCCAGG CCGTCAC TTC CTGAACGGCG CGACCAGGGG CTTGATCGTC 26760 TTGAACACGG CCTCACGCAG CGTCCGCACG GGCGCGGCGA CGAGGTGATC GAACGCGAGC 26820 GTCATCCCGC TCACCCGCTG GGGTGCGACG TCGCTGCGGA TCTTGAACGA TTCGACCACC 26880 TCGATATCGG AAACCAGCCG CCCCTTGATG CGGTTGATGA CATTCTCGCC ATGCACCACC 26940 TGCAGCCATA CCGGCCGCCC GGCGACCTGG GTGATCTTCC ACTTCTGGCC CAGCTCATGA 27000 TGGGGCTTGG CCCAGATCGT CTCGACGCTG GCGAGATCGC GCTCGACCAG CGAGGTGAAC 27060 GGATTGCTGT GGTCCGCAGC GGTGTAGAGC CGGCCCTGGC GCATCGCGAT GCCCTGGGTG 27120 AAGTTCAGCA CCGTCTGTGC CGGCGCATCC TTCGCCGCGG CCTGCACCCG TGCCACGAAG 27180 TCGTTCGAAA GCGCGTCGTC ATTGTCCAGC CGCGTGGTGA CGATCAGCTG CTCGCCGGGC 27240 GTCGCCAGCG CCTTCACGTC GTCCGCGATC ATCGCCTTGT CGAACATCGC GACGTAGCGC 27300 GGCGTGAAGT TGTAGATCTG CCGATCGCGC TCGATCCGCT CGCGGAACTC GGCGGGGGTG 27360 TCCTTGTCGA AGTAGATGAG CCAGTGGAAG TTGCGCTCGG TCTGGCCCGC GATGCTCGGC 27420 AGGCAGAACT GCTCGAACAG CCCGAAACGG CGGTCGAGCC AACCCGGCGA ATTGCGGATC 27480 GCCACCTCGC GGCCCGGGCT GGCGATGTTG AAGCGCGTCA GGATCACGTG AAGCATCGGT 27540 TCGATCAGCC CCGGTCTAGC AAAACGAAGA AAGCCCGGCC GCTACAACGG CCTTGTTCGA 27600 ACAACGCGCA AGAAACAGGG TACACGCGAA CGGCACGTTC GTCTTCGCCC ACCCCGCTGG 27660 TTGCCGCCAT TCCCACGAAC GGTTACGGGA TATTCCGGAA CTGGGCAACC GGGGATTGCT 27720 GCACTGCGCA ATGACACGCG GCCGGAATGA CAAACGGCTT GCCGCCCGCG CCCCCCGCGC 27780 CTAACCCTCC GCCCGTGCCC GACGCCCGTC CCGATCGCAT TGCCACCGGC CTGGCGCTTC 27840 GCCTGTTCGC CATTGCCTGC CTGTCGACCA TGTCGGCGCT CATCAAGATG TCGGAACTGC 27900 GCGGCGCCTC GCTGATCGAG ACGATGTTCC ACCGCCAGCT CTGGGCGGTG CCGCTGGTCA 27960 CCTTGTGGGT GGTGATGGGC CCGGGGCTCA AGTCGCTCAA GACGCAGCGC TTCGGCGCGC 28020 ATGTCTGGCG CACCGCGGTG GGCCTCACCG GCATGATCTT CACCTTCGGC GCGGTGATCC 28080 TGCTGCCCCT GGCCGAGGCG CAGACCTTCC AGTTCACCGT GCCCATCTTC GCCACGCTGC 28140 TCGGCGCGCT GATCCTCGGC GAGCCGACCG GCCGGCATCG CTGGGGCGCA GTGATCGTCG 28200 GCTTCCTCGG CGTGCTGATC GTCGTCCAGC CGGGCCGGGA AGCCATTCCG ATCTTCGGCG 28260 CCTTCGTCGG GCTGATGGCG GCGTTGTTCG TCGCCATCGT CGCGATCACG CTGCGGCAGA 28320 TCACCCGCAC CGAAAGCGCC GGCACCACCG TCTTCTGGTT CTCGCTGCTC TCGGTGCCCG 28380 TGCTCGGCGC CA TCTACGCG TTCAACTTCC GTCCGCACGA TGCCGAGACC TGGGCGATCC 28440 TCATCGCCAC AGGACTGGTG GGCGGCGTCG GCCAGCTGGC GCTGACCGGT GCGATGCGCT 28500 TCGCCCCCGT CTCGGCGGTG GTACCGATGG ACTATTCGGG GCTGATCTGG GCGACGCTCT 28560 ACGGCTGGCT GCTGTTCGAC GTGTTCCCGA CCTTCTCGAC CTGGCTCGGT GCGCCGGTGA 28620 TCATCGCCAG CGGGCTCTAC ATCGTCTATC GCGAGCAGAA GCTGGCCCGC GGCCAGGCTA 28680 GCTACGCCGA AACGCCACTA TGAGGTTGTT GGCGGGCATC GCCACCCGCC GATCGAACAC 28740 CAGGCCTTGC GCCCCCGCCG CCGCGATCAC CTCGTCCAGC AAGCGCAGCC CCCAGGCAGG 28800 ATCC 28804

