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A New Pathway for N-Acetylglucosamine Production:
Coupling a Catabolic Enzyme,
Glucosamine-6-P Deaminase, with a
Biosynthetic Enzyme, Glucosamine-6-P N-Acetyltransferase



A metabolic pathway for production of N-acetylglucosamine (GlcNAc) has been engineered in Escherichia coli by over-expressing E. coli glucosamine (GlcN) synthase (GlmS) and Saccharomyces cerevisiae GlcN-6-P acetyltransferase (GNA1). GlmS catalyzes the synthesis of GlcN-6-P from fructose-6-P and glutamine. GNA1 converts GlcN-6-P into GlcNAc-6-P, which is then dephosphorylated and secreted into the medium. In the present work, we evaluated over-expression of E. coli GlcN deaminase (NagB) as an alternative to GlmS for the production of GlcNAc. NagB is typically a catabolic enzyme that hydrolyzes GlcN-6-P to fructose-6-P and ammonium. Its biosynthetic reaction is kinetically very unfavorable. In a glmS deletion strain normally requiring GlcN, NagB over-expression resulted in the synthesis of GlcN-6-P sufficient to support cell growth. No significant amount of GlcN could be detected in the growth medium. When NagB was over-expressed concurrently with GNA1, GlcNAc was produced at 24 g/liter in shake flasks, a level comparable to strains over-expressing both GlmS and GNA1. This indicated that the over-expression of GNA1 played a critical role in determining the direction and efficiency of the reaction catalyzed by NagB. The data demonstrated that biosynthetic pathways could be successfully engineered to use kinetically unfavorable enzyme reactions by coupling with efficient downstream reactions.