Metabolic Engineering Of Aspergillus Niger To Produce N-acetylglucosamine

Glc NAc is widely used in the field of food and health care,cosmetics industry,biological medicine and so on,Glc NAc has a very broad application prospect.At present,the production methods of Glc NAc mainly include chemical hydrolysis,enzymatic hydrolysis and microbial fermentation.Compared with the other two methods,Glc NAc production by microbial fermentation has the advantages of high efficiency,high yield and low environmental pollution.In this paper,used from Aspergillus niger ATCC 1015 S469(including Tet-on::Cre/lox P system)was used as the starting strain to construct a recombinant Aspergillus niger strain to synthesize Glc NAc.The following is the main research content and results of this subject,which is divided into the following six parts:(1)The gene encoding the Glc NAc uptake transporter ngt A was firstly knocked out to obtain a Glc NAc uptake and utilization-deficient strain,which was beneficial to the accumulation of extracellular Glc NAc(2)On this basis,a complete synthesis pathway of Glc NAc in A.niger was constructed by expressing the gene yqa B encoding haloacid dehalogenase-like phosphatase from Escherichia coli,and the synthesis of Glc NAc was successfully achieved in A.niger with a titer of1.78 g/L.(3)By co-overexpressing the glucosamine-6-phosphate acetyltransferase gene gna A and the Glc N6 P synthase gene gfa A,the Glc NAc biosynthesis pathway was enhanced,and the accumulation of Glc NAc was increased to 3.64 g/L.(4)In order to increase the intracellular supply of Glc NAc synthesis precursor GlcNAc6 P,attenuated expressions of glucosamine-6-phosphate deaminase gene(nag B)and acetylglucosamine-6-phosphate deacetylase gene(nag A)in Glc NAc6 P catabolic pathway was achieved by RNA interference.The Glc NAc yield was further increased to 4.03 g/L.(5)In order to reduce the competition of fructose 6-phosphate between the glycolytic pathway and Glc NAc synthesis pathway,attenuated expressions of phosphofructokinase gene pfk A was achieved by RNA interference,and the final titer of Glc NAc increased to 4.61g/L.(6)In order to further evaluate the fermentation performance of the recombinant A.niger strain,the large-scale fermentation experiment of the recombinant strain was carried out in a 2-L fermentation tank.In the 120 h fermentation cycle,glucose consumption rate was faster after 24 h,and glucose was almost exhausted at 120 h.After 36 h,Glc NAc began to accumulate significantly.After 120 h of fermentation,the Glc NAc yield of the recombinant strain was 10.8 g/L,and the conversion of glucose was 0.108 g/g.This study lays the foundation for the industrial production of Glc NAc in A.niger.