Activation of cryptic biosynthetic gene clusters by fungal artificial chromosomes to produce novel secondary metabolites.

In 2017, we reported the discovery of Berkeleylactone A (BPLA), a novel, potent antibiotic produced exclusively in co-culture by two extremophilic fungi, and , which were isolated from the Berkeley Pit, an acid mine waste lake, in Butte, Montana. Neither fungus synthesized BPLA when grown in axenic culture. Recent studies suggest that secondary metabolites (SMs) are often synthesized by enzymes encoded by co-localized genes that form "biosynthetic gene clusters" (BGCs), which might remain (inactive) under various fermentation conditions.

Terpenoid balance in unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize due to cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into . Here, we demonstrate a new twist to FAC utility wherein heterologous expression of a FAC in altered endogenous terpene biosynthetic pathways.

Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds.

Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs).

Identification of the First Diketomorpholine Biosynthetic Pathway Using FAC-MS Technology.

Filamentous fungi are prolific producers of secondary metabolites with drug-like properties, and their genome sequences have revealed an untapped wealth of potential therapeutic leads. To better access these secondary metabolites and characterize their biosynthetic gene clusters, we applied a new platform for screening and heterologous expression of intact gene clusters that uses fungal artificial chromosomes and metabolomic scoring (FAC-MS). We leverage FAC-MS technology to identify the biosynthetic machinery responsible for production of acu-dioxomorpholine, a metabolite produced by the fungus, Aspergilllus aculeatus.

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

The benzodiazepine benzomalvin A/D is a fungally derived specialized metabolite and inhibitor of the substance P receptor NK1, biosynthesized by a three-gene nonribosomal peptide synthetase cluster. Here, we utilize fungal artificial chromosomes with metabolomic scoring (FAC-MS) to perform molecular genetic pathway dissection and targeted metabolomics analysis to assign the in vivo role of each domain in the benzomalvin biosynthetic pathway. The use of FAC-MS identified the terminal cyclizing condensation domain as BenY-C and the internal C-domains as BenZ-C and BenZ-C.

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

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, the recalcitrant genetics, cryptic expression, and unculturability of these fungi prevent scientists from systematically exploiting these gene clusters and harvesting their products. As heterologous expression of fungal BGCs is largely limited to the expression of single or partial clusters, we established a scalable process for the expression of large numbers of full-length gene clusters, called FAC-MS.

Fungal artificial chromosomes for mining of the fungal secondary metabolome.

Background: With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30-80 kb in size, breakthrough techniques are needed to characterize this SM wealth.

Results: Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E.