Engineering the ribosomal DNA in a megabase synthetic chromosome

Weimin Zhang, Guanghou Zhao, Zhouqing Luo, Yicong Lin, Lihui Wang, Yakun Guo, Ann Wang, Shuangying Jiang, Qingwen Jiang, Jianhui Gong, Yun Wang, Sha Hou, Jing Huang, Tianyi Li, Yiran Qin, Junkai Dong, Qin Qin, Jiaying Zhang, Xinzhi Zou, Xi HeLi Zhao, Yibo Xiao, Meng Xu, Erchao Cheng, Ning Huang, Tong Zhou, Yue Shen, Roy Walker, Yisha Luo, Zheng Kuang, Leslie A. Mitchell, Kun Yang, Sarah M. Richardson, Yi Wu, Bing-Zhi Li, Ying-Jin Yuan, Huanming Yang, Jiwei Lin, Guo-Qiang Chen, Qingyu Wu, Joel S. Bader, Yizhi Cai, Jef D. Boeke, Junbiao Dai*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

113 Citations (Scopus)


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.

Original languageEnglish
Article numbereaaf3981
Pages (from-to)1-7
Number of pages8
Issue number6329
Publication statusPublished - 10 Mar 2017
Externally publishedYes


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