HK1191964A - 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 gum and preparation method thereof

The application is a divisional application with the application date of 2006, 10, 31, the application number of 200680048801.9 and the name of high-viscosity diutan gum and a preparation method thereof.

Technical Field

The present invention describes diutan (diutan) polysaccharides that exhibit increased viscosity characteristics compared to previously produced polysaccharides of the same type of repeating units. This improved diutan polysaccharide was prepared by producing a derivative of Sphingomonas sp ATCC53159 which contains a multicopy broad-spectrum host plasmid in which the biosynthetic genes for the diutan polysaccharide were cloned. The plasmid is capable of producing multiple gene copies for such polysaccharide synthesis in the host Sphingomonas strain. The method provided in this way not only allows an increased yield of the target diutan polysaccharide, but also allows the production of diutan polysaccharides with improved physical properties (higher viscosities as described above). Such diutan polysaccharides have proven to be particularly useful as viscosifiers (viscoresins) in oil field applications and cement materials. The invention also includes inventive methods for producing such improved diutan polysaccharides and novel cloned genes required for producing the improved diutan in such methods. In addition, the invention includes engineered novel strains of Sphingomonas containing the desired DNA sequences.

Background

Polysaccharides or gums are generally used to thicken or gel aqueous solutions and are generally divided into two categories: thickeners and gelling agents. Typical thickeners include: starch, xanthan gum, diutan gum, welan gum (welan gum), guar gum, carboxymethyl cellulose, alginate, methyl cellulose, karaya gum, and tragacanth gum. Common gelling agents include gelatin, gellan gum, starch, alginate, pectin, carrageenan, agar and methyl cellulose.

For many years, certain polysaccharides, or more specifically biogums, such as xanthan gum, gellan gum, welan gum, and diutan gum, have been produced by microbial fermentation. These biogums exhibit different characteristics, such as viscosity modifying ability that allows them to be used in many different fields. Gelling agents for food products, such as confectionery gels (confectionery jelly), jams and jellies, confectionery gels, frostings and dairy products, as well as components of the microbiological culture medium belong to this list. In addition, thickeners may be used in many end-use applications to improve the viscosity of the target fluid. Of particular interest are those gels that can alter the viscosity of subterranean and/or underwater petroleum fluids to facilitate collection of such fluids, although many other different end uses may exist (e.g., including cement production). Different biogums have been produced from different bacterial sources, such as xanthan gum from Xanthomonas campestris (Xanthomonas campestris), gellan gum from Sphingomonas elodea, Welan gum from Sphingomonas sp.sphingomonas ATCC31555, and diutan gum from Sphingomonas sp.ATCC 53159 (S-657). These strains have been genetically modified in the past in an attempt to significantly alter the gum material produced by the fermentation process described above. Such modifications can result in changes such as removal of acyl groups, resulting in different gum materials exhibiting different physical properties. The types of these genetic modifications are generally: the composition of the target bio-gel may be ultimately altered by altering gene expression within the host organism, or the yield of the target bio-gel may be increased by introducing plasmids that individually exhibit gene amplification (e.g., U.S. Pat. Nos. 5,854,034, 5,985,623, and 6,284,516 to Pollock et al and U.S. Pat. No. 6,709,845 to Pollock et al).

Diutan gum (also known as heteropolysaccharide) S-657 was prepared by fermenting the strain sphingomonas sp ATCC53159, which exhibits thickening, suspending and stabilizing properties in aqueous solution. Diutan typically appears as a hexamer repeat unit consisting of 4 sugars in the backbone (glucose-glucuronic acid-glucose-rhamnose) and the side chains of two rhamnose residues linked to one of the glucose residues. Structural details of diutan gum are found in Chowdhury, T.A., B.Lindberg, U.Lindquist and J.Baird, Carbohydrate Research164(1987) 117-122. Diyoutan shows two acetyl substituents per repeat unit, see Diltz et al, Carbohydrate Research331(2001) 265-270. Both references are incorporated herein by reference in their entirety. Details of the preparation of diutan gum can be found in U.S. patent No. 5,175,278, which is incorporated herein by reference in its entirety. Diutan can be prepared from Sphingomonas strains using standard fermentation techniques, for example, using carbohydrate sources (non-limiting examples are glucose, maltose, and the like), nitrogen sources, and other salts.

Certain industries require physical characteristics imparted by various wild-type diutan biogums, particularly their viscosity modifying properties and/or water retention properties. Unfortunately, the cost-effectiveness of producing diutan has proven to be low. Furthermore, these cost issues currently preclude the widespread use of diutan because this biogum exhibits insufficient viscosity to replace other cheaper but effective biogums (e.g., xanthan gum). Therefore, there is a need to provide a method that can produce at least such effective diutan at a lower cost and/or to provide a way to produce a diutan-type bio-gum that exhibits significant improvements in physical properties. At present, the only description of the production of any type of related gellan polysaccharides (sphingans) (no specificity has been demonstrated for diutan) involves higher yields (see Pollock et al, supra). There has not been any way to discuss or reasonably suggest a method to produce higher molecular weight diutan gum that would exhibit improvements in viscosity measurements by this production method.

Brief description of the invention

It is now known that amplification of certain newly isolated DNA sequences used for diutan biosynthesis in the host Sphingomonas microorganism not only increases diutan gum production, but also produces diutan gum exhibiting increased viscosity characteristics. Thus, this novel DNA sequence (introduced into the host microorganism by any well-known method, such as, but not limited to, the use of plasmids) provides the desired results sought by the diutan synthesis method. The obvious advantage of this method of amplifying these genes using plasmids is that the incorporation of such isolated DNA sequences into the diutan synthesis process is relatively simple. Another advantage is the ability to produce such a high viscosity performance of the target diutan gum, while possibly also increasing fermentation production efficiency (if desired).

