CN109294966A - A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu - Google Patents

A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu Download PDF

Info

Publication number
CN109294966A
CN109294966A CN201811256384.8A CN201811256384A CN109294966A CN 109294966 A CN109294966 A CN 109294966A CN 201811256384 A CN201811256384 A CN 201811256384A CN 109294966 A CN109294966 A CN 109294966A
Authority
CN
China
Prior art keywords
leu
recombinant bacterium
corynebacterium glutamicum
ala
gly
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.)
Pending
Application number
CN201811256384.8A
Other languages
Chinese (zh)
Inventor
张伟国
钱和
王颖妤
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.)
Jiangnan University
Original Assignee
Jiangnan University
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 Jiangnan University filed Critical Jiangnan University
Priority to CN201811256384.8A priority Critical patent/CN109294966A/en
Publication of CN109294966A publication Critical patent/CN109294966A/en
Pending legal-status Critical Current

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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
    • 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
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01009—Leucine dehydrogenase (1.4.1.9)

Landscapes

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

Abstract

The invention discloses a kind of Corynebacterium glutamicum recombinant bacterium of high yield L-Leu and its construction methods, belong to genetic engineering field.Present invention application gene engineering method, replacing NADP dependent form branched-chain amino acid transaminase in Corynebacterium glutamicum is from Bacillus sphaericus NAD dependent form leucine dehydrogenase LeuDH, building it is new, efficient L-Leu route of synthesis and to solve NADH accumulation in Corynebacterium glutamicum L-Leu producing strains excessive, the disadvantage of NADPH supply deficiency enhances L-Leu synthesis capability in recombinant bacterial strain and improves the ability of strain for accumulating NADPH.Recombinant bacterium is tested through shake flask fermentation, and L-Leu accumulation reaches 16.7gL‑1, maximum specific growth rate 0.23gL‑1·h‑1, higher than the 13.2gL of starting strain‑1And 0.18gL‑1·h‑1.L-Leu route of synthesis in the invention successful modification Corynebacterium glutamicum, improves the unbalanced disadvantage of co-factor intracellular, provides new thinking for breeding L-Leu Producing Strain.

