Deqiang Zhu1, Xiaobei Zhan2, Jianrong Wu1, Minjie Gao1, Zhongsheng Zhao1. 1. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. 2. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China. xbzhan@yahoo.com.
Abstract
OBJECTIVE: To develop a strategy for producing N-acetyl-D-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate. RESULT: An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 ± 0.06 g Neu5Ac l-1 after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 ± 0.04 g l-1. In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 ± 0.08 g l-1. CONCLUSION: A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.
OBJECTIVE: To develop a strategy for producing N-acetyl-D-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate. RESULT: An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 ± 0.06 g Neu5Ac l-1 after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 ± 0.04 g l-1. In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 ± 0.08 g l-1. CONCLUSION: A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.