CN106497844A - One plant is produced the tartaric genetic engineering bacteriums of L and its construction method and application - Google Patents

One plant is produced the tartaric genetic engineering bacteriums of L and its construction method and application Download PDF

Info

Publication number
CN106497844A
CN106497844A CN201611141246.6A CN201611141246A CN106497844A CN 106497844 A CN106497844 A CN 106497844A CN 201611141246 A CN201611141246 A CN 201611141246A CN 106497844 A CN106497844 A CN 106497844A
Authority
CN
China
Prior art keywords
gene
acid
fermentation
pfl
genetic engineering
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201611141246.6A
Other languages
Chinese (zh)
Other versions
CN106497844B (en
Inventor
潘春
马江锋
姜岷
万屹东
芮新生
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CHANGMAO BIOCHEMICAL ENGINEERING Co Ltd
Original Assignee
CHANGMAO BIOCHEMICAL ENGINEERING Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CHANGMAO BIOCHEMICAL ENGINEERING Co Ltd filed Critical CHANGMAO BIOCHEMICAL ENGINEERING Co Ltd
Priority to CN201611141246.6A priority Critical patent/CN106497844B/en
Publication of CN106497844A publication Critical patent/CN106497844A/en
Application granted granted Critical
Publication of CN106497844B publication Critical patent/CN106497844B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01037—Malate dehydrogenase (1.1.1.37)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01054—Formate C-acetyltransferase (2.3.1.54), i.e. pyruvate formate-lyase or PFL

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

The invention discloses one plant is produced the tartaric genetic engineering bacteriums of L, preserving number is CCTCC NO：M2016455.Present invention also offers the construction method of the bacterial strain, the bacterial strain is related in wild-type e. coli key gene pyruvate formate-lyase gene in acetone acid decomposition approachpflAnd lactate dehydrogenase geneldh, and oxaloacetic acid downstream decomposition pathway key gene malate dehydrogenase enzyme coding genemdhKnockout, while one section of gene order of synthetic, import and knock outpfl、ldh、mdhHigh vigor expression is carried out in the escherichia coli of gene.The present invention discloses above-mentioned bacterial strains prepare the application in L tartaric acid in fermentation.Present invention achieves adopt renewable biomass resources to prepare the tartaric routes of L for fermenting raw materials completely, route green, the environmental protection.

