Engineering direct fructose production in processed potato tubers by expressing a bifunctional alpha-amylase/glucose isomerase gene complex

Manipulation of starch biosynthesis/degradation and formation of novel mole cules in storage organs of plants through genetic engineering is an attract ive but technically challenging goal. We report here, for the first time, t hat starch was degraded and glucose and fructose were produced directly whe n crushed potato tubers expressing a starch degrading bifunctional gene wer e heated for 45 minutes at 65 degrees C. To achieve this, we have construct ed a fusion gene encoding the thermostable enzymes: alpha-amylase (Bacillus stearothermophilus) and glucose isomerase (Thermus thermophilus). The chim eric gene was placed under the control of the granule-bound-starch synthase promoter. This enzymatic complex produced in transgenic tubers was only ac tive at high temperature (65 degrees C). More than 100 independent transgen ic potato plants were regenerated. Molecular analyses confirmed the stable integration of the chimeric gene into the potato genome. The biochemical an alyses performed on young and old tubers after high temperature treatment ( 65 degrees C) revealed an increase in the formation rate of fructose and gl ucose by a factor of 16.4 and 5.7, respectively, in the transgenic tubers a s compared to untransformed control tubers. No adverse discernible effect o n plant development and metabolism including tuber formation and starch acc umulation was observed in the transgenic plants before heat treatment. Our results demonstrate that it is possible to replace starch degradation using microbial enzymes via a system where the enzymes are produced directly in the plants, but active only at high temperature, thus offering novel and vi able strategies for starch-processing industries. (C) 2000 John Wiley & Son s, Inc.