MXPA01002475A - Bacterial strains for the production of 2-keto-l-gulonic acid - Google Patents

Abstract

The present invention relates to novel bacterial strains useful for the production of 2-keto-L-gulonic acid. The present invention further relates to the use of these strains for the production of 2-keto-L-gulonic acid by fermentative conversion of L-sorbose. The present invention further relates to the use of these novel bacterial strains for the production of pyrroloquinoline quinone and a nontoxic lipopolysaccharide. Also described is the strains of the present invention transformed by a vector.

Description

BACTERIAL CEPAS FOR THE PRODUCTION OF 2-CETO-L-GULONIC ACID BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to novel bacterial strains useful for the production of 2-keto-L-gulonic acid. The present invention also relates to the use of these strains for the production of 2-keto-L-gulonic acid by fermentative conversion of L-sorbose. The present invention also relates to the use of those novel bacterial strains for the production of pyrroloquinoline quinone and a non-toxic lipopolysaccharide. The strains of the present invention transformed by a vector are also described.

BACKGROUND INFORMATION 2-keto-L-gulonic acid ("2-KLG") is a significant intermediate in the preparation of L-ascorbic acid (vitamin C), an essential nutrient. 2-KLG has been synthesized in the past on an industrial scale using the Reichstein method. ' { Helvética Chimi ca Acta 17: 311 (1934)). This method, however, has a number of disadvantages for the commercial application, including the use Ref: 127532 of large quantities of solvents and the involvement of a number of complex reaction steps. Consequently, as an alternative to the Reichstein method, a number of processes have been developed that employ one or more microorganisms to produce 2-KLG by fermentation. U.S. Patent No. 2,421,611, for example, describes a method involving the microbial oxidation of D-glucose 5-keto-D-gluconic acid, followed by chemical or microbial reduction to L-idonic acid and subsequent microbial oxidation to 2-KLG. Japanese Patent Publications Nos. 39-14493, 53-25033, 56-15877 and 59-35290, for example, describe similar processes involving the microbial oxidation of D-glucose to 2,5-diketo-D-gluconic acid. followed by microbial or chemical reduction to 2-KLG. These methods, however, also suffer from a number of disadvantages that reduce their utility in the commercial production of 2-KLG. For example, the steps of chemical reduction in these methods (ie the reduction of 5-keto-D-gluconic acid to L-idonic acid and 2,5-diketo-D-gluconic acid to 2-KLG) are accompanied by problems with control of the stereochemistry of the reduction (thereby producing D-gluconic acid and 2-keto-D-gluconic acid, respectively, as by-products) which, in turn, reduces the yield of 2-KLG. Alternatively, when this reduction is effected by one or more microorganisms, an excess of sugar is required to provide a source of energy for the reduction, which also reduces the yield of 2-KLG. In view of these problems, an alternative route has been used for the fermentative production of 2-KLG, which only involves the oxidation of L-sorbose to 2-KLG via an intermediary of sorbosone. A number of processes using this route have been developed that employ a wide range of microorganisms of the Glucono-acter genera, such as Gluconoba cter oxydans (US Pat. 4,935,359; 4,960,695; 5,312,741; and 5,541,108), Pseudogluconobacter, such as Pseudogl uconobacter saccharoketogenes (U.S. Patent No. 4, 877, 735; European Patent No. 221 707), Pseudomonas, such as Pseudomonas sorbosoxidans (U.S. Patent Nos. 4,933,289 and 4,892,823), and mixtures of microorganisms of these and other genera, such as Acetobacter, Bacillus, Serra tia, Mycoba cterium, and Streptomyces. (U.S. Patent Nos. 3,912,592; 3,907,639; and 3,234,105). These processes, however, suffer from certain disadvantages that limit their usefulness for the commercial production of 2-KLG. For example, the processes referenced above that employ G. oxydans also require the presence of an additional "aid" microbial strain, such as Bacillus mega terium, or commercially unattractive amounts of yeast or growth components. yeast derivatives to produce sufficiently high levels of 2-KLG for commercial use. In a similar way, processes employing Pseudogluconobacter may require supplemented medium are expensive and unusual rare earth salts or the presence of a support strain, such as B. mega terium, and / or the presence of yeast to achieve commercially adequate 2-KLG concentrations and the efficient use of the sorbose substrate. Other processes employing Pseudomonas sorbosoxidans also include commercially unattractive amounts of yeast or yeast extract in the medium. Pyrroloquinolin quinone (PQQ) (2,7,9-tricarboxy-lH-pyrrolo [2, 3-f] quino-lin-4,5-dione) was initially isolated from cultures of methylotrophic bacteria (using methanol) and more Later it was found to be present in many animal tissues. The structure of the PQQ is as follows: PQQ can be a novel vitamin since it is essential for normal growth and development. When fed to animals as a supplement, the PQQ prevents oxidative changes that would commonly occur. In addition, PQQ increases the synthesis of nerve growth factor in mouse astrogial cells and has potential for a therapeutic role in the brain. (Bishop, et al., "Pyrroioquinoline Quinone: A Novel Vitamin", Nutri tion Reviews 56: 281-293 (1998) Organic chemical synthesis is the conventional means of producing PQQ, however, organic chemical synthesis has numerous disadvantages For example, chemical synthesis is uneconomical and time-consuming because the synthesis requires multiple and sometimes complicated reaction steps and produces low yields.Therefore, the need to overcome the disadvantages of synthetic chemical techniques for the production of PQQ it has been partially met by the use of bacterial strains for the production of PQQ (US Patents Nos. 4,944,382 and 5,344,768) However, the need still exists for strains of microorganisms that produce more efficient and economical PQQ 5 Lipopolysaccharide (LPS) ) is an amphipathic molecule which is a component of the cell wall of many gram negative bacteria. has been implicated in much of the pathophysiology associated with gram-negative infections in humans and animals. The LPS of Rhodoba character Sphaeroides is non-toxic and has various uses as an immunomodular and antitumor agent. However, there are several disadvantages associated with the production of non-toxic LPS through Rhodoba cter sphaeroides, for example, the inconvenience of cultivating phototrophically. 5 BRIEF DESCRIPTION OF THE INVENTION The present invention provides strains of microorganisms which efficiently produce 2- keto-L-guiónico acid. Another embodiment of the present invention is directed to strains for the production of 2-keto-L-gulonic acid in cooperation with aid strains. A further embodiment of the present invention provides a method for producing PQQ.

Another embodiment of the present invention is to provide a method for producing a non-toxic LPS. A further embodiment of the present invention is to provide the bacterial strains of the present invention transformed by a vector, and a method for the transformation of the bacterial strains by a vector. These and other embodiments are achieved by the methods of the present invention, which, in a first embodiment, is directed to a culture of any of the strains of microorganisms ADM 291-19 (NRRL B-30035), ADM 62A-12A (NRRL B-30037N), ADM 266-13B (NRRL B-30036), or mutants thereof. Other features and advantages of the present invention will be set forth in the detailed description of the following preferred embodiments, and in part will be apparent from the description or may be learned by practice of the invention. Those advantages of the invention will become reality and will be achieved by the methods particularly indicated in the written description and the claims thereof. It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to provide additional explanation of the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a description of the RiboPrint® standards of bacterial strains capable of producing 2-KLG from L-sorbose, the RiboPrint1® standards were obtained from the following bacterial strains: ADM X6L (NRRL B-21627), ADM 291-19 (NRRL B-30035), ADM 266-13B (NRRL B-30036), ADM 62A-12A (NRRL B-30037N), DSM 4025C (a reisolation of the component strain of the colony small from mixed culture tank DSM 4027, US Patent No. 4,935,359), Pseudogl uconoba cter sa ccharoketogenes strain IFO 14484 and Pseudomonas sorbosoxidans strain IFO 14502.

DETAILED DESCRIPTION OF THE INVENTION In a first embodiment, the present invention is directed to a biologically pure culture of a microorganism strain having characteristics that identify it from a strain selected from the group consisting of ADM 291-19 (NRRL B-3035), ADM 64A-12A (NRRL B-30037N), ADM 266-13B (NRRL B-30036), or mutants thereof. The strains of the microorganism of the present embodiment are capable of producing 2-KLG from L-sorbose by fermentation in pure culture, ie, in the absence of one or more strains of additional microorganisms.

In a further embodiment, the strains of the microorganism of the present invention and strain ADM X6L (NRRL B-21627, US Patent No. 5, -834,231) are capable of producing PQQ from a suitable carbon source. Strains ADM 291-19 and ADM 266-13B were deposited at the Agricultural Research Service Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, USA, on June 18, 1998, under the provisions of the Budapest Treaty and they were assigned access numbers NRRL B-30035 and NRRL B-30036, respectively. The strain ADM 62A-12A-was deposited in the NRRL on August 25, 1998 and assigned the accession number NRRL B-30037N. The characteristics of strains NRRL B-30035, NRRL B-30037N, and NRRL B-30036 include, but are not limited to: (1) Cell morphology - gram negative; it can be variable gram in old crops; thin sticks or coccobacilli; cells that appear alone and in pairs; they can be pleiomorphic; they can form short chains or cells of irregular length; they do not form spores; (2) Colony morphology - punctiform, convex, entire, smooth, butorous and translucent; of khaki or brown coloration in older colonies in some media; (3) Pigment - the colonies produce a diffusible brown pigment, especially on nutrient-rich media containing calcium carbonate and with fructose as the source of carbon. (4) Physiological characteristics: (a) catalase: positive; (b) oxidase: positive (c) gelatinase: negative; (5) Characteristics of the crop: (a) Infusion of Brain and Heart Infusion: growth; (b) growth occurs on liquid DM basal medium (Table 5), without NaCl, or the basal DM medium solidified on agar; (c) does not form films or rings within 24 hours at rest in broth of glucose or mannitol culture at pH in the range of 4.0-5.0; (d) growth in DM liquid basal medium (Table 5), without NaCl, or the basal DM medium solidified in agar, occurs at 4 ° C but not at 37 ° C. The optimum growth temperature is between 25 ° C-30 ° C in DM liquid basal medium (Table 5), without NaCl, or in basal DM medium solidified in agar; (e) the pH optimum for growth in DM basal medium (Table 5), without NaCl, or the basal medium solidified in agar, is between pH 7.0 and pH 8.0; (6) Antibiotic resistance - sensitive to amikacin, aumentin (amoxicillin -more clavulonic acid), ampicillin, cefazolin, cefoxitin, ceftacidin, ceftiofur, cefalotin, enrofloxacin, florfenicol, gentamicin, imepenem, kanamycin, sarafloxicin, tetracycline, ticarcillin, and tilmicosin , weight resistant to tribissen (aumentin plus sulfamethozole), according to what is determined by the minimum inhibitory concentration (MIC), based on the concentrations of antibiotics "physiologically attainable, using the commercial" Pasco "system, and (7) RiboPrint Analysis ®: The RiboPrint® is an automated ribotyping system that generates and analyzes genetic fingerprints of bacteria.The patterns of genetic fingerprints are standardized digital representations of the genetic data of each sample.The patterns obtained by this method are useful to differentiate not only between organisms of different species, but also in different strains of the same species The RiboPrint® standards for strains NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B) and a number of comparative strains that known to be capable of producing 2-KLG from L-sorbose are described in Figure 1. The uniqueness of the bacterial strains can be demonstrated from their RiboPrint® patterns in cases where those patterns are different. In cases where two strains give RiboPrint® patterns that are not distinguishable, the RiboPrint® data are inclusive and other methods are required to show the uniqueness of the strains. One such method is the reassociation of DNA, in which the degree of relationship of the strains is estimated on the entire bacterial chromosome. This can be effected by reciprocal, quantitative cross-hybridization of the chromosomal DNA of the two strains. In the case of strains NRRL B-30035 (ADM 291-19) and NRRL B-30036 (ADM 266-13B), the results of such studies showed a chromosomal similarity of less than 70%, a result that, in the taxonomy Modern bacterial is often associated with strains belonging to separate species (Wayne, LG et al., In t J System, Bacteriol 37: 463-464 (1987)). Thus, the DNA reassortment data unequivocally show that strains NRRL B-30035 (ADM 291-19) and NRRL B-30036 (ADM 266-13B) are unique and different from each other. The ADM X6L bacterial strain (NRRL B-21627) and mutants thereof that produce 2-KLG from L-sorbose via fermentation are described in U.S. Patent No. 08 / 834,231, filed November 10, 1998 and the Application US Serial No. 08 / 893,598, filed July 11, 1997, respectively. The strain and its mutants described in this application are distinct from those of the present invention, as can be seen from a comparison of their RiboPrint standards shown in Figure 1. In addition to the biologically pure strains NRRL-B-30035 ( ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B) may also be mutant employees thereof for the production of 2-KLG, provided that these mutants are also capable of producing 2-KLG from L-sorbose. The strains of microorganisms of the present invention, and the mutants thereof, and strains NRRL B-21627 (ADM X6L), and mutants thereof, can also be used for the production of PQQ, provided that the mutants are also capable of producing PQQ. As used herein, a "biologically pure" strain is meant to mean the strain separated from materials with which it is normally associated in nature. Note that a strain associated with other strains, or with compounds or materials not normally found in nature, is still defined as "biologically pure". A monoculture of a particular strain is, of course, "biologically pure". As used herein, a mutant of a given strain of the present invention is derived from one of the strains of the present invention, namely, NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A ) or NRRL B-30036 (ADM 266-13B) or the microorganism strain NRRL B-21627 (ADM X6L). Illustrative examples of suitable methods for preparing the mutants of "strains of microorganisms of the invention include, but are not limited to: mutagenesis by irradiation with ultraviolet light or X-rays, or by treatment with a chemical mutagen such as nitrosoguanidine (N -methyl-N '-nitro-N-nitrosoguanidine), methyl methanesulfonate, nitrogenated mustard and the like; gene integration techniques, such as those mediated by insertion elements or transposons or by homologous recombination of linear or circular DNA transformation molecules; and transduction mediated by bacteriophages. These methods are well known in the art and are described, for example in J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Coid Spring Harbor, New York (1972); J.H. Miller, A Short Course on Genetic Bacterial, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1992); M. Singer and P. Berg, Genes and genomes, University Science Books, Mili Valley, California (1991); J. Sambrook, E.F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); P.B. Kaufman et al. , Manual of Molecular and Celular Methods in Biology and Medicine, CRC Press, Boca Raton, Florida (1995); Methods in Biology and Molecular Biotechnology in Plants, B.R. Glick and J.E. Thompson, eds., CRC Press, Boca Raton, Florida (1993); and P.F. Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press,. New York, NY (1989). A mutant that produces 2-KLG may or may not have the same biological characteristics of identification of the mother or progeny strain, as long as the mutant produces 2-KLG. A mutant that produces PQQ also may or may not have the same biological characteristics of identification of the parent or progenicora strain, as long as the mutant producesPQQ Mutant strains derived from the organisms of the invention NRRL B-30035 (ADM 2991-19), NRRL B-30037N (ADM 62A-12A), or NRRL B-30036 (ADM 266-13B), or mutants derived from NRRL B -21627 (ADM X6L) using known methods are then preferably selected or separated with their potential for the improved production of 2-KLG and / or PQQ or by other desirable properties related to their utility in the production of 2-KLG from L -sorbose, and / or its utility in the production of PQQ. In accordance with the present invention, a microorganism strain or a mutant thereof of the invention is contacted with L-sorbose for a sufficient time and then the accumulated 2-KLG is isolated. Preferably, the microorganism strain is cultured in a natural or synthetic medium containing L-sorbose for a period of time to produce 2-KLG and accumulated 2-KLG which is subsequently isolated. Alternatively, a preparation derived from cells in the microorganism strain can be contacted with L-sorbose for a sufficient time and the accumulated 2-KLG can then be isolated. In accordance with the present invention, a microorganism strain or a mutant thereof of the invention is cultured in a culture medium comprising a carbon source and a nitrogen source. The source of carbon may be various sugar alcohols, polyols, aldol sugars or keto sugars, including but not limited to arabinose, cellobiose, fructose, glucose, glycerol, inositol, lactose, maltose, mannitol, mannose, rhamnose, raffinose, sorbitol, sorbose, sucrose, trehalose, pyruvate, succinate or methylamine or other substances that can be determined by a person skilled in the art. The medium preferably contains a polyol or aldol sugar, and even more preferably, mannitol, inositol, sorbose, glycerol, sorbitol, lactose and arabinose as the carbon source at a concentration of 0.1% to 20.0% by weight. All the carbon source can be added to the medium before starting the culture, or it can be added step by step or continuously during cultivation.

