In Vivo Multienzyme Complex Coconstruction of N-Acetylneuraminic Acid Lyase and N-Acetylglucosamine-2-epimerase for Biosynthesis of N-Acetylneuraminic Acid

Metabolic channeling enables efficient transfer of the intermediates by forming a multienzyme complex. To leverage the metabolic channeling for improved biosynthesis, we coexpressed N-acetylneuraminic acid lyase from C. glutamicum ATCC 13032 (CgNal) and N-acetylglucosamine-2-epimerase from Anabaena sp. CH1 (anAGE) in Escherichia coli and used the whole cell to synthesize N-acetylneuraminic acid (Neu5Ac) from N-acetylglucosamine (GlcNAc) and pyruvate. To get the multienzyme complex, polycistronic plasmid with high levels of CgNal and anAGE expression was constructed by tuning the orders of the genes. The Shine-Dalgarno (SD) sequence and aligned spacing (AS) distance were optimized. The E. coli Rosetta harboring the polycistronic plasmid pET-28a-SD₂-AS₁-CgNal-SD-AS-anAGE increased the production of Neu5Ac by 58.7% to 92.5 g/L in 36 h by whole-cell catalysis and by 21.9% up to 112.8 g/L in 24 h with the addition of Triton X-100.