| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | March 30, 1999 |
| PATENT TITLE |
Method for producing 2-keto-L-gulonic acid |
| PATENT ABSTRACT |
An expression vector containing both a DNA encoding an L-sorbose dehydrogenase and a DNA encoding an L-sorbosone dehydrogenase; a transformant having an ability to produce 2-keto-L-gulonic acid (hereinafter 2KLGA) at high yields from D-sorbitol, which is prepared by transforming, with said expression vector, a microorganism capable of producing L-sorbose at high yields from D-sorbitol, which has no or low 2KLGA-decomposing activity or a host microorganism having, in addition to the above-mentioned properties, no or low L-idonic acid-producing activity; and a process for producing 2KLGA, which comprises culturing said transformant in a medium containing D-sorbitol. According to the present invention, 2KLGA useful for the production of L-ascorbic acid can be produced with ease and in larger amounts by a single operation of culture. -------------------------------------------------------------------------------- Inventors |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | October 3, 1997 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED | Balbas et al. (1990) Methods in Enzymology, vol. 185, pp. 14-37. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. An expression vector comprising a DNA encoding an L-sorbose dehydrogenase and a DNA encoding an L-sorbosone dehydrogenase, wherein the L-sorbose dehydrogenase has the amino acid sequence of SEQ ID NO: 1 and the L-sorbosone dehydrogenase has the amino acid sequence of SEQ ID NO: 2. 2. The expression vector of claim 1, wherein the DNA encoding the L-sorbose dehydrogenase and the DNA encoding the L-sorbosone dehydrogenase are constructed such that transcriptions occur successively from one promoter, or a transcription of each DNA occurs under the control of a different promoter. 3. The expression vector of claim 1, wherein the promoter is a promoter region of the L-sorbosone dehydrogenase gene or a part thereof having a promoter activity. 4. The expression vector of claim 1, wherein the promoter is derived from Escherichia coli. 5. The expression vector of claim 1, wherein the promoter is Escherichia coli promoter tufB, .lambda. PL, trp or tac. 6. The expression vector of claim 1, which is suitable for transforming a host microorganism capable of producing L-sorbose at high yields from D-sorbitol, which has no or low 2-keto-L-gulonic acid-decomposing activity. 7. The expression vector of claim 1, which is suitable for transforming a host microorganism capable of producing L-sorbose at high yields from D-sorbitol, which has no or low 2-keto-L-gulonic acid-decomposing activity, and no or low L-idonic acid-producing activity. 8. The expression vector of claim 1, wherein the DNA encoding an L-sorbose dehydrogenase and a DNA encoding an L-sorbosone dehydrogenase are obtained from a bacterium belonging to the genus Gluconobacter. 9. The expression vector of claim 1, wherein the DNA encoding an L-sorbose dehydrogenase and a DNA encoding an L-sorbosone dehydrogenase are obtained from Gluconobacter oxydans. 10. The expression vector of claim 1, wherein the DNA encoding the L-sorbose dehydrogenase and the DNA encoding the L-sorbosone dehydrogenase are DNAs encoding said enzymes derived from Gluconobacter oxydans T-100. 11. A transformant obtained by transforming a host cell with the expression vector of claim 1. 12. A transformant which produces a 2-keto-L-gulonic acid from D-sorbitol, which is obtained by transforming a host microorganism, which produces L-sorbose from D-sorbitol, with the expression vector of claim 1. 13. A transformant, (1) which produces 37 to 91 mg/ml of 2-keto-L-gulonic acid from D-sorbitol; and (2) which is obtained by transforming a host microorganism, which produces L-sorbose from D-sorbitol, with the expression vector of claim 1. 14. The transformant of claim 13, which produces 42 to 91 mg/ml of 2-keto-L-gulonic acid from D-sorbitol. 15. The transformant of claim 13, which produces 56 to 91 mg/ml of 2-keto-L-gulonic acid from D-sorbitol. 