JP5364377B2 - High viscosity diutane gum and production method - 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

Detailed Description of the Invention

(Technical field of the invention)
The present invention describes the production of diutan polysaccharides, which consist of repeating units of the same type, exhibiting increased viscosity properties compared to previously produced polysaccharides. Such improved diutane polysaccharides can be obtained through the production of a derivative of Sphingomonas sp. ATCC 53159 that encapsulates a multi-copy broad range host plasmid in which the gene for biosynthesis of the diutane polysaccharide has been cloned. Generate. This plasmid provides the ability to generate multiple copies of the gene for synthesis of such polysaccharides within the host Sphingomonas strain. In such a manner, not only merely increased production of the target diutane polysaccharide, but also a method for its production of improved physical properties (referred to as higher viscosity as described above) is also provided. Such diutane polysaccharides have proven to be particularly useful in oil field applications and as possible viscosifiers in cement materials. Also encompassed within the scope of the present invention are the methods of the present invention that produce such improved diutane polysaccharides, as well as the new cloned genes that are included in such methods and that are necessary to produce improved diutane. Is included. In addition, newly engineered Sphingomonas strains containing the required DNA sequences are also encompassed within the scope of the present invention.

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

  Some polysaccharides, or more specifically, biogum, such as xanthan, gellan, welan, and diutane, have long been produced through fermentation from microorganisms. Such biogum exhibits various characteristics that have enabled its use on many different applications, such as the ability to modify viscosity. Included on such application lists include food grade ingredients such as confectionery jelly, jams and jelly, dessert gels, icing, and dairy gelling agents, and microbial media. In addition, thickeners are used in countless end-use applications to modify the viscosity of the target liquid. Of particular interest is the ability of such gums to give them modifications in viscosity to facilitate underground and / or underwater oil mining, but many other different possible end uses (as an example Including production of cement). Different biogum from different microbial sources such as Xanthomonas campestris to xanthan gum, Sphingomonas elodea to gellan gum, Sphingomonas bacterium ATCC 31555 to welan gum, and Sphingomonas bacterium ATCC 53159 to diutan gum (S-657) Has been generated. Genetic modification of such strains has been made in the past to achieve significant changes in the resulting gum material produced through the fermentation methods described above. Such modifications have allowed changes such as removal of acyl groups to create different gum materials that exhibit different physical properties. In general, such genetic modifications are those that ultimately change the composition of the target biogum through altered gene expression in the host organism, or that increase the yield of the target biogum through the introduction of a plasmid that exhibits only gene amplification (eg, US Pat. Nos. 5,854,034, 5,985,623, and 6,284,516 to Pollock et al. And US Pat. No. 6,709,845 to Pollock individuals). .

  Diutane gum (also known as heteropolysaccharide S-657) is prepared by fermentation of Sphingomonas bacterial strain ATCC 53159 and exhibits thickening, suspension, and stabilization properties in aqueous solution. Diutane generally exhibits a hexameric repeating unit consisting of four saccharides of the backbone (glucose-glucuronic acid-glucose-rhamnose) and two rhamnose residue side chains attached to one of the glucose residues. Details of the structure of diutan gum may be found in a paper by Chowdhury, T. A., B. Lindberg, U. Lindquist and J. Baird, Carbohydrate Research 164 (1987) 117-122. Diutane was shown in Diltz et al., Carbohydrate Research 331 (2001) 265-270 to have two acetyl substituents per repeating unit. Both of these references are hereby incorporated by reference in their entirety. Details for preparing diutan gum are found in US Pat. No. 5,175,278, which is hereby incorporated by reference in its entirety. Diutane is produced from Sphingomonas strains by utilizing standard fermentation techniques such as techniques using carbohydrate raw materials (glucose, maltose, etc. as non-limiting examples), nitrogen raw materials, and additional salts. Can do.

  The physical characteristics obtained by such diutane biogum in the wild type are desired by certain industries, especially with regard to their viscosity modifying properties and / or moisture retention characteristics. Unfortunately, diutane has proven difficult to produce cost-effectively. In addition, such cost issues are currently detrimental to the widespread use of diutane, because the degree of viscosity exhibited by such biogum is less expensive but effective. This is because it is not sufficient to replace (for example, xanthan gum as an example). In that regard, providing a method for producing such effective diutane at a lower cost, and / or a method for producing a diutane-type biogum that exhibits significant improvement in physical properties thereon It was an established need to provide To date, the only statement about the production of all types of related sphingans (specifically nothing has been demonstrated for diutane) is only for higher yields (in the above-mentioned Pollock et al. Patent). . No discussion or reasonable suggestion has been made about any way to provide a method for producing higher molecular weight improved diutane gum that exhibits any improvement in viscosity measurement by such production methods.

(Brief description of the invention)
Amplification of certain new isolated DNA sequences for biosynthesis of diutane within the host Sphingomonas bacterium not only allows increased production of diutane gum from the host, but also increased viscosity. It has now been recognized that it also produces diutan gum that exhibits properties. Thus, such novel DNA sequences, which are introduced into the host organism by any well-known method, such as, but not limited to, plasmids, have achieved the desired results that have been pursued for the diutane synthesis method. provide. A distinctive advantage for such utilization of these genes amplified on plasmids is their relatively simple nature of incorporating such isolated DNA sequences into the process of diutane synthesis. Another advantage is the ability to produce such higher viscosity characteristics for the target diutan gum while at the same time potentially increasing fermentation production efficiency if necessary.