Thus, the present invention includes diutan gum that exhibits improvements in many different viscosity tests. Wherein: i) an intrinsic viscosity higher than 150, preferably higher than 155, more preferably higher than 160 dL/g; ii) seawater 3rpm viscosity above 35, preferably above 37, more preferably above 40, most preferably above 42 dial reading (dialreading); iii) viscosity of seawater at 0.3rpm above 35,000, preferably above 39,000, more preferably above 40,000, most preferably above 41,000 centipoise (cP); PEG low shear rate viscosity is higher than 3500, preferably higher than 3700, more preferably higher than 3900, most preferably higher than 4000 cP. As defined by the above terms, the present invention also includes a method of producing such diutan gum by introducing a specific gene cluster into a host sphingosine microorganism and fermenting the microorganism to produce diutan gum. In addition, the invention includes specific DNA sequences and any vectors (e.g., plasmids) to provide multiple copies of genes or to utilize stronger promoters to enhance expression of such genes, and the like. Also included are genetically modified sphingosine strains containing multiple copies of the diutan biosynthetic genes identified by this unique isolated DNA sequence.

Another aspect of the present invention provides a method of producing diutan gum, the method comprising:

introducing a coding sequence for at least one diutan biosynthetic enzyme into a diutan-producing sphingomonas host microorganism;

culturing the host microorganism under fermentation conditions such that the host microorganism produces diutan gum exhibiting at least one of the following characteristics:

a) intrinsic viscosity greater than 150 dL/G;

b) the viscosity of seawater at 3rpm is higher than 35 dial plate reading;

c) viscosity of seawater at 0.3rpm higher than 35,000 centipoise; and

d) the polyethylene glycol dispersant has a low shear rate viscosity greater than 3500 centipoise.

Finding this unique isolated DNA sequence requires at least one diutan biosynthetic enzyme, dspsg polymerase. In another possible embodiment, the diutan biosynthetic enzyme includes a dspsg polymerase and a glucose-1-phosphate thymidine transferase; dTDP-6-deoxy-glucose-3-5-epimerase; dTDP-D-glucose-4, 6-dehydratase; and dTDP-6-deoxy-L-mannose-dehydrogenase. In yet another possible embodiment, the diutan biosynthetic enzyme comprises dspsg polymerase and rhamnosyltransferase IV; beta-1, 4-glucuronyltransferase II; glucosyl-prenyl phosphotransferase I; and glucosyltransferase III. In yet another possible embodiment, the diutan biosynthetic enzymes include dspsg polymerase and the polysaccharide export proteins dspsd, dspsc and dspse. In yet another possible embodiment, the diutab biosynthetic enzyme includes rhamnosyltransferase IV; beta-1, 4-glucuronyltransferase II; glucosyl-prenyl phosphotransferase I; glucosyltransferase III; glucose-1-phosphate thymidine transferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4, 6-dehydratase; and dTDP-6-deoxy-L-mannose-dehydrogenase. The process of the invention and the diutan biosynthetic enzymes belonging to the products of the invention are generally chosen from: a polymerase; a lyase enzyme; rhamnosyltransferase IV; beta-1, 4-glucuronyltransferase II; glucosyltransferase III; a polysaccharide export protein; a secreted protein; glucosyl-prenyl phosphotransferase I; glucose-1-phosphate thymidine transferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4, 6-dehydratase; dTDP-6-deoxy-L-mannose-dehydrogenase and combinations thereof. The invention also includes isolated nucleic acid molecules (other than DNA that may be present on a target chromosome) that encode at least one diutan biosynthetic enzyme as shown in seq id no: 5. 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43; or encodes enzymes 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 and 43 that are at least 95% identical to seq id no below.

In certain embodiments, the nucleic acid molecule can encode a diutan polymerase.

In certain embodiments, the nucleic acid molecule can encode a diutan polymerase and a polysaccharide export protein.

In certain embodiments, the nucleic acid molecule can encode diutan polymerase and rhamnosyltransferase IV; glucosyl-prenyl phosphotransferase I; beta-1, 4-glucuronyltransferase II; and glucosyltransferase III.

In certain embodiments, the nucleic acid molecule can encode diutan polymerase and glucose-1-phosphate thymidine transferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4, 6-dehydratase; and dTDP-6-deoxy-L-mannose-dehydrogenase.

In certain embodiments, the nucleic acid molecule can encode a diutan polymerase; a lyase enzyme; rhamnosyltransferase IV; beta-1, 4-glucuronyltransferase II; glucosyltransferase III; a polysaccharide export protein; a secreted protein; glucosyl-prenyl phosphotransferase I; glucose-1-phosphate thymidine transferase; dTDP-6-deoxy-D-glucose-3-5-epimerase; dTDP-D-glucose-4, 6-dehydratase; and dTDP-6-deoxy-L-mannose-dehydrogenase.

In certain embodiments, the nucleic acid molecule can comprise the nucleic acid sequence shown in SEQ ID NO. 1.

In a further aspect of the invention there is provided a method of preparing a gellan gum-like polysaccharide having a relatively long average polymer length, the method comprising:

introducing a coding sequence for at least one gellan polysaccharide polymerase into a gellan polysaccharide producing sphingomonas host microorganism;

culturing said host microorganism under fermentation conditions such that said host microorganism produces a gellan gum-like polysaccharide gum having an average polymer length that is longer than that produced by said sphingosine microorganism prior to introduction of said coding sequence.

Thus, the present methods (and products made therefrom) relate to gellan gum-based polysaccharide gums, particularly diutan-type, including but not limited to: s88, S60, and S657.

As mentioned above, the present invention is the culmination of the development and enables the introduction of multiple copies of specific DNA sequences into certain strains of Sphingomonas to increase the biosynthetic production of high viscosity diutan polysaccharides. Engineered bacteria containing these genes for enhanced yield produce significantly higher amounts of diutan polysaccharide than non-engineered bacteria, while producing the high viscosity characteristics described above.

According to the present invention, the DNA sequences introduced into the host microorganism (in any well-known form, such as a non-limiting example or a plasmid) can be isolated, recovered and cloned by techniques well known in the art, thereby giving rise to the above-described yield-increasing and viscosity-increasing properties (without wishing to be bound to any particular scientific theory, but it is believed that this is via an increase in molecular weight range properties). Multiple copies of the DNA are then delivered into bacteria of the genus Sphingomonas (by means of plasmids, other known means) or the gene expression is increased by means of suitable, for example stronger promoters. After insertion of the target bacteria, the production of diutan can be determined by fermenting the engineered bacteria and comparing the yields based on the quantity and quality produced. The yield increase and viscosity increase (amount) can be determined simultaneously by comparing the yield of diutan obtained by the method of the invention with that of the wild type diutan-producing strain (ATCC 53159).

Detailed Description

The following terms related to the present invention are used throughout the specification, and have the meanings as follows:

the term "Sphingomonas" is used throughout the specification to refer to gram-negative bacterial strains of the genus Sphingomonas.

The term "increased producer" or "increased production" is used throughout the specification to describe engineered bacteria containing multiple copies of a DNA sequence isolated from the same strain that produces significantly higher (at least about 5% by weight higher) diutan polysaccharide as compared to the same strain of wild-type bacteria.

The term "isolated" is used to describe DNA that has been removed from a microorganism and subjected to at least some degree of purification, i.e., one or more purification steps, which DNA may be cleaved with restriction enzymes and cloned or inserted into a plasmid vector, or 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 "insertion" is used throughout the specification to describe the process and results (by way of non-limiting example, with the aid of a plasmid) of transferring a DNA segment isolated from chromosomal DNA of a strain of Sphingomonas producing diutan into a strain of Sphingomonas. Such isolated DNA may be first introduced (again without limiting possibilities) into the desired plasmid (in this case pLAFR3) using techniques well known in the art and then transferred, for example by conjugation or migration, into the recipient Sphingomonas bacterium. After insertion into a recipient Sphingomonas bacterium, the plasmid containing the relevant DNA sequence is replicated in the recipient cell to obtain several (at least two, and typically 4-10) copies of the DNA segment required to produce a high viscosity (still believed to be in the high molecular weight range) diutan polysaccharide. It is generally effective to transfer the plasmid vector into a recipient bacterium using conjugation or migration. Purified DNA may also be used to electroporate or chemically transform competent cells. Other vectors or phages may be used to transfer the DNA into the host cell. In the diutan-producing recipient Sphingomonas, there is no need to maintain the DNA segment on a plasmid (or other well-known delivery vehicle). It is customary to introduce additional several copies of a segment of DNA into a bacterial chromosome so that each generation of the segment can be replicated by the same mechanism by which the bacterial DNA is replicated. Alternatively, stronger promoter elements can be used to increase gene expression.

The term "gene amplification" is used to mean, for example, increasing the copy number of a gene by cloning the target gene on a multicopy plasmid (e.g., 4-10 copies) or inserting multiple copies of the gene (e.g., 4-10 copies) into the bacterial genome, or increasing gene expression by modifying a promoter element. These and other methods can increase the amount of protein encoded.

Throughout the specification the term "biosynthesis" is used to describe the biological production or synthesis of diutan by bacteria of the genus Sphingomonas. Diutan polysaccharide is synthesized from a single carbohydrate unit by a series of steps regulated by a number of bacterial enzymes.

The relevant DNA sequences that may be incorporated into the recipient bacterium in any selected form (e.g., still preferably in a plasmid form, but not necessarily) encode genetic information known to be beneficial or necessary for increased production and increased molecular weight of the diutan polysaccharide. Furthermore, without being bound to a particular scientific theory, while it is believed that the particular DNA sequences of the present invention (e.g., in plasmid pS 8) induce, rather than merely increase, production, the number of repeat units polymerized within each polymer of diutan itself is also increased. Thus, it is believed that this increase in repeating units results in the viscosity characteristics provided by diutan gum being unexpectedly high. Since an increase in intrinsic viscosity is detected, an increase in molecular weight can be assumed, which has a power law relationship. Thus, it is known that the intrinsic viscosity of linear polymers (such as diutan gum) is actually proportional to molecular weight in that relationship.

The relevant DNA sequences, which are the basis of the method of the invention and which result in a diutan polysaccharide with increased viscosity, are isolated using standard techniques and methods. Thus, a strain of spokeslem producing diutan can be grown by standard methods and these sequences produced therefrom. DNA extraction can then be performed, for example, by first centrifuging and resuspending the bacterial cells and then eluting the DNA through a purification column. After purification is complete, the isolated DNA may be digested with restriction endonucleases, cloned into the desired plasmid or other delivery vector, and then transferred to the recipient strain. Other techniques known in the art may be employed without specific limitation.

In the present invention, the cloning of DNA is dependent on conventional techniques and methods standard in the art. It should be noted that any method may be used to clone a DNA segment of the present invention, and the present invention is not limited to, for example, the use of plasmid cloning vectors. For example, the DNA fragment can be cloned by inserting a phage vector.

