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

Abstract

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-SD-AS-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.

Reconstruction of a Cofactor Self-Sufficient Whole-Cell Biocatalyst System for Efficient Biosynthesis of Allitol from d-Glucose

“…44 Furthermore, high local concentrations of the metabolites maintained by the complexes are beneficial for unstable compounds. 45 Sucrose hydrolysis often requires splitting of water molecules and breaking glycosidic bonds of the disaccharides, and sucrose in molasses can be hydrolyzed via the enzyme (sucrase or invertase) or the chemical (acid or alkali) treatment processes. 46 Compared with the enzyme-catalyzed hydrolysis, the acid-catalyzed sucrose hydrolysis pretreatment has advantages due to its potential to reduce the production cost, and low pH would facilitate the breaking of glycosidic bonds.…”

Enzymatic production of N-acetylneuraminic acid: advances and perspectives

“…The European Union, USA, and Chinese Food and Drug Administrations have approved NeuAc as a novel nutritional supplement [1]. As NeuAc has attracted much attention, there is a vital need for large-scale production of NeuAc through cost-effective and reliable methods [10]. By 2024, the worldwide NeuAc market is expected to have grown at a compound annual growth rate (CAGR) of 12.19%, with a market value of USD 15 million [11].…”

“…NeuAc can also be chemically synthesized using N-acetyl-mannosamine (Man-NAc) and acrylic acid as substrates and indium as a catalyst in an acidic alcohol solution [17]. Under alkaline conditions (pH [9][10][11], NeuAc can also be formed by the condensation of oxaloacetate and N-acetylglucosamine (GlcNAc), preceded by decarboxylation [18]. Asymmetric synthesis of NeuAc from non-sugar precursors and d-mannose has also been reported [19].…”

“…Three different methods have been reported to perform whole-cell catalysis for NeuAc synthesis: multicellular coupling biosynthesis and direct and indirect precursor single-cell biosynthesis [1]. For whole-cell catalytic systems, resistance faced by end products and substrates to cross cell membranes often results in a low yield (less than 60%) [10]. This method is especially suitable for multi-enzyme processes, which necessitates the use of costly cofactors.…”

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

“…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 …”