β-carotene is a terpenoid compound with important medical, food and industrial values [1]. Traditional physical extraction methods are time-consuming and cumbersome, and the cultivation of β-carotene-containing crops requires geographical location and growth cycle. Chemical synthesis of β-carotene may be toxic, limiting its application [2]. Microbial fermentation for the synthesis of β-carotene is currently the most promising synthetic method [3]. Saccharomyces cerevisiae (S. cerevisiae), a recognized food-safe grade microorganism with the advantages of easy molecular manipulation, rapid growth and good tolerance, has been adapted for the production of a wide range of terpenoids including β-carotene [4].

In this project, we targeted the β-carotene synthesis pathway, preferentially selected the synthesis pathway genes, arranged and combined the candidate gene species sources, constructed the corresponding strains, tested the β-carotene yield of the resulting strains, and obtained the optimal gene combinations for the construction of a high-yielding β-carotene S. cerevisiae to assist in the commercial production of β-carotene.

The results of our experiments can be categorized as follows:

1. Construction of integration plasmids

2. Integration of target genes into the yeast genome

• Obtain the integrating fragment by enzymatic cleavage.
• Integrate the target fragment into the yeast genome by CRISPR/Cas9 technology to obtain yeast strains with three gene combinations.

3. Qualitative and quantitative analysis of yeast strains

• Identify the expression of target genes on the yeast genome by qPCR.
• Shake flask culture of three yeast strains, extract β-carotene content determination.

We have researched the literature to find some key genes in the β-carotene synthesis pathway and intend to categorize them into the following three combinations (Table 1). Among the genes, CrtE encodes GGPP synthetase. FPP forms GGPP under the action of GGPP synthetase. Two GGPP molecules will then form octahydrolycopene under the action of octahydrolycopene synthetase which is encoded by CrtB. The octahydrolycopene dehydrogenase that is encoded by CrtI then converts the octahydrolycopene into lycopene. At last, lycopene β-ring enzyme encoded by CrtY will catalyze lycopene and finally form β-carotene. CrtYB, on the other hand, encodes a bifunctional gene that functions as both CrtB and CrtY. The combinations we designed all contain genes for the pathway to synthesize β-carotene: CrtE, CrtI, CrtYB, (CrtY and CrtB).

Table 1 Genes for β-carotene biosynthesis from different species sources

Firstly, we obtained seven 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 (Figure 1A). 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 plasmid 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 seven integration plasmids (Figure 1B-H).

Figure 1 The construction results of the seven integration plasmids.

(A) Amplification results of target fragments.

(B-H) Transformation, colony PCR and sequencing results of the integration plasmid.

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). By agarose gel electrophoresis analysis, we confirmed that all integration fragments had the expected molecular weight size (Figure 2).

Figure 2 Electrophoresis gel results of integration fragments.

Subsequently, we recovered these integration fragments in combination with the corresponding gRNA plasmids (X-2-XII-5-gRNA-HYG, XI-2-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. Considering the need to integrate three or four fragments into the yeast genome, we adopted a strategy of integrating in two rounds in order to improve the integration efficiency (Table 2).

Table 2 Integration strategies for three yeast strains.

After the first round of integration, we used yeast colony PCR to verify whether the target fragments were successfully integrated into the yeast genome. For the transformants whose PCR results showed the expected bands, we prepared them into competent cells and performed the second round of integration to introduce the remaining target genes. Subsequently, we verified the success of the second round of integration by colony PCR (Figure 3).

Figure 3 Colony PCR results of three yeast strains.

Finally, we cultured the transformants identified as positive by PCR on medium containing appropriate resistance for 48 to 72 hours. The yeast colonies from group 1 showed a distinct orange color, followed by group 2, while those from group 3 showed only a pale-yellow color (Figure 4). These differences tentatively suggest that yeast strains with different gene combinations differ in their ability to biosynthesize β-carotene.

Figure 4 Plate culture results of three yeast strains.

To determine the transcript levels of target genes integrated into the yeast genome, we used quantitative PCR (qPCR) technology. First, we extracted total RNA from recombinant S. cerevisiae and reverse transcribed the RNA into cDNA using a reverse transcription kit, followed by qPCR analysis. In this experiment, we chose the ACT1 gene as the internal reference gene and used the 2-ΔΔCt method to calculate the relative expression of the target gene.

To assess the reliability of the qPCR reaction, we performed standard curve analysis for each primer pair. We plotted standard curves to determine the amplification efficiency of the primers using the logarithmic value of the dilution of the template cDNA series as the X-axis and the corresponding Ct value as the Y-axis. The results showed that the correlation coefficient R2 values of all standard curves were greater than 0.99. The R2 value reflects the linear relationship of the data, and the closer the value is to 1, the better linear relationship is and the higher the confidence of the data is. The PCR amplification efficiencies “e” (i.e., slopes) were all in the range of 90%

Figure 5 Standard curves for primer amplification efficiency.

(a) ACT1: e=105.97%, (b) PaCrtY: e=92.75%, (c) PagCrtB: e=110.8%, (d) PaCrtE: e=106.54%,

(e) BtCrtI: e=106.94%, (f) XdCrtI: e=93.64%, (g) XdCrtE: e=106%, (h) XdCrtYB: e=90.99%.

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 group 1; 0.769~2.305-fold in group 2; and 0.771~1.709-fold in group 3 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 3-5).

Table 3 Gene expression analysis of yeast strain Group 1.

Table 4 Gene expression analysis of yeast strain Group 2.

Table 5 Gene expression analysis of yeast strain Group 3.

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 in group 1 produced the highest concentration of β-carotene at 31.47 mg/L; group 2 had a concentration of 18.24 mg/L; and group 3 had the lowest concentration of 13.37 mg/L (Figure 6).

Figure 6 β-carotene production by three yeast strains.

(A) Cultures of three yeast strains (after 48h of incubation). (B) Standard curve of β-carotene.

(C) β-carotene yield of the three yeast strains.

Through literature research, we speculated the following possible reasons for the differences in β-carotene production capacity of these recombinant yeast strains:

• Adaptation of gene origin: genes from X. dendrorhous may be more suitable for expression in S. cerevisiae, whereas genes from P. ananatis or P. agglomerans may be more suitable for expression in E. coli. This host specificity may result from differences in codon usage preferences, post-translational modification mechanisms, or protein folding environments [9].
• 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 [10].

These results indicate that we successfully established three β-carotene-producing strains of S. cerevisiae. Among them, the Group 1 (XdCrtE+XdCrtI+XdCrtYB) had the highest β-carotene production. This confirms the feasibility of our synthetic biology-based idea to construct yeast cell factories with high β-carotene production, which can contribute to the commercial production of β-carotene.

In order to further increase carotenoid production, we consider that future work could be improved in the following ways.

• Systematically optimize fermentation parameters, including temperature, pH, and dissolved oxygen levels.
• Investigate the impact of various carbon and nitrogen sources on β-carotene yield.
• Implement fed-batch and continuous fermentation strategies to improve productivity.

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