Monoterpene biosynthesis pathway construction in Escherichia coli.
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.
Carter
Ora A
OA
Institute of Biological Chemistry, and Plant Physiology Program, Washington State University, Pullman, WA 99164-6340, USA.
Peters
Reuben J
RJ
Croteau
Rodney
R
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism; D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D005798:Genes, Bacterial / Q000235:genetics; D045782:Hemiterpenes / Q000378:metabolism; D019753:Intramolecular Lyases / Q000235:genetics / Q000378:metabolism; D000077222:Limonene; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D039821:Monoterpenes; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism*
D000081005:Cyclohexane Monoterpenes; D053138:Cyclohexenes; D029968:Escherichia coli Proteins; D045782:Hemiterpenes; D039821:Monoterpenes; D009943:Organophosphorus Compounds; D010940:Plant Proteins; D011994:Recombinant Proteins; D013729:Terpenes; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; C006923:carvone; D003577:Cytochrome P-450 Enzyme System; D000077222:Limonene; D006899:Mixed Function Oxygenases; D000429:Alcohol Oxidoreductases; C121536:carveol dehydrogenase; C120916:limonene-6-hydroxylase; D009251:NADPH-Ferrihemoprotein Reductase; D019883:Alkyl and Aryl Transferases; D019753:Intramolecular Lyases; C040265:pinene cyclase I