Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system

Summary 13‐Lipoxygenase (13‐LOX) and 13‐hydroperoxide lyases (13‐HPL) are the key enzymes for the production of the ‘green note’ compounds hexanal, (3 Z )‐ and (2 E )‐hexenal in plant tissues. To produce high levels of 13‐LOX and 13‐HPL enzymatic activities for a biocatalytic process to generate C 6 ‐aldehydes on a large scale, soya bean 13‐LOX ( GmVLXC ) and watermelon 13‐HPL ( ClHPL ) genes were expressed in Nicotiana benthamiana using a viral vector system mediated by agroinfiltration. The N. benthamiana leaves produced high activity of watermelon HPL, but not GmVLXC 13‐LOX. In addition, all leaves treated with bacterial suspension displayed a high activity of 9‐LOX, indicating that the internal tobacco 9‐LOX gene was highly induced through agroinfiltration because of wounding. GmVLXC and ClHPL transcripts could be detected in the corresponding transformed tobacco leaves by real‐time RT‐PCR analysis but the expression level of ClHPL was 24‐fold higher than that of GmVLXC . Western blot analysis showed that LOX was present in all tobacco leaves which were treated with bacterial suspensions, but not in the untreated wild‐type control. This result confirms that internal 9‐LOX was highly induced by agroinfiltration. The highest levels of ClHPL activity under optimal infiltration conditions were 80 times the HPL activity of wild‐type plants or plants transformed with control vector. A large amount of hexanal was formed when linoleic acid was incubated with extracts from N. benthamiana leaves over‐expressing ClHPL in combination with GmVLXC ‐expressing yeast extracts. One gram of ClHPL ‐expressing N. benthamiana leaves (fresh weight) could produce 17 ± 0.4 mg hexanal from 50 mg linoleic acid after 30 min.