  Accordingly, the present invention includes diutane gum that exhibits improvements in several different viscosity measurements. For these measurements: i) an intrinsic viscosity greater than 150 dL / g, preferably greater than 155, more preferably greater than 160; ii) greater than 35 dial scale value of seawater 3 rpm, preferably greater than 37, more preferably A viscosity greater than 40, and most preferably greater than 42; iii) greater than 35,000 centipoise (cP) of seawater 0.3 rpm, preferably greater than 39,000, more preferably greater than 40,000, and most preferably Includes a viscosity greater than 41,000; and a viscosity greater than 3500 cP at a PEG low shear rate, preferably greater than 3700, more preferably greater than 3900, and most preferably greater than 4000. The present invention also introduces any of the above terms by introducing specific clusters of genes into the host Sphingomonas organism, allowing fermentation of the organism to produce the resulting diutan gum. Comprehensively includes methods for producing such diutane gum, as defined in The invention further encompasses any vector (eg, a plasmid) that provides multiple copies of the gene or increased expression of the gene through the use of a specific DNA sequence and a stronger promoter. In addition, genetically modified strains of Sphingomonas that contain multiple copies of the diutane biosynthetic gene defined by such unique isolated DNA sequences are also encompassed comprehensively.

  It has been discovered that such a unique isolated DNA sequence requires at least one diutane biosynthetic enzyme that is a DpsG polymerase. In another possible embodiment, such diutane biosynthetic enzymes include DpsG polymerase, as well as 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 yet another possible embodiment, such diutane biosynthetic enzymes are DpsG polymerase, and rhamnosyltransferase IV; beta-1,4-glucuronosyltransferase II; glucosyl-isoprenyl phosphate transferase I; and Contains glucosyltransferase III. In yet another possible embodiment, such diutane biosynthetic enzymes include dpsG polymerase and the polysaccharide efflux proteins dspD, dspC, and dspE. In yet another possible embodiment, such diutane biosynthetic enzymes are rhamnosyltransferase IV; beta-1,4-glucuronosyltransferase II; glucosyl-isoprenyl phosphate transferase I; glucosyltransferase 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 Shall be included. In general, the diutane biosynthetic enzymes within the methods and products of the present invention are: polymerase; lyase; rhamnosyltransferase IV; beta-1,4-glucuronosyltransferase II; glucosyltransferase III; polysaccharide excretion protein; Glucosyl-isoprenyl phosphate transferase I; glucose-1-phosphate thymidylyl transferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4,6-dehydratase; It may be selected from the group consisting of dTDP-6-deoxy-L-mannose-dehydrogenase, and combinations thereof. As further encompassed by the present invention, SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 (in addition to DNA that may be present on the target chromosome) 27, 29, 31, 33, 35, 37, 39, 41, and 43, or at least one diutane biosynthetic enzyme, or SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, There are isolated nucleic acid molecules that encode enzymes that are at least 95% identical to 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43.

Thus, the methods of the present invention (and the products produced thereby) relate to sphingan gums, particularly the diutane type, including but not limited to S88, S60, and S657.
As noted above, the present invention can provide a biosynthetic product with increased viscosity of a highly viscous diutane polysaccharide in which a specific DNA sequence introduced in multiple copies within certain Sphingomonas strains. That is the accumulation of development and recognition. Genetically engineered bacteria containing such genes for increased production produce significantly higher amounts of diutane polysaccharide compared to unengineered bacteria and create the resulting high viscosity properties To do.

  According to the present invention (by no means relying on a specific scientific theory, but by what is believed to be an increase in molecular weight range properties), the production increases and viscosity properties increase (all well-known forms, A DNA sequence introduced into a host organism (eg, again as a non-limiting example) can be isolated, recovered, and cloned by techniques readily available in the art. The DNA is then delivered into a bacterium of the genus Sphingomonas in the form of multiple copies (via plasmids, other known methods), or increased expression of the gene through an appropriate, eg, stronger promoter. Is done. After insertion into the target bacterium, the production of diutane can be determined by fermenting the genetically engineered bacterium and comparing the yield with respect to the amount produced and the quality produced. Both increased production and increased viscosity can be determined by comparing the production of diutane via the method of the invention in comparison to a wild type diutane producing strain (ATCC 53159).

(Detailed description of the invention)
The following terms are used throughout this specification in connection with the present invention and shall have the meanings set forth below:
The term “Sphingomonas” is used throughout this specification to refer to strains of gram-negative bacteria from the genus Sphingomonas.

  The terms “increased production strain” or “increased production” are used throughout this specification to be significantly higher (greater than at least about 5% by weight based on weight) compared to wild-type bacteria of the same strain. Used to describe a genetically engineered bacterium that contains multiple copies of a DNA sequence isolated from the same strain that produces a diutane polysaccharide.

  The term “isolated” refers to DNA that has been removed from a microorganism and has been subjected to at least some purification, ie, one or more purification steps, and cleaved or cleaved with restriction enzymes and cloned into multiple copies. Or DNA that can be inserted into a plasmid vector, or otherwise inserted or incorporated into a bacterium.

