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<h4>This year, we focus on an important chemical organic synthesis raw material – acrylic acid. We hope to | <h4>This year, we focus on an important chemical organic synthesis raw material – acrylic acid. We hope to | ||
− | build an | + | build an cell factory to achieve "all green" production of acrylic acid efficiently, so |
we choose glycerol as the raw material for microbial cell factories to produce bulk chemical products. | we choose glycerol as the raw material for microbial cell factories to produce bulk chemical products. | ||
− | Glycerol has the advantage of being cheap and | + | Glycerol has the advantage of being cheap and environmentally-friendly, and allaying the pressure on |
the by-product waste in the production of biodiesel. In addition, compared to glucose, xylose and | the by-product waste in the production of biodiesel. In addition, compared to glucose, xylose and | ||
other carbohydrate substrates, glycerol metabolism can produce higher reducing power. | other carbohydrate substrates, glycerol metabolism can produce higher reducing power. | ||
<br><br> Complex synthetic pathway, vague synthetic mechanism and low efficiency of the synthesis, are the | <br><br> Complex synthetic pathway, vague synthetic mechanism and low efficiency of the synthesis, are the | ||
− | current shortcomings of acrylic biosynthesis method. How to | + | current shortcomings of acrylic biosynthesis method.How to come up with a short and efficient acrylic biosynthetic pathway to build a highly efficient acrylic acid biosynthetic factory is the key to success! This is also |
− | + | ||
the entry point for our project this year. | the entry point for our project this year. | ||
− | <br><br> In the previous experiments, we further demonstrated the new function of ceaS2 enzyme, which can | + | <br><br> |
− | + | <center><img src="https://static.igem.org/mediawiki/2017/5/59/NPU-formerAA.png" class="img-responsive"></center> | |
− | + | <center><h4>Overview of existing and hypothetical metabolic pathways for biosynthesis of acrylate from sugars. </h4></center> | |
+ | |||
+ | <br> In the previous experiments, we had further demonstrated the new function of ceaS2 enzyme, which can catalyze the production of acrylic acid with DHAP (dihydroxy acetone phosphate) or G3P (glyceraldehyde 3-phosphate) as substrate. Because acrylic acid is not the main product of ceaS2 enzyme, the catalytic | ||
effect of wild-type ceaS2 enzyme is very weak and the yield of acrylic acid is only 1mg / L. So we | effect of wild-type ceaS2 enzyme is very weak and the yield of acrylic acid is only 1mg / L. So we | ||
carried out engineering modification of this enzyme to improve the catalytic effect of the core part. | carried out engineering modification of this enzyme to improve the catalytic effect of the core part. | ||
We used the AEMD (Auto Enzyme Mutation Design) platform to identify mutational sites and screened | We used the AEMD (Auto Enzyme Mutation Design) platform to identify mutational sites and screened | ||
− | for high catalytic efficiency by HPLC (High Performance Liquid Chromatography | + | for high catalytic efficiency by HPLC (High Performance Liquid Chromatography) and HTS (High throughput screening) of the ceaS2 mutant. |
− | + | ||
<br><br> In the selection of the chassis organisms, we chose E. coli which is a kind of classic chassis organism | <br><br> In the selection of the chassis organisms, we chose E. coli which is a kind of classic chassis organism | ||
in prokaryote and Saccharomyces cerevisiae which is the most easily manipulated chassis organism | in prokaryote and Saccharomyces cerevisiae which is the most easily manipulated chassis organism | ||
in eukaryotes. | in eukaryotes. | ||
− | <br><br> The GDC (GlyDH-DAK- | + | |
+ | <center><img src="https://static.igem.org/mediawiki/2017/8/85/System.png" class="img-responsive"></center> | ||
+ | <center><h4> E.coli - V.S - S.cerevisiae </h4></center> | ||
+ | |||
+ | <br><br> The GDC (GlyDH-DAK-ceaS2) pathway was designed in order to improve the ability of chassis cells | ||
to convert glycerol to DHAP and finally synthesize acrylic acid. We also added NOX (NADH dehydrogenase) | to convert glycerol to DHAP and finally synthesize acrylic acid. We also added NOX (NADH dehydrogenase) | ||
− | and CAT (Catalase) to this pathway to provide the required reducing power for | + | and CAT (Catalase) to this pathway to provide the required reducing power for GlyDH by two layers |
of substrate circulation. Ultimately, GNCDC (GlyDH-NOX-CAT-DAK-ceaS2) is our desired new biosynthetic | of substrate circulation. Ultimately, GNCDC (GlyDH-NOX-CAT-DAK-ceaS2) is our desired new biosynthetic | ||
pathway of acrylic acid. | pathway of acrylic acid. | ||
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and to analyze the stability and robustness of the new route, we asked SCAU-China to help us did | and to analyze the stability and robustness of the new route, we asked SCAU-China to help us did | ||
the work of metabolic flow modeling. We analyzed the changes of carbon flux of the two key intermediates, | the work of metabolic flow modeling. We analyzed the changes of carbon flux of the two key intermediates, | ||
− | DHAP and G3P, before and after joining the new pathway. The modeling results show that the new | + | DHAP and G3P, before and after joining the new pathway. The modeling results show that the new pathway |
can increase the carbon flow of DHAP and G3P, thereby contributing to the increase in the production | can increase the carbon flow of DHAP and G3P, thereby contributing to the increase in the production | ||
of acrylic acid. | of acrylic acid. | ||
+ | |||
+ | <div class="col-md-12" style="padding-top:30px"> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/1/10/%E5%A4%A7%E8%82%A0%E8%B7%AF%E5%BE%84%E5%9B%BE.png" class="img-responsive"> | ||
+ | <center> <h4> the GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) pathway for E.coli </h4></center> | ||
+ | </div> | ||
+ | <div class="col-md-6"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/b/b0/%E9%85%B5%E6%AF%8D%E8%B7%AF%E5%BE%84%E5%9B%BE.png" class="img-responsive"> | ||
+ | <center> <h4> the GNDC(GlyDH-NOX-DAK-ceaS2) pathway for S.cerevisiae </h4></center> | ||
+ | </div> | ||
+ | </div> | ||
+ | </h4> | ||
+ | <h4> | ||
<br><br> We used whole cell catalysis to carry out the initial acrylic acid synthesis "fermentation" process. | <br><br> We used whole cell catalysis to carry out the initial acrylic acid synthesis "fermentation" process. | ||
The advantage of whole cell catalysis is that the intracellular complete multi-enzyme system can | The advantage of whole cell catalysis is that the intracellular complete multi-enzyme system can | ||
achieve the cascade reaction of enzyme, so as to make up the deficiency of cascade reaction in reaction | achieve the cascade reaction of enzyme, so as to make up the deficiency of cascade reaction in reaction | ||
− | which only | + | which only uses pure enzyme and improves the catalytic efficiency. While eliminating the complex process |
in enzyme purification, it is easier to carry out the reaction and lower production costs. | in enzyme purification, it is easier to carry out the reaction and lower production costs. | ||
<br><br> We optimized the reaction process, selected the carbon source, buffer, temperature, pH, reaction | <br><br> We optimized the reaction process, selected the carbon source, buffer, temperature, pH, reaction | ||
time and other conditions to optimize the production process of the cell factory. | time and other conditions to optimize the production process of the cell factory. | ||
− | <br><br> We also made <a href="https://2017.igem.org/Team:NPU-China/Hardware"> | + | <center><img src="https://static.igem.org/mediawiki/2017/6/67/Production2.png" class="img-responsive"></center> |
+ | <br><br> We also made Hardware<a href="https://2017.igem.org/Team:NPU-China/Hardware">(Click Here)</a> to simulate the industrial production process of acrylic acid! | ||
+ | |||
+ | </h4> | ||
</div> | </div> |
Latest revision as of 03:34, 2 November 2017