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- Title
Development of a Plant Viral-Vector-Based Gene Expression Assay for the Screening of Yeast Cytochrome P450 Monooxygenases.
- Authors
Hanley, Kathleen; Nguyen, Long V.; Khan, Faizah; Pogue, Gregory P.; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B.; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L.
- 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 ω-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, ~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.
- Publication
Assay & Drug Development Technologies, 2003, Vol 1, p147
- ISSN
1540-658X
- Publication type
Article
- DOI
10.1089/154065803321537863