集胞藻pcc6803中与乙醇胁迫相关的转运蛋白slr0982的鉴定分析-identification and analysis of transport protein slr 0982 related to ethanol stress in polycystis pcc 6803.docxVIP

摘要在最近兴起的合成生物学研究中，光合蓝细菌经常被用于构建生产化学品和 生物燃料如乙醇的工程菌。然而，其中限制乙醇产量的一个重要因素就是蓝细菌 底盘对乙醇耐受性很低。转运蛋白是一种介导离子、小分子或者大分子物质跨膜 转运的蛋白质，经常被发现与对外界胁迫的耐受性相关。在本研究中，为了鉴定 与乙醇耐受相关的转运蛋白，我们利用同源重组原理对集胞藻 PPC 6803 中 58 个编码转运蛋白的基因进行了逐一敲除，通过筛选各突变株在乙醇胁迫下的表型，发现编码 ABC 转运蛋白基因的突变株△slr0982 在含有 1.5%(v/v)乙醇的培养基中，与野生株相比生长速率较慢，而互补株?slr0982/pXT-slr0982 生长速率几乎与野生株相同，表明集胞藻 PPC 6803 中的基因 slr0982 与乙醇耐受相关。为了探索 该基因与乙醇耐受相关的机制，利用代谢组学并结合生物信息学分析，对野生株和乙醇敏感株△slr0982 进行了研究。通过 WGCNA 网络分析，我们鉴定出 4 个与基因 slr0982 敲除相关的代谢模块，其中包括中心代谢物蔗糖和左旋焦谷氨酸，可能在细胞对抗乙醇毒性过程中起重要作用。本研究第一次在集胞藻 PPC 6803 中报道与乙醇耐受相关的转运蛋白，这可能为进一步研究溶剂耐受机制提供一个 有用的靶点。此外，利用代谢组学结合网络分析的方法为更准确地揭示集胞藻 PPC 6803 的乙醇耐受机制提供了重要的视点。关键词：基因敲除 乙醇耐受 转运蛋白 代谢组学 集胞藻ABSTRACTCyanobacteria have been engineered to produce biofuel ethanol through recent synthetic biology efforts. However, one major challenge to the cyanobacterial systems for high-efficiency ethanol production is their low tolerance to the ethanol toxicity. With a major goal to identify novel transporters involved in ethanol tolerance, we constructed gene knockout mutants for 58 transporter-encoding genes of Synechocystis sp. PCC 6803 and screened their tolerance change under ethanol stress. The efforts allowed discovery of a mutant of slr0982 gene encoding an ATP-binding cassette transporter which grew poorly in BG-11 medium supplemented with 1.5% (v/v) ethanol when compared with the wild type, and the growth loss could be recovered by complementing slr0982 in the?slr0982 mutant, suggesting that slr0982 is involved in ethanol tolerance in Synechocystis. To decipher the tolerance mechanism involved, a comparative metabolomic and network-based analysis of the wild type and the ethanol-sensitive?slr0982 mutant was performed. The analysis allowed the identification of four metabolic modules related to slr0982 deletion in the ?slr0982 mutant, among which metabolites like sucrose and L-pyroglutamic acid which might be involved in ethanol tolerance, were found important for slr0982 deletion in the ?slr0982 mutant. This study