CN104480055A - Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli - Google Patents

Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli Download PDF

Info

Publication number
CN104480055A
CN104480055A CN201410693426.XA CN201410693426A CN104480055A CN 104480055 A CN104480055 A CN 104480055A CN 201410693426 A CN201410693426 A CN 201410693426A CN 104480055 A CN104480055 A CN 104480055A
Authority
CN
China
Prior art keywords
escherichia coli
bacterium
coli strain
plasmid
recombinant escherichia
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
CN201410693426.XA
Other languages
Chinese (zh)
Inventor
李申
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.)
TIANJIN SHENZONG TECHNOLOGY Co Ltd
Original Assignee
TIANJIN SHENZONG TECHNOLOGY 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 TIANJIN SHENZONG TECHNOLOGY Co Ltd filed Critical TIANJIN SHENZONG TECHNOLOGY Co Ltd
Priority to CN201410693426.XA priority Critical patent/CN104480055A/en
Publication of CN104480055A publication Critical patent/CN104480055A/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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric
    • 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
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/01—Hydro-lyases (4.2.1)
    • C12Y402/0103—Glycerol dehydratase (4.2.1.30)

Abstract

The invention relates to a method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli and protects a recombinant Escherichia coli strain, a construction method of the recombinant Escherichia coli strain and a method of synthesizing the poly 3-hydracrylic acid by using the recombinant Escherichia coli strain by virtue of a biological method. The method has the beneficial effects that in the metabolic pathway of biologic synthesis of P3HP with glycerol serving as a substrate, NAD+, serving as coenzyme, can generate a great deal of NADH when used for catalyzing 3-hydroxy-propionaldehyde to produce 3HP; the oxidation-reduction disequilibrium in a recombinant strain, caused by increase of NADH is capable of inhibiting further increase of the yield of P3HP, and NADH can be consumed in the process that the recombinant Escherichia coli strain metabolizes glycerol to generate 1,3-PDO, and therefore synthesis of 1,3-PDO is introduced into the synthesis pathway of P3HP, the in-vivo reducing power of the strain can be balanced, the plasmid loss rate is effectively reduced, and the plasmid stability is kept.

