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

Abstract

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.

Armochaetoglobins A–J: Cytochalasan Alkaloids from Chaetomium globosum TW1-1, a Fungus Derived from the Terrestrial Arthropod Armadillidium vulgare

“…A comparison of the spectroscopic data for 2 and 1 indicated The relative configuration of 2 was in agreement with that of 1 based on the similar NOESY spectra and NMR patterns, as well as biosynthetic considerations. 8,23,24 As shown in Figure S4 (SI), the Cotton effects in the experimental ECD spectrum of 2 were consistent with those in the calculated ECD curves. Therefore, the absolute configuration of 2 was deduced with the exception of C-16.…”

Targeted Disruption of Transcriptional Regulators inChaetomium globosumActivates Biosynthetic Pathways and Reveals Transcriptional Regulator-Like Behavior of Aureonitol

“…Regarding CHGG_01239, we reported elsewhere recently that it is responsible for the biosynthesis of 7. 50 LaeA Deletion Leads to No New Activation of Biosynthetic Gene Cluster. We also attempted to investigate the effect of deleting the laeA gene, which codes for a wellknown global regulator controlling expression of secondary metabolite biosynthetic gene clusters in A. nidulans.…”

Effective Use of Heterologous Hosts for Characterization of Biosynthetic Enzymes Allows Production of Natural Products and Promotes New Natural Product Discovery

“…Thus, we hypothesize that a Diels-Alder reaction is involved in the biosynthesis of Sch210972. A similar mechanism has also been proposed for the PKS-NRPS-mediated formation of the core scaffolding structure of prochaetoglobosin I (2), the precursor of chaetoglobosin A (3) (see the next section for details) also from C. globosum 33,34) (Chart 3A), cytochalasin E from another fungus Aspergillus clavatus NRRL 1 (Chart 3B), and equisetin in Fusarium heterosporum. 35) Most interestingly, targeted disruption of cghA from the Sch210972 biosynthetic gene cluster in C. globosum led to the formation of an exo form of the product 5 as determined by NMR spectroscopy and X-ray crystallography, in addition to the endo product 1, which is the sole product isolated from the wild type C. globosum.…”

“…By preparing multiple knockout strains, we were able to determine the function of three genes encoding for redox enzymes, one flavin-containing monooxygenase (FMO) (CHGG_01242-2) and two cytochrome P450 oxygenases (P450s) (CHGG_01242-1 and CHGG_01243) 34) as illustrated in Chart 4. We also identified four products, prochaetoglobosin IV (6), 33,43) 20-dihydrochaetoglobosin A (7), 44) cytoglobosin D (8), 45) and chaetoglobosin J (9), 37) as intermediates formed by the redox enzymes during the biosynthesis of 3 from 2 based on the chemical structures of those compounds.…”

“…Those findings reiterate the critical role oxygenating enzymes, such as FMOs and P450s, play in introducing chemical diversity into natural products. 34,46) …”