Additive effects of the feed-back insensitive bacterial aspartate kinase and the Brazil nut 2S albumin on the methionine content of transgenic narbon bean (Vicia narbonensis L.)

Abstract

So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean (Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial aspartate kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals.