| Acetic acid is one of the main inhibitors in lignocellulosic hydrolysate,and is also one of the by-products of ethanol fermentation.High concentration acetic acid in the fermentation medium represses cell growth,metabolism and fermentation efficiency of Saccharomyces cerevisiae,which is widely used for cellulosic ethanol production.Meanwhile,acetic acid can induce ROS accumulation in S.cerevisiae,which results in oxidative damage of intracellular protein,DNA,phospholipids,and cell membranes.Therefore,improvement of acetic acid tolerance is one of the key targets for improving cellulosic ethanol production efficiency of S.cerevisiae.Currently,various methods have been employed to improve acetic acid tolerance of S.cerevisiae,one important finding in our previous study is that supplementation of zinc sulfate in the fermentation medium improved cell growth and ethanol fermentation performance of S.cerevisiae under acetic acid stress condition.We therefore proposed that zinc status exerts important effects on acetic acid stress tolerance.However,the molecular mechanisms is still unclear.To explore the underlying mechanism of zinc sulfate protection against acetic acid stress,transcriptomic and proteomic analysis were performed.Yeast cell samples grown with or without 0.03 g/L ZnSO4.7H2 O addition were collected at the log phase in the presence of 10 g/L acetic acid.Transcription levels of 212 genes were significantly changed with zinc sulfate addition,which contained 50 upregulated genes and 162 downregulated genes.Zinc sulfate addition changed expression levels of in a total of 116 proteins,which contained 54 upregulated proteins and 62 downregulated proteins.The changed genes and proteins are related to carbon metabolism,amino acid biosynthesis,energy metabolism,vitamin biosynthesis and stress responses.In a total,28 genes showed differential expression both in transcriptomic and proteomic data,indicating that zinc sulfate affects gene expression at posttranscriptional and posttranslational levels.We propose that zinc sulfate addition results in global changes in gene transcription and protein synthesis under acetic acid stress,leading to enhanced antioxidant efficiency and improved energy production,which benefits cell growth and metabolism.Almost no overlap was found in the transcriptomic and proteomic data,indicating that zinc status exerts effect on cellular metabolisms in post-transcription and post-translational levels.Based on the transcriptomic and proteomic analysis,four genes,namely PET18,SAM2,RNY1 and YIL002W-A were selected for further studies.The results showed that overexpression of RNY1 and YIL002W-A endowed S.cerevisiae improved growth and fermentation performance in the presence of acetic acid.As the function of gene YIL002W-A is still not clear,this gene was further studied.GFP-encoding gene was inserted in the chromosomal location of this gene,and fluorescence analysis revealed that the expression level of gene YIL002W-A was highly upregulated in response to acetic acid,which indicates that YIL002W-A is an acetic acid responsive gene.Meanwhile,gene YIL002W-A was proved to be regulated by transcription factors Yap1 p and Stb5 p,which was deduced from the results that the expression level of YIL002W-A was significantly reduced when the Yap1 p or Stb5 p binding sites in YIL002W-A were mutated.Comparative transcriptomic analysis was performed using YIL002W-A deletion mutant and the wild type strain S288 c,and the results revealed that genes involved in cell division were down-regulated by YIL002W-A deletion.We propose that overexpression of YIL002W-A enhanced cell division,leading to improved cell growth and stress tolerance.The results in this study provide basis for further exploration of the molecular mechanisms underlying improved stress tolerance by zinc status,as well as development stress tolerant yeast strains to improve cellulosic ethanol production. |
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