BACKGROUND OF THE INVENTION

This application is a continuation-in-part application of U.S. Patent Application Serial No. 844,435 filed March 26, 1986.

This invention relates to polysaccharide polymers. In particular, it relates to xanthan-based polysaccharide polymers, defined here as polymers structurally similar to xanthan gum and produced by components of the xanthan bios nthetic pathway, including non-acetylated, non-pyruvylated, non-acetylated and non-pyruvylated, low pyruvylated and fully pyruvylated xanthan (pentamer) gums, and polytetramer gums, both acetylated and non- acetylated .

Xanthan gum is produced by bacteria of the genus Xanthomonas , in particular by microorganisms of the species X. campestris. Xanthan gum is a widely used product due to its un¬ usual physical properties, i.e., its extremely high specific viscosity and its pseudoplasticity. It is commonly used in foods as a thickening agent and in secondary or tertiary oil re¬ covery as a mobility control and profile modification agent, as well as in petroleum drilling fluids.

Chemically, xanthan gum is an anionic hetero- polysaccharide. The repeating unit of the polymer is a pentamer composed of five sugar moieties, specifically two glucose, one glucuronic acid and two mannose moieties. These sugar residues are arranged such that the glucose moieties form the backbone of the polymer chain, with side chains of mannose-glucuroniσ acid- mannose residues generally extending from alternate glucose moieties. Usually, this basic structure is specifically acetylated and pyruvylated, as described, for example, by Janson, P.E., Kenne, L. , and Lindberg, B. , in Carbohydrate Re¬ search, 4^:275-282 (1975) and Melton, L.D., Minot, L. , Rees, D.A., and Sanderson, G.R., in Carbohydrate Research, 46:245-257 (1976), each of which is specifically incorporated herein by reference. The extent of acetylation and pyruvylation is known to vary. The structure of xanthan gum is depicted below:

SUBSTITUTE SHEET

In spite of the broad utility of naturally-occurring xanthan gum, there are some situations where its physical prop¬ erties become limiting. In particular, in secondary or tertiary oil recovery it is not uncommon for the temperature of the oil bearing reservoir and the salt concentrations in the reservoir brine to be higher than are optimal for xanthan solutions. When these conditions occur, xanthan can precipitate, flocculate and/or lose its viscosity. Therefore, new viscosifying products which perform well at various conditions encountered during oil recovery, such as high temperature and high salt concentrations would be desirable.

The present invention discloses a family of xanthan- based polysaccharides having improved properties relative to naturally-occurring xanthan gum. Modifications of xanthan gum have been previously described. For example, Bradshaw _et al . (Carbohydrate Polymers, 3_: 23-38 (1983)) describe methods for preparing chemically-modified xanthan gum which are deacetylated or depyruvylated. Various means of chemically deacetylating xanthan gum produced by Xanthomonas campestris also are described in U.S. Patent Nos . 3,000,790 and 3,054,689. To date, the sole method utilized for these deacetylation processes has been through chemical removal of the acetate moieties from nor-- mally acetylated xanthan gum. It has been found that chemical processes for deacetylating xanthan gums result in a number of undesirable side effects and may cause hydrolysis of the glycosidic backbone, resulting in an irreversible change in the conformation of the molecule and lowered molecular weight.

Some of the rheological properties of deacetylated xanthan in aqueous media are known. See, e.g., Tako and Nakamura, Agriσ. Biol. Chem. 48:2987-2993 (1984) and U.S. Pat- ■ ents Nos. 3,000,790 and 3,054,689. Also, a method of increasing the viscosity of an aqueous solution using a deacetylated poly¬ saccharide is described in U.S. Patent No. 3,096,293. Thus, a method for obtaining non-acetylated xanthan which does not cause untoward side effects has been sought.

Xanthan gum can be chemically depyruvylated as well, as described by Holzwarth and Ogletree in Carbo. Res. 76:277-280 (1979). This chemical method of depyruvylation also can alter

the xanthan polymeric unit and/or cause hydrolysis of the glycosidic backbone. While a strain of X. campestris has been described in U.S. Patent No. 4,296,203 which produces non- pyruvylated xanthan gum, this non-pyruvylated gum was either fully acetylated or deactylated using chemical means. The pres¬ ent inventors believe that a xanthan polysaccharide that is both non-acetylated and non-pyruvylated will have improved rheologiσal and viscosifying properties and have therefore sought such a gum along with a microbial method of manufacturing it.

Additionally, the extent of acetylation of the inter¬ nal mannose on the xanthan side chain and the extent of the pyruvylation of the terminal mannose may vary. The present inventors believe that a fully acetylated and/or fully pyruvylated xanthan will have improved rheological properties for certain oil recovery purposes .

Moreover, the present inventors have identified poly- saccharides which are based on alterations of the normal xanthan pentamer building block. These polymers exhibit improved rheological properties over normal xanthan gum with respect to shear rate, their ability to tolerate salinity and their re¬ sponse to temperature as it affects their viscosifying prop¬ erties. These altered polysaσcharides include the polytetramer which is depicted below and the non-acetylated polytetramer.

These polysaccharides also include the acetylated and non- acetylated polytrimer described by Vandersliσe et al . in co-pending United States Patent Application Serial No. 762,878,

entitled "A Polysaccharide Polymer Made By Xanthomonas," filed August 6, 1985, which is specifically incorporated herein by reference .

SUMMARY OF THE INVENTION

An object of the present invention is to provide a family of polysaccharide polymers which are better viscosifiers of water than naturally-occurring xanthan gum. Another object of the present invention is to provide a family of poly¬ saccharide polymers having improved rheological properties over naturally-occurring xanthan gum at elevated temperatures and/or in the presence of salts and which members possess other desired properties .

