Alanine production by Escherichia coli transformed with alanine dehydrogenase and formate dehydrogenase genes

Abstract

Alanine dehydrogenase (EC 1.4.1.1) catalyzes the NAD[superscript +]-dependent reversible oxidative deamination of L-alanine to form ammonia, pyruvate, and NADH. The enzyme is important as a catalyst for the synthesis of alanine and its derivatives. Moreover, it is therefore applicable to diagnosis of malignant hematopoietic disease. The application of this enzyme to industrial production of L-alanine has been hampered by the cost of coenzymes. A multienzyme reaction system for simultaneous coenzyme regeneration has been proposed to overcome this problem. Alanine dehydrogenase from Aeromonas hydrophila has high activity and high substrate specificity, so it is suitable for L-alanine production. Subsequently, the aladh gene was cloned into E. coli JM109 by using plasmid vector pUC18. To enhance the enzyme activity and regeneration of NADH in a single cell by co-existence of aladh and fdh in E. coli BL21(DE3) host cell, two methods were performed 1) cloning of heterologous gene of aladh and fdh in a high expression vector pET-17b (pETAF and pETFA) and 2) co-transformation of plasmids containing aladh and fdh gene under T7 promoter by two systems using plasmid vector pET-17b and pSY343 (pETAlaDH/pSYFDH) or plasmid vector pMPM-K3 and pET-17b (pMPMAlaDH/pETFDH), respectively. The heterologous gene expression clones and cotransformed clones had alanine dehydrogenase and formate dehydrogenase activities higher than those of their original clones (pUCAlaDH and pUCFDH). However, their activities were less than those of the single gene clones (pETAlaDH, pMPMAlaDH, pETFDH) except for the AlaDH of heterologous gene expression clones which were the same as that expressed by pETAlaDH. Production of alanine by various recombinant clones were not significantly different with about 50% yield and ratio of D:L form about 1.6:1