Increasing expression of P450 and P450-reductase proteins from monocots in heterologous 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 forms 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 sequences from wheat. The first attempts at expressing these cDNAs in yeast and tobacco led to levels of protein, which were extremely low, often not even detectable. The wheat P450 cDNAs were efficiently transcribed, but no protein or activity was found. Wheat coding sequences, like those of other monocots, 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 single PCR megaprimer designed to comply with codon usage of the host was attempted. It was sufficient to relieve translation inhibition and to obtain good levels of protein expression. The same strategy also resulted in a dramatic increase in protein expression in tobacco. A basis for the success of such a partial recoding strategy, much easier and cheaper than complete recoding of the cDNA, is proposed.
Batard
Y
Y
Département d'Enzymologie Cellulaire et Moléculaire, Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique FRE 2161, 28 rue Goethe, Strasbourg Cedex, 67083, France.
Hehn
A
A
Nedelkina
S
S
Schalk
M
M
Pallett
K
K
Schaller
H
H
Werck-Reichhart
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics*; D017343:Genes, Plant / Q000235:genetics; D005916:Globulins / Q000235:genetics; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics; D014908:Triticum / Q000201:enzymology* / Q000235:genetics; D015003:Yeasts / Q000235:genetics
D003062:Codon; D017931:DNA Primers; D005916:Globulins; D010940:Plant Proteins; D011994:Recombinant Proteins; C094238:TAR1 protein, Colocasia esculenta; D003577:Cytochrome P-450 Enzyme System; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase