Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus Eugenio Alcalde a,1 , Humberto R. Medina a , M. Mar Herrador b , Alejandro F. Barrero b , Enrique Cerdá-Olmedo a,⇑ a Departamento de Genética, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain b Departamento de Química Orgánica, Instituto de Biotecnología, Universidad de Granada, Fuente Nueva s/n, E-18071 Granada, Spain article info Article history: Received 1 November 2015 Received in revised form 11 January 2016 Accepted 20 January 2016 Available online xxxx Keywords: Phycomyces blakesleeanus Phycomycetaceae Mucorales Bioconversion Cyclofarnesoids Methylhexanoids b-Carotene Apocarotenoids abstract The oxidative cleavage of b-carotene in the Mucorales produces three fragments of 18, 15, and 7 carbons, respective heads of three families of apocarotenoids: the methylhexanoids, the trisporoids, and the cyclo- farnesoids (named after their 1,6-cyclofarnesane skeleton). The apocarotenoids are easily recognized because they are absent in white mutants unable to produce b-carotene. In cultures of Phycomyces bla- kesleeanus we detected thirty-two apocarotenoids by LC, UV absorbance, and MS. With additional IR and NMR we identified two methylhexanoids and the eight most abundant cyclofarnesoids. Four of them were previously-unknown natural compounds, including 4-dihydrocyclofarnesine S, the most abundant cyclofarnesoid in young cultures. We arranged the apocarotenoids of the Mucorales in a scheme that helps classifying and naming them and suggests possible metabolites and biosynthetic pathways. We propose specific biosynthetic pathways for cyclofarnesoids and methylhexanoids based on structural comparisons, the time course of appearance of individual compounds, and the bioconversion of b-apo- 12-carotenol, an early precursor, to three more oxygenated cyclofarnesoids by the white mutants. Some of the reactions occur spontaneously in the increasingly acidic culture media. Mating increased the contents of methylhexanoids and cyclofarnesoids by ca. threefold in young cultures and ca. twelve- fold in old ones (five days); cyclofarnesine S, the most abundant cyclofarnesoid in old cultures, increased over one hundredfold. We found no differences between the sexes and no activity as sexual pheromones, but we suggest that methylhexanoids and cyclofarnesoids could mediate species-specific physiology and behavior. Ó 2016 Elsevier Ltd. All rights reserved. 1. Introduction The sexual cycle of Phycomyces blakesleeanus Bgff., Blakeslea tris- pora Thaxter, and other fungi of the order Mucorales (Blakeslee, 1904) begins when hyphae of different sex, (+) and (), grow together under appropriate conditions (‘‘mated cultures”). Sex- specific diffusible signals induce nearby hyphal tips of the opposite sex to develop into zygophores, the initial structures of the sexual cycle. This was the first case of a pheromone exchange reported for any organism (Burgeff, 1924). The active molecules are believed to be apocarotenoids, derived from fragments of b-carotene, because they are missing in mutants that cannot synthesize b-carotene (Sutter, 1975). Sexual interaction is accompanied in many strains by an increased accumulation of b-carotene that is exploited by industry (Cerdá-Olmedo, 1989; Ciegler, 1965; Iturriaga et al., 2000). Trispo- ric acid C, a metabolite of b-carotene found in mated cultures of Blakeslea, stimulates carotene production in single cultures (Austin et al., 1970; Caglioti et al., 1966). It was the first of the tris- poroids, a family of C 18 compounds and their C 19 methyl esters (Schimek and Wöstemeyer, 2009; Sutter, 1987; van den Ende, 1979). The discovery in Phycomyces of the cleavage of b-carotene into C 18 , C 15 , and C 7 fragments justified the recognition of two additional families of apocarotenoids: the cyclofarnesoids (with the carbon skeleton of 1,6-cyclofarnesane) and the methylhex- anoids (Polaino et al., 2010). An oxygenase, the product of gene carS, first cleaves b-carotene into C 15 and C 25 fragments (Medina et al., 2011; Tagua et al., 2012), and another one, the product of http://dx.doi.org/10.1016/j.phytochem.2016.01.013 0031-9422/Ó 2016 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. E-mail addresses: eugenalc@gmail.com (E. Alcalde), hurmedina@gmail.com (H.R. Medina), mmar@ugr.es (M.M. Herrador), afbarre@ugr.es (A.F. Barrero), eco@ us.es (E. Cerdá-Olmedo). 1 Present address: School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, UK. Phytochemistry xxx (2016) xxx–xxx Contents lists available at ScienceDirect Phytochemistry journal homepage: www.elsevier.com/locate/phytochem Please cite this article in press as: Alcalde, E., et al. Cyclofarnesoids and methylhexanoids produced from b-carotene in Phycomyces blakesleeanus. Phyto- chemistry (2016), http://dx.doi.org/10.1016/j.phytochem.2016.01.013