Description

A kind of method utilizing recombination bacillus coli to synthesize poly-3-hydroxy-propionic acid
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of method utilizing recombination bacillus coli to synthesize poly-3-hydroxy-propionic acid.
Background technology
Along with improving constantly of living standards of the people, the research about biodegradable and green material receives much attention.This kind of material not only degradable, and there is good biocompatibility, therefore in the field such as environment, biological medicine, there is wide application prospect.Poly-3-hydroxy-propionic acid [P-(3-HP)] is a kind of Biodegradable polymer, it not only has good biological degradability and biocompatibility, and the P of high molecular (3-HP) has very high physical strength and the excellent properties such as tensile strength (>400MPa) and larger elongation at break.Thus cause the attention of the researchist of some developed countries in the world, but report still less about the study on the synthesis of P (3-HP) in the world so far.
Polyhydroxyalkanoate (Polyhydroxyalkanoates, PHAs) be the Biodegradable material developed rapidly nearly two more than ten years, being the general name of the linear polyester that a class of synthesis in microbe is made up of 3-hydroxy fatty acid, is as the macromolecule polyester that the reserve substance of carbon source and the energy is formed when in cell, carbon nitrogen source is uneven.From Lemoigne after Late Cambrian poly butyric ester (Poly-hydroxybutyricacid, PHB) in bacillus megaterium (Bacillusmegaterium), have and determined can be used to polymerization generation PHA more than 150 kinds of monomers.Poly-3-hydroxy-propionic acid [Poly (3-hydroxypropionate), P3HP is a kind of novel biodegradable plastic, there is excellent biomaterial character and mechanical property, as high mechanical strength and tensile strength, high extension at break amount, biological degradability, biocompatibility, water insoluble, nontoxic, piezoelectricity, thermoplasticity etc.At present, the chemical synthesis process of poly-3-hydroxy-propionic acid is based on beta-propiolactone ring-opening polymerization, but beta-propiolactone is expensive and be carcinogenic substance, limits the application of the method.The research of biosynthesizing P3HP is also at the early-stage.In most biology, 3-hydroxy-propionic acid (3-Hydroxypropionate, 3HP) is not the compound that native metabolic pathway produces, and therefore synthesizing P3HP needs in substratum, to add the compound relevant to 3HP structure, as 3-HP, vinylformic acid etc.These precursor substances have cytotoxicity, can the growth of T suppression cell and expensive, are not suitable for large-scale industrial production.For addressing this problem, investigator constructs respectively with the pathways metabolism of cheap carbon source (glucose and glycerine) for substrate synthesis P3HP, after fermentation ends, acetone-chloroform extraction process is utilized to obtain P3HP, and by nmr for the determination polymer purity.Meanwhile, gel permeation chromatography is utilized to record the number-average molecular weight (Mn) of P3HP and weight-average molecular weight (Mw) is respectively 1.2 × 10 5da and 1.78 × 10 5da.But take glucose as the route of synthesis of the P3HP of substrate, output only has 13mg/L, and is the route of synthesis of substrate with glycerine, although the optimization of condition by fermentation, output is also far from reaching the requirement of suitability for industrialized production.This is on the one hand owing to being that substrate is produced in the process of P3HP with glycerine, and 3-HPA is reduced to the NADH that produces in the process of 3-hydroxyl propionyl coenzyme A cannot regeneration NAD+ in time, causes NAD+ deficiency in cell, finally have impact on the synthesis of P3HP.Meanwhile, also lose relevant in a large number with the plasmid carrying synthetic gene in fermenting process.
Summary of the invention
