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Article summary:
1. This article describes a glyco-recoding strategy for replacing major non-essential polysaccharide gene clusters in K-12 Escherichia coli with orthogonal glycosylation components for both biosynthesis of heterologous glycan structures and site-specific glycan conjugation to target proteins.
2. The native enterobacterial common antigen (ECA) and O-polysaccharide (O-PS) antigen loci were systematically replaced with ∼9–10 kbp of synthetic DNA encoding Campylobacter jejuni enzymes required for asparagine-linked (N-linked) protein glycosylation.
3. Compared to E. coli cells carrying the same glycosylation machinery on extrachromosomal plasmids, glyco-recoded strains attached glycans to acceptor protein targets with equal or greater efficiency while exhibiting markedly better growth phenotypes and higher glycoprotein titers.
Article analysis:
This article provides a detailed description of a novel approach to engineering bacterial genomes by adding, removing or editing large segments of genomic DNA in order to expand the range of functions that an organism can perform. The authors describe a “glyco-recoding” strategy whereby major non-essential polysaccharide gene clusters in K-12 Escherichia coli are replaced with orthogonal glycosylation components for both biosynthesis of heterologous glycan structures and site-specific glycan conjugation to target proteins. The authors provide evidence that this approach is effective in producing high levels of product titers and efficient attachment of glycans to acceptor proteins, while also providing a more stable route for chemical diversification of proteins in vivo than plasmid based systems.
The article is well written and provides clear descriptions of the methods used as well as the results obtained from experiments conducted using this approach. The authors provide sufficient detail about their methodology, which allows readers to evaluate the trustworthiness and reliability of their claims. Furthermore, they cite relevant literature throughout the article, which further adds credibility to their work.
The only potential bias present in this article is that it does not explore any potential risks associated with this approach or discuss any possible drawbacks or limitations that may be encountered when using it. Additionally, there is no discussion about alternative approaches that could be used instead or how this approach compares to other existing methods for engineering bacterial genomes.
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