A novel 4-hydroxycoumarin biosynthetic pathway Benye Liu Æ Torben Raeth Æ Till Beuerle Æ Ludger Beerhues Received: 24 March 2009 / Accepted: 3 September 2009 / Published online: 15 September 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Coumarin forms in melilotoside (trans-ortho- coumaric acid glucoside)-containing plant species upon cell damage. In moldy melilotoside-containing plant material, trans-ortho-coumaric acid is converted by fungi to 4-hydroxycoumarin, two molecules of which spontaneously combine with formaldehyde to give dicoumarol. Dicouma- rol causes internal bleeding in livestock and is the forerunner of the warfarin group of medicinal anticoagulants. Here, we report 4-hydroxycoumarin formation by biphenyl synthase (BIS). Two new BIS cDNAs were isolated from elicitor- treated Sorbus aucuparia cell cultures. The encoded isoen- zymes preferred ortho-hydroxybenzoyl (salicoyl)-CoA as a starter substrate and catalyzed a single decarboxylative condensation with malonyl-CoA to give 4-hydroxycouma- rin. When elicitor-treated S. aucuparia cell cultures were fed with the N-acetylcysteamine thioester of salicylic acid, 4-hydroxycoumarin accumulated in the culture medium. Incubation of the BIS isoenzymes with benzoyl-CoA and malonyl-CoA resulted in the formation of 3,5-dihydroxy- biphenyl which is the precursor of the phytoalexins of the Maloideae. Keywords 4-Hydroxycoumarin Á Biphenyl synthase Á Type III polyketide synthase Á Salicylic acid Á Salicoyl-NAC Á Sorbus aucuparia Introduction Coumarins (1,2-benzopyrones; Fig. 1) are widely distrib- uted secondary products, which are subdivided into simple coumarins, furanocoumarins, and pyranocoumarins (Murray 1995; Matern et al. 1999; Bourgaud et al. 2006). The latter classes are further grouped into linear and angular compounds. The coumarin skeleton is also present as a partial structure of coumestans which, however, are isoflavonoid derivatives. Coumarin itself is well-known for its pleasant vanilla-like odour and contributes to the smell of new-mown hay (Bourgaud et al. 2006). It is commonly not present as a genuine constituent of intact plant tissue but liberated upon injury of cells. Its precursor is melilotoside, the glucoside of trans-2-coumaric acid (Fig. 1), which accumulates in vacuoles and undergoes either photochemical or enzymatic trans/cis isomerization (Murray et al. 1982; Brown 1986; Matern et al. 1999). Upon cell damage, the cis-glucoside is cleaved by extra- vacuolar b-glucosidase activity and free cis-2-coumaric acid spontaneously lactonizes to give coumarin. Involve- ment of trans-2,4-dihydroxycinnamic acid results in the formation of 7-hydroxycoumarin (umbelliferone). The para-hydroxylation of cinnamic acid was found to pre- cede the ortho-hydroxylation reaction (Murray et al. 1982). The direct cyclization of 4-coumaric acid via spi- rodienone or quinol intermediates may be an alternative mechanism (Matern 1991). The formation of the more complex carbon frameworks of furanocoumarins and pyranocoumarins proceeds via the regiospecific attach- ment of a prenyl side chain ortho to the phenol group of umbelliferone (Matern et al. 1999; Bourgaud et al. 2006). The dimethylallyl group then cyclizes with the phenol group to give either five-membered furan or six-membered pyran heterocycles. B. Liu Á T. Raeth Á T. Beuerle Á L. Beerhues (&) Institut fu ¨r Pharmazeutische Biologie, Mendelssohnstraße 1, 38106 Braunschweig, Germany e-mail: l.beerhues@tu-bs.de B. Liu Key Laboratory of Photosynthesis and Environmental Molecular Physiology, Institute of Botany, The Chinese Academy of Sciences, Nanxincun 20, Haidian District, Beijing 100093, China 123 Plant Mol Biol (2010) 72:17–25 DOI 10.1007/s11103-009-9548-0