Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides

Abstract

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides, converting four herbicides of this class (f luometuron, linuron, chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to f luometuron, linuron, and diuron, and as a ring-methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10-mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants.Cytochrome P450 monooxygenases (P450s) are ubiquitous hemoproteins present in microorganisms, plants, and animals. Composed of a large and diverse group of isozymes, P450s mediate a great array of oxidative reactions. Most of these activities support biosynthetic processes such as phenylpropanoid, fatty acid, and terpenoid biosynthesis. Others metabolize natural products (e.g., the monoterpene camphor) or play a role in xenobiotic detoxification (1). In a typical P450-catalyzed reaction, one atom of molecular oxygen (O 2 ) is incorporated into the substrate, and the other atom is reduced to water by using NADPH. For most eukaryotic P450s, NADPH͞cytochrome P450 reductase, a membrane-bound flavoprotein, transfers the necessary two electrons from NADPH to the P450 (2).After the initial report of Frear et al. (3), who demonstrated the metabolism of monuron by a mixed-function oxidase located in a microsomal fraction of cotton seedlings, an extensive body of evidence has accumulated supporting the involvement of P450s in the metabolism and detoxification of numerous herbicides (1, 2). Differential herbicide-metabolizing P450 activities are believed to represent one of the mechanisms that enables certain crop species to be more tolerant of a particular herbicide than other crop or weedy species. Although the association of P450s with herbicide metabolism has been investigated for several decades, reports on the successful cloning and expression of P450 cDNAs encoding herbicide-metabolizing isozymes are relatively recent. In one example, constructs using the rat CYP1A1 cDNA conferred chlortoluron resistance in tobacco by enhancing herbicid...

Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke

“…There are few reports on the engineering of herbicide metabolism with plant genes (Jepson et al, 1996;Siminszky et al, 1999), but the rather scarce data available do not indicate how large increases in herbicide tolerance have been achieved. CYP76B1, a gene inducible by chemical stress isolated from Jerusalem artichoke, was previously shown to metabolize herbicides of the class of phenylurea with high turnover rates (Robineau et al, 1998).…”

“…In vivo metabolism was assayed with excised leaves from the wild-type and transgenic tobacco plants fed with 0.84 kBq [phenyl-U-14 C] chlortoluron or 1.55 kBq [phenyl-U-14 C] isoproturon dissolved in water, as described by Siminszky et al (1999). Herbicide and metabolites were quantified by TLC analysis of methanol extracts.…”

Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

“…CYP76B1 from Helianthus tuberosus was, however, found to metabolize herbicides belonging to the class of phenylurea (Robineau et al, 1998;Didierjean et al, 2002), but its physiological function was not reported. Other P450s from soybean (Glycine max; CYP71A10; Siminszky et al, 1999) or tobacco (Nicotiana tabacum; CYP71A11 and CYP81B1; Yamada et al, 2000) were also reported to metabolize phenylurea, but their physiological function was not investigated.…”

Identification of the transcriptionally active cytochrome P450 repertoire in Coffea arabica

“…This is an important step to confirm the accuracy of the information obtained by bioinformatic analyses (Figure 2). In Figure 2, we observed that the CaCYP71 and CaCYP76 subfamilies grouped in the same clade, which may suggest that they share similar functions (e.g., herbicide detoxification; Siminszky et al, 1999;Swaminathan, et al, 2009). The same occurred for CaCYP81 and CaCYP82, which are putatively related to glucosinolate biosynthesis (Cabello-Hurtado et al, 1998;Tholl et al, 2011).…”