As used herein, "a preparation derived from cells" is intended to mean any and all cell extracts of the culture broths of a strain or a mutant thereof of the invention, cells dried with acetone, cells immobilized on or within supports, such as polyacrylamide gel, K-carrageenan, calcium alginate and the like, and similar preparations. An illustrative example of such a procedure involves adding L-sorbose and CaC03 in a suitable aqueous buffer, such as 2- (N-methylmorpholino) ethanesulfonic acid (pH 6.5, 0.5 M), to an aqueous extract of the microorganism strain in a flask with agitation. This reaction preferably proceeds at a pH in the range of 6.0 to 8.0 at a temperature in the range of 20 ° C to 40 ° C for about 1 to 100 hours. The concentration of L-sorbose should be from about 0.1 to 10% w / v, more preferably from about 0.3 to 6% (w / v) and the amount of the preparation derived from the cells of the NRRL B strains -30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A) or NRRL B-30036 (ADM 266-13B) or a mutant thereof should be from about 1 to 30 mg / ml. After stirring for a sufficient period of time under empirically determined temperature and pH conditions to maximize the yield of 2-KLG, the accumulated 2-KLG can be isolated by conventional methods.

The medium used herein can be solid or liquid, synthetic (ie, man-made) or natural, and contains sufficient nutrients for the culture of the microorganism strain of the invention. Preferably, the medium used is a liquid medium, more preferably a synthetic liquid medium. The different modalities of the method (which as used herein, is synonymous with processes) of the present invention, the initial material, L-sorbose, may be present in the medium before the introduction of a microorganism know of the invention or it may be added to the medium after the introduction of the strain, either all at once at the beginning or continuously or in parts during the course of the crop, or it can be generated in itself by fermentative conversion of D-sorbitol. The amount of L-sorbose can be determined empirically by one skilled in the art, but is at least sufficient for the strain of the microorganism to produce at least about 40 g / L 2 -KLG. Preferably, the L-sorbose comprises from 3 to 30% (weight / volume) of the culture medium, more preferably from 5 to 20%. In a preferred embodiment of the present invention, the initial L-sorbose material is generated in itself by fermentative conversion of D-sorbitol using a suitable microorganism or mixture of microorganisms.

Any microorganism or mixture of microorganisms that can convert D-sorbitol to L-sorbose in the presence of NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A) or NRRL B-30036 (ADM 266-13B ) or a mutant thereof and which does not adversely affect its ability to convert L-sorbose to 2-KLG can be employed. Preferably, the microorganism used is a strain of Gluconobacter oxydans, most preferably G. oxydans strain ATCC 621 or G. oxydans strain IFO 3293. According to this preferred embodiment of the present invention, the initial material of D- sorbitol may be present in the medium before the production of one or more of the microorganisms or it may be added to the medium after the introduction of one or more of the microorganisms, either all at once at the start or continuously or in parts during the course of cultivation. The natural or synthetic culture medium used in the embodiments of the invention described above and subsequently also contains a source of nitrogen, suitable inorganic salts, and, as appropriate, traces of various nutrients, growth factors and the like suitable for culture. of the microorganism strain, and may also contain at least one supplemental carbon source. The amount of each of those additional ingredients to be employed is preferably selected to maximize the production of 2-KLG and / or PQQ and / or LPS. Such amounts can be determined empirically by one skilled in the art according to the different methods and techniques known in the art. In a particularly preferred embodiment of the present invention, the culture medium used for the production of 2-KLG contains approximately 10% (weight / volume) of L-sorbose, approximately 3% (weight of dry solids / volume) of macerated corn liquor, and approximately 0.2% (weight / volume) of MgSO4 »7H20 with pH controlled using NH4OH, Ca (OH) 2 or CaC03. In a particularly preferred embodiment of the present invention, the culture medium used for the production of PQQ contains about 5 to 40 g / L of mannitol, glucose, sorbose or inositol, preferably 10 to 20 g / L of mannitol. , glucose, sorbose or inositol and the culture is carried out at a temperature between 0 ° C to 40 ° C, preferably 2 ° C to 35 ° C, and more preferably 20 ° C to 35 ° C. C. The pH of the medium is generally from 6 to 9, preferably from 6.5 to 8.0. The culture time is generally 20 to 150 hours, preferably 20 to 50 hours. In the present embodiment, the PQQ accumulates in the cells and / or culture medium. An illustrative example of the medium for the production of PQQ from ADM X6L (NRRL B-21627) is DM Basal Medium (Table 5), pH 6.0-7.8. In the case of strains ADM 62A-12A, 266-13B, and 291-19, Basal Medium DM without NaCl is used. Instead of mannitol, any other sugar alcohols or polyols such as myo-inositol, sorbose and glucose can be used. The medium to be used in the preparation of the inoculum may contain additional components as appropriate, such as peptone, N-ZAmine, enzymatic soy hydrolyzate, additional yeast extract, malt extract, supplemental carbon sources and various vitamins. Illustrative examples of suitable supplemental carbon sources include, but are not limited to: other carbohydrates, such as glucose, fructose, mannitol, starch or starch hydrolyzate, cellulose hydrolyzate and molasses; organic acids, such as acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, and fumaric acid; and alcohols, such as glycerol, inositol, mannitol and sorbitol. Illustrative examples of suitable nitrogen sources include, but are not limited to: ammonia, including gaseous ammonia and aqueous ammonia; ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; urea; nitrate or nitrite salts, and other nitrogen containing materials, including amino acids such as pure or crude preparations, meat extract, peptone, fish meal, fish hydrolyzate, macerated corn liquor, casein hydrolyzate, soy cake hydrolyzate, yeast extract, dry yeast, ethanol-yeast distillate, soybean meal, cottonseed meal, and the like. Illustrative examples of suitable inorganic salts include, but are not limited to: salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper, molybdenum, tungsten and other trace elements, and phosphoric acid. Illustrative examples of traces of appropriate nutrients, growth factors, and the like, include, but are not limited to: coenzyme A, pantothenic acid, pyridoxine-HCl, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide, DL- acid 6, 8-thioctic, folic acid, vitamin Bi2, other vitamins, amino acids such as cysteine and hydroxyproline, bases such as adenine, uracil, guanine, thymine and cytosine, sodium thiosulfate, p- or r-aminobenzoic acid, niacinamide, nitriloacetate, and the like, either as pure or partially purified chemical compounds or as they are present in natural materials. The culture of the microorganism strain of the invention can be carried out using any immersion fermentation techniques known to those skilled in the art, such as the low-agitation, traditional air-raised designs, or in agitated culture. The culture conditions employed, including temperature, pH, aeration rate, agitation speed, culture duration, and the like, can be determined empirically by an expert to maximize the production of 2-KLG and / or PQQ. The selection of specific culture conditions depends on factors such as the microorganism strain of the particular invention used, composition and type of medium, culture technique and similar considerations. In a particularly preferred embodiment of the present invention, when using the pure strains NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B) or NRRL B-21627 (ADM X6L) or a mutant thereof, the cultivation takes place at a temperature in the range of 22 ° C to 35 ° C, preferably of about 30 ° C and to a pH in the range of 6.0 to 8.0, preferably in the range of 6.0 to 7.5, more preferably from about 6.5 to 7.5. The culture conditions employed may, of course, be varied by known methods at different points in time during cultivation, as appropriate, to maximize production of 2-KLG and / or PQQ. After cultivation for a sufficient period of time, such as, for example, from 10 to 150 hours, the 2- KLG and / or PQQ that has accumulated in the cells and / or culture broth is isolated according to any of the known methods including ion exchange chromatography, gel filtration, solvent extraction, affinity chromatography, or any combination thereof. Any method that is suitable with the conditions used by the crop can be used; Illustrative examples for suitable methods for recovering 2-KLG are described in U.S. Patent Nos. 5,474,924; 5,312,741; 4,960,695; 4,935,359; 4,877,735; 4,933,289; 4,892,823; 3,043,749; 3,912,592; 3,907,639 and 3,234,105. Illustrative examples of suitable methods for recovering PQQ are described in U.S. Patent Nos. 4,994,382 and 5,344,768. According to one such method for the removal of PQQ, solid-liquid separation, such as filtration and / or centrifugation, is applied to the culture broth to effect the removal of the cells. Either the supernatant, which is the liquid portion that results after the removal of the cells, or the culture broth, which contains the cells, can be used in additional recovery steps. The recovery of PQQ from the supernatant or the culture broth is effected, for example, by ion exchange chromatography, gel filtration, solvent extraction or affinity chromatography.

