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

• Published in: Biotechnology journal 2019-07, Vol.14 (7), p.e1800682-n/a
• Main Authors: Zhao, Lin, Tian, Rongzhen, Shen, Qingyang, Liu, Yanfeng, Liu, Long, Li, Jianghua, Du, Guocheng
• Format: Article
• Language: English
• Subjects: Acetoin - metabolism; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Biocatalysis; Escherichia coli - genetics; Metabolic Engineering - methods; N-Acetylneuraminic Acid - analysis; N-Acetylneuraminic Acid - metabolism; N‐acetylneuraminic acid; N‐terminal coding sequence; pyruvate degradation; Pyruvic Acid - metabolism; Staphylococcus hominis - genetics; whole‐cell biocatalysis
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201800682

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. N‐acetylneuraminic acid (NeuAc) is a common sialic acid that has a wide range of applications as a nutraceutical and pharmaceutical. A generally regarded as safe (GRAS) bacterium Bacillus subtilis is developed as a whole‐cell biocatalyst for efficient NeuAc production for the first time. This work lays the foundation for producing food‐grade NeuAc with GRAS strain.