非模式圆红冬孢酵母的CRISPR系统建立和适配机制及其代谢工程应用

CRISPR systems played important roles in many genetic applications, such as gene editing and transcriptional regulation. However, most studies are carried in host with clear genetic background and plenty of genetic tools. It is thus important to find an efficient way to develop CRISPR systems in non-model organisms that lack of genetic tools and clear genetic information, such as the non-model oleaginous yeast Rhodosporidium toruloides with great biotechnological potentials. Here, as a case study, we propose a strategy to develop CRISPR-based platforms in R. toruloides. In our method, biological units, such as stable expression plasmids and RNA Pol III promoters, will be explored and optimized that can be used in CRISPR systems. Then, with the help of a fully automated platform, BioFoundry., combinatorial expression of these units will be carried out in high-throughput manner to optimize the efficiency of different CRISPR systems for gene editing, regulation, and screening. Lessons from the high-throughput data in combination with multivariate data analysis (MVDA) will facilitate the understanding of the mutual adaptation between different units, which could be used a guide to develop CRISPR systems in other non-model organisms. As a demonstration, we will apply these new strategies and tools to engineer fatty acid metabolism in R. toruloides, with a focus on the production of a fatty acid derivative product, fatty acid ethyl esters (FAEEs).

目前，基于CRISPR系统的基因组编辑和调控技术因其操作简单高效成为研究热点，然而相关研究仍集中于遗传背景清晰且遗传工具较为丰富的宿主。如何将CRISPR系统延伸到遗传工具匮乏甚至遗传背景模糊的非模式生物（如具有广泛工业应用前景的非模式产油菌株圆红冬孢酵母），成为了一个有待挖掘的研究领域。为此，本项目以在圆红冬孢酵母中开发CRISPR系统为研究模型，挖掘和优化相关生物元件，并借助本团队前期建立的全自动化工作平台BioFoundry对CRISPR系统中各元件的表达策略进行高通量测试和筛选，构建可在圆红冬孢酵母中进行基因缺失、转录调控和基因组水平转录双向筛选的CRISPR系统。拟利用高通量测试及多变量数据分析方法，解析CRISPR系统中各个元件之间的适配性，指导CRISPR系统的优化及推广。本项目还将利用开发的新技术调控圆红冬孢酵母脂肪酸代谢，进而构建高产脂肪酸乙酯的工程菌株，展现其应用潜力。

合成生物学工具及其生物元件的缺乏是限制非模式生物的代谢工程应用的一个主要瓶颈。本研究在非模式圆红冬孢酵母中挖掘了多种基本的生物元件，同时建立并优化CRISPR系统可实现对任意基因表达水平的调控或单碱基编辑。这些元件挖掘和工具开发既为在众多遗传信息和工具匮乏的非模式生物中开发CRISPR系统提供指导和示范意义，又可为促进圆红冬孢酵母发展成为新型脂肪酸细胞工厂提供有力的技术支持。与此同时，我们还构建了高产脂肪酸乙酯和蜡酯的圆红冬孢酵母细胞工厂，其中脂肪酸乙酯产量达到9.97g/L，为文献发表时酵母中的最高产量，在菌株的优化过程中还揭示了影响脂肪酸合成代谢的关键基因，这些示例研究和发现可促进圆红冬孢酵母的代谢工程改造从而发展新的脂肪酸合成细胞工厂。

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