A Universal Fungal Transposase System for Increasing Natural Product and Protein Titers

项目总结。真菌天然产物(NPs)和酶因其独特和多样化而受到重视 生物活动。旗舰NPs在农业方面的收入高达数亿美元，在 全球每年的药品市场，表明了不同国家对真菌分子的广泛需求 工业。到2030年，全球所有微生物NPs的市场预计将超过3000亿美元。真菌 酶占全球酶市场的一半以上，预计到2031年将超过100亿美元。 尽管我们对化学和酶多样性的广度的计算理解最近取得了进展 在真菌基因组中编码的现有合成生物学表达工具的局限性阻碍了快速访问 到新的和改进的产品。目前开发新真菌产品的瓶颈特别是在 菌株工程的步骤，因为通常观察到，即使是异源菌株也不能产生足够的产物 像分子检测或功能筛选这样基本的东西，更不用说扩大到工业上了 制作。 这项研究建议旨在简化和缩短产生稳定、高滴度的开发时间 将真菌合成生物学工具的最新进展与已证明的 真核转座酶技术。这种方法将克服现有方法的局限性，这些方法 导致低效价性能。Leap-InTM和PiggyBacTM转座酶产品使哺乳动物发生了革命性的变化 通过稳定整合多个菌株快速设计高效价生产菌株 通过剪切和粘贴机制复制完整的转座子有效载荷，而不考虑大小或序列内容 在整个基因组中多次优先进入活性染色质。有了这项技术，生产 稳定产生抗体的细胞系现在只需要几个月的时间，而不是一年或更长时间。不同于任何事情 目前市场上，拟议的真菌转座酶工程平台将利用这些优势 通过确保有效载荷，直接解决异源菌株性能的数量和质量限制 保持完整、稳定，并尽可能在整个基因组中转录活跃。 支持这一目标的具体目标是：(1)建立一个针对异源真菌转座酶的工具包 (2)通过真菌转座酶激活的多重整合过表达NP BGC。这些 AIMS将解决使哺乳动物细胞工程技术适应 困难的丝状真菌，并评估其工程改良高滴度菌株的能力。这项建议可以 使用成熟的分子技术和利用已知的报告和NP控制完成：荧光素酶 作为评估基于拷贝数的转录过度表达的单一酶产物，以及NP BGC 已证实在模型中异源表达的咪唑喹和梨酸酰胺的编码 有机体尼杜拉曲霉。

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.