Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco

Abstract

A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. The ldcgene from Hafnia alvei was therefore (a) placed under the control of the 1' promoter of the bidirectional Tr promoter from Agrobacterium tumefaciens Ti-plasmid, and (b) cloned behind the rbcS promoter from potato fused to the coding region of the rbcS transit peptide. Both ldc constructs, introduced into Nicotiana tabacum with the aid of A. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of the rbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3-1% of dry mass.

Increased production of serotonin by suspension and root cultures ofPeganum harmala transformed with a tryptophan decarboxylase cDNA clone fromCatharanthus roseus

“…When overexpressed enzymes were supplied with substrates, increased levels of the reaction products were found which were sometimes even unknown for the engineered plant (Hain et aL, 1990;Songstad et al, 1990;Herminghaus et aL, 1991;Cahoon et aL, 1992;Goddijn, 1992). However, in all these cases the reaction products were either not further metabolized or the further fate of the overproduced metabolite was not investigated.…”

Manipulating secondary metabolism in culture

“…Untransformed plants contained barely detectable cadaverine levels whereas this group of transformants contained cadaverine at 0.3-1 % dry weight in leaf tissue. As might be expected, root tissues did not synthesise cadaverine presumably due to the lack of a differentiated chloroplast in these tissues (Herminghaus et al, 1991).…”

“…The gene for lysine decarboxylase (LDC), from the bacterium Hafnia alvei, was introduced into N tabacum, using a disarmed binary vector/leaf disc transformation procedure (Herminghaus et at., 1991). The gene was engineered such that in one group of transformants protein would accumulate in the cytoplasm while a second group of transformants were generated with the gene fused to a DNA sequence encoding a chloroplast-targeting amino acid sequence (see Robinson, 1991).…”

Cadaverine

“…The tobacco transgenic with ldc gene elevated expression observed with bidirection to promoters ligated with rbcs promoter of potato. 186 A significant elevation (0.3-1% of dry mass) found in Cad. Pincus et al 187 opined that Cad might be employed in developing Anti HIV drugs, which needs urgent focus of the scientist for the well being of larger society at present.…”