| Neu5Ac has extensive application in the field of pharmaceutical and chemical. As a vital anti-influenza drug precursor, Neu5 Ac is one of important raw materials for synthetic drugs which could anti adhesion, treat inflammation and cancer. Production of Neu5 Ac by enzymatic conversion or whole cell biocatalysis has been predominant technology. A two-step enzymatic catalytic strategy involving AGE and Nan A has been adopted to produce Neu5 Ac using Glc NAc as a substrate, however the low yield and high cost lead to the difficulty for large-scale production. Therefore, development of a more-efficient biocatalyst to improve Neu5 Ac yield become an important and scientific issue. Here we developed an efficient E. coli biocatalyst for Neu5 Ac synthesis. The main results were shown as below:(1) The recombinant plasmids p ET28a-slr and p ET28a-nan A were constructed, the slr gene from Synechocystis sp. PCC 6803 was codon-optimized using software of E. coli Codon Usage Analyzer 2.1 and chemically synthesized. Which were then transformed into E. coli BL21(DE3). Whole-cell catalysis by coupling these two recombinant strains was investigated and biocatalysis conditions for Neu5 Ac production were optimized. A yield of Neu5 Ac, in which 236.9 mmol·L-1 Neu5 Ac was produced using 600 mmol·L-1 Glc NAc and 1600 mmol·L-1 pyruvate as substrates. And a Neu5 Ac productivity of 1.22 g·L-1·h-1 with 39.5% conversion yield from Glc NAc could be obtained.(2) Nag E and Nan T protein, which are responsible for the transport of Glc NAc and Neu5 Ac, were engineered and the effects on the Neu5 Ac production were evaluated. A maximum yield of Neu5 Ac, 260.0 mmol·L-1, was produced by an overall whole-cell biocatalysis process using E. coli △NE/p ET28a-slr and E. coli △NTE/p ET28a-nan A. The Neu5 Ac productivity of 1.34 g·L-1·h-1 and Glc NAc conversionrate of 43.3% were obtained.(3) Different promoters were used for overexpression of AGE and Nan A and transformed into E. coli BL21(DE3). SDS-PAGE, enzyme activity analysis and catalysis experiments demonstrated that the recombinant strain E. coli/p ET28a-(T7-nan A)-(tac-slr) showed the more potential for Neu5 Ac synthesis. Using this recombinant strain, optimization of biocatalysis conditions resulted in a higher yield of Neu5 Ac, in which 298.8 mmol·L-1 Neu5 Ac was produced with a Neu5 Ac productivity of 1.93 g·L-1·h-1 and a yield of 49.8% from Glc NAc was achieved. 600 mmol·L-1 Glc NAc and 1600 mmol·L-1 pyruvate as substrates were found to be the optimal concentration for Neu5 Ac production.(4) An N-acetylneuraminate lyase gene(shnal) from Staphylococcus hominis was cloned. Then recombinant plasmid of p ET28a-(T7-shnal)-(tac-slr) was constructed and transformed into E. coli BL21(DE3) host cells. Using this recombinant strain, optimization of biocatalysis conditions resulted in the a yield of Neu5 Ac, in which 312.5 mmol·L-1 Neu5 Ac was produced using 600 mmol·L-1 Glc NAc and 1600 mmol·L-1 pyruvate as substrates. And a higher Neu5 Ac productivity of 8.10 g·L-1·h-1 and a yield of 52.1% from Glc NAc was achieved.(5) Moreover, to improve both the catalytic efficiency of AGE from Glc NAc to Man NAc and the Neu5 Ac conversion yield, several AGE mutant strains were constructed by mutagenesis techniques. Using the novel recombinant strain of E. coli △NTE/p ET28a-(T7-shnal)-(tac-slr A), 351.8 mmol·L-1 Neu5 Ac was obtained with a yield of 58.6% from Glc NAc.(6) Neu5 Ac was isolated from the reaction solution with a recovery of 72.5%. Under the same condition of HPLC, it has the same peak time compared with the Neu5 Ac from Sigma. The purification of Neu5 Ac were simple and efficient.In summary, the efficient biocatalyst of E. coli △NTE/p ET28a-(T7-nan A)-(tac-slr A) were constructed and the high efficient Neu5 Ac produciton process was developed in this study. |
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