Plasmid Preparation and Construction of Expression Strains.

Native hyaA containing a C-terminal His₆ tag (hyaA.his₆) and hyaB genes in a pUC18 plasmid (Addgene; plasmid 50004) was used as a template for mutagenesis (52). Cysteines at positions 76, 79, 576, and 579 in hyaB were mutated to TAG using the QuikChange II Site-Directed Mutagenesis protocol (Agilent Technologies). Flanking regions (50 bp) homologous to the surrounding hyaA-hyaB gene sequence were added to both ends of the successful plasmids in preparation for recombination. Primers were acquired from Keck Biotechnology Resource and DNA sequencing was performed at the Keck DNA Sequencing Facility at Yale University. The resulting TAG variants were used to replace the hyaA and hyaB genes in the genome of ME6 cells (E. coli K-12 Δgor ΔselABC Δfdh strain) via recombination (53) to generate strains NK157 to NK160 (SI Appendix, Table S2).