16. A transformant obtained by transforming a host cell with the expression vector of claim 1, wherein the transformant produces an undetectable quantity of L-idonic acid as measured by absorbance at 210 nm. 17. A process for producing a 2-keto-L-gulonic acid, comprising culturing the transformant of claim 11 in a medium containing D-sorbitol, thereby producing 2-keto-L-gulonic acid, and harvesting the 2-keto-L-gulonic acid. 18. A process for producing a 2-keto-L-gulonic acid, comprising culturing the transformant of claim 16 in a medium containing D-sorbitol, thereby producing 2-keto-L-gulonic acid, and harvesting the 2-keto-L-gulonic acid. 19. The host microorganism of claim 16, which belongs to the genus Gluconobacter or Acetobacter. 20. A process for producing a 2-keto-L-gulonic acid, comprising culturing the transformant of claim 11, in a medium containing D-sorbitol, and harvesting the 2-keto-L-gulonic acid from the obtained culture. 21. A process for producing a 2-keto-L-gulonic acid, comprising culturing the transformant of claim 13 in a medium containing D-sorbitol, and harvesting the 2-keto-L-gulonic acid from the culture. 22. A method of transforming a host microorganism, comprising: culturing a host microorganism in a culture medium containing D-mannitol; placing the cultured host cell in a solution containing an expression vector comprising a DNA encoding an L-sorbose dehydrogenase and a DNA encoding an L-sorbosone dehydrogenase, wherein the L-sorbose dehydrogenase has the amino acid sequence of SEQ ID NO:1 and the L-sorbosone dehydrogenase has the amino acid sequence of SEQ ID NO:2; and subjecting the cultured host cell to electroporation. -------------------------------------------------------------------------------- Description -------------------------------------------------------------------------------- TECHNICAL FIELD The present invention relates |
| PATENT DESCRIPTION |
TECHNICAL FIELD The present invention relates to a method for producing 2-keto-L-gulonic acid (hereinafter also referred to as 2KLGA), which is a precursor of L-ascorbic acid, efficiently and with ease by genetic engineering. The present invention also relates to a series of expression systems involved in the efficient production of 2KLGA. BACKGROUND ART The 2KLGA is a key intermediate in the synthesis of L-ascorbic acid. For industrial production, 2KLGA is chemically synthesized from D-sorbitol by oxidation according to the Reichstein's method. Meanwhile, many microorganisms inclusive of the microorganisms belonging to the genus Gluconobacter are known to convert D-sorbitol to 2KLGA through an enzymatic oxidation. The microorganisms belonging to the genus Gluconobacter have been improved by genetic engineering using conjugal transfer and transposon. However, because of the low production of 2KLGA by these microorganisms, they have not been utilized in the industrial production yet. Accordingly, there has been a desire for a more efficient and simplified method for the production of 2KLGA. It is also well known that, in secondary metabolite production by microorganisms such as that of 2KLGA, a mere insertion of a gene (group) responsible for the biosynthesis of a substance into a plasmid and culture of the cells of microorganism, which have been recombined with this plasmid, does not necessarily result in an improved production of the desired substance, but rather, may degrade the productivity ›Thomas, D. I. et al., J. Gen. Microbiol., 137, pp. 2331-2337 (1991)!. DISCLOSURE OF THE INVENTION An object of the present invention is to provide an expression vector containing both a DNA encoding L-sorbose dehydrogenase (hereinafter SDH) and a DNA encoding L-sorbosone dehydrogenase (hereinafter SNDH), a transformant having an ability to produce 2KLGA at high yields from D-sorbitol, which has been transformed with said expression vector, and an efficient and simplified process for producing 2KLGA, which comprises culturing said transformant. In an attempt to accomplish the above-mentioned objects, the present inventors have conducted intensive studies to succeed in obtaining an expression vector having the above-mentioned preferable property, and found that 2KLGA can be efficiently produced from D-sorbitol by a series of culture systems which comprise introducing said expression vector into a host microorganism capable of producing L-sorbose at high yields and having low 2KLGA-decomposing activity, or a host microorganism having, in addition to the above-mentioned properties, low L-idonic acid-producing activity, which