Monocyclic Carotenoids Research Articles

Identification of a novel monocyclic carotenoid and prediction of its biosynthetic genes in Algoriphagus sp. oki45

Bacteria belonging to the genus Algoriphagus have been isolated from various sources, such as Antarctic sea ice, seawater, and sediment, and some strains are known to produce orange to red pigments. However, the pigment composition and biosynthetic genes have not been fully elucidated. A new red-pigmented Algoriphagus sp. strain, oki45, was isolated from the surface of seaweed collected from Senaga-Jima Island, Okinawa, Japan. Genome comparison revealed oki45’s average nucleotide identity of less than 95% to its closely related species, Algoriphagus confluentis NBRC 111222 T and Algoriphagus taiwanensis JCM 19755 T. Comprehensive chemical analyses of oki45’s pigments, including 1H and 13C nuclear magnetic resonance and circular dichroism spectroscopy, revealed that the pigments were mixtures of monocyclic carotenoids, (3S)-flexixanthin ((3S)-3,1′-dihydroxy-3′,4′-didehydro-1′,2′-dihydro-β,ψ-caroten-4-one) and (2R,3S)-2-hydroxyflexixanthin ((2R,3S)-2,3,1′-trihydroxy-3′,4′-didehydro-1′,2′-dihydro-β,ψ-caroten-4-one); in particular, the latter compound was new and not previously reported. Both monocyclic carotenoids were also found in A. confluentis NBRC 111222 T and A. taiwanensis JCM 19755 T. Further genome comparisons of carotenoid biosynthetic genes revealed the presence of eight genes (crtE, crtB, crtI, cruF, crtD, crtYcd, crtW, and crtZ) for flexixanthin biosynthesis. In addition, a crtG homolog gene encoding 2,2ʹ-β-hydroxylase was found in the genome of the strains oki45, A. confluentis NBRC 111222 T, and A. taiwanensis JCM 19755 T, suggesting that the gene is involved in 2-hydroxyflexixanthin synthesis via 2-hydroxylation of flexixanthin. These findings expand our knowledge of monocyclic carotenoid biosynthesis in Algoriphagus bacteria.Key points• Algoriphagus sp. strain oki45 was isolated from seaweed collected in Okinawa, Japan.• A novel monocyclic carotenoid 2-hydroxyflexixanthin was identified from strain oki45.• Nine genes for 2-hydroxyflexixanthin biosynthesis were found in strain oki45 genome.Graphical

Optimization of Rhodococcus erythropolis JCM3201T Nutrient Media to Improve Biomass, Lipid, and Carotenoid Yield Using Response Surface Methodology.

The oleaginous bacterium Rhodococcus erythropolis JCM3201T offers various unique enzyme capabilities, and it is a potential producer of industrially relevant compounds, such as triacylglycerol and carotenoids. To develop this strain into an efficient production platform, the characterization of the strain's nutritional requirement is necessary. In this work, we investigate its substrate adaptability. Therefore, the strain was cultivated using nine nitrogen and eight carbon sources at a carbon (16 g L-1) and nitrogen (0.16 g L-1) weight ratio of 100:1. The highest biomass accumulation (3.1 ± 0.14 g L-1) was achieved using glucose and ammonium acetate. The highest lipid yield (156.7 ± 23.0 mg g-1DCW) was achieved using glucose and yeast extract after 192 h. In order to enhance the dependent variables: biomass, lipid and carotenoid accumulation after 192 h, for the first time, a central composite design was employed to determine optimal nitrogen and carbon concentrations. Nine different concentrations were tested. The center point was tested in five biological replicates, while all other concentrations were tested in duplicates. While the highest biomass (8.00 ± 0.27 g L-1) was reached at C:N of 18.87 (11 g L-1 carbon, 0.583 g L-1 nitrogen), the highest lipid yield (100.5 ± 4.3 mg g-1DCW) was determined using a medium with 11 g L-1 of carbon and only 0.017 g L-1 of nitrogen. The highest carotenoid yield (0.021 ± 0.001 Abs454nm mg-1DCW) was achieved at a C:N of 12 (6 g L-1 carbon, 0.5 g L-1 nitrogen). The presented results provide new insights into the physiology of R. erythropolis under variable nutritional states, enabling the selection of an optimized media composition for the production of valuable oleochemicals or pigments, such as rare odd-chain fatty acids and monocyclic carotenoids.

