CN109112156A - A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application - Google Patents

Abstract

Translated fromChinese

一种生产3‑羟基丙酸的重组假单胞菌、其构建方法及其应用，属于基因工程领域和发酵工程领域，所述重组宿主菌为假单胞菌，表达编码乙酰辅酶A羧化酶的基因accABCD和编码丙二酰辅酶A还原酶的基因mcr。制备方法为：获得重组载体pUCPK‑AM；转化到宿主感受态细胞中。本发明通过构建外源表达3‑羟基丙酸生产途径，获得的菌株能够在以乙酸为唯一碳源的培养基中生产3‑羟基丙酸，可以降低3‑羟基丙酸生物制造成本，并为工农业废弃乙酸的利用提供一种可行的研究思路与方向，具有重要的经济价值和社会意义。

A recombinant Pseudomonas for producing 3-hydroxypropionic acid, a construction method and applications thereof, belonging to the fields of genetic engineering and fermentation engineering, wherein the recombinant host is Pseudomonas and expresses coding acetyl-CoA carboxylase The gene accABCD and the gene mcr encoding malonyl-CoA reductase. The preparation method is as follows: obtaining a recombinant vector pUCPK-AM; and transforming it into a host competent cell. In the present invention, by constructing an exogenous expression 3-hydroxypropionic acid production pathway, the obtained strain can produce 3-hydroxypropionic acid in a medium with acetic acid as the sole carbon source, which can reduce the biological production cost of 3-hydroxypropionic acid, and is effective for the production of 3-hydroxypropionic acid. The utilization of industrial and agricultural waste acetic acid provides a feasible research idea and direction, which has important economic value and social significance.

Description

A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application

Technical field

The present invention relates to molecular biology, genetic engineering field, and in particular to a kind of recombination for producing 3- hydracrylic acid is falseMonad, its construction method and its application.

Background technique

3- hydracrylic acid is the green material for the acrylic acid that synthesis has high added economic value, is widely used in baby's paperIn urine pants, surface covering, paint and adhesives industry.In recent years, the demand of acrylic acid is continuously increased.Traditional chemical methodProduction 3- hydracrylic acid has the characteristics that process is complicated, environmental pollution at high cost and easy, and microbial method production 3- hydracrylic acidThese factors can be effectively avoided.Biology base 3- hydracrylic acid is mainly obtained using glycerol as substrate by microbial fermentation at present, raw materials glycerine price is larger by Biodiesel influence on development.Under the premise of China does not strive the policy on ground with grain and oil, biologyDiesel oil industry raw material resources are insufficient, cost is excessively high, bring limitation to Material synthesis 3- hydracrylic acid is used glycerol as.CauseThis, solves the competitiveness that raw material sources and cost problem are produced for improving Chinese biological base 3- hydracrylic acid, realizes biology baseThe sustainable development of 3- hydracrylic acid is most important.Acetic acid is in China's abundance, the waste water of industrial or agricultural discharge rich in a large amount ofAcetic acid；The conversion of synthesising biological gas, a carbonizable substance methanol and methane conversion, sewage sludge anaerobic digestion etc. can also generate largelyAcetic acid.Compared with other raw materials, acetic acid raw material is cheap.If being used for the conjunction of biology base 3- hydracrylic acid using acetic acid as raw materialAt can be greatly lowered production cost, improve the competitiveness of biology base 3- hydracrylic acid, realize biology base 3- hydracrylic acidSustainable development.Mode microorganism Escherichia coli (E.coli), which has been reported, can use acetic acid synthesis 3- hydracrylic acid,But the concentration of tolerable acetic acid is low (< 8g/L), cell slow growth when bacterial metabolism acetic acid.

Summary of the invention

The object of the present invention is to provide the recombination of the high production 3- hydracrylic acid of the tolerance of a kind of pair of substrate acetic acid is false singleBorn of the same parents bacterium and its construction method, and the method using recombination pseudomonad 3-hydroxyl ethylformic acid fermentation production.

As the first aspect of the present invention, a kind of construction method of recombination pseudomonad for producing 3- hydracrylic acid is provided,The construction method is one of following method:

Method 1: acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr are building up to originalOn nuclear expression carrier, then prokaryotic expression carrier is imported in pseudomonad, building obtains recombination pseudomonad；

Method 2: acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr will be integrated intoBuilding obtains recombination pseudomonad in pseudomonad genome.

