蓝细菌脂肪醛去甲酰加氧酶与醛脱氢酶参与脂肪烃降解的代谢途径解析及生理功能研究

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	蓝细菌脂肪醛去甲酰加氧酶与醛脱氢酶参与脂肪烃降解的代谢途径解析及生理功能研究
其他题名	无
	乔月
导师	王纬华 ; 吕雪峰
	2017-05
学位授予单位	中国科学院大学；中科院青岛生物能源与过程研究所
学位授予地点	北京；青岛
学位专业	生物工程
关键词	脂肪烃蓝细菌脂肪醛去甲酰加氧酶醛脱氢酶活性氧自由基
摘要	石油是重要的能源和化学工业品原料，主要成分包括烷烃、环烷烃以及芳香烃等烃类。随着石油化学工业的发展，石油烃污染对生态环境造成严重破坏。光能自养微生物蓝细菌能够降解石油烃的主要成分脂肪烃，但降解途径尚未得到鉴定。脂肪烃生物合成在蓝细菌中普遍存在，合成途径已经得到鉴定，主要包括两个关键酶：脂酰-酰基载体蛋白（acyl carrier protein，ACP）还原酶（acyl-ACP reductase， AAR）与脂肪醛去甲酰加氧酶（aldehyde deformylating oxygenase，ADO）。体外酶学实验发现蓝细菌脂肪烃合成关键酶ADO能够催化从脂肪烃到脂肪醛的反应，而蓝细菌醛脱氢酶（Aldehyde Dehydrogenase，ADH）能够催化从脂肪醛到脂肪酸的反应。上述反应与需氧微生物中从脂肪烃到脂肪酸的降解反应是类似的。本论文围绕蓝细菌中ADO和ADH参与的脂肪烃降解途径开展体外酶学表征及生理功能研究，取得如下主要结果：（1）ADO能够催化不同链长的脂肪醛底物生成少一个碳原子的脂肪烃、脂肪醇和脂肪醛，铁离子在ADO反应中扮演着关键的角色；（2）ADO-ADH酶系能够催化以Cn脂肪醛为底物，经Cn-1脂肪烃-脂肪醇-脂肪醛，生成Cn-1脂肪酸的反应；（3）高光培养条件下多株蓝细菌高产烃突变株生长明显变差，光合色素含量下降，出现漂白现象，且膜脂过氧化程度升高。在高光下能快速生长的聚球藻UTEX 2973细胞抽提物中检测到十四醛的存在。从上述结果推测蓝细菌高产烃突变株出现上述表型可能的原因是高光强条件下，蓝细菌胞内活性氧自由基水平升高，过量表达的ADO行使烷烃单加氧酶功能将胞内的脂肪烃转化为脂肪醛，而相应的ADH活性不能完全匹配，导致脂肪醛积累对细胞造成毒害。本研究丰富了我们对光能自养微生物降解脂肪烃机制的理解，对蓝细菌脂肪烃的高效合成也具有借鉴作用。
其他摘要	Petroleum hydrocarbon pollution can result in serious damage to the ecological environment. As photoautotrophic microorganisms, cyanobacteria can not only synthesize alkanes but also degrade them. So far, alkane biosynthesis pathways were identified in cyanobacteria but the degradation pathway is still unknown. A two-step alka(e)ne biosynthetic pathway consisting of acyl- acyl carrier protein (ACP) reductase (AAR), and aldehyde deformylating oxygenase (ADO) has been identified in cyanobacteria. Interestingly, it has recently been shown that ADO, the key enzyme in biosynthesis, is capable of catalyzing incorporation of an oxygen atom into the alkane to yield alcohol and aldehyde. Aldehyde dehydrogenase (ADH) in cyanobacteria can mediate the oxidation of aldehyde into the corresponding fatty acids. In aerobic alkane degradation pathways, alkane is converted to alcohol, and subsequently oxidized to the corresponding aldehyde and fatty acid. In vitro activity of ADO/ADH enzyme and the roles of ADO and ADH in alkane degradation were investigated in this study. The main results are as follows: (1) ADO can convert Cn fatty aldehyde with different carbon chain length to Cn-1 alkane, alcohol and aldehyde, and ferrous ion plays an important role in ADO reactions. (2) ADO-ADH enzyme system can catalyze Cn aldehyde to Cn-1 alcohol, aldehyde, and then to the Cn-1 fatty acid. (3) Under high light condition, growth of Synechocystis sp. PCC 6803 wild type strain is much better than high-alkane content mutants. Photobleaching of photosynthetic pigments was also observed in high-alkane content mutant LX56 under high light condition. Enhanced level of membrane lipid peroxidation in LX56 mutant indicated oxidative damage to lipids under high light. It is speculated that the high content of alkanes can be converted to aldehyde by overexpressed ADO. Aldehyde can be accumulated due to deficiency of ADH enzyme activity relative to monooxygenase activity of ADO in high-alkane content mutants under high light. These studies help to understand alkane biodegradation pathways in photoautotrophic microorganisms. Approaches and strategies will also be provided for improving alkane production in cyanobacteria.
作者部门	微生物代谢工程团队
学科领域	工学
公开日期	2017-07-01
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/337004/9969
专题	微生物代谢工程研究组
作者单位	中国科学院大学；中科院青岛生物能源与过程研究所
推荐引用方式 GB/T 7714	乔月. 蓝细菌脂肪醛去甲酰加氧酶与醛脱氢酶参与脂肪烃降解的代谢途径解析及生理功能研究[D]. 北京；青岛. 中国科学院大学；中科院青岛生物能源与过程研究所,2017.

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