The present invention is for solving the problems of the technologies described above, the synthetic gene of glycerol dehydratase and incitant thereof is integrated in coli strain, and dhaT gene, make the coli strain after restructuring have biosynthesizing and gather 3-hydroxy-propionic acid and 1, the ability of ammediol, the technical scheme of employing is as follows:
A kind of recombinant escherichia coli strain, genome of E.coli is integrated the synthetic gene of glycerol dehydratase and incitant thereof, and dhaT gene, and the coli strain after restructuring has the ability that biosynthesizing gathers 3-hydroxy-propionic acid and 1,3-PD.
Described dhaT gene is with Klebsiella pneumonia genomic dna for template, obtains with primer ZG-440 and ZG-441 clone.
Utilize a construction process for described recombinant escherichia coli strain, step is as follows:
Step 1: the structure of plasmid pET21 (A): with Klebsiella pneumonia genomic dna for template, clone dhaT gene with primer ZG-440 and ZG-441, dhaT fragment is connected after HindIII, XhoI enzyme is cut with plasmid pWQ02;
Step 2: the structure of plasmid pRE112 (B): with the genome of E.coliBL21 for template, utilize primer ZG-268 and ZG-269 respectively, primer ZG-270 and ZG-271 amplify fragment prpR gene both sides homologous fragment, again with two amplified fragments for template, utilize primer ZG-268 and ZG-271 to amplify fragment prpR; After restriction enzyme SacI and NheI enzyme cut prpR, be connected to the plasmid pRE112 after restriction enzyme SacI and XbaI enzyme cutting, be built into recombinant plasmid pRE112 (A); Recycling restriction enzyme XhoI and XbaI enzyme cutting recombinant plasmid pRE112 (A) and pWQ04, cut glue and reclaim plasmid pRE112 (A) and dhaB-gdrAB fragment, after carrying out enzyme company, build recombinant plasmid pRE112 (B);
Step 3: first time restructuring: with E.coli/pRE112 (B) for plasmid donor bacterium, to treat that plasmid recipient bacterium is done in sudden change bacterial classification E.coliBL21 (DE3); After donor bacterium and recipient bacterium are activated, respectively get 1.5mL, centrifugal rear sterilized water is resuspended washes 2 times, to remove microbiotic, again with respectively getting 100 μ L after the resuspended bacterial sediment of sterilized water of 500 μ L and mixing, the resuspended thalline of 1%DAP of centrifugal rear use 50 μ L, coats non-resistant flat board, cultivates 12h in 37 DEG C; The thalline collected on flat board is resuspended in sterilized water; By resuspended bacterium liquid dilution 10 -1-10 -4doubly, each multiple is all got 30 μ L and is coated on paraxin flat board, cultivates 24h in 37 DEG C; PCR verifies mono-clonal bacterium colony, mark positive bacteria;
Step 4: second time restructuring: by the once restructuring positive monoclonal of mark access non-resistant LB test tube, cultivate 15h in 37 DEG C; By bacterium liquid dilution 10 -1-10 -6doubly, each multiple is all got 20 μ L and is coated on 10% sucrose plate, cultivates 12h for 37 DEG C; Select the mono-clonal bacterium colony on sucrose plate, draw paraxin flat board and 10% sucrose plate respectively, cultivate 10h for 37 DEG C; Choose growth on sucrose plate and the dull and stereotyped bacterium do not grown of paraxin is cooked PCR checking, mark positive bacteria; Draw sucrose plate and be separated mono-clonal 2 times, guarantee that secondary recombinant bacterium is pure; PCR verifies correct and in paraxin LB test tube, detects the bacterium do not grown, in-80 DEG C of preservations the most at last.
Utilize described recombinant escherichia coli strain to synthesize a method for poly-3-hydroxy-propionic acid, it is characterized in that, step is as follows:
Step 1: described recombinant escherichia coli strain is activated in LB substratum;
Step 2: after activation, the 500mL proceeding to 1/5 liquid amount is with in the triangle shaking flask of baffle plate and carries out substratum fermentation, inoculum size 1%, add 100mg/L sodium ampicillin and 34mg/L paraxin simultaneously, 37 DEG C, 200r/min cultivation, OD600 adds IPTG (Isopropyl β-D-1-Thiogalactopyranoside) when reaching 0.6-1.0, and 0.03g/L vitamin B12 culture temperature is adjusted to 30 DEG C; Cultivate 72h;
