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

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.

Exploiting the Feedstock Flexibility of the Emergent Synthetic Biology Chassis Vibrio natriegens for Engineered Natural Product Production

“…Our efforts demonstrated that V. natriegens heterologously produced violacein (and deoxyviolacein) in both rich media and in minimal media containing the six carbon sources tested. In rich medium, the production of violacein with V. natriegens (~13 mg·L −1 ) was similar to the production observed in E. coli (~15 mg·L −1 ) when using a plasmid containing the same RBS as pVio [21], although higher production has been demonstrated in other optimized systems [23,25,69,70]. Interestingly, production was high when grown in mannitol or glycerol.…”

Strategies and Challenges for the Development of Industrial Enzymes Using Fungal Cell Factories

“…Synthetic biology approaches can also be used to engineer strains for improved yield and productivity; it provides an opportunity to engineer biology in a more standardized and rational fashion, and several examples using a synthetic yeast genome have been described (Liu et al 2018). One of these approaches is the rewiring of metabolic flux by deleting genes that negatively affect enzyme production.…”

Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

“…By enabling controllable chromosome rearrangements between the designed loxP sites, a synthetic yeast can delete, duplicate and reorder many of its genes, allowing in vivo selection for desirable traits such as increased alkali tolerance [62]. Alternatively, the system can be coupled to heterologously expressed genes allowing the rapid optimisation of pathways [63].…”