Production of N-Acetyl-d-neuraminic Acid by Whole Cells Expressing Bacteroides thetaiotaomicron N-Acetyl-d-glucosamine 2-Epimerase and Escherichia coli N-Acetyl-d-neuraminic Acid Aldolase.

Abstract

N-Acetyl-d-neuraminic acid (Neu5Ac) is a potential baby nutrient and the key precursor of antiflu medicine Zanamivir. The Neu5Ac chemoenzymatic synthesis consists of N-acetyl-d-glucosamine epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc) and aldolase-catalyzed condensation between ManNAc and pyruvate. Herein, we cloned and characterized BT0453, a novel AGE, from a human gut symbiont Bacteroides thetaiotaomicron. BT0453 shows the highest soluble fraction among the AGEs tested. With GlcNAc and sodium pyruvate as substrates, Neu5Ac production by coupling whole cells expressing BT0453 and Escherichia coli N-acetyl-d-neuraminic acid aldolase was explored. After 36 h, a 53.6% molar yield, 3.6 g L-1 h-1 productivity and 42.9 mM titer of Neu5Ac were obtained. Furthermore, for the first time, the T7- BT0453-T7- nanA polycistronic unit was integrated into the E. coli genome, generating a chromosome-based biotransformation system. BT0453 protein engineering and metabolic engineering studies hold potential for the industrial production of Neu5Ac.