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 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.
Highlights
Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression
The regulatory elements, selected recombinase and target pathway DNA are mixed in a single reaction, and the recombinase will integrate the regulatory elements into the target recombination site to generate a combinatorially assembled pathway library
Synthetic biology has been widely used in the design, modification and assembly of genetic elements, significantly speeding up the metabolic engineering process, and succeeding in the production of several important metabolites
Summary
Exogenous pathway optimization and chassis engineering are two crucial methods for heterologous pathway expression. 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. Site-specific recombinase Cre has been widely applied to gene editing and genome engineering in vivo[18,19] and the loxP site has been engineered for better integration efficiency and cassette exchange purposes[17,20,21,22]. In the synthetic yeast genome Sc2.0 project (www.syntheticyeast.org), the SCRaMbLE system (Synthetic Chromosome Rearrangement and Modification by loxP-mediated Evolution) was designed to introduce genomewide loxPsym sites and can generate a massive number of genome permutations upon induction[32,33]. With continuing successful synthesis and construction of these synthetic chromosomes[18,34,35,36,37], the synthetic yeast is becoming a very attractive platform for metabolic engineering
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