毕赤巴斯德酵母中法尼基焦磷酸的代谢调控研究

Farnesyl pyrophosphate (FPP) is an important precursor for terpenoids biosynthesis and engineering FPP supply is essential to increase the microbial production of terpenoids, which is considered a feasible approach for sustainable production of bio-chemicals and biofuels. As an important chemical raw material, methanol can be produced in large quantities from coal, natural gas, coke oven gas and other raw materials. The use of microorganisms to convert methanol into high-value compounds can realize the cleanly utilization of coal resources. And the methylotrophic yeast Pichia pastoris can use methanol as sole carbon source for high density fermentation. However, the methanol metabolism toward FPP biosynthesis is significantly different with that of glucose-based FPP biosynthesis. Meanwhile, the FPP accumulation always hindered by the synthesis of ergosterol, fatty acids and other metabolites. This project aims to elucidate the metabolic flux distribution of FPP in P. pastoris. And the biosynthesis efficiency of FPP will be increased through down-regulating the genes relating in the competition pathways, up-regulating key gene expression in FPP synthetic pathways, and constructing heterogeneous FPP synthesis pathway. The research results will elucidate the metabolic distribution of FPP when using methanol as the sole carbon source by P. pastoris and construct a platform strain with high supply of FPP, which will provides a new way to convert methanol into high-value isoprenoid-derived chemicals.

法尼基焦磷酸（Farnesyl pyrophosphate, FPP）是重要的萜类化合物合成前体，调控其生物合成效率是提高微生物萜类物质产量的有效手段。作为重要的化工原料，甲醇可以通过煤炭、天然气、焦炉气等原料进行大量生产，利用微生物将甲醇高效转化为高值化合物可实现煤炭资源的洁净化及多样性利用。甲醇酵母（Pichia pastoris）能够以甲醇为唯一碳源进行高密度大规模发酵，然而其代谢过程与以葡萄糖为碳源时差异显著，同时麦角固醇、脂肪酸等物质的生成也将削弱FPP的积累。本项目拟分析P. pastoris利用甲醇生产FPP的的代谢规律，通过抑制FPP代谢支路，强化FPP供给，构建异源FPP合成途径，提高FPP生物合成效率。研究结果将阐明P. pastoris利用甲醇合成FPP的代谢分配规律，获得FPP高效合成平台菌株，为甲醇高效转化高值化学品提供新途径。

法尼基焦磷酸（Farnesyl pyrophosphate,FPP）是重要的萜类化合物合成前体，调控其生物合成效率是提高微生物萜类物质产量的有效手段。作为重要的化工原料，甲醇可以通过煤炭、天然气、焦炉气等原料进行大量生产，利用微生物将甲醇高效转化为高值化合物可实现煤炭资源的洁净化及多样性利用。毕赤酵母（Pichia pastoris）已被广泛用于生产蛋白质，并作为一种生产化学品的底盘细胞工厂引起了人们的注意。然而，合成生物学工具的缺乏使Pichia pastoris代谢重组方面面临挑战。本研究通过构建基于CRISPR-Cas9的毕赤酵母基因组编辑平台，对重组机制进行了广泛的改造，将同源重组(HR)效率提高了54倍以上，特别是将多个片段同时组装的效率提高了13.5倍。研究还发现，相较于酿酒酵母（Saccharomyces cerevisiae），Pichia pastoris的同源重组关键基因RAD52被强烈抑制。毕赤酵母基因编辑系统实现了高效无缝的基因敲除、基因组整合和多基因组装，阳性率为68 -90%。且以改造的高同源重组效率的Pichia pastoris为底盘微生物，通过整合AgBIS基因成功将甲醇转化为高值化学品没药烯，同时通过强化MVA途径，提高了没药烯前体物质法尼基焦磷酸的供给，使没药烯产量提高了45.2倍。本研究不仅扩大了Pichia pastoris作为遗传工具用于化工生产的范围，也对其他非传统酵母的工程改造研究提供了新的思路。