Description

The genetic engineering bacterium of one plant of product L-TARTARIC ACID and its construction method and application
Technical field
The invention belongs to gene engineering technology field, and in particular to one plant using glucose or xylose fermentation for producing L-TARTARIC ACID Genetic engineering bacterium and its construction method and application.
Background technology
L-TARTARIC ACID is widely present in the various plants fruit of nature, in wherein ripe Fructus Vitis viniferae L-TARTARIC ACID content compared with Many, which is a kind of purposes natural organic acidss widely, mainly as food additive and medical resolving agent be applied to food, The field such as medicine and chemical industry, it was reported that L-TARTARIC ACID can be additionally used in the preparation of nano material and change as fuel economy Property agent and antiwear additive.According to incompletely statistics, L-TARTARIC ACID market whole world year in 2000 consumes about 20,000 tons, is mainly used in eating Conduct industry.
At present, the production of L-TARTARIC ACID mainly has 4 kinds of methods, including extraction method, chemical resolution method, enzyme process and fermentation-urge Change method, current extraction method is limited output limited by raw material, and chemical resolution method is eliminated substantially due to a large amount of uses of solvent.Enzyme Method is the topmost production method of current L-TARTARIC ACID, and production efficiency is high, but the method relies on the cis epoxy succinic of fossil feedstock Acid, is therefore faced with the problem for relying on fossil resource.Fermentation-catalysis method solves the problems, such as raw material well, with glucose etc. Saccharic is raw material, but glucose fermentation first need to be converted into 5- keto-D-gluconic acids by the way of fermentation by its production process, L-TARTARIC ACID is catalyzed and synthesized using metallic catalyst further, is caused the method overall catalytic efficiency still low, is needed technology Break through.And increased the operability difficulty for amplifying production process by the way of two-step catalysis, and production cost is greatly improved.
If can be by the metabolic engineering to bacterial strain so as to one-step fermentation can be passed through with saccharics such as glucoses as raw material Method produces L-TARTARIC ACID, not only solves the problem of raw material, and its relatively simple operation mode will effectively improve which and produce effect Rate reduces cost, more manufacturing feasibility.
The present invention is reconstructed the metabolism network of bacterial strain with wild-type e. coli as starting strain, suppresses carbon flow flow direction breast The by-products such as acid, formic acid, while strengthening the metabolic fluxes for flowing to L-TARTARIC ACID, and block the catabolic pathway of L-TARTARIC ACID, lead to Cross serial genes operation, build obtain can one-step fermentation prepare the genetic engineering bacterium of L-TARTARIC ACID, the innovative works are not See any report, with significant novelty.
Content of the invention
It is an object of the invention to, overcome the defect of prior art, there is provided provide one plant and L- winestones are produced using monosaccharide fermentation The genetic engineering bacterium of acid.
For achieving the above object, the present invention is adopted the following technical scheme that：
The genetic engineering bacterium of one plant of product L-TARTARIC ACID that fermented using monosaccharide, its Classification And Nomenclature are colon bacillus (Escherichia coli) CM-MA-184, is preserved in China typical culture collection center on 5th in September in 2016 CCTCC, deposit number is：CCTCC NO：M 2016455.
Another object of the present invention is to providing the construction method of said gene engineering bacteria CM-MA-184.
The construction method of the genetic engineering bacterium CM-MA-184 comprises the steps：
(1) with wild type colon bacillus (Escherichia coli) K12 as starting strain, using RED restructuring skills Three sections of genes in art traceless knockout colon bacillus K12, respectively the key gene acetone acid in acetone acid decomposition approach (EcoGene registration numbers are for formate lyase gene pfl (EcoGene registration numbers be EG10701), lactate dehydrogenase gene ldh EG13186) and oxaloacetic acid downstream decompose pathway key gene malate dehydrogenase enzyme coding gene mdh (EcoGene registration numbers For EG10576).
(2) phosphoenolpyruvic acid of high vigor simple substance grain coexpression catalytic phosphatase enol pyruvic acid to oxaloacetic acid Carboxylation kinases (encoding gene is pck) and catalysis oxaloacetic acid are to L-TARTARIC ACID dehydratase (encoding gene be ttd1, ttd2)；Tool Body is, by aforementioned three sections of gene chemical synthesis, one section of objective gene sequence pck-ttd1-ttd2, its nucleotide sequence such as SEQ ID NO： Shown in 1, objective gene sequence is imported simple substance grain, obtain recombiant plasmid, three sections will have been knocked out in recombiant plasmid steps for importing (1) The K12 bacterial strain competence of gene, the positive transformant of acquisition are the genetic engineering bacterium of the present invention.Wherein, pck nucleotide sequences Such as SEQ ID NO：Shown in 2, ttd1 nucleotide sequences such as SEQ ID NO：Shown in 3, ttd2 nucleotide sequences such as SEQ ID NO：4 Shown.
