A Universal Fungal Transposase System for Increasing Natural Product and Protein Titers - PROJECT SUMMARY. Fungal natural products (NPs) and enzymes are valued for their unique and diverse bioactivities. Flagship NPs command hundreds of millions of dollars in agricultural and tens of billions in pharmaceutical markets per year globally, illustrating a broad appetite for fungal molecules across disparate industries. Global markets for all microbial NPs are projected to exceed $300 billion (USD) by 2030. Fungal enzymes represent over half the worldwide enzyme market, which is projected to exceed $10 billion by 2031. Despite recent progress in our computational understanding of the breadth of chemical and enzymatic diversity encoded in fungal genomes, limitations in existing synthetic biology expression tools have stymied rapid access to new and improved products. The current bottleneck in developing novel fungal products is specifically at the strain engineering step as it is routinely observed that heterologous strains do not produce enough product, even for something as fundamental as molecular detection or functional screening, let alone scaleup for industrial production. This research proposal seeks to simplify and shorten development timelines for generating stable, high titer heterologous production strains by integrating recent advances in fungal synthetic biology tools with proven eukaryotic transposase technologies. This approach would overcome limitations seen with existing methods that result in low titer performance. Leap-InTM and piggyBacTM transposase products have revolutionized mammalian cell culture industries by rapidly engineering high titer production-worthy strains by stably integrating multiple copies of an intact transposon payload, regardless of size or sequence content, by a cut-and-paste mechanism multiple times throughout the genome preferentially into active chromatin. With this technology, the production of stable antibody producing cell lines now only takes a few months rather than a year or more. Unlike anything on the market currently, the proposed Fungal Transposase engineering platform will leverage these benefits to directly address quantitative and qualitative limitations in heterologous strain performance by ensuring payloads remain intact, stable, and as transcriptionally active throughout the genome as possible. Supporting this objective are the following specific aims: (1) Build a Fungal Transposase toolkit for heterologous overexpression, and (2) Overexpress NP BGCs by Fungal Transposase-enabled multiple integrations. These aims will address the technical challenges inherent to adapting a mammalian cell engineering technology to the difficult filamentous fungi and assess its capacity for engineering improved high titer strains. This proposal can be completed using established molecular techniques and leveraging known reporter and NP controls: luciferase as a single enzyme product to evaluate copy number-based transcriptional overexpression, and NP BGCs encoding imizoquin and pestalamide which have been demonstrated to heterologously express in the model organism Aspergillus nidulans.