Preferably, method 1 specifically includes: by acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductaseGene mcr is building up on Escherichia coli-pseudomonad shuttle plasmid pUCPK, then obtained recombinant plasmid pUCPK-AM is usedPUCPK-AM converts pseudomonad, obtains recombination pseudomonad ZAP-AM1.

Preferably, the pseudomonad is ATCC13867.

As a second aspect of the invention, the recombination pseudomonad of the building as described in above-mentioned construction method is provided.

As the third aspect of the present invention, application of the recombination pseudomonad in 3-hydroxyl ethylformic acid fermentation production is provided.

Preferably, the fermentation is to carry out aerobic fermentation by raw material of acetic acid.

Preferably, fermentative medium formula used are as follows: 100mmol/L phosphate buffer, 5g/L sodium acetate, 0.25g/L sulphurSour magnesium, 1g/L ammonium chloride, 1g/L yeast extract；Biotin 2.2mg/L, sodium bicarbonate 20mmol/L, the pH=of phosphate buffer7.0。

Preferably, the fermentation condition are as follows: seed liquor is inoculated by 50mL hair with the inoculum concentration of OD600=0.1 after inoculationIn ferment culture medium, ferment 24 hours under the conditions of 37 DEG C of temperature, 250rpm.

Beneficial effects of the present invention: recombination pseudomonad of the invention is high to the tolerance of substrate acetic acid, can be used for scaleMetaplasia produces 3- hydracrylic acid；Bacterium is produced with recombination pseudomonad production 3- hydracrylic acid of the invention, CO can be fixed₂, improve 3-Hydracrylic acid carbon yield and substrate Atom economy reduce raw materials for production cost and the addition of fermentation and acid process alkalinity neutralization reagentCost.

Detailed description of the invention

Fig. 1 is the metabolic pathway for producing 3- hydracrylic acid in the embodiment of the present invention using recombination pseudomonad.

Specific embodiment:

Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially fromIn the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged toRange.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art, such asThe molecular cloning experiment handbooks such as Sambrook, or the condition according to product description suggestion.

Escherichia coli used in the following embodiment-pseudomonad shuttle plasmid pUCPK is building early period.

Embodiment 1 is overexpressed acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reduction in pseudomonadEnzyme gene mcr

Using the genome of pseudomonad (Pseudomonas sp.ATCC13867) as template, design primer carries out PCR pointsIt Kuo Zeng not accA (SEQ ID NO.1), accB (SEQ ID NO.2), accC (SEQ ID NO.3), accD (SEQ IDNO.4), over-lap PCR is carried out, then to obtain a gene cluster of accADBC.With the gene of photosynthetic Chloronema Dubinina and Gorlenko thermophilic in NCBIGroup information inquires the nucleotide sequence of mcr gene, after then carrying out codon optimization, sends to gene chemical synthesis company synthesis mcr baseBecause of (mcr gene order is as shown in SEQ ID NO.5), the gene of synthesis is subjected to PCR amplification.By Escherichia coli-pseudomonadShuttle plasmid pUCPK carries out digestion with XbaI and HindIII, and mcr segment is connected on pUCPK, the recombinant plasmid life of acquisitionEntitled pUCPK-M；This plasmid carries out digestion with BamHI and EcoRI, and accABDBC segment is connected on pUCPK-M, acquisitionRecombinant plasmid is named as pUCPK-AM.PUCPK-AM is transferred to pseudomonad by electrotransformation using electroporation apparatus (Bole)In, electrode conditions are 25 μ F, 200 Ω, 180V (electric shock cup width is 2mm).On the kanamycins LB plate containing 30mg/LScreening obtains recombinant bacterium, is named as ZAP-AM1.

Embodiment 2 utilizes recombination pseudomonad 3-hydroxyl ethylformic acid fermentation production

Pseudomonad wild strain ZAP and recombinant bacterium ZAP-AM1 are incubated overnight on LB plate respectively.It is fresh from thisSingle colonie is inoculated into the 50mL triangular flask containing 10mL seed culture medium on plate, 37 DEG C, 250rpm culture 12 hours, gainedSeed liquor OD600 value is 3-4.

The formula of seed culture medium Luria-Bertani (LB) are as follows: 10g/L tryptone, 5g/L yeast extract, 10g/L chlorineChange sodium.

Seed liquor is inoculated into 50mL fermentation medium with the inoculum concentration of OD600=0.1 after inoculation, fermentation uses250mL triangular flask, control 37 DEG C of temperature, 250rpm, fermentation time 24 hours.

