Enabling Plant Natural Product Biosynthesis by Debugging Heterologous Protein Expression in Yeast

通过调试酵母中的异源蛋白表达实现植物天然产物的生物合成

• 批准号: 10335983
• 负责人: Mark Alan Blenner
• 金额: $ 39.55万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10335983
• 关键词: Address; Agriculture; Alkaloids; Anabolism; Biochemical; CRISPR/Cas technology; Chemistry; Engineering; Enzymes; Failure; Fatty Alcohols; Flavonoids; Genes; Genetic Engineering; Goals; Health; Human; Libraries; Lipids; Microbe; Natural Products; Omega-3 Fatty Acids; Pathway interactions; Plants; Production; Property; Proteins; Research; Structure; System; Terpenes; Therapeutic; Therapeutic Effect; Time; Universities; Yarrowia lipolytica; Yeasts; base; bioactive natural products; cofactor; cost; genome editing; improved; lipid biosynthesis; microbial; novel; promoter; protein expression; response; synthetic biology; tool; Address; Agriculture; Alkaloids; Anabolism; Biochemical; CRISPR/Cas technology; Chemistry; Engineering; Enzymes; Failure; Fatty Alcohols; Flavonoids; Genes; Genetic Engineering; Goals; Health; Human; Libraries; Lipids; Microbe; Natural Products; Omega-3 Fatty Acids; Pathway interactions; Plants; Production; Property; Proteins; Research; Structure; System; Terpenes; Therapeutic; Therapeutic Effect; Time; Universities; Yarrowia lipolytica; Yeasts; base; bioactive natural products; cofactor; cost; genome editing; improved; lipid biosynthesis; microbial; novel; promoter; protein expression; response; synthetic biology; tool

PROJECT SUMMARY The Blenner Research Group at Clemson University is broadly focused on using genetic engineering and synthetic biology to improve microbial systems for applications in human health, sustainability, and national defense. Our research is aimed at developing the capabilities to accurately and precisely control the properties of oleaginous yeast – Yarrowia lipolytica. This yeast has naturally evolved the capability to produce a significant amount of lipid. As such, they are useful for producing biochemicals derived from lipids, such as omega-3 fatty acids and fatty alcohols. As oleaginous yeast are considered non-conventional and in many cases non-model systems, there were few genetic engineering tools available to manipulate these systems. The Blenner Research Group has invested significant time and energy building finely-tuned promoters, inducible promoters, temporal controlled promoters, and a suite of CRISPR-Cas9 technologies for rapid genome editing. Significant progress in lipid and oleochemical production was due to Y. lipolytica's innate high flux capacity through intermediates and cofactors needed for lipid biosynthesis. Our broad goal over the next five years are to increase the diversity of products that are made in oleaginous yeast. There are over 200,000 known natural products structures and natural products make up the majority of therapeutic compounds. Many bioactive natural products are derived from plants; however, isolation from plants is uncertain, can be extremely costly, and in some cases, agriculture cannot provide enough material. Several classes of plant natural products, including terpenoids, flavonoids, and alkaloids, are biosynthesized using fundamental building blocks from high flux pathways in Y. lipolytica. As such, we hypothesize oleaginous yeast are ideally suited for high titer natural product biosynthesis. This proposal focuses on addressing a common problem across all natural product heterologous pathways – difficulty expressing foreign proteins. We seek to identify known and novel responses to common mode of heterologous protein expression failure. Once specific genes associated with each modes of failure are identified, these can serve as indicators to identify a new gene's mode of failure. These can also be used to help identify solutions to improve expression based on the mode of failure. The resulting improvements can be stacked into platform strains and used to more easily screen natural product pathway libraries. We will focus on flavonoids due to their diverse chemistry and known therapeutic effects; however, we expect these platform strains to be useful for making other natural products as well.

项目摘要 克莱姆森大学的布伦纳研究小组广泛地致力于使用基因工程和 合成生物学以改善用于人类健康，可持续性和国家应用的微生物系统 防御。我们的研究旨在开发准确，精确控制属性的能力 省酵母 - Yarrowia脂溶剂。这种酵母自然发展了产生重要的能力 脂质量。因此，它们对于生产源自脂质的生化物，例如omega-3脂肪 酸和脂肪醇。由于卵酵母被认为是非常规的，在许多情况下，非模型 系统，很少有基因工程工具可以操纵这些系统。 Blenner研究 集团已经投入了大量的时间和能源建设精心调整的启动子，诱导启动子，临时性的 受控启动子和一套CRISPR-CAS9技术用于快速基因组编辑。取得重大进展 在脂质和油化学生产中，是由于Y. lipolytica的先天高通量通过中间体引起的 脂质生物合成所需的辅助因子。我们未来五年的广泛目标是增加多样性 用油脂酵母制造的产品。有超过200,000种已知的天然产品结构， 天然产品构成了大多数治疗化合物。许多生物活性天然产品是派生的 来自植物；但是，从植物中隔离是不确定的，可能非常昂贵，在某些情况下，农业 无法提供足够的材料。几类植物天然产品，包括萜类，类黄酮和 生物碱是使用Y.脂溶剂中高通量途径的基本构建块进行生物合成的。像这样， 我们假设肠酵母非常适合高滴度天然产物生物合成。这个建议 着重于解决所有天然产品异源途径的常见问题 - 困难 表达异物。我们试图确定对异源模式的已知和新颖回应 蛋白质表达衰竭。一旦确定了与每种失败模式相关的特定基因，这些都可以 用作确定新基因失败方式的指标。这些也可以用来帮助确定解决方案 根据失败方式改善表达。由此产生的改进可以堆叠到平台中 应变，并用于更容易筛选天然产品途径库。由于类黄酮，我们将专注于它们 多样化的化学和已知理论效应；但是，我们希望这些平台应变对 也制作其他天然产品。