The identification of the recovered PQQ is made by comparing with pure standard (Fluka Product No. 64682), using, for example, paper chromatography, thin layer chromatography, gel permeation chromatography, elemental analysis, such as mass spectrometry, spectroscopy nuclear magnetic resonance, absorption spectroscopy or high-performance liquid chromatography (CLAP), or a combination thereof. The quantitative analysis of the PQQ can be done using a variant of suppression of the D-glucose dehydrogenase activity of Pseudomonas aeruginosa (Ameyama et al., FEBS Lett 130: 119-183 (1981)) and E. coli (Ameyama et al. al., Agri., Biol. Chem. 49: 1221-1231 (1985)), UV absorption spectrum (Dekker et al., Eur. J. Biochem. 125: 69-73 (1982)), CLAP, gel permeation combined with mass spectrometry or infrared spectroscopy with Fourier transformation (FTIR). According to one such method to recover 2-KLG, at least the microorganisms are removed from the culture broth by known methods, such as centrifugation or filtration, and the resulting solution concentrated in va cuo. The crystalline 2-KLG is then recovered by filtration and, if desired, purified by recrystallization. Similarly, 2-KLG can be recovered using known methods such as the use of ion exchange resins, solvent extraction, precipitation, desalination or the like. When the 2-KLG recovered as a free acid, it can be converted to a salt, as desired, with sodium, potassium, calcium, ammonium or similar cations using conventional methods. Alternatively, when the 2-KLG is recovered as a salt it can be converted to its free form or to a different salt using conventional methods. In an alternative embodiment of the present invention, a microorganism of the invention is cultured in culture mixed with one or more aid strains. As used herein, "aid strain" is intended to mean a strain of a microorganism that increases the amount of 2-KLG and / or PQQ produced in the process of the invention. Suitable aid strains can be determined empirically by one skilled in the art. Illustrative examples of suitable helper strains include, but are not limited to, members of the following genera: Aureoba cterium (preferably L. lichens or A. saperdae), Corybacteriumum (preferably C. ammoniagenes or C. glutamicum), Bacillus, Brevibacterium um (preferably B. linens or B. flavum), Pseudomonas, Proteus, In terobacter, Ci trobacter, Erwinia, Xan thomonas and Flavoba cteri um. Preferably, the help strain is Corynebacterium gumta cum ATCC 21544.

