CN1746292A - Gene for coding, 1,3-propylene glycol reductase in E.aero strain - Google Patents

Abstract

A coded 1,3-propylene-glycol redox enzyme gene of Eaero strain high-yielding mutant strain of aerogens 1.3-PD with number CGMCC 0532. The total length of the dhaT gene is 1164bp which codes 387 amino acid proteins, it begins with ATG initial codon and ends with TGA condon. The molecular weight of expressed protein is 43Kda and enzyme activity is 39muM/mg.

Description

Encode 1 in a kind of E.aero bacterial strain, the gene of ammediol oxydo-reductase

Technical field

The present invention relates to fields such as genetically engineered, enzyme engineering, microbiology, biological chemistry, be specifically related to a kind of high yield 1, ammediol (is called for short 1, (the Enterobacter aerogenes of enteroaerogen 3-PD), being abbreviated as E.aero) bacterial strain (is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center January 16 calendar year 2001, preserving number is CGMCC 0532, the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City), also relate in this bacterial strain and encode 1, the gene of ammediol oxydo-reductase (dhaT).

Background technology

1, the 3-PD Production by Microorganism Fermentation can be divided into: single stage method and two step method.Single stage method is meant with the primverose to be that substrate is converted into 1 through engineering bacteria, 3-PD; Two step method is meant with the primverose to be that substrate is converted into glycerine through yeast earlier, then is that substrate is converted into 1,3-PD through the anaerobion fermentation again with glycerine.1, the research of 3-PD Production by Microorganism Fermentation starts from phase early 1990s, during this in main research two step method transformation of glycerol be 1, the 3-PD strain selection, up to now, have been found that and to produce 1, the microorganism strains of 3-PD has: Klebsiella, Citrobacter, Clostridium Lactobacillus, Enterobacter, Ilyobacter and Pelobacter, Klebsiella pneumoniae (being called for short K.pneu) and Clostridium butyricum (being called for short C.but) are the maximum bacterial strain (Barbirato of research, F., 1995; Slaugh, 1995), these microorganisms are 1 with transformation of glycerol, need 2 step enzymic catalytic reactions during 3-PD.The first step, glycerol dehydratase is 3-hydroxy propanal (3-HPA) and 1 molecular water (1) with transformation of glycerol; In second step, the 3-hydroxy propanal is 1, and the effect of 3-PD oxydo-reductase forms 1,3-PD (2) down.

(1)

(2)

In this reaction process, form 1,3-PD is no longer by metabolism, but assembles at the nutrient solution camber.The glycerine disproportionation forms 1, and 3-PD is under anaerobic, and glycerine is as sole carbon source and lack and finish under other the situation of external source reducing equivalent acceptor.Under these conditions, also have path arranged side by side in the time of the glycerine disproportionation, they are: glycerol dehydrogenase is (with NAD ⁺Or NADP ⁺Coupling) glycerine is oxidized to Protosol (DHA) (3), then Protosol is converted into phosphodihydroxyacetone (DHAP) (4) under Protosol is kinase catalytic; Then DHAP enters glycolytic pathway.With 1, the 3-PD generation pass is compared, and this approach can

(3)

(4)

Carbon source and energy are provided and produce NADH.After the mid-90, mainly lay particular emphasis on research 1, the 3-PD mechanism of production, make up single stage method with gene engineering method and produce 1,3-PD high yield engineering bacteria is in the structure stage at present.

In recent years, along with the continuous development of molecular biology theory, the continuous maturation of genetic engineering technique, Production by Microorganism Fermentation 1, the research of 3-PD genetic engineering technique begins.Tong.H (1991) and Daniel.R (1995) etc. discover that K.pneu can grow and produce 1 on glycerinated substratum, 3-PD is owing to have the dha regulon on its gene.The E.coli that does not have dha regulon system owing to there is not the external source electron acceptor(EA), can not can not produce 1,3-PD by anaerobic growth on glycerine.They have made up K.pneu ATCC25955 gene library in E.coli AG1, make E.coli AG1 can be on glycerine and Protosol anaerobic growth, and filter out high yield 1, the bacterial strain of 3-PD, find that from isolated pTC1 recombinant plasmid (total length 42.5kb) this plasmid has four kinds of enzyme genes that the dha regulon is instructed: glycerol dehydratase (dhaB), 1, ammediol oxydo-reductase (dhaT), glycerol dehydrogenase (dhaD) and Protosol kinases (dhaK).These four kinds of enzymes all are the existence inductive whether that is subjected to glycerine.Frank A.Skraly (1998) etc. discovers, in the n DNA of K.pneu, controls 1, and the gene that 3-PD produces mainly contains dhaT and dhaB:dhaB gene---encoding glycerol dehydratase; DhaT gene---coding 1, the 3-PD oxydo-reductase, in fact dhaT transcribes (Fig. 1) by reaching under the control of two different promotors in different directions with dhaB.

K.pneu dna fragmentation nucleotide sequence analysis (Fig. 1) shows, contain dhaB (3a, 4a, 4) and dhaT (2) gene ORF s in this fragment, but dhaT and dhaB gene transcription direction is opposite.

1, during the 3-PD batch fermentation is produced, 1, the maximum production of 3-PD is 50～60g/L, and in the cultured continuously of routine, C.but can only obtain K.pneu bacterial strain 1, half of 3-PD maximum production (being approximately 48.75g/L), but because the former is a pathogenic bacteria and being eliminated gradually, in view of the security of C.but bacterial strain, it is to have the bacterial strain that potentiality are used in industry most abroad.But the C.but bacterial strain produces 1, and the output of 3-PD is lower, needs screening to have security and 1, the bacterial strain of 3-PD high yield.

Summary of the invention

The purpose of this invention is to provide a kind of new have security and 1, the bacterial strain of 3-PD high yield, this bacterial strain is an isolating enteroaerogen (E.aero) from the mud of freshwater lake, Shenyang, through physics, the independent mutagenesis of chemical process and physics and chemically composited mutagenesis, a strain 1.3-PD high productive mutant that selects, this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and preserving number is CGMCC 0532.1 of this bacterial strain, 3-PD output have surpassed the throughput of C.but bacterial strain, under the most adaptable method, CGMCC 0532 bacterial strain 1 in the 30L fermentor tank, 3-PD output is 48.56g/L, and this bacterial strain is for holding concurrently gas, is that a strain gets a good eye and is worth and the bacterial strain of application potential.

Another object of the present invention provides a kind of isolating dhaT gene, and it derives from above-mentioned enteroaerogen 1.3-PD high productive mutant CGMCC 0532 bacterial strain.After measured, this dhaT gene is shown in SEQ ID NO:1, and total length is 1164bp, is to begin with the ATG initiator codon, and the TGA terminator codon finishes.387 amino acid whose protein of this dhaT genes encoding, shown in SEQ IDNO:2, expressed protein molecular weight is 43KDa, expressed proteinic enzyme work is 39 μ M/mg.

Particularly, the invention provides following technical scheme:

A kind of production 1 is provided, and ammediol (1, enteroaerogen 3-PD) (Enterobacter aerogenes is abbreviated as E.aero) bacterial strain, its preserving number is CGMCC 0532.

A kind of separated coding 1 is provided, the gene of ammediol oxydo-reductase, it has the nucleotide sequence shown in SEQ ID NO:1.

A kind of nucleotide sequence coded 1 by shown in the SEQ ID NO:1 is provided, the ammediol oxydo-reductase, it has the aminoacid sequence shown in SEQID NO:2.

A kind of separated coding 1 is provided, and the gene of ammediol oxydo-reductase, its coding have the aminoacid sequence shown in SEQ ID NO:2.

A kind of coding 1 that has is provided, and the gene of ammediol oxidoreductase activity, its one or several Nucleotide for the gene of the aminoacid sequence shown in the coding SEQ ID NO:2 replace, lack, replace or insert the gene that is then obtained.

A kind of recombinant vectors is provided, and it comprises above-mentioned gene.

Provide a kind of by said gene or by above-mentioned recombinant vectors transformed host cells.

Description of drawings

Fig. 1: expression K.pneu dna fragmentation nucleotide sequence analysis figure, in this fragment, contain dhaB (forming) and dhaT (shown in 2) gene ORF s by 3 fragment genes shown in 3a, 4a and 4, dhaT and dhaB gene transcription direction are opposite.

Fig. 2: expression CGMCC 0532 strain gene group DNA electrophorogram, wherein M represents DNA standard λ-Hin d III (Kb), A represents CGMCC 0532 strain gene group DNA.

