CN108004274B - Method for producing acrylic acid by fermentation of saccharomyces cerevisiae - Google Patents
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Abstract
The invention relates to a method for producing acrylic acid by saccharomyces cerevisiae fermentation, which takes glycerol as a raw material and saccharomyces cerevisiae as host bacteria and is completed by catalysis of a plurality of enzymes in the host bacteria. The invention has the beneficial effects that: 1) the compound with low cost is used as the raw material, and the cost of the raw material of the process is low. 2) The method is based on the technical foundation of modifying the microorganism, carries out the catalytic synthesis of the biological enzyme in the microorganism body, avoids the large-scale extraction, separation and purification processes, and is easy to control from the aspects of production cost and environmental friendliness. 3) The process has the advantages of smaller scale and quantity of production equipment than other methods, simple operation and easy industrial transformation. 4) The process only has a separation and purification process in the last step of production, the product purification process is simple, and the product quality is higher than that of the general method in purity and content. 5) No waste liquid is discharged in the synthesis process, the method is environment-friendly, pollution-free and sustainable in production.
Description
Technical Field
The invention relates to a biosynthesis method of acrylic acid, in particular to a method for producing acrylic acid by fermentation of saccharomyces cerevisiae.
Background
The acrylic acid molecular formula: c3H4O2(ii) a English name: acrylic acid; CAS: 79-10-7; molecular weight: 72.06, respectively; physical properties: mixing with water, and dissolving in ethanol and diethyl ether to obtain colorless liquid with pungent odor; melting point of 13 deg.C, boiling point of 141 deg.C, flash point of 54 deg.C, and densityIt was 1.05 g/cm.
Acrylic acid is an important organic synthetic raw material and synthetic resin monomer, and is a vinyl monomer having a very high polymerization rate. Is the simplest unsaturated carboxylic acid, consisting of one vinyl group and one carboxyl group. Pure acrylic acid is a colorless clear liquid with a characteristic pungent odor. It is miscible with water, alcohol, ether and chloroform. Most of them are used for producing acrylic esters such as methyl acrylate, ethyl acrylate, butyl acrylate, hydroxyethyl acrylate, etc. Acrylic acid and acrylic ester can be homopolymerized and copolymerized, and the polymer is used in the industrial departments of synthetic resin, synthetic fiber, super absorbent resin, building material, paint and the like. Acrylic acid is used as a bulk chemical with market demand ranking of nearly 20, and acrylic acid and polymers thereof are widely applied to the aspects of industrial production, aerospace, sewage treatment, medical materials, nano materials and the like. With the development of science and technology, people increasingly demand green and environment-friendly materials, so that functional materials using acrylic acid as organic synthetic raw materials and synthetic resin monomers are more and more favored by people.
The industrial production of acrylic acid mainly adopts the propylene oxidation method. Propylene reacts with oxygen to generate acrolein, and the acrolein is deoxidized to generate acrylic acid, so that the conversion rate is high. Despite the many advantages of this process, the raw materials rely on traditional fossil energy, which is highly polluting, energy intensive and non-sustainable. The synthesis of acrylic acid by the semi-biosynthesis or the total biosynthesis is also currently advantageous. The semi-biosynthesis method of acrylic acid is a method of converting petrochemical raw materials such as acrylonitrile and acrylamide into acrylic acid by using microorganisms.
Although the yield of acrylic acid is high, the raw materials of acrylonitrile and acrylamide are higher than that of acrylic acid in cost, and the industrial production of the acrylic acid is limited. The acrylic acid whole biological method is a method for producing acrylic acid by directly utilizing biomass such as saccharides and the like by microorganisms through fermentation. The existing acrylic acid biological total synthesis method has the defects of complex synthesis route, unclear synthesis mechanism and extremely low synthesis efficiency. The invention aims to solve the technical problems and provides a method for biologically synthesizing acrylic acid, which has mild reaction and high synthesis efficiency.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides the method for producing the acrylic acid by fermenting the saccharomyces cerevisiae, which has the advantages of mild reaction, high synthesis efficiency, high yield, no pollution and short production period.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for producing acrylic acid by saccharomyces cerevisiae fermentation is characterized in that glycerol is used as a raw material, saccharomyces cerevisiae is used as host bacteria, and the synthesis is completed through catalysis of a plurality of enzymes in the host bacteria;
the host bacterium is constructed and obtained according to the following method:
1) optimizing codons of genes GlyDH and DAK according to the codon preference of saccharomyces cerevisiae, and then constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 by using a bidirectional promoter to obtain a vector 1, wherein the expression vector YCplac33 has ampicillin resistance of escherichia coli and a selection marker URA gene of the saccharomyces cerevisiae; constructing a heterologous glycerol metabolic pathway in saccharomyces cerevisiae, wherein glycerol can be converted into dihydroxyacetone by an enzyme expressed by a GlyDH gene, and the dihydroxyacetone is converted into dihydroxyacetone phosphate by an enzyme expressed by a DAK gene;
2) optimizing the ceaS2 gene and the NOX gene according to the codon preference of the saccharomyces cerevisiae, and then simultaneously constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 to obtain a vector 2, wherein the expression vector YCplac33 has an ampicillin resistance gene of escherichia coli and a screening marker Leu gene of the saccharomyces cerevisiae;
the enzyme expressed by the ceaS2 gene has the function of converting dihydroxyacetone phosphate or glyceraldehyde 3-phosphate into acrylic acid, and since GlyDH enzyme needs NADH to catalyze the conversion of glycerol into dihydroxyacetone, NADH is converted into NAD +. In order to increase the supply of NADH and thus the efficiency of the heterologous glycerol metabolic pathway constructed in 1), a heterologous NADH circulation module is constructed in Saccharomyces cerevisiae, i.e. by introducing a NOX gene, the enzyme expressed by the NOX gene is capable of reducing NAD + to NADH;
3) knocking out a PDC1 gene, a DLD1 gene, a GPD1 gene and a GPD2 gene in the Saccharomyces cerevisiae BY4741 to obtain a modified Saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 strain; the aim is to reduce consumption of core intermediate products, namely dihydroxyacetone phosphate and glyceraldehyde 3-phosphate by other metabolic pathways;
4) and (3) simultaneously transferring the vectors 1 and 2 into the modified saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 described in the step 3), screening positive clones according to screening markers ura and Leu genes of the saccharomyces cerevisiae, then extracting a saccharomyces cerevisiae genome, and further carrying out PCR verification.
