Error-prone PCR was used to introduce random mutations in the E. coli glmS gene. Mutants that produced GlcN at higher levels were screened by a cross-feeding plate assay. These mutants showed significantly reduced sensitivity to GlcN-6-P. Different strains with an integrated expression cassette of mutant glmS genes were grown in shake flasks in M9A supplemented with glucose. GlcN production was induced by the addition of 0.2 mM IPTG at the time of inoculation. One of the best mutants, 2123-54, produced 20-fold more GlcN than the control.
- Wild type E. coli GlmS is inhibited by GlcN-6-P. Activity was 80% inhibited at 6 mM GlcN-6-P.
- GlmS mutant resistant to product inhibition produced higher levels of GlcN than the control strain expressing the wild-type E. coli enzyme.