It is also an object of the present invention to pro¬ vide an _in vitro method for obtaining certain of these products and microorganisms having the ability to produce members of this family of polysaccharide polymers _in vivo. A further object of the present invention is to provide processes for preparing mem¬ bers of this family of polysaccharides by aerobically fermenting microorganisms having the ability to produce the various poly¬ saccharide polymers.

Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may ixe learned by practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalities and com¬ binations particularly pointed out in the appended claims.

To achieve the objects and in accordance with the pur¬ poses of the invention, as embodied and broadly described here¬ in, there is provided a composition comprising a polysaccharide polymer having a D-glucose: D-mannose: D-glucuronic acid ratio of about 2:1:1, wherein the D-glucose moieties are linked in a beta-[l,4] configuration, the D-mannose moieties are linked in an alpha-[l,3] configuration, generally to alternate glucose moieties, and the D-glucuronate moieties are linked in a beta-[l,2] configuration to the mannose moieties. This poly¬ saccharide polymer is herein termed "polytetramer" because it consists of a repeating tetra er unit: glucose-glucose-mannose- glucuronic acid. There is also provided a polytetramer composi¬ tion, as described above, wherein at least 90%, preferably 95% and most preferably 100% of the mannose moieties are acetylated at the 6-0 position as well as a polytetramer which is non-acetylated.

To further achieve the objects and in accordance with the purposes of the present invention, there is provided a com¬ position comprising xanthan gum wherein the mannose moieties are not acetylated. This gum is hereinafter referred to as "non- acetylated xanthan." A second structure is referred to herein where the terminal mannose moieties of xanthan gum are not pyruvylated. This gum will be referred to as "non-pyruvylated xanthan." Moreover, there has been disclosed a gum which is neither acetylated nor pyruvylated. This polymer is hereinafter referred to as "non-acetylated, non-pyruvylated xanthan gum." Also, the present invention relates to a xanthan gum herein referred to as "fully acetylated xanthan gum," wherein at least 90% of the internal mannose moieties are acetylated, preferably 95% and more preferably 100%, are acetylated. Also, the present invention relates to a xanthan gum wherein at least 90% of the terminal mannose moieties, preferably 95% and more preferably 100%, are pyruvylated, herein referred to as "fully-pyruvylated xanthan gum."

This invention also contemplates processes for the production of the polysaccharide polymers described above. The polysaccharide polymers of this invention can be made generally by genetic manipulations of the microbial biosynthetic pathways which lead to the production of polysaccharides. In particular, microbial pathways for the production of xanthan gum may be ma¬ nipulated to create an _in vivo or _in vitro system for the pro¬ duction of an altered polymeric unit. Thus, systems can be cre¬ ated through the use of regulatable Acetylase and Ketalase genes, in particular, to create polysaccharides which are acetylated or pyruvylated to varying degrees. For example, it is contemplated that xanthan gum which is 10%, 20%, 30%, 40%, or 50% can be synthesized as well as xanthan which is 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% pyruvylated. Microorganisms which produce the present polysaccharide polymers _in vivo and methods of using these polysaccharide polymers are also dis¬ closed.

Various strains of X. campestis described more fully hereinbelow have been deposited with the American Type Culture Collection, Rockville, Maryland, on March 21, 1986. These

strains are X921, X1006, X934 and X1231 and have been deposited under Acccession Nos. 53472, 53473, 53474 and 67344, respective¬ ly.

It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate vari¬ ous embodiments of the invention and, together with the descrip¬ tion, serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 depicts the presumed pathway of xanthan gum biosynthesis. Abbreviations used are: Glu=Glucose; GluA=Glucu- ronic acid; Man=Mannose; Glu-Glu=Cellobiose; P=Phosphate; PP=Pyrophosρhate; C55=Isoprenoid Lipid Carrier; PEP=Phosphoenol- pyruvate; AcCoA=Acetylcoenzyme A; I-V=Glycosyltransferases; UDP=Uridine 5 '-diphosphate; and GDP=Guanosine 5 '-Diphosphate.

FIGURE 2 depicts a viscosity comparison between wild- type and non-pyruvylated gums.

FIGURE 3 depicts a viscosity comparison between non- acetylated and chemically-deacetylated gums.

FIGURE 4 depicts the approximate physical location of . 3 TnK12 insertion mutations within the cloned gum gene cluster DNA of recombinant plasmid pRK290-H336. This figure also shows the approximate locations of Spel restriction endonuσlease cleavage sites in pRK290-H336. DETAILED DESCRIPTION OF THE INVENTION

Reference vvill now be made in detail to the presently preferred embodiments of the present invention which, together with the following examples, serve to explain the principles of the invention.

The polysaccharide polymers of the present invention have been described in detail above. These polysaccharide poly¬ mers can be produced _in vitro with a cell-free enzyme system or can be produced ^Lri vivo by growing cells of an appropriate mu¬ tant strain. Other means of preparing the polysaccharide poly¬ mers are also described below.

In Vitro Polysaccharide Synthesis

The basic method relating to the use of a cell-free systems to make non-variant xanthan gum is described by Ielpi, L., Couso, R.O., and Dankert, M.A. in FEBS Letters 130: 253-256 (1981), specifically incorporated herein by reference. It has been found that a modified version of this method may be employed to create the variant polysaccharides of this inven¬ tion.

For this novel, modified method, the _irt vitro cell- free system is prepared generally by lysing cells of a micro¬ organism of the genus Xanthomonas, preferably Xanthomonas campestris, in the presence of a suitable buffer, preferably including EDTA, and obtaining the appropriate biosynthetiσ enzymes which are able to subsequently process exogenously added substrates. Alternate means of lysis may be used, including but not limited to sonication, French Pressure cell, detergent treatment, enzyme treatment and combinations thereof.

