Design And Construction Of Designer Chromosome V Of Saccharomyces Cerevisiae

Design and synthesis of genome enable modifying and understanding natural biological systems by “writing” the genome information and chemically synthesizing designer genomes from scratch.Synthesis of viral and prokaryotic genomes has laid the groundwork for the field of de novo genome synthesis,which largely deepens our understanding of biology and expands our abilities of genome design.Whereas prokaryotic genomes are typically circular and lack telomeres,with a relatively simple structure,almost all natural eukaryotic chromosomes are linear and flanked by telomeres,carrying more information and with more complex structure.More efforts are needed to design and construct a eukaryotic genome."Synthetic Yeast Genome"(Sc2.0)international cooperation aims to modify and construct the whole yeast genome,and this study focuses on the design,building,testing,debugging and redesign of chromosome V of Saccharomyces cerevisiae.Accoding to Sc2.0 principles,we designed 536,024–base pair synthetic yeast chromosome V(synV)on the basis of the complete nucleotide sequence of native yeast chromosome V [576,874 base pairs(bp)].Major edits during synV design included deleting two subtelomere regions,20 tRNA genes,30 transposons/Ty elements,and 10 introns and inserting 176 loxPsym sites;additional base changes included 62 TAG/TAA stop-codon swaps and 339 synonymous recodings to introduce PCRTags derived from wtV.Overall,the synV designed sequence has a total 62,450-bp nucleotide changes compared with wtV,accounting for about 10.83% of wtV and is shorter than the wtV by 7.62%,enhancing the stability and flexibility of chromosome V.Several approaches were developed to complete the syn V chromosome construction,including multilevel modular DNA assembly,iterative chromosome replacement and meiotic recombination-mediated parallel chromosome construction.The native chromosome V was replaced by synV in 17 steps of minichunk(2?4-kb,263 in total)incorporation and two rounds of universal telomere cap(UTC)replacement.The two semi-synV chromosomes,parallel constructed,were assembled by meiotic recombination,producing a hybrid chromosome carrying 73.75% synV.The growth defect is one of the key problem affecting the genome synthesis.A PCRTag mapping strategy was developed to identify the defect targets precisely,which was debugged.In total,the synV sequence was redesigned and updated twice,improving the design of Sc2.0 genome.Perfect matching of an assembled physical sequence to a specified designed sequence is crucial to verify design principles in genome synthesis.Several approaches were developed to fix sequence alterations on synV,including a multiplex intergrative cotransformation-mediated genome editing approach and a large DNA duplication removal strategy.In 24 steps,the synV was corrected to perfectly match the designed sequence by removing 35,888-bp nucleotides and correcting 3,333-bp nucleotides.SynV strains exhibit high fitness under a variety of culture conditions,compared with that of wild-type V strains.The “perfect” synV systematic evaluated the underlying Sc2.0 design principles,and supported “Genome Project-write”(GP-write)in design,function test and technique improment.Ring chromosome derivatives were constructed by design in S.cerevisiae,in which hundreds of distinguishable PCRTags can be used to track the chromosome changes during meiosis and mitosis easily.Ring chromosome can extend design principles to provide a model with which to study genomic rearrangement,ring chromosome evolution,cancer,aging,and human ring chromosome disorders.