瘤胃细菌H1纤维素酶合成的群感效应调控研究

It is generally believed that microbial cellulase syntheses are induced by the substrate cellulose, while repressed by the catabolite cellobiose. However, we found that the cellulases production in a cellulytic bacterium, Cellulosilyticum ruminicola H1, a strain we previously isolated from yak rumen, requires culturing the bacterium on cellobiose to gain higher cell biomass. Preliminary assays showed that small extracellular secretes of the bacterium stimulated the lower cell-density culture to produce higher yield of cellulases, as well as elevated the expression of the main cellulase-encoding genes. These imply quorum sensing regulation is involving in the cellulase synthesis in strain H1. On the basis of the draft genome sequence of strain H1, we have determined the primary cellulases and their encoding genes. According to the protein architectures of the dominant cellulases, we concluded that this anaerobic cellulytic bacterium employs the fungi-like free-enzyme system in cellulose degradation. In the proposed study, we are going to determine whether quorum sensing mechanism involves in the regulation of celluase synthesis in strain H1 through achieving the following three aims. Aim one is to obtain the small extracellular molecules, and resolve the chemical structures; and to determine them acting as quorum sensing signals. Aim two is to determine the quorum sensing regulation circuit(s) and the regulated genes including cellulase genes and others. Aim three is to study the regulation mechanisms of the quorum sensing regulators. The outcome of this study would gain new insights into the diverse mechanisms of microbial cellulase synthesis, as well as provide new ideas for promoting microbial cellulase yield in industries.

普遍认为，微生物的纤维素酶合成受底物纤维素的诱导，而被代谢产物纤维二糖抑制。但我们在对居瘤胃降解纤维细菌H1的研究中，发现需要将其在纤维二糖中培养获得高密度细胞时，才能产生高活性的纤维素酶；初步分析发现，菌株H1的小分子分泌物可提高低密度细胞的纤维素酶活，及主要纤维素酶基因的表达。这暗示菌株H1的纤维素酶表达合成依赖细胞密度，即受群感效应调控。目前已完成了H1菌株的基因组序列分析，并鉴定了其主要的纤维素酶系组成及特点，发现该菌的纤维素酶以真菌样的自由酶系为主。本研究拟在前期发现和研究的基础上，通过化学、分子生物学手段为主，转录组技术为辅，探讨菌株H1的纤维素酶合成与群感效应的关系；鉴定菌株H1的群感效应信号分子及其调控系统基因；揭示群感效应所调控的纤维素酶及其相关基因；分析调控蛋白的调控机制。该研究将揭示微生物纤维素酶合成调控的新机制，为获得高产量的微生物纤维素酶提供指导。

微生物合成纤维素酶的诱导机制是理解和提高其纤维素酶产量的前提。普遍接受的理论是纤维素的直接水解产物纤维二糖抑制纤维素酶基因表达。但我们的前期研究发现瘤胃纤维细菌降解H1需要在纤维二糖中培养获得高密度细胞时，才能产生高活性的纤维素酶。因此，本项目开展厌氧纤维素降解细菌的纤维素酶诱导表达机制，以其为获得高产量的微生物纤维素酶提供指导。本研究首先分析了菌株H1的纤维素、半纤维素及降解产物纤维二糖培养物的转录组。结果发现：生长早期高表达的基因主要编码核糖体蛋白和氨基酸代谢，而晚期高表达是编码丙酮酸甲酸裂解酶(PFL) 及蛋白酶基因。说明早期细胞物质的合成活跃，而生长晚期厌氧代谢特有途径可能发挥重要的能量代谢作用。在滤纸培养物中各类纤维素酶基因均在早期上调表达, 说明这些基因的表达主要受底物诱导，而不依赖生长时期。纤维素不仅诱导纤维素酶而且诱导纤维小体、及甘露聚糖酶基因；酶谱结合蛋白组分析支持转录组结果。并且在0.5%纤维二糖存在时抑制菌株H1降解滤纸。发现纤维二糖具有浓度依赖的、对纤维素酶合成的双向调控模式，即低浓度（0.05%）纤维二糖显著诱导细菌H1的关键纤维素外切酶基因、及纤维小体的关键蛋白“脚手架蛋白”基因的表达；而高浓度（0.5%）的纤维二糖则抑制纤维素酶基因的表达及其纤维素酶的合成。之外，还发现木糖对细菌H1阿魏酸酯酶FaeI的诱导在转录后水平，并鉴定了细菌H1的群感效应调控的双组份信号系统AgrCA，推测其调控纤维素酶的表达。本工作的研究结果提供了提高细菌纤维酶产量的依据，并揭示了浓度相关的产物反馈抑制/诱导的双重调控机制。研究结果已整理成文投稿Appl Environ Microbiol. 项目的实施过程培养了两名研究生。