American Cockroach (Periplaneta americana) Synthesizes Carotenoids from the Precursor [14C]Mevalonic Acid Pyrophosphate

The level of an important carotenoid (-carotene) in the gut of Periplaneta americana depends on the content of the carotenoid in food: a carotenoid-fortified diet causes accumulation of -carotene up to 10 g/g wet weight, while on a carotenoid-deficient diet the level of this substance is low (0.7 g/g wet weight). In the eye, in contrast to the gut, a constant level of -carotene (1.3-1.4 g/g wet weight) is found regardless of the diet. This phenomenon remained unchanged over three years of feeding of the cockroaches with the carotenoid-deficient diet, suggesting that P. americana produces carotenoids by de novo biosynthesis. This suggestion was confirmed in experiments using intraperitoneal injection of the exogenous carotenoid biosynthesis precursor [¹⁴C]mevalonic acid pyrophosphate followed by extraction of carotenoid and chromatographic purification of the labeled product. Injection of 3.4 nmoles [¹⁴C]mevalonic acid pyrophosphate transiently increased the -carotene content in eyes on days 2 and 4 after injection of the label. Purification of radiolabeled carotenoids from eye and gut by the transfer of carotenoids into a less polar solvent, alkaline hydrolysis (saponification), and chromatography on alumina and cellulose columns decreased the specific radioactivity to a constant level that cannot be further decreased by repeated chromatography. The elution profile of these purified preparations of -carotene after chromatography is characterized by coincidence of symmetric peaks of count and absorption. We suggest that to create the optimal carotenoid concentration in the eye, P. americana uses two biochemical mechanism: 1) it accumulates carotenoids in reserve in the gut when abundant supplies of carotenoids are available in the diet; 2) it synthesizes carotenoids de novo when its food is deficient in these compounds.     

青霉PT95菌固态发酵产生类胡萝卜素的研究

本文对青霉Ｐｅｎｉｃｉｌｌｉｕｍｓｐ．ＰＴ９５菌株在固态发酵条件下菌核内产生类胡萝卜素进行了初步研究。结果表明,在３种固态发酵培养基中,玉米粉培养基（ＳＭＡ）比麸皮２基和棉籽壳培养基更适合于ＰＴ９５菌株固态发酵产生类胡萝卜素。为了增加菌核干重和提高类胡萝卜素产率,ＳＭＡ中需要添加氮源、碳源和植物油。在所度的各种氮、碳源中,以硝酸钠和麦芽糖效果最佳。通过我试验确定了在培养基盐溶液中添加硝酸钠３ｇ／Ｌ     

Carotenoids and fatty acids in red yeasts Sporobolomyces roseus and Rhodotorula glutinis

Rhodotorula glutinis and Sporobolomyces roseus, grown under different aeration regimes, showed differential responses in their carotenoid content. At higher aeration, the concentration of total carotenoids increased relative to biomass and total fatty acids in R. glutinis, but the composition of carotenoids (torulene > beta-carotene > gamma-carotene > torularhodin) remained unaltered. In contrast, S. roseus responded to enhanced aeration by a shift from the predominant beta-carotene to torulene and torularhodin, indicating a biosynthetic switch at the gamma-carotene branch point of carotenoid biosynthesis. The overall levels of total carotenoids in highly aerated flasks were 0.55 mol-percent and 0.50 mol-percent relative to total fatty acids in R. glutinis and S. roseus (respectively), and 206 and 412 microg g(-1) dry weight (respectively).     

Production of canthaxanthin by <Emphasis Type="Italic">Haloferax alexandrinus</Emphasis> under non-aseptic conditions and a simple,rapid method for its extraction

The production of carotenoids from Haloferax alexandrinus strain TM(T) was investigated at various concentrations of NaCl (10-25%) in culture media under non-aseptic conditions. PCR and dot blot hybridization assays were employed to monitor the growth of Hfx. alexandrinus in the culture under aseptic and non-aseptic conditions. The amplified PCR products of 16S rDNA from Hfx. alexandrinus grown under aseptic conditions were used as specific probes, which bound with amplified PCR products of 16S rDNA dots from both aseptic and non-aseptic conditions (20-25% NaCl). The results indicated that contamination of the culture was precluded at high NaCl concentrations (20-25%). Therefore, it is not necessary to perform asepsis during the biotechnological processes of carotenoid production by Hfx. alexandrinus. A 1-l-scale cultivation of the cells in flask cultures under non-aseptic conditions produced 3.12+/-0.5 g dry weight, 6.34+/-2.5 mg total carotenoids and 2,156.67+/-0.1 microg canthaxanthin. Further experiments in a batch fermenter, under non-aseptic conditions, also demonstrated increases in the biomass concentration and carotenoid production. When grown in a standard growth medium at 25% NaCl, the cells of Hfx. alexandrinus lysed spontaneously in fresh water and hence carotenoids could be extracted directly from the cells without any mechanical disintegration. These results demonstrate the feasibility and simplicity of commercial production of carotenoids using Hfx. alexandrinus.     

Production of carotenoids byPhaffia rhodozyma grown on media composed of corn wet-milling co-products

Summary Natural isolates of the carotenoid-producing yeastPhaffia rhodozyma were analyzed for their ability to grow and to produce carotenoids in culture media composed exclusively of co-products of corn wet-milling for fuel ethanol production. FiveP. rhodozyma strains were tested for biomass produced (dry weight) and carotenoid yield. Six co-products were examined, ranging in cost from approximately $0.02 per kg to $0.11 per kg, all less expensive than conventional or agricultural growth substrates previously tested. The three co-products allowing the greatest accumulation of biomass and carotenoids byP. rhodozyma were thin stillage (TS), corn condensed distiller's solubles (CCDS) and corn gluten feed (CGF). Of the medium compositions tested, 10–15% CGF, 70% TS and 6–8% CCDS generally allowed maximum carotenoid production. Cultures grown in these three media produced up to 65%. 148% and 104% of the carotenoid yield per ml of yeast extract/malt extract (YM) cultures, respectively. Under the conditions tested, this was at an approximate medium cost of $0.67 per g carotenoids for CCDS and $0.73 per g for CGF as compared to $385.00 per g for YM. These results indicate that certain co-products of corn wet-milling can serve, at the appropriate concentration, as efficient, economical substrates for growth and carotenoid production byPhaffia rhodozyma.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Torularhodin and torulene are the major contributors to the carotenoid pool of marine Rhodosporidium babjevae (Golubev)

A carotenoid-producing yeast strain, isolated from the sub-arctic, marine copepod Calanus finmarchicus, was identified as Rhodosporidium babjevae (Golubev) according to morphological and biochemical characteristics and phylogenetic inference from the small-subunit ribosomal RNA gene sequence. The total carotenoids content varied with cultivation conditions in the range 66–117 μg per g dry weight. The carotenoid pool, here determined for the first time, was dominated by torularhodin and torulene, which collectively constituted 75–91% of total carotenoids under various regimes of growth. β-Carotene varied in the range 5–23%. A high-peptone/low-yeast extract (weight ratio 38:1) marine growth medium favoured the production of torularhodin, the carotenoid at highest oxidation level, with an average of 63% of total carotenoids. In standard yeast medium (YM; ratio 1.7:1), torularhodin averaged 44%, with increased proportions of the carotenes, torulene and β-carotene. The anticipated metabolic precursor γ-carotene (β,ψ-carotene) constituted a minor fraction (≤8%) under all conditions of growth.     

