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From Concept to Process:
Metabolic Engineering for Production of Glucosamine
and N-Acetylglucosamine

 

Abstract

Metabolic engineering and microbial fermentation offer advantageous alternatives to production of numerous chemicals which are traditionally manufactured by chemical synthesis or extracted from raw materials. Developing an academically interesting concept into an industrial process is a challenging task. It often requires creative approaches to overcome different bottlenecks.

Metabolic engineering for glucosamine production in E. coli first started with a straightforward strategy: inactivating genes involved in glucosamine transport and catabolism, and over-expressing the biosynthetic gene (glucosamine synthase, GlmS). This resulted in a 15-fold increase in glucosamine concentration, but the titer remained below 100 mg/L.

Over-expression of a GlmS mutant resistant to product inhibition led to a level of 18 g/L. Fast degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further titer improvement. N-acetylglucosamine was identified as the alternative fermentation product since it is stable, non-inhibitory and readily hydrolyzed to glucosamine. Therefore, the glucosamine synthesis pathway was extended to N-acetylglucosamine by over-expressing a yeast glucosamine-6-P N-acetyltransferase (GNA1). Using a simple and low-cost fermentation process, strains over-expressing GlmS and GNA1 produced N-acetylglucosamine at concentrations greater than 110 g/L.