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 col i 44277 through a primer-directed polymerase chain reaction and cloned into pUC19 between the sites EcoRI and SalI. The sequence analysis showed the c loned 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. Th e four base substitutions did not change the amino acid sequence. Based on the preliminary expression experiment and computer-aided analysis of the ge ne structure, six bases were mutated in order to achieve high-level express ion. 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 t he total bacterial proteins, which was 850-fold higher than that of the wil d strain. Under the optimal inducing condition, the enzyme yield reached 10 0 U/l cell culture. Using the partially purified recombinant enzyme, the sy nthesis of CMP-NeuAc was carried out in 90% yield. (C) 2000 Elsevier Scienc e B.V. All rights reserved.