集胞藻PCC 6803 FabG在PHA合成中的功能研究

项目名称： 集胞藻PCC 6803 FabG在PHA合成中的功能研究

项目编号： No.31500294

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 生物科学

项目作者： 刘颖慧

作者单位： 中国科学院大连化学物理研究所

项目金额： 20万元

中文摘要： 聚羟基脂肪酸酯（PHA）是石油基材料和能源的潜在替代物。利用蓝藻光合固碳合成PHA是PHA可持续生产的重要手段。光合自养蓝藻集胞藻PCC 6803中β-酮脂酰-酰基载体蛋白还原酶（SpFabG）是脂肪酸合成途径关键酶，同时由于其具有β-酮脂酰-CoA还原酶活性而可能参与脂肪酸代谢与PHA合成调控，但目前并无明确的实验证据，且其作用方式并不清楚。本项目在体外克隆、表达、纯化SpFabG，考察其作为β-酮脂酰-CoA还原酶的酶反应动力学特征，揭示该酶促反应的热力学倾向和底物选择性；同时在phaB缺失株中验证SpFabG参与PHB和mcl-PHA合成的功能，并通过考察PHB在合成和分解过程中fabG的转录水平以及代谢物含量的变化，推测SpFabG在参与PHB代谢、调节碳流分配的作用，最后通过超表达和敲降实验证实其作用方式，从而为利用蓝藻固定CO2光合自养生产PHA、提高PHA产量奠定理论基础。

中文关键词： 集胞藻；β-酮脂酰-酰基载体蛋白还原酶；聚羟基脂肪酸酯；合成；调控

英文摘要： Polyhydroxyalkanoates (PHA) are potential alternatives of petroleum-based materials and energy. Photosynthetic carbon fixation for PHA biosynthesis in cyanobacteria is an important strategy for sustainable production of PHA. The β-ketoacyl-ACP reductase of photoautotrophic cyanobacteria Synechocystis sp. PCC 6803 (SpFabG) is a key enzyme for fatty acid synthesis, and also it possesses β-ketoacyl-CoA reductase activity, which could entitle it to play regulatory roles in fatty acid metabolism and PHA synthesis. However, hitherto no specific experimental evidence has been found regarding to this putative regulatory function as well as that how it works. In this proposal, the SpFabG is cloned, expressed and purified in vitro for the investigation of its enzymatic reaction dynamics as a β-ketoacyl-CoA reductase to reveal the thermodynamic tendency of the enzymatic reaction and its substrate selectivity. Meanwhile, the participation of SpFabG in the PHB and mcl-PHA synthesis is verified in the phaB knock-out mutant. Then by characterizing the fabG transcript level and metabolites contents during PHB synthesis and mobilization, the functions of SpFabG for the regulation of PHB metabolism and carbon partitioning will be revealed. Finally, the pattern it works will be validated via fabG over-expression and knock-down experiments. This project will provide novel insights into the application of cyanobacteria for PHA production via photoautotrophic CO2 fixation and the enhancement of PHA productivity in cyanobacteria.

英文关键词： Synechocystis; FabG;polyhydroxyalkanoates; synthesis;regulation