Generally, to produce the variant polysaccharides of the present invention, a lysate of a microorganism possessing the enzymes required to assemble the desired polysaccharide is incubated with the appropriate substrates, which, depending on the gum desired, may include UDP-glucose, GDP-mannose, UDP-glucuronic acid, acetyl-CoA and phosphoenolpyruvate. The choic.e of substrates is dependent on the polysaccharide which it is desired to produce. For example, a non-acetylated poly¬ saccharide is obtained by eliminating acetyl-CoA as a substrate. Similarly, a non-pyruvylated gum is obtained by eliminating phosphoenolpyruvate as a substrate. Chemical and/or enzymatic treatment of the cell lysates in order to deplete endogeneous substrates will be evident to one skilled in the art.

In addition, cell-free systems may be create from mu¬ tant organisms deficient in one or more of the enzymes of the xanthan biosynthetic pathway set forth in Figure 1. Such mutant-derived cell lysates would produce the variant gums described herein, either due solely to the mutation or due to the mutation in combination with a withheld substrate. For example, a cell-free system created from a mutant culture lacking Transferase V would produce polytetramer while the same

_g_

cell-free system, when no acetyl-CoA was present, would produce non-acetylated polytetramer.

The biosynthetic process may, in one embodiment, be monitored by the incorporation of radiolabeled substrates into the polymeric units. Other methods may also be used to allow identification of the biosynthetic intermediates that are known to those of ordinary skill in the art. In particular, chromatographic methods have been developed to separate and to identify the oligosaccharide intermediates. These include thin layer chromatography and high-performance liquid chroma ograph .

The cell-free biosynthesis of xanthan has been found to be a time-dependent, sequential process that is dependent on the addition of all three specific sugar nucleotides. The back¬ ground of non-specific incorporation of labeled substrate is minimal and does not interfere with the detection of the xanthan-specific polymer in the gum fraction.

The involvement of lipid carriers, specifically isoprenoid pyrophosphate, has been shown in several poly¬ saccharide biosynthetic pathways. Additionally, the involvement of pyrophosphoryl-linked lipid carrier in xanthan biosynthesis has been demonstrated. Thus, the xanthan biosynthetic interme¬ diates have been found to be recoverable in the organic soluble- fraction with these carrier lipids. The recovered oligo¬ saccharide can subsequently be freed from the carrier lipid by mild acid hydrolysis, for example, pH 2 for 20 minutes at 90°C and dephosphorylated with alkaline phosphatase for analysis.

Using these methods for recovery of intermediate prod¬ ucts, it has been discovered that, under _in vitro conditions, certain lysates of X. campestris mutants will produce non- acetylated or non-pyruvylated xanthan gum even in the presence • of all substrates required for non-variant gum synthesis. In light of the teachings herein, these methods will enable one skilled in the art to identify cell lysates which produce other altered polysaccharides, for example, a mutant cell lysate which produces polytetramer gum.

In Vivo Polysaccharide Synthesis

The development of the cell-free synthesis process for' the polysaccharides described above demonstrated that various

Xanthomonas campestris cells have all the enzymes necessary to synthesize acetylated or non-acetylated polytetramer, non- acetylated and/or non-pyruvylated xanthan gum and fully- acetylated xanthan gum. However, to use whole cells to synthe¬ size polytetramer _in vivo, a means for blocking xanthan gum synthesis at Reaction V (see Figure 1) would be required. More¬ over, in order for the whole cells to synthesize non-acetylated polytetramer, means for blocking the acetylation reaction (see Figure 1) as well as reaction V would be required.

Furthermore, for whole cells to synthesize non- acetylated xanthan gum, a means of blocking the acetylation step during xanthan gum synthesis would be required. Additionally, for the whole cells to synthesize non-acetylated, non- pyruvylated xanthan gum, a means of blocking xanthan gum synthe¬ sis at both the acetylation and pyruvylation steps would be re¬ quired. In one embodiment of the present invention, mutagenesis was employed to alter some of the genes responsible for these various reactions .

Transposons, including but not limited to TnlO and Tn903, can be used to mutagenize Xanthomonas campestris . These transposons, in one embodiment, confer resistance to tetracycline and kanamycin, respectively. Transposons have the ability to insert themselves into genes wherein they cause muta¬ tions b interrupting the coding sequence. The transposons can be introduced into Xanthomonas campestris on various vectors, including on so-called suicide vectors, such as pRK2013. Vector pRK2013, as described by Ditta, G. , Corbin, D. and Helinski, D.R. in Proc. Natl . Acad. Sci. U.S.A., T_-.7347-7351 (1980), spe¬ cifically incorporated herein by reference, has the ability to transfer itself into non-enteric bacteria, such as Xanthomonas campestris, but cannot replicate in that host. Thus, if the suicide vector is introduced into a population of Xanthomonas campestris cells and that population is subsequently challenged with either tetracycline or kanamycin, the Individuals which survive are those in which one of the transposons has inserted into the genome of Xanthonomas campestris. Survivors of such a challenge can be screened for those which have lost the ability to make xanthan gum. Such mutants may appear less mucoid than wild-type Xanthomonas campestris.

In other embodiments of the invention, other means of mutagenesis can be employed to generate mutants which have lost the ability to make xanthan gum or that do not acetylate and/or pyruvylate the gums they produce. Such means will readily occur to one skilled in the art, and include, without limitation, irradiation, recombinant DNA technology (in particular, as described in United States Patent Application Serial No. 842,944 of Capage e_t al_. , entitled "Recombinant-DNA Mediated Production of Xanthan Gum," filed March 26, 1986, and incorporated specifi¬ cally herein by reference, and in Example 1 below) and chemical mutagen treatment. Examples of such mutagenesis procedures have been described by Miller, J.H. in Experiments in Molecular Genetics (1972); Davis, R.W. , Bostein, D. and Roth, J.R. in Advanced Bacterial Genetics (1980); and Maniatis, T., Fritsch, E.F. and Sambrook, J. in Molecular Cloning (1982), Cold Spring Harbor.