Wherein, described expression plasmid is pTrc99a plasmids.
A further object of the present invention is said gene engineering bacteria in the application for producing L-TARTARIC ACID.Especially anti-in electrochemistry Answer the application that L-TARTARIC ACID is prepared in device using monosaccharide fermentation.
Provide a kind of application that L-TARTARIC ACID is specifically prepared in electrochemical reactor using monosaccharide fermentation in the present invention Method, electrochemical reactor anode fill fermentation medium, and its formula is nicotinic acid 0.1mM；Citric acid 3.0g/L；Na2HPO4·7H2O 3.00g/L；KH2PO48.00g/L；(NH4)2HPO420.00g/L；NH4Cl 10g/L；(NH4)2SO45g/L；MgSO4·7H2O 1.00g/L；CaCl2·2H2O 10.0mg/L；ZnSO4·7H2O 0.5mg/L；CuCl2·2H2O 0.25mg/L；MnSO4·H2O 2.5mg/L；CoCl2·6H2O 1.75mg/L；H3BO30.12mg/L；Al2(SO4)3·xH2O 1.77mg/L；Na2MoO4·2H2O 0.5mg/L；Fe (III) citrate 16.1mg/L, dimethyl diaminophenazine chloride 0.1mM, solvent are water, and it is 7.0 to adjust pH with ammonia after sterilizing, Wherein 60g/L glucoses are added after individually sterilizing；
Cathodic Composition is：The phosphate buffer of 100mM, pH=6.8, NaCl 6g/L.
Wherein, seed liquor incubation is as follows：
(S1) bacterium solution preserved in cryopreservation tube is transferred in LB culture medium from cryopreservation tube for 1～2% by volume fraction, is had Oxygen 10～12h of culture；
(S2) it is 1～2% LB culture medium for being transferred to seed fermentation tank by the culture fluid after processing through S1 by volume fraction In；
(S3) thalline OD is treated600During to 2.5～4, culture fluid 5～10% is seeded to electrochemical reactor by volume Anode.
Wherein, in seed liquor incubation, in step (S1) and (S2), cultivation temperature is 35～37 DEG C.
Wherein, adopt anaerobic fermentation pattern in step (S3), during need logical carbon dioxide to maintain anaerobic environment.
Wherein, the anaerobic fermentation process of anode maintains temperature at 30～32 DEG C, and incubation pH ammonia is adjusted to 6.8～ 7.0.
Beneficial effect：
The genetic engineering bacterium that the present invention builds can realize that one-step fermentation prepares L-TARTARIC ACID, innovatively establish L- wine The production method of stone acid one-step fermentation, has thoroughly broken away from the problem for depending on petroleum-based feedstock, it is achieved that completely using renewable Biomass resource (such as glucose, xylose etc.) prepares the route of L-TARTARIC ACID for fermenting raw materials, route green, the environmental protection.
Description of the drawings
Fig. 1 is the bacterium colony PCR proof diagrams of E.coli K12 (Δ pfl) mutant for having knocked out pfl genes；
Fig. 2 is that the bacterium colony PCR of E.coli K12 (Δ pfl, the Δ ldh) mutant for having knocked out pfl genes and ldh genes is tested Card figure；
Fig. 3 is that the E.coli K12 (Δ pfl, Δ ldh, Δ mdh) for having knocked out pfl genes, ldh genes and mdh genes dash forward The bacterium colony PCR proof diagrams of variant；
Fig. 4 is the building process schematic diagram of the recombiant plasmid based on expression vector pTrc99a.
Biomaterial of the present invention, its Classification And Nomenclature are colon bacillus (Escherichia coli) CM-MA- 184, China typical culture collection center CCTCC is preserved within 5th in September in 2016, deposit number is：CCTCC NO：M 2016455, preservation address is：China. Wuhan. Wuhan University.
Specific embodiment
According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that real Apply the content described by example and be merely to illustrate the present invention, and should not also without limitation on sheet described in detail in claims Invention.
In embodiment, starting strain Escherichia coli K12 used are purchased from ATCC (NO.53678)；PIJ773 used Plasmid and pKD46 plasmids by Britain John En Nisi centers (John Innes Centre, Norwich Research Park, Colney, Norwich NR47UH, UK) provide；PTrc99a plasmids used are purchased from Addgene companies.
Embodiment 1
This example demonstrates that being knocked out after tri- gene of pfl, ldh and mdh using RED recombinant techniques, recombinant bacterium and starting strain Escherichia coli K12 when bacterium colony PCR is identified, the difference of electrophoresis pattern, illustrate trigenic knock out successfully, which is big Cause process is as follows：
(1) with pIJ773 plasmids as template, pKD46 is restructuring enzyme induction expression plasmid, using RED recombinant techniques, seamless Knock out tri- gene of pfl, ldh and mdh, pfl, ldh and mdh trigenic knockout order not influence technique effect；
Gene knockout step and process refer to " Gust B, Kieser T, Chater K (2002) PCR targeting system in Streptomyces coelicolor A3(2).The John Innes Foundation,Norwich”.
(2) Escherichia coli K12 are adopted for control strain, three pairs of primer pair gene knockout processes is respectively adopted In positive bacteria carry out bacterium colony PCR identifications, verify that the primer sequence adopted during tri- gene of pfl, ldh and mdh is as follows：
(I) pfl genes are verified：Forward primer 5 '-GAGGATGCGGAAAAAATTCAACTC-3 ',
Downstream primer 5 '-TAGTAGTGCAAATGTCCTAATACGG-3 '；
PCR system (20uL systems)：H2O 13.3uL, Buffer 2uL, dNTP 1.6uL, MgCl21.6uL, upstream and downstream draw The each 0.5uL of thing, Ex-Taq 0.5uL, toothpick point take single bacterium colony；
