Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Nat Commun. 2017 May 4;8:15187. doi: 10.1038/ncomms15187.
Genome-scale engineering is indispensable in understanding and engineering microorganisms, but the current tools are mainly limited to bacterial systems. Here we report an automated platform for multiplex genome-scale engineering in Saccharomyces cerevisiae, an important eukaryotic model and widely used microbial cell factory. Standardized genetic parts encoding overexpression and knockdown mutations of >90% yeast genes are created in a single step from a full-length cDNA library. With the aid of CRISPR-Cas, these genetic parts are iteratively integrated into the repetitive genomic sequences in a modular manner using robotic automation. This system allows functional mapping and multiplex optimization on a genome scale for diverse phenotypes including cellulase expression, isobutanol production, glycerol utilization and acetic acid tolerance, and may greatly accelerate future genome-scale engineering endeavours in yeast.
基因组规模的工程在理解和工程微生物中是不可或缺的，但目前的工具主要限于细菌系统。在这里，我们报告了一个在酿酒酵母中进行多路基因组规模工程的自动化平台，酿酒酵母是一种重要的真核模型和广泛使用的微生物细胞工厂。通过 CRISPR-Cas 技术，这些遗传元件可以使用机器人自动化的方式，以模块化的方式从全长 cDNA 文库中一步构建出超过 90%的酵母基因的过表达和敲低突变的标准化遗传元件。该系统允许对包括纤维素酶表达、异丁醇生产、甘油利用和乙酸耐受性在内的各种表型进行基因组规模的功能映射和多路优化，这可能会极大地加速未来在酵母中的基因组规模工程努力。
