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 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 nepetalactol production, which is vital for subsequent MIAs biosynthesis. In the present study, we focused on engineering of cell factories to enhance the production of geraniol, 8-hydroxygeraniol, and 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 , precursor and cofactor supply engineering, diploid engineering, and dual subcellular compartmentalization engineering, we achieved the highest ever reported production of 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 cell factories for sustainable production of MIAs and other plant natural products.

中文翻译：

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毕赤酵母代谢工程过量生产顺反荆芥内酯


单萜吲哚生物碱（MIA）是一组具有高价值药用特性的植物源性天然产物。然而，由于植物提取方面的挑战，它们的临床应用受到限制。微生物生产已成为满足 MIA 临床需求的一种有前景的策略。荆芥内酯的生物合成途径作为所有 MIA 的通用环烯醚萜支架，已被成功鉴定和重建。然而，构建用于荆芥内酯生产的高产平台菌株仍然存在瓶颈和挑战，这对于后续的MIA生物合成至关重要。在本研究中，我们重点关注细胞工厂的工程设计，以提高香叶醇、8-羟基香叶醇和荆芥内酯的产量。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，我们在两个区室中获得了相当的香叶醇滴度。通过蛋白质工程，我们发现G8H或CPR截短都会增加8-羟基香叶醇的产量，过氧化物酶体和胞质途径菌株分别增加47.8倍和14.0倍。此外，通过前体和辅因子供应工程、二倍体工程和双亚细胞区室化工程的动态控制相结合，我们利用补料分批发酵实现了有史以来最高的荆芥内酯产量，滴度为 4429.4 mg/L。 5升生物反应器。我们预计我们的系统代谢工程策略将促进细胞工厂的发展，以可持续生产 MIA 和其他植物天然产品。