Overview

Our project aimed to increase vitamin B2 production in the baker’s yeast (Saccharomyces cerevisiae) to produce vitamin B2-enriched food. We overexpressed ADE4, RIB1, and RIB7 (all combinations) in S. cerevisiae S288C. By doing so, we found that the more genes got overexpressed, the more vitamin B2 was produced. We successfully created engineered yeast that produced significantly more vitamin B2 than the WT. We have achieved the following contributions.

 

Introduced a part collection for the overexpression of vitamin B2 in S. cerevisiae

This year, we introduced 13 new parts, all contributed to the overproduction of vitamin B2 in S. cerevisiae, and made up a part collection. BBa_K4883000, BBa_K4883001, BBa_K4883002, BBa_K4883003 BBa_K4883005, BBa_K4883007, BBa_K4883009 are combinations of ADE4, RIB1, and RIB7, and BBa_K4883004, BBa_K4883006, BBa_K4883008, BBa_K4883010, BBa_K4883011, BBa_K4883012, BBa_K4883013 are expression cassettes of the combinations. In the future, the iGEM community can use our part collection for vitamin B2 production. Future teams can also expand our part collection by testing and stacking more vitamin B2-related genes. See Parts for more details.

Name	Type	Description	Length
BBa_K4883000	Basic	ADE4	1530 bp
BBa_K4883001	Basic	RIB1	1035 bp
BBa_K4883002	Basic	RIB7	732 bp
BBa_K4883003	Composite	RIB1-PTV-ADE4	2631 bp
BBa_K4883004	Composite	Ptef1-RIB1-PTV-ADE4-Tcyc1	3303 bp
BBa_K4883005	Composite	RIB1-ERBV-1-RIB7	1827 bp
BBa_K4883006	Composite	Ptef1-RIB1-ERBV-1-RIB7-Tcyc1	2499 bp
BBa_K4883007	Composite	RIB7-PTV-ADE4	2328 bp
BBa_K4883008	Composite	Ptef1-RIB7-PTV-ADE4-Tcyc1	3000 bp
BBa_K4883009	Composite	RIB1-ERBV-1-RIB7-PTV-ADE4	3423 bp
BBa_K4883010	Composite	Ptef1-RIB1-ERBV-1-RIB7-PTV-ADE4-Tcyc1	4095 bp
BBa_K4883011	Composite	Ptef1-ADE4-Tcyc1	2202 bp
BBa_K4883012	Composite	Ptef1-RIB1-Tcyc1	1707 bp
BBa_K4883013	Composite	Ptef1-RIB7-Tcyc1	1404 bp

 

Built a vitamin B2 overproduction strain of S. cerevisiae as future chassis

To find the best combination of genes in terms of vitamin B2-overexpression, we tested all the combinations of ADE4, RIB1, and RIB7. The results showed that the more genes got overexpressed, the more vitamin B2 was produced. The strain co-overexpressing RIB1, RIB7, and ADE4 increased vitamin B2 production by 193% in liquid YPD media, and by 91% in steamed buns (p < 0.0001), which was the highest among strains. The future teams can use our engineered yeast as chassis for more improvements. See Engineering Success for more details.

 

 

Built an overexpression system for S. cerevisiae

To overexpress ADE4, RIB1, and RIB7, we constricted recombinant vectors, including pCEV-G4-ADE4, pCEV-G4-RIB1, pCEV-G4-RIB7, pCEV-G4-RIB1-ADE4, pCEV-G4-RIB7-ADE4, pCEV-G4-RIB1-RIB7, and pCEV-G4-RIB1-RIB7-ADE4. We proved that the overexpression system based on pCEV-G4-Km is potent for S. cerevisiae. We confirmed that multigene co-overexpression can be achieved by dividing genes by 2A peptides. Future teams can utilize the overexpression system and similar vector designs for their projects. See Engineering Success for more details.

 

 

PCR troubleshooting

When we tried to use pCEV-G4-RIB7-PTV-ADE4-Km as the template to amplify RIB7-PTV-ADE4 by PCR, gel electrophoresis showed no bands. We had to adjust the PCR program to get the fragment we needed. After several failed PCRs, we did some research and tried touchdown PCR from 68°C to 52°C. Although the gel electrophoresis results were still smeared, bands at around 2400 bp were identifiable. The fragments were successfully collected through proper gel extraction, enabling the construction of pCEV-G4-RIB1-ERBV-1-RIB7-PTV-ADE4-Km. Our experience showed how to troubleshoot PCR, and future teams can adopt a similar strategy. See Engineering Success for more details.

 

Explained China’s regulations on GM microorganisms as food ingredients

We did literature research to find the regulations and the approval process for engineered organisms and new food raw materials in China. To conclude, food made with GMOs has to go through two rounds of assessment. The safety of the GMOs will be evaluated first by the State Commission for the Safety of Agricultural Genetically Modified Organisms and the Office for the Safety Management of Agricultural Genetically Modified Organisms. After approval, the Chinese National Center for Food Safety Risk Assessment will evaluate the safety of GMOS as food ingredients. We clarified the approval process for GMOs as food ingredients in China for future teams. See Human Practices for more details.