Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome

Abstract

Here, we report the successful design, construction, and characterization of a 770-kilobase synthetic yeast chromosome II (synII). Our study incorporates characterization at multiple levels-including phenomics, transcriptomics, proteomics, chromosome segregation, and replication analysis-to provide a thorough and comprehensive analysis of a synthetic chromosome. Our Trans-Omics analyses reveal a modest but potentially relevant pervasive up-regulation of translational machinery observed in synII, mainly caused by the deletion of 13 transfer RNAs. By both complementation assays and SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution), we targeted and debugged the origin of a growth defect at 37°C in glycerol medium, which is related to misregulation of the high-osmolarity glycerol response. Despite the subtle differences, the synII strain shows highly consistent biological processes comparable to the native strain.

Fig. 1. Structure variation repair through chromosome breakage.

(A) I-SceI mediated repair strategy for synII structure variations. The donor fragment was designed to carry a URA3 cassette (yellow) and I-SceI recognition site (red), with both ends overlapping the structural variation sequences observed in synII (yeast_chr02_9.01). The donor fragment was integrated into synII between the two tandem repeats through homologous recombination, then an episomal plasmid pRS413-pGal1-I-SceI was transformed into the cell. A double strand break at the I-SceI site was induced in galactose medium, and the homologous recombination of two partial chromosomes of synII eliminated the duplication. (B) Structure variations in megachunks L and T and their corresponding donor sequence design. In megachunk L, a copy of chunk L2-L4 was observed following the original L2-L4 sequence that generated a tandem-duplication. The donor fragment was inserted directly between two duplications. In megachunk T, a complicated variation involving sequence of chunk T4-T5 was inserted into synII between chunk T4 and T5 and the donor fragment was inserted to remove the complex variation. (C) Deep sequencing read depth analysis revealed the successful sequential removal of duplications. The starting synthetic chromosome synII (yeast_chr02_9.01) has both duplication regions, and after the first round of repair at megachunk L, the resulting chromosome synII (yeast_chr02_9.02) has only one duplication. The finished chromosome synII (yeast_chr02_9.03) was obtained after the second repair at the megachunk T region.

Fig. 2. Phenotypic profiling of synII.

(A) Phenotype tests of synII on different media. 10-fold serial dilutions of overnight cultures of synII and wild-type (BY4741 and BY4742) strains were used for plating. From left to right: YPD at 25°C, 30°C, and 37°C; SC at 25°C, 30°C, and 37°C; low pH YPD (pH 4.0) and high pH YPD (pH 9.0); YPEG; SC+6-Azauracil; YPD+Benomyl; YPD+Camptothecin; YPD+Hydroxyurea; YPD+Cycloheximide (10 μg/ml, 2 hrs pretreatment); YPD+H₂O₂ (1 mM, 2 hrs pretreatment); YPD+Sorbitol; YPD+MMS, (YPD, yeast extract peptone dextrose; YPEG, yeast extract peptone glycerol ethanol; MMS, methyl methane sulfone; SC, synthetic complete). (B) Growth curves of synIIA-R, synIIR-Y and synII strains compared with those of BY4741 and BY4742 strains in YPD at 30°C. (C) Cell cycle comparison between synII and BY4741. Images show cell morphology at different stages during the cell cycle after release from G1 block. DNA staining is shown in blue; CEN2-GFP in G1, S, G2 and M phase are shown in green. Graphs show the percentage of synII cells with separated CEN2-GFP dots, metaphase spindles and anaphase spindles during the cell cycle. For each time point at least 200 cells were counted. The inset numbers of 0.99 indicate the overall ratio of metaphase to anaphase cells throughout the time course for synII and BY4741 strains. (D) synII (red) and BY4741 (black) replication time expressed as relative copy number by deep sequencing.

Fig. 3. synII strain Trans-Omics profile (BY4741 as reference) demonstrates that the synthetic chromosome design has minimal impact on cell physiology, despite a modest up-regulation of translational machinery triggered by tRNAs removal.

(A-D) Identified dysregulated genetic features at (A) transcriptome level, (B) proteome level and (C and D) metabolome level (Metabolic and lipid profiling in LC-MS positive mode respectively) of synII cells, compared to BY4741 cells. The detailed methods of these comparisons are described in the materials and methods section of the SOM. The total number of differentially expressed (p-value < 0.001) features in transcriptome, proteome and metabolome are also presented as well. Up-regulated and down-regulated features are labeled in red and green respectively. (E) Enriched pathways and the co-expression profile revealed by transcriptome and proteome in yeast GO terms. Up-regulated features are labeled in red and down-regulated features are labeled in green. (F) RNAseq analysis of synII with/without tRNA array. By adding back the tRNA array of synII, the up-regulation of translational functions is greatly mitigated. For E and F, significance level is indicated by heatmap color intensities and symbol sizes. Up-regulated and down-regulated features are labeled in red and green respectively.