In this part, we validate how ceaS2 works as expected and how we construct GAACF1.0.
1.Verification of Synthesis of acrylic acid catalyzed by ceaS2 enzyme
1.1 construction of pET-28a-ceaS2 plasmid：
First, we constructed pET-28a-ceaS2 plasmid with pET-28a plasmid skeleton as vector.
1.2 Expression and Purification of ceaS2 Protein：
We transferred the pET-28a-ceaS2 plasmid constructed into E.coli BL21 (DE3) strain and induced E. coli
expression of ceaS2 protein by IPTG. Harnessing the His protein tag from the pET-28a plasmid skeleton,
we used the affinity chromatography nickel column to separate and purify the ceaS2 protein and identified
it by 12% SDS-PAGE electrophoresis. The results were as follows:
Purified ceaS2 after Ni-NTA affinity chromatography
(M, protein marker (from top to bottom is 25、35、48、63、75、100、135、180 kDa);Lane1, precipitation samples
in the cell lysates; 2, supernatant samples in the cell lysates; 3, supernatant flow through Ni-NTA
affinity chromatography; 4, 50 mM imidazole eluent; 5, 100 mM imidazole eluent; 6, 200 mM imidazole
eluent; 7, 300 mM imidazole eluent)
According to the information in the Uniprot database, the ceaS2 protein has 573 amino acids and
the molecular weight of the protein is 62.34 kDa. In Figure, electrophoretic display of recombinant
protein molecular weight is consistent with the theoretical molecular weight of the protein, which
leads to the conclusion that this is the expression of our target protein. The results show that
ceaS2 has good expression and high purity after Ni-NTA affinity chromatography.
1.3 ceaS2 enzymatic reaction:
In order to ensure the reliability and accuracy of the experiment, we first examined the concentration
of the purified protein. Proteins were quantified using the Thermol Scientific BCA Protein Quantification
Kit. The standardized curve of protein concentration of OD562 interval measured.
Standardized curve of protein concentration
The purified and quantified protein was used for enzymatic activity reaction. The control group
was not add ceaS2 enzyme, and the protein buffer was used to make up the volume. The reaction system
was mixed and reacted at 30 ° C for 10 h.
1.4 Determination of Acrylic Acid by Liquid Chromatography
We used high performance liquid chromatography (HPLC) to determine the reaction solution. Determination conditions and parameters:
87H chromatographic column, 5 mM H2SO4 mobile phase, flow rate 0.6 mL / min, UV absorbance 210 nm.
Figure for the liquid phase determination results.
Identification function of ceaS2 by liquid chromatogram
（The black line is acrylic acid standard, green line is control, blue line is reaction of D-G3P
as substrate, red line is reaction DHAP as substrate）
The liquid chromatogram results show that the acrylic acid standardized peak time was in 19.304
minutes presenting a single and satisfying peak shape. The samples in the control group did not correspond
with the treatment group. D-G3P and DHAP reaction group samples both had peaks, in line with the
standard sample of acrylic acid. Therefore, we can initially determine that ceas2 is able to catalyze
D-G3P and DHAP to generate acrylic acid.
1.5 Determination of Acrylic Acid by Liquid Chromatography-Mass Spectrometry (LC-MS)
For the reliable and persuasive results, we also carried out the determination of LC-MS samples. The results are shown below.
LC-MS of ceaS2 generated acrylic acid
（From top to bottom is acrylic acid standard, control, ceas2 reaction by DHAP as substrate, secondary
mass spectrum of acrylic acid）
According to the liquid chromatogram, the control group had no peaks, and the retention time
and molecular weight of the sample group were consistent with the standard sample of acrylic acid.
Also, the molecular ion peaks and fragment ion peaks in the secondary mass spectrum of the sample
also fully demonstrated that ceas2 catalyzed DHAP or ceaS2 to produce the acrylic acid.
2. GAACF 1.0: de novo synthesis of acrylic acid
2.1 A New Approach to Acrylic Biosynthesis
G3P are common secondary products of the E. coli central metabolic pathway. By introducing the ceaS2
enzyme directly into the chassis cells, we can construct a new pathway of acrylic biosynthesis based
on any carbon source. DHAP and G3P are the carbon flow nodes that E. coli glycerol metabolic pathway
must pass through, according to which we designed a new approach to acrylic acid biosynthesis based
on glycerol.
The new pathway, as shown below
The acrylic biosynthetic pathway based on E. coli glycerol aerobic metabolic pathway
This new approach is the shortest compared to other known acrylic pathways, where only three enzymes
are needed to achieve the synthesis of acrylic acid from glycerol, so this pathway not merely has
a better malleability, but also a more promising development prospect.
2.2 Realization of de novo synthesis of acrylic acid
We use the whole cell catalytic method to verify whether the new pathway works in E.coli. After
inducing the expression of ceaS2 enzyme in E. coli, the reaction system was prepared and carried
out. The reaction solution was filtered and the acrylic acid content was determined by high performance
liquid chromatography (HPLC). The results are as follows:
Primitive peak figure of liquid phase, with standard control sample. Shown as follows
Whole Cell Catalytic Acrylic de novo Synthesis of Liquid Chromatography
From the peak chromatogram, it can be seen that the goal of de novo synthesis of acrylic acid from
the cells have been realized.
We have achieved the prototype of the glycerol-based acrylic cell factory based on the E. coli BL21
(DE3) strain as the chassis cell.
Whole Cell Catalytic Acrylic de novo Synthesis of Liquid Chromatography