GATA transcription factor WC2 regulates the biosynthesis of astaxanthin in yeast Xanthophyllomyces dendrorhous.

Astaxanthin is a type of carotenoid widely used as powerful antioxidant and colourant in aquaculture and the poultry industry. Production of astaxanthin by yeast Xanthophyllomyces dendrorhous has attracted increasing attention due to high cell density and low requirements of water and land compared to photoautotrophic algae. Currently, the regulatory mechanisms of astaxanthin synthesis in X. dendrorhous remain obscure. In this study, we obtained a yellow X. dendrorhous mutant by Atmospheric and Room Temperature Plasma (ARTP) mutagenesis and sequenced its genome. We then identified a putative GATA transcription factor, white collar 2 (XdWC2), from the comparative genome data and verified that disruption of the XdWC2 gene resulted in a similar carotenoid profile to that of the ARTP mutant. Furthermore, transcriptomic analysis and yeast one‐hybrid (Y1H) assay showed that XdWC2 regulated the expression of phytoene desaturase gene CrtI and astaxanthin synthase gene CrtS. The yeast two‐hybrid (Y2H) assay demonstrated that XdWC2 interacted with white collar 1 (XdWC1) forming a heterodimer WC complex (WCC) to regulate the expression of CrtI and CrtS. Increase of the transcriptional levels of XdWC2 or CrtS in the wild‐type strain did not largely modify the carotenoid profile, indicating translational and/or post‐translational regulations involved in the biosynthesis of astaxanthin. Overexpression of CrtI in both the wild‐type strain and the XdWC2‐disrupted strain apparently improved the production of monocyclic carotenoid 3‐hydroxy‐3′, 4′‐didehydro‐β, ψ‐carotene‐4‐one (HDCO) rather than β‐carotene and astaxanthin. The regulation of carotenoid biosynthesis by XdWC2 presented here provides the foundation for further understanding the global regulation of astaxanthin biosynthesis and guides the construction of astaxanthin over‐producing strains.

Separation of the Glycosylated Carotenoid Myxoxanthophyll from Synechocystis Salina by HPCCC and Evaluation of Its Antioxidant, Tyrosinase Inhibitory and Immune-Stimulating Properties

Global demand for natural pigments has increased in the past few years. Myxoxanthophyll, a glycosylated monocyclic carotenoid, is a pigment that occurs naturally in cyanobacteria but no scalable isolation process has been developed to obtain it from its natural source to date. In this study, myxoxanthophyll was isolated from unicellular cyanobacterium Synechocystis salina (S. salina) using high-performance countercurrent chromatography (HPCCC), where the lower phase of the biphasic solvent system composed of n-heptane–ethanol–water (2:4:4, v/v/v) was used as a mobile phase, whereas its upper phase was the stationary phase. For the HPCCC isolation, a multi-injection method was developed, and four consecutive sample injections (70 mg each) were performed, obtaining, in total, 20 mg of myxoxanthophyll, which was finally purified with high-performance liquid chromatography (HPLC). Overall, a final myxoxanthophyll yield of 15 mg (98% purity) was obtained. The target pigment showed a weak antioxidant and tyrosinase inhibitory effect, and exhibited immune-stimulating properties by activating human granulocytes. The results presented here form a basis for the large-scale production of myxoxanthophyll, and show the potential benefits of this pigment for human health.

In vitro study of the carotenoid-cleavage enzyme from Staphylococcus pasteuri TS-82 revealed substrate specificities and generation of norisoprenoid flavors.

