蓝细菌产脂肪烃种质资源的筛选和产烃关键基因分子进化的研究

Concerns about energy security have intensified interest in new sources of transportation fuels. Renewable aliphatic hydrocarbons are promising alternatives as they are same to the major constituents of gasoline, diesel, and jet fuel. Hydrocarbons biosynthesis pathway in most cyanobacteria, consists of an acyl-acyl carrier protein reductase and an aldehyde decarbonylase, has recently been identified. This significant discovery promotes researches on aliphatic hydrocarbons production of cyanobacteria which are abundant in natural resources. However, to date, there are still no reports on screening cyanobacterial germplasm for more significant strains and genes. So the main purposes of this study, based on collecting a large number of cyanobacterial strains, show as follows: (i) Try to illustrate the relationship between cyanobacterial morphology and hydrocarbon patterns by analyzing the physiological characters and hydrocarbons profiles (chain length, branched or not, saturation) of sixty representative cyanobacterial strains, meanwhile find out which have higher hydrocarbons synthesis abilities. (ii) Degenerated primers will be designed based on the conserved amino acid sequences of key genes (acyl-acyl carrier protein reductase and an aldehyde decarbonylase) involved in biosynthesis pathway of aliphatic hydrocarbon and the two genes of significant strains will be cloned. We also attempt to discuss the molecular evolution of these two genes widespread in cyanobacteria and gain a clear idea of the internal relationship among cyanobacterial morphology, hydrocarbon patterns and key hydrocarbon biosynthesis genes. Our study aims at providing high-quality cyanobacterial strains and significant genes resources for future biosynthesis of aliphatic hydrocarbon.

脂肪烃是汽油、柴油和航空煤油的主要成分。可再生脂肪烃因其燃料性能和与现存发动机设备的兼容性被认为是化石燃料的最佳替代品。蓝细菌脂肪烃合成途径的解析为产烃研究奠定了坚实基础，这一途径涉及脂酰载体蛋白还原酶和脂肪醛脱羰基酶两个关键酶。蓝细菌种类丰富，目前尚无针对蓝细菌产烃进行系统筛选的报道，本项目在大量收集蓝细菌种质资源的基础上，拟针对蓝细菌产烃特性和产烃关键基因开展以下研究：一，测定约60株蓝细菌的基本生理指标，分析各菌株的产烃特性，探讨菌株形态特征与产烃链长、饱和度、分支情况等之间的关系，筛选得到产烃量较高的菌株；二，基于产烃关键基因的保守性设计简并引物，克隆特色菌株的产烃关键基因，并分析其分子进化关系，试图解析蓝细菌形态特征、产烃特性和产烃关键基因之间的关联性。本项目的目标是为未来利用蓝细菌合成脂肪烃类生物燃料提供优质的种质资源并基于蓝细菌产烃关键路径获得有价值的基因资源。

脂肪烃在蓝细菌中广泛存在，其合成前体是脂肪酸代谢的中间物。目前在蓝细菌中已鉴定了Aar/Ado和Ols两条脂肪烃合成途径。前者涉及脂酰载体蛋白还原酶和脂肪醛脱甲酰加氧酶，后者由末端烯烃合成酶（类聚酮合成酶I）催化。本项目收集了60余株蓝细菌种质资源，基于其生理特征、产烃特性、产烃关键基因克隆，并结合文献调研，将蓝细菌的脂肪烃组成划分为六个主要的类别，发现细菌形态特征与其产烃特性存在一定关联：支链烷烃仅在丝状蓝细菌中，而单细胞蓝细菌鲜有支链烷烃的存在。本研究基于全基因测序，获得了9株产烃蓝细菌基因组草图。克隆获得了其产烃关键基因全长序列，并在大肠杆菌和蓝细菌中进行了功能验证。基于16S rDNA和Aar/Ado途径关键酶的分子进化分析，发现Aar较之Ado在蓝细菌进化中更为保守。此外，本项目鉴定了17株可以合成末端烯烃的蓝细菌。