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 ZM4 produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. However, the genetic and physiological basis of the ZM4 response to various industrially-relevant stresses is poorly understood. In this study, the dynamics of ZM4 oxygen stress responses were elucidated by characterizing the transcriptomic and metabolomic profiles of aerobic and anaerobic fermentations using whole-genome microarray analysis and gas chromatography-mass spectrometry. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Under aerobic conditions, much less ethanol was observed with other end-products produced, including acetate, lactate, and acetoin. In the early exponential phase, no genes were detected as being significantly differentially expressed between aerobic and anaerobic conditions via microarray analysis. However, microarray analysis of the stationary phase cultures revealed that 166 genes were significantly differentially expressed by more than two-fold. Quantitative-PCR validated the expression values for seventeen genes from different categories of stationary phase microarray data. Transcripts for Entner-Doudoroff pathway genes and gene pdc, encoding a key enzyme leading to the ethanol production, were at least 30-fold more abundant under anaerobic conditions at stationary phase based on quantitative-PCR results. Expression of stress response genes was found to be greater under oxygen stress conditions. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 under anaerobic conditions contained lower levels of amino acids, glucose and ED pathway intermediates, whereas metabolites such as ribitol, trehalose, myristic acid, glyceric acid, glucose 6-phosphate, mannose 6-phosphate, and 4-hydroxybutanoic acid were more abundant than under aerobic conditions. Transcriptomic and metabolomic data were consistent with faster glucose consumption and greater ethanol production under anaerobic conditions and suggested gene targets for deletion and improved fermentation. Keywords: Time course/ Stress Response

Project description:Zymomonas mobilis ZM4 produces near theoretical yields of ethanol with high specific productivity and recombinant strains are able to ferment both C-5 and C-6 sugars. However, the genetic and physiological basis of the ZM4 response to various industrially-relevant stresses is poorly understood. In this study, the dynamics of ZM4 oxygen stress responses were elucidated by characterizing the transcriptomic and metabolomic profiles of aerobic and anaerobic fermentations using whole-genome microarray analysis and gas chromatography-mass spectrometry. In the absence of oxygen, ZM4 consumed glucose more rapidly, had a higher growth rate, and ethanol was the major end-product. Under aerobic conditions, much less ethanol was observed with other end-products produced, including acetate, lactate, and acetoin. In the early exponential phase, no genes were detected as being significantly differentially expressed between aerobic and anaerobic conditions via microarray analysis. However, microarray analysis of the stationary phase cultures revealed that 166 genes were significantly differentially expressed by more than two-fold. Quantitative-PCR validated the expression values for seventeen genes from different categories of stationary phase microarray data. Transcripts for Entner-Doudoroff pathway genes and gene pdc, encoding a key enzyme leading to the ethanol production, were at least 30-fold more abundant under anaerobic conditions at stationary phase based on quantitative-PCR results. Expression of stress response genes was found to be greater under oxygen stress conditions. GC-MS analysis of stationary phase intracellular metabolites indicated that ZM4 under anaerobic conditions contained lower levels of amino acids, glucose and ED pathway intermediates, whereas metabolites such as ribitol, trehalose, myristic acid, glyceric acid, glucose 6-phosphate, mannose 6-phosphate, and 4-hydroxybutanoic acid were more abundant than under aerobic conditions. Transcriptomic and metabolomic data were consistent with faster glucose consumption and greater ethanol production under anaerobic conditions and suggested gene targets for deletion and improved fermentation. Keywords: Time course/ Stress Response A total of 24 samples were analyzed. These consisted of two times points, (3 hours and 26 hours) under two conditions (anaerobic and aerobic). There were 3 biological replicates and two dye swaps. Each microarray containted one to two probes per predicted coding sequence.

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Genome-scale transcription–translation mapping reveals novel features of Zymomonas mobilis promoters and transcription units

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