Dataset Information

Phenotypic and genomic analysis of Zymomonas mobilis ZM4 mutants with enhanced ethanol tolerance.

ABSTRACT: Zymomonas mobilis ZM4 is an ethanol-producing microbe that is constitutively tolerant to this solvent. For a better understanding of the ethanol tolerance phenomenon we obtained and characterized two ZM4 mutants (ER79ap and ER79ag) with higher ethanol tolerance than the wild-type. Mutants were evaluated in different ethanol concentrations and this analysis showed that mutant ER79ap was more tolerant and had a better performance in terms of cell viability, than the wild-type strain and ER79ag mutant. Genotyping of the mutant strains showed that both carry non-synonymous mutations in clpP and spoT/relA genes. A third non-synonymous mutation was found only in strain ER79ap, in the clpB gene. Considering that ER79ap has the best tolerance to added ethanol, the mutant alleles of this strain were evaluated in ZM4 and here we show that while all of them contribute to ethanol tolerance, mutation within spoT/relA gene seems to be the most important.

SUBMITTER: Carreon-Rodriguez OE

PROVIDER: S-EPMC6444122 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Phenotypic and genomic analysis of <i>Zymomonas mobilis</i> ZM4 mutants with enhanced ethanol tolerance.

Carreón-Rodríguez Ofelia E OE Gutiérrez-Ríos Rosa María RM Acosta José L JL Martinez Alfredo A Cevallos Miguel A MA

Biotechnology reports (Amsterdam, Netherlands) 20190321

<i>Zymomonas mobilis</i> ZM4 is an ethanol-producing microbe that is constitutively tolerant to this solvent. For a better understanding of the ethanol tolerance phenomenon we obtained and characterized two ZM4 mutants (ER79ap and ER79ag) with higher ethanol tolerance than the wild-type. Mutants were evaluated in different ethanol concentrations and this analysis showed that mutant ER79ap was more tolerant and had a better performance in terms of cell viability, than the wild-type strain and ER7 ...[more]

PMID: 30984572

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Project description:Background: Growth in the global population and industrial activities has increased world energy consumption. Bioethanol is considered as an alternative renewable energy source. Among various ethanol-producing microbes, Zymomonas mobilis ZM4 has received special attention due to its higher ethanol yield and tolerance. Advances in genetic engineering are particularly important for developing microorganisms with improved ethanol production. However, the variety of factors involved in the response to high concentrations of ethanol makes it difficult to devise genetic engineering strategies to generate alcohol tolerant strains. For a better understanding of the ethanol tolerance phenomenon, we obtained and characterized two Z. mobilis ZM4 mutants (ER79ap and ER79ag) with increased ethanol tolerance. Results: Mutants were obtained using a strain adaptive evolution method in mini-fermentors and sequential transfers to higher ethanol concentrations. Mutations were identified by Illumina genomic sequencing. Strain ER79ap possesses three point mutations in the following genes: SpoT/RelA, which synthesizes and degrades the alarmone ppGpp; clpB, encoding a disgregase; and clpP, a component of the Clp protease. In contrast, strain ER79ag has four mutations in the subsequent genes: spoT/relA; rimO; clpP; and in a gene encoding a hypothetical protein with a CBS domain. Transcript profiles of ZM4 and ER79ap were obtained with microarray analysis and identified 126 genes in ZM4 and 148 genes in ER79ap that were differentially expressed in response to ethanol. Conclusions: Both mutants carry mutations in clpP and relA/spoT genes, suggesting that these genes are responsible of their enhanced ethanol tolerance. Transcript profile analysis of the ZM4 and ER79ap showed that they share a set of forty genes that are differentially expressed under ethanol stress and this set may correspond to those that are crucial for the ethanol response. The expression profiles indicate that ethanol induces a major reprograming of transcription that involves changes in the cell membrane, protein synthesis, and in some metabolic pathways, especially those involved in the amino acid metabolism. Our data suggest that clpP and in particular the relA/spoT gene can be targets for bioengineering ethanol tolerance.

Project description:Background Zymomonas mobilis ZM4 is a capable ethanologenic bacterium with high ethanol productivity and ethanol tolerance. Previous studies indicated that several stress-related proteins and changes in the ZM4 membrane lipid composition may contribute to ethanol tolerance. However, the molecular mechanisms of its ethanol stress response have not been elucidated fully. Methodology/Principal Findings In this study, ethanol stress responses were investigated using systems biology approaches. Medium supplementation with an initial 47 g/L (6% v/v) ethanol reduced Z. mobilis ZM4 glucose consumption, growth rate and ethanol productivity compared to that of untreated controls. A proteomic analysis of early exponential growth identified about one thousand proteins, or approximately 55% of the predicted ZM4 proteome. Proteins related to metabolism and stress response such as chaperones and key regulators were more abundant in the early ethanol stress condition. Transcriptomic studies indicated that the response of ZM4 to ethanol is dynamic, complex and involves many genes from all the different functional categories. Most down-regulated genes were related to translation and ribosome biogenesis, while the ethanol-upregulated genes were mostly related to cellular processes and metabolism. Transcriptomic data were used to update Z. mobilis ZM4 operon models. Furthermore, correlations among the transcriptomic, proteomic and metabolic data were examined. Among significantly expressed genes or proteins, we observe higher correlation coefficients when fold-change values are higher. Conclusions Our study has provided insights into the responses of Z. mobilis to ethanol stress through an integrated “omics” approach for the first time. This systems biology study elucidated key Z. mobilis ZM4 metabolites, genes and proteins that form the foundation of its distinctive physiology and its multifaceted response to ethanol stress. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 at different time points of 6, 10, 13.5, and 26h post-inoculation with 6% (v/v) treatment compred to that of control without ethanol supplementation. Two biological replicates for treatment and control condition.

Dataset Information

Phenotypic and genomic analysis of Zymomonas mobilis ZM4 mutants with enhanced ethanol tolerance.

Publications

Phenotypic and genomic analysis of <i>Zymomonas mobilis</i> ZM4 mutants with enhanced ethanol tolerance.

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