Cunningham Francis X, Gantt Elisabeth
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Photosynth Res. 2007 May;92(2):245-59. doi: 10.1007/s11120-007-9210-0. Epub 2007 Jul 17.
Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, beta-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.
类胡萝卜素是植物、藻类和蓝细菌光合机构中不可或缺的色素，许多细菌和真菌也能产生类胡萝卜素。通过使用积累类胡萝卜素的大肠杆菌菌株作为异源宿主，在体内对原本难以研究的膜相关途径酶进行功能测定，极大地促进了对陆地植物光合膜中发挥作用的类胡萝卜素生物化学途径的阐明。同样的实验方法特别适合于发现和表征尚未确定的酶，这些酶可导致藻类细胞光合膜中的类胡萝卜素、非绿色植物组织中发现的多种类胡萝卜素，以及植物组织中源自类胡萝卜素的无数风味和香气化合物。本文展示了一组适合在大肠杆菌中生产多种类胡萝卜素的质粒。以开花植物夏侧金盏花为例，描述了如何使用这些产类胡萝卜素的大肠杆菌来鉴定编码类胡萝卜素和类异戊二烯生物合成酶的cDNA，表征这些cDNA编码的酶，以及生产用作酶底物和参考标准的特定类胡萝卜素。已鉴定出编码夏侧金盏花九种不同类胡萝卜素和类异戊二烯合成酶的cDNA，并验证了其产物的酶活性。新描述的那些cDNA包括编码八氢番茄红素合酶、β-胡萝卜素羟化酶、1-脱氧-D-木酮糖-5-磷酸合酶、异戊烯基二磷酸异构酶和香叶基香叶基二磷酸合酶的cDNA。
