| DESCRIPTION |
D-glucosamine is a nutraceutical compound with applications in the treatment of human osteoarthritic conditions. It is currently produced by the acid hydrolysis of chitin, a process limited by the availability of raw materials, such as crab shells. We are developing a novel fermentation process to produce glucosamine by E. coli metabolic engineering. First generation glucosamine-overproducing strains were generated by deleting genes that encode key enzymes involved in the transport and metabolism of glucosamine and over-expressing the glmS gene. The glmS gene encodes glucosamine-6-phosphate synthase, which converts fructose-6-phosphate and glutamine to glucosamine-6-phosphate. Such strains produced about 0.4 g/L of glucosamine in shake flasks, representing a 100-fold increase with respect to the wild type strain. Since the GlmS enzyme is subject to strong product inhibition, the cloned glmS gene was mutagenized by error prone PCR and mutant variants were screened in a plate assay for glucosamine overproduction. The best variant produced about 6 g/L glucosamine in shake flasks and about 12 g/L in fermentors. By further strain and fermentation process development, glucosamine production in a simple mineral salt medium with glucose and ammonium feed has reached levels approaching the target for a competitive manufacturing process
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