毕赤巴斯德酵母中乙酰辅酶A代谢调控

Acetyl-CoA plays an essential role in central metabolism and serves as a precursor for the production of a wide range of industrially very interesting products such as fatty acid derivatives and isoprenoids. Thus, increasing its supply could be a feasible key approach to increase the biosynthesis. There is relative rich coal resources in our country, however, the current coal resource utilization process is rough and causes large pollution. Methanol, derived from coal, is a good feedstock for chemical industry and methanol biotransformation would provide an alternative route for clean utilization of coal. Pichia pastoris is ideal host for methanol transformation due to its fast growth and high efficiency in methanol utilization. However, as a super factory for protein production, its high amino acid biosynthesis may by shunt the metabolic flux from acetyl-coA flux, which limits the downstream biosynthesis capacity for acetyl-coA derived chemicals and biofuels. This proposal will elucidate the metabolic flux distribution between amino acid and acetyl-CoA biosynthesis by using 13C based flux analysis and identify the bottleneck of acetyl-CoA supply. Then the amino acid biosynthesis will be dynamically downregulated and alternative efficient acetyl-CoA biosynthetic pathways will be constructed and optimized, for enhanced acetyl-CoA supply. This study will reveal the regulation mechanisms of acetyl-coA and amino acid/protein biosynthesis and construct a platform strain with high supply of acetyl-coA, which will facilitate the establishing a bio-manufacturing process to bio-transform methanol and other single-carbon feedstocks.

乙酰辅酶A是多种生物化学品及生物燃料的前体，调控其生物合成是提高微生物转化效率的有效手段。甲醇作为重要化工中间体，可以通过煤炭、天然气等转化大量获得。构建甲醇生物转化过程有望实现煤资源洁净利用。甲醇酵母Pichia pastoris生长迅速，易于大规模高密度培养，是甲醇生物转化的理想宿主。然而，P. pastoris作为蛋白质高效生产细胞工厂，蛋白质和氨基酸合成能力强，可能会削弱乙酰辅酶A生物合成代谢流。本项目拟利用代谢流分析P. pastoris中乙酰辅酶A及氨基酸代谢规律，阐明其蛋白质高效合成的代谢基础。动态弱化氨基酸合成，构建及优化乙酰辅酶A生物合成途径，强化乙酰辅酶A供给，从而增加生物合成效率。研究成果将阐明P. pastoris乙酰辅酶A及氨基酸代谢分配规律，获得乙酰辅酶A高效合成平台菌株，实现甲醇生物转化为脂肪酸衍生物等高值化学品，为甲醇等一碳资源洁净利用探索新途径。

甲醇作为重要化工中间体，可以通过煤炭、天然气等转化大量获得。构建甲醇生物转化过程有望实现煤资源洁净利用。甲醇酵母毕赤巴斯德酵母Pichia pastoris生长迅速，易于大规模高密度培养，是甲醇生物转化的理想宿主。乙酰辅酶A是多种生物化学品及生物燃料的前体，调控其生物合成是提高微生物转化效率的有效手段。本项目拟系统探索毕赤酵母中乙酰辅酶A代谢规律，构建及优化乙酰辅酶A生物合成途径，强化乙酰辅酶A供给，从而增加脂肪酸生物合成效率。.本项目执行过程中，建立了基于CRISPR-Cas9的基因编辑工具，并且强化了其同源重组效率，同源重组效率提高了27倍；实现了基因无缝敲除、基因整合以及多篇段整合，多片段同源重组效率提高了13.5倍。利用该高效遗传操作平台，系统鉴定到了46个能用于整合外源基因的中性位点；随后，我们构建了脂肪酸合成的底盘菌株，并且构建了系列乙酰辅酶A生物合成途径，包括基于柠檬酸裂解酶的线粒体-胞浆穿梭途径、丙酮酸脱氢酶及磷酸乙酮醇酶途径等，脂肪酸产量达到了435 mg/L，酯酰辅酶A强化后脂肪酸产量提高了1.6倍。.该项目成果阐明了毕赤酵母中同源重组和非同源重组的竞争机制，并建立了高效遗传操作平台，为拓展毕赤酵母细胞工厂提供了技术支持；阐明了毕赤酵母中乙酰辅酶A的调控机制，主要是从甲醇到乙酰辅酶A的合成是限速步骤，所构建的乙酰辅酶A高产菌株将为合成其它乙酰辅酶A衍生化合物提供技术指导；首次在毕赤酵母中实现了甲醇高效生物转化合成脂肪酸衍生物。