Key Laboratory of Biomass Chemical Engineering of Ministry of Education & National Key Laboratory of Biobased Transportation Fuel Technology, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, 310027, China.
ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou, 310000, China.
Metab Eng. 2024 Jul;84:83-94. doi: 10.1016/j.ymben.2024.06.007. Epub 2024 Jun 17.
Monoterpene indole alkaloids (MIAs) are a group of plant-derived natural products with high-value medicinal properties. However, their availability for clinical application is limited due to challenges in plant extraction. Microbial production has emerged as a promising strategy to meet the clinical demands for MIAs. The biosynthetic pathway of cis-trans nepetalactol, which serves as the universal iridoid scaffold for all MIAs, has been successfully identified and reconstituted. However, bottlenecks and challenges remain to construct a high-yielding platform strain for cis-trans nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of Pichia pastoris cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and cis-trans nepetalactol. By targeting the biosynthetic pathway from acetyl-CoA to geraniol in both peroxisomes and cytoplasm, we achieved comparable geraniol titers in both compartments. Through protein engineering, we found that either G8H or CPR truncation increased the production of 8-hydroxygeraniol, with a 47.8-fold and 14.0-fold increase in the peroxisomal and cytosolic pathway strain, respectively. Furthermore, through a combination of dynamical control of ERG20, precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of cis-trans nepetalactol, with a titer of 4429.4 mg/L using fed-batch fermentation in a 5-L bioreactor. We anticipate our systematic metabolic engineering strategies to facilitate the development of P. pastoris cell factories for sustainable production of MIAs and other plant natural products.
单萜吲哚生物碱（MIAs）是一组具有高药用价值的植物衍生天然产物。然而，由于植物提取方面的挑战，它们在临床上的应用受到限制。微生物生产已成为满足 MIAs 临床需求的一种有前途的策略。顺式-反式新薄荷醇的生物合成途径已被成功鉴定并重建，顺式-反式新薄荷醇是所有 MIAs 的通用环烯醚萜支架。然而，构建高产顺式-反式新薄荷醇的平台菌株仍然存在瓶颈和挑战，这对于随后的 MIAs 生物合成至关重要。在本研究中，我们专注于工程酿酒酵母细胞工厂，以提高香叶醇、8-羟基香叶醇和顺式-反式新薄荷醇的产量。通过靶向过氧化物酶体和细胞质中从乙酰辅酶 A 到香叶醇的生物合成途径，我们在两个隔室中都实现了可比的香叶醇滴度。通过蛋白质工程，我们发现 G8H 或 CPR 截断都增加了 8-羟基香叶醇的产量，过氧化物体和细胞质途径菌株分别增加了 47.8 倍和 14.0 倍。此外，通过 ERG20 的动力学控制、前体和辅助因子供应工程、二倍体工程和双亚细胞区室化工程的组合，我们使用 5-L 生物反应器中的分批补料发酵实现了顺式-反式新薄荷醇的最高产量，达到 4429.4 mg/L。我们预计我们的系统代谢工程策略将有助于开发毕赤酵母细胞工厂，以可持续生产 MIAs 和其他植物天然产物。
