基于抗逆基因适配性优化的耐温型鞘氨醇单胞菌的构建与耐热机制解析

Welan gum is a microbial polysaccharide of gellan gum family with broad applications. Sphingomonas sp. is the main fermentation strain for gellan gum polysaccharide production. There are shortcomings of low fermentation temperature and pigment production, resulted in high energy consumption in a viscous fermentation process. A mutant welan gum producing strain S. sp.HT-1 with heat tolerance was breeding by ARTP mutation technology. The genome sequence alignment with wild type strain showed that gene mutant was occurred in some heat shock protein (HSP), which enhanced the HSP stability under higher temperature. In this project, based on the investigation of relationship between HSP structure and activity of extreme microorganisms, the adaptive Sphingomonas HSP proteins will be obtained by artificial optimization. Furthermore, the recombinant strain, with crtB defection and HSP modular assembly integration, will be constructed using CRISPR-Cas9 technology. Then, the influence of HSP protein on welan gum synthesis pathway and the molecular mechanism of HSP protein on cell heat tolerance will be studied. Furthermore, process engineering for improving the performance of welan gum production will be conducted. At the end, the expected results will provide a useful reference of tolerance improvement for industrial microorganisms.

威兰胶是结冷胶家族的重要成员，在石油化工、食品医药等领域具有重要应用价值。鞘氨醇单胞菌（Sphingomonas sp.）是生产结冷胶类多糖的主要菌株，该菌发酵温度偏低、产色素，导致高粘发酵体系能耗较高，分离纯化有机溶剂用量大。前期通过ARTP选育得到一株耐热型菌株S. sp.HT-1，发现部分热休克蛋白（HSP）发生了基因突变，增强了HSP高温下的稳定性。本项目拟在此基础上，研究极端微生物HSP蛋白的构效关系，人工优化获得高效的鞘氨醇单胞菌适配性HSP蛋白；采用CRISPR-Cas9技术获得产色素基因缺失，同时植入HSP模块化组装的重组抗逆菌株；从基因表达和代谢分析入手，研究HSP蛋白对威兰胶合成途径的影响规律，解析HSP蛋白对细胞温度耐受性增强的分子机制；并通过过程工程手段提高重组菌的威兰胶发酵水平，预期结果将为工业微生物的抗逆改造提供有益借鉴。

威兰胶是结冷胶家族的重要成员，在石油化工、食品医药等领域具有重要应用价值。鞘氨醇单胞菌（Sphingomonas sp.）是生产结冷胶类多糖的主要菌株，该菌发酵温度偏低、产色素、发酵后期传质效率低，导致高粘发酵体系能耗较高，分离纯化有机溶剂用量大，生产成本高。前期工作中获得了一株由ARTP选育的耐热型菌株S. sp.HT-1。本项目在此基础上，通过比较基因组学研究确定耐温型菌株在环境胁迫下上调的关键基因—热休克蛋白，阐明了耐热机制，并从极端微生物中挖掘更高效的抗逆元件，人工优化获得高效的鞘氨醇单胞菌适配性抗逆元件—全局转录调节因子（IrrE）；通过基因敲除整合手段沉默了色素合成途径，并获得了稳定遗传的无色素耐热型重组菌株；从基因表达和代谢分析阐明抗逆机制，研究表明全局转录调节因子（IrrE）能上调包括外部环境刺激响应蛋白基因、蛋白激酶基因、代谢和生长相关蛋白基因、鞭毛合成和运动相关基因等，增强菌株环境耐受性。针对生产过程中传质效率低等实际问题，在无色素耐热型重组菌的基础上进行了非包裹型菌株研究及耐氧元件的开发与应用，成功获得多重抗逆型威兰胶菌株，最终可在40 C高温，自然pH条件下获得27.21 ± 0.18 g/L威兰胶产量，同时从鞘氨醇菌的分子应答机制上解析了抗逆原理。本项目成果将为工业微生物的抗逆改造提供有益借鉴，有效降低工业生产成本，具有巨大经济意义。