The formula of fermentation medium includes: 100mmol/L phosphate buffer (pH7.0), 5g/L sodium acetate, 0.25g/L sulphurSour magnesium, 1g/L ammonium chloride, 1g/L yeast extract；Biotin 2.2mg/L, sodium bicarbonate 20mmol/L.

As shown in Figure 1, acetic acid generates intermediate product acetyl coenzyme A in microbial body intracellular metabolite, acetyl coenzyme A is first in secondMalonyl coenzyme A is generated under the action of acyl coenzyme A carboxylase (ACC), malonyl coenzyme A further passes through malonyl coenzyme AThe catalysis of reductase (MCR) generates 3- hydracrylic acid.

After fermentation 24 hours, after measured, wild strain ZAP does not produce 3- hydracrylic acid, and recombinant bacterium ZAP-AM produces the 3- of 5mMHydracrylic acid.

Sequence table

<110>Jiangsu Normal University

<120>a kind of recombination pseudomonad for producing 3- hydracrylic acid, its construction method and its application

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 951

<212> DNA

<213>pseudomonad (Pseudomonas sp. ATCC13867)

<400> 1

atgaacccga attttctcga tttcgaacag cccattgccg acctgcacgc caagatcgaa 60

gaactgcgcc tggttggcaa cgacaacgcg ctgaacatca ccgacgaaat ctcccgtctg 120

caggagaaga gcaaggcgct gaccgagaac atcttcggca acctgaccag ctggcagatc 180

gcccagctcg cgcgccatcc gcgtcgcccc tacaccctgg actacatcga gcacatcttc 240

ggcgagttcg aagagctgca cggcgaccgt catttcgccg atgatgcggc catcgttggc 300

ggcgttgccc gcctcgacga gcaaccggtg atgatcatcg gtcaccagaa gggtcgcgaa 360

gtccgcgaga aggtgcgccg caacttcggc atgccgcgtc cggaaggcta ccgcaaggcc 420

tgccgcctga tggaaatggc cgaacgcttc aagatgccga tcctgacctt catcgacacc 480

cccggcgcct acccgggcat cgatgccgaa gagcgtggtc agagcgaggc gattgcctgg 540

aacctgcgcg tcatggcgcg cctgaagacc ccgatcatcg ccaccgttat cggcgagggc 600

ggttccggcg gcgcgctggc cattggcgtg tgcgaccagc tgaacatgct gcagtattcc 660

acctacgcgg tgatctcccc ggaaggctgc gcctcgattc tctggcgtac cgccgagaag 720

gcgccggaag cggccgaggc gatgggcatc accgccaacc gcctgaagga tctgggcatt 780

gtcgacagca tcatccccga gccgctgggc agcgctcacc gcgacccggc cgcgatgtcc 840

cagtcgatcc gcaaggccct gctcggccag ctggatgttc tcaagcaact gagcaccgaa 900

gagctgctgg cgcgccgcta cgagcgcctg atgagctacg gcgtcgcctg a 951

<210> 2

<211> 468

<212> DNA

<213>pseudomonad (Pseudomonas sp. ATCC13867)

<400> 2

atggacatcc gtaaagtcaa gaaactgatc gaactgctcg aagaatccgg catcgacgaa 60

ctggagatca aagagggcga agagtccgta cgcatcagcc gtcacagcaa gaccgccgcc 120

cagccggtct acgccgccgc cccggcctac gccccggctc ccgtcgcagc cgctccggtc 180

gctgccgccg cagccccgac cgccgaagcc gctccggccg caccggtgct caacggcaac 240

gccgtgcgct cgccgatggt cggcaccttc tatcgcgccg cctcgccgac ctccgccaac 300

ttcgtcgaag tcggccagag cgtgaagaaa ggcgacatcc tgtgcatcgt cgaagccatg 360

aagatgatga accacatcga ggccgagacc agcggcgtga tcggtcaagt cctcgtggag 420

aacggccagc cggtcgagtt cgaccagccc ctgttcacca tcgtttaa 468

<210> 3

<211> 1350

<212> DNA

<213>pseudomonad (Pseudomonas sp. ATCC13867)

