iGEM 2024 — Team foshan-greatbay

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Notebook
![](https://static.igem.wiki/teams/5419/biguiyuan/2-2sx.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/290.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/2x33.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/12.png)

CONTENT

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7-8

Day 9-13

Day 14-18

Day 19-23

Day 24-26

Day 27-30

Day 1

Task:

1. Laboratory safety training.
2. Preparation of LB and YPD solid medium.
3. Autoclaving, pouring of plates, shaking of bacterial solutions.

![](https://static.igem.wiki/teams/5419/day-1.jpg)

Day 2

Task:

1. Extraction of plasmids.
2. PCR amplification of target genes.
3. Electrophoresis to verify PCR results
4. Cut the gel and recover the DNA.

![](https://static.igem.wiki/teams/5419/day-2-1.jpg)
![](https://static.igem.wiki/teams/5419/day-2-2.jpg)

Day 3

Task:

1. Double cleavage and ligation of the gene fragment to the plasmid backbone
2. Transformation to E. coli DH5α competent cells.

![](https://static.igem.wiki/teams/5419/day-3-1.jpg)
![](https://static.igem.wiki/teams/5419/day-3-2.jpg)

Day 4

Task:

1. Colony PCR identification of positive transformants.
2. Positive transformants with correct colony PCR results were picked for overnight incubation.

![](https://static.igem.wiki/teams/5419/day-4-1.jpg)
![](https://static.igem.wiki/teams/5419/day-4-2.jpg)

Day 5

Task:

1. Integrative plasmid digestion to obtain target fragments
2. Agarose electrophoresis for identification and recovery of target fragment gels.

![](https://static.igem.wiki/teams/5419/day-5.jpg)

Day 6

Task: transformation: first round of integration

1. Yeast Cell Expansion Cultures.
2. Preparation of Receptor Yeast Cells.
3. Saccharomyces cerevisiae LiAc transformation.

![](https://static.igem.wiki/teams/5419/day-6.jpg)

Day 7-8

Task: first round of integration (fail)

1. PCR identification of yeast transformant colonies
2. Expanded culture of yeast transformants with target bands.

Day 9-13

Task: Repeat the first round of integration

1. PCR identification of yeast transformant colonies.
2. Expanded culture of yeast transformants with target bands.

![](https://static.igem.wiki/teams/5419/day-9-13.jpg)

Day 14-18

Task: second round of integration

1. Preparation of Receptor Yeast Cells.
2. Saccharomyces cerevisiae LiAc transformation.
3. PCR identification of yeast transformant colonies.
4. Expanded culture of yeast transformants with target bands.

![](https://static.igem.wiki/teams/5419/day-14-18.jpg)

Day 19-23

Task:

1. Learning the principles of qPCR and how to operate it.
2. Analysis of gene expression in recombinant yeast strains by qPCR.

![](https://static.igem.wiki/teams/5419/day-19-23.jpg)

Day 24-26

Task:

1. Shake flask culture of three yeast strains.
2. Ultrasonic crushing of yeast cells.
3. Preparation of β-carotene standard curve.
4. Determination of β-carotene content by microplate reader.

![](https://static.igem.wiki/teams/5419/day-24-26.jpg)

Day 27-30

Task:

1. Analyzing experimental data.
2. Writing wiki materials.

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Engineering
![](https://static.igem.wiki/teams/5419/biguiyuan/2-2sx.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/290.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/2x33.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/12.png)

CONTENT

Overview

Cycle 1: PaCrtE + BtCrtI + PaCrtY + PagCrtB

Cycle 2: PaCrtE + BtCtrI + XdCrtYB

Cycle 3: XdCrtE + XdCrtI + XdCrtYB

Reference

Overview

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 3: XdCrtE + XdCrtI + XdCrtYB

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).

![](https://static.igem.wiki/teams/5419/engi/figure-1.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-2.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-3.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-4.jpg)

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.

![](https://static.igem.wiki/teams/5419/engi/table-1-n.png)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-5.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-6.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-7.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-8.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-9.jpg)

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

Table 2 Gene expression analysis of yeast strain.

