Improved production of lycopene and β-carotene by Blakeslea trispora with oxygen-vectors

Lycopene and β-carotene production were increased when oxygen-vectors, n-hexane and n-dodecane, were added to cultures of Blakeslea trispora because of the enhanced dissolved oxygen concentrations. With 1% (v/v) n-hexane or n-dodecane added in the medium, lycopene production was 51% or 78% higher and β-carotene production was 44% or 65% higher than that of the control, respectively. The highest lycopene and β-carotene production, 533 mg l⁻¹and 596 mg l⁻¹, were obtained when 1% (v/v) n-dodecane and 0.1% (w/v) Span 20 were added together, which were 2.1-fold and 1.8-fold of the control, respectively.     

Stimulation of β-carotene synthesis by hydrogen peroxide in Blakeslea trispora

-Carotene synthesis was increased from a negligible amount to 152 mg (g-dry cells)^–1 and H₂O₂ was accumulated up to 16.7 M during 2.5 day-culture of Blakeslea trispora. When cells were cultivated in 250 ml flasks containing various volumes (25–150 ml) of the medium, not only H₂O₂ accumulation but also -carotene synthesis increased as culture volume decreased. Addition of H₂O₂ (10 M) to the 1.5-day old cultures of B. trispora resulted in 46% higher -carotene synthesis than that without addition. All these results indicate that -carotene biosynthesis is stimulated by H₂O₂ in B. trispora.     

Oxidative stress response of Blakeslea trispora induced by H2O2 during β-carotene biosynthesis

The cellular response of Blakeslea trispora to oxidative stress induced by H₂O₂ in shake flask culture was investigated in this study. A mild oxidative stress was created by adding 40 μm of H₂O₂ into the medium after 3 days of the fermentation. The production of β-carotene increased nearly 38 % after a 6-day culture. Under the oxidative stress induced by H₂O₂, the expressions of hmgr, ipi, carG, carRA, and carB involving the β-carotene biosynthetic pathway all increased in 3 h. The aerobic metabolism of glucose remarkably accelerated within 24 h. In addition, the specific activities of superoxide dismutase and catalase were significantly increased. These changes of B. trispora were responses for reducing cell injury, and the reasons for increasing β-carotene production caused by H₂O₂.     

Effect of oxygen transfer rate on β-carotene production from synthetic medium by Blakeslea trispora in shake flask culture

The effect of oxygen transfer rate (OTR) on β-carotene production by Blakelsea trispora in shake flask culture was investigated. The results indicated that the concentration of β-carotene (704.1 mg/l) was the highest in culture grown at maximum OTR of 20.5 mmol/(l h). In this case, the percentage of zygospores was over 50.0% of the biomass dry weight. On the other hand, OTR level higher than 20.5 mmol/(l h) was found to be detrimental to cell growth and pigment formation. To elucidate the effect of oxidative stress on β-carotene synthesis, the accumulation of hydrogen peroxide during fermentation under different OTRs was determined. A linear response of β-carotene synthesis to the level of H₂O₂ was observed, indicating that β-carotene synthesis is stimulated by H₂O₂. However, there was an optimal concentration of H₂O₂ (2400 μM) in enhancing β-carotene synthesis. At a higher concentration of H₂O₂, β-carotene decreased significantly due to its toxicity.     

Increased β-carotene synthesis in Blakeslea trispora under strong alkaline culture condition

Summary A high yield of -carotene, 48 mg/l, was obtained by cultivation of a strain of Blakeslea trispora under a strong alkaline culture condition (pH 10), which was significantly higher than those obtained under milder pH conditions (3.26–7.80 mg/l at pH 4 to 8). The formation of small pellets at pH 10 was observed, whereas a single large cluster was formed for the pHs ranged from 4 to 8. Span 20 at 0.1 to 1% in the culture medium changed cellular morphology to micro-pellets regardless of the culture pH. The same trend of -carotene production, depending on the alkalinity of the culture broth, was also observed in the presence of 1% of Span 20 yielding much higher productivity. The amounts of -carotene yielded at pH 10 and 11 were 92.0 and 100.9 mg/l, respectively, which were about two-times higher than those obtained at pH 4 to 9.     

Comparative effects of β-carotene and fucoxanthin on retinol deficiency induced oxidative stress in rats

This study aimed at comparing antioxidant potential of fucoxanthin (FUCO) with β-carotene in relieving lipid peroxidation (Lpx) caused by retinol deficiency (RD) in rats. RD rats (n = 45) were fed a dose of either β-carotene (0.81 μmol) or FUCO (0.83 μmol). Plasma and liver lipid peroxide levels and activity of antioxidant enzymes catalase (CAT) and glutathione transferase (GST) were measured for 8 h. Results revealed that RD increased (P < 0.05) Lpx in plasma and liver by 34.3% and 19.4%, while the CAT activity in plasma (89%) and liver microsomes (91%) and GST in liver homogenate (31%) and liver microsomes (30%) were decreased (P < 0.05) compared to control (rats fed basal diet). FUCO suppressed (P < 0.05) the Lpx level by 7–85% (plasma) and 24–72% (liver) as compared to β-carotene (51–76%, 33–65%) over a period of 8 h. The activity of CAT in plasma and liver microsomes was higher (P < 0.05) in FUCO (90–95%, 85–93%) and β-carotene (87–96%, 79–91%) groups as compared to RD group. Similarly, the activity of GST in liver and its microsomes was also elevated (P < 0.05) in FUCO (44–51%, 22–51%) and β-carotene (19–54%, 30–43%) groups as compared to RD group. Results demonstrate that FUCO has greater potential than β-carotene in modulating Lpx, CAT, GST in plasma and liver of RD rats.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Spectroscopy analysis for simultaneous determination of lycopene and β-carotene in fungal biomass of Blakeslea trispora

Blakeslea trispora is a good alternative source for producing such carotenoids as lycopene and β-carotene. The objective of this research was to elaborate a method for the simultaneous determination of lycopene and β-carotene in Blakeslea trispora products using a usual UV-vis spectrophotometer. The standard solutions of the mixture of different concentrations of β-carotene and lycopene were measured with the UV-vis method and correlation formula for the extinction coefficients of 1% standard solution of lycopene in the solvent (hexane) and the ratios of the optical densities at the character peaks of 470 and 502 nm was elaborated. This gives a possibility to calculate the concentrations of lycopene and β-carotene in the mixture. The prediction quality of the UV-vis method was sufficient and the obtained results were very close to the ones, being measured with the HPLC technique. The proposed method can be used for both routine industrial work and academic research, providing the rapid analysis for simultaneous measurements of lycopene and β-carotene.     

Strain selection for β-carotene production by Dunaliella

Four strains of Dunaliella were grown at 25°C and pH 8±0.5, with continous illumination at 200 W/m². Their maximum specific growth rates ranged from 0.093 day^-1 to 0.234 day^-1, nitrate yields from 3.0 to 7.8 g cells/g NaNO₃ and lipid contents from 3% to 6% of the dry wt, with carotenes 50 to 80% of the lipids. Of the carotenes, -carotene made up 7 to 19%; all-trans--carotene 32 to 52% and 9-cis--carotene 29 to 55%. There are, therefore, considerable intra-specific differences between strains of Dunaliella.     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Molecular cloning and functional expression of two key carotene synthetic genes derived from Blakeslea trispora into E. coli for increased β-carotene production

Blakeslea trispora is used commercially to produce β-carotene. Isopentenyl pyrophosphate isomerase (IPI) and geranylgeranyl pyrophosphate synthase (GGPS) are key enzymes in the biosynthesis of carotenoids. The cDNAs of genes ipi and carG were cloned from the fungus and expressed in Escherichia coli. Greater GGPS activity was needed in the engineered E. coli when IPP activity was increased. The introduction of GGPS and IPI increased the β-carotene content in E. coli from 0.5 to 0.95?mg/g dry wt.     

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> during β-carotene production in submerged fermentation

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during β-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of β-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during β-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased β-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.     

Thymoquinone ameliorates chemical induced oxidative stress and β-cell damage in experimental hyperglycemic rats

The present study was aimed to investigate the effect of thymoquinone (TQ) on pancreatic insulin levels, tissue antioxidant and lipid peroxidation (LPO) status in streptozotocin (STZ) nicotinamide (NA) induced diabetic rats. Diabetes was induced in experimental rats by a single intraperitoneal (i.p) injection of STZ (45 mg/kg b.w) dissolved in 0.1 mol/L citrate buffer (pH 4.5), 15 min after the i.p administration of NA (110 mg/kg b.w). Diabetic rats exhibited increased blood glucose with significant decrease in plasma insulin levels. The activities of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and the levels of low-molecular weight antioxidants Vitamin C, Vitamin E and reduced glutathione (GSH) were decreased while increases in the levels of lipid peroxidation markers were observed in liver and kidney tissues of diabetic control rats as compared to control rats. In addition, diabetic rats showed an obvious decrease in pancreatic insulin levels. Administration of TQ (80 mg/kg b.w) to diabetic rats for 45 days significantly reversed the damage associated with diabetes. Biochemical findings were supported by histological studies. These results indicated that TQ exerts a protective action on pancreatic beta cell function and overcomes oxidative stress through its antioxidant properties.     

Retinal production from β-carotene by β-carotene 15,15′-dioxygenase from an unculturable marine bacterium

The conversion of β-carotene to retinal by a recombinant β-carotene 15,15′-dioxygenase (Blh protein) from an unculturable marine bacterium was optimized in aqueous solution. Toluene was optimal solvent for the dissolution of β-carotene and the optimal solution for the conversion reaction contained 2.4% (w/v) Tween 20, 0.15 U enzyme/ml, and 350 mg β-carotene/l. Under these conditions, the enzyme produced 181 mg retinal/l after 20 h. This is the highest reported value for the retinal concentration from β-carotene.     

The protective effect of peroxiredoxin II on oxidative stress induced apoptosis in pancreatic β-cells

Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.