Synthetic yeast chromosome XI design provides a testbed for the study of extrachromosomal circular DNA dynamics.

We describe construction of the synthetic yeast chromosome XI (synXI) and reveal the effects of redesign at non-coding DNA elements. The 660-kb synthetic yeast genome project (Sc2.0) chromosome was assembled from synthesized DNA fragments before CRISPR-based methods were used in a process of bug discovery, redesign, and chromosome repair, including precise compaction of 200 kb of repeat sequence. Repaired defects were related to poor centromere function and mitochondrial health and were associated with modifications to non-coding regions. As part of the Sc2.0 design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show that these sites can facilitate induced extrachromosomal circular DNA (eccDNA) formation, allowing direct study of the effects and propagation of these important molecules. Construction and characterization of synXI contributes to our understanding of non-coding DNA elements, provides a useful tool for eccDNA study, and will inform future synthetic genome design.

N-Terminal Modification of Gly-His-Tagged Proteins with Azidogluconolactone.

Site-specific protein modifications are vital for biopharmaceutical drug development. Gluconoylation is a non-enzymatic, post-translational modification of N-terminal HisTags. We report high-yield, site-selective in vitro α-aminoacylation of peptides, glycoproteins, antibodies, and virus-like particles (VLPs) with azidogluconolactone at pH 7.5 in 1 h. Conjugates slowly hydrolyse, but diol-masking with borate esters inhibits reversibility. In an example, we multimerise azidogluconoylated SARS-CoV-2 receptor-binding domain (RBD) onto VLPs via click-chemistry, to give a COVID-19 vaccine. Compared to yeast antigen, HEK-derived RBD was immunologically superior, likely due to observed differences in glycosylation. We show the benefits of ordered over randomly oriented multimeric antigen display, by demonstrating single-shot seroconversion and best virus-neutralizing antibodies. Azidogluconoylation is simple, fast and robust chemistry, and should accelerate research and development.

Total synthesis of a eukaryotic chromosome: Redesigning and SCRaMbLE-ing yeast.

A team of US researchers recently reported the design, assembly and in vivo functionality of a synthetic chromosome III (SynIII) for the yeast Saccharomyces cerevisiae. The synthetic chromosome was assembled bottom-up from DNA oligomers by teams of students working over several years with researchers as the first part of an international synthetic yeast genome project. Embedded into the sequence of the synthetic chromosome are multiple design changes that include a novel in-built recombination scheme that can be induced to catalyse intra-chromosomal rearrangements in a variety of different conditions. This system, along with the other synthetic sequence changes, is intended to aid researchers develop a deeper understanding of how genomes function and find new ways to exploit yeast in future biotechnologies. The landmark of the first synthesised designer eukaryote chromosome, and the power of its massively parallel recombination system, provide new perspectives on the future of synthetic biology and genome research.