Fig. 3: the restriction enzyme mapping of expression plasmid vector pMD18-T.

Fig. 4: the restriction enzyme mapping of expression expression vector pMAL-c2X.

Fig. 5: the restriction enzyme mapping of the integrative gene expression vector pMC58 that expression makes up.

Fig. 6: expression SDS-PAGE electrophorogram, wherein A represents inducing cell, and B represents the fusion rotein of purifying, and C represents the dhaT albumen of purifying, and D represents not inducing cell, M represents protein standard (kDa).

Embodiment

Embodiment 1:CGMCC 0532 strain gene group DNA extracts

After CGMCC 0532 bacterial strain activation of the present invention, be seeded in the 8ml LB liquid nutrient medium (concentration of component is 1% (W/V) peptone, 0.5% (W/V) yeast extract, 1% (W/V) NaCl) 30 ℃ of overnight incubation.With the liquid seeds of above-mentioned overnight incubation, the inoculum size by 3～5% is inoculated in the 200ml LB liquid nutrient medium, and 30 ℃ are cultured to logarithmic growth mid-term.With above-mentioned bacterium liquid centrifugal 10000rpm under 4 ℃, 10～15min abandons supernatant liquor.The thalline of collecting suspends with the 2ml sterile pure water, and under 4 ℃, centrifugal 5000rpm, 10min abandons supernatant liquor, repeats this operation secondary.In the thalline of collecting, add 400 μ l sterile pure water, vibration suspends on the earthquake device, adds 100 μ l N,O-Diacetylmuramidases (10mg/ml), makes final concentration reach 2mg/ml, in 37 ℃ of thermostatic water-circulator baths, constant temperature 30min.The SDS that adds 30 μ l 20% makes final concentration reach 1%.Add proteolytic enzyme 60 μ l (20mg/ml), make final concentration reach 2mg/ml.Add sterile pure water, make final volume reach 600 μ l.Add 1 μ lRNaseA (40mg/ml), 37 ℃ of water bath with thermostatic control 3h.Adopt phenol/chloroform/primary isoamyl alcohol (25: 24: 1) solution-treated three times.Use 20 μ l sterile pure water or TE solution (concentration of component is 100Mm Tris-HCl, 10mM EDTA) dissolving again, promptly obtain the genomic dna solution of CGMCC 0532 bacterial strain.

Purity and integrity detection to CGMCC 0532 strain gene group DNA are as follows: get above-mentioned 5 μ l dna solutions and be diluted to 200 μ l, measure the OD value of 260nm, 280nm with ultraviolet spectrophotometer, and with 1% agarose gel electrophoresis at 260～280V, the integrity of rapid detection CGMCC 0532 bacterial strain DNA under the voltage of 30mA.Experimental result, OD ₂₆₀/ OD ₂₈₀=1.9＞1.8, illustrate and extract highly purified CGMCC 0532 strain gene group DNA; Electrophoresis result shows that CGMCC 0532 strain gene group DNA that extracts is complete (Fig. 2).In Fig. 2, M represents DNA standard λ-Hind III (Kb), and A represents CGMCC 0532 strain gene group DNA.

Embodiment 2:dhaT gene PCR clone

1.PCR method clone dhaT gene

1.1 primer design is with synthetic

DhaT gene (shown in SEQ ID NO:3) according to the K.pneu bacterial strain, design and synthesize the Oligonucleolide primers that is used for dhaT gene amplification, designed Hind III restriction enzyme site at 5 of upstream primer ' end, 5 of downstream primer ' end has designed EcoR I restriction enzyme site.

The PCR primer:

dhaT F 5′- AAG CTT ATG AGC TAT CGT ATG TTT GAT TAT CTG-3′(SEQ ID NO：4)

dhaT R 5′- G AAT TCT CAG AAT GCC TGG CGG AAA AT-3′(SEQ ID NO：5)

1.2PCR clone dhaT gene

Use LA Taq ^TM, pyrobest Taq, EX Taq ^TM(available from precious biotechnology (Dalian) company limited, production code member is respectively test kit: DRR002A or DRR002B; DR005A or DR005B; DRR001A or DRR001B or DRR001C), be template with CGMCC 0532 strain gene group DNA, dhaTF/dhaTR is a primer, makes PCR.

