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Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods
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Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods
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Journal Article
Rapid pathway prototyping and engineering using in vitro and in vivo synthetic genome SCRaMbLE-in methods
2018
Overview
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.
Pathway optimization and chassis engineering are usually carried out in a step-wise and trial-and-error manner. Here the authors present ’SCRaMbLE-in’ that combines in-vitro pathway rapid prototyping with in-vivo genome integration and optimization.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 45/44
/ 49/23
/ 82/80
/ 82/83
/ beta Carotene - biosynthesis
/ Chassis
/ Chromosomes, Fungal - chemistry
/ Gene Expression Regulation, Fungal
/ Genetic Engineering - methods
/ Genomes
/ Genotype
/ High-Throughput Nucleotide Sequencing
/ Humanities and Social Sciences
/ Metabolic Networks and Pathways - genetics
/ Methods
/ Saccharomyces cerevisiae - genetics
/ Saccharomyces cerevisiae - metabolism
/ Science
/ Yeast
