This project targets the β-carotene synthesis pathway, selects key genes of the β-carotene synthesis pathway from different species sources and combines them with the aim of constructing yeast strains that can produce β-carotene at high yields. We carried out three engineering cycles following the logic of DBTL. In each cycle, we designed different gene combinations, integrated the genes into the genome of Saccharomyces cerevisiae (S. cerevisiae) by CRISPR/Cas9 technology, and examined the β-carotene yield of the resulting strains to obtain the optimal combination of genes for the construction of a high-yielding β-carotene, which would help in the commercial production of β-carotene.

The outline of the three cycles is as follows:

• Cycle 1: PaCrtE + BtCrtI + PaCrtY + PagCrtB
• Cycle 2: PaCrtE + BtCtrI + XdCrtYB
• Cycle 1: PaCrtE + BtCrtI + PaCrtY + PagCrtB

• Cycle 1: PaCrtE + BtCrtI + PaCrtY + PagCrtB

Design 1

In the first round of DBTL, we selected β-carotene synthesis pathway genes from different microbial sources, including CrtE gene from Pantoea ananatis (PaCrtE), CrtI gene from Blakeslea trispora (BtCrtI), CrtY gene from Pantoea ananatis (PaCrtY), and CrtB gene from Pantoea agglomerans (PagCrtB) [1-3]. We constructed the plasmids by placing these 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 to obtain a recombinant yeast strain capable of producing β-carotene (Figure 1).

Figure 1 Design diagrams of (A) pX-2-PaCrtE, (B) pXII-5-BtCrtI, (C) pXI-2-PaCrtY, and (D) pX-3-PagCrtB integration plasmids.

Build 1

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, X-3, XI-2, and XII-5 integration site genes, respectively) and obtained the plasmids by enzymatic ligation. Finally, we transformed the enzyme-ligation product into Escherichia coli (E. coli) DH5α competent cells, and the colony PCR and sequencing results showed that we successfully constructed four integration plasmids (Figure 2).

Figure 2 The construction results of the (A) pX-2-PaCrtE, (B) pXI-2-PaCrtY, (C) pXII-5-BtCrtI, and (D) pX-3-PagCrtB.

We reserved NotI digestion sites upstream and downstream of the integration fragment, respectively. Thus, 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 X-3-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.

Test 1

We used quantitative PCR (qPCR) technique to determine the transcript levels of target genes integrated into the yeast genome. We selected the ACT1 gene as an internal reference gene and used the 2-ΔΔCt method to calculate the relative expression of the target gene. The primer amplification efficiency standard curves showed R2 values of all standard curves were greater than 0.99, and the amplification efficiencies “e” ranged between 90% and 110%, which indicated that this qPCR amplification has great reproducibility and accuracy (Figure 4).

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

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 0.771~1.709-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).

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 the strain produced β-carotene at a concentration of 13.37 mg/L. (Figure 5).

Figure 5 β-carotene production in yeast strain.

Learn 1

Preliminary analyses indicated that this gene combination produces low levels of β-carotene. We hypothesized that this could be due to the inefficient expression of the selected heterologous genes in the yeast cells, resulting in a limitation of β-carotene biosynthesis. Thus, we decided to replace PaCrtY and PagCrtB with the CrtYB fusion gene from Xanthophyllomyces dendrorhous (X. dendrorhous) (XdCrtYB) [4] in the next round of DBTL to assess whether this change could improve β-carotene production.

• Cycle 2: PaCrtE + BtCtrI + XdCrtYB

Design 2

In the second round of DBTL, we used a new combination of genes, including PaCrtE, BtCrtI, and XdCrtYB. We followed the strategy of the first round by placing these genes under the regulation of the GAP promoter and the CYC terminator, and added integration sites upstream and downstream of the expression cassette for integration into the yeast genome by the CRISPR/Cas9 technology (Figure 6).

Figure 6 Design diagrams of (A) pX-2-PaCrtE, (B) pXII-5-BtCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

Build 2

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 7).

Figure 7 The construction results of the (A) pX-2-PaCrtE, (B) pXII-5-BtCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

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 8).

Figure 8 Construction results of yeast strain.

(A) Strategy for constructing yeast strain.

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

Test 2

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 9). 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 2).

Figure 9 Standard curves for primer amplification efficiency of (A) PaCrtE, (B) BtCrtI, and (C) XdCrtYB.

Table 2 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 10).

Figure 10 β-carotene production in yeast strain.

Learn 2

Compared with the first round, the recombinant yeast strain constructed this cycle increased the β-carotene production by about 1.4-fold, confirming the effectiveness of our gene replacement strategy. To further increase the β-carotene yield, we plan to replace PaCrtE and BtCrtI with homologous genes from X. dendrorhous in the third round of DBTL as well.

• Cycle 3: XdCrtE + XdCrtI + XdCrtYB

Design 3

In the third round of DBTL, we used all the genes from X. dendrorhous (XdCrtE, XdCrtI, and XdCrtYB) and constructed the corresponding three integration plasmids [4]. The construction strategy remained consistent with the previous two rounds (Figure 11).

Figure 11 Design diagrams of (A) pX-2-XdCrtE, (B) pXII-5-XdCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

Build 3

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 12).

Figure 12 The construction results of the (A) pX-2-XdCrtE, (B) pXII-5-XdCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

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 13).

Figure 13 Construction results of yeast strain.

(A) Strategy for constructing yeast strain.

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

Test 3

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 14). 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 3).

Figure 14 Standard curves for primer amplification efficiency of (A) XdCrtE, (B) XdCrtI, and (C) XdCrtYB.

Table 3 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 15).

Figure 15 β-carotene production in yeast strain.

Learn 3

This round achieved remarkable results, β-carotene production was successfully increased to 31.47 mg/L, which is 2.35-fold higher than the constructed strains of the first cycle, and 1.73-fold higher than that of the second cycle. This result strongly supports our gene replacement strategy. Through in-depth analyses of these results and in conjunction with literature research, we have summarized the following key factors that may contribute to the improvement of β-carotene production in S. cerevisiae.

. Choice of gene source: genes from X. dendrorhous showed better expression efficiency and function in S. cerevisiae [4].

. Protein engineering: XdCrtYB is a fusion protein that may spatially favor sequential catalytic reactions and improve metabolic efficiency [5].

These findings provide a valuable reference for similar metabolic engineering projects in the future. However, we also recognize that there are many aspects that deserve further exploration, such as:

. Optimizing gene expression levels: attempts could be made to use promoters of different strengths or inducible promoters to regulate the expression of each gene.

. Increasing precursor supply: Increasing the supply of isopentenyl pyrophosphate (IPP) by overexpressing key enzymes in the MVA pathway could be considered.

. Reducing metabolic burden: the use of genomic integration site selection or copy number optimization can be explored to balance metabolic burden and yield.

. Optimization of fermentation conditions: systematic optimization of culture conditions, including parameters such as temperature, pH, and dissolved oxygen level, may further improve yields.

These optimization strategies are expected to provide useful guidance and inspiration for future research teams

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[2] Zhao J, Li Q, Sun T, et al. Engineering central metabolic modules of Escherichia coli for improving beta-carotene production. Metab Eng, 2013, 17: 42–50.

[3] Wang JF, Liu XJ, Liu RS, et al. Optimization of the mated fermentation process for the production of lycopene by Blakeslea trispora NRRL 2895 (+) and NRRL 2896 (−). Bioprocess Biosyst Eng, 2012, 35(4): 553-564.

[4] Verwaal R, Wang J, Meijnen JP, et al. High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous. Appl Environ Microbiol, 2007, 73(13): 4342–4350.

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