Three GNA1 coding sequences from yeast and a higher plant were inserted into pET24d(+), forming T7lac-GNA1 expression cassettes. Recombinant plasmids were transformed into E. coli strain 7107-18, which contained an integrated expression cassette of T7lac-glmS*54. Resulting strains with GNA1 expression vectors were grown for 23 hours in a simple mineral medium (M9B) supplemented with 40 g L-1 glucose, 10 g L-1 ribose, and 5 g L-1 yeast extract. Cultures were induced with 0.2 mM IPTG at the time of inoculation.
- The GlcN production pathway was modified for GlcNAc production by expressing a heterologous glucosamine-6-P acetyltransferase (GNA1), which converts glucosamine-6-P into N-acetylglucosamine-6-P.
- GlcNAc were produced at high levels in the growth medium.