Rapidly Improving Prodeoxyviolacein Production Via SCRaMbLE

Variations in gene and protein levels are the basis of cellular phenotypic diversity which is a hallmark of strain evolution.Genome rearrangements can cause multiple genetic variations,including gene copy number variations(CNVs)and genome structure variations(SV).This study used the strategy of Synthetic Chromosome Recombination and Modification by LoxP-Mediated Evolution(SCRaMbLE)to achieve the continuous evolution of synthetic yeast haploid rapidly.We established a complete research process based on the optimization of genome rearrangement conditions,and improved the scale of gene rearrangement to chromosomal levels.Prodeoxyviolacein(PDV)is a light green intermediate product in the path of violacein.In this study,the prodeoxyviolacein synthesis pathway(vioA,vioB,vioE)was constructed and integrated into the synV&ring_synV yeast chromosomes.Subsequently,the Cre/LoxPsym genome rearrangement system with precise control elements was established in both strains.In the first round of SCRaMbLE,we determined the relationship between rearrangement time and lethality as well as the ratio of dominant phenotypes preliminarily.Iterative SCRaMbLE was successfully implemented and strains with continuous increase in the yield of prodeoxyviolacein were obtained.After 5 rounds of SCRaMbLE,the yield of this secondary metabolite was increase to 4.04 times and 5.07 times comparied to the initial synV&ring_synV yeast haploid strains.In this study,the second-generation sequencing technology was used to perform the genome-wide sequencing on the high-yeild strains or some growth-deficient strains.The results showed multiple gene deletions and gene duplications in synV&ring_synV chromosome,but higher frequency of gene duplications in ring_synV.Through the analysis of the gene mutations,we have discovered the target gene mutations associated with the prodeoxyviolacein production,such as the deletion of YER151 C which can increase the production of by approximately 1.2 times in synV yeast hapliod.This study extends the application of the SCRaMbLE,and for the first time generates the copy number variations(CNVs)rapidly in synthetic yeast haploid through iterative SCRaMbLE which lay the theoretical research foundation of the relationship between the genome structure and genetic function.