Reactions of a crude enzyme extracted from S. pasteuri TS-82 to cleave carbon-carbon bonds in bicyclic and monocyclic carotenoid substrates were investigated. Dependencies of enzyme activities on processing temperature and pH were investigated and non-volatile and volatile breakdown products were characterized. The crude enzyme showed a maximum activity with zeaxanthin, followed in decreasing order by β-carotene, canthaxanthin, astaxanthin, and β-apo-8'-carotenal. The optimum pH value of the enzyme was 3.0 for both bicyclic and monocyclic substrates, whereas the optimum temperature of the enzyme was substrate specific at 60°C for C40 carotenoids and 50°C for β-apo-8'-carotenal. Liquid Chromatography-Mass Spectra (LC-MS) and Gas Chromatography- Mass Spectra (GC-MS) indicated that the crude enzyme was able to catalyze substrates with cleavage at 9-10 and 9'-10' double bonds with C13 norisoprenoids being the main volatile reaction products in each case. Astaxanthin is a major source for α,β-dihydro-β-ionone.

Structure of a novel monocyclic carotenoid, 3″-hydroxy-2′-isopentenylsaproxanthin ((3R,2′S)-2′-(3-hydroxy-3-methylbutyl)-3′, 4′-didehydro-1′, 2′-dihydro-β, ψ-carotene-3, 1′-diol), from a flavobacterium Gillisia limnaea strain DSM 15749

Abstract Gillisia limnaea strain DSM 15749 (=R-8282 T , =LMG21470,=CIP108418) is a Gram-negative, strictly aerobic, psychrophilic, and non-photosynthetic bacterium that belongs to the family Flavobacteriaceae , which was isolated from a microbial mat in Lake Fryxell, Antarctica ( Van Trappen et al., 2004 ). The bacterium shows yellow colored colony, but the carotenoid composition has never been determined yet. In this study, we elucidated the structure of carotenoids produced by the strain DSM 15749. G. limnaea strain DSM 15749 synthesized a novel monocyclic-type carotenoid, 3″-hydroxy-2′-isopentenylsaproxanthin ((3 R , 2′ S )-2′-(3-hydroxy-3-methylbutyl)-3′, 4′-didehydro-1′, 2′-dihydro-β, ψ-carotene-3, 1′-diol), as well as (3 R , 3′ R )-zeaxanthin. Furthermore, another monocyclic carotenoid, 2′-isopentenylsaproxanthin, was also detected in the strain DSM 15749. 3″-Hydroxy-2′-isopentenylsaproxanthin is “Chimera”-like unique structures, in that one end group forms the same structure of zeaxanthin, and the other end group is the same as bacterioruberin which is produced by several extremophilic bacteria and archaea. This structure involved the hydroxy derivative of 2′-isopentenylsaproxanthin. The present study is the first report about the carotenoids produced by the genus Gillisia , of the family Flavobacteriaceae .

Identification of a novel carotenoid, 2'-isopentenylsaproxanthin, by Jejuia pallidilutea strain 11shimoA1 and its increased production under alkaline condition.

Carotenoids are a class of naturally occurring pigment, carrying out important biological functions in photosynthesis and involved in environmental responses including nutrition in organisms. Saproxanthin and myxol, which have monocyclic carotenoids with a γ-carotene skeleton, have been reported to show a stronger antioxidant activity than those with β-carotene and zeaxanthin. In this research, a yellow-orange bacterium of strain 11shimoA1 (JCM19538) was isolated from a seaweed collected at Nabeta Bay (Shizuoka, Japan). The 16S rRNA gene sequence of strain 11shimoA1 revealed more than 99.99 % similarity with those of Jejuia pallidilutea strains in the family Flavobacteriaceae. Strain 11shimoA1 synthesized two types of carotenoids. One of them was (3R, 3'R)-zeaxanthin with dicyclic structure and another was identified as (3R, 2'S)-2'-isopentenylsaproxanthin, a novel monocyclic carotenoid with pentenyl residue at C-2' position of saproxanthin, using FAB-MS, (1)H NMR, and CD analyses. Culturing strain 11shimoA1 in an alkaline medium at pH 9.2 resulted in a markedly increased in production of 2'-isopentenylsaproxanthin per dry cell weight, but a decreased in zeaxanthin production as compared to their respective production levels in medium with pH 7.0. These carotenoids are likely to play some roles in the adaptation of the bacterium to the environmental conditions.