After step 3:72h, collected by centrifugation thalline, with sterilized water and 95% ethanol purge bacterial sediment; Lyophilize obtains dry cell weight to constant weight;
Step 4: at ambient temperature, the acetone solvent mixing, washing of 10 times of biomasses is added once in cold dry mycelium, after evaporation drying, dry mycelium is wrapped and is placed in apparatus,Soxhlet's, add the chloroform of 10 times of biomasses, 90 DEG C of extraction 4h, leave standstill cooling after extraction, filter, Aspirate supernatant must clarify poly-3-hydroxy-propionic acid solution;
Step 5: use Rotary Evaporators to be steamed by chloroform at 45 DEG C, the precipitate bottom distilling flask is the 3-hydroxy-propionic acid polymkeric substance that extraction purification obtains, and 60 DEG C of dry 12h, eliminate residual chloroform completely, obtains 3-hydroxy-propionic acid.
Described in step 1, LB substratum consists of: Tryptones 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, pH7.0.The substratum carrying out described in step 2 fermenting consists of: glycerine 20g/L, (NH 4) 2sO4 3g/L, glucose 3g/L, KH 2pO 41.5g/L, KCl 1.9g/L, citric acid 1g/L, Trisodium Citrate 1g/L, FeSO 47H 2o 0.138g/L, micro-1mL/L, VITMAIN B1 0.045g/L, pH7.0.Described trace element consists of: (NH 4) 6mo 7o 244H 2o0.37g/L, H 3bO 42.47g/L, MnCl 24H 2o 1.58g/L, ZnSO 47H 2o 0.29g/L, CuSO 45H 2o 0.25g/L.
Suicide vector refer to can copy in a kind of host but in another host not reproducible plasmid, when entering host cell, or not reproducible and being eliminated, or being integrated on karyomit(e), copying together with karyomit(e).Utilize this feature of suicide vector, the genetically deficient of structure or the DNA fragmentation of sudden change are cloned into suicide vector, utilize the homologous fragment at missing gene two ends, the integration site of location suicide plasmid.By bonding method, the DNA built is imported recipient bacterium.Utilize homology DNA fragmentation that the principle of recombinating can occur, build accurate genetically deficient or mutant strain.The suicide plasmid pRE112 used in this experiment has two selection markers: positive selection markers chloramphenicol resistance gene and negative selection markers sucrose levanase (Levansucrase) sacB gene.
PrpR is the gene dividing decorrelation in intestinal bacteria with propionic acid, its disappearance both can not have an impact to whole intracellular metabolic reaction, can also avoid causing P3HP production declining because propionic acid decomposes, therefore we select prpR fragment as the insertion point of glycerol dehydrase gene dhaB123 and cofactor gdrAB thereof.Utilize PCR method Successful amplification to go out prpR gene upstream and downstream homology arm fragment, insert dhaB123 and gdrAB gene wherein, and be connected into pRE112 suicide plasmid carrier and successfully construct recombinant plasmid pRE112 (B).Plasmid pRE112 (B) is converted in E.coli bacterial strain, engages with E.coli BL21, by chlorampenicol resistant LB plate screening recombinant bacterium.Now, there occurs first time restructuring bacterial chromosome on carry the pRE112 whole piece sequence comprising chloramphenicol resistance gene, thus can at paraxin grow on plates.As long as obtain resistance bacterium to be a recombinant bacterium, PCR verifies a recombinant bacterium.
SacB genes encoding sucrose levanase, this enzyme energy catalysing sucrose is hydrolyzed into glucose and fructose, and fructose is aggregated into the Polylevulosan of high molecular.But there is potential toxic action to cell in the accumulation of high molecular Polylevulosan, can cause necrocytosis.One time recombinant bacterium contains sacB gene, cannot stablize under containing the condition of sucrose and go down to posterity, and restructuring can lose suicide vector gradually, therefore chooses growth on sucrose plate and bacterium that paraxin flat board does not grow is cooked PCR checking, can obtain aimed strain.
