WO2014116042A1 - Souche recombinée pour la production de 2,3-butanediol, comprenant (a) une lactate déshydrogénase inactivée et (b) un régulateur de saccharose inactivé - Google Patents

Souche recombinée pour la production de 2,3-butanediol, comprenant (a) une lactate déshydrogénase inactivée et (b) un régulateur de saccharose inactivé Download PDF

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WO2014116042A1
WO2014116042A1 PCT/KR2014/000677 KR2014000677W WO2014116042A1 WO 2014116042 A1 WO2014116042 A1 WO 2014116042A1 KR 2014000677 W KR2014000677 W KR 2014000677W WO 2014116042 A1 WO2014116042 A1 WO 2014116042A1
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butanediol
genus
sucrose
strain
producing
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Korean (ko)
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이진원
오민규
정무영
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서강대학교 산학협력단
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Priority to US14/785,607 priority Critical patent/US9994875B2/en
Publication of WO2014116042A1 publication Critical patent/WO2014116042A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • Recombinant strain for producing 2,3-butanediol comprising (a) inactivated lactate dehydrogenase and (b) inactivated sucrose modulator
  • the present invention was made by the task number 10035578 under the support of the Ministry of Knowledge Economy of Korea, the research management specialized organization of the task is the Korea Industrial Technology Evaluation and Management Institute, the research project name is “industrial technology development project”, the research title is "2, 3-butane Development of technology for discovering strains for diol production and optimization of metabolic circuits ", The lead organization is Sogang University Industry-Academic Cooperation Group, and the research period is 2010. 04. 01 ⁇ 2015. 03. 31.
  • the present invention relates to a recombinant strain for the production of 2,3-butanediol comprising (a) an inactivated lactate dehydrogenase and (b) an inactivated sucrose modulator.
  • Biorefinery development can produce environmentally friendly compounds that can reduce dependence on petroleum resources.
  • the production of microorganisms of 2, 3-butanediol which is a flat compound, has recently attracted attention due to the high applicability of the industry (Ji et al., 2011). Therefore, some bacteria, such as Krebsiella pneumoniae (Ma et al., 2009), Serratia Marcenses ( 2010) and Enterobacter aerogenes (Jung et al., 2012), 3-butanedai is being developed as an industrial strain for production.
  • FTS-dependent transport introduces sucrose by phosphorylation by EII scr identified in the crab forla pneumoniae scr operon (Sprenger & Lengeler, 1988).
  • EII scr identified in the crab forla pneumoniae scr operon
  • the non-PTS permease found in E. coli csc operon transfers sucrose into cells without chemical modulation (Bockmann et al., 1992).
  • operons are fructokinase (ScrK), sucrose-specific envelope membrane (ScrY) ⁇ PTS ⁇ transport protein (ScrA), sucrose-6-phosphate hydrolase (ScrB) and sucrose It consists of 50RF of dependent regulators (ScrR) (Fig. Lb).
  • the present inventors have tried to develop a strain and a carbon source that can reduce the production cost of 2, 3-butanedi.
  • a recombinant strain comprising an inactivated lactate dehydrogenase and an inactivated sucrose regulator was developed and cultured in a culture medium containing molasses as a carbon source.
  • This invention was completed by confirming showing 2, 3- butanediol production efficiency.
  • Another object of the present invention is to provide a method for producing 2,3-butanediol.
  • Another object of the present invention is to provide 2,3-butanedi.
  • the invention comprises ( a ) an inactivated lactate dehydrogenase and (b) an inactivated sucrose regulator.
  • an inactivated lactate dehydrogenase and (b) an inactivated sucrose regulator.
  • the present inventors have tried to develop a strain and a carbon source that can reduce the production cost of 2,3-butanediol.
  • a recombinant strain comprising an inactivated lactate dehydrogenase and an inactivated sucrose regulator was developed to produce molasses.
  • the basic strategy of the present invention is to inactivate the lactate dehydrogenase and sucrose modulators found in strains for producing 2,3-butanedi.
