MXPA99002383A - Deficient mutants in l-sorbosa reductase modified geneticame - Google Patents

Abstract

A genetically modified microorganism derived from a microorganism belonging to the genus Gluconobacter or Acetobacter that is characterized because its biological activity to reduce L-sorbose is substantially annulled by recombination of a gene of the same type.

Description

DYNAMIC MOTTORS IN - SCRBOSA REDOCTASE GEITHICALLY MODIFIED Description of the invention: The present invention relates to new genetically modified L-sorbose reductase-deficient mutants of a microorganism belonging to the genus Gluconobacter or Acetobacter which have advantages in their use for the production of L-sorbose by fermentation, as well as an improvement in the production process of vitamin C. The production of vitamin C has been carried out by means of the Reichstein method, which involves a fermentation process for the conversion of D-sorbitol to L-sorbose by means of a microorganism belonging to the genus Gl uconobacter or Acetobacter, as a single biological stage. Said conversion to L-sorbose is one of the key stages for the efficiency in the production of vitamin C. It has been observed, however, that the product, L-sorbose, is consumed after the consumption of the substrate, D-sorbitol. , during oxidative fermentation with said microorganism. The explanation for this phenomenon has been that L-sorbose is reduced by NADPH-dependent L-sorbose reductase (hereinafter occasionally referred to as SR) present in the cytosol (see: Sugisawa et al., Agrie. Biol. Chem 55: 2043-2049, 1991). It has been described that, in Gluconobacter, D-sorbitol is converted to D-fructose, which can be incorporated into the pentose pathway and subsequently metabolized to C02 (Shinjoh et al., Agrie. Biol. Chem., 54: 2257-2263, 1990). Said pathways that consume the product as well as the substrate can ultimately cause lower vitamin C productivity. filed by Nogami et al., in Japanese Patent Application Kodai No. 51054/1995. The authors subject the microorganisms of Gl uconobacter oxydans and Gl uconobacter su-boxydans to conventional chemical mutagenesis and isolate the mutant strains whose capacity to use D-sorbitol as the only assimilable carbon source is reduced. By applying said mutant to the fermentation for the production of L-sorbose, they observe an improvement of more than 2-3% in productivity compared to the productivity of the parental strain. One of the disadvantages often observed in mutant strains produced by conventional mutagenesis is backmowing which overrides the improved characteristics of the mutant strains during the course of the fermentation or subculture of the mutant, which ultimately results in a decrease of vitamin C productivity. Accordingly, a stable blocked mutant for L-sorbose reductase is desirable. In one aspect, the present invention provides a novel genetically modified microorganism derived from a microorganism belonging to the genus Gluconobacter or Acetobacter which is characterized in that the biological activity thereof to reduce L-sorbose is substantially abolished by genetic engineering. And preferably it possesses said biological activity at 10% less than the original microorganism; said activity is from 0.07 to 0.02 units per mg of protein or less according to the definition of activity described below (for example, in Example 1 (iii)). The gene of the genetically modified microorganism of the present invention can be a carrier of at least one mutation obtained by interrupting, adding, inserting, deleting and / or replacing a nucleotide or nucleotides within the region required for the formation of L-sorbose. active reductase in cells of the microorganism. In a preferred embodiment of the genetically modified microorganism of the present invention, said mutation can be caused by gene disruption within the region required for the formation of active L-sorbose reductase. Said interruption may contain at least one fragment of interfering DNA selected from the group consisting of a transposon, a cassette of antibiotic resistance gene and any DNA sequence that does not allow the host microorganism to form active L-sorbose reductase. In another embodiment of the present invention, said mutation can be produced by mutagenesis with the help of site-specific mutagenesis. Said mutagenesis can be carried out in the region required for the formation of active L-sorbose reductase, which region can include a structural gene for L-sorbose reductase and expression control sequences, such as a promoter, an operator, ternate, repressor encoded by DNA, activator and the like. Another aspect of the present invention provides the use of an L-sorbose reductase gene of a microorganism belonging to the genus Gluconobacter or Acetobacter in the production of the genetically modified microorganism described above, wherein said gene is characterized in that it encodes the amino acid sequence of the gene. L-sorbose reductase described in SEQ ID No. 2 or its functional equivalents containing insertions, deletions, additions and / or substitutions of one or more amino acids in said SEQ ID No. 2. Another aspect of the present invention provides an efficient method for producing L-sorbose reductase by fermentation of a microorganism in a suitable medium, comprising the use of the genetically modified microorganisms of the present invention described above. In connection with this production method of L-sorbose re-ductase, the present invention also provides an efficient process for the production of vitamin C containing a fermentation step for the production of L-sorbose reductase which is characterized in that said fermentation it is carried out using the genetically modified microorganism of the present invention as described above. The present invention provides a new genetically modified microorganism derived from a microorganism belonging to a microorganism belonging to the genus Gluconobacter or Acetobacter, characterized in that the biological activity thereof for the reduction of L-sorbose is substantially canceled by genetic engineering .

Such a genetically engineered microorganism may be a microorganism belonging to the genus Gluconobacter or Acetobacter, which includes Gluconobacter albi-dus, Gluconobacter capsulatus, Gluconobacter cerinus, Glucosebacter di oxyacetonicus, Gluconobacter gl uconi cus, Gluconobacter industri us, Gl uconobacter melanogenus (IFO 3293 and FERM P-8386 [National Institute of Bioscience and Human Technology, Japan]), Gl uconobacter nonoxygl uconi cus, Gluconobacter oxydans, Gluconobacter oxydans subsp. sphaericus, Gairtn ± acter roseus, Gl uconobacter rubiginosus, Gl uconobacter suboxydans (IFO 3291), Acetobacter xylinum [commercially available from the Institute of Fermentation, Osaka, Japan, (IFO) as IFO 3288], Acetobacter pasteurianus, Acetobacter aceti, Acetobacter hansenii, and Acetobacter liguefaci ens (IFO 12388; ATCC 14835). For information on the strains see also European Patent Application Publ. No. (EPA) 213591 and 518136. Gluconobacter suboxydans IFO 3291 has been deposited in the form of a mixture with Gluconobacter oxydans DSM4025 as FERM BP-3813 and Gl uconobacter melanogenus IFO 3293 as FERM BP-8256. Additional details may be known from EP 518136. For Gluconobacter suboxydans (IFO 3291) and Gluconobacter melanogenus (IFO 3293) see also EP 728 840 (US Patent Application No. 08/606 807). In order to nullify the biological activity of said microorganism in terms of the ability to reduce L-sorbose, the present invention involves genetic engineering to identify the region of the microorganism gene required for the formation of active L-sorbose reductase.

