Studies Of Responses Of Yeast To Inhibitors During Lignocellulosic Ethanol Production Using Technologies For Systems Biology

The end product ethanol and inhibitors produced during lignocellulose pretreatment exhibit severe toxcities to Saccharomyces cerevisiae, which limits the production of ethanol during fermentation. In order to break through it, it is the efficient approach to increase yeast tolerance to these inhibitory substances. In this study, the responses of yeast to inhibitors and the differences of strains with different tolerance were investigated by using technologies for systems biology.Genome-wide transcriptional responses to furfural and acetic acid were investigated in S. cerevisiae using microarray analysis. Furfural downregulated the expression of genes related to metabolism of folic acid, spermidine and spermine, while it upregulated the expression of genes related to oxidative stress response. Acetic acid downregulated the expression of genes encoding mitochondrial ribosomal proteins specifically and upregulated the expression of genes related to metabolism of several amino acids.The transcriptomic differences of the parent strain and the tolerant strains to furfural and acetic acid, obtained by adaptation, were studied using genome-wide microarray. In the tolerant strain to furfural, the transcripts of genes involved in cell wall synthesis and signaling related to cell wall were at low level, which might lead to thinner cell wall; HMG1, inducing the formation of karmellae, was at high level in the tolerant strain to furfural. These differences might enhance furfural conversion and improve tolerance to furfural. In the tolerant strain to acetic acid, the transcripts of genes were at low level, which are related to mitochondria and autophagy, and function in apoptosis. In addition, the transcripts of genes related to pheromone response signaling in a type haploid strain were at high level.Transcriptomic, metabolomic and proteomic differences of homogenous diploid and haploid yeast strains with different tolerance to ethanol in the presence of ethanol with varied concentrations were studied by employing microarray, GC-TOF/MS and two-dimensional electrophoresis. At transcriptome level, ethanol induced the expression of genes related to lipid synthesis regulation, including chain length of fatty acid, sterol uptake, and inositol synthesis, indicating that lipid might play important roles in cellular responses to ethanol. Ethanol repressed the transcription of genes involved in synthesis of amino acid, purine synthesis and metabolism of cofactors, indicating inhibition of ethanol on cell growth and viability. The transcripts at low level in haploid strains were mainly related to electron transport, transport and metabolism of metal, and cell wall. Cluster analysis of metabolic profiling showed that diploid strain and a type haploid strain were similar, and that the divergence among the strains with ethanol stress was smaller in the presence of ethanol. Membrane components and the intermediate metabolites of N metabolism and C metabolism were different in diploid and haploid strains in the presence of ethanol. At proteome level, the levels of proteins related to ethanol metabolism in diploid were higher than that in haploids under the absence of ethanol, suggesting that diploid have higher glycolytic activity than haploid. Some proteins involved in stress responses were downregulated in diploid, but they were upregulated in haploids under ethanol stresses, indicating the different mechanisms of ethanol tolerance in diploid and haploid strains.