CN105602978A - Method for producing L-proline by means of fermentation by aid of genome-modified recombinant Escherichia coli - Google Patents

Method for producing L-proline by means of fermentation by aid of genome-modified recombinant Escherichia coli Download PDF

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Publication number
CN105602978A
CN105602978A CN201610034446.5A CN201610034446A CN105602978A CN 105602978 A CN105602978 A CN 105602978A CN 201610034446 A CN201610034446 A CN 201610034446A CN 105602978 A CN105602978 A CN 105602978A
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prob
proline
genome
kinase gene
glutamic acid
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张震宇
姚动邦
王晓姣
魏照辉
黄建华
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Jiangnan University
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Jiangnan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • 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
    • C12P13/24—Proline; Hydroxyproline; Histidine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02011—Glutamate 5-kinase (2.7.2.11)

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

Abstract

The invention discloses a method for producing L-proline by means of fermentation by the aid of genome-modified recombinant Escherichia coli. Gene-modified bacteria of the recombinant Escherichia coli carry gamma-glutamyl kinase genes (proB2A) with point mutation, and proB500 in genomes is replaced by proB2500. The gamma-glutamyl kinase genes (proB2A) with the point mutation are acquired from recombinant plasmids with the genes by means of enzyme digestion, the recombinant plasmids are preserved in a laboratory, and appropriate primers are designed according to the proB2A which is used as a template, so that the proB2500 can be acquired by means of PCR (polymerase chain reaction). The invention further discloses application of the Escherichia coli to producing the L-proline.

