Manuela Albrecht

Novel hydroxycarotenoids with improved antioxidative properties produced by gene combination in Escherichia coli.

We have used combinatorial biosynthesis to synthesize novel lipophilic carotenoids that are powerful cellular antioxidants. By co-expressing three different carotenoid desaturases in combination with a carotenoid hydratase, a cyclase, and a hydroxylase on compatible plasmids in Escherichia coli, we synthesized four novel carotenoids not previously detected in biological material or chemically synthesized. Their identification was based on their relative retention times on HPLC, spectroscopic properties, molecular weights, number of hydroxy groups, and 1H-NMR spectra. The carotenoids were designated as 1-HO-3′, 4′-didehydrolycopene, 3, 1′-(HO)2-γ-carotene, 1,1′-(HO)2-3, 4, 3′, 4′-tetradehydrolycopene, and 1, 1′-(HO)2-3, 4-didehydrolycopene. These novel acyclic derivatives differ from structurally related compounds by extension of the conjugated polyene chain as well as additional hydroxy groups at position C-1′. We determined their antioxidative activity in a liposome-membrane model system, which showed that their ability to protect against photooxidation and radical-mediated peroxidation reactions was linked to the length of the conjugated double-bond system and the presence of a single hydroxy group. The protection of membrane degradation was superior to the related 1-HO and 1, 1′-(HO)2 lycopene derivatives, making them interesting pharmaceutical candidates.

The biotechnological potential and design of novel carotenoids by gene combination in Escherichia coli

Carotenoids are antioxidants with considerable pharmaceutical potential. More than 600 carotenoid structures are known but their availability is limited owing to practical difficulties associated with chemical synthesis and isolation from microorganisms or plant tissue. To overcome some of these problems, heterologous expression of carotenoid genes in Escherichia coli can be used for the synthesis of rare derivatives or even of novel carotenoids. Novel and rare carotenoids can be obtained by combining carotenoid genes from different host species in E. coli.

Molecular cloning and functional expression in E. coli of a novel plant enzyme mediating ξ‐carotene desaturation

We have cloned a cDNA from the plant Capsicum annuum which encodes a novel enzyme mediating the dehydrogenation of ξ‐carotene and neurosporene to lycopene when expressed in E. coli cells accumulating ξ‐carotene or neurosporene. This enzyme is unable to dehydrogenate either phytoene or lycopene. The deduced amino acid sequence suggests that this cDNA encodes a polypeptide whose mature size is ca. 59 kDa and which is synthesized as a precursor with a NH2‐terminal extension resembling transit peptides for plastid targeting. Sequence comparison reveals 33–35% similarity with previously cloned plant or cyanobacterial phytoene desaturases. In contrast, only limited sequence similarity is found with a ξ‐carotene desaturase from the cyanobacterium Anabaena.

Light-Stimulated Carotenoid Biosynthesis during Transformation of Maize Etioplasts Is Regulated by Increased Activity of Isopentenyl Pyrophosphate Isomerase

Light-stimulated carotenoid biosynthesis associated with the transformation of etioplasts to chloroplasts was investigated after dark-grown maize (Zea mays) seedlings were transferred into light. These studies focused on the enzymes of the pathway to detect those enzyme activities that were stimulated in the light and thus that were responsible for increased biosynthesis of carotenoids. In preliminary experiments, norflurazon, an inhibitor of phytoene desaturase, was used to prevent phytoene being further metabolized to carotenoids. Light-dependent stimulation of phytoene accumulation indicated that the light-regulated steps are located in the pathway leading to phytoene synthesis. The use of the 14C- labeled precursors mevalonic acid, isopentenyl pyrophosphate, and farnesyl pyrophosphate pointed to increased activity of an enzyme involved in the biosynthetic steps between isopentenyl pyrophosphate and farnesyl pyrophosphate. Determination of the activities of all five enzymes of the pathway involved in the sequence from mevalonic acid to phytoene revealed that the only enzyme activity stimulated by light was isopentenyl pyrophosphate isomerase. Over a 3-h period of illumination, this enzyme activity, like carotenoid biosynthesis, was stimulated 2.8-fold.