Analysis and Identification of Astaxanthin and its Carotenoid Precursors from Xanthophyllomyces dendrorhous by High-Performance Liquid Chromatography

This study presents an HPLC method for simultaneous analysis of astaxanthin and its carotenoid precursors from Xanthophyllomyces dendrorhous. The HPLC method is accomplished by employing a C18 column and the mobile phase methanol/water/acetonitrile/ dichloromethane (70:4:13:13, v/v/v/v). Astaxanthin is quantified by detection at 480 nm. The carotenoid precursors are identified by LC-APCI-MS and UV-vis absorption spectra. Peaks showed in the HPLC chromatogram are identified as carotenoids in the monocyclic biosynthetic pathway or their derivatives. In the monocyclic carotenoid pathway, 3,3'-dihydroxy-beta,psi-carotene-4,4'-dione (DCD) is produced through gamma-carotene and torulene.

Characterization of the rice carotenoid cleavage dioxygenase 1 reveals a novel route for geranial biosynthesis

Carotenoid cleavage products--apocarotenoids--include biologically active compounds, such as hormones, pigments and volatiles. Their biosynthesis is initiated by the oxidative cleavage of C-C double bonds in carotenoid backbones, leading to aldehydes and/or ketones. This step is catalyzed by carotenoid oxygenases, which constitute an ubiquitous enzyme family, including the group of plant carotenoid cleavage dioxygenases 1 (CCD1s), which mediates the formation of volatile C(13) ketones, such as beta-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic and acyclic carotenoids. Recently, it was reported that plant CCD1s also act on the C5-C6/C5'-C6' double bonds of acyclic carotenes, leading to the volatile C(8) ketone 6-methyl-5-hepten-2-one. Using in vitro and in vivo assays, we show here that rice CCD1 converts lycopene into the three different volatiles, pseudoionone, 6-methyl-5-hepten-2-one, and geranial (C(10)), suggesting that the C7-C8/C7'-C8' double bonds of acyclic carotenoid ends constitute a novel cleavage site for the CCD1 plant subfamily. The results were confirmed by HPLC, LC-MS and GC-MS analyses, and further substantiated by in vitro incubations with the monocyclic carotenoid 3-OH-gamma-carotene and with linear synthetic substrates. Bicyclic carotenoids were cleaved, as reported for other plant CCD1s, at the C9-C10 and C9'-C10' double bonds. Our study reveals a novel source for the widely occurring plant volatile geranial, which is the cleavage of noncyclic ends of carotenoids.

Conversion of the lycopene monocyclase of Myxococcus xanthus into a bicyclase

Depending on the cyclized hydrocarbon backbone ends, carotenoids can be acyclic, monocyclic, or bicyclic. Lycopene cyclases are the enzymes responsible for catalyzing the formation of cyclic carotenoids from acyclic lycopene. Myxococcus xanthus is a bacterium that accumulates monocyclic carotenoids such as a glycoside ester of myxobacton. We show here that this bacterium possesses a cyclase belonging to the group of the heterodimeric cyclases CrtYc and CrtYd. These two individual proteins are encoded by crtYc and crtYd, which are located in the carotenogenic carA operon of the carB-carA gene cluster, and the presence of both is essential for the cyclization of lycopene. CrtYc and CrtYd from M. xanthus form a heterodimeric cyclase with beta-monocyclic activity, which converts lycopene into monocyclic gamma-carotene, but not into bicyclic beta-carotene like most beta-cyclases. This is an unusual case where two different proteins constitute a lycopene cyclase enzyme with monocyclic activity. We were able to convert this lycopene monocyclase into a lycopene bicyclase enzyme producing beta-carotene, by fusing both proteins with an extra transmembrane domain. The chimeric protein appears to allow a proper membranal disposition of both CrtYc and CrtYd, to perform two cyclization reactions, while a hybrid without the extra transmembrane helix performs only one cyclization.

Rare carotenoids, (3R)-saproxanthin and (3R,2′S)-myxol, isolated from novel marine bacteria (Flavobacteriaceae) and their antioxidative activities

We isolated three orange or yellow pigment-producing marine bacteria, strains 04OKA-13-27 (MBIC08261), 04OKA-17-12 (MBIC08260), and YM6-073 (MBIC06409), off the coast of Okinawa Prefecture in Japan. These strains were classified as novel species of the family Flavobacteriaceae based on their 16S rRNA gene sequence. They were cultured, and the major carotenoids produced were purified by chromatographic methods. Their structures were determined by spectral data to be (3R)-saproxanthin (strain 04OKA-13-27), (3R,2'S)-myxol (strain YM6-073), and (3R,3'R)-zeaxanthin (strains YM6-073 and 04OKA-17-12). Saproxanthin and myxol, which are monocyclic carotenoids rarely found in nature, demonstrated significant antioxidative activities against lipid peroxidation in the rat brain homogenate model and a neuro-protective effect from L-glutamate toxicity.

