Molecular breeding of carotenoid biosynthetic pathways.

The burgeoning demand for complex, biologically active molecules for medicine, materials science, consumer products, and agrochemicals is driving efforts to engineer new biosynthetic pathways into microorganisms and plants. We have applied principles of breeding, including mixing genes and modifying catalytic functions by in vitro evolution, to create new metabolic pathways for biosynthesis of natural products in Escherichia coli. We expressed shuffled phytoene desaturases in the context of a carotenoid biosynthetic pathway assembled from different bacterial species and screened the resulting library for novel carotenoids. One desaturase chimera efficiently introduced six rather than four double bonds into phytoene, to favor production of the fully conjugated carotenoid, 3, 4,3',4'-tetradehydrolycopene. This new pathway was extended with a second library of shuffled lycopene cyclases to produce a variety of colored products. One of the new pathways generates the cyclic carotenoid torulene, for the first time, in E. coli. This combined approach of rational pathway assembly and molecular breeding may allow the discovery and production, in simple laboratory organisms, of new compounds that are essentially inaccessible from natural sources or by synthetic chemistry.

Spin-label studies on phosphatidylcholine-polar carotenoid membranes: effects of alkyl-chain length and unsaturation.

Spin-labeling methods were used to study the structure and dynamic properties of phosphatidylcholine (PC)-dihydroxycarotenoid membranes as a function of phospholipid alkyl chain length, alkyl chain saturation, temperature and mol fraction of carotenoids. (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin increase order and decrease motional freedom of the lipid alkyl chains in fluid-phase PC membranes. The effect of carotenoids decreases as the alkyl chain length of saturated PC increases. (2) The abrupt changes of spin-label motion observed at the main-phase transition of the saturated PC membranes are broadened and shifted to lower temperatures. At a carotenoid concentration of 10 mol%, they disappear for short-chain PC membranes (12-14 carbons), but are still observed for long-chain PC membranes (18-22 carbons). (3) In fluid-phase PC membranes possessing short alkyl chains (12-14 carbons), the activation energy of the rotational diffusion of 16-doxylstearic acid spin label (16-SASL) is significantly lower at a carotenoid concentration of 10 mol%. The difference decreases as the alkyl-chain length increases. (4) The presence of unsaturated alkyl chains greatly reduces the effects of carotenoids on the mobility of the polar headgroups as observed with tempocholine dipalmitoylphosphatidic acid ester and on the order of alkyl chains near the polar headgroup region as observed with 5-doxylstearic acid spin label (5-SASL). The effect of unsaturation is, however, moderate in the membrane center as shown with 16-SASL. Also, the effect of carotenoids on the order and motion of the rigid and highly anisotropic molecules dissolved in the PC membranes is significantly greater in saturated PC membranes.

Co-oxidation of carotenes requires one soybean lipoxygenase isoenzyme.

The type II lipoxygenase (optimum pH 6.5) from soybeans was purified and separated into two fractions either by chromatography on DEAE-Sephadex or by isoelectric focusing. In the presence of linoleic acid and oxygen both fractions co-oxidise canthaxanthine or beta-carotene as effectively as a combination of these fractions. Oxygenation of linoleic acid and co-oxidation of canthaxanthine by type II lipoxygenase is stimulated by 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid but not by 13-hydroxy-cis-9,trans-11-octadecadienoic acid or 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid.

Orientation of xanthophylls in phosphatidylcholine multibilayers.

Oriented multibilayers of dimyristoyl phosphatidylcholine (DMPC) modified with violaxanthin or zeaxanthin were examined by X-ray diffractometry and linear dichroism. It appears that pigment molecules and the normal to the bilayer plane form an angle of 24-25 degrees. It was also observed that rather small concentrations of added xanthophylls (molar fraction up to 3%) increase the pigmented bilayer thickness by a value of about 2 A as compared with that of the pure DMPC bilayer. The observed nonzero linear dichroism at normal incidence of light suggests the possibility of nonhomogeneous orientation of transition dipoles in the plane of the bilayer.

Effect of polar carotenoids on the oxygen diffusion-concentration product in lipid bilayers. An EPR spin label study.

The oxygen diffusion-concentration product was determined in phosphatidylcholine (PC) bilayers from oxygen broadening of the spin label EPR spectra. The use of fatty acid spin labels makes it possible to do structural and oximetric measurements with the same sample. We find that polar carotenoids, zeaxanthin and violaxanthin, increase ordering of hydrocarbon chains in saturated (dimyristoyl-PC) and unsaturated (egg yolk PC) membranes and also significantly decrease the oxygen diffusion-concentration product in the hydrocarbon region of these membranes. At 25 degrees C in the presence of 10 mol% of carotenoids, the product is about 30% smaller than in pure PC membranes. Intercalation of carotenoids decreases the oxygen diffusion-concentration product in the central part of the bilayer and has little effect on the product in the polar head group region. In contrast, cholesterol molecules significantly reduce the product on and near the membrane surface, and do not change it (saturated PC) or increase it (unsaturated PC) in the middle of the bilayer (Subczynski, W.K., Hyde, J.S. and Kusumi, A. (1989) Proc. Natl. Acad. Sci. USA 86, 4474-4478). The decrease of oxygen diffusion-concentration product may be a mechanism of carotenoid protective activity, which should be effective in plant and animal cells in the light as well as in the dark.

The P-700-chlorophyl alpha-protein complex and two major light-harvesting complexes of Acrocarpia paniculata and other brown seaweeds.

Acrocarpia paniculata thylakoids were fragmented with Triton X-100 and the pigment-protein complexes so released were isolated by sucrose density gradient centrifugation. Three main chlorophyll-carotenoid-protein complexes with distinct pigment compositions were isolated. (1) A P-700-chlorophyll a-protein complex, with a ratio of 1 P-700: 38 chlorophyll a: 4 beta-carotene molecules, had similar absorption and fluorescence characteristics to the chlorophyll-protein complex 1 isolated with Triton X-100 from higher plants, green algae and Ecklonia radiata. (2) an orange-brown complex had a chlorophyll a : c2 : fucoxanthin molar ratio of 2 : 1 : 2. this complex had no chlorophyll c1 and contained most of the fucoxanthin present in the chloroplasts. This pigment complex is postulated to be the main light-harvesting complex of brown seaweeds. (3) A green complex had a chlorophyll a : c1 : c2 : violaxanthin molar ratio of 8 : 1 : 1. This also is a light-harvesting complex. the absorption and fluorescence spectral characteristics and other physical properties were consistent with the pigments of these three major complexes being bound to protein. Differential extraction of brown algal thylakoids with Triton X-100 showed that a chlorophyll c2-fucoxanthin-protein complex was a minor pigment complex of these thylakoids.

Effects of polar carotenoids on dimyristoylphosphatidylcholine membranes: a spin-label study.

Spin labeling methods were used to study the structure and dynamic properties of dimyristoylphosphatidylcholine (DMPC) membranes as a function of temperature and the mole fraction of polar carotenoids. The results in fluid phase membranes are as follows: (1) Dihydroxycarotenoids, zeaxanthin and violaxanthin, increase order, decrease motional freedom and decrease the flexibility gradient of alkyl chains of lipids, as was shown with stearic acid spin labels. The activation energy of rotational diffusion of the 16-doxylstearic acid spin label is about 35% less in the presence of 10 mol% of zeaxanthin. (2) Carotenoids increase the mobility of the polar headgroups of DMPC and increase water accessibility in that region of membrane, as was shown with tempocholine phosphatidic acid ester. (3) Rigid and highly anisotropic molecules dissolved in the DMPC membrane exhibit a bigger order of motion in the presence of polar carotenoids as was shown with cholestane spin label (CSL) and androstane spin label (ASL). Carotenoids decrease the rate of reorientational motion of CSL and do not influence the rate of ASL, probably due to the lack of the isooctyl side chain. The abrupt changes of spin label motion observed at the main phase transition of the DMPC bilayer are broadened and disappear at the presence of 10 mol% of carotenoids. In gel phase membranes, polar carotenoids increase motional freedom of most of the spin labels employed showing a regulatory effect of carotenoids on membrane fluidity. Our results support the hypothesis of Rohmer, M., Bouvier, P. and Ourisson, G. (1979) Proc. Natl. Acad. Sci. USA 76, 847-851, that carotenoids regulate the membrane fluidity in Procaryota as cholesterol does in Eucaryota. A model is proposed to explain these results in which intercalation of the rigid rod-like polar carotenoid molecules into the membrane enhances extended trans-conformation of the alkyl chains, decreases free space in the bilayer center, separate the phosphatidylcholine headgroups and decreases interaction between them.

Light-harvesting systems of brown algae and diatoms. Isolation and characterization of chlorophyll a/c and chlorophyll a/fucoxanthin pigment-protein complexes.

The present study examined the protein associations and energy transfer characteristics of chlorophyll c and fucoxanthin which are the major light-harvesting pigments in the brown and diatomaceous algae. It was demonstrated that sodium dodecyl sulfate (SDS)-solubilized photosynthetic membranes of these species when subjected to SDS polyacrylamide gel electrophoresis yielded three spectrally distinct pigment-protein complexes. The slowest migrating zone was identical to complex I, the SDS-altered form of the P-700 chlorophyll a-protein. The zone of intermediate mobility contained chlorophyll c and chlorophyll a in a molar ratio of 2 : 1, possessed no fucoxanthin, and showed efficient energy transfer from chlorophyll c to chlorophyll a. The fastest migrating pigment-protein zone contained fucoxanthin and chlorophyll a, possessed no chlorophyll c, and showed efficient energy transfer from fucoxanthin to chlorophyll a. It is demonstrated that the chlorophyll a/c-protein and the chlorophyll a/fucoxanthin-protein complexes are common to the brown algae and diatoms examined, and likely share similar roles in the photosynthetic units of these species.

Comparison of the effects of inserted C40- and C50-terminally dihydroxylated carotenoids on the mechanical properties of various phospholipid vesicles.

We have measured the extent of incorporation of zeaxanthin (C40) and decaprenozeaxanthin (C50) in unilamellar vesicles of dimyristoylphosphatidylcholine (n-C14) and dipalmitoylphosphatidylcholine (n-C16). The incorporation is larger when the molecular length of the carotenoid corresponds to the thickness of the phospholipid bilayer. Stereochemically pure 2,3-di-O-phytanyl-sn-glycero-1-phosphocholine was prepared by modification of the polar heads of the phospholipids of Halobacterium halobium. Vesicles of this branched-chain ether phospholipid incorporate poorly the carotenoids, whereas egg lecithin vesicles incorporate them better. Osmotic swelling and water permeability of vesicles, with or without carotenoids, were measured in a stopped-flow, light-scattering system. The reinforcing effect (lower permeability and higher rigidity) of carotenoids at 1.5 mol% incorporation into diphytanylphosphatidylcholine vesicles is comparable to that of 5 mol% cholesterol; however, carotenoids have no measurable effect on the egg lecithin vesicles. These results imply that the reinforcement of the membrane depends on a subtle adjustment of the phospholipid-carotenoid system.

Light-harvesting complexes of brown algae. Biochemical characterization and immunological relationships.

The pigment composition of the light-harvesting complexes isolated from several brown algae belonging to different orders has been analysed by reverse-phase HPLC. Relative to whole chloroplasts, they were markedly enriched in Chl c, fucoxanthin and violaxanthin and conversely depleted in Chl a. The relative molar proportions of the 4 main pigments (Chl a/Chl c/fucoxanthin/violaxanthin) ranged from 100:18:76:6 to 100:30:107:17. The protein moiety of LH complexes of all the species studied were composed of one or two main polypeptide components in the range of 19-22 kDa. These polypeptide subunits were arranged in polymeric particles about 240 kDa in Laminaria saccharina. A polyclonal antibody raised against the LH polypeptide of Fucus serratus has been tested on LH apoproteins of other Chromophytes and Chlorophytes. Phylogenic implications of these results are discussed.

The protective function of the xanthophyll cycle in photosynthesis.

The rapid conversion of the carotenoid violaxanthin to zeaxanthin via antheraxanthin (xanthophyll cycle) in potato leaves exposed at 23 degrees C to a strong white light of 2000 microE.m-2.s-1 was associated with a slight inhibition of photosynthetic electron transport (as estimated from chlorophyll fluorescence measurements) and a low lipid peroxidation (as estimated from ethane measurements). When the xanthophyll cycle was blocked by dithiothreitol (3 mM) or low temperature (3 degrees C), photoinhibition of electron transport was exacerbated and pronounced lipid peroxidation occurred concomitantly. Accumulation of zeaxanthin and antheraxanthin in potato leaves by a non-photoinhibitory light treatment at 23 degrees C (900 microE.m-2.s-1 for 1 h) considerably reduced the level of lipid peroxidation during subsequent light stress at 3 degrees C. The presented results indicate that one of the functions of the xanthophyll cycle could be the protection of thylakoid membranes against lipid peroxidation, suggesting that zeaxanthin and antheraxanthin synthesized in strong light are present as free pigments in the membrane lipid bilayer.

Epoxidation of zeaxanthin and antheraxanthin reverses non-photochemical quenching of photosystem II chlorophyll a fluorescence in the presence of trans-thylakoid delta pH.

The xanthophyll cycle apparently aids the photoprotection of photosystem II by regulating the nonradiative dissipation of excess absorbed light energy as heat. However, it is a controversial question whether the resulting nonphotochemical quenching is soley dependent on xanthophyll cycle activity or not. The xanthophyll cycle consists of two enzymic reactions, namely deepoxidation of the diepoxide violaxanthin to the epoxide-free zeaxanthin and the much slower, reverse process of epoxidation. While deepoxidation requires a transthylakoid pH gradient (delta pH), epoxidation can proceed irrespective of a delta pH. Herein, we compared the extent and kinetics of deepoxidation and epoxidation to the changes in fluorescence in the presence of a light-induced thylakoid delta pH. We show that epoxidation reverses fluorescence quenching without affecting thylakoid delta pH. These results suggest that epoxidase activity reverses quenching by removing deepoxidized xanthophyll cycle pigments from quenching complexes and converting them to a nonquenching form. The transmembrane organization of the xanthophyll cycle influences the localization and the availability of deepoxidized xanthophylls is to support nonphotochemical quenching capacity. The results confirm the view that rapidly reversible nonphotochemical quenching is dependent on deepoxidized xanthophyll.

Antioxidant activities of carotenes and xanthophylls.

The purpose of this study was to assess the relative antioxidant activities of a range of carotenes and xanthophylls through the extent of their abilities to scavenge the ABTS(.+) radical cation. The results show that the relative abilities of the carotenoids to scavenge the ABTS(.+) radical cation are influenced by the presence of functional groups with increasing polarities, such as carbonyl and hydroxyl groups, in the terminal rings, as well as by the number of conjugated double bonds.

Consumption of carotenoids in photosensitized oxidation of human plasma and plasma low-density lipoprotein.

Human plasma and plasma low-density lipoprotein (LDL) were exposed to photoirradiation in the presence of methylene blue (water-soluble photosensitizer) or 12-(1-pyrene)dodecanoic acid (P-12, lipid-soluble photosensitizer). In methylene-blue-sensitized photooxidation of human plasma and LDL, endogenous carotenoids and tocopherols were consumed with the accumulation of cholesteryl ester hydroperoxides (CE-OOH). Xanthophylls (zeaxanthin and lutein) decreased faster than lycopene and carotenes in the case of human plasma. In P-12-sensitized photooxidation of human plasma and LDL, the decrease rate of xanthophylls was slower than that of lycopene and carotenes. A lower level of beta-carotene exerted the effective inhibition of lipid peroxidation and retarded the oxidative loss of alpha-tocopherol, when the phosphatidylcholine liposomes containing these two lipid-soluble antioxidants were subjected to methylene blue- or P-12-sensitized photooxidation. These results suggest that antioxidant activity of carotenoids in photosensitized oxidation (Type II) of human plasma LDL depends on the site of singlet oxygen (1O2) to be generated and that carotenoids can protect tocopherols from the oxidative loss by 1O2 in the plasma.

FAD is a further essential cofactor of the NAD(P)H and O2-dependent zeaxanthin-epoxidase.

In chloroplasts of plants the xanthophyll cycle is suggested to function as a protection mechanism against photodamage. Two enzymes catalyze this cycle. One of them, violaxanthin de-epoxidase, transforms violaxanthin (Vio) to zeaxanthin (Zea) via antheraxanthin (Anth) and is bound to the lumenal surface of the thylakoid vesicles, when being in its active state. The other enzyme, Zea-epoxidase, is responsible for the backward reaction (Zea-->Anth-->Vio) and is active at the stromal side of the thylakoid. For the epoxidation of Zea this enzyme requires NAD(P)H and O2 as cosubstrates. Using isolated thylakoid membranes we found that FAD enhances the epoxidase activity (decrease of apparent Km for NAD(P)H and two-fold increase of Vmax). The flavin functions as a third cofactor which is partially lost during the isolation procedure of thylakoids. Other flavins, such as FMN or riboflavin are without effect. The involvement of FAD in the enzymatic reaction is also demonstrated by the inhibitory action of diphenyleneiodoniumchloride (DPI) (IC50 = 2.3 microM), a compound that blocks the reoxidation of reduced flavins within enzymes. The Zea-epoxidase is a multi-component enzyme system which can be classified as FAD-containing, NAD(P)H- and O2-dependent monooxygenase that is able to epoxidize 3-hydroxy beta-ionone rings of xanthophylls in the 5,6 position.

Abscisic acid induced protection against photoinhibition of PSII correlates with enhanced activity of the xanthophyll cycle.

The exogenous application of abscisic acid (ABA) to barely seedlings resulted in partial protection of the PSII photochemistry against photoinhibition at low temperature, the effect being most pronounced at 10(-5) M ABA. This was accompanied by higher photochemical quenching (qP) in ABA-treated leaves. A considerable increase (122%) in the amount of total carotenoids and xanthophylls (antheraxanthin, violaxanthin and zeaxanthin) was also found in the seedlings subjected to ABA. The activity of the xanthophyll cycle measured by the epoxidation state of xanthophylls under high-light treatment was higher in ABA-treated plants compared with the control. This corresponds to a higher value (0.411) of non-photochemical quenching (qNP) observed in ABA-treated than in control (0.306) leaves.

Novel amplification of non-photochemical chlorophyll fluorescence quenching following viral infection in Chlorella.

In higher plants non-photochemical dissipation of excess light, trapped by the pigment pool of photosystem II, prevents photodamage to the photosynthetic apparatus. We report here that an algal virus infecting Chlorella strain Pbi induces non-photochemical quenching of photosystem II fluorescence, indicating enhanced loss of absorbed light energy from photosystem II. This phenomenon occurs soon after the establishment of the virus infection cycle and is observed at low irradiance (20 micromol quanta m-2 s-1). At low light, infection associated non-photochemical quenching is not linked to extensive conversion of violaxanthin to antheraxanthin and zeaxanthin. However, such conversion occurs rapidly (2-10 min) in infected cells under conditions of high irradiance (100-300 micromol quanta m-2 s-1). Under similar conditions uninfected Chlorella cells do not display significant changes in non-photochemical quenching.

Diurnal and seasonal variation of five carotenoids measured in human serum.

We studied within-person variation over time in serum concentrations of five carotenoids. In a diurnal study involving 33 subjects, only the 1700 h blood samples demonstrated carotenoid concentrations different from the original 0800 values. Correlations between serum concentrations of the same carotenoids drawn 1 d apart ranged from 0.93 to 0.98. In a seasonal study involving 29 subjects, no systematic trends were observed for serum concentrations of these carotenoids. Correlations between concentrations of the same carotenoids drawn 1 y apart ranged from 0.57 to 0.82. Concentrations of different carotenoids within an individual tended to be correlated with each other. Obtaining one blood sample from subjects is a relatively imprecise way to estimate their usual serum concentrations of carotenoids. If an epidemiological study was to be based on only one determination of serum carotenoids, within-person variability in serum concentrations would attenuate true regression coefficients by 4-13% and would increase the required numbers of study subjects by 19-65%.

Essential fatty acids, plasma cholesterol, and fat-soluble vitamins in subjects with age-related maculopathy and matched control subjects.

A matched-control study of plasma retinol, alpha-tocopherol, carotenoid, and cholesterol concentrations and the polyunsaturated fatty acid content of plasma and erythrocyte phospholipids was undertaken in 65 elderly patients with age-related maculopathy and 65 control subjects matched for age and sex. Despite the high statistical power of the study and large variations between subjects in the variables under consideration, no significant differences were noted between patients and control subjects. However, several statistically significant differences were noted between male and female subjects independent of their classification with maculopathy or as controls and age: plasma cholesterol, total phospholipids, alpha-tocopherol, and beta-cryptoxanthin concentrations were higher in females than in males. The mean plasma cholesterol concentration for the upper tertile of the whole sample was 7.6 mmol/L. Plasma concentrations of total carotenoids, alpha-carotene, and beta-carotene, but not alpha-tocopherol, were significantly lower in smokers than in non-smokers. The results of this study do not provide any evidence in favor of changing the dietary intake of polyunsaturated fatty acids or fat-soluble vitamins to protect against age-related maculopathy.

Effects of palm carotenoids in rat hepatic cytochrome P450-mediated benzo(a)pyrene metabolism.

Using benzo(a)pyrene (BaP) metabolism as a probe for chemical carcinogenesis, in vitro and in vivo effects of palm-oil carotenoid [beta-carotene (BC), alpha-carotene (AC), or canthaxanthin (CTX)] on BaP metabolism in the rat hepatic cytochrome P450-mediated monooxygenase system were studied. Apparent Michaelis-Menten constants (Km) for formation of the precursor carcinogen, 7,8-dihydrodiol BaP, were found to be 14.4 (BC), 1.74 (AC), and 0.7 (CTX) mumol/L. The order of anticarcinogenic strength established in this study was BC much greater than AC greater than CTX. Increased formation of the detoxification intermediate, 3-hydroxy BaP, with increased carotenoid concentration was observed. The order of detoxification strength was BC greater than AC = CTX. The presence of carotenoids in vivo inhibited BaP metabolism. Using 9,10-dihydrodiol BaP as an indicator for inhibition, the order of the antioxidative activity was palm oil (with carotenoids) greater than BC greater than CTX greater than palm oil (without carotenoids). BC and AC may selectively modify the rat-liver microsomal enzymes, thus providing a biochemical mechanism for the inhibitory effect of palm carotenoids on chemical carcinogenesis.