Combinatorial Generation of Complexity by Redox Enzymes in the Chaetoglobosin A Biosynthesis

Redox
enzymes play a central role in generating structural complexity
during natural product biosynthesis. In the postassembly tailoring
steps, redox cascades can transform nascent chemical scaffolds into
structurally complex final products. Chaetoglobosin A (1) is biosynthesized by a hybrid polyketide synthase–nonribosomal
peptide synthetase. It belongs to the chaetoglobosin family of natural
products, comprising many analogs having different degrees of oxidation
introduced during their biosynthesis. We report here the determination
of the complete biosynthetic steps leading to the formation of 1 from prochaetoglobosin I (2). Each oxidation
step was elucidated using Chaetomium globosum strains
carrying various combinations of deletion of the three redox enzymes,
one FAD-dependent monooxygenase, and two cytochrome P450 oxygenases,
and in vivo biotransformation of intermediates by heterologous expression
of the three genes in Saccharomyces cerevisiae. Five
analogs were identified in this study as intermediates formed during
oxidization of 2 to 1 by those redox enzymes.
Furthermore, a stereochemical course of each oxidation step was clearly
revealed with the absolute configurations of five intermediates determined
from X-ray crystal structure. This approach allowed us to quickly
determine the biosynthetic intermediates and the enzymes responsible
for their formation. Moreover, by addressing the redox enzymes, we
were able to discover that promiscuity of the redox enzymes allowed
the formation of a network of pathways that results in a combinatorial
formation of multiple intermediate compounds during the formation
of 1 from 2. Our approach should expedite
elucidation of pathways for other natural products biosynthesized
by many uncharacterized enzymes of this fungus.

氧化还原酶（Redox enzymes）在天然产物的生物合成过程中，对构建分子结构复杂性发挥核心作用。在组装后修饰阶段，氧化还原级联反应可将新生的化学骨架转化为结构复杂的终产物。球毛壳甲素A（1，Chaetoglobosin A）由聚酮合酶-非核糖体肽合成酶（polyketide synthase–nonribosomal peptide synthetase）杂合体系催化生物合成，隶属于球毛壳甲素类天然产物家族，该家族包含多种在生物合成过程中被引入不同氧化修饰程度的类似物。本研究阐明了从前球毛壳甲素I（2，prochaetoglobosin I）生成1的完整生物合成路径。研究通过两类实验手段解析每一步氧化反应：其一，使用携带三种氧化还原酶、一种黄素腺嘌呤二核苷酸（FAD）依赖型单加氧酶以及两种细胞色素P450加氧酶不同缺失组合的球毛壳（Chaetomium globosum）菌株；其二，将上述三个基因在酿酒酵母（Saccharomyces cerevisiae）中异源表达，实现中间体的体内生物转化。本研究共鉴定出5种类似物，它们是上述氧化还原酶介导2氧化生成1过程中形成的关键中间体。此外，通过解析5种中间体的绝对构型（基于X射线晶体结构（X-ray crystal structure）），本研究明确了每一步氧化反应的立体化学进程。该研究策略使得我们能够快速确定生物合成中间体及其对应的催化酶类。此外，通过针对氧化还原酶的研究，我们发现这类酶的底物宽泛性促成了一条代谢通路网络，使得在2转化为1的过程中，多种中间体化合物以组合方式生成。本研究的策略将有助于加速阐明该真菌中大量未表征酶所参与的其他天然产物的生物合成通路。