Genome-wide analysis reveals Hsf1 maintains high transcript abundance of target genes controlled by strong constitutive promoter in Saccharomyces cerevisiae

Abstract

AbstractBackgroundIn synthetic biology, the strength of promoter elements is the basis for precise regulation of target gene transcription levels, which in turn increases the yield of the target product. However, the results of many researches proved that excessive transcription levels of target genes actually reduced the yield of the target product. This phenomenon has been found in studies using different microorganisms as chassis cells, thus, it becomes a bottleneck problem to improve the yield of the target product.ResultsIn this study, promotersPGK1pandTDH3pwith different strengths were used to regulate the transcription level of alcohol acetyl transferase encoding geneATF1. The results demonstrated that the strong promoterTDH3pdecreased the production of ethyl acetate. The results of Real-time PCR proved that the transcription level ofATF1decreased rapidly under the control ofTDH3p, and the unfolded protein reaction was activated, which may be the reason for the abnormal production caused by the strong promoter. RNA-sequencing analysis showed that the overexpression of differential geneHSP30increased the transcriptional abundance ofATF1gene and production of ethyl acetate. Interestingly, deletion of the heat shock protein family (e.g., Hsp26, Hsp78, Hsp82) decreased the production of ethyl acetate, suggesting that the Hsp family was also involved in the regulation ofATF1gene transcription. Furthermore, the results proved that the Hsf1, an upstream transcription factor of Hsps, had a positive effect on alleviating the unfolded protein response and that overexpression of Hsf1 reprogramed the pattern ofATF1gene transcript levels. The combined overexpression of Hsf1 and Hsps further increased the production of ethyl acetate. In addition, kinase Rim15 may be involved in this regulatory pathway. Finally, the regulation effect of Hsf1 on recombinant strains constructed by other promoters was verified, which confirmed the universality of the strategy.ConclusionsOur results elucidated the mechanism by which Rim15–Hsf1–Hsps pathway reconstructed the repression of high transcription level stress and increased the production of target products, thereby providing new insights and application strategies for the construction of recombinant strains in synthetic biology.