[Brief description of drawings]

FIG. 1 34 kbp isolated from the chromosomal DNA of Sphingomonas strain S88 (ATCC Deposit No. 31554).
FIG. 3 is a schematic diagram of a site obtained by digesting a nucleotide-based DNA sequence with a restriction enzyme. Many DNA sequences shown in FIG. 1 were inserted into Sphingomonas bacterium, and the ability of these Sphingomonas strains to increase sphingan production was examined.
The restriction sites of the next several enzymes are also shown in FIG. 1 (and also in FIG.
3, 8, 9 and 10). B (BamHI), Bg
(Bg1II), E (EcoRI), H (HindII
I) and S (SalI). The spsB area shown in FIG.
Corresponds to the DNA sequence encoding the protein SpsB.

FIG. 2 is a schematic illustration of the site of a DNA sequence isolated from the chromosomal DNA of Sphingomonas strain S60 (ATCC Deposit No. 31461) (about 28 kbp units) digested with restriction enzymes. The restriction sites of this DNA sequence are shown in Figure 2 as B, E and H sites. The sgeB region corresponds to the DNA sequence encoding the protein SgeB.

FIG. 3 is a schematic diagram of the restriction enzyme sites of the DNA sequence (approximately 33 kbp units) isolated from the chromosomal DNA of Sphingomonas strain NW11 (ATCC Deposit No. 53272). The restriction sites of this sequence are shown in Figure 3 as the E, H, B, Bg and S sites. The snwB region is the protein Snw
Corresponds to the DNA sequence encoding B.

FIG. 4 is a schematic diagram of the chemical structure of a representative monosphingan polysaccharide produced according to the present invention.

FIG. 5 is a structural map of plasmids pSEB24 and pSEB26 detailed in Examples 6 and 12 of the present application. Mc
sI is the following restriction site: EcoRI, SmaI, Ba
A multi-cloning site containing mHI, SAII, PstI and HindIII (clockwise from top). Similarly, McsII is also HindIII, PstI,
SalI, XbaI, BamHI, SmaI, SstI
And EcoRI (clockwise from top). O
riT is the origin of zygotic translocation, OriV is the origin of widespread host replication, and ori is the origin of replication starting from pUC12 and 13.

FIG. 6: 34 kb isolated from the chromosomal DNA of Sphingomonas strain S88 (ATCC Deposit No. 31554)
It is a schematic diagram of a restriction enzyme cleavage site of a DNA segment of p nucleotide units. Restriction sites for the following several enzymes: B (BamHI), Bg (BglII), E
(EcoRI), H (HindIII) and S (Sal
I) is shown in FIG. 1 (and also in FIGS. 2 and 3). The sps shown in FIG. 6 (and also in FIG. 1)
The B region corresponds to the DNA sequence encoding the protein SpsB. Other "sp's involved in sphingan biosynthesis
The "s" genes are capital letters G, S, R, Q, I, K, L,
They are designated J, F, D, C and E. rhsACBD is the name of the gene indicating the position on the map of the gene involved in the synthesis of the sphingan precursor dTDP- (L) rhamnose. Genes 32, 26, 31 and 34 are open reading frames (ORFs) for translation that have not yet been identified, and at
The rDB gene encodes a transport function independent of sphingan synthesis. “Sec” indicates a gene required for secretion of sphingans, and “Trace” indicates a gene encoding an enzyme that transfers a sugar from a nucleotide-sugar precursor to a sphingan when a repeating subunit of sphingan is assembled. ing.

