RESUMEN
The Arabidopsis thaliana gene encoding CYP71A16 is part of the gene cluster for the biosynthesis and modification of the triterpenoid marneral. Previous investigations of A. thaliana have revealed that CYP71A16 catalyzes marneral oxidation, while it also can accept marnerol as substrate. The aim of the present study was to investigate functional properties of CYP71A16 in vitro. For this purpose, heterologous expression of a N-terminally modified version of CYP71A16 was established in Escherichia coli, which yielded up to 50mgL-1 recombinant enzyme. The enzyme was purified and activity was reconstituted in vitro with different redox partners. A heterologous bacterial redox partner system consisting of the flavodoxin YkuN from Bacillus subtilis and the flavodoxin reductase Fpr from E. coli clearly outperformed the cytochrome P450 reductase ATR2 from A. thaliana in supporting the CYP71A16-mediated hydroxylation of marnerol. Substrate binding experiments with CYP71A16 revealed a dissociation constant KD of 225µM for marnerol. CYP71A16 catalyzed the hydroxylation of marnerol to 23-hydroxymarnerol with a KM of 142µM and a kcat of 3.9min-1. Furthermore, GC/MS analysis revealed an as of yet unidentified overoxidation product of this in vitro reaction. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Bacillus subtilis/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas de Escherichia coli/metabolismo , Ferredoxina-NADP Reductasa/metabolismo , Flavodoxina/metabolismo , Triterpenos/metabolismo , Secuencia de Aminoácidos , Arabidopsis/química , Proteínas de Arabidopsis/genética , Bacillus subtilis/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ferredoxina-NADP Reductasa/genética , Flavodoxina/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Hidroxilación , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.