基于生物催化剂构建的细胞耐溶剂表型分子机制研究

Biocatalysis is a green technology compared with traditional chemistry and chemical process. However, industrial application of whole-cell biocatalysis is usually hindered by the toxicity of most organic solvents. The organic-solvent-tolerant (OST) mechanisms of microbial cells have been investigated since the first OST strain P. putida was reported in 1989. However, The OST mechanisms of microorganisms are not completely understood so far. In this study, proteomic analysis will be applied to analyze total cellular protein samples of OST adaptive mutants, Pseudomonas putida JUCT1 and Escherichia coli JUCT609, to identify OST associated proteins and to further understand its machinery. Additionally, global transcription machinery engineering (gTME) in combining with DNA microarray will be applied to improve organic-solvent tolerance of non-OST Escherichia coli strains, and the underlying mechanism of the OST phenotype will also be elucidated. Therefore, this study is important for understanding the microbial OST mechanisms, and will provide molecular basis and feasible approach for constructing Escherichia coli as well as other Gram-negative OST strains with independent intellectual property for industrial whole-cell biocatalysis applications.

生物催化是最绿色的技术之一，而有机溶剂对于微生物细胞的毒性往往成为限制整体细胞生物催化剂工业化应用的最大障碍之一。有关微生物细胞耐溶剂性的分子机制是国际上的研究热点，相关机制尚待建立和完善。本课题拟从耐溶剂驯化菌株Pseudomonas putida JUCT1和Escherichia coli JUCT609出发，一方面采用蛋白质组学方法，进行全基因组的表达水平分析，鉴定与细胞耐溶剂性相关的基因并研究其作用机制；另一方面采用全局转录机制调控手段，并结合基因芯片技术，研究与细胞耐溶剂表型相关的全局转录调控机制。基于以上耐溶剂性相关基因和全局转录调控机制的研究，建立和完善微生物细胞溶剂耐受性的分子机制，为构建包括大肠杆菌和其它革兰氏阴性菌的耐溶剂性整体细胞生物催化剂提供理论依据和有效途径。因此，本课题对开发有实际应用价值和自主知识产权的耐溶剂型整体细胞生物催化剂具有重要意义。

从自然界筛选获得的恶臭假单胞菌（Pseudomonas putida）出发，驯化得到一株能够在60%（V/V）的环己烷中生长的菌株JUCT1。通过蛋白组学分析，从22个表达量差异均超过50%的蛋白质中，鉴定获得可显著提高大肠杆菌溶剂耐受性的3-羟基异丁酸脱氢酶编码基因mmsB。通过对全局转录因子σ70的编码基因rpoD进行随机突变构建突变文库，结合高通量筛选，获得σ70优良突变株C9和B8，分别能使大肠杆菌JM109耐受69%（v/v）环己烷和2%（v/v）丁醇。采用基因芯片分析方法研究B8基因组的转录水平差异。通过基因芯片数据分析，得到329个转录水平相差2倍以上的差异基因，包括197个上调基因和132个下调基因。揭示了相关基因通过调控细胞膜不饱和脂肪酸的合成、膜蛋白的合成、细胞膜脂肪酸组分等方面对提高微生物溶剂耐受性的作用机制。本研究对大肠杆菌中溶剂耐受性相关基因的作用机制进行了深入探究，为基因工程改造微生物提高其溶剂耐受性提供了理论依据。