Lichenyuan (Talk | contribs) |
Lichenyuan (Talk | contribs) |
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weak, and acrylic acid production is only 1mg / L. So it is necessary to improve the catalytic | weak, and acrylic acid production is only 1mg / L. So it is necessary to improve the catalytic | ||
effect of this core factor, ceaS2 enzyme. | effect of this core factor, ceaS2 enzyme. | ||
− | <br> The gene of ceaS2 enzyme consists of 1719 deoxynucleotides and the protein sequence consists | + | <br><br> The gene of ceaS2 enzyme consists of 1719 deoxynucleotides and the protein sequence consists |
of 573 amino acids. We need to use bioinformatics to analyze and simulate, in order to help us | of 573 amino acids. We need to use bioinformatics to analyze and simulate, in order to help us | ||
decide the correct proposal. | decide the correct proposal. | ||
− | <br> We constructed ceaS2 enzyme mutants using the AEMD (Auto Enzyme Mutation Design) platform. We | + | <br><br> We constructed ceaS2 enzyme mutants using the AEMD (Auto Enzyme Mutation Design) platform. We |
constructed the ceaS2 wild-type sequence on pET-28a plasmid. We used pET-28a-ceaS2 plasmid as | constructed the ceaS2 wild-type sequence on pET-28a plasmid. We used pET-28a-ceaS2 plasmid as | ||
a template to create point mutation, and then transformed the plasmid into BL21. Then, we did | a template to create point mutation, and then transformed the plasmid into BL21. Then, we did | ||
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reduction force for GLY DH through the two layers of substrate level cycle. At last, we construct | reduction force for GLY DH through the two layers of substrate level cycle. At last, we construct | ||
a new pathway for acrylic acid synthesis- GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) | a new pathway for acrylic acid synthesis- GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) | ||
− | <br> The genes of GlyDH and DAK were constructed on two MCS (multiple cloning sites) on the backbone | + | <br><br> The genes of GlyDH and DAK were constructed on two MCS (multiple cloning sites) on the backbone |
of pCDFDuet-1 plasmid. NOX and CAT were constructed on two MCSs on the backbone of pETDuet-1 | of pCDFDuet-1 plasmid. NOX and CAT were constructed on two MCSs on the backbone of pETDuet-1 | ||
plasmid. ) (质粒图注释) | plasmid. ) (质粒图注释) | ||
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acrylic acid. Escherichia coli and Saccharomyces cerevisiae are two model organisms which can | acrylic acid. Escherichia coli and Saccharomyces cerevisiae are two model organisms which can | ||
be easily modified in the prokaryotic and eukaryotic. | be easily modified in the prokaryotic and eukaryotic. | ||
− | <br> Therefore, in the choice of the chassis organism, we tested two organisms, E. coli MG1655 and | + | <br><br> Therefore, in the choice of the chassis organism, we tested two organisms, E. coli MG1655 and |
Saccharomyces cerevisiae BY4741. BY4741 has a great ability to metabolize glycerol. According | Saccharomyces cerevisiae BY4741. BY4741 has a great ability to metabolize glycerol. According | ||
to GAACF1.0, we used the YCPlac33 plasmid with URA defect screening marker as the backbone and | to GAACF1.0, we used the YCPlac33 plasmid with URA defect screening marker as the backbone and | ||
used the pTDH3 constitutive promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid. | used the pTDH3 constitutive promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid. | ||
− | <br> 【S.C图+路径图+质粒图】 We confirmed the proposal can make S.cerevisiae produce acrylic acid, but the | + | <br><br> 【S.C图+路径图+质粒图】 We confirmed the proposal can make S.cerevisiae produce acrylic acid, but the |
yield is low, so we decided to optimize it. | yield is low, so we decided to optimize it. | ||
<br> First, according to GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) in E.coli, we added NOX to the pathway(the | <br> First, according to GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) in E.coli, we added NOX to the pathway(the | ||
CAT enzyme is active in S.cerevisiae). So we designed a pathway, GNDC(GlyDH-NOX -DAK-ceaS2), | CAT enzyme is active in S.cerevisiae). So we designed a pathway, GNDC(GlyDH-NOX -DAK-ceaS2), | ||
for S.cerevisiae. | for S.cerevisiae. | ||
− | <br> 【新途径+质粒图】 The genes of GlyDH and DAK were constructed on the backbone of YCPlac33 plasmid with | + | <br><br> 【新途径+质粒图】 The genes of GlyDH and DAK were constructed on the backbone of YCPlac33 plasmid with |
URA marker. We used the ADH1 promoter and TGPD1 terminator for GlyDH, the PGK1 promoter and the | URA marker. We used the ADH1 promoter and TGPD1 terminator for GlyDH, the PGK1 promoter and the | ||
tPFK1 terminator for DAK. NOX and ceaS2 were constructed on the backbone of the other YCPlac33 | tPFK1 terminator for DAK. NOX and ceaS2 were constructed on the backbone of the other YCPlac33 | ||
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engineering bacteria, it is a waste of time and reagents to use the traditional fermentation | engineering bacteria, it is a waste of time and reagents to use the traditional fermentation | ||
method. We used whole cell catalysis to carry out the reaction for acrylic acid production | method. We used whole cell catalysis to carry out the reaction for acrylic acid production | ||
− | <br> After the enzyme is expressed, the bacteria solution will be centrifuged and concentrated 10 | + | <br><br> After the enzyme is expressed, the bacteria solution will be centrifuged and concentrated 10 |
times with buffer before the reaction. Therefore, we optimized the reaction process, selected | times with buffer before the reaction. Therefore, we optimized the reaction process, selected | ||
the carbon source, Buffer, temperature, pH, reaction time and other conditions to optimize the | the carbon source, Buffer, temperature, pH, reaction time and other conditions to optimize the |
Revision as of 13:17, 1 November 2017