Expression of a Ketolase Gene Mediates the Synthesis of Canthaxanthin in Synechococcus Leading to Tolerance Against Photoinhibition, Pigment Degradation and UV-B Sensitivity of Photosynthesis¶

Abstract The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed. Therefore, the cyanobacterium Synechococcus was transformed with a foreign β-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin. This diketo carotenoid is absent in the original strain. Most of the newly formed canthaxanthin was located in the thylakoid membranes. The endogenous β-carotene hydroxylase was unable to interact with the ketolase. Therefore, only traces of astaxanthin were found. The transformant was treated with strong light (500 or 1200 μmol m−2 s−1) and with UV-B radiation. In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 μmol m−2 s−1 and UV-B radiation of 6.8 W m−2. Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 μmol m−2 s−1, which was substantial in the nontransformed control, was prevented. These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation. These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

Physiological analysis of transgenic Arabidopsis thaliana plants expressing one nitrilase isoform in sense or antisense direction

Summary The biosynthesis of IAA in Arabidopsis thaliana can proceed via different pathways, which involve indole-3-acetonitrile (IAN) as an intermediate. The enzyme nitrilase, which converts IAN to the auxin indole-3-acetic acid (IAA), might be a key enzyme in this biosynthetic pathway. To elucidate the role of nitrilase during the development of the plant, we have investigated transgenic Arabidopsis plants, carrying one of the four nitrilase isoforms so far identified in Arabidopsis in either sense or antisense direction. The plants have been analyzed for their phenotype, auxin content, nitrilase transcript and protein amounts, and nitrilase activity. Analysis of free IAA content in seedlings did not reveal any prominent differences between the different lines, whereas the total IAA content was slightly increased in the line overexpressing NIT2 (sNIT2). In two of the antisense lines (aNIT1 and aNIT2) the total auxin content was about 75% lower than in the wild type, while IAN was increased in these two antisense lines by about 2-fold. In sNIT2 plants the IAN content was slightly decreased compared with the wild type. When IAN was added to the medium, a strong increase in IAA content was found in sNIT2 seedlings (ca. 22-fold), which was much lower in wild type and antisense plants. In mature plants levels of free IAA were decreased by about 25% and 50% in the antisense lines aNIT2 and aNIT1, respectively, whereas the IAA content in the other lines did not differ much. sNIT2/aNIT2 plants were further characterized because it was shown by Northern and Western analysis as well as enzyme activity measurements that sNIT2 plants overexpress nitrilase constitutively. The results with these plants have shown that i) both nitrilase 1 and nitrilase 2 accept the same substrates, and ii) overexpression of nitrilase does not lead to a visible phenotype because in vivo IAN might be the limiting factor in these plants. Application of IAN leads to an increase in total nitrilase protein as shown by Western blotting. Using transgenic lines with promoter-GUS fusions for each NIT gene, it was shown that i) NIT1 is highly expressed in seedlings and ii) NIT2 is strongly induced (21-fold) after IAN application.

Development of high-performance liquid chromatographic systems for the separation of radiolabelled carotenes and precursors formed in specific enzymatic reactions

Abstract High-performance liquid chromatographic (HPLC) systems were developed to separate radiolabelled carotenes and precursors formed in the following carotenogenic enzyme reactions: geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase(s), lycopene cyclase and β-carotene hydroxylase. Separations were carried out on reversed- (C 18 ) and normal-phase columns, using mobile phase mixtures of acetonitrile, methanol and hexane with appropriate modifiers. An isocratic mode of elution was employed throughout, although in several instances isocratic steps were necessary to achieve the desired resolution. The methods developed are specific for each enzyme reaction, resolving substrate from its reaction products and any interfering radiolabelled compounds, thus permitting reliable and accurate determination of enzyme activities. The new separation systems will facilitate further studies on the characterization of these proteins.

Accumulation of Colorless Carotenes and Derivatives during Interaction of Bleaching Herbicides with Phytoene Desaturation

Colorless carotenoids were accumulated, analyzed and quantified in heterotrophically- grown Scenedesmus cultures treated with the bleaching herbicide norflurazon. By optical, IR and mass spectroscopy 15-cis as well as all-trans phytofluene, 15-cis phytoenes and traces of its all-trans isomer were identified. Furthermore, a phytoene epoxyde and several hydroxyphy- toenes were assigned. Comparing the concentrations of these phytoene derivatives in hetero-trophic cultures grown in darkness with those in light-grown autotrophic cells, an exchange of phytoene derivatives was observed. Levels of phytoene were much higher in the dark whereas concentrations of hydroxyphytoenes dominated in the light. Other herbicides like fluridone, diflufenican, and difunon accumulate the same colorless carotenoids including phytoene epoxyde and various hydroxyphytoenes. For all herbicides an almost constant hydroxyphytoene to phytoene epoxyde ratio was observed indicating an interrelated formation of both oxygenated phytoene derivatives which can be influenced by light conditions of the cultures.