小蛋白介导的梭菌溶剂合成调控机制研究

Small proteins (SPs), which generally contain less than 100 amino acid residues, exist in all three domains of life and are “widespread, powerful and novel”. However, as the “dark matter” in proteins, they have not been well studied yet, and to date, our knowledge on SPs was quite limited. In our previously study, we discovered a novel small protein (SP) that is unknown in function but can significantly increase solvents production. It was named Hssp01. The following study indicates that Hssp01 may act on the downstream genes via some interacting proteins and then greatly affect solvents formation. Thus, the underlying mechanism is worth exploring. In this study, we will analyze the alternations in transcriptional profile which was caused by Hssp01 inactivation in C. acetobutylicum by using transcriptome, so as to identify the downstream genes that are influenced by Hssp01, and simultaneously, find the Hssp01’s interacting proteins by using bacterial two-hybrid and Pull-down methods. On this basis, we will further study the relationship between the Hssp01’s interacting proteins and the downstream genes, which will enable us to dissect the action mechanism and the metabolic network of Hssp01. An universality analysis will also be performed. The research will, for the first time, reveal a kind of new SPs that can greatly influence solvents formation in clostridia as well as the relevant action mechanism, which may broaden and deepen our knowledge on SPs, and moreover, provide new clues for metabolic engineering of clostridia.

生物体内存在一类氨基酸残基数少于100的小蛋白，其具有“分布广、作用大、机制新”的特点。但作为蛋白质中的“暗物质”，目前小蛋白的功能研究仍很薄弱，对其认知有限。申请人前期在重要工业微生物—产溶剂梭菌中发现了一个功能未知的小蛋白（命名为Hssp01），其缺失可显著提升梭菌的溶剂合成能力，进一步的研究暗示Hssp01通过互作蛋白作用于下游基因而影响溶剂合成，其作用机制值得探究。本项目拟通过比较转录谱分析，鉴定受Hssp01影响的、与溶剂合成相关的功能基因集群，并采用细菌双杂交和Pull-down技术获取Hssp01的互作蛋白。在此基础上，研究这些互作蛋白与基因集群的作用及关联，最终阐明Hssp01的作用机制及受其影响的代谢网络，并分析普适性。本研究将揭示梭菌中一个与溶剂合成调控相关的新型小蛋白及其作用的新机制，从而拓宽和加深人们对微生物小蛋白的认识，同时为梭菌的代谢工程设计和改造提供新的思路。

短链非编码RNA (sncRNA) 在微生物中发挥重要的代谢调控作用，其功能研究受到广泛关注。但受限于分子技术手段，产溶剂梭菌中的这类研究较为滞后，相关报道极少。我们近期利用mariner转座子插入突变技术构建了产溶剂梭菌模式菌——丙酮丁醇梭菌的随机突变体文库 (超过3万个突变体)，并将该突变体库用于功能筛查，包括生长和产物合成。在完成近600个突变株表型分析后，有幸获得了一株在玉米淀粉发酵时总溶剂产量达到26-27 g/L 的高产菌株，但在以葡萄糖发酵时该突变株的生长和产量则显著降低，暗示转座子插入在该突变株的关键功能基因上。我们随后利用反向PCR 技术定位了引起溶剂高产的功能基因 (CAC2384)。该基因包含198个核苷酸。后续的进一步深入研究发现，该基因上游存在一段DNA序列可编码一个功能性sRNA (sr8384），正是sr8384表达的改变造成了上述突变株的表型变化。进一步的研究发现，sr8384是一个多效调控因子，它可以通过直接或间接的方式控制大量基因的表达，对sr8384进行优化表达后可以显著提升丙酮丁醇梭菌的生长和产溶剂性能。更为重要的是，我们在另一株重要的产溶剂梭菌—拜氏梭菌中也鉴定得到了一个与sr8384序列相似度很高的sRNA (sr8889)，且强化sr8889的表达同样可以提升拜氏梭菌的生长和溶剂产物的合成能力，这表明这类短链非编码RNA可能在产溶剂梭菌中普遍存在并具有重要的应用价值。本研究不仅拓展和加深了我们对sncRNA在产溶剂梭菌中功能的认识，而且为这类工业菌的改造提供了新的思路。