Description

A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu
Technical field
The present invention relates to a kind of Corynebacterium glutamicum recombinant bacterium of high yield L-Leu and its construction methods, belong to gene work Journey field.
Background technique
L-Leu also known as leucine, its chemical name is alpha-amido-γ-methylvaleric acid, alpha-amido isocaproic acids, with L- Valine, l-Isoleucine are same to be had methyl chains branched structure and is referred to as branched-chain amino acid.All due to people and mammal It itself cannot synthesize these three amino acid and external source supply must be relied on, therefore three also belongs to essential amino acid.The bright ammonia of L- Acid has different physiological roles, is widely used in food additives, pharmaceutical industry, cosmetic industry and animal feed additive row Industry.Meanwhile it being also widely used in clinical as compound amino acid intravenous fluid.Therefore, breeding height with independent intellectual property rights L-Leu synthesis, low byproducts build-up production bacterial strain, realize Sustainable Development of Enterprises be of great significance.
The production method of L-Leu mainly has extraction method, and chemical synthesis adds precursor substance fermentation method, direct fermentation Method etc..Microbe fermentation method reaction condition is mild, environmental-friendly, stable product quality, is the main production process of L-Leu. L-Leu producing strains are mostly that random mutation obtains, and usually use Corynebacterium glutamicum, brevibacterium lactofermentum and brevibacterium flavum As host strain, mutagenic treatment selects auxotroph or Amino acid analogue resistant mutant strain, is adjusted with releasing metabolism Feedback repression and inhibition during section, achieve the purpose that excess accumulation L-Leu.However, chemical mutagenesis screening amino acid generates Bacterium has many problems, such as generates unnecessary mutation, generates by-product, and thalli growth is slowly etc..
Leucine dehydrogenase (Leucine dehydrogenase, LeuDH) is a kind of oxidoreducing enzyme of NAD dependent form, Its reversible catalysis L-Leu and the reaction of other branched-chain amino acids generate corresponding ketone acid and the like, can be used for measuring branch Chain fatty acid and its ketone acid analog, while ketone acid reduction can be catalyzed and prepare the amino acid such as L-Leu and S-Leucine.It should Enzyme has higher expression in bacillus, is primarily involved in the internal metabolism of branched-chain l-amino acids.Sanwal and Zink in Leucine dehydrogenase is found and purified in the Bacillus cereus of bacillus first within 1961, hereafter Bacillus Sphaerius, Bacillus stearothermophlius, Clostridium thermoaceticum etc. produce LeuDH bacterial strain It is screened out in succession.The enzyme molecular weight about 40kDa is made of six same subunits, and it is co-factor that when enzyme reaction, which needs NADH,. However, the enzyme is but not present as main branched-chain amino acid production strain Corynebacterium glutamicum.There is document to point out in glutamic acid Heterogenous expression LeuDH in bar bacterium, can change the supply of co-factor NADPH in Valine route of synthesis, to be conducive to Valine synthesis.Therefore, screening obtains using NADH as the leucine dehydrogenase of co-factor being to improve L-Leu to generate mycetocyte The interior unbalanced key of co-factor.
Metabolic engineering provides the breeding method of an effective alternative classic mutagenesis.But existing pass through is metabolized way The L-Leu production bacterium of diameter transformation still remains low output, the high problem of by-products content.
Summary of the invention
To solve the above problems, the present invention will be with NADH in spherical bacillus (B.sphaericus) for the first time The LeuDH of co-factor is introduced into the L-Leu producing strains of Corynebacterium glutamicum, improves L-Leu in recombinant bacterial strain Yield, and by-products content is effectively reduced.
The first purpose of the invention is to provide a kind of Corynebacterium glutamicum recombinant bacterium, the recombinant bacterium is bright in production L- The heterogenous expression leucine dehydrogenase in bacillus source in the Corynebacterium glutamicum of propylhomoserin.
In one embodiment of the invention, the production L-Leu producing strains include Corynebacterium glutamicum and its Asia Kind, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, cognate rod Bacterium, wherein the present invention relates to L-Leu producing strains be Corynebacterium glutamicum ATCC13032 bacterial strain or its by mutagenesis sieve Obtained C.glutamicum XQ-9 is selected, and C.glutamicum XQ-9 is methionine and isoleucine deficiency, has α- Thiazolealanine, butyrine, sulphaguanidine and leucine hydroxamate resistance.
In one embodiment of the invention, the bacillus be Bacillus sphaericus, bacillus cereus or Bacillus stearothermophilus.
In one embodiment of the invention, the leucine dehydrogenase is following any:
(1) amino acid sequence (nucleotide sequence such as SEQ ID NO.2) as shown in SEQ ID NO.1；
(2) there is 80% or more homology with amino acid sequence shown in SEQ ID NO.1, and have leucine dehydrogenase living Property.
In one embodiment of the invention, the insertion position of the leucine dehydrogenase is branched-chain amino acid transaminase Encoding gene ilvE gene coded sequence among, realize that excalation ilvE encoding gene expresses leucine dehydrogenase simultaneously.
It in one embodiment of the invention, is using pK18mobsacB as expression vector.
A second object of the present invention is to provide a kind of construction methods of recombinant bacterium, specifically:
(1) building of recombinant expression carrier pDXW-8-leuDH
By amino acid sequence leuDH segment as shown in SEQ ID NO.1 with after identical digestion with restriction enzyme C.glutamicum-E.coli shuttle expression plasmid pDXW-8 (disclosing in CN101693901A) is connected construction recombination plasmid pDXW-8-leuDH；