The cloned DNA sequence can then be introduced into a Sphingomonas strain using a plasmid or other delivery vector. The genetically modified Sphingomonas strain can then be used to produce diutan by fermentation. The medium suitable for fermentation is essentially an aqueous medium, which typically contains a carbon source, such as carbohydrates including glucose, lactose, sucrose, maltose or maltodextrin; nitrogen sources, such as inorganic ammonium, organic nitrates, organic amino acids or protein substances, such as hydrolyzed yeast, soya flour or casein, distillery waste liquor concentrates or corn steep liquor; and inorganic salts. Various fermentation media support the production of diutan according to the invention.

The fermentation broth may contain various levels of carbohydrate, but is typically between about 1-10% by weight (preferably 2-8%) of the fermentation medium. The carbohydrate may be added first and then fermented, or may be added during fermentation. The nitrogen content of the aqueous medium is from about 0.01% to about 0.4% by weight. One carbon or nitrogen source may be used, and a mixture of these carbon and nitrogen sources may also be used. Inorganic salts useful for fermenting sphingomonas bacteria are salts containing sodium, potassium, ammonium, nitrate, calcium, phosphate, sulfate, chloride, carbonate and similar ions. It is also preferred to include trace metals such as magnesium, manganese, cobalt, iron, zinc, copper, molybdenum, iodide, and borate.

The fermentation may be carried out at a temperature in the range of between about 25 ℃ and 40 ℃, preferably between about 27 ℃ and 35 ℃. The inoculum can be prepared by standard methods of volume enlargement, including shake flask culture and small scale submerged stirred fermentation. The medium from which the inoculum is prepared 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. Multiple seed stages (seed stages) may be employed to achieve the desired seed volume. Typical inoculation volumes range from about 0.5% to about 10% of the total volume of the final fermentation.

The fermentation vessel may be equipped with a stirrer to stir the contents. The vessel may also be equipped with automatic pH and bubble control means. The production medium may be added to the vessel and suitably sterilized by heating. Alternatively, the carbohydrate or carbon source may be sterilized separately and added to the vessel. The cultured seed broth can be added to the cooled medium (typically at a preferred fermentation temperature of about 27 ℃ to about 35 ℃) and the fermentation broth agitated for about 48 to about 110 hours, thereby producing a high viscosity broth. The diutan polysaccharide is recovered from the broth by standard methods such as precipitation with an alcohol, typically isopropanol.

Preferred embodiments of the invention

The following examples are provided to illustrate the invention. The description of the embodiments should not be mistaken for limiting the scope of the invention in any way.

DNA sequence isolation/plasmid Generation

To first isolate and determine the appropriate sequence for the inventive results described above, a gene library of the ATCC53159 microorganism was constructed as follows: chromosomal DNA of Sphingomonas ATCC53159 was isolated and partially digested with Sau3AI restriction endonuclease. DNA fragments in the range of 15-50kb were purified on agarose gel and ligated into the BamHI digested cosmid cloning vector pLAFR3 (according to Staskawicz et al, "Molecular characterization of cloned avirulent genes of P.syringae soybean-pathologically induced population 0and population 1" (Molecular characterization of cloned avirulent genes from 0and race1of Pseudomonas syringae pv. Glycinea), J.bacteriology.1987.169:5789-94) isolated from E.coli (Escherichia coli) strain JZ279(Harding et al, "Genetic and physical analysis of the gene clusters necessary for biosynthesis of xanthan in P.brassicae"), Genetic and physical analysis of the gene clusters necessary for biosynthesis of xanthan in P.brassicae.2854. J.19854. microbial infection J.19861. 19854). The ligation reaction (product) was packaged into lambda phage particles (using a gegapack III Gold packaging extract (Gigapack III Gold packaging extract), sterla gene company (Stratagene), rahoya, ca) and transfected into Library Efficiency (Library Efficiency) e.coli DH5 α MCR cells (Life Technologies, rockville, maryland). Approximately 10,000 tetracycline-resistant colonies were pooled to form a gene library. The sequences of the library were then isolated. This work carried out in this example involved the isolation of specific genes for polysaccharide biosynthesis from a microorganism of Sphingomonas ATCC 53159.

These genes for polysaccharide biosynthesis were identified by complementation with polysaccharide synthesis-deficient mutants, in particular those mutants in which the first step, i.e.glucosyltransferase I, is blocked. Since a transferase I-deficient mutant of ATCC53159 was not originally obtained, genes for diutan polysaccharide biosynthesis were identified by complementation with transferase I-deficient mutants of Sphingomonas elodea and Xanthomonas campestris. Plasmid pLAFR3 can be transferred from its E.coli host to other gram-negative bacteria by means of triparental conjugation (according to Ditta et al, "Broad-host DNA cloning System for gram-negative bacteria: construction of GenBank of Rhizobium meliloti" (Broad host DNA cloning system for gram-negative bacteria: construction of gene bank of Rhizobium meliloti), Proc. Natl. Acad. Sci.1980.77:7347-51.) using helper plasmids that provide IncP transfer function. The estimated RK2 type plasmid copy number per chromosome in E.coli is 5-7 (Figurski et al, "inhibition of ColE1 replication characteristics by IncP-I plasmid RK2in vitro constructed hybrid plasmids" (Suppression of genetic replication properties by the Inc P-I plasmids RK2in hybrid plasmids controlled in vitro), J.MoI.biol.1979133: 295-.