  The term “sequence” is used to describe a particular segment of DNA identified by its nucleotide unit. The term “inserted” is used throughout this specification to transfer a DNA segment isolated from the chromosomal DNA of a diutane-producing Sphingomonas strain (through a plasmid as one non-limiting example) into the Sphingomonas strain. Used to describe the process and results of Such isolated DNA, once again as a non-limiting possibility, is first introduced into the desired plasmid (here, pLAFR3) by well-known techniques in the art, eg, the recipient's sphingo. It may be transferred by conjugation or mobilization into Monas bacteria. After insertion into the recipient's sphingomonas bacterium, the plasmid containing the relevant DNA sequence is replicated in the recipient's cells to give a high viscosity (again considered to be in the high molecular weight range) dilute polysaccharide. Obtain several (at least 2 and usually 4-10) copies of the DNA segment necessary for increased production of. It is generally effective to use conjugation or mobilization to transfer the plasmid vector into the recipient bacterium. Electroporation or chemical transformation of competent cells with purified DNA may also be used. Other vectors or bacteriophages can be used to transfer the DNA into the host cell. It is not necessary to maintain the DNA segment on a plasmid (or other well-known delivery vector) in the recipient's diutane-producing Sphingomonas. Introducing additional copies of a DNA segment into the bacterial chromosome is a routine procedure, so that the segment is replicated with each generation by the same mechanism that replicates the bacterial DNA. Alternatively, increased expression of the gene may be achieved by using stronger promoter elements.

  The term “gene amplification” refers to, for example, by cloning a target gene onto a multicopy plasmid (eg, 4 to 10 copies) or by inserting multiple (eg, 4 to 10) copies of a gene into the bacterial genome. Used to refer to either an increased copy of the gene or an increased expression of the gene by modification of a promoter element to increase gene expression. Both of these methods and other methods can result in increased amounts of the encoded protein.

  The term “biosynthesis” is used throughout this specification to describe the biological production or synthesis of diutane by Sphingomonas bacteria. Diutane polysaccharides are synthesized from individual carbohydrate units in a series of steps controlled by a number of bacterial enzymes.

  In any selected form (eg, again, but preferably, but not necessarily, in the form of a plasmid), the relevant DNA sequences incorporated into the recipient's bacteria will have increased production and increased molecular weight. It encodes genetic information known to be beneficial or essential for the biosynthesis of diutane polysaccharides. In addition, however, certain DNA sequences of the present invention (eg, in plasmid pS8) do not rely on specific scientific theory, but do not only induce increased production, but also individual polymers of diutane itself. It is believed that an increase in the number of repeating units polymerized within is also induced. As a result, such an increase in repeat units is believed to produce the resulting high viscosity characteristics surprisingly provided by diutan gum. The increase in molecular weight was assumed by the increase in intrinsic viscosity measurements related to molecular weight due to power law relevance. For linear polymers (such as diutane gum), it is known that the intrinsic viscosity is thus essentially proportional to the molecular weight.

  Isolation of related DNA sequences based on the method of the present invention and producing diutan polysaccharides of increased viscosity is accomplished via standard techniques and methods. Such sequences may therefore be generated from a diutane-producing Sphingomonas strain cultured using standard manufacturing methods. The DNA can then be extracted, for example, through initial centrifugation and resuspension of the bacterial cells, followed by subsequent elution of the DNA through a purification column. After purification is complete, the isolated DNA can be digested with restriction endonucleases, cloned into the desired plasmid or other delivery vector, and then transferred to the recipient strain. Other techniques as are known in the art can also be used without limitation.

  Cloning of the DNA of the present invention relies on common techniques and methods that are standard in the art. It should be mentioned in particular that any method may be used to clone the DNA segment according to the invention, and the invention is not limited to the use of eg plasmid cloning vectors. For example, the DNA fraction may be cloned by insertion into a bacteriophage vector.

  The cloned DNA sequence can then be introduced into the Sphingomonas strain via a plasmid or other delivery vector. The genetically modified Sphingomonas strain can then be used to produce diutane by fermentation. Basically, suitable media for fermentation are carbon sources such as carbohydrates including glucose, lactose, sucrose, maltose, or maltodextrin, nitrogen sources such as inorganic ammonium, inorganic nitrates, organic amino acids or proteinaceous materials, For example, an aqueous medium containing hydrolyzed yeast, soy flour or casein, distiller's solubles or corn leachate, and inorganic salts. A wide variety of fermentation media will support the production of diutane according to the present invention.

  Carbohydrates can be included in the fermentation broth in varying amounts, but usually between about 1 and 10% (preferably 2-8%) of the weight of the fermentation medium. Carbohydrates may be added before or during fermentation. The amount of nitrogen may range from about 0.01% to about 0.4% of the weight of the aqueous medium. A single carbon material or nitrogen material may be used, but a mixture of these materials may be used as well. Inorganic salts found for use in fermenting Sphingomonas contain sodium, potassium, ammonium, nitrate, calcium, phosphate, sulfate, chloride, carbonate, and similar ions There is salt. Trace elements such as magnesium, manganese, cobalt, iron, zinc, copper, molybdenum, iodide and borates may also be included as advantageous.

