Discovery and biosynthesis of α-diazocarbonyl-containing natural products

Abstract

Microbes perform a variety of complex and synthetically challenging chemical transformations under environmentally benign conditions, including the production of structurally unusual natural products. Discovery and biosynthetic characterization of enzymes involved in microbial natural product biosynthesis may enable new biocatalytic and metabolic engineering approaches to overcome limitations in synthetic chemistry. In addition, identification of new biosynthetic enzymes facilitates genome mining approaches to discover novel natural products with potentially interesting biological activity. A rare subset of natural products contains a highly reactive diazo functional group which is synthetically attractive and often confers potent biological activity. Despite the biological and synthetic utility of diazo-containing metabolites, their biosynthesis remains poorly understood. In this work we discovered new biosynthetic logic for diazo formation which we leveraged to discover diazo-containing molecules through genome mining and reactivity-based screening. Elucidation of the biosynthesis of these molecules revealed an unprecedented diazo-forming metalloenzyme with promising biocatalytic applications. Chapter 2 describes the discovery and characterization of the azaserine biosynthetic gene cluster which encodes biological production of the synthetically enabling ⍺-diazoester functional group. Characterization of the azaserine biosynthetic pathway revealed a novel strategy for diazo production through iterative hydrazine oxidation. Further, our collaborator Dr. Jing Huang (Keasling and Hartwig labs) engineered a biosynthetic pathway for unnatural carbene transfer which utilized the azaserine biosynthetic gene cluster to biologically generate the key ⍺-diazoester carbene donor. Chapter 3 describes the development of a reactivity-based screening workflow using strained cyclooctynes to discover new diazo-containing natural products. Genome mining for iterative hydrazine oxidation machinery revealed putative diazo-producing organisms which were prioritized for reactivity-based screening. This approach revealed two previously unappreciated ⍺-diazoketones, 4-diazo-3-oxobutanoic acid (DOBA) and diazoacetone (DAC), from pathogenic Nocardia with potentially interesting biological roles. Finally, Chapter 4 describes the discovery and characterization of the DOBA and DAC biosynthetic gene cluster. Biochemical characterization confirmed iterative hydrazine oxidation logic and revealed a novel diazo-forming metalloenzyme that catalyzes a biologically unprecedented hydrazone N-oxidation with significant biocatalytic potential. Additionally, discovery of this biosynthetic gene cluster now enables biological production of the known carbene transfer reagent diazoacetone which expands the potential scope of engineered biosynthetic pathways for carbene transfer.