Archives of Biochemistry and Biophysics 454 (2006) 123–136 www.elsevier.com/locate/yabbi 0003-9861/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2006.08.006 The diverse sesquiterpene proWle of patchouli, Pogostemon cablin, is correlated with a limited number of sesquiterpene synthases  Fabienne Deguerry a , Laurence Pastore a , Shuiqin Wu b , Anthony Clark a , Joseph Chappell b , Michel Schalk a,¤ a Firmenich SA, Biotechnology Department, Corporate R and D Division, P.O. Box 239, CH-1211 Geneva 8, Switzerland b Plant Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, KY 40546-0312, USA Received 26 May 2006, and in revised form 2 August 2006 Available online 23 August 2006 Abstract Pogostemon cablin (patchouli), like many plants within the Lamiaceae, accumulates large amounts of essential oil. Patchouli oil is unique because it consists of over 24 diVerent sesquiterpenes, rather than a blend of diVerent mono-, sesqui- and di-terpene compounds. To determine if this complex mixture of sesquiterpenes arises from an equal number of unique sesquiterpene synthases, we developed a RT-PCR strategy to isolate and functionally characterize the respective patchouli oil synthase genes. Unexpectedly, only Wve terpene syn- thase cDNA genes were isolated. Four of the cDNAs encode for synthases catalyzing the biosynthesis of one major sesquiterpene, includ- ing a -curcumene synthase, two germacrene D synthases, and a germacrene A synthase. The Wfth cDNA encodes for a patchoulol synthase, which catalyzes the conversion of FPP to patchoulol plus at least 13 additional sesquiterpene products. Equally intriguing, the yield of the diVerent in vitro reaction products resembles quantitatively and qualitatively the proWle of sesquiterpenes found in patchouli oil extracted from plants, suggesting that a single terpene synthase is responsible for the bulk and diversity of terpene products produced in planta. © 2006 Elsevier Inc. All rights reserved. Keywords: Patchouli; Patchoulol; Terpenes; Sesquiterpenes; Terpene synthases; Heterologous expression; Functional characterization Patchouli oil is obtained by steam distillation of the leaves of Pogostemon cablin (patchouli), a plant from the Lamiaceae family, and is widely appreciated for its char- acteristic pleasant and long lasting woody, earthy, cam- phoraceous odor. Patchouli oil is hence an important ingredient in many Wne fragrance products like perfumes, as well as in soaps and cosmetic products [1]. The compo- sition of the patchouli oil is complex like many essential oils, but distinct because it consists largely of sesquiter- penes [2–4]. The sesquiterpene (¡)-patchoulol (Fig. 1) is the major constituent and is the primary component responsible for the typical patchouli note [5]. The oil contains a large number of other sesquiterpenes hydrocar- bons such as -/-/-patchoulenes, -bulnesene, -guaiene and seychellene, with structures clearly related to (¡)- patchoulol and sesquiterpenes with unrelated structures like trans--caryophyllene, -humulene and -curcumene (Fig. 1). At present, patchouli plants are the only commercial source of patchoulol and cost-eVective synthetic routes for enantiomeric pure patchoulol have yet to be devel- oped [5,6]. However, recent progress in biotechnology and gene technology suggests that the biosynthetic production of complex molecules like terpenes in surrogate hosts is promising [7,8]. A prerequisite for the engineering of a (¡)-patchoulol biosynthetic platform is of course to have the genes encoding for the enzymes responsible for patc- houlol biosynthesis. Sesquiterpenes are biosynthesized from the ubiquitous intermediate farnesyl-diphosphate  The GenBank Accession Nos. for the genes isolated in this study are AY508726, AY508727, AY508728, AY508729, AY508730 and DQ355151. * Corresponding author. Fax: +41 22 780 33 34. E-mail address: michel.schalk@Wrmenich.com (M. Schalk).