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枯草芽孢杆菌氧化还原感应全局调控因子Rex对乙偶姻合成的影响
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国家自然科学基金(31500065);宁夏回族自治区科技创新团队项目(KJT2017001);山东省重大科技创新工程项目(2019JZZY020605);生物基材料与绿色造纸国家重点实验室开放基金(KF201907)


Effect of global redox-sensing transcriptional regulator Rex on acetoin synthesis of Bacillus subtilis
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    摘要:

    【背景】枯草芽孢杆菌体内含有一种可响应胞内氧化还原水平的因子,称之为氧化还原感应全局调控因子Rex (由基因ydiH编码)。Rex可通过感知辅酶NADH/NAD+水平的变化来调节胞内氧化还原平衡。【目的】研究Rex对枯草芽孢杆菌乙偶姻合成和辅因子代谢的相关性。【方法】利用比较转录组挖掘乙偶姻和2,3-丁二醇可逆转化过程中显著差异的基因,并通过Cre/lox基因敲除技术敲除ydiH、acuA (乙酰AcsA)和acoC (二氢脂酰胺乙酰转移酶)。随后,利用实时荧光定量PCR (RT-qPCR)技术分析敲除菌株中乙偶姻相关基因的转录水平。【结果】通过发酵实验发现,敲除ydiH会在一定程度上抑制菌体的生长速率,但发酵前期乙偶姻单位细胞产量和底物转化率都得到了显著提高;敲除acuA和acoC后,对乙偶姻合成、菌体生长和糖耗速率均影响不大;敲除ydiH后,与乙偶姻合成相关基因alsR (alsSD的正转录调控因子)、alsS (α-乙酰乳酸合成酶)、alsD (α-乙酰乳酸脱羧酶)和bdhA (2,3-丁二醇脱氢酶)的转录水平显著上调。【结论】枯草芽孢杆菌氧化还原感应全局调控因子Rex通过抑制与乙偶姻相关基因的转录水平影响乙偶姻合成。本研究首次报道了枯草芽孢杆菌中Rex和乙偶姻合成的相关性,为探索Rex如何通过调控相关基因的转录来影响胞内氧化还原稳态奠定了基础,也为提高枯草芽孢杆菌工业化生产强度和底物转化率提供了借鉴。

    Abstract:

    [Background] The global redox-sensing transcriptional regulator Rex (encoded by the ydiH gene) of Bacillus subtilis can regulate intracellular redox balance by sensing NADH/NAD+ levels. [Objective] To study the effect of Rex on the synthesis of acetoin through regulation of cofactor pools in B. subtilis. [Methods] RNA-seq was used to identify the transcriptional diversity of genes that were involved in the bioprocess of reversible transformation between acetoin and 2,3-butanediol, and ydiH, acuA (acetyl AcsA) and acoC (dihydrolipoamide acetyltransferase) were knocked out by Cre/lox gene knockout technology. Subsequently, the transcriptional levels of acetoin-related genes in the knockout strains was analyzed by real-time fluorescence quantitative PCR (RT-qPCR). [Results] Fermentation results indicated that blocking of ydiH could inhibit biomass of B. subtilis to a certain extent, but acetoin yield of unit cell and substrate conversion rate were significantly increased in early stage of fermentation. However, knockout of acuA and acoC had little effect on acetoin synthesis, biomass and glucose consumption rate. After knocking out ydiH, the transcriptional levels of alsR (positive transcriptional regulator of alsSD), alsS (α-acetolactate synthase), alsD (α-acetolactate decarboxylase) and bdhA (2,3-butanediol dehydrogenase), involved in acetoin synthesis, were significantly up-regulated. [Conclusion] Global redox-sensing transcriptional regulator Rex in B. subtilis affect the synthesis of acetoin by down regulation of the transcription levels of acetoin synthesis related genes. This study first reported the correlation between Rex and the synthesis of acetoin in B. subtilis, laying the foundation for exploring how Rex affects intracellular redox balance by regulating the transcription of related genes, and providing reference for improving the industrial production intensity and substrate conversion rate of B. subtilis.

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韩如梦,柳鑫燕,林文萱,李翔飞,杨套伟,徐美娟,邵明龙,张显,饶志明. 枯草芽孢杆菌氧化还原感应全局调控因子Rex对乙偶姻合成的影响[J]. 微生物学通报, 2020, 47(11): 3494-3503

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  • 在线发布日期: 2020-11-06
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