Metabolic engineering for synthesis of aryl carotenoids in Rhodococcus

Rhodococcus erythropolis naturally synthesizes monocyclic carotenoids: 4-keto-gamma-carotene and gamma-carotene. The genes and the pathway for carotenoid synthesis in R. erythropolis were previously described. We heterologously expressed a beta-carotene desaturase gene (crtU) from Brevibacterium in Rhodococcus to produce aryl carotenoids such as chlorobactene. Expression of the crtU downstream of a chloramphenicol resistance gene on pRhBR171 vector showed higher activity than expression downstream of a native 1-deoxyxylulose-5-phosphate synthase gene (dxs) on pDA71 vector. Expression of the crtU in the beta-carotene ketolase (crtO) knockout Rhodococcus host produced higher purity chlorobactene than expression in the wild-type Rhodococcus host. Growth of the engineered Rhodococcus strain in eight different media showed that nutrient broth yeast extract medium supplemented with fructose gave the highest total yield of chlorobactene. This medium was used for growing the engineered Rhodococcus strain in a 10-l fermentor, and approximately 18 mg of chlorobactene was produced as the almost exclusive carotenoid by fermentation.

Novel β-carotene ketolases from non-photosynthetic bacteria for canthaxanthin synthesis

We reported previously that the Rhodococcus erythropolis strain AN12 synthesizes the monocyclic carotenoids 4-keto gamma-carotene and gamma-carotene. We also identified a novel lycopene beta-monocyclase in this strain. Here we report the identification of the rest of the carotenoid synthesis genes in AN12. Two of these showed apparent homology to putative phytoene dehydrogenases. Analysis of Rhodococcus knockout mutants suggested that one of them ( crtI) encodes a phytoene dehydrogenase, whereas the other ( crtO) encodes a beta-carotene ketolase. Expression of the beta-carotene ketolase gene in an Escherichia coli strain which accumulates beta-carotene resulted in the production of canthaxanthin. In vitro assays using a crude extract of the E. coli strain expressing the crtO gene confirmed its ketolase activity. A crtO homologue (DR0093) from Deinococcus radiodurans R1 was also shown to encode a beta-carotene ketolase, despite its sequence homology to phytoene dehydrogenases. The Rhodococcus and Deinococcus CrtO ketolases both catalyze the symmetric addition of two keto groups to beta-carotene to produce canthaxanthin. Even though this activity is similar to the CrtW-type of ketolase activity, the CrtO ketolases show no significant sequence homology to CrtW-type ketolases. The presence of six conserved regions may be a signature for the CrtO-type of beta-carotene ketolases.

1-Hydroxy monocyclic carotenoid 3,4-dehydrogenase from a marine bacterium that produces myxol

A crtD (1-HO carotenoid 3,4-dehydrogenase gene) homolog from marine bacterium strain P99-3 included in the gene cluster for the biosynthesis of myxol (3 ′,4 ′-didehydro-1 ′,2 ′-dihydro-β,ψ-carotene-3,1 ′,2 ′-triol) was functionally identified. The P99-3 CrtD was phylogenetically distant from the other CrtDs. A catalytic feature was its high activity for the monocyclic carotenoid conversion: 1 ′-HO-torulene (3 ′,4 ′-didehydro-1 ′,2 ′-dihydro-β,ψ-caroten-1 ′-ol) was prominently formed from 1 ′-HO-γ-carotene (1 ′,2 ′-dihydro-β,ψ-caroten-1 ′-ol) in Escherichia coli with P99-3 CrtD, indicating that this enzyme has been highly adapted to myxol biosynthesis. This unique type of crtD is a valuable tool for obtaining 1 ′-HO-3 ′,4 ′-didehydro monocyclic carotenoids in a heterologous carotenoid production system.

