Construction and expression of a polycistronic plasmid encoding N-acetylglucosamine 2-epimerase and N-acetylneuraminic acid lyase simultaneously for production of N-acetylneuraminic acid

• Published in: Bioresource technology 2013-02, Vol.130, p.23-29
• Main Authors: Sun, Wujin, Ji, Wenyan, Li, Nan, Tong, Peng, Cheng, Jian, He, Ying, Chen, Yong, Chen, Xiaochun, Wu, Jinglan, Ouyang, Pingkai, Xie, Jingjing, Ying, Hanjie
• Format: Article
• Language: English
• Subjects: Acetylglucosamine - metabolism; Anabaena - enzymology; Anabaena - genetics; Biocatalysis; Biological and medical sciences; Biotechnology; Carbohydrate Epimerases - genetics; Carbohydrate Epimerases - metabolism; Catalysis; Escherichia coli; Fundamental and applied biological sciences. Psychology; Inclusion bodies; N-acetylglucosamine 2-epimerase; N-acetylneuraminic acid; N-Acetylneuraminic Acid - biosynthesis; N-acetylneuraminic acid lyase; Oxo-Acid-Lyases - genetics; Oxo-Acid-Lyases - metabolism; Plasmids - chemical synthesis; Plasmids - genetics; Plasmids - metabolism; Synechocystis - enzymology; Synechocystis - genetics
• ISSN: 0960-8524; 1873-2976
• DOI: 10.1016/j.biortech.2012.12.042

► Encoding enzymes for two reactions in one plasmid simplified fermentation process. ► Activities of two types of N-acetylglucosamine 2-epimerase were compared. ► Intact E. coli cells avoid adding ATP to activate N-acetylglucosamine 2-epimerase. ► Lowering N-acetylglucosamine 2-epimerase expression level improved its solubility. ► High production of 61.3g/l N-acetylneuraminic acid. 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.