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Directed Evolution of GlmS Enzyme to
Reduce Product Inhibition

 

GlmS is inhibited by GlcN-6-P. Activity was inhibited to about 20% in the presence of 6 mM GlcN-6-P. 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 (Figure 2).

Different strains with an integrated expression cassette of mutant glmS genes were grown in shake flasks in M9A supplemented with glucose and induced with IPTG. One of the best mutants, 2123-54, produced 6 g/L GlcN in the growth medium. This is 20 fold higher than the control strain expressing the wild-type E. coli gene (Figure 3).
  

Figure 2. Reduced Sensitivity to Product Inhibition in glmS Mutants

Product Inhibition in glmS Mutants

Figure 3. GlcN Production Using Different glmS Mutants

GlcN Production

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