Construction,Debugging And Scrambleing Of Synthetic Yeast Chromosomes

Design and construction of an extensively modified yeast genome is a direct means to interrogate the integrity,comprehensiveness,and accuracy of the knowledge amassed by the yeast community to date.The international synthetic yeast genome project(Sc2.0)aims to build an entirely designer,synthetic Saccharomyces cerevisiae genome.The synthetic genome is designed to increase genome stability and genetic flexibility while maintaining cell fitness near that of the wild type.SCRaMbLE(Synthetic Chromosome Recombination and Modification by LoxP Mediated Evolution)is a genome restructuring technique that can be used in synthetic genomes of Sc2.0,which contains hundreds to thousands of strategically positioned loxPsym sites.Here,we chemically synthesized yeast chromosome X,synX,designed to be 707,459 base pairs.A high-throughput mapping strategy called pooled PCRTag mapping(PoPM)was developed to identify unexpected bugs during chromosome assembly.Several bugs were identified and corrected,including a growth defect mapping to a specific synonymously recoded PCRTag sequence in the essential FIP1 ORF and the effect of introducing a loxPsym site that unexpectedly altered the the promoter function of a nearby gene,ATP2.In addition,meiotic crossover was employed to repair the massive duplications and rearrangements in the synthetic chromosome.The debugged synX strain exhibited high fitness under a variety of conditions tested and in competitive growth with the wild-type strain.We describe a collection of heterozygous diploid strains produced by mating haploid synthetic Sc2.0 strains to haploid native parental strains.We subsequently show that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid synthetic strains.Two specific strains were used as illustrative examples to demonstrate that SCRaMbLE in heterozygous diploids can be successfully applied to a virtually infinite variety of hybrid strain backgrounds and generate both industrially relevant gains in phenotype and new biological knowledge.We also describe an in vitro SCRaMbLE system,driven by purified Cre recombinase in a test tube in the presence of loxPsym site-encoding DNA.We demonstrate two strategies using the in vitro SCRaMbLE system,top-down and bottom-up methods.Using the ?-carotene pathway in yeast as an example,we demonstrate the two strategies can be used for pathway engineering and optimization.Our results indicated that in vitro SCRaMbLE is a special,effective and straightforward method for DNA library construction.