The part we contributed to: K2809002
Since we want our proteins being transported to the extracellular space, a secretion signal was needed. Therefore, we have chosen the alpha-factor mating secretion sequence that has been used by the Broad Run School iGEM Team 2015. They express this secretion signal in the S.cerevisiae to secrete a functional amylase, which was similar to our experimental design.
However, as the project progressed, we speculated that the secretion rate of proteins did not meet expectations, which may be a reason that our the results of the function assay were not ideal. The literature published by JH Bae and his team introduced a fusion partner that can enhance the secretion rate, and this inspired us. According to this literature, the scientist inserted a modified HL peptide right after the MF alpha-factor to promote secretion. The results indicated that the modified HL 28 peptide can efficiently increase the secretion rate when working with the MF alpha pre-pro peptide. This fusion partner also includes an affinity chromatography tag, which will convince the future purification step of the proteins. We may implement this change in our subsequent works.
Amino acid sequence of HL28: EDEDGDDEYATEETLSHHHHHHGDDDDK
Figure. HL28 usage example
In our project, we designed to enable the yeast to secrete the protein which originally existed only inside the yeast and verified the feasibility of this design through experiments. In this section, we turn our experience into instruction for iGEM teams that need to use this strategy in the future.
The first key point is to choose a suitable promoter based on your own project. Here are some conditions that need to be concerned when selecting.
1. The gene sequence compatibility serves as a crucial factor regarding the success of gene expression as well as the concentration of exogenous protein the yeast produces. In our design, both the promoter and secretory sequence originated from yeast, that is, MFalpha factor, TPS1, and PGK1 promoter are of outstanding function in their original strain. The proximal species genetic crossing can optimize the success rate.
2. Another key point when considering assay success and obviousness is the efficiency of the gene. The promoters and secretory factors we utilized are contributory to high-level protein secretion. TPS1, PGK1 promoter has great expression under certain circumstances, MF alpha factor enhances the vesicles transportation within yeast cells. Thus, by combining PGK1, TPS1 with MFalpha respectively we can build a mighty strain to produce enzymes exogenously.
3. We consider another novel strategy to make the final yeast strain achieve final functions by complementing the function pathway each enzyme realizes. Chit42 produces chitooligosaccharide by sacrificing yeast to lysate cell walls and exposes chitin to endochitinase. TPS1 promoter has ethanol inducible nature. When ethanol level increases to 3% (v/v), and peaks at 8% (v/v), it initiates the expression of the gene it regulates. Thus, the TPS1 promoter is chosen to ensure the COS can be produced ultimately. Besides, the other two enzymes constitutively express enzymes throughout yeast life, and this complementation of two promoters and the consideration of producing product induced under different circumstances can achieve a series of secretion purposes at different time points. Consider the expression amount of the protein you need.
ADE2 is a non-essential gene of Saccharomyces cerevisiae. It encodes AIR-carboxylase which catalyzes the sixth step of the purine biosynthetic pathway. And the null mutant displays adenine auxotrophy forms red colonies and is sensitive to purine analogs. We flank two ADE2 complementary sites at the end of our inserts of interest. Due to the homologous recombination of SC yeast, they can automatically delete this marker and positive cloned yeast having inserts incorporated will grow into a red colony on the plate. Such a way of screening yeast by using the ADE2 site is in analogy with the blue-white screening strategy of bacteria, however, using different genes on another creature.
- Bae, Jung-Hoon et al. “A novel fusion partner for enhanced secretion of recombinant proteins in Saccharomyces cerevisiae.” Applied microbiology and biotechnology vol. 100,24 (2016): 10453-10461. doi:10.1007/s00253-016-7722-2
- Qian Li, Xing-Qing Zhao, Alan K chang, etc. (2012) ethanol-induced yeast flocculation directed by the promoter of TPS1 encoding trehalose-6-phosphate synthase 1 for efficient ethanol production.MBE 14: 1-8
- Wang, Z., Bai, X., He, X., & Zhang, B. (2014). Secretion expression of SOD1 and its overlapping function with GSH in brewing yeast strain for better flavor and anti-aging ability. Journal of Industrial Microbiology & Biotechnology, 41(9), 1415-24. doi:http://dx.doi.org.libezproxy.um.edu.mo/10.1007/s10295-014-1481-9