巴斯德毕赤酵母甲醇表达调控因子1促进甲醇高效转化机制研究

Methanol is considered to be a promising C-1 substrate. Although it can be used as a carbon source by Pichia pastoris (P. pastoris), the efficiency is far from satisfactory. Therefore, down this line, the potential capacity for P. pastoris to produce natural products is not yet fully realized. Methanol expression regulator 1 (Mxr1p) has been tentatively identified as an important transcription factor for methanol metabolism in P. pastoris. Detailed elucidation of its function will be beneficial for understanding and improving many defects in methanol application (e.g. low utilization rate of carbon source), changing energy source, improving methanol utilization and increasing type and concentration of intracellular intermediates. It also assumed to play an important role in guiding us to engineer other novel one-carbon nutrient microorganisms. Based on these reasons and the applicant's years’ ground-digging work in P. pastoris, this project intends to explore the key genes and related networks regulated by Mxr1p in methanol metabolism, with consideration of energy supply and material balance through transcriptional regulation and protein interaction. All above attempts aim at reducing CO2 production, improving methanol utilization rate, and constructing a methanol high-efficient utilization strain. Then the adaptive laboratory evolutionary strategy will be adopted to optimize the growth profile. The project will reveal the mechanism of Mxr1p promoting methanol high-efficient utilization, and lay a foundation for the subsequent efficient utilization of C-1 unit and the synthesis of natural products.

甲醇被认为是一类极具前景的一碳单位，巴斯德毕赤酵母虽然以甲醇作为碳源但其甲醇利用率低且对天然产物表现出有限的生产能力。甲醇表达调控因子1(Mxr1p)是毕赤酵母中甲醇代谢的重要转录因子，详尽阐明其在甲醇代谢调控中的功能有助于改良甲醇使用中诸多缺陷、转变代谢所需能量来源、提高甲醇利用率及胞内中间产物种类与浓度，并对其他一碳营养型微生物的构建具有重要的指导作用。基于上述原因及申请人对巴斯德毕赤酵母多年研究，本项目拟通过转录调控及蛋白互作手段，从能量供给与物质平衡角度发掘并调控甲醇代谢过程中受Mxr1p调控的关键基因及相关网络，从而减少CO2生成，提高甲醇利用率，构建甲醇高效利用型巴斯德毕赤酵母，然后结合实验室适应性进化手段，优化细胞生长状态，进一步提高甲醇转化效率。项目将解析巴斯德毕赤酵母重要转录因子Mxr1p促进甲醇高效利用的机制，为后续一碳单位的高效利用及天然产物的合成奠定基础。

甲醇被认为是一类极具前景的一碳单位，巴斯德毕赤酵母虽然以甲醇作为碳源但其甲醇利用率低且对天然产物表现出有限的生产能力。甲醇表达调控因子1(Mxr1p)是毕赤酵母中甲醇代谢的重要转录因子，详尽阐明其在甲醇代谢调控中的功能有助于改良甲醇使用中诸多缺陷、转变代谢所需能量来源、提高甲醇利用率及胞内中间产物种类与浓度，并对其他一碳营养型微生物的构建具有重要的指导作用。在本课题中，我们首先阐述了受Mxr1p调控的甘油转运体(Gt1)同时受到MAPK/HOG 途径的调控,进而参与细胞对于外界胁迫的响应；同时通过对转录组数据分析，解析出与Mxr1p类似转录因子Sut2的在麦角固醇代谢中的重要作用，而麦角固醇作为重要的细胞膜组分，其可以参与到细胞对于甲醇以及甲醇有害代谢物的胁迫相应途径；Gt1作为甘油转运体在甲醇代谢过程中受到Mxr1p的调控，通过对转录组数据分析以及同源序列比对，我们发现并鉴定了锌脂转运蛋白Zrt1同样参与到甲醇代谢进程中。除此以外，通过对毕赤酵母以及其他甲醇代谢通路的分析，并结合区室化等策略，成功将模式生物，酿酒酵母，转变为能够在甲醇作为唯一碳源的基础培养基中生长的甲基营养型微生物，随后通过实验室适应性进化以及基因组测序，转录组测序等发现乙醛酸-丝氨酸途径对于提高细胞对于甲醇的利用以及提高细胞对于有害代谢物耐受性具有重要作用，这为后续构建其他一碳营养型微生物提供了重要的理论依据。