P450脂肪酸脱羧酶OleTJE的催化机理与代谢工程研究

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	P450脂肪酸脱羧酶OleTJE的催化机理与代谢工程研究
	房博
导师	李盛英，张伟
	2016-05
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	生物工程
关键词	生物燃料 P450脂肪酸脱羧酶 Oletje 还原伴侣蛋白催化机理
摘要	为了解决能源稀缺的世界性难题，寻找能够替代化石燃料的可再生清洁能源成为当务之急。由生物储能物质脂肪酸衍生而来的脂肪烃类生物燃料被认为是最理想的石油类燃料替代品。在众多脂肪烃生物合成途径中，由来自于咸海鲜球菌Jeotgalicoccus sp. ATCC 8456的P450酶OleTJE催化的脂肪酸单步脱羧途径，被认为是最简单可行的一种。利用基因工程、酶工程和代谢工程手段，定向改造这一脂肪烃生物合成途径，对开发工业化生产生物燃料新方法具有重要的意义。 P450酶OleTJE作为CYP152家族的成员，以H2O2作为氧供体和电子供体，不仅能够催化不同链长的脂肪酸发生脱羧反应，生成相应链长的α-烯烃，而且能够催化脂肪酸发生羟基化反应生成α-和β-羟基脂肪酸。除此之外，OleTJE还能够分别以O2、NAD(P)H作为氧供体和电子供体，在还原伴侣蛋白的支撑下，催化脂肪酸发生上述两种反应。为了解析OleTJE反应类型选择性的分子机制，本研究首先通过结构分析对OleTJE关键位点氨基酸残基进行了定点突变，系统分析了这些突变体的产烃能力，确定了Arg245、Ile170和His85这三个位点在决定产物分布和催化机理方面的重要性；其次，结合动力学分析，以及一系列异源还原伴侣对OleTJE催化功能的支撑作用，对OleTJE的具体催化机理进行了较为深入的研究，获得了体外高效支撑OleTJE脱羧活性的还原伴侣组合Fdx_0698和FdR_2658。这些工作为今后通过定向酶工程改造来提高OleTJE的整体产烃能力提供了基础理论支撑。与此同时，为了提高以OleTJE作为细胞核心产烃元件生成中等链长脂肪烯烃的产率，从而尽可能契合航空燃料的物理化学特性，本研究还通过代谢工程手段将OleTJE脱羧与大肠杆菌脂肪酸合成和代谢途径结合，利用基因工程方法异源表达植物来源的硫酯酶，定向改变α-烯烃产物的链长。摇瓶发酵显示该法成功提高了C11 α-烯烃在总烯烃中的比例，达到58.2%。综上，P450脂肪酸脱羧酶OleTJE的催化机理研究以及产烃途径的代谢工程改造不仅具有重要的理论意义，而且将为未来实现微生物合成α-烯烃的工业化应用打下基础。
其他摘要	With the increasing energy resource scarcity becoming a global concern, it’s imperative to seek renewable biofuel alternatives for the traditional unsustainable fossil fuels. The fatty acid-derived hydrocarbons have been recognized as the most ideal alternatives. Among the biosynthetic pathways of aliphatic hydrocarbons, the P450 fatty acid decarboxylase OleTJE identified from Jeotgalicoccus sp. ATCC 8456 is able to catalyze one-step decarboxylation reaction of fatty acids to terminal olefins, representing the most simple and direct biosynthetic pathway of bio-hydrocarbons. Thus, it’s of great significance to modify this pathway via genetic engineering, enzyme engineering and metabolic engineering approaches to develop new methods for the industrial production of biohydrocarbons. As a member of CYP152 family, P450 OleTJE could utilize H2O2 as the oxidant and electron donor to catalyze the decarboxylation and hydroxylation of fatty acids, to produce the main product α-alkenes, and the side products α-hydroxy and β-hydroxy fatty acids, respectively. Moreover, OleTJE is also able to carry out the above two catalytic reactions using O2 as oxygen donor and two electrons transported from NAD(P)H by redox partner proteins. Herein, to explore the molecular mechanism of OleTJE reaction type selectivity, we performed systematic mutagenesis analysis on some of the key residues based on structural analysis. Upon evaluation of the catalytic activity and alkene productivity of the mutants, three residues, Arg245, Ile170 and His85, were identified to be important in regulating OleTJE catalysis and products distribution. In addition, enzyme kinetics studies and analysis of the supportive effects of heterologous redox partners on the decarboxylation activity of OleTJE were carried out to further understand the catalytic mechanism of the enzyme. A redox partner protein pair of Fdx_0698 and FdR_2658 was found to highly support the decarboxylation reaction of OleTJE in vitro. Together, these studies will provide theoretical basis for future directed enzyme engineering towards improved alkene yield. Meanwhile, in order to improve the medium-chain length alkene production catalyzed by OleTJE in Escherichia coli to meet the bio-jet fuel properties, we have combined the synthetic and metabolic pathways of fatty acids with the decarboxylation catalyzed by OleTJE via genetic and metabolic engineering. In this study, we overexpressed the thioesterases from plants in E. coli and obtained 1-undecene accounted for 58.2% of the total alkene titers by shake-flask fermentation. In summary, our studies on the unique catalytic mechanism of OleTJE and the metabolic engineering of α-alkene synthetic pathways in E. coli not only provide great theoretical significance, but also lay the foundation for future industrial production of α-alkenes in microbes.
作者部门	酶工程团队
学科领域	生物工程
公开日期	2020-12-01
学位类型	硕士 ; 学位论文
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9776
专题	酶工程研究组
作者单位	中国科学院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	房博. P450脂肪酸脱羧酶OleTJE的催化机理与代谢工程研究[D]. 北京. 中国科学院大学,2016.

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