Metabolic Engineering Escherichia Coli For N-acetyl Neuraminic Acid Production

N-acetylneuraminic acid(NeuAc), as a predominant type of the sialic acid family, plays an important role in life activities. Owing to its many physiological functions, NeuAc can used as a precursor to manufacture the essential drugs of antiviral drugs, anti-cancer drugs, etc. Generally, there are some methods to prepare NeuAc such as extraction from natural sources, chemical synthesis and enzymatic synthesis, whole cell biological catalysis. However some drawbacks still hamper NeuAc production in scale. Recently, fermentation process has been paid on more attention for NeuAc production. In this study, systematic metabolic engineering was employed for Escherichia coli to overproducing NeuAc yield from glucose. The main results showed as below:Firstly, the genes negatively affecting NeuAc synthesis including nanT, nagE and yhbJ were deleted sequentially according to metabolic pathways analysis for NeuAc synthesis. Then, GNA1 gene encoding glucosamine-6-phosphate acetyltransferase from Saccharomyces cerevisiae EBY100 and AGE gene encoding N-acetyl-D-glucosamine-2-epimerase from Synechocystis sp. PCC 6803 were integrated into nagA-nagB and lacI gene loci in E. coli genome, respectively. The resulting strain K12 TEJBAI was obtained, in which NeuAc synthesis pathway from glucose was constructed and foreign genes were constitutively expressed without addition of inducer. NeuAc accumulation in the recombinant strain K12 TEJBAI reached 0.24 g·L-1 in shake flask.Secondly, in order to reduce by-product yield, acetate kinase gene(ack A), phosphate acetyltransferase(pta), lactate dehydrogenase(ldh A) and pyruvate oxidase(poxB) were knocked out sequentially, the resulting strain K12 TEJBAIAa, K12 TEJBAIAal and K12 TEJBAIAalp were obtained, which reduced the acetate and lactate secretion. Deletion of ack A-pta in the strain K12 TEJBAIAa enhanced the NeuAc accumulation to 0.64 g·L-1, while the acetate yield reduced by 21%. The NeuAc titer in K12 TEJBAIAal strain reached 0.76 g·L-1, and no lactate was detected in the culture. The acetate concentration reached 0.73 g·L-1, increased 19%. The production of NeuAc reached 0.85 g·L-1 in K12TEJBAIAalp(2.58-fold of K12TEJBAI), which achieved 1.02 g·L-1 NeuAc after adding 14 mmol·L-1 pyruvate. The plasmid p KK-glmS1 with four mutant sites was constructed, the NeuAc titer in K12TEJBAIAalp/pKK-glm S1 improved to 0.95 g·L-1, 13% increased comparing to K12 TEJBAIAalp, the yield dramatically increased to 1.16 g·L-1 after adding pyruvate. As can be seen, glmS gene with mutant sites was more beneficial to NeuAc yield.Finally, shnal gene encoding NeuAc aldolase(cloned from Staphylococcus hominis) was integrated to nanA gene loci of K12 TEJBAIAalp by Red recombination, generating K12 TEJBAIAalps, the NeuAc yield reached 0.87 g·L-1 in shake flask, which showed that shnal gene was more beneficial to NeuAc production. The NeuAc accumulation reached 3.12 g·L-1 in fed-batch fermentation, meanwhile, GlcNAc, ManNAc and pyruvate were also detected, which indicated that the fermentation level of the resulting strain should be further improved by metabolic engineering and fermentation process optimization.