PCR conditions：95 DEG C, 10 minutes；(94,45 seconds, 54,45 seconds, 72,100 seconds, 30 circulations)；72,10 minutes.
(II) ldh genes are verified：Forward primer 5 '-CTTAAATGTGATTCAACATCACTG-3 ',
Downstream primer 5 '-CGTAACAGCAATTTTGTCATTAC-3 '；
PCR system (20uL systems)：H2O 13.3uL, Buffer 2uL, dNTP 1.6uL, MgCl21.6uL, upstream and downstream draw The each 0.5uL of thing, Ex-Taq 0.5uL, toothpick point take single bacterium colony；
PCR conditions：95 DEG C, 10 minutes；(94,45 seconds, 52,45 seconds, 72,100 seconds, 30 circulations)；72,10 minutes.
(III) mdh genes are verified：Forward primer 5 '-CTTGCGTGACTACACATTCTTGAG-3 ',
Downstream primer 5 '-AAGGGATCGTGGTTAATGAAGTGT-3 '；
PCR system (20uL systems)：H2O 13.3uL, Buffer 2uL, dNTP 1.6uL, MgCl21.6uL, upstream and downstream draw The each 0.5uL of thing, Ex-Taq 0.5uL, toothpick point take single bacterium colony；
PCR conditions：95 DEG C, 10 minutes；(94,45 seconds, 55,45 seconds, 72,100 seconds, 30 circulations)；72,10 minutes.
The electroresis appraisal figure of bacterium colony PCR is successively such as Fig. 1, Fig. 2, shown in Fig. 3, Fig. 1 E.coli K12 (Δ pfl) mutant Bacterium colony PCR verifies that the bacterium colony PCR of Fig. 2 E.coli (Δ pfl, Δ ldh) mutant is verified, Fig. 3 E.coli (Δ pfl, Δ ldh, Δ mdh) mutant bacterium colony PCR checking.
Embodiment 2
This example demonstrates that build that there is high activity PEP carboxylase kinases and L-TARTARIC ACID dehydratase weight The process of group bacterial strain, its substantially process such as Fig. 4, including：
(1) a section such as SEQ ID NO of synthetic by the way of chemosynthesis：Sequence shown in 1, in the sequence of synthesis Downstream is respectively provided with EcoR I and Xba I restriction enzyme sites, and successively containing from bacillus subtilises phosphoric acid enol form propanone Sour carboxylation kinase gene (pck genes, its nucleotide sequence such as SEQ ID NO：Shown in 2) and derive from colibacillary L- winestones (ttd1, ttd2 gene, ttd1, ttd2 gene are respectively two of L-TARTARIC ACID dehydrase gene to the sequence of sour dehydrase gene Subunit, has knocked out in two subunits and has obtained ttd1, ttd2 gene after original one section of sequence, and its nucleotide sequence is respectively such as SEQ ID NO：3、SEQ ID NO：Shown in 4).The chemosynthesis of sequence are completed by Jin Sirui companies.
(2) gene order for synthesizing and expression plasmid pTrc99a are obtained with EcoR I and Xba I double digestions, connection respectively Recombiant plasmid pTrc99a-pck-ttd1-ttd2.
2nd, plasmid pTrc99a-pck-ttd1-ttd2 is imported E.coli (Δ pfl, Δ ldh, Δ mdh) senses in embodiment 1 State, the positive transformant of acquisition is received to be the new structure bacterial strain of the present invention.
Wherein, competent construction method, concrete steps for importing, positive transformant obtaining step include：
A () is inoculated with E.coli (Δ pfl, Δ ldh, Δ mdh) single bacterium colony in 5mL LB culture fluid, 37 DEG C, 200r/min is trained Foster 8h or overnight.
B () is added to 2.5mL culture fluid in the 2L triangular flasks equipped with 500mL LB culture fluid, 37 DEG C, and 200r/min is cultivated, An OD is measured every 20min600Value.
C () works as OD600For 0.3～0.4 when, inoculum is placed in ice-water bath 10～15min of cooling rapidly, then It is transferred in the 1L centrifuge bottles of pre-cooling, in 4 DEG C, 5,000r/min centrifugation 20min precipitate the water dissolution with 5mL pre-coolings.
D () adds 500mL icy waters, resuspended and mix, and by step (c) repeated centrifugation 1 time, outwells supernatant immediately, With remaining liquid again suspension cell.
E () takes 50 μ L competent cells, plasmid pTrc99a-pck-ttd1-ttd2 about 50～100ng, electric shock condition：200 Ω, 25 μ F, shock voltage 1.25kV, shock by electricity 4～5ms of time, is rapidly added the 1mL SOC of pre-cooling after electric shock, 37 DEG C, 200r/ Min cultivates 1h, is applied to the plate screening positive transformant with amicillin resistance afterwards.
3rd, the mode preserved with glycerol preserves positive transformant, and its process includes：
A () takes the positive transformant single bacterium colony that can be grown on the flat board of amicillin resistance with toothpick, 5mL LB are trained Nutrient solution, 37 DEG C, 200r/min culture 8h or overnight.
B () is added to 2.5mL culture fluid in the 2L triangular flasks equipped with 500mL LB culture fluid, 37 DEG C, and 200r/min is cultivated, An OD is measured every 20min600Value.
C () works as OD600For 0.8～1.2 when, mix with the sterilized water that 500mL glycerol contents are 30%, after 1.5mL subpackages Preserve in -80 DEG C of cryopreservation tubes.
Embodiment 3
This example demonstrates that the new recombination bacillus coli for building fermentation in electrochemical reactor prepares answering for L-TARTARIC ACID With sweat includes：
(S1) cryopreservation tube preserved in Example 2, takes bacterium solution for 1～2% from cryopreservation tube by volume fraction and is transferred to LB In culture medium, aerobic 10～12h of culture；
(S2) culture fluid for obtaining S1 is 1～2% to be transferred in the LB culture medium of seed fermentation tank by volume fraction；