FIG. 7: Isolated from chromosomal DNA of Sphingomonas strain S198 (ATCC Deposit No. 31853) (approximately 4
FIG. 3 is a schematic diagram of a restriction enzyme cleavage site of a DNA segment (of 2 kbp unit). In FIG. 7, the restriction sites of this DNA sequence are indicated as H (HindIII) and E (EcoRI). The order of closely spaced parts in parentheses is unknown. Cosmid clone c2, c
The outward spread of 3, c4, c5 and subclone L242 is indicated by a broken line. The boxed "B" region corresponds to the DNA sequence encoding the protein that complements the variant of the S88 gene spsB.

FIG. 8: Isolated from chromosomal DNA of Sphingomonas strain S7 (ATCC Deposit No. 21423) (approximately 42 k
FIG. 3 is a schematic representation of restriction enzyme cleavage sites for DNA segments (in bp units). In FIG. 8, the restriction sites of this DNA sequence are H (HindIII), E (EcoRI) and B.
It is displayed as (BamHI). The outward spread of cosmid clones c1, c2, c3, and c6 is
Shown by broken lines. Clones c2 and c3 extend beyond the region on the right side of FIG. Similarly, clone c6 extends to the left. The "B" area surrounded by a square is
It corresponds to the DNA sequence encoding the protein that complements the variant of the S88 gene spsB.

FIG. 9 is a schematic diagram of gene clusters used for Sphingan S-88 synthesis. The gene name is at the bottom of the map,
Boundary, planned function, and restriction enzyme cleavage site (B for BamHI, E for EcoRI, H for HindII
I) is shown. Towards the top of the figure, the natural Sps ^- mutation name and position, S
ps ⁺ or Sps ^- complementary results, and plasmid and S
Figure 8 shows the Sps phenotype due to a specific insertion mutation on chromosome 88. pZ167, pZ168, pZ180, pZ202 and pZ206 with the insertion of mini-Tn10kan are in the c2 segment cloned into the plasmid pRK311 and the two deletion strains ΔTn
It was introduced into either 493 or ΔTn495. Similarly, all transposon insertions into other plasmids were made in the c3 segment, and the deletion strain ΔTn
358 and 365. mini-Tn10kan
Insertion accuracy is ± relative to the sequenced restriction sites.
The accuracy of the graph is 50 bp and ± 100 bp. In addition,
At the time of filing a foreign document, this figure was one figure together with FIG. 10. However, due to the size, in Japanese writing, the upper part of the original figure is shown.

FIG. 10 is a schematic diagram of gene clusters used for Sphingan S-88 synthesis. At the top of the map are gene names, boundaries, putative functions, and restriction enzyme cleavage sites (B for BamHI, E for EcoRI, H for HindI).
II) is shown. Cloned fragments (c1Δ3, c2, c
3, c4, c5 and c6), the subcloned segment (named according to the restriction enzyme used and the approximate length of the kbp unit), and the deletion that occurred on the S88 chromosome (each line indicates the presence of DNA). (Shown) is shown below the genetic map. When applying for a foreign document, this figure
Although it was one figure together with FIG. 9, due to the size,
In Japanese, the lower part of the original figure is shown.

FIG. 11: Alignment of the deduced amino acid sequences of SpsB and glycosyl-IP transferases. The number on the right is the number of rightmost amino acid residues in each sequence. For each sequence, the galactosyl-IP transferase is Sp
Directly above the sB sequence, the glycosyl-IP transferase is shown below the SpsB sequence. The following gene products are shown on the right. ExoYn, a kind of Rhizobium ( Rhizobium sp. ) NGR234
(Gray, et al., 1990, J. Bacte.
riol. , 172: 193). CpsD, S.I. S. agalactiae (Ruvent
s, et al. , 1993, Mol. Microbi
ol. , 8: 843). RfbP, S. Enterica L
T2 (Jiang, et al., 1991, Mol.
Microbiol. , 5: 695). GumD, X.
Campestris B1459S-4L (Capage,
et al. , 1987, International
Patent WO / 05938). Pss4, R.I. R. leguminosarum b
v. vica strain VF39 (GenBank Deposit No. M93042). Pss2, R.I. Legumino salam b
v. phaseoli (Borthakur, et a
l. , 1988, Mol. Gen. Genet. , 21
3: 155). About symbols. | Indicates the same amino acid of SpsB and galactosyl-IP transferase or glycosyl-IP transferase shown above and below, respectively. : Is the next related amino acid, ie IFVWML,
Conservative amino acid substitutions based on the ST, QNED and HKR groups are shown. The underlined sequence is a continuous segment of about 20 hydrophobic amino acids.

