An Effective Strategy for Exploring Unknown Metabolic Pathways by Genome Mining

被引:42
作者
Castillo, Dorianne A. [1 ]
Kolesnikova, Mariya D. [1 ]
Matsuda, Seiichi P. T. [1 ,2 ]
机构
[1] Rice Univ, Dept Chem, Houston, TX 77005 USA
[2] Rice Univ, Dept Biochem & Cell Biol, Houston, TX 77005 USA
关键词
ARABIDOPSIS-THALIANA; OXIDOSQUALENE CYCLASE; GENE CLUSTERS; SECONDARY METABOLITES; FUNCTIONAL GENOMICS; CYTOCHROME P450S; NATURAL-PRODUCTS; BIOSYNTHESIS; IDENTIFICATION; NMR;
D O I
10.1021/ja401535g
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Plants allocate an estimated 15-25% of their proteome to specialized metabolic pathways that remain largely uncharacterized. Here, we describe a genome mining strategy for exploring such unknown pathways and demonstrate this approach for triterpenoids by functionally characterizing three cytochrome P450s from Arabidopsis thaliana. Building on proven methods for characterizing oxidosqualene cyclases, we heterologously expressed in yeast known cyclases with candidate P450s chosen from gene clustering and microarray coexpression patterns. The yeast cultures produced mg/L amounts of plant metabolites in vivo without the complex phytochemical background of plant extracts. Despite this simplification, the product multiplicity and novelty overwhelmed analytical efforts by MS methods. HSQC analysis overcame this problem. Side-by-side HSQC comparisons of crude P450 extracts against a control resolved even minor P450 products among similar to 100 other yeast metabolites spanning a dynamic range of >10 000:1. HSQC and GC-MS then jointly guided purification and structure determination by classical NMR methods. Including our present results for P450 oxidation of thalianol, arabidiol, and marneral, the metabolic fate for most of the major triterpene synthase products in Arabidopsis is now at least partially known.
引用
收藏
页码:5885 / 5894
页数:10
相关论文
共 74 条
[1]  
Bak Soren, 2011, Arabidopsis Book, V9, pe0144, DOI 10.1199/tab.0144
[2]   Activation of camalexin biosynthesis in Arabidopsis thaliana in response to perception of bacterial lipopolysaccharides: a gene-to-metabolite study [J].
Beets, Caryn Ann ;
Huang, Ju-Chi ;
Madala, Ntakadzeni Edwin ;
Dubery, Ian .
PLANTA, 2012, 236 (01) :261-272
[3]   Discovery of New Modules in Metabolic Biology Using ChemoMetabolomics [J].
Bocobza, Samuel ;
Willmitzer, Lothar ;
Raikhel, Natasha V. ;
Aharoni, Asaph .
PLANT PHYSIOLOGY, 2012, 160 (03) :1160-1163
[4]   A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene [J].
Cankar, Katarina ;
van Houwelingen, Adele ;
Bosch, Dirk ;
Sonke, Theo ;
Bouwmeester, Harro ;
Beekwilder, Jules .
FEBS LETTERS, 2011, 585 (01) :178-182
[5]   CYP79F1 and CYP79F2 have distinct functions in the biosynthesis of aliphatic glucosinolates in Arabidopsis [J].
Chen, SX ;
Glawischnig, E ;
Jorgensen, K ;
Naur, P ;
Jorgensen, B ;
Olsen, CE ;
Hansen, CH ;
Rasmussen, H ;
Pickett, JA ;
Halkier, BA .
PLANT JOURNAL, 2003, 33 (05) :923-937
[6]   Conversion of a C-20 2,3-oxidosqualene analog to tricyclic structures with a five-membered C-ring by lanosterol synthase. Further evidence for a C-ring expansion step in sterol biosynthesis [J].
Corey, EJ ;
Cheng, HM .
TETRAHEDRON LETTERS, 1996, 37 (16) :2709-2712
[7]   MOLECULAR-CLONING, CHARACTERIZATION, AND OVEREXPRESSION OF ERG7 THE SACCHAROMYCES-CEREVISIAE GENE ENCODING LANOSTEROL SYNTHASE [J].
COREY, EJ ;
MATSUDA, SPT ;
BARTEL, B .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (06) :2211-2215
[8]  
Croteau R., 2000, Biochemistry & molecular biology of plants, V24, P1250, DOI DOI 10.1201/B11003-3
[9]   The secondary metabolism of Arabidopsis thaliana:: growing like a weed [J].
D'Auria, JC ;
Gershenzon, J .
CURRENT OPINION IN PLANT BIOLOGY, 2005, 8 (03) :308-316
[10]   Unusually cyclized triterpenes: occurrence, biosynthesis and chemical synthesis [J].
Domingo, Victoriano ;
Arteaga, Jesus F. ;
Quilez del Moral, Jose F. ;
Barrero, Alejandro F. .
NATURAL PRODUCT REPORTS, 2009, 26 (01) :115-134