集胞藻pcc6803中双组份反应调控蛋白slr1037对丁醇耐受机制的研究-study on the tolerance mechanism of two-component reaction regulator slr 1037 to butanol in polycystis pcc 6803.docx

摘要丁醇由于其独特的化学性质已成为极具潜力的二代生物能源。近年来，在“微 生物细胞工厂”蓝细菌中，运用合成生物学的方法构建生物燃料丁醇的生产系统 已经引起研究者的广泛关注。然而丁醇对宿主细胞具有一定的毒性，致使蓝藻对 于生物燃料的耐受性非常低，从而限制了其作为可再生宿主的经济实用性。蛋白组学研究发现，在丁醇处理条件下蓝细菌集胞藻PCC 6803中部分双组 份信号转导系统（TCSTSs）得到了不同程度的调控。为了进一步分析双组份信 号转导系统对集胞藻PCC 6803应对丁醇胁迫的调控机制，我们首先构建了丁醇- 响应的TCSTSs基因敲除突变株并对其进行耐受性分析，发现编码反应调控蛋白 的slr1037基因与蓝细菌的丁醇耐受相关。有趣的是，突变体Δslr1037在其它胁迫 条件下与野生株的生长相似，这表明slr1037对蓝细菌的丁醇耐受性调控具有专一 性。其次，我们运用iTRAQ LC-MS/MS蛋白质组学技术和Real Time RT-PCR进一 步分析了Slr1037蛋白可能参与的调控网络。分析结果表明，slr1037基因敲除后， 与光合成、糖酵解/糖异生等中心代谢相关的蛋白及与胁迫应答功能相关的谷氧 还蛋白、多肽甲硫氨酸亚砜还原酶和葡萄糖基甘油磷酸合成酶都普遍下调，这说 明Slr1037在抵抗丁醇胁迫时能调控广泛的细胞功能。此外，对slr1037过表达后 进行表型生长分析表明，丁醇胁迫下过表达菌株的生长明显优于野生型，进一步 说明slr1037与集胞藻的丁醇耐受性相关。本研究揭示了第一个直接与丁醇耐受性相关的信号转导蛋白Slr1037，这对 通过全局转录工程来提高集胞藻对丁醇的耐受性提供了可能的基因靶点。关键词：丁醇胁迫 反应调控蛋白 蛋白质组学 集胞藻ABSTRACTButanol is a promising biofuel with better chemical properties, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts as “microbial factories” for its production. However, butanol is always toxic to cell and cyanobacteria have very low tolerance to biofuels, limiting the economic viability of biofuels production from these renewable producing systems.In previous studies, we have found that a few two-component signal transduction systems (TCSTSs) were differentially regulated in Synechocystis sp. PCC 6803 after butanol treatment. To explore regulatory mechanisms of butanol tolerance, in this work, by constructing gene knockout mutants of the butanol-responsive TCSTS genes and conducting tolerance analysis, we uncovered that an orphan slr1037 gene encoding a novel response regulator was involved in butanol tolerance in Synechocystis. Interestingly, the Δslr1037 mutant grew similarly to the wild type under several other stress conditions tested, which suggests that its regulation on butanol tolerance is specific. Using a quantitative iTRAQ LC-MS/MS proteomics approach coupled with real-time reverse transcription PCR, we further determined the possible butanol-tolerance regulon reg