Description

A kind of method of the recombination bacillus coli fermenting and producing L-PROLINE that utilizes genome manipulation
Technical field
A method of utilizing the recombination bacillus coli fermenting and producing L-PROLINE of genome manipulation, belongs to microbial geneEngineering field.
Background technology
L-PROLINE (L-Proline) is a kind of imino acid, and L-PROLINE is the important amino of synthesized human proteinOne of acid, it is a kind of nonpolar amino acid, in the time that it is incorporated in protein, destroys in protein thereby form teritary amideα spiral and β-pleated sheet chip architecture, have been widely used at field tools such as medicine, chemical industry, food, agriculturals.
Along with the development of the raising of China's medical and health level and chemical synthesis industry, agricultural, to the need of L-PROLINEAsk also growing, good application prospect that the research of L-PROLINE production technology is paid close attention to widely. L-PROLINEProduction method mainly contain three kinds: chemical method, hydrolysis extraction method and microbe fermentation method. Wherein, by chemical method and hydrolysisExtraction method is produced L-PROLINE process complexity, and benefit is low, and cost is high, and microbe fermentation method is low with its cost, pollutes less impurityFew, the advantages such as purity height are day by day occupied an leading position in industrial production.
In microbial body, the production process of L-PROLINE is that the glutamic acid being formed by KG, swashs through glutamic acidUnder enzyme (proB coding) catalysis, provide phosphate group to form glutamy phosphoric acid by ATP, again at glutamte dehydrogenase (proA coding)Under effect, the γ-carboxyl reduction of glutamic acid is become to glutamic acid-γ-semialdehyde, then spontaneous cyclisation forms 5-membered ring compounds △ ' twoHydrogen pyrroles-5-carboxylic acid, then form proline by pyrrolin reductase (proC coding) catalysis. Wherein proline can feed back and press downGamma-Glutamate kinase processed, and proB and proA express as an operon proBA, and proBA is mutated intoproB2After A, can reduce the feedback inhibition of proline, produce in a large number L-PROLINE. By proB2500 replace recombination bacillus coliProB500 in genome, can further improve the accumulation of L-PROLINE in microorganism, and this proterties can be withGoing down to posterity of microorganism and stable existence.
Summary of the invention
The present invention is by building the recombination bacillus coli of suitable genome manipulation, this paddy of overexpression in Escherichia coliPropylhomoserin kinases, and can, taking glucose as carbon source in fermentation medium, directly obtain a large amount of L-PROLINEs, before commercial ApplicationScape is wide.
Of the present invention by building suitable recombination bacillus coli genetic modification bacterium fermenting and producing L-PROLINE, its spyLevy and be, cut and obtain the gamma-glutamic acid kinase gene proB with point mutation by enzyme2A is connected with plasmid pET28a, builds heavyGroup plasmid pET28a-proB2A, the proB then PCR being obtained2500 replace in recombination bacillus coli genomeProB500, the recombination bacillus coli of the genome manipulation building can be in suitable culture medium, under suitable condition of cultureFermenting and producing L-PROLINE.
The gamma-glutamic acid kinase gene proB with point mutation in the present invention2A enzyme from the existing carrier in laboratory is cutObtain fragment proB2The 500th, according to proB2A is template, designs suitable primer, obtains by PCR.
Escherichia coli of the present invention can be E.coliBL21 (DE3), E.coliBL21 (DE3) Δ putA andE.coliJM109 etc.
Expression vector in the present invention, can be independently duplicated in host cell, for example have pET-28a, pKYP10,PUC19, pAMP, pBR322 etc.
In the present invention for make to have the gamma-glutamic acid kinase gene of point mutation can be in recombinant bacterium stable existence tableReach, use fragment proB2500 replace the proB500 on recombinant bacterium genome
The present invention can be applied in the production of L-PROLINE, cultivate the culture medium of recombinant bacterium to contain microorganism can be in order toWith carbon source, nitrogenous source, inorganic salts etc., can be natural medium, can be also synthetic media.
Carbon source in the present invention in fermentation medium, has glucose, starch, sucrose, glycerine etc.
Nitrogenous source in the present invention in fermentation medium can be that the ammonium salt classes such as ammonium chloride, ammonium sulfate, ammonium phosphate or other containNitrogen compound, can also have the organic nitrogen sources such as yeast extract, peptone, tryptone, corn steep liquor, dregs of beans.
Inorganic salts in the present invention in fermentation medium, have dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, chlorinationCalcium etc.
The cultivation of recombinant bacterium need to shake or stir to maintain the required amount of oxygen of thalli growth. Cultivation temperature is at 20-37DEG C, incubation time 12-72 hour.
Method of the present invention is the recombination bacillus coli fermenting and producing L-PROLINE by genome manipulation, has wideProspects for commercial application.
Brief description of the drawings
Fig. 1 is recombinant vector pET28a-proB2A design of graphics.
Fig. 2 recombinant vector pET28a-proB2Single double digestion electrophoretogram of A; M:DL10000DNAladderMaker; 1:Plasmid is through XhoI single endonuclease digestion, pET28a-proB2A size is 7723bp; 2: plasmid, through XhoI, Hind III double digestion, obtains twoBar band pET28a size is 5348bp, proB2A size is 2375bp.
Fig. 3 is proB2PCR proof diagram after 500 proB500 that replace on recombinant bacterium genome; M:DL5000DNALadderMaker; 1: former bacterium contrast, stripe size is 612bp; 2: be fragment Kan-proB2500 replace proB500 successBacterium, stripe size is 2132bp.
Detailed description of the invention
General explanation: in detailed description of the invention, enzyme used is all bought from TaKaRa company, sanprep pillar matterGrain DNA extraction agent box and DNA gel reclaim kit all purchased from the raw work in Shanghai, and concrete operations are saying according to kit completelyBright.
LB culture medium: Tryptone10g/L, Yeastextract5g/L, NaCl10g/L, pH7.0-7.2.
Fermentation medium: Glucose20g/L, Peptone8g/L, K2HPO41g/L，NaCl2g/L，MgSO40.2g/L，FeSO41mM，CaCl20.015g/L，(NH4)2SO410g/L。
Kan resistant panel: 1%Tryptone, 0.5%Yeastextract, 1%NaCl, Kanamycinsulfate50μg/mL。
5 × KCM buffer solution: 0.5MKCl, 0.15MCaCl2，0.25MMgCl2。
The assay method of L-PROLINE: after zymotic fluid is centrifugal, get supernatant, suitably after dilution, get 1mL in 10mL test tube,Add 1mL glacial acetic acid, (the phosphoric acid solution 40mL that gets glacial acetic acid 60mL, 2mol/L, adds after shaking up, to add 1mL acid ninhydrine2.5g ninhydrin, 70 DEG C of heating water baths dissolve, and preserve cooled reagent with brown reagent bottle. Wherein 2mol/L phosphoric acid solutionBe configured to: the H that takes 9.22g85%3PO4, add water to 40mL. ) shake up rear boiling water bath 1h, then add glacial acetic acid 2mL, shake upRear cold water is cooling, then measures the absorbance at 515nm wavelength place.
Embodiment 1: the gamma-glutamic acid kinase gene (proB with point mutation2A) acquisition
By the pET28a-Ptrp2-proB that contains of the freezing preservation in laboratory2The bacterial strain of A activates, in 37 DEG C, and 220rpmCultivate 12-16 hour, the bacterium liquid of getting after cultivation extracts plasmid according to sanprep pillar DNA extraction agent box description, usesXho I and Hind III double digestion glue reclaim and obtain proB2A, then processes plasmid pET28a by Xho I and Hind III, by enzyme processingAfter fragment proB2A, the plasmid pET28a connection of spending the night under the condition of 16 DEG C with T4DNA ligase, turns the connecting fluid of 10 μ LEnter in JM109 competence, the single bacterium colony cultivation extraction plasmid after picking transforms carries out enzyme and cuts checking, verifies correct recombinant plasmidBe pET28a-proB2A。
Embodiment 2: fragment proB2500 acquisition
According to the recombinant plasmid pET28a-proB building in embodiment 12A is template, design pair of primers P1 (SEQIDNO:2), P2 (SEQIDNO:3), pcr amplification obtains genes of interest proB2500。
ProB in present embodiment2500 gene orders are shown in SEQIDNO:1
Embodiment 3: genome proB2The structure of 500 innovation bacterium
Design pair of primers P3 (SEQIDNO:4), P4 (SEQIDNO:5), be template according to pKD4 plasmid, pcr amplificationObtain Kan fragment to be merged, by the fragment proB obtaining in itself and embodiment 22500 merge, and utilize Red recombination systemBy fragment proB2500 with genome on proB500 replace, verify that correct bacterial strain is genome proB2500 replaceTransformation bacterium BL21 (DE3) Δ putA-proB2500。
Embodiment 4: contain proB2A recombination bacillus coli genome proB2The structure of 500 innovation bacterium
The genome proB that embodiment 3 is obtained2500 innovation bacterium BL21 (DE3) Δ putA-proB2500 makeChange and turn competence, by the recombinant plasmid pET28a-proB obtaining in embodiment 12A is transferred to BL21 (DE3) Δ of makingputA-proB2In 500 competent cells, the single bacterium colony cultivation extraction plasmid after picking transforms carries out enzyme and cuts checking, and checking is correctBacterial strain be and contain proB2A recombination bacillus coli genome proB2500 innovation bacterium BL21 (DE3) Δ putA-proB2500/pET28a-proB2A。
Embodiment 5: the fermentation test of recombination bacillus coli genome manipulation bacterium
Seed liquor is cultivated: will in embodiment 4, obtain BL21 (DE3) Δ putA-proB2500/pET28a-proB2A is at KanFlat lining out, chooses single colony inoculation to containing in corresponding antibiotic LB culture medium, at 37 DEG C, under the condition of 220rpm, cultivates8h。
Fermented and cultured: by the proline fermentation medium in 6% inoculum concentration access 250mL shaking flask, in rotary shaking table30 DEG C, 220rpm are cultivated 24h, then get zymotic fluid and detect L-PROLINE concentration. The assay method of L-PROLINE refers to embodimentGeneral explanation. Fermentation results shows, recombination bacillus coli acquisition BL21 (DE3) Δ putA-proB2500/pET28a-proB2AL-PROLINE output reach 400.52mg/L.

