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Involvement of ergosterol contents in the tolerance to vanillin in Saccharomyces cerevisiae

ABSTRACT: The vanillin tolerance Saccharomyces cerevisiae was screened and compared intracellular ergosterol levels with several laboratory yeast strains, to study potential relationship between ergosterol contents and vanillin tolerance. S. cerevisiae NBRC1950 was selected as a vanillin tolerant strain. Its ergosterol contents were higher than those of laboratory strains. The results of DNA microarray and quantitative RT-PCR analysis showed that 5 genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5 and ERG7) were up-regulated in NBRC 1950 compared with strain X2180, suggested that high expressions of genes involved in ergosterol biosynthesis may cause for the high ergosterol content in strain NBRC 1950. S. cerevisiae HX strain, which was a high ergosterol content strain derived from X2180, became more tolerant to vanillin compared with the parental strain. It is suggested that high ergosterol contents may be in part responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin.

ORGANISM(S): Schizosaccharomyces pombe Saccharomyces cerevisiae

PROVIDER: GSE13755 | GEO | 2009/06/29

SECONDARY ACCESSION(S): PRJNA109415

REPOSITORIES: GEO

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Project description:The vanillin tolerance Saccharomyces cerevisiae was screened and compared intracellular ergosterol levels with several laboratory yeast strains, to study potential relationship between ergosterol contents and vanillin tolerance. S. cerevisiae NBRC1950 was selected as a vanillin tolerant strain. Its ergosterol contents were higher than those of laboratory strains. The results of DNA microarray and quantitative RT-PCR analysis showed that 5 genes involved in ergosterol biosynthesis (ERG28, HMG1, MCR1, ERG5 and ERG7) were up-regulated in NBRC 1950 compared with strain X2180, suggested that high expressions of genes involved in ergosterol biosynthesis may cause for the high ergosterol content in strain NBRC 1950. S. cerevisiae HX strain, which was a high ergosterol content strain derived from X2180, became more tolerant to vanillin compared with the parental strain. It is suggested that high ergosterol contents may be in part responsible for vanillin tolerance. These findings provide a biotechnological basis for the molecular engineering of S. cerevisiae with increased tolerance to vanillin. Experiment Overall Design: Total RNA was extracted from cells in YPD media with shakin by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix userâs manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome 2.0 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in duplicate independently. Statistical analysis after data acquisition and normalization of expression data were performed using GeneSpring ver.7.3.1 (Agilent Technologies, Palo Alto, CA, USA) based on the gene expression data from two independent experiments. After data transformation to GeneSpring, per-chip normalization to the 50th percentile was performed, and per-gene normalization to the specific samples (X2180 samples) was applied to the per-chip normalized data. Quality control was performed based on experimental confidence levels (each condition in which all samples were present or marginal) and on statistical confidence levels (condition in which P-value of T-test comparisons between X2180 and NBRC 1950 was less than 0.05).

Project description:One of the key issues affecting yields of biovanillin in cell factories such as Escherichia coli is product toxicity, the mechanisms of which are poorly understood. To identify targets for engineering improved strains, we have studied mechanisms of vanillin toxicity in E. coli using a two-pronged apparoach: (i) a global proteomic analysis supported by multiple physiological experiments and mutant analyses (ii) adaptive laboratory evolution (ALE) of vanillin tolerance combined with genome sequencing. We identified 147 proteins that exhibited a significant change in abundance in response to vanillin. Upregulated proteins included enzymes capable of converting aldehydes to potentially less toxic compounds; pentose phosphate pathways enzymes, fumarase C and enzymes of the glyoxylate shunt; proteins involved in several key stress responses, in particular oxidative stress; proteins mediating uptake and processing of metal ions. 1H-NMR confirmed that E. coli detoxifies vanillin by reduction to vanillyl alcohol; the aldehyde reductases YqhD and DkgA were highly upregulated by vanillin and the purified enzymes reduced vanillinin an NADPH dependent manner. Vanillin caused accumulation of reactive oxygen species and activated an oxidative stress response through SoxRS, OxyR and MarA pathways. Slow vanillin dependent copper (II) to copper (I) reduction lead to upregulation of the copA gene and growth in the presence of vanillin was hypersensitive to inhibition by copper ions. RT-PCR and mutant growth data suggested AcrD and AaeAB as potential vanillin efflux systems. Vanillin-tolerant strains isolated by ALE had distinct non-synonymous SNPs in the citrate synthase gene gltA, in addition to strain specific mutations in cpdA, rob and marC. One strain had a large ~10 kb deletion including the marRAB region. Purifed variant GltA enzymes all showed higher activity due to a lowered Km for oxaloacetate. Our data provide new understanding and novel gene targets for engineering vanillin-tolerant strains of E. coli, including deletion of the efflux pumps eamA, acrA, and acrB, enhancing oxidative stress defences and NADPH production, improving copper homeostasis and increasing citrate synthase activity using variant enzymes.

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Involvement of ergosterol contents in the tolerance to vanillin in Saccharomyces cerevisiae

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