title: Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.
abstract: Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.
authors.affiliation: Large Scale Biology Corporation, 3333 Vaca Valley Parkway, Vacaville, CA 95688, USA. kathy.hanley@lsbc.com
authors.forename: Kathleen
authors.initials: K
authors.lastname: Hanley
authors.forename: Long V
authors.initials: LV
authors.lastname: Nguyen
authors.forename: Faizah
authors.initials: F
authors.lastname: Khan
authors.forename: Gregory P
authors.initials: GP
authors.lastname: Pogue
authors.forename: Fakhrieh
authors.initials: F
authors.lastname: Vojdani
authors.forename: Sanjay
authors.initials: S
authors.lastname: Panda
authors.forename: Franck
authors.initials: F
authors.lastname: Pinot
authors.forename: Vincent B
authors.initials: VB
authors.lastname: Oriedo
authors.forename: Lada
authors.initials: L
authors.lastname: Rasochova
authors.forename: Mani
authors.initials: M
authors.lastname: Subramanian
authors.forename: Barbara
authors.initials: B
authors.lastname: Miller
authors.forename: Earl L
authors.initials: EL
authors.lastname: White
mesh_terms: D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D042926:Cytochrome P-450 CYP4A / Q000096:biosynthesis / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D005822:Genetic Vectors; D008861:Microsomes / Q000201:enzymology; D010641:Phenotype; D018515:Plant Leaves / Q000201:enzymology / Q000648:ultrastructure; D012328:RNA Viruses / Q000235:genetics*; D014026:Nicotiana / Q000201:enzymology* / Q000648:ultrastructure; D014158:Transcription, Genetic; D025062:Yarrowia / Q000201:enzymology*
chemical_list: D003577:Cytochrome P-450 Enzyme System; D042926:Cytochrome P-450 CYP4A