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

TLDR

Exogenous pathway optimization and chassis engineering are essential for heterologous expression, yet they are typically performed step‑wise and trial‑and‑error. The authors present SCRaMbLE‑in, a recombinase‑based combinatorial approach designed to address both challenges simultaneously. SCRaMbLE‑in combines an in‑vitro recombinase toolkit that rapidly prototypes and diversifies pathway gene expression with an in‑vivo genome‑reshuffling system that integrates assembled pathways into a synthetic yeast genome while inducing extensive chromosomal rearrangements, using optimized loxP mutant pairs and high‑throughput sequencing to map genotype‑to‑phenotype relationships. Using SCRaMbLE‑in, β‑carotene and violacein pathways were successfully assembled, diversified, and integrated, demonstrating the method’s rapid, efficient, and universal capability to accelerate the engineering biology cycle.

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.