Automated discovery of stress-responsive natural products from anaerobic gut fungi in extreme environments

Automated discovery of stress-responsive natural products from anaerobic gut fungi in extreme environments at  Fungal Genetics Conference, Pacific Grove, CA, Asilomar Conference Grounds

Abstract: 

Strict anaerobes from extreme environments harbor extensive but largely untapped biosynthetic potential. Yet natural products from these organisms remain poorly characterized because they are difficult to cultivate at scale and many of their biosynthetic pathways are not organized as contiguous gene clusters. In anaerobic gut fungi (AGF; Neocallimastigomycota), components of nonribosomal peptide synthetases, polyketide synthases, and associated tailoring enzymes are often distributed across chromosomes or embedded within broader stress-response networks, limiting the effectiveness of standard genome-mining approaches. To systematically uncover stress-responsive biosynthetic activity, we developed an automated anaerobic workflow that couples environmental perturbation with microbial co-culture. Leveraging the NSF ExFAB BioFoundry’s liquid-handling systems enclosed within an anaerobic chamber, AGF are exposed to gradients of pH, temperature, nutrient availability, and co-cultures with rumen bacteria such as Fibrobacter succinogenes. Time-resolved samples generate both nucleic acid and metabolite datasets suitable for transcriptomic and metabolomic profiling. To sensitively detect activation of distributed biosynthetic pathways, we employ a multiplex probe-based RT-qPCR library targeting conserved NRPS, PKS, and tailoring domains, enabling rapid screening of hundreds of conditions. Conditions exhibiting transcriptional activation are prioritized for untargeted LC–MS/MS, molecular networking, and functional assays using fungal supernatants against representative rumen microbes. This integration of expression signatures, metabolite families, and microbial interaction effects reveals stress-dependent biosynthetic logic that is not predictable from genome architecture alone. Together, this platform provides a scalable strategy to discover stress-responsive natural products in anaerobes, decode how environmental and microbial cues regulate fragmented biosynthetic pathways, and build a mechanistic framework for engineering resilient microbial communities.