resulted in the completion of the present invention. Accordingly, the present invention relates to an expression vector containing both a DNA encoding SDH (hereinafter also referred to as SDH gene) and a DNA encoding SNDH (hereinafter also referred to as SNDH gene). The present invention also relates to a microorganism capable of producing L-sorbose at high yields from D-sorbitol and having no or low 2KLGA-decomposing activity, or a host microorganism having, in addition to the above-mentioned properties, no or low L-idonic acid-producing activity. This microorganism is useful as a host into which the above-mentioned expression vector is introduced. The expression vector of the present invention includes not only that having an SDH gene and an SNDH gene on one vector, but also a pair of vectors separately having each gene. The present invention further relates to a transformant which is a microorganism having the above-mentioned expression vector introduced therein, and which has an ability to produce 2KLGA at high yields from D-sorbitol. In addition, the present invention relates to a process for producing 2KLGA, which comprises harvesting 2KLGA from a culture obtained by culturing said transformant in a medium containing D-sorbitol. The present invention moreover relates to an expression vector, a host, a plasmid and a transformation method useful for efficiently and easily producing 2KLGA from D-sorbitol by genetic recombination. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a restriction enzyme map of plasmid pUC18SD180. FIG. 2 is a restriction enzyme map of plasmid pUC19SD5. FIG. 3 shows restriction enzyme maps of plasmids pF2, pF3 and pF4. FIG. 4 shows the construction of shuttle vector pFG5B. FIG. 5 shows the construction of shuttle vector pFG14A. FIG. 6 shows the construction of shuttle vector pFG15A. FIG. 7 shows a restriction enzyme map of plasmid pSD5-RIBg. FIG. 8 shows the construction of expression vector pSDH145. FIG. 9 shows the construction of expression vector pSDH155. FIG. 10 shows the construction of expression vector pSD33. FIG. 11 shows the construction of expression vector pSD34. FIG. 12 shows the construction of expression vector pSDH155-NC. FIG. 13 shows the construction of expression vector pSDH155-NN. FIG. 14 shows the construction of expression vector pSDH165-NN. FIG. 15 shows the construction of expression vector pSDH-tufB1. DETAILED DESCRIPTION OF THE INVENTION (1) Expression vector The expression vector of the present invention contains both a DNA encoding SDH and a DNA encoding SNDH. The expression vector of the present invention is a DNA molecule incorporating an SDH gene and an SNDH gene in an expressionable state, which encompasses any vehicle capable of autoreplication in a host microorganism or capable of being integrated into a genome of host microorganism. Preferred are expression vectors suitable for transforming a host microorganism capable of producing L-sorbose at high yields from D-sorbitol and having no or low 2KLGA-decomposing activity, or a host microorganism having, in addition to the above-mentioned properties, no or low L-idonic acid-producing activity, into a microorganism capable of producing 2KLGA from D-sorbitol at high yields. Such expression vector is exemplified by vectors preferably containing, at least, a promoter having a high promoter activity in a microorganism capable of producing L-sorbose at high yields from D-sorbitol, an SDH gene, an SNDH gene, an autoreplicatable unit, and optionally a terminator region, which can be stably present in said microorganism. More preferred are those suitable for transforming a host microorganism belonging to the genus Gluconobacter or Acetobacter which is capable of producing L-sorbose at high yields from D-sorbitol, and which has no or low 2KLGA-decomposing activity, or a host microorganism having, in addition to the above-mentioned properties, no or low L-idonic acid producing activity, into a microorganism capable of producing 2KLGA from D-sorbitol at high yields. The use, as such expression vector, of a shuttle vector containing, in addition to an autoreplicatable unit which functions stably in a host microorganism, an autoreplicatable unit of a microorganism, such as Escherichia coli, suitable for cloning of said expression vector, is particularly advantageous. A