SCRaMbLE Of Haploid And Aneuploid Yeast Genomes For Tolerance Study

Genomic variation drives evolution of strains.Compared with traditional artificial DNA variation technology,genomic rearrangement can generate diverse structural variations rapidly.An inducible genomic rearrangement(SCRaMbLE)system was constructed by introducing a large number of lox Psym sites in the synthetic yeast genome.This study used the SCRaMbLE system for alkali tolerance and thermotolerance study of haploid and aneuploid yeast strains.The genome rearrangement system was used to explore alkali tolerance in synthetic haploid yeast.Haploid yeast strains harboring either one(synV)or two(synV and synX)synthetic chromosomes were subjected to SCRaMbLE.Seven evolved strains with increased alkali tolerance at p H 8.0 were generated through multiple independent SCRaMbLE experiments.Whole genome sequencing revealed several complex structural variations.A common deletion region of YER161C-YER164 W in alkali-tolerant strains was mapped by comparative genomic analysis.Knock-out experiments confirmed that deletion of YER161C(SPT2)in this region can enhance the alkali tolerance of yeasts.In this study,aneuploid yeasts containing synthetic chromosomes were constructed and subjected to SCRaMbLE for thermotolerance study.Based on the kar1?15 mutant gene,an efficient synthetic chromosome transfer technique was established.Chromosomes of syn?,synV and synX were successfully transferred for the construction of synthetic aneuploid yeast strains.Evolved aneuploid strains with thermotolerance were obtained by SCRaMbLE,and a model for gene dose study was established.This study enriches the understanding of the alkali tolerance and thermotolerance of yeasts,and provides a new idea for the correlation analysis of structural variation and phenotype.