Glyco-recoded Escherichia coli: Recombineering-based genome editing of native polysaccharide biosynthesis gene clusters
| Autor: | Aravind Natarajan, Laura E. Yates, Margaret E. Hale, Mingji Li, Dominic C. Mills, Matthew P. DeLisa |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
Glycan Glycosylation Bioengineering Computational biology Bacterial genome size Biology medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology Recombineering Campylobacter jejuni 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins 010608 biotechnology Extrachromosomal DNA Escherichia coli medicine 030304 developmental biology Gene Editing 0303 health sciences Polysaccharides Bacterial Glycome genomic DNA chemistry Multigene Family biology.protein Biotechnology |
| Zdroj: | Metabolic Engineering. 53:59-68 |
| ISSN: | 1096-7176 |
| DOI: | 10.1016/j.ymben.2019.02.002 |
| Popis: | Recombineering-based redesign of bacterial genomes by adding, removing or editing large segments of genomic DNA is emerging as a powerful technique for expanding the range of functions that an organism can perform. Here, we 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. Specifically, 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. 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. Overall, our results define a convenient and reliable framework for bacterial glycome editing that provides a more stable route for chemical diversification of proteins in vivo and effectively expands the bacterial glycoengineering toolkit. |
| Databáze: | OpenAIRE |
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