TERPENE: Titer Enhancement via Refactored Precursor pathways for Natural Produce Discovery

I. PROJECT SUMMARY The abundance of available genome sequence information across the tree of life coupled to advances in DNA synthesis and genetic engineering tools have enabled innovative approaches to genome-guided natural product discovery1. Unfortunately, the success-rate of any individual approach (e.g., heterologous expression, regulator engineering, promoter replacement) is still low. Also, existing approaches are challenging to integrate into high- throughput natural product (NP) discovery pipelines. Each biosynthetic gene cluster targeted for activation/NP- discovery requires a bespoke set of genetic reagents (plasmids, sgRNAs, etc.) that must be introduced to the host genome using transgenesis pipelines that have not changed in the past thirty years. Put another way, plasmid creation and transgenesis protocols stand as major bottlenecks that prevent research groups from leveraging the abundant strain and genome sequence resources for high-throughput, genetics-enabled NP discovery. We propose a simple but radical shift in classical approaches by using universal genetic reagents and high- throughput transgenesis to invert the role of strain engineering in NP drug discovery campaigns. In this MIRA proposal we will elaborate on this approach, provide preliminary data to support its effectiveness, and describe plans to expand the technology from a proof-of-concept towards a new paradigm for NP drug discovery. The first innovation in our approach is a shift from bespoke genetic reagents towards universal ‘performance- enhancing plasmids’. These plasmids will encode the expression of transgenes designed to increase secondary metabolism in any recombinant Streptomyces host. Example transgenes include (i) conserved pleiotropic regulators, (ii) mutant RNA polymerases and ribosomal proteins, (iii) the bldA tRNA, (iv) pathways to overproduce precursor pools and/or posttranslational modifications to support secondary metabolism, and (v) combinations of the above elements. This circumvents a major bottleneck in genetic engineering. The second innovation is the development of a novel Streptomyces transgenesis pipeline that incorporates single-pot transgenesis, outgrowth, and NP discovery. By applying this approach in multi-well plates, we will be able to create and screen hundreds of transgenic Streptomyces strains (i.e., the same performance-enhancing plasmid in hundreds of unique genomic contexts). LC-MS/MS coupled with molecular networking will identify novel, over-produced NPs that will be structurally characterized from clonal cultures using traditional scale-up and analytical chemistry approaches. We have a solid foundation to begin work in scaling this approach for finding novel antiviral terpenoids from curated collections of actinobacteria. We will present plans to scale this in future years toward other biological activities (e.g., antibacterial, anticancer) and NP biosynthetic classes (e.g., nucleosides, aminoglycosides, polyketides). Project Number: 1R35GM161283-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Michael Smanski | Institution: UNIVERSITY OF MINNESOTA, MINNEAPOLIS, MN | Award Amount: $423,500 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award B Study Section[MRAB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11258771