Preferably, the GlyDH and DAK genes of step 1) are synthesized according to the amino acid sequences of the corresponding genes in Geobacillus stearothermophilus and Pichia pastoris, respectively.
Preferably, the ceaS2 and NOx genes in step 2) are synthesized based on the amino acid sequences of the corresponding genes of Streptomyces clavuligerus and Geobacillus stearothermophilus, respectively.
A method for producing acrylic acid by saccharomyces cerevisiae fermentation comprises the following steps:
1) optimizing codons of genes GlyDH and DAK according to the codon preference of saccharomyces cerevisiae, and then constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 by using a bidirectional promoter to obtain a vector 1, wherein the expression vector YCplac33 has ampicillin resistance of escherichia coli and a selection marker URA gene of the saccharomyces cerevisiae;
2) optimizing the ceaS2 gene and the NOX gene according to the codon preference of the saccharomyces cerevisiae, and then simultaneously constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 to obtain a vector 2, wherein the expression vector YCplac33 has an ampicillin resistance gene of escherichia coli and a screening marker Leu gene of the saccharomyces cerevisiae;
3) knocking out a PDC1 gene, a DLD1 gene, a GPD1 gene and a GPD2 gene in the Saccharomyces cerevisiae BY4741 to obtain a modified Saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 strain;
4) simultaneously transferring the vectors 1 and 2 into the modified saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 described in 3), screening positive clones according to screening markers ura and Leu genes of the saccharomyces cerevisiae, then extracting a saccharomyces cerevisiae genome, and further carrying out PCR verification;
5) culturing saccharomyces cerevisiae host bacteria, and performing induced expression;
6) adding a glycerol substrate into the expressed bacterial liquid, and continuing to react for 20-26 h;
7) extracting, separating and purifying the acrylic acid in the reaction liquid.
Preferably, the addition amount of glycerol in step 6) is: 10g/L-100g/L, the reaction temperature is as follows: 25-35 ℃.
The genes referred to in this patent are GlyDH, DAK, NOX and ceaS2 (see Table 1).
The difference between the preparation of acrylic acid by the invention and the prior art is that:
1) the compound with low cost is used as the raw material, and the cost of the raw material of the process is low.
2) The method is based on the technical foundation of modifying the microorganism, carries out the catalytic synthesis of the biological enzyme in the microorganism body, avoids the large-scale extraction, separation and purification processes, and is easy to control from the aspects of production cost and environmental friendliness.
3) The process has the advantages of smaller scale and quantity of production equipment than other methods, simple operation and easy industrial transformation.
4) The process only has a separation and purification process in the last step of production, the product purification process is simple, and the product quality is higher than that of the general method in purity and content.
5) The synthesis process has no waste liquid discharge, is environment-friendly, has no pollution, and can be used for sustainable production.
Drawings
FIG. 1 is a scheme of the acrylic acid biosynthesis scheme of the present invention;
FIG. 2(a) is a plasmid map of YCplac33-GlyDH-DAK in the present invention;
FIG. 2(b) is a YCplac33-ceaS2-NOX plasmid map in accordance with the present invention;
FIG. 3 is a liquid phase result graph of a synthetic sample according to the present invention;
FIG. 4 is a mass spectrum result chart of a synthesized sample of the present invention, wherein FIG. 4(a) is a chromatogram of LC-MS analysis of an acrylic acid standard, FIG. 4(b) is a chromatogram of LC-MS analysis of a control sample, FIG. 4(c) is a chromatogram of LC-MS analysis of a strain culture reaction solution after modification, and FIG. 4(d) is a mass spectrum of LC-MS analysis of a strain culture reaction solution after modification.
