食品级毕赤酵母鼠李糖诱导系统高效表达机制的研究

With respect to the prokaryote expression systems commonly used in the production of food enzymes, food-grade and rhamnose-inducible Pichia expression system presented the several advantages including high expression level of target proteins, high secretory capabilities for heter-ologous proteins while secreting only low amounts of endogenous proteins, typical eukaryotic post translation modifications like glycosylation, and many other advantages. It was useful to enhance production of food enzymes in Pichia by disclose the mechanism of high efficient expression.. The widely used Pichia expression system based on methanol inducible AOX1 promoter was not suitable to production of food enzymes as the use of toxic and flammable methanol can cause a considerable safety risk in industrial processes. Thereby, tightly controlled and strong inducible promoters with induction via food-grade compound were in demand. It was reported that expression of genes involved in rhamnose metabolism were strictly induced by rhamnose and rhamnose-induced promoters presented excellent in driving the expression of target proteins. In our previous study, L-rhamnose utilization related genes including four catabolic genes and one regulatory gene were indentified in P. pastoris. Using β-galactosidase and green fluorescent protein as reporters, the promoter of one rhamnose utilization related gene designated as P0075 performed well in driving efficient production of heterologous proteins. Additonally, one mutant P. pastoris strain GS115m grew slowly compared with the wild strain using rhamnose a sole carbon source, and simutaneously biomass of P. pastoris GS115m was much lower than that of the wild strain. However, it was noted that β-galactosidase activity in supernatant of P. pastoris GS115m was enhanced over 60% compared with wild strain at the duration of 48~120 h, and the production of β-galactosidase by P. pastoris GS115m was advantageous over P. pastoris GS115. According to the above results, the strain P. pastoris GS115m was an excellent host for production of heterologous proteins. In this project, characterization of P0075 will be intensively assayed as follows. Binding of regulatory factor RhaR to P0075 was detected by an electrophoretic mobility shift assay (EMSA), and the binding sites were indentified via DNase I footprinting experiment. Subsequently, a functional promoter library was generated based on mutagenesis of P0075, and improved promoters were screen for developing expression vectors in P. pastoris GS115m. In addition, mechamnism of high-efficiency protein expression of P. pastoris GS115m was elucidated via comparative transcriptome analysis of P. pastoris GS115m and the wild strain. . Based on the improved rhamnose-inducible promoter and the excellent expression host P. pastoris GS115m, a Pichia expression system with rhamnose as inducer was developed. By using food grade rhamnose instead of the toxic compound methanol as the inducer, the expression system would be an excellent candidate for driving the production of food-grade and therapeutically important recombinant proteins. Subsequently, several parameters such as rhamnose concentration, fermentation time and dissolved oxygen were optimized to enhance the production of target proteins. Several food enzymes such as chymosin, pectase and pectin esterase were expressed using the expression system.

相对于普遍用于食品酶生产的原核生物表达系统，食品级毕赤酵母鼠李糖诱导表达系统具有表达量高、内源分泌性杂蛋白少、蛋白翻译后修饰等诸多优势，进一步解析系统的高效表达机制对于提升食品酶的安全、高效生产具有重要现实意义。在挖掘了鼠李糖诱导表达基因LRA3的强启动子P0075和鼠李糖代谢相关高产菌株基础上，申请人将应用基因敲除和基因回补、凝胶阻滞实验和DNase 足迹实验，揭示鼠李糖代谢途径中调控因子RhaR调控LRA3高效表达的机制，同时解析P0075的结构特征；应用关键位点饱和突变的方法改良P0075，筛选启动效率显著提高的启动子，并构建高效表达载体；应用比较转录组学分析，鉴定原始菌株和高产菌株显著差异性表达的基因，阐明高产菌株调控外源蛋白高效表达的机制。在此基础上，建立一套具有自主知识产权的、由可食用的鼠李糖作为诱导剂的食品级毕赤酵母高效表达系统，实现食品酶在毕赤酵母中安全、高效生产。

前期建立的毕赤酵母鼠李糖诱导表达系统可满足安全性要求极高的重组蛋白（食品级和医药级）生产，并通过随机突变获得的毕赤酵母工程菌株以鼠李糖为碳源时重组蛋白表达水平相对于亲本菌株明显提高。本研究从多个层面、多个维度上剖析了工程菌株高效表达系统机制：鼠李糖代谢关键基因表达水平的降低引发了宿主轻度碳源饥饿，宿主通过增强鼠李糖代谢相关基因的转录和表达（除LRA4）以缓解饥饿；由于鼠李糖代谢关键基因LRA3的启动子PLRA3转录强度提高，其调控外源基因转录能力随之增高，进而导致重组蛋白表达水平显著提升。为进一步优化表达系统，阐明了鼠李糖诱导强启动子PLRA3的结构特征。凝胶阻滞实验（EMSA）确认调控因子RhaR可与PLRA3结合，足迹试验（DNaseⅠ footprinting）揭示结合位点为5´-TAAACTGAAA-3´，表明调控因子RhaR通过结合PLRA3特定位点进而调控LRA3表达。通过CR-RT-PCR方法确定了PLRA3转录起始位点位于翻译起始位点上游22 bp处，为序列5´-TACCCCAGA-3´的第2个A。应用生物信息学预测了PLRA3的核心启动子、基础转录作用元件TATA盒及可能的顺式作用元件。基于以上信息，通过易错PCR构建了PLRA3随机突变体库，以乳糖酶LacB为报告基因进行高通量筛选，获得到转录活性提高25%的改良型启动子。基于工程菌株和改良的启动子进一步优化了鼠李糖诱导表达系统，进一步提高了重组蛋白表达水平。此外，证实了PLRA3在鼠李糖存在时高强度转录、在无鼠李糖时呈低水平渗漏表达，进而建立了基于PLRA3的毕赤酵母无痕敲除系统，可用于毕赤酵母底盘细胞开发，以进一步提高重组蛋白表达水平和在毕赤酵母中开展合成生物学以生产附加值高的次级代谢产物。