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
Large Scale Biology Corporation, 3333 Vaca Valley Parkway, Vacaville, CA 95688, USA.
Assay Drug Dev Technol. 2003 Feb;1(1 Pt 2):147-60. doi: 10.1089/154065803321537863.
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.
开发用于异源表达细胞色素P450单加氧酶的基因发现工具一直都极具难度。活性测定工作强度大，且不适用于平行筛选。此外，生化确认需要共表达同源P450还原酶或互补的异源活性。植物病毒基因表达系统已被用于多种生物体。在本研究中，我们描述了一种使用RNA载体表达系统从表型上筛选细胞色素P450依赖性脂肪酸ω-羟化酶活性的方法。从基因组DNA中扩增出参与正构烷烃同化的解脂耶氏酵母CYP52基因家族成员，克隆到植物病毒基因表达载体中，并用作确定异源表达的模型系统。用表达酵母CYP52基因（YlALK1 - YlALK7）的病毒载体感染的植物在接种的植物叶片上表现出明显的坏死病变表型。在阴性对照构建体上未检测到表型。使用薄层色谱和气相色谱/质谱法确认，接种YlALK3、YlALK5和YlALK7的植物均催化了月桂酸的末端羟基化。基于植物的细胞色素P450表型筛选在正构烷烃诱导的解脂耶氏酵母植物病毒表达文库上进行了测试。在植物上测试了1025个随机文库克隆的一个子集，包括YlALK1 - YlALK7构建体。所有YlALK基因构建体在随机筛选中均获得阳性评分。对使用表型筛选获得阳性评分的克隆进行核苷酸测序后，约5%被认为适合进一步的生化分析。本报告说明了基于植物的系统在异源细胞色素P450单加氧酶表达及基因功能鉴定方面的实用性。