Detailed Description
The technical solution of the present invention is further specifically described below by way of specific examples in conjunction with the accompanying drawings. The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
A method for producing acrylic acid by saccharomyces cerevisiae fermentation is shown in figure 1, and the synthesis method is completed by using glycerol as a raw material and saccharomyces cerevisiae as a host bacterium and catalyzing a plurality of enzymes in the host bacterium; the host bacterium is constructed and obtained according to the following method:
1) optimizing codons of genes GlyDH and DAK according to the codon preference of saccharomyces cerevisiae, and then constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 by using a bidirectional promoter to obtain a vector 1, wherein the expression vector YCplac33 has ampicillin resistance of escherichia coli and a selection marker URA gene of the saccharomyces cerevisiae as shown in a figure 2 (a); constructing a heterologous glycerol metabolic pathway in saccharomyces cerevisiae, wherein glycerol can be converted into dihydroxyacetone by an enzyme expressed by a GlyDH gene, and the dihydroxyacetone is converted into dihydroxyacetone phosphate by an enzyme expressed by a DAK gene;
2) optimizing the ceaS2 gene and the NOX gene according to the codon preference of the saccharomyces cerevisiae, and then simultaneously constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 to obtain a vector 2, wherein the expression vector YCplac33 has an ampicillin resistance gene of escherichia coli and a screening marker Leu gene of the saccharomyces cerevisiae as shown in a figure 2 (b);
the enzyme expressed by the ceaS2 gene has the function of converting dihydroxyacetone phosphate or glyceraldehyde 3-phosphate into acrylic acid, and since GlyDH enzyme needs NADH to catalyze the conversion of glycerol into dihydroxyacetone, NADH is converted into NAD +. In order to increase the supply of NADH and thus the efficiency of the heterologous glycerol metabolic pathway constructed in 1), a heterologous NADH circulation module is constructed in Saccharomyces cerevisiae, i.e. by introducing a NOX gene, the enzyme expressed by the NOX gene is capable of reducing NAD + to NADH;
3) knocking out a PDC1 gene, a DLD1 gene, a GPD1 gene and a GPD2 gene in the Saccharomyces cerevisiae BY4741 to obtain a modified Saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 strain; the aim is to reduce consumption of core intermediate products, namely dihydroxyacetone phosphate and glyceraldehyde 3-phosphate by other metabolic pathways;
4) and (3) simultaneously transferring the vectors 1 and 2 into the modified saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 described in the step 3), screening positive clones according to screening markers ura and Leu genes of the saccharomyces cerevisiae, then extracting a saccharomyces cerevisiae genome, and further carrying out PCR verification.
Specifically, in this example, the GlyDH and DAK genes in step 1) were synthesized according to the amino acid sequences of the corresponding genes in Geobacillus stearothermophilus and Pichia pastoris, respectively.
Specifically, in this example, the ceaS2 and the NOX gene in step 2) were synthesized based on the amino acid sequences of the corresponding genes of Streptomyces clavuligerus and Geobacillus stearothermophilus, respectively.
A method for producing acrylic acid by saccharomyces cerevisiae fermentation comprises the following steps:
1) optimizing codons of genes GlyDH and DAK according to the codon preference of saccharomyces cerevisiae, and then constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 by using a bidirectional promoter to obtain a vector 1, wherein the expression vector YCplac33 has ampicillin resistance of escherichia coli and a selection marker URA gene of the saccharomyces cerevisiae;
2) optimizing the ceaS2 gene and the NOX gene according to the codon preference of the saccharomyces cerevisiae, and then simultaneously constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 to obtain a vector 2, wherein the expression vector YCplac33 has an ampicillin resistance gene of escherichia coli and a screening marker Leu gene of the saccharomyces cerevisiae;
3) knocking out a PDC1 gene, a DLD1 gene, a GPD1 gene and a GPD2 gene in the Saccharomyces cerevisiae BY4741 to obtain a modified Saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 strain;
4) simultaneously transferring the vectors 1 and 2 into the modified saccharomyces cerevisiae BY 4741-delta PDCl-delta DLD 1-delta GPD 1-delta GPD2 described in 3), screening positive clones according to screening markers ura and Leu genes of the saccharomyces cerevisiae, then extracting a saccharomyces cerevisiae genome, and further carrying out PCR verification;
5) culturing saccharomyces cerevisiae host bacteria, and performing induced expression;
6) adding a glycerol substrate into the expressed bacterial liquid, and continuing to react for 20-60h, preferably 36-48 h;
7) extracting, separating and purifying the acrylic acid in the reaction liquid.
Preferably, the addition amount of glycerol in step 6) is: 10g/L to 100g/L, preferably 60 g/L to 80g/L, and the reaction temperature is as follows: 25-35 ℃, preferably 30 ℃.
The liquid phase result graph and the mass spectrum result graph of the sample synthesized by the present invention are shown in fig. 3 and fig. 4(a) - (d), respectively.
The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.
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<110> Jiaxing Xin Beilai Biotech Ltd
JIAXING SYNBIOLAB BIOTECHNOLOGY Co.,Ltd.
