KR20090016655A - High viscosity diutan gums and methods of producing - Google Patents

Abstract

The present invention describes the production of a diutan polysaccharide exhibiting increased viscosity properties as compared with previously produced polysaccharide of the same type of repeating units. Such an improved diutan polysaccharide is produced through the generation of a derivative of Sphingomonas sp. ATCC 53159 that harbors a multicopy broad-host-range plasmid into which genes for biosynthesis of diutan polysaccharide have been cloned. The plasmid provides the capability within the host Sphingomonas strain to produce multiple copies of genes for such polysaccharide synthesis. In such a manner, a method of not just increased production of the target diutan polysaccharide, but also production of a diutan polysaccharide of improved physical properties (of the aforementioned higher viscosity) thereof is provided. Such a diutan polysaccharide has proven particularly useful as a possible viscosifier in oilfield applications and within cement materials. The inventive methods of production of such an improved diutan polysaccharide, as well as the novel cloned genes required to produce the improved diutan within such a method, are also encompassed within this invention. Additionally, the novel engineered Sphingomonas strain including the needed DNA sequence is encompassed within this invention.

Description

HIGH VISCOSITY DIUTAN GUMS AND METHODS OF PRODUCING

The present invention describes the preparation of diutane polysaccharides which exhibit increased viscosity properties compared to previous prepared polysaccharides having the same type of repeat units. This improved diutan polysaccharide is a Sphingomonas species that contains a broad host-wide multicopy plasmid in which the gene for biosynthesis of diutan polysaccharide is cloned. sp . ) Through the production of derivatives of ATCC 53159. The plasmid provides the ability to produce multiple copies of genes for such polysaccharide synthesis in a host sphingomonas strain. In this way, a method of increasing the production of the target diutan polysaccharide is provided, as well as a method of producing the diutan polysaccharide whose physical properties (high viscosity characteristics described above) are improved. These diutan polysaccharides have proven to be particularly useful as thickeners that can be used in cement materials and in the field of oilfield. Also included in the present invention are methods according to the present invention for producing such improved diutan polysaccharides, and novel cloned genes necessary for producing improved diutan using this method. In addition, new genetically engineered sphingomonas strains comprising the required DNA sequences are also included in the present invention.

Polysaccharides or gums are mainly used to thicken or gel aqueous solutions and are often classified into two groups, thickeners and gelling agents. Typical thickeners include starch, xanthan gum, diutan gum, wellan gum, guar gum, carboxymethylcellulose, alginate, methylcellulose, gum karaya and gum tragacanth. Common gelling agents are gelatin, gellan gum, starch, alginate, pectin, carrageenan, agar and methylcellulose.

Some polysaccharides, or more specifically, biogum, such as xanthan, gellan, wellan and diutan, have been produced through fermentation from microorganisms for many years. Such biogum exhibits a number of properties, such as the ability to change viscosity, which has been used in many different applications. Included in this list are gelling agents for foods such as confectionery jelly, jams and jellies, dessert gels, sugaring and dairy products, as well as components of microbial media. Thickeners are also utilized in numerous end use applications to change the viscosity of the target liquid. There may be many other different possible end uses (including cement production, for example), but the ability of the gums to make viscosity changes in underground and / or underwater petroleum liquids to facilitate their flocculation is of particular interest. . Different biogum is made from different bacterial sources, for example Xanthan gum Xanthomonas campestris ), gellan gum from Sphingomonas elodea , wellan gum from sphingomonas species ATCC 31555, and diutan gum (S-657) from sphingomonas species ATCC 53159. Genetic engineering of these strains has been undertaken in the past to make significant changes to the synthetic gum materials prepared through the fermentation process described above. This manipulation causes a change, such as the removal of acyl groups, resulting in different gum materials exhibiting different physical properties. In general, these genetic manipulations include types that ultimately alter the composition of the target biogum through altered gene expression in the host organism, or types that increase the yield of the target biogum through the introduction of plasmids representing only gene amplification (Pollock). US Pat. Nos. 5,854,034, 5,985,623 and 6,284,516, and US Pat. No. 6,709,845 to Pollock.

Diutan gum (also known as heteropolysaccharide S-657) is prepared through fermentation of strain sphingomonas species ATCC 53159 and shows thickening, suspending and stabilizing properties in aqueous solution. Diutan generally exhibits hexameric repeating units consisting of the four sugars of the main chain (glucose-glucuronic acid-glucose-rhamnose), and the side chains of two rhamnose residues attached to one of the glucose moieties. A detailed description of the diutan gum structure can be found in Chowdhury, TA, B. Lindberg, U. Lindquist and J. Baird, carbohydrate Research 164 (1987) 117-122. Di wootan are each repeating unit the literature as having two acetyl substituent have been described in [Diltz et al., Carbohydrate Research 331 (2001) 265-270]. Both of these references are incorporated herein by reference in their entirety. A detailed description of the preparation of diutan gum can be found in US Pat. No. 5,175,278, which is incorporated herein by reference in its entirety. Diutane can be prepared from sphingomonas strains, for example, using standard fermentation techniques using carbohydrate sources (including but not limited to glucose, maltose and the like), nitrogen sources and additional salts.

Physical properties imparted by wild type diutan biogum are particularly desirable for certain industries in terms of viscosity change properties and / or moisturizing properties. Unfortunately, Diutan has proven difficult to manufacture cost-effectively. In addition, this cost problem currently has a disadvantageous effect on the widespread use of diutan, where the viscosity seen by biogum is insufficient to replace other inexpensive but effective biogum (eg, xanthan gum). Because. Thus, there has been a clear need, at least, to provide a method for producing an effective diutan at low cost, and / or a method for producing a biogum of the diutan type which is also significantly improved in physical properties. To date, methods of making any type of related sphingan have been mentioned only in terms of yield improvement (specifically, without any demonstration of diutan) (mentioned in the Pollock et al. Patents described above). There was no discussion or appropriate proposal for any method of providing a method for producing high molecular weight, improved diutan gum, which showed any improvement in viscosity measurement by the above production method.