![](https://static.igem.wiki/teams/5419/engi/table-2-n.png)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-10.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-11.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-12.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-13.jpg)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-14.jpg)

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

Table 3 Gene expression analysis of yeast strain.

![](https://static.igem.wiki/teams/5419/engi/table-3-n.png)

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).

![](https://static.igem.wiki/teams/5419/engi/figure-15.jpg)

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

Reference

[1] Zhu FY, Lu L, Fu S, et al. Targeted engineering and scale up of lycopene overproduction in Escherichia coli. Proc Biochem, 2015, 50(3): 341–346.

[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.

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Experiments
![](https://static.igem.wiki/teams/5419/biguiyuan/2-2sx.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/290.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/2x33.png)
![](https://static.igem.wiki/teams/5419/biguiyuan/12.png)

CONTENT

1. Preparation of Solid Culture Media

2. Seed Liquid Culture

3. Plasmid Extraction

4. Target Gene Amplification by PCR

5. Agarose Gel Electrophoresis

6. DNA Gel Extraction

7. Restriction Enzyme Digestion

8. DNA Ligation

9. Transformation of E. coli DH5α

10. Colony PCR of E. coli

11. Integrative Plasmid Digestion

12. Saccharomyces cerevisiae Transformation

13. Yeast Colony PCR

14. Gene Expression Analysis of Recombinant Yeast Strains

15. β-Carotene Concentration Determination

1. Preparation of Solid Culture Media

###### 1.1 LB Medium

###### Materials:

* 1% (w/v) tryptone
* 1% (w/v) NaCl
* 0.5% (w/v) yeast extract
* 1.5% (w/v) agar
* Ampicillin (100 μg/mL)

###### Procedure:

(1) Weigh 1% (w/v) tryptone, 1% (w/v) NaCl, 0.5% (w/v) yeast extract, and 1.5% (w/v) agar in a 250 mL conical
flask and dissolve in deionized ddH2O.

(2) Autoclave the mixture at 121°C for 20 min.

(3) Allow the solution to cool to 50-60°C.

(4) Add ampicillin to a final concentration of 100 μg/mL. Gently mix the contents.

(5) Aseptically pour the medium into culture plates and allow to solidify.

(6) Store the plates at 4°C overnight.

###### 1.2 YPD Medium

###### Materials:

* 2% (w/v) tryptone
* 2% (w/v) yeast extract
* 2% (w/v) glucose
* 1.5% (w/v) agar
* Kanamycin (50 μg/mL)
* Ampicillin (100 μg/mL)

###### Procedure:

(1) Weigh 2% (w/v) tryptone, 2% (w/v) yeast extract, 2% (w/v)

glucose, and 1.5% (w/v) agar in a 250 mL conical flask and dissolve in deionized ddH2O.

(2) Autoclave the glucose solution at 116°C for 15 min.

(3) Allow the solution to cool to 50-60°C.

(4) Add kanamycin and ampicillin to a final concentration of 50 μg/mL and 100 μg/mL, respectively. Gently
mix the contents.

(5) Aseptically pour the medium into culture plates and allow to solidify.

(6) Store the plates at 4°C overnight.

2. Seed Liquid Culture

Inoculate strains containing the following plasmids into 5 mL of liquid LB medium supplemented with 100 μg/mL
ampicillin:

*p-XdCrtE**pX-2-vector*

*p-PaCrtE* *pX-3-vector*

*p-PagCrtB**pXI-2-vector*

*p-XdCrtYB* *pXII-5-vector*

*p-PaCrtY**X-2-XII-5-gRNA-HYG*

*p-XdCrtI* *XI-2-gRNA-HYG*

*p-BtCrtI**X-3-XI-2-gRNA-HYG*

3. Plasmid Extraction

###### Materials and Equipment:

* RNase A
* Buffer SP1, SP2, and SP3
* Wash Solution
* 100% ethanol
* Miniprep columns
* Collection tubes
* LB bacterial culture
* Centrifuge
* Vortex mixer

###### Procedure:

(1) Prepare buffers:

a) Add RNase A to Buffer SP1.

b) Add indicated volume of 100% ethanol to Wash Solution.

c) Check Buffer SP2 and SP3 for precipitation.