Although mutants can first be chosen which appear less mucoid than wild-type organisms, those desired generally retain the ability to make some polysaccharide. Cell-free extracts of each of the xanthan mutants can be prepared and tested as noted above by the addition of different combinations of substrates and analysis of the resultant products.

Alternatively, appropriate mutants can be detected by assaying the culture broth of each mutant for the presence of the desired polysaccharide, e.g., polytetramer gum which is acetylated or non-acetylated or xanthan gum which is non- acetylated, non-acetylated and non-pyruvylated, low pyruvate or fully-acetylated. Thus, mutants can be found which appear to be blocked at various positions of the xanthan gum pathway. A mu¬ tant of Xanthomonas campestris which produces non-acetylated xanthan gum (X1006), a mutant which produces non-pyruvylated gum (X921), a mutant which produces xanthan gum containing low lev¬ els of pyruvylation (less than 5% of the terminal mannose X934), and a mutant which produces non-acetylated, non-pyruvylated xanthan gum (X1231 (p41KS22)) have each been placed on deposit at the American Type Culture Collection, Rockville, Maryland, under Accession Nos. 53472, 53473, 53474 and 67344, respective¬ ly.

It is not beyond the scope of the invention to employ enzyme inhibitors of wild-type Transferase V, Acetylase and Ketalase to arrive at the same products. Still other alterna¬ tives for producing this family of polysaccharides are contem¬ plated, including enzymatic and chemical degradation of natural xanthan gum.

The mutants can be grown under conditions generally known in the art for growth of wild-type Xanthomonas campestris . For example, they can be grown on suitable assimilable carbon sources such as glucose, sucrose, maltose, starch, invert sugar, complex carbohydrates such as molasses or corn syrup, various organic acids and the like. Mixtures of carbon sources can also be employed. The concentration of carbon source supplied is often between 10 and 60 grams per liter. Also necessary for growth are an assimilable source of organic or inorganic nitro¬ gen, generally between about 0.1 and 10.0 grams per liter, and minerals, the choice of which are easily within the skill of the art. Examples of suitable nitrogen sources are ammonium salts, nitrate, urea, yeast extract, peptone, or other hydrolyzed pro- teinaceous materials or mixtures thereof. Examples of suitable minerals include phosphorus, sulfur, potassium, sodium, iron, magnesium; these are often added with a chelating agent such as EDTA or citric acid.

Optimal temperatures for growth of Xanthomonas campestris generally are between 18°C and 35°C, preferably between about 27°C and 30°C. Xanthomonas campestris cells are grown aerobically by supplying air or oxygen so that an adequate level of dissolved oxygen is maintained, for example, above about 10% of saturation. Preferably, the level is kept above about 20%. The pH often is maintained at about 6.0 to 8.0, preferably at about 6.5 to 7.5.

The polysaccharides of the present invention can be recovered from fermentation broths by a suitable means. Precip¬ itation with isopropanol, ethanol, or other suitable alcohol readily yields the polysaccharides of this invention. Gener¬ ally, alcohols are added to a concentration of about 50 to 75%, on the basis of volume, preferably in the presence of potassium chloride, sodium chloride or other salt. Alternatively, the polymers can be recovered from the broth by ultra-filtration.

Non-pyruvylated xanthan gums are superior to xanthan as viscosifiers of aqueous mediums in particular applications. The viscosity of solutions of non-acetylated xanthan and non- pyruvylated xanthan are retained at conditions of high tempera¬ ture and/or high salinity. The products of this invention are thus ideally suited for use in secondary and tertiary oil recov¬ ery.

Mobility control solutions for use in enhanced oil re¬ covery may also be prepared from the variant polysaccharide polymers disclosed herein. Solutions of the polysaccharide polymers at concentrations of from about 50 to about 3000 ppm are appropriate for such mobility control solutions. Other known additives may also be used in combination with these solu¬ tions to further enhance oil recovery. Such additives include, for example, surfactants, alkaline agents or metal or organic crosslinking agents.

The polysaccharide polymers, like xanthan gum, can also be used as thickening agents in foods, cosmetics, medicinal formulations, paper sizings, drilling muds, printing inks, and the like and as a gelling agent. In addition, they can be used to reduce frictional drag of fluid flow in pipes.

EXAMPLE 1

This example shows the methods of mutagenesis and screening employed to generate X« campestris mutant strains having defects in Acetylase or Ketalase activity.

The genes encoding the enzymes of xanthan gum biosynthesis have been shown to comprise a set of clustered genes on the X- campestris chromosome. This "gum gene cluster" has been described in detail by Capage et al . Segments of gum gene DNA have been cloned on plasmid vectors such as pMW79 as detailed in Capage e_t al .

Regionally-directed mutagenesis was performed upon subcloned portions of the gum DNA carried in plasmid pMW79. These cloned DNA segments were mutagenized in vivo with transposons and in vitro, by using recombinant DNA technology to generate insertion, deletion, and substitution mutations within the cloned X. campestris DNA. In order to study the phenotypes conferred by these mutations, the plasmids carrying the

mutations were transferred back into X. campestris and subse¬ quently recombinants were identified in which the plasmid-borne, mutated gene had been inserted in the chromosome via homologous recombination. The tetracycline resistance encoded by TnlO affords a convenient selective system for movement of mutations from a plasmid into the chromosome.