<400> 3

atgttggaaa aagtactgat cgccaaccgc ggcgaaatcg cgctgcgcat cctgcgcgcg 60

tgcaaggagc tgggcatcaa gacggtggct gtgcattcga ccgccgaccg cgaactgatg 120

cacctgtcgc tggccgacga agcggtctgc atcggtccgg ccccggctgc gcagtcctac 180

ctgcacatcc cggcgatcat cgccgcggcc gaggtcaccg gtgccgtggg tatccatccc 240

ggctacggct tcctcgccga gaacgccgac ttcgccgagc aggtcgagcg ctcgggcttc 300

accttcatcg gcccgagcgc cgacgtcatc cgcctgatgg gtgacaaggt gtccgccaag 360

gacgccatga agaaagccgg cgtgccgacc gtaccgggct ccgacggccc gctgcccgaa 420

gacgaagaga ccgcgctggc gatcgcccgc gaggttggct acccggtgat catcaaggcc 480

gccggtggcg gcggtggtcg cggcatgcgc gtcgtgcacc acgaggaaga cctgatcaag 540

tccgccaagc tgacccgtac cgaagcgggc gcggccttcg gcaactcgat ggtctacctg 600

gagaagttcc tgaccaaccc gcgtcacgtg gaagtccagg tgctctccga cggccagggc 660

aacgccatcc acctgggcga ccgcgactgc tccctgcagc gtcgtcacca gaaggtactg 720

gaagaagccc cagccccggg catcgacgag aaggcccgcg aagaagtgct ggcccgctgc 780

gtccaggcct gcatcgagat cggctatcgt ggcgccggca ccttcgagtt cctctacgag 840

aacggccgct tctacttcat cgagatgaac acccgcgtcc aggtggagca tccggtgtcg 900

gagatggtca ccggtatcga catcgtcaag gaaatgctca gcatcgcctc gggcaacaag 960

ctgtcgatcc gccaggaaga cgtggtcatc cgtggccatg cgctggaatg ccggatcaat 1020

gccgaagacc cgaagacctt catgccgagc cctggcaagg tcaagcactt ccacgcgccg 1080

ggcggcaacg gcgtgcgcgt cgattcgcac ctgtacagcg gctacgccgt accgccgaac 1140

tatgactcgc tggtgggcaa ggtcatcacc tacggcaagg accgagccga agccctggcg 1200

cgcatgcgca atgccctgga cgagctgatc gtcgacggca tcaagaccaa taccgagctg 1260

cacaaggatc tggtccgcga caaggagttc tgcaaaggtg gcgtgaacat ccactacctg 1320

gagaagaagc tgggcatgga caagcactaa 1350

<210> 4

<211> 876

<212> DNA

<213>pseudomonad (Pseudomonas sp. ATCC13867)

<400> 4

atgagcaact ggctggtaga caagctgatc ccttccatca tgcgttccga agcgaagaag 60

agctcggttc cggaaggcct gtggcacaag tgcccgtcct gcgaggcggt gctgtaccgt 120

cccgagctgg aaaagaccct ggacgtctgc ccgaagtgcg atcaccacat gcgcatcggc 180

gcccgtgccc gcctggacat cttcctcgat gaagagggtc gcgaagagct tggcgccgag 240

ctggagccgg tggatcgcct gaagttccgc gacagcaaga agtacaagga ccgcctgagc 300

gccgcgcaga aggacaccgg cgagaaggat gcgctgatct ccatgagcgg caagctgatg 360

ggcatgccgg tggtggccag cgccttcgag ttctccttca tgggcggctc gatgggctcc 420

atcgtcggcg agcgtttcgt gcgcgccgcc aactacgcct tggaaaaccg ttgcccgatg 480

atctgcttct ccgcttcggg cggtgcgcgc atgcaggaag cgctgatttc cctgatgcag 540

atggccaaga cctccgccgc gctggcgcgc ctgcgcgaag aaggcattcc gttcatctcc 600

gtgctgaccg acccggtcta cggcggcgtt tccgccagcc tggcgatgct cggcgacgtg 660

atcgtcggcg agccgcgcgc cctgatcggc ttcgccggcc cgcgggtgat cgagcagacc 720

gtgcgcgaga agctgcccga aggcttccag cgtagcgagt tcctgctgga gcacggtgcc 780

atcgacatga tcgtccaccg ttcggaaatg cgtcagcggc tcgccagcct gctggccaag 840

ttcaccaaca ctccaagtac cgcgctcgca ggatga 876

<210> 5

<211> 3660

<212> DNA

<213>thermophilic photosynthetic Chloronema Dubinina and Gorlenko (Chloroflexus aurantiacus)

