Biochi et Biophysica A~ta ELSEVIER Biochimica ct Bi;~phy~ica A~.ta 12gt~~ltJ~+~) 20~ 2<Jg Carotenoid biosynthesis in wild type and mutant strains of Mucor circinelloides Paul D. Fraser a Maria Jose Ruiz-Hidalgo b Maria Angeles Lopez-Matas b. Maria Isabel Alvarez ~, Arturo P. Eslava ~', Peter M. Bramley "'" Dirision of Biochemislr3.'. School o1" Bioh~gwul Scietwes. Royal Itolhmav. Uni~ er~itv of London. Egham. Surrey TW20 OEX. UK I, Area de Gen~tica. Departamemo de Microhiologia y Gen~t~ca. Fdificio Departamentol. I +m+ ersidad de Salamanca. E.37007. Salalnaltt'd. Spa#l Received 6 July 1995; revised 17 October t995: accepted 15 No,~ember 1995 Abstract Carotenoid biosynthesis in wild typ~ Mucorcircinelloides has been investigated and the biochemical characteri.sation of the MS I and MS9 mutant strains, impaired in carotenoid formation, carded out. In liquid cultures, all strains produced c~rotenoids (mainly ~camtene. but also ~'-caxotene. lycopene and ~-carotene) at the onset of stationary phase of growth. Carotenogenesis was light dependent. !, liquid cultures carotenoid formation in wild type was affected by diphcnylamine, which prevented desaturation, nicotine, resulting in reduced carotenoid levels, but CPTA caused an increase in the total carotenoid content but a reduced /3-carotene level, w~th ihe accumulation of lycopene and 7-carotene. The mutant strains MSI and MS9 contained only 5.0 and 11.5% of wild type carotenoid levels, respectively. Cell extracts of light-grown mycelia, incubated with 3(R)-[2-t~C] mevalonic acid. produced ,~-carotene. but incorporations into caxotenoids were substantially reduced in the cell extracts of MS i and MS9. A~aly,~is of prenyl diphosphate intermediate,~ indicated that. compared to wild type. geranylgeranyl diphosphate accumulated in MSI. MS9 extracts produced a larger amount of prenyl phosphates and a more even distribution of radioactivity from mevalopic acid into farnesyl and gera, bylgeranyl diphosphates. Squalene and long chain prenyl phosphates were formed by the cell extracts of all strains. It is proposed that the MSI strain posses~s a mutation in a gene responsible for phytoene formation, whilst a regulatory mutation, affecting prenyl transfcra.~ activities, has occurred in Msg. Keywords: Carotenoid: lsoprenoid: Mutant: Mucar circinelloidex 1. Introduction Carotenoids, an abundant group of pigments found dis- tributed throughout nature, are isoprenoids and therefore related to other terpenoids such as sterols, gibberellins and dolichols. All these compounds are derived from meval- onic acid (MVA), the first specific terpenoid precursor. which is converted into the basic isoprene unit. isopentenyl diphosphate, IDP. Conches-:: ttorts between IDP and atlylic diphosphates give rise to farnesyl diphosphate, (F-'DE C ~) and geranylgeranyl diphosphate (GGDP, C:0). Dimerisa- Abbreviations: AMO 1018. 2'-isopropyl-4'-(triethylammonium chlo- ride)-5'-methylphenyl piperidine-l-carboxylate, CPTA. 2-(4+chlo- rphenylthio)-trielhylammooium chloride: DPA, diphenylamine: FDP. rar- ncsyl diphosphate; GGDP. geranylgeranyl diphosphate. IDP, isopentenyl diphosphate; MVA, mevalonic acid " Corresponding author. Fax: +44 1784 434326: E-mail: p.bramley@vms.rhbnc.ac.uk. 0304-4165/96/$15.00 ~ 1996 Elsevier Science B.V. All righls reserved SSDI 0304-4165(95 )001 69-7 tion of FDP yields squalene, the first committed step in steroi biosynlhesis, whilst the head to head condensation of two GGDP molecules forming phytoene (C m) is the firr,t unique step in carotenoid biosynthesis. In most carotenogenic organisms phytoene is desaturated to ly- copene via phytofluene, ~'-carotene and neurosporene. Typ- ically+ iycopcne undergoes cyclisation to form fl-carotene via monocyclic ?-carotene [ i]. ~-Carotene is tht r)rincipal carotene found in fungi and many non-photosynthetic prokaryotes [2], although it is oxidised to xanthophylls in some fungi, e.g. Gibberella [3]. A number of fungal mutants affected in carotenoid biosynthesis have been isolated, with those of Phycomyces blake~leeanus being the most widely studied [4]. The Phycomyces mutants typically accumulate phytoene e~" ly- copene, or have regulatory mutations which cause either the over-accumulation or negSgible production of carotenoids [4]. However, very. few fungal mutants are defective in phytoene formation.