One such mutant strain (X1006) carried a TnlO inser¬ tion that was found to cause inactivation of the Acetylase activity. This mutant strain was characterized as described in Example 2 and 3, and found to produce a polysaccharide that was non-acetylated. A second mutant strain was constructed by the in vitro insertion of a fragment of DNA containing the tetracycline resistance gene of TnlO into a restriction site within the gum gene cluster. This mutant strain (X921) was found to be defective in the Ketalase activity. As found by the methods of Examples 2 and 3 , this mutant produced xanthan that was non-pyruvylated .

A third mutant strain (X934) was also found that greatly reduces the ketalase activity. This mutant strain pro¬ duces xanthan gum that has a very low level of pyruvylation: 1-5% of the level of pyruvylation found in normal xanthan.

The mutant strain X934 was found as described below. . In preliminary experiments designed to study recombination between plasmid-borne X. campestris DNA and the X. campestris chromosome, the plasmid pTX655 was used as a model system. This plasmid carries a TnlO insert in the middle of a 2.3 kb X. campestris Pstl fragment cloned in plasmid RSF1010. This inser¬ tion of TnlO causes the Gum- defect in the mutant strain X655 as described by Capage e_t jtl . and Vanderslice e_t jil_. The experi¬ ment was to mobilize pTX655 with plasmid pRK2013 and transfer it from E. coli into X. campestris by selecting for transfer of the tetracycline resistance encoded by TnlO. The initial results of this mating were anomalous and suggested that TnlO did not express tetracycline resistance efficiently in X. campestris when carried on the plasmid, but that the drug resistance was more efficiently expressed when TnlO was carried in the chromo¬ some of X. campestris . This phenomenon has also been described - for TnlO in E. coli. There, it has been shown that strains

carrying one copy of TnlO inserted in the chromosome are resis¬ tant to significantly higher concentrations of tetracycline than are strains carrying TnlO on a multicopy plasmid. The selection of Tet ^r X. campestris out of the above mating resulted in a high frequency (0.5 per recipient) of progeny which grew very poorly (i.e., only small, watery colonies) on tetracycline. After pro¬ longed incubation, a large fraction of the colonies (25%) pro¬ duced sectors of more vigorously growing cells. More than 50% of these sectors appeared to be Gum- in morphology. These prob¬ ably result from recombination between the plasmid-borne DNA containing the TnlO insertion and the chromosomal wild type DNA.

When the TnlO is recombmed i .nto the chromosome, hi.gh-level Tetr is obtained and the vigorously growing sector is observed. When these Gum-, Tet sectors were picked and restreaked on tetracycline, they grew well and displayed a characteristic Gum- morphology. Gum +, Tetr isolates were also characterized. Some of these strains (in particular X934) were found to contain the entire plasmid pTX655 inserted into the chromosome of X. campestris via homologous recombination. The chromosomal struc¬ ture of the X934 strain was determined by Southern blot hy¬ bridization of the chromosomal DNA which shows that the plasmid sequences exist in a chromosomally integrated form.

EXAMPLE 2

This example shows how the altered polysaccharides of the present invention can be prepared _in vitro. For instance, it shows how non-acetylated and/or non-pyruvylated xanthan gum was prepared _in vitro.

Preparation of Lysates

Xanthomonas campestris B1459 S4-L or S4-L mutants described in Examples 1 and 5 were grown in YM (yeast-malt medi¬ um) supplemented with 2% (w/v) glucose as described by Jeanes, A. et al . in U.S. Department of Agriculture, ARS-NC-51, pp. 14 (1976), specifically incorporated herein by reference. Cultures were grown to late log phase at 30°C. The cells were harvested by centrifugation and washed with cold Tris-HCl, 70mM, pH 8.2 with lOmM EDTA and were freeze-thawed three times by a procedure similar to Garcia, R.C. e_t aT. described in European Journal of

Biochemistry 43_:93-105 (1974), specifically incorporated herein by reference. This procedure ruptured the cells, as was evi¬ denced by the increased viscosity of the suspensions and the complete loss of cell viability (one of 10 survivors) after this treatment. The freeze-thawed lysates were frozen in ali- quots at -80°C. Protein concentration was determined with BIO RAD assay (BIO RAD Laboratories, Richmond, California) and was found to be 5 to 7 mg cell protein per ml of lysate.

Biosynthetic Assay Procedure

As described by Ielpi, L. , Cσuso, R.O., and Dankert, M.A. in FEBS Letters 130:253-256 (1981), specifically incorpo¬ rated herein by reference, an aliquot of freeze-thawed lysate (equivalent to 300 to 400 ug protein), DNAase I (10 ug/ml), and gCl _p (8 mM) were preincubated at 20°C for twenty minutes. An equal volume of 70 mM Tris-HCl, pH 8.2, with the desired ra- diolabeled sugar nucleotides (UDP-glucose, GDP-mannose and UDP-glucuronic acid) were added and incubated at 20°C. Ra- diolabeled phosphoenol pyruvate and acetyl coenzvme A were added when desired as described in Ielpi _et _aJL . , supra, and Ielpi, L. , Couso, R.O., and Dankert, M.A. Biochem. Biophys . Res. Comm. 102:1400-1408 (1981) and Ielpi, L. , Couso, R.O., and Dankert, M.A., Biochem. Intern. 6 _^ :323-333 (1983), both of which are spe¬ cifically incorporated herein by reference. At various times, the reactions were stopped by dilution with 4°C buffer. The samples were centrifuged and the pellets were washed two times with buffer. The supernatants were combined, carrier xanthan (100 ug) was added, and the xanthan plus synthesized polymer were precipitated with ethanol (60%)-KCl (0.8%) . The precipi¬ tated polymer was resuspended in water and reprecipitated two more times to remove unincorporated label. Radioactivity incor¬ porated into the precipitate (termed the gum fraction) was determined in a liquid scintillation counter and the data were processed to obtain incorporation in terms of picomoles of the radiolabeled components.

