Chemogenomic Response Mechanism Of Saccharomyces Cerevisiae To Cell Wall Antagonists

Saccharomyces cerevisiae is an important industrial biological host.It is also a representative model organism for studying eukaryotic cell gene function and cell metabolism.Cell wall is one of the important targets of antifungal drugs.The study of yeast cell wall regulation provides a reference for other pathogenic fungi.In this study,Chemogenomic was used to determine the sensitive and resistant strains of cell wall antagonists CFW and SDS,and the global regulatory networks responding to cell wall antagonists were analyzed by bioinformatics method.The chitin content in the cell wall of the mutant strains was determined,and the activation of CWI pathway was studied.The specific research contents and results are as follows:(1)Establishment of high-throughput screening systemCell wall related genes FKS1,BCK1,CHS3,CRZ1 were knocked out using Cre/lox P and CRISPR/cas9 gene editing system.Then a high-throughput screening method for 96 well inoculated needles was established.After tolerance test,the concentration for high-throughput screening of CFW sensitive and resistant strains,and SDS sensitive and resistant strains were selected.(2)Genome wide screening and cell process analysis of CFW sensitive or resistant strainsBased on the yeast knock-out library,485 strains with enhanced sensitivity to CFW and 52 strains with enhanced resistance to CFW were selected.As expected,in addition to the cell wall related genes,a number of new genes have been identified.Cellular response processes were mainly involved in cell wall component synthesis and MAPK signaling pathway,RNA degradation or t RNA thiolation or ribosome composition of protein synthesis process,RIM101 pathway and endocytosis,autophagy,vacuole acidification,cysteine and methionine metabolism pathway.The finding of genes involved in cysteine and methionine metabolism may indicate a new potential toxic mechanism in eukaryotic cells in response to calcofluor white.In addition,a group of new transcripts or putative transcription factors Aca1,Arg81,Flo8,Stp1,Plm2 and Ace2 were also screened out.In addition,it was found for the first time that cysteine could relieve the pressure of cell wall antagonists CFW and CR.(3)Genome wide screening and cell process analysis of SDS sensitive or resistant strains730 Sensitive mutants and 77 resistant mutants were identified through screening of SDS sensitive and resistant strains.Among the sensitive mutants,mitochondrial gene expression;the mitogen-activated protein kinase signaling pathway;the metabolic pathways were found to be enriched.Additionally,a set of transcription factors related to SDS responses,such as Yap1,Pdr1,Swi4 and Crz1,were identified.Then the correlation between SDS,CFW and CR was analyzed.Among the resistant mutants,disruption of ribosome biogenesis and translation alleviated SDS-induced cytotoxicity.This was also confirmed by the Pichia pastoris ribosomal protein deletion library test.The strong correlation with the SDS-resistant phenotype emphasized the importance of SDS resistance-related gene set for the study of ribosome biogenesis and translation.(4)Analysis of cell wall components and cell wall integrity pathway activation analysis of interfering agent-sensitive strainsThe established high-efficiency method for determination of chitin contents showed the correlation between the absolute content of chitin determined by classical enzymolysis.The relative content of chitin in 327 cell wall antagonists SDS,CFW,CR cross-sensitive strains was determined,and it was found that the cell wall chitin content of 77 gene deletion mutant strains increased by more than 60%,of which 24 strains were involved in cell wall synthesis.PCWP1-EGFP and PSED1-EGFP sensors that can respond to the activation and deletion of the CWI pathway were established.48 Deletion mutant strains with increased chitin content were tested.Among them,the fluorescence values of both sensors of 10 deletion mutant strains were increased by more than 50%,involving the central metabolic gene Pro2 and protein metabolism genes MOT2,SHP1,vesicle transport genes GET2,SPC72,CTF4,transcription related gene DEF1,unknown function genes YLR358 C,YJR018W,FYV6.The deletion of these genes has been discovered for the first time to activate the CWI pathway.In this study,Chemogenomics was used to analyze the global regulatory network of response to cell wall antagonists,which broadened the understanding of response mechanism of cell wall antagonists and regulation of cell wall integrity,and provided good basic data for the study of fungal cell wall synthesis mechanism.