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Synthetic Yeast Chromosome

Summary and opinion

The successful synthesis of the Mycoplasma mycoides genome in 2010 was the first time a living cell contained a lab-made genome. The bacterium, “Synthia,” was touted as the first synthetic life form. The genome was a technical proof-of-concept, demonstrating that large-scale DNA synthesis is possible. Though encoded in the Synthia genome were a few names and quotes (one of which led to the threat of a breach of copyright lawsuit), there was no genomic novelty that could address basic research questions.

The synthetic Saccharomyces cerevisiae chromosome III, or synIII, embodies a different scientific approach. The authors integrated the goals of developing new technical approaches with establishing an experimental system that can be interrogated for useful data from many angles. Their thoughtful in silico design for a streamlined chromosome, containing alterations such as removal of introns, was successfully made a reality by an army of Johns Hopkins undergraduates. The chromosome-wide alterations do not appear to affect fitness in laboratory growth conditions.

synIII is a novel approach in the field of synthetic biology. Precedents for multiplexed, chromosome scale manipulations are scant, owing to the complexity of making iterative genome alterations. Through its integrated recombination sites, synIII could be the first of a new type of experimental genomics in the spirit of the genetic screen. What genomes (combinations of genes) can give rise to a phenotype, the most obvious being viability? The main text of the paper reports an extremely high rate of recombination at the MAT locus under induction of their engineered recombinases in diploid yeast. Recombinase activity was sufficiently high that Cre was toxic to haploid cells due to excessive gene deletion. An optimized system that uses a weaker Cre variant could be immensely useful in generating libraries of mutant, viable yeast lacking specific genes.

Figure 1

The synIII chromosome is derived from the S. cerevisiae chromosome III, but with deliberate changes. Figure 1 shows examples of the changes made across the chromosome. First, all TAG stop codons were changed to TAA. The elimination of one of the 64 codons from the genome could be useful in future applications, such as introducing of a fully orthogonal tRNA carrying a non-standard amino acid. The authors also integrated loxPsym recombination sites (sym for symmetrical) flanking genes that are singly non-essential. The loxPsym sites could allow future researchers to discover large combinations of non-essential genes. Other genome changes include deletions of introns and tRNA genes as well as the incorporation of markers (PCRTags) that distinguish synIII from the endogenous chromosome III.

Figure 2

Undergraduates at Johns Hopkins University were responsible for the bulk of synIII assembly. Because in vitro DNA synthesis produces short ssDNA oligos, the assembly of a complete chromosome required multiple stages of hierarchical assembly. In the first step, overlapping oligos were amplified by PCR to produce ~750-bp dsDNA Building Blocks. Building Blocks were then assembled into Minichunks by homologous recombination in yeast, facilitated by short overlaps between adjacent Building Blocks built into their design.

To generate the fully assembled synIII, they used homologous recombination to swap segments of the endogenous yeast chromosome III with their synthetic chromosome. By alternating between the selectable markers LEU2 and URA3, they incorporated synIII segments in a stepwise fashion along the length of chromosome III. The successful integration of each set of Minichunks is associated with flipping between Leu⁻/Ura⁺ and Leu⁺/Ura⁻ phenotypes.

Figure 3

To ensure that the endogenous chromosome III was fully converted to synIII by their homologous recombination scheme, the authors used PCRTags (markers) unique to either the wild type chromosome or synIII. Using gDNA extracted from wild type and synIII strains, they found that the PCRTags only generated a PCR product when matched with their cognate strain. The result indicates that the wild type chromosome was successfully converted to synIII in the engineered strain. By running the gDNA on a gel, they confirmed the smaller length of synIII relative to III by increased migration distance through the gel matrix. Genome sequencing (not shown in the figure) indicated almost complete replacement of the wild type chromosome with synIII sequence, except for 10 sites, mostly single bases. Additionally, they tested growth phenotypes for synIII and the assembly intermediate synIIIL under different culturing conditions. They found no differences in colony morphology or titer between the two engineered strains and wild type under the various growth conditions.

Figure 4

A whole genome transcriptome analysis found differential expression of only two genes present in both synIII and wild type, indicating that the chromosomes are functionally similar. The authors also monitored the stability of synIII over 125 generations using 30 replicate strains by tracking their PCRTags, finding no deletions. They also measured the frequency of conversion to the MATa mating type from MATα, an indication of chromosome III loss due to the location of MAT on chromosome III. Consistent with their PCRTag result, there was no significant difference in mating type conversion rates between synIII and wild type. In the final experiment of the figure, they expressed Cre recombinase to introduce random deletions using the loxPsym sites included in synIII, a process they called SCRaMbLE. In heterozygotes carrying the MATα allele on synIII and the MATa allele on wild type III, almost half of the yeast became mating type a. The wild type diploid heterozygotes are normally non-mating. Expression of Cre in MAT heterozygotes carrying only wild type chromosomes failed to produce mating diploids, as expected. PCRTag mapping confirmed that mating diploids carrying Cre-edited synIII lost the MATα locus, allowing the yeast to adopt the a mating phenotype.

Reference:

Annaluru N and Muller H et al. 2014. Total synthesis of a functional designer eukaryotic chromosome. Science 344:55-58.

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