文档介绍：ARTICLE IN PRESS
Metabolic Engineering 7 (2005) 201–214
ate/ymben
Metabolic engineering of Escherichia coli for industrial production of
glucosamine and N-acetylglucosamine
Ming-De DengÃ,David K. Severson,Alan D. Grund,Sarah L. Wassink,
Richard P. Burlingame1,Alan Berry 2,Jeffrey A. Running,Candice A. Kunesh,
Linsheng Song,Thomas A. Jerrell,Reinhardt A. Rosson
Bio-Technical Resources, 1035 South 7th Street, Manitowoc, WI 54220, USA
Received 17 September 2004; accepted 8 February 2005
Available online 23 March 2005
Abstract
Glucosamine and N-acetylglucosamine are currently produced by extraction and acid hydrolysis of chitin from shellfish waste.
Production could be limited by the amount of raw material available and the product potentially carries the risk of shellfish protein
contamination. Escherichia coli was modified by metabolic engineering to develop a fermentation process. Over-expression of
glucosamine synthase (GlmS) and inactivation of catabolic genes increased glucosamine production by 15 fold,reaching 60 mg l À1.
Since GlmS is strongly inhibited by glucosamine-6-P,GlmS variants were generated via error-prone PCR and screened. Over-
expression of an improved enzyme led to a glucosamine titer of 17 g lÀ1. Rapid degradation of glucosamine and inhibitory effects
of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation pro-
duct, N-acetylglucosamine,is stable,non-inhibitory to the host and readily hydrolyzed to glucosamine under acidic conditions.
Therefore,the glucosamine pathway was extended to N-acetylglucosamine by over-expressing a heterologous glucosamine-6-P
N-acetyltransferase. Using a simple and low-cost fermentation process developed for this strain,over 110 g l À1 of
N-acetylglucosamine was produced.
r 2005 Elsevier Inc. All rights reserved.
Keywords: Glucosamine; N-acetylglucosamine; Esherichia coli; glmS gene; GNA1 gene; Metabolic engineering; Fermentation; L