CcpA介导的嗜热氧杆菌碳代谢物阻遏机制解析及解除研究

Thermoanaerobacterium has gained great attention over the past decade years, motivated by their broader substrate spectra and higher ethanol/hydrogen yields. Carbon catabolite repression (CCR), defined as the control process that microorganisms preferentially utilize a rapidly metabolizable carbon source (normally glucose), along with inhibition of some gene expression and enzyme activities related to catabolism of non-preferred carbon resources, is observed in the bacterium. Although the physiological outcome is similar, the global mechanisms underlying CCR are completely different in different bacteria. In the research on Thermoanaerobacterium, the emphasis so far lies on the strain isolation and metabolic engineering. In this project, we attempt to elucidate the control mechanism of CCR in Thermoanaerobacterim. The concern will be focused on: 1) identification of carbon catabolite protein A (CcpA), which is a global transcriptional regulator functioning at the core of carbon catabolite repression; 2) construction of a mutant with ccpA deletion in the Thermoanaerobacterium chromosome; 3) construction of metabolite pathway and gene coexpression network through transcriptome sequencing and real-time PCR; 4) elimination of glucose repression on xylose utilization. These results could enhance our understanding on how CcpA influences the expression profile of central and carbohydrate-specific metabolic genes during CCR control in other industrial bacterium. Based on the mechanism insights, researchers will further give a feasible strategy for improving simultaneous utilization of mixed sugars, which is essential for commercial exploitation of lignocelluloses for the production of biofuels.

嗜热厌氧杆菌因具有广泛的底物谱和较高的底物转化率而引起科学家的关注。申请者研究发现该菌株有明显的碳代谢物阻遏(CCR)现象，即葡萄糖的存在会抑制其它糖类代谢。嗜热厌氧杆菌的研究多集中在菌株筛选和代谢途径改造，生化研究几乎空白。而CCR调控过程的复杂性，使得解析该微生物CCR机制、鉴定CCR调控关键因子势在必行。本课题以CCR的核心调控单元CcpA为切入点，克隆、鉴定T.aotearoense的CcpA编码基因，沉默CcpA，通过转录组测序和Real-Time PCR分析突变前后基因表达差异，对差异基因进行Pathway分析，建立差异基因共表达网络；同时在蛋白水平检测糖代谢酶活变化，以解析该类微生物的CCR机制，并设计验证CCR抑制的解除方案。由此获得的CCR调控机理及其解除方案，将为理解和降低其它工业微生物CCR抑制这一复杂而具有普遍性现象提供新的思路和方法。推动第二代生物燃料产业化进程。

本项目针对嗜热厌氧杆菌的碳代谢物阻遏（CCR）的科学问题，提出从CCR抑制关键蛋白CcpA入手，解析其机制的研究思路。研究通过功能互补实验、EMSA实验以及分子相互作用等检测方法（手段）结果表明，从目标菌株中获得的CcpA蛋白承担着该菌株的CCR调节作用，此外还鉴定了CCR相关功能蛋白HPr和HPrP。进一步的，本研究通过突变手段获得了碳耐受菌株，并通过转录组测序进一步解析了耐受菌株的分子机理，为后续该菌株的工业化应用提供了很好的基础和借鉴。在本项目资助下，申报人还展开了多项围绕该菌株的科研工作，如构建高产光学纯L-乳酸工程菌、克隆表达鉴定多个纤维素降解酶。项目结束时，申报人以通讯作者身份发表了SCI 论文7篇（第一标注），其中中科院分区一区论文4篇。另有一篇综述Pubmed文章（第一作者）被评为Most Cited Articles（他引已达67次）。申请发明专利7项，其中1项已授权，另有1项PCT专利（已进入国家审核阶段）。出版译著1部（科学出版社）。项目承担人职称晋升为教授，博士生导师。培养博士生1名，硕士3名。

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