The aid strain is preferably incubated in an appropriate medium under suitable conditions for a sufficient amount of time until a sufficient population culture is obtained. This aid strain inoculum can then be introduced into the culture medium for the production of 2-KLG and / or PQQ either separately or in combination with the microorganism strain of the invention, i.e., a mixed inoculum. Preferably, for the production of 2-KLG, the ratio of the amount of the aid strain to the amount of the strain NRRL-B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A- 12A), 0 NRRL B-30036 (ADM 266-13B) is in the range of 10: 1 to 1: 10,000. Preferably, for the production of PQQ, the ratio of the amount of the aid strain in relation to the amount of strain NRRL B-21627 (ADM X6L) is in the range of 10: 1 to 1:10, 000 Another embodiment of the present invention is directed to the novel microorganism strains described above which are useful in fermentation processes for the production of 2-KLG. A further embodiment of the invention provides a method for isolating a non-toxic lipopolysaccharide (LPS) from NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B) or NRRL B-21627 (ADM X6L) or a mutant thereof. In the context of this embodiment, a mutant is defined as a strain derived from one of the strains of the present invention that produces a non-toxic lipopolysaccharide. The LPS can be purified from the strains of the present invention by any of the methods described in, for example, Strittmater et al. , "Nontoxic Lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023," J. .Bacteriol. 155: 153-158 (1983), Galanos, C. et al. , "A novel method for the extraction of R lipopolysaccharides", Eur. J. Biochem. 9: 245-249 (1969), and Qureshi et al. , "Position of the Ester Group in the Skeleton of Lipid A of Lipopolysaccharides obtained from Salmonella typhimurium" J. Biol. Chem. 258: 12941-12951 (1983). One such method for the production and purification of LPS from NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B) or NRRL B- 21627 (ADM X6L) involves cultivating in a medium comprising 1% Difone's Soytone solution, 1% Difco's Yeast, 0.5% Difco's Malt Extract, 0.5% NaCl, 0.25% K2HP04, 2% mannitol or another suitable carbon source, pH 7.8. Suitable carbon sources can be selected from the group comprising glycerol, mannitol, sorbitol, inositol, glucose and fructose. In the case of ADM guests 62A-12A, 266-13B, and 291-19, media without NaCl are used. Tryptic Soy Broth (Difco) adjusted to pH 7.8 with NaOH could also be used. The cell mass can be grown in liquid media, or in a surface culture on media solidified with 1.3% Bac.to Difco Agar. The wet bacteria are then washed at least once with n-butanol with a content of about 0.1 to 5% acetic acid. The bacteria are then further washed with ethanol, acetone and ether and then dried, for example, in vacuo. The cells are then subjected to extraction with phenol-chloroform-petroleum ether and the obtained LPS are optionally treated again with phenol-chloroform-petroleum ether. The present invention also relates to strains of the present invention transformed with vectors which optionally include at least one marker gene. Recombinant constructs can be introduced into the bacterial strains of the present invention using well known techniques such as transduction, transfection, conjugation, and electroporation or other transformation methods. The vector can be, for example, a phage, plasmid, cosmid or a minichromosome. As defined herein, "host" and "host cells" are synonymous with the cells of the microorganism strains of the present invention. The polynucleotides of interest can be attached to a vector that contains a selectable marker for propagation in the host. A plasmid vector can be introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with charged lipid. Preferred are vectors comprising control regions of action in the cis position for a polynucleotide of interest. The appropriate trans position acting factors can be supplied by the host, supplied by a complementation vector, or delivered by the vector itself upon introduction into the host. In certain preferred embodiments in this regard, the vectors provide a specific expression, which may be inducible, mutant specific and / or condition specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to handle, such as temperature, nutritional additives or chemical additives. Other suitable environmental factors will be readily apparent to those skilled in the art. Expression vectors useful in the present invention include chromosomal, episomal vectors, for example, vectors derived from plasmids, bacteriophages, and vectors derived from combinations thereof, such as cosmids and phagemids. A DNA insert of interest should be operably linked to an appropriate promoter that is preferably a promoter derived from the host. The expression constructs will also contain sites for the initiation of transcription termination, and in the region described, a ribosomal binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation start codon appropriate for the host at the start and a stop codon appropriately placed at the end of the polypeptide to be translated. According to the indicated, the expression vectors will preferably include at least one marker capable of being selected or separated. Such markers include the genes for resistance to amikacin, augmentin (amoxicillin plus clavulonic acid), ampicillin, cefazolin, cefoxitin, ceftazidime, ceftiofur, cephalothin, chloramphenicol, enrofloxacin, erythromycin, florfenicol, gentamicin, imipenem, kanamycin, penicillin, sarafloxicin, spectinomycin. , streptomycin, tetracycline, ticarcillin, or tilmicosin. Preferred markers include ampicillin, chloramphenicol, erythromycin, kanamycin, penicillin, spectinomycin, streptomycin and / or tetracycline. Other suitable markers will be readily apparent to those skilled in the art. A preferred vector is pMF 1014-a (M. T. Follettie, "DNA Technology for Corynebacterium um gl utamicum: Isolation and Facetization of Biosynthetic Amino Acid Genes", Ph.D. Dissertation Massachusetts Institute of Technology, Cambridge. Massachusetts (1989)), which comprises the pSRl-a replicon and a determinant of kanamycin resistance. Specifically, pMF 1014-a comprises the pSRl replicon (Archer, JA et al, J. Gen. Mi crobiol 139: 1753-1759 (1993)), and the pSRl-a mutation that allows the reproductive maintenance of the plasmid in hosts of E. coli (Follettie Dissertation, 1989), and the kanamycin resistance gene derived from Tn903 from plasmid pUC4K (Taylor, LA et al, Nuclei c Acids Res. 1 6: 358 (1988)). The present invention provides the strains of the present invention, or mutants thereof, which comprise pMF1014-a. The present invention provides a biologically pure culture of strain of microorganism NRRL B-21627 or a mutant thereof, comprising pMF1014-a. The introduction of the construct into the host cells can be effected by transfection with calcium phosphate, transfection mediated by DEAE-dextran. Transfection mediated by cationic lipid, electroporation and other methods of transformation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, "Basic Methods in Molecular Biology," (1986). The methods used and described herein are well known in the art and are described more particularly, for example, in J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1972); J.H. Miller, A Brief Course on Bacterial Genetics, Cold Spring Harbor Laboratory Press. Cold Spr: -ng Harbor, New York (1992); M. Singer and P. Berg, Genes and Genomes, University Science Books, Mili Valley, California (1991); J. Sambrook, E.F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); P.B. Kaufman et al, Manual of Molecular and Celular Methods in Biology and Medicine, CRC Press, Boca Raton, Florida (1995); Methods in Biology and Molecular Biotechnology in Plantas, B.R. Glick and J.E. Thompson, eds., CRC Press, Boca Raton, Florida (1993); P.F. Smith-Keary, Molecular Genetics of Escherichia coli, The Guilford Press, New York, NY (1989); Plasmids: An All Practical Me. 2nd Edition, Hardy, K.D., ed., Oxford University Press, New York, NY (1993); Vectors; Da Cough Essentials, Gacesa, P., and Ramji, D.P., eds., John Wiley & Sons Pub. New York, NY (1994); Guide for Electroporation and Electrofusion, Chang, D., et al, eds., Academic Press, San Diego, CA (1992); Promiscuous Plasmids of Gram Negative Bacteria, Thomas, C.M., ed., Academic Press, London (1989); The Biology of Plasmids, Summers, D.K., Blackwell Science, Cambridge, MA (1996); DNA Comprehension and Gene Cloning: A Guide for the Curious, Drica, K., ed., John Wiley and Sons Pub., New York, NY (1997); Vectors: A Molecular Cloning Vector Study and Its Uses, Rodriguez, R.L., et al, eds., Butterworth, Boston, MA (1988); Bacterial Conjugation, Clewell, D.B., ed., Plenum Press, New York, NY (1993); Del Solar, G., al, "Reproduction and control of circular bacterial plasmids", Microbiol. Mol. Biol. Rev. 62: 434-464 (1998); Meijer, W.J., al, "Circular Bacillus subtilis plasmids: complete nucleotide sequences and gene analysis of pTA1015, pTA1040, pTA1050 and pTAlOdO, and comparison with related plasmids of gram positive bacteria", FEMS Microbiol Rev. 21: 37-368 (1998); Khan, S.A., "Cyclic reproduction of bacterial plasmids", Microbiol. Mol. Biol. Rev. 61: 442-455 (1997); Baker, RL., "Expression of protein using fusion and cleavage of ubiquitin", Curr. Opin. Biotechnol 7: 541-546 (nineteen ninety six); Makrides, S.C., "Strategies for achieving high-level expression of genes in Escherichia coli", Microbiol. Rev. 60: 512-538 (1996): Alonso, J.C., al, "Site-specific recombination in thetata positive gram reproduction plasmids" FEMS Microbiol. Lett. 142: 1-11 (1996); Miroux, B., et al, "Overproduction of protein in Escherichia coli: mutant hosts that allow the synthesis of some membrane protein and globular protein at high levels", J. Mol. Bíol. 260: 289-298 (1996); Kurland, C.G., and Dong, H., "Bacterial growth inhibited by protein overproduction", Mol. Microbiol. 21: 1-4 (1996); Saki, H., and Komano, T., "Reproduction of Plasmid DNA from Wide Host Intervals IncQ in Gram Negative Bacteria" Biosci. Biotechnol. Biochem. 60: 311-382 (1996); Deb, J.K., and Nath, N., "Corynebacteria plasmids," FEMS Microbiol. Lett. 175: 11-20 (1999); Smith, G.P., "Filamentous phages as cloning vectors". Biotechnol 10: 61-83 (1988); Espinosa, M .. el al, "Cyclic reproduction of plasmids and their control" FEMS Microbiol. Lett. 130: 111-120 (1995); Lanka, E., and Wilkins. B.M., "Reaction of DNA processing in bacterial conjugation" Ann. Rev. Biochem. 64: 141-169 (1995); Dreiseikelmann, B., "Translocation of DNA through bacterial membranes" Microbiol. Rev. 58: 293-316 (1994); Nordstrom, K., and Wagner, E.G., "Kinetic aspects of the control of plasmid reproduction by antisense RNA", Trends Biochem. Sci. 19: 294-300 (1994); Frost, L.S., et al, "Analysis of the sequence of genetic products of the sex factor transfer region F", Microbiol. Rev. 5S: 162-210 (1994); Drury, L., "Transformation of bacteria by electroporation," Methods Mol. Biol. 55: 249-256 (1996); Dower, W.J., "Electroporation of bacteria: a general method for genetic transformation", Genet. Eng. 12: 215-295 (1990); Na, S., et al. , "The factors that affect the efficiency of the transformation of coryneform bacteria by electroporation", Chin. J. Biotechnol. 11: 193-198 (1995); Pansegrau, W., "Covalent association of the trand gene product of the RP4 plasmid with the 5'-terminal nucleotide at the site of the relaxation notch" J. Biol. Chem. 265: 10637-10644 (1990); and Bailey J.E., "Host-vector interactions in Escherichia coli", Adv. Biochem. Eng. Biotechnol. 48: 29-52 (1993). The following examples are illustrative and are not intended to limit the scope of the invention as defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations may be made in the methods of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention as long as they fall within the appended claims and their equivalents.

