title: Construction and expression of a polycistronic plasmid encoding N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase simultaneously for production of N-acetylneuraminic acid.
abstract: Synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetylglucosamine (GlcNAc) and pyruvate was carried out by constructing and expressing a polycistronic plasmid encoding an N-acetylglucosamine 2-epimerase (AGE) gene and an N-acetylneuraminic acid lyase (Nal) gene simultaneously. Nal from Escherichia coli K12 and AGEs from Synechocystis sp. PCC 6803 (snAGE) and Anabaena sp. CH1 (anAGE) were used. And four polycistronic plasmids were constructed in which the positions of AGE gene differed with respect to Nal gene. Among these plasmids, pET-28a-Nal-anAGE with anAGE gene located next to Nal gene caused the production of the highest amount of Neu5Ac, generating 61.3g/L in 60h by whole-cell catalysis without the addition of ATP as AGE activator. And pET-28a-Nal-anAGE lowered anAGE's expression level, allowing it to fold properly. Thus, an inclusion-body-free E. coli strain capable of producing Neu5Ac by whole-cell catalysis with high yield and low cost was constructed in the present study.
authors.affiliation: State Key Laboratory of Materials-Oriented Chemical Engineering, National Engineering Technique Research Center for Biotechnology, College of Biotechnology and Pharmaceutical Engineering, Nanjing University of Technology, Nanjing, China.
authors.forename: Wujin
authors.initials: W
authors.lastname: Sun
authors.forename: Wenyan
authors.initials: W
authors.lastname: Ji
authors.forename: Nan
authors.initials: N
authors.lastname: Li
authors.forename: Peng
authors.initials: P
authors.lastname: Tong
authors.forename: Jian
authors.initials: J
authors.lastname: Cheng
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.forename: Yong
authors.initials: Y
authors.lastname: Chen
authors.forename: Xiaochun
authors.initials: X
authors.lastname: Chen
authors.forename: Jinglan
authors.initials: J
authors.lastname: Wu
authors.forename: Pingkai
authors.initials: P
authors.lastname: Ouyang
authors.forename: Jingjing
authors.initials: J
authors.lastname: Xie
authors.forename: Hanjie
authors.initials: H
authors.lastname: Ying
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D017033:Anabaena / Q000201:enzymology / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004926:Escherichia coli; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis*; D007652:Oxo-Acid-Lyases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000138:chemical synthesis / Q000235:genetics* / Q000378:metabolism; D046939:Synechocystis / Q000201:enzymology / Q000235:genetics
chemical_list: D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D002238:Carbohydrate Epimerases; C100033:UDP acetylglucosamine-2-epimerase; D019158:N-Acetylneuraminic Acid; D000117:Acetylglucosamine