Group: Foshan-GreatBay iGEM 2024

New Improved Part: BBa_K5419002 (pX-2-XdCrtE), BBa_K5419007 (pXII-5-XdCrtI), and BBa_K5419009 (pXI-2-XdCrtYB)

Existing Part: BBa_K2407309 (CrtI), BBa_K530000 (CrtYB)

Summary:

To construct Saccharomyces cerevisiae (S. cerevisiae) strain with high β-carotene production, we added new composite parts (BBa_K5419002, BBa_K5419007, and BBa_K5419009). At the same time, experimental data were also added to the existing parts (BBa_K2407309 (CrtI), BBa_K530000 (CrtYB)) that included:

1. Construction of the integration plasmids coding XdCrtE, XdCrtI, and XdCrtYB, respectively.
2. Integration of the genes by CRISPR/Cas9 technology into S. cerevisiae.
3. Analysis of the expression levels of the genes, and the testing of the combination of these genes for the production of β-carotene.

Documentation:

a. Usage and Biology

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 [1]. CrtYB gene, on the other hand, encodes a bifunctional gene that functions as both CrtB and CrtY (Figure 1). For the species origin of the genes, we chose CrtE, CrtI, and CrtYB genes from Xanthophyllomyces dendrorhous (X. dendrorhous), which are more suitable for expression in S. cerevisiae [2].

Figure 1 Construction of β-carotene biosynthesis pathway in S. cerevisiae [1].

b. Characterization/Measurement

(1) Construction Design

We constructed the plasmids by placing the genes under the regulation of a strong constitutive GAP promoter and a CYC terminator, respectively. Integration sequences were added upstream and downstream of the expression cassettes in order to integrate the target genes into the genome of S. cerevisiae using the CRISPR/Cas9 system (Figure 2).

Figure 2 Design diagrams of (A) pX-2-XdCrtE, (B) pXII-5-XdCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

(2) 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, XI-2, and XII-5 integration site genes, respectively) 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 three integration plasmids (Figure 3).

Figure 3 The construction results of the (A) pX-2-XdCrtE, (B) pXII-5-XdCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

(3) Integration of target genes into the yeast genome

We reserved NotI digestion sites upstream and downstream of the integration fragment, respectively. After successfully obtaining the integration plasmids, 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 4).

Figure 4 Construction results of yeast strain.

(A) Strategy for constructing yeast strain.

(B) Transformation plate and (C) colony PCR results of the yeast strain.

(4) Measurement: Quantitative analysis

We used quantitative PCR (qPCR) technique to determine the transcript levels of target genes integrated into the yeast genome. The ACT1 gene was selected as an internal reference gene and the 2-ΔΔCt method was used to calculate the relative expression of the target gene. The primer amplification efficiency standard curves showed that this qPCR amplification has great reproducibility and accuracy (Figure 5). 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 0.843~1.796-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 5 Standard curves for primer amplification efficiency of (A) XdCrtE, (B) XdCrtI, and (C) XdCrtYB.

Table 1 Gene expression analysis of yeast strain.

Finally, we quantified the β-carotene production of the three 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 the strain produced β-carotene at a concentration of 31.47 mg/L. (Figure 6).

Figure 6 β-carotene production in yeast strain.

c. Learn

Based on our experimental results and literature research, it is advisable to refer to the following two recommendations when producing β-carotene in S. cerevisiae strains:

• Adaptation of gene origin: genes from X. dendrorhous may be more suitable for expression in S. cerevisiae. This host specificity may result from differences in codon usage preferences, post-translational modification mechanisms, or protein folding environments [3].
• Functional properties of the enzyme: XdCrtBY is a bifunctional enzyme that may be spatially more favorable for successive catalytic reactions. This bifunctional property could allow the product of the first reaction to bind faster to the second catalytic site, thus accelerating the whole reaction process and increasing the efficiency of carotenoid production [4].

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

[4] Tokuhiro K, Muramatsu M, Ohto C, et al. Overproduction of geranylgeraniol by metabolically engineered Saccharomyces cerevisiae. Appl Environ Microbiol, 2009, 75(17): 5536–5543.