A scalable platform to identify fungal secondary metabolites and their gene clusters

Abstract

The genomes of filamentous fungi contain up to ~90 biosynthetic gene clusters (BGCs), encoding diverse secondary metabolites, an enormous reservoir of untapped chemical potential. However, recalcitrant genetics, cryptic expression, and unculturability prevent the systematic exploitation of these gene clusters and harvesting of their products. With heterologous expression of fungal BGCs largely limited to expression of single or partial clusters, we established a scalable process for expression of large numbers of full-length gene clusters, called FAC-MS. Using Fungal Artificial Chromosomes (FACs) with Metabolomic Scoring (MS) we screened 56 secondary metabolite BGCs from diverse fungal species for expression in A. nidulans. Fifteen new metabolites were discovered and confidently assigned to their BGCs. A new macrolactone, valactamide A, and its hybrid PKS-NRPS gene cluster were characterized extensively using this integrated platform. Regularizing access to fungal secondary metabolites at an unprecedented scale stands to revitalize drug discovery platforms with renewable sources of natural products.

Biosynthesis of alkylcitric acids inAspergillus nigerinvolves both co-localized and unlinked genes

“…Multiple approaches have been devised over the past decades to identify and produce fungal SMs [17][18] . More recent approaches take advantage of the wealth of information garnered through whole genome sequencing and genome annotation to accelerate novel compound discovery 12,15,[19][20] . The accumulated body of genomic information from many different organisms has allowed for the bioinformatic prediction of SM gene clusters.…”

Interrogation of Benzomalvin Biosynthesis Using Fungal Artificial Chromosomes with Metabolomic Scoring (FAC-MS): Discovery of a Benzodiazepine Synthase Activity

“…Domain deletions were made in the same manner as previously reported for whole gene deletions. 7 Briefly, to delete the benY -C or benZ -C 2 domain, a pair of polymerase chain reaction (PCR) primers of the kanamycin resistance gene (21 bases forward and 18 bases reverse shown in Table S2 as lowercase letters) with an additional 50 bp homology sequence (uppercase letters shown in Table S2) flanking the appropriate domain of either benY -C or benZ -C 2 were used to generate the GalK-PCR or Kan-PCR product, respectively, each of which was gel-purified. FAC DNA was transformed into Red/ET-inducible E. coli strain SW012.…”

“…Metabolite peak areas were integrated, normalized relative to the total ion current, and averaged across biological quadruplicate metabolite extracts to determine metabolite abundances. 7 Error bars for metabolite abundances were derived from the standard deviation.…”

“…Recently, we reported fungal artificial chromosomes with metabolomic scoring (FAC-MS) as a robust new platform for the discovery of fungal specialized metabolites and their biosynthetic gene clusters. 7 FAC-MS enables specialized metabolite discovery through high-throughput capture of intact fungal biosynthetic gene clusters from genomic DNA in an Escherichia coli – Aspergillus nidulans shuttle vector called a FAC, 8 followed by gene cluster heterologous expression in A. nidulans and detection and analysis of small molecule products by untargeted metabolomics and cheminformatics using high-resolution mass spectrometry. Because the FAC serves as a shuttle vector, FAC genetic recombineering in E. coli can be subsequently used for facile “gene cluster editing” of biosynthetic gene clusters, followed by transformation into A. nidulans to enable rapid targeted metabolomic and genetic validation of gene cluster–metabolite relationships.…”

Activation of silent secondary metabolite gene clusters by nucleosome map-guided positioning of the synthetic transcription factor VPR-dCas9

“…They include promoter replacements in cis, overexpression of cluster-specific or global activators in trans, deletion of global regulators, interference with chromatin-based silencing or trans-expression of whole BGCs in a heterologous host and approaches based on the variation of cultivation conditions (i.e. "OSMAC") (Bode et al, 2002;Brakhage and Schroeckh, 2011;Chiang et al, 2010;Clevenger et al, 2017;Connolly et al, 2013;Gacek-Matthews et al, 2016;Gerke et al, 2012;Gerke and Braus, 2014;Hansen et al, 2015;Keller, 2019;Lyu et al, 2020;Niehaus et al, 2016b;Strauss and Reyes-Dominguez, 2011;Studt et al, 2016;Westphal et al, 2019;Wiemann et al, 2013;Wiemann et al, 2018). Promoter exchanges seem only feasible with small predicted clusters or clusters that harbour a clear candidate of BGC-specific transcription factor (TF) that can be overexpressed and converted to an active state.…”