Part:BBa_K4883012

Ptef1-RIB1-Tcyc1

This is a complete expression cassette consisting of a strong promoter Ptef1, a RIB1 gene, and a CYC1 terminator.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1702
  Illegal BamHI site found at 836
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 856
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 176
  

Usage and Biology

To mitigate vitamin B2 deficiency, we planned to make vitamin B2-enriched bread with engineered yeast, Saccharomyces cerevisiae. Overexpression of RIB1 (BBa_K4883001) should lead to the overproduction of vitamin B2 in S. cerevisiae. Ptef1 (BBa_K2407300) has been proven one of the strongest promoters of S. cerevisiae, so we used it for overexpression (Partow et al., 2010). A CYC1 terminator was also included to form the complete expression cassette.

Characterization

2023 Hangzhou-SDG Team characterized this part with vitamin B2 production

Vitamin B2 Production Tests in Liquid YPD Media

The WT S. cerevisiae S288C and the strain overexpressing RIB1 were inoculated into YPD media, and cultured at 30 ℃, 180 rpm. The riboflavin concentrations in the supernatants were measured using high-performance liquid chromatography (HPLC).

Figure 1. Vitamin B2 contents in liquid YPD media containing engineered S. cerevisiae strains for 72h.

Results showed that overexpression of RIB1 increased vitamin B2 production by 46% after 72h in YPD.

Vitamin B2 Production Tests in Steamed Buns To determine whether our engineered yeasts can help to make vitamin-B2-enriched food, we made steamed buns using our yeasts in the lab. The riboflavin concentrations in the buns were measured using HPLC.

Figure 2. Vitamin B2 contents in steamed buns leavened by engineered S. cerevisiae strains.

Results showed that overexpression of RIB1 increased vitamin B2 production by 17% in steamed buns (p > 0.05).

References

Partow, S.; Siewers, V.; Bjørn, S.; Nielsen, J.; Maury, J. Characterization of Different Promoters for Designing a New Expression Vector in Saccharomyces Cerevisiae. Yeast 2010, 27 (11), 955–964. DOI:10.1002/yea.1806.