Summary of the Invention

The inventors have found that amplifying certain novel isolated DNA sequences for diutan biosynthesis in a host sphingomonas organism not only increases the production of diutan gum derived from this sequence, but also exhibits increased viscosity characteristics. It was found to produce Utan gum. This novel DNA sequence, which is introduced into the host organism via any well known method such as, but not limited to, a plasmid, provides the desired results and is used in the diutane synthesis method. This particular advantage of using the amplified genes in the plasmid is that it is relatively simple to integrate such isolated DNA sequences into the diutane synthesis process. Another advantage is the ability to generate high viscosity characteristics in the target diutan gum, potentially increasing fermentation production efficiency if necessary.

Thus, the present invention provides improvements, in particular, in many different viscosity measurements

i) intrinsic viscosity greater than 150 dL / g, preferably greater than 155 dL / g, more preferably greater than 160 dL / g;

ii) a salt water 3 rpm viscosity of greater than 35, preferably greater than 37, more preferably greater than 40 and most preferably greater than 42 when measuring dialel viscosity;

iii) 0.3 rpm viscosity of seawater above 35,000 centipoise (cP), preferably above 39,000 cP, more preferably above 40,000 cP, most preferably above 41,000 cP; And PEG low shear viscosity above 3500 cP, preferably above 3700 cP, more preferably above 3900 cP, and most preferably above 4000 cP.

Diutan gum that represents. In addition, the present invention provides the above-described diutan gum by introducing a specific bundle of genes into a host sphingomonas organism, as defined in any of the foregoing sections, and obtaining a diutan gum produced by fermentation in the organism. It includes how to do it. The invention also encompasses certain DNA sequences and any vectors (such as plasmids) for providing multiple copies of a gene or for enhancing expression of a gene using stronger promoters and the like. In addition, genetically modified strains of sphingmonas containing plural copies of the diutan biosynthetic gene defined by the unique isolated DNA sequence are also included.

Such unique isolated DNA sequences have been found to require one or more diutan biosynthetic enzymes, ie DpsG polymerases. In another possible embodiment, the diutan biosynthetic enzyme is selected from DpsG polymerase and glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; And dTDP-6-deoxy-L-mannose-dehydrogenase. In another possible embodiment, the diutan biosynthetic enzyme comprises DpsG polymerase and rhamnosyl transferase IV; Beta-1,4-glucuronosyl transferase II; Glucosyl isoprenylpoate transferase I; And glucosyl transferase III. In another possible embodiment, the diutane biosynthetic enzyme comprises dpsG polymerase and the polysaccharide transport protein dpsD, dpsC and dpsE. In another possible embodiment, the diutan biosynthetic enzyme is selected from rhamnosyl transferase TV; Beta-1,4-glucuronosyl transferase II; Glucosyl isoprenylpoate transferase I; Glucosyl transferase III; Glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; And dTDP-6-deoxy-L-mannose-dehydrogenase. In general, the diutan biosynthetic enzymes of the process according to the invention and the diutan biosynthetic enzymes in the products according to the invention are polymerases; Lyase; Rhamnosyl transferase IV; Beta-1,4-glucuronosyl transferase II; Glucosyl transferase III; Polysaccharide transport protein; Secreted protein; Glucosyl-isoprenylphosphate transferase I; Glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase and combinations thereof. The present invention is one as shown in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43. Or more diutan biosynthesis enzymes, or SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43 and 95 It further comprises an isolated nucleic acid molecule (in addition to DNA that may be present on the target chromosome) encoding at least% identical enzymes.

Thus, the process according to the invention (also the product produced by this process) relates to sphingum gums, in particular diutan types, including but not limited to S88, S60 and S657.

As described above, the present invention has been accomplished by discovering and realizing that a specific DNA sequence introduced into a plurality of copies within a particular sphingomonas strain can increase the biosynthetic production of high viscosity diutan polysaccharides. Genetically engineered bacteria containing genes for increased production produce significantly higher amounts of diutan polysaccharides than nongenic engineered bacteria, resulting in the high viscosity properties of the aforementioned results.

According to the present invention, host organisms (any well-known form, such as also, in order to obtain the above-mentioned increase in production and increase in viscosity properties (not supported by any particular scientific theory, but through which the molecular weight range properties are believed to increase) DNA sequences introduced into plasmids, by way of non-limiting example, can be isolated, recovered and cloned by techniques readily available in the art. The DNA is then transferred to a plurality of copyings (via plasmids, in other known ways) to sphingomonas bacteria, or the expression of the gene is increased via a suitable, e.g., more potent, promoter. After insertion into the target bacterium, the production of diutan can be measured by fermenting the genetically engineered bacterium and comparing the yields in terms of amount produced and quality produced. Both increased production and viscosity increase can be measured by comparing the production of diutan over the wild type diutan producing strain (ATCC 53159) via the method according to the invention.

Brief description of the drawings

1 is a plot of isolated genes for diutan gum biosynthesis. Presumptive or known genes are indicated. Segments inserted into different plasmids are also indicated.

2 is a graph of the improvement in intrinsic viscosity measurement achieved by the diutan biogum material according to the invention.

details

The following terms will be used throughout this specification in connection with the present invention and have the meanings specified below:

The term "sphingmonas" is used throughout this specification to refer to strains of Gram-negative bacteria from the genus Sphingomonas.

The term "increased producer" or "increased production" is used throughout this specification to produce significantly more (more than about 5% more on a weight / weight basis) diutan polysaccharide compared to wild-type bacteria of the same strain. Used to describe genetically engineered bacteria containing multiple copies of DNA sequences isolated from strains.

The term “isolated” is intended to be recovered from a microorganism and subjected to some degree of purification, ie one or more purification steps, cleaved or cleaved by restriction enzymes, cloned into a plurality of copies, inserted into a plasmid vector or inserted into or incorporated into a bacterium. Used to describe DNA that can be.

