Metabolism of phenylalanine and biosynthesis of styrene in Penicillium camemberti Yves Pagot 1 *, Jean-Marc Belin 2 , Florence Husson 2 and Henry-Eric Spinnler 3 1 UMR de Microbiologie et Ge ´ne ´tique Mole ´culaire, Institut National Agronomique Paris-Grignon, UMR INRA 537, URA CNRS 1925, F-78850 Thiverval-Grignon France 2 Laboratoire de Microbioologie, UMR INRA 1283 Ecole Nationale Supe ´rieure de Biologie Applique ´e a ` la. Nutrition et a ` l’Alimentation, 1, Esplanade Erasme, F-21000 Dijon, France 3 UMR de Ge ´nie et Microbiologie des Proce ´de ´s et Alimentaires, Institut National Agronomique Paris-Grignon, UMR INRA GMPA, F-78850 Thiverval-Grignon, France Received 26 October 2005 and accepted for publication 28 September 2006 The occurrence of styrene in food may be an important aroma defect (celluloid odour), even at very low concentrations (Miltz et al. 1980) causing consumer rejection and is therefore a problem for the food industry. We examined the biosynthetic pathway leading to styrene formation by Penicillium camemberti using labelled compounds. As styrene is strongly hydrophobic and vol- atile, we first had to develop a continuous extraction process. Using resins XAD2 it was reasonable to suspect phenylalanine (Phe) as the precursor. The addition of Phe marked with 13 C on the ring provokes the accumulation of labelled styrene. The enzyme activities involved were also tentatively measured. Styrene appears to be synthesized from phenylalanine by phenylalanine ammonia-lyase activity followed by a decarboxylation catalysed by a cinnamic acid decarboxylase. Keywords : Styrene production, Phenylalanine catabolism, Penicillium camemberti. The occurrence of styrene (vinylbenzene, CAS N8 100-42- 5) in a wide variety of foods has been studied (Maarse & Vischer, 1992 ; Steele et al. 1994). Its concentration is highly variable in foodstuffs, ranging from 0 (milk, tomatoes) to 40 000 ppb (cinnamon). The origin of styrene in foods is not yet clearly documented. It can originate from packaging (Lickly et al. 1995 ; Tawfik & Huyghebaert, 1998), normal metabolism of the raw product or microbial metabolism. It can be formed, for example, during the fermentation of grapes (Steele et al. 1994), by micro-organisms during the ripening or storage of mould-ripened cheeses (Adda et al. 1989; Spinnler et al. 1992) or by decomposition of chemi- cally related food additives by micro-organisms (Sato et al. 1988; Shimada et al. 1992). Several possible precursors of styrene in foods have been proposed : carotenoids ( Johnson et al. 1969, Adda et al. 1989), long chain hydrocarbons, fatty acids and esters (Ducruet, 1980, Taylor & Mottram, 1990), 2-phenyl-ethanol (Maarse & Vischner, 1989, 1990, 1991), aldehydes (Min et al. 1977, Maarse & Vischner, 1991), glucose, phenyl- alanine (Ducruet 1980, Westphal & Cieslik, 1981) and cinnamic acid (Sato et al. 1988), but none has been proven to be the direct precursor. During ripening or storage of mould ripened cheese, a flavour defect characterized by a typical celluloid flavour sometimes appears. This defect originates from the pro- duction of styrene by Penicillium strains. The ability of fungi to perform the bioconversion of cinnamic acid into styrene is well known (Clifford et al. 1969, Shimida et al. 1992, Middelhoven et al. 1995). This reaction is catalyzed by a cinnamic acid decarboxylase (CAD ; Finkel et al. 1962, Zeyen et al. 1995). The presence of phenyl- alanine ammonia-lyase (PAL) in fungi (Takac et al. 1995) able to catalyze the conversion of phenylalanine into cin- namic acid could then explain the production of styrene by microorganisms during cheese ripening. In order to elucidate the metabolic pathway lead- ing to styrene formation, the metabolism of 14 C and 13 C phenylalanine by the fungus Pen. camemberti was studied. Enzyme activities PAL and cinnamate decarboxylase (CDC) were monitored. Material and Methods Chemicals Czapek medium was purchased from Difco laboratories (Detroit, MI, USA). Solvents were obtained from Carlo *For correspondence ; e-mail : pagot@grignon.inra.fr Journal of Dairy Research (2007) 74 180–185. f Proprietors of Journal of Dairy Research 2007 180 doi:10.1017/S0022029906002251 First published online 12 February 2007 Printed in the United Kingdom