Difference between revisions of "Part:BBa K5419000"

Construction Design

We constructed the plasmid by placing the gene under the regulation of a strong constitutive GAP promoter and a CYC terminator, respectively. Integration sequence was added upstream and downstream of the expression cassette to integrate the target gene into the genome of S. cerevisiae using the CRISPR/Cas9 system (Figure 1).

Figure 1 Design diagrams of pX-2-PaCrtE.

Engineering Principle

In the S. cerevisiae, CrtE gene encodes GGPP synthase, in the presence of which FPP forms GGPP. The two GGPP molecules then form octahydrodicarbons via the CrtB gene-encoded octahydrodicarbon synthase. Then, octahydro lycopene dehydrogenase encoded by CrtI gene converts octahydro lycopene to lycopene. Finally, the CrtY-encoded lycopene β-cyclase will catalyze lycopene and eventually form β-carotene. CrtYB gene, on the other hand, encodes a bifunctional gene that functions as both CrtB and CrtY [1].

Experimental Approach

(1) Construction of integration plasmids

Firstly, we obtained the target gene expression frames (GAP promotor-gene-CYC terminator) by PCR amplification, and agarose gel electrophoresis results showed that we succeeded in obtaining these fragments. Next, we double-digested the target fragment and the vector (containing the S. cerevisiae X-2 integration site genes) and obtained the plasmid by enzymatic ligation. Finally, we transformed the enzyme-ligation product into E. coli DH5α competent cells, and the colony PCR and sequencing results showed that we successfully constructed the plasmid (Figure 2).

Figure 2 The construction results of the pX-2-PaCrtE plasmid.

(2) Integration of target genes into the yeast genome

After successfully obtaining the integration plasmids (pX-2-PaCrtE, pXII-5-BtCrtI, and pXI-2-XdCrtYB), we used NotI restriction endonuclease to obtain the complete destination fragment (containing the sequence upstream of the integration site, the GAP promoter, the target gene, the CYC terminator and the sequence downstream of the integration site). Subsequently, we recovered these integration fragments in combination with the corresponding gRNA plasmids (X-2-XII-5-gRNA-HYG and XI-2-gRNA-HYG) and introduced them into the modified S. cerevisiae 1974 strain (which had pre-integrated the Cas9 gene) by a modified lithium acetate transformation method. After two rounds of integration, we used yeast colony PCR to verify that the target fragments were successfully integrated into the yeast genome (Figure 3).

Figure 3 Construction results of yeast strain. (A) Strategy for constructing yeast strain. (B) Transformation plate and (C) colony PCR results of the yeast strain.

Measurement: Quantitative analysis

We used the quantitative PCR (qPCR) technique to determine the transcript levels of target genes integrated into the yeast genome. The primer amplification efficiency standard curves showed that this qPCR amplification has great reproducibility and accuracy (Figure 4). Subsequently, we analyzed the expression of the target genes in the recombinant yeast strains. The results showed that the expression of the target gene was increased 0.769~2.305-fold in this group compared to the control. These results confirmed that the target genes had been successfully integrated into the yeast genome and could be efficiently transcribed (Table 1).

Figure 4 Standard curves for primer amplification efficiency of (A) PaCrtE, (B) BtCrtI, and (C) XdCrtYB.

Table 1 Gene expression analysis of yeast strain.

Finally, we quantified the β-carotene production of the recombinant yeast strains. We plotted a standard curve using a series of β-carotene standards with concentration gradients and established a linear regression equation for calculating β-carotene concentration. After extraction and analysis, we found that this strain produced β-carotene at a concentration of 18.24 mg/L (Figure 5).

Figure 5 β-carotene production in yeast strain.

Reference

[1] WANG Rui-zhao, PAN Cai-hui, WANG Ying, XIAO Wen-hai, YUAN Ying-jin. Design and Construction of high β-carotene Producing Saccharomyces cerevisiae[J]. China Biotechnology, 2016, 36(7): 83-91.