Team:NPU-China/Model

Abstract

Acrylic acid is a bulk chemical raw material, which is widely used in many fields because of its excellent polymerization capacity, such as paint, glue, and even mobile phone screen protective film. The average annual market demand of acrylic acid is up to 8 million tons, and the market value is nearly 10 billion US dollars. It has broad market prospect. At present, acrylic acid is made from propylene (which is obtained by petroleum cracking) after multi-step treatment. The production process causes pollution, high energy consumption and it is unsustainable.
This year, we aim to use a green and environmentally friendly carbon source, glycerol to achieve all green production of acrylic acid. Compared to traditional chemical synthesis methods, Synbio is green and sustainable, and glycerol is cheaper than ethylene.

We construct cell factory based on 4 levels, which are—

Core Part

We use ceaS2 enzyme as the core part, but acrylic acid is a byproduct of ceaS2 enzyme, the wild type 's catalytic effect is very weak, whose production is only 1mg/L. So we hope to improve the catalytic effect of ceaS2 enzyme.
We designed ceaS2 enzyme mutants via the AEMD(Auto Enzyme Mutation Design) platform and screened for better-worked ceaS2 mutants by HPLC(High Performance Liquid Chromatography) and HTS(High throughput screening).

System

Respectively, E. coli and S. cerevisiae are the two sorts of model organisms that are most convenient to operate in the prokaryote and eukaryote. Therefore, in terms of our choice of the chassis organisms, we have them both tested, which were E. coli MG1655 and S. cerevisiae BY4741 individually.

Pathway

We need to design two different metabolic pathways for two different chassis organisms and propose different optimization schemes for them.We introduced the ceaS2 enzyme exogenously on the basis of the glycerol metabolism of the two bacteria, so that it could produce the target product acrylic acid using the intermediates G3P and DHAP.Besides having finished the construction of the pathways, we also reconstructed and optimized the original metabolic pathway to increase the carbon flux rate of the designed pathway and reduce the loss of bypass carbon flux.

Production

All of the previous processes are to build the engineering strains which have a high production of acrylic acid that we need. In the subsequent fermentation, we also need to determine the best parameters of the engineering strain.
Therefore, we selected the carbon source, Buffer, temperature, pH and other conditions to optimize the cell production process.