Production of N-acetyl-d-neuraminic Acid by Recombinant Single Whole Cells Co-expressing N-acetyl-d-glucosamine-2-epimerase and N-acetyl-d-neuraminic Acid Aldolase

Abstract

N-acetyl-D-neuraminic acid (Neu5Ac) is a costly precursor for many drugs such as anti-influenza antivirals. In a previous study, a whole-cell process for Neu5Ac production was developed using a combination of two Escherichia coli cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine-2-epimerase (bage) and E. coli N-acetyl-D-neuraminic acid aldolase (nanA), respectively. In this study, we constructed a bAGE and NanA co-expression system to improve Neu5Ac production. Two recombinant E. coli strains, E. coli BL21 (DE3) pET-bage-nanA (HA) and E. coli BL21 (DE3) pET-bage-2nanA (HAA), synchronously expressing bAGE and NanA were used as biocatalysts to generate Neu5Ac from N-acetyl-D-glucosamine (GlcNAc) and pyruvate. The HA biocatalysts produced 187.5 mM Neu5Ac within 8 h. The yield of GlcNAc was 15.6%, and the Neu5Ac production rate was 7.25 g/L/h. The most active HAA biocatalysts generated 412.6 mM Neu5Ac and a GlcNAc yield of 34.4%. HAA achieved a Neu5Ac production rate of 15.9 g/L/h, which surpassed those for all reported Neu5Ac production processes so far. The present study demonstrates that using recombinant E. coli cells synchronously expressing bAGE and NanA as biocatalysts could potentially be used in the industrial mass production of Neu5Ac.

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

“…The Neu5Ac yield could be further improved by adjusting the ratio of AGE and aldolase, strain engineering via glucosamine synthase gene variant glmS *72 and GlcN-6-P N -acetyltransferase gene chromosomal knock-in, , AGE and NanA protein engineering, − Neu5Ac metabolic engineering, use of irreversible Neu5Ac synthatase, and other microbial hosts . Improvement of BT0453 catalytic properties via directed evolution is ongoing in our laboratory.…”

“…Since the characterization of human renin binding protein (hRnBP) as an AGE, 15 a variety of AGEs were discovered, 10,16−19 and some of them, by coupling with the Escherichia coli aldolase, were used in the production of Neu5Ac. 20−23 Though as much as 34.4% yield and 412.6 mM Neu5Ac was obtained, 23 the process is inferior to the enzymatic catalysis process with 77% yield and 625 mM Neu5Ac. 13 The reported AGEs thus far are of low solubility which might be the bottleneck for the Neu5Ac yield improvement.…”

“…Contrast to the pure enzyme-based biocatalysis, whole cell chemoenzymatic biotransformation by using the recombinant enzyme-expressing strains circumvents the enzyme purification steps and simplifies the production process. Since the characterization of human renin binding protein (hRnBP) as an AGE, a variety of AGEs were discovered, ,− and some of them, by coupling with the Escherichia coli aldolase, were used in the production of Neu5Ac. − Though as much as 34.4% yield and 412.6 mM Neu5Ac was obtained, the process is inferior to the enzymatic catalysis process with 77% yield and 625 mM Neu5Ac …”

One-pot biosynthesis of N-acetylneuraminic acid from chitin via combination of chitin-degrading enzymes, N-acetylglucosamine-2-epimerase, and N-neuraminic acid aldolase

“…Although many innovative engineering strategies have been developed and applied to the de novo synthesis of Neu5Ac, the current Neu5Ac titer of de novo synthesis is still low, causing difficulties in industrial applications. In enzymatic catalysis, N-acetyl-D-glucosamine as a substrate can be efficiently catalyzed into Neu5Ac via cascade catalysis of two enzymes: N-acetylglucosamine-2-epimerase (AGE) catalyzes the reversible reaction of N-acetyl-D-glucosamine (GlcNAc) to N-acetyl-D-manosamine (ManNAc), subsequently, N-acetyl-neuraminic acid aldolase (NanA) catalytic synthesis of Neu5Ac from ManNAc and pyruvate ( Lee et al, 2004 ; Kao et al, 2018 ). Compared with biological fermentation the enzyme catalysis method has many advantages such as simplicity, mild catalytic conditions, and high conversion rates.…”

Metabolic Engineering of Escherichia coli for Increased Bioproduction of N-Acetylneuraminic Acid

“…However, the enzymatic synthesis of NeuAc requires the addition of cofactor ATP in the catalytic process, and the purification steps of the enzyme are complicated and time-consuming, which limit its large-scale industrial application. In addition to natural extraction, chemical synthesis, and enzymatic synthesis methods, whole-cell catalysis is a common strategy used for the synthesis of NeuAc. − Whole-cell catalysis omits the enzymatic purification step and does not require ATP but requires exogenous addition of the substrates, pyruvate, and N -acetylglucosamine (GlcNAc), as precursors during the reaction process, which increases the production cost, and the pyruvate in the reaction waste liquid causes environmental pollution. , Therefore, the direct de novo synthesis of NeuAc using cheap carbon sources, such as glycerol or glucose, as substrates without adding any precursors is a good choice for industrial production.…”