RESUMEN
The sesquiterpenoid (+)-nootkatone is a highly demanded and highly valued aroma compound naturally found in grapefruit, pummelo or Nootka cypress tree. Extraction of (+)-nootkatone from plant material or its production by chemical synthesis suffers from low yields and the use of environmentally harmful methods, respectively. Lately, major attention has been paid to biotechnological approaches, using cell extracts or whole-cell systems for the production of (+)-nootkatone. In our study, the yeast Pichia pastoris initially was applied as whole-cell biocatalyst for the production of (+)-nootkatone from (+)-valencene, the abundant aroma compound of oranges. Therefore, we generated a strain co-expressing the premnaspirodiene oxygenase of Hyoscyamus muticus (HPO) and the Arabidopsis thaliana cytochrome P450 reductase (CPR) that hydroxylated extracellularly added (+)-valencene. Intracellular production of (+)-valencene by co-expression of valencene synthase from Callitropsis nootkatensis resolved the phase-transfer issues of (+)-valencene. Bi-phasic cultivations of P. pastoris resulted in the production of trans-nootkatol, which was oxidized to (+)-nootkatone by an intrinsic P. pastoris activity. Additional overexpression of a P. pastoris alcohol dehydrogenase and truncated hydroxy-methylglutaryl-CoA reductase (tHmg1p) significantly enhanced the (+)-nootkatone yield to 208mg L(-1) cell culture in bioreactor cultivations. Thus, metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-nootkatone from simple carbon sources.
Asunto(s)
Proteínas de Arabidopsis , Ingeniería Metabólica , Pichia , Sesquiterpenos/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Cupressus/enzimología , Cupressus/genética , Hyoscyamus/enzimología , Hyoscyamus/genética , Pichia/enzimología , Pichia/genética , Sesquiterpenos PolicíclicosRESUMEN
ß-Thujaplicin is a wood monoterpene and tropolone compound with a unique conjugated 7-membered ring. Because of its strong antifungal and antitumor activities, ß-thujaplicin is used in several fields. The biosynthesis pathway of ß-thujaplicin has not yet been elucidated. Using Cupressus lusitanica cell cultures in a radioisotope feeding experiment, our group previously demonstrated that geranyl pyrophosphate (GPP) is the starting material of ß-thujaplicin biosynthesis. The results of our previous terpene synthase assay suggested that terpinolene is the first olefin terpenoid intermediate from GPP to ß-thujaplicin, although there was no experimental evidence of this at that time. In the present study, we fed deuterium-labeled terpinolene to cultured C. lusitanica cells to determine whether terpinolene is an intermediate metabolite of ß-thujaplicin biosynthesis. A gas chromatography-mass spectroscopy analysis of the cell extracts from labeled terpinolene cultures revealed a peak of labeled ß-thujaplicin that was not observed after treatment with non-labeled terpinolene. The identification of labeled ß-thujaplicin was also performed by mass spectrum assignment. The outcome indicated that terpinolene is indeed an intermediate metabolite of ß-thujaplicin biosynthesis. To the best of our knowledge, there has been no prior report that tropolone compounds are biosynthesized via a terpene biosynthesis system, and our results thus suggest the existence of a novel biosynthetic pathway that produces the conjugated 7-membered ring.
Asunto(s)
Cupressus/metabolismo , Monoterpenos/metabolismo , Terpenos/metabolismo , Tropolona/análogos & derivados , Tropolona/metabolismo , Extractos Celulares , Células Cultivadas , Cupressus/enzimología , Monoterpenos Ciclohexánicos , Deuterio/metabolismo , Cromatografía de Gases y Espectrometría de MasasRESUMEN
Pollen is an important trigger of allergic diseases. Recent studies have shown that ragweed pollen NAD(P)H oxidase generates reactive oxygen species (ROS) and plays a prominent role in the pathogenesis of allergies in mouse models. Here, we demonstrated that allergenic pollen grains showed NAD(P)H oxidase activity that differed in intensity and localization according to the plant families. The activity occurred at the surface or in the cytoplasm in pollen of grasses, birch, and ragweed; in subpollen particles released from ragweed pollen; and at the inner surface or in the cytoplasm but not on the outer wall, which was sloughed off after the rupture, of pollen of Japanese cedar and Japanese cypress. The activity was mostly concentrated within insoluble fractions, suggesting that it facilitates the exposure of tissues to ROS generated by this enzyme. The extent of exposure to pollen-generated ROS could differ among the plant families.
Asunto(s)
Alérgenos/inmunología , NADPH Oxidasas/metabolismo , Polen/enzimología , Polen/inmunología , Especies Reactivas de Oxígeno/metabolismo , Animales , Cryptomeria/enzimología , Cryptomeria/inmunología , Cupressus/enzimología , Cupressus/inmunología , Hipersensibilidad/enzimología , Hipersensibilidad/inmunología , Ratones , Nitroazul de Tetrazolio/química , Nitroazul de Tetrazolio/metabolismo , Oxidación-Reducción , Superóxido Dismutasa/metabolismoRESUMEN
Elicitor treatment initiates defense responses in cultured Cupressus lusitanica cells. In order to investigate the defense mechanism with a yeast extract elicitor, we carried out solid-phase microextraction coupled with gas chromatography for monoterpene analysis. Ten hydrocarbon monoterpenes, including high amounts of sabinene and limonene, were detected in the gas phase of the elicitor-treated cell cultures. Six oxidized monoterpenes including beta-thujaplicin were also detected in the ether extract of the cells and the medium. Time-course profiles of volatile monoterpenes showed that one group of hydrocarbon monoterpenes was maximized on the second day after elicitation, while the other group was maximized on the third day. There were no oxidized monoterpenes that are structurally related to sabinene and limonene in the gas phase or cell extracts, suggesting that these compounds are produced exclusively for emission. Other monoterpenes, which are produced during later stages of elicitation, are metabolized into more complex compounds such as oxidized monoterpenes, including beta-thujaplicin. Although terpinolene synthase was the principal monoterpene synthase in these cell cultures, terpinolene was detected only as a minor compound in the gas phase. The time course for terpinolene synthase activity coincided with beta-thujaplicin biosynthesis. Thus, most of the terpinolene is metabolized rapidly to oxidized terpenes such as beta-thujaplicin rather than emitted.