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

项目摘要 克莱姆森大学的Blenner研究小组主要致力于利用基因工程， 合成生物学，以改善微生物系统在人类健康，可持续性和国家 防御我们的研究旨在开发精确控制性能的能力 解脂耶氏酵母。这种酵母自然进化出了产生显著的 量的脂质。因此，它们可用于生产衍生自脂质的生物化学品，例如ω-3脂肪酸， 酸和脂肪醇。由于产油酵母被认为是非传统的，在许多情况下非模型 系统，很少有基因工程工具可用于操纵这些系统。Blenner研究 集团已经投入了大量的时间和精力建设微调启动子，诱导型启动子，时间 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 在油脂和油脂化学生产中的作用是由于Y. Lipolytica通过中间体的先天高通量能力 和脂质生物合成所需的辅因子。我们未来五年的总体目标是增加 产油酵母制造的产品。有超过200，000种已知的天然产物结构， 天然产物构成治疗化合物的大部分。许多具有生物活性的天然产物是从 然而，从植物中分离是不确定的，可能非常昂贵，在某些情况下，农业 不能提供足够的材料。几类植物天然产物，包括萜类化合物、类黄酮和 生物碱，是使用来自Y中高通量途径的基本构件生物合成的。解脂因此，在本发明中， 我们假设产油酵母理想地适用于高滴度天然产物的生物合成。这项建议 重点是解决所有天然产物异源途径的共同问题-困难 表达外源蛋白。我们试图确定已知的和新的反应，共同模式的异源性 蛋白表达失败。一旦确定了与每种失效模式相关的特定基因， 作为识别新基因失效模式的指标。这些也可以用来帮助确定解决方案， 根据故障模式改进表达。由此产生的改进可以叠加到平台中， 菌株，并用于更容易地筛选天然产物途径文库。我们将重点关注黄酮类化合物， 不同的化学性质和已知的治疗效果;然而，我们期望这些平台菌株可用于 也生产其他天然产品。

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.