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

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.

项目总结 克莱姆森大学的Blenner研究小组广泛地专注于使用基因工程和 合成生物学，以改善微生物系统在人类健康、可持续发展和国家 防守。我们的研究旨在发展准确和精确地控制性能的能力 产油酵母菌--解脂亚罗维菌。这种酵母菌自然进化出了产生显著 脂类的量。因此，它们可用于生产从脂肪中提取的生物化学物质，如omega-3脂肪。 酸和脂肪醇。因为含油酵母被认为是非常规的，在许多情况下是非典型的 在这些系统中，几乎没有可用来操纵这些系统的基因工程工具。布莱纳研究中心 集团已投入大量时间和精力打造精心打造的促进剂、诱导型促进剂、临时性促进剂 受控启动子，以及一套用于快速基因组编辑的CRISPR-CAS9技术。重大进展 脂肪和油脂化学的产生是由于解脂耶尔森菌通过中间体的天然高通量能力 以及脂质生物合成所需的辅因子。我们在未来五年的总体目标是增加生物多样性 用含油酵母制成的产品。有超过200,000种已知的天然产物结构和 天然产品构成了治疗化合物的大部分。许多具有生物活性的天然产物都是从 与植物隔离；然而，与植物隔离是不确定的，可能代价极其高昂，在某些情况下，还可能与农业隔离 无法提供足够的材料。几类植物天然产物，包括萜类、黄酮类和 生物碱，是使用解脂耶尔森菌高通量途径的基本构建块生物合成的。因此， 我们推测含油酵母非常适合于高滴度天然产物的生物合成。这项建议 重点解决所有天然产物异源途径中的一个共同问题-困难 表达外源蛋白。我们试图确定已知的和新的对常见的异源模式的反应 蛋白质表达失败。一旦确定了与每种故障模式相关的特定基因，这些基因就可以 作为识别新基因失败模式的指示器。这些信息还可用于帮助确定解决方案 根据失败模式提高表达能力。由此产生的改进可以堆叠到平台中 菌株，并用于更容易地筛选天然产物途径文库。我们将重点研究黄酮类化合物，因为它们 不同的化学成分和已知的治疗效果；然而，我们希望这些平台菌株对 也可以制造其他天然产品。