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli.

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28 degrees C showed better production of torulene than 37 degrees C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2xYT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and beta-carotene, in E. coli. In contrast, glycerol-containing LB, 2xYT, TB, and MR media enhanced torulene production. Overexpression of dxs, dxr, idi and/or ispA, individually and combinatorially, enhanced torulene production up to 3.1-3.3 fold. High torulene production was observed in a high dissolved oxygen level bioreactor in TB and MR media containing glycerol. Lycopene was efficiently converted into torulene during aerobic cultures, indicating that the engineered torulene synthesis pathway is well coordinated, and maintains the functionality and integrity of the carotenogenic enzyme complex.

Metabolic engineering of the astaxanthin-biosynthetic pathway of Xanthophyllomyces dendrorhous.

This review describes the different approaches that have been used to manipulate and improve carotenoid production in Xanthophyllomyces dendrorhous. The red yeast X. dendrorhous (formerly known as Phaffia rhodozyma) is one of the microbiological production systems for natural astaxanthin. Astaxanthin is applied in food and feed industry and can be used as a nutraceutical because of its strong antioxidant properties. However, the production levels of astaxanthin in wild-type isolates are rather low. To increase the astaxanthin content in X. dendrorhous, cultivation protocols have been optimized and astaxanthin-hyperproducing mutants have been obtained by screening of classically mutagenized X. dendrorhous strains. The knowledge about the regulation of carotenogenesis in X. dendrorhous is still limited in comparison to that in other carotenogenic fungi. The X. dendrorhous carotenogenic genes have been cloned and a X. dendrorhous transformation system has been developed. These tools allowed the directed genetic modification of the astaxanthin pathway in X. dendrorhous. The crtYB gene, encoding the bifunctional enzyme phytoene synthase/lycopene cyclase, was inactivated by insertion of a vector by single and double cross-over events, indicating that it is possible to generate specific carotenoid-biosynthetic mutants. Additionally, overexpression of crtYB resulted in the accumulation of beta-carotene and echinone, which indicates that the oxygenation reactions are rate-limiting in these recombinant strains. Furthermore, overexpression of the phytoene desaturase-encoding gene (crtI) showed an increase in monocyclic carotenoids such as torulene and HDCO (3-hydroxy-3',4'-didehydro-beta,-psi-carotene-4-one) and a decrease in bicyclic carotenoids such as echinone, beta-carotene and astaxanthin.

Chromatographic performance on a C 30-bonded stationary phase of monohydroxycarotenoids with variable chain length or degree of desaturation and of lycopene isomers synthesized by various carotene desaturases

Selectivity towards geometric isomers is a superior feature of a C 30 polymeric stationary phase. Therefore, lycopene isomers synthesized in Escherichia coli transformants by catalysis of divers carotene desaturases were separated on this stationary phase. Due to their spectral characteristics and by co-chromatography with nuclear magnetic resonance-characterized carotene standards, some of them could be identified. Most of the lycopene isomers were cyclized by lycopene cyclase yielding mainly 9 Z, 13 Z and all- E β-carotene. In contrast, 7,9,7′,9′ Z prolycopene is accumulating since it cannot be converted by this enzyme. Finally several acyclic hydroxycarotenoids with a chain of 30, 40 and 45 carbon atoms differing in the length of the polyene chain from 9 to 13 were separated on the C 30 stationary phase. Longer retention times were observed when the length of the molecule increased and also when the conjugated double bond system was extended. Corresponding monocyclic carotenoids were less retained on the C 30 stationary phase and derivatives with an ϵ-ionone end group eluted earlier than with a β-end group.

