Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome

Hugh D. Goold (), Heinrich Kroukamp (), Paige E. Erpf, Yu Zhao, Philip Kelso, Julie Calame, John J. B. Timmins, Elizabeth L. I. Wightman, Kai Peng, Alexander C. Carpenter, Briardo Llorente, Carmen Hawthorne, Samuel Clay, Niël Wyk, Elizabeth L. Daniel, Fergus Harrison, Felix Meier, Robert D. Willows, Yizhi Cai, Roy S. K. Walker, Xin Xu, Monica I. Espinosa, Giovanni Stracquadanio, Joel S. Bader, Leslie A. Mitchell, Jef D. Boeke, Thomas C. Williams, Ian T. Paulsen () and Isak S. Pretorius ()
Additional contact information
Hugh D. Goold: Woodbridge Road
Heinrich Kroukamp: Macquarie University
Paige E. Erpf: Macquarie University
Yu Zhao: NYU Langone Health
Philip Kelso: Macquarie University
Julie Calame: Macquarie University
John J. B. Timmins: Macquarie University
Elizabeth L. I. Wightman: Macquarie University
Kai Peng: Macquarie University
Alexander C. Carpenter: Macquarie University
Briardo Llorente: Macquarie University
Carmen Hawthorne: Macquarie University
Samuel Clay: Macquarie University
Niël Wyk: Macquarie University
Elizabeth L. Daniel: Macquarie University
Fergus Harrison: Macquarie University
Felix Meier: Macquarie University
Robert D. Willows: Macquarie University
Yizhi Cai: 131 Princess Street
Roy S. K. Walker: Macquarie University
Xin Xu: Macquarie University
Monica I. Espinosa: Macquarie University
Giovanni Stracquadanio: The University of Edinburgh
Joel S. Bader: Johns Hopkins University
Leslie A. Mitchell: NYU Langone Health
Jef D. Boeke: NYU Langone Health
Thomas C. Williams: Macquarie University
Ian T. Paulsen: Macquarie University
Isak S. Pretorius: Macquarie University

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5’ UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.

Date: 2025
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DOI: 10.1038/s41467-024-55318-3

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