Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods

Zhouqing Luo, Yun Wang, Nhan T Pham, Laura Tuck, Irene Pérez-Pi, Longying Liu, Yue Shen, Chris French, Manfred Auer, Jon Marles-Wright, Junbiao Dai, Yizhi Cai

    Research output: Contribution to journalArticlepeer-review


    Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. The two methods are normally carried out step-wise and in a trial-and-error manner. Here we report a recombinase-based combinatorial method (termed "SCRaMbLE-in") to tackle both challenges simultaneously. SCRaMbLE-in includes an in vitro recombinase toolkit to rapidly prototype and diversify gene expression at the pathway level and an in vivo genome reshuffling system to integrate assembled pathways into the synthetic yeast genome while combinatorially causing massive genome rearrangements in the host chassis. A set of loxP mutant pairs was identified to maximize the efficiency of the in vitro diversification. Exemplar pathways of β-carotene and violacein were successfully assembled, diversified, and integrated using this SCRaMbLE-in method. High-throughput sequencing was performed on selected engineered strains to reveal the resulting genotype-to-phenotype relationships. The SCRaMbLE-in method proves to be a rapid, efficient, and universal method to fast track the cycle of engineering biology.

    Original languageEnglish
    Article number1936
    JournalNature Communications
    Issue number1
    Early online date22 May 2018
    Publication statusPublished - 2018

    Research Beacons, Institutes and Platforms

    • Manchester Institute of Biotechnology


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