FIG. 12: S. Enterica (Jiang, et a
l. , 1991, Mol. Microbiol. , 5:
695), and X. Campestris (Koeplin,
et al. , 1993, J .; Bacteriol. , 1
75: 7786-7792).
1 shows an alignment of dTDP-L-rhamnose biosynthetic enzymes. The symbols are the same as in FIG. It should be noted that at the time of filing a foreign document, this figure was one figure together with FIG. 13 to FIG. 15, but due to the size, in the Japanese document, a part of the original figure is shown.

FIG. 13: S. Enterica (Jiang, et a
l. , 1991, Mol. Microbiol. , 5:
695), and X. Campestris (Koeplin,
et al. , 1993, J .; Bacteriol. , 1
75: 7786-7792),
1 shows an alignment of dTDP-L-rhamnose biosynthetic enzymes. The symbols are the same as in FIG. At the time of filing a foreign document, this figure was a single figure including FIGS. 12 and 14 to 15. However, due to the size of the figure, a part of the original figure is shown in Japanese. There is.

FIG. 14: S. Enterica (Jiang, et a
l. , 1991, Mol. Microbiol. , 5:
695), and X. Campestris (Koeplin,
et al. , 1993, J .; Bacteriol. , 1
75: 7786-7792), and
1 shows an alignment of dTDP-L-rhamnose biosynthetic enzymes. The symbols are the same as in FIG. At the time of filing a foreign document, this figure was a single figure including FIGS. 12 to 13 and FIG. 15. However, due to the size of the figure, a part of the original figure is shown in Japanese. There is.

FIG. 15: S. Enterica (Jiang, et a
l. , 1991, Mol. Microbiol. , 5:
695), and X. Campestris (Koeplin,
et al. , 1993, J .; Bacteriol. , 1
75: 7786-7792), and
1 shows an alignment of dTDP-L-rhamnose biosynthetic enzymes. The symbols are the same as in FIG. At the time of filing a foreign document, this figure was one figure together with FIG. 12 to FIG. 14, but due to its size, a part of the original figure is shown in the Japanese document.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical indication (C12N 15/09 ZNA C12R 1:01) (C12N 1/21 C12R 1:01) (C12N 1 / 21 C12R 1:19) (C12P 19/04 C12R 1:01) (72) Inventor Thomas J. Pollack United States, 92121 California, San Diego, Rusk Boulevard 6650, Sweet B-102 Shin-Etsu Bio Inc. In Tid (72) Inventor Motohide Yamazaki United States, 92121 San Diego, California, Rusk Boulevard 6650, Sweetby 102 Shin-Etsu Bio Inc. In (72) Inventor Linda Thorne United States, 92121 California A, San Diego, Rusk Boulevard 6650, Sweet Bee-102 Shin-Etsu Bio Incorporated (72) Inventor Marshall Mikolayjack USA, 92121 California, San Diego, Rusk Boulevard 6650, Sweet Bee -102 Shin-Etsu Bio Inc. (72) Inventor Richard W. Armentlaut United States, 92121 California, San Diego, Rusk Boulevard 6650, Sweet B -102 Shin-Etsu Bio Inc.