The gene library of ATCC53159 chromosomal DNA in E.coli was transferred into a non-mucinous mutant (GPS2) of Sphingomonas elodea with ATCC31461 by triparental conjugation, and tetracycline and chloramphenicol resistance was selected. The helper plasmid used was pRK2013 (in e.coli strain JZ 279), which contains a narrow host replication origin but shows the trans acting function (trans acting function) required for migration of pLAFR 3. Plasmid pRK2013 does not replicate in Sphingomonas strains. The bacterium sphingomonas elodea ATCC31461 produces the polysaccharide gellan gum. Gellan gum and diutan polysaccharide both have identical tetrasaccharide repeat units consisting of [ → 4) - α -L-rhamnose- (1 → 3) - β -D-glucose- (L → 4) - β -D-glucuronic acid- (L → 4) - β -D-glucose- (1 → ]. However, diutan also contains a side chain of two rhamnose molecules linked to one of the glucose residues, which side chain is modified with acetyl groups, whereas gellan has no side chain sugars and is modified with acetyl and glyceryl groups. Mutant GPS2 is deficient in the first step of polysaccharide biosynthesis, i.e., the transfer of glucose-1-phosphate from UDP-D-glucose to bacterial prenyl (bactoprenyl) phosphate lipid carriers by glucosyltransferase I. Polysaccharide (mucus) -producing colonies were isolated from tetracycline selection plates against a non-mucus colony. It is presumed that the clone that restored polysaccharide production comprised the ATCC53159 gene encoding glucosyltransferase I plus about 20-25kb of contiguous DNA. Plasmid DNA was isolated from 8 cosmid GPS2 transconjugants and transferred by electroporation to E.coli strain DH5 α (Life technologies). The plasmid was isolated from E.coli to obtain enough DNA for the restriction endonuclease HindIII/EcoRI (to cut either side of the BamHI restriction endonuclease site in the polylinker) to double digest, and the inserted DNA was cut out from the vector. The size of the inserted DNA in the clones was determined by gel electrophoresis. The final sequences of several plasmids were determined by sequencing starting from primers specific for the plasmid sequences flanking the BamHI site of the vector. Sequences were analyzed by comparing sequences in a computer database using BLASTX. Similarly, the ATCC53159 gene library was transferred into a transferase I deficient rifampicin resistant non-slime mold Xanthomonas campestris mutant (CXC109) by triparental conjugation (e.g., as described in the Harding et al reference above), and tetracycline and rifampicin resistance were selected. Xanthomonas campestris produces xanthan gum polysaccharide, and transferase I transfers glucose-1-phosphate from UDP-D-glucose to the bacterial prenyl phospholipide carrier also initiates its synthesis (Ielpi et al, "polyprenol-linked pentasaccharide repeat units that sequentially assemble and polymerize xanthan gum polysaccharide in Xanthomonas campestris" (Sequential analysis and polymerization of the polyprenol-linked pentasaccharide repeat unit of the xanthane polysaccharide), J.bacteriology.1993.175: 2490-. Plasmids were purified from the mucus transconjugants as described above and the terminal sequences were determined as described above.

S657DNA cloned in plasmids pS8 and pX6 was completely sequenced using double-strand shotgun sequencing by Rake Technologies Inc. (Lark Technologies Inc., Houston, TX). These sequences were analyzed to identify genes for diutan biosynthesis. Gene function was assigned based on homology to other genes in the database, particularly published genes for S-88 gellan polysaccharide (e.g., as described in the' 516Pollock et al patent, supra), GeneBank accession No. U51197, and gellan (GenBank AY217008 and AY220099) biosynthesis. Genes encoding transferases for the four sugars in the backbone and 4 genes for dTDP-rhamnose synthesis were identified. The genes for polysaccharide secretion are based on homology with genes for biosynthesis of other polysaccharides. Both genes encode proteins homologous to proteins involved in protein secretion. Two genes are presumed to encode a polymerase and a lyase. The insert in plasmid pX6 contained 17 genes, including the gene dpsB encoding transferase I (the first step in the start of diutan synthesis), the gene for secretion and 4 genes for dTDP-rhamnose synthesis, but lacking the genes for transferase II, III and IV, and the putative genes for polymerase and lyase. Plasmid pS8 contains 20 genes of the dps gene cluster, including the genes for all 4 backbone glycosyltransferases, four genes for dTDP-rhamnose synthesis, genes for polysaccharide secretion, including the putative genes for polymerase and ligase, but lacking the genes of unknown function, orf6 and orf 7. Plasmid pS6 contains the genes for secretion and 4 glycosyltransferases, but does not contain all the genes for dTDP-rhamnose synthesis or the gene for polymerase. Plasmid pX4 contained only a small portion of the dps region, but contained 4 genes reported by Pollock et al for dTDP-rhamnose synthesis and the gene encoding transferase I, sufficient to result in an increased polysaccharide production in sphingosine strains.

Strain production

The four plasmids described above were then introduced into Sphingomonas sphingomonas ATCC53159 by triparental conjugation as described above, resulting in new S657 engineered strains (S657/pS8, S657/pS6, S657/pX6 and S657/pX 4). Fermentation was then carried out as described above, thereby producing a biogel substance as described below. All 4 plasmids had a beneficial effect on diutan production; however, the pS8 plasmid also unexpectedly increased the viscosity of diutan greatly and increased its molecular weight. The DNA sequence (26278bp) of pS8 (DNA sequence No. 1) is provided, and the genes encoded are listed in table form in table 1 below. The insert DNA in plasmid pS8 contained part of the genes DpsG to rmlD and the genes dpsS and orf 7.

The following gene list is essentially a list of genes represented by the DNA sequence of the insert in plasmid pS 8.

