Lv Yongkun, Edwards Harley, Zhou Jingwen, Xu Peng
Department of Chemical, Biochemical and Environmental Engineering , University of Maryland Baltimore County , Baltimore , Maryland 21250 , United States.
Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology , Jiangnan University , Wuxi , Jiangsu China.
ACS Synth Biol. 2019 Mar 15;8(3):568-576. doi: 10.1021/acssynbio.8b00535. Epub 2019 Feb 8.
Conventional plasmid-based gene expression tends to introduce genetic instability and gene copy number variations that lead to degenerated production. The limited number of auxotrophic markers in Yarrowia lipolytica also restricts our ability to perform iterative genetic modifications and manipulate long gene clusters. To overcome these limitations, we combined the high recombination efficiency of the Cre-loxP system and the high integration rate of 26s rDNA, and developed a versatile framework to iteratively integrate multicopy metabolic pathways in Y. lipolytica. We demonstrated the efficient genome integration of a plant-derived flavonoid pathway at random sites with multiple copies. Transient expression of Cre recombinase enabled efficient marker removal and allowed for the next round of genome integration. Investigating the recombination events demonstrated that the iterative integration is happening at sufficiently high rates (more than 80%) without disrupting the previous integration. Both the flavonoid precursor pathway and the plant-derived cytochrome c P450 enzymes were functionally integrated to improve flavonoid and hydroxylated flavonoid production. The engineered strains produced 71.2 mg/L naringenin, 54.2 mg/L eriodyctiol, and 48.1 mg/L taxifolin. The reported work provides a versatile platform to iteratively integrate functional gene clusters at high copy numbers. This work may streamline and expand our capability to build efficient microbial cell factories for high-value natural products and commodity chemical production in Y. lipolytica.
基于传统质粒的基因表达往往会引入遗传不稳定性和基因拷贝数变异，从而导致产量下降。解脂耶氏酵母中营养缺陷型标记数量有限，这也限制了我们进行迭代基因改造和操纵长基因簇的能力。为了克服这些限制，我们将Cre-loxP系统的高重组效率和26s rDNA的高整合率相结合，开发了一个通用框架，用于在解脂耶氏酵母中迭代整合多拷贝代谢途径。我们证明了植物来源的类黄酮途径在多个随机位点的高效基因组整合。Cre重组酶的瞬时表达实现了有效的标记去除，并允许进行下一轮基因组整合。对重组事件的研究表明，迭代整合以足够高的速率发生（超过80%），而不会破坏先前的整合。类黄酮前体途径和植物来源的细胞色素c P450酶都在功能上进行了整合，以提高类黄酮和羟基化类黄酮的产量。工程菌株产生了71.2 mg/L的柚皮素、54.2 mg/L的圣草酚和48.1 mg/L的紫杉叶素。所报道的工作提供了一个通用平台，用于以高拷贝数迭代整合功能基因簇。这项工作可能会简化并扩展我们构建高效微生物细胞工厂以生产解脂耶氏酵母中高价值天然产物和商品化学品的能力。