<120> method for producing acrylic acid by fermentation of saccharomyces cerevisiae
<130> 20182018
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gtccactgtg tgccgaacat gctccttcac tattttaaca tgtggaatta attctcatgt 4860
ttgacagctt atcatcgaac tctaagaggt gatacttatt tactgtaaaa ctgtgacgat 4920
aaaaccggaa ggaagaataa gaaaactcga actgatctat aatgcctatt ttctgtaaag 4980
agtttaagct atgaaagcct cggcattttg gccgctccta ggtagtgctt tttttccaag 5040
gacaaaacag tttctttttc ttgagcaggt tttatgtttc ggtaatcata aacaataaat 5100
aaattatttc atttatgttt aaaaataaaa aataaaaaag tattttaaat ttttaaaaaa 5160
gttgattata agcatgtgac cttttgcaag caattaaatt ttgcaatttg tgattttagg 5220
caaaagttac aatttctggc tcgtgtaata tatgtatgct aaagtgaact tttacaaagt 5280
cgatatggac ttagtcaaaa gaaattttct taaaaatata tagcactagc caatttagca 5340
cttctttatg agatatatta tagactttat taagccagat ttgtgtatta tatgtattta 5400
cccggcgaat catggacata cattctgaaa taggtaatat tctctatggt gagacagcat 5460
agataaccta ggatacaagt taaaagctag tactgttttg cagtaatttt tttctttttt 5520
ataagaatgt taccacctaa ataagttata aagtcaatag ttaagtttga tatttgattg 5580
taaaataccg taatatattt gcatgatcaa aaggctcaat gttgactagc cagcatgtca 5640
accactatat tgatcaccga tatatggact tccacaccaa ctagtaatat gacaataaat 5700
tcaagatatt cttcatgaga atggcccagc gatatatgcg gtgtgaaata ccgcacagat 5760
gcgtaaggag aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg 5820
aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg 5880
caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 5940
ccagtgaatt cgagctctgt caacgcagat taagcacacc ctaaaacttg aataatgcaa 6000
attccatagc ttagtttaat caaggcgcca ttattcctca gttgaaaagt atatctaatg 6060
gttaggatta gaaatatgtt cattgctcat tgttatgtgt atcatatcgt acaaaaatta 6120
tataaagaaa taataatgaa aaagagtaaa acaaagacaa atagacaaaa atagaggata 6180
gaaaaagaaa aacaacagat acataaataa gtcttatgac aaaaaaacga ttgtataaaa 6240
attaaagtgc atgccatttt tacctccttt tgcttaactt aaacttttca ttgcaatcat 6300
gatagacaaa gcaccgaaac cacctggttg gaagtcgtag ttaactggaa cttcgatcaa 6360
gaatggtcta cccaattcag caccctttct caaagcagcc aacaattctt ctctgttagt 6420
agctctagta gcgtcaacac cgttagcttc agccaaagca acgaagtcaa caccaccgaa 6480
cttaacagct gggtcgtgag atctgtggtg accgatgttt tggtacaatt cgatcaaacc 6540
gttagtgtcg ttgttaacaa caacagtaac gattggcaag ttcaatctag cgatagtttc 6600
caagtcagaa gagttagagt ggaaaccacc gtcaccagcg atcaagaaag ttggttggtc 6660
tggtctagcc atttgagcac cgatagcagc tgggataccg taaccgaaag aagaacaacc 6720
agcagaagtc aagaaaccga atggttggtc agctctagcg aacaaaacac cgtagtgtct 6780
gaagaaaccg atgtcagaaa cgatagtacc ttcacctggt tcagcagctt cttccataac 6840
agtgttcata gagtcgataa cttggtgaac tctcataccg tcttcgtaag tttctgggtc 6900
agccaagaat tcagcgattc tagctctcaa tggttcgatg tcgtgtcttt gcttagcacc 6960
gaaagaagca gtagcagttt cgaagtgttc aacgaaagcc aaaacgtcag taacaacgtc 7020
aacgtctggt ctgtaaactc ttgggattgg gttaacagtt ggagagattc taacagtctt 7080
cttttcgata cccttttgcc acatagatgg tctcaagtct tcagcgtagt cgtaaccaac 7140
agtcaaaacc aagtcaactg gagcgaacat agtttgcaaa gctgggaagt tcaagatacc 7200
gtccatgtaa ccagtaacag caccgtagtt caattcgtga ccaactggca aaacaccctt 7260
agcgatgtaa gtagtgataa ctgggatgtt caatctttca gccaaagctc tgatagctgg 7320
aacagcacca gatctgatag cagcagcacc aacaaccaaa actgggtgct tagcttcagc 7380
caacaaagca gcagcttggt cagcagcctt ttgccaaccg tcagcaacaa caccaactgg 7440
cttagctgga gtgttagctg gtgggtttgg aacagtagtg tcgatacctt cagaagaacc 7500
caacaagtca actggcaaag agatgaaaga tggaccaact ggttcagtca tagcagcgtt 7560
aacagcagag tcaaccaagt cagtgatttc gtgtggtctt tgcaattcaa cagcgtactt 7620
agacattgga gcaacgatag caacagagtc caaacattgg tgagtgtcgt ttgggaagat 7680
gtcgtgagat tcagattgag cagccaaagc gataactgga gatctgtcca aaacagaagt 7740
agcgatacca gtagacaagt tagtcatacc tggacccaaa gtagcccaac aagcttgtgg 7800
tctaccagtg attctagcca aaacgtcagc agcaacacca gcagtgaatt cgtgtctagt 7860
caaaacgaag tcgatacctt caacttcgtc gaacaagata gaagcagctt ctctaccaac 7920
aacaccgaaa accttaccaa caccgtggtc tctcaatcta gacaacaaag cgtgagcagc 7980
agttggctta ccagatggag cagtagaaac tctagacatt ttgtttgttt atgtgtgttt 8040
attcgaaact aagttcttgg tgttttaaaa ctaaaaaaaa gactaactat aaaagtagaa 8100
tttaagaagt ttaagaaata gatttacaga attacaatca atacctaccg tctttatata 8160
cttattagtc aagtagggga ataatttcag ggaactggtt tcaacctttt ttttcagctt 8220
tttccaaatc agagagagca gaaggtaata gaaggtgtaa gaaaatgaga tagatacatg 8280
cgtgggtcaa ttgccttgtg tcatcattta ctccaggcag gttgcatcac tccattgagg 8340
ttgtgcccgt tttttgcctg tttgtgcccc tgttctctgt agttgcgcta agagaatgga 8400
cctatgaact gatggttggt gaagaaaaca atattttggt gctgggattc tttttttttc 8460
tggatgccag cttaaaaagc gggctccatt atatttagtg gatgccagga ataaactgtt 8520
cacccagaca cctacgatgt tatatattct gtgtaacccg ccccctattt tgggcatgta 8580
cgggttacag cagaattaaa aggctaattt tttgactaaa taaagttagg aaaatcacta 8640
ctattaatta tttacgtatt ctttgaaatg gcagtattga taatgataaa ctcgaactga 8700