Inhibition of lipid synthesis and glucose-6-phosphate dehydrogenase in rat skin by dehydroepiandrosterone

Lipid synthesis from acetate-1-(14)C by rat skin was inhibited 44-56% by 2.5 x 10(-4) m dehydroepiandrosterone (DHA) in vitro with or without addition of glucose in the incubation medium. This inhibition affected all the lipid fractions examined (hydrocarbons, sterols, sterol esters, tri-, di- and monoglycerides, fatty acids, and polar lipids) and could be reversed by NADPH. DHA also inhibited lipid synthesis from glucose-U-(14)C and the formation of (14)CO(2) from glucose-1-(14)C, indicating interference with pentose cycle activity. Experiments with the 105,000 g supernatant fluid of rat skin homogenates demonstrated considerable activities of malic enzyme (ME) (12.6 nmoles of NADPH generated per min per mg of protein), of glucose-6-phosphate dehydrogenase (G6PD), and of 6-phosphogluconate dehydrogenase (6PGD) (17.5 nmoles of NADPH generated per min per mg of protein). G6PD was inhibited 98% by 2.5 x 10(-4) m dehydroepiandrosterone, while 6PGD and ME were not affected. It can be estimated from these data that the pentose cycle may contribute 41-57% of the NADPH needed for lipid synthesis in rat skin; the remainder of the necessary NADPH is presumably supplied by malic enzyme.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Adaptation to sub- and supraoptimal temperatures of inbred maize lines differing in origin with regard to seedling development and photosynthetic traits

Six inbred lines of maize ( Zea mays L.) from cool temperate regions (C) and from warm regions (W) were grown at 14, 22, 30 and 38°C up to the same physiological age, the full expansion of the third leaf. Generally, plants developed smaller shoot dry weights and leaf areas at extreme temperatures. The shoot:root ratio was lowest at 22°C and highest at 30°C. Most lines had a minimum for specific leaf dry weight at 30°C, but W lines had a second lower minimum at 14°C. Phosphofructokinase activity scarcely reacted to temperature between 22° and 38°C; at 14°C one C line and all W lines had rather low activities. Generally, the chlorophyll content increased steeply from 14 to 22°C and decreased somewhat from 30 to 38°C. In C lines the carotenoid level decreased from 14 to 38°C. No uniform temperature response was found for PEP carboxylase activity, but the highest activity was mostly attained at 38°C. RuBP carboxylase activity increased considerably from 14 to 22°C and remained comparatively constant at higher temperatures. The highest activity of NADP malate dehydrogenase was found at 22°C, with a decrease up to 38°C and with second lowest values at 14°C. C lines possessed larger leaf areas, shoot dry weights and higher shoot:root ratios than W lines at 14 and 22°C, and higher specific leaf dry weights over the whole temperature range. The genotypic pattern of shoot dry weight at 14°C corresponded reasonably well with that of phosphofructokinase activity. A better adaptation of C lines to suboptimal temperatures was mostly clearly indicated for photosynthetic traits which have a well proven relationship with the chloroplast membranes: chlorophyll, carotenoids and RuBP carboxylase. The least distinct effects of origin were observed at 38°C; a tendency prevailed for a better performance of C lines with regard to phosphofructokinase, carotenoids, RuBP carboxylase and NADP malate dehydrogenase.     

Increased carotenoid production in Xanthophyllomyces dendrorhous G276 using plant extracts

The red yeast Xanthophyllomyces dendrorhous (previously named Phaffia rhodozyma) produces astaxanthin pigment among many carotenoids. The mutant X. dendrorhous G276 was isolated by chemical mutagenesis. The mutant produced about 2.0 mg of carotenoid per g of yeast cell dry weight and 8.0 mg/L of carotenoid after 5 days batch culture with YM media; in comparison, the parent strain produced 0.66 mg/g of yeast cell dry weight and a carotenoid concentration of 4.5 mg/L. We characterized the utilization of carbon sources by the mutant strain and screened various edible plant extracts to enhance the carotenoid production. The addition of Perilla frutescens (final concentration, 5%) or Allium fistulosum extracts (final concentration, 1%) enhanced the pigment production to about 32 mg/L. In a batch fermentor, addition of Perilla frutescens extract reduced the cultivation time by two days compared to control (no extract), which usually required five-day incubation to fully produce astaxanthin. The results suggest that plant extracts such as Perilla frutescens can effectively enhance astaxanthin production.     

Accumulation of astaxanthin and lutein in<Emphasis Type="Italic"> Chlorella zofingiensis</Emphasis> (Chlorophyta)

When grown photoautotrophically, Chlorella zofingiensis strain CCAP 211/14 accumulates a significant amount of valuable carotenoids, namely astaxanthin and lutein, of increasing demand for use as feed additives in fish and poultry farming, as colorants in food, and in health care products. Under standard batch-culture conditions, this microalgal strain exhibits high values of both growth rate (about 0.04 h^–1) and standing cell population (over 10¹¹ cells l^–1, or 7 g dry weight l^–1). Lutein, in a free (unesterified) form, was the prevalent carotenoid during early stages of cultivation (over 0.3 pg cell^–1, equal to 4 mg g^–1 dry weight, or 20 mg l^–1 culture), whereas esterified astaxanthin accumulated progressively, to reach a maximum (over 0.1 pg cell^–1, equal to 1.5 mg g^–1 dry weight, or 15 mg l^–1 culture) in the late stationary phase. A differential response of lutein and astaxanthin accumulation was also recorded with regard to the action of some environmental and nutritional factors. C. zofingiensis CCAP 211/14 represents a unique model system for analyzing the differential regulation of the levels of primary (lutein) and secondary (astaxanthin) carotenoids. Relevant also from the biotechnological viewpoint, this photosynthetic organism, with outstanding attributes for fast photosynthetic growth and carotenoid accumulation, might prove most valuable for its application to the mass production of either or both lutein and astaxanthin.     

Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.     

Sclerotia growth and carotenoid production of Penicillium sp PT95 during solid state fermentation of corn meal

Using corn meal as fermentation substrate, the effect of some factors, fermentation time and supplementation of saccharide and nitrogen sources as well as vegetable oil, on the sclerotia growth and carotenoid production of Penicillium sp PT95 during solid state fermentation were studied. When PT95 strain was grown on the amended medium by supplementing of 3g NaNO3, 10g maltose and 2.5g soybean oil per liter of salt solution to basal medium for 20 days, the dry sclerotia weight and carotenoid yield reached 9.70 g and 5260 g / 100 g of substrate, respectively. Without supplementation only 5.36g dry sclerotia and 2149g carotenoid / 100 g of substrate was attained. © Rapid Science Ltd. 1998     

Carotenoids of clam,coral and nudibranch zooxanthellae in aposymbiotic culture

The carotenoids of unialgal cultures originating from symbiotic zooxanthellae of two molluscan (Tridacna crocea, a giant clam, and Pteraeolidia ianthine a nudibranch) and one cnidian (Pseudopterogorgia bipinnata, a gorgonian coral) host have been analysed by HPLC or TLC procedures combined with several spectroscopic techniques including MS and NMR. A high total carotenoid content (0.45-0.63% of the dry wt) was obtained. The carotenoid pattern with C₃₇-norcarotenoids (peridinin and pyrrhoxanthin) comprising around 80% of total carotenoids, and β,β-carotene (2%), the ailenic dinoxanthin (3–4%) and the acetylenic diatoxanthin (1–3%) and diadinoxanthin (7–9%) representing minor C₄₀-carotenoids, corresponds to that of peridinin-producing free-living dinoflagellates. Supplementary ¹H NMR and ¹³C NMR data are reported for peridinin and pyrrhoxanthin. A polar, minor carotenoid, P447, was partly characterized as containing a disaccharide glycosidically bound to an allenic carotenoid aglycone. Re-evaluation of previous reports suggests the wide-spread occurrence of related carotenoid disaccharides in Dinophyceae for which they are considered a new chemosystematic marker.     

Exploitation of heterosis for carotenoid content in African marigold (<Emphasis Type="Italic">Tagetes erecta</Emphasis> L.) and its correlation with esterase polymorphism

African marigold (Tagetes erecta L.), a major source of carotenoids, is also grown as a cut flower and a garden flower in addition to being grown for its medicinal values. We studied gene action, combining ability and heterosis, aiming at genetic improvement of T. erecta for enhanced carotenoid content in petals, and report for the first time that heterosis can be exploited for total carotenoids and its commercially important fractions. Total content of carotenoids and lutein appears to be governed by dominance (or non-additive) gene action, while content of xanthophyll esters is governed by both additive and dominance (or non-additive) gene actions. Specific combining ability variance was predominant for all the three traits. General and specific combining abilities and heterosis were highly significant. Heterobeltiosis was also positive. General combining ability (GCA) variances were not significantly correlated to performance per se. There was also no correlation between performance per se of normal petalled pollen parents and the performance of crosses made between male-sterile (female) and male-fertile (pollen) parents. These findings suggest that carotenoid content should not be the only criterion considered in the selection of parental lines. Studies on esterase in seeds and peroxidase in seedlings revealed a relatively high level of polymorphism in esterase with a total of 14 isoforms, whereas peroxidase showed low polymorphism. Similarity indices between different parental combinations, calculated based on seed esterase polymorphism, showed a significant negative correlation (r = -0.479, P = 0.05) with heterosis for carotenoid content. This indicates that the selection of parents with wider variation in their esterase profiles may possibly be exploited for genetic enhancement of carotenoids in T. erecta.     

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background

As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.

Methology/Principal Findings

In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.

Conclusions

The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.     

Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

Studies in Carotenogenesis. Some Observations on Carotenoid Synthesis in Two Varieties of Euglena gracilis.

Euglena gracilis v. bacillaris and E. gracilis v. fuscopunctata produce the same three carotenoids, β-carotene, lutein and neoxanthin in approximately the same relative amounts. Lutein is the major pigment and β-carotene represents about 10–15% of the total. The concentration of carotenoids in E. gracilis v. bacillaris is very high, reaching 700 mg./100 g. dry weight.
Streptomycin (0.02%, w/v) and darkness reduced growth of E. gracilis v. bacillaris by about 50% and carotenoid synthesis by about twenty times. Diphenylamine (1/70,000) reduced growth and carotenoid synthesis equally (about 50%). In no case was the synthesis of more saturated polyenes (the phytofluene series) stimulated.
The nature of the eye spot pigment is discussed.     

Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions

The microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in salmon feed. This study was made to discover optimal conditions for biomass and astaxanthin production in H. pluvialis from Steptoe, Nevada (USA), cultured in batch mode. Growth was carried out under autotrophic (with NaNO3, NH4Cl and urea) and mixotrophic conditions (with 4, 8, 12 mM sodium acetate) under two photon flux densities (PFD) (35 and 85 mumol m-2 s-1). The carotenogenesis was induced by 1) addition of NaCl (0.2 and 0.8%), 2) N-deprivation and 3) high PFD (150 mumol m-2 s-1). Total carotenoids were estimated by spectrophotometry and total astaxanthin by HPLC. Ammonium chloride was the best N-source for growth (k = 0.7 div day-1, 228-258 mg l-1 and 2.0 x 10(5)-2.5 x 10(5) cells ml-1 at both PFD, respectively). With increasing acetate concentration, a slight increment in growth occurred only at 85 mumol m-2 s-1. Light was the best inductive carotenogenic factor, and the highest carotenoid production (4.9 mg l-1, 25.0 pg cell-1) was obtained in cultures pre-grown in nitrate at low light. The NaCl caused an increase in carotenoid content per cell at increasing salt concentrations, but resulted in a high cell mortality and did not produce any increment in carotenoid content per volume compared to cultures grown at 150 mumol m-2 s-1. The highest carotenoid content per cell (22 pg) and astaxanthin content per dry weight (10.3 mg g-1) (1% w/w) were obtained at 85 mumol m-2 s-1 with 0.8% NaCl.