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. ) (质粒图注释)
【E.coli新路径图(含旧路径部分),区别主要途径和还原力模块+质粒图】
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 URA defect screening marker as the backbone and
used the pTDH3 constitutive promoter and tPFK1 constitutive terminator to construct ceaS2 plasmid.
【S.C图+路径图+质粒图】 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.
【新途径+质粒图】 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. (质粒图注释)
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.
【筛选条件组合表,分为E.coli和S.C的】
PS. We also made Hardware
(Click Here) to simulate the industrial production process of acrylic acid!