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A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes.

ABSTRACT: The tolerant yeast Candida glycerinogenes, with high D-xylonate and low-pH tolerances, was used as the host for D-xylonate production at low pH in this study. A low-pH inducible promoter, pGUKd, was engineered using the core promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) combined with the upstream activating sequence of the promoter of the guanylate kinase gene (pGUK1) that had substituted pH-responsive TF binding sites. The recombinant cells that expressed GFP from the hybrid promoter pGUKd displayed dramatically increased fluorescence intensity at pH 2.5, thus verifying that pGUKd is a low-pH inducible promoter. The promoter pGUKd was then used to express the D-xylose dehydrogenase gene xylB, resulting in increased expression levels of xylB at low pH. The recombinant protein exhibited higher specific activities under lower pH conditions and produced 38 g/l D-xylonate at pH 2.5. This rate is much higher than that produced by fermentation at pH 5.5. These results suggest that the novel hybrid promoter pGUKd functions to direct the production of D-xylonate at low pH, and we provide a candidate genetic tool for the stress tolerant yeast C. glycerinogenes.

SUBMITTER: Ji H

PROVIDER: S-EPMC5736338 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes.

Ji Hao H Lu Xinyao X Zong Hong H Zhuge Bin B

Bioengineered 20170504 6

The tolerant yeast Candida glycerinogenes, with high D-xylonate and low-pH tolerances, was used as the host for D-xylonate production at low pH in this study. A low-pH inducible promoter, pGUKd, was engineered using the core promoter of the glyceraldehyde-3-phosphate dehydrogenase gene (pGAP) combined with the upstream activating sequence of the promoter of the guanylate kinase gene (pGUK1) that had substituted pH-responsive TF binding sites. The recombinant cells that expressed GFP from the hyb ...[more]

PMID: 28471311

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Project description:Ethanol fermentations can be prematurely halted as Saccharomyces cerevisiae faces adverse conditions, such as acidic pH, presence of acetic acid, and supraoptimal temperatures. The knowledge on yeast responses to these conditions is essential to endowing a tolerant phenotype to another strain by targeted genetic manipulation. In this study, physiological and whole-genome analyses were conducted to obtain insights on molecular responses which potentially render yeast tolerant towards thermoacidic conditions. To this end, we used thermotolerant TTY23, acid tolerant AT22, and thermo-acid tolerant TAT12 strains previously generated by adaptive laboratory evolution (ALE) experiments. The results showed an increase in thermoacidic profiles in the tolerant strains. The whole-genome sequence revealed the importance of genes related to: H+, iron, and glycerol transport (i.e., PMA1, FRE1/2, JEN1, VMA2, VCX1, KHA1, AQY3, and ATO2); transcriptional regulation of stress responses to drugs, reactive oxygen species and heat-shock (i.e., HSF1, SKN7, BAS1, HFI1, and WAR1); and adjustments of fermentative growth and stress responses by glucose signaling pathways (i.e., ACS1, GPA1/2, RAS2, IRA2, and REG1). At 30 °C and pH 5.5, more than a thousand differentially expressed genes (DEGs) were identified in each strain. The integration of results revealed that evolved strains adjust their intracellular pH by H+ and acetic acid transport, modify their metabolism and stress responses via glucose signaling pathways, control of cellular ATP pools by regulating translation and de novo synthesis of nucleotides, and direct the synthesis, folding and rescue of proteins throughout the heat-shock stress response. Moreover, the motifs analysis in mutated transcription factors suggested a significant association of SFP1, YRR1, BAS1, HFI1, HSF1, and SKN7 TFs with DEGs found in thermoacidic tolerant yeast strains. KEY POINTS: • All the evolved strains overexpressed the plasma membrane H+ -ATPase PMA1 at optimal conditions • Tolerant strain TAT12 mutated genes encoding weak acid and heat response TFs HSF1, SKN7, and WAR1 • TFs HSF1 and SKN7 likely controlled the transcription of metabolic genes associated to heat and acid tolerance.

Project description:BackgroundProduction of D-xylonate by the yeast S. cerevisiae provides an example of bioprocess development for sustainable production of value-added chemicals from cheap raw materials or side streams. Production of D-xylonate may lead to considerable intracellular accumulation of D-xylonate and to loss of viability during the production process. In order to understand the physiological responses associated with D-xylonate production, we performed transcriptome analyses during D-xylonate production by a robust recombinant strain of S. cerevisiae which produces up to 50 g/L D-xylonate.ResultsComparison of the transcriptomes of the D-xylonate producing and the control strain showed considerably higher expression of the genes controlled by the cell wall integrity (CWI) pathway and of some genes previously identified as up-regulated in response to other organic acids in the D-xylonate producing strain. Increased phosphorylation of Slt2 kinase in the D-xylonate producing strain also indicated that D-xylonate production caused stress to the cell wall. Surprisingly, genes encoding proteins involved in translation, ribosome structure and RNA metabolism, processes which are commonly down-regulated under conditions causing cellular stress, were up-regulated during D-xylonate production, compared to the control. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with stress, including stress induced by organic acid treatment or production. Quantitative PCR analyses of selected genes supported the observations made in the transcriptomic analysis. In addition, consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain, compared to the control.ConclusionsThe production of organic acids has a major impact on the physiology of yeast cells, but the transcriptional responses to presence or production of different acids differs considerably, being much more diverse than responses to other stresses. D-Xylonate production apparently imposed considerable stress on the cell wall. Transcriptional data also indicated that activation of the PKA pathway occurred during D-xylonate production, leaving cells unable to adapt normally to stationary phase. This, together with intracellular acidification, probably contributes to cell death.

Dataset Information

A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes.

Publications

A synthetic hybrid promoter for D-xylonate production at low pH in the tolerant yeast Candida glycerinogenes.

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