Typical methodologies for this purpose are known as gene disruption with transposons or cassette gene selection marker and site-specific mutagenesis. As described below, the method of gene disruption is useful for identifying the target gene of the microorganism, as well as for blocking gene function. Once the mentioned target region of the gene has been identified, conventional mutagenesis can also be applied by treating the target DNA fragment with for example a chemical mutagen, ultraviolet radiation and the like. Gene disruption can be carried out by introducing an interfering DNA fragment into the chromosomal DNA of the microorganism with the aid of transposon mutagenesis, introduction of a gene cassette that carries a selection marker, such as a gene. of antibiotic resistance, DNA sequences that abolish the formation of active L-sorbose reductase, (a) Transposon mutagenesis: Transposon mutagenesis is known as a powerful tool for genetic analysis (P. Gerhardt et al., "Methods for General and Molecular Bacteriology ", Chapter 17, Transposon Mutagenesis, American Society for Microbiology). This method uses transposable elements that are defined segments of DNA that possess the unique ability to move (transpose) to new sites within the genome of host organisms. The transposition process is independent of the classical homologous recombination system of the organism. The insertion of a transposable element into the genome at a new genomic site does not require extensive DNA homology between the ends of the element and its target site. Transpomble elements have been identified in a wide variety of prokaryotic and eukaryotic organisms, in which they can cause mutation mutations, chromosomal rearrangements, and new patterns of expression or genetic insertion in the coding region or regulatory sequences of resident genes and operons. Prokaryotic transposable elements can be roughly divided into three different classes. Class I consists of simple elements such as insertion sequences (IS elements), which have a length of approximately 800 to 1500 bp. IS elements usually consist of a gene that encodes an enzyme required for transposition (this is, transposase), flanked by DNA sequences with terminal repeats that serve as a substrate for the transposase. The IS elements were initially identified in the lactose and galactose use operons of enteric bacteria, in which the elements were found to cause mutations or polar insertions that are often unstable. Class II consists of co-located transposable elements. Members of this class are also referred to as transposons or Tn elements. Transposons in prokaryotes have been identified as a class of complex transposable elements, often containing simple IS elements (or parts thereof) as direct or inverted repeats at their ends, behaving formally as IS elements but carrying additional genes not related to transposition functions, such as antibiotic resistance genes, heavy metal resistance or pathogenicity determining genes. The insertion of a transposon into a genetic locus or replicon (phage) in particular is termed by the use of two-point aobles, for example lacZ:: Tn5 or? :: Tn5. Class III includes "transposable" bacteriophages such as Mu and its derivatives. Mu phage is both a virus and a transposon. It is known that it can be integrated into multiple sites in the host chromosome, thereby frequently causing mutations. Transposon mutagenesis using the transposable elements described has the following characteristics: (i) Said mutation generally leads to inactivation of the gene, and the resulting null mutation is relatively stable. (iL) Transposons introduce new genetic and physical markers at the target locus, such as antibiotic resistance genes, new endonuclease restriction sites, and unique DNA sequences that can be identified by genetic means, DNA-DNA hybridization or genetic analysis. heteroduplexes by electron microscopy. Genetic markers are useful for the mapping of mutated loci as well as for the scrutiny of mutants. (iü) Transposons can generate a variety of genomic rearrangements, such as deletions, inversions, translocations or duplications, and can be used to introduce specific genes into target bacteria. Various transposons are known in the field, such as Tn3, Tn5, Tn7, Tn9, TnlO, Mu phage and the like. Among them, Tn5 is known to have almost no insertion specificity, and is relatively small in size. Tn5 is also one of the most frequently used transposable elements derived directly from sources, such as pfd-Tn5 [American Type Culture Collection, USA]. (ATCC) ATCC 77330) or pCHRdl (ATCC 37535)]. For the purpose of their use in random mutagenesis in the practice of the present invention, Tn5 is preferred. A variety of Tn5 derivatives, termed mini-Tn5s, which consist of 19 bp of the inverted terminal repeats required for transposition coupled to resistance to antibiotics or other selectable marker genes are also useful for the present invention. Such mini-Tn5s are inserted into a suicide vector, in addition to the Tn5 transposase (tnp), to construct an effective suicide Tn5 mutagenesis system. Additional information on working methods with Tn5 transposons can be obtained from the following references: P. Gerhardt et al., "Methods for General and Molecular Bacteriology", Chapter 17, Transposon Mutagenesis: American Society for Microbiology, 1994; K.N. Timmis et al., "Mini-Tn5 transpo-are derivatives for insertion mutagenesis, pro-oter pro-bing, and chromosomal insertion of cloned DNA in Gra -negative Eubacteria", J. Bacteriology, 172: 6568-6572, 1990. The information which describes how Tn5 can be derived from pdf-Tn5 (see explanation on pfd-Tn5 (ATCC 77330)) can be obtained from the ATCC home page on the Internet http://www.atcc.org / catalog / recomb.html. According to these data, the suicide plasmid pfd-Tn5 can be introduced into E. coli as well as other Gram-negative bacteria by electroporation (see references for the recommended conditions). The plasmid itself can be used as a Tn5 donor. Additionally, a vector Tn5 can be constructed by introducing a pfd-Tn5 plasmid into the recipient E. coli with any plasmid that is to be used, selecting transformants that show Km 'r and resistance of the target plasmid, isolating plasmids from the transformants, transforming E. coli with the isolated plasmids, and selecting the transformants for Km resistance and plasmid markers to obtain an E. coli strain carrying the target plasmid with Tn5. The fundamental idea of this protocol is also available in "region-directed mutagenesis by Tn5", in P. Gerhardt et al. (Previously mentioned). Random mutagenesis with transposons involves the introduction of a transposon into a bacterial target cell by transformation, transduction, mating by conjugation or electroporation using suicide plasmids or phage vectors. The resulting mutants can be screened with the aid of the marker transported in the transposon. The transposition of the transposon into the genome of the recipient bacterium can be detected after the loss of the vector used by segregation. For the introduction of transposons into microorganisms of the genus Gluconobacter or Acetobacter, so-called suicide vectors are commonly used, including a derivative of phage