Production of Carotenoids from Cultivated Seaweed.

Cladosiphon (C.) okamuranus, a brown alga endemic to the Nansei Islands, Japan, has been conventionally ingested as food. Nowadays, it is a major aquatic product of the Okinawa Prefecture with an annual production of around 20,000 tons. The life cycle of C. okamuranus comprises the macroscopic sporophyte (algal body) generation and the microscopic gametophyte generation. The germlings in the latter generation can proliferate when floating in seawater. This floating form has been exploited in techniques involved in the commercial production of C. okamuranus seedlings.Brown algae contain fucoxanthin, a carbonyl carotenoid known to have anticancer, anti-obesity, and antidiabetic effect in addition to the anti-oxidation effect. We found that the fucoxanthin content of cultivated floating form of C. okamuranus discoid germlings becomes up to 50 times that of the mature alga. Since the discoid germlings repeatedly grow like microorganisms, although they are large algae, they are utilized to produce fucoxanthin. We optimize the culture conditions by changing the temperature, light intensity, photoperiod, light wavelength, and nutrient salt conditions for optimal fucoxanthin productivity. The cultivation has been successful to industrial plant scale, culminating in the use of 1 ton of cultivating medium.In brown algal cells, fucoxanthin is primarily found bound to the photosynthetic pigment-protein complexes known as fucoxanthin-chlorophyll protein (FCP). Consequently cultivated floating form of C. okamuranus also shows high content of FCP. Isolation and characterization of pigments bound to the FCP were determined precisely, and ultrafast spectroscopies were applied to elucidate the photosynthetic function of fucoxanthin bound to the pigment-protein complexes. This cultivation method has also been applied to the other edible brown algae. We found that the optimal cultivation conditions as well as the yields of fucoxanthin and FCP highly depend on the species.The floating form cultivation was also applied to a large-sized edible green alga, Codium intricatum, which is uniquely producing a carbonyl carotenoid, siphonaxanthin. This has several anti-disease effects and is also a primal photosynthetic pigment which is found bound to photosynthetic antenna complex usually called siphonaxanthin-chlorophyll protein (SCP). We are working on the improvement of productivity, scale-up of production, and development of cultivation technology of new macro algae.

Molecular Structures and Functions of Chlorophylls-a Esterified with Geranylgeranyl, Dihydrogeranylgeranyl, and Tetrahydrogeranylgeranyl Groups at the 17-Propionate Residue in a Diatom, Chaetoceros calcitrans.

The 17-propionate ester group of chlorophyll(Chl)-a in some oxygenic phototrophs was investigated using HPLC. Chls-a esterified with partially dehydrogenated forms of a phytyl group were found in fully grown cells of a diatom, Chaetoceros calcitrans: geranylgeranyl (GG), dihydrogeranylgeranyl (DHGG), and tetrahydrogeranylgeranyl (THGG). Chls-a bearing such esterifying groups were reported to be found only in greening processes of higher plants, and thus these Chls-a have been thought to be biosynthetic precursors for phytylated Chl-a. Their molecular structures were unambiguously determined using 1H and 13C NMR spectroscopy and mass spectrometry. In particular, the positions of C═C double bonds in DHGG were identified at C2═C3, C6═C7, and C14═C15, and those in THGG were determined to be at C2═C3 and C14═C15. Notably, the present DHGG was different from the previously determined DHGG of bacteriochlorophyll-a in purple bacteria (C2═C3, C10═C11, and C14═C15). Moreover, thylakoid membranes as well as fucoxanthin-chlorophyll-a/c proteins called FCPs were isolated from the diatom, and their Chl-a compositions were analyzed. Chls-a esterified with GG, DHGG, and THGG were detected by HPLC, indicating that such Chls-a were not merely biosynthetic precursors, but photosynthetically active pigments.

Photoprotection vs. Photoinhibition of Photosystem II in Transplastomic Lettuce (Lactuca sativa) Dominantly Accumulating Astaxanthin.

Transplastomic (chloroplast genome-modified; CGM) lettuce that dominantly accumulates astaxanthin grows similarly to a non-transgenic control with almost no accumulation of naturally occurring photosynthetic carotenoids. In this study, we evaluated the activity and assembly of PSII in CGM lettuce. The maximum quantum yield of PSII in CGM lettuce was <0.6; however, the quantum yield of PSII was comparable with that in control leaves under higher light intensity. CGM lettuce showed a lower ability to induce non-photochemical quenching (NPQ) than the control under various light intensities. The fraction of slowly recovering NPQ in CGM lettuce, which is considered to be photoinhibitory quenching (qI), was less than half that of the control. In fact, 1O2 generation was lower in CGM than in control leaves under high light intensity. CGM lettuce contained less PSII, accumulated mostly as a monomer in thylakoid membranes. The PSII monomers purified from the CGM thylakoids bound echinenone and canthaxanthin in addition to ß-carotene, suggesting that a shortage of ß-carotene and/or the binding of carbonyl carotenoids would interfere with the photophysical function as well as normal assembly of PSII. In contrast, high accumulation of astaxanthin and other carbonyl carotenoids was found within the thylakoid membranes. This finding would be associated with the suppression of photo-oxidative stress in the thylakoid membranes. Our observation suggests the importance of a specific balance between photoprotection and photoinhibition that can support normal photosynthesis in CGM lettuce producing astaxanthin.

Characterization of the intramolecular transfer state of marine carotenoid fucoxanthin by femtosecond pump-probe spectroscopy.

Fucoxanthin, containing a carbonyl group in conjugation with its polyene backbone, is a naturally occurring pigment in marine organisms and is essential to the photosynthetic light-harvesting function in brown alga and diatom. Fucoxanthin exhibits optical characteristics attributed to an intramolecular charge transfer (ICT) state that arises in polar environments due to the presence of the carbonyl group. In this study, we report the spectroscopic properties of fucoxanthin in methanol (polar and protic solvent) observed by femtosecond pump-probe measurements in the near-infrared region, where transient absorption associated with the optically allowed S2 (1(1)B u (+) ) state and stimulated emission from the strongly coupled S1/ICT state were observed following one-photon excitation to the S2 state. The results showed that the amplitude of the stimulated emission of the S1/ICT state increased with decreasing excitation energy, demonstrating that the fucoxanthin form associated with the lower energy of the steady-state absorption exhibits stronger ICT character.

Light-dependent conformational change of neoxanthin in a siphonous green alga, Codium intricatum, revealed by Raman spectroscopy.

Siphonous green algae, a type of deep-sea green algae, appear olive drab and utilize blue-green light for photosynthesis. A siphonous green alga, Codium (C.) intricatum, was isolated from Okinawa prefecture in Japan, and a clonal algal culture in filamentous form was established. The major light-harvesting antenna was analogous to the trimeric LHCII found in higher plants, but the C. intricatum complex contained an unusual carbonyl carotenoid siphonaxanthin. Culture conditions were optimized to achieve high siphonaxanthin content in intact lyophilized filamentous bodies. Interestingly, the carotenoid composition was different when cultured under high irradiance: all-trans neoxanthin was accumulated in addition to the normal 9'-cis form in whole cell extract. Resonance Raman spectra of intact filamentous bodies, cultured under high- and low-light conditions, confirmed the accumulation of all-trans neoxanthin under high irradiance conditions. A plausible function of the presence of all-trans neoxanthin will be discussed in relation to the regulation against high light stress.

Characterization of the Ultraviolet-B Absorption Band of Carotenoids Using Solvent-dependent Shifts in Steady-State and Transient Absorption Spectra.

The versatile functions of carotenoids in biological systems are associated with the extended π-electron conjugation system. Strong visible absorption resulting from the optically allowed S2 (1Bu+) state and the low-lying optically forbidden S1 (2Ag-) state examined. Carotenoids also exhibit an absorption band in the ultraviolet-B region; however, the origin of this band (hereafter referred to as Suv state) is not well characterized. The Suv state is a candidate for the destination level of the well-known S1 → Sn transient absorption; however, an obvious energy mismatch has been observed. In this study, we examined the steady-state and picosecond transient absorption spectra of lycopene in various solvents. The Suv absorption of carotenoids with diverse conjugation lengths was also examined. The dependence of the energies on solvent polarizability and conjugation length revealed that both Suv and Sn are the "second" Bu+ state. The absorption spectrum for lycopene at 200 K revealed an additional vibrational band, which may be the vibrational origin of the S0 → Suv band. Considering the slow vibrational relaxation of the 2Ag- state, the S1 → Sn transition may represent the 2Ag- (v = 1) → 2Bu+ (v = 0) transition, and the energetic contradiction can be resolved.

Structure-based validation of recombinant light-harvesting complex II.

Light-harvesting complex II (LHCII) captures sunlight and dissipates excess energy to drive photosynthesis. To elucidate this mechanism, the individual optical properties of pigments in the LHCII protein must be identified. In vitro reconstitution with apoproteins synthesized by Escherichia coli and pigment-lipid mixtures from natural sources is an effective approach; however, the local environment surrounding each pigment within reconstituted LHCII (rLHCII) has only been indirectly estimated using spectroscopic and biochemical methods. Here, we used cryo-electron microscopy to determine the 3D structure of the rLHCII trimer and found that rLHCII exhibited a structure that was virtually identical to that of native LHCII, with a few exceptions: some C-terminal amino acids were not visible, likely due to aggregation of the His-tags; a carotenoid at the V1 site was not visible; and at site 614 showed mixed occupancy by both chlorophyll a and b molecules. Our observations confirmed the applicability of the in vitro reconstitution technique.

New insights into the structure of the reaction centre from Blastochloris viridis: evolution in the laboratory.

Newly determined crystal structures of the photosynthetic RC (reaction centre) from two substrains of the non-sulfur purple bacterium Blastochloris viridis strain DSM 133, together with analysis of their gene sequences, has revealed intraspecies evolutionary changes over a period of 14 years. Over 100 point mutations were identified between these two substrains in the four genes encoding the protein subunits of the RC, of which approximately one-fifth resulted in a total of 16 amino acid changes. The most interesting difference was in the M subunit where the change from a leucine residue to glycine in the carotenoid-binding pocket allowed NS5 (1,2-dihydroneurosporene) to adopt a more sterically favoured conformation, similar to the carotenoid conformation found in other related RCs. The results of the present study, together with a high rate of mutations in laboratory bacterial cultures described recently, suggest that bacteria evolve faster than has been generally recognized. The possibility that amino acid changes occur within protein sequences, without exhibiting any immediately observable phenotype, should be taken into account in studies that involve long-term continuous growth of pure bacterial cultures. The Blc. viridis RC is often studied with sophisticated biophysical techniques and changes such as those described here may well affect their outcome. In other words, there is a danger that laboratory-to-laboratory variation could well be due to different groups not realising that they are actually working with slightly different proteins. A way around this problem is suggested.

Squaraine dye/Ti3C2Tx MXene organic-inorganic hybrids for photocatalytic hydrogen evolution.

MXenes, a new family of 2D nanostructured materials, have been widely studied in the field of artificial photosynthesis due to their outstanding physicochemical properties. In this work, a series of 2,4-bis[4-(N,N-dibutylamino)phenyl] squaraine (SQ) derivatives with different number of hydroxyl groups were hybridized with Ti3C2Tx MXene nanosheets, and the organic-inorganic hybrid photocatalysts were applied for water-splitting hydrogen evolution. The mass ratios of SQ@Ti3C2Tx were optimized to 4 wt% for each SQ, and the best hydrogen evolution reaction (HER) rate of 28.6 µmol h-1 g-1 was achieved by SQ-3 with four OH groups. The photocatalytic ability of the hybrid comes from the outstanding light harvesting of SQ dye, sufficient active sites of Ti3C2Tx, and efficient separation and transfer of the photogenerated charges via heterojunction between SQ aggregates and Ti3C2Tx. This work firstly demonstrates an example of SQ sensitizer combined with MXene for hydrogen generation, which provides a new insight to further explore the MXene-based hybrid nanomaterials for water splitting hydrogen evolution.

Kaempferia galanga L. extract and its main component, ethyl p-methoxycinnamate, inhibit the proliferation of Ehrlich ascites tumor cells by suppressing TFAM expression.

Kaempferia galanga L. shows anti-cancer effects; however, the underling mechanism remains unclear. In this study, we explored the underlying mechanism of the anti-cancer effects of Kaempferia galanga L. Kaempferia galanga L. rhizome extracts (KGEs) suppressed Ehrlich ascites tumor cell (EATC) proliferation by inhibiting S-phase progression. The main component of KGE is ethyl p-methoxycinnamate (EMC), which exhibits the same anti-proliferative effect as KGE. Furthermore, EMC induced the downregulation of cyclin D1 and upregulation of p21. EMC also decreased the expression of mitochondrial transcription factor A (TFAM) but did not significantly change mitochondrial DNA copy number and membrane potential. Phosphorylation at Ser62 of c-Myc, a transcription factor of TFAM, was decreased by EMC treatment, which might be due to the suppression of H-ras expression. These results indicate that EMC is the active compound responsible for the anti-cancer effect of KGE and suppresses EATC proliferation by regulating the protein expression of cyclin D1 and p21; TFAM may also regulate the expression of these genes. In addition, we investigated the anticancer effects of KGE and EMC in vivo using EATC bearing mice. The volume of ascites fluid was significantly increased by intraperitoneal administration of EATC. However, the increase in the volume of ascites fluid was suppressed by oral administration of EMC and KGE. This study provides novel insights into the association between the anti-cancer effects of natural compounds and TFAM, indicating that TFAM might be a potential therapeutic target.