Expression of a bi-functional and thermostable amylopullulanase in transgenic rice seeds leads to autohydrolysis and altered composition of starch

Overexpression of bacterial-derived starch metabolic enzymes in plant starch storage organs represents a valuable strategy for improving starch quality, bioprocessing and nutritional value. Transgenic rice seeds producing a thermostable and bifunctional starch hydrolase, amylopullulanase (APU) from Thermoanaerobacter ethanolicus 39E, were generated. Starch in these seeds could be hydrolyzed with optimal temperatures between 85 and 95degreesC, which resulted in complete conversion of starch into soluble sugars and production of protein-enriched flour within a few hours. By expressing various levels of APU, rice seeds containing reduced amounts of amylose, which is an important factor affecting starch quality, were obtained without a significant impact on grain yield. Elevation in granule-bound pullulanase activity correlates with the reduction of amylose in developing APU-containing rice seeds. APU was found to be localized within amyloplasts and in cell walls, which could be the result of overexpression of APU with a signal peptide. This study establishes novel approaches to alter starch properties, accelerate bioprocessing of starch and production of protein-enriched flour from rice seeds, and could significantly impact the industrial and food uses of cereals.