  The fermentation can be carried out at a temperature between about 25 and 40 ° C, preferably in a temperature range of about 27 ° C and 35 ° C. The inoculum can be prepared by standard methods of volume scale up, including shake flask culture and small scale submerged stirred fermentation. The medium for preparing the inoculum can be the same as the production medium, or in any one of several standard media well known in the art, such as Luria broth or YM media. There can also be. One or more inoculum stages may 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 contain a stirrer for stirring the contents. The container may also have automatic pH control and foam control. The production medium can be added to the container and sterilized in situ by heating. Alternatively, the carbohydrate or carbon source may be sterilized separately before addition. The pre-grown inoculum culture is added to a chilled medium (generally 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 broth can be produced. Diutane polysaccharides can be recovered from the broth by standard precipitation methods using alcohol, typically isopropyl alcohol.

(Preferred embodiment of the invention including detailed description of the drawings)
The following examples are provided to illustrate the present invention. The description of the examples should in no way be construed as limiting the scope of the invention in any way.

DNA Sequence Isolation / Plasmid Generation To perform initial isolation and determine the appropriate sequence to obtain the results of the invention described above, a gene library of ATCC 53159 organism was constructed as follows: Chromosomal DNA was isolated from Sphingomonas bacterium ATCC 53159 and partially digested with Sau3AI restriction endonuclease. A DNA fragment ranging from 15 to 50 kb was purified from an agar gel and isolated from E. coli strain JZ279, BamHI digested cosmid cloning vector pLAFR3 (Staskawicz, et al., “Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syrinae pv. Glycinea ”, J. Bacteriology. 1987, 169: 5789-94) (Harding, et al.,“ Genetic and physical analysis of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris ", According to J. Bacteriology. 1987. 169: 2854-61). The ligation reaction was packaged into λ phage particles (using Gigapack III Gold packaging extract from Stratagene, La Jolla, Calif.) And transfected into Library Efficiency E. coli DH5α MCR cells (Life Technologies, Rockville, MD). Approximately 10,000 tetracycline resistant colonies were pooled to form a gene library. Individual sequences were then isolated from this library. The studies performed in this case involved the isolation of specific genes for polysaccharide biosynthesis from the Sphingomonas ATCC 53159 organism.

  Such genes for polysaccharide biosynthesis are typically identified by complementation with mutations that are deficient in polysaccharide biosynthesis, particularly mutations that are blocked in the first step, glycosyltransferase I. Since the ATCC 53159 transferase I-deficient mutation was not available for the first time, the gene for diutane polysaccharide biosynthesis was identified using the complementation of Sphingomonas erodea and Xanthomonas campestris transferase I-deficient mutations. Plasmid pLAFR3 is (according to Ditta et al., “Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti”, Proc. Natl. Acad. Sci. 1980 77: 7347-51) It can be transferred from its E. coli host to other Gram negative bacteria by triple parental mating using a helper plasmid that provides IncP transfer function. RK2-type plasmids have an estimated copy number of 5-7 per chromosome in E. coli (Figurski et al., “Suppression of ColE1 replication properties by the Inc P-1 plasmid RK2 in hybrid plasmids constructed in vitro”, J Mol. Biol. 1979 133: 295-318).

  A gene library of ATCC 53159 chromosomal DNA in E. coli was transferred into a non-mucoid mutant (GPS2) of Sphingomonas erodea ATCC 31461 by triple parental conjugation and selected for tetracycline resistance and streptomycin resistance. The helper plasmid used was pRK2013 (in E. coli strain JZ279), which contains the origin of replication of the narrow host but exhibits the trans-acting function necessary to mobilize pLAFR3. Plasmid pRK2013 was not replicated in Sphingomonas strains. Sphingomonas erodea ATCC 31461 produces the polysaccharide gellan. Both gellan and diutane polysaccharides are [→ 4] -α-L-rhamnose- (1 → 3) -β-D-glucose- (1 → 4) -β-D-glucuronic acid- (1 → 4). Having the same tetrasaccharide repeating unit composed of -β-D-glucose (1 →), but diutane also contains a side chain composed of two rhamnose molecules attached to one of the glucose residues; While modified by acetyl, gellan has no side chain sugars and is modified with acetyl and glyceryl Mutant GPS2 is UDP-D in the first step of polysaccharide biosynthesis, ie by glucosyltransferase I enzyme. -Deficiency in glucose-1-phosphate transfer from glucose to bactoprenyl phosphate lipid carrier from tetracycline selective plate Production (mucoid) colonies were isolated from a background of non-mucoid colonies, clones that retained polysaccharide production probably contain the ATCC 53159 gene encoding glucosyltransferase I, plus approximately 20-25 kb of flanking DNA. Plasmid DNA was isolated from eight mucoid GPS2 conjugants and transferred to E. coli strain DH5α (Life Technologies) by electroporation The plasmid was isolated from E. coli and the restriction endonuclease HindIII / EcoRI (polylinker) Sufficient DNA was obtained for double digestion in which either side of the BamHI restriction endonuclease site was cleaved) and the insert DNA was excised from the vector.The size of the insert DNA in the clones was determined by gel electrophoresis. Several plasmids The final sequence of was determined by sequencing from primers specific for the plasmid sequence adjacent to the BamHI site of the vector, and this sequence was analyzed using BLASTX by comparison with sequences in a computer database. Two of the plasmids, pS8 and pS6, are shown in Figure 1. Similarly, an ATCC 53159 gene library was obtained from a rifampicin-resistant non-mucoid X. campestris mutant (CXC109) that is defective in transferase I (eg Harding et al., (See above), transferred through a triple parental mating and selected for tetracycline resistance and rifampicin resistance X. campestris produced xanthan polysaccharide, the synthesis of which was also derived from UDP-D-glucose by the transferase I enzyme. Bactoprenyl Initiated by transfer of glucose-1-phosphate to an oxylipid carrier (Ielpi et al., “Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris”, J. Bacteriology. 1993. 175: 2490-500). The plasmid was purified from the mucoid conjugate and the final sequence was determined as described above. Two of these plasmids, pX6 and pX4, are represented in FIG.

