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

用于提高天然产物和蛋白质滴度的通用真菌转座酶系统

• 批准号: 10760459
• 负责人: NANCY P KELLER
• 金额: $ 29.53万
• 依托单位: VARIGEN BIOSCIENCES CORPORATION
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-05 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10760459
• 关键词: Acceleration; Address; Adoption; Agriculture; Antibody-Producing Cells; Aspergillus; Aspergillus nidulans; Biological Assay; Cell Culture Techniques; Cell Line; Chemicals; Chromatin; Cloning; Cyclosporine; DNA; Data; Desire for food; Detection; Development; Disparate; Engineering; Ensure; Enzymes; Event; Excision; Fungal Genome; Fungi Model; Fusarium; Future; Gene Cluster; Genetic Transcription; Genome; Genome Stability; Genomics; Goals; Growth; Health; Human; Industrialization; Industry; Infrastructure; Lipase; Lovastatin; Luciferases; Maintenance; Mammalian Cell; Marketing; Methods; Modernization; Molds; Molecular; Natural Products; Neurospora; Paste substance; Penicillium; Performance; Personal Satisfaction; Pharmacologic Substance; Phase; Phenotype; Planets; Play; Population; Positioning Attribute; Production; Proteins; Reagent; Reporter; Research Proposals; Role; Serial Passage; Services; Shuttle Vectors; Site; Small Business Innovation Research Grant; System; Techniques; Technology; Testing; Time; Transposase; Trichoderma; cellular engineering; cost; flexibility; genomic locus; improved; industrial production; innovation; luminescence; manufacture; microbial; model organism; next generation; novel; overexpression; pressure; promoter; screening; synthetic biology; timeline; tool

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.

项目摘要。真菌天然产品（NP）和酶因其独特而多样化而受到重视 生物活性。旗舰NPS指挥数亿美元的农业和数十亿美元 全球制药市场每年在全球范围内说明真菌分子的广泛胃口 行业。所有微生物NP的全球市场预计到2030年将超过3000亿美元（USD）。真菌 酶代表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广度的计算理解最近取得了进展 在真菌基因组中编码，现有的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。当前开发新型真菌产品的瓶颈专门用于 应变工程步骤通常观察到异源菌株不会产生足够的产品，即使 对于类似于分子检测或功能筛选的基本的东西，更不用说工业规模 生产。 该研究建议旨在简化和缩短开发时间表，以产生稳定的高滴定时间 通过将真菌合成生物学工具的最新进展与经过验证 真核转座酶技术。这种方法将通过现有方法来克服限制 导致低滴度性能。 Leap-Intm和PiggyBactm转座酶产品已彻底改变了哺乳动物 通过稳定整合多个，通过迅速设计高滴度生产菌株，通过迅速设计高滴度的培养行业 完整的转座有效载荷的副本，无论大小或序列内容如何，​​都可以通过剪切机制 在整个基因组中多次优先进入活性染色质。有了这项技术，生产 现在，产生细胞系的稳定抗体仅需几个月而不是一年或更长时间。不像任何东西 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 直接通过确保有效载荷来直接解决异源应变性能的定量和定性限制 在整个基因组中保持完整，稳定并具有转录活性。 支持此目标的是以下特定目的：（1）为异源构建真菌转座酶工具包 过表达，（2）通过真菌转座酶启用多个集成的过表达NP BGC。这些 目标将解决将哺乳动物细胞工程技术改编为固有的技术挑战 困难的丝状真菌并评估其工程能力提高了高滴水菌株。该提议可以 使用已知的分子技术和利用已知报告基因和NP对照组完成：荧光素酶 作为单个酶产品，用于评估基于拷贝数的转录过表达，而NP BGCS 编码Imizoquin和Pestalamide已证明在模型中表达异源 有机曲霉尼杜兰人。