(2) acquisition of expression cassette Ptac-leuDH-rrnBT1T2
Using pDXW-8-leuDH as template, PCR is carried out, acquisition contains strong promoter Ptac's and terminator rrnBT1T2 Ptac-leuDH-rrnBT1T2 expression cassette；
The nucleotide sequence of the strong promoter Ptac and terminator rrnBT1T2 are respectively SEQ ID in sequence table Shown in NO.3 and SEQ ID NO.4.
(3) building of integrating vector pK18mobsacB- △ ilvE::leuDH
Using C.glutamicum ATCC13032 or C.glutamicum XQ-9 genome as template design primer, carry out PCR, obtaining has the PCR product of identical restriction enzyme at 3 ' ends and 5 ' ends respectively, will be with above-mentioned PCR product respectively and linearly Change the connected construction recombination plasmid pK18mobsacB- △ ilvE of pK18mobsacB suicide type carrier；It then will be by PCR after purification Product Ptac-leuDH-rrnBT1T2 is connect with pK18mobsacB- △ ilvE, obtains recombinant plasmid pK18mobsacB- △ ilvE::leuDH；
The △ ilvE segment is spliced by the left homology arm and right homology arm of branched-chain amino acid transaminase, nucleotide Sequence is respectively in sequence table shown in SEQ ID NO.5 and SEQ ID NO.6.
(4) recombinant bacterial strain C.glutamicum XQ- △ ilvE::leuDH or C.glutamicum ATCC13032- △ The building of ilvE::leuDH
By the electroporated C.glutamicum XQ-9 of plasmid pK18mobsacB- △ ilvE::leuDH or glutamic acid rod Bacillus C.glutamicum ATCC13032, screening obtain recombinant bacterium C.glutamicum XQ-9- △ ilvE::leuDH or Corynebacterium glutamicum C.glutamicum ATCC13032- △ ilvE::leuDH.
In one embodiment of the invention, the branched-chain amino acid aminotransferase gene sequence and C.glutamiucm The ilvE gene order in the source ATCC13032 it is consistent (amino acid sequence as shown in SEQ ID NO:7, nucleotide sequence such as SEQ Shown in ID NO:8).
Third object of the present invention is to provide the method for the recombinant bacterium fermenting and producing L-Leu, the method be by The recombinant bacterium single colonie is seeded to liquid seed culture medium, and 28-29 DEG C, 80-120rmin-1Cultivate 10-14h；With 5-15% Seed culture fluid is forwarded to fermentation medium by inoculum concentration, and 28-30 DEG C, 80-120rmin-1Cultivate 60-80h.
In one embodiment of the invention, the seed culture medium (gL-1): glucose 30-35, corn pulp 30- 40, yeast extract 5-15, (NH4)2SO45-15, KH2PO42-4, MgSO4·7H2O 0.5-1.5, MnSO4·4H2O 0.05- 0.15, sodium citrate 5-15, urea 2-10, L-Methionine 0.5-1.5, thiamine 0.0004-0.0008, biotin 0.0002- 0.0006, CaCO3 15-25。
In one embodiment of the invention, the fermentation medium (gL-1): glucose 80-120, corn pulp 20- 30, (NH4)2SO410-20, CH3COONH410-20, KH2PO42-4, MgSO4·7H2O 0.5-1.5, MnSO4·4H2O 0.05-0.15, sodium citrate 2-4, urea 2-5, L-Methionine 0.5-1.5, Pidolidone 0.5-1.5, l-Isoleucine 0.05- 0.15, hydrochloric acid betaine salt 1-2, biotin 0.0003-0.0008, thiamine 0.0005-0.0010, CaCO3 30-50。
Fourth object of the present invention is to provide the recombinant bacterium answering in feed industry, medical industry or food industry With.
The beneficial effects of the present invention are: the present invention is synthesized by producing L-Leu in bacterium in the L-Leu of Corynebacterium Insertion encodes base in bacillus with the LeuDH of NAD dependent form at the site pathway key enzyme TA encoding gene ilvE Because of expression cassette, efficient L-Leu route of synthesis is constructed in the L-Leu production bacterium of Corynebacterium, obtains high yield The recombinant bacterial strain that L-Leu and by-products content reduce.
Detailed description of the invention
Fig. 1: L-Leu metabolic pathway and its key gene are generated by glucose
Abbreviation explanation: AHAS, acetolactate synthase；AHAIR, acetolactic acid restore isomerase；DHAD, dihydroxy acid dehydration Enzyme；IPMS, isopropylmalate synthetase；IPMI, isopropylmalate isomerase；IPMD, isopropylmalate dehydrogenase； TA, branched-chain amino acid transaminase；
Fig. 2: the LeuDH expression in C.gluatmicum XQ
Swimming lane explanation: M swimming lane is protein molecular weight standard Marker；No. 1 swimming lane is C.gluatmicum XQ；No. 2 swimming Road is C.gluatmicum XQ- △ ilvE::leuDH；
Fig. 3: starting strain and recombinant bacterial strain TA and LeuDH enzyme activity determination；
Fig. 4: starting strain and recombinant bacterial strain fermenting and producing L-Leu fermentation process curve
Figure explanation: ■ ▼ ◆-starting strain C.gluatmicum XQ fermentation process curve ● ▲-recombinant bacterial strain C.gluatmicum XQ-9- △ ilvE::leuDH fermentation process curve；
Fig. 5: starting strain and recombinant bacterial strain fermentation liquid by-product measurement result.
Specific embodiment
The present invention will be further explained below with reference to the attached drawings and specific examples, so that those skilled in the art can be with It more fully understands the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention.
In the present invention, bacterium germination is C.glutamicum ATCC13032 or C.gluatmicum XQ-9 out；Wherein, Bacterium germination C.gluatmicum XQ-9 is that while having α-thiazolealanine and α-ammonia for methionine and isoleucine deficiency out The bacterial strain of base butyric acid resistance can be used to produce L-Leu.
The monitoring of the quantification and qualification and thalli growth situation of substrate and product: glucose is examined in real time in fermentation liquid It surveys and passes through SBA-40B bio-sensing analysis-e/or determining.Leucine product real-time monitoring is measured using amino acid determining instrument.Bacterium solution is dense Degree measurement: pipette samples bacterium solution dilutes certain multiple with distilled water, using distilled water as blank control, using spectrophotometer OD is measured in 1cm light path562nm。