Pl and plasmids of narrow host range. The phage vectors Pl and the plasmid vectors can be transferred by infection and transformation, conjugation pairing or electroporation, as appropriate, into the recipient cells, in which these vectors preferably lack the suitable origins for the receptors. The choice of a suicide vector and a transposon to be used depends on various criteria including phage sensitivity, the intrinsic resistance to antibiotics of the recipient cell, the availability of a gene transfer system including transformation, conjugation pairing, electroporation or infection to introduce the transposon carrier vector into E. coli. One of the preferable vectors for use in the present invention is phage Pl (ATCC 25404) which injects its DNA into a microorganism belonging to the genus Gluconobacter or Acetobacter, although this DNA is incapable of replication and will be lost by segregation. Said phage Pl of Tn5 (Pl :: Tn5) can be used in the form of a phage lysate which can be prepared by lysing carrier E. coli of Pl :: Tn5 in accordance with known procedures (see for example "Methods for General and Molecular Bac"). -tepology "Chapter 17, Transposon Mutagenesis, American Society for Microbiology or US Patent 5082785, 1992). The other preferable suicide vectors that may be used in the present invention are suicide plasmid vectors, which may be based on replicons derived from plasmid RP4 or its relative RK2 (ATCC 37125), carriers of the same transfer by conjugation of broad host spectrum of sites and mobilization functions but with a narrow range of host in terms of origin of replication. These vectors can be mobilized at a high frequency from E. coli to a microorganism belonging to the genus Gluconobacter or Acetobacter, but can not be stably maintained in the recipient cells. These vectors contain, in addition to Tn5, the mobilization site of type IncP (mob; op T) and are based on cloning vectors customary in E. coli, such as for example pACYC177 (ATCC 37031), pACYC184 (ATCC 37033) and pBR325 (Bolivar F., 1978, Gene 4: 121-136; pBR322 (ATCC 31344)), none of which can replicate in non-enteric bacteria (pSUP series, Simon R. et al., 1983, Bio / Technology 1: 784-791). Plasmids of the pSUP type can be mobilized by biparental pairing experiments by providing the trans transference function from an integrated copy on the chromosome of the IncP RP4 plasmid in the donor strain itself (eg strain S17-1) or in experiments of tri-parental mating providing the transfer functions from plasmid pRK2013 (ATCC 37159) vehicled by a non-donor helper non-donor E. coli strain. The receptor cell that will receive the transposon can be selected by the marker carried by the element, for example resistance to certain antibiotics. When Tn5 is used as a transposon, the Kmr or Nmr r ~ marker can be used normally. In addition to the Km & or Nmr, genetic markers of Tcr Gm, Spr, Apr, Cpf and the like can be used as alternative marker genes. Other carrier transposons of directly viewable gene products such as those encoded by lacZ, l uxAB or phoA can also be used. These Tn5 derivatives are especially useful if the target bacterial strain possesses an intrinsic resistance to the antibiotics normally used to select for Tn5 (kanamicin, neomycin, bleomycin and streptomycin) or if a secondary mutagenesis of a carrier strain must be carried out. of a derivative of Tn5 (P. Gerhardt et al., "Methods for General and Molecular Bacteriology", Chapter 17, Transposon Mutagenesis, American Society for Microbiology). (b) Region-specific mutagenesis A potent extension of the Tn5 mutagenesis protocol involves the use of gene replacement techniques to replace the wild-type gene in the bacterial strain ori-gmal with a well-characterized Tn5-mutated analogue vehicled by a plasmid in E. coli. When a wild-type locus has been cloned, region-specific mutagenesis with Tn5 or its derivatives or gene cassettes carrying selection markers can be used efficiently to inactivate the genes and target operons vehicuiados per the cloned region. For this purpose, the Tn5 itself or its derivatives mentioned above or any gene cassette with selectable markers can be used, for example Kmr gene cassette vehiculated by pUC4K (Pharmacia, Uppsala, Sweden). This approach is very efficient when it is desired to inactivate the gene of interest, for example the gene of a mutant strain developed from the parental strain from which it was cloned at the time of the wild-type gene. The only limitation in these types of replacement experiments is the length of the homologous sequences necessary for the double cross-recombination event; homologous sequences of 0.5 kb to 5 kb in length are preferably required at both ends. The preferred vectors for region-specific mutagenesis are the same as those useful for transposon mutagenesis: the aforementioned plasmid and phage vectors. (c) Site-specific mutagenesis When a wild-type gene is cloned and its nucleotide sequence is determined, site-specific mutagenesis can also be used to inactivate the target genes. Oligonucleotide primers including nucleotide addition and deletion are used for reading frame shift, or nucleotide substitution for the introduction of different stop codons or codons for the mutation of the genes of interest. Mutagenesis with primers that include mutations can usually be carried out in E. coli with any commercial site-specific mutagenesis kit. This type of mutagenesis is useful if mutagen-sis driven by Tn5 or by region-specific cassette causes a polar effect affecting downstream or upstream gene expression. The preferred vectors for the introduction of the mutated gene into the target microorganism are the same as those useful for transposon mutagenesis. In this case, the desired mutant can be assayed by biological assay, for example enzymatic or immunological screening to detect deficiency in the target gene proauct. Instead, the mutated gene can be labeled with a selectable marker gene described above at the point where it does not affect gene expression downstream or upstream to facilitate selection of gene replacement. The mutant with the interrupted target gene can be obtained more easily by the combination of biological assay plus marker selection. The mutant deficient in L-sorbose reductase can generally be selected as follows: 3,000 to 10,000 mutants are transposon-bound to an L-sorbose product assay as a substrate to select the mutant that does not convert L-sorbose to D-sorbitol. The first screen can preferably be carried out in microtitre plates with the reaction mixture containing L-sorbose. The formation of the product, D-sorbitol, is detected first by TLC with a suitable development solvent; Candidates are selected that form an undetectable amount of D-sorbitol. Next, the candidate mutants are subjected to an L-sorbose reductase activity assay as exemplified in Example 1 of the present invention to confirm the deficiency in L-sorbose reductase. To confirm that the deficient mutant actually carries a transposon, colony or Southern-type hybridization is usually performed with a labeled DNA fragment containing the transposon used as the probe by standard methods (Molecular Cloning, a Laboratory Manual, second edition, Maniatis, T. et al., 