Difference between revisions of "Team:NPU-China/Proofofconcept"

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.