Use of response surface methodology to optimise carotenogenesis in the microalga,Haematococcus pluvialis

The factors controlling biomass production and the synthesis of astaxanthin esters in the microalga Haematococcus pluvialis (CCAP 34/7) have been investigated using a statistical approach employing response surface methodology (RSM). The culture conditions required for optimal growth and carotenogenesis in this alga are very different. Of particular importance is the photon flux density: for growth the optimum is 50–60 μmol m⁻² s⁻¹ whereas the optimum for astaxanthin synthesis is much higher at ∼-1600 μmol m⁻² s⁻¹. The addition of low levels of NaCl to the medium also stimulates to a small extent synthesis of astaxanthin, but photon flux density remains the overriding factor. The optimal temperature for this strain is quite low at 14–15 °C. RSM has been shown to be a rapid and effective technique leading to the optimisation of algal culture conditions. This statistical approach can be applied readily to the majority of microalgae and their products.     

HPLC-analysis of algal pigments: comparison of columns,column properties and eluents

The effects of columns (Nucleosil C₁₈ODS, MZ-PAH, YMC-PACK C₃₀), column properties (inner diameters of 4 mm, 3 mm and 2 mm, pore-width 10 nm and 30 nm) and eluents (methanol, acetonitrile, acetone, water) were tested on the separation of algal pigments. The length of columns was 250 mm and particle size was 5 μm. Flow rates and gradients were adjusted to optimize peak separation; remaining chromatographic conditions were kept constant. The resolution of chromatographic systems was tested with pigment standards and various algal cultures. Total flow rate and retention times decreased with decreasing inner diameter, whereas pressure, sensitivity and peak-width increased. Pore width had negligible effects on the chromatographic separation of pigments under the test conditions. Only with acetonitrile as eluent were all the taxonomically important pigments resolved adequately: zeaxanthin (Cyanophyceae), lutein (Chlorophyceae), fucoxanthin (Bacillariopyceae), alloxanthin (Cryptophyceae), peridinin (Dinophyceae).     

Algal Carotenoids 51. Secondary carotenoids 2.Haematococcus pluvialis aplanospores as a source of (3S, 3′S)-astaxanthin esters

Aplanospores ofHaematococcus pluvialis MUR 145 contained 0.7% carotenoids (dry wt. basis) consisting of β,β-carotene (5% of total carotenoid), echinenone (4%), canthaxanthin (4%), (3S,3′S)-astaxanthin diester (34%), (3S,3′S)-astaxanthin monoester (46%), (3S,3′S)-astaxanthin (1%) and (3R,3′R,6′R)-lutein (6%). The astaxanthin esters were examined by TLC and HPLC and VIS,¹H NMR and mass spectra recorded. Their chirality was determined by the camphanate method (Vecchi & Müller, 1979) after anaerobic hydrolysis. The tough cell wall of the aplanospores required enzymatic treatment prior to pigment extraction. The potential use of this microalga as a feed ingredient in aquaculture is discussed briefly.     

Chemical induction of β-carotene biosynthesis

Marsh white seedless grapefruit were treated with the 2-diethylaminoethanol esters of the following acids: benzoic, phenylacetic, hydrocinnamic, 4-phenylbutyric, 5-phenylvaleric, valeric, hexanoic, heptanoic, octanoic, nonanoic, 5-chlorovaleric, cyclohexanecarboxylic, phenoxyacetic, p-chlorophenoxyacetic, 3-phenoxypropionic, cinnamic and p-chlorocinnamic. Several of these esters, in particular the hexanoate, 4-phenylbutyrate and cinnamate, caused the accumulation of large amounts of β-carotene. The effects of the hexanoate and of 2-phenoxytriethylamine, which causes only lycopene accumulation, were studied as functions of time. The hexanoate caused the rapid accumulation of lycopene during the first day. The amount of lycopene then began to decrease and that of β-carotene increased until, after 14 days, β-carotene was the major pigment. 2-Phenoxytriethylamine caused rapid lycopene accumulation during the first day and a slow steady increase afterwards. Thus, the mode of action of the β-carotene inducers may be similar to that of the lycopene inducers except that the former are probably rapidly hydrolysed by the esterase(s) in the flavedo, so that they no longer inhibit the cyclase(s), and β-carotene is accumulated at the expanse of lycopene.     

Effects of Visible and Ultraviolet Light on Carotene-deficient Mutants of Crypthecodinium cohnii

Synopsis.
The ability of carotenoids to protect a heterotrophic dinoflagellate, Crypthecodinium cohnii , from photodynamic damage by sunlight in the presence of an exogenous dye was demonstrated. Wild-type C. cohnii and 2 carotcnoid-deficient mutants were plated on agar-solidified media and exposed to natural sunlight. The wild-type strain, which synthesizes γ–carotene and β–carotene, had the lowest mortality. Mutant car 17 which accumulates mainly §–carotene had an intermediate mortality rate while the albino mutant, car 3, which contains only phytoene, lost viability most rapidly. Wild-type cells treated with diphenylaminc, a carotenogenic inhibitor, were killed at the same rate as mutant car 3. The survival rate of mutants on exposure to sunlight was dependent upon the chromophore length of the accumulated carotenoids. The 3 strains showed no difference in rate of mortality when exposed to ultraviolet light. Protection from sunlight by accumulation of carotenes may be an important ecological factor for this species whose natural habitat is tidepools.     

Characterization of carotenes in a combination of a C18 HPLC column with isocratic elution and absorption spectra with a photodiode-array detector

Carotenes have attracted much attention in recent years for their biological function in processes such as photosynthesis. The characterization of carotenes is difficult, however, because they consist of only carbon and hydrogen atoms, without oxygen. In the present study, we systematically examined the chemical structures of more than 30 carotenes, including most of the carotenes found in phototrophic organisms, and observed their elution order using a Novapak C₁₈ HPLC column with simple isocratic elution. The elution order of the carotenes was C₃₀, C₄₀,C₄₅ then C₅₀. The C₄₀ carotenes with fewer conjugated double bonds (N) had longer retention times. With respect to the end groups, the carotenes eluted in the following order: φ, Ψ, ∈ then β end groups. Furthermore, absorption spectra in the HPLC eluent used were recorded with a photodiode-array detector. A greater N value was associated with a longer absorption maximum wavelength. Since the conjugated end groups (φ and β) influenced the absorption spectra and the non-conjugated end groups (Ψ and ∈) did not, the number of conjugated end groups (zero, one and two) was clearly distinguishable. Therefore, the chemical structures of carotenes can be easily determined by a combination of the HPLC retention times and the absorption spectra. This revised version was published online in June 2006 with corrections to the Cover Date.     

高产虾青素红发夫酵母选育研究进展

虾青素是 60 0多种类胡萝卜素中的一种 ,具有抗氧化 ,抗肿癌和增强免疫力等许多重要的生理和生物学功能 ,在水产养殖、食品和医药等领域应用前景广阔。综述了虾青素的生物来源、生物转化途径以及高产虾青素红发夫酵母菌株的选育。     

Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods

The growth and carotenoid production of Phaffia rhodozyma in fed-batch cultures with different feeding methods and grown at specific growth rates similar to the batch culture was compared. With constant feeding, exponential feeding, DO-stat and pH-stat fed-batch cultures of Phaffia rhodozyma, the highest biomass (17.4 g/l) and lowest carotenoid content (307 g/g cell) of Phaffia rhodozyma was from the DO-stat fed-batch culture. The lowest biomass (14.7 g/l) and highest carotenoid content (412 g/g cell) was from the exponential, fed-batch culture.     

Metabolic engineering of the terpenoid biosynthetic pathway of Escherichia coli for production of the carotenoids β-carotene and zeaxanthin

Metabolic engineering of the early non-mevalonate terpenoid pathway of Escherichia coli was carried out to increase the supply of prenyl pyrophosphates as precursor for carotenoid production. Transformation with the genes dxs for over-expression of 1-deoxy-d-xylulose 5-phosphate synthase, dxr for 1-deoxy-d-xylulose 5-phosphate reductoisomerase and idi encoding an isopentenyl pyrophosphate stimulated carotenogenesis up to 3.5-fold. Co-transformation of idi with either dxs or dxr had an additive effect on ß-carotene and zeaxanthin production which reached 1.6 mg g^–1 dry wt.     

Spirilloxanthin is released by detergent from Rubrivivax gelatinosus reaction center as an aggregate with unusual spectral properties

A preparation containing spirilloxanthin has been isolated from Rubrivivax gelatinosus SC2, a mutant devoid of the reaction center-associated tetraheme cytochrome c, after solubilisation of membranes with lauryl-di-methyl-amine oxide. It was purified by ammonium sulfate precipitation and gel filtration, and analyzed by SDS-gel electrophoresis. Spirilloxanthin was shown to be aggregated in large particles (apparent M_W > 600 kDa) and was not associated with a specific protein. This aggregate was characterized by absorption, circular dichroism and resonance Raman spectroscopies. The absorption spectrum contained two UV bands at 370 and 300 nm, and did not present the visible bands of spirilloxanthin, which however reappeared when spirilloxanthin was extracted from the aggregate with organic solvents. Resonance Raman spectra indicated that at least four different populations of spirilloxanthin were present in the preparation as a mixture of different trans and cis configurations. These properties are similar to those described for a so-called carotenoprotein solubilized with sodium dodecyl sulfate from Rhodospirillum rubrum membranes [Schwenker et al. (1974) Biochim Biophys Acta 351: 246-260; Kito et al. (1983) Photochem Photobiophys 5: 209-217]. We further observed absorption spectra of pure spirilloxanthin dissolved in mixtures of water, polar solvents and detergent, in the absence of protein, resembling those of the.aggregate. We conclude that the aggregate is not a carotenoprotein, but rather an artefact due to the release of spirilloxanthin from the reaction center, leading to the isomerization and association of spirilloxanthin molecules in a detergent particle. We propose the same interpretation for the complex isolated from Rhodospirillum rubrum.