甲醇酵母正交/交互生物器件库设计与精细表达控制

Methanol, a byproduct of the fossil fuel industry, can be produced inexpensively from natural gas, or renewably through reduction of carbon dioxide by hydrogen, and it shows potential to serve as a high-volume, low-cost feedstock for production of pharmaceuticals and chemicals. Engineered strains with methanol utilization pathway usually shows weak methanol consumption abilities and low biosynthetic rates, while most natural methylotrophs are generally known to lack well-developed genetic tools for carrying out extensive biosynthetic pathways. Pichia pastoris (Komagataella pastoris) is a methylotrophic yeast that can utilize methanol as the sole carbon source for its growth and compound biosynthesis. Moreover, a wide range of genetic tools are available for P. pastoris, and many expression vectors have been commercialized. Nonetheless, this chassis host is still lack of suitable and sufficient biological elements and devices for fine tuning expression control on pathway nodes and pathway balancing. This project aims to exploit biological elements from transcription regulation system of AOX1 promoter and construct universally used constitutive and induced device libraries, which are used to re-construct the AOX1 promoter transcription system. Then with the combination use of RNA-seq and CRISPR-dCas9/RNAi, a strict regulation upstream device for low/none leakage expression control of the strong transcription regulation device will be constructed. Finally, we will use lovastatin biosynthetic pathway as a test case, for its fine tuning expression control with various modes involving different combinations of device libraries, which is to evaluate their application results, decrease intermediates accumulation and improve final products titre and develop the potential use of this methylotrophic chassis host.

甲醇来源广泛、价格低廉，可作为碳一底物用于医药、化工产品的生物合成。在非天然甲醇菌株中构建甲醇代谢途径，甲醇利用和产物合成的效率较低；而天然甲醇菌株大多遗传背景不清楚且遗传操作工具缺乏，难以构建及优化复杂的异源途径。巴斯德毕赤酵母可利用甲醇作为单一碳源，遗传背景较清晰，有多种商业化载体，但该底盘宿主尚缺少合适、足量的元器件用于途径节点的精细表达控制和途径优化平衡。本课题拟针对毕赤酵母AOX1启动子转录调控系统发掘生物学元件，并构建通用的组成型和诱导型器件库(正交/交互)，重塑AOX1启动子转录调控系统，同时结合RNA-seq和CRISPR-dCas9/RNAi开发无/低渗漏表达的严谨调控型器件用于控制强表达器件。最后以洛伐他汀生物合成途径为对象，采用正交/交互器件精准控制各个途径节点以评估不同器件库及组合的应用效果，以此降低中间体累积，促进终产物合成效率，发掘和拓展该甲醇宿主的应用潜力。

甲醇来源广泛、价格低廉，可作为优质的碳一底物用于医药、化工产品的生物合成。在非天然甲醇菌株中构建甲醇代谢途径，甲醇利用和产物合成的效率较低；而天然甲醇菌株大多遗传背景不清楚且遗传操作工具缺乏，难以构建及优化复杂的异源途径。巴斯德毕赤酵母可利用甲醇作为单一碳源，遗传背景较清晰，但该底盘宿主尚缺少合适、足量的元器件用于途径节点的精准表达控制和途径优化平衡。.本项目针对巴斯德毕赤酵母PAOX1启动子转录调控系统发掘生物学元件，重塑启动子转录调控系统，构建了组成型cTRDL和甲醇诱导型iTRDL人工转录器件库，器件总量288个，强度覆盖PAOX1启动子的16%~520%；进一步结合RNA-seq和CRISPR-dCas9/RNAi开发低渗漏表达的严谨调控型器件SRUD用于控制强表达器件，具有高强度、灵活可控、可编程的优质性能，为毕赤酵母基因表达提供了新的调控策略，为毕赤酵母细胞工厂的拓展应用提供了新的平台体系。进一步，开发无标记多基因编辑工具，以降血脂药物中间体为对象，理性设计选择器件库的不同器件组合精准控制各个途径节点，优化不同器件库及组合，降低中间体累积，实现终产物合成能力提升3倍以上。在此基础上，基于cTRDL和iTRDL开发了拓展型乙醇响应型器件，开发了碳源、前体、诱导剂三位一体的新型生物合成系统，实现了辛伐他汀 (及洛伐他汀中间体) 产量高达3.2 g/L，并可拓展和提升其他植物源黄酮类分子的生物合成。研究成果对于拓展该甲醇酵母蛋白表达及小分子化学品生物合成的应用范畴、促进甲醇及其他相关碳一底物的生物转化利用具有重要意义。

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