aaaagcgtgt tttttattca aaatgattct aactccctta cgtaatcaag gaatcttttt 8760
gccttggcct ccgcgtcatt aaacttcttg ttgttgacgc taacattcaa cgctagtatt 8820
atacttacat atagtagatg tcaagcgtag gcgcttcccc tgccggctgt gagggcgcca 8880
taaccaaggt atctatagac cgccaatcag caaactacct ccgtacattc atgttgcacc 8940
cacacattta tacacccaga ccgcgacaaa ttacccataa ggttgtttgt gacggcgtcg 9000
tacaagagaa cgtgggaact ttttaggctc accaaaaaag aaagaaaaaa tacgagttgc 9060
tgacagaagc ctcaagaaaa aaaaaattct tcttcgacta tgctggaggc agagatgatc 9120
gagccggtag ttaactatat atagctaaat tggttccatc accttctttt ctggtgtcgc 9180
tccttctagt gctatttctg gcttttccta tttttttttt tccatttttc tttctctctt 9240
tctaatatat aaattctctt gcattttcta tttttctctc tatctattct acttgtttat 9300
tcccttcaag gttttttttt aaggagtact tgtttttaga atatacggtc aacgaactat 9360
aattaactaa acactagtac catggaagct actttgccag ttttggacgc taagactgct 9420
gctttgaaga gaagatctat cagaagatac agaaaggacc cagttccaga aggtttgttg 9480
agagaaatct tggaagctgc tttgagagct ccatctgctt ggaacttgca accatggaga 9540
atcgttgttg ttagagaccc agctactaag agagctttga gagaagctgc tttcggtcaa 9600
gctcacgttg aagaagctcc agttgttttg gttttgtacg ctgacttgga agacgctttg 9660
gctcacttgg acgaagttat ccacccaggt gttcaaggtg aaagaagaga agctcaaaag 9720
caagctatcc aaagagcttt cgctgctatg ggtcaagaag ctagaaaggc ttgggcttct 9780
ggtcaatctt acatcttgtt gggttacttg ttgttgttgt tggaagctta cggtttgggt 9840
tctgttccaa tgttgggttt cgacccagaa agagttagag ctatcttggg tttgccatct 9900
cacgctgcta tcccagcttt ggttgctttg ggttacccag ctgaagaagg ttacccatct 9960
cacagattgc cattggaaag agttgttttg tggagataag cttgttgtct acaaattata 10020
aaatagtttt ttataatcaa ccattaaata attttgtaca cttataataa cactttattt 10080
ctcgttccaa ttatgcgatg catgtgcctt ccgtttagag tgagcattat caaatataaa 10140
gtcaaatcct tttggcattt attttgattg aaacccagtc tgtccttaac tcttttgcct 10200
atcaaacgct gtaggtcact cttggtacgg tacagcggat agcgctattt attcagcaag 10260
agaagcaaga ttaggcgact acaatagatc aaaattttag gatcctctag agtcgacctg 10320
caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 10380
gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 10440
atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 10500
cctgtcgtgc cag 10513
<210> 3
<211> 10763
<212> DNA
<213> Artificial sequence (unknown)
<400> 3
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 60
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 120
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 180
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 240
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 300
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 360
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 420
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 480
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 540
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 600
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 660
tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 720
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 780
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 840
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 900
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 960
atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 1020
cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 1080
ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 1140
ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 1200
agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 1260
agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 1320
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 1380
cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 1440
gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 1500
tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 1560
tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 1620
aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 1680
cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 1740
cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 1800
aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 1860
ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 1920
tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 1980
ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 2040
acgaggccct ttcgtcttca agaattagct tttcaattca attcatcatt ttttttttat 2100
tctttttttt gatttcggtt tctttgaaat ttttttgatt cggtaatctc cgaacagaag 2160
gaagaacgaa ggaaggagca cagacttaga ttggtatata tacgcatatg tagtgttgaa 2220
gaaacatgaa attgcccagt attcttaacc caactgcaca gaacaaaaac atgcaggaaa 2280
cgaagataaa tcatgtcgaa agctacatat aaggaacgtg ctgctactca tcctagtcct 2340
gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg tgcttcattg 2400
gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc caaaatttgt 2460
