Conversion of the lycopene monocyclase of <Emphasis Type="Italic">Myxococcus xanthus</Emphasis> 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.     

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-gamma-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.     

A single five-step desaturase is involved in the carotenoid biosynthesis pathway to beta-carotene and torulene in Neurospora crassa

Phytoene desaturase Al-1 from Neurospora crassa was expressed in Escherichia coli and an active enzyme was isolated which catalyzed the stepwise introduction of up to five double bonds into the substrate phytoene. The major reaction products were 3, 4-didehydrolycopene and lycopene. Several of the desaturation intermediates, zeta-carotene, neurosporene, and lycopene, were also accepted as a substrate by Al-1. In contrast to the structurally related bacterial enzymes, the cofactor involved in the dehydrogenation reaction was NAD for Al-1. In situ competition with a neurosporene- and lycopene-converting hydratase and cyclase indicated that these enzymes can divert intermediates of the desaturation sequence. Based on the in vitro and in vivo results, the organization of the phytoene desaturase from N. crassa was proposed as an assembly of identical protein units which are responsible for the multistep reaction. However, the spatial arrangement should be loose enough to allow an exchange of individual intermediates in both directions in and out of this complex. Since gamma-carotene is not accepted as a substrate by Al-1, the formation of torulene must proceed exclusively by the cyclization of 3,4-didehydrolycopene.     

Sll0254 (CrtL(diox)) is a bifunctional lycopene cyclase/dioxygenase in cyanobacteria producing myxoxanthophyll

Upon depletion of Sll0254 in Synechocystis sp. strain PCC 6803, cyclized carotenoids were replaced by linear, relatively hydrophilic carotenoids, and the amount of the two photosystems decreased greatly. Full segregants of the sll0254 deletion in Synechocystis were not obtained, implying that this gene is essential for survival, most likely to allow normal cell division. The N-terminal half of Sll0254 has limited similarity to the family of lycopene cyclases, has an additional dehydrogenase motif near the N terminus, and is followed by a Rieske 2Fe-2S center sequence signature. To test whether Sll0254 serves as a lycopene cyclase in Synechocystis, the corresponding gene was expressed in Escherichia coli strains that can produce lycopene or neurosporene. In the presence of Sll0254 these linear carotenoids were converted into cyclized, relatively hydrophilic pigments, with masses consistent with the introduction of two hydroxyl groups and with spectra indicative of only small changes in the number of conjugated double bonds. This suggests that Sll0254 catalyzes formation of oxygenated, cyclized carotenoids. We interpret the appearance of the hydroxyl groups in the carotenoids to be due to dioxygenase activity involving the Rieske 2Fe-2S center and the additional dehydrogenase domain. This dioxygenase activity is required in the myxoxanthophyll biosynthesis pathway, after or concomitant with cyclization on the other end of the molecule. We interpret Sll0254 to be a dual-function enzyme with both lycopene cyclase and dioxygenase activity and have named it CrtL(diox).     

Carotenoids in a Corynebacterineae, Gordonia terrae AIST-1: carotenoid glucosyl mycoloyl esters

We isolated a strain of Corynebacterineae from surface seawater from the Inland Sea of Japan. This strain, AIST-1, was determined to be a strain of Gordonia terrae based on its 16S rRNA gene sequence. The colony was red-colored, and the pigments were identified to be carotenoid derivatives. The structures of two major carotenoids were (2'S)-deoxymyxol 1'-glucoside, a dihydroxyl derivative of gamma-carotene with 12 conjugated double bonds, and (2'S)-4-ketodeoxymyxol 1'-glucoside. Their glucosyl acyl esters and mycoloyl esters were also identified. While these carotenoid moieties have been found in only a few other bacteria, the carotenoid mycoloyl esters are novel carotenoid derivatives. The type strain of G. terrae NBRC 10016T also contained the same carotenoids, but the composition of the two carotenoid glucosides was low and the total carotenoid content was less than one tenth of that of strain AIST-1.     