  S657 DNA cloned into plasmids pS8 and pX6 was fully sequenced by double-stranded shotgun sequencing at Lark Technologies Inc., (Houston, TX). These sequences were analyzed to identify genes for diutane biosynthesis (represented in FIG. 1). The function of the gene is described in GenBank accession number U51197, which is a published gene for biosynthesis of S-88 sphingan (eg, the aforementioned '516 Pollock et al. Patent), and gellan (GenBank AY217008). And AY220099)). The genes encoding transferases for the four sugars of the backbone and four genes for dTDP-rhamnose synthesis were identified (FIG. 1). The genes for polysaccharide secretion were based on homology with genes for biosynthesis of other polysaccharides. Two genes encode proteins that are homologous to proteins involved in protein secretion. Two genes putatively encode polymerase and lyase. The insert of plasmid pX6 contains 17 genes including the gene dpsB encoding transferase I (initiating the first step of diutane synthesis), a gene for secretion, and four genes for dTDP-rhamnose synthesis. Does not include genes for II, III and IV, and putative genes for polymerase and lyase. Plasmid pS8 contains genes for polysaccharide secretion, including 20 genes in the dps gene cluster containing genes for all four backbone sugar transferases, 4 genes for dTDP-rhamnose synthesis, and putative genes for polymerase and lyase. However, the genes of orf6 and orf7, which are genes whose functions are not known, are not included. Plasmid pS6 contains genes for secretion and four sugar transferases, but does not have all genes for dTDP-rhamnose synthesis or genes for polymerase. Plasmid pX4 contains only a small portion of the dps region but is sufficient to effect increased production of polysaccharides in Sphingomonas strains, a gene encoding transferase I reported by Pollock et al., And dTDP-rhamnose Contains 4 genes for synthesis.

Strain Generation Next, the four plasmids described above were transformed into Sphingomonas strain ATCC No. 1 by triple parental conjugation as described above. And introduced into 53159 to form new S657 genetically engineered strains (S657 / pS8, S657 / pS6, S657 / pX6, and S657 / pX4). The fermentation was then followed as described above to produce a biogum material as described below. All four plasmids had a beneficial effect on diutane productivity; however, the pS8 plasmid surprisingly also provided a very large increase in diutane viscosity and an increase in molecular weight. The DNA sequence (26278 bp) (DNA SEQ ID NO: 1) of the DNA sequence of pS8 is provided, and the encoded genes are listed in the form of a chart in the following Table 1 and FIG. The inserted DNA of plasmid pS8 contains the gene passed from dpsG to rmD, and parts of genes dpsS and orf7.

  The following gene table is basically a list of genes represented by the DNA sequence for insertion in the plasmid pS8 provided in FIG.

Diutane production by the genetically engineered plasmid-containing Sphingomonas strain S657 compared to the S657 wild type strain without the diutane production plasmid was performed in the same liquid medium in an Applikon 20L fermentor with agitation and aeration. 3 fermentations were performed and determined. For plasmid-containing strains, 5 mg / L antibiotic tetracycline was added throughout the fermentation to ensure plasmid retention. When it was necessary to control the pH, KOH was added. Two inoculum stages were used with 1-6% inoculum transfer. The medium used for fermentation contained corn syrup, an assimilable nitrogen source, and salt as carbohydrate sources. Nutrients that can be used for fermentation are well known in the art and are carbohydrates such as glucose, sucrose, maltose, or maltodextrins, nitrogen sources such as inorganic nitrogen (eg ammonium or nitrate), organic nitrogen (eg Amino acids, hydrolyzed yeast extract, soy protein, or corn leachate), and addition salts containing, for example, chloride, phosphate, sulfate, calcium, copper, iron, magnesium, potassium, sodium, or zinc.

  As a measure of the resulting diutane production, broth viscosity and precipitated fibers were determined. The viscosity of the fermentation broth was measured with a Brookfield viscometer running at spindle # 4, 60 rpm. The results are shown in Table 2. At the end of the fermentation, the broth was treated with the introduction of the well-known glucoamylase enzyme to hydrolyze any remaining oligosaccharides from corn syrup. The produced diutane gum was then precipitated from an aliquot of broth with 2 volumes of isopropyl alcohol. The fiber was collected by filtration and dried. In Table 2, the term DWY refers to the precipitable total dry weight yields of biogum after hydrolysis of excess oligosaccharides from corn syrup.