ttactaaaaa cacatgtgga tatcttgact gatttttcca tggagggcac agttaagccg 2520
ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa atttgctgac 2580
attggtaata cagtcaaatt gcagtactct gcgggtgtat acagaatagc agaatgggca 2640
gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt gaagcaggcg 2700
gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt gtcatgcaag 2760
ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa gagcgacaaa 2820
gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga aggttacgat 2880
tggttgatta tgacacccgg tgtgggttta gatgacaagg gagacgcatt gggtcaacag 2940
tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt tggaagagga 3000
ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa agcaggctgg 3060
gaagcatatt tgagaagatg cggccagcaa aactaaaaaa ctgtattata agtaaatgca 3120
tgtatactaa actcacaaat tagagcttca atttaattat atcagttatt acccaattct 3180
catgtttgac agcttatcat cgatcgtcca actgcatgga gatgagtcgt ggcaagaata 3240
ccaagagttc ctcggtttgc cagttattaa aagactcgta tttccaaaag actgcaacat 3300
actactcagt gcagcttcac agaaacctca ttcgtttatt cccttgtttg attcagaagc 3360
aggtgggaca ggtgaacttt tggattggaa ctcgatttct gactgggttg gaaggcaaga 3420
gagccccgag agcttacatt ttatgttagc tggtggactg acgccagaaa atgttggtga 3480
tgcgcttaga ttaaatggcg ttattggtgt tgatgtaagc ggaggtgtgg agacaaatgg 3540
tgtaaaagac tctaacaaaa tagcaaattt cgtcaaaaat gctaagaaat aggttattac 3600
tgagtagtat ttatttaagt attgtttgtg cacttgcctg caagcctttt gaaaagcaag 3660
cataaaagat ctaaacataa aatctgtaaa ataacaagat gtaaagataa tgctaaatca 3720
tttggctttt tgattgattg tacaggaaaa tatacatcgc agggggttga cttttaccat 3780
ttcaccgcaa tggaatcaaa cttgttgaag agaatgttca caggcgcata cgctacaatg 3840
acccgattct tgctagcctt ttctcggtct tgcaaacaac cgccggcagc ttagtatata 3900
aatacacatg tacatacctc tctccgtatc ctcgtaatca ttttcttgta tttatcgtct 3960
tttcgctgta aaaactttat cacacttatc tcaaatacac ttattaaccg cttttactat 4020
tatcttctac gctgacagta atatcaaaca gtgacacata ttaaacacag tggtttcttt 4080
gcataaacac catcagcctc aagtcgtcaa gtaaagattt cgtgttcatg cagatagata 4140
acaatctata tgttgataat tagcgttgcc tcatcaatgc gagatccgtt taaccggacc 4200
ctagtgcact taccccacgt tcggtccact gtgtgccgaa catgctcctt cactatttta 4260
acatgtggaa ttaattctca tgtttgacag cttatcatcg aactctaaga ggtgatactt 4320
atttactgta aaactgtgac gataaaaccg gaaggaagaa taagaaaact cgaactgatc 4380
tataatgcct attttctgta aagagtttaa gctatgaaag cctcggcatt ttggccgctc 4440
ctaggtagtg ctttttttcc aaggacaaaa cagtttcttt ttcttgagca ggttttatgt 4500
ttcggtaatc ataaacaata aataaattat ttcatttatg tttaaaaata aaaaataaaa 4560
aagtatttta aatttttaaa aaagttgatt ataagcatgt gaccttttgc aagcaattaa 4620
attttgcaat ttgtgatttt aggcaaaagt tacaatttct ggctcgtgta atatatgtat 4680
gctaaagtga acttttacaa agtcgatatg gacttagtca aaagaaattt tcttaaaaat 4740
atatagcact agccaattta gcacttcttt atgagatata ttatagactt tattaagcca 4800
gatttgtgta ttatatgtat ttacccggcg aatcatggac atacattctg aaataggtaa 4860
tattctctat ggtgagacag catagataac ctaggataca agttaaaagc tagtactgtt 4920
ttgcagtaat ttttttcttt tttataagaa tgttaccacc taaataagtt ataaagtcaa 4980
tagttaagtt tgatatttga ttgtaaaata ccgtaatata tttgcatgat caaaaggctc 5040
aatgttgact agccagcatg tcaaccacta tattgatcac cgatatatgg acttccacac 5100
caactagtaa tatgacaata aattcaagat attcttcatg agaatggccc agcgatatat 5160
gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc gccattcgcc 5220
attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca 5280
gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg gtaacgccag ggttttccca 5340
gtcacgacgt tgtaaaacga cggccagtga attcgagctc tgtcaacgca gattaagcac 5400
accctaaaac ttgaataatg caaattccat agcttagttt aatcaaggcg ccattattcc 5460
tcagttgaaa agtatatcta atggttagga ttagaaatat gttcattgct cattgttatg 5520
tgtatcatat cgtacaaaaa ttatataaag aaataataat gaaaaagagt aaaacaaaga 5580
caaatagaca aaaatagagg atagaaaaag aaaaacaaca gatacataaa taagtcttat 5640
gacaaaaaaa cgattgtata aaaattaaag tgcatgccat ttttacctcc ttttgcttaa 5700
cttaaacttt tcattgcaat catttacaac ttagtttcag acttgaagta agcgtcagtg 5760
ataccagaga tcaaagcagc caaaccgata gcacctgggt ctggcaaacc accttcagat 5820
tcgaattgct tgaattcttc ttcagcaacg taagaagctc taccgaactt agcttccaac 5880
tttctagtag cttcagcacc gtcgtgagca gccttgttag ccaacttcaa gtccttagac 5940
ttagcgaatt ccttaacgaa tggttccaaa gcgtcgatca aagttctgtc accagttcta 6000
gctctagtgt acttgaacaa agattgcaaa gcagcttgca aagaaccaga gatagtttcc 6060
aaagtcaaag tgtaagcacc ttcagacaat tccttttcct tcaaagactt agccaaagca 6120
gagatgaaga tagagtacaa accaccagaa gtaccaccca tagcagtttc aacgatgtca 6180
gtgatttgaa ccaaagactt aacaccgtcc ttcaagtcca acttaccttc agccaaagcc 6240
ttcaagatag cgttagaacc gttagccaaa gtttcaccac agtcaccgtc accagcaaca 6300
gtgtcgtaca aagtgatctt tggttcctta gagataacct tcttaacacc agattccaac 6360
aagtcagcgt acaacttagc gtcaacagaa accttagaag aagagtcacc ttcgtcgatt 6420
tctggagcag caacgatgaa gttgtcaact ctaccagacc agtcagagat gttagagttc 6480
