Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae.
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.
Knust
B
B
Carlsberg Laboratory, Department of Physiology, Copenhagen Valby, Denmark.
von Wettstein
D
D
D001483:Base Sequence; D001709:Biotechnology; D004247:DNA / Q000235:genetics; D007887:Fabaceae / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008084:Lipoxygenase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D010946:Plants, Medicinal; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D007527:Isoenzymes; D004247:DNA; D008084:Lipoxygenase