Pathway Engineering of Bacillus subtilis for Enhanced N‐Acetylneuraminic Acid Production via Whole‐Cell Biocatalysis

Abstract

N‐acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications in nutraceuticals and pharmaceuticals. However, low production efficiency and high environmental pollution associated with traditional extraction and chemical synthesis methods constrain the supply of NeuAc. Here, a biological approach is developed for food‐grade NeuAc production via whole‐cell biocatalysis by the generally regarded as safe (GRAS) bacterium Bacillus subtilis (B. subtilis). Promoters for controlling N‐acetylglucosamine 2‐epimerase (AGE) and NeuAc adolase (NanA) are optimized, yielding 32.84 g L−1 NeuAc production with a molar conversion rate of 26.55% from N‐acetylglucosamine (GlcNAc). Next, NeuAc production is further enhanced to 46.04 g L−1, which is 40.2% higher than that of the strain with promoter optimization, by expressing NanA from Staphylococcus hominis instead of NanA from Escherichia coli. To enhance the expression level of ShNanA, the N‐terminal coding sequences of genes with high expression levels are fused to the 5′‐end of the ShNanA gene, resulting in 56.82 g L−1 NeuAc production. Finally, formation of the by‐product acetoin from pyruvate is blocked by deleting the alsS and alsD genes, resulting in 68.75 g L−1 NeuAc production with a molar conversion rate of 55.57% from GlcNAc. Overall, a GRAS B. subtilis strain is demonstrated as a whole‐cell biocatalyst for efficient NeuAc production.

Recent advances on N-acetylneuraminic acid: Physiological roles, applications, and biosynthesis

“…Neu5Ac can also be synthesized by chemical methods that require complex protection, deprotection, reduction, and oxidation steps. But the chemical methods are limited by their low production efficiency, unsatisfactory stereoselectivity, and high environmental pollution [ 71 ]. In recent decades, biosynthesis, which is associated with good application potential and environmentally friendly characteristics, has become an indispensable approach for large-scale production of Neu5Ac.…”

Terminal-Enhanced Polymerization in the Biosynthesis of Polysialic Acid

“…The central region of KPS (region II) contains six key genes, namely, neuD, neuB, neuA, neuC, neuE, and neuS, which direct the biosynthesis, activation, and polymerization of Neu5Ac [12]. Neu5Ac synthase (neuB) [9,13] catalyzes the energetic reaction of ManNAc and phosphoenolpyruvate (PEP) to form Neu5Ac [14], and Neu5Ac aldolase (nanA) catalyzes the reversible cleavage of Neu5Ac to ManNAc and pyruvate [15][16][17]. N-acylneuraminate cytidylyltransferase (neuA), a bifunctional enzyme with cytidine 5-monophosphate-sialic acid synthase (CMP-Neu5Ac) [18,19], catalyzes the generation of free sialic acid to CMP-Neu5Ac, thus generating a candidate sialic acid donor for all known sialic acid transferases; neuD is a Neu5Ac 7-O-(or 9-O)-acetyltransferase that acetylates residues in the seven or nine positions of Neu5Ac [20]; and neuS encodes an α-2,8 sialyltransferase, responsible for polymerizing the homopolymer of Neu5Ac in an α-2,8 glycosidic bond to the end of adjacent Neu5Ac to form PSA.…”

Biosynthesis of Guanidinoacetate by Bacillus subtilis Whole-Cell Catalysis

“…As a typical industrial model microorganism of food safety grade, B. subtilis has a clear genetic background and mature gene manipulation technology [12]. Due to its non-pathogenicity and strong protein synthesis ability, it is very suitable for GAA biocatalysis [13].…”