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: 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!
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!