The quantification and qualification of by-product: by-product mainly examines or check organic acid and amino acid in fermentation liquid.For organic The measurement of acid is measured using high performance liquid chromatograph (HLPC)；Measurement for amino acid, is measured using amino acid determining instrument. Standard sample and the respective appearance time of conversion fluid and peak face are measured using high performance liquid chromatograph or amino acid determining instrument respectively Product, it can qualitative and quantitative detection is carried out to test substance in conversion fluid.
The acquisition of embodiment 1:B.sphaericus LeuDH encoding gene leuDH expression cassette
According to B.sphaericus leuDH gene order in GenBank, it is separately added into its gene upstream and downstream restricted Restriction endonuclease EcoR I and Kpn I restriction enzyme site sequence, while Corynebacterium glutamicum SD being added at its upstream and identifies sequence GAAAGGAGATATACC, and the sequence combined is submitted into general biosystem (Anhui) Co., Ltd and is synthesized, it obtains Recombinant plasmid pUC57-leuDH containing target gene.Then, using restriction enzyme EcoR I and Kpn I digested plasmid pUC57-leuDH.LeuDH segment is then recycled using plastic recovery kit.By leuDH segment with through identical restriction enzyme The connected construction recombination plasmid pDXW-8-leuDH of C.gluatmicum-E.coli shuttle expression plasmid pDXW-8 after digestion.Most Afterwards, using pDXW-8-leuDH as template, Ptac- F and Ptac- R is that primer (table 1) carries out PCR, obtains Ptac-leuDH-rrnBT1T2 Expression cassette.
Primer sequence needed for table 1.PCR is expanded (underscore is restriction enzyme site)
TA encoding gene ilvE replaces with B.sphaericus LeuDH coding in embodiment 2:C.gluatmicum XQ-9 Gene leuDH
Using C.gluatmicum XQ-9 genome as template, respectively with ilvE-L-F, ilvE-L-R and ilvE-R-F, IlvE-R-R is primer PCR, and obtain has the PCR of identical restriction enzyme to produce in the 3 ' of ilvE-L and the 5 ' of ilvE-R respectively Object.It will be with above-mentioned PCR product respectively at the connected construction recombination plasmid pK18mobsacB- △ of linearisation pK18mobsacB carrier ilvE；Then by PCR product P after purificationtac- leuDH-rrnBT1T2 is connect with pK18mobsacB- △ ilvE, and the two is simultaneously With Xba I digestion, digestion products are connected by cohesive end, obtain recombinant plasmid pK18mobsacB- △ ilvE::leuDH.
By the electroporated C.gluatmicum XQ- of the correct plasmid pK18mobsacB- △ ilvE::leuDH of above-mentioned verifying Through LBG+Km solid medium culture under 9,30 DEG C of condition of culture, screening obtains first time homologous recombination transformant.Then, by Homologous recombination transformant is transferred to containing cultivating in 10% sucrose solids culture medium and in 30 DEG C, coerces secondary recombination screening, Scribing line separation and the multiple transformants of picking are finally carried out on LBG plate.Conversion subgenom is extracted, with target gene ilvE's It verifies primer ilvE-F and ilvE-R and carries out PCR product sequencing identification.It is final to obtain purpose recombinant bacterial strain C.gluatmicum XQ-9-△ilvE::leuDH。
Using similar method, building obtains recombinant bacterial strain C.gluatmicum ATCC13032- △ ilvE::leuDH.
Expression of the embodiment 3:LeuDH in C.gluatmicum XQ-9
Respectively by starting strain C.gluatmicum XQ-9 and recombinant bacterial strain C.gluatmicum XQ-9- △ ilvE:: LeuDH is seeded to liquid LBG culture medium, and thallus is collected after IPTG induction after ultrasonic disruption, supernatant SDS-PAGE electrophoresis, Detect the specific band (Fig. 2) of a molecular weight about 40kDa, in the same size with the target protein of report, explanation derives from LeuDH in B.sphaericus can be expressed correctly in corynebacterium glutamicum.
Embodiment 4: recombinant bacterium C.gluatmicum XQ-9- △ ilvE::leuDH and starting strain C.gluatmicum LeuDH and TA enzyme activity determination in XQ-9
The measurement of LeuDH: taking the strain of cryovial preservation to be inoculated in LBG fluid nutrient medium, and 30 DEG C of shaken cultivations are stayed overnight, and In 10000rmin-1Thalline were collected by centrifugation.Then, thallus is suspended in 100mmolL-1Glycine-NaOH(pH 9.5) In buffer and ultrasonic disruption prepares crude enzyme liquid.LeuDH enzyme activity determination: NADH has maximum light absorption value, LeuDH at 340nm Vigor is calculated by the variation of NADH light absorption value at 340nm in detection reaction process.Enzyme reaction system: 100mmolL- 1Glycine-NaOH (pH 9.5) buffer, 200mmolL-1NH4Cl, 10mmolL-1Ketoisocaproate, 0.2molL- 1NADH and crude enzyme liquid；Reaction temperature: 30 DEG C；Reaction time: >=300s.Enzyme-activity unit (U) is defined as under the above-described reaction conditions Enzyme amount needed for 1 μm of ol NADH oxidation of catalysis per minute.After measured, recombinant bacterium has LeuDH enzyme activity, and starting strain is not With LeuDH enzyme activity, as a result as shown in Figure 3.
The measurement of TA: taking the strain of cryovial preservation to be inoculated in LBG fluid nutrient medium, and 30 DEG C of shaken cultivations are stayed overnight, and in 10000r·min-1Thalline were collected by centrifugation.Then, thallus is suspended in 100mmolL-1Tris-HCl (pH 9.0) buffer In and ultrasonic disruption prepare crude enzyme liquid.TA enzyme activity determination: using glutamic acid as amino group donor, it is bright that ketoisocaproate amination generates L- The accumulation of propylhomoserin calculates.Enzyme reaction system: 100mmolL-1Tris-HCl (pH 9.0) buffer, 0.25mmolL-1Phosphorus Sour -5 '-pyridoxals, 5mmolL-1Ketoisocaproate, 10mmolL-1Pidolidone and crude enzyme liquid；Reaction temperature: 30 DEG C；Reaction Time: >=300s；21% perchloric acid is added after reaction and terminates reaction.Enzymatic reaction solution neutralized by 5mol KOH solution and from Heart processing, high performance liquid chromatography detection L-Leu production quantity.Enzyme-activity unit (U) is defined as under the above-described reaction conditions per minute Enzyme amount needed for catalysis generates 1 μm of ol L-Leu oxidation.After measured, recombinant bacterium no longer has a TA enzyme activity, and starting strain With TA enzyme activity, as a result as shown in Figure 3.
