CN109112156A - A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application - Google Patents
A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application Download PDFInfo
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- CN109112156A CN109112156A CN201811092228.2A CN201811092228A CN109112156A CN 109112156 A CN109112156 A CN 109112156A CN 201811092228 A CN201811092228 A CN 201811092228A CN 109112156 A CN109112156 A CN 109112156A
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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Abstract
A kind of recombination pseudomonad producing 3- hydracrylic acid, its construction method and its application, belong to genetic engineering field and field of fermentation engineering, the recombinant host bacterium is pseudomonad, expresses the gene accABCD of encoding acetyl CoA carboxylase enzyme and the gene mcr of coding malonyl coenzyme A reductase.The preparation method comprises the following steps: obtaining recombinant vector pUCPK-AM；It is transformed into host's competent cell.The present invention passes through building heterogenous expression 3- hydracrylic acid production ways, the bacterial strain of acquisition can produce 3- hydracrylic acid in the culture medium using acetic acid as sole carbon source, 3- hydracrylic acid biology manufacturing cost can be reduced, and a kind of feasible Research Thinking and direction are provided for the utilization that acetic acid is discarded in industrial or agricultural, there is important economic value and social effect.
Description
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 false
Monad, 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 paper
In urine pants, surface covering, paint and adhesives industry.In recent years, the demand of acrylic acid is continuously increased.Traditional chemical method
Production 3- hydracrylic acid has the characteristics that process is complicated, environmental pollution at high cost and easy, and microbial method production 3- hydracrylic acid
These 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, biology
Diesel oil industry raw material resources are insufficient, cost is excessively high, bring limitation to Material synthesis 3- hydracrylic acid is used glycerol as.Cause
This, solves the competitiveness that raw material sources and cost problem are produced for improving Chinese biological base 3- hydracrylic acid, realizes biology base
The 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 of
Acetic acid；The conversion of synthesising biological gas, a carbonizable substance methanol and methane conversion, sewage sludge anaerobic digestion etc. can also generate largely
Acetic 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 material
At can be greatly lowered production cost, improve the competitiveness of biology base 3- hydracrylic acid, realize biology base 3- hydracrylic acid
Sustainable 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 single
Born 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 original
On 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 into
Building obtains recombination pseudomonad in pseudomonad genome.
Preferably, method 1 specifically includes: by acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase
Gene mcr is building up on Escherichia coli-pseudomonad shuttle plasmid pUCPK, then obtained recombinant plasmid pUCPK-AM is used
PUCPK-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 sulphur
Sour magnesium, 1g/L ammonium chloride, 1g/L yeast extract；Biotin 2.2mg/L, sodium bicarbonate 20mmol/L, the pH=of phosphate buffer
7.0。
Preferably, the fermentation condition are as follows: seed liquor is inoculated by 50mL hair with the inoculum concentration of OD600=0.1 after inoculation
In 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 scale
Metaplasia 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 reagent
Cost.
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 from
In 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 to
Range.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art, such as
The 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 pseudomonad
Enzyme gene mcr
Using the genome of pseudomonad (Pseudomonas sp.ATCC13867) as template, design primer carries out PCR points
It Kuo Zeng not accA (SEQ ID NO.1), accB (SEQ ID NO.2), accC (SEQ ID NO.3), accD (SEQ ID
NO.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 NCBI
Group information inquires the nucleotide sequence of mcr gene, after then carrying out codon optimization, sends to gene chemical synthesis company synthesis mcr base
Because of (mcr gene order is as shown in SEQ ID NO.5), the gene of synthesis is subjected to PCR amplification.By Escherichia coli-pseudomonad
Shuttle plasmid pUCPK carries out digestion with XbaI and HindIII, and mcr segment is connected on pUCPK, the recombinant plasmid life of acquisition
Entitled pUCPK-M；This plasmid carries out digestion with BamHI and EcoRI, and accABDBC segment is connected on pUCPK-M, acquisition
Recombinant 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/L
Screening 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 this
Single colonie is inoculated into the 50mL triangular flask containing 10mL seed culture medium on plate, 37 DEG C, 250rpm culture 12 hours, gained
Seed 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 chlorine
Change sodium.
Seed liquor is inoculated into 50mL fermentation medium with the inoculum concentration of OD600=0.1 after inoculation, fermentation uses
250mL 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 sulphur
Sour 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 second
Malonyl coenzyme A is generated under the action of acyl coenzyme A carboxylase (ACC), malonyl coenzyme A further passes through malonyl coenzyme A
The 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 5mM
Hydracrylic 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 is
One of column method:
Method 1: acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr are building up to protokaryon table
Up 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 cell
Building 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 exist
In the method 1 specifically includes: by acetyl-coA carboxylase gene accADBC and malonyl coenzyme A reductase gene mcr structure
It is built on Escherichia coli-pseudomonad shuttle plasmid pUCPK, obtained recombinant plasmid pUCPK-AM, is then turned with pUCPK-AM
Change 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, feature
It 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 2
Recombinate 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 slow
Fliud flushing, 5g/L sodium acetate, 0.25g/L magnesium sulfate, 1g/L ammonium chloride, 1g/L yeast extract；Biotin 2.2mg/L, sodium bicarbonate
20mmol/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 inoculation
Seed liquor is inoculated into 50mL fermentation medium by inoculum concentration, under the conditions of 37 DEG C of temperature, 250rpm, is fermented 24 hours.
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