Increasing expression of P450 and P450-reductase proteins from monocots inheterologous systems

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant form s of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules. We report here the isolation and functional characterization of the first P450 and P450 reductase coding se quences from wheat, The first attempts at expressing these cDNAs in yeast a nd tobacco led to levels of protein, which mere extremely low, often not ev en detectable. The wheat P450 cDNAs were efficiently transcribed, but no pr otein or activity was found. Wheat coding sequences, like those of other mo nocots, are characterized by a high GC content and by a related strong bias of codon usage, different from that observed in yeast or dicots. Complete recoding of genes being costly, the reengineering their 5'-end using a sing le PCR megaprimer designed to comply with codon usage of the host was attem pted. It was sufficient to relieve translation inhibition and to obtain goo d levels of protein expression. The same strategy also resulted in a dramat ic increase in protein expression in tobacco. A basis for the success of su ch a partial recoding strategy, much easier and cheaper than complete recod ing of the cDNA, is proposed. (C) 2000 Academic Press.