Team:NPU-China/Description

This year, we focus on an important chemical organic synthesis raw material – acrylic acid. We hope to 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. 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 other carbohydrate substrates, glycerol metabolism can produce higher reducing power.

Complex synthetic pathway, vague synthetic mechanism and low efficiency of the synthesis, are the 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.

Overview of existing and hypothetical metabolic pathways for biosynthesis of acrylate from sugars.

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 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 for high catalytic efficiency by HPLC (High Performance Liquid Chromatography) and HTS (High throughput screening) of the ceaS2 mutant.

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 eukaryotes.

E.coli - V.S - S.cerevisiae

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) 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 pathway of acrylic acid.

In order to determine the effect of the new route on the yield of acrylic acid before the wet experiment 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, 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 of acrylic acid.

the GNCDC(GlyDH-NOX-CAT-DAK-ceaS2) pathway for E.coli

the GNDC(GlyDH-NOX-DAK-ceaS2) pathway for S.cerevisiae

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 achieve the cascade reaction of enzyme, so as to make up the deficiency of cascade reaction in reaction 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.

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.

We also made Hardware(Click Here) to simulate the industrial production process of acrylic acid!