A metabolic pathway for high level production of N-acetylglucosamine has been engineered in Escherichia coli by overexpressing E. coli glucosamine synthase (GlmS) and Saccharomyces cerevisiae glucosamine-6-phosphate acetyltransferase (GNA1). GlmS catalyzes the synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. GNA1 converts glucosamine-6-phosphate into N-acetylglucosamine, which is dephosphorylated and secreted into the growth medium. In the present work, E. coli glucosamine-6-phosphate deaminase (NagB) was evaluated as an alternative to GlmS for the production of glucosamine and N-acetylglucosamine. NagB is a catabolic enzyme that converts glucosamine-6-phosphate to fructose-6-phosphate and ammonia. The reverse biosynthetic reaction forming glucosamine-6-phosphate is kinetically unfavorable. In a glmS deletion strain requiring glucosamine supplement to survive and grow, NagB overexpression resulted in the synthesis of glucosamine-6-phosphate. This supported cell growth, but little or no glucosamine accumulated in the medium. Overexpression of both NagB and GNA1 resulted in production of N-acetylglucosamine at levels comparable to strains overexpressing both GlmS and GNAL This indicates that the overexpression of GNA1 played a critical role in determining the direction and efficiency of the reaction catalyzed by NagB. These data demonstrate that a catabolic enzyme can be utilized in a biosynthetic pathway by coupling with an efficient downstream reaction. (c) 2006 Elsevier Inc. All rights reserved.