Heterologous expression of piperideine-6-carboxylate dehydrogenase and lysine-6-dehydrogenase genes for L-pipecolic acid production

Abstract

L-aminoadipic acid (L-AAA) is a non-natural protein amino acid which is widely used as an important intermediate for systhesis of drugs such as methotrexate derivatives carcinostatic agent and as precursor for -lactam antibiotics. To produce L-AAA, an Escherichia coli containing heterologous genes of lysine-6-dehydrogenase (LysDH) and piperideine-6-carboxylate dehydrogenase (P6CDH) was constructed in this study. The gene encoding P6CDH (pcd) from Pseudomonas putida ADH3 was sequenced and cloned into E. coli BL21(DE3) using pET-17b vector. The optimum condition for pcd expression was induction with 0.4 mM IPTG for 4 hours. The recombinant enzyme was purified 5.36 fold to homogeneity with a 32% yield. The molecular mass of enzyme subunit was determined to be about 54 kDa by SDS-PAGE. Optimum pH and temperature for the enzyme reaction were 8.07 and 40 °C, respectively. The enzyme was stable in pH ranging from 7.0 to 8.5. The apparent Km values for acetaldehyde and NAD+ were 1.18 and 0.24 mM, respectively. Then, the pcd gene was co-expressed with lysdh gene, encoding LysDH from Acromobacter denitrificans K-1 and transformed into E. coli BL21(DE3) using pET- 17b. The highest production of L-aminoadipic acid, approximately 25 mM, was obtained by induction of the recombinant clone with 0.2 mM IPTG for 4 hours and then 0.05 g of cell wet weight was incubated in the reaction mixture consisted of 200 mM L-lysine in 200 mM Tris-HCl buffer, pH 8.5 for 24 hours.