All patents and publications referred to herein are expressly incorporated by reference.

EXAMPLES EXAMPLE 1: ISOLATION OF CEPA NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B). A. ORIGIN, ENRICHMENT AND SEPARATION OF SOIL SAMPLES. The environmental specimens were subjected to microbial enrichment in shake flasks. The resulting mixed cultures were separated to identify those that contained at least one strain of microorganism capable of producing 2-KLG from L-sorbose. Samples of wet soil, sand, sediment, fruit, berries, humus and other environmental specimens were collected from the U.S. Each specimen was immediately stored in a cold, ventilated, moist container. The enrichments were started by adding one gram of soil or specimen to 30 mL of Medium A (Table 1) in a shake flask, with baffles, of 250 mL, followed by incubation with shaking at 30 ° C, 230 rpm, for approximately 2 hours. days . To select or separate enrichments by fermentation, 0.5 to 0.75 mL of each enrichment was transferred to a 250 mL baffled flask containing 30 mL of fresh Medium B (Table 1). These flasks were shaken at 30 ° C, 230 rpm for approximately 3 days, after which portions of the fermentations of the mixed culture were analyzed to determine the content of 2-KLG, and were cryogenically preserved. For preservation, 2.0 mL of each culture was mixed with 1.0 mL of 40% glycerol in sterile water, then stored at -70 ° C. The flasks were selected or separated for the production of 2-KLG using thin layer chromatography on 150 plates of Silica Gel Whatman LK5, 250 mm thick (Catalog No. 4855-820). The plates were stained with 5 μL of centrifuged culture broth, and developed for 5-6 hours in solvent (157 mL of n-propanol, 39 mL of deionized water, 4 mL of 1% phosphoric acid, 0.4 mL of acetic acid). glacial). The plates were air dried and then sprayed with 0.125 g of tetrazolium blue chloride dissolved in 25 mL of methanol and 25 mL of 6N sodium hydroxide, after which they were baked at 60 ° C for 5 minutes. Sorbose and 2-KLG were visualized as purple spots on the finished plates, and were identified by comparison with a standard containing 10 g / L each of 2-KLG and L-sorbose.

The production of 2-KLG was quantified by CLAP.

The samples were prepared by 1:10 dilution in mobile phase, followed by filtration through 0.45 μm porous membranes. The mobile phase contained 1.1 mL of ACS-grade sulfuric acid diluted to 4.0 L using Milli-Q water. Samples of 100 μm each were loaded into two columns of A inex inexhaustive HPX-87H 2 mm x 300 mm x 7.8 mm (BioRad) arranged in series to provide a total column length of 600 mm, preceded by a protective column of the same resin. The column was run at 55 ° C using a flow rate of 0.6 mL / min. L-sorbose and 2-KLG were detected using a Water Model No. 410 differential refractometer, and were identified by comparing with a standard containing 2-KLG and L-sorbose. Thirty-three (33) of the mixed culture fermentations produced 2-KLG, in amounts ranging from 1.8 to 9.3 g / L. Mixed culture fermentations of which strains NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B) were subsequently isolated (Example IB) produced 6.9 g / L, 9.3 g / L, and 5.4 g / L of 2-KLG, respectively.

B. ISOLATION AND TEST OF MONOCULTIVES Pure cultures of microorganisms capable of producing 2-KLG were isolated from L-sorbose, either in monoculture or in culture mixed with other microorganisms, of the enrichments described above. Eleven enrichments of mixed culture were chosen in Example IA on the basis of their superior 2-KLG production. These were thawed and diluted at 10-fold serial increments using Medium A, after which 0.1 mL of each dilution was dispersed on the surface of an agar plate of Medium A. The plates were incubated at 30 ° C for 24 hours. hours, then examined under an amplification of 8 to 40 times. Attention was needed to the smaller colonies, which grew more slowly, to recover the 2-KLG-producing strains from the dilution plates. Several examples of each type and size of colony were selected and subcultured on fresh Medium A plates, after which the dilution plates were returned at 30 ° C for 24 hours. Additional slow-growing colonies were selected from the dilution plates and subcultured after a second incubation period. Each strain was purified thoroughly for 1-3 cycles on any of the Medium A plates or in PYM plates (10 g / L of peptone, 10 g / L of yeast extract, 0.5 g / L of glycerol, 30 g / L). L of mannitol, 20 g / L of agar). The pure strains were cryogenically preserved at -70 ° C in PYM liquid medium containing 20% glycerol. A total of 118 pure strains of the eleven enrichment mixtures were recovered. The 118 new strains were tested for their potential to convert L-sorbose to 2-KLG in shake flasks. To take into account the possibility that the production of 2-KLG may require the combined activity of two or more microorganisms, each new isolate was tested in combination in pairs with all the strains that originated from the same enrichment, as well as the pure culture . To prepare the inoculum, each strain was grown on PYM agar for 24 hours, after which a large ring of cells was suspended in sterile buffer containing 50 mM sodium phosphate, 0.4% sodium chloride, and 0.05% mannitol , pH 7.2. For each pure strain or paired strain test, a flask with baffles, 250 mL containing 30 mL of Medium C (Table 1) was inoculated with 0.2 mL of cell suspension from each of the relevant strains. These flasks were shaken at 30 ° C, 230 rpm for 24 hours, after which 1.0 mL was transferred to 30 mL of fermentation medium D (Table 1). The fermentation flasks were shaken at 30 ° C, 230 rpm for three days, then the broth was analyzed to determine the content of 2-KLG and the sorbose content using TLC and CLAP. The flasks containing the strains NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B) showed a total pattern of higher levels of 2-KLG. than the flasks that did not contain those strains. These strains were individualized for subsequent studies, as candidates producing 2-KLG.

Table 1: Means used in Example 1 The glucose, macerated corn, iron sulfate and calcium carbonate were adjusted to pH 7.9, then autoclaved for 20 minutes, the remaining ingredients were adjusted to pH 6.3, then sterilized by filtration. The finished medium had a pH in the range of 7.1-7.4. The Yeast Nitrogen Base was the product Difco # 0335-15-9.

EXAMPLE 2: PRODUCTION OF 2-KLG FROM L-SORBOSE BY STRAINS NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B) IN MATRACES WITH AGITATION. For each producing strain, a fresh culture ring grown on agar medium was inoculated in a 250 mL baffled flask, containing 20 mL of Medium A or B seeded (Table 2), which was stirred at 30 ° C for 22-24 hours at 240 rpm. Two mL of the sowing content was used to inoculate 25 mL of fermentation medium C or D (Table 2) in a shake flask, with baffles, of 250 mL, and the flasks were shaken for 72-92 hours at 30 ° C, 240 rpm. The broth was later removed and analyzed by CLAR. The results of the production of 2-KLG are shown in Table 3.

Table 2: Means used in Examples 2 and 3 Table 2: Means used in Examples 2 and 3 (continued) Table 2: Means used in Examples 2 and 3 (continued) Soluble soy was a liquid residual fraction of soybean processing. The amount was expressed as grams of dry solids per liter of medium. , Table 3: Production of 2-KLG from L-sorbose by pure cultures in shake flasks Table 3: Production of 2-KLG from L-sorbose by pure cultures in shake flasks (continued) * Yield was expressed as grams of 2-KLG produced per 100 grams of initial L-sorbose in the reaction.

