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

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• 作者：Wujin Sun ; Wenyan Ji ; Nan Li ; Peng Tong ; Jian Cheng ; Ying He ; Yong Chen ; Xiaochun Chen ; Jinglan Wu ; Pingkai Ouyang ; Jingjing Xie ; ^{xiej@njut.edu.cn} ; Hanjie Ying ; ^{yinghanjie@njut.edu.cn} ; ^{cxc_1981@sohu.com}
• 关键词：Nal ; N-acetylneuraminic acid lyase ; AGE ; N-acetylglucosamine 2-epimerase ; snAGE ; N-acetylglucosamine 2-epimerase from Synechocystis sp. PCC 6803 ; anAGE ; N-acetylglucosamine 2-epimerase from Anabaena sp. CH1 ; GlcNAc ; N-acetylglucosamine ; ManNAc ; N-acetyl-D
• 刊名：Bioresource Technology
• 出版年：2013
• 出版时间：February, 2013
• 年：2013
• 卷：130
• 期：Complete
• 页码：23-29
• 全文大小：521 K

文摘

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 61.3 g/L in 60 h 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.