Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin–hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.

已知担子菌可生物合成许多生物学上令人感兴趣的化合物，包括萜类化合物。但是，众所周知，它们很难操作。以前，我们确定了编码负责lagopodins的生物合成酶的基因簇，cuparene型的倍半萜醌类天然产物Coprinopsis灰霉病。在这项研究中，我们试图通过在灰葡萄孢中过表达萜烯环化酶基因cop6来确定lagopodin B和1的生物合成途径的细节，从而提高lagopodin B（1）和相关途径产物的生产力。随机整合的COP6到基因组中KU70缺陷型C.菌菌株导致1的产量增加约2.4倍。然而，整合的COP6到我们指定作为表达升压区域（EBA）的染色体内的高度转录位置导致了〜14倍更大的生产的1。此外，EBA整合菌株使我们能够分离出以前未检测到的产物2，我们确定是已知的化合物，羟基糖蛋白B。这一发现扩展了我们对lagopodin-hitoyol生物合成途径的理解，并允许我们推测相关的同二聚化合物lagopodin C的生物合成的可能机制。我们的结果证明了其潜力。靶向EBA将关键的生物合成基因整合到基因组中，以增强难于获得的化合物的生产，以研究担子菌和其他复杂的真核生物中复杂的次级代谢产物的生物合成。