Abstract:

Saccharomyces cerevisiae, one of the most commonly used cell factories for industrial biotechnology, is widely employed for mass production of bio-based chemicals and value-added compounds. Due to complicated cellular metabolism and regulatory network of biological systems, genome evolution is generally required for engineering with complicated phenotypes, which are coordinated and regulated by multiple genes. To achieve rapid evolution of S. cerevisiae genome, this study employed Clustered Regularly Interspaced Short Palindromic Repeats Interference (CRISPRi) to regulate the expression of genes closely related to chromosome replication and maintenance, such as MSH2, TSA1, RAD27, and CLB5, known as mutator genes. By designing guide RNAs (gRNAs) with differential repression efficiency, four mutators: MSH2 mutator mainly for point mutations and small InDels (MM), TAS1 mutator mainly for point mutations, small InDels, and structural variants (TM), RAD27 mutator mainly for small InDels and structural variants (RM), and CLB5 mutator mainly for structural variants and aneuploidy chromosomes (CM) were constructed to control genome mutation rates and types (e.g. point mutation, small InDels, structural variants, and aneuploid chromosomes). These mutators were used for continuous evolution of a series of industrially relevant phenotypes, such as isobutanol tolerance, xylose utilization, and β-carotene biosynthesis. Interestingly, TM was more efficient for evolving isobutanol tolerance, but TM and CM were preferred for evolving β-carotene overproduction, and all mutators were verified to have comparable performance for evolving xylose utilization with yeast strains. We also discovered that the effectiveness of mutators was dependent on the phenotype to be evolved. To address challenges in the evolution of phenotypes without pre-determined knowledge on mutation rates and types, a mixed mutator (MTRC) was constructed to rapidly evaluate the mutator-phenotype relationship. Finally, a combinatorial mutator (MTRC*2) were constructed to explore synergistic interactions among various mutators for the continuous genome evolution of S. cerevisiae. The established mutators can not only be used for constructing robust yeast cell factories, but also be further developed as a generally applicable genome evolution tool.

Key words: mutators, Saccharomyces cerevisiae, continuous genome evolution, CRISPR Interference, mutator genes

摘要：

酿酒酵母是工业生物技术最常用的底盘细胞之一，被广泛应用于生物基化学品和高附加值产品的大规模生产。鉴于生物体系代谢和调控网络的复杂性，由多基因协同控制的复杂生理性状的改造通常需要采取全基因组进化来实现。为实现酿酒酵母基因组的快速进化，本研究采用CRISPR干扰技术（CRISPRi）调控与染色体复制和稳定性相关基因（MSH2、TSA1、RAD27和CLB5，即增变基因）的表达水平，构建了能够调控酿酒酵母基因组突变率和突变类型的基因增变器。利用基因增变器提高酿酒酵母的β-胡萝卜素合成水平、木糖利用效率和异丁醇耐受性。此外，构建了混合基因增变器和多重基因增变器，进一步探究了不同表型基因组进化所需的最佳突变类型以及不同突变类型之间的协同进化机制。本研究不仅可用于创建高性能酿酒酵母细胞工厂，还有可能发展一个具有普遍适用性的基因组连续进化策略。

关键词: 基因增变器, 酿酒酵母, 基因组连续进化, CRISPR干扰, 增变基因