Embodiment 5: recombinant bacterium C.gluatmicum XQ-9- △ ilvE::leuDH and starting strain C.gluatmicum XQ-9 fermentation produces L-Leu
Culture medium: 1. seed culture medium (gL-1): glucose 30, corn pulp 30-40, yeast extract 5-10, ammonium sulfate 5, lemon Lemon acid sodium 10, urea 2, KH2PO4·3H2O 2, MgSO4·7H2O 0.5, MnSO4·H2O 0.02, methionine 0.4, biotin 0.0003, thiamine 0.0004, CaCO320, pH 7.3-7.5,121 DEG C of 20min.2. fermentation medium (g L-1): glucose 100, corn pulp 20-30, ammonium sulfate 15, ammonium acetate 15, sodium citrate 2, urea 2-3, KH2PO4·3H2O2, MgSO4·7H2O 0.5, MnSO4·H2O 0.06, methionine 0.7, isoleucine 0.06, glutamic acid 0.5, hydrochloric acid betaine salt 1, biotin 0.0004, thiamine 0.0006, CaCO330, pH 7.3-7.5,115 DEG C of 10min；
By above-mentioned verified recombinant bacterium C.gluatmicum XQ-9- △ ilvE::leuDH and starting strain C.gluatmicum XQ-9 carries out shake flask fermentation experiment respectively.Picking one expires ring paddy ammonia from the slant medium of fresh activation Sour bar bacterium (bacterium germination and recombinant bacterium out) is inoculated into 50mL liquid amount 500mL shake-flask seed culture medium, 4 layers of gauze sealing, and 30 ℃100r··min-1Seed liquor is accessed the culture of 50/500mL shake flask fermentation by inoculum concentration 6% by reciprocal shaker culture 16h Base, 30 DEG C of 100rmin-1Reciprocal shaker culture 72h；Time segment measures L-Leu, residual sugar and its biomass, as a result Compared with starting strain, as a result as shown in Figure 4.It can apparently find out the yield of L-Leu by 13.2gL from Fig. 4-1It mentions It is raised to 16.7gL-1, illustrate that heterogenous expression can be improved the production of L-Leu from the leucine dehydrogenase of bacillus Amount.In addition, recombinant bacterial strain and starting strain byproducts build-up situation (including organic acid and amino acid), as a result such as table 2 and Fig. 5 institute Show.
The concentration of free heteroacid in 2 starting strain of table and recombinant bacterial strain fermentation liquid
Recombinant bacterium is tested through shake flask fermentation, and L-Leu accumulation reaches 16.7gL-1, maximum specific production rate is 0.23g·L-1·h-1, higher than the 13.2gL of starting strain-1And 0.18gL-1·h-1。
Using identical method, compares recombinant bacterial strain C.gluatmicum ATCC13032- △ ilvE::leuDH and set out The case where bacterial strain C.gluatmicum ATCC13032 fermentation produces L-Leu.The results show that recombinant bacterium C.gluatmicum The L-Leu accumulation of ATCC13032- △ ilvE::leuDH reaches 12.2gL-1, maximum specific production rate is 0.18gL-1·h-1, higher than the 9.1gL of starting strain-1And 0.12gL-1·h-1。
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention Protection scope within.Protection scope of the present invention is subject to claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>the Corynebacterium glutamicum recombinant bacterium and its construction method of a kind of high yield L-Leu
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 364
<212> PRT
<213> leuDH
<400> 1
Met Glu Ile Phe Lys Tyr Met Glu Lys Tyr Asp Tyr Glu Gln Leu Val
1 5 10 15
Phe Cys Gln Asp Glu Ala Ser Gly Leu Lys Ala Ile Ile Ala Ile His
20 25 30
Asp Thr Thr Leu Gly Pro Ala Leu Gly Gly Ala Arg Met Trp Thr Tyr
35 40 45
Ala Thr Glu Glu Asn Ala Ile Glu Asp Ala Leu Arg Leu Ala Arg Gly
50 55 60
Met Thr Tyr Lys Asn Ala Ala Ala Gly Leu Asn Leu Gly Gly Gly Lys
65 70 75 80
Thr Val Ile Ile Gly Asp Pro Phe Lys Asp Lys Asn Glu Glu Met Phe
85 90 95
Arg Ala Leu Gly Arg Phe Ile Gln Gly Leu Asn Gly Arg Tyr Ile Thr
100 105 110
Ala Glu Asp Val Gly Thr Thr Val Thr Asp Met Asp Leu Ile His Glu
115 120 125
Glu Thr Asn Tyr Val Thr Gly Ile Ser Pro Ala Phe Gly Ser Ser Gly
130 135 140
Asn Pro Ser Pro Val Thr Ala Tyr Gly Val Tyr Arg Gly Met Lys Ala
145 150 155 160
Ala Ala Lys Glu Ala Phe Gly Thr Asp Met Leu Glu Gly Arg Thr Ile
165 170 175
Ser Val Gln Gly Leu Gly Asn Val Ala Tyr Lys Leu Cys Glu Tyr Leu
180 185 190
His Asn Glu Gly Ala Lys Leu Val Val Thr Asp Ile Asn Gln Ala Ala
195 200 205
Ile Asp Arg Val Val Asn Asp Phe Gly Ala Thr Ala Val Ala Pro Asp
210 215 220
Glu Ile Tyr Ser Gln Glu Val Asp Ile Phe Ser Pro Cys Ala Leu Gly
225 230 235 240
Ala Ile Leu Asn Asp Glu Thr Ile Pro Gln Leu Lys Ala Lys Val Ile
245 250 255
Ala Gly Ser Ala Asn Asn Gln Leu Gln Asp Ser Arg His Gly Asp Tyr
260 265 270
Leu His Glu Leu Gly Ile Val Tyr Ala Pro Asp Tyr Val Ile Asn Ala
275 280 285
Gly Gly Val Ile Asn Val Ala Asp Glu Leu Tyr Gly Tyr Asn Arg Glu
290 295 300
Arg Ala Leu Lys Arg Val Asp Gly Ile Tyr Asp Ser Ile Glu Lys Ile
305 310 315 320
Phe Glu Ile Ser Lys Arg Asp Ser Ile Pro Thr Tyr Val Ala Ala Asn
325 330 335
Arg Leu Ala Glu Glu Arg Ile Ala Arg Val Ala Lys Ser Arg Ser Gln
340 345 350
Phe Leu Lys Asn Glu Lys Asn Ile Leu Asn Gly Arg
355 360
<210> 2
<211> 1095
<212> DNA
<213> leuDH
<400> 2
atggaaatct tcaagtatat ggaaaagtat gattatgaac aattggtatt ttgccaagac 60
gaagcatctg ggttaaaagc gattatcgct atccatgaca caacacttgg accagcatta 120
ggtggtgctc gtatgtggac ctacgcgaca gaagaaaatg cgattgagga tgcattaaga 180
ttagcacgcg ggatgacata taaaaatgca gctgctggtt taaaccttgg cggtggaaaa 240
acggtcatta ttggggaccc atttaaagat aaaaacgaag aaatgttccg tgctttaggt 300
cgtttcattc aaggattaaa cggtcgctat attaccgctg aagatgttgg tacaaccgta 360
acagatatgg atttaatcca tgaggaaaca aattacgtta caggtatatc gccagcgttt 420
ggttcatcgg gtaatccttc accagtaact gcttatggcg tttatcgtgg catgaaagca 480
gcggcgaaag aagcatttgg tacggatatg ctagaaggtc gtactatatc ggtacaaggg 540
ctaggaaacg tagcttacaa gctttgcgag tatttacata atgaaggtgc aaaacttgta 600
gtaacagata ttaaccaagc ggctattgat cgtgttgtca atgattttgg cgctacagca 660
gttgcacctg atgaaatcta ttcacaagaa gtcgatattt tctcaccgtg tgcacttggc 720