Claims (74)

[Claims] 1. A DNA sequence isolated from the DNA of a sphingomonas genus producing sphingans, wherein when a large number of copies of said DNA sequence are incorporated into a sphingomonas genus serving as a recipient bacterium, said recipient bacterium is An isolated DN that encodes beneficial or indispensable genetic information when a sphingomonas bacterium belonging to the above-mentioned recipient strain, which produces a large-scale sphingan polysaccharide-producing bacterium, synthesizes sphingan
A sequence. 2. ATCC31554, ATCC3146
1, ATCC31853, ATCC21423, ATC
C31555, ATCC31961, ATCC5315
9. The DNA sequence according to claim 1, isolated from a bacterial strain of the genus Sphingomonas selected from the group consisting of 9 and ATCC 53272. 3. ATCC31554, ATCC3146
1. The DNA sequence according to claim 1, which is isolated from a bacterial strain of the genus Sphingomonas selected from the group consisting of 1. ATCC53272. 4. Sphingomonas strain ATCC3155
4. The DNA sequence according to claim 3 isolated from 4. 5. S88clΔ3, S88c2, and S8
The DNA sequence according to claim 4, which has a restriction enzyme map of a segment selected from 8c3. 6. The DNA sequence according to claim 5, which is a part of the segment. 7. A sphingomonas strain ATCC3146
A DNA sequence according to claim 3 isolated from 1. 8. The DNA sequence according to claim 7, which has a restriction enzyme map of a segment selected from the group consisting of S60c1, S60c2, and S60c3. 9. A DNA sequence according to claim 8 which is a fragment of said sequence. 10. Sphingomonas strain ATCC532
The DNA sequence of claim 3 isolated from 72. 11. The DNA sequence according to claim 1, which has a segment restriction map selected from the group consisting of NWc2.1 and NWc2.2. 12. The DNA sequence according to claim 11, which is a fragment of the segment. 13. The DNA sequence according to claim 1, wherein the sphingan is represented by the following general formula. Embedded image In the formula, Glc is glucose, GlcA is glucuronic acid, Rha is rhamnose, Man is mannose, and X is rhamnose or mannose. Z
Is attached to Glc residue 2 and Z is α-L-Rha-
It is (1--6) -α-L-Rha, α-L-Man, or α-L-Rha. W is attached to Glc residue 1 and W is β-D-Glc- (1--6) -α-D-Gl
c or α-L-Rha. The subscripts v and y are 0, 0.33, 0.5, 0.67, or 1. The reducing end of the polymer is on the side of the X residue of the main chain, v and y
When is 0, W and Z do not exist on the main chain. 14. A DNA sequence according to claim 1, containing a gene encoding a glycosyltransferase enzyme. 15. The glycosyltransferase enzyme is a glycosyl IP-transferase.
The DNA sequence according to 4. 16. The DNA sequence according to claim 1, which contains a rhamnose operon or a rhamnose gene. 17. A method for isolating a DNA sequence from a DNA of a genus Sphingomonas that produces a donor sphingan, which sequence is useful or indispensable when a donor sphingan-producing bacterium biosynthesizes sphingan. Encoding the genetic information, inserting the sequence into the recipient strain,
The recipient incorporates multiple copies of the sequence,
A method for producing a sphingan polysaccharide in large scale by manipulating a bacterium derived from a strain of the genus Sphingomonas containing the production amount of sphingan polysaccharide related to the donor bacterium under the same fermentation condition. . 18. The bacterium inserting the DNA sequence is A
TCC31554, ATCC31461, ATCC31
853, ATCC 21423, ATCC 31555, A
TCC31961, ATCC53159, ATCC53
18. The method of claim 17, which is a member of the Sphingomonas strain selected from the group consisting of 272. 19. The bacterium that inserts the DNA sequence is A
TCC31554, ATCC31461, and ATCC
The method according to claim 17, which is a member of the Sphingomonas strain selected from the group consisting of 53272. 20. The DNA sequence is ATCC3155.
4, ATCC31461, ATCC31853, ATC
C21423, ATCC31555, ATCC3196
18. The method of claim 17, isolated from a Sphingomonas strain selected from the group consisting of 1, ATCC53159, and ATCC53272. 21. The DNA sequence is ATCC3155.
18. The method of claim 17, isolated from a Sphingomonas strain selected from the group consisting of 4, ATCC31461, and ATCC53272. 22. The method of claim 18, wherein the DNA sequence was isolated from Sphingomonas strain ATCC31554. 23. The DNA sequence is S88clΔ3, S