The term "sequence" is used to describe a specific segment of DNA identified by a nucleotide unit. The term "inserted" is used throughout the present specification to describe the process and results of delivering (eg, by way of non-limiting example, via a plasmid) DNA fragments isolated from the chromosomal DNA of a diutan producing sphingmonas strain to the sphingmonas strain. Used for. The isolated DNA is first introduced into a preferred plasmid (here pLAFR3), which is also well known in the art as a non-limiting example, and then to be delivered to the receptor sphingomonas bacteria, for example by conjugation or activation. Can be. After insertion into the receptor sphingomonas bacterium, the plasmid containing the relevant DNA sequence can be used to increase the production of several (more than two, typically 4-10) high-viscosity (also thought to be high molecular weight ranges) diutane polysaccharides. Replication will be done at the receptor cell to provide) DNA fragments. It is generally effective to use conjugation or maneuver to deliver plasmid vectors to receptor bacteria. In addition, chemical transformation or electroporation of competent cells using purified DNA may be used. Other vectors or bacteriophages can be used to deliver DNA to the host cell. There is no need to maintain DNA segments in plasmids (or other well-known delivery vectors) in the receptor diutan producing sphingmonas. It is common to introduce additional copies of the DNA segments into the bacterial chromosomes so that the segments are replicated every time by the same mechanism of replicating the bacterial DNA. Alternatively, increased gene expression can be achieved using stronger promoter elements.

The term "gene amplification" refers to an enhanced gene copy, for example, by cloning a multicopy plasmid (such as 4-10) into a target gene or inserting multiple copies of the gene (such as 4-10) into the bacterial genome, or Alternatively, it is used to indicate enhanced gene expression by changing promoter elements to enhance gene expression. Both of these and other methods can increase the amount of encoded protein.

The term "biosynthesis" is used throughout this specification to describe the biological production or synthesis of diutan by sphingmonas bacteria. Diutan polysaccharides are synthesized from individual carbohydrate units in a series of steps controlled by a number of bacterial enzymes.

Relevant DNA sequences incorporated into any selected form (e.g., again, but not necessarily, but not necessarily, in the form of plasmids), may contain genetic information known to be beneficial or essential for enhanced production and biosynthesis of increased molecular weight diutan polysaccharides. Coding In addition, however, certain DNA sequences according to the present invention (such as those within plasmid pS8), while not supporting specific scientific theories, not only increase production but also increase the number of repeat units polymerized in the individual polymers of the diutan itself. I think that. As a result, the increase in repeat units as described above is surprisingly believed to generate the high viscosity properties provided by diutan gum. The molecular weight increase has been assumed to be due to the measured increase in intrinsic viscosity related to the molecular weight, by a power law relationship. Thus, for linear polymers (such as diutan gum), it is known that the intrinsic viscosity is substantially proportional to the molecular weight in that respect.

Isolation of the relevant DNA sequences which are the basis of the method according to the invention and which produce diutane polysaccharides of increased viscosity is accomplished by standard techniques and methods. Thus, such sequences can be produced from Diutan producing Sphingmonas strains cultured using standard procedures. The extraction of DNA can then be carried out, for example, by centrifugation and resuspension of the initial bacterial cell, followed by elution of the DNA through subsequent purification columns. After purification is complete, the isolated DNA can be digested with restriction endonucleases, cloned into the desired plasmid or other transfer vector, and then transferred to the receptor strain. Other techniques as known in the art can be used without limitation.

DNA cloning in the present invention is in accordance with general techniques and methods that have become standard in the art. It is noted that a number of methods can be used to clone DNA fragments according to the invention, and the invention is not limited to, for example, using plasmid cloning vectors. For example, the DNA fragment can be inserted into a bacteriophage vector and cloned.

The cloned DNA sequence can then be introduced into the sphingmonas strain via a plasmid or other transfer vector. The genetically modified sphingomonas strain can then be used to produce diutan by fermentation. Basically, a medium suitable for fermentation is generally a carbohydrate, nitrogen source, for example inorganic ammonium, inorganic nitrates, organic amino acids or proteinaceous materials, including carbon sources such as glucose, lactose, sucrose, maltose or maltodextrin, Such as hydrolyzed yeast, soy flour or casein, distiller's soluble or corn immersion water and inorganic salts. A wide variety of fermentation media will be supported for the preparation of the diutan according to the invention.

Carbohydrates may be included in the fermentation broth in varying amounts, but are typically included in about 1-10% by weight (preferably 2-8% by weight) of the fermentation broth. Carbohydrates may be added prior to fermentation or alternatively during fermentation. The amount of nitrogen may range from about 0.01% to about 0.4% by weight of the aqueous medium. A single carbon source or nitrogen source can also be used as a mixture of these sources. Inorganic salts used to ferment sphingomonas bacteria are in particular salts containing sodium, potassium, ammonium, nitrate, calcium, phosphate, sulfate, chloride, carbonate and similar ions. Trace metals such as magnesium, manganese, cobalt, iron, zinc, copper, molybdenum, iodide and borate salts may also be advantageously included.

Fermentation may be carried out at a temperature in the range of about 25 ° C. to 40 ° C., preferably in the range of about 27 ° C. to 35 ° C. Inoculum can be prepared by standard methods of volume scale-up, including shaking flask culture and small scale in-situ fermentation. The medium for preparing the inoculum may be the same as the production medium or may be any of several standard media well known in the art, such as Luria broth or YM medium. More than one seed stage can be used to obtain the desired volume for inoculation. Typical inoculation volumes range from about 0.5% to about 10% of the total final fermentation volume.

The fermentation vessel may comprise a stirrer for stirring the contents. The vessel may also be equipped with automatic pH and foaming regulators. The production medium can be added to the container and sterilized by heating in place. Alternatively, carbohydrate or carbon sources can be sterilized separately before addition. Pre-incubated inoculation culture can be added to the cooled medium (typically at a preferred fermentation temperature of about 27 ° C. to about 35 ° C.), and the stirred culture is fermented for about 48 hours to about 110 hours, High viscosity broths can be created. Diutane polysaccharides can be recovered from the broth as a standard method of precipitation using alcohols, generally isopropanol.

Including a detailed description of the drawings

Preferred of the present invention Embodiment

The following examples are provided to illustrate the invention. The description of the embodiments should not be construed as limiting the scope of the invention in any manner.

