蓝细菌末端烯烃合成的分子机制及关键酶的分子进化研究

Hydrocarbons are the major constituents of gasoline, diesel, and jet fuel. Cyanobacteria are one of only a few types of organisms that are capable of direct producing hydrocarbons on the planet. Two cyanobacterial hydrocarbon synthetic pathways,ADO/AAR and OLS pathway, have been identified recently. The former comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (AAR) and aldehyde deformylating oxygenase (ADO), which occupies a majority of cyanobacteria. The latter involves a modular type I polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (OLS), which exists only in a small number of cyanobacterial strains yet with higher teminal olefin yield. Hence, it is of great significance to reveal the synthetic mechanism of terminal olefin for biofuel production．The ols gene encodes a large multidomain protein with homology to type I polyketide synthases (PKS), and substrates binding domain in the N-terminal of OLS appears to be fatty acyl-AMP ligases (FAAL) based on bioinformatics analysis. In this study, seventeen cyanobacterial strains that are capable of producing terminal alkene of different chain length have been identified, and a series of mutants of Synechococcus sp. PCC 7002 which are relevant to OLS substrates supply have been constructed. Domains confer the hydrocarbon chain length specificity of OLS pathway will be investigated and substrates for terminal olefin synthesis will be verified by functional domain replacement in vivo, fatty acid feeding, gene deletion and enzymatic assays in vitro. We will set up rapid detection methods for terminal olefin producing cyanobacterial strains and OLS pathways based on GC-MS analysis and degenerate PCR. Diversity of chain length of the terminal olefin and OLS modular functional domain will be analyzed after screening for a large number of terminal olefin producing strains. We also seek to reveal the molecular evolution of OLS based on cyanobacterial 16S rDNA phylogeny and hydrocarbon pathway distribution.

脂肪烃是汽油、柴油和航空煤油的主要成分。在蓝细菌中已鉴定ADO/AAR和OLS两条脂肪烃合成途径。前者存在于多数蓝细菌中，代谢机制相对清晰。后者仅在少数蓝细菌中发现，该途径产烃量高，作用机制有待解析。OLS具有I型聚酮合成酶(PKS)特征，由8个模块化结构域组成，其N端结合碳链底物的结构域是解析末端烯烃合成机制的关键，生物信息学分析显示该结构域具有脂酰AMP连接酶特征。本项目已鉴定17株可合成不同链长末端烯烃的蓝细菌，并在聚球藻PCC 7002中构建了系列可能涉及OLS底物供应的突变株。基于此，拟采用结构域置换、脂肪酸饲喂、基因敲除、原核表达、酶活分析及质谱鉴定方法，解析蓝细菌末端烯烃合成的底物来源及链长控制机制，并建立以GC-MS和简并PCR相结合的快速筛选末端烯烃合成菌株和克隆关键基因的方法。最后基于获得的关键基因序列，结合生物信息学分析和生化证据，解析OLS的分子进化机制。

脂肪烃是重要的能源化合物。蓝细菌是自然界少数可以直接合成脂肪烃的物种之一。Ado/Aar和Ols途径是目前已被鉴定的蓝细菌脂肪烃合成途径。与Ado/Aar相比，Ols末端烯烃合成途径虽仅在少数蓝细菌中发现，但此类菌株的产烃量却更高，因此本项目的研究重点是蓝细菌Ols末端烯烃途径。取得的主要研究结果总结如下：(1)蓝细菌末端烯烃组成和含量层面：证实蓝细菌至少可以合成三种链长(C15、C17和C19)的末端烯烃，并发现了一株高产末端烯烃蓝细菌; (2)蓝细菌ols基因结构层面：发现末端烯烃合成酶编码基因可划分为三种基本的类型。类型I为FAAL-ACP与其他结构域在同一CDS中，类型II为CDS1（FAAL-ACP）和CDS2为嵌套基因，类型III为CDS1与CDS2之间有7-19bp不等长度的间隔序列；(3) Ols的作用机制层面：发现蓝细菌Ols途径底物活化结构域具有典型的脂酰AMP连接酶特征，并基于脂肪酸饲喂试验证实脂肪酸底物链长组成与脂肪烃链长组成相关，但是脂肪酸含量与脂肪烃产量之间无明显的关联关系；(4) Ols途径产烃特性与分子进化层面：发现ols的独特基因结构跟其脂肪烃的独特性并没有直接的关联，末端烯烃链长特异性与Ols分子进化之间存在一定关联性。本项目较为系统的阐明了蓝细菌末端烯烃类能源化合物的多样性及其分布特征，并初步解析了末端烯烃合成途径的底物活化机制，为未来脂肪烃类生物燃料的定向合成奠定了基础。