1989). A mutant of this type was isolated as described in Example 1 of the present invention. The transposon mutant is useful to additionally identify the target gene of L-sorbose reductase and to determine the nucleotide sequence of the region marked with the transposon. The DNA fragment inserted by a transposon can be cloned into any cloning vector of E. coli, preferably pUCld, pUC19, pBluescript II (Stratagene Cloning Systems, CA, USA) and its derivatives, selecting the transformants that show both phenotypes of the selection markers of both the vector and the transposon. The nucleotide sequences adjacent to the transposon can be determined for example by a chain termination method (Sanger F.S. et al., Proc. Nati, Acad. Sci. USA, 74: 5463-5467, 1977). The resulting nucleotide sequences may be partial sequences whose reading frame may be difficult to determine. Once the nucleotide sequences have been determined, they can be subjected to a homology search performed on the databases of protein and / or nucleotide sequences using genetic analysis programs, for example BLASTP search (Lip an et al., J. Mol. Biol., 215: 403-410, 1990). If homologous sequences are found, their amino acid sequences can be aligned to find consensus sequences that are conserved among the homologous proteins. Consistent with the consensus sequences, oligonucleotide primers can be synthesized and used to amplify the partial DNA of the target gene by polymerase chain reaction (PCR). In addition to the consensus sequences, any amino acid sequence, which can be determined after adjusting the reading frame by alignment of the homologous proteins, can be used to design the PCR primers. The partial gene resulting from the PCR can be used as a probe to obtain the complete target gene through Southern and colony hybridization. Southern hybridization reveals the size of the DNA fragment containing the target gene and a minigenes library containing DNA fragments of suitable size can be constructed. The mini-library can be subsequently selected with the partial gene as a probe by hybridization in colony to obtain the complete target gene. The complete nuclectic sequence of the target gene can be determined to identify its open reading frame. The region inserted by a transposon may be a regulatory gene that controls the expression of the structural gene of L-sorbose reductase; the regulatory gene may also be the target to interrupt the L-sorbose reductase gene. The cloned DNA fragment containing the complete or partial L-sorbose reductase gene of the target microorganism can be used to interrupt the L-sorbose reductase gene of the target microorganism. In Figs. 4 and 5 a schematic procedure and mechanism for interruption are presented, respectively. The DNA fragment is first cloned into an E. coli vector such as pBluescppt II SK. Next, a genetic cassette carrying a selection marker such as a Kmr gene is inserted into the target L-sorbose reductase gene to prevent it from forming active L-sorbose reductase. The fragment of resulting DNA with the interrupted L-sorbose reductase gene is recloned into a suicide vector such as pSUP202. The suicide carrier plasmid of the interrupted gene can be mutated in the recipient microorganism by any method of gene transfer including conjugation matching as described above. The selection of the target mutant generated by a double cross-linked recombination event can be carried out by isolating the colonies expressing the selection marker gene (for example Kmr) and characterizing their chromosomal DNA by blotting on Southern blotting. The L-sorbose reductase deficiency of the candidate mutant is confirmed to prove that it does not show detectable L-sorbose reductase enzymatic activity. A mutant of this type was isolated as described in Example 5 of the present invention, being called strain SR3. The non-assimilation of L-sorbose can be examined using any medium containing 1-500 g / 1 of D-sorbitol for fermentation of L-sorbose by measuring the concentration of L-sorbose once it has been converted from D-sorbitol into the means of fermentation. Instead, the medium containing L-sorbose can be used to confirm the non-assimilation of L-sorbose ba or the fermentation conditions. The mutants provided by the present invention can be cultured in an aqueous medium supplemented with suitable nutrients under aerobic conditions. The culture can be carried out at a pH between about 3.0 and 9.0, preferably between about 5.0 and 8.0. Although the time of cultivation varies depending on the pH, temperature and nutrient medium used, usually from 1 to 6 days will yield favorable results. A preferred temperature range for carrying out the culture is from about 13 ° C to 45 ° C, preferably from about 18 ° C to 42 ° C.

The culture medium is usually required to contain nutrients such as assimilable carbon sources, digestible nitrogen sources and inorganic substances, vitamins, trace elements and the rest of the growth promotion factors. Glycerol, D-glucose, D-mannitol, D-fructose, D-arabitol, D-sorbitol, L-sorbose and the like can be used as assimilable carbon sources. Various organic or inorganic substances can also be used as nitrogen sources, such as yeast extract, meat extract, peptone, casein, corn liquor, urea, amino acids, nitrates, ammonium salts and the like. Magnesium sulfate, potassium phosphate, ferrous and ferric chlorides, calcium carbonate and the like can be used as inorganic substances. The mutant deficient in L-sorbose reductase of the present invention can be used for the fermentative oxidation of D-sorbitol, and it is expected to improve the yield in the production of vitamin C by increasing the L-sorbose usable for the condensation reaction stage. - L-sorbose to produce diacetone-L-sorbose. As will be apparent to those skilled in the art, the mutant deficient in L-sorbose reductase of the present invention may be applied to any process for the production of vitamin C that includes L-sorbose as a reaction medium. The present invention is further illustrated with the Examples described below with reference to the accompanying drawings, which contain what is described below: Figure 1 shows the nucleotide sequences upstream and downstream of the Tn5-inserted region of the DNA Chromosome of the mutant deficient in L-sorbose reductase, 26-9A derived from G. melanogenus IFO 3293. Figure 2 shows the oligonucleotide primers used for PCR cloning of the SR gene of G. suboxydans IFO 3291. Figure 3 illustrates the map restriction fragment of 8.0 kb EcoRV fragment carrying the gene of L-sorbose reductase of G. suboxydans IFO 3291 (upper) and its increased region near the L-sorbose reductase (lower) gene showing the ORFs that can find Figure 4 is a schematic for the construction of a suicide plasmid for the interruption of the L-sorbose reductase gene in G. suboxydans IFO 3291. Figure 5 illustrates a schematic mechanism for the interruption of the L-sorbose reductase gene in G. suboxydans IFO 3291. Figure 6 shows the graphs showing the fermentation profiles of strain SR3 and G. suboxydans IFO 3291 in sorbitol medium 8% no. 5. Figure 7 shows the graphs that account for the fermentation profiles of strain SR3 and G. suboxydans IFO 3291 in a sorbitol 2% - SCM medium.