Cell lysates of X1006 did not incorporate carbon-14 acetate from ι[-14C] acetyl CoA into the gum fraction of the in vitro system. Cell lysates of S4-L did produce _in vitro gum ra¬ diolabeled with C ¹⁴ c] acetate. Similarly, cell lysates of X921

did not incorporate [14C] pyruvate into the gum fraction while

S4-L cell lysates did incorporate radiolabeled pyruvate from phosphoenol [ 14C] pyruvate into the gum fraction of the _in vitro system. Thus, X1006 was identified as a mutant strain with a defect in the gene for Acetylase and X921 as a mutant strain with a defect in the gene for Ketalase. Lysates of these strains produced non-acetylated xanthan and non-pyruvylated xanthan, respectively, _Ln vitro.

It has also been shown that, by withholding sub¬ strates, X. campestris B1459 S4-L lysates produce altered poly¬ saccharides in vitro. For example, cell lysates of S4-L did produce non-acetylated, non-pyruvylated xanthan gum in vitro when the endogenous acetyl-CoA and phosphoenolpyruvate were depleted .

A mutation in the gene for Transferase V would result in the production of polytetramer. This phenotype would be dem¬ onstrated by the _in vitro method described above. The gum frac¬ tion would reveal a polysaccharide composed of glucose, mannose and glucuronic acid in molar ratios of 2:1:1. The tetrameric intermediate hydrolyzed from the lipid carrier would also be detected by its mobility on TLC and its molar ratio of sugars.

EXAMPLE 3

This example demonstrates the use of the Aσetylase- deficient strain, X1006, to produce non-acetylated gum in vivo. This example also demonstrates the _in vivo production of the non-pyruvylated gum from the Ketalase-minus strain, X921, and xanthan gum with a reduced level of pyruvylation from the strain X934.

The three mutant strains described above and S4-L were grown overnight in broth with two percent glucose at 30°C. The gum was harvested by removing cells by centrifugation, precipitating the gum from the supernatant by addition of 2-propanol or ethanol with 0.5 to 1% potassium chloride. The gum precipitate was recovered by centrifugation and resuspended. The procedure was repeated. The resuspended gum was dialysed against water. A sample of each polysaccharide was acid hydrolyzed and analyzed by HPLC using a BIO RAD HPX-87H column.

Xanthan components were quantitated by the injection of stan¬ dards of known concentration.

The HPLC analysis of the hydrolyzed gums showed that X921 produced xanthan gum without pyruvate. Strain X1006 pro¬ duced xanthan gum with no acetate. Strain X934 produced a gum that contained pyruvate at a level of 1-5% that of S4-L gum.

EXAMPLE 4

This example describes methods and strategies that could be employed to construct mutants of X. campestris that will produce xanthan that is both non-acetylated and non- pyruvylated.

Mutant strains of X. campestris lacking Ketalase (X921) or Acetylase (X1006) activity have been described. One could use microbial genetics and recombinant DNA methods as described by Vanderslice _et ^1_. and Capage _et al. to introduce a Ketalase defect and an Acetylase defect into a single strain of X. campestris . This double-mutant strain would produce xanthan gum that was both non-acetylated and non-pyruvylated.

Methods for producing insertion mutations into cloned gum gene DNA carried on plasmid vectors have been described by Capage e_t aT. One could envision using a plasmid carrying the insertion mutation that gave rise to mutant strain X921 as a substrate for a second round of mutagenesis _in vivo or in vitro. This second round of mutagenesis could employ any of a number of transposable elements that would readily occur to one skilled in the art or any of an equally obvious number of DNA restriction fragments containing selectable markers. A set of plasmids carrying two insertions mutations could be used in the gene re¬ placement technique of Capage _et _al_. to transfer the plasmid- borne mutations into the chromosome of X. campestris. Phenotypes of the resultant strains can be analyzed by the in vivo and _in vitro techniques described by Vanderslice _et ^1_. and Capage et _al _^ ., supra, in Example 2. These analyses will reveal doubly mutant strains that are blocked in both the Ketalase and Acetylase activity. These double mutant strains will produce xanthan that is simultaneously non-pyruvylated and non- acetylated.

EXAMPLE 5

This example describes procedures to obtain a X. campestris mutant which produces polytetramer.

As described above and in Vanderslice _et al . , supra, a xanthan-related polysaccharide with a truncated side chain, polytrimer, has already been obtained. This mutant was charac¬ terized by _in vivo and in vitro methods described herein. Using the methods described herein and in the referenced patent appli¬ cations, one versed in the art can create mutant strains of X. campestris which make polytetramer gum described above.

Identification of these mutant strains can be achieved using _in vitro methods or by analysis of the polysaccharides produced _in vivo, as described herein and in Capage e_t al.. , supra.

Once the polytetramer-producing mutant has been obtained, additional mutational steps, such as those described in Examples 1 and 4 and generally known to those versed in the art, can be carried out to generate mutant strains which produce nonacetylated polytetramer.

EXAMPLE 6

This example discusses cloning the acetylase gene and the Ketalase gene onto vectors to ensure that the poly¬ saccharides described herein are fully acetylated, fully pyruvylated, or both.

The X. campestris stains X1006 and X921 have mutations in the genes for acetylase and ketalase, respectively, as described in Examples 2 and 3. These mutants were created employing the methods described in Example 1. It is possible for one skilled in the art to employ methods described in Betlach j2t _al_. supra, to recover native DNA restriction frag¬ ments containing the Acetylase or Ketalase genes. That is, plasmids with DNA restriction fragments containing the Acetylase or Ketalase genes interrupted by a drug resistance marker can be used to probe lambda genomiσ libraries to obtain native DNA se¬ quences for the Acetylase or Ketalase gene. In another embodi¬ ment, the genes for the acetylase and ketalase enzymes have already been cloned onto the plasmid pRK290-H336, ATCC Accession

No. , as described by Capage e_t ^1_. , supra, and other plasmids described therein. It is a simple matter for those skilled in the art to subclone the Acetylase and Ketalase genes themselves from these plasmids.

