Construction of a 10rolGLP-1-expressing glucose-lowing Saccharomyces cerevisiae by CRISPR/Cas9 technique

Abstract:

To develop a novel glucose-lowering biomedicine with potential benefits in the treatment of type 2 diabetes, we used the 10rolGLP-1 gene previously constructed in our laboratory and the CRISPR/Cas9 genome editing technique to create an engineered Saccharomyces cerevisiae strain. The gRNA expression vector pYES2-gRNA, the donor vector pNK1-L-PGK-10rolGLP-1-R and the Cas9 expression vector pGADT7-Cas9 were constructed and co-transformed into S. cerevisiae INVSc1 strain, with the PGK-10rolGLP-1 expressing unit specifically knocked in through homologous recombination. Finally, an S. cerevisiae strain highly expressing the 10rolGLP-1 with glucose-lowering activity was obtained. SDS-PAGE and Western blotting results confirmed that two recombinant strains of S. cerevisiae stably expressed the 10rolGLP-1 and exhibited the desired glucose-lowering property when orally administered to mice. Hypoglycemic experiment results showed that the recombinant hypoglycemic S. cerevisiae strain offered a highly hypoglycemic effect on the diabetic mouse model, and the blood glucose decline was adagio, which can avoid the dangerous consequences caused by rapid decline in blood glucose. Moreover, the body weight and other symptoms such as polyuria also improved significantly, indicating that the orally hypoglycemic S. cerevisiae strain that we constructed may develop into an effective, safe, economic, practical and ideal functional food for type 2 diabetes mellitus treatment.