caacctggag cagaagtctt gtggtccaag aacttcaaga tgtccttgtc accagtctta 6540
gtagcgttca acaaagtgat agagaaacct ggaccgtcca aagaagtagt gaaagtacca 6600
gtgaagattc taactggctt gatagagtac ttagaagcca attggtcaac aacgatgttt 6660
tggatagcgt acaattccaa aacagaagta ccacccaagt tgttgatcaa caaaacaact 6720
tcgtcgttct tgtcgaattg aacgtagttt ctgtccttgt cagtagtaga caacaagtat 6780
tccaacaatt cagcaaccaa ttcgtcaaca gttgggattg gagaagactt cttgatacct 6840
ggttcgttgt ggatacccat accgatttcg aattcgtcgt gcttcaaaac ttcgtaaccg 6900
tgttcgtcgt cagagtcgtc ttcttcttgc ttgttagctc tagctgggat agtaacgtgg 6960
tccaaagaag caccgatagt aaccaagtta gcaacaacct tttcaccgaa agtaaccaat 7020
tggtgcaatt ccaagttgtc cttttgcaag taagccttag cacccaagat cttgtgaacc 7080
aaagaagtac cagccaaacc tcttctacca accaaaccgt tcttagcctt accaacagaa 7140
acgtcgtctt gaacgatcaa caattcagcg ttcaaacctt cagccttagc cttttcagca 7200
gccaaaccga agtgcaagat gtcaccagtg tagttcttaa cgatgatcaa agtacccttc 7260
ttagatggct tagccttgat agcagagaag atttgcttag tagatggaga agcgaagatg 7320
aaaccagcaa cagcagcgtc caacaaaccg tccttagtaa cgaaaccagc gtgcaatggt 7380
tcgtgaccag aaccaccacc agagatcaaa gtgatcttgt cttcttggtt ttcagcagag 7440
ataacaactc tttcagattc gatcaatcta acgtgtgggt tagattgaca caaaccagcc 7500
aagtgagaca aaaccaagtc cttcttgtag tcccagtgct tagaagacat tgtttttata 7560
tttgttgtaa aaagtagata attacttcct tgatgatctg taaaaaaaga gaaaaagaaa 7620
gcatctaaga acttgaaaaa actacgaatt agaaaagacc aaatatgtat ttcttgcatt 7680
gaccaattta tgcaagttta tatatatgta aatgtaagtt tcacgaggtt ctactaaact 7740
aaaccacccc cttggttaga agaaaagagt gtgtgagaac aggctgttgt tgtcacacga 7800
ttcggacaat tctgtttgaa agagagagag taacagtacg atcgaacgaa ctttgctctg 7860
gagatcacag tgggcatcat agcatgtggt actaaaccct ttcccgccat tccagaacct 7920
tcgattgctt gttacaaaac ctgtgagccg tcgctaggac cttgttgtgt gacgaaattg 7980
gaagctgcaa tcaataggaa gacaggaagt cgagcgtgtc tgggtttttt cagttttgtt 8040
ctttttgcaa acaaatcacg agcgacggta atttctttct cgataagagg ccacgtgctt 8100
tatgagggta acatcaattc aagaaggagg gaaacacttc ctttttctgg ccctgataat 8160
agtatgaggg tgaagccaaa ataaaggatt cgcgcccaaa tcggcatctt taaatgcagg 8220
tatgcgatag ttcctcactc tttccttact cacgagtaat tcttgcaaat gcctattgtg 8280
cagatgttat aatatctgtg cgtcttgagt tgaagtcagg aatctaaaat aacatgtagg 8340
tggcggaggg gagatataca atagaacaga taccagacaa gacataatgg gctaaacaag 8400
actacaccaa ttacactgcc tcattgatgg tggtacataa cgaactaata ctgtagccct 8460
agacttgata gccatcatca tatcgaagtt tcactaccct ttttccattt gccatctatt 8520
gaagtaataa taggcgcatg caacttcttt tctttttttt tcttttctct ctcccccgtt 8580
gttgtctcac catatccgca atgacaaaaa aatgatggaa gacactaaag gaaaaaatta 8640
acgacaaaga cagcaccaac agatgtcgtt gttccagagc tgatgagggg tatctcgaag 8700
cacacgaaac tttttccttc cttcattcac gcacactact ctctaatgag caacggtata 8760
cggccttcct tccagttact tgaatttgaa ataaaaaaaa gtttgctgtc ttgctatcaa 8820
gtataaatag acctgcaatt attaatcttt tgtttcctcg tcattgttct cgttcccttt 8880
cttccttgtt tctttttctg cacaatattt caagctatac caagcataca atcaactatc 8940
tcatatacaa tggctgctga aagagttttc atctctccag ctaagtacgt tcaaggtaag 9000
aacgttatca ctaagatcgc taactacttg gaaggtatcg gtaacaagac tgttgttatc 9060
gctgacgaaa tcgtttggaa gatcgctggt cacactatcg ttaacgaatt gaagaagggt 9120
aacatcgctg ctgaagaagt tgttttctct ggtgaagctt ctagaaacga agttgaaaga 9180
atcgctaaca tcgctagaaa ggctgaagct gctatcgtta tcggtgttgg tggtggtaag 9240
actttggaca ctgctaaggc tgttgctgac gaattggacg cttacatcgt tatcgttcca 9300
actgctgctt ctactgacgc tccaacttct gctttgtctg ttatctactc tgacgacggt 9360
gttttcgaat cttacagatt ctacaagaag aacccagact tggttttggt tgacactaag 9420
atcatcgcta acgctccacc aagattgttg gcttctggta tcgctgacgc tttggctact 9480
tgggttgaag ctagatctgt tatcaagtct ggtggtaaga ctatggctgg tggtatccca 9540
actatcgctg ctgaagctat cgctgaaaag tgtgaacaaa ctttgttcaa gtacggtaag 9600
ttggcttacg aatctgttaa ggctaaggtt gttactccag ctttggaagc tgttgttgaa 9660
gctaacactt tgttgtctgg tttgggtttc gaatctggtg gtttggctgc tgctcacgct 9720
atccacaacg gtttcactgc tttggaaggt gaaatccacc acttgactca cggtgaaaag 9780
gttgctttcg gtactttggt tcaattggct ttggaagaac actctcaaca agaaatcgaa 9840
agatacatcg aattgtactt gtctttggac ttgccagtta ctttggaaga catcaagttg 9900
aaggacgctt ctagagaaga catcttgaag gttgctaagg ctgctactgc tgaaggtgaa 9960
actatccaca acgctttcaa cgttactgct gacgacgttg ctgacgctat cttcgctgct 10020
gaccaatacg ctaaggctta caaggaaaag cacagaaagt aaatttattg gagaaagata 10080
acatatcata ctttccccca cttttttcga ggctcttcta tatcatattc ataaattagc 10140
attatgtcat ttctcataac tactttatca cgttagaaat tacttattat tattaaatta 10200
atacaaaatt tagtaaccaa ataaatataa ataaatatgt atatttaaat tttaaaaaaa 10260
aaatcctata gagcaaaagg attttccatt ataatattag ctgtacacct cttccgcatt 10320
ttttgagggt ggttacaaca ccactcattc agaggctgtc ggcacagttg cttctagcat 10380
ctggcgtccg tattccgata cgccttgtct ccaactcaat acagtcggta gctgaacgag 10440
