Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine.
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(-1). 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 l(-1). Rapid degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further improvement. An alternative fermentation product, 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(-1) of N-acetylglucosamine was produced.
Deng
Ming-De
MD
Bio-Technical Resources, 1035 South 7th Street, Manitowoc, WI 54220, USA. mddeng@biotechresources.com
Severson
David K
DK
Grund
Alan D
AD
Wassink
Sarah L
SL
Burlingame
Richard P
RP
Berry
Alan
A
Running
Jeffrey A
JA
Kunesh
Candice A
CA
Song
Linsheng
L
Jerrell
Thomas A
TA
Rosson
Reinhardt A
RA
D000117:Acetylglucosamine / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods; D005944:Glucosamine / Q000096:biosynthesis* / Q000235:genetics; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000235:genetics* / Q000378:metabolism*; D007218:Industrial Microbiology / Q000379:methods*; D016297:Mutagenesis, Site-Directed; D010865:Pilot Projects; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D005944:Glucosamine; D000117:Acetylglucosamine