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 successfully created engineered yeast that produced significantly more vitamin B2 than the WT, and the test results showed that the more genes got overexpressed, the more vitamin B2 was produced. Our parts are combinations of ADE4, RIB1, and RIB7 and expression cassettes of them.

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

Part Collection

All 13 new parts contribute to the overproduction of vitamin B2 in S. cerevisiae. Therefore, they make up a part collection.

To study the roles of ADE4, RIB1, and RIB7, the interactions between genes, and to find the best combination of genes in terms of vitamin B2-overexpression, we tested all the combinations of them. 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.

Figure 1. A. Vitamin B2 contents in liquid YPD media containing engineered S. cerevisiae strains for 72h. B. Vitamin B2 contents in steamed buns leavened by engineered S. cerevisiae strains. *: p < 0.01; **: p < 0.001.

The results from the vitamin B2 production tests confirmed that all genes contributed to the production of vitamin B2. And in general, the more genes got overexpressed, the more vitamin B2 was produced. Results showed that 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.

In the future, the iGEM community can expand our part collection by testing and stacking more vitamin B2-related genes.