(2) Equilibrate column:

a) Place Miniprep column in collection tube.

b) Add 500 μL Buffer S, centrifuge at 12,000 ×g for 1 min.

c) Discard filtrate, return column to collection tube.

(3) Harvest bacterial cells:

a) Centrifuge 1.5-5 mL overnight LB culture at 8,000 ×g for 2 min.

b) Discard supernatant.

(4) Resuspend pellet in 250 μL Buffer SP1, vortex thoroughly.

(5) Add 250 μL Buffer SP2, mix by inverting 10 times.

(6) Add 350 μL Buffer SP3, mix immediately by inverting 10 times.

(7) Centrifuge at 12,000 ×g for 10 min.

(8) Transfer supernatant to column, centrifuge at 8,000 ×g for 30 sec.

(9) Discard filtrate, return column to collection tube.

(10) Add 500 μL Wash Solution, centrifuge at 9,000 ×g for 30 sec.

(11) Repeat step 10.

(12) Centrifuge empty column at 9,000 ×g for 1 min.

(13) Transfer column to clean 1.5 mL microcentrifuge tube.

(14) Add 50 μL ddH2O to center of membrane.

(15) Incubate for 1 min at room temperature.

(16) Centrifuge at 9,000 ×g for 1 min to elute DNA.

4. Target Gene Amplification by PCR

###### Materials and Equipment:

* PrimeStar Max Premix (2×)
* Forward and reverse primers (10 μM each)
* DNA template
* Nuclease-free water
* Thermal cycler
* PCR tubes

###### Procedure:

*Reagent**Volume (μL)**Procedure*

*2×PrimeStar Max Premix..**25**Pre-denaturation: 98 ℃ for 2 min*

*Primer F (10 μM)**1* *Annealing: Tm for 15 sec*

*Primer R (10 μM)**1**Denaturation: 98 ℃ for 15 sec*

*DNA template**1 ng* *Extension: 72 ℃, 30 sec/kb*

*ddH2O* *Back to step 1 (30 times)*

*Total**up to 50**Final elongation: 72 ℃ for 2 min*

*10 ℃ hold*

5. Agarose Gel Electrophoresis

###### Materials and Equipment:

* Electrophoresis equipment
* 1×TAE buffer
* Agarose (0.8% w/v)
* YeaRed™ nucleic acid dye
* DNA loading buffer
* DNA marker
* UV transilluminator

###### Procedure:

###### (1) Preparation of 1×TAE solution

Dilute 50×TAE concentrates to a working concentration (1×), i.e., add 20 mL of the 50× TAE concentrates mix
to 980 mL of ddH2O.

###### (2) Preparation of 0.8% DNA agarose gel

a) Weigh 0.64 g agarose powder using a balance.

b) Pour 80 mL 1× TAE solution using a measuring cylinder.

c) Add the 1× TAE solution and agarose powder into a conical flask and mix them well.

d) Cover the conical flask and heat it in a microwave until all powder is dissolved completely.

e) Cool agarose solution to about 60 °C and add YeaRed™ nucleic acid gel stain to a final concentration of
0.1 μL/mL.

f) Pour gel solution into gel tray and insert well comb. Wait for 20-30 min until the gel is solidified.

###### (3) Electrophoresis conditions

a) Remove the comb and place the solidified agarose gel into the horizontal electrophoresis bath.

b) Pour the TAE solution into the bath, and make sure the solution cover the surface of the agarose gel

c) Prepare DNA samples by mixing the 6× DNA loading buffer with PCR products at a ratio of 1: 5.

d) Load 5 μL DNA marker and 3.5 μL PCR products into wells of solidified agarose gel.

e) Run gel electrophoresis at 150 V in 1× TAE buffer until sufficient migration (at about 25-30 min).

f) Visualize DNA bands under UV light.

6. DNA Gel Extraction

###### Materials and Equipment:

* Gel extraction kit
* Buffer B2
* Wash Solution
* Elution Buffer
* Heating block (50°C)
* Microcentrifuge

###### Procedure:

(1) Excise DNA band from agarose gel.

