Abstract
Genome-scale engineering and custom synthetic genomes are reshaping the next generation of industrial yeast strains. The Cre-recombinase-mediated chromosomal rearrangement mechanism of designer synthetic Saccharomyces cerevisiae chromosomes, known as SCRaMbLE, is a powerful tool which allows rapid genome evolution upon command. This system is able to generate millions of novel genomes with potential valuable phenotypes, but the excessive loss of essential genes often results in poor growth or even the death of cells with useful phenotypes. In this study we expanded the versatility of SCRaMbLE to industrial strains, and evaluated different control measures to optimize genomic rearrangement, whilst limiting cell death. To achieve this, we have developed RED (rapid evolution detection), a simple colorimetric plate-assay procedure to rapidly quantify the degree of genomic rearrangements within a post-SCRaMbLE yeast population. RED-enabled semi-synthetic strains were mated with the haploid progeny of industrial yeast strains to produce stress-tolerant heterozygous diploid strains. Analysis of these heterozygous strains with the RED-assay, genome sequencing and custom bioinformatics scripts demonstrated a correlation between RED-assay frequencies and physical genomic rearrangements. Here we show that RED is a fast and effective method to evaluate the optimal SCRaMbLE induction times of different Cre-recombinase expression systems for the development of industrial strains.
Highlights
Specialized strains of the yeast Saccharomyces cerevisiae are harnessed by industry for the production of food and beverages, pharmaceuticals, chemical building blocks and fuel
An example of the capability for genome rearrangement was the rapid generation of semi-synthetic heterozygous diploid strains containing a single copy of the synthetic chromosomes synV and synX, with significantly improved thermotolerance at 42 ◦C after a single round of SCRaMbLE [9], whereas a similar increase in thermotolerance using ALE took over 300 generations [10]
We have developed rapid evolution detection (RED), a simple colorimetric plate-assay procedure to determine the degree of genomic rearrangements within SCRaMbLEd diploid yeast populations
Summary
Specialized strains of the yeast Saccharomyces cerevisiae are harnessed by industry for the production of food and beverages, pharmaceuticals, chemical building blocks and fuel. SCRaMbLE was used to optimize the biosynthetic pathway for improved violacein yields, demonstrating that this strategy could potentially be applied to optimize the production of any metabolite [7] These SCRaMbLEd strains contained only one synthetic chromosome in a haploid genome context; it is conceivable that strains harboring more synthetic DNA, with more loxP recombinase recognition sites, could produce greater genomic diversity with associated novel phenotypes of interest. SCRaMbLEing in diploid cells overcomes some of the limitations associated with rapid haploid cell death, it abolishes the use of viability as a simple output to gauge the degree of genome scrambling in the population It increases the complexity of the bioinformatic analysis of these genomes due to the high sequence similarity between equivalent synthetic and non-synthetic genomic regions. We have showed that RED could be generally applied to semi-synthetic industrial strains to rapidly evaluate the frequency of genomic rearrangements in a SCRaMbLEd population, which allows the fine-tuning and selection of optimal SCRaMbLE conditions for strain library generation
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