Applications of Fungal Indole Alkaloid Biosynthetic Machinery in Biocatalysis

Abstract

The prenylated fungal indole alkaloids are a diverse class of natural products, highly sought after due to their valuable biological properties and fascinating structures. Specifically, significant effort has been devoted to the elucidation of bicyclo[2.2.2]diazaoctane biosynthesis in fungi to understand the unique transformations native biocatalysts use to generate the diazaoctane scaffold and perform downstream tailoring events. Genome mining and sequence annotation of the biosynthetic gene clusters (BGCs) of the malbrancheamides (mal), paraherquamides (phq), notoamides (not), brevianamides (bvn) and other related fungal indole alkaloids has enabled the identification of the requisite gene products to build these complex natural products. The biosynthetic enzymes in these fungal pathways characterized to date include non-ribosomal peptide synthetases (NRPSs) that couple tryptophan and proline building blocks, prenyltransferases (PTs) that catalyze C2 reverse prenylation of the tryptophan indole, bifunctional reductases/“Diels-Alderases” that catalyze formation of the [2.2.2]diazaoctane core, and additional various tailoring biocatalysts including a halogenase, flavin monooxygenases (FMOs), cytochromes P450, and additional PTs. This dissertation first describes the characterization of three FMOs within the notoamide (NotI and NotI') and paraherquamide (PhqK) biosynthetic pathways that catalyze stereoselective epoxidations followed by spontaneous semipinacol rearrangement and spirocyclization, and the repurposing of several biocatalysts’ native reactivity for applications in pharmaceutical diversification and natural products synthesis. A unique late-stage halogenase, MalA, natively dichlorinates premalbrancheamide to install halogens critical for malbrancheamide’s calmodulin inhibitory activity. Chapter 3 describes the exploration of MalA’s broad substrate scope on a library of complex drug fragments provided by the Novartis Institutes for Biomedical Research (NIBR), and the use of this biocatalyst in achieving orthogonal site selectivity compared to traditional chemical halogenation methods. Additionally, chapter 4 details the results of the high-resolution crystal structures of C2 reverse PT NotF that were solved, revealing the structural basis for its broad substrate scope. In collaboration with the Sigman Lab at the University of Utah, we developed a multivariate linear regression (MLR) model that explains the rates of NotF with a panel of sterically and electronically differentiated tryptophanyl diketopiperazines (DKPs). We were further able to use our model and an induced fit docking protocol to predict and explore active site 1mutations that increased rate on substrates that were slowly turned over by the wild-type. This work also enabled access to the privileged 3- hydroxypyrroloindoline scaffold, and culminated in the first stereoselective synthesis of the marine fungal secondary metabolite (–)-eurotiumin A using a diastereoselective PT- FMO biocatalytic cascade. In Chapter 5, we were inspired by a recent total synthesis of (+)-brevianamides A and B in which a dehydrogenated hydroxypyrroloindoline, dehydrobrevianamide E, served as a key intermediate. Given the diastereoselectivity of BvnB and our ability to couple pyrroloindoline formation with NotF-catalyzed prenyltransfer, we sought to include both biocatalysts in an artificial biosynthetic gene cluster along with four other enzymes for the fermenative construction of dehydrobrevianamide E and subsequent conversion to (+)-brevianamides A and B. Chapter 5 details these efforts to combine cyclodipeptide synthase NascA, cyclodipeptide oxidases DmtD2/DmtE2, kinases PhoN and IPK, PT NotF, and FMO BvnB to selectively access this key intermediate in vivo.