Engineering the ribosomal DNA in a megabase synthetic chromosome.
Weimin ZhangGuanghou ZhaoZhou-Qing LuoYicong LinLihui WangYakun GuoAnn WangShuangying JiangQingwen JiangJianhui GongYun WangSha HouJing HuangTianyi LiYiran QinJunkai DongQin QinJiaying ZhangXinzhi ZouXi HeLi ZhaoYibo XiaoMeng XuErchao ChengNing HuangTong ZhouYue ShenRoy S K WalkerYisha LuoZheng KuangLeslie A MitchellKun YangSarah M RichardsonYi WuBing-Zhi LiYing-Jin YuanHuanming YangJiwei LinGuo-Qiang ChenQingyu WuJoel S BaderYizhi CaiJef D BoekeJun-Biao DaiPublished in: Science (New York, N.Y.) (2017)
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 two-step method, specified by successive megachunk integration and meiotic recombination-mediated 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.