Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond

Abstract

INTRODUCTION Total synthesis of designer chromosomes and genomes is a new paradigm for the study of genetics and biological systems. The Sc2.0 project is building a designer yeast genome from scratch to test and extend the limits of our biological knowledge. Here we describe the design, rapid assembly, and characterization of synthetic chromosome VI (synVI). Further, we investigate the phenotypic, transcriptomic, and proteomic consequences associated with consolidation of three synthetic chromosomes–synVI, synIII, and synIXR—into a single poly-synthetic strain. RATIONALE A host of Sc2.0 chromosomes, including synVI, have now been constructed in discrete strains. With debugging steps, where the number of bugs scales with chromosome length, all individual synthetic chromosomes have been shown to power yeast cells to near wild-type (WT) fitness. Testing the effects of Sc2.0 chromosome consolidation to uncover possible synthetic genetic interactions and/or perturbations of native cellular networks as the number of designer changes increases is the next major step for the Sc2.0 project. RESULTS SynVI was rapidly assembled using nine sequential steps of SwAP-In (switching auxotrophies progressively by integration), yielding a ~240-kb synthetic chromosome designed to Sc2.0 specifications. We observed partial silencing of the left- and rightmost genes on synVI, each newly positioned subtelomerically relative to their locations on native VI. This result suggests that consensus core X elements of Sc2.0 universal telomere caps are insufficient to fully buffer telomere position effects. The synVI strain displayed a growth defect characterized by an increased frequency of glycerol-negative colonies. The defect mapped to a synVI design feature in the essential PRE4 gene ( YFR050C ), encoding the β7 subunit of the 20 S proteasome. Recoding 10 codons near the 3′ end of the PRE4 open reading frame (ORF) caused a ~twofold reduction in Pre4 protein level without affecting RNA abundance. Reverting the codons to the WT sequence corrected both the Pre4 protein level and the phenotype. We hypothesize that the formation of a stem loop involving recoded codons underlies reduced Pre4 protein level. Sc2.0 chromosomes (synI to synXVI) are constructed individually in discrete strains and consolidated into poly-synthetic (poly-syn) strains by “endoreduplication intercross.” Consolidation of synVI with synthetic chromosomes III (synIII) and IXR (synIXR) yields a triple-synthetic (triple-syn) strain that is ~6% synthetic overall—with almost 70 kb deleted, including 20 tRNAs, and more than 12 kb recoded. Genome sequencing of double-synthetic (synIII synVI, synIII synIXR, synVI synIXR) and triple-syn (synIII synVI synIXR) cells indicates that suppressor mutations are not required to enable coexistence of Sc2.0 chromosomes. Phenotypic analysis revealed a slightly slower growth rate for the triple-syn strain only; the combined effect of tRNA deletions on different chromosomes might underlie this result. Transcriptome and proteome analyses indicate that cellular networks are largely unperturbed by the existence of multiple synthetic chromosomes in a single cell. However, a second bug on synVI was discovered through proteomic analysis and is associated with alteration of the HIS2 transcription start as a consequence of tRNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-syn strain “omics” analyses. CONCLUSION Analyses of phenotypes, transcriptomics, and proteomics of synVI and poly-syn strains reveal, in general, WT cell properties and the existence of rare bugs resulting from genome editing. Deletion of subtelomeres can lead to gene silencing, recoding deep within an ORF can yield a translational defect, and deletion of elements such as tRNA genes can lead to a complex transcriptional output. These results underscore the complementarity of transcriptomics and proteomics to identify bugs, the consequences of designer changes in Sc2.0 chromosomes. The consolidation of Sc2.0 designer chromosomes into a single strain appears to be exceptionally well tolerated by yeast. A predictable exception to this is the deletion of tRNAs, which will be restored on a separate neochromosome to avoid synthetic lethal genetic interactions between deleted tRNA genes as additional synthetic chromosomes are introduced. Debugging synVI and characterization of poly-synthetic yeast cells. ( A ) The second Sc2.0 chromosome to be constructed, synVI, encodes a “bug” that causes a variable colony size, dubbed a “glycerol-negative growth-suppression defect.” ( B ) Synonymous changes in the essential PRE4 ORF lead to a reduced protein level, which underlies the growth defect. ( C ) The poly-synthetic strain synIII synVI synIXR directs growth of yeast cells to near WT fitness levels.