Nature's ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF-SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF-SH didomains. Further analysis of the representative genomes from each of the clades identified 28 -type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature's rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature's biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.

The guangnanmycins (GNMs) belong to a small group of natural products featuring a 1-aminocyclopropane-1-carboxylic acid (ACC) moiety. While extensively studied in plants, ACC biosynthesis in bacteria remains poorly understood. Here we report inactivation of gnmY in vivo and biochemical characterization of GnmY in vitro, assigning GnmY as the first bacterial free ACC synthase that catalyzes the synthesis of ACC from Sadenosyl methionine. ACC is activated by GnmS and subsequently incorporated into the GNM scaffold by the GNM hybrid nonribosomal peptide synthetase−polyketide synthase system in GNM biosynthesis. GnmS exhibits relaxed substrate specificity, exploitation of which allowed the incorporation of 1-aminocyclobutane-1-carboxylic acid (ACBC) into the GNM scaffold to produce a GNM analogue with a cyclobutane ring at C-17. This study provides new insights into ACC biosynthesis in bacteria. GnmY and GnmS might be portable to engineer other ACC/ACBC-containing natural products.

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A new natural mycotoxin was isolated from the fermentation broth of Trichoderma sp. Jing-8 and the structure was determined as alternariol 1'-hydroxy-9-methyl ether (1), together with twelve known compounds. The structures were elucidated on the basis of their 1D, 2D NMR spectra and mass spectrometric data. Compounds 1, 8 and 9 indicated inhibitions against germination of the seeds of cabbage with MICs < 3 μg/mL. The compound 1 showed the antibacterial activity against Bacillus subtilis and Staphylococcus aureus with MICs at 64 μg/mL. Compound 1 and 3 showed significant DPPH radical-scavenging activities with IC at 12 μg/mL, respectively. The OH at C-1' in compound 1 decreased the cytotoxicity of these mycotoxins. A primary structure-activity relationship about the alternariol derivatives was discussed. Compounds 2-7 and 8 were the first time to be isolated from the Trichoderma.