(S3) thalline OD is treated600During to 2.5～4, culture fluid 5～10% is seeded to electrochemical reactor by volume Anode, anode fill fermentation medium, and the formula of the fermentation medium is：Nicotinic acid 0.1mM；Citric acid 3.0g/L；Na2HPO4· 7H2O 3.00g/L；KH2PO48.00g/L；(NH4)2HPO420.00g/L；NH4Cl 10g/L；(NH4)2SO45g/L； MgSO4·7H2O 1.00g/L；CaCl2·2H2O 10.0mg/L；ZnSO4·7H2O 0.5mg/L；CuCl2·2H2O 0.25mg/ L；MnSO4·H2O 2.5mg/L；CoCl2·6H2O 1.75mg/L；H3BO30.12mg/L；Al2(SO4)3·xH2O 1.77mg/ L；Na2MoO4·2H2O 0.5mg/L；Fe(III)citrate 16.1mg/L；Dimethyl diaminophenazine chloride 0.1mM, solvent are water, use after sterilizing It is 7.0 that ammonia adjusts pH, and wherein 60g/L glucoses are added after individually sterilizing.
Wherein, in seed liquor incubation, in step (S1) and (S2), cultivation temperature is 35～37 DEG C.In step (S3) Using anaerobic fermentation pattern, during need logical carbon dioxide to maintain anaerobic environment.The anaerobic fermentation process of anode maintains temperature At 30～32 DEG C, incubation pH ammonia is adjusted to 6.8～7.0 to degree.The formula of electrochemical reaction appts negative electrode is：Phosphate Buffer (100mM, pH 6.8), NaCl 6g/L.
Recombinant bacterial strain CM-MA-184 the results are shown in Table after anaerobic fermentation 48h in common fermentation tank and electrochemical reactor 1.
Fermentation and acid situation under the conditions of 1 recombinant bacterial strain different fermentations of table
Sequence table
<110>Nanjing University of Technology
<120>The genetic engineering bacterium of one plant of product L-TARTARIC ACID and its construction method and application
<130> xb16121201
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 3968
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 1
ttgacgtttt gttgcctatt gtccgaatgt taagtgttaa tggtgggaaa tctgggaaag 60
ttgccccctg gaatgtgtga taattgccca aatctgaacc caatggccat ggacggggaa 120
tgaactgtcg ggggacggtt gaggttaatt cttgaaacca cccccaaaat aggctattaa 180
aacgggtgct ctcatattaa agaaagtgtg tagatgcgtg tgggcagggg gtaggtccac 240
tggtaatgac aaatgtgtcc gttgtctcac ctaaagtttt aactagttct gtatctgaaa 300
gctacgctag ggggcgagaa ctctgtcgaa tgacacaaaa tctggagaag taatgactac 360
tgctgcaatc aggggccttc agggtgaggc gccgaccaag aataaggaac tgctgaactg 420
gatcgcagac gccgtcgagc tcttccagcc tgaggctgtt gtgttcgttg atggatccca 480
ggctgagtgg gatcgcatgg cggaggatct tgttgaagcc ggtaccctca tcaagctcaa 540
cgaggaaaag cgtccgaaca gctacctagc tcgttccaac ccatctgacg ttgcgcgcgt 600
tgagtcccgc accttcatct gctccgagaa ggaagaagat gctggcccaa ccaacaactg 660
ggctccacca caggcaatga aggacgaaat gtccaagcat tacgctggtt ccatgaaggg 720
gcgcaccatg tacgtcgtgc ctttctgcat gggtccaatc agcgatccgg accctaagct 780
tggtgtgcag ctcactgact ccgagtacgt tgtcatgtcc atgcgcatca tgacccgcat 840
gggtattgaa gcgctggaca agatcggcgc gaacggtagc ttcgttaagt gcctccactc 900
cgttggtgct cctttggagc caggccagga agacgttgca tggccttgca acgacaccaa 960
gtacatcacc cagttcccag agaccaagga aatttggtcc tacggttccg gctacggcgg 1020
aaacgcaatt ctggcaaaga agtgctacgc actgcgtatc gcatctgtca tggctcgcga 1080
agaaggctgg atggctgagc acatgctcat cctgaagctg atcaacccag agggcaaggc 1140
gtaccacatc gcagcagcat tcccatctgc ttgtggcaag accaacctcg ccatgatcac 1200
tccaaccatc ccaggctgga ccgctcaggt tgttggcgac gacattgctt ggctgaagct 1260
gcgcgaggac ggcctctacg cagttaaccc agaaaacggt ttcttcggtg ttgctccagg 1320
caccaactac gcatctaacc caatcgcgat gaagaccatg gaaccaggca acaccctgtt 1380
caccaacgtg gcactcaccg acgacggcga catctggtgg gagggcatgg atggcgacgc 1440
ccccgctcac ctcattgact ggaagggtaa cgactggacc ccagagtccg acgaaaacgc 1500
tgctcaccct aactcccgtt actgcgtagc aatcgaccag tccccagcag cagcacctga 1560
gttcaacgac tgggaaggcg tcaagatcga cgcaatcctc ttcggtggac gtcgcgcaga 1620
caccgtccca ctggttactc agacctacga ctgggagcac ggcaccatgg ttggtgcact 1680
gctcgcatcc ggtcagaccg cagcttccgc agaagcaaag gtcggcacac tccgccacga 1740
cccaatggca atgctcccat tcattggcta caacgctggt gaatacctgc agaactggat 1800
tgacatgggc aacaagggtg gcgacaagat gccatccatc ttcctggtca actggttccg 1860
ccgtggcgaa gatggacgct tcctgtggcc tggcttcggc gacaactccc gcgttctgaa 1920
gtgggtcatc gaccgcatcg aaggccgcgt tggcgcagac gagaccgttg ttggacacac 1980
cgctaaggct gaagacctcg acctcgacgg cctcgacacc ccaattgagg atgtcaagga 2040
agcactgacc gctcctgcag agcagtgggc gaacgacgtt caagacaacg ccgagtacct 2100
cactttcctc ggaccacgtg ttcctgcaga ggttcacagc cagttcgatg ctctgaaggc 2160
ccgcatttca gcagctcacg cttaaagttc acgcttaaga actgctaaat aacaagaaag 2220
gctcccgaaa gggagccttt cttgtcgtta agcgatgaat tcctcaaaac ctcagtgctt 2280
tttaaacacc aacaccaagt tacttaccgc gaattctcgg agcactggga ctttaaccat 2340
ccaccaggcc caatacgggt ggtagcgggg aaaagcggca accaattccg cattgcccac 2400
ggaggctccc cattccagcc cctcccggca ggacacatta aacagtgaca tgatgagcga 2460
aagtaataag caacaggcag tgaataagtt gacagagatt gtcgctaact ttaccgccat 2520
gatttctacc cgaatgcctg atgacgtggt ggataaacta aaacagctaa aggatgccga 2580
aacgtcgtcg atggggaaaa ttatctacca tacgatgttc gacaacatgc aaaaagcgat 2640
tgacctgaat cgtcctgcct gtcaggacac cggggagatt atgttcttcg ttaaagtcgg 2700
ttcccgcttc ccactgcttg gcgagctgca aagcatactc aaacaagccg tggaagaggc 2760
aaccgtcaaa gcgccactac gtcacaatgc ggtagaaatt tttgacgaag taaacaccgg 2820
caaaaatacc ggtagcggcg taccgtgggt cacctgggac atcatccccg acaatgacga 2880
tgcggaaatc gaagtttaca tggcaggcgg cggctgcacg ctacctggcc gctcgaaagt 2940
gttaatgccg tcagaaggct acgaaggcgt ggtgaaattc gtcttcgaaa atatctccac 3000
cctcgccgta aacgcctgtc caccggtact ggtgggcgtg ggcatcgcca cctcggtgga 3060
aaccgccgcc gtactctcgc gtaaagccat tttgcgcccg attggcagcc gccatcccaa 3120
tccaaaagcg gcagaactgg agctacgcct ggaagaagga ctcaaccgtc tggggattgg 3180
tccacaaggg ctgaccggca acagttcagt gatgggcgta catatcgaat ctgccgcccg 3240
ccatccgtca accatcggcg ttgctgtctc taccggctgc tgggcgcatc gtcgcggcac 3300
gctgctggtt catgccgatc tcacctttga aaatctgtct cacacccgga gcgcgttaat 3360
gaaaaagatc ctgacaaccc cgatcaaagc tgaagatctg caagatattc gcgtcggcga 3420