gcaattttaa atgacgaaac gattccgcaa ttaaaagcaa aagttattgc tggttctgct 780
aataaccaac tacaagattc acgacatgga gattatttac acgagctagg cattgtttat 840
gcacctgact atgtcattaa tgcaggtggt gtaataaatg tcgcggacga attatatggc 900
tataatcgtg aacgagcgtt gaaacgtgta gatggtattt acgatagtat tgaaaaaatc 960
tttgaaattt ccaaacgtga tagtattcca acatatgttg cggcaaatcg tttggcagaa 1020
gaacgtattg ctcgtgtagc gaaatcgcgt agtcagttct taaaaaatga aaaaaatatt 1080
ttgaacggcc gttaa 1095
<210> 3
<211> 107
<212> DNA
<213>promoter Ptac sequence
<400> 3
catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat catcggctcg 60
tataatgtgt ggaattgtga gcggataaca atttcacaca ggaaaca 107
<210> 4
<211> 187
<212> DNA
<213>terminator rrnBT1T2 sequence nucleotide sequence
<400> 4
agatctccgc ggcttaagct gcagaagctt ctgttttggc ggatgagaga agattttcag 60
cctgatacag attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg 120
cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc 180
cgatggt 187
<210> 5
<211> 483
<212> DNA
<213> ilvE-L
<400> 5
caagcctagc cattcctcaa aaccgtgaga cgaaattggc tattcatccc ataaaatggg 60
gctgactagt gtatctgtca ggtagcaggt gtaccttaaa atccatgacg tcattagagt 120
tcacagtaac ccgtaccgaa aatccgacgt cacccgatcg tctgaaggaa attcttgccg 180
caccgaagtt cggtaagttc ttcaccgacc acatggtgac cattgactgg aacgagtcgg 240
aaggctggca caacgcccaa ttagtgccat acgcgccgat tcctatggat cctgccacca 300
ccgtattcca ctacggacag gcaatttttg agggaattaa ggcctaccgc cattcggacg 360
aaaccatcaa gactttccgt cctgatgaaa acgccgagcg tatgcagcgt tcagcagctc 420
gaatggcaat gccacagttg ccaaccgagg actttattaa agcacttgaa ctgctggtag 480
acg 483
<210> 6
<211> 529
<212> DNA
<213> ilvE-R
<400> 6
tgggatacga agtagaagag cgaaagatca ccaccaccga gtgggaagaa gacgcaaagt 60
ctggcgccat gaccgaggca tttgcttgcg gtactgcagc tgttatcacc cctgttggca 120
ccgtgaaatc agctcacggc accttcgaag tgaacaacaa tgaagtcgga gaaatcacga 180
tgaagcttcg tgaaaccctc accggaattc agcaaggaaa cgttgaagac caaaacggat 240
ggctttaccc actggttggc taaatcaacc ggttttaaga ccccgctgca ttaaaccctg 300
atttattgca gcggggtttt tgcgttgaca agctcttatg agacgtaggg ggtggaagca 360
ggggtaggac gtgtccagcc caagtggcat gctggtttct gtacaaaacg taggtgactt 420
taaggagaat gcagtggctg aaaatgggca aaaagttgcg gtcgtaactg gtggatcaag 480
cggaattggc gcagcttcag ctcgagcatt ggcagctgac ggttggaaa 529
<210> 7
<211> 367
<212> PRT
<213> ilvE
<400> 7
Met Thr Ser Leu Glu Phe Thr Val Thr Arg Thr Glu Asn Pro Thr Ser
1 5 10 15
Pro Asp Arg Leu Lys Glu Ile Leu Ala Ala Pro Lys Phe Gly Lys Phe
20 25 30
Phe Thr Asp His Met Val Thr Ile Asp Trp Asn Glu Ser Glu Gly Trp
35 40 45
His Asn Ala Gln Leu Val Pro Tyr Ala Pro Ile Pro Met Asp Pro Ala
50 55 60
Thr Thr Val Phe His Tyr Gly Gln Ala Ile Phe Glu Gly Ile Lys Ala
65 70 75 80
Tyr Arg His Ser Asp Glu Thr Ile Lys Thr Phe Arg Pro Asp Glu Asn
85 90 95
Ala Glu Arg Met Gln Arg Ser Ala Ala Arg Met Ala Met Pro Gln Leu
100 105 110
Pro Thr Glu Asp Phe Ile Lys Ala Leu Glu Leu Leu Val Asp Ala Asp
115 120 125
Gln Asp Trp Val Pro Glu Tyr Gly Gly Glu Ala Ser Leu Tyr Leu Arg
130 135 140
Pro Phe Met Ile Ser Thr Glu Ile Gly Leu Gly Val Ser Pro Ala Asp
145 150 155 160
Ala Tyr Lys Phe Leu Val Ile Ala Ser Pro Val Gly Ala Tyr Phe Thr
165 170 175
Gly Gly Ile Lys Pro Val Ser Val Trp Leu Ser Glu Asp Tyr Val Arg
180 185 190
Ala Ala Pro Gly Gly Thr Gly Asp Ala Lys Phe Ala Gly Asn Tyr Ala
195 200 205
Ala Ser Leu Leu Ala Gln Ser Gln Ala Ala Glu Lys Gly Cys Asp Gln
210 215 220
Val Val Trp Leu Asp Ala Ile Glu His Lys Tyr Ile Glu Glu Met Gly
225 230 235 240
Gly Met Asn Leu Gly Phe Ile Tyr Arg Asn Gly Asp Gln Val Lys Leu
245 250 255
Val Thr Pro Glu Leu Ser Gly Ser Leu Leu Pro Gly Ile Thr Arg Lys
260 265 270
Ser Leu Leu Gln Val Ala Arg Asp Leu Gly Tyr Glu Val Glu Glu Arg
275 280 285
Lys Ile Thr Thr Thr Glu Trp Glu Glu Asp Ala Lys Ser Gly Ala Met
290 295 300
Thr Glu Ala Phe Ala Cys Gly Thr Ala Ala Val Ile Thr Pro Val Gly
305 310 315 320
Thr Val Lys Ser Ala His Gly Thr Phe Glu Val Asn Asn Asn Glu Val
325 330 335
Gly Glu Ile Thr Met Lys Leu Arg Glu Thr Leu Thr Gly Ile Gln Gln
340 345 350
Gly Asn Val Glu Asp Gln Asn Gly Trp Leu Tyr Pro Leu Val Gly
355 360 365
<210> 8
<211> 1104
<212> DNA
<213> ilvE
<400> 8
atgacgtcat tagagttcac agtaacccgt accgaaaatc cgacgtcacc cgatcgtctg 60
aaggaaattc ttgccgcacc gaagttcggt aagttcttca ccgaccacat ggtgaccatt 120
gactggaacg agtcggaagg ctggcacaac gcccaattag tgccatacgc gccgattcct 180
atggatcctg ccaccaccgt attccactac ggacaggcaa tttttgaggg aattaaggcc 240
taccgccatt cggacgaaac catcaagact ttccgtcctg atgaaaacgc cgagcgtatg 300
cagcgttcag cagctcgaat ggcaatgcca cagttgccaa ccgaggactt tattaaagca 360
cttgaactgc tggtagacgc ggatcaggat tgggttcctg agtacggcgg agaagcttcc 420
ctctacctgc gcccattcat gatctccacc gaaattggct tgggtgtcag cccagctgat 480
gcctacaagt tcctggtcat cgcatcccca gtcggcgctt acttcaccgg tggaatcaag 540
cctgtttccg tctggctgag cgaagattac gtccgcgctg cacccggcgg aactggtgac 600
gccaaatttg ctggcaacta cgcggcttct ttgcttgccc agtcccaggc tgcggaaaag 660
ggctgtgacc aggtcgtatg gttggatgcc atcgagcaca agtacatcga agaaatgggt 720
ggcatgaacc ttgggttcat ctaccgcaac ggcgaccaag tcaagctagt cacccctgaa 780
ctttccggct cactacttcc aggcatcacc cgcaagtcac ttctacaagt agcacgcgac 840
ttgggatacg aagtagaaga gcgaaagatc accaccaccg agtgggaaga agacgcaaag 900
tctggcgcca tgaccgaggc atttgcttgc ggtactgcag ctgttatcac ccctgttggc 960
accgtgaaat cagctcacgg caccttcgaa gtgaacaaca atgaagtcgg agaaatcacg 1020
atgaagcttc gtgaaaccct caccggaatt cagcaaggaa acgttgaaga ccaaaacgga 1080
tggctttacc cactggttgg ctaa 1104
<210> 9
<211> 25
<212> DNA
<213>artificial sequence
<400> 9
gctctagaca tcataacggt tctgg 25
<210> 10
<211> 24
<212> DNA
<213>artificial sequence
<400> 10
gctctagaac catcggcgct acgg 24
<210> 11
<211> 27
<212> DNA
<213>artificial sequence
<400> 11
tcccccgggc aagcctagcc attcctc 27
<210> 12
<211> 27
<212> DNA
<213>artificial sequence
<400> 12
gctctagacg tctaccagca gttcaag 27
<210> 13
<211> 29
<212> DNA
<213>artificial sequence
<400> 13
gctctagatg ggatacgaag tagaagagc 29
<210> 14
<211> 27
<212> DNA
<213>artificial sequence
<400> 14
acgcgtcgac tttccaaccg tcagctg 27
<210> 15
<211> 15
<212> DNA
<213>artificial sequence
<400> 15
gtgtatctgt caggt 15
<210> 16
<211> 15
<212> DNA
<213>artificial sequence
<400> 16
ttagccaacc agtgg 15