DNA  Sequence Separation / Plasmid Preparation

In order to perform initial isolation and determine sequences suitable for the results of the invention described above, a genetic library of ATCC 53159 organisms was prepared as follows: Chromosome DNA was isolated from sphingomyosis ATCC 53159 and Sau 3AI restriction endo Partially digested with nuclease. DNA fragments ranging from 15-50 kb were purified from agarose gels and bound to the cosmid cloning vector pLAFR3 digested with Bam HI (Staskawicz, et al, "Molecular characterization of cloned avirulence genes from race 0 and race 1). of Pseudomonas syrinae pv. Glycinea ", J. Bacteriology 1987. 169: . Conforms to 5789-94), Escherichia coli (Escherichia coli ) was isolated from strain JZ279 (Harding, et al., "Genetic and physical analysis of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris ", J.Bacteriology 1987. 169:. . 2854-61) combined reactants λ phage particles (from packaged using a Gigapack III Gold packaging extract from California, La Jolla material Stratagene), E. coli and Library Efficiency (E . coli) DH5αMCR cells (were transfected by Life Technologies) in the United States Rockville, Md material. Approximate were pooled 10,000 tetracycline-resistant colonies to form a gene library, was isolated the individual sequences from the library, in which case Work carried out included isolating specific genes for polysaccharide biosynthesis from the sphingmonas ATCC 53159 organism.

Such genes for polysaccharide biosynthesis are generally identified by the complementarity of mutations that are defective in polysaccharide synthesis, especially those blocked in the first step (glycosyl transferase I). Since the transferase I lacking mutations of ATCC 53159 are not available initially, the complementarity of the transferase I lacking mutations of Sphingmonas elodea and xanthomonas campestris is used to identify genes for diutan polysaccharide biosynthesis. It was. Plasmid pLAFR3 can be transferred from its E. coli host to other Gram-negative bacteria by tri-parental conjugation using a helper plasmid that provides IncP delivery function [Ditta, et al., "Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti ", according to Proc. Natl. Acad. Sci. 1980. 77 : 7347-51.]. The RK2 plasmid has an estimated copy number of 5 to 7 per chromosome in this coli (Figurski et al.," Suppression of ColE1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro ", J. MoI. Biol. 1979 133 : 295-318.).

The gene library of ATCC 53159 chromosomal DNA in this coli is transferred to the nonmucoid mutation (GPS2) of S. eldeaea ATCC 31461 by triad conjugation, which is selected for tetracycline and streptomycin resistance. The helper plasmid used was pRK2013 (in this E. coli strain JZ279) which contained a narrow host range of origin of replication but exhibited the trans-functional function required to activate pLAFR3. Plasmid pRK2013 did not replicate in Sphingmonas strains. S. Elodea ATCC 31461 produces polysaccharide gellans. Both the gellan and the diutan polysaccharide are [→ 4) -α-L-rhamnose- (1 → 3) -β-D-glucose- (1 → 4) -β-D-glucuronic acid- (1 → 4 have the same tetrasaccharide repeating unit consisting of) -β-D-glucose- (1 →], however, the diutan also comprises a side chain of two rhamnose molecules attached to one of the glucose residues and is denatured by acetyl On the other hand, the gellans do not have side chain sugars and are denatured by acetyl and glyceryl The mutant GPS2 is the first step in polysaccharide biosynthesis, ie glucose-1 phosphate is UDP-D-glucose by the glucosyl transferase I enzyme. Defects in the delivery to the bactoprenyl phosphate lipid carrier at .. In tetracycline selection plates, polysaccharide production (mucoid) colonies were isolated from the background of non-mucolytic colonies. Coding Laze I Contains ATCC 53159 gene and approximately 20-25 kb of contiguous DNA. Plasmid DNA was isolated from eight mucinous GPS2 transconjugants and transferred to this E. coli strain DH5α (Life Technologies) by electroporation. The plasmid was isolated from this coli to obtain sufficient DNA for double digestion using restriction endonuclease Hind III / Eco RI (cutting both sides of the Bam HI restriction endonuclease site in the polylinker) to insert the insert DNA. The size of the insert DNA in the clone was determined by gel electrophoresis The terminal sequence of some plasmids was determined by sequencing from primers specific for the plasmid sequence flanking the Bam HI site of the vector. Sequences were analyzed by comparison with sequences in a computer database using BLASTX, two plasmids pS8 and pS6 are shown in FIG. And Similarly, ATCC 53159 gene libraries, Article 3 through the junction, transferase I (CXC109) (which will be the same as in the literature, such as Harding described above) is devoid rifampicin resistance X boiling liquid. It was delivered to Campestris mutants and selected for resistance to tetracycline and rifampicin. X. Campestris produces xanthan polysaccharides, the synthesis of which is also initiated by the transfer of glucose-1-phosphate from UDP-D-glucose to bactoprenyl phosphate lipid carriers by transferase I enzymes ( Ielpi et al., "Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthodmonas compestris ", J. Bacteriology. 1993. 175 : 2490-500). The plasmid was purified from the mucin-transfected strain and the terminal sequence was determined as described above. Two plasmids pX6 and pX4 are shown in FIG. .

S657 DNA cloned from plasmids pS8 and pX6 were fully sequenced by double stranded shotgun sequencing at Lark Technologies Inc. (Houston, TX). These sequences were analyzed to identify genes for diutan biosynthesis (shown in FIG. 1). Gene function is characterized by other genes in the database, particularly published genes for biosynthesis between S-88 sphinxes (e.g., in the patents of '516 Pollock et al., Supra), GenBank Accession No. U51197 and Gellan (GenBank AY217008 and AY220099) Same sex was specified as a reference. The gene was found to encode transferases for four sugars of the backbone and four genes for dTDP-rhamnose synthesis (FIG. 1). Genes for the secretion of polysaccharides were based on homology to genes for biosynthesis of other polysaccharides. Both genes encode proteins that are homologous to proteins involved in protein secretion. Both genes presumably encode polymerase and lyase. The insert in plasmid pX6 contains 17 genes, including the gene dpsB encoding transferase I (which initiates the first step in diutan synthesis), the gene for secretion and four genes for dTDP- rhamnose synthesis. One lacks genes for transferases II, III and IV and putative genes for polymerases and lyases. Plasmid pS8 is a group of dps gene bundles containing genes for the secretion of polysaccharides, including all four backbone sugar transferases, four genes for dTDP-rhamnose synthesis, and putative genes for polymerase and lyase. Contains dog genes, but lacks genes of unknown function, orf6 and orf7 . Plasmid pS6 contains a gene for secretion and four sugar transferases, but no genes for polymerase or all of the genes for dTDP-rhamnose synthesis. Plasmid pX4 contains only a small portion of the dps region, but by genes encoding transferase I and Pollock et al., Described as sufficient to increase the production of polysaccharides in sphingomonas strains, for dTDP-rhamnose synthesis It contains four genes.

Strain manufacturing

The four plasmids described above were then used to form the novel S657 genetically engineered strains (S657 / pS8, S657 / pS6, S657 / pX6, and S657 / pX4) via the triad conjugation as described above. Introduced in 53159. It was then fermented as described above to produce a biogum material as noted below. While all four plasmids had a beneficial effect on diutan productivity, the pS8 plasmid also surprisingly provided a very large increase in diutane viscosity and an increase in molecular weight. The DNA sequence of pS8 (26278 bps) (DNA sequence 1) is provided, and the encoded genes are listed in Table 1 below, which is shown in diagram form in FIG. 1. Insertion DNA in plasmid pS8 includes genes from dpsG to rmlD and some genes dpsS and orf7 .

The gene table below is basically a list of genes represented by DNA sequences for insertion into plasmid pS8, as shown in FIG.

pS8  Gene on plasmid insert start End designation Explanation 2 ^*  1054  dpsS homologous with gelS (partially)   2738  1113 C  dpsG  Putative polymerase   4895  2898 C  dpsR  Putative lyase   5093  6031  dpsQ  Putative Rhamnosyl Transferase IV   7082  6111 C  dpsI  Unknown   7121  8167  dpsK  Beta-1,4-glucuronosyl transferase II   8164  9030  dpsL  Glucosyl Transferase III  10467  9079 C  dpsJ  Unknown  11076  12374  dpsF  Unknown  12389  13306  dpsD  Putative polysaccharide transport protein  13341  14687  dpsC  Putative polysaccharide transport protein  14687  15394  dpsE  Putative polysaccharide transport protein  15405  16286  dpsM  Putative polysaccharide transport protein  16270  16968  dpsN  Putative polysaccharide transport protein  18454  17060 C  atrD  Putative secreted protein  20637  18451 C  atrB  Putative secreted protein  21229  22641  dpsB  Glucosyl-Isprenylphosphate Transferase I  22757  23635  rmlA  Glucose-1-phosphate thymidylyl transferase  23632  24198  rmlC  dTDP-6-deoxy-D-glucose-3-5-epimerase  24202  25263  rmlB  dTDP-D-glucose-4,6-dehydratase  25263  26129  rmlD  dTDP-6-deoxy-L-mannose-dehydrogenase 26277 ^*  26146 C  orf7  (Partial) unknown function ^{* No} first in-frame codon, start codon

Diutan  Produce

Diutane production using the genetically engineered plasmid containing Sphingomonas S657 strain, compared to the S657 wild type strain without plasmid, was measured by carrying out three sets of fermentations with stirring and aeration in the same liquid medium in an Applikon 2 O L fermentor. . To the plasmid containing strain, antibiotic tetracycline was added at 5 mg / L during fermentation to ensure preservation of the plasmid. KOH was added as needed for pH adjustment. Two inoculation steps were performed using 1% to 6% inoculum inoculation. The medium used for fermentation contained corn syrup as a carbohydrate source, an assimilable nitrogen source and salts. Nutrients that can be used for fermentation are well known in the art and include carbohydrates such as glucose, sucrose, maltose or maltodextrin, nitrogen sources such as inorganic nitrogen such as ammonium or nitrate, organic nitrogen such as amino acids, Hydrolyzed yeast extract, soy protein, or corn steep water, and additional salts containing, for example, chloride, phosphate, sulfate, calcium, copper, iron, magnesium, potassium, sodium or zinc.

As a measure of the resulting diutane yield, broth viscosity and precipitated fibers were measured. The viscosity of the fermentation broth was measured through a Brookfield viscometer operated at 60 rpm with spindle # 4, the results of which are shown in Table 2. At the end of the fermentation, the broth was treated with the well known introduction of glucoamylase enzymes to hydrolyze any residual oligosaccharides derived from corn syrup. The produced diutan gum was then precipitated from one aliquot of broth using two volumes of isopropyl alcohol. The fibers were collected in a filter and dried. In Table 2, the term DWY refers to the total dry weight yield of biogum that can be precipitated after hydrolysis of excess oligosaccharides derived from corn syrup.

Obviously, the material produced is higher in yield due to the plasmids pX4, pX6, pS6 or pS8 carrying additional copies of the gene for diutane biosynthesis present therein. However, with the pS8 plasmid, the broth viscosity did not significantly increase, compared to the increase in dry weight yield, which showed that in addition to the increase in the amount of diutane produced, some factors influence viscosity.

Fermentation of Plasmid-Containing Strains DWY Strain Conduct # 1 Conduct # 2 Conduct # 3 Average % increase  S657 34.3 32.2 33.9 33.5 ----  S657 / pS8 37.1 35.4 35.9 36.1 8.0%  S657 / pX6 38.4 37.6 33.5 36.5 9.1%  S657 / pS6 37.6 12.3%  S657 / pX4 36.4 8.8% Bros  Viscosity Strain Conduct #l Conduct # 2 Conduct # 3 Average % increase  S657 5150 4950 5550 5217 ----  S657 / pS8 6650 6850 6850 6783 30.0%  S657 / pX6 5400 6250 5125 5592 7.2%  S657 / pS6 6675 28.0%  S657 / pX4 5525 5.9%

Obviously, the material produced was higher in yield due to any of the four plasmids present therein, while the pS8 and pS6 plasmids surprisingly significantly increased the broth viscosity, indicating a high production quality. The quality of the resulting diutan gum product, ie the viscosity, was then measured.

In the application test Diutan Rheology

The diutan gum samples were then analyzed in terms of potentially beneficial uses within two different zones: dielectric additive for oil recovery and cement additive for moisturizing and rapid installation.

The oilfield industry is based on what is referred to as a "sea water viscosity (SWV)" test as an acceptable method of performance for oil recovery gums. This test is basically a measure of the effectiveness of the gum to increase the viscosity at brine conditions of water (eg to repeat recovery from the seabed).

Predicting the likelihood of a gum produced as a suitable viscosity modifier for oil recovery applications is generally acceptable in view of the change in viscosity of the test seawater formulation. This "Synthetic Seawater" formulation is prepared by mixing 419.53 grams of sea salt (ASTM D-1141-52) in 9,800 grams of DI water. For seawater viscosity testing, 0.86 grams of sample gum is added to 307.0 g of Synthetic Seawater and mixed for 35 minutes at approximately 11,500 rpm in a Fann Multimixer (Model 9B5, part number N5020). At the end of 35 minutes, the solution is cooled to approximately 26 ° C. and then the viscosity is measured. For 3-rpm readings, the sample is placed in the Fann sample platform (Fann Model 35 A; Torsion Spring MOC 34/35 F0.2b; Bob B1; Rotor R1), turning the motor at low speed and setting the gear shifter to an intermediate position To adjust the speed to 3 rpm. The reading is then stabilized and the shear stress values are read from the dial and recorded as SWV 3 rpm dial reading (DR). For 0.3-rpm readings, the viscosity is measured using a Brookfield viscometer (Brookfield LV DV-II or DV-II viscometer with LV-2C spindle). The speed of the spindle is set at 0.3 rpm, after the spindle has been rotated for at least 6 minutes, the viscosity is recorded as SWV-0.3 rpm reading and expressed in centipoise (cP). For cement applications, the PEG LSRV test (testing low shear viscosity using polyethylene glycol as a dispersant as outlined below) provides the industry with an indication of the effectiveness of the performance of viscosity modifiers. This test measures the viscosity of a 0.25% solution of biogum in Standard Tap Water (STW). STW is prepared by adding 10.0 grams of NaCl and 1.47 grams of CaCl ₂ .2H ₂ O to 10 liters of deionized water. For viscosity measurements, 0.75 grams of biogum is added to 4.5 grams polyethylene glycol 200 (CAS 25322-68-3) in a 400 ml beaker and fully dispersed. 299 grams of STW are then added to the beaker and mixed for approximately 4 hours using a low-pitched propeller stirrer at 800 ± 20 rpm. After 4 hours of mixing, the beaker was placed in a 25 ° C. water bath and allowed to settle for approximately 30 minutes. The viscosity is then measured using a Brookfield LV viscometer with a 2.5+ torque spring (or equivalent instrument, such as Model DVE 2.5+) at 3 rpm (first rotating the spindle for 3 minutes) using the LV 1 spindle. This is referred to as centipoise (cP).

The produced diutan sample was tested in this manner; The results were as follows:

From plasmid-containing strains Diutan Rheology Strain SWV 3 rpm DR SWV -0.3 rpm cP PEG LSRV cP Conduct # 1 Conduct # 2 Conduct # 3 Conduct # 1 Conduct # 2 Conduct # 1 Conduct # 2 Conduct # 3  S657 Wild Type 25 26 22 24400 28600 2820 3150 2280  S657 / pS8 42 43 47 41500 38800 4720 4980 4920  S657 / pX6 25 29 26 25000 29100 2860 3400 3270  S657 / pS6 --- --- 22 --- --- --- --- 2270  S657 / pX4 --- --- 24.5 --- --- --- --- 2950  SWV = viscosity in seawater LSRV = low shear rate viscosity

Surprisingly, there is a marked increase in viscosity exhibited by the diutan gum according to the invention produced by some genetically engineered plasmid containing strains. But most surprisingly, the viscosity increase of SWV at 3 rpm for the pS8 strain was 80%, while the same assay conducted for the pX6 strain was only 9.6% compared to the wild type results. There was no significant increase in plasmids pS6 and pX4. Likewise, a low SWV rpm test showed an increase of 51.5% for wild type for pS8 type, whereas an increase of only more than 2% for pX6. Finally, the polyethylene glycol LSRV test showed a pS8 result of more than 77% increase in viscosity compared to wild-type gum, compared to less than 16% increase for pX6 diutan, 7.2% increase for pX4 and significant increase for plasmid pS6. Showed that there was no. In addition, a very surprising result during this period is that diutan gum production is greatly improved by using the necessary gene sequences exemplified in the pS8 plasmid as a way of introducing sequences in the target diutan producing bacteria.

Thus, the diutan according to the invention produced through the introduction of pS8 showed a surprisingly increased viscosity measurement in all three calculations, especially compared to the variants produced by wild type and pX6 plasmids. Thus, it was expected that this new diutane would work very well under typical dielectric conditions and in cement applications.

Rheology  Basic description of the improvement

The above examples showed that Diutan from S657 / pS8 strain showed a significant increase in rheological parameters. Thus, this substantial increase in seawater and PEG low shear viscosity measurements cannot be seen solely due to increased productivity, since the pX6 strain also showed similar (if not higher) yield results. In addition, in the previous examples illustrated in Table 2, the dry weight yield (alcohol precipitable) increased to 8.0%, while the rheological parameters increased significantly more for the S657 / pS8 strain (52-80). %). Basic studies were performed to demonstrate that rheological improvements were obtained using strain S657 / pS8 in wild type strains.

Intrinsic viscosity is a well known technique in polymer science for estimating the molecular weight of large molecules (C. Tanford, 1961. Physical Chemistry of Macromolecules . John Wiley & Sons, New York. The intrinsic viscosity is obtained by plotting the reduced viscosity (viscosity based on the concentration) relative to the solution concentration, and extrapolating the linear regression of the data to the zero concentration (y-intercept of the plot). Surprisingly, the produced black showed an increase in intrinsic viscosity as shown in the table below.

Five diutan samples, namely two samples from the wild-type strain (Control 1, Control 2) and three samples from the S657 / pS8 strain (Sample 1, Sample 2, Sample 3), were subjected to intrinsic viscosity, neutral sugar and organic acid. The analysis was evaluated. These samples were purified by alcohol precipitation, rehydration, treatment with hypochlorite, treatment with glucoamylase, treatment with lysozyme, and finally treatment with protease (performed in sequential order). These were then recovered in a 4: 1 CBM: broth ratio, dried and ground. CBM is an azeotropic isopropyl alcohol / water mixture comprising up to 82% by weight isopropyl alcohol.

Samples were tested for water content by performing as follows: In general, two 0.7 gram aliquots of the samples were tested using a Mettler HB 43 halogen moisture balance. The results from the two tests were then averaged and the results were used for moisture control.

After obtaining moisture data, a 0.2% solution of gum was prepared in 0.01 M NaCl based on controlled moisture. For three tests, a total of 200 grams of 0.2% solution was prepared. The gum was weighed on an analytical balance to the maximum approximation of the third decimal place and added to the water weighed to the maximum approximation to the third decimal place. Samples were stirred for 2 hours using a 2.5 inch diameter propeller mixer at 1000 rpm in a 400 ml long beaker.

Following initial hydration, each sample was diluted to 0.02% with 0.01 M NaCl. This was done by weighing 20 grams of 0.2% solution into a 400 ml beaker and then adding 180 ml of diluent as well. The diluted sample was mixed for an additional 30 minutes. The final dilution last used to measure the intrinsic viscosity was prepared from this sample. Each diutan sample was evaluated at concentrations of 0.004%, 0.008%, 0.010% and 0.012%.

Viscosity measurements were performed using the VilasticA® VE System. Prior to the measurement, Vilastic was adjusted to an error of less than 2.0% with water. Samples were measured using a Timer program (running at 2 Hz), one strain and a shear rate of approximately 12 1 / sec (all at a constant temperature of 23 ° C.). The measurements were taken five times and averaged for each sample. The averaged viscosity data was then used to calculate the intrinsic viscosity. 2 and Table 4 below provide the final results of the tests.

Comparison of Diutans Based on Intrinsic Viscosity Calculations Diutan samples Measured solid Intrinsic viscosity S657 control group 1 93.76 138.3 S657 control group 2 92.42 143 S657 / pS8 Sample 1 91.7 170.7 S657 / pS8 Sample 2 91.4 162.2 S657 / pS8 Sample 3 91.94 162.8

The results show that the S657 / pS8 strain consistently produces diutan with significantly higher intrinsic viscosity, in fact the average decreasing viscosity of the strain according to the invention was 165.2, whereas the control group was 140.7 (all similar measured solids). Level). This result shows that the diutane produced by S657 / pS8 has a higher molecular weight than the wild type control.

FIG. 2 is a graph of this trend showing a constant and high intrinsic viscosity measured with similar solids content between the control and the strain according to the invention.

In order to confirm that the high viscosity diutan gum derived from S657 / pS8 has the same composition as the diutan derived from the wild type strain, the composition was measured by testing for neutral sugars and organic acids. Purified samples used for intrinsic viscosity measurements were used for neutral sugar analysis. Aliquots of each purified sample were hydrolyzed to constituent sugars using trifluoroacetic acid (100 ° C./-18 h). The hydrolyzate neutral sugar was quantified by high performance anion exchange chromatography using pulsed current detection. The hydrolyzate organic acid was quantified by high performance ion exclusion chromatography using chemically inhibited conductivity detection. Table 5 below summarizes the results from the neutral sugar analysis. As can be seen, the neutral sugar profile for the S657 / pS8 strain is nearly identical to the neutral sugar profile for the S657 wild type strain. Although both results differ from the theoretical values, these results show that the structure of the repeating unit of the diutan gum prepared using pS8 is identical to that of the wild type, and any increase in the viscosity imparted by the pS8 material is long chain, Due to high molecular weight.

Neutral sugar and organic acid analysis for pS8 and wild type (control) Diutan strains Strain % Rhamnos % Glucose % Acetate Sample 1 S657 / pS8 32 19 8.9 Sample 2 S657 / pS8 32 19 8.2 Sample 3 S657 / pS8 32 17 8.6 Control group 1 S657 Wild Type 30 18 8.6 Control group 1 S657 Wild Type 33 20 8.7 Average S657 / pS8 32 18.3 8.6 Average S657 Wild Type 31.5 19 8.65 Theoretical value --- 46 30 8

Thus, the significantly improved seawater viscosity and PEG low shear viscosity of the diutan produced by the S657 / pS8 genetically engineered strain is due to the increase in molecular weight or length of the diutan molecule, ie the presence of more repeat units per molecule. It is not due to changes in the utane molecular composition and thus to the change in the repeating structure itself. Improved rheology may not be due to only an increase in the amount of diutan produced. Some of the bundles of genes for cloned diutan biosynthesis evaluated four different plasmids, pS6, pS8, pX4 and pX6, all of which showed a slight increase in productivity, with only plasmid pS8 recovered. There was a surprisingly high increase in the rheological parameters of.

Comparing the genes for cloned diutan biosynthesis in the tested plasmids suggests that the gene responsible for the increased molecular weight is likely the gene dpsG , because this gene is present in pS8 and not in other plasmids. . The gene dpsG encodes a hydrophobic membrane protein with strong homology with other membrane proteins involved in polysaccharide synthesis. Some proteins have homology with proteins for polymerases, enzymes that promote the binding of repeat units to form high molecular weight polysaccharides. The homologous gene gelG in S60 was considered to act as a polymerase for gellan synthesis (Harding, NE et al. 2004. "Organization of genes required for gellan polymerase biosynthesis in Sphingomonas) elodea ATCC31461 "J. Ind Microbiol Biotech 31 :...... 70-82 Sa-Correia, I. et al 2002." Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461:. Genes, enzymes and exopolysaccharide production engineering "J. Ind . Microbiol . Biotechnol . 29 : 170-176.) Homologues of dpsG were also isolated from sphingomonas strains ATCC 31554 and ATCC 21423 producing polysaccharides S88 and S7 (Pollock et al., US Pat. No. 5,854,034, No. 5,985,623 and 6,284,516, and Pollock, TJ US Pat. No. 6,709,845. Thus, it is likely that additional copies of the gene for polymerase will affect increasing the molecular length of the diutan molecule. It cannot be excluded that other genes in the bundle may be needed with dpsG to achieve the observed increase in viscosity, as candidates for genes encoding sugar transferases I, II, III and IV dpsB , dpsL , dpsK and dpsQ, special Is the first party of the repeating unit may be a gene dpsB encoding a transferase I to be added to the lipid carrier. Other important gene, when it is amplified by the multi-copy plasmid gene it has been found to increase the xanthan molecular gumB and gumC Homologous to dpsD , dpsC and dpsE All genes cloned in plasmid pS8 may be required to achieve dramatic viscosity increases.

