GSE160256
Base editing of general transcription factor Spt15 to enhance yeast stress tolerance
We aimed to explore the application of the Target-AID base editor in genomic in situ protein engineering by generating nonsynonymous mutations. A general transcription factor Spt15 (TATA-box binding protein) gene of Saccharomyces cerevisiae was selected as a target. Based on computational and experimental scanning mutagenesis of the Spt15 gene as well as flask-fermentation screening, three stress-tolerant Spt15 mutant strains (A140G, P169A and R238K) and two stress-sensitive Spt15 mutant strains (S118L and L214V) were obtained. To validate the regulatory mechanisms underlying these different Spt15 mutants, genome-wide transcriptome analysis by RNA sequencing was carried out to quantify global transcription changes in the Spt15 mutant strains compared to the wild type strain at the same culture conditions including the unstressed normal condition as well as hyperosmotic and thermal stress conditions. Results uncover the impacts of the Spt15 point mutations on global transcriptional regulation in response to hyperosmotic and thermal stresses, and provide insight into the applicability of the Target-AID base editor in genomic in situ protein engineering to alter yeast stress tolerance. Overall design: Six strains including three stress tolerant mutants Spt15-A140G, Spt15-P169A and Spt15-R238K, two stress sensitive mutants Spt15-S118L and Spt15-L214V, as well as the wild type strain BY4741 were subjected to transcriptome analysis using RNA sequencing on Illumina platform using 150-bp paired-end sequencing. Flask fermentation of these six strains were carried out in biological duplicates at hyperosmotic or thermal stress conditions as well as the unstressed normal condition, respectively. We identified differential expressions in comparisons of the SPT15 mutant strains versus the wild type strain BY4741 at each culture condition.