Lysine Accumulation in Transgenic Tobacco Expressing Dihydrodipicolinate Synthase of Escherichia coli

Abstract

The dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) gene from E. coli was introduced into tobacco to examine the ability and role of chimeric DHDPS in the biosynthesis of lysine in higher plants. Southern blotting and genetic analysis of six transformed tobacco plants showed that they contained on an average one to three copies. Seeds at T 1 generation of K 11 T-9 showed a 3 resistant: 1 sensitive segregation ratio in the lysine-resistance test with addition of 2 mM lysine. The DHDPS purified from the heterozygous and homozygous K 11 T-9 required ca. 1 mM and ca. 2 mM lysine for 50 % inhibition of enzyme activity, respectively, but only 20 μM lysine in wild type tobacco. The DHDPS activity in the heteroand homozygous K 11 T-9 plant leaves increased ca. 34- and 59-fold as compared with wild type tobacco. The significant increase in the level of free lysine was observed in heterozygous K 11 T-9 (25-fold) and homozygous K 11 T-9 (55-fold) as compared with wild type plants. In transgenic plants, a highly significant correlation was found between the content of free lysine and the level of DHDPS activity. The accumulation levels of lysine were sufficiently high to inhibit the activity of the lysine-sensitive aspartate kinase (AK; EC 2.7.2.4) in vivo , i.e. 49 % and 68 % in heterozygous and homozygous K 11 T-9 plants, respectively, as compared with wild type plants. However, the inhibition of AK activity was not influenced by lysine accumulation in all of the transformed plants, although threonine and methionine contents decreased in homozygous K 11 T-9. The present results showed an important role of the E. coli DHDPS gene in biosynthesis of lysine in higher plants, and introduction of the lysine-insensitive DHDPS gene may confer agronomically useful levels of essential amino acid concentrations in a number of crops and vegetable species.