shuttle vector containing a marker gene, such as an antibiotic resistant gene, is more advantageous. Examples of preferable shuttle vector in the present invention include pFG14A and pFG14B which are combined plasmids of pHSG298 and pF3, and pFG15A and pFG15B which are combined plasmids of pHSG298 and pF4. The marker gene to be used here is exemplified by drug resistant genes such as kanamycin resistant gene, ampicillin resistant gene, chloramphenicol resistant gene and hygromycin resistant gene, auxotrophic gene and enzyme gene such as lacZ. The promoter to be used for the expression vector of the present invention is not particularly limited as long as it has a promoter activity to transcribe SNDH and/or SDH gene in a host microorganism. A promoter having a high promoter activity to transcribe SNDH and/or SDH gene in a host microorganism capable of producing L-sorbose at high yields from D-sorbitol, is particularly preferable. As such promoter, a promoter region of SNDH gene and a part thereof having a promoter activity are exemplified. Specific examples thereof include a promoter region of SNDH gene derived from Gluconobacter oxydans T-100, which is included in the Sequence Listing, SQ I:No. 5, nucleotides 1-1040, and a part thereof having a promoter activity. Examples of the promoter include promoters derived from Escherichia coli, such as tufB, .lambda.PL, trp, tac, lacUV5, lap, lac, ompA, phoA, recA and rrnB promoters, with preference given to tufB, .lambda.PL, trp and tac promoters. The sequences other than -35 region and -10 region may be appropriately selected to be suitable for the construction of plasmid. The autoreplicatable unit is a DNA compound capable of replicating the DNA sequence belonging thereto in a host cell, and may include autoreplicatable unit derived from natural plasmid, artificially modified plasmid (e.g. DNA fragment prepared from natural plasmid) and synthetic plasmid. The autoreplicatable unit in the present invention can be appropriately selected according to the microorganism to be used as a host. Preferred is an autoreplicatable unit derived from a plasmid obtained from a microorganism of the same kind with the host. For example, when the host is a microorganism belonging to the genus Gluconobacter, a plasmid derived from the genus Gluconobacter is preferably used. The plasmid containing such autoreplicatable unit is 1-100 kb, preferably 1.gtoreq.10 kb in size, and advantageously has a useful restriction enzyme recognition site. The useful restriction recognition enzyme site here means that wherein a cleavage site of a certain restriction enzyme is limited, which does not lose the activity of replica table unit when cleaved by said restriction enzyme or when a DNA sequence is inserted in this site. Accordingly, it is advantageous in that cleavage and insertion of optional DNA at said site can be manipulated freely. Examples of such plasmid include plasmid pF3 derived from Gluconobacter IAM12138 and plasmid pF4 derived from Gluconobacter T-100. The autoreplicatable unit of a microorganism which is suitable for cloning is not subject to any particular limitation as long as it is a replica table unit of a plasmid derived from a microorganism generally used for cloning in the field of genetic engineering. Preferable examples include replica table units derived from plasmids pBR322, pUC18, pHSG298, pHSG396, pACYC184 and pACYC177, and artificially modified plasmids thereof (e.g., DNA fragment obtained from a suitable restriction enzyme treatment of pBR322) derived from E. coli, and replica table units derived from yeast 2.mu. plasmid derived from yeast. The mode of presence of SDH gene and SNDH gene in the expression vector of the present invention is not particularly limited as long as it allows expression of SDH and SNDH. For example, the expression vector of the present invention may contain said gene in a mode wherein the transcription and translation of SDH gene and SNDH gene are controlled by different expression systems, or wherein the both genes are controlled by a set of expression systems. The expression vector of the former mode is exemplified by an expression vector wherein respective genes are constructed such that a transcription of each gene occurs under the control of a different promoter, and the expression vector of the latter mode