Claims (7)

1. the recombination bacillus coli construction method of the genome manipulation of a strain fermenting and producing L-PROLINE is, will have point mutationGamma-glutamic acid kinase gene (proB2A) be connected to plasmid pET28a above, construction recombination plasmid pET28a-proB2A, then willThe proB that PCR obtains2500 replace the proB500 in recombination bacillus coli genome, the recombination bacillus coli gene buildingTransformation bacterium can produce L-PROLINE in suitable culture medium.
2. carrier claimed in claim 1, multicopy plasmid are including, but not limited to pET-28a, pUC19, pKYP10 etc.
3. proB500 claimed in claim 1 is not the full sequence of gamma-glutamic acid kinase gene proB, but proB risesBeginning codon starts 500bp fragment backward.
4. proB claimed in claim 12500 is not the gamma-glutamic acid kinase gene proB with point mutation2Whole ordersRow, but proB2Initiation codon starts 500bp fragment backward.
5. method claimed in claim 1, wherein Escherichia coli used are that proline decomposes deficient strain BL21 (DE3)ΔputA。
6. the method for enhancing L-PROLINE biosynthesis system activity claimed in claim 1, is characterized in that being subject to by importingGamma-glutamic acid kinase gene (the proB that proline feedback inhibition significantly reduces2A), increase this γ-paddy ammonia in recombinant microorganismThe copy number of acid kinase gene, and use proB2500 proB500 that replace in genome realize.
The production method of 7.L-proline, is characterized in that the recombinant microorganism described in claim 1,5 to cultivate on culture medium,Using the culture obtaining, thalline or their handled thing as enzyme source, under glucose exists, in aqueous medium, make grapeSugar is converted into L-PROLINE, then extracts the L-PROLINE generating from this aqueous medium.
CN201610034446.5A 2016-01-19 2016-01-19 Method for producing L-proline by means of fermentation by aid of genome-modified recombinant Escherichia coli Pending CN105602978A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112111469A (en) * 2020-11-23 2020-12-22 中国科学院天津工业生物技术研究所 Gamma-glutamyl kinase mutant and application thereof
CN113564093A (en) * 2021-09-28 2021-10-29 天津工微生物科技有限公司 Escherichia coli and application thereof in high-yield preparation of D-proline

Citations (1)

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CN104561072A (en) * 2014-11-19 2015-04-29 江南大学 Method for fermentation production of L-proline by utilizing recombinant Escherichia coli

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CN104561072A (en) * 2014-11-19 2015-04-29 江南大学 Method for fermentation production of L-proline by utilizing recombinant Escherichia coli

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112111469A (en) * 2020-11-23 2020-12-22 中国科学院天津工业生物技术研究所 Gamma-glutamyl kinase mutant and application thereof
CN113564093A (en) * 2021-09-28 2021-10-29 天津工微生物科技有限公司 Escherichia coli and application thereof in high-yield preparation of D-proline
CN113564093B (en) * 2021-09-28 2021-12-10 天津工微生物科技有限公司 Escherichia coli and application thereof in high-yield preparation of D-proline

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Application publication date: 20160525