Construction of a synthetic Saccharomyces cerevisiae pan-genome neo-chromosome

Abstract

The Synthetic Yeast Genome Project (Sc2.0) represents the first foray into eukaryotic genome engineering and a framework for designing and building the next generation of industrial microbes. However, the laboratory strain S288c used lacks many of the genes that provide phenotypic diversity to industrial and environmental isolates. To address this shortcoming, we have designed and constructed a neo-chromosome that contains many of these diverse pan-genomic elements and which is compatible with the Sc2.0 design and test framework. The presence of this neo-chromosome provides phenotypic plasticity to the Sc2.0 parent strain, including expanding the range of utilizable carbon sources. We also demonstrate that the induction of programmable structural variation (SCRaMbLE) provides genetic diversity on which further adaptive gains could be selected. The presence of this neo-chromosome within the Sc2.0 backbone may therefore provide the means to adapt synthetic strains to a wider variety of environments, a process which will be vital to transitioning Sc2.0 from the laboratory into industrial applications.

Fig. 1. Construction of a yeast pan-genome neo-chromosome.

a Schematic representation of the circular PGNC construct (PGNC^circ). b Iterative assembly of the PGNC via homologous recombination and using alternating selectable markers. c Confirmation of the PGNC assembly using PCR reactions that span each inter-chunk junction. Identical PCR results were obtained across at least two repetitions and during sub-assembly d Production of three linear PGNC variants using the telomerator (PGNC^lin1, PGNC^lin2 and PGNC^lin3). Yellow boxes define the position of the centromeric-plasmid backbone. Red boxes denote positions of synthetic telomeric repeat sequences added by the telomerator process. e Microplate growth kinetics of the four PGNC variants (PGNC^circ, PGNC^lin1, PGNC^lin2 and PGNC^lin3) compared to the BY4742 (WT) strain. Data are presented as mean values +/− SD, based upon values obtained from three independent biological replicates. Source data are provided as a Source Data file.

Fig. 2. Mitotic stability of the PGNC in the absence of selection.

a Populations were established for each four PGNC variants (PGNC^circ, PGNC^lin1, PGNC^lin2 and PGNC^lin3) and serially passaged eight times for ~50 generations of total growth. b Differential plating of 96 colonies for phenotypic assessment of loss of the PGNC element. c Results of passaging experiments (96 colonies per replicate) for each of the PGNC strains after both 25 and 50 generations. d Results of passaging experiments (96 colonies per replicate) for each of the PGNC strains containing additional ARS sequences after both 25 and 50 generations. All graphs are presented as mean values +/− SD, based upon values obtained from three independent biological replicate passaging experiments. Source data are provided as a Source Data file.

Fig. 3. The PGNC element expands the phenotypic repertoire of BY4742.

a BioLog results for the carbon-source utilization plates, PM1 and PM2A. Values are presented as log₂ normalized (PGNC:WT) observations of maximal respiration rate, with increased growth rates highlighted in green. b Growth kinetics for PGNC strains with palatinose as a sole carbon source. c Identifying the palatinose-utilizing region of the PGNC via 24 h end-point growth assays of intermediate PGNC elements. d Growth kinetics for PGNC strains with melibiose as a sole carbon source. e Identifying the melibiose-utilizing region of the PGNC via 24 h end-point growth assays of intermediate PGNC elements. All growth kinetic data are presented as mean values +/− SD, based upon values obtained from three independent biological replicates. Source data are provided as a Source Data file.

Fig. 4. SCRaMbLE induced genetic and phenotypic diversification of the PGNC.

a The methodology for SCRaMbLE-induced selection for increased growth on melibiose. b Growth kinetics of individual isolates from Cre-expressing and control populations. Data are presented as mean values +/− SD, based upon values obtained from three independent biological replicates. c Locations of clone-specific SCRaMbLE-induced recombination between loxPsym sites within the PGNC element. d Structural variation adjacent to ORF21. e SCRaMbLE-induced copy number variation within the melibiose-selected isolates (yellow, isolates from the Cre-expressing populations; green, isolates from the control populations). Source data are provided as a Source Data file.