Ring synthetic chromosome V SCRaMbLE

Abstract

Structural variations (SVs) exert important functional impacts on biological phenotypic diversity. Here we show a ring synthetic yeast chromosome V (ring_synV) can be used to continuously generate complex genomic variations and improve the production of prodeoxyviolacein (PDV) by applying Synthetic Chromosome Recombination and Modification by LoxP-mediated Evolution (SCRaMbLE) in haploid yeast cells. The SCRaMbLE of ring_synV generates aneuploid yeast strains with increased PDV productivity, and we identify aneuploid chromosome I, III, VI, XII, XIII, and ring_synV. The neochromosome of SCRaMbLEd ring_synV generated more unbalanced forms of variations, including duplication, insertions, and balanced forms of translocations and inversions than its linear form. Furthermore, of the 29 novel SVs detected, 11 prompted the PDV biosynthesis; and the deletion of uncharacterized gene YER182W is related to the improvement of the PDV. Overall, the SCRaMbLEing ring_synV embraces the evolution of the genome by modifying the chromosome number, structure, and organization, identifying targets for phenotypic comprehension.

Fig. 1

Ring_synV SCRaMbLE to generate genomic and phenotypic variation. The ring_synV (orange circle) was divided into 170 segments (green arrows) by loxPsym sites, in which genes were demonstrated in purple and blue arrows. a The PDV biosynthesis pathway was incorporated in YEL063C (CAN1) of ring_synV by homologous recombination to generate the initial PDV producer strain yWJS1. b The SCRaMbLE system of yWJS1 functioned and SCRaMbLEd colonies with diversity of PDV improvement were isolated when exposed to galactose and estradiol. c Genomic SV analysis of SCRaMbLEants revealed chromosome number variation, CNV and novel SV. d Colonies with increased PDV production were selected when transformed novel SVs into the initial strain

Fig. 2

Continuous improvement of PDV production and generation of SVs. a Fold changes of PDV production in the top 10 SCRaMbLEd strains. b Phenotypic analysis of SCRaMbLEants and fold changes of PDV production after each round of SCRaMbLE. Initial strain, yWJS1; first cycle SCRaMbLEant, yWJS067; second cycle, yWJS168; third cycle, yWJS184; forth cycle, yWJS249; fifth cycle, yWJS321. c Boxplots showing WGS read cover depth, which indicated recurrent chromosomal aneuploidies by SCRaMbLEing the ring_synV in haploid yeast strain. The strains yWJS044 (aneuploid chromosomes: I, III, ring_synV and VI), yWJS047, and yWJS067 were generated by SCRaMbLEing initial strain yWJS1. The strains yWJS184 (aneuploid chromosome: XIII) and yWJS321 (aneuploid chromosomes: XII and XIII) were generated by SCRaMbLEing yWJS067. The coverage depth stands for the chromosome CN that were determined from WGS result. The aneuploid chromosomes were marked in red, and ring_synV was marked in purple. The centre line in each box denotes the median of read coverage depth, the upper bound of box denotes the upper quartile and the lower bound denotes the lower quartile, the upper whisker denotes the maximum read coverage depth and the lower whisker denotes the minimum read coverage depth. d CNV heatmap of ring_synV. Read depth-based CN estimations (loxPsym divided segments) are indicated by color (scale provided to the right). Values are averages from three experiments, and error bars denote s.d. Strain information was listed in Supplementary Data 3

Fig. 3

Intrachromosomal interactions in the SCRaMbLEd ring_synV. a CIRCOS diagrams showing the interchromosomal SV fusions for the SCRaMbLEants yWJS067, yWJS184 and yWJS321, and interacting partners with each line representing an interaction. Chromosomes are plotted across the circle. b The SCRaMbLEd ring_synV (yWJS067) chromosome aligned to the reference. The rearrangement events of inversion, duplication, deletion, and translocation in SCRaMbLEd ring_synV chromosome (yWJS067, bottom) are shown as diagrammatic interpretations with the reference ring_synV chromosome (top)

Fig. 4

Functional novel SVs that may correlate with PDV production improvement. a Schematics showing novel SV that may correlate with PDV production improvement. The left DNA segment (orange arrow) and right DNA segment (blue) are divided by loxPsym sites (green diamond). The DNA sequence was amplified by using primers designed (black arrow) and transformed into yWJS1 and assembled with linearized pRS413 backbone plasmid. We isolated transformants with virtual darker colors by selection from 11 novel SVs than the initial strain. b Fold change of PDV production improvement related to novel SV structures that are quantitated by HPLC. Values are averages from three experiments, and error bars denote s.d