The process that microbial metabolism glycerine generates 1,3-PDO can consume NADH, in the route of synthesis of P3HP, therefore introduces the synthesis of 1,3-PDO, can balance reducing power in thalline.By the homologous recombination technique of suicide vector System-mediated, the synthetic gene of glycerol dehydratase and incitant thereof is incorporated in genome of E.coli, effectively can reduces the Loss Rate of plasmid, keep plasmid stability.In order to determine that the homologous recombination mutant strain of coexpression dhaT has promoter action to P3HP output, be that sole carbon source ferments with glycerine, experimental result shows, after E.coliBL21 (B) fermentation containing pWQ02 and pWQ04, P3HP is 0.2g/L, accounts for 6.5% of dry cell weight; After the homologous recombination mutant strain fermentation of coexpression dhaT gene, P3HP is 0.8g/L, 14.6%, the P3HP output accounting for dry cell weight for the former 4 times.
The invention has the beneficial effects as follows: be in the pathways metabolism of substrate biosynthesizing P3HP with glycerine, NAD+ can produce a large amount of NADH as coenzyme while catalysis 3-HPA produces 3HP.The recombinant bacterium vivo oxidation reduction dysequilibrium that the rising of NADH causes is that restriction P3HP output improves further, recombinant escherichia coli strain metabolism glycerine of the present invention generates 1, the process of 3-PDO can consume NADH, therefore in the route of synthesis of P3HP, 1 is introduced, the synthesis of 3-PDO, can balance reducing power in thalline.By the homologous recombination technique of suicide vector System-mediated, the synthetic gene of glycerol dehydratase and incitant thereof is incorporated in genome of E.coli, the Loss Rate of plasmid can be effectively reduced, keep plasmid stability.
Accompanying drawing explanation
Fig. 1 is recombination bacillus coli take glycerine as the pathways metabolism reaction equation that substrate synthesis gathers 3-hydroxy-propionic acid.
Embodiment
The technique means realized to make the present invention, creation characteristic, reaching object and effect is easy to understand, below the specific embodiment of the present invention being described further, but not limiting protection scope of the present invention.
Embodiment 1
A kind of recombinant escherichia coli strain, genome of E.coli is integrated the synthetic gene of glycerol dehydratase and incitant thereof, and dhaT gene, and the coli strain after restructuring has the ability that biosynthesizing gathers 3-hydroxy-propionic acid and 1,3-PD.
Described dhaT gene is with Klebsiella pneumonia genomic dna for template, obtains with primer ZG-440 and ZG-441 clone.
Utilize a construction process for described recombinant escherichia coli strain, step is as follows:
Step 1: the structure of plasmid pET21 (A): with Klebsiella pneumonia genomic dna for template, clone dhaT gene with primer ZG-440 and ZG-441, dhaT fragment is connected after HindIII, XhoI enzyme is cut with plasmid pWQ02;
Step 2: the structure of plasmid pRE112 (B): with the genome of E.coliBL21 for template, utilize primer ZG-268 and ZG-269 respectively, primer ZG-270 and ZG-271 amplify fragment prpR gene both sides homologous fragment, again with two amplified fragments for template, utilize primer ZG-268 and ZG-271 to amplify fragment prpR; After restriction enzyme SacI and NheI enzyme cut prpR, be connected to the plasmid pRE112 after restriction enzyme SacI and XbaI enzyme cutting, be built into recombinant plasmid pRE112 (A); Recycling restriction enzyme XhoI and XbaI enzyme cutting recombinant plasmid pRE112 (A) and pWQ04, cut glue and reclaim plasmid pRE112 (A) and dhaB-gdrAB fragment, after carrying out enzyme company, build recombinant plasmid pRE112 (B);
