RESUMO
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.
Assuntos
Evolução Molecular Direcionada/métodos , Escherichia coli/enzimologia , Glucosamina/biossíntese , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Acetilglucosamina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular/métodos , Estabilidade Enzimática/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/química , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Cinética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Análise de Sequência de Proteína , SolubilidadeRESUMO
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.