The one PCR reaction system: dNTP (each 0.5mM) 8 μ l, Taq archaeal dna polymerase 0.5 μ l, 10 * LA damping fluid, 5 μ l, genomic dna 2 μ l, primer dhaT R (0.5 μ M) 0.5 μ l, primer dhaT F (0.5 μ M) 0.5 μ l adds sterile pure water to 50 μ l.

Reaction conditions: initial melting temperature(Tm) is 94 ℃, and the time is 1min; In each repetend subsequently, to unwind at 98 ℃ of 10s, annealing extends in 72 ℃ of 1～2min at 50～55 ℃ of 30～45s, amounts to 30～35 circulations.After reaction finished, reaction system was kept at 4 ℃.

The 2nd PCR reaction system: dNTP (each 0.5mM) 8 μ l, LATaq archaeal dna polymerase 0.5 μ l, 10 * LA damping fluid, 5 μ l, genomic dna 2 μ l, primer dhaT R (0.5 μ M) 0.5 μ l, primer dhaT F (0.5 μ M) 0.5 μ l adds sterile pure water to 50 μ l.

Reaction conditions: initial melting temperature(Tm) is 94 ℃, and the time is 1min; In each repetend subsequently, to unwind at 98 ℃ of 10s, annealing extends in 72 ℃ of 1min at 55 ℃ of 30s, amounts to 30 circulations.After reaction finished, reaction system was kept at 4 ℃.

1.3PCR the dhaT gene that amplifies directly checks order

With dhaT seqF and dhaT seqR is that primer checks order.

dhaT seqF5′-CCG ATT TCC CGT ATA TGG-3′(SEQ ID NO：6)

dhaT seq R5′-TGG TGT CTT TAG GGT TTG-3′(SEQ ID NO：7)

The sequence that records is as shown in SEQ ID NO:1.

2.dhaT gene clone is to pMD18-T carrier and order-checking

2.1dhaT gene inserts segmental adjustment

A. be template with above-mentioned PCR product, dhaT F/dhaT R is a primer, 800 μ l LA Taq dna polymerase reaction system pcr amplification dhaT genes.

B. the PCR product is concentrated with ethanol, 25 μ l sterile pure water dissolution precipitations, adding 25 μ l 3M NaAc and 2.5 times of volumes does not have water-cooled ethanol, in-80 ℃ of refrigerators, preserve 20min, 4 ℃ of centrifugal 13500rpm 15～20min abandon supernatant liquor, will precipitate part and dissolve with TE solution or sterile pure water.

C. fragment reclaims: dna fragmentation reclaims with NaI after agarose gel electrophoresis separates again.

D. fragment purification: adopt ethanol, polyoxyethylene glycol or Virahol purifying.

2.2 connect

Dna fragmentation behind the purifying and enzyme being cut (Hind III/EcoR I) pMD18-T carrier (available from precious biotechnology (Dalian) company limited, production code member is: D504A or D504B or D504C, this product structure such as Fig. 3) after refining connects with dna ligase.(available from precious biotechnology (Dalian) company limited, production code member is: D2050) to use dna ligation kit among the present invention.

Reaction system is: pMD18-T 1 μ l, DNA 3 μ l connect damping fluid 5 μ l, add sterile pure water to 10 μ l.

Reaction conditions is: 16 ℃, and 1h.

2.3 transform

(available from precious biotechnology (Dalian) company limited, production code member is: D3050) to e. coli jm109 will to connect the whole thermal transitions of liquid (or electric shock transforms).

2.4 check

With primer RV-M on the pMD18-T carrier (shown in SEQ ID NO:8) and PCR primer dhaT R, adopt PCR method to test.

PCR checks first reaction system to be: dNTP 4 μ l, and LA damping fluid 2.5 μ l, primer RV-M 0.5 μ l, primer dhaTR0.5 μ l, LATaq 0.5 μ l, bacterium liquid 10 μ l add sterile pure water to 25 μ l.Reaction conditions: initial melting temperature(Tm) is 94 ℃, and the time is 1min; In each repetend subsequently, to unwind at 98 ℃ of 10s, annealing extends in 72 ℃ of 1min at 55 ℃ of 30s, amounts to 30 circulations.After reaction finished, reaction system was kept at 4 ℃.