Step 3: first time restructuring: with E.coli/pRE112 (B) for plasmid donor bacterium, to treat that plasmid recipient bacterium is done in sudden change bacterial classification E.coliBL21 (DE3); After donor bacterium and recipient bacterium are activated, respectively get 1.5mL, centrifugal rear sterilized water is resuspended washes 2 times, to remove microbiotic, again with respectively getting 100 μ L after the resuspended bacterial sediment of sterilized water of 500 μ L and mixing, the resuspended thalline of 1%DAP of centrifugal rear use 50 μ L, coats non-resistant flat board, cultivates 12h in 37 DEG C; The thalline collected on flat board is resuspended in sterilized water; By resuspended bacterium liquid dilution 10 -1-10 -4doubly, each multiple is all got 30 μ L and is coated on paraxin flat board, cultivates 24h in 37 DEG C; PCR verifies mono-clonal bacterium colony, mark positive bacteria;
Step 4: second time restructuring: by the once restructuring positive monoclonal of mark access non-resistant LB test tube, cultivate 15h in 37 DEG C; By bacterium liquid dilution 10 -1-10 -6doubly, each multiple is all got 20 μ L and is coated on 10% sucrose plate, cultivates 12h for 37 DEG C; Select the mono-clonal bacterium colony on sucrose plate, draw paraxin flat board and 10% sucrose plate respectively, cultivate 10h for 37 DEG C; Choose growth on sucrose plate and the dull and stereotyped bacterium do not grown of paraxin is cooked PCR checking, mark positive bacteria; Draw sucrose plate and be separated mono-clonal 2 times, guarantee that secondary recombinant bacterium is pure; PCR verifies correct and in paraxin LB test tube, detects the bacterium do not grown, in-80 DEG C of preservations the most at last.
Utilize described recombinant escherichia coli strain to synthesize a method for poly-3-hydroxy-propionic acid, it is characterized in that, step is as follows:
Step 1: described recombinant escherichia coli strain is activated in LB substratum;
Step 2: after activation, the 500mL proceeding to 1/5 liquid amount is with in the triangle shaking flask of baffle plate and carries out substratum fermentation, inoculum size 1%, add 100mg/L sodium ampicillin and 34mg/L paraxin simultaneously, 37 DEG C, 200r/min cultivation, OD600 adds IPTG (Isopropyl β-D-1-Thiogalactopyranoside) when reaching 0.6-1.0, and 0.03g/L vitamin B12 culture temperature is adjusted to 30 DEG C; Cultivate 72h;
After step 3:72h, collected by centrifugation thalline, with sterilized water and 95% ethanol purge bacterial sediment; Lyophilize obtains dry cell weight to constant weight;
Step 4: at ambient temperature, the acetone solvent mixing, washing of 10 times of biomasses is added once in cold dry mycelium, after evaporation drying, dry mycelium is wrapped and is placed in apparatus,Soxhlet's, add the chloroform of 10 times of biomasses, 90 DEG C of extraction 4h, leave standstill cooling after extraction, filter, Aspirate supernatant must clarify poly-3-hydroxy-propionic acid solution;
Step 5: use Rotary Evaporators to be steamed by chloroform at 45 DEG C, the precipitate bottom distilling flask is the 3-hydroxy-propionic acid polymkeric substance that extraction purification obtains, and 60 DEG C of dry 12h, eliminate residual chloroform completely, obtains 3-hydroxy-propionic acid.
Described in step 1, LB substratum consists of: Tryptones 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, pH7.0.The substratum carrying out described in step 2 fermenting consists of: glycerine 20g/L, (NH 4) 2sO 43g/L, glucose 3g/L, KH 2pO 41.5g/L, KCl 1.9g/L, citric acid 1g/L, Trisodium Citrate 1g/L, FeSO 47H 2o 0.138g/L, micro-1mL/L, VITMAIN B1 0.045g/L, pH7.0.Described trace element consists of: (NH 4) 6mo 7o 244H 2o0.37g/L, H 3bO 42.47g/L, MnCl 24H 2o 1.58g/L, ZnSO 47H 2o 0.29g/L, CuSO 45H 2o 0.25g/L.
Method of the present invention is described by specific embodiment.Those skilled in the art can use for reference the links such as each component proportion of content appropriate change of the present invention and realize other object corresponding, its relevant change does not all depart from content of the present invention, all similar replacements and change will become apparent to those skilled in the art that and be all deemed to be included within scope of the present invention.