aaaactcttg ataagctggg gaatggttct tggaagtagt ataggcgcca gctaggatct 10500
ttctcttccc agccccgttc tgtcaggtag aactcgtcca attgagacgg gatcctctag 10560
agtcgacctg caggcatgca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa 10620
attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 10680
ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 10740
agtcgggaaa cctgtcgtgc cag 10763
Claims (5)
1. A method for producing acrylic acid by saccharomyces cerevisiae fermentation is characterized in that glycerol is used as a raw material, saccharomyces cerevisiae is used as host bacteria, and the method is completed by catalysis of a plurality of enzymes in the host bacteria;
the host bacterium is constructed and obtained according to the following method:
1) optimizing codons of genes GlyDH and DAK according to the codon preference of saccharomyces cerevisiae, and then constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 by using a bidirectional promoter to obtain a vector 1, wherein the expression vector YCplac33 has ampicillin resistance of escherichia coli and a selection marker URA gene of the saccharomyces cerevisiae;
2) optimizing the ceaS2 gene and the NOX gene according to the codon preference of the saccharomyces cerevisiae, and then simultaneously constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 to obtain a vector 2, wherein the expression vector YCplac33 has an ampicillin resistance gene of escherichia coli and a screening marker Leu gene of the saccharomyces cerevisiae;
3) knocking out a PDC1 gene, a DLD1 gene, a GPD1 gene and a GPD2 gene in the Saccharomyces cerevisiae BY4741 to obtain a modified Saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 strain;
4) and (3) simultaneously transferring the vectors 1 and 2 into the modified saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 described in the step 3), screening positive clones according to screening markers ura and Leu genes of the saccharomyces cerevisiae, then extracting a saccharomyces cerevisiae genome, and further carrying out PCR verification.
2. The method for producing acrylic acid by fermentation of Saccharomyces cerevisiae according to claim 1, wherein the GlyDH and DAK genes in step 1) are synthesized according to the amino acid sequences of the corresponding genes in Geobacillus stearothermophilus and Pichia pastoris, respectively.
3. The method for producing acrylic acid by fermentation of Saccharomyces cerevisiae according to claim 1, wherein in step 2) ceaS2 and NOX genes are synthesized based on the amino acid sequences of the corresponding genes of Streptomyces clavuligerus and Geobacillus stearothermophilus, respectively.
4. The method for producing acrylic acid by saccharomyces cerevisiae fermentation according to claim 1, wherein the synthetic method comprises the following steps:
1) optimizing codons of genes GlyDH and DAK according to the codon preference of saccharomyces cerevisiae, and then constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 by using a bidirectional promoter to obtain a vector 1, wherein the expression vector YCplac33 has ampicillin resistance of escherichia coli and a selection marker URA gene of the saccharomyces cerevisiae;
2) optimizing the ceaS2 gene and the NOX gene according to the codon preference of the saccharomyces cerevisiae, and then simultaneously constructing the two genes in a saccharomyces cerevisiae expression vector YCplac33 to obtain a vector 2, wherein the expression vector YCplac33 has an ampicillin resistance gene of escherichia coli and a screening marker Leu gene of the saccharomyces cerevisiae;
3) knocking out a PDC1 gene, a DLD1 gene, a GPD1 gene and a GPD2 gene in the Saccharomyces cerevisiae BY4741 to obtain a modified Saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 strain;
4) simultaneously transferring the vectors 1 and 2 into the modified saccharomyces cerevisiae BY 4741-delta PDC 1-delta DLD 1-delta GPD 1-delta GPD2 described in 3), screening positive clones according to screening markers ura and Leu genes of the saccharomyces cerevisiae, then extracting a saccharomyces cerevisiae genome, and further carrying out PCR verification;
5) culturing saccharomyces cerevisiae host bacteria, and performing induced expression;
6) adding a glycerol substrate into the expressed bacterial liquid, and continuing to react for 20-60 h;
7) extracting, separating and purifying the acrylic acid in the reaction liquid.
5. The method for producing acrylic acid by saccharomyces cerevisiae fermentation according to claim 4, wherein the glycerol is added in the step 6) in an amount of: 10g/L-100g/L, the reaction temperature is as follows: 25-35 ℃.
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