Example 1 Isolation of a Tn5 mutant deficient in L-sorbose reductase from a derivative of G. melanosenus IFO 3293 (i) Mutagenesis by Tn5 Transposon mutagenesis was performed (Manning RF et al., USP 5082785) to construct a strain deficient in L-sorbose reductase from a strain L42-9 producing 2-keto-L-gulonic acid. Any strain that produces L-sorbose from D-sorbitol and assimilates L-sorbose by the L-sorbose reductase of the present invention can be used obtained from G. melanogenus IFO 3293 by multistage mutations with chemical mutagens including NTG and ICR170, ultraviolet irradiation, etc. Inoculated E. coli W3110 carrying Pl :: Tn5 maintained on LB agar plates containing 30 μg / ml kanamycin in 5 ml of Pl medium (LB supplemented with MgSO4 • 7 0.01 M H2O and 10 μg / ml thymine) ) and was grown at 30 ° C overnight. 1 ml of this culture was transferred to 100 ml of the same medium in a 500 ml Erlenmeyer flask and grown to an OD550 of about 0.09 at 30 ° C for 95 min. The resulting culture was chilled on ice for 10 min and centrifuged at 3500 rpm at 4 ° C for 20 min. The cells were suspended in 25 ml of Pl medium and the cell suspension was transferred to a 300 ml flask and incubated at 42 ° C for 20 min without agitation. When the lysis of the cells was observed keeping them at 37 ° C for 90 min, the cells were used adding 0.5 ml of chloroform. The mixture was vigorously vortexed, maintained at room temperature for 10 min, and centrifuged at 10,000 rpm at 4 ° C for 15 min. After transferring the supernatant to a sterile glass bottle with a screw cap, an additional 0.5 ml of chloroform was added again and the lysate was kept at 4 ° C. The recipient cell, L42-9 grown on an agar plate No. 4 consisting of 0.5% glycerol, 0.5% yeast extract (Difco) and MgSO4 • 7H20 0.5% was inoculated into a tube which contained 5 ml of medium No. 4 and was grown in a tube agitator at 30 ° C overnight. 1 ml of the culture was transferred to 30 ml of the same medium in a 500 ml Erlenmeyer flask and cultivated at 30 ° C for 3 h. The culture was centrifuged for 15 min. The resulting pellet was suspended in 1.8 ml of a 100 M MC buffer consisting of 100 mM MgSO4 • 7 H20 and 100 mM CaCl2. 0.1 ml of the cell suspension was mixed with 0.1 ml of the phage solution diluted 10 times with 10 mM MC buffer in the tube, and placed at 30 ° C for 60 min. After adding 0.8 ml of medium No. 4, the tube was incubated at room temperature for 2 h. The suspension of infected cells of 0.2 ml volume was spread on No. 4 medium agar plates containing 100 μg / ml of kanamycin, and incubated at 27 ° C for 5 days. (ii) Screening of mutants deficient in L-sorbose reductase by assay of the product Strains with Tn5 (Kp >r) grown on agar plates 4 km at 27 ° C for 4 days were suspended in 50 μl of citrate-Na buffer solution, 0.15M HP04 (pH 8.0) containing 4% L-sorbose. well of a 96-well microtiter plate and incubating at 30 ° C for 24 h without agitation. The D-sorbitol converted from L-sorbose was analyzed by thin layer chromatography (TLC) using 1 μl of the sample. TLC analysis was performed using a TLC plate of Kieselgel 60 F254 Merck; ethyl acetate / isopropyl alcohol / acetic acid / H20 = 10: 6: 3: 5: 3 solvent; and spray reagents of 0.5% solution of KIO4 and a tetrabase reagent prepared by mixing saturated 2N acetate with tetrabase and MnS04 • 4-6 H20 15% (1: 1 by volume). By TLC analysis, a mutant named 26-9A was obtained as a mutant candidate deficient in L-sorbose reductase. Strain 26-9A produced a small amount of D-sorbitol from L-sorbose, while the remainder of mutants Tn5 and L42-9 parental produced a significant amount of D-sorbitol. (iii) Determination of the enzymatic activity L-sorbose reductase. Cells grown in 8% sorbitol medium - No. 5 consisting of D-sorbitol 8%, yeast extract 1.5% (Oriental Yeast Co., Osaka, Japan), MgS04 • 7H20 0.25%, glycerol 0.05% and CaC03 1.5% (production grade) at 30 ° C for 2 days, and washed twice with 0.3% NaCl solution. The cell paste was suspended in 10 mM KH2P04-K2HP0 buffer (pH 7.0) and passed through a pressure homogenizer twice. After centrifugation to remove the intact cells, the supernatant was centrifuged at 100,000 x g for 60 min. The resulting supernatant was collected as a source of L-sorbose reductase. The activity of L-sorbose reductase was determined by photometric analysis in the presence of NADPH (T. Sugi-sawa et al., Agrie. Biol. Chem. 55: 2043-2049, 1991). The reaction mixture contained 5 mg / ml of L-sorbose, 0.4 mg / ml of NADPH in 50 mM of 10 mM KH2P04-K2HP04 buffer (pH 7.0) and 10 μl of enzyme solution. The change in absorption resulting from substrate dependent oxidation of NADPH was followed at 340 nm with a Kontron UVIKON 810 spectrophotometer. One unit of reductase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmole of NADP per minute. . The L-sorbose reductase activity of strain 26-9A and its parental L42-9 were determined as described above, and were found to be less than 0.01 and 0.21 units / mg protein, respectively. (iv) Colony hybridization The introduction of the Tn5 fragment into the chromosome of strain 26-9A was confirmed by colony blot hybridization with ColEl :: Tn5 DNA labeled with 2P as a probe.

