Overexpression of hydroperoxide lyase gene in Nicotiana benthamiana using a viral vector system.
13-Lipoxygenase (13-LOX) and 13-hydroperoxide lyases (13-HPL) are the key enzymes for the production of the 'green note' compounds hexanal, (3Z)- and (2E)-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.
Huang
Fong-Chin
FC
Technische Universität München, Biomolecular Food Technology, Freising, Germany.
Studart-Witkowski
Claudia
C
Schwab
Wilfried
W
D000446:Aldehyde-Lyases / Q000235:genetics*; D000447:Aldehydes / Q000378:metabolism; D029773:Citrullus / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D008084:Lipoxygenase; D030821:Plants, Genetically Modified / Q000201:enzymology / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013025:Glycine max / Q000201:enzymology*; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics*
D000447:Aldehydes; D018749:RNA, Plant; D003577:Cytochrome P-450 Enzyme System; C117433:13-lipoxygenase; D008084:Lipoxygenase; D000446:Aldehyde-Lyases; C054540:hydroperoxide lyase