Abstract:In this study，Aspergillus niger was used as the chassis cell，and the uptake transporter ngtA，the encoding gene of N-acetylglucosamine（GlcNAc），was firstly knocked out to obtain a defective strain for absorbing and utilizing GlcNAc，which was beneficial to the accumulation of extracellular GlcNAc.On this basis，a complete synthesis pathway of GlcNAc in A.niger was constructed by expressing the encoding gene yqaB of haloacid dehalogenase-like phosphatase from Escherichia coli，and the synthesis of GlcNAc was successfully achieved in A.niger with a yield of 1.78 g/L.By co-overexpressing the glucosamine-6-phosphate acetyltransferase gene gnaA and the GlcN6P synthase gene gfaA，the GlcNAc biosynthesis pathway was enhanced，and the synthesis level of GlcNAc increased to 3.64 g/L.To increase the intracellular supply of GlcNAc6P，the synthesis precursor of GlcNAc，attenuated expressions were performed to the glucosamine-6-phosphate deaminase gene nagB and acetylglucosamine-6-phosphate deacetylase gene nagA in GlcNAc6P catabolic pathway by RNA interference.The yield of GlcNAc further increased to 4.03 g/L.To reduce the competition of fructose 6-phosphate between the glycolytic pathway and GlcNAc synthesis pathway of A.niger，the phosphofructokinase gene pfkA was weakened，and the final yield of GlcNAc increased to 4.61 g/L.