TABLE 1

First in frame codon, absence of start codon

Diutan production

Three fermentations were performed in an Applikon20L fermentor with the same liquid medium while stirring and aeration to determine the yield of dioutan by the plasmid-containing engineered S657 Sphingomonas strain relative to the plasmid-free S657 wild-type strain. For the plasmid containing strains, the entire fermentation process in 5mg/L tetracycline antibiotic to ensure plasmid maintenance. KOH was added as needed to control pH. Two sub-stages are adopted, and the inoculation transfer amount is 1-6%. The medium used for the fermentation contains corn syrup as a carbohydrate source, an assimilable nitrogen source and salts. Nutrients useful for fermentation are well known in the art and include carbohydrates such as glucose, sucrose, maltose or maltodextrins; nitrogen sources, for example inorganic nitrogen such as ammonium or nitrate, organic nitrogen such as amino acids, hydrolyzed yeast extract, soy protein or corn steep liquor; and other salts containing, for example, chlorine, phosphate, calcium, copper, iron, magnesium, potassium, sodium, or zinc.

The broth viscosity and precipitated fiber were measured to examine the resulting yield of diutan. The viscosity of the fermentation broth was measured using a Brookfield viscometer, spindle No. 4, 60rpm and the results are shown in Table 2. At the end of the fermentation, the broth is treated by well-known methods of introducing glucoamylase to hydrolyze any remaining corn syrup oligosaccharides. The diutan gum produced in the broth samples was then precipitated with two volumes of isopropanol. The fibers were collected with a filter and dried. In Table 2, the term DWY denotes the total dry weight yield of precipitable biogum after hydrolysis of the remaining corn syrup oligosaccharides.

The resulting material yields were significantly higher for plasmids pX4, pX6, pS6 or pS8 carrying additional copies of the diutan biosynthesis gene. However, the broth viscosity of the pS8 plasmid was unexpectedly increased substantially compared to the increase in dry weight yield, indicating that certain factors other than increasing diutan production affect viscosity.

TABLE 2

Fermentation of plasmid-containing strains

The yield of material obtained from any of the four plasmids described above was significantly higher, while the broth viscosity of plasmids pS8 and pS6 was unexpectedly high, thus also indicating a high quality of product. The mass, i.e. the viscosity, of the resulting diutan gum product was then determined.

Rheology of diutan in application test

These samples of diutan gum were then analyzed for potential beneficial use in two different areas: an oil field additive for crude oil recovery and a cement additive for water retention and rapid formulation.

The oilfield industry relies on a test known as "seawater viscosity" (SWV) to estimate the acceptable performance of crude oil recovery gums. This test is essentially an indicator of the effectiveness of the glue to increase the viscosity of seawater (e.g., repeated recovery from the seabed).

Generally accepted according to the test seawater preparationViscosity improvement was used to predict the feasibility of the resulting gum as a suitable viscosity modifier for crude oil recovery purposes. This "synthetic seawater" formulation was prepared by mixing 419.53 grams of sea salt (ASTM D-1141-52) in 9800 grams of deionized water. For the seawater viscosity test, 0.86 grams of the sample gum was added to 307.0 grams of synthetic seawater and mixed with a pan-mixer (Fann Multimixer) (model 9B5, part No. N5020) at about 11,500rpm for 35 minutes. At the end of 35 minutes, the solution was cooled to about 26 ℃ before the viscosity was measured. For the 3-rpm reading, the sample was placed on a pan's sample stage (pan's, model 35A; torsion spring MOC34/35 F0.2b; pendulum (Bob) B1; rotor R1), the motor was turned to low speed and the transmission was set to a neutral position to set the speed to 3 rpm. The reading was then stabilized and the shear stress value was read from the meter and recorded as the SWV3rpm Dial Reading (DR). For the 0.3-rpm reading, the viscosity was measured using a Brookfield viscometer (Brookfield LV DV-II or DV-II viscometer, equipped with an LV-2C spindle). The spindle speed was set at 0.3rpm, and the viscosity was recorded as SWV-0.3rpm reading and expressed in centipoise (cP) after the spindle was rotated for at least 6 minutes. For cement applications, the PEGLSRV test (described below, low shear rate viscosity using polyethylene glycol as a dispersant) provides the industry with an indication of the performance efficacy of viscosity additives. This test measures the viscosity of a 0.25% Standard Tap Water (STW) solution of biogel. 10.0 g NaCl and 1.47 g CaCl were added₂·2H₂The STW was prepared by adding O to 10 liters of deionized water. For viscosity measurements, 0.75 grams of the biogel was added to 4.5 grams of polyethylene glycol 200(CAS25322-68-3) in a 400-mL beaker and thoroughly dispersed. Next, 299 g of STW was added to the beaker and mixed at 800. + -. 20rpm for about 4 hours using a low-pitched paddle stirrer. After mixing for 4 hours, the beaker was placed in a water bath at 25 ℃ and left to stand for about 30 minutes. The viscosity was then measured using a Brookfield LV viscometer (or equivalent instrument, e.g., DEV2.5+ model) equipped with a 2.5+ torsion spring at 3rpm using an LV1 spindle, which was rotated for 3 minutes before measuring, expressed in centipoise (cP).

The diutan samples produced above were tested in this manner with the results shown below:

TABLE 3

Rheology of diutan containing plasmid strains

SWV = viscosity in seawater

LSRV = low shear rate viscosity

Unexpectedly, some engineered strains containing plasmids produced the diutan gum of the present invention which exhibited limited viscosity increase. Most unexpectedly, however, the increase in 3rpmSWV viscosity of the pS8 strain was 80%, while the same analysis performed on the pX6 strain resulted in only 9.6% more than the wild-type. There was no significant increase in plasmids pS6 and pX 4. Similarly, the lower SWVrpm test showed that pS8 type was 51.5% higher than wild type, whereas pX6 was only 2% higher. Finally, the polyethylene glycol LSRV test showed that pS8 caused a viscosity increase of more than 77% compared to the wild type glue, whereas pX6 diutan increased less than 16%, pX4 increased 7.2%, and plasmid pS6 did not increase significantly. The highly unexpected results in these cases again show that by using the exemplified desired gene sequence in the pS8 plasmid, e.g., one way of introducing such a sequence into a diutan-producing target bacterium, the (viscous) great improvement is consistent with diutan gum production.

