Research On Production Of N-Acetyl-Neuraminic Acid By Micoorganism Fermentation

N-Acetyl-Neuraminic Acid(Neu5Ac) is widely distributed in many organisms, which is usually found at the end of non-reducing oligosaccharide. It has been known to play an important role in many processes of life activities, and has been applied in numerous fields. In this article, Bacillus subtilis with a Generally Recognized as Safe(GRAS) status was engineered and first applied to the production of N-Acetyl-Neuraminic Acid. An artificial biosynthetic pathway from glycerol to Neu5Ac has been designed and constructed through introducing and over-expressing the enzyms related to the Neu5Ac in Bacillus subtilis. The strain with the highest yield and most stability was chosen from a series of engineering strains, and then the components of medium and fermentation conditions were optimized.This paper focuses on the construction of the recombinant plasmid, Firstly, neuB gene encoding N-acetyl neuraminic acid synthetase and neuC gene encoding N-acetylglucosamine 2-epimerase were cloned into the pHT01 shuttle plasmid of Escherichia coli-Bacillus subtilis to obtain pHT01-neuBC, which was then transformed into Bacillus subtilis 164 and Bacillus subtilis 168 through electrotransformation. In order to acquire a higher yield of Neu5AC, glmS gene encoding Glucosamine-6-P synthase was cloned into pHT01-neuBC to get pHT01-neuBC-glmS which was transformed into Bacillus subtilis168.Through verifying the fermentation properties of Neu5AC yield and corresponding stability, we finally found the Bacillus subtilis168/ pHT01-neuBC with the highest yield and stability.After optimizing the medium components including carbon source, nitrogen source, growth factor, inorganic salt and the fermentation conditions including the concentration of isopropyl-β-d-thiogalactoside(IPTG), the added time of IPTG, and the pH. The optimum conditions were determined, they are glycerol 20 g·L-1, tryptone 3 g·L-1, yeast extract 1 g·L-1, MgSO4·7H2O 1 g·L-1, and IPTG was added to ensure the final concentration (0.2 mmol·L-1) when the Optical Density 600 was 0, and the initial pH value can keep at the starting level.Unoptimized and optimized culture medium were compared by shaking flask fermentation of Bacillus subtilis 168/pHT01-neuBC (50 mL medium in 250 mL shaking flask), and the maximum yield of Neu5Ac were 0.76 g·L-1 and 1.36 g·L-1 respectively, indicating that the optimization of medium components and fermentation conditions can make a 78.9% improvement of the Neu5Ac yield. Furthermore, the maximum yield of Neu5Ac in the 5L bioreactor with optimized medium (2 L medium are contained,37℃ was 1.95 g·L-1, which was measured at 72 h.This is the first report regarding the pathway engineering of B.subtilis for microbial production of Neu5Ac, and provides a good starting point for further metabolic engineering to achieve the industrial production of Neu5Ac by a generally regarded as safe strain.