Monocyclic carotenoid biosynthetic pathway in the yeast Phaffia rhodozyma ( Xanthophyllomyces dendrorhous)

The biosynthetic pathway of monocyclic carotenoids in the yeast Phaffia rhodozyma was studied by identifying carotenoids, applying inhibitors of carotenoid synthesis, and analyzing the carotenoids in carotenogenic mutants. Two carotenoids, torulene and 3,3′-dihydroxy-β,φ-carotene-4,4′-dione (DCD), were identified from the yeast. Piperonyl butoxide inhibited dehydrogenation of carotenes and caused accumulation of neurosporene, lycopene, γ-carotene, and β-zeacarotene. Yellow mutants of P. rhodozyma produced mainly β-carotene, which is a dicyclic carotene produced from neurosporene through lycopene. The yellow mutants grown with 0.01% triethylamine (TEA) or 2 mM 2-methylimidazole (MI) produced significantly increased amounts of torulene, which is a monocyclic carotene produced from neurosporene through β-zeacarotene. When red strains of P. rhodozyma were grown with TEA or MI, they produced increased amounts of β-zeacarotene, torulene, and 3-hydroxy-3′,4′-didehydro-β,φ-carotene-4-one (HDCO) (all monocyclic). DCD, accumulated especially in old cultures, was also increased in MI or TEA cultures coincident with the increases in HDCO. We propose the following monocyclic carotenoid pathway in P. rhodozyma: neurosporene→β-zeacarotene→γ-carotene→torulene→HDCO→DCD.

Structural investigation of nonpolar sulfur cross-linked macromolecules in petroleum

A novel hexane-soluble nonpolar macromolecular fraction (NPMF) has been found to occur in substantial amounts (up to 32%) in sulfur-rich crude oils and a rock extract. It is highly aliphatic and has a molecular weight culminating at several thousand mass units, as proven by spectroscopic and molecular weight studies. C-S bond hydrogenolysis of NPMF with Raney nickel as a catalyst yields high proportions of aliphatic hydrocarbons in which long linear, acyclic polyisoprenoid and carotenoid chains usually predominate (except in one case) over polycyclic structures, such as steroids and hopanoids. Hence, NPMF consists mainly of macromolecules composed of low molecular weight hydrocarbon subunits cross-linked with sulfide bridges. Use of deuterated Raney nickel indicated in one case (Rozel Point oil) that the long chains and some hopanoids are multiattached to the macromolecular network, whereas other structural subunits, such as steroids or gammacerane, are essentially monoattached. Detailed structural determinations of the hydrocarbon “building blocks” of NPMF give information on their origin and the mode of formation of these macromolecules in the subsurface. Indeed, most of the building blocks can be related to algal (e.g., long linear chains, steroids, β-carotene, and related carotenoids) or bacterial (e.g., acyclic and monocyclic carotenoids, long-chain acyclic isoprenoids) precursors which essentially exist in living organisms as monounsaturated or polyunsaturated species or are easily transformed into such species by diagenetic processes (e.g., steroids). It appears that these alkenes or polyenes become selectively trapped into a macromolecular network by reaction with inorganic sulfur species produced by bacteria in a kind of natural, low-temperature, vulcanization process. This process could start at early diagenesis already in the water column or eventually continue in the bottom sediment. Although its exact nature is yet unknown, it seems likely that the cross-linking reaction can be initiated by the cleavage of sulfur species in a radical type mechanism. The alkanes formed upon desulfurization of NPMF usually represent much higher amounts than the free alkanes of the samples and show a dramatically different composition. They may deliver very useful, complementary information in studies related to source and palaeoenvironment.

Carotenoids from the aerobic photosynthetic bacterium, Erythrobacter longus: ?-Carotene and its hydroxyl derivatives

About 20 different carotenoids were found in a strictly aerobic photosynthetic bacterium, Erythrobacter longus. All the carotenoids except the highly polar ones were identified as C40-skeletal carotenoids, which could be devided into three groups: (1) bicyclic carotenoids: β-carotene and its hydroxyl derivatives; β-cryptoxanthin, zeaxanthin, caloxanthin and nostoxanthin, (2) monocyclic carotenoids: rubixanthin, bacteriorubixanthin and bacteriorubixanthinal, which was a unique cross-conjugated carotenal, and (3) acyclic carotenoids: anhydrorhodovibrin and spirilloxanthin. Bacteriorubixanthinal and zeaxanthin were the major components. (3R)-3-Hydroxy-β-ionone has rarely been found in carotenoids of purple photosynthetic bacteria, while the acyclic carotenoids have been found exclusively in photosynthetic bacteria. Thus, this bacterium is interesting in its composition of carotenoids.