Title of Invention

GENETICALLY ENGINEERED L-SORBOSE REDUCTASE-DEFICIENT MUTANTS

Abstract

ABSTRACT 288/MAS/99 "Genetically engineered L-sorbose reductase-deficient mutants" The present invention relates to a genetically engineered microorganism derived from a microorganism belonging to the genes Gluconobacter or Acetobacter comprising a gene encoding a protein having L-sorbose reductase activity characterized in that, said gene has a stably integrated mutation and where in said genetically engineered microorganism is deficient in the conversion of L-sorbose to D-sorbitol and wherein the L-sorbose reductase activity within said genetically engineered microorganism is less than 10% of the amount of the activity of the wild-type microorganism.

Full Text

The present invention relates to nove! genetically engineered L-sorbose reductase-deficient mutants of a microorganism belonging to the genus Gluconobacfer or Acetobacwr which are of advantage in the use for the production of L-sorbose by fermentation as well as in improvement of vitamin C produciion process. The production of vitamin C has been conducted by Reichstein method which involves a fermentation process for the conversion from D-sorbitol to L-sorbose by a microorganism belonging to the genus Gliicouubacter or Acaobader, as a sole biological step. The said con\'ersion to L-sorbose is one of the key steps for the efficacy of vitamin C pj-oduction. Il was. however, observed thai the product. L-sorbose, was consumed after the consumption of the substiale. D-sorbitol, duiing the oxidative fcrmentalion with the said microorganism. This phenomenon was understood that L-sorbose was reduced by NADPH-linked L-sorbose reductase (hereinafter occasionally refcn'ed to as SR) present in ihc cytosol (See: Sugisawa ct al.. Agric. Biol. Chem, 55: 2043-2049, 1991). It was reported thai, in (Ihiconohaciei; D-sorbitol was converted to D-fructose which could be incorporated into the pentose pathway and further metabolized to C02 (Shinjoh el al., Agric. Biol. Chem. 54: 2257-226."^. 1990). Such pathways consuming the product as well as the substrate might have caused less productivity of vitamin C ultimately. An improvement of L-sorbose production was repoilcd by Nogami et al. in Japanese Patent Application Kokai No. 51054/1995. They subjected the micro-organisms of Gluconohaacr oxydans and Gluconobacler s'ubo.xyclans to conventional chemical mutagenesis and isolated the mutant strains whose ability of utilizing D-sorbitol as a sole assimilable carbon source was reduced. By applying such mutant to the fermentation for L-sorbose produciion, they observed more than 2~3 % improvement of the productivity in comparison with the productivity of the parent strain. One of the disadvantages which is often observed in mutant strains produced by the conventional mutagenesis is back mutation which nullifies the improved characteristics of the mutant strains during the course of fermentation or subculture of the mutant, which would result in decreased productivity of vitamin C ultimately. Therefore a stably blocked mutant oi L-sorbose reductase is desired. AB/So 5.2.99 In one aspect, the present invention provides a novel genetically engineered microorganism derived from a microorganism belonging to the genus Gliicoiiabacler or Acelobacier which is characterized in that the biological activity thereof for reducing L-sorbose is substantially nullified by means of genetic engineering. And preferably it has the said biological activity less than 10% of that of the original microorganism; the said activity is 0.07 to 0.02 units/mg protein or less according to the definition of the activity described belo%v (e.g. in Example 1 (iii)). The gene of the genetically engineered claim microorganism of the present invention may carry at least one mutation wiih the aid of disruption, addition, insertion, deletion and/or substitution of nucleotide(s) within the region required for the formation of active L-sorbose reductase in cells of the microorganism. In one preferable embodiment of ihe genciically engineered microorganism of the present invention, the said mutation may be caused by the gene disruption within the region required for the formation of active L-sorbose reductase. Such disruption may i^.. :ontain at least one interfering DNA fragment selected from the group consisting of a iransposon, an unlibioiics resistant gene cassette and any DNA sequences which prevent ihe host mici'oorganism from the formation of active L-sorbose I'cductase. In another embodiment of the present invention, the said mutation may be produced by mutagenesis with ihe aid of sile-dircctcd mutagenesis. Such mutagenesis can 3e effected in the region required for the formation o^ active L-sorbose reductase, which region may include a structural gene of L-sorbose reductase and expression control sequences, such as promoter, operator, terminator, DNA encoding repressor, activator and :he like. Another aspect of the present invention provides the use of an L-sorbose reductase gene of a microorganism belonging to Ihe genus Glticonobacler or Acelobacier in producing the genetically engineered microorganism as described above, in which the 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 equivalents containing insertion, deletion, addition and/or substitution of one or more amino acid(s) in said SEQ ID NO: 2, A further aspect of the present invention provides an efficient method for producing L-sorbose by the fermentation of a microorganism in an appropriate medium, which comprises use of the genetically engineered microorganism of the present invention described above- In connection to this L-sorbose production method, the present invention also provides an efficient vitamin C production process containing a fermentation step for the production of L-sorbose which is characterized in that the said fermentation is carried out by using the genetically engineered microorganism of the present invention as described above. mutagenesis, inlroduction of a gene cassette cairying a selection marker, such as antibiotics resistant gene, DNA sequences which nullify the fomiation of active L-sorbose reductase. (a) Transposon mutagenesis: Transposon mutagenesis is known as a potent tool for genetic analysis (P. Gerhardt et al,. ..Methods for General and Molecular Bacteriology" Chapter 17, Transposon Mutagenesis; American Society for Microbiology), This method utilizes a Iransposable elements which are distinct DNA segments having the unique capacity to move (transpose! to new sites within the genome of the host organisms. The transposition process is independent of the classical homologous recombination system of the organism. The insertion of a transposahle element mio a new genomic site docs not require extensive DNA homology between the ends of the element and its target site, Transposablc elements have been found in a wide variety of prokaryotic and cukaryotic organisms, where they can cause null mutations, chromosome rearrangements, and novel patterns of gene expression on insertion in ihc coding region or regulatory sequences of resident genes and operons. Prokaryolic transposablc elements can be roughly divided into three different classes. Class 1 consists of simple elements such as insertion sequences (iS elements), which arc approximately 800 to \.^Q{) bp in length. IS elements normally consist of a gene encoding an enzyme required for transposition (i.e. transpos:ise). Hanked by terminally repeated DNA sequences which serve as substrate for the transposase. IS elements were initially identified m the lactose and galactose utilization operons of enteric bacteria, where the elements were found to cause often unstable, polar mutation on insertion. Class II consists of composite transposablc elements. The members of this class are also referred to as transposons or Tn elements. Transposons in prokaryotes have been identified as a class of complex transposablc elements, often containing simple IS elements (or parts thereof) as direct or inverted repeats at their termini, behaving formally like IS elements but carrying additional genes unrelated to transposition functions, such as antibiotic resistance, heavy-metal resistance or pathogenicity determinant genes. The insertion of a transposon into a particular genetic locus or repHcon (phage) is designated by using a double colon, e.g. !acZ::Tn5 or ?t::Tn5. Class II! includes „transposabIe" bacteriophages, such as Mu and its relatives. Phage Mu is both a virus and a transposon. It is known that it can integrate at multiple sites in the host chromosome, thereby frequently causing mutations. The iransposon mutagenesis uiilizing the above iransposable elements is known to possessihe following chaniclerislics: (i) Such mutalion generally leads to inactivation of the gene, und ihe resulting null mutation is rchilively stable. (UJTransposons introduce new genetic and physical markers into the target locus, such as antibiotic resistance genes, new restriction endonuclease cleavage sites, and unique DNA sequences which can be identified by genetic means, DNA-DNA hybridization, or electron microscopic heterodupiex analysis. The genetic markers are useful for mapping the mutated loci as well as screening the mutants. {ifpTransposons can generate a variety of genomic rearrangements, such as deletions, inversions, translocations, or duplications, and can be used to introduce specific genes into the target bacteria, A variety of tianspc^ons are known in Ihe art. such as Tn3, TnS, Tn7, Tn9, TnlO, phage Mu and the like. Among them, Tn5 is known to have almost no insertion specificity, and its size is relatively small. Tn5 is also one of the most frequently used Iransposable elements which is readily derived from the sources, such as pfd-Tn5 [American Type Culture Colleclion, USA (ATCC) ATCC 77330 or pCMRSI (ATCC 37535)]. I'or the purpose of use in the random mutagenesis in ihc praclicc of the present invention, Tn5 is preferred. A variety of Tn5 derivatives, designated Mini-Tn5s, which consists of 19 bp of ihe Tn5 inverted repeals required for transposition coupled to antibiotic resistance or other sclcclablc marker genes are also useful for the present invention. Such Mini-TnSs are inserted into a suicide vector, in addition to the Tn5 transposase (iiip), to construct an efficient suicide Tn5 mutagenesis system. Further information how to work with Tn5 transposons can be taken 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 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal inerslion of cloned DNA in Gram-negative Eubacteria, J. Bacteriology, 172:6568-6572, 1990. Information describing how Tn5 can be derived from pfd-Tn5 [see explanation of pfd-Tn5 {ATCC 77330), are obtainable from the ATCC Home Page on Internet, http://wvvw.atcc.org;catalogy,recomb.h{ml]. Accordingly the suicide ptasmid pfd-Tn5 can be introduced into E. coli as well as other Gram negative bacterium by electroporation (see the reference for recommended conditions). The plasmid itself can be used as a Tn5 donor. In addition, one can construct a suicide Tn5 vector by introducing pfdTn5 plasmid into the recipient E. coli with any plasmid which one wants to use, selecting transformats showing Km'and resistance of the target plasmid, isolating plasmids from the trans form ants, transfoiTning E. coli with the isolated plasmids, and selecting for Km and plasmid-marker-resistant transformants to obtain E. coli strain cairying the target plasmid with Tn5. Concept of this protocol is also available in "region-directed Tn5 mutagenesis" in P, Gerhardt ct al. (mentioned above). Random mutagenesis with iransposon invohes the introduction of a transposon into a target bacterial ceil via transformation, transduction, conjugal mating or electroporalion b\ using suicide plasmid or phage vectors. The resulting mutants may be I screened with the aid of the marker carried by the transposon. Transposition of the transposon into the genome of the recipient bacterium can be detected after the vector used has been lost by segregation. For the introduction of transposons into a microorganisms of the genus Gliicoiiohacler OT AcfKihach'i; so-called suicide vectors mcluding a derivative of phage PI and narrow-hosl-range plasmids arc commonly used. The phage PI vectors and the plasmid vectors can be transferred by infection and by transformation, conjugal mating or electroporation. respectively, into the recipient cells, wherein these vectors preferably lack the appropriate origins of recipients. The choice of suicide vector and transposon to be I used depends on criteria including phage sensitivity, intrinsic antibiotic resistance of the recipient cell, the availability of a gene transfer system including transformation, conjugal transfer, eleclioporation, or infection to introduce transposon-can'ying vector into E. coli. One of the preferable vectors for use in the present invention is phage PI {ATCC25404) which injects its DNA into a microorganism belonging to the genus Gluconobacter ox Acelobaacr, however, this DNA will be unable to replicate and will be lost by segregation. Such PI phage can'ying Tn5 (Pl::Tn5} can be used in the form of phage lysate which may be prepared by lysing E. coli caiTying Pl::Tn5 in accordance with known procedures (see: e.g. Methods for General and Molecular Bacteriology" Chapter 17, Transposon Mutagenesis; American Society for Microbiology or US patent 5082785, 1992). The other preferable suicide vectors which can be used in the present invention are plasmid suicide vectors, which may be based on replicon derived from plasmid RP4, or its relative RK2 (ATCC37125), can'ying the same broad-host-range conjugal transfer of mobilization functions and sites but a narrow-hosl-range origin of replication. These vectors can be mobilized at a high frequency from E. coli to a microorganism belonging to the genus Gluconobacwr or Acetobucter but cannot be stably maintained in the recipient only limitalion in these types of replacement experiments is the length of homologous sequences needed for the double-crossover recombination event; 0.5 - 5 kb long homologous sequences at both ends are preferably required. The preferable vectors for the region-directed mutagenesis are the same as those useful for transposon mutagenesis; above mentioned suicide phage and plasmid vectors. (c) Site-dirccled mutagenesis When a wild-type gene is cloned and its nucleotide sequence is deiennined, site-directed mutagenesis can also be used to inactivate the target genes. Oligonucleotide primers including addition and deletion of nucleotides for frame-shift, or substitution of nucleotides for introduction of .stop codons or different codons are used for mutating genes of interest. The mutagenesis with the primers including mutation(s) can be i:onducted usually in E. coli with any commercial site-directed mutagenesis kits, This-type mutagenesis is useful if Tn5- or region directed-cassetie-mutagenesis causes polar effect affecting gene expression of downstream or upstream. The preferable vectors for introducing the mutated gene into the target microorganism are the same as those useful for transposon mutagenesis. In this case, aimed mulani can be isolated hy biological assay, e. g.. enzymological or immunological screening to detect deficiency of the target gene product. In.stead, the mutated gene can be tagged with selection marker gene described above at the site not affecting gene c.vpression of downstream or upstream to facilitate the selection of the gene replacement. One can obtain the target gene disruptant more easily by combination oi biological assay with marker-selection. The L-sorbosc rcductase-deficicnt mutant can be selected generally as follows: 3,000 to 10,000 transposon mutants are subjected to the product assay with L-sorbose as the substrate to select the mutant which docs not convert L-sorbose to D-sorbitol. The first screening can be done preferably in microtiter plates with the reaction mixture containing L-sorbose. The fonnation of the product, D-sorbitol, is firstly detected by TLC with appropriate developing solvent; candidates fonning undetectable amount of D-sorbitol are selected. Then, the candidate mutants are subjected to assay of L-sorbose reductase activity as exemplified in the Example 1 of the present invention to confirm L-sorbose reductase-deficiency. For confirming that the deficient mutant really canies transposon, colony- or Southern-hybridization is usually conducted with iabeled-DNA fragment containing the transposon used as the probe by the standard methods (Molecular cloning, a laboratory manual second edition, Maniatis T., et al., 1989). Such a niulanl was isolated as described in Example 1 of the present invention. The iransposon muiant is useful for further identifying the target L-sorbose reductase gene and determining its nucleotide sequence of the region tagged with the Iransposon. The DNA fragment inserted by a iransposon can be cloned into any E. coli cloning vectors, preferably pUClS, pUC19, pBluescript II (Straiagene Cloning Systems, CA, USA) and iheir relatives, by selecting transformants showing both phenotypes of selection markers of the vector and the iransposon. The nucleotide sequences adjacent to the Iransposon are able to be determined by e. g., a chain termination method (Sanger F, S,. et al., Proc. Nail. Acad. Sci. USA 74:5463-5467,1977). The resulting nucleotide sequences may be partial sequences whose reading frame might be difficult to be determined. Once the nucleotide sequences were determined, they can be subjected to homology search performed wiih nucleotide and/or protein sequence data ba.ses by using genetic analysis program, c, g., BLASTP search (Lipman et al., J. Mol. Biol. 215: 403-410, 1990). II' any homologous sequences arc found, their amino acid