The de novo design and synthesis of yeast chromosome XIII facilitates investigations on aging

Abstract

In the era of synthetic biology, design, construction, and utilization of synthetic chromosomes with unique features provide a strategy to study complex cellular processes such as aging. Herein, we successfully construct the 884 Kb synXIII of Saccharomyces cerevisiae to investigate replicative aging using these synthetic strains. We verify that up-regulation of a rRNA-related transcriptional factor, RRN9, positively influence replicative lifespan. Using SCRaMbLE system that enables inducible whole-genome rearrangement on synXIII, we obtain 20 SCRaMbLEd synXIII strains with extended lifespan. Transcriptome analysis reveal the expression of genes involve in global protein synthesis is up-regulated in longer-lived strains. We establish causal links between genotypic change and the long-lived phenotype via reconstruction of some key structural variations observed in post-SCRaMbLE strains and further demonstrate combinatorial effects of multiple aging regulators on lifespan extension. Our findings underscore the potential of synthetic yeasts in unveiling the function of aging-related genes.

Fig. 1. Design, construction, and characterization of synXIII.

a Overview of synXIII construction with the indicated designer features. The gray reprensents the native chromosome XIII, and the orangered represents the synthetic chromosome; The right table listed the difference between the chrXIII, synXIII and final constructed synXIII. b Native chromosome XIII replacement with synthetic chunks. On average 6~7 chunks were used to replace the native segments of chromosome XIII (gray). 9 and 6 step-by-step replacements were carried out in parallel from the left arm and right arm respectively with iterative selectable markers (LEU2 or URA3) through homologous recombination in yeast with homologous regions around 500 bp ~1 kb junctions (blue) produced by PCR using ligated chunks as template was designed to have 500 bp overlapping regions with two adjacent chunks to improve the replacement efficiency. I-SceI mediated synXIIIA-I and synXIIIJ-O integration. An I-SceI site (yellow) was introduced on both semi-synthetic chromosomes. After mating, I-SceI induction was performed to generate double strain breaks on both semi-synthetic chromosomes. 30 kb homologous region on both semi-synthetic chromosomes enabled the integration of complete synXIII.

Fig. 2. Characterization of synXIII.

a Scanning Electron Microscopy (SEM) images of BY4741 and synXIII strains. Scale bars: 5.00 μm. Source data are provided as a Source data file. b Growth curve analysis of BY4741 and synXIII. Each strain was analyzed with three biological replicates (n = 3), and each biological replicate consisted of three technical replicates. The error bands represent a total of nine replicates at each time point. Data are presented as mean values ±SD. Source data are provided as a Source data file c Doubling times of corresponding strains in YPD medium, each strain was conducted with three replicates (n = 3). Each replicate represents the mean doubling time of three technical replicates. Data are presented as mean values ±SD from three independent replicates. Source data are provided as a Source data file. d Cellular viability responses of synXIII upon exposure to normal and stress conditions. 10-fold serial dilutions of overnight cultures of synXIII and wild-type (BY4741 and BY4742) strains were used for plating. Conditions include: YPD at 30 °C; SC at 30 °C; YPD + Benomyl; YPD + H₂O₂ (1 mM, 2 h pretreatment); YPD + methyl methane sulfone (MMS, 0.01% v/v); YPD + Camptothecin (0.1 μg/mL); YPD + Rapamycin (0.2 μg/mL); YPD + Cycloheximide (10 μg/mL, 2 h pretreatment); YPD + Sorbitol (1 M); YPD, yeast extract peptone dextrose; YPEG, yeast extract peptone glycerol ethanol; SC, synthetic complete. e Survive curves of synXIII and BY4741. X-axis presents the number of daughter cells generated from the mother cell. Each strain was measured with three replicates (n = 3) and 40 cells were monitored for each replicate. Source data are provided as a Source data file. f Identified dysregulated genes of synXIII strain at the transcriptome level compared with BY4741. The P value was calculated by DESeq2 (v1.30.1). The differentially expressed genes were assessed at genome-wide significance P < 7.56 × 10⁻⁶ for 5% family-wise error rate based on 6607 genes with at least one mapped read and also corresponding to log₂ (foldchange) ≥1 or ≤−1. Source data are provided as a Source data file. g Identified dysregulated genetic features of synXIII strain at the proteome level compared with BY474. The P value was calculated by IQuant (v2.0.1) in two-sided T-test. Source data are provided as a Source data file. f, g the up and down-regulated genes are labeled in red and green respectively.