Example 2 Cloning and Sequencing of Nucleotides from the Tn5 Inserted Region New Tn5 mutants were reconstructed using pSUP202 (Apr mrTcr; Simon R. et al., BIO / TECHNOL., 1: 784-791, 1983) with the DNA fragment containing Tn5 to confirm that the deficiency in L-sorbose reductase of 26-9A is caused by the insertion of Tn5, not by a different mutation that has occurred simultaneously in a position different from the DNA 26-9A with respect to the position of the insertion of Tn5. Southern blot hybridization of several DNA fragments of the strain 26-9A chromosome revealed that the 13 kb EcoRV fragment containing a complete Tn5 was of sufficient length (more than at least 1 kb) of DNA on both sides of the insertion of Tn5 for a recombination by double crossing. The EcoRV fragment was cloned into pSUP202 to produce pSR02, which was then introduced into G. melanogenus IFO 3293 to obtain strains KmrCms, 3293EV-1 and 3293EV-9. Analysis by Southern blot hybridization of strains 3293EV-1 and 3293EV-9 revealed that both strains contained Tn5 without the vector portion pSUP202 as well as strain 26-9A (data not shown), indicating that it had occurred a mutation by double crossing over. The deficiency in L-sorbose reductase activity in strains 3293EV-1 and 3293EV-9 was examined by product assay and by a photomictic enzymatic assay as performed for strain 26-9A; the new Tn5 mutants also produced undetectable levels of L-soroose and showed an L-sorbose reductase activiaad below 0.01 units / mg of cytosol protein, while G. melanogenus IFO 3293 showed an L-sorbose reductase activity. 0.20 units / mg of cytosol protein. It should be concluded that the insertion of Tn5 at 26-9A causes deficiency in L-sorbose reductase activity. The nucleotide sequence of the Tn5-inserted region of pSR02 was determined by the dideoxis chain termination method (Sanger et al., Proc. Nati, Acad. Sci. USA, 74: 5463-5467, 1977) (Fig. ). The analysis of the region inserted with Tn5 by homology search with the BLASTP program (Lipman DJ et al., J. Mol. Biol., 215; 403-410, 1990) revealed that the region encodes a poly-peptide that possesses a homology with polypeptides belonging to the family of mannitol dehydrogenase (MDH). One of the members, mannitol 2-dehydrogenase (EC 1.1.1.67) of Rhodobacter spnaeroides (Schneider K.-H. et al., J. Gen. Microbiol., 1993) catalyzes the NAD-dependent oxidation of mannitol to fructose. Gluconobacter L-sorbose reductase catalyzes not only the reduction of L-sorbose and D-fructose to produce D-sorbitol and D-mannitol in the presence of NADPH, but also the oxidation of D-sorbitol and D-mannitol to produce L -sorbose and D-fructose in the presence of NADP (Sugisawa et al., ibid.). Homology analyzes indicated that the polypeptide encoded by the interrupted Tn5 gene of strain 26-9A is itself an L-sorbose reductase gene, not its regulatory gene.

Example 3 PCR Cloning The partial gene of L-sorbose reductase from G. suboxydans IFO 3291 was cloned by PCR amplification using a group of primers synthesized in accordance with the amino acid sequences SEQ ID No. 3 and SEQ ID No. 4 shown in Figure 2; the degenerate primers were synthesized taking into account the use of Gl uconobacter codon. The PCR yielded a product of approximately 300 bp. It was obtained by analysis by Southern hybridization and in colony using this fragment amplified by PCR as probe the complete gene of L-sorbose reductase of G. suboxydans IFO 3291 in an EcoRV fragment of 8.0 kb. Example 4 Determination of the nucleotide sequence of the gene of L-sorbose reductase of G. subox-vdans IFO 3291 The complete nucleotide sequence of the L-sorbose reductase gene was determined from an EcoRV fragment of 8.0 kb containing the gene of L-sorbose reductase from G. suboxydans IFO 3291. The nucleotide sequence and the resulting amino acid sequence is shown in SEQ ID No. 1 and SEQ ID No. 2. The restriction map of the EcoRV fragment of 8, 0 kb is shown in Figure 3. The SR gene and two open reading frames, found downstream of the L-sorbose reductase gene and encoding polypeptides having homologies with the DnaJ-like protein, are in the opposite direction. and ferrodoxin. No relevant ORF was found upstream of the L-sorbose reductase gene that would likely form an operon with the L-sorbose reductase gene. It was therefore considered that the interruption of the L-sorbose reductase gene did not affect the expression of the adjacent genes. Example 5 Construction of a disrupted L-sorbose reductase gene from G. suboxydans IFO 3291 As can be seen from Figure 4, the plasmids pUC4K, pSR03-l and pSUP202 are the plasmids used as starting material. pUC4K is a source of a cassette of a Km resistance gene and is available from Pharmacia (Uppsala, Sweden, Code: 27-4958-01). Plasmid pSR03-l is a derivative of the commercially available vector pBluescript II SK (Stratagene, CA, USA).; Cat. No.-go: 212205 and 212206) carrier of the EcoRV fragment obtained in Example 3. The vector pSUP202 is a derivative of pBR325 carrying a fragment containing a mob site, and, as is well known, pBR325 is a derivative of pBR322. Although pBR325 itself does not appear to be commercially available, an alternative material, for example pBR322 (ATCC 31344), pACYC177 (ATCC 37031) or pACYC184 (ATCC 37033) is considered to be directly usable by persons skilled in the art, such as and as described on the pSUP series by Simon R., et al., Bio / Technology, 1983, 1: 784-791. The construction of a plasmid containing a mob site is also described by Simon R. et al. (Ibid.). Figure 4 shows the scheme for the construction of a vector directed to the L-sorbose reductase gene, pSUP202-SR:: Km. The 8.0 kb EcoRV fragment was cloned into the EcoRI site of the vector pBluescript SK (Ating-Mees MA, et al., Methods in Enzymology, 216: 483-95, Academic Press, London, 1992) to produce pSR03-l . A cassette with kanamycin resistance (Km cassette) was inserted into an EcoRI site in the cloned L-sorbose reductase gene to obtain pSR04-l. This L-sorbose reductase gene interrupted with the Km cassette was subcloned into a suicide vector pSUP202. The resulting pSR05-2, pSUP202-SR:: Km (Kmr), was then introduced into G. suboxydans IFO 3291 to obtain a null mutant of L-sorbose reductase (Kmr). The desired gene disruption was finally confirmed by Southern hybridization analysis. An eschematic mechanism for interruption is illustrated in Figure 5. In this way, a mutant deficient in L-sorbose reductase SR3 was obtained. The mutant directed to the L-sorbose reductase gene, SR3, and G. suboxydans IFO 3291 showed an L-sorbose reductase activity below 0.02 and 0.681 units / mg protein, respectively. EXAMPLE 6 Fermentation profile of SR3 strain in 8% sorbitol medium - No. 5 Growth and assimilation profiles of L-sorbose from strain SR3 and G. suboxydans IFO 3291 were evaluated with 8% sorbitol medium - No. 5 in a 500 ml Erlenmeyer flask (Fig. 6). Strain SR3 and G. suboxydans IFO 3291 were able to convert 80 g / L of D-sorbitol to L-sorbose in 24 h (data not shown). G. suooxydans IFO 3291 assimilated most of the converted L-sorbose in 48 h. In contrast, strain SR3 scarcely used L-sorbose until the third day; The remaining L-sorbose was more than 70 g / 1. Strain SR3 and G. subo-xydans IFO 3291 showed an OD600 (cell growth) of 6, 4 and 24 after 6 days, respectively. Example 7 Fermentation profile of the ce..c. "-a SR3 in the sorbitol medium 2% - SCM The growth and assimilation profiles of L-sorbose of the SR3 strain and G. suboxydans IFO 3291 evaluated with the sorbitol 2% - SCM medium in a 500 ml Erlenmeyer flask (Fig. 7). Strains SR3 and G. suboxydans IFO 3291 were able to convert 20 g / L of D-sorbitol to L-sorbose in 12 h. G. suboxydans IFO 3291 assimilated half of the converted L-sorbose in 23 h. In contrast, strain SR3 poorly used L-sorbose. Strain SR3 and G. suboxydans IFO 3291 showed an OD600 (cell growth) of 2.5 and 5.9 after 23 h, respectively.

