Bacterial glmS and yeast GFA1 gene coding sequences were inserted into a pET vector and transformed into an E. coli strain (genotype: Δnag, manXYZ, DE3). Strains containing a free-replicating plasmid or an integrated expression cassette were grown in shake flasks under IPTG-induced conditions at 30°C for 26 hours in a defined mineral salt medium (M9A) supplemented with glucose.
- Significant amounts of GlcN were detected in the growth medium.
- The highest levels of enzyme activity and GlcN production were seen with the Bacillus glmS gene, which encodes an enzyme more resistant to inhibition by GlcN-6-P than the E. coli homologue.
- GlcN production was higher with an integrated expression cassette than with a free-replicating plasmid.