The method according to claim 22, having a restriction enzyme map of a segment selected from 88c2 and S88c3. 24. The method of claim 23, wherein the DNA sequence is a fragment of the segment. 25. The method of claim 18, wherein said DNA sequence was isolated from Sphingomonas strain ATCC31461. 26. The DNA sequences are S60c1 and S60.
The method according to claim 25, which has a restriction enzyme map of a segment selected from the group consisting of c2 and S60c3. 27. The method of claim 22, wherein the DNA sequence is a fragment of the segment. 28. The method of claim 18, wherein said DNA sequence was isolated from Sphingomonas strain ATCC53272. 29. The DNA sequence comprises a segment NWc
3. A restriction map of 2.1 or NWc2.2.
9. The method according to 8. 30. The method of claim 29, wherein the DNA sequence is a fragment of the segment. 31. The method of claim 17, wherein the sphingan has the general formula: Embedded image In the formula, Glc is glucose, GlcA is glucuronic acid, Rha is rhamnose, Man is mannose, and X is rhamnose or mannose. Z
Is attached to Glc residue 2 and Z is α-L-Rha-
It is (1--6) -α-L-Rha, α-L-Man, or α-L-Rha. W is attached to Glc residue 1 and W is β-D-Glc- (1--6) -α-D-Gl
c or α-L-Rha. The subscripts v and y are 0, 0.33, 0.5, 0.67, or 1. The reducing end of the polymer is on the side of the X residue of the main chain, v and y
When is 0, W and Z do not exist on the main chain. 32. The method of claim 17, which contains a gene encoding a glycosyltransferase enzyme. 33. The glycosyltransferase enzyme is a glycosyl IP-transferase.
3. The method according to 2. 34. The method according to claim 17, which comprises a rhamnose operon or a rhamnose gene. 35. A bacterium derived from a strain of the genus Sphingomonas, wherein the bacterium encodes DNA information useful or indispensable for biosynthesis of sphingan polysaccharide.
It contains multiple copies of the sequence, and the DNA sequence is isolated from chromosomal DNA or plasmid DNA of Sphingomonas sp. Is a bacterium that becomes a mass-producing bacterium of sphingan polysaccharide. 36. ATCC 31554, ATCC 314
61, ATCC31853, ATCC21423, AT
CC31555, ATCC31961, ATCC531
59. The bacterium of claim 35, which is derived from a Sphingomonas strain selected from the group consisting of 59 and ATCC 53272. 37. ATCC31554, ATCC314
36. The bacterium according to claim 35, which is derived from a Sphingomonas strain selected from the group consisting of 61 and ATCC 53272. 38. The DNA sequence is ATCC3155.
4, ATCC31461, ATCC31853, ATC
C21423, ATCC31555, ATCC3196
36. The bacterium of claim 35, isolated from a Sphingomonas strain selected from the group consisting of 1, ATCC53159, and ATCC53272. 39. The strain is ATCC31554, AT
The bacterium according to claim 35, which is selected from the group consisting of CC31461 and ATCC53272. 40. The sphingomonas strain AT
The bacterium according to claim 35, which is CC31554. 41. The DNA sequence is S88clΔ3, S
The bacterium according to claim 35, which has a restriction enzyme map of a segment selected from 88c2 and S88c3. 42. The bacterium of claim 35, wherein the DNA sequence is a fragment of the segment. 43. The bacterium of claim 35, wherein the strain is ATCC31461. 44. The DNA sequences are S60c1 and S60.
36. The bacterium according to claim 35, which has a restriction enzyme map of a segment selected from the group consisting of c2 and S60c3. 45. The bacterium of claim 35, wherein the DNA sequence is a fragment of the segment. 46. The bacterium of claim 35, wherein said strain is ATCC53272. 47. The DNA sequence comprises a segment NWc
4. It has a restriction enzyme map of 2.1 or NWc2.2.
The bacterium according to 5. 48. The DNA sequences are S60c1 and S60.
36. The bacterium according to claim 35, which has a restriction enzyme map of a segment selected from the group consisting of c2 and S60c3. 49. The bacterium according to claim 35, wherein the sphingan is represented by the following general formula. Embedded image In the formula, Glc is glucose, GlcA is glucuronic acid, Rha is rhamnose, Man is mannose, and X is rhamnose or mannose. Z
Is attached to Glc residue 2 and Z is α-L-Rha-