List of sequences INFORMATION FOR SEQ ID NUM. NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1458 base pairs (3) TYPE: nucleic acid (C) STRING: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) ( iii) ORIGINAL SOURCE: ORGANISM: Gl uconobacter suboxydans CEPA: IFO 3291 (iv) CHARACTERISTICS: KEY CHARACTERISTICS: CDS POSITION: 1. . 1458 METHOD SEQUENCING: E ATGATC? CGC? CGAAACCC7 C ?? G7CTCTT CCCGCCGCTG TGCAGGCTCC GCCCTATG? C 60 ATCAATGGGA TCAAACCGGG GA rCGTGCAT TTTGGCGTGG GAAACTTCTT CCGGGCCCAT 120 GAGGCTTTC? CGTTGA? CA GATCCTCAAG GACGATCCG? ACTGGGGAAT CATCGGCGTT 180 GGTCTG? CGG GTAGCG? CAG GTC ?? AGAAG AAGGCCGAGG AATTCAAGAA GCAGGACTGC 240 CTCTTTTCCC TGACCGAAAC GGCTCCGTCC GGCAAGAGC? CGGTTCGTGT TATGGGCGCG 300 CTGAGGGATT ACCTTTTGGC TCCTGCCGAT CCGGAAGCCG TGCTGAAGCA TCTCGCTGAC 360 CCGGGAATCC GTATCGTTTC CATGACAATC ACGGAAGGCG GTTACAACAT TAACGAGACG 420 ACAGGTGAGT TCGATCTTGA GAACAAGGCG GTTCAGCAGG ATCTGAAGAC ACCCGAAACG 480 CCGTCCACAA TCTTTGGATA TGTTGTGGAA GGACTGCGCC GCCGCCGTGA CGCAGGTGGC 540 AAGGCCTTCA CGATCATGTC CTGCGATAAT CTGCGGCATA ACGGTAATGT CGCCCGCAAG 600 GCATTTCTGG GATACGCGAA GGCCCGTGAT CCGGAACTGG CCAAGTGGAT TGAAGAGAAC 660 GCGACGTTCC CAAATGGCAT GGTTGATCGC ATCACGCCGA CCGTTTCTGC TGACATTGCG 720 AAGAAGCTCA ACGAAGCCAG TGGCCTGCAC GACGACCTGC CGCTCGTTGC AGAAGACTTT 780 CATCAGTGGG TGCTGGAAGA CAGCTTTGCT GATGGCCGGC CTGCGCTGGA AAAGGCCGGA 840 GTGCAGTTCG TTGGGGATGT GACGGACTAC GAGCATGTAA AAATCCGCAT GCTGAATGCT 900 GGTCACATCA TGCTCTGCTT CCCGGCTGTT CTGGCAGGAT TTGAAAATGT CGATCATGCC 960 CTTGCTGATC CCGATCTACG GCGTATCCTC GAGAACTTCC TGAACAAAGA CGTCATCCCG 1020 ACCCTGAAGG CACCGCCGGG CATGACGCTG GAAGGCTATC GGGACAGCGT GATCAGCCGT 1080 TTCTCGAATC CGGCCATGGC GGATCAGACA TTGCGTATTT CCGGGGACGG GAGCTCGAAG 1140 ATCCAGGTCT TCTGGACGGA AACGGTCCGC AAGGCTTTTG AGGGCAA.GCG CGATCTGTCC 1200 CGCATTGCTT TTGGTATGGC ATCCTACCTG GAAATGCTGC GCGGTAAGG? TGAAACGGGT 1260 GGCACCT? CG AGCCATTCGA GCCGACTTTT GGTGACAACC ATAAGACTCT GGCCA? GGC? 1320 GATGATTTTG AGAGCGCGCT CAA.GCTGCCA GCGTTCGATG CCTGGCGCGA 7C7GGAGACG 13 SO TCCGGGCTGA ACAACAAGGT TGTGGAGCTT CGCAAGATTA TCCGCGAGAA. GGGCGTCAAG 1440 GCTGCCCTTC CGGCCTGA 1458 INFORMATION FOR SEQ ID No. NUM. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 485 residues (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) ORIGINAL SOURCE: ORGANISM: Gl uconobacter suboxydans CEPA: IFO 3291 (iv) CHARACTERISTICS: KEY FEATURES: mature peptide POSITION: 1.485 METHOD SEQUENCING: E Met Lie Thr His Glu Thr Leu Lys Ser Leu Pro Wing Gly Val Gln 10 15 Wing Pro Pro Tyr Asp lie Asn Gly lie Lys Pro Gly lie Val His 20 25 30 Phe Gly Val Gly Asn Phe Phe Arg Ala His Glu Ala Phe Tyr Val 35 40 45 Glu Gln lie Leu Lys Asp Asp Pro Asn Trp Gly lie lie Gly Val 50 55 60 Gly Leu Thr Gly Ser Asp Arg Ser Lys Lys Lys Wing Glu Glu Phe 65 70 75 Lys Lys Gln Asp Cys Leu Phe Ser Leu Thr Glu Thr Ala Pro Ser 80 85 90 Gly Lys Ser Thr Val Arg Val Met Gly Ala Leu Arg Asp Tyr Leu 95 100 105 Leu Ala Pro Ala Asp Pro Glu Ala Val Leu Lys His Leu Ala Asp 110 115 120 Pro Gly lie Arg He Val Ser Met Thr He Thr Glu Gly Gly Tyr 125 130 135 Asn He Asn Glu Thr Thr Gly Glu Phe Asp Leu Glu Asn Lys Wing 140 145 150 Val Gln Gln Asp Leu Lys Thr Pro Glu Thr Pro Ser Thr He Phe 155 160 165 Gly Tyr Val Val Glu Gly Leu Arg Arg Arg Arg Asp Wing Gly Gly 170 175 180 Lys Ala Phe Thr He Met Ser Cys Asp Asn Leu Arg His Asn Gly 185 190 195 Asp Val Ala Arg Lys Ala Phe Leu Gly Tyr Ala Lys Ala Arg Asp 200 205 210 Pro Glu Leu Wing Lys Trp He Glu Glu Asn Wing Thr Phe Pro Asn 215 220 225 Gly Met Val Asp Arg He Thr Pro Thr Val Ser Wing Asp He Wing 230 235 240 Lys Lys Leu Asn Glu Wing Ser Gly Leu His Asp Asp Leu Pro Leu 245 250 255 Val Ala Glu Asp Phe His Gln Trp Val Leu Glu Asp Ser Phe Ala 260 265 270 Asp Gly Arg Pro Ala Leu Glu Lys Ala Gly Val Gln Phe Val Gly 275 280? R Asp Val Thr Asp Tyr Glu His Val Lys He Arg Met Leu Asn Ala 290 295 300 Gly His He Met Leu Cys Phe Pro Wing Val Leu Wing Gly Phe Glu 305 310 315 Asn Val Asp His Wing Leu Wing Asp Pro Asp Leu Arg Arg He Leu 320 325 330 Glu Asn Phe Leu Asn Lys Asp Val He Pro Thr Leu Lys Ala Pro 335 340 345 Pro Gly Met Thr Leu Glu Gly Tyr Arg Asp Ser Val He Ser Arg '350 355 360 Phe Ser Asn Pro Wing Met Wing Asp Gln Thr Leu Arg He Ser Gly 365 370 375 Asp Gly Be Ser