【摘要】 由于细胞代谢和调控网络的复杂性,尤其是对于多基因调控的复杂性状和遗传工具有限的生物系统而言,基因组进化在微生物细胞工厂的构建中起着至关重要的作用。基因组进化通过人为创造多样化性状以及功能筛选的迭代循环,在实验室中模拟且加速自然进化的过程,从而快速获得满足目标需求的进化突变体。酿酒酵母是代谢工程中重要的底盘细胞,全基因组进化是对其进行系统性改造的最有效合成生物学手段之一。本文总结了基因组进化在构建高效的酿酒酵母细胞工厂中的技术进展和应用,包括基因组改组、转座子插入诱变和全局转录机制工程(gTME)等基于随机突变的非理性基因组进化以及诸如酵母寡核苷酸介导的基因组工程(YOGE),真核基因组多重位点自动改造技术(eMAGE)、RNAi辅助的基因组进化方法 (RAGE)以及基于CRISPR体系的基因组规模改造技术(CHAnGE、MAGIC和MAGESTIC)等可示踪的半理性基因组进化,并简要介绍了基因组进化面临的挑战和高通量筛选方法的发展前景。更多还原

【Abstract】 Due to our limited knowledge of the complicated cellular networks, genome evolution has played critical roles in the construction and optimization of microbial cell factories, especially for those complex traits regulated by multigenes and for organisms with few genetic engineering tools. Directed genome evolution mimics natural evolution in the laboratory via iterative rounds of genetic diversification and functional screening or selection to isolate evolved mutants with the desirable phenotypes. Genome evolution has been found to be one of the most effective synthetic biology tools for systematic modification and optimization of Saccharomyces cerevisiae, one of the most important chassises inmetabolic engineering. This review summarized the advances and applications of genome evolution techniques in the construction and optimization of efficient S. cerevisiae cell factories. Firstly, random mutagenesis based genome evolution strategies, including chemical/physical mutagenesis, genome shuffling, transposon mediated mutagenesis, global transcriptional machinery engineering, recombinase mediated mutagenesis, as well as adaptive laboratory evolution, are introduced. Then, the recently developed trackable genome-scale engineering techniques, including YOGE(yeast oligomediated genome engineering), e MAGE(eukaryotic multiplex automated genome engineering), RAGE(RNAi-assisted genome evolution), CHAn GE(CRISPR/Cas9-and homology-directed-repair-assisted genome-scale engineering),MAGIC(multi-functional genome-wide CRISPR system), and MAGESTIC(multiplexed accurate genome editing with short, trackable, integrated cellular barcodes), are discussed in details. In addition, the applications of these irrational and semi-rational genome evolution techniques in engineering yeast cell factories to expand substrate utilization, enhance product formation, and improve cellular properties, are also presented. Finally, the challenges and future directions of genome evolution, particularly when in combination with the high-throughput screening methodologies, are prospected.更多还原