  The resulting material results in higher yields using plasmids pX4, pX6, pS6, or pS8, which carry additional copies of the genes for diutane biosynthesis clearly shown in the table. However, when using the pS8 plasmid, an unexpectedly high increase in broth viscosity was observed compared to an increase in dry weight yield, and in addition to the increase in the amount of diutane produced, some factor affected the viscosity. Showed that.

  Using any of the four plasmids shown in the table, clearly higher yields of the resulting material were observed, whereas the plasmids of pS8 and pS6 were unexpectedly high in broth viscosity. An increase was possible, thus showing a high quality of product on it. The quality, i.e. viscosity, of the resulting diutane gum product was then determined.

Diutane hydrodynamics in application testing. These diutane gum samples are then added to two different areas: oil field additives for oil recovery, and cement additives for moisture retention and rapid set-up, The potential beneficial uses in were analyzed.

  The oil field industry relies on a method called “seawater viscosity (SWV)” testing as an estimate of acceptable performance for oil recovery gums. Such a test is basically an indicator of the effectiveness of the gum to increase the viscosity under saline conditions of water (eg to replicate recovery from the seabed).

Predicting the availability of the resulting gum as a suitable viscosity modifier for petroleum recovery purposes is generally accepted in terms of modifying the viscosity of a laboratory seawater preparation. Such a “synthetic seawater” preparation is produced by mixing 419.53 grams of sea salt (ASTM D-1141-52) in 9800 grams of deionized water. For seawater viscosity testing, 0.86 g of sample gum is added to 307.0 g of synthetic seawater and mixed in a Fann Multimixer (Model 9B5, part number N5020) at approximately 11,500 rpm for 35 minutes. At the end of 35 minutes, the solution is cooled to approximately 26 ° C. and the viscosity is measured. For the 3-rpm reading, place the sample on the Fann sample platform (Fann model 35A; Torsin spring MOC 34/35 F0.2b; Bob B1; Roter R1), rotate the motor at low speed and shift gears Adjust the speed to 3 rpm by setting to a medium position. Then, the scale is stabilized by leaving it as it is, and the shear stress value is read from the dial and recorded as the SWV 3 rpm dial scale value (dial reading, DR). For the 0.3 rpm scale value, the viscosity is measured using a Brookfield viscometer (Brookfield LV DV-II or DV-II viscometer with LV-2C spindle). The spindle speed was set at 0.3 rpm and the spindle was allowed to rotate for at least 6 minutes and allowed to stand, after which the viscosity was recorded as a scale value of SWV-0.3 rpm and expressed in centipoise (cP). For cement applications, the PEG LSRV test (low shear rate viscosity using polyethylene glycol as a dispersant as outlined below) gives an indication of the effectiveness of the viscosity modifier's performance for the industry. provide. Such a test measures the viscosity of a 0.25% solution of biogum in standard tap water (STW). The 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 measurement, 0.75 grams of biogum is added to 4.5 grams of polyethylene glycol 200 (CAS 25322-68-3) in a 400 mL beaker and dispersed thoroughly. 299 grams of STW is then added to the beaker and mixed for approximately 4 hours using a low pitch propel stirrer at 800 ± 20 rpm. After a 4 hour mixing time, the beaker was placed in a 25 ° C. water bath and allowed to stand for approximately 30 minutes. Next, using a Brookfield LV viscometer with a 2.5+ torque spring (or equivalent device, eg, model DVE 2.5+), rotate the spindle for 3 minutes using the LV1 spindle at 3 rpm and centipoise After displaying (cP), the viscosity was measured. The diutane sample produced above was examined in this way and the results were as follows:

  Unexpectedly, a clear increase was observed in the viscosity exhibited by the diutane gum of the present invention produced by some of the engineered plasmid-containing strains. Most surprisingly, however, the 3 rpm SWV viscosity increase was 80% for the pS8 strain, whereas the same analysis performed for the pX6 strain only surpassed the wild-type result by 9.6%. there were. There was no significant increase in plasmids pS6 and pX4. Similarly, the lower SWV rpm test revealed a 51.5% increase over the wild type for the pS8 type, but only a 2% increase for pX6. Finally, the polyethylene glycol LSRV test showed that the pS8 result was less than 16% increase for pX6 diutane and 7.2% increase for pX4 and no significant increase for plasmid pS6. An increase in viscosity of over 77% was observed over the wild type gum. Again, quite unexpected results in these respects, as one way of introducing the required gene sequence into the target diutane-producing bacterium, through the use of such an example sequence into the pS8 plasmid, It represents a dramatic improvement given to the production of diutan gum.

  Thus, the diutane of the present invention produced through the introduction of pS8 shows a surprisingly increased viscosity measurement at all three measurement counts, especially compared to wild type and pX6 plasmid production variants. It was. It was therefore expected that such new diutane would work very well under typical oil field conditions and in cement applications.

Basic explanation for improvement of fluid dynamics The previous examples have shown that diutane from strain S657 / pS8 shows a significant increase in the parameters of fluid dynamics. Therefore, such a substantial increase in seawater and PEG low shear rate viscosity measurements is due to an increase in productivity only, as the pX6 strain also shows similar, if not large, yield results. I can't let you. In fact, in the previous examples shown in Table 2, the dry weight yield (alcohol-precipitable substance) increased by 8.0%, while the hydrodynamic parameters increased significantly more for the S657 / pS8 strain. (52-80%). Basic research continued to explain why hydrodynamic improvements were obtained using strain S657 / pS8 over wild type strains.