is exemplified by an expression vector wherein SDH gene and SNDH gene are constructed such that transcriptions occur successively from one promoter. The expression vector of the present invention may contain plural SDH genes and/or SNDH genes. The DNA encoding SDH and the DNA encoding SNDH to be used in the present invention may be any as long as they encode enzymes having an SDH activity and enzymes having an SNDH activity, and are exemplified by cytoplasmic or membrane-bound SDH and SNDH, and co-enzyme dependent or independent SDH and SNDH. Preferred are a gene encoding an enzyme derived from a microorganism and a gene encoding a mutant thereof. Examples of the microorganism include those belonging to the genus Gluconobacter or Acetobacter, specifically Gluconobacter oxydans T-100 (FERM BP-4188). The membrane-bound SDH derived from Gluconobacter oxydans T-100 is exemplified by an enzyme characterized by the following (1)-(3): (1) an ability to catalyze the conversion of L-sorbose into L-sorbosone, (2) a molecular weight of 58,000 daltons (SDS-PAGE), and (3) an N-terminal amino acid sequence of Thr-Ser-Gly-Phe-Asp-Tyr-Ile-Val-Val-Gly-Gly-Gly-Ser-Ala-(SEQ ID NO:6). Said SDH gene is more preferably a DNA encoding a protein having an amino acid sequence depicted in Sequence Listing, SQ:ID No. 1 to be mentioned later, and most preferably a DNA having a nucleotide sequence depicted in Sequence Listing, SQ:ID No. 3. The cytoplasmic SNDH derived from Gluconobacter oxydans T-100 is exemplified by an enzyme characterized by the following (1)-(3): (1) an ability to catalyze the conversion of L-sorbosone into 2-keto-L-gulonic acid, (2) a molecular weight of 50,000 daltons (SDS-PAGE), and (3) an N-terminal amino acid sequence of Asn-Val-Val-Ser-Lys-Thr-Val-Xaa-Leu (SEQ ID NO:7, Xaa being an unidentified amino acid). Said SNDH gene is more preferably a DNA encoding a protein having an amino acid sequence depicted in Sequence Listing, SQ:ID No. 2 to be mentioned later, and most preferably a DNA having a nucleotide sequence depicted in Sequence Listing, SQ:ID No. 4. Examples of suitable expression vector in the present invention are pSDH145 and pSDH155 (FERN BP-4522). The expression vector of the present invention can be also prepared by a conventional method (e.g. digestion with restriction enzyme, ligation using T4 DNA ligase) using, if necessary, a suitable DNA fragment, by linking the above-mentioned expression system, DNA encoding SDH and DNA encoding SNDH, which are circularly linked with an adequate autoreplicatable unit. (2) Host microorganism The microorganism to be used as a host in the present invention is a microorganism capable of producing L-sorbose at high yields from D-sorbitol, and has no or low 2KLGA-decomposing activity. Preferably, it has, in addition to the above-mentioned properties, no or low L-idonic acid-producing activity. That is, it is important in the present invention that the host has high conversion efficiency of D-sorbitol into L-sorbose; has low L-sorbose-metabolizing activity or even if it metabolizes, the activity to metabolize L-sorbose into a substance outside the metabolic pathway from L-sorbose into 2KLGA is very weak; and has low 2KLGA-decomposing activity. Preferably, such microorganism can grow at a high D-sorbitol concentration of preferably not less than 5%, more preferably not less than 15%, and can convert D-sorbitol into L-sorbose at a nearly 100% efficiency. Examples of such microorganism include microorganisms belonging to the genus Gluconobacter or Acetobacter, such as Gluconobacter oxydans G624 (FERN BP-4415) and Gluconobacter oxydans NB6939. (3) Transformant The transformant of the present invention can be prepared by introducing the above-mentioned expression vector into a host cell. The transformant of the present invention can be also prepared by introducing SDH gene and SNDH gene into different expression vectors, and incorporating them into the cell of a host microorganism. The method for preparing the transformant is not particularly limited, and can be appropriately determined according to the host microorganism. For example, when a microorganism belonging to the genus Gluconobacter or Acetobacter is used as a host, electroporation ›Dower, W. J., Miller, J. F. and Ragsdale, C. W., Nucleic Acid Res. Vol. 16, p. 6127 (1988)! is advantageously