(2) Add three volume Buffer B2 to gel slice.

(3) Incubate at 50°C for 5-10 min until gel is dissolved.

(4) Transfer mixture to spin column, centrifuge at 8,000 ×g for 30 sec.

(5) Discard flow-through.

(6) Add 500 μL Wash Solution, centrifuge at 8,000 ×g for 30 sec.

(7) Repeat step 6.

(8) Centrifuge empty column at 8,000 ×g for 2 min.

(9) Place column in new 1.5 mL microcentrifuge tube.

(10) Add 30 μL ddH2O to column membrane.

(11) Incubate at room temperature for 1 min.

(12) Centrifuge at 8,000 ×g for 1 min to elute DNA.

7. Restriction Enzyme Digestion

###### Materials and Equipment:

* AscI and PmeI restriction enzymes
* 10×CutSmart buffer
* Plasmid DNA and target fragment
* Nuclease-free water
* Thermal cycler
* Gel extraction kit

###### Procedure:

*Reagent* *Volume (μL)*

*10×Cutsmart buffer**5*

*Plasmid DNA and target fragment**2 μg*

*AscI**2*

*PmeI**2*

*ddH2O* *up to 50*

*37℃, 3-4 h; 65℃, 10 min*

8. DNA Ligation

###### Materials:

* T4 DNA Ligase
* 10×T4 DNA Ligase buffer
* Vector and insert DNA
* Nuclease-free water

###### Procedure:

*Reagent*  *Volume (μL)*

*10×T4 DNA Ligase buffer**1*

*Vector or insert DNA**1 μg*

*T4 DNA Ligase**0.5*

*ddH2O**up to 10*

*16°C, 16 h; 65°C 10 min*

9. Transformation of E. coli DH5α

###### Materials and Equipment:

* Plasmid DNA
* Competent E. coli DH5α cells
* Ice bath
* 42°C water bath
* Antibiotic-free liquid medium
* Solid medium with antibiotics
* L-shaped spreader
* Incubator (37°C)

###### Procedure:

(1) Add 2.5 μL plasmid DNA to 50 μL competent E. coli DH5α cells.

(2) Incubate on ice for 30 min.

(3) Heat shock at 42°C for 90 sec.

(4) Incubate on ice for 2 min.

(5) Add 900 μL antibiotic-free liquid medium.

(6) Incubate at 37°C, 200 rpm for 1 h.

(7) Centrifuge at 3,000 rpm for 5 min.

(8) Resuspend pellet in 100 μL medium.

(9) Spread on solid medium with appropriate antibiotics.

(10) Incubate plates at 37°C overnight.

10. Colony PCR of E. coli

###### Materials and Equipment:

* 2× Hieff Ultra-Rapid II HotStart PCR Master Mix
* Forward and reverse primers (10 μM each)
* Bacterial colonies
* Nuclease-free water
* Thermal cycler

###### Procedure:

(1) Pick 8 well-isolated colonies from the transformation plate onto a new LB agar plate, and incubate at 37
℃ for 6 h.

(2) Pick the colonies, resuspend them in 10 μL of sterile water, and the resuspension is lysed at 98 °C for
10 min.

(3) Centrifuge the tubes for 5 min to pellet the cell debris.

(4) Add 1 μL of the supernatant as a DNA template and followed by PCR amplification according to the
following table:

*Reagent**Volume (μL)**Procedure*

*2×Hieff® ® Ultra-Rapid II HotStart PCR Master Mix**5**Pre-denaturation: 95 ℃ for 3 min*

*Primer F (10 μM)**0.4* *Denaturation: 95 ℃ for 15 sec*

*Primer R (10 μM)**0.4**Annealing: Tm for 20 sec*

*DNA template**1**Extension: 72 ℃, 10 sec/kb*

*ddH2O* *3.2**Back to step 1 (30 times)*

*Total**up to 10**Final elongation: 72 ℃ for 2 min
10 ℃ hold*

*10 ℃ hold*

(5) After amplification, analyze 3.5 μL of the PCR reaction by agarose gel electrophoresis. The procedure is
as previously described.

(6) Inoculate the positive transformants in an LB culture medium containing antibiotics at 200 rpm, 37 ℃
overnight.

