Team:NPU-China/Design

Introduction

The essence of biochemical synthesis is the catalytic reaction with enzyme as the catalyst. Creating new biochemical reactions is an important research direction of synthetic biology.

ceaS2, whose full name is N2-(2-carboxyethyl)arginine synthase2, is a kind of enzyme in Streptomyces clavuligerus. The mentor of our team, Jiang Huifeng, has confirmed the new functions of ceaS2 with the help of TPP (Thiamine pyrophosphate) and magnesium ions. ceaS2 enzyme can catalyze the production of acrylic acid with DHAP (dihydroxy acetone phosphate) or G3P (glyceraldehyde 3-phosphate) as substrate.

Cell factory of acrylic acid (GAACF) 1.0:

DHAP and G3P are the central metabolic secondary products which can be easily found in various organisms. They are the carbon flow nodes that must be passed in the glycerol metabolic pathway in most organisms. ceaS2 enzyme being the core part, it is possible to create a new pathway to synthesize acrylic acid based on glycerol metabolic pathway in organisms and construct a cell factory with a high yield of acrylic acid.

First, we took E. coli BL21 (DE3) as the chassis cells and constructed engineering bacteria carrying the gene of ceaS2 enzyme with pET-28a plasmid as the vector. We constructed a new pathway to synthesize acrylic acid from any carbon source by transforming ceaS2 directly into the chassis cells. This new approach is the shortest compared to other pathways. Take the glycerol metabolic pathway of E. coli as an example, we only need three enzymes to achieve the synthesis of acrylic acid from glycerol. So this pathway has stronger malleability and broader development prospects.

Through the whole cell catalysis and HPLC (High Performance Liquid Chromatography), the results show that the engineering bacteria can use glycerol as carbon source to produce acrylic acid. However, the yield of the cell factory 1.0 is not high, only about 1mg / L.

It is known that acrylic acid can not be metabolized in the cell, so we analyzed the possible reasons as the following:
1. The activity and the catalytic efficiency of wild type ceaS2 is low.
2. The low carbon flow rate of glycerol metabolic pathway in E. coli leads to the low concentration of DHAP and G3P.
3. Acrylic acid is toxic to the chassis cells.
4. The reaction conditions such as carbon source, pH, temperature and reaction time are not suitable.
Based on the analyzing results, we have made improvements and built a new cell factory.

Cell factory of acrylic acid (GAACF) 2.0:
We built a new cell factory of acrylic acid through the four part: CO-PART, SYSTEM, PATHWAY, PRODUCTION!

Core Part

Acrylic acid is a byproduct of ceaS2 enzyme, the catalytic effect of wild type ceaS2 enzyme is very weak, and acrylic acid production is only 1mg / L. So it is necessary to improve the catalytic effect of this core factor, ceaS2 enzyme.

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 decide the correct proposal.

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 a template to create point mutation, and then transformed the plasmid into BL21. Then, we did the whole cell catalysis to get the products. Finally, we screened for ceaS2 mutants with high catalytic efficiency by HPLC (High Performance Liquid Chromatography) and HTS (High throughput screening) .

Pathway

The carbon flow rate of the glycerol metabolic pathway is low. In order to solve the problem, we need reconstruction and optimization of the original metabolic pathway.

RE-Construction：We designed the GDC (GlyDH-DAK-ceaS2) pathway to produce acrylic acid from glycerol. In this pathway, GlyDH(Glycerol dehydrogenase) can efficiently convert Glycerol into DHA(1,3-Dihydroxyacetone). Then DAK (Dihydroxyacetone kinase) converts DHA into DHAP. Finally, ceaS2 converts DHAP into acrylic acid. In addition, because GlyDH depends on NAD+, we added two reduction models, NOX (NADH dehydrogenase )and CAT(Catalase), to the pathway, with the purpose of providing the required 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)


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 plasmid.

System

The choice of the chassis organism is vital to the efficiency of the cell factory. Acrylic acid may do damage to the cell membrane. So we need to choose an organism which has high tolerance of acrylic acid. Escherichia coli and Saccharomyces cerevisiae are two model organisms which can be easily modified in the prokaryotic and eukaryotic.

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 to GAACF1.0, we used the YCPlac33 plasmid with LEU defect screening marker as the backbone and used the pTDH3 constitutive promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid.

We confirmed the proposal can make S.cerevisiae produce acrylic acid, but the yield is low, so we decided to optimize it.
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), for S.cerevisiae.

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 tPFK1 terminator for DAK. NOX and ceaS2 were constructed on the backbone of the other YCPlac33 plasmid. We replaced URA marker with Leu marker to screen for two plasmids easily. We used the TEF2 promoter and tRPS2 terminator for GlyDH, the same promoter and terminator as the original pathway for ceaS2.

Production

To make the engineering bacteria produce acrylic acid, it takes two stages. First, bacteria must grow and express the enzyme, then use carbon source to synthesize acrylic acid. To screen for 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

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 the carbon source, Buffer, temperature, pH, reaction time and other conditions to optimize the production process of the cell factory.

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