sequences can be aligned 10 find any consensus sequences which are conserved between the homologous proteins. According lo ihe consensus sequences, oligonucleotide primers can be synthesized and used to amplify the partial DNA of the target gene by polymerase chain reaction (PCR). Besides the consensus sequences, any amino acid secjucnccs, which are determined after adjusting the reading frame by the alignment of homologous proteins, can be used for designing the PCR primers. The resulting PCR-bom partial gene can be used as a probe to obtain the whole target gene through Southern- and colony-hybridization. Southern-hybridization reveals size of the DNA fragment containing the target gene and one can construct a mini-gene library containing the DNA fragments with the aimed size. The mini-library can then be screened with the partial gene as the probe by colony-hybridization to obtain the whole target gene. Then complete nucleotide 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 controlling the expression of the structure gene of L-sorbose reductase; the regulatory gene can also be the target for disrupting L-sorbose reductase gene. Cloned DNA fragment containing the panial or whole L-sorbose reductase gene of the target microorganism can be used for disrupting the L-sorbose reductase gene of the target microorganism. A schematic procedure and mechanism for the disruption are illustrated in Fig- 4 and 5, respectively. The DNA fragment is first cloned into E. coli vector such as pBluescript II SK. Then a gene cassette cairying a selection marker such as Km'^gene is inserted into ihe target L-sorbose reductase gene not lo form active L-sorbose reductase. The resulting DNA fragment with disixipted L-sorbose reductase gene is recloned on a suicide vector such as pSUP202. The suicide plasmid carrying disrupted gene can be introduced into the recipient microorganism by any gene transfer methods including conjugal mating as described above. Selection of the target mutant generated by double crosso\er recombination event can be done by isolating colonies expressing the selection marker gene (e.g., Km^ and characterizing its chromosomal DNA by Southern-blot hybridization. The L-sorbose reductase deficiency of the candidate mutant is confirmed not to show detectable enzyme activity of L-soi'bose reductase. Such ;i muiant was isolated as described in E.xample 5 of the present invention as the strain SR3. Non-assimilation of L-sorbose can be examined with any media containing 1-500 g/L of D-sorbilol for L-sorbose fermentation by chasing the concentration of L-sorbosc once con\ertcd from D-sorbilol in the Icrmentation broth. Instead, L-sorbosc-conlaining mcdmni can also be used for confirming non-assimilation of L-sorbose under fermentation conditions. The mulants provided in the present invention may be cultured in an aqueous medium supplemented wiUi iippvopvimc miwiciils under acvoVtic conditions. The culliviilion may be conducted at pH between about 3.0 and 9.0, preferably between about 5.0 and 8.0. While the culti\ation period varies depending upon pH, temperature and nutrient medium used, usually 1 to 6 days will bring about favorable results. A preferred temperature range forcan'ying out the cultivation is from about 13°C to 45°C preferably from about IS^C lo 42°C, It is usually required that the culture medium contains such nutrients as assimilable carbon sources, digestible nitrogen sources and inorganic substances, vitamins, trace elements and the other growth promoting factors. As assimilable carbon sources, glycerol, D-glucose, D-mannitol, D-fructose, D-arabitol, D-sorbitol, L-sorbose, and the like can be used. Various organic or inorganic substances may also be used as nitrogen sources, such as yeast extract, meat extract, peptone, casein, com sleep liquor, urea, amino acids, nitrates, ammonium salts and the like. As inorganic substances, magnesium sulfate, potassium phosphate, feirous and fenic chlorides, calcium carbonate and the like may be used. The L-sorbose reductase-deficienl mutant of the present invention can be applied to the fermentative oxidation of D-sorbitoi and is expected to improve the yield of vitamin C by increasing L-sorbose usable for the step of condensation reaction of L-sorbose to produce diacetone-L-sorbose. As it is apparent for those skilled in the ait, the L-sorbose reductase-deficient mutant of the present invention may be applied to any processes for vitamin C production which include L-sorbose as a reaction intermediate. The present invention is further illustrated with Examples described below by refening to the attached drawings which show as described below: Figure I illustrates the nucleotide sequences upstream and downstream of Tn5-inseried region of the chrotnosomal DNA of L-sorbose reductase-deficient mutant, 26-9A derived from G. mdaiuigcuus IFO 3293. Figure 2 illustrates oligonucleotide primers used for PCR cloning of SR gene from G. suhoxyikms \?0?