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

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

Research output: Contribution to journalArticlepeer-review

Abstract

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 rapid 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
Number of pages12
JournalNature Communications
Volume9
Issue number1
DOIs
Publication statusPublished - 22 May 2018

Keywords

  • Genomic engineering
  • Metabolic engineering
  • Synthetic biology

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