Carter Ora A, Peters Reuben J, Croteau Rodney
Institute of Biological Chemistry, and Plant Physiology Program, Washington State University, Pullman, WA 99164-6340, USA.
Phytochemistry. 2003 Sep;64(2):425-33. doi: 10.1016/s0031-9422(03)00204-8.
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.
将四个编码从C(5)类异戊二烯前体异戊烯基二磷酸和二甲基烯丙基二磷酸生物合成留兰香单萜酮(-)-香芹酮连续步骤的基因导入大肠杆菌。在细菌宿主中对这些基因进行诱导型过表达，使得能够产生近5毫克/升的途径中间体(-)-柠檬烯，其大部分分泌到培养基中，因此未检测到下游步骤的产物(-)-香芹醇和(-)-香芹酮。对途径酶和中间体的分析表明，由香叶基二磷酸合酶和柠檬烯合酶催化的初始步骤的通量受到宿主中C(5)类异戊二烯前体可用性的严重限制。用(-)-柠檬烯进行补料研究以克服通量不足，证明了柠檬烯-6-羟化酶和香芹醇脱氢酶产生终产物香芹酮的功能能力；然而，摄取和转运限制极大地损害了这些转化的效率。
