Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp Strain X514 via Isotopomer-Assisted Metabolite Analysis
Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp Strain X514 via Isotopomer-Assisted Metabolite Analysis
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DOI：
10.1128/aem.00715-09
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发表时间：
2009-08-01
影响因子：
4.4
通讯作者：
Tang, Yinjie J.
中科院分区：
文献类型：
--
作者：
Feng, Xueyang;Mouttaki, Housna;Tang, Yinjie J.
嗜热厌氧细菌属。菌株X514能够在高温条件下将C-5和C-6糖发酵成乙醇和其他代谢物，在生物技术方面具有巨大的潜力。本研究以葡萄糖或丙酮酸为碳源和能源，通过C-13标记示踪实验对菌株X514的中心代谢进行了研究。X514生长在最低限度的培养基上，因此包含了所有大分子构建块的完整生物合成途径。基于基因组注释和氨基酸的同位素分析，可以对X514的中心代谢途径进行三个观察。首先，在发酵生长条件下，X514的磷酸戊糖氧化途径不起作用，三元酸循环不完整。第二，X514具有(Re)型柠檬酸合成酶活性，但没有发现与最近发现的克鲁维梭菌(Clostridium Kluyveri)的(Re)型柠檬酸合成酶同源的基因。第三，X514中的异亮氨酸是由乙酰辅酶A和丙酮酸通过柠檬酸途径得到的，而不是由苏氨酸通过苏氨酸解氨酶合成的。柠檬酸合成酶基因(CIMA[Teth514_1204])的功能已被酶活性测定所证实，而细胞内柠檬酸的存在已被质谱仪检测到。这项研究证明了结合C-13辅助代谢物分析、酶分析和代谢物检测的优点，不仅可以检查基因组序列注释，还可以发现新的酶活性。
Thermoanaerobacter sp. strain X514 has great potential in biotechnology due to its capacity to ferment a range of C-5 and C-6 sugars to ethanol and other metabolites under thermophilic conditions. This study investigated the central metabolism of strain X514 via C-13-labeled tracer experiments using either glucose or pyruvate as both carbon and energy sources. X514 grew on minimal medium and thus contains complete biosynthesis pathways for all macromolecule building blocks. Based on genome annotation and isotopic analysis of amino acids, three observations can be obtained about the central metabolic pathways in X514. First, the oxidative pentose phosphate pathway in X514 is not functional, and the tricarboxylic acid cycle is incomplete under fermentative growth conditions. Second, X514 contains (Re)-type citrate synthase activity, although no gene homologous to the recently characterized (Re)-type citrate synthase of Clostridium kluyveri was found. Third, the isoleucine in X514 is derived from acetyl coenzyme A and pyruvate via the citramalate pathway rather than being synthesized from threonine via threonine ammonia-lyase. The functionality of the citramalate synthase gene (cimA [Teth514_1204]) has been confirmed by enzymatic activity assays, while the presence of intracellular citramalate has been detected by mass spectrometry. This study demonstrates the merits of combining C-13-assisted metabolite analysis, enzyme assays, and metabolite detection not only to examine genome sequence annotations but also to discover novel enzyme activities.