PCR checks second reaction system to be: dNTP 8 μ l, and 2X GC damping fluid 25 μ l, primer RV-M 0.5 μ l, primer dhaTR0.5 μ l, LA Taq 0.5 μ l, bacterium liquid 10 μ l add sterile pure water to 50 μ l.Reaction conditions: check the condition of first reaction system identical with PCR.

2.5 plant bacterium

The PCR check has the bacterium colony picking of purpose band a small amount of, implants in the 3ml LB substratum, and 37 ℃ of 160～200rpm shaking culture are spent the night.

2.6 plasmid extracts

Concrete operation method adopts " molecular cloning experiment guide ", second edition, Sa nurse Brooker etc., 1998.The alkaline lysis of introducing carries out.

2.7 order-checking

Sequencing primer RV-M (shown in SEQ ID NO:8) and M with the pMD18-T carrier _13-47(shown in SEQ ID NO:9) checks order.Through recording, the result that order-checking of pMD18-T carrier and dhaT gene PCR product directly check order is identical.

Embodiment 3:dhaT genetic expression

Made up integrative gene expression vector pMC58 (structure is seen Fig. 5) with pMAL-c2X plasmid (purchase the company in New England Biolabs, production code member is: E8000S, structure is seen Fig. 4).Sequencing result shows that the dhaT gene on the pMC58 expression vector is a total length dhaT gene.

In 1 liter of LB substratum, the inoculum size by 1% inserts the culture of the e. coli jm109 that contains pMC58, adds IPTG and makes final concentration reach 0.4～0.8mM, the fusion rotein that the fusion gene that malE gene and dhaT gene constitute is expressed; Show that through the SDS-PAGE electrophoresis fusion protein molecule amount is 85KDa, express electrophoresis result (seeing Fig. 6, the B band).A among Fig. 6: inducing cell, B: the fusion rotein of purifying, C: the dhaT albumen of purifying, D: inducing cell not, Mh (kDa): 200,160,97,4,66,45, ML (kDa): 97,4,66,45,31,21.5,14.5.

The proteinic isolation and purification of embodiment 4:dhaT genetic expression

With the warm protein purification system purifying of the pMAL-c2X dhaT albumen available from New England Biolabs, the fusion protein molecule amount that the fusion gene that malE gene and dhaT gene constitute is expressed is 85KDa; The fusion rotein of purifying is cut through the FactorXa enzyme, separates the protein that has obtained dhaT genetic expression through ion exchange chromatography, shows that through the SDS-PAGE electrophoresis protein molecular weight is 43KDa (seeing Fig. 6, the C band).

The proteinic enzyme activity determination of embodiment 5:dhaT genetic expression

In the time of 25 ℃, spectrophotometer method is measured 365nm, and 1, the 3-PD oxydo-reductase is converted into 1 with the trihydroxy-propionic aldehyde, the consumption of the initial NADH of 3-PD.Enzyme work is defined as, and per minute NADH consumes micromole's number.

Experimental result shows: the proteinic enzyme work of dhaT genetic expression is 39 μ M/mg.

Sequence table

＜110〉Chi Naiyu

＜120〉encode 1 in a kind of E.aero bacterial strain, the gene of ammediol oxydo-reductase

＜130〉1, the ammediol oxydo-reductase

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Claims (7)

1. produce 1 for one kind, and ammediol (1, enteroaerogen 3-PD) (Enterobacter aerogenes is abbreviated as E.aero) bacterial strain, its preserving number is CGMCC 0532.

2. separated coding 1, the gene of ammediol oxydo-reductase, it has the nucleotide sequence shown in SEQ ID NO:1.

3. one kind by 1 of the described genes encoding of claim 2, the ammediol oxydo-reductase, and it has the aminoacid sequence shown in SEQ ID NO:2.

4. separated coding 1, the gene of ammediol oxydo-reductase, its coding have the aminoacid sequence shown in SEQ ID NO:2.

5. one kind has coding 1, the gene of ammediol oxidoreductase activity, and it replaces, lacks, replaces for one or several Nucleotide of the described gene of claim 4 or inserts the gene that is afterwards obtained.

6. recombinant vectors, it comprises claim 4 or 5 described genes.

7. one kind by claim 4 or 5 described genes or by the described recombinant vectors transformed host cells of claim 5.

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