Claims (7)

1. a recombinant escherichia coli strain, it is characterized in that, genome of E.coli is integrated the synthetic gene of glycerol dehydratase and incitant thereof, and dhaT gene, coli strain after restructuring has the ability that biosynthesizing gathers 3-hydroxy-propionic acid and 1,3-PD.
2. a kind of recombinant escherichia coli strain according to claim 1, is characterized in that, described dhaT gene is with Klebsiella pneumonia genomic dna for template, obtains with primer ZG-440 and ZG-441 clone.
3. a construction process for recombinant escherichia coli strain described in claim 1, is characterized in that, step is as follows:
Step 1: the structure of plasmid pET21 (A): with Klebsiella pneumonia genomic dna for template, clone dhaT gene with primer ZG-440 and ZG-441, dhaT fragment is connected after HindIII, XhoI enzyme is cut with plasmid pWQ02;
Step 2: the structure of plasmid pRE112 (B): with the genome of E.coliBL21 for template, utilize primer ZG-268 and ZG-269 respectively, primer ZG-270 and ZG-271 amplify fragment prpR gene both sides homologous fragment, again with two amplified fragments for template, utilize primer ZG-268 and ZG-271 to amplify fragment prpR; After restriction enzyme SacI and NheI enzyme cut prpR, be connected to the plasmid pRE112 after restriction enzyme SacI and XbaI enzyme cutting, be built into recombinant plasmid pRE112 (A); Recycling restriction enzyme XhoI and XbaI enzyme cutting recombinant plasmid pRE112 (A) and pWQ04, cut glue and reclaim plasmid pRE112 (A) and dhaB-gdrAB fragment, after carrying out enzyme company, build recombinant plasmid pRE112 (B);
Step 3: first time restructuring: with E.coli/pRE112 (B) for plasmid donor bacterium, to treat that plasmid recipient bacterium is done in sudden change bacterial classification E.coliBL21 (DE3); After donor bacterium and recipient bacterium are activated, respectively get 1.5mL, centrifugal rear sterilized water is resuspended washes 2 times, to remove microbiotic, again with respectively getting 100 μ L after the resuspended bacterial sediment of sterilized water of 500 μ L and mixing, the resuspended thalline of 1%DAP of centrifugal rear use 50 μ L, coats non-resistant flat board, cultivates 12h in 37 DEG C; The thalline collected on flat board is resuspended in sterilized water; By resuspended bacterium liquid dilution 10 -1-10 -4doubly, each multiple is all got 30 μ L and is coated on paraxin flat board, cultivates 24h in 37 DEG C; PCR verifies mono-clonal bacterium colony, mark positive bacteria;
Step 4: second time restructuring: by the once restructuring positive monoclonal of mark access non-resistant LB test tube, cultivate 15h in 37 DEG C; By bacterium liquid dilution 10 -1-10- 6doubly, each multiple is all got 20 μ L and is coated on 10% sucrose plate, cultivates 12h for 37 DEG C; Select the mono-clonal bacterium colony on sucrose plate, draw paraxin flat board and 10% sucrose plate respectively, cultivate 10h for 37 DEG C; Choose growth on sucrose plate and the dull and stereotyped bacterium do not grown of paraxin is cooked PCR checking, mark positive bacteria; Draw sucrose plate and be separated mono-clonal 2 times, guarantee that secondary recombinant bacterium is pure; PCR verifies correct and in paraxin LB test tube, detects the bacterium do not grown, in-80 DEG C of preservations the most at last.
4. a method for the poly-3-hydroxy-propionic acid of recombinant escherichia coli strain synthesis described in claim 1, it is characterized in that, step is as follows:
Step 1: described recombinant escherichia coli strain is activated in LB substratum;
Step 2: after activation, the 500mL proceeding to 1/5 liquid amount is with in the triangle shaking flask of baffle plate and carries out substratum fermentation, inoculum size 1%, add 100mg/L sodium ampicillin and 34mg/L paraxin simultaneously, 37 DEG C, 200r/min cultivation, OD600 adds IPTG (Isopropyl β-D-1-Thiogalactopyranoside) when reaching 0.6-1.0, and 0.03g/L vitamin B12 culture temperature is adjusted to 30 DEG C; Cultivate 72h;
After step 3:72h, collected by centrifugation thalline, with sterilized water and 95% ethanol purge bacterial sediment; Lyophilize obtains dry cell weight to constant weight;
Step 4: at ambient temperature, the acetone solvent mixing, washing of 10 times of biomasses is added once in cold dry mycelium, after evaporation drying, dry mycelium is wrapped and is placed in apparatus,Soxhlet's, add the chloroform of 10 times of biomasses, 90 DEG C of extraction 4h, leave standstill cooling after extraction, filter, Aspirate supernatant must clarify poly-3-hydroxy-propionic acid solution;
Step 5: use Rotary Evaporators to be steamed by chloroform at 45 DEG C, the precipitate bottom distilling flask is the 3-hydroxy-propionic acid polymkeric substance that extraction purification obtains, and 60 DEG C of dry 12h, eliminate residual chloroform completely, obtains 3-hydroxy-propionic acid.
5. recombinant escherichia coli strain synthesis gathers the method for 3-hydroxy-propionic acid according to claim 4, and it is characterized in that, described in step 1, LB substratum consists of: Tryptones 10.0g/L, yeast extract 5.0g/L, NaCl 10.0g/L, pH 7.0.
6. recombinant escherichia coli strain synthesis gathers the method for 3-hydroxy-propionic acid according to claim 4, and it is characterized in that, the substratum carrying out described in step 2 fermenting consists of: glycerine 20g/L, (NH 4) 2sO 43g/L, glucose 3g/L, KH 2pO 41.5g/L, KCl 1.9g/L, citric acid 1g/L, Trisodium Citrate 1g/L, FeSO 47H 2o 0.138g/L, micro-1mL/L, VITMAIN B1 0.045g/L, pH 7.0.
7. recombinant escherichia coli strain synthesis gathers the method for 3-hydroxy-propionic acid according to claim 6, and it is characterized in that, described trace element consists of: (NH 4) 6mo 7o 244H 2o 0.37g/L, H 3bO 42.47g/L, MnCl 24H 2o 1.58g/L, ZnSO 47H 2o 0.29g/L, CuSO 45H 2o 0.25g/L.
CN201410693426.XA 2014-11-26 2014-11-26 Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli Pending CN104480055A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201410693426.XA CN104480055A (en) 2014-11-26 2014-11-26 Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410693426.XA CN104480055A (en) 2014-11-26 2014-11-26 Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli

Publications (1)

Publication Number Publication Date
CN104480055A true CN104480055A (en) 2015-04-01

Family

ID=52754664

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201410693426.XA Pending CN104480055A (en) 2014-11-26 2014-11-26 Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli

Country Status (1)

Country Link
CN (1) CN104480055A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950642A (en) * 2016-07-20 2016-09-21 清华大学 Glycerol dehydratase gene resistant to oxygen and glycerin and application thereof
CN106834322A (en) * 2017-03-14 2017-06-13 中国科学院南海海洋研究所 The application of antitoxin SO_1445 or its encoding gene in stability of the plasmid in host cell is improved
CN107119003A (en) * 2017-04-28 2017-09-01 中国科学院青岛生物能源与过程研究所 A kind of utilization glucan synthesizes recombinant bacterium and its construction method and the application of 3 hydracrylic acids

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105950642A (en) * 2016-07-20 2016-09-21 清华大学 Glycerol dehydratase gene resistant to oxygen and glycerin and application thereof
CN106834322A (en) * 2017-03-14 2017-06-13 中国科学院南海海洋研究所 The application of antitoxin SO_1445 or its encoding gene in stability of the plasmid in host cell is improved
CN106834322B (en) * 2017-03-14 2020-09-04 中国科学院南海海洋研究所 Application of antitoxin SO _1445 or encoding gene thereof in improving stability of plasmid in host cell
CN107119003A (en) * 2017-04-28 2017-09-01 中国科学院青岛生物能源与过程研究所 A kind of utilization glucan synthesizes recombinant bacterium and its construction method and the application of 3 hydracrylic acids
CN107119003B (en) * 2017-04-28 2020-04-24 中国科学院青岛生物能源与过程研究所 Recombinant bacterium for synthesizing 3-hydroxypropionic acid by utilizing glucan and construction method and application thereof

Similar Documents

Publication Publication Date Title
Nangle et al. Valorization of CO2 through lithoautotrophic production of sustainable chemicals in Cupriavidus necator
Singh et al. Progress and challenges in producing polyhydroxyalkanoate biopolymers from cyanobacteria
Kumar et al. Extending the limits of Bacillus for novel biotechnological applications
Jiang et al. Efficient polyhydroxyalkanoates production from a waste-activated sludge alkaline fermentation liquid by activated sludge submitted to the aerobic feeding and discharge process
Somleva et al. PHA bioplastics, biochemicals, and energy from crops
Muthuraj et al. Recent developments in short-and medium-chain-length Polyhydroxyalkanoates: Production, properties, and applications
Aldor et al. Metabolic engineering of a novel propionate-independent pathway for the production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Salmonella enterica serovar typhimurium
Orita et al. Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions
Van‐Thuoc et al. Polyester production by halophilic and halotolerant bacterial strains obtained from mangrove soil samples located in N orthern V ietnam
Zhou et al. Production of 3-hydroxypropionate homopolymer and poly (3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli
Meng et al. Production of poly (3-hydroxypropionate) and poly (3-hydroxybutyrate-co-3-hydroxypropionate) from glucose by engineering Escherichia coli
Obulisamy et al. Polyhydroxyalkanoates from extremophiles: a review
Guleria et al. Polyhydroxyalkanoates production from domestic waste feedstock: A sustainable approach towards bio-economy
Brown et al. Rhodopseudomonas palustris: A biotechnology chassis
Wübbeler et al. New pathways for bacterial polythioesters
CN103703137B (en) The method being manufactured useful chemicals by p-phthalic acid potassium salt
Bhola et al. Established and emerging producers of PHA: Redefining the possibility
Yamaguchi et al. Production of poly (3-hydroyxybutylate) by a novel alginolytic bacterium Hydrogenophaga sp. strain UMI-18 using alginate as a sole carbon source
CN104480055A (en) Method of synthesizing poly 3-hydracrylic acid by utilizing recombinant Escherichia coli
CN103403157A (en) Phenylpyruvate reductase and method for manufacturing optically-active phenyllactic acid and 4-hydroxyl-phenyllactic acid using same enzyme
CN114807206A (en) Bacterial strain for synthesizing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) and construction method and application thereof
Zhang et al. Effective production of Poly (3-hydroxybutyrate-co-4-hydroxybutyrate) by engineered Halomonas bluephagenesis grown on glucose and 1, 4-Butanediol
Esposito et al. Enhanced production of biobased, biodegradable, Poly (3-hydroxybutyrate) using an unexplored marine bacterium Pseudohalocynthiibacter aestuariivivens, isolated from highly polluted coastal environment
Goswami et al. Valorization of organic wastes using bioreactors for polyhydroxyalkanoate production: Recent advancement, sustainable approaches, challenges, and future perspectives
CN102517303A (en) Recombination blue-green alga for producing lactic acid as well as preparation method and applications thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20150401