Abstract
Synthetic biology tools, such as modular parts and combinatorial DNA assembly, are routinely used to optimise the productivity of heterologous metabolic pathways for biosynthesis or substrate utilisation, yet it is well established that host strain background is just as important for determining productivity. Here we report that in vivo combinatorial genomic rearrangement of Saccharomyces cerevisiae yeast with a synthetic chromosome V can rapidly generate new, improved host strains with genetic backgrounds favourable to diverse heterologous pathways, including those for violacein and penicillin biosynthesis and for xylose utilisation. We show how the modular rearrangement of synthetic chromosomes by SCRaMbLE can be easily determined using long-read nanopore sequencing and we explore experimental conditions that optimise diversification and screening. This synthetic genome approach to metabolic engineering provides productivity improvements in a fast, simple and accessible way, making it a valuable addition to existing strain improvement techniques.
Highlights
Synthetic biology tools, such as modular parts and combinatorial DNA assembly, are routinely used to optimise the productivity of heterologous metabolic pathways for biosynthesis or substrate utilisation, yet it is well established that host strain background is just as important for determining productivity
The diversity of outcomes seen just with this short synthetic DNA region highlights how millions of unique genotypes could be produced by SCRaMbLE of synthetic chromosomes within a population of cells. We reasoned that this would offer a huge potential phenotype space that could be exploited for strain improvement, and for generating strains with altered genetic backgrounds that provide a benefit for heterologous pathways of industrial relevance
SCRaMbLE generates a strain with improved biosynthesis yield
Summary
Synthetic biology tools, such as modular parts and combinatorial DNA assembly, are routinely used to optimise the productivity of heterologous metabolic pathways for biosynthesis or substrate utilisation, yet it is well established that host strain background is just as important for determining productivity. The recent arrival of Saccharomyces cerevisiae yeast strains where natural chromosomes are replaced by designed, synthetic chromosomes[9,10] offers a radically new form of genome diversification that can be induced in vivo and leads to combinations of genes being deleted or altered in their expression This is afforded by the Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that has been designed into the synthetic sequence of chromosomes produced by the Sc2.0 project[11]. The diversity of outcomes seen just with this short synthetic DNA region highlights how millions of unique genotypes could be produced by SCRaMbLE of synthetic chromosomes within a population of cells We reasoned that this would offer a huge potential phenotype space that could be exploited for strain improvement, and for generating strains with altered genetic backgrounds that provide a benefit for heterologous pathways of industrial relevance. When haploid synthetic yeast strains expressing heterologous pathways are subject to a short SCRaMbLE process with no applied selective pressure, diverse genetic backgrounds are quickly produced that significantly improve the productivity of multiple heterologous metabolic pathways of industrial interest
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