(7) Extracted the integrated plasmids as described above.

11. Integrative Plasmid Digestion

###### Materials and Equipment:

* NotI restriction enzyme
* 10× CutSmart buffer
* Plasmid DNA
* Nuclease-free water
* Thermal cycler
* Agarose gel electrophoresis equipment

###### Procedure:

*Reagent**Volume (μL)*

*10×Cutsmart buffer**5*

*Plasmid DNA and target fragment**2 μg*

*NotI**2*

*ddH2O**up to 50*

*37℃, 3-4 h; 65℃, 10 min*

12. Saccharomyces cerevisiae Transformation

###### Materials:

* S. cerevisiae strain (1974-Cas)
* YPD and 2×YPD media
* Sterile water
* Integrated fragment
* gRNA plasmid
* Salmon sperm ssDNA (10 mg/mL)
* LiAc, PEG, and other transformation reagents
* Selection plates (Amp+Kan)

###### Procedure:

###### (1) Preparation of Yeast Culture

a. Pick a single colony and inoculate it into a test tube containing 3 mL of YPD liquid medium.

b. Incubate overnight at 30°C with shaking at 240 rpm.

c.Dilute the overnight culture 10-fold and measure OD600. The OD600 should be greater than 0.2.

d. Inoculate a 250 mL Erlenmeyer flask containing 25 mL of 2×YPD medium with the overnight culture to an
initial OD600 of 0.2.

e. Incubate at 30°C with shaking at 240 rpm until the OD600 reaches 0.8-1.0.

###### (2) Preparation of Competent Cells

a. Centrifuge the culture at 3,000 rpm for 5 min at 20°C to collect the cells.

b. Resuspend the cell pellet in 25 mL of sterile water.

c.Centrifuge at 3,000 rpm for 5 min at 20°C. Repeat this washing step twice.

d. Resuspend the cell pellet in 1 mL of sterile water and transfer to a 1.5 mL microcentrifuge tube.

e. Centrifuge at 12,000 rpm for 30 sec and discard the supernatant to obtain competent yeast cells.

f. Preheat a water bath to 42°C and a heat block to 100°C.

###### (3) Transformation Procedure

a. Prepare carrier DNA by heating 2 mg/L salmon sperm ssDNA at 100°C for 5 min, then immediately place on
ice.

b. Add the following components to microcentrifuge tubes containing competent cells in the order and amount
specified in the table below.

*Reagent**Volume (μL)*

*LiAc（1 M）**240 μL*

*Primer F (10 μM)**36 μL*

*Salmon sperm ssDNA（2 g/L）**50 μL*

*DNA+H2O**34 μL*

c. Heat-shock the mixture at 42°C for 15 min.

d. Centrifuge at 12,000 rpm for 30 sec and discard the supernatant.

e. Add 1 mL of YPD liquid medium and resuspend the cell pellet.

f. Seal the tube with parafilm.

g. Incubate in a shaking incubator at 30°C and 240 rpm for 2 h to allow cell recovery.

h. Centrifuge at 3,000 rpm for 4 min.

i. Discard 600 μL of the supernatant and resuspend the cells in the remaining liquid.

j. Spread 200 μL of the cell suspension evenly onto a selective solid medium plate.

k. Incubate the plates inverted at 30°C for two days.

13. Yeast Colony PCR

###### Materials and Equipment:

* Yeast Lysis Buffer
* Neutralization Buffer
* 2×Yeast Colony PCR Mix
* Thermal cycler

###### Procedure:

(1) Add 10 µL of Yeast Lysis Buffer to a PCR tube.

(2) Transfer 0.2-1 mm of yeast monoclone from a sterile suction head into the PCR tube containing Yeast Lysis
Buffer.

(3) Mix thoroughly by gentle pipetting or vortexing, then centrifuge briefly to collect the liquid at the
bottom of the tube.

(4) Incubate at 95°C in a thermal cycler for 5 min to fully release yeast DNA.

