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 Sac...
Saved in:
| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-01-01
|
| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-024-55318-3 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1832585585921884160 |
|---|---|
| author | 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 |
| author_facet | 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 |
| author_sort | Hugh D. Goold |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-ac0d8dbbcd894c7a9517722842a34dd6 |
| institution | Kabale University |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-ac0d8dbbcd894c7a9517722842a34dd62025-01-26T12:42:10ZengNature PortfolioNature Communications2041-17232025-01-0116111410.1038/s41467-024-55318-3Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosomeHugh D. Goold0Heinrich Kroukamp1Paige E. Erpf2Yu Zhao3Philip Kelso4Julie Calame5John J. B. Timmins6Elizabeth L. I. Wightman7Kai Peng8Alexander C. Carpenter9Briardo Llorente10Carmen Hawthorne11Samuel Clay12Niël van Wyk13Elizabeth L. Daniel14Fergus Harrison15Felix Meier16Robert D. Willows17Yizhi Cai18Roy S. K. Walker19Xin Xu20Monica I. Espinosa21Giovanni Stracquadanio22Joel S. Bader23Leslie A. Mitchell24Jef D. Boeke25Thomas C. Williams26Ian T. Paulsen27Isak S. Pretorius28New South Wales Department of Primary Industries, Elizabeth Macarthur Agriculture Institute, Advanced Gene Technology Centre, Woodbridge RoadSchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversityInstitute for Systems Genetics, NYU Langone HealthSchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversityManchester Institute of Biotechnology, The University of Manchester, 131 Princess StreetSchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Biological Sciences, The University of EdinburghDepartment of Biomedical Engineering, Johns Hopkins UniversityInstitute for Systems Genetics, NYU Langone HealthInstitute for Systems Genetics, NYU Langone HealthSchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversitySchool of Natural Sciences, and ARC Centre of Excellence in Synthetic Biology, Macquarie UniversityAbstract 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.https://doi.org/10.1038/s41467-024-55318-3 |
| spellingShingle | 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 Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome Nature Communications |
| title | Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome |
| title_full | Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome |
| title_fullStr | Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome |
| title_full_unstemmed | Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome |
| title_short | Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome |
| title_sort | construction and iterative redesign of synxvi a 903 kb synthetic saccharomyces cerevisiae chromosome |
| url | https://doi.org/10.1038/s41467-024-55318-3 |
| work_keys_str_mv | AT hughdgoold constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT heinrichkroukamp constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT paigeeerpf constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT yuzhao constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT philipkelso constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT juliecalame constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT johnjbtimmins constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT elizabethliwightman constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT kaipeng constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT alexanderccarpenter constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT briardollorente constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT carmenhawthorne constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT samuelclay constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT nielvanwyk constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT elizabethldaniel constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT fergusharrison constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT felixmeier constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT robertdwillows constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT yizhicai constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT royskwalker constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT xinxu constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT monicaiespinosa constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT giovannistracquadanio constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT joelsbader constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT leslieamitchell constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT jefdboeke constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT thomascwilliams constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT iantpaulsen constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome AT isakspretorius constructionanditerativeredesignofsynxvia903kbsyntheticsaccharomycescerevisiaechromosome |