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

Abstract

In the past few years, there has been impressive progress in elucidating the mechanism of biosynthesis of various natural products accomplished through the use of genetic, molecular biological and biochemical techniques. Here, we present a comprehensive overview of the current results from our studies on fungal natural product biosynthetic enzymes, including nonribosomal peptide synthetase and polyketide synthasenonribosomal peptide synthetase hybrid synthetase, as well as auxiliary enzymes, such as methyltransferases and oxygenases. Specifically, biosynthesis of the following compounds is described in detail: (i) Sch210972, potentially involving a Diels-Alder reaction that may be catalyzed by CghA, a functionally unknown protein identified by targeted gene disruption in the wild type fungus; (ii) chaetoglobosin A, formed via multi-step oxidations catalyzed by three redox enzymes, one flavin-containing monooxygenase and two cytochrome P450 oxygenases as characterized by in vivo biotransformation of relevant intermediates in our engineered Saccharomyces cerevisiae; (iii) ( )-ditryptophenaline, formed by a cytochrome P450, revealing the dimerization mechanism for the biosynthesis of diketopiperazine alkaloids; (iv) pseurotins, whose variations in the Cand O-methylations and the degree of oxidation are introduced combinatorially by multiple redox enzymes; and (v) spirotryprostatins, whose spiro-carbon moiety is formed by a flavin-containing monooxygenase or a cytochrome P450 as determined by heterologous de novo production of the biosynthetic intermediates and final products in Aspergillus niger. We close our discussion by summarizing some of the key techniques that have facilitated the discovery of new natural products, production of their analogs and identification of biosynthetic mechanisms in our study.

A comparative genomics study of 23 Aspergillus species from section Flavi

“…The section Flavi strains were cultivated as three-point cultures on CYA and YES media for 7 days in the dark at 30°C; subsequently three plugs (6 mm inner diameter) were taken across the colony, 800 µL of isopropanol ethyl acetate (1:3 v/v) with 1% formic acid was added and ultrasonicated for 1 h. The liquid sample was transferred to another tube and evaporated; after this 300 µL of methanol was added to dissolve the pellets and the samples were ultrasonicated for 20 min [88][89][90] . Samples were then centrifuged at max g-power for 2-3 min, and afterward 150 µL of the supernatant was transferred to HPLC vials [91][92][93][94][95][96][97] . Ultra-high-performance liquid chromatography-diode array detection-quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOFMS) was performed on an Agilent Infinity 1290 UHPLC system equipped with a diode array detector.…”

Metabolism of steroidal lactones by the fungus Corynespora cassiicola CBS 161.60 results in a mechanistically unique intramolecular ring-D cyclization resulting in C-14 spiro-lactones

“…2 and Fig. 3), especially as spiro-carbons are a rare metabolic phenomenon within xenobiotic pathways [21,22]. The unique bimodal base catalysed transformation initiates after 48 hours incubation with hydroxylation at C-14 alpha, a position on the steroid nucleus at the trans C/D ring junction (Fig.…”

Molecular Genetic Characterization of an Anthrabenzoxocinones Gene Cluster in Streptomyces Sp. FJS31-2 for the Biosynthesis of BE-24566B and Zunyimycin Ale

“…Starter units vary in different aromatic polyketide biosynthesis pathways, leading to various polyketide backbones [12,13]. Enzymes, such as ketoreductases [14,15], oxygenases [16,17], cyclases [18,19], methyltransferases [20], halogenase [21,22], and glycosyltransferases [23], function on the skeleton to form aromatic structures or provide novel biological activities once the polyketide chains are formed by PKSs [24].…”