EXAMPLE 3: PRODUCTION OF 2-KLG FROM L-SORBOSE BY MIXED CROPS COMPRISED OF PRODUCER STRAINS NRRL B-30035 (ADM 291-19), OR NRRL B-30037N (ADM 62A-12A), OR NRRL B-30036 (ADM 266-13B), IN COCULTATION WITH A SECOND ORGANISM. For each producing strain, a fresh culture ring grown on agar medium was inoculated into a 250 mL baffled flask containing 20 mL of Medium A (Table 2), followed immediately by inoculation with 100 μL. of a frozen culture of Aureobacterium um liquefaciens strain X6S. The flasks were shaken at 240 rpm for 22-24 hours at 30 ° C. Two mL of this culture was transferred to a 250 L baffled flask containing 25 mL of Medium C, which was then stirred for 72-92 hours at 30 ° C, 240 rpm. The broth was later removed and analyzed by CLAR. The results of the production of 2-KLG are shown in Table 4.

Table 4: Production of 2-KLG from L-sorbose by cultures mixed in flasks * Yield was expressed as grams of 2-KLG produced per 100 grams of initial L-sorbose in the reaction EXAMPLE 4: PRODUCTION OF PQQ BY STRAINS NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B), and NRRL B-21627 (ADM X6L) ) IN MATRACES WITH AGITATION. Strain NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B), or NRRL B-21627 (ADM X6L), inoculated in 10 ml of DM Basal Medium (Table 5), pH 7.8 and stirred at 300 rpm, and 30 ° C, until a maximum optical density was reached at a wavelength of 600 nm. In the case of ADM 62A-12A, 266-13B, and 291-19 means were used without NaCl. 5 ml of this culture were transferred to 500 ml of fresh medium in a 2 L baffled flask, which was inoculated with shaking at 300 rpm, 30 ° C, for a sufficient time to reach a maximum optical density at one length 600 nm. To determine the amount of PQQ in the medium, a sample was removed at a predetermined time and centrifuged to obtain a supernatant. The supernatant was analyzed according to the methods of U.S. Patent Nos. 4,994,382 and / or 5,344,768 or by gel permeation chromatography coupled with mass spectrometry.

EXAMPLE 5: EXTRACTION OF A NON-TOXIC LIPOPOLISACARIDE FROM NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B) and NRRL B-21617 (NRRL B-21617) ADM X6L) NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B) or NRRL B-21617 (ADM X6L) was grown in medium which comprised 1% Difone Soytone, 1% Difco Yeast Extract, 0.5% Difco Malt Extract, 0.5% NaCl, 0.25% K2HP04, 2% Mannitol, 2% Jpyo-Inositol, or 2% glucose, or another suitable carbon source, pH 7.8 and stirred at 300 rpm, 30 ° C. In the case of NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), NRRL B-30036 (ADM 266-13B), media out NaCl were used. The cells were then harvested and then washed three times water. 120 g of wet bacteria were then washed three times, each time 600 ml of n-butanol containing about 1% acetic acid. The bacteria were further washed ethanol, acetone and ether (each, three times 600 ml) and dried in vacuo. The dried bacteria were placed in a centrifugation vessel and an extraction mixture (200 ml) was added. The extraction mixture contains liquid phenol (90 g of dry phenol + 11 ml of water), chloroform and petroleum ether (e.g., 40-60 ° C) in a volume ratio of 2: 5: 8, respectively. This mixture is monophasic when the phenol used is dry. If water is present in the original phenol preparation, the mixture is cloudy and can be rinsed by adding solid phenol. The suspension is then homogenized for 2 minutes cooling, so that the temperature remains between 5 ° and 20 ° C. It does not mean that this treatment breaks the bacteria, but that it is to obtain them in a fine suspension. If the bacteria are already finely suspended, stirring the mixture for a few minutes is sufficient. Sometimes the suspension is very viscous after homogenization. In this case, more extraction mixture is added. The bacteria are then centrifuged (5000 rev / min, 15 min) and the supernatant containing the lipopolysaccharide is filtered through filter paper in a spherical flask. The bacterial residue is extracted once more the same amount of extraction mixture, stirred and centrifuged as above and the supernatant is added to the first extract. The extraction could be repeated a third time. The combined supernatants have a slightly yellow to dark brown color. The petroleum ether and the chloroform are then completely removed on a rotary evaporator at 30-40 ° C (or at high vacuum to less than 0 ° C). If the remaining phenol crystallizes now, enough water is added to dissolve it. The solution is transferred into a cold centrifuge container and water is added dropwise until the lipopolysaccharide precipitates. The addition of water stops when the lipopolysaccharide begins to settle after the mixture is allowed to stand for 1 to 2 minutes. Although the precipitation of lipopolysaccharide is complete long before the phenol is saturated water, care must be taken not to add too much water as this causes the formation of two phases. The precipitated lipopolysaccharide is then centrifuged (3000 rev / min, 10 min), the supernatant is decanted, and the tube is allowed to stand for 2 to 3 minutes flipped down. It is then cleaned internally filter paper. The precipitate is washed two to three times small portions of 80% phenol (approximately 5 ml) and the inside of the tube is cleaned filter paper before decanting the supernatant. Finally, the precipitate is washed three times ether to remove any remaining phenol, and dried in vacuo. The lipopolysaccharide is removed in distilled water (50 ml), heated to 45 ° C, and vacuum is carefully applied to remove the air. It is then stirred for a few minutes, which results in a viscous solution, sometimes very viscous. Viscosity can be reduced by placing the solution in an ultravibrator for 5 minutes. The lipopolysaccharide solution is centrifuged once at high speed (100,000 x g, 4 h). The resulting sediment is clear and transparent, so that sometimes it is difficult to recognize until the supernatant is decanted. The lipopolysaccharide is redissolved in water and lyophilized.

Table 5: Defined Basal Medium (DM) for the characterization of the isolate Table 5: Defined Basal Medium (DM) for the characterization of the isolate (CONTINUED) Table 5: Defined Basal Medium (DM) for the characterization of the isolate (Continued) EXAMPLE 6: TRANSFORMATION OF THE STRAIN WITH A VECTOR The bacterial host strain ADM X6L was transformed by electroporation with the plasmid vector pMF1014-a, which comprises the pSRl-a replicon and a kanamycin resistance determinant. The plasmid pMF1014-a was then re-isolated from the resulting ADM X6L transformant and subsequently used to transform an E. coli host. The example demonstrates the transformation of the ADM strains with a vector, the selection of transformants by expression of kanamycin resistance in the ADM host, maintenance of the plasmid as an extrachromosomal element in the ADM host, and the use of pMF1014-a as a launch vector of the new E. Co i / ADM strain. In the ADM of plasmid pMF1014- (MT Follettie, "DNA technology for Corynebacterium glutamicum: isolation and characterization of amino acid biosynthetic genes", Ph.D. Dissertation, MIT, USA, 1989) DNA was isolated using the materials and procedures provided in the "Wizard Plus Midipreps" DNA Purification System (Promega), of a 50 ml overnight culture of E. coli DH5aMCR / pMFl014-a grown in Luria broth (1% Tripcox Difco, 0.5% Difco Yeast Extract, 0.5% NaCl) with 50 μg / ml kanamycin sulfate.

To prepare competent ADM X6L host cells, a single ADM X6L colony was inoculated in 10 ml of X6L Medium (1% Difco Soytone, 1% Difco Yeast Extract, 0.5% Difco Malt Extract, 0.5% of NaCl, 0.25% of K2HP04, 2% of mannitol, pH 7.8) and stirred at 300 rpm, 30 ° C, until 0.8 absorption units were reached at a D? 6oo-In the case of ADM guests 62A- 12A, 266-13B, and 291-19, X6L Medium without NaCl was used. Five ml of this culture was transferred to 500 ml of fresh X6L medium in a 2 L baffled flask, which was incubated with shaking at 300 rpm, 30 ° C, for a sufficient time to reach 1.0 absorption units at a time. DOÉOO • The mature crop was cooled rapidly, and a temperature of 2-4 ° C was maintained during the subsequent steps. The cells were harvested by centrifugation and washed at the resuspension sites with 500 ml of ice-cooled water followed by recentrifugation. The sediment from the second wash was suspended in 40 ml of 10% glycerol cooled on ice, mixed, and recentrifuged. The volume of this sediment was estimated, and the sediment was suspended in an equivalent volume of 10% glycerol cooled with ice. The suspension of competent cells with the resulting transformation was divided into aliquots in microcentrifuge tubes, 40 μL per tube, and stored at -80 ° C.