used, since the methods generally used for transformation show low transformation efficiency. This electroporation may be carried out by a method routinely used in the field of genetic engineering, or upon modification as appropriate according to the host microorganism to be transformed. The present invention also relates to a method for producing a competent cell suitable for the above-mentioned electroporation. The competent cell suitable for the above-mentioned electroporation is preferably prepared by culture of a host microorganism belonging to the genus Gluconobacter or Acetobacter, which is capable of producing L-sorbose at high yields from D-sorbitol, and has no or low 2KLGA-decomposing activity, or a host microorganism which has, in addition to the above-mentioned properties, no or low L-idonic acid-producing activity, in a medium containing D-mannitol. The transformant thus produced has an ability to produce 2KLGA at high yields from D-sorbitol as a starting material. In the present invention, whether or not the host has been correctly transformed can be examined by determining a selection marker that the transformant has, such as a gene having resistance to antibiotics such as resistance to kanamycin, and enzyme genes, such as auxotrophic gene and LacZ gene, or by determining SDH activity and SNDH activity that the transformant has. (4) Process for producing 2KLGA The 2KLGA of the present invention is obtained by culturing the above-mentioned transformant having an expression vector in a medium containing D-sorbitol, and harvesting 2KLGA from the obtained culture. While the preferable composition of the nutrient medium to be used varies depending on the host, it generally contains D-sorbitol and may contain carbon sources such as D-mannitol, D-glucose, D-fructose, L-sorbose and glycerol. It is preferable that it further contain inorganic or organic nitrogen sources (e.g., ammonium sulfate, ammonium chloride, hydrolysate of casein, yeast extract, polypeptone, Bactotrypton, beef extract and corn steep liquor). If desired, other nutritious sources such as inorganic salts (e.g., disodium hydrogenphosphate, sodium dihydrogenphosphate, dipotassium hydrogenphosphate, potassium dihydrogenphosphate, magnesium chloride, magnesium sulfate, calcium carbonate and calcium chloride), vitamins (e.g., vitamin B.sub.1), and antibiotics (e.g., ampicillin, kanamycin, tetracyclin and chloramphenicol) may be added to the medium. When a microorganism belonging to the genus Gluconobacter is used as a host, a medium containing D-sorbitol, yeast extract and calcium carbonate may be used. The concentration of D-sorbitol in the medium is generally 1-30%, preferably 5-20%. The transformant is cultured under the conditions which permit production of 2KLGA at high yields, according to the host microorganism to be used. For example, when a microorganism belonging to the genus Gluconobacter is used, pH is generally 5.5-8.5, preferably 7-7.5, culture temperature is generally 18.degree.-40.degree. C., preferably 20.degree.-30.degree. C., and culture time is generally 20-170 hours. The 2KLGA thus produced is generally contained in solution fractions in the culture. Thus, 2KLGA can be purified by obtaining a culture filtrate by filtration or centrifugation of the culture and purification of the culture filtrate by a method generally used for purification, such as column chromatography on a suitable adsorbent and crystal precipitation. Commercially available plasmid, restriction enzyme, enzyme such as T4 DNA ligase and other substances used in the following Examples were used according to the instructions of the suppliers. Cloning of DNA, culture of transformant, recovery of 2KLGA from the obtained culture are well-known to those skilled in the art, or can be known from published literatures. Gluconobacter oxydans T-100 (FERM BP-4188), Gluconobacter oxydans G624 (FERM BP-4415), Gluconobacter oxydans GA-1 (G624-pSDH155) (FERM BP-4522) and Gluconobacter oxydans N952 (NB6939-pSDH155) (FERM BP-4580) have been internationally deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan. According to the present invention, 2KLGA useful for the production of L-ascorbic acid can be produced with ease and in larger amounts by a single operation of culture |
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