(5) Add 10 µL of Neutralization Buffer and mix well. Use this mixture as the DNA template for PCR detection
and followed by PCR amplification according to the following table:

*Reagent**Volume (μL)**Procedure*

*2×Hieff® ® Ultra-Rapid II HotStart PCR Master Mix**5**Pre-denaturation: 95 ℃ for 3 min*

*Primer F (10 μM)**0.4* *Denaturation: 95 ℃ for 15 sec*

*Primer R (10 μM)**0.4**Annealing: Tm for 20 sec*

*DNA template**1**Extension: 72 ℃, 10 sec/kb*

*ddH2O* *3.2**Back to step 1 (30 times)*

*Total**up to 10**Final elongation: 72 ℃ for 2 min
10 ℃ hold*

*10 ℃ hold*

(8) Inoculate the positive transformants in an YPD culture medium containing antibiotics at 220 rpm, 30 ℃.

(9) The positive yeast cells were expanded in culture, prepared again as a sensory state using the method
described above, and transformed by LiAc method

14. Gene Expression Analysis of Recombinant Yeast Strains

###### (1) RNA Extraction from Yeast Cells

Culture yeast strains in YPD medium and harvest cells at the logarithmic growth phase. Extract total RNA
using the S. cerevisiae RNA extraction kit (TRNzol Universal Total RNA Extraction Reagent, DP424) following
the manufacturer's protocol:

a. Resuspend 5×10⁶ - 1×10⁷ cells in 1 mL TRNzol Universal reagent.

b. Incubate the homogenized sample at room temperature for 5 min to ensure complete dissociation of
nucleoprotein complexes.

c. Add 0.2 mL chloroform per 1 mL of TRNzol Universal reagent used. Cap the tube securely and shake
vigorously for 15 sec. Incubate at room temperature for 3 min.

d. Centrifuge at 12,000 rpm for 15 min at 4°C. The mixture will separate into three phases: a lower red
organic phase, an interphase, and a colorless upper aqueous phase containing RNA.

e. Transfer the aqueous phase (approximately 500 µL) to a new microcentrifuge tube.

f. Add an equal volume of isopropanol to the aqueous phase, mix well, and incubate at room temperature for 10
min.

g. Centrifuge at 12,000 rpm for 10 min at 4°C. Discard the supernatant.

h. Wash the RNA pellet with 1 mL of 75% ethanol (prepared with RNase-free water).

i. Centrifuge at 10,000 rpm for 5 min at 4°C. Carefully remove the ethanol without disturbing the pellet.

j. Air-dry the RNA pellet at room temperature for 2-3 min.

k. Resuspend the RNA in 50 µL of RNase-free water, depending on the experimental requirements.

###### (2) Quantitative PCR

###### Materials and Equipment:

* RNA template
* Primers specific to the target gene
* SYBR Green
* qPCR Master Mix
* Nuclease-free water
* 96-well PCR plate
* qPCR machine

###### Procedure:

a. Add the reaction reagents listed below to a sterile nuclease-free tube placed on ice.

*Reagent**Volume (μL)*

*2×RT Master Mix**10*

*Transcript Enzyme**2*

*RNA**≤ 5 µg*

*Primer F (10 μM)**2*

*Primer F (10 μM)**2*

*Total* *up to 20*

b. Mix gently and incubate at 25°C for 10 min.

c. Incubate at 50°C for 30 min.

d. Stop the reaction at 85°C for 5 min and immediately cool the sample on ice.

e. Analyze the amplification plots to determine the Ct (cycle threshold) values for each sample.

f. Use the Ct values to quantify gene expression or DNA copy number.

g. Normalize the target gene expression to a reference gene (ACT1) and use the 2-ΔΔCt method for analysis.

15. β-Carotene Concentration Determination

###### (1) Preparation of β-Carotene Standard Curve

a. Prepare a 50 mg/L β-carotene stock solution by dissolving 1.25 mg of β-carotene standard in 10 mL of
n-hexane and diluting to 25 mL in a volumetric flask.

b. Prepare a dilution series of 2.5, 5, 7.5, 10, 15, 20, and 25 mg/L from the stock solution.

c. Use n-hexane as a blank control.

d. Add 200 µL of each standard (in triplicate) to wells of a microplate.

e. Measure absorbance at 450 nm using a plate reader.

f. Plot the standard curve with absorbance values on the y-axis and β-carotene concentration (mg/L) on the
x-axis.

