title: Directed evolution and characterization of Escherichia coli glucosamine synthase.
abstract: Glucosamine synthase (GlmS) converts fructose-6-phosphate to glucosamine-6-phosphate. Overexpression of GlmS in Escherichia coli increased synthesis of glucosamine-6-P, which was dephosphorylated and secreted as glucosamine into the growth medium. The E. coli glmS gene was improved through error-prone polymerase chain reaction (PCR) in order to develop microbial strains for fermentation production of glucosamine. Mutants producing higher levels of glucosamine were identified by a plate cross-feeding assay and confirmed in shake flask cultures. Over 10 mutants were characterized and all showed significantly reduced sensitivity to inhibition by glucosamine-6-phosphate. Ki of mutants ranged from 1.4 to 4.0 mM as compared to 0.56 mM for the wild type enzyme. Product resistance resulted from single mutations (L468P, G471S) and/or combinations of mutations in the sugar isomerase domain. Most overexpressed GlmS protein was found in the form of inclusion bodies. Cell lysate from mutant 2123-72 contained twice as much soluble GlmS protein and enzyme activity as the strain overexpressing the wild type gene. Using the product-resistant mutant, glucosamine production was increased 60-fold.
authors.affiliation: Bio-Technical Resources, South 7th Street, Manitowoc, WI 54220, USA. mddeng@biotechresources.com
authors.forename: Ming-De
authors.initials: MD
authors.lastname: Deng
authors.forename: Alan D
authors.initials: AD
authors.lastname: Grund
authors.forename: Sarah L
authors.initials: SL
authors.lastname: Wassink
authors.forename: Susan S
authors.initials: SS
authors.lastname: Peng
authors.forename: Kathleen L
authors.initials: KL
authors.lastname: Nielsen
authors.forename: Brian D
authors.initials: BD
authors.lastname: Huckins
authors.forename: Richard P
authors.initials: RP
authors.lastname: Burlingame
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005944:Glucosamine / Q000031:analogs & derivatives / Q000096:biosynthesis* / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D012995:Solubility
chemical_list: D000117:Acetylglucosamine; D001426:Bacterial Proteins; D005944:Glucosamine; D019298:Glucose-6-Phosphate; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D050505:Mutant Proteins; C001293:glucosamine 6-phosphate; C000731191:component S, glutamate mutase protein, Bacteria