Monoterpene biosynthesis pathway construction in Escherichia coli |
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Authors: | Carter Ora A Peters Reuben J Croteau Rodney |
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Affiliation: | Institute of Biological Chemistry, and Plant Physiology Program, Washington State University, Pullman, WA 99164-6340, USA. |
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Abstract: | Four genes encoding sequential steps for the biosynthesis of the spearmint monoterpene ketone (-)-carvone from the C(5) isoprenoid presursors isopentenyl diphosphate and dimethylallyl diphosphate were installed in Escherichia coli. Inducible overexpression of these genes in the bacterial host allowed production of nearly 5 mg/l of the pathway intermediate (-)-limonene, which was mostly excreted to the medium such that products of the downstream steps, (-)-carveol and (-)-carvone, were not detected. Assay of pathway enzymes and intermediates indicated that flux through the initial steps catalyzed by geranyl diphosphate synthase and limonene synthase was severely limited by the availability of C(5) isoprenoid precursors in the host. Feeding studies with (-)-limonene, to overcome the flux deficiency, demonstrated the functional capability of limonene-6-hydroxylase and carveol dehydrogenase to produce the end-product carvone; however, uptake and trafficking restrictions greatly compromised the efficiency of these conversions. |
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Keywords: | Escherichia coli Gene overexpression Spearmint monoterpenes Carvone biosynthesis Geranyl diphosphate synthase Limonene synthase Limonene-6-hydroxylase Carveol dehydrogenase |
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