###### (2) β-Carotene Extraction and Quantification

a. To the 0.2 mL cell suspension, add 200 µL of sterile water, 0.5 mm glass beads, and 1 mL of chloroform.

b. Disrupt cells by vortexing for 3 min, followed by a 1-min ice bath. Repeat this cycle 3-4 times.

c. Centrifuge at 4°C for 1 min and collect the supernatant in a 5 mL centrifuge tube.

d. Repeat the extraction process 3-4 times until the cell pellet is colorless.

e. Evaporate the collected supernatant under a stream of nitrogen.

f. Resuspend the dried extract in 1 mL of hexane.

g. Measure the β-carotene concentration using the prepared standard curve and calculate the yield.

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Results
![](https://static.igem.wiki/teams/5419/biguiyuan/2-2sx.png)
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CONTENT

Overview

Construction of integration plasmids

Integration of target genes into the yeast genome

Qualitative and quantitative analysis of yeast strains

Conclsion

Future plans

References

Overview

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

pX-2-PaCrtE

pX-2-XdCrtE

pX-3-PagCrtB

pXI-2-PaCrtY

pXI-2-XdCrtYB

pXII-5-BtCrtI

pXII-5-XdCrtI

###### 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.

Construction of integration plasmids

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

![](https://static.igem.wiki/teams/5419/res/t2.jpg)

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).

![](https://static.igem.wiki/teams/5419/res/figure-1.jpg)

**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.

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

![](https://static.igem.wiki/teams/5419/res/figure-2.jpg)

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.

![](https://static.igem.wiki/teams/5419/res/table-2-n.jpg)

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).

![](https://static.igem.wiki/teams/5419/res/figure-3.jpg)

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.

![](https://static.igem.wiki/teams/5419/res/figure-4.jpg)

Figure 4 Plate culture results of three yeast strains.

Qualitative and quantitative analysis of 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%, indicating that the qPCR system has good amplification efficiency. These results indicated that our qPCR system has great reproducibility and accuracy (Figure 5).

![](https://static.igem.wiki/teams/5419/res/figure-5.jpg)

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.

![](https://static.igem.wiki/teams/5419/res/table-3-n.png)

Table 4 Gene expression analysis of yeast strain Group 2.

![](https://static.igem.wiki/teams/5419/res/table-4-n.png)

Table 5 Gene expression analysis of yeast strain Group 3.

![](https://static.igem.wiki/teams/5419/res/table-5-n.png)

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).

![](https://static.igem.wiki/teams/5419/res/figure-6.jpg)

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].

Conclsion

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.

Future plans

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.

References

[1] Grune T, Lietz G, Palou A, Ross AC, Stahl W, Tang G, Thurnham D, Yin SA, Biesalski HK. β-Carotene Is an Important Vitamin A Source for Humans. J Nutr. 2010 Dec;140(12):2268S-2285S.

[2] Mata-Gómez LC, Montañez JC, Méndez-Zavala A, Aguilar CN. Biotechnological production of carotenoids by yeasts: an overview. Microb Cell Fact. 2014 Jan 21;13:12.

[3] Nielsen J, Keasling JD. Engineering Cellular Metabolism. Cell. 2016 Mar 10;16

[4] Guo Q, Peng QQ, Li YW, Yan F, Wang YT, Ye C, Shi TQ. Advances in the metabolic engineering of *Saccharomyces cerevisiae* and Yarrowia lipolytica for the production of β-carotene. Crit Rev Biotechnol. 2024 May;44(3):337-351.

[5] 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.

[6] Zhu FY, Lu L, Fu S, et al. Targeted engineering and scale up of lycopene overproduction in Escherichia coli. Proc Biochem, 2015, 50(3): 341–346.

[7] 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.

[8] 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.

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

[10] Tokuhiro K, Muramatsu M, Ohto C, et al. Overproduction of geranylgeraniol by metabolically engineered *Saccharomyces cerevisiae*. Appl Environ Microbiol, 2009, 75

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