<400> 5

atgtctggta ccggtcgtct ggcaggtaag atcgcactga tcaccggcgg tgctggtaac 60

atcggttctg agctgactcg tcgtttcctg gcagagggtg caactgtcat catcagcggt 120

cgtaaccgcg caaagctgac cgcactggca gagcgtatgc aagcagaggc aggtgtacct 180

gcaaagcgta tcgacctgga ggtgatggac ggtagcgacc ctgtagcagt gcgtgcaggt 240

atcgaggcaa tcgtggctcg tcatggtcag atcgacatcc tggtcaacaa cgcgggcagc 300

gcaggtgcac aacgtcgtct ggctgaaatc ccactgactg aggcagagct gggtcctggt 360

gcagaagaga ctctgcatgc aagcatcgca aacctgctgg gcatgggttg gcacctgatg 420

cgtatcgcag cacctcacat gcctgtcggt agcgcagtca tcaacgtctc taccatcttc 480

tcccgtgctg agtactacgg ccgtatcccg tacgtcaccc ctaaggctgc tctgaacgct 540

ctgtcccagc tggctgcacg tgagctgggt gcacgtggta tccgtgttaa tacgatcttc 600

ccgggcccta tcgagagcga ccgtatccgt accgtgttcc agcgtatgga ccagctgaaa 660

ggccgtccag agggtgacac tgcacaccac ttcctgaaca ccatgcgtct gtgccgtgca 720

aacgaccagg gtgcactgga gcgtcgtttc ccaagcgtcg gtgacgtagc agacgcagct 780

gtattcctgg caagcgcaga gtctgcagct ctgtctggtg agactatcga ggtcacgcac 840

ggtatggagc tgccagcatg ctctgagacc tctctgctgg cacgtactga cctgcgtact 900

atcgacgcat ctggtcgtac gactctgatc tgcgctggtg accagatcga ggaggtgatg 960

gcactgaccg gtatgctgcg tacctgcggt tctgaggtga tcatcggctt ccgttccgca 1020

gcggcgctgg cacaattcga acaggcggtc aacgaatccc gccgtctggc aggcgctgat 1080

ttcactccac caatcgctct gccactggac ccacgtgatc cagctactat cgatgcggtc 1140

ttcgactggg ctggtgaaaa caccggtggt atccacgcag ctgtaatcct gccagctacc 1200

agccacgaac cggctccgtg tgtaatcgaa gtggacgacg aacgtgtgct gaacttcctg 1260

gccgacgaaa tcaccggcac gatcgtgatc gccagccgtc tggctcgtta ctggcaatcc 1320

caacgtctga ccccgggtgc tcgtgctcgt ggtccgcgtg ttattttcct gtctaacggt 1380

gctgaccaaa acggcaacgt gtacggccgt attcagtccg ccgctatcgg tcagctgatc 1440

cgtgtgtggc gtcacgaagc tgaactggac taccagcgtg ctagcgctgc tggtgaccac 1500

gtgctgccgc cggtttgggc taatcaaatc gtgcgtttcg ctaaccgtag cctggaaggt 1560

ctggaattcg cgtgcgcttg gactgctcag ctgctgcaca gccagcgtca tatcaacgaa 1620

atcaccctga acatcccggc gaacatcagc gcgaccaccg gtgctcgtag cgcttctgtg 1680

ggttgggctg aatccctgat cggtctgcac ctgggtaaag tggctctgat taccggcggt 1740

agcgctggta tcggtggtca gatcggtcgt ctgctggctc tgtctggcgc tcgtgtaatg 1800

ctggctgctc gcgatcgtca taaactggaa cagatgcagg cgatgatcca gtccgaactg 1860

gccgaagtgg gctacaccga cgtggaagac cgcgtgcaca tcgctccggg ttgtgacgtt 1920

agctccgaag ctcagctggc tgacctggtt gaacgtactc tgtccgcttt cggtaccgtg 1980

gactacctga tcaacaatgc gggcatcgcg ggtgtggaag aaatggttat cgacatgccg 2040

gttgaaggct ggcgccacac cctgttcgcg aacctgatct ccaactactc cctgatgcgc 2100

aaactggcgc cgctgatgaa aaaacagggc tccggctaca tcctgaacgt ttcctcctac 2160

ttcggcggcg aaaaagacgc ggcgatcccg tacccgaacc gcgcggatta cgcggtttcc 2220

aaagccggcc agcgcgctat ggcggaagtt ttcgcgcgtt tcctgggccc ggaaattcag 2280

attaacgcga ttgcgccggg cccggttgaa ggtgatcgcc tgcgtggtac tggtgaacgt 2340

ccgggtctgt tcgctcgtcg tgcccgtctg attctggaaa acaaacgcct gaacgaactg 2400

cacgccgccc tgattgcggc cgcccgtact gatgaacgtt ctatgcacga actggttgaa 2460

ctgctgctgc cgaacgatgt tgcggccctg gaacagaacc cggccgcccc gactgcgctg 2520

cgtgaactgg cccgtcgttt tcgttctgaa ggcgatccgg cggcgtctag cagctccgcg 2580

ctgctgaatc gttctattgc ggccaaactg ctggcccgtc tgcataatgg cggctatgtt 2640

ctgccggcgg atattttcgc caatctgccg aatccgccgg atccgttttt cactcgtgcg 2700

cagattgatc gtgaagcccg taaagttcgc gatggcatta tgggcatgct gtacctgcag 2760