Lys He Gln Val Phe Trp Thr Glu Thr Val Arg 380 385 390 Lys Ala Phe Glu Gly Lys Arg Asp Leu Ser Arg He Ala Phe Gly 395 400 405 Met Wing Being Tyr Leu Glu Met Leu Arg Gly Lys Asp Glu Thr Gly 410 415 420 Gly Thr Tyr Glu Pro Phe Glu Pro Thr Phe Gly Asp Asn His Lys 425 430 435 Thr Leu Ala Lys Ala Asp Asp Phe Glu Be Ala Leu Lys Leu Pro 440 445 450 Wing Phe A.sp Wing Trp Arg Asp Leu Glu Thr Ser Gly Leu Asn Asn 455 460 465 Lys Val Val Glu Leu Arg Lys He He Arg Glu Lys Gly Val Lys 470 475 80 Ala Ala Leu Pro Ala 485 INFORMATION FOR SEQ ID No. NUM. 3: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 residues (B) TYPE: amino acid (C) TOPOLOGY: linear (II) TYPE OF MOLECULE: peptide (m) ORIGINAL SOURCE: ORGANISM: Gl uconobacter melanogenus CEPA: IFO 3293 (ív) CHARACTERISTICS: KEY FEATURES: peptide METHOD SEQUENCING: E Go Glu Gly Gly Tyr 5 8 INFORMATION FOR SEQ ID No. NUM. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 residues (B) TYPE: amino acid (C) TOPOLOGY: linear (n) TYPE OF MOLECULE: peptide (III) ORIGINAL SOURCE: ORGANISM: Gl uconobacter melanogenus CEPA: IFO 3293 (ív) CHARACTERISTICS: KEY FEATURES: peptide METHOD SEQUENCING: E Phe Pro Asn Gly Met Val Asp Arg 1 5 8 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 (11)

1. R E I V I N D I C A C I O N E S Having described the invention as above, the contents of the following are declared as property: 1. A genetically modified microorganism derived from a microorganism belonging to the genus Gluconobacter or Acetobacter which is characterized in that the biological activity thereof to reduce L-sorbose is substantially abolished by recombination of a gene thereof.
2. 2. The genetically modified microorganism of confirapyrrha in rEavipSc-ci n 1, cv *? * 9-? VBrln p-rtjt »passe said biological LvJLdsd to reduce L-sorbose in 10% less than that of the microorganism original 3. The genetically modified microorganism according to claim 2, characterized in that the gene thereof carries at least one mutation with the aid of addition, insertion, deletion and / or substitution of a nucleotide or nucleotides within the region required for the formation of active L-sorbose reductase. Four . The genetically modified microorganism according to claim 3, characterized in that said mutation is caused by an interruption within the region required for the formation of active L-sorbose reductase. 5. The genetically modified microorganism according to claim 4, characterized in that the interruption contains at least one interfering DNA fragment selected from the group consisting of a transposon, a cassette of antibiotic resistance gene and DNA sequences that prevent the Active L-sorbose reductase formation by the host microorganism. 6. The genetically modified microorganism according to claim 3, characterized in that said mutation is induced by mutagenesis with the aid of genetic engineering technology. 7. The genetically modified microorganism according to claim 3, characterized in that said region required for the formation of active L-sorbose reductase is found in the DNA sequence selected from the group consisting of a structural gene encoding L-sorbose reductase and the derived expression control sequences. 8. The use of an L-sorbose reductase gene of a microorganism belonging to the genus Gluconobacter or Aceto-bacterium in the production of the genetically modified microorganism defined in any of claims 1 to 7, wherein said gene is characterized in that it encodes the amino acid sequence of L-sorbose reductase described in SEQ ID No. 2, or its functional equivalent containing insertion, deletion, addition and / or substitution of one or more amino acids in said SEQ ID No. 2. 9. A method for producing L-sorbose by fermentation of a pdcxoorganism in a medium, suitable characterized in that it comprises the use of said genetically modified microorganism defined in any of claims 1 to 7. 10. Ui pétxx to pr rir vitaiüna C, cacadbari2ack > PT ** u? N »n i- > a fermentation step for the production of L-sorbose, characterized in that the process comprises the use of a genetically modified microorganism defined in any of claims 1 to 7. 11. Vitamin C produced by the method defined in claim 10