Two μL of a cold solution containing 140 μg / ml of purified pMF104-a DNA in water was added to 40 μL of cold competent ADM X6L cells, and mixed. The cell-DNA mixture was transferred to a pre-cooled electroporation cuvette (1 mm cuvette, Catalog No. 940-00-100-5, Eppendorf Scientific, Inc.), quickly transferred to an electroporation device "BioRad Gene Pulser II "and was boosted at 1.5 kV, 25 μF, 200 ohms. Immediately after the pulse, 1 ml of X6L medium was added at room temperature to the boosted cells, and the mixture was transferred to a sterile 10 ml test tube and incubated with shaking at 300 rpm, 30 ° C. After two hours of incubation to allow expression of kanamycin resistance, 1.04 ml of cell suspension was removed and microcentrifuged for 2 minutes at 14,000 rpm. 0.9 ml of supernatant was removed, the cell pellet was suspended in the remaining supernatant, and the cell suspension was dispersed on a petri dish of Medium X6L containing 20 μg / ml kanamycin and 1.3% Bacto Agar from Difco. The plate was incubated for 2 days at 30 ° C. Twenty ADM X6L transformant colonies resistant to kanamycin were obtained by this procedure. The X6L transformants maintained the plasmid pMF1014-a as an extrachromosomal element. To demonstrate this, the plasmid DNA was isolated from the X6L transformants using the procedure set forth above for E. coli, except that the transformed X6L cells were grown in X6L Medium containing 40 μg / ml kanamycin. The plasmid DNA isolated from the X6L transformants had the same size as the original pMF1014-a plasmid, as demonstrated by agarose gel electrophoresis. The plasmid isolated from the X6L transformant still had the kanamycin resistance gene and E. coli reproduction determinants. To demonstrate this, competent E. coli cells were prepared by the method of Letterberg and Cohen (J. Bact. 119: 1072-1074, 1964), and transformed with plasmid DNA from the X6L transformants using the D.A. Morrison (J. Bact. 132: 349-351, 1977). The E. coli cells transformed by this method acquired resistance to kanamycin, and showed the presence of a plasmid having the same size as the original plasmid pMF1014-a. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (2)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. A biologically pure culture of a microorganism strain, characterized in that it comprises identification characteristics of the strain selected from the group consisting of NRRL B-30035 (ADM 291-19), NRRL B-30037N (ADM 62A-12A), and NRRL B-30036 (ADM 266-13B) or a mutant thereof derived from the strain. 2. The biologically pure culture according to claim 1, characterized in that it comprises the microorganism strain NRRL B-30035 (ADM 291-19) or a mutant thereof derived from the strain. 3. The biologically pure culture according to claim 1, characterized in that it comprises the microorganism strain NRRL B-30037N (ADM 62A-12A) or a mutant thereof derived from the strain. 4. The biologically pure culture according to claim 1, characterized in that it comprises the microorganism strain NRRL B-30036 (ADM 266-13B) or a mutant thereof derived from the strain. 5. A method for the production of 2-keto-L-gulonic acid, characterized in that it comprises cultivating the strain according to claim 1 in a medium comprising L-sorbose for a sufficient time for the L-sorbose to be converted to 2-keto-L-gulonic acid; and recover 2-keto-L-gulonic acid. 6. The method according to claim 5, characterized in that it also comprises converting the acid
2. 2-keto-L-gulónico to ascorbic acid or a salt thereof. The method according to claim 6, characterized in that the strain is capable of producing at least about 40 g / L of 2-keto-L-gulonic acid to ascorbic acid or to a salt thereof. The method according to claim 5, characterized in that the cultivation is carried out at a pH of about 6.0 to 8.0 9. The method according to claim 5, characterized in that the cultivation is carried out at a temperature of approximately 22 ° C. up to about 35 ° C. The method according to claim 5, characterized in that the microorganism is cultivated in pure culture. The method according to claim 5, characterized in that the microorganism strain is cultured in a mixed culture comprising at least one additional microorganism strain. 12. The method according to claim 11, characterized in that the additional microorganism strain is a member of a genus selected from the group consisting of Aureoba cterium, Corybacterium, Ba cillus, Brevibacterium, Pseudomonas, Proteus, In terobacter, Ci troba cter, Erwinia, Xan thomonas and Flavobacteri um. The method according to claim 5, characterized in that the L-sorbose is generated by fermentative conversion of D-sorbitol. 14. The method according to the claim 13, characterized in that L-sorbose is generated by the fermentative conversion of D-sorbitol using Gluconobacter oxydans. 15. The method according to claim 14, characterized in that the Gl uconobacter oxydans of the strain ATCC 621 or IFO strain 3293 or a mutant thereof. 16. A method for the production of 2-keto-L-gulonic acid, characterized in that it comprises culturing the strain according to claim 1 in culture mixed with a microorganism strain capable of converting D-sorbitol to L-sorbose in a medium containing D-sorbitol, for a sufficient time for the D-sorbitol to be converted to 2-keto-L-gulonic acid; and recover 2-keto-L-gulonic acid. 17. The method according to claim 16, characterized in that the additional microorganism strain is a member of the genus Gluconobacter or Acetobacter. 18. The method according to claim 17, characterized in that the additional microorganism strain is either Gluconobacter oxydans ATCC 621 or Gluconoba cter oxydans IFO 3293 or mutants thereof. 19. A method for isolating PQQ, characterized in that it comprises: cultivating the strain according to claim 1 in culture medium comprising glucose, sorbose, glycerol, mannitol, sorbitol or inositol; and recover the PQQ. 20. A method for isolating PQQ, characterized in that it comprises: culturing the microorganism strain NRRL B-21627 or a mutant thereof in culture medium comprising glucose, sorbose, glycerol, mannitol, sorbitol or inositol; and recover the PQQ. 21. The method according to the claim 19 or claim 20, characterized in that the microorganism strain or mutant thereof is cultured in a mixed culture comprising at least one additional strain. 22. A method for isolating a non-toxic lipopolysaccharide, characterized in that it comprises culturing the strain according to claim 1 in a medium comprising glycerol, glucose, fructose, mannitol, sorbitol or inositol and recovering the lipopolysaccharide. 23. A method for isolating a non-toxic lipopolysaccharide, characterized in that it comprises culturing the microorganism strain KRRL B-21627 in medium comprising glycerol, glucose, fructose, mannitol, sorbitol or inositol and recovering the lipopolysaccharide. 24. The culture according to claim 1, characterized in that the strain comprises a vector. 25. The culture according to claim 24, characterized in that the vector is pMF 1014-a. 26. A biologically pure culture of microorganism strain MRRL-B-21627 or a mutant thereof, characterized in that it comprises pMF 1014-a. 27. The culture according to claim 24, characterized in that the vector comprises a marker gene. 28. Cultivation in accordance with the claim 27, characterized in that the marker gene comprises a nucleotide sequence that operates to direct the synthesis of a protein that confers resistance to an antibiotic in a host cell. 29. Cultivation in accordance with the claim 28, characterized in that resistance to an antibiotic comprises resistance to ampicillin, chloramphenicol, erythromycin, kanamycin, spectinomycin, streptomycin and tetracycline. 30. The culture according to claim 24, characterized in that the vector comprises (a) an exogenous terminator of transcription; (b) an exogenous promoter; and (c) a discrete series of restriction endonuclease recognition sites, the series is between the promoter and the terminator. 31. A method for transforming the strain according to claim 1, characterized in that it comprises inserting a vector into the strain.