Identification of acetic acid sensitive strains through biosensor-based screening of a Saccharomyces cerevisiae CRISPRi library

Abstract

Background Acetic acid tolerance is crucial for the development of robust cell factories for conversion of lignocellulosic hydrolysates that typically contain high levels of acetic acid. Screening mutants for growth in medium with acetic acid is an attractive way to identify sensitive variants and can provide novel insights into the complex mechanisms regulating the acetic acid stress response. Results An acetic acid biosensor based on the Saccharomyces cerevisiae transcription factor Haa1, was used to screen a CRISPRi yeast strain library where dCas9-Mxi was set to individually repress each essential or respiratory growth essential gene. Fluorescence-activated cell sorting led to the enrichment of a population of cells with higher acetic acid retention. These cells with higher biosensor signal were demonstrated to be more sensitive to acetic acid. Biosensor-based screening of the CRISPRi library strains enabled identification of strains with increased acetic acid sensitivity: strains with gRNAs targeting TIF34, MSN5, PAP1, COX10 or TRA1. Conclusions This study demonstrated that biosensors are valuable tools for screening and monitoring acetic acid tolerance in yeast. Fine-tuning the expression of essential genes can lead to altered acetic acid tolerance.

Saccharomyces cerevisiae strains performing similarly during fermentation of lignocellulosic hydrolysates show pronounced differences in transcriptional stress responses

“…Almost all of the 10 most differently expressed genes of the LBCM strains ( Fig. 6A ) have been previously reported to be important for tolerance to hydrolysate or inhibitors therein [ BNA6 ( 60 ), YHB1 ( 61 ), COX10 ( 62 – 65 ), SCW4 ( 2 , 65 ), TOP1 ( 2 , 66 ), UPS3 ( 2 , 65 ), SOP4 ( 2 , 65 , 66 ), SFP1 ( 64 ), SSM4 ( 66 ), STB4 ( 2 , 65 ), SER33 ( 65 ), TMT1 ( 65 ), FAT3 ( 65 ), IMA1 ( 65 ), MAL13 ( 2 , 65 ), and MAL11 ( 67 )] or osmotic and oxidative stress tolerance [ DOG2 ( 68 )]. Still, their mechanistic role in this context is often still to be elucidated.…”

Biosensor-Assisted Engineering for Diverse Microbial Cellular Physiologies

“…Mormino et al focused on characterizing genes related to acetate tolerance in S. cerevisiae. 45 They constructed a biosensor that included a synthetic transcription factor, BM3R1-Haa1-mTurquoise, which specifically binds to acetate and induces the expression of mCherry. The biosensor was integrated into a genome-wide CRISPRi library.…”