Enzymatic production of N-acetylneuraminic acid: advances and perspectives

Abstract

N-Acetyl-d-neuraminic acid (NeuAc), a well-known and well-studied sialic acid, is found in cell surface glycolipids and glycoproteins, where it performs a variety of biological functions. The use of NeuAc as a nutraceutical for infant brain development and as an intermediate for pharmaceutical production demands its production on an industrial scale. Natural extraction, chemical synthesis, enzymatic synthesis, and biosynthesis are the methods used for NeuAc production. Among these methods, enzymatic synthesis using N-acetyl-glucosamine (GlcNAc) 2-epimerase (AGE) for epimerization and N-acetyld-neuraminic acid lyase (NAL) for aldol condensation, has been reported to produce NeuAc with high production efficiency. In this review, we discuss advances in the two-step enzymatic synthesis of NeuAc using pyruvate and GlcNAc as substrates. The major challenges in producing NeuAc with high yield are highlighted, including multiple parameter-dependent processes, undesirable reversibility, and diminished solubility of AGEs and NALs. Further, different strategies applied to overcome the limitations of the two-step enzymatic production are discussed, such as pyruvate concentration and temperature shift during the process to increase conversion yield, use of mathematical and computational simulations for process optimization, enzyme engineering to make enzymes highly efficient, and the use of tags and chaperones to increase enzyme solubility. We suggest future directions and the strategies that can be followed to improve enzymatic synthesis of NeuAc.

Engineering of Synthetic Multiplexed Pathways for High-Level N-Acetylneuraminic Acid Bioproduction

“…All NanA enzymes derived from E. coli have a K m value of approximately 3 mM for the cleavage of NeuAc and a K m value of approximately 200 mM for ManNAc for the condensation of ManNAc and pyruvate for NeuAc synthesis. 26 In addition, NanA may catalyze NeuAc analogues synthesis with other substrates and pyruvate, such as N-glycolylneuraminic acid (NeuGc) or other derivatives. 27 Therefore, although NanA derived from E. coli can also obtain a higher yield and conversion rate in the whole cell catalysis process with the addition of excess substrates, it is not suitable for In particular, when NemNeuC is expressed under a strong promoter (stronger than the P5 promoter), the colony undergoes apoptosis on the LB plate.…”

“…All NanA enzymes derived from E. coli have a K m value of approximately 3 mM for the cleavage of NeuAc and a K m value of approximately 200 mM for ManNAc for the condensation of ManNAc and pyruvate for NeuAc synthesis . In addition, NanA may catalyze NeuAc analogues synthesis with other substrates and pyruvate, such as N -glycolylneuraminic acid (NeuGc) or other derivatives .…”

Improved N‐acetylneuraminic acid bioproduction by optimizing pathway for reducing intermediate accumulation

“…Both the AGE and NanE pathways require N ‐acetylglucosamine‐6‐phosphate (GlcNAc6P) as a precursor. However, N ‐acetylglucosamine (GlcNAc) is synthesized from GlcNAc6P by dephosphorylation of endogenous phosphatase and is then transported to the medium, resulting in intermediate accumulation (Hussain et al, 2022; Liu et al, 2014; Zhang et al, 2021). In the NeuC pathway, all precursors except N ‐acetylmannosamine (ManNAc) are phosphorylated or coupled with uridine diphosphate and cannot be transported out of the cell, resulting in less accumulation of intermediate metabolites (Zhang et al, 2021).…”

Shift of the reaction equilibrium at high pressure in the continuous synthesis of neuraminic acid

“…In this study, the rate expressions from Groher et al are used [ 8 ]. So far, different approaches have been attempted to increase the overall position of an equilibrium by using additional enzymes [ 12 ], different temperatures or high concentrations of the substrates [ 13 ].…”