  • the lactate dehydrogenase is a triose phosphate isomerase, and is an enzyme that catalyzes the reaction of converting pyruvic acid into lactate by reducing NADH (Nicotinamide adenine dinucleotide) in glycolysis.
  • the sucrose modulator is one of the LacI-GalR family, a transcriptional inhibitor that suppresses the expression of ScrB encoding sucrose hydrolase, and a sucrose operon repressor and Lac I family transcription. It is synonymous with Lacl family transcriptional regulator.
  • Lactate dehydrogenase and sucrose modulators to be inactivated have been found and identified in various strains.
  • lactate dehydrogenase of Enterobacter aerogenes of SEQ ID NO: 1 and sucrose modulators of Enterobacter aerogenes of SEQ ID NO: 2 are illustrated, but various lactate dehydrogenases And sucrose adjusters are included in the present invention.
  • lactate dehydrogenase is GenBank accession number CP_002824 (/ Z ⁇ e aerogenes), accession number K_015663 (En terobacter aerogenes), accession number NC_018522 (/ i7e / «/ e // pneumonia), accession number NC_011283 7eZ?
  • accession number NC_017540 (/ e /? 5 / e // 3 pneumoniae)
  • accession number NC_012731 (/ i7eZ «/ e // s pneumoniae)
  • accession number NC_016612 (A7 ⁇ / e // oxytoca)
  • accession number i8106 (Iebs / ella oxytoca) ⁇
  • the disclosed lactate dehydrogenases may be subject to inactivation of the invention, and the sucrose modulator is GenBank accession number CP_002824 3 ⁇ 4 eOtoc: er aerogenes), Access number
  • the disclosed sucrose modulators may be subject to inactivation in the present invention.
  • W 201 Endobacter aerogenes
  • accession number NC_01852 accession number NC_01852
  • accession number NC_011283 accession number NC_011283
  • accession number j ⁇ 2 accession number NC OlSlOe VeZ ⁇ / e / oxytoca
  • the term "inactivation”, referring to lactate dehydrogenase and sucrose modulators, refers to any variation (eg, deletion, substitution, or loss of function of lactate dehydrogenase and sucrose modulators). Additional variation).
  • the deletion of the lactate dehydrogenase gene and the sucrose modulator gene include all, partial or complete deletions of the lactate dehydrogenase or sucrose modulator coding sequences.
  • the inactivated lactate dehydrogenase is a deletion, substitution or addition mutation in the nucleotide sequence encoding the lactate dehydrogenase.
  • the inactivated sucrose modulator is a deletion, substitution or addition mutation occurred in the nucleotide sequence encoding the sucrose modulator.
  • Such mutation of the lactate dehydrogenase or sucrose regulator coding sequence can be carried out through various mutagenesis methods known in the art. For example, PCR mutagenesis, cassette mutagenesis (Sambrook, J. et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (200D), ⁇ Red recombination method (Jung et al., 2012, Datsenko and Wanner, Proc. Natl. Acad. Sci.
  • the inactivated lactate dehydrogenase is one in which a deletion mutation occurs in the nucleotide sequence encoding the lactate dehydrogenase.
  • the inactivated sucrose modulator is a deletion mutation occurred in the nucleotide sequence encoding the sucrose modulator.
  • said deletion mutations result in deletion mutations via a red lambda recombination method.
  • the recombinant strain for producing 2,3-butanediol additionally generates a deletion, substitution or additional mutation in the nucleotide sequence encoding wabG.
  • a deletion mutation occurs in the nucleotide sequence encoding wabG.
  • Recombinant for producing 2,3-butanedi comprising (a) inactivated lactate dehydrogenase and (b) inactivated sucrose regulator of the present invention
  • the strain can be applied to various strains that produce 2, 3-butanedi.
  • the strain is genus Aeromonas C4er ⁇ /?
  • Bacillus genus Brevibacillus ( ⁇ e / Z c /// i / s) genus Corynebacterium (Coynebacteriu) genus , Enterobacter ieOto ⁇ er) in Klebsiella 7e / e //), Lactobacillus a C ofec /// "s) in Lactococcus ( ⁇ 3 0C0" S) in Leukonostock ( ⁇ g / CiMCiS C ) In Oenocaucus in Pedao Caucus (/ 3 ⁇ 4 // £ a?: C / s) in Lao Ultella 0 / e // 3), Serratia (5err / 3), Streptococcus (Yre o?
  • the strain is of the genus Enterobacter or of the genus Klebsiella, and according to certain embodiments of the present invention, enterobacter aerogenes ( ⁇ e / OZ c aerogenes) or crab forla pneumoniae / e / / pneumoniae).
  • the strain for producing 2,3-butanediol of the present invention uses sugarcane molasses as a carbon source.
  • the present invention when culturing in a culture medium containing sugarcane molasses as a carbon source of the strain for producing 2, 3-butanedi of the present invention, glucose, other carbon sources (eg, fructose or sucrose)
  • glucose, other carbon sources eg, fructose or sucrose
  • the 2,3-butanediol efficiency and productivity are excellent compared to the case of culturing in a culture medium containing.
  • it provides a method for producing 2,3-butanediol comprising culturing the strain of the present invention in a culture medium.
  • the medium in the culturing step of the method for producing 2,3-butane iol of the present invention includes any medium used for culturing Enterobacter strains, and basically includes a carbon source, a nitrogen source, and an energy source.
  • the carbon source used in the method of the present invention may be a variety of carbohydrates, according to one embodiment of the present invention, molasses, glucose, sucrose or fructose, according to another embodiment of the present invention, molasses.
  • Nitrogen source used in the method of the present invention is an organic nitrogen source is used, according to one embodiment of the present invention, yeast extract and peptone, according to another embodiment of the present invention, yeast extract.
  • the energy source used in the process of the invention is used simultaneously with a carbon source and a nitrogen source.
  • the culture medium may further include a trace element source and an essential amino acid source in addition to a carbon source, a nitrogen source and an energy source.
  • a trace element source KCl (NH 4) S0 4 , Na 2 S0 4, MgS0 4 - 7H 2 0, ZnCl 2, FeCl 3 ⁇ 6H 2 0, MnCl 2 ⁇ 4H 2 0, CuCl 2 ⁇ 2H 2 0 and 3 ⁇ 4B0 3
  • the trace element source KCl include casamino acid as the essential amino acid source.
  • the pH of the sugarcane molasses used as a carbon source in the present invention is very low at pH 4.94, the pH should be adjusted during the culturing process for normal strain growth.
  • the culture medium has an optimal pH of pH 5.5 to pH 7.0.
  • the culture temperature is 30 ° C to 40 ° C, according to one embodiment of the present invention, the culture temperature is 33 ° C to 39 ° C, according to another embodiment of the present invention ;
  • the incubation temperature is 35 ° C to 38 ° C.
  • Method for producing 2, 3-butanedi of the present invention is carried out through various methods of culturing the recombinant strain of the present invention.
  • the culture is batch fermentation (Batch ferment at ion) or fed-batch fermentation (Fed one batch fermentat ion).
  • the term 'batch fermentation' refers to a fermentation that proceeds to the first medium only without treatment to add or remove the medium during fermentation
  • 'feed fermentation' means fermentation that proceeds with addition and removal of medium during fermentation.
  • 2,3-butanes per 0.329 g / g improved by at least 10% compared to the cultivation of wild-type strains when the 2, 3-butanediol preparation of the present invention is carried out by batch fermentation.
  • the present invention provides 2,3-butanedi produced by the production method of the present invention.
  • 2,3-butanedi' refers to the yield of 2,3-butanediol per gram of sugar added to the medium as a carbon source
  • '2,3-butanediol productivity' refers to 1 hour It means 2,3-butanedi produced in L. Since 2,3-butanediol of the present invention is 2,3-butanediol prepared by the above production method, the common content between the two is omitted in order to avoid excessive complexity of the present specification. ⁇ effect ⁇
  • the present invention provides a strain for producing 2,3-butanediol.
  • the present invention can economically produce 2,3-butaneiol using an inexpensive carbon source.
  • La shows the PTS and non-PTS sugar transporter sucrose catabolic pathways of bacteria.
  • Lb shows the gene sequence of three types of intestinal bacterial genomes.
  • ScrA is an ⁇ sucrose transfer protein
  • CscB is a sucrose-specific permease
  • CscA is a sucrose-6-phosphate hydrolase
  • ScrK and CscK are fructokinase
  • Pgm is phosphoglucomutase
  • ScrY is outer membrane number Crossporin
  • ScrR and CscR represent genes of sucrose metabolism inhibitors.
  • FIG. 2 shows cultures of (a) glucose, (b) fructose, (c) sucrose, or (d) sugarcane molasses of wild type (closed rectangle), EMY-0K open circle) and EMY-68 (cross). One 2,3-butanediol production is shown. Error bars are the standard deviation for three experiments.
  • Figure 3 shows sugars for (a) wild type, (b) glucose (closed circle), sucrose (cross), fructose (open triangle) and total sugar (closed rectangle) of EMY-01 and (c) EMY-68. The result of the batch fermentation of consumption is shown.
  • FIG. 4 shows the fermentation results of sugar cane molasses for concentrations of ⁇ (plus), 2, 3-butanedi (open circle), ethanol (open diamond), acetoin (open square) and total sugar (closed square).
  • Indicates. 5A to 5D show the yields of 2 and 3-butanedies of KMK-01 (closed square), KM-02 (open circle) and KMK-08 (x table),
  • FIG. 5A is glucose
  • FIG. 5B is Fructose
  • FIG. 5C is a result of flask culture using sucrose and FIG. 5D with molasses as the only carbon source.
  • 6A-6C show MK-0K in flask culture 6A), KMK-02
  • Enterobacter aerogenes strains were derived from wild type strains (KCTC 2190).
  • EMY-01 was prepared by deleting the lactate dehydrogenase (LdhA) of Enterobacter aerogenes KCTC2190 using the ⁇ red recombination method (Jung et al., 2012).
  • SCRR a gene encoding the sucrose regulator of the Lacl family in the genome of EMY-01, was deleted by a similar method and named EMY-68.
  • the scrR_F F_fw and scrR_F F_rv primers were used to delete ScrR and confirmed by colony PCR (Polymerase Chain React ion) with scrR_con_A and scrR ⁇ con ⁇ B primers.
  • the strains, plasmids and primers used in the present invention are shown in Table 1.
  • the underlined sequence matches the scrR sequence of Enterobacter aerogenes.
  • Red lambda recombination was used to remove the scrR gene from Enterobacter aerogenes.
  • gamma and beta were transformed into enterobacter aerogenes prepared with competent cells to prevent degradation of linear genes and enhance the effect of homologous recombinat ion.
  • An ampicillin resistant pKM208 vector with a tac promoter was used in the present invention and transformation was confirmed.
  • a linear gene for homologous recombination was constructed, and a pKD4 vector with an FRT-kanamycin-FRT cassette was used as a template.
  • enterobacter aerobics was confirmed that the transformed pKM208 vector was incubated at 30 ° C.
  • the pKM208 vector was a temperature sensitive vector and was incubated at 30 ° C because its activity was lost above 37 ° C.
  • OD600 was about 0.1 hours after incubation
  • 1 mM of IPTG (Isopropylthio-p-galactoside) induced red lambda recombinase expression of pKM208.
  • 0D600 was about 0.6, the produced linear genes were transformed into competent cells for electroporation.
  • kanamycin was spread on LB solid medium to which 12.5 wg / mi was added and incubated for 12 hours.
  • Two primers were prepared to confirm the success of homologous recombination.
  • Two to 24 base pairs homologous to both sides of lactate dehydrogenase were named scrR_con_A and _B. When colony PCR was performed using these two primers, homologous recombination was confirmed through the length of the PCR product.
  • FLP recombinase production vector pSClOl ori, cI857, (Datsenko & pCP20
  • scrR_con_A CCGCTTCTTTGCGGATTAT beam 3 ⁇ 4 scrR_con_B GCGCTCATTCATGAAAAATTC beam balm culture medium and culture conditions
  • Fermentation medium of the present invention is 3 g H 2 P0 4) 6.8 g Na 2 HP0 4 , 0.75 g KCl, 5.35 g (NH 4 ) S0 4> 0.28 g Na 2 S0 4 , 0.26 g MgS0 4 ⁇ 7H 2 0, 0.42 g citric acid, 5 g yeast extract, 10 g casamino acid, and 34.2 g / L ZnCl 2 , 2.7 g / L FeCl 3 ⁇ 6H 2 0, 10 g / L MnCl 2 .4H 2 0, 0.85 g / L CuCl 2 0.3 containing 2H 2 0 and 0.31 g / LH 3 B0 3 ml microelement solution (Jung et al., 2012).
  • sugar such as sucrose, glucose fructose, sucrose or sugar cane molasses
  • sugar cane molasses were added to the culture medium as the only carbon source.
  • Glucose, fructose, sucrose and other media components were purchased from Sigma (USA).
  • the sugarcane molasses used in the present invention is native to Brazil and was used by Samyang Genex Bio.
  • Sugar composition of sugarcane molasses was determined to be 87 g / L fructose, 81 g / L glucose and 387 g / L sucrose.
  • Sugar cane molasses is very low (pH 4.94).
  • the initial pH of the medium containing sugarcane molasses was adjusted to pH 6.14 by the addition of 5 M NaOH.
  • the flask was sealed with a si li stopper and incubated at 250 rpm and 37 ° C. for 12 hours in a 250 ml flask containing 50 id medium in a microaerobic environment.
  • the sucrose catabolism gene cluster of Krebssiella pneumoniae is inhibited by ScrR, a sucrose regulator of the Lacl family (Reid & Abratt, 2005).
  • ScrR a sucrose regulator of the Lacl family
  • deletion of the sc / vP gene can improve sucrose utilization.
  • the scrR gene was searched by the Blast (Basic Local Alignment Search Tool; BLAST) sequence of ScrR protein (GenBank Accession No. ⁇ _004593287) of crab forla pneumoniae in Enterobacter aerogenes KCTC 2190 genome (Shin et al., 2012 and Mortlock, 1982).
  • the sequence of the protein found in Enterobacter aerogenes (GenBank accession number YP 304593287) is 92% homologous to the sequence of the ScrR protein of Krebsiella pneumoniae. As can be seen in Table 1, primers were designed to delete sc /? Genes from genome DNA.
  • EMY-68 showed almost the same phenotype as EMY-01 in culture under glucose or fructose. this is. This means that there is no effect of scrR deletion on this carbon source.
  • cell growth of EMY-01 and production of 2,3-butanediol did not increase significantly in the presence of sucrose or sugarcane molasses, whereas EMY-68 was significantly higher compared to wild type and EMY-01.
  • Cross consumption and 2, 3-butanedi showed production capacity (FIG. 2).
  • EMY-68 consumed 57.65 g / L sucrose for 12 hours and produced 18.05 g / L of 2,3-butanediol.
  • Sucrose is a dimer of glucose and fructose, but the scrR deletion only affected sucrose metabolism, not fructose.
  • Enterobacter aerogenes saw extracellular fructose through the mult i component phosphotransferase system (PTS) to convert intracellular fructose-1-phosphate to use fructose (Ferenci & Kornberg, 1973; Kelker et al. , 1970).
  • Fructose -1-phosphate is phosphorylated to fructose-1,6-2 phosphate and is consumed by glycolysis.
  • some of the fructose of sucrose is hydrolyzed by fructokinase and converted to fructose-6-phosphate (Fig. La).
