Optimization Of Whole-cell Biocatalytic Process For N-Acetylneuraminic Acid Pmduction And Study On The Preparation Of Polysialic Acid By Microbial Fermentation

N-Acetylneuraminic Acid (Neu5Ac) has important medical value in the treatment of influenza, the neurological disease, inflammation and cancer etc, especially in the treatment of flu (including H5N1and H1N1in2009). The conventional methods of production have some shortcomings, such as high cost and low yield. So the available resource of Neu5Ac can not meet the large-scale demand of medical industry. Whole-cell catalysis was used for the production of Neu5Ac with N-acetyl glucosamine (GIcNAc) and pyruvate as the substrate. Fermentation condition of engineered strain E.coli AnanTEK/pNA and subsequent biological transformation condition were studied. By conditions optimization, the production of Neu5Ac was improved and91.05g/L of Neu5Ac was obtained. The process can be used for industrial large-scale production of Neu5Ac in terms of convenience, efficiency and economy.In the process of production, it was found that part of the pyruvate was used for the metabolism of cells, In order to reduce the bypass metabolic pathway of pyruvate and ensure high production strength for Neu5Ac, genes ldhA, poxB, aceE, plfB, meaB were knockout through the gene knockout technology. It was found that the mutant strains which without IdhA or plfB gene have certain effect for reducing the bypass metabolic pathway of pyruvate, but not remarkable. P1phage transduction was used to construct mutant strain E.coli ABA, which conbined the mutant ofâ–³plfB and AldhA, and mutant strain E.coli ABA was constructed. With0.2M pyruvate and0.8M GlcNAc as substrates, the results of bioconversion showed that the amount of pyruvate used in metabolic pathways decreased by24.96%, while Neu5Ac production increases by7.11%.Polysialic acid (PSA) is a polymer of sialic acid linked with a-2,8or a-2,9glycosidic (ketosidic) bonds. PSA has many advantages, such as poor immunogenicity, biodegradable and so on, which is considered as the most ideal material used in the control-release drugs and scaffolds in biomedical applications. In addition, various sialo-products have been derivated by means of treating PSA with degradation methods or enzymatic catalysis, and these derivatives can subsequently be used in pharmaceutical, food and health-care industries. Bacterial fermentation is the only way to produce PSA up to now. E. coli SA9can produce polysialic acid. With the E. coli SA9genome as a template, the gene of CMP-Neu5Ac synthetase was cloned, the gene codon of N-acetylneuraminate7-O(or9-O)-acetyltransferase and sialytransferase were optimized, and then constructed a series of expression vectors. In order to strengthen the synthesis pathway of polysialic acid, these vectors were transformed into E. coli SA9to over-expression. After expressing, the recombinant bacteria were fermented in shake flasks and0.97mg/mL of polysialic acid were obtained. Compared with the original strains, the production was increased by about150%. It is verified that this procedure can achieve the purpose of strengthen polysialic acid synthesis pathway, and proved that the N-acetylneuraminate7-O(or9-O)-acetyltransferase is a key enzyme for polysialic acid synthetic pathway.