Promoters for high-efficiency expression and proinsulin secretion in Saccharomyces cerevisiae

INTRODUCTION. The lack of systematic research comparing promoter activity in Saccharomyces cerevisiae limits approaches to improving the efficiency of recombinant human proinsulin biosynthesis. This study compared the activity of constitutive promoters under standardised technological conditions to identify the most productive regulatory elements for application in industrial strains. AIM. This study aimed to compare the influence of TEF1 , ADH2 , ALD4 , TDH3 ( GPD ), and TPI1 promoters on recombinant proinsulin expression in Saccharomyces cerevisiae and rank their effectiveness for selecting a highly productive promoter. METERIALS AND METHODS. S. cerevisiae YS3 diploid strain obtained by haploid cells fusion was used for proinsulin expression. The vectors based on the pRS425 plasmid consisted of synthetic promoters ( TPI1, TEF1, ADH2, ALD4, TDH3 ) and signal sequence. Plasmids were electroporated in the cell. Comparative expression analysis was performed by cultivating S. cerevisiae YS3 in flasks and bioreactors using YPD media. Proinsulin and ethanol concentration was measured using spectrophotometry, HPLC, and gas chromatography. The expression was confirmed using HPLC-MS. Statistical analysis included variance and correlation analysis. RESULTS. Strains S. cerevisiae YS3/pF1145 ( TPI1 ), YS3/pF1157 ( TEF1 ), YS3/pF1199 ( ADH2 ), YS3/pF1200 ( ALD4 ), and YS3/pF1201 ( TDH3 ) were obtained, then expressions of recombinant protein were compared. The strain YS3/pF1201 that had proinsulin expression controlled by the promoter TDH3 showed a maximum productivity of 15.51±0.57 mg/L. When fermented in laboratory bioreactors, the strain YS3/pF1201 achieved a productivity of 139.17 mg/L and a biomass of 154.5 mg/L after 72 hours of cultivation. CONCLUSIONS. Analysis of experimental data ranked the promoters for proinsulin expression in S. cerevisiae cells by their effectiveness: TDH3 ≈ ALD4 > ADH2 > TEF1 > TPI1 . During the fermentation, TDH3 promoter showed significantly higher productivity, which was 6.1 times more than in the classical strain with the TPI1 promoter for proinsulin expression.