The introduction of multi-gene metabolic pathways is generally the first step for the construction of microbial cell factories and plays an essential role in metabolic engineering and synthetic biology. Here, we developed a “PCR & Go” system for facile integration and assembly of multi-gene pathways into the chromosome of Saccharomyces cerevisiae. The core component of the “PCR & Go” system was an expression chassis, where eight promoter/terminator pairs were pre-installed into the yeast chromosome and PCR amplified gene fragments could be inserted directly for functional expression. In combination with the CRISPR/Cas9 system and a gRNA plasmid library, the β-carotene (three genes), zeaxanthin (four genes), and astaxanthin (five genes) biosynthetic pathways were integrated and assembled into the yeast genome with an efficiency of ~93, ~85, and 69%, respectively, using PCR amplified gene fragments with ~40 bp homology arms in a single step. Therefore, the “PCR & Go” system can be used for fast construction of yeast cell factories harboring multi-gene pathways with high efficiency and flexibility.

中文翻译：

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PCR & Go：用于轻松整合多基因生物合成途径的预装表达底盘

多基因代谢途径的引入一般是构建微生物细胞工厂的第一步，在代谢工程和合成生物学中起着至关重要的作用。在这里，我们开发了一个“PCR & Go”系统，用于将多基因途径轻松整合和组装到酿酒酵母的染色体中。“PCR&Go”系统的核心部件是表达底盘，其中8对启动子/终止子预装在酵母染色体中，可以直接插入PCR扩增的基因片段进行功能表达。结合CRISPR/Cas9系统和gRNA质粒文库，将β-胡萝卜素（三个基因）、玉米黄质（四个基因）和虾青素（五个基因）的生物合成途径整合并组装到酵母基因组中，效率为~ 93, ~85, 和 69%，分别使用 PCR 扩增的基因片段，在一个步骤中具有 ~40 bp 同源臂。因此，“PCR&Go”系统可以高效、灵活地快速构建多基因通路的酵母细胞工厂。