The model ethanologenic bacterium Zymomonas mobilis has many advantages for diverse biochemical production. Although the impact of temperature especially high temperature on the growth and ethanol production of Z. mobilis has been reported, the transcriptional profiles of Z. mobilis grown at different temperatures have not been systematically investigated. In this study, Z. mobilis wild-type strain ZM4 was used to study the effect of a broad range of temperatures of 24, 30, 36, 40, and 45 °C on cell growth and morphology, glucose utilization and ethanol production, as well as the corresponding global gene expression profiles using RNA-Seq-based transcriptomics. In addition, a recombinant Z. mobilis strain expressing reporter gene EGFP (ZM4_EGFP) was constructed to study the effect of temperature on heterologous protein expression at different temperatures. Our result demonstrated that the effect of temperature on the growth and morphology of ZM4 and ZM4_EGFP were similar. The biomass of these two strains decreased along with the temperature increase, and an optimal temperature range is needed for efficient glucose utilization and ethanol production. Temperatures lower or higher than normal temperature investigated in this work was not favorable for the glucose utilization and ethanol production as well as the expression of exogenous protein EGFP based on the results of flow cytometry and Western blot. Temperature also affected the transcriptional profiles of Z. mobilis especially under high temperature. Compared with ZM4 cultured at 30 °C, 478 genes were up-regulated and 481 genes were down-regulated at 45 °C. The number of differentially expressed genes of ZM4 cultured at other temperatures (24, 36 or 40 °C) was relatively small though compared with those at 30 °C. Since temperature usually increases during the fermentation process, and heat tolerance is one of the important robustness traits of industrial strains, candidate genes related to heat resistance based on our RNA-Seq result and literature report were then selected for genetics study using the strategies of plasmid overexpression of candidate gene or replacement of the native promoter of candidate gene by an inducible Ptet promoter. The genetics studies indicated that ZMO0236, ZMO1335, ZMO0994, operon groESL, and cspL, which encodes Mrp family chromosome partitioning ATPase, flavoprotein WrbA, an uncharacterized protein, chaperonin Cpn10 and GroEL, and an exogenous cold shock protein, respectively, were associated with heat tolerance, and recombinant strains over-expressing these genes can improve their heat tolerance. Our work thus not only explored the effects of temperature on the expression of exogenous gene EGFP and endogenous genes, but also selected and confirmed several genes associated with heat tolerance in Z. mobilis, which provided a guidance on identifying candidate genes associated with phenotypic improvement through systems biology strategy and genetics studies for other microorganisms.

中文翻译：

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研究广泛的温度范围对运动发酵单胞菌ZM4的生理和转录谱的影响，用于耐高温重组菌株的开发

模型产乙醇细菌运动发酵单胞菌 (Zymomonas mobilis) 具有多种生化生产的优势。尽管已经报道了温度特别是高温对运动发酵单胞菌生长和乙醇生产的影响，但尚未系统研究运动发酵单胞菌在不同温度下的转录谱。在本研究中，运动发酵单胞菌野生型菌株 ZM4 用于研究 24、30、36、40 和 45 °C 的广泛温度范围对细胞生长和形态、葡萄糖利用和乙醇生产的影响，如以及使用基于 RNA-Seq 的转录组学的相应全局基因表达谱。此外，重组 Z. 构建表达报告基因 EGFP (ZM4_EGFP) 的 mobilis 菌株以研究温度对不同温度下异源蛋白表达的影响。我们的结果表明温度对 ZM4 和 ZM4_EGFP 的生长和形态的影响是相似的。这两种菌株的生物量随着温度的升高而下降，需要一个最佳温度范围来有效利用葡萄糖和乙醇生产。根据流式细胞术和蛋白质印迹的结果，在这项工作中研究的低于或高于常温的温度不利于葡萄糖的利用和乙醇的产生以及外源蛋白 EGFP 的表达。温度也影响运动发酵单胞菌的转录谱，尤其是在高温下。与30°C培养的ZM4相比，478个基因上调，481个基因在45°C下下调。与30°C相比，在其他温度（24、36或40°C）下培养的ZM4差异表达基因的数量相对较少。由于发酵过程中温度通常会升高，而耐热性是工业菌株重要的健壮性状之一，根据我们的RNA-Seq结果和文献报道，选择与耐热性相关的候选基因进行遗传学研究，使用质粒策略候选基因的过度表达或用诱导型 Ptet 启动子替换候选基因的天然启动子。遗传学研究表明，ZMO0236、ZMO1335、ZMO0994、操纵子 groESL 和 cspL，其编码 Mrp 家族染色体分区 ATPase，黄素蛋白 WrbA，一种未表征的蛋白质，伴侣蛋白 Cpn10 和 GroEL，以及外源性冷休克蛋白，分别与耐热性相关，过表达这些基因的重组菌株可以提高它们的耐热性。因此，我们的工作不仅探索了温度对外源基因EGFP和内源基因表达的影响，而且还筛选并确认了运动发酵单胞菌耐热性相关的几个基因，为鉴定与表型改善相关的候选基因提供了指导。其他微生物的系统生物学策略和遗传学研究。过表达这些基因的重组菌株可以提高它们的耐热性。因此，我们的工作不仅探索了温度对外源基因EGFP和内源基因表达的影响，而且还筛选并确认了运动发酵单胞菌耐热性相关的几个基因，为鉴定与表型改善相关的候选基因提供了指导。其他微生物的系统生物学策略和遗传学研究。过表达这些基因的重组菌株可以提高它们的耐热性。因此，我们的工作不仅探索了温度对外源基因EGFP和内源基因表达的影响，而且还筛选并确认了运动发酵单胞菌耐热性相关的几个基因，为鉴定与表型改善相关的候选基因提供了指导。其他微生物的系统生物学策略和遗传学研究。