  Intrinsic viscosity is a well-known technique in polymer science for inferring the molecular weight of polymers (C. Tanford, 1961. Physical Chemistry of Macromolecules. John Wiley & Sons, New York). Intrinsic viscosity is obtained by plotting the reduced viscosity (viscosity normalized to concentration) against the concentration of the solution and extrapolating the linear regression of the data to zero concentration (y-intercept of the plot). Surprisingly, the resulting gum showed an increase in intrinsic viscosity as shown below in the table below.

  Five diutane samples, two from the wild type strain (Control 1, Control 2) and three from the S657 / pS8 strain (Sample 1, Sample 2, Sample 3), intrinsic viscosity, neutral sugar, and The analysis of organic acids was evaluated. The three samples were purified by alcohol precipitation, rehydration, treatment with hypochlorite, treatment with glucoamylase, treatment with lysozyme, and finally treatment with protease (in this order). They were then recovered at a 4: 1 CBM: broth ratio, dried and ground. CBM is an azeotropic isopropyl alcohol / water mixture containing ˜82% by weight of isopropyl alcohol.

  Samples were examined for moisture content by performing the following procedure: Two samples, typically 0.7 grams aliquots, were examined using a Mettler HB 43 halogen moisture balance. The results of the two trials were then averaged and these results were used for moisture correction.

  After obtaining moisture data, a 0.2% solution of gum was prepared in 0.01 M NaCl based on moisture correction. In these trials, a total of 200 grams of 0.2% solution was prepared. The gum was weighed on an analytical balance in the nearest 10/1000 increments and added to the water weighed in approximately 1/1000 increments. The sample was stirred for 2 hours at 1000 rpm in a 400 ml vertical beaker using a 2.5 inch diameter propeller stirrer.

  Following initial hydrolysis, each sample was diluted to 0.02% using 0.01 M NaCl. This operation was performed by weighing 20 grams of 0.2% solution in a 400 ml beaker and then adding 180 ml of diluent. The diluted sample was mixed for an additional 30 minutes. The final dilution used to finally determine the intrinsic viscosity was prepared from this sample. Each diutane sample was evaluated for the following concentrations: 0.004%, 0.008%, 0.010% and 0.012%.

  Viscosity measurements were made using a Vilastic® VS system. Prior to measurement, Vilastic was calibrated with water to give an error of less than 2.0%. The samples were all measured at a constant temperature of 23 ° C. using a timer program, 2 Hz, 1 strain, and a shear rate of approximately 121 / sec. Each sample was measured 5 times and averaged. The intrinsic viscosity was then calculated using the average viscosity data. FIG. 2 and Table 4 below provide the final results of these attempts.

  These results show that the S657 / pS8 strain consistently produced diutane with significantly higher intrinsic viscosity; in fact, at all similar measured solid levels, the average reduced viscosity for the strain of the invention was 165. The control was 140.7 compared to 2. This finding indicates that diutane produced by S657 / pS8 has a higher molecular weight than the wild type control.

FIG. 2 graphically illustrates these trends, showing that consistently higher intrinsic viscosities were measured at similar solids content between the control and the strains of the present invention.
To determine whether the higher viscosity diutane gum from S657 / pS8 has the same composition as the wild-type diutane, the composition was determined by examining for neutral sugars and organic acids. The purified sample used for intrinsic viscosity measurement was used for analysis of neutral sugars. An aliquot of each purified sample was hydrolyzed to the sugar component by hydrolysis with trifluoroacetic acid (100 ° C./˜18 hours). The neutral sugars of the hydrolyzate were quantified by fast anion exchange chromatography with amperometric detection. Hydrolyzate organic acids were quantified by fast ion evacuation chromatography with chemically suppressed conductivity detection. Table 5 summarizes the results of neutral sugar analysis. As shown in the table, the neutral sugar profile for the S657 / pS8 strain is nearly identical to the neutral sugar profile of the S657 wild type strain. Both results differ from the theoretical values, but these results show that the structure of the repeat unit of diutan gum produced using pS8 is the same as that of the wild type and any viscosity obtained with the material pS8. The increase is also due to longer chains, indicating higher molecular weight.

  Thus, the greatly improved seawater viscosity and PEG low shear rate viscosity of diutane produced by genetically engineered strains of S657 / pS8 increases the molecular weight or length of the diutane molecule, i.e., per molecule. It is due to a larger number of repeating units, not due to a change in its composition, and therefore not due to a change in the repeating structure itself. This improved hydrodynamics cannot simply be attributed to the increased amount of diutane produced. Four plasmids with different parts of the cloned gene cluster for biosynthesis, ie pS6, pS8, pX4, and pX6, were evaluated and all showed some increase in productivity, but only plasmid pS8 Showed an unexpected and very high increase in the hydrodynamic parameters of the recovered diutane product.