Claims (10)

1. a kind of Corynebacterium glutamicum recombinant bacterium, which is characterized in that the recombinant bacterium is the glutamic acid rod in production L-Leu The heterogenous expression leucine dehydrogenase in bacillus source in bacillus.
2. recombinant bacterium according to claim 1, which is characterized in that the Corynebacterium glutamicum of the production L-Leu is egg Propylhomoserin and isoleucine deficiency, while there is the Corynebacterium glutamicum of α-thiazolealanine and butyrine resistance.
3. recombinant bacterium according to claim 1, which is characterized in that the bacillus is Bacillus sphaericus, waxy Bacillus or bacillus stearothermophilus.
4. recombinant bacterium according to claim 1, which is characterized in that the leucine dehydrogenase is following any:
(1) amino acid sequence is as shown in SEQ ID NO.1；
(2) there is 80% or more homology with amino acid sequence shown in SEQ ID NO.1, and there is leucine dehydrogenase activity.
5. recombinant bacterium according to claim 1, which is characterized in that the insertion position of the leucine dehydrogenase is branch ammonia Among the encoding gene ilvE gene coded sequence of base acid transaminase, realize that excalation ilvE encoding gene expresses bright ammonia simultaneously Acidohydrogenase.
6. recombinant bacterium according to claim 1, which is characterized in that the expression is carried using pK18mobsacB as expression Body.
7. the construction method of the described in any item recombinant bacteriums of claim 1~6.
8. a kind of method of fermenting and producing L-Leu, which is characterized in that the method is any described using claim 1~6 Recombinant bacterium.
9. the application of any recombinant bacterium of claim 1-6.
10. application according to claim 9, which is characterized in that the application be apply feed industry, medical industry or In food industry.
CN201811256384.8A 2018-10-26 2018-10-26 A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu Pending CN109294966A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201811256384.8A CN109294966A (en) 2018-10-26 2018-10-26 A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811256384.8A CN109294966A (en) 2018-10-26 2018-10-26 A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu

Publications (1)

Publication Number Publication Date
CN109294966A true CN109294966A (en) 2019-02-01

Family

ID=65157945

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811256384.8A Pending CN109294966A (en) 2018-10-26 2018-10-26 A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu

Country Status (1)

Country Link
CN (1) CN109294966A (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110551670A (en) * 2019-09-19 2019-12-10 天津科技大学 Genetically engineered bacterium for producing L-leucine and application thereof
CN110643648A (en) * 2019-10-25 2020-01-03 江南大学 Method for producing L-leucine by efficiently utilizing starch
CN112708650A (en) * 2020-12-31 2021-04-27 河南巨龙生物工程股份有限公司 Method for shortening cytidine fermentation lag phase
JP2023503077A (en) * 2019-12-06 2023-01-26 シージェイ チェイルジェダン コーポレーション Novel branched-chain amino acid aminotransferase mutant and method for producing leucine using the same
US11866737B2 (en) 2019-08-29 2024-01-09 Tianjin University Of Science And Technology 2-isopropylmalate synthetase and engineering bacteria and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1702168A (en) * 2005-06-06 2005-11-30 无锡晶海氨基酸有限公司 L-leucine high-yield bacterium and fermentation method using the same for L-leucine production
CN103710375A (en) * 2013-12-30 2014-04-09 江南大学 Novel plasmid for gene modification of Corynebacterium glutamicum and application thereof
CN105441501A (en) * 2015-12-30 2016-03-30 江南大学 High-yield L-leucine strain and application of L-leucine strain in production of L-leucine with fermentation method
CN106661571A (en) * 2014-07-11 2017-05-10 住友化学株式会社 Oxidase, polynucleotide that codes for same, and uses for both

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1702168A (en) * 2005-06-06 2005-11-30 无锡晶海氨基酸有限公司 L-leucine high-yield bacterium and fermentation method using the same for L-leucine production
CN103710375A (en) * 2013-12-30 2014-04-09 江南大学 Novel plasmid for gene modification of Corynebacterium glutamicum and application thereof
CN106661571A (en) * 2014-07-11 2017-05-10 住友化学株式会社 Oxidase, polynucleotide that codes for same, and uses for both
CN105441501A (en) * 2015-12-30 2016-03-30 江南大学 High-yield L-leucine strain and application of L-leucine strain in production of L-leucine with fermentation method

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MICHAEL VOGT ET AL.: "Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes", 《MICROBIAL BIOTECHNOLOGY》 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11866737B2 (en) 2019-08-29 2024-01-09 Tianjin University Of Science And Technology 2-isopropylmalate synthetase and engineering bacteria and application thereof
CN110551670A (en) * 2019-09-19 2019-12-10 天津科技大学 Genetically engineered bacterium for producing L-leucine and application thereof
CN110551670B (en) * 2019-09-19 2020-09-25 天津科技大学 Genetically engineered bacterium for producing L-leucine and application thereof
CN110643648A (en) * 2019-10-25 2020-01-03 江南大学 Method for producing L-leucine by efficiently utilizing starch
JP2023503077A (en) * 2019-12-06 2023-01-26 シージェイ チェイルジェダン コーポレーション Novel branched-chain amino acid aminotransferase mutant and method for producing leucine using the same
JP7378621B2 (en) 2019-12-06 2023-11-13 シージェイ チェイルジェダン コーポレーション Novel branched chain amino acid aminotransferase mutant and leucine production method using the same
CN112708650A (en) * 2020-12-31 2021-04-27 河南巨龙生物工程股份有限公司 Method for shortening cytidine fermentation lag phase

Similar Documents

Publication Publication Date Title
CN109294966A (en) A kind of the Corynebacterium glutamicum recombinant bacterium and its construction method of high yield L-Leu
AU756507B2 (en) L-glutamic acid-producing bacterium and method for producing L-glutamic acid
AU2009234625B2 (en) Mutant microorganisms having high ability to produce putrescine and method for producing putrescine using the same
CN100529094C (en) Process for prodn of L-Thr
JPH05304969A (en) Production of amino acid by fermentation method
CN108913642B (en) Escherichia coli genetic engineering bacteria and application thereof in synchronous production of L-tryptophan and L-valine through fermentation
CN114752544B (en) Method for producing gamma-aminobutyric acid by one-step method and strain construction thereof
CN114752589B (en) Glutamic acid decarboxylase mutant and application thereof in production of gamma-aminobutyric acid
CN109402034A (en) Only produce recombinant bacterium and its application of a kind of branched-chain amino acid
CN109722459B (en) 5-aminolevulinic acid high-yield strain and preparation method and application thereof
EP3023493B1 (en) A modified ornithine decarboxylase protein and a use thereof
CN114634918B (en) D-amino acid oxidase mutant, engineering bacteria and application
EP1346026B1 (en) Microorganism producing 5&#39;-inosinic acid and process for producing 5&#39;-inosinic acid using the same
WO2018077159A1 (en) Method for modifying amino acid attenuator and use of same in production
CN109402038A (en) The recombinant bacterium and its construction method of a kind of high yield L-PROLINE and application
CN101952418B (en) Process for producing (2S,3R,4S)-4-hydroxy-L-isoleucine
CN110331153A (en) A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application
JP2000106869A (en) L-glutamic acid-producing bacterium and production of l-glutamic acid
CN108504617A (en) A kind of Escherichia coli recombinant strain and its construction method of high-yield L-lysine
Ikeda et al. Screening of L-isoleucine producers among ethionine resistant mutants of L-threonine producing bacteria
JP2000189169A (en) L-glutamate productive bacterium and production of l- glutamic acid
CN108441525A (en) The Corynebacterium glutamicum and its construction method that a kind of lysine production improves
DE102004009454A1 (en) Fermentative production of L-amino acids, especially methionine, useful e.g. in animal nutrition, by culturing bacteria in which components of the methionine uptake system are suppressed
Pavlov et al. Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor
Kawahara et al. Stimulatory effect of glycine betaine on L-lysine fermentation

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20190201