tgtgatctac ctgaccggta cgctggtgac ctgccgcgac gtttgtcacc gccgtttgat 3480
cgaactgaaa cgtccgatcc cttacgatct caacggcaaa gcgattttcc acgctggccc 3540
catcgtgcgc aaaaacggcg acaaatggga gatggtctcc gtcggcccga caaccagtat 3600
gcgtatggaa agttttgaac gtgaatttat tgagcagacc ggcgtgaaac tggtggttgg 3660
caaaggtggt atggggccgc tgaccgaaga aggctgccag aaattcaagg cgctacatgt 3720
gattttcccg gcaggctgcg cggtgctggc ggcaacccag gtggaagaga ttgaagaagt 3780
gcactggaca gagctcggaa tgccggagtc actgtgggtc tgccgggtca aagagttcgg 3840
cccgctgatt gtctctattg atacccacgg caacaacctg atagccgaaa acaaaaagct 3900
gttcgccgaa cgccgcgatc ccatcgtgga agagatctgc gagcacgtcc attacatcaa 3960
atagatct 3968
<210> 2
<211> 2449
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 2
ttgacgtttt gttgcctatt gtccgaatgt taagtgttaa tggtgggaaa tctgggaaag 60
ttgccccctg gaatgtgtga taattgccca aatctgaacc caatggccat ggacggggaa 120
tgaactgtcg ggggacggtt gaggttaatt cttgaaacca cccccaaaat aggctattaa 180
aacgggtgct ctcatattaa agaaagtgtg tagatgcgtg tgggcagggg gtaggtccac 240
tggtaatgac aaatgtgtcc gttgtctcac ctaaagtttt aactagttct gtatctgaaa 300
gctacgctag ggggcgagaa ctctgtcgaa tgacacaaaa tctggagaag taatgactac 360
tgctgcaatc aggggccttc agggtgaggc gccgaccaag aataaggaac tgctgaactg 420
gatcgcagac gccgtcgagc tcttccagcc tgaggctgtt gtgttcgttg atggatccca 480
ggctgagtgg gatcgcatgg cggaggatct tgttgaagcc ggtaccctca tcaagctcaa 540
cgaggaaaag cgtccgaaca gctacctagc tcgttccaac ccatctgacg ttgcgcgcgt 600
tgagtcccgc accttcatct gctccgagaa ggaagaagat gctggcccaa ccaacaactg 660
ggctccacca caggcaatga aggacgaaat gtccaagcat tacgctggtt ccatgaaggg 720
gcgcaccatg tacgtcgtgc ctttctgcat gggtccaatc agcgatccgg accctaagct 780
tggtgtgcag ctcactgact ccgagtacgt tgtcatgtcc atgcgcatca tgacccgcat 840
gggtattgaa gcgctggaca agatcggcgc gaacggtagc ttcgttaagt gcctccactc 900
cgttggtgct cctttggagc caggccagga agacgttgca tggccttgca acgacaccaa 960
gtacatcacc cagttcccag agaccaagga aatttggtcc tacggttccg gctacggcgg 1020
aaacgcaatt ctggcaaaga agtgctacgc actgcgtatc gcatctgtca tggctcgcga 1080
agaaggctgg atggctgagc acatgctcat cctgaagctg atcaacccag agggcaaggc 1140
gtaccacatc gcagcagcat tcccatctgc ttgtggcaag accaacctcg ccatgatcac 1200
tccaaccatc ccaggctgga ccgctcaggt tgttggcgac gacattgctt ggctgaagct 1260
gcgcgaggac ggcctctacg cagttaaccc agaaaacggt ttcttcggtg ttgctccagg 1320
caccaactac gcatctaacc caatcgcgat gaagaccatg gaaccaggca acaccctgtt 1380
caccaacgtg gcactcaccg acgacggcga catctggtgg gagggcatgg atggcgacgc 1440
ccccgctcac ctcattgact ggaagggtaa cgactggacc ccagagtccg acgaaaacgc 1500
tgctcaccct aactcccgtt actgcgtagc aatcgaccag tccccagcag cagcacctga 1560
gttcaacgac tgggaaggcg tcaagatcga cgcaatcctc ttcggtggac gtcgcgcaga 1620
caccgtccca ctggttactc agacctacga ctgggagcac ggcaccatgg ttggtgcact 1680
gctcgcatcc ggtcagaccg cagcttccgc agaagcaaag gtcggcacac tccgccacga 1740
cccaatggca atgctcccat tcattggcta caacgctggt gaatacctgc agaactggat 1800
tgacatgggc aacaagggtg gcgacaagat gccatccatc ttcctggtca actggttccg 1860
ccgtggcgaa gatggacgct tcctgtggcc tggcttcggc gacaactccc gcgttctgaa 1920
gtgggtcatc gaccgcatcg aaggccgcgt tggcgcagac gagaccgttg ttggacacac 1980
cgctaaggct gaagacctcg acctcgacgg cctcgacacc ccaattgagg atgtcaagga 2040
agcactgacc gctcctgcag agcagtgggc gaacgacgtt caagacaacg ccgagtacct 2100
cactttcctc ggaccacgtg ttcctgcaga ggttcacagc cagttcgatg ctctgaaggc 2160
ccgcatttca gcagctcacg cttaaagttc acgcttaaga actgctaaat aacaagaaag 2220
gctcccgaaa gggagccttt cttgtcgtta agcgatgaat tcctcaaaac ctcagtgctt 2280
tttaaacacc aacaccaagt tacttaccgc gaattctcgg agcactggga ctttaaccat 2340
ccaccaggcc caatacgggt ggtagcgggg aaaagcggca accaattccg cattgcccac 2400
ggaggctccc cattccagcc cctcccggca ggacacatta aacagtgac 2449
<210> 3
<211> 909
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 3
atgatgagcg aaagtaataa gcaacaggca gtgaataagt tgacagagat tgtcgctaac 60
tttaccgcca tgatttctac ccgaatgcct gatgacgtgg tggataaact aaaacagcta 120
aaggatgccg aaacgtcgtc gatggggaaa attatctacc atacgatgtt cgacaacatg 180
caaaaagcga ttgacctgaa tcgtcctgcc tgtcaggaca ccggggagat tatgttcttc 240
gttaaagtcg gttcccgctt cccactgctt ggcgagctgc aaagcatact caaacaagcc 300
gtggaagagg caaccgtcaa agcgccacta cgtcacaatg cggtagaaat ttttgacgaa 360
gtaaacaccg gcaaaaatac cggtagcggc gtaccgtggg tcacctggga catcatcccc 420
gacaatgacg atgcggaaat cgaagtttac atggcaggcg gcggctgcac gctacctggc 480
cgctcgaaag tgttaatgcc gtcagaaggc tacgaaggcg tggtgaaatt cgtcttcgaa 540
aatatctcca ccctcgccgt aaacgcctgt ccaccggtac tggtgggcgt gggcatcgcc 600
acctcggtgg aaaccgccgc cgtactctcg cgtaaagcca ttttgcgccc gattggcagc 660
cgccatccca atccaaaagc ggcagaactg gagctacgcc tggaagaagg actcaaccgt 720
ctggggattg gtccacaagg gctgaccggc aacagttcag tgatgggcgt acatatcgaa 780
tctgccgccc gccatccgtc aaccatcggc gttgctgtct ctaccggctg ctgggcgcat 840
cgtcgcggca cgctgctggt tcatgccgat ctcacctttg aaaatctgtc tcacacccgg 900
agcgcgtta 909
<210> 4
<211> 610
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<400> 4
atgaaaaaga tcctgacaac cccgatcaaa gctgaagatc tgcaagatat tcgcgtcggc 60
gatgtgatct acctgaccgg tacgctggtg acctgccgcg acgtttgtca ccgccgtttg 120
atcgaactga aacgtccgat cccttacgat ctcaacggca aagcgatttt ccacgctggc 180
cccatcgtgc gcaaaaacgg cgacaaatgg gagatggtct ccgtcggccc gacaaccagt 240
atgcgtatgg aaagttttga acgtgaattt attgagcaga ccggcgtgaa actggtggtt 300
ggcaaaggtg gtatggggcc gctgaccgaa gaaggctgcc agaaattcaa ggcgctacat 360
gtgattttcc cggcaggctg cgcggtgctg gcggcaaccc aggtggaaga gattgaagaa 420
gtgcactgga cagagctcgg aatgccggag tcactgtggg tctgccgggt caaagagttc 480
ggcccgctga ttgtctctat tgatacccac ggcaacaacc tgatagccga aaacaaaaag 540
ctgttcgccg aacgccgcga tcccatcgtg gaagagatct gcgagcacgt ccattacatc 600
aaatagatct 610