  A comparison of the genes for diutane biosynthesis cloned in the tested plasmids suggests that the most likely gene responsible for the increase in molecular weight is the gene dpsG. This is because this gene is present in pS8 but not in other plasmids. The gene dpsG encodes a hydrophobic membrane protein having strong homology with other membrane proteins involved in polysaccharide synthesis. A part of this protein has homology with a protein related to polymerase, which is an enzyme that catalyzes the linking of repeating units to form a high molecular weight polysaccharide. The homologous gene gelG in S60 is presumed to function as a polymerase for gellan synthesis (Harding, NE et al. 2004. “Organization of genes required for gellan polysaccharide 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). Homologs of dpsG have also been isolated from Sphingomonas strains ATCC 31554 and ATCC 21423, which produce the polysaccharides S88 and S7 (Pollock et al. US Pat. Nos. 5,854,034, 5,985,623). And 6,284,516, and Pollock TJ US Pat. No. 6,709,845). It is therefore very likely that an additional copy of the gene for the polymerase may have the effect of increasing the molecular length of the diutane molecule. It cannot be excluded that other genes in the gene cluster of diutane biosynthesis may be required in combination with dpsG to achieve the observed increase in viscosity. As likely candidates, genes dpsB, dpsB, dpsL, dpsK and dpsQ encoding sugar transferases I, II, III and IV, especially gene dpsB encoding transferase I which adds the first sugar of the repeating unit to the lipid carrier. Would be mentioned. Other important genes appear to be dpsD, dpsC and dpsE, which are homologous to the genes gumB and gumC, which have been shown to increase xanthan molecular weight when amplified on multicopy plasmids. There is also the possibility that all genes cloned in plasmid pS8 may be necessary to achieve a dramatic increase in viscosity.

  While the invention has been described and disclosed in connection with certain preferred embodiments and practices, it is in no way intended to limit the invention to these particular embodiments, but rather to the appended claims and their equivalents. It is intended to encompass structural equivalents and all alternative embodiments and modifications that may be defined by the scope of

(Deposit)
The following bacterial strains were identified on October 21, 2005, in accordance with the Budapest Treaty for the International Recognition of the Deposit of Microorganisms, on 10801 University Boulevard, Manassas, Va. 20110, Deposited at the Patent Depository of the American Type Culture Collection:
Sphingomonas strain S657 containing plasmid pS8.

FIG. 1 is a diagram of isolated genes for diutane gum biosynthesis. Indicates a putative or known gene. Segments inserted into different plasmids are also shown. FIG. 2 is a graph representing the improved intrinsic viscosity measurement achieved with such a diutane biogum material of the present invention.

Claims (17)

Sphingomonas strain ATCC No. 1 comprising a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO: 1. Diutane gum produced using 53159 and exhibiting an intrinsic viscosity greater than 150 deciL / g.   2. Diutane gum according to claim 1 exhibiting an intrinsic viscosity greater than 155 deciL / g.   3. Diutane gum according to claim 2, exhibiting an intrinsic viscosity greater than 160 deciL / g. Sphingomonas strain ATCC No. 1 comprising a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO: 1. Diutane gum produced using 53159 and having a viscosity of 3 rpm seawater greater than the 35 dial scale value.   The diutan gum according to claim 4, which exhibits a viscosity of 3 rpm of seawater that is larger than a 37 dial scale value.   6. Diutan gum according to claim 5, showing a viscosity of seawater 3 rpm greater than the 40 dial scale value.   The diutan gum according to claim 6, which shows a viscosity of seawater 3 rpm which is larger than a 42 dial scale value. Sphingomonas strain ATCC No. 1 comprising a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO: 1. Diutane gum produced using 53159, showing a viscosity of seawater 0.3 rpm greater than 35,000 cp.   9. Diutane gum according to claim 8, exhibiting a viscosity of seawater 0.3 rpm greater than 38,000 cp.   10. Diutane gum according to claim 9, exhibiting a viscosity of seawater 0.3 rpm greater than 40,000 cp.   11. Diutan gum according to claim 10, exhibiting a viscosity of seawater 0.3 rpm greater than 41,000 cp. Sphingomonas strain ATCC No. 1 comprising a plasmid comprising the nucleic acid sequence set forth in SEQ ID NO: 1. Are produced using 53159, diutan gum showing the viscosity of the low shear rate in the presence of polyethylene glycol dispersing agent have greater than 3500Cp. Shows the viscosity of the low shear rate in the presence of polyethylene glycol dispersing agent have greater than 3700Cp, diutan gum of claim 12. Shows the viscosity of the low shear rate in the presence of polyethylene glycol dispersing agent have greater than 3900Cp, diutan gum of claim 13. Shows the viscosity of the low shear rate in the presence of polyethylene glycol dispersing agent have greater than 4000 cp, diutan gum of claim 14. A method for producing diutan gum, comprising:
The host diutan producing Sphingomonas organism strain ATCC No. Introducing a plasmid containing the nucleic acid sequence set forth in SEQ ID NO: 1 into 53159;
Culturing the host organism under fermentation conditions, whereby the host organism has the following characteristics:
a) Intrinsic viscosity greater than 150 dL / g;
b) 35 Viscosity seawater 3rpm not greater than dial scale values;
c) 35,000 centipoise size than has the viscosity of seawater 0.3 rpm; and d) the viscosity of the low shear rate in the presence of 3500 centipoise than have a size of polyethylene glycol dispersing agent a step of producing a diutan gum indicative of at least one Including methods.   An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 1.