深海真菌Phomopsis lithocarpus FS508产生具有抗肿瘤活性的tenellone-大环内酯缀合异二聚体石果素A-G。新型分子间狄尔斯-阿尔德 (DA) 酶的缺乏阻碍了新型生物活性异二聚体的开发。本研究首次发现了一种新型单功能分子间DA酶g7882。 g7882 的缺失导致石果品 A 的消失和前体水平的增加。 g7882的过表达显着提高了石果品A的产量。 g7882DA的体外功能也通过使用tenellone B作为底物的生化反应得到证实。此外，第41簇和第81簇（lit簇）中PKS的KS模块的敲除消除了石果苷的产生，这首次解释了FS508中DA酶介导的异二聚体石果苷的生物合成过程。此外，去除簇 41 中的新型乙酰转移酶 GPAT 和簇 81 中的氧化还原酶异戊烯基转移酶导致 P. lithocarpus 中石果苷 A 的减少。 gpat在P. lithocarpus FS508中的过表达显着提高了石果品A的产量，并产生了新的tenellone衍生物石果卡罗G。本研究为新型异二聚体的生物合成提供了新的DA酶工具，并为阐明石果品的生物合成逻辑提供了生化线索。 lithocarpins.版权所有 © 2024。由 Elsevier B.V. 出版

The deep-sea fungus Phomopsis lithocarpus FS508 produces tenellone-macrolide conjugated hetero-dimer lithocarpins A-G with anti-tumor activities. The deficiency of new intermolecular Diels-Alder (DA) enzymes hindered the development of new bioactive hetero-dimers. A novel single-function intermolecular DA enzyme, g7882, was initially discovered in this study. The deletion of g7882 led to the disappearance of lithocarpin A and an increase in precursor levels. and the overexpression of g7882 significantly improved lithocarpin A yield. The in vitro function of g7882DA was also confirmed by biochemical reaction using tenellone B as a substrate. Additionally, the knockout of KS modules of PKS in cluster 41 and cluster 81 (lit cluster) eliminated the production of lithocarpins, which firstly explains the biosynthetic process of hetero-dimer lithocarpins mediated by DA enzyme in FS508. Furthermore, the removal of a novel acetyltransferase GPAT in cluster 41 and the oxidoreductase, prenyltransferase in cluster81 resulted in the reduction of lithocarpin A in P. lithocarpus. The overexpression of gpat in P. lithocarpus FS508 improved the yield of lithocarpin A significantly and produced a new tenellone derivative lithocarol G. This study offers a new DA enzyme tool for the biosynthesis of novel hetero-dimer and biochemical clues for the biosynthetic logic elucidation of lithocarpins.Copyright © 2024. Published by Elsevier B.V.