Cloning and overexpression of a cytidine 5′-monophosphate N-acetylneuraminic acid synthetase from Escherichia coli

The gene for cytidine 5′-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase (EC2.7.7.43) was amplified from the total DNA of Escherichia coli 44277 through a primer-directed polymerase chain reaction and cloned into pUC19 between the sites EcoRI and SalI. The sequence analysis showed the cloned DNA was the same as the published neuA except for a codon missing for Asp230 and four base substitutions at the sites 153, 438, 618, and 687. The four base substitutions did not change the amino acid sequence. Based on the preliminary expression experiment and computer-aided analysis of the gene structure, six bases were mutated in order to achieve high-level expression. The gene mutated by PCR was cloned into the expression vector pET15b. Upon the induction of IPTG, the recombinant enzyme was shown to be 26% of the total bacterial proteins, which was 850-fold higher than that of the wild strain. Under the optimal inducing condition, the enzyme yield reached 100 U/l cell culture. Using the partially purified recombinant enzyme, the synthesis of CMP-NeuAc was carried out in 90% yield.