Fig. 3. Synthetic RRN9 exhibits higher expression and promotes lifespan extension.

a Lifespan measurement of intermediate strains with three replicates (n = 3), synXIII-O and synXIII-N. The arrow presents the direction of construction. Source data are provided as a Source data file. b The lifespan measurement of all intermediate strains of synthetic megachunk N (highlight in orange) compared to synXIII-O. Synthetic megachunk N was segmented into N1~N6 chunks, which was integrated respectively using synXIII-O as the host strain. N2 chunk carried six synthetic genes (RSN2, PPA2, PRP24, TMA23, RRN9, URA10) which were integrated in the synXIII-O host strain respectively. Data are presented as mean values ±SD from three independent replicates (n = 3). Source data are provided as a Source data file. c The expression level of synthetic and native RRN9 gene measured by RT-PCR (normaried to native RRN9 in synXIII-O). The red dashed line represents the expression level of native RRN9 gene in BY4741. Each strain was conducted with three replicates (n = 3). Each replicate represents the expression level of three technical replicates. Data are presented as mean values ±SD from three independent experiments. The blue and gray bars present the synthetic and native PCRTags respectively. Source data are provided as a Source data file. d Dissection of the effect of synonymous recoding by PCRTags in RRN9 gene to its gene expression measured by RT-PCR, each strain was conducted with three replicates (n = 3). Each replicate represents the expression level of three technical replicates. Data are presented as mean values ±SD from three independent replicates. The synthetic and wildtype PCRTags are presented in blue and gray respectively. The right panel presents the corresponding sequence of synthetic and wildtype PCRtags. Source data are provided as a Source data file. e Replicative aging measurement of synXIII-O and BY4741 strains with wildtype and synthetic PCRTag2 with three independent replicates (n = 3). Data are presented as mean values ±SD from three independent replicates. Source data are provided as a Source data file. f Survival curves of the strains with increased doses of RRN9. The Ycz402 and Ycz403 strains express pRS416 (centromere-based empty vector) and pRS416-RRN9 in BY4741 respectively. Each lifespan measurement was performed with three replicates (n = 3) and 40 cells were monitored for each replicate. The statistical confidence is calculated by one-sided T-test, and error bars represent standard division. Source data are provided as a Source data file.

Fig. 4. Screening and characterizing of long-lived strains from SCRaMbLEd synXIII strains.

a Schematic illustration of SCRaMbLE and screening process for long replicative aging strains. The HSP104 fused to eGFP (HSP104-eGFP) acts as a reporter for replicative lifespan in yeast. The URA3 reporter was randomly integrated into regions positioned between two loxPsym sites allowing for SCRaMbLEant selection by 5-FOA. SCRaMbLEants with lower fluorescence intensity were selected by FACS, followed by lifespan measurement using a microfluidic device. b Lifespan extension of 20 long-lived SCRaMbLEd strains compared to parental strain synXIII Ycz063, replicative lifespan measurement of each strain was conducted with three replicates (n = 3), each replicate was conducted with 40 cells. Each data presentation is derived from comparing the value of each sample with each control value respectively, generating nine data. Data are presented as mean values ±SD from three independent replicates. Source data are provided as a Source data file. c Transcriptome profiling of lifespan extended strains compared to synXIII strain Ycz063. The heatmap represents the statistical significance of Gene Ontology (GO) enrichment of differentially expressed genes. The number in the brackets indicates the Pearson correlation between the statistical significance and lifespan extension. red, upregulated; blue, down-regulated; green: relative lifespan compared to initial synXIII. Source data are provided as a Source data file.

Fig. 5. Genome-wide analysis of 20 lifespan-extended SCRaMbLEants.

a Schematic map of structural variations in 20 long-lived SCRaMbLEants. Y-axis shows the relative aging extension of each SCRaMbLEant in comparison to parental synXIII strain that of represented by color scale. The red rectangles indicate the loci of aging regulators on chromosome synXIII. Source data are provided as a Source data file. b Different variations were individually constructed in synXIII, and the lifespan was measured for each variant along with the calculation of the rate of lifespan change with three replicates (n = 3). In the figures, the upward arrow indicates an increase, the downward arrow indicates a decrease. The blue color indicates the sequence inverted, the orangered color indicates the sequence duplicated, and the vanished sequence is indicative of the deletion. Source data are provided as a Source data file.