It is (1--6) -α-L-Rha, α-L-Man, or α-L-Rha. W is attached to Glc residue 1 and W is β-D-Glc- (1--6) -α-D-Gl
c or α-L-Rha. The subscripts v and y are 0, 0.33, 0.5, 0.67, or 1. The reducing end of the polymer is on the side of the X residue of the main chain, v and y
When is 0, W and Z do not exist on the main chain. 50. The bacterium of claim 35, which contains a gene encoding a glycosyltransferase enzyme. 51. The glycosyltransferase enzyme is a glycosyl IP-transferase.
The bacterium according to 0. 52. The bacterium according to claim 35, which contains a rhamnose operon or a rhamnose gene. 53. A method for increasing the production of sphingans by Sphingomonas spp. 1) Useful for sphingan biosynthesis of 1) from a Sphingomonas strain that is a sphingan-producing bacterium and is a donor bacterium to a Sphingomonas sp. Incorporating multiple copies of a DNA sequence encoding essential genetic information, 2) incorporating multiple copies of the DNA sequence isolated in step 1 into a recipient sphingomonas, 3) from step 2 Culturing the resulting bacteria in a fermentation broth to produce a sphingan, and 4) isolating the sphingan from step 3. 54. The bacterium in which the DNA sequence is inserted is A
TCC31554, ATCC31461, ATCC31
853, ATCC 21423, ATCC 31555, A
TCC31961, ATCC53159, and ATCC
54. The method of claim 53, which is a member of the Sphingomonas strain selected from the group consisting of 53272. 55. The bacterium in which the DNA sequence is inserted is A
TCC31554, ATCC31461, and ATCC
54. The method of claim 53, which is a member of the Sphingomonas strain selected from the group consisting of 53272. 56. The DNA sequence is ATCC3155.
4, ATCC31461, ATCC31853, ATC
C21423, ATCC31555, ATCC3196
54. The method of claim 53, isolated from a Sphingomonas strain selected from the group consisting of 1, ATCC53159, and ATCC53272. 57. The DNA sequence is ATCC3155.
54. The method of claim 53, isolated from a Sphingomonas strain selected from the group consisting of 4, ATCC31461, and ATCC53272. 58. The method of claim 53, wherein the DNA sequence was isolated from Sphingomonas strain ATCC31554. 59. The DNA sequence is S88clΔ3, S
59. The method according to claim 58, which has a restriction enzyme map of a segment selected from 88c2 and S88c3. 60. The method of claim 59, wherein the DNA sequence is a fragment of the segment. 61. The method of claim 53, wherein said DNA sequence was isolated from Sphingomonas strain ATCC31461. 62. The DNA sequence comprises segment S60c
The method according to claim 61, which has a restriction enzyme map of 1, S60c2, or S60c3. 63. The method of claim 62, wherein the DNA sequence is a fragment of the segment. 64. The method of claim 53, wherein said DNA sequence was isolated from Sphingomonas strain ATCC53272. 65. The DNA sequence comprises a segment NWc
7. A restriction map of 2.1 or NWc2.2 is included.
The method according to 4. 66. The method of claim 65, wherein the DNA sequence is a fragment of the segment. 67. The method of claim 53, wherein the sphingan has the general formula: Embedded image In the formula, Glc is glucose, GlcA is glucuronic acid, Rha is rhamnose, Man is mannose, and X is rhamnose or mannose. Z
Is attached to Glc residue 2 and Z is α-L-Rha-
It is (1--6) -α-L-Rha, α-L-Man, or α-L-Rha. W is attached to Glc residue 1 and W is β-D-Glc- (1--6) -α-D-Gl
c or α-L-Rha. The subscripts v and y are 0, 0.33, 0.5, 0.67, or 1. The reducing end of the polymer is on the side of the X residue of the main chain, v and y
When is 0, W and Z do not exist on the main chain. 68. The method of claim 53, wherein said DNA sequence encodes a glycosyltransferase enzyme. 69. The glycosyltransferase enzyme is a glycosyl IP-transferase.
9. The method according to 8. 70. The method of claim 53, which comprises the rhamnose operon or rhamnose gene. 71. Prior to said step 1 of said incorporating, said DNA
54. The method further comprises the step of isolating the sequence.
The method described in. 72. ATCC69735, ATCC697
44, ATCC31853, ATCC21423, and ATCC319196, a biologically pure culture of a Sphingomonas microbial organism selected from the group consisting of: 73. Escherichia coli ATCC69732, A
Biologically pure cultures of TCC69733, or ATCC69734. 74. A DNA sequence substantially corresponding to the sequence shown in SEQ ID NO: 3.