  • Crab forla pneumoniae strains have genes that can use sucrose, and these genes are operon (ScrA, EI I transport protein for sucrose; ScrB, sucrose ⁇ 6 ⁇ phosphate hydrolase! ScrK, f ructokinase; ScrY, outer membrane sucrose porin). It also has a scrR gene that controls the expression of this operon. Therefore, scrR was deleted through the lambda red recombination method in order to increase the sucrose and molasses utilization in the crab forla pneumoniae strain. In addition, the lactate dehydrogenase was removed in the same way to enjoy lactate production, a major by-product, and to prevent acidification of the medium.
  • Klebsiella pneumoniae strain belongs to two classes of pathogens, there are limitations in utilizing industrialization. Because pathogenicity is known to be caused by extracellular capsules, the wabG gene involved in the formation of external capsules was removed in the same way. Therefore, a total of three mutant strains were developed from the Crab forla pneumoniae strains, and Kb 01 for the Klebsiella pneumoniae ( ⁇ wabG) and Kepsiella pneumoniae (AwabG A.ldhA) Monia (AwabGAldhAAscrR) was named KMK-08 and comparative experiments were conducted on the production of 2,3-butanedie using sucrose and molasses. Culture medium and culture conditions
  • the culture of the Krebsiella pneumoniae mutant strain of the present invention was applied in the same manner as in the culture medium and culture conditions, ⁇ conditions of Example 1. result
  • KMK-08 showed a 10% decrease in 2,3-butanediol production when glucose or fructose was used as the only carbon source compared to the parent strain.
  • sucrose was used as the carbon source, it produced almost the same 2,3-butanediol as the parent strain, and when molasses was the carbon source, it showed an ability to produce 2,3-butanedial which was increased by about 32% compared to the parent strain. All.
  • the sugar cane molasses was used as a carbon source by culturing Enterobacter aerogenes KCTC2190 and Krebsciella pneumoniae metabolized to produce 2,3-butanediol.
  • IdhA and scrR-deleted Enterobacter aerogenes were fermented with sugarcane molasses as a carbon source, they produced 72.89 g / L of 2,3-butanediol at 36 hours and showed an efficiency per 0.36 g product / g.

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Abstract

Cette invention concerne une souche recombinée pour la production de 2,3-butanediol, comprenant (a) une lactate déshydrogénase inactivée et (b) un régulateur de saccharose inactivé. Selon la présente invention, il est possible de produire du 2,3-butanediol de manière économique à l'aide d'une source de carbone bon marché, l'efficacité et la productivité dudit 2,3-butanediol étant remarquables comparées à celles obtenues à l'aide d'une souche de type sauvage.
PCT/KR2014/000677 2013-01-25 2014-01-23 Souche recombinée pour la production de 2,3-butanediol, comprenant (a) une lactate déshydrogénase inactivée et (b) un régulateur de saccharose inactivé WO2014116042A1 (fr)

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KR10-2013-0008816 2013-01-25
KR20130008816 2013-01-25
KR1020130083817A KR101542879B1 (ko) 2013-01-25 2013-07-16 (a) 불활성화 락테이트 디하이드로제나제 및 (b) 불활성화 수크로즈 조절자를 포함하는 2,3-부탄다이올 생성용 재조합 균주
KR10-2013-0083817 2013-07-16

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KR20120104765A (ko) * 2011-03-14 2012-09-24 서강대학교산학협력단 2, 3-부탄다이올 생산량 향상을 위한 락테이트 디하이드로겐에이즈가 제거 된 돌연변이 엔테로박터 에어로진스

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US20100112655A1 (en) * 2008-09-29 2010-05-06 Butamax(Tm) Advanced Biofuels Llc Enhanced pyruvate to 2,3-butanediol conversion in lactic acid bacteria
KR20120104765A (ko) * 2011-03-14 2012-09-24 서강대학교산학협력단 2, 3-부탄다이올 생산량 향상을 위한 락테이트 디하이드로겐에이즈가 제거 된 돌연변이 엔테로박터 에어로진스

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