Project description:Production of D-xylonate in the yeast S. cerevisiae represents an example of bioprocess development for more sustainable production of value-added chemicals from cheap raw material or waste. Previously it was shown that the production of D-xylonate led to its significant intracellular accumulation and to dramatic loss of viability during the production process. In order to identify the physiological or pathological responses associated with D-xylonate production, we performed a time-course transcriptome analysis of D-xylonate production in yeast cultivated in a bioreactor. Comparison of the transcriptomes of D-xylonate producing strain with control strain showed considerably higher expression in the xylonate producing strain of the genes controlled by the cell wall integrity pathway (CWI) and of some genes previously identified as upregulated in response to the organic acid stress. Surprisingly, also genes encoding proteins involved in translation, ribosome structure and RNA metabolism – the processes commonly found to be down-regulated under virtually every condition causing cellular stress – were upregulated during the D-xylonate production. The overall transcriptional responses were, therefore, very dissimilar to those previously reported as being associated with diverse stresses including the organic acid treatment and production. In addition, it was observed that the consumption of ethanol was slower and the level of trehalose was lower in the D-xylonate producing strain. Validation experiments including Slt2 kinase phosphorylation profiles and the quantitative PCR analyses of selected gene showed remarkably good match with our findings and confirmed the observations made in the transcriptome analysis. The production of organic acids has a major impact on the physiology of yeast cells. There is, however, very limited overlap at the transcriptional level in responses to treatment or production of different acids. The loss of viability, observed during production and accumulation of D-xylonate, seems to be caused by erroneous interpretation of environmental signals causing a failure in entering the stationary phase and eventually leading to depletion of scarce resources by the affected cells. This, together with intracellular acidification, inevitably results in cell death.
Project description:The production of biofuels in photosynthetic microalgae and cyanobacteria is considered a promising alternative to the generation of fuels from fossil resources. However, to be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria to forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Here we report the results of a monitoring experiment, in which the transcriptome-wide response to continuous ethanol production in the unicellular model cyanobacterium Synechocystis sp. PCC6803 was examined using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) EtOH d-1 M-BM-1 0.002 and 0.0303% (v/v) d-1 M-BM-1 0.002 were obtained over 18 consecutive days, measuring biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation in the producer strains and the development of a bleaching phenotype. Absorption spectroscopy indicated in particular a down-regulation of light harvesting capacity. Microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an about 4 fold reduction in cpcB (sll1577) and an about 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional operon discoordination in the cpcBA operon leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, Western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. We conclude that the changes in gene expression upon induction of long term ethanol production in Synechocystis sp. PCC6803 are highly specific. in particular we did not observe a comprehensive stress response contributing to a complex phenotype as might have been expected. Gene expression of Synechocystis sp. PCC 6803 WT (#621) and the isogenic ethanol producing strain #309 was monitored at 4, 7, 11 and 18 days after induction of ethanol production by copper depletion. Each condition was sampled in biological duplicates
Project description:Clostridium thermocellum is a leading candidate organism for implementing a consolidated bioprocessing (CBP) strategy for biofuel production due to its native ability to rapidly consume cellulose and its existing ethanol production pathway. C. thermocellum converts cellulose and cellobiose to lactate, formate, acetate, H2, ethanol, amino acids, and other products. Elimination of the pathways leading to products such as H2 could redirect carbon flux towards ethanol production. Rather than delete each hydrogenase individually, we targeted a hydrogenase maturase gene (hydG), which is involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes by assembling the active site. This functionally inactivated all three Fe-Fe hydrogenases simultaneously, as they were unable to make active enzymes. In the ∆hydG mutant, the [NiFe] hydrogenase-encoding ech was also deleted to obtain a mutant that functionally lacks all hydrogenase. The ethanol yield increased nearly 2-fold in ∆hydG∆ech compared to wild type, and H2 production was below the detection limit. Interestingly, ∆hydG and ∆hydG∆ech exhibited improved growth in the presence of acetate in the medium. Transcriptomic and proteomic analysis reveal that genes related to sulfate transport and metabolism were up-regulated in the presence of added acetate in ∆hydG, resulting in altered sulfur metabolism. Further genomic analysis of this strain revealed a mutation in the bi-functional alcohol/aldehyde dehydrogenase adhE gene, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities, whereas the wild type strain can only utilize NADH. This is the exact same adhE mutation found in ethanol-tolerant C. thermocellum strain E50C, but ∆hydG∆ech is not more ethanol tolerant than the wild type. Targeting protein post-translational modification is a promising new approach to target multiple enzymes simultaneously for metabolic engineering.
Project description:Although the relationship between phenotypic plasticity and evolutionary dynamics has attracted large interest, very little is known about the contribution of phenotypic plasticity to adaptive evolution. In this study, we analyzed phenotypic and genotypic changes in E. coli cells during adaptive evolution to ethanol stress. To quantify the phenotypic changes, transcriptome analyses were performed. We previously obtained 6 independently evolved ethanol tolerant E. coli strains, strains A through F, by culturing cells under 5% ethanol stress for about 1000 generations and found a significantly larger growth rate than the parent strains (Horinouchi et al, 2010, PMID: 20955615). To elucidate the phenotypic changes that occurred during adaptive evolution, we quantified the time-series of the expression changes by microarray analysis. Starting from frozen stocks obtained at 6 time points (0, 384, 744, 1224, 1824 and 2496 hours) in laboratory evolution, cells were cultured under 5% ethanol stress, and mRNA samples were obtained in the exponential growth phase for microarray analysis.
Project description:Although the relationship between phenotypic plasticity and evolutionary dynamics has attracted large interest, very little is known about the contribution of phenotypic plasticity to adaptive evolution. In this study, we analyzed phenotypic and genotypic changes in E. coli cells during adaptive evolution to ethanol stress. To quantify the phenotypic changes, transcriptome analyses were performed.
Project description:we aimed to screen candidate kinase genes under the stress of phenolic aldehydes during ethanol fermentation for Zymomonas mobilis ZM4
Project description:The production of biofuels in photosynthetic microalgae and cyanobacteria is considered a promising alternative to the generation of fuels from fossil resources. However, to be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria to forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Here we report the results of a monitoring experiment, in which the transcriptome-wide response to continuous ethanol production in the unicellular model cyanobacterium Synechocystis sp. PCC6803 was examined using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) EtOH d-1 ± 0.002 and 0.0303% (v/v) d-1 ± 0.002 were obtained over 18 consecutive days, measuring biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (~40%) delay in biomass accumulation in the producer strains and the development of a bleaching phenotype. Absorption spectroscopy indicated in particular a down-regulation of light harvesting capacity. Microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an about 4 fold reduction in cpcB (sll1577) and an about 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional operon discoordination in the cpcBA operon leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, Western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. We conclude that the changes in gene expression upon induction of long term ethanol production in Synechocystis sp. PCC6803 are highly specific. in particular we did not observe a comprehensive stress response contributing to a complex phenotype as might have been expected.
Project description:Raw milk usually needs to be refrigerated at 4℃ before production, and microbial contamination during this process can cause spoilage of refrigerated raw milk. However, there is currently almost no research on plateau characteristic milk (yak milk and cattle-yak milk) (YM and CYM). In this study, significant differences in bacterial communities and their succession patterns were found between YM and CYM during 4℃ refrigeration, while Lactococcus, Pseudomonas, and Serratia revealed significant differences between the two groups of milk by Ancom2 analysis. And the microbial community in CYM exhibits higher network complexity and tighter interactions. The enzymes exhibiting stronger correlations identified in YM are more abundant than those in CYM predicted by PICRUSt2. Through metabolomics methods, the metabolome profiles between CYM and YM during refrigeration were significantly different. With the extension of storage time, the relative abundances of lipids and lipid-like molecules, and organic nitrogen compounds were decreased, the relative abundances of organic acids and derivatives was increased (YM is more obvious than CYM). Moreover, 8 metabolites gradually increases with the extension of storage time, and has a good correlation with Psychropliles growth. These metabolites have the potential to serve as markers of plateau characteristic milk deterioration. The results will provide a theoretical basis for improving the quality and safety of plateau characteristic milk and its products.