Thus, the diutan of the invention produced by the introduction of pS8 showed a surprising improvement in viscosity measurements for all three counts, particularly compared to the wild type and the species produced by the pX6 plasmid. Therefore, it is estimated that this new diutan would work very well in typical oilfield conditions and cement applications.

Fundamental explanation for rheological improvements

The above examples show a significant increase in the rheological parameters of diutan from strain S67/pS 8. Thus, this substantial increase in seawater and PEG low shear rate viscosity measurements cannot be attributed solely to yield increase, since pX6 strain also showed the same yield results, if not greater. Indeed, in the above examples shown in table 2, the dry weight yield (alcohol precipitable material) of the S657/pS8 strain increased by 8%, while the rheological parameter increase was more significant (52-80%). A basic study was conducted to explain why strain S657/pS8 gave a rheological improvement over the wild-type strain.

Intrinsic viscosity is a well-known technique for inferring the molecular weight of Macromolecules in polymer science (c.tanford, 1961. physicochemical of Macromolecules, Physical Chemistry of Macromolecules) john wiley parent-son (john wiley & Sons, new york.) intrinsic viscosity (y-intercept in the figure) can be obtained by plotting reduced viscosity (viscosity normalized to concentration) against solution concentration and extrapolating the linear regression of the data to 0 concentration.

Five samples of diutan were evaluated for intrinsic viscosity, neutral sugars and analyzed for organic acids, two from the wild type strain (control 1, control 2) and three from the S657/pS8 strain (sample 1, sample 2, sample 3). These samples were purified by the following steps: alcohol precipitation, rehydration, treatment with hypochlorite, treatment with glucoamylase, treatment with lysozyme and final treatment with protease (in this order). The diutan was then recovered in a 4:1 CBM to broth ratio, dried and ground. CBM is an isopropyl alcohol/water azeotrope containing about 82% isopropyl alcohol by weight.

The moisture content of each sample was tested as follows: two 0.7 gram sample specimens were typically tested using a MettlerHB43 halogen moisture balance (halogen moisture balance). The results of the two tests were then averaged and the water content was corrected using these results.

After obtaining the moisture data, a 0.2% gum solution was prepared with 0.01m nacl according to the corrected moisture. For these tests, a total of 200 grams of 0.2% solution was prepared. The gel, which is closest to ten thousandths of a weight, is weighed on an analytical balance and added to the closest thousandths of the weighed water. Each sample was stirred in a 400ml tall form beaker (tall form shaker) for two hours at 1000rpm with a 2.5 inch diameter paddle mixer.

After initial hydration, each sample was diluted to 0.02% with 0.01m nacl. 20 grams of the 0.2% solution was weighed into a 400ml beaker and then 180ml of the diluent was added to achieve the dilution. The diluted sample was mixed for an additional 30 minutes. Finally, a final dilution for determining the intrinsic viscosity is prepared from the sample. Each diutan sample was evaluated at the following concentrations: 0.004%, 0.008%, 0.010% and 0.012%.

By usingThe VE system measures viscosity. Vilastic was corrected to an error of less than 2.0% with water before testing. Each sample was tested using a 2Hz timer program, a stress of 1 and a shear rate of about 121/sec, all at 23 ℃. Each sample was tested 5 times and the average was determined. The average viscosity data is then used to calculate the intrinsic viscosity. Table 4 below provides the final results of these tests.

TABLE 4

These results indicate that strain S657/pS8 consistently produced diutan with a significantly higher intrinsic viscosity; in fact, the average reduced viscosity of the strains of the invention was 165.2, while the control was 140.7, and the measured solids levels were similar on average. These results indicate that S657/pS8 produced diutan with a higher molecular weight than the wild-type control.

To determine whether the higher viscosity diutan gum of S657/pS8 is the same as the diutan composition of the wild type strain, the composition was determined by testing for neutral sugars and organic acids. Neutral sugar analysis utilized purified samples for intrinsic viscosity detection. Aliquots of each purified sample were hydrolyzed (100 ℃ C./about 18 hours) with trifluoroacetic acid to the component sugars. The neutral sugar of the hydrolysate is quantitatively determined by high-efficiency anion exchange chromatography and pulse current meter detection. The hydrolysis product organic acid is quantitatively determined by chemical inhibition conductivity detection through high-efficiency ion exchange chromatography. Table 5 summarizes the results of the neutral sugar analysis. As shown, the neutral sugar profile of the S657/pS8 strain was almost the same as that of the S657 wild-type strain. Although both results differ from theoretical, these results indicate that the structure of the repeating units of diutan gum produced using pS8 is the same as that produced by the wild type, with pS8 species resulting in any increase in viscosity due to longer chains, i.e. higher molecular weight.

