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 van 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; Isak S. Pretorius

doi:10.1038/s41467-024-55318-3

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 van Wyk, Elizabeth L. Daniel, Fergus Harrison, Felix Meier, Robert D. Willows, Yizhi Cai, Roy S. K. WalkerXin Xu, Monica I. Espinosa, Giovanni Stracquadanio, Joel S. Bader, Leslie A. Mitchell, Jef D. Boeke, Thomas C. Williams, Ian T. Paulsen^*, Isak S. Pretorius^*

^*Corresponding author for this work

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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.

Original language	English
Article number	841
Pages (from-to)	1-14
Number of pages	14
Journal	Nature Communications
Volume	16
Issue number	1
DOIs	https://doi.org/10.1038/s41467-024-55318-3
Publication status	Published - 20 Jan 2025

Bibliographical note

Copyright the Author(s) 2025. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.

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10.1038/s41467-024-55318-3

Publisher version (open access)Final published version, 1.55 MBLicence: CC BY

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Goold, H. D., Kroukamp, H., Erpf, P. E., Zhao, Y., Kelso, P., Calame, J., Timmins, J. J. B., Wightman, E. L. I., Peng, K., Carpenter, A. C., Llorente, B., Hawthorne, C., Clay, S., van Wyk, N., Daniel, E. L., Harrison, F., Meier, F., Willows, R. D., Cai, Y., ... Pretorius, I. S. (2025). Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome. Nature Communications, 16(1), 1-14. Article 841. https://doi.org/10.1038/s41467-024-55318-3

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title = "Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome",

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.",

author = "Goold, {Hugh D.} and Heinrich Kroukamp and Erpf, {Paige E.} and Yu Zhao and Philip Kelso and Julie Calame and Timmins, {John J. B.} and Wightman, {Elizabeth L. I.} and Kai Peng and Carpenter, {Alexander C.} and Briardo Llorente and Carmen Hawthorne and Samuel Clay and {van Wyk}, Ni{\"e}l and Daniel, {Elizabeth L.} and Fergus Harrison and Felix Meier and Willows, {Robert D.} and Yizhi Cai and Walker, {Roy S. K.} and Xin Xu and Espinosa, {Monica I.} and Giovanni Stracquadanio and Bader, {Joel S.} and Mitchell, {Leslie A.} and Boeke, {Jef D.} and Williams, {Thomas C.} and Paulsen, {Ian T.} and Pretorius, {Isak S.}",

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Goold, HD , Kroukamp, H , Erpf, PE, Zhao, Y, Kelso, P , Calame, J, Timmins, JJB, Wightman, ELI, Peng, K, Carpenter, AC, Llorente, B, Hawthorne, C, Clay, S, van Wyk, N, Daniel, EL, Harrison, F , Meier, F , Willows, RD, Cai, Y, Walker, RSK, Xu, X, Espinosa, MI, Stracquadanio, G, Bader, JS, Mitchell, LA, Boeke, JD, Williams, TC , Paulsen, IT & Pretorius, IS 2025, 'Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome', Nature Communications, vol. 16, no. 1, 841, pp. 1-14. https://doi.org/10.1038/s41467-024-55318-3

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AU - Calame, Julie

AU - Timmins, John J. B.

AU - Wightman, Elizabeth L. I.

AU - Peng, Kai

AU - Carpenter, Alexander C.

AU - Llorente, Briardo

AU - Hawthorne, Carmen

AU - Clay, Samuel

AU - van Wyk, Niël

AU - Daniel, Elizabeth L.

AU - Harrison, Fergus

AU - Meier, Felix

AU - Willows, Robert D.

AU - Cai, Yizhi

AU - Walker, Roy S. K.

AU - Xu, Xin

AU - Espinosa, Monica I.

AU - Stracquadanio, Giovanni

AU - Bader, Joel S.

AU - Mitchell, Leslie A.

AU - Boeke, Jef D.

AU - Williams, Thomas C.

AU - Paulsen, Ian T.

AU - Pretorius, Isak S.

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

Abstract

Bibliographical note

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Sculpting a masterpiece: synthesis and evolution of minimal yeast genomes

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

Abstract

Bibliographical note

Access to Document

Other files and links

Projects

Sculpting a masterpiece: synthesis and evolution of minimal yeast genomes

Cite this