Investigation of Ty1 sites for multi-copy integration of a Brazzein expression system using CRISPR/CAS9 in Saccharomyces cerevisiae

Obesity and its comorbidities have become a global concern as a result of widespread climbing obesity rates. Sugar, particularly sucrose is thought to play a dominant causative role in the observed trends, and many believe reducing sucrose intake to be a key solution. A new generation of protein-based sweeteners have been isolated from fruits of plants native to tropical climates. One of these sweet-tasting proteins, brazzein, is a promising alternative sweetener. The aim of this project is to utilize recent advances in recombinant DNA technology and yeast engineering to construct a Saccharomyces cerevisiae expression system capable of producing high titres of the brazzein protein.
Results: A gene cassette was constructed with the PTEF1 promoter, an ORF encoding an αMF secretory signal fused to the N-terminus of brazzein and the TCYC1 terminator.
Brazzein expression cassettes were integrated at varying copy number into Ty1-LTRs in Saccharomyces cerevisiae via a CRISPR/Cas9 mediated approach. Quantitative PCR demonstrated that one strain (DVT20) was estimated to contain approximately 6 copies of the brazzein cassette. Tricine-SDS PAGE analysis showed a protein whose levels increased with copy number but was larger than the expected size of brazzein. The protein may be brazzein with an un-cleaved αMF secretory peptide.
Conclusion: CRISPR/Cas9 was used to successfully used to integrate multiple copies of brazzein at Ty1 LTRs.