Heterozygous diploid and interspecies SCRaMbLEing

Abstract

SCRaMbLE (Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution) is a genome restructuring technique that can be used in synthetic genomes such as that of Sc2.0, the synthetic yeast genome, which contains hundreds to thousands of strategically positioned loxPsym sites. SCRaMbLE has been used to induce rearrangements in yeast strains harboring one or more synthetic chromosomes, as well as plasmid DNA in vitro and in vivo. Here we describe a collection of heterozygous diploid strains produced by mating haploid semisynthetic Sc2.0 strains to haploid native parental strains. We subsequently demonstrate that such heterozygous diploid strains are more robust to the effects of SCRaMbLE than haploid semisynthetic strains, rapidly improve rationally selected phenotypes in SCRaMbLEd heterozygous diploids, and establish that multiple sets of independent genomic rearrangements are able to lead to similar phenotype enhancements. Finally, we show that heterozygous diploid SCRaMbLE can also be carried out in interspecies hybrid strains.

Fig. 1

Construction and testing of heterozygous diploid strains. a Experimental workflow. A S. cerevisiae strain bearing one or more synthetic chromosomes is mated to a S. cerevisiae or S. paradoxus strain with a “wild-type” genome. The resultant heterozygous diploid cells can be selected for, SCRaMbLEd, and tested for tolerance of a variety of environmental and chemical conditions. Strains showing increased fitness have their phenotype verified and can be analyzed with whole genome sequencing to determine the sets of SCRaMbLE events responsible for a given phenotype. b SCRaMbLE of haploid and heterozygous diploid synVsynX yeast was induced by adding 1 µM β-estradiol to culture media for 6 h. Heterozygous diploid S288C-synVsynX strains demonstrate a lesser degree of SCRaMbLE-mediated lethality at both 30 °C and 37 °C compared to haploid synVsynX strains. c Heterozygous diploid strains incorporating a variety of S. cerevisiae “wild-type” genomes are robust to SCRaMbLE. Additionally, heterozygous diploid strains containing two synthetic chromosomes can be SCRaMbLEd without appreciable loss in viability compared to strains containing one synthetic chromosome

Fig. 2

SCRaMbLE of Y12-synX rapidly improves thermotolerance. a Serial dilution assay comparing the growth of two independent SCRaMbLEd isolates of Y12-synX (yYW166 and yYW167) with the non-SCRaMbLEd parent strain (yMS423) and a Y12 diploid strain (yYW207) under various temperature conditions, as well as in YPGE. b Average sequencing depth per segment along synX of yMS423, yYW166, and yYW167. Deletions (boxes A, B, C, E, F, G, H, I, and J), as well as a duplication (box D) are highlighted. c BY4743, a Y12/Y12 diploid, yMS423, yYW166, yYW167, and yMS423∆ were all grown in liquid YPD overnight and diluted to a starting A₆₀₀ of 0.1 in fresh YPD. These strains were then cultured in a 96-well plate reader with shaking at 30 °C or 42 °C. Optical density measurements were taken every 10 min and used to calculate doubling time. Error bars shown are mean and standard deviation from four technical replicates. One-way ANOVA with multiple comparisons was used to assess difference between each sample and yMS423 (***p < 0.001, **p < 0.01). Variance between the groups was determined to be similar

Fig. 3

SCRaMbLE of CBS5829-syn(V)X improves caffeine tolerance. a Serial dilution assay comparing the growth of SCRaMbLEd S. paradoxus CBS5829-synX (yMS637, yMS638, yMS639, yMS640, yYW184, and yYW185) or CBS5829-synVsynX (yYW186, yYW187, yYW195, and yYW196) strains to their non-SCRaMbLEd CBS5829-synX parent (yMS521) on high caffeine YPD plates. b The POL32 gene with 500 bp upstream/300 bp downstream sequence was cloned from BY4741 into the episomal plasmid pRS416 and the resulting plasmid pRS416-POL32 transformed into yMS521. These strains were compared via serial dilution assay to the SCRaMbLEd strain yYW185 transformed with pRS416, yMS521 transformed with pRS416, or BY4743 transformed with pRS416 on SC–Ura + 4 mg/mL caffeine. c yMS521 transformed with pRS416, yMS521 transformed with pRS416-POL32, yYW184 transformed with pRS416, and yYW185 transformed with pRS416 were all grown in liquid SC–Ura media overnight and then diluted to a starting A₆₀₀ of 0.1 in either SC–Ura + 4 mg/mL or 5 mg/mL caffeine and cultured in a 96-well plate reader with shaking. Optical density measurements were taken every 10 min and used to calculate doubling time. Error bars shown are mean and standard deviation from four technical replicates. One-way ANOVA with multiple comparisons was used to assess difference between yMS521 + pRS416 and other samples (****p < 0.0001). Variance between the groups was determined to be similar