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.
Carlsberg Laboratory, Department of Physiology, Copenhagen Valby, Denmark.
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Knust
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von Wettstein
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