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.