Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae |
| |
Authors: | Birgitt Knust Diter von Wettstein |
| |
Institution: | (1) Department of Physiology, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark;(2) Research Laboratory of Danisco A/S, Danish Distillers, Copenhagen S., Denmark |
| |
Abstract: | Summary Lipoxygenases (EC 1.13.11.12) catalyse the oxygenation of polyunsaturated fatty acids such as linoleic and arachidonic acid into reactive cis/trans hydroperoxidiene intermediates, which then serve as substrates for other enzymes leading to the production of a variety of secondary metabolites. In order to explore the characteristics of the individual lipoxygenase isoenzymes in more detail larger amounts of the pure enzymes are needed and their production in a heterologous host is therefore desirable. Full-length cDNAs encoding pea-seed lipoxygenase isoenzymes 2 and 3 were expressed in Saccharomyces cerevisiae with the aid of yeast-Escherichia coli shuttle vectors. Expression of the cDNA for lipoxygenase 2 under the control of the constitutive phosphoglycerate kinase (PGK) gene promoter yielded significant amounts of active enzyme inside the cell, both with yeast transformants carrying the cDNA gene on high-copy-number plasmids or integrated in chromosome V. Addition of the yeast invertase signal sequence in front of the pea lipoxygenase 3 yielded secreted active pea-seed lipoxygenase in the medium, but large amounts of inactive lipoxygenase 3 remained inside the yeast cell. Expression of the LOX3 cDNA can be achieved either constitutively with the PGK promoter or inducibly with the GAL1 promoter.
Correspondence to: B. Knust |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|