Engineering the ribosomal DNA in a megabase synthetic chromosome

We designed and synthesized a 976,067-base pair linear chromosome, synXII, based on native chromosome XII in Saccharomyces cerevisiae. SynXII was assembled using a twostep method, specified by successive megachunk integration and meiotic recombinationmediated assembly, producing a functional chromosome in S. cerevisiae. Minor growth defect " bugs" detected in synXII, caused by deletion of tRNA genes, were rescued by introducing an ectopic copy of a single tRNA gene. The ribosomal gene cluster (rDNA) on synXII was left intact during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, which can be used to determine species identity, were swapped to generate a Saccharomyces synXII strain that would be identified as Saccharomyces bayanus by standard DNA barcoding procedures.National Natural Science Foundation of China [31471254, 21621004, 21390203]; Chinese Ministry of Science and Technology [2012CB725201, 2012AA02A708]; Ph.D. Programs Foundation of Ministry of Education of China [20110002120055]; Research Fund for the Doctoral Program of Higher Education of China [20120002110022]; Tsinghua University Initiative grant [2011Z02296]; NSF [MCB-1026068, MCB-1158201, MCB-1445545]SCI(E)ARTICLE6329,SI35