cgcatgccga cggaattcga tgttgcgatg gcgaccgttt attatctggc ggatcgcaac 2820

gtttccggcg aaaccttcca cccgtccggc ggcctgcgct atgaacgcac cccgaccggc 2880

ggcgaactgt ttggcctgcc gtctccggaa cgtctggcgg aactggttgg ctctactgtt 2940

tatctgattg gcgaacacct gacggaacac ctgaacctgc tggcgcgtgc gtatctggaa 3000

cgttacggcg cgcgccaggt tgttatgatt gttgaaacgg aaaccggcgc cgaaacgatg 3060

cgccgcctgc tgcacgatca cgttgaagcc ggccgcctga tgactattgt tgcgggcgat 3120

cagattgaag cggccattga tcaggccatt acccgctatg gccgcccggg tccggttgta 3180

tgtactccgt ttcgcccgct gccgactgta ccgctggtag gccgcaaaga ttctgattgg 3240

tccactgttc tgtctgaagc ggaatttgcg gaactgtgtg aacaccagct gacccaccat 3300

tttcgcgtag cgcgcaaaat tgcgctgtct gatggcgcgt ctctggcgct ggtaaccccg 3360

gaaaccaccg cgacctctac caccgaacag tttgcgctgg cgaactttat taaaacgacc 3420

ctgcatgcct ttacggcgac cattggcgta gaatctgaac gcacggcgca gcgcattctg 3480

attaaccagg tagatctgac ccgccgcgcg cgcgccgaag aaccgcgcga tccgcatgaa 3540

cgccagcagg aactggaacg ctttattgaa gccgttctgc tggtaaccgc cccgctgccg 3600

ccggaagccg atacccgcta tgcgggccgc attcatcgcg gccgcgcgat taccgtttaa 3660

Claims (8)

1. a kind of construction method for the recombination pseudomonad for producing 3- hydracrylic acid, which is characterized in that under the construction method isOne of column method:

Method 1: acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr are building up to protokaryon tableUp on carrier, then prokaryotic expression carrier is imported in pseudomonad, building obtains recombination pseudomonad；

Method 2: acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr are integrated into false unit cellBuilding obtains recombination pseudomonad in bacterium genome.

2. a kind of construction method of recombination pseudomonad for producing 3- hydracrylic acid according to claim 1, feature existIn the method 1 specifically includes: by acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr structureIt is built on Escherichia coli-pseudomonad shuttle plasmid pUCPK, obtained recombinant plasmid pUCPK-AM, is then turned with pUCPK-AMChange pseudomonad, obtains recombination pseudomonad ZAP-AM1.

3. a kind of construction method of recombination pseudomonad for producing 3- hydracrylic acid according to claim 1 or 2, featureIt is, the pseudomonad is ATCC13867.

4. being constructed by a kind of construction method for the recombination pseudomonad for producing 3- hydracrylic acid of any of claims 1 or 2Recombinate pseudomonad.

5. application of recombination pseudomonad described in claim 4 in 3-hydroxyl ethylformic acid fermentation production.

6. application according to claim 5, which is characterized in that the fermentation is to carry out aerobic fermentation by raw material of acetic acid.

7. application according to claim 6, which is characterized in that fermentative medium formula used are as follows: 100mmol/L phosphoric acid is slowFliud flushing, 5g/L sodium acetate, 0.25g/L magnesium sulfate, 1g/L ammonium chloride, 1g/L yeast extract；Biotin 2.2mg/L, sodium bicarbonate20mmol/L, wherein the pH=7.0 of phosphate buffer.

8. application according to claim 6, which is characterized in that the fermentation condition are as follows: with OD600=0.1 after inoculationSeed liquor is inoculated into 50mL fermentation medium by inoculum concentration, under the conditions of 37 DEG C of temperature, 250rpm, is fermented 24 hours.