Pseurotin A is biosynthesized by a polyketide synthase�nonribosomal peptide synthetase hybrid megasynthase. The pseurotin family of natural products is comprised of many analogs that are generated due to C- and O-methylations, and different degrees of oxidation introduced by redox enzymes during the biosynthesis. The redox enzymes, FAD-dependent and cytochrome P450 oxygenases, play a central role in generating structural complexity during natural product biosynthesis. We present here the elucidation of the biosynthetic steps leading to the formation of pseurotin A from the formation of backbone core via a predominant precursor, azaspirene. The deficiency of the hybrid megasynthase allowed us to identify as pseurotin biosynthetic cluster. Each methylation and oxidation step was elucidated using strains carrying some of deletion in the methyltransferase, FAD-dependent monooxygenase and cytochrome P450 oxygenase. Thirteen analogs were identified in this study as intermediates formed during oxidization of azaspirene to pseurotins by those redox enzymes. This approach allowed us to quickly determine the biosynthetic intermediates and the enzyme responsible for the formation of each intermediate. Moreover, the FAD-dependent monooxygenase could play a critical role of an epoxidation followed by two different types of diol formations as well as the C-methylation in the core structure as a bifunctional enzyme. By addressing the redox enzymes we were able to discover that the promiscuity of the redox enzymes allowed the formation of a network of pathways that results in a combinatorial biosynthesis of unnatural intermediates during the formation of pseurotin A from azaspirene. Our approach should expedite elucidation of pathways for other natural products biosynthesized by many enzymes encoded in the wide range of gene clusters present in the fungus Aspergillus fumigatus.

Functional analysis of biosynthetic enzyme from fungus

247th National Meeting (2014)

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