Many works have been done on the production of polysialic acid(PSA),which were focused on the fermentation and the purification of PSA without emphasis on the strengthening of synthetic pathway of PSA.In this study,an efficient process for enhanced production of PSA by co-overexpressing genes of N-acetylneuraminate(Neu5Ac)7-O(or 9-O)-acetyltransferase(NeuD),CMP-Neu5Ac synthetase(NeuA)and alpha-Neu5Ac alpha-2,8-sialyltransferase(NeuS)using E.coli SA9 as a host strain was developed.To improve the efficiency of the engineered strain,the key enzyme of the biosynthetic pathway of PSA was overexpressed and deleting of competing pathways of intermediate Neu5Ac.The results showed that NeuD played an important role in the biosynthesis of PSA.11.85 g/L PSA was obtained by fed-batch cultivations with strain E.coli SA9ΔnanA/pbla-DA,and the molecular weight was 113.33 KDa.The production of PSA was increased 36%compared with the original strain(8.71 g/L).The high productivity of our process should make it a promising cost-effective resource for PSA.