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).     

Use of metabolic stimulators and inhibitors for enhanced production of beta-carotene and lycopene by Blakeslea trispora NRRL 2895 and 2896

This work investigates the potential of metabolic stimulators, firstly to enhance the production of beta-carotene, and later use of inhibitors of lycopene cyclase so as to accumulate lycopene in the fermentation medium. Various non-ionic surfactants, natural oils, stimulators such as amino-acids, tricarboxylic acid cycle (TCA) intermediates, vitamin A and antibiotics were investigated for improved production of beta-carotene using the zygomycete fungus Blakeslea trispora. Span 20 at 0.2% increased the beta-carotene production from 139 mg/l to 318 mg/l. Examination of the mycelial morphology of the B. trispora with span 20 showed a shorter mycelial length, which allowed a well-dispersed growth of B. trispora. Supplementation of the medium with 1000 ppm vitamin A acetate gave highest concentration of beta-carotene (830+/-6 mg/l). Several chemical inhibitors such as imidazole, pyridine, triethylamine, piperidine, and nicotinic acid were then evaluated to block the biosynthesis at lycopene. Piperidine at 500 ppm gave a 7.76-fold improvement, and produced high titers of lycopene (775+/-5 mg/l) in a medium supplemented with vitamin A acetate.     

Dietary supplementation with beta-carotene, but not with lycopene, inhibits endothelial cell-mediated oxidation of low-density lipoprotein.

Carotenoids may protect low-density lipoprotein from oxidation, a process implicated in the development of atherosclerosis. Our previous studies showed that in vitro enrichment of low-density lipoprotein (LDL) with beta-carotene protected it from cell-mediated oxidation. However, in vitro enrichment with either lutein or lycopene actually enhanced oxidation of the LDL. In the present studies we have examined the impact of LDL carotenoid content on its oxidation by human aortic endothelial cells (EaHy-1) in culture, comparing the effects of in vivo supplementation with in vitro enrichments. The beta-carotene content in human LDL was increased three- to sixfold by daily supplementation with 15 mg beta-carotene for 4 weeks, and the lycopene content of LDL in other individuals was increased two- to threefold by ingestion of one glass (12 ounce) of tomato juice daily for 3 weeks. LDL isolated from these healthy, normolipidemic donors not taking supplemental carotenoid was incubated at 0.25 mg protein/ml with EaHy-1 cells in Ham's F-10 medium for up to 48 h. Following dietary beta-carotene supplementation, LDL oxidation (as assessed by formation of lipid hydroperoxides) was markedly inhibited, to an even greater extent than was observed for LDL enriched in vitro with beta-carotene (that resulted in an 11- to 12-fold increase in LDL beta-carotene). No effect on cell-mediated oxidation was observed, however, for LDL enriched in vivo with lycopene. Thus, beta-carotene appears to function as an antioxidant in protecting LDL from cell-mediated oxidation although lycopene does not. The fact that the three- to sixfold enrichments of LDL with beta-carotene achieved by dietary supplementation were more effective in inhibiting oxidation than the 11- to 12-fold enrichments achieved by an in vitro method suggests that dietary supplementation is a more appropriate procedure for studies involving the enrichment of lipoprotein with carotenoids.     

Increased beta-carotene production in recombinant Escherichia coli harboring an engineered isoprenoid precursor pathway with mevalonate addition

When pT-LYCm4 containing lycopene synthetic genes was co-transformed with pSUcrtY or pSHcrtY containing crtY gene of Pantoea ananatis (P. ananatis) or Pantoea agglomerans (P. agglomerans), beta-carotene productions of 36 and 35 mg/L were obtained, respectively. No lycopene was detected in the beta-carotene production culture. pT-HB, constructed by addition of P. ananatis crtY gene into pT-LYCm4, was used for co-transformation with pSdxs and pSSN12Didi, which increased isopentenyl diphosphate and dimethylallyl diphosphate synthesis. beta-Carotene production significantly increased 1.5-fold (51 mg/L) with the amplification of the dxs gene through pSdxs and 4-fold (135 mg/L) with the mevalonate bottom pathway of pSSN12Didi in the presence of 3.3 mM mevalonate. The pT-DHB, constructed by integrating the dxs gene into pT-HB, was used for cotransformation of Escherichia coli (E. coli) harboring pSSN12Didi, resulting in beta-carotene production of 141 mg/L. Recombinant E. coli harboring pT-DHB and pSSN12Didi was used to maximize beta-carotene production by adjusting the available amounts of glycerol, a carbon source, and mevalonate, the precursor of the mevalonate bottom pathway. When recombinant E. coli was given 16.5 mM mevalonate and 2.5% (w/v) glycerol, beta-carotene production of 503 mg/L in concentration and 49.3 mg/g DCW in content was obtained at 144 h, which was the highest level of carotenoid production in E. coli ever reported in the literature.     

Ergosterol production from molasses by genetically modified <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Ergosterol is an economically important metabolite produced by fungi. Recombinant Saccharomyces cerevisiae YEH56(pHXA42) with increased capacity of ergosterol formation was constructed by combined overexpression of sterol C-24(28) reductase and sterol acyltransferase in the yeast strain YEH56. The production of ergosterol by this recombinant strain using cane molasses (CM) as an inexpensive carbon source was investigated. An ergosterol content of 52.6 mg/g was obtained with 6.1 g/l of biomass from CM medium containing 60 g/l of total sugar in 30 h in shake flask. The ergosterol yield was enhanced through the increasing cell biomass by supplementation of urea to a concentration of 6 g/l in molasses medium. Fermentation was performed in 5-l bioreactor using the optimized molasses medium. In batch fermentation, the effect of agitation velocity on ergosterol production was examined. The highest ergosterol yield was obtained at 400 rpm that increased 60.4 mg/l in comparison with the shake flask culