Abstract
BackgroundRedundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. The synthetic chromosome rearrangement and modification by loxP-mediated evolution (SCRaMbLE) system is a unique feature implanted in the synthetic yeast genome (Sc2.0), which is proposed as an effective tool for genome minimization. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction.ResultsWe develop a method termed SCRaMbLE-based genome compaction (SGC) and demonstrate that a synthetic chromosome arm (synXIIL) can be efficiently reduced. The pre-introduced episomal essential gene array significantly enhances the compacting ability of SGC, not only by enabling the deletion of nonessential genes located in essential gene containing loxPsym units but also by allowing more chromosomal sequences to be removed in a single SGC process. Further compaction is achieved through iterative SGC, revealing that at least 39 out of 65 nonessential genes in synXIIL can be removed collectively without affecting cell viability at 30 °C in rich medium. Approximately 40% of the synthetic sequence, encoding 28 genes, is found to be dispensable for cell growth at 30 °C in rich medium and several genes whose functions are needed under specified conditions are identified.ConclusionsWe develop iterative SGC with the aid of eArray as a generic yet effective tool to compact the synthetic yeast genome.
Highlights
A genome encodes both core functions that are essential for viability and accessory functions conferring better adaptation to different and changing niches
The URA3 reporter is integrated into the synthetic chromosome. 5-FOA is used to select strains that have lost regions flanking the URA3 reporter during SCRaMbLE (Fig. 1)
81 genes are encoded in the synthetic sequence, including 16 essential and 65 nonessential genes (Additional file 2: Table S1)
Summary
A genome encodes both core functions that are essential for viability and accessory functions conferring better adaptation to different and changing niches. Through eliminating sequences that are nonessential for growth under optimal conditions in the laboratory, genome compaction studies greatly facilitate our understanding of the core principles of life, and have the potential to create workhorses for biotechnology [1, 2]. Craig Venter and colleagues across 20 years of creative works [4,5,6,7] These led to the generation of the 531 kbp JCVI-syn3.0 genome (473 genes), a synthetic genome smaller than any known in nature capable of supporting independent growth [8]. Redundancy is a common feature of genomes, presumably to ensure robust growth under different and changing conditions. Genome compaction, removing sequences nonessential for given conditions, provides a novel way to understand the core principles of life. As the Sc2.0 project is nearing its completion, we have begun to explore the application of the SCRaMbLE system in genome compaction
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