Enhancement of Production of D-Glucosamine in Escherichia Coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc

d-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that d-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The d-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of d-glucosamine by a factor of 2.16. Moreover, for further increasing d-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of d-glucosamine in E. coli BL21 glmU-Δgpa′, E. coli BL21ΔmurQ′, and E. coli BL21ΔnanE′ were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.