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.

Project description:Zymomonas mobilis is an excellent ethanologenic bacterium. Biomass pretreatment and saccharification provides access to simple sugars, but also produces inhibitors such as acetate and furfural. Our previous work has identified and confirmed the genetic change of a 1.5-kb deletion in the sodium acetate tolerant Z. mobilis mutant (AcR) leading to constitutively elevated expression of a sodium proton antiporter encoding gene nhaA, which contributes to the sodium acetate tolerance of AcR mutant. In this study, we further investigated the responses of AcR and wild-type ZM4 to sodium acetate stress in minimum media using both transcriptomics and a metabolic labeling approach for quantitative proteomics the first time. Proteomic measurements at two time points identified about eight hundreds proteins, or about half of the predicted proteome. Extracellular metabolite analysis indicated AcR overcame the acetate stress quicker than ZM4 with a concomitant earlier ethanol production in AcR mutant, although the final ethanol yields and cell densities were similar between two strains. Transcriptomic samples were analyzed for four time points and revealed that the response of Z. mobilis to sodium acetate stress is dynamic, complex and involved about one-fifth of the total predicted genes from all different functional categories. The modest correlations between proteomic and transcriptomic data may suggest the involvement of posttranscriptional control. In addition, the transcriptomic data of forty-four microarrays from four experiments for ZM4 and AcR under different conditions were combined to identify strain-specific, media-responsive, growth phase-dependent, and treatment-responsive gene expression profiles. Together this study indicates that minimal medium has the most dramatic effect on gene expression compared to rich medium followed by growth phase, inhibitor, and strain background. Genes involved in protein biosynthesis, glycolysis and fermentation as well as ATP synthesis and stress response play key roles in Z. mobilis metabolism with consistently strong expression levels under different conditions. A sixteen array study using total RNA recovered from wild-type cultures of Zymomonas mobilis subsp mobilis ZM4 and acetate-tolerant mutant AcR at different time points of 130, 148, 166, and 190h post-inoculation with 10g/L sodium acetate (NaAc) treatment were carried out to investigate the expression differences between ZM4 and AcR. Two biological replicates for treatment or control condition.

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.

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Comparison of gene expression and mutant fitness in Zymomonas mobilis ZM4

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