>29\. Figure 3 illustrates Restriction map of 8,0 kb EcoRV fragment carrying L-sorbosc reductase gene of G. siibo.wckius iFO 3291 (upper) and its enlarged region near the L-sorbose reductase gene (lower) showing ORFs found. Figure 4 is a scheme for the construction of a suicide plasmid for disruption of L-sorbose reductase gene in G. suboxydans IFO 3291. Figure 5 illustrates a schematic mechanism for the disruption of L-sorbose reductase gene of G. suboxydans IFO 3291. Figure 6 shows the graphs illustrating the fermentation profiles of strain SR3 and G. suboxydans IFO 3291 in 8% sorbitol-No. 5 medium. Figure 7 shows the graphs illustrating the fermentation profiles of strain SR3 and G. suboxydans IFO 3291 in 2% sorbitol-SCM medium. Example 2. Cloning and nucleotide sequencing of Tn5-inserted region New Tn5-mut:ints were re-constructed by using pSUP202 (Ap^'-Cm'Tc''; SimonR. et al., BIO/TECHNOL., 1: 784-791, 1983) with ihe DNA fragment containing Tn5 lo confirm that L-sorbose reductase deficiency of 26-9A was caused by the Tn5 insertion, noi by different mutaiion simultaneously occuired on the different position of 26-9A DNA from the position of the Tn5 insertion. Southcm-bloi hybridization of various DNA fragments o( strain 26-9A chromosome revealed that 13 kb EcoRV fragment containing a whole Tn5 had enough length (more than at least 1 kb) of DNA at both sides of the Tn5 insertion point for a double-crossing o\'er recombination. The Ecu RV fragment was cloned in pSUP202 lo produce pSR02, which was then introduced into G. iiielanogeiuis IFO 3293 to give KnfCm* strains, 3293EV-1 and 3293EV-9. Southern-blot hybridization analysis of the strains 3293EV-1 and 3293EV-9 j-evealed that both strains contained Tn5 without pSUP2()2 \ector poilion as strain 26-9A did (data not shown), indicating that homologous recombination iiy a double crossover was occuired. Deficiency of L-sorbose reductase acli\ii\ m the stiains 3293EV-1 and 3293EV-9 was examined by a product assay and a photometric enzyme assay as performed for strain 26-9A; new Tn5 mutants also produced undcleciahle L-stirbose and showed L-soi'bosc reductase acti\'ily below 0.01 unit/mg cytosol protein, while Ci. ))\i'hiiu)i;cnns IFO 3293 showed L-sorbose reductase activity of 0.20 unils/mg cytosol protein. It should be concluded thai the Tn5 insertion in 26-9A caused L-sorbose reductase deficiency. The nucleotide sequence of Tn5-inserted region on pSR02 was determined by Ihe dideoxy chain termination method (Sanger I-. el al., Proe. Natl. Acad. Sei, USA,, 74: 5463-5467, 1977) (Fig, !). Analysis of the Tn5-insertcd region by the homology search with BLASTP program (Lipman D. J. et al., J. Mol. Biol. 215: 403-410, 1990) revealed that the region encodes a polypeptide having a homology with polypeptides belonging to mannitol dehydrogenase (MDH) family. One of the member, mannitol 2-dehydrogenase (EC 1.1.1.67) of RJiodobacler sphaeroides (Schneider K.-H. el al, J. Gen. Microbiol., 1993) catalyzes the NAD-dependent oxidation of mannitol into fructose. L-Sorbose reductase of Glucoiwbaacr 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 Ihe oxidation of D-sorbitol and D-mannitol to produce L-sorbose and D-fructose in the presence of NADP {Sugisawa T. el al., ibid). The homology analysis indicated thai polypeptide encoded by Tn5-disrupted gene of strain 26-9A is L-sorbose reductase gene itself, not its regulatory gene. Example 3. PCR cloning. Partial L-sorbose reeduetase gene of G. suboxydans IFO 3291 was cloned by PCR amplification with a set of primers synthesized according to the amino acid sequences Figure 4 shows the scheme for the construction of a L-sorbose reductase gene targeting vector, pSUP202-SR::Km. Eco RV fragment of 8.0 kb was cloned in the Eco RI site of pBluescript 11 SK vector (Alting-Mees M. A. et al,, Methods in enzymology 216: 11 Example 7. Ftrmenlation prolllc of strain SR3 in 2% sorbilol-SCM medium Growlh and L-sorbose-assimilalion profiles of