Functional assembly of the foreign carotenoid lycopene into the photosynthetic apparatus of Rhodobacter sphaeroides, achieved by replacement of the native 3-step phytoene desaturase with its 4-step counterpart from Erwinia herbicola

Photosynthetic organisms synthesize a diverse range of carotenoids. These pigments are important for the assembly, function and stability of photosynthetic pigment-protein complexes, and they are used to quench harmful radicals. The photosynthetic bacterium Rhodobacter sphaeroides was used as a model system to explore the origin of carotenoid diversity. Replacing the native 3-step phytoene desaturase (CrtI) with the 4-step enzyme from Erwinia herbicola results in significant flux down the spirilloxanthin pathway for the first time in Rb. sphaeroides. In Rb. sphaeroides, the completion of four desaturations to lycopene by the Erwinia CrtI appears to require the absence of CrtC and, in a crtC background, even the native 3-step enzyme can synthesize a significant amount (13%) of lycopene, in addition to the expected neurosporene. We suggest that the CrtC hydroxylase can intervene in the sequence of reactions catalyzed by phytoene desaturase. We investigated the properties of the lycopene-synthesizing strain of Rb. sphaeroides. In the LH2 light-harvesting complex, lycopene transfers absorbed light energy to the bacteriochlorophylls with an efficiency of 54%, which compares favourably with other LH2 complexes that contain carotenoids with 11 conjugated double bonds. Thus, lycopene can join the assembly pathway for photosynthetic complexes in Rb. sphaeroides, and can perform its role as an energy donor to bacteriochlorophylls.     

Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to β-carotene when expressed in Escherichia coli . The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene β-cyclase activity when expressed in E. coli . A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re-channeling of linear carotenoids into β-cyclic carotenoids in C. annuum ripening fruits.     

Identification of carotenoids in Erwinia herbicola and in a transformed Escherichia coli strain

The yellow pigments of Erwinia herbicola Eho 10 and of a transformed Escherichia coli LE392 pPL376 have been identified as carotenoids. HPLC separation, spectra and in some cases mass spectroscopy demonstrated the presence of phytoene (15-cis isomer), beta-carotene (all-trans, 9-cis and 15-cis), beta-cryptoxanthin ( = 3-hydroxy beta-carotene), zeaxanthin (3,3'-dihydroxy beta-carotene) and corresponding carotene glycosides. In addition, lycopene and gamma-carotene accumulated in the presence of the inhibitor 2-(4-chlorophenylthio)-triethylamine.HCl. Carotenoid content in the transformed E. coli was two-fold higher than in E. herbicola. The pattern of the carotenoids was similar in the two organisms. Inactivation of the katF gene in E. coli resulted in an 85% lowering of carotenoid formation, as did the addition of 0.5% glucose to the medium. Suppression of carotenoid formation by inactivation of the katF gene lowered, but did not abolish, the protection offered by carotenoids against inactivation by alpha-terthienyl plus near-ultraviolet light (320-400 nm).     

Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

Heterologous production of two unusual acyclic carotenoids, 1,1'-dihydroxy-3,4-didehydrolycopene and 1-hydroxy-3,4,3',4'-tetradehydrolycopene by combination of the crtC and crtD genes from Rhodobacter and Rubrivivax

Acyclic hydroxy carotenoids were produced from lycopene and 3,4-didehydrolycopene in Escherichia coli by combining different carotenogenic genes including the carotene hydratase gene crtC and the carotene 3,4-desaturase gene crtD. The genes originated either from Rhodobacter species or Rubrivivax gelatinosus. It was shown that the product of crtD from Rubrivivax unlike the one from Rhodobacter is able to convert 1-HO-3,4-didehydrolycopene to 1-HO-3,4,3',4'-tetradehydrolycopene (=3,4,3',4'-tetradehydro-1,2-dihydro-psi,psi-caroten-1-ol). Thus, only when the desaturase from Rubrivivax is expressed can this novel carotenoid be obtained. In the presence of crtC from Rubrivivax, another carotenoid 1,1'-(HO)(2)-3,4-didehydrolycopene (=3,4-didehydrolycopene-1,2,1',2'-tetrahydro-psi,psi-caroten-1,1'-diol) not found in a non-transgenic organism before is formed in E. coli. Its accumulation under these conditions and its absence when crtC from Rubrivivax is replaced by the corresponding gene from Rhodobacter is discussed. The function of the different crtC and crtD genes in the pathway leading to the individual carotenoids is outlined. Since 1,1'-(HO)(2)-3,4-didehydrolycopene could not be produced in substantial amounts and 1-HO-3,4,3',4'-tetradehydrolycopene has not been described before, their structural characteristics were determined for the definite assignment of their identity. This included spectral properties, determination of relative molecular mass as well as the number of hydroxy groups by mass spectroscopy and NMR spectroscopy for 1,1'-(HO)(2)-3,4-didehydrolycopene.     

Amplification of HMG-CoA reductase production enhances carotenoid accumulation in Neurospora crassa

Neurospora crassa, a filamentous fungus, naturally produces the carotenoids lycopene and neurosporaxanthin. To increase the carbon flux through the carotenoid biosynthetic pathway, the 1658-bp region of the HMG1 gene encoding the catalytic domain (cHMG1) of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase of Saccharomyces cerevisiae was expressed in N. crassa under control of the strong, constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and the inducible alcohol dehydrogenase (alcA) promoter. Overexpressing cHMG1 under control of the GPD promoter increased lycopene and neurosporaxanthin production 6- and 1.5-fold, respectively, relative to the wild-type strain. Over-expression of cHMG1 under control of the alcA promoter increased production of lycopene and neurosporaxanthin 3- and 2-fold, respectively.     

Carotenoid Pigments of Mycobacterium kansasii

Partitioned between aqueous methanol and petroleum ether, the unsaponifiable pigments of Mycobacterium kansasii were all epiphasic. Thin-layer chromatography of these carotenoids showed that M. kansasii formed at least nine pigments. These pigments were identified by their chromatographic properties and spectral characteristics as phytoene, zeta-carotene, neurosporene, lycopene, leprotene, gamma-carotene, delta-carotene, alpha-carotene, and beta-carotene. Three additional pigmented spots on thin-layer chromatography found in trace amounts were possibly degradation products of the major carotenoids.     

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

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°C showed better production of torulene than 37°C while initial culture pH had no significant effect on torulene production. Glucose-containing LB, 2×YT, TB and MR media significantly repressed the production of torulene, and the other carotenoids lycopene, tetradehydrolycopene, and -carotene, in E. coli. In contrast, glycerol-containing LB, 2×YT, 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.     

Functional analysis of the beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation.

Carotenoids with cyclic end groups are essential components of the photosynthetic membranes in all plants, algae, and cyanobacteria. These lipid-soluble compounds protect against photooxidation, harvest light for photosynthesis, and dissipate excess light energy absorbed by the antenna pigments. The cyclization of lycopene (psi, psi-carotene) is a key branch point in the pathway of carotenoid biosynthesis. Two types of cyclic end groups are found in higher plant carotenoids: the beta and epsilon rings. Carotenoids with two beta rings are ubiquitous, and those with one beta and one epsilon ring are common; however, carotenoids with two epsilon rings are rare. We have identified and sequenced cDNAs that encode the enzymes catalyzing the formation of these two rings in Arabidopsis. These beta and epsilon cyclases are encoded by related, single-copy genes, and both enzymes use the linear, symmetrical lycopene as a substrate. However, the epsilon cyclase adds only one ring, forming the monocyclic delta-carotene (epsilon, psi-carotene), whereas the beta cyclase introduces a ring at both ends of lycopene to form the bicyclic beta-carotene (beta, beta-carotene). When combined, the beta and epsilon cyclases convert lycopene to alpha-carotene (beta, epsilon-carotene), a carotenoid with one beta and one epsilon ring. The inability of the epsilon cyclase to catalyze the introduction of a second epsilon ring reveals the mechanism by which production and proportions of beta,beta- and beta, epsilon-carotenoids may be controlled and adjusted in plants and algae, while avoiding the formation of the inappropriate epsilon,epsilon-carotenoids.     

Chemistry,distribution, and metabolism of tomato carotenoids and their impact on human health

Recent epidemiological studies have suggested that the consumption of tomatoes and tomato-based food products reduce the risk of prostate cancer in humans. This protective effect has been attributed to carotenoids, which are one of the major classes of phytochemicals in this fruit. The most abundant carotenoid in tomato is lycopene, followed by phytoene, phytofluene, zeta-carotene, gamma-carotene, beta-carotene, neurosporene, and lutein. The distribution of lycopene and related carotenoids in tomatoes and tomato-based food products has been determined by extraction and high-performance liquid chromatography-UV/Visible photodiode array detection. Detailed qualitative and quantitative analysis of human serum, milk, and organs, particularly prostate, have revealed the presence of all the aforementioned carotenoids in biologically significant concentrations. Two oxidative metabolites of lycopene, 2,6-cyclolycopene-1,5-diols A and B, which are only present in tomatoes in extremely low concentrations, have been isolated and identified in human serum, milk, organs (liver, lung, breast, liver, prostate, colon) and skin. Carotenoids may also play an important role in the prevention of age-related macular degeneration, cataracts, and other blinding disorders. Among 25 dietary carotenoids and nine metabolites routinely found in human serum, mainly (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, lycopene, and their metabolites were detected in ocular tissues. In this review we identified and quantified the complete spectrum of carotenoids from pooled human retinal pigment epithelium, ciliary body, iris, lens, and in the uveal tract and in other tissues of the human eye to gain a better insight into the metabolic pathways of ocular carotenoids. Although (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their metabolites constitute the major carotenoids in human ocular tissues, lycopene and a wide range of dietary carotenoids have been detected in high concentrations in ciliary body and retinal pigment epithelium. The possible role of lycopene and other dietary carotenoids in the prevention of age-related macular degeneration and other eye diseases is discussed.     

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.     

Slr1293 in Synechocystis sp. strain PCC 6803 Is the C-3',4' desaturase (CrtD) involved in myxoxanthophyll biosynthesis

When grown at high light intensity, more than a quarter of the total carotenoids in the unicellular cyanobacterium Synechocystis consists of myxoxanthophyll, a polar carotenoid glycoside. The biosynthetic pathway of myxoxanthophyll is unknown but is presumed to involve a number of enzymes, including a C-3',4' desaturase required to add one double bond to generate 11 conjugated double bonds in the monocyclic myxoxanthophyll. A candidate for this desaturase is Slr1293, which was identified by genome similarity searching. To determine whether Slr1293 is a desaturase recognizing neurosporene and lycopene, slr1293 was expressed in Escherichia coli strains accumulating neurosporene or lycopene. Confirming such a desaturase function for Slr1293, these E. coli strains accumulated 3',4'-didehydroneurosporene and 3',4'-didehydrolycopene, respectively. Indeed, deletion of slr1293 in Synechocystis provides further evidence that Slr1293 is a desaturase recognizing neurosporene: In the slr1293 deletion mutant, neurosporene was found to accumulate and was further processed to produce neurosporene glycoside. Neurosporene hereby becomes a primary candidate to be the branch point molecule between carotene and myxoxanthophyll biosynthesis in this cyanobacterium. The slr1293 gene was concluded to encode a C-3',4' desaturase that is essential for myxoxanthophyll biosynthesis, and thus it was designated as crtD. Furthermore, as Slr1293 appears to recognize neurosporene and to catalyze the first committed step on the myxoxanthophyll biosynthesis pathway, Slr1293 plays a pivotal role in directing a portion of the precursor pool for carotenoid biosynthesis toward myxoxanthophyll biosynthesis in Synechocystis sp. strain PCC 6803.     

Isorenieratene biosynthesis in green sulfur bacteria requires the cooperative actions of two carotenoid cyclases

The cyclization of lycopene to γ- or β-carotene is a major branch point in the biosynthesis of carotenoids in photosynthetic bacteria. Four families of carotenoid cyclases are known, and each family includes both mono- and dicyclases, which catalyze the formation of γ- and β-carotene, respectively. Green sulfur bacteria (GSB) synthesize aromatic carotenoids, of which the most commonly occurring types are the monocyclic chlorobactene and the dicyclic isorenieratene. Recently, the cruA gene, encoding a conserved hypothetical protein found in the genomes of all GSB and some cyanobacteria, was identified as a lycopene cyclase. Further genomic analyses have found that all available fully sequenced genomes of GSB encode an ortholog of cruA. Additionally, the genomes of all isorenieratene-producing species of GSB encode a cruA paralog, now named cruB. The cruA gene from the chlorobactene-producing GSB species Chlorobaculum tepidum and both cruA and cruB from the brown-colored, isorenieratene-producing GSB species Chlorobium phaeobacteroides strain DSM 266^T were heterologously expressed in lycopene- and neurosporene-producing strains of Escherichia coli, and the cruB gene of Chlorobium clathratiforme strain DSM 5477^T was also heterologously expressed in C. tepidum by inserting the gene at the bchU locus. The results show that CruA is probably a lycopene monocyclase in all GSB and that CruB is a γ-carotene cyclase in isorenieratene-producing species. Consequently, the branch point for the synthesis of mono- and dicyclic carotenoids in GSB seems to be the modification of γ-carotene, rather than the cyclization of lycopene as occurs in cyanobacteria.     

Chemical induction of poly-cis carotenoid biosynthesis

A new class of synthetic bioregulators is reported which cause the accumulation of poly-cis carotenoids in the flavedo of Marsh white seedless grapefruit. The compounds tested were all secondary amines: dibenzylamine, substituted dibenzylamines (4-F; 4-Cl; 4-Br; 2-, 3-and 4-Me; 4-NO₂; 4-CN; 4-Cl, 4′-Me; 4-Me, 4′-NO₂), N-benzyl phenethylamine and N-benzyl 2-naphthalenemethylamine. The most effective, 4-chlorodibenzylamine, caused the accumulation of 74μgg/g dry wt of poly-cis carotenoids. Prolycopene was the predominant pigment but substantial amounts of proneurosporene, poly-cis-γ-carotenes and other cis carotenes were also present. The mode of action of these new bioregulators is probably gene derepression, the same as that of the lycopene inducers. However, the secondary amines probably derepress a recessive gene governing the biosynthesis of poly-cis carotenoids; whereas, the lycopene inducers derepress the dominant gene that gives rise to the normal all-trans carotenoids. The new compounds did not seem to inhibit the cyclase(s), as the lycopene inducers do.     

Biosynthesis of Carotenoids in Brevibacterium sp. KY-4313

The biosynthesis of 4-keto and 4,4′-diketo carotenoids in Brevibacterium sp. KY-4313 was studied. Echinenone and canthaxanthin were isolated from the cultures grown on a medium containing several n-alkanes. When glutathione was added to the bacterial cultures, the formation of canthaxanthin was inhibited while β-carotene and its hydroxy derivatives accumulated. It is suggested that these 4-hydroxy compounds, isocryptoxanthin, isozeaxanthin, and 4-hydroxy-4′-keto-β-carotene, are intermediates in the biosynthesis of canthaxanthin. In the presence of 2-(4-chlorophenylthio)-triethylamine hydrochloride or nicotine, lycopene and neurosporene accumulated. The β-carotene level decreased slightly but β-zeacarotene remained unchanged. β-carotene and its derivatives were resynthesized upon removal of the inhibitors. It was concluded that cyclization can take place at either the neurosporene or lycopene level in Brevibacterium sp. KY-4313.