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

项目摘要。真菌天然产物和酶因其独特性和多样性而受到重视 生物活性旗舰NP在农业领域拥有数亿美元的收入，在农业领域拥有数百亿美元的收入。 全球每年的制药市场，说明了不同的真菌分子的广泛需求 行业预计到2030年，所有微生物NP的全球市场将超过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。 尽管我们最近在化学和酶多样性的广度的计算理解方面取得了进展， 在真菌基因组中编码，现有合成生物学表达工具的局限性阻碍了快速获取 到新的和改进的产品。目前开发新型真菌产品的瓶颈具体在于 菌株工程化步骤，因为常规观察到异源菌株不能产生足够的产物，甚至 对于像分子检测或功能筛选这样的基础性工作， 生产 这项研究提案旨在简化和缩短开发时间表，以产生稳定，高滴度 通过整合真菌合成生物学工具的最新进展， 真核生物转座酶技术。这种方法将克服现有方法的局限性， 导致低滴度性能。Leap-InTM和piggyBacTM转座酶产品彻底改变了哺乳动物 通过稳定整合多个高滴度生产价值菌株， 完整转座子有效载荷的拷贝，无论大小或序列内容如何，通过剪切和粘贴机制 在整个基因组中多次优先进入活性染色质。有了这项技术， 稳定的抗体生产细胞系现在只需要几个月，而不是一年或更长时间。不像任何 在目前的市场上，拟议的真菌转座酶工程平台将利用这些好处， 通过确保有效载荷，直接解决异源菌株性能的定量和定性限制 在整个基因组中保持完整、稳定和尽可能的转录活性。 支持这一目标的具体目标是：（1）建立一个真菌转座酶工具包， 过表达，和（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，并评估其工程化改进的高滴度菌株的能力。该提案可以 使用已建立的分子技术并利用已知的报告基因和NP对照：荧光素酶 作为单一酶产物，以评估基于拷贝数的转录过表达，以及NP BGC 编码咪唑喹和培他酰胺，已证明其在模型中异源表达 构巢曲霉

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.