TABLE 5

Neutral sugar and organic acid analysis of pS8 and wild-type (control) Diyoutan strains

	Bacterial strains	Rhamnose%	Glucose%	Acetate salt%
					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 1	S657 wild type	30	18	8.6
					Control 1	S657 wild type	33	20	8.7
Mean value of	S657/pS8	32	18.3	8.6
					Mean value of	S657 wild type	31.5	19	8.65
Theoretical value	---	46	30	8

Thus, the dramatic increase in seawater viscosity and PEG low shear rate viscosity of diutan produced by the S657/pS8 engineered strain is due to the increase in molecular weight or length of the diutan molecules, i.e., more repeat units per molecule, rather than due to a change in its composition and therefore not due to a change in the repeat structure itself. This improvement in rheology (parameter) is not simply due to the increased yield of diutan. Although evaluation of the 4 plasmids pS6, pS8, pX4 and pX6 cloned with different portions of the diutan biosynthetic gene cluster showed an increase in production, only plasmid pS8 showed an unexpected and very high increase in the rheological parameters of the recovered diutan product.

Comparison of the diutan biosynthesis genes cloned in the test plasmids suggested that the gene most likely to result in an increase in molecular weight was the gene dpsG, since this gene was present in pS8 and not in the other plasmids. The gene dpsG encodes a hydrophobic membrane protein with strong homology to other membrane proteins involved in polysaccharide synthesis. A portion of the protein has homology to a polymerase protein that catalyzes the attachment of repeat units to form a high molecular weight polysaccharide. It has been speculated that the homologous gene gelG in S60 functions as a Gellan gum synthesizing polymerase (Harding, N.E. et al 2004. "Genes required for Gellan gum polysaccharide biosynthesis in Sphingomonas histolytica ATCC 31461." (Organization of Genes requiring for Gellan gum polysaccharide biosynthesis in Gellanthus in Sphingomonas elodea ATCC31461) J.Ind.Microbiol.Biotech.31: 70-82. Sa-Correa, I.et al 2002. "Genes, enzymes and exopolysaccharides engineering in Sphingomonas pargylcospira minosa ATCC 31461. (Gellanthus gum biosynthesis in Sphingomonas Paucimobilis ATCC31461: Genes, enzymes and exopolysaccharides engineering J.170 Biotechnology.176). Homologues of dpsG have also been isolated from the Sphingomonas strains ATCC31554 and ATCC21423, which produce polysaccharides S88 and S7 (Pollock et al, U.S. Pat. Nos. 5,854,034, 5,985,623 and 6,284,516; Pollock, T.J. U.S. Pat. No. 6,709,845). Thus, the extra copies of the polymerase gene are likely to have an effect on the increase in molecular length of the diutan molecule. It cannot be excluded that dpsG may need to be combined with other genes in the diutan biosynthesis gene cluster to achieve the observed viscosity increase. Possible candidates are the genes dpsB, dpsL, dpsK and dpsQ encoding the glycosyltransferases I, II, III and IV, in particular the gene dpsB encoding the transferase I which adds the first saccharide of the repeat unit to the lipid carrier. Other important genes may be dpsD, dpsC and dpsE, which are homologous to the genes gumB and gumC, which when amplified on multicopy plasmids show an increase in xanthan molecular weight. Achieving a large increase in viscosity may require cloning all of the genes in plasmid pS 8.

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 those specific embodiments, but rather, is intended to cover structural equivalents and all other embodiments and modifications as are within the scope of the appended claims and equivalents thereof.

Preservation of

According to the budapest treaty on international recognition of the preservation of microorganisms for Patent procedures, the following bacterial strains were deposited at 21.10.2005 by the Patent depositary department of the american type culture collection (university avenue 10801, 20110, ma, va):

sphingomonas strain S657 containing plasmid pS 8.

Claims (16)

1. A diutan gum exhibiting an intrinsic viscosity of greater than about 150deciL/g up to about 170.7 deciL/g.

2. The diutan gum of claim 1 exhibiting an intrinsic viscosity of greater than about 155deciL/g up to about 170.7 deciL/g.

3. The diutan gum of claim 2 exhibiting an intrinsic viscosity of greater than about 160deciL/g up to about 170.7 deciL/g.

4. A diutan gum exhibiting a seawater 3rpm viscosity of greater than about 35 dial reading up to about 47 dial reading.

5. The diutan gum of claim 4 exhibiting a seawater 3rpm viscosity of greater than about 37 dial readings up to about 47 dial readings.

6. The diutan gum of claim 5 exhibiting a seawater 3rpm viscosity of greater than about 40 dial readings up to about 47 dial readings.

7. The diutan gum of claim 6 exhibiting a seawater 3rpm viscosity of greater than about 42 dial readings up to about 47 dial readings.

8. A diutan gum exhibiting a seawater 0.3rpm viscosity of greater than about 35,000cp to up to about 41,500 cp.

9. The diutan gum of claim 8 exhibiting a seawater 0.3rpm viscosity of greater than about 38,000cp up to about 41,500 cp.

10. The diutan gum of claim 9 exhibiting a seawater 0.3rpm viscosity of greater than about 39,000cp up to about 41,500 cp.

11. The diutan gum of claim 10 exhibiting a seawater 0.3rpm viscosity of greater than about 40,000cp up to about 41,500 cp.

12. The diutan gum of claim 11 exhibiting a seawater 0.3rpm viscosity of greater than about 41,000cp up to about 41,500 cp.

13. A diutan gum exhibiting a low shear rate viscosity of greater than about 3500cp up to about 4980cp in the presence of a polyethylene glycol dispersant.

14. The diutan gum of claim 13 exhibiting a low shear rate viscosity of greater than about 3700cp up to about 4980cp in the presence of a polyethylene glycol dispersant.

15. The diutan gum of claim 14 exhibiting a low shear rate viscosity of greater than about 3900cp up to about 4980cp in the presence of a polyethylene glycol dispersant.

16. The diutan gum of claim 15 exhibiting a low shear rate viscosity of greater than about 4000cp up to about 4980cp in the presence of a polyethylene glycol dispersant.