β-carotene is a terpenoid compound with important medical, food and industrial values [1]. 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 Saccharomyces cerevisiae (S. cerevisiae) strain that can produce β-carotene at high yields. As a result, we offer the following new parts that have been designed, manufactured and functionally verified, as well as adding new functional data to some of the already existing components.

Table 1 Part contributions in this project.

Part 1- BBa_K5419001 (XdCrtE)

Profile

Name: XdCrtE

Base Pairs: 946 bp

Origin: Xanthophyllomyces dendrorhous (X. dendrorhous); synthesized

Properties:

It is an enzyme that regulates β-carotene through a pathway usage and biology. CrtE code GGPP synthetase. GGPP synthetase can produce phytoene under the action of phytoene synthase. After continuous dehydrogenation reaction, phytoene forms lycopene, under action of several enzymes, it can product β-carotene [2].

Part 2- BBa_K5419004 (PagCtrB)

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Name: PagCtrB

Base Pairs: 888 bp

Origin: Pantoea Gavini; synthesized

Properties:

CrtB is a key enzyme in the carotenoid synthesis pathway. The activity of CrtB enzyme directly affects the synthesis efficiency and types of carotenoids, which in turn affects the health and viability of organisms. ‌In addition,‌ because carotenoids have antioxidant, ‌anti-inflammatory and other biological activities, ‌CrtB research also has potential application value in the development of new drugs and health products [3].

Part 3- BBa_K5419006 (BtCrtI)

Profile

Name: BtCrtI

Base Pairs: 1749 bp

Origin: Blakeslea trispora; synthesized

Properties:

It is an enzyme that regulates β-carotene through a pathway. It is also the primary rate-limiting enzyme in the β-carotene synthesis pathway downstream of lycopene. It can catalyze multi-step continuous dehydrogenation to produce lycopene and other valuable products [4].

Part 4- BBa_K5419010 (pCas9-G418-Backbone)

Profile

Name: pCas9-G418-Backbone

Base Pairs: 10949 bp

Origin: Artificial gene synthesis

Properties:

It is a synthetic gene that codes for Cas9. It encodes the Cas9 protein that can combine with gRNA, gRNA then leads Cas9 protein to a specific site of genomes of the yeasts and cut out the gene.

Figure 1 Plasmid map of pCas9-G418-Backbone

Part 5- BBa_K5419011 (X-2-XII-5-gRNA-Backbone)

Profile

Name: X-2-XII-5-gRNA-Backbone

Base Pairs: 6379 bp

Origin: Artificial gene synthesis

Properties:

It encodes gRNA which can combine with Cas9 protein in the yeast and can also combine with X-2 and XII-5 sites of yeast's genome. Then Cas9 protein can cut out a length of DNA.

Figure 2 Plasmid map of X-2-XII-5-gRNA-Backbone.

Part 6- BBa_K5419011 (XI-2-gRNA-Backbone)

Profile

Name: XI-2-gRNA-Backbone

Base Pairs: 6008 bp

Origin: Artificial gene synthesis

Properties:

It encodes gRNA that can combine with Cas9 protein and guide Cas9 protein to the XI-2 site, and then Cas9 protein cut a length of DNA.

Figure 3 Plasmid map of XI-2-gRNA-Backbone.

Part 7- BBa_K5419012 (X-3-XI-2-gRNA-Backbone)

Profile

Name: X-3-XI-2-gRNA-Backbone

Base Pairs: 6379

Origin: Artificial gene synthesis

Properties:

It encodes gRNA that can combine with Cas9 protein and guide Cas9 protein to the X-3 and XI-2 gene sites, and then Cas9 protein cut a length of DNA.

Figure 4 Plasmid map of X-3-XI-2-gRNA-Backbone.

Group 1:

Table 2 Part contributions in group 1.

Summary

We added two new composite parts (BBa_K5419003, BBa_K5419005), add new experimental data to the existing parts (BBa_I742151 (CtrE), BBa_I742154 (CrtY)), and based on this, constructed new composite parts (BBa_K5419000, BBa_K5419008). The added experimental data included the integration of the genes, the analysis of the expression levels of the genes, and the testing of the combination of these genes for the production of β-carotene.

Table 2 Integration strategies for three yeast strains.

Construction Design

We constructed expression cassettes 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 to obtain a recombinant yeast strain capable of producing β-carotene (Figure 5).

Figure 5 Construction of the integration plasmids.

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 (Figure 6).

Figure 6 Construction of β-carotene biosynthesis pathway in S. cerevisiae [5].

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, 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 7).

Figure 7 The construction results of the (A) pX-2-PaCrtE, (B) pXI-2-PaCrtY, (C) pXII-5-BtCrtI, and (D) pX-3-PagCrtB.

(2) Integration of target genes into the yeast genome

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 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.

Measurement: Quantitative analysis

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 9).

Figure 9 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 3).

Table 3 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 10).

Figure 10 β-carotene production in yeast strain.

Group 2:

Table 4 Part contributions in group 2.

Summary

We added new composite parts (BBa_K5419003, BBa_K5419005, and BBa_K5419009) to verify whether these genes can produce β-carotene in yeast. At the same time, experimental data were also added to the existing parts (BBa_I742151 (CtrE), and BBa_K530000 (CrtYB)) that included the integration of the genes, the analysis of the expression levels of the genes, and the testing of the combination of these genes for the production of β-carotene.

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 11).

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

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 (Figure 6).

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, 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-PaCrtE, (B) pXII-5-BtCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

(2) Integration of target genes into the yeast genome

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.

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 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 0.769~2.305-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 5).

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

Table 5 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 15).

Figure 15 β-carotene production in yeast strain.

Group 3:

Table 6 Part contributions in group 3.

Summary

To further increase the production of β-carotene in the yeast cells, 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: the integration of the genes, the analysis of the expression levels of the genes, and the testing of the combination of these genes for the production of β-carotene.

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 16).

Figure 16 Design diagrams of (A) pX-2-XdCrtE, (B) pXII-5-XdCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

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 (Figure 6).

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, 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 17).

Figure 17 The construction results of the (A) pX-2-XdCrtE, (B) pXII-5-XdCrtI, and (C) pXI-2-XdCrtYB integration plasmids.

(2) Integration of target genes into the yeast genome

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 18).

Figure 18 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 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 19). 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 7).

Figure 19 Standard curves for primer amplification efficiency of (A) XdCrtE, (B) XdCrtI, and (C) XdCrtYB.

Table 7 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 20).

Figure 20 β-carotene production in yeast strain.

Through literature research, we speculated the following possible reasons for the differences in β-carotene production capacity of these recombinant yeast strains. These can be used as a reference for other teams doing similar studies

• 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 [6].
• 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 [7].

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[2] Sandmann G, Misawa N. New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species[J]. FEMS Microbiol Lett,1992,69(3):253-257.

[3] Li Chunji, Li Bingxue, Han Xiaori. Advances in phytoene dehydrogenase-A review[J]. Acta Microbiologica Sinica, 2016, 56(11): 1680-1690.

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[5] 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.

[6] Puigbo P, Bravo IG, Garcia-Vallve S. CAIcal: a combined set of tools to assess codon usage adaptation. Biol Direct, 2008, 3: 38.

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