The native DNA sequences obtained by either method can then be inserted onto plasmids capable of replication in X. campestris, for instance pMW79, using the methods revealed in Betlach e_t a_L_. and Capage _et al . Expression of the Acetylase and/or Ketalase gene can be controlled by modifying the existing DNA sequence or inserting regulatable promoters upstream from the gene. Such techniques are well known to those versed in mo¬ lecular biology and genetics. Similarly, the plasmids onto which the genes are inserted can be high copy number plasmids. As revealed in Capage e_t al_ . , the xanthan biosynthetic enzymes are present in low amounts in X- campestris . Insertion of the plasmids described above into X. campestris, and growth of cul¬ tures under appropriate conditions for expression of the plasmid-borne genes, will result in synthesis of much greater numbers of the Acetylase and/or Ketalase enzymes than the other xanthan biosynthetic enzymes. Overexpression of the Acetylase should cause the xanthan polysaccharide to be fully acetylated, i.e., all internal mannose residues acetylated. Similarly, overexpression of the Ketalase should cause xanthan poly¬ saccharide to be fully pyruvylated on the terminal mannose.

It should be evident to one skilled in the art that full acetylation and full pyruvylation of xanthan can be achieved by the methods described above. Furthermore, full acetylation of polytetramer, and full acetylation of polytrimer (described in Vanderslice e_t _al. , supra) can be achieved employing the methods described herein.

EXAMPLE 7

This example demonstrates the economic and technical advantages of a non-pyruvylated polysaccharide produced by a genetically modified Xanthomonas campestris for viscosifying water at high temperatures .

Xanthan gum, a natural product of Xanthomonas campestris, is an effective viscosifier of water for use in, for

example, enhanced recovery of petroleum. These viscosity appli¬ cations frequently necessitate xanthan gum to be applied at high temperature and in saline brines. Xanthan gums are effective viscosifiers even at high temperature (for example, 75° to 100°C), although their viscosity is substantially reduced over that at lower temperature (for example, 25° to 60°C).

The inventors have discovered a new and novel poly¬ saccharide produced by a genetically modified strain of Xanthomonas campestris (strain X921) that produced a viscosity at high temperature equal to that of wild-type xanthan gum pro¬ duced from the S4-L parent strain (strain X237). This xanthan gum has the normal pentameric xanthan structure but, because of the genetic modification, contains no pyruvate moiety on the terminal mannose. This novel polysaccharide has low viscosity at fermentation temperature near 30°C which will result in sub¬ stantial cost savings and processing conveniences. The cost to produce pyruvylated xanthan gum is high primarily because the polymer' s high viscosity requires great energy input for agita¬ tion, aeration and cooling.

Figure 2 shows a viscosity comparison of the novel non-pyruvylated xanthan gum to a wild-type xanthan gum produced by the unmodified parent. The wild-type gum shows high viscosi¬ ty at low temperature but the viscosity decreases rapidly with increasing temperature. The non-pyruvylated gum, on the other hand, has lower viscosity at low temperature, but retains a vis¬ cosity at high temperature essentially equivalent to the wild- type xanthan gum. The viscosities reported in Figure 2 were recorded at a shear rate of 8 s~ in a concentric cylinder viscometer on solutions of 1000 ppm active polymer solids in 5000 ppm NaCl brine.

EXAMPLE 8

This example demonstrates the advantages of a non- acetylated xanthan produced by a genetically modified Xanthomonas campestris for use as a visosifying agent for aque¬ ous solutions .

Figure 3 compares the viscosities of chemically deacetylated commercial xanthan and its parent compound with

those of a non-acetylated xanthan polysaccharide made from a genetically manipulated Xanthomonas campestris (strain X1006) and of the xanthan gum made from the wild-type X* campestris parent (strain X237). The viscosities were obtained at a shear rate of 8 s for 1000 ppm polymer concentration in 50,000 ppm NaCl brine. Viscosities were measured over the temperature range of 25° to about 80°C.

Figure 3 demonstrates that chemical deacetylation of xanthan results in a loss of viscosifying power over the entire temperature range. However, elimination of acetylation by genetic means results in substantially increased viscosity com¬ pared to the wild-type xanthan gum. Thus, non-acetylated xanthan gum produced by a mutant strain of X* campestris is an improved polysaccharide compared to the xanthan itself and com¬ pared to deactetylated xanthan gum produced by chemical methods.

Example 9

This example describes the methods used to construct a double mutuant strain of X. campestris that produces non-acetylated, non-pyruvylated xanthan.

Capage e_t al. have described the cloning of a gene cluster from X. campestris that contains genes that direct the biosynthesis of xanthan gum. They also described the isolation of chromosomal deletion mutations in X. campestris that elimi¬ nate all or varying portions of this gene cluster. One such deletion mutant, strain X1231, lacks all of the X* campestris DNA that is carried on the recombinant plasmid pRK290-H336. Thus, strain X1231 does not synthesize xanthan. When pRK290-H336 is transferred into strain X1231, the ability to synthesize xanthan is restored. Capage _et al . also described methods of isolating and characterizing insertion mutations of transposon TnK12 within the cloned gum gene DNA carried on pRK290-H336. Two such mutant plasmids are pRK290-H336.22 and pRK290-H336.41. The approximate locations of the TnK12 inserts on each of these plasmids is shown in Figure 4. When pRK290-H336.22 is transferred into X1231, the resultant strain produces non-acetylated xanthan. When pRK290-H336.41 is trans¬ ferred into X1231, non-pyruvylated xanthan is produced. These two plasmid mutants have been used to construct, by means on _in

vitro recombination, a double mutant plasmid that directs syn¬ thesis of non-acetylated, non-pyruvylated xanthan when carried in the deletion strain X1231.

