甲酸诱导毕赤酵母表达外源蛋白与代谢调控机制研究

Non-methanol carbon source induction of Komagataella phaffii (K. phaffii) is an important topic. The major issue of current non-methanol induction K. phaffii strains is low expression level. Based on high expression level of heterologous protein expression from K. phaffii by formate induction combined with sorbitol in our previous work, this research builds a mechanism of K. phaffii expression by formate induction through the relationship between formate and the transcript factor, upstream active sequence, expression level of K. phaffii. Then metabolism of K. phaffii is reconstructed to synthesize formate through carbon dioxide reduction for higher formate concentration in vivo. The culture strategy of K. phaffii in the condition of supplemental carbon source is optimized for NADH generation without repression of K. phaffii through energy charge monitoring. The branch pathway of supplemental carbon source is blocked through gene knockout with result of high formate metabolic flux. The synergistic effect of carbon dioxide and supplemental carbon source is investigated by techniques of C13 label and mass spectrometry. The mechanism of high protein expression level from K. phaffii in the condition of formate and supplemental carbon source is clarified, establishing an efficient K. phaffii expression system without methanol. This system maintains the strict expression pattern and high expression level, with additional advantage of carbon dioxide utilization, which has an important influence on fundamental research and applied technology of K. phaffii.

非甲醇诱导是毕赤酵母研究的重要方向。但是目前非甲醇诱导系统的主要缺点是表达强度不高。本研究在甲酸可以诱导毕赤酵母，甲酸联合山梨醇使毕赤酵母高效表达外源蛋白的基础上，利用基因敲除和转录组学技术分析甲酸浓度、转录因子、启动子上调序列对毕赤酵母表达的影响，阐明甲酸诱导毕赤酵母的调控机制；重构毕赤酵母代谢途径利用胞内还原二氧化碳合成甲酸，提高胞内甲酸浓度；优化辅助碳源使其提供NADH而不抑制毕赤酵母表达，敲除辅助碳源的分支代谢以增强甲酸代谢，分析生理指标并解释辅助碳源显著提高表达强度的机理，用C13标记结合质谱技术解析二氧化碳和辅助碳源的协同作用，阐明二氧化碳还原为甲酸联合辅助碳源诱导毕赤酵母高效表达外源蛋白的机理，建立高效非甲醇诱导的毕赤酵母系统。该系统保持了毕赤酵母原有的诱导表达严谨性，还具有高表达强度，而且可以利用二氧化碳，对基础研究和工业应用都有重要意义。

毕赤酵母（Komagataella phaffii、原名Pichia pastoris）是一种高效表达重组蛋白的甲基营养型微生物。由于毕赤酵母传统上高效表达外源蛋白需要甲醇诱导，甲醇的毒性致使毕赤酵母表达的食品用酶和药物蛋白受到人们担忧；而且甲醇具有爆炸性，在工业上大量使用危险较大。近年来研究表明较为安全的甲酸可以诱导PAOX1。为阐明甲酸诱导毕赤酵母PAOX1的机理，本项目调控顺式作用元件、转录因子，优化辅助碳源使外源蛋白的表达量提高42%；RNA-seq分析揭示了能量代谢为甲酸诱导PAOX1差异的主要瓶颈，并利用强化辅酶转换的方式予以证明；重构毕赤酵母的甲酸代谢途径，解析了其代谢流向，使毕赤酵母能利用甲酸同时为诱导物和碳源，实现了逆生理代谢，使外源蛋白的表达量提高1倍。最后，建立了能还原CO2 为甲酸，表达强度为传统甲醇诱导系统的1.7倍，进而诱导外源蛋白表达的环境友好、能可持续发展的高效毕赤酵母表达系统。本项目的结果对毕赤酵母高效、环境友好地表达外源蛋白具有重要意义。