While the present invention has been described and described in connection with certain preferred embodiments and examples, it is in no way intended to limit the invention to those specific embodiments, but rather to the extent of the appended claims and their equivalents. It is intended to cover structural equivalents as may be defined and all alternative embodiments and modifications.

submission

The following bacterial strains were produced by the American Type Culture Collection of the Boulevard 10801, Manassas University, Virginia, USA, dated October 21, 2005, in accordance with the Budapest Treaty for International Approval of Microbial Deposits. Deposited to Patent Depository:

Sphingmonas strain S657 containing plasmid pS8.

Claims (34)

Diutan gum exhibiting an intrinsic viscosity above 150 dL / g. The diutan gum of claim 1 having an intrinsic viscosity of greater than 155 dL / g. The diutan gum of claim 2 having an intrinsic viscosity of greater than 160 dL / g. Diutan gum showing a seawater 3 rpm viscosity of greater than 35 when measuring dialy viscosity. The diutan gum according to claim 4, which exhibits a salt water viscosity of greater than 37 when measured by dial viscosity. The diutan gum according to claim 5, which exhibits a viscosity of more than 40 seawater 3 rpm when the dial viscosity is measured. The diutan gum according to claim 6, which exhibits a seawater viscosity of greater than 42 rpm when the dial viscosity is measured. Diutan gum showing 0.3 rpm viscosity of seawater above 35,000 cp. The diutan gum according to claim 8 which exhibits 0.3 rpm viscosity of seawater above 35,000 cp. The diutan gum according to claim 9 which exhibits 0.3 rpm viscosity of seawater above 38,000 cp. The diutan gum of claim 10, which exhibits 0.3 rpm viscosity of seawater above 40,000 cp. The diutan gum according to claim 11, which exhibits 0.3 rpm viscosity of seawater above 41,000 cps. Diutan gum exhibiting a low shear rate viscosity above 3,500 cps in the presence of polyethylene glycol dispersant. The diutan gum of claim 13, wherein the gum has a low shear rate of greater than 3,700 cps in the presence of a polyethylene glycol dispersant. The diutan gum of claim 14, wherein said gum exhibits a low shear rate viscosity of greater than 3,900 cps in the presence of a polyethylene glycol dispersant. The diutan gum of claim 15, having a low shear viscosity of greater than 4000 cps in the presence of a polyethylene glycol dispersant. Introducing a coding sequence for at least one diutan biosynthetic enzyme into a diutan producing host Sphingomonas organism; And Culturing the host organism under fermentation conditions, wherein the host organism a) inherent viscosity greater than 150 dL / g, b) 3 rpm viscosity of seawater above 35 when the dial viscosity is measured; c) 0.3 rpm viscosity of seawater above 35,000 cp, and d) low shear viscosity above 3,500 cps in the presence of polyethylene glycol dispersant Producing diutan gum that exhibits at least one of the properties of Including, Diutan gum manufacturing method. The method of claim 17, wherein the at least one diutan biosynthetic enzyme is DpsG polymerase. 18. The method of claim 17, wherein the one or more diutan biosynthetic enzymes comprise DpsG polymerase and glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; And dTDP-6-deoxy-L-mannose-dehydrogenase. The method of claim 17, wherein the one or more diutan biosynthetic enzymes comprise DpsG polymerase and rhamnosyl transferase IV; Glucosyl-isoprenylphosphate transferase I; Beta-1,4-glucuronosyl transferase II; And glucosyl transferase III. 18. The method of claim 17, wherein the one or more diutan biosynthetic enzymes comprise DpsG polymerase and the polysaccharide foreign transport proteins DpsD, DpsC, and DpsE. 18. The method of claim 17, wherein the one or more diutan biosynthetic enzymes comprise rhamnosyl transferase IV; Beta-1,4-glucuronosyl transferase II; Glucosyl transferase III; Glucose-1-phosphate thymidylyltransferase; Glucosyl-isoprenylphosphate transferase I; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; And dTDP-6-deoxy-L-mannose-dehydrogenase. 18. The method of claim 17, wherein the one or more diutan biosynthetic enzymes are selected from polymerases; Lyase; Rhamnosyl transferase IV; Beta-1,4-glucuronosyl transferase II; Glucosyl transferase III; Polysaccharide transport protein; Secreted protein; Glucosyl-isoprenylphosphate transferase I; Glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase and combinations thereof. Introducing a coding sequence for one or more sphingan polymerase enzymes into a sphinggan producing host sphingomonas organism; Culturing the host organism under fermentation conditions, wherein the host organism produces sphingan gum with a longer average polymer length than that produced by sphingomonas organisms prior to introduction of the coding sequence. Method of producing a sphingan gum having a long average polymer length, comprising. The method of claim 24, wherein the sphingan gum is S88. The method of claim 24, wherein the sphingan gum is S60. The method of claim 24, wherein the sphingan gum is S657. One or more diutan biosynthesis as shown in SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 Enzyme, or at least 95% identical to SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43 Nucleic acid molecule encoding a protein. 29. The isolated nucleic acid molecule of claim 28, which encodes diutan polymerase. The isolated nucleic acid molecule of claim 28, which encodes a diutan polymerase and a polysaccharide transport protein. The method of claim 28, further comprising diutan polymerase and rhamnosyl transferase IV; Glucosyl-isoprenylphosphate transferase I; Beta-1,4-glucuronosyl transferase II; And glucosyl transferase III. The method of claim 28, further comprising diutan polymerase and glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; And dTDP-6-deoxy-L-mannose-dehydrogenase. The method of claim 28, further comprising diutan polymerase; Lyase; Rhamnosyl transferase IV; Beta-1,4-glucuronosyl transferase II; Glucosyl transferase III; Polysaccharide transport protein; Secreted protein; Glucosyl-isoprenylphosphate transferase I; Glucose-1-phosphate thymidylyltransferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; And dTDP-6-deoxy-L-mannose-dehydrogenase. The isolated nucleic acid molecule of claim 28, comprising the nucleic acid sequence according to SEQ ID NO: 1.