Claims (9)

1. one plant product L-TARTARIC ACID genetic engineering bacterium, it is characterised in that its Classification And Nomenclature be colon bacillus （Escherichia coli）CM-MA-184, preserving number are CCTCC NO：M2016455.
2. the construction method of genetic engineering bacterium described in claim 1, it is characterised in that comprise the steps：
（1）With wild type colon bacillus K12 as starting strain, tri- sections of genes of pfl, ldh and mdh in K12 bacterial strains are knocked out；
（2）By SEQ ID NO：2、SEQ ID NO：3、SEQ ID NO：Three sections of gene orders shown in 4 are using chemosynthesis Mode synthesizes, and obtains objective gene sequence, its gene order such as SEQ ID NO：Shown in 1；
（3）Objective gene sequence is imported pTrc99a plasmids, recombiant plasmid is obtained；
（4）By recombiant plasmid steps for importing（1）Middle knockout pfl, the K12 bacterial strain competence of ldh and mdh genes obtain positive turning Beggar.
3. application of the genetic engineering bacterium described in claim 1 in L-TARTARIC ACID is produced.
4. application according to claim 3, it is characterised in that prepare L- wine using monosaccharide fermentation in electrochemical reactor Stone acid.
5. application according to claim 4, it is characterised in that electrochemical reactor anode fills fermentation medium, its formula For nicotinic acid 0.1mM；3.0 g/L of citric acid；Na2HPO4∙7H2O 3.00 g/L；KH2PO48.00 g/L；(NH4)2HPO4 20.00 g/L；NH4Cl 10 g/L；(NH4)2SO45 g/L；MgSO4∙7H2O 1.00 g/L；CaCl2∙2H2O 10.0 mg/L； ZnSO4∙7H2O 0.5 mg/L；CuCl2∙2H2O 0.25 mg/L；MnSO4∙H2O 2.5 mg/L；CoCl2∙6H2O 1.75 mg/ L；H3BO30.12 mg/L；Al2(SO4)3∙xH2O 1.77 mg/L；Na2MoO4∙2H2O 0.5 mg/L；Fe(III) 16.1 mg/L of citrate, dimethyl diaminophenazine chloride 0.1mM, solvent are water, and it is 7.0 to adjust pH with ammonia after sterilizing, wherein 60g/L Fructus Vitis viniferaes Add after sugared individually sterilizing；
Cathodic Composition is：The phosphate buffer of 100mM, pH=6.8,6 g/L of NaCl.
6. application according to claim 5, it is characterised in that the seed liquor incubation for fermenting is as follows：
(S1) bacterium solution preserved in cryopreservation tube is transferred in LB culture medium from cryopreservation tube for 1 ~ 2% by volume fraction, aerobic is trained Support 10～12h；
(S2) culture fluid after processing through S1 is transferred in the LB culture medium of seed fermentation tank for 1 ~ 2% by volume fraction；
(S3) thalline OD is treated600During to 2.5 ~ 4, by culture fluid 5 ~ 10% anode for being seeded to electrochemical reactor by volume.
7. application according to claim 6, it is characterised in that in step (S1) and (S2), cultivation temperature are 35 ~ 37 DEG C.
8. application according to claim 6, it is characterised in that in step (S3) adopt anaerobic fermentation pattern, during lead to Carbon dioxide maintains anaerobic environment.
9. application according to claim 6, it is characterised in that the anaerobic fermentation process of anode maintains temperature at 30 ~ 32 DEG C, Incubation pH ammonia is adjusted to 6.8～7.0.
CN201611141246.6A 2016-12-12 2016-12-12 One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application Active CN106497844B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611141246.6A CN106497844B (en) 2016-12-12 2016-12-12 One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611141246.6A CN106497844B (en) 2016-12-12 2016-12-12 One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application

