Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects

Thomas C Williams; Heinrich Kroukamp; Xin Xu; Elizabeth L I Wightman; Briardo Llorente; Anthony R Borneman; Alexander C Carpenter; Niel Van Wyk; Felix Meier; Thomas R V Collier; Monica I Espinosa; Elizabeth L Daniel; Roy S K Walker; Yizhi Cai; Helena K M Nevalainen; Natalie C Curach; Ira W Deveson; Timothy R Mercer; Daniel L Johnson; Leslie A Mitchell; Joel S Bader; Giovanni Stracquadanio; Jef D Boeke; Hugh D Goold; Isak S Pretorius; Ian T Paulsen

doi:10.1016/j.xgen.2023.100379

Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects

Cell Genom. 2023 Nov 9;3(11):100379. doi: 10.1016/j.xgen.2023.100379. eCollection 2023 Nov 8.

Authors

Thomas C Williams^{1

2}, Heinrich Kroukamp¹, Xin Xu¹, Elizabeth L I Wightman¹, Briardo Llorente^{1

2

3}, Anthony R Borneman^{4

5}, Alexander C Carpenter^{1

2}, Niel Van Wyk^{1

6}, Felix Meier¹, Thomas R V Collier¹, Monica I Espinosa¹, Elizabeth L Daniel¹, Roy S K Walker^{1

7

8}, Yizhi Cai^{7

9}, Helena K M Nevalainen¹, Natalie C Curach^{1

10}, Ira W Deveson^{11

12}, Timothy R Mercer^{11

12

13}, Daniel L Johnson^{1

4}, Leslie A Mitchell¹⁴, Joel S Bader¹⁵, Giovanni Stracquadanio¹⁶, Jef D Boeke^{14

17

18}, Hugh D Goold^{1

19}, Isak S Pretorius¹, Ian T Paulsen^{1

3}

Affiliations

¹ School of Natural Sciences, ARC Centre of Excellence in Synthetic Biology, Macquarie University, Sydney, NSW, Australia.
² CSIRO Synthetic Biology Future Science Platform, Canberra, ACT 2601, Australia.
³ The Australian Genome Foundry, Sydney, NSW, Australia.
⁴ The Australian Wine Research Institute, Adelaide, SA 5064, Australia.
⁵ School of Agriculture, Food & Wine, Faculty of Sciences, University of Adelaide, Adelaide, SA 5005, Australia.
⁶ Department of Microbiology and Biochemistry, Hochschule Geisenheim University, Geisenheim, Germany.
⁷ School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.
⁸ School of Engineering, Institute for Bioengineering, The University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK.
⁹ Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
¹⁰ Bioplatforms Australia, Research Park Drive, Macquarie University, Macquarie Park, NSW 2109, Australia.
¹¹ St Vincent's Clinical School, University of New South Wales, Sydney, NSW 2010, Australia.
¹² The Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
¹³ Australian Institute for Bioengineering and Nanotechnology, University of Queensland, Brisbane, QLD, Australia.
¹⁴ Institute for Systems Genetics, NYU Langone Health, New York, NY 10016, USA.
¹⁵ Department of Biomedical Engineering, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
¹⁶ Institute of Quantitative Biology, Biochemistry, and Biotechnology, SynthSys, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, UK.
¹⁷ Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY 10016, USA.
¹⁸ Department of Biomedical Engineering, NYU Tandon School of Engineering, Brooklyn, NY 11201, USA.
¹⁹ New South Wales Department of Primary Industries, Orange, NSW 2800, Australia.

Abstract

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Keywords: SCRaMbLE; TAR1; adaptive laboratory evolution; directed evolution; synthetic biology; synthetic genome; yeast.