The jLn vitro strategy for generating a non-acetylated, non-pyruvylated double mutuant plasmid is as follows. A kanamycin-sensitive (Kan ) derivative of pRK290-H336.41 was gen¬ erated by doing a partial Hindlll digestion of the plasmid, ligating the digestion products at very low DNA concentrations (to promote intramolecular ligation), and then screening

__7 tetracycline-resistant (Tet ) transformants for kanamycin sensi- tivity. Plasmid DNAs were then prepared from Tetr, Kans iso¬ lates and analyzed by restriction endonuclease digestion and agarose gel electrophoresis . There are only three HindiII sites in pRK290-H336.41, and two of these occur within TnK12 and bracket the Kan gene. Thus, a high proportion of the deletions generated in the partial digestion were deleted for the Kan gene whereas the rest of the plasmid was retained intact. The Kan plasmid still carries an insertion (1 kb) in the ketalase gene and thus was expected to yield non-pyruvylated gum. This plasmid is termed p41KS. The next step was to clone the large Spel fragment of the non-acetylated mutant plasmid pRK290-H336.22 into p41KS. As shown in Figure 4, each plasmid contains three Spel sites at positions 758, 771, and 11,716 within the DNA sequence of Capage e_t _al_. The 10.9 kb Spel frag¬ ment carries the Tnkl2 insertion of pRK290-H336.22. The small (13 bp) Spel fragment lies entirely within a tRNA gene which which is nonessential for X. campestris growth and xanthan pro¬ duction. Thus, deletion of this small Spel segment in the pro¬ cess of the double mutant construction ought not to affect xanthan biosynthesis. Plasmids p41KS and pRK290-H336.22 were purified and digested to completion with Spel and a ligation was performed. In this ligation, p41KS/SpeI was ligated in lOx molar excess with H336.22/SpeI . Thus, when recombinants con- taining the Kanr Spel fragment of H336.22 were selected, they should most often be associated with the Spel vector fragment of p41KS. We performed transformations with these ligations and obtained Kan transformants. The plasmids carried by these transformants were analyzed to identify the recombinants of

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interest. The desired recombinant plasmid was readily identi¬ fied among the Kan ^r transformants. This recombinant plasmid, termed p41KS22, contains the p41KS-derived insertion in the ketalase gene and the H336.22-derived TnK12 insertion within the acetylase gene. Appropriate restriction digestion analysis con¬ firmed the presence of both insertion mutations and, further¬ more, showed that the Spel fragment containing the TnKl2 muta¬ tion had been inserted in the correct orientation.

Plasmid p41KS22 was subsequently transferred into a series of X* campestris strains via conjugation. The large Gum deletion strain X1231 was among the recipients. This deletion lacks all of the gum gene DNA carried on p41KS22; therefore, X1231 carrying p41KS22 should produce non-acetylated, non-pyruvylated xanthan. The plasmid transferred efficiently into X1231, and the resultant phenotype was clearly mucoid but significantly less so than a wild-type control. Polysaccharide produced by X1231 carrying p41KS22 was prepared and analyzed. This polymer contained glucose, mannose, and glucuronic acid but no detectable acetate or pyruvate, demonstrating that X1231 (p41KS22) does produce the expected non-acetylated, non-pyruvylated gum.

Example 10

This example describes the construction and properties of a double mutant plasmid that combines an Acetylase mutation and a Transferase IV mutation.

Capage et al . described methods for isolating and characterizing transposon TnK12 insetions within the cloned gum gene DNA carried by plasmid pRK290-H336. One mutant plasmid carrying such an insertion is pRK290-H336.6. The approximate location of the TnK12 insertion in this plasmid is shown in Fig¬ ure 4. When present in the deletion strain X1231, this plasmid directs the synthesis of polytrimer gum as a result of the in- sertional inactivation of Transferase IV. Using procedures analogous to those described in Example 9, a double mutant plasmid was constructed that combines this Transferase IV defect with the Acetylase mutation carried in pRK290-H336.22. A kanamycin-sensitive derivative of pRK290-H336.6 was dervied by deletion of the Hindlll fragment of TnKl2. This plasmid, p6KS,

is analogous to the Kan plasmid p41KS and still retains 1 kb of TnK12 inserted within the Transferase IV gene. Subsequently, the large TnK12-containing Spel fragment of pRK290-H336.22 was ligated into ^pel-digested p6KS plasmid DNA as described in Example 9 and the double mutant plasmid p6KS22 was obtained. This plasmid carries insertion mutations in Transferase IV and the Acetylase. When transferred into deletion strain X1231, it ought to direct the synthesis on non-acetylated polytrimer. However, in several independent plasmid transfer experiments, no transfer of p6KS22 into strain X1231 was detected, although the plasmid was successfully transferred at high frequency into other recipients, including both Gum- and Gum strains. This tesult suggests that the presence of p6KS22 in strain X1231 is lethal, probably as a direct result of production of non-acetylated polytrimer gum. Capage _et al. described three other lethal mutations within the gum gene cluster and concluded that these lethal mutations cause the accumulation of a toxic intermediate in xanthan biosynthesis. Accumulation of non-acetylated polytrimer could potentially be toxic if this polysaccharide cannot be secreted by the transport system that normally secretes xanthan.

It is to be understood that application of the teach¬ ings of the present invention to a specific microorganism will be within capabilities of one having ordinary skill in the art in light of the teachings contained herein.

It will be apparent to those skilled in the art that various modifications and variations can be made in the pro¬ cesses and products of the present invention. Thus, it is in¬ tended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

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