strain SR3 and G. subuxydans IFO 3291 were evaluated with 2% sorbitol-SCM medium in 500 ml Erlenmeyer flask (Fig. 7), Strain SR3 and G. suboxyduns IFO 3291 could conveit 20 g/L D-sorbitol to L-sorbose within 12 hr. G. suboxydans IF03291 assimilated half of the converted L-sorbose in 23 hr. On the other hand, strain SR3 hardly utilized L-sorbose. Strain SR3 and G. suboxydans IFO 3291 showed OD600 (cell growth) of 2.5 and 5.9 after 23 hr, respectively. We claim: 1. A genetically engineered microorganism derived from a microorganism belonging to the genes Gluconobacter or Acetobacter comprising a gene encoding a protein having L-sorbose reductase activity characterized in that, said gene has a stably integrated mutation and where in said genetically engineered microorganism is deficient in the conversion of L-sorbose to D-sorbitol and wherein the L-sorbose reductase activity within said genetically engineered microorganism is less than 10% of the amount of the activity of the wild-type microorganism. 2. The genetically engineered microorganism according to claim 1, in which the gene thereof carries at least one mutation with the aid of disruption, addition, insertion, deletion and/or substitution within a region required for the formation of active L-sorbose reductase. 3. The genetically engineered microorganism according to claim 2, in which the disruption contains at least one interfering DNA fragment selected from the group consisting of a transposon, an antibiotic resistant gene cassette and DNA sequences which prevent the host microorganism from formation of active L-sorbose reductase. 4. The genetically engineered microorganism according to claims 2 or 3, in which the said region required for the formation of active L-sorbose reductase lies in the DNA sequence selected from the group consisting of a structural gene encoding L-sorbose reductase and the expression control sequences therefor. 5. The genetically engineered microorganism according to any one of claims 1 to 4 wherein the gene encoding a protein having L-sorbose reductase activity is selected from the group consisting of SEQ ID NO: 1, a fragment of SEQ NO: 1 encoding a polypeptide having L-sorbose reductase activity and a polynucleotide encoding SEQ ID NO: 2. 6. A process for producing L-sorbose comprising : (a) fermenting the genetically engineered microorganism according to claim 1 in an appropriate medium containing D-sorbitol, or a source of D-sorbitol, and (b) recovering the L-sorbose produced in step (a) by a known method. 7. The process for the production of a genetically engineered microorganism according to claim 1 comprising: (a) providing a microorganism of the genus Gluconobacter or Acetobacter comprising a gene encoding a protein having L-sorbose reductase activity, and (b) introducing a mutation in said polynucleotide to result in a biological activity for reducing L-sorbose which is less than 10% of the amount of the activity of the wild-type microorganism. 8. The process for the production of a genetically engineered microorganism according to claim 1 comprising: (a) providing a microorganism of the genus Gluconobacter or Acetobacter comprising a gene encoding a protein having L-sorbose reductase activity, (b) genetically engineering said microorganism by introducing a stable mutation into said gene. (c) determining the L-sorbose reductase activity of the microorganism comprising the mutated gene obtained in step (b), (d) selecting a microorganism based on step (c) in which L- sorbose reductase activity is less than 10% of the wild-type microorganism. 9. The process according to claim 7 or 8 wherein the microorganism is genetically engineered by gene disruption, addition, insertion, deletion and/or substitution within Pa region required for the formation of active L-sorbose reductase. 10. The process according to any one of claims 7 to 9 wherein the gene coding for L-sorbose reductase is selected from the group consisting of SEQ ID NO: 1, a fragment of SEQ ID NO: 1 encoding a polypeptide having L-sorbose reductase activity and a polynucleotide encoding SEQ ID NO: 2. 11. A process for producing vitamin C comprising : (a) fermenting a microorganism as produced by a process according to any one of claims 7 to 10 in an appropriate medium containing D-sorbitol or a source of D-sorbitol, and (b) converting the L-sorbose produced in step (a) into vitamin C by a known method.

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