Publications (2)

Publication Number Publication Date
CN106497844A true CN106497844A (en) 2017-03-15
CN106497844B CN106497844B (en) 2019-05-21

Family

ID=58329849

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611141246.6A Active CN106497844B (en) 2016-12-12 2016-12-12 One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application

Country Status (1)

Country Link
CN (1) CN106497844B (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481681A (en) * 2008-02-27 2009-07-15 杭州宝晶生物化工有限公司 Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria
CN102703419A (en) * 2012-05-21 2012-10-03 中国科学院青岛生物能源与过程研究所 Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid
CN103614398A (en) * 2013-08-22 2014-03-05 杭州宝晶生物化工有限公司 Coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications
CN103882000A (en) * 2014-03-17 2014-06-25 中国科学院过程工程研究所 Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof
CN105018443A (en) * 2015-07-30 2015-11-04 浙江大学 Epoxide hydrolase mutant and preparation method thereof
CN105861463A (en) * 2016-04-12 2016-08-17 南京工业大学 Epoxysuccinate hydrolase, and carrier and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481681A (en) * 2008-02-27 2009-07-15 杭州宝晶生物化工有限公司 Method for producing D(-)-tartaric acid or salt thereof by using gene engineering bacteria
CN102703419A (en) * 2012-05-21 2012-10-03 中国科学院青岛生物能源与过程研究所 Cellulose immobilized cis epoxy succinic acid hydrolase and method for preparing tartaric acid
CN103614398A (en) * 2013-08-22 2014-03-05 杭州宝晶生物化工有限公司 Coding gene of cis-form epoxy succinic acid hydrolase, polypeptides coded thereby and related applications
CN103882000A (en) * 2014-03-17 2014-06-25 中国科学院过程工程研究所 Cis-epoxysuccinate hydrolase immobilization method and immobilized enzyme thereof
CN105018443A (en) * 2015-07-30 2015-11-04 浙江大学 Epoxide hydrolase mutant and preparation method thereof
CN105861463A (en) * 2016-04-12 2016-08-17 南京工业大学 Epoxysuccinate hydrolase, and carrier and application thereof

Also Published As

Publication number Publication date
CN106497844B (en) 2019-05-21

Similar Documents

Publication Publication Date Title
CN101240259B (en) Novel constructed high-yield fumaric acid gene engineering bacterium and method for producing fumaric acid thereby
CN105296411A (en) Genetically engineered bacterium producing L-aspartic acid through monosaccharide fermentation, and construction method and application thereof
CN106434510B (en) One plant of fermentation produces the genetic engineering bacterium of L-Aspartic acid
CN104312987A (en) Biosynthesis method of glucuronic acid and glucuric acid
Singhvi et al. Nanoparticle-associated single step hydrogen fermentation for the conversion of starch potato waste biomass by thermophilic Parageobacillus thermoglucosidasius
CN105462949A (en) Glutamic acid decarboxylase mutant and preparation method thereof and application
CN104911117A (en) Method for producing glucaric acid by improving yeast fermentation by use of fusion expression
CN110029068A (en) The aspergillus niger strain and application of high yield organic acid under a kind of low dissolved oxygen condition
CN104080918B (en) Method for producing glucaric acid
CN109652434B (en) Recombinant bacterium for producing succinic acid by using glycerol as substrate and construction method and application thereof
CN107881140A (en) The Leuconostoc mesenteroides mutant strain of one plant height production mannitol and its application process
CN107227284A (en) A kind of restructuring streptococcus zooepidemicus for the micromolecule hyaluronic acid that ferments
CN104630100A (en) Reconstructed Klebsiella pneumoniae and application of reconstructed Klebsiella pneumoniae in production of R-acetoin
CN110144318B (en) Pigment-free low-molecular-weight welan gum production strain and construction method and application thereof
CN108220323A (en) Utilize the oleaginous yeast engineered strain construction method and engineering bacteria of methanol and application
CN106497844B (en) One plant of genetic engineering bacterium for producing L-TARTARIC ACID and its construction method and application
KR20110012170A (en) Host cells transformed with hydrogenase or nitrogenase of rhodobacter sphaeroide and method for preparing hydrogen using the same
CN103898089B (en) Strain capable of highly producing L-alanine and tolerant to tap water and construction method thereof
CN111304138B (en) Recombinant escherichia coli for producing beta-carotene and construction method and application thereof
CN106834128A (en) One plant of genetic engineering bacterium and its construction method and application that beta Alanine is produced using glucose fermentation
CN106916776A (en) The engineering bacteria and construction method of overexpression phosphoglucomutase gene and UDPglucose pyrophosphorylase gene
CN106636151A (en) Engineering bacterium for over-expressing glycosyltransferase gene and construction method
CN112725372A (en) Multi-enzyme complex carrier for improving expression of welan gum and recombinant bacterium for high-yield welan gum
CN106676118A (en) Bacillus licheniformis gene engineering bacterium with high polysaccharide flocculant yield and construction method for same
CN114456964B (en) Recombinant yarrowia lipolytica for high yield of stigmasterol, construction method thereof, fermentation medium for producing stigmasterol and application

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant