Construction of alanine racemase gene knockout mutants of Escherichia coli BL21(DE3) by using a group II intron

Abstract

The L-form of amino acid is widely used in food and health care industry. L-alanine is currently used as a food sweetener and for pharmaceutical application. To improve the L-alanine production as well as coenzyme regeneration, alanine dehydrogenase gene (aladh) and formate dehydrogenase gene (fdh) were introduced into Escherichia coli BL21 (DE3) by 2 methods 1) cloning of heterologous gene of aladh and fdh in high expression vector pET-17b and 2) co-transformation of aladh gene and fdh gene using vectors a) pET-17b for aladh gene and pSY343 for fdh gene and b) pMPM-K3 for aladh gene and pET-17b for fdh gene. However, the production of alanine by various recombinatnt clones were not signigicantly different with ratio of D:L form about 1.6:1 because of the activity of alanine racemase. In E. coli, there are 2 types of alanine racemase encoding by alr gene and dadX gene. The alr gene encodes the constitutively expressed biosynthetic enzyme. The catabolic dadX gene, encodes a second alanine racemase isozyme whose expression is subjected to induction by L-alanine. For further L-alanine production, the alanine racemase gene(s) in E. coli BL21 (DE3) must be disrupted. In this work, group II intron was used to inactivate alanine racemase genes. In all constructed mutants, alr gene knockout mutant, dadX gene knockout mutant as well as dadX and alr genes knockout mutant, group II intron inserted only at the target gene. At 20[superscript th] of subculture, the alr gene knockout mutant and dadX gene knockout mutant still had intron insertion. Alanine racemase activity of alr gene knockout mutant and the dadX gene knockout mutant were lower than that of wild type 3.3 and 1.1 times, respectively. The dadX and alr genes knockout mutant showed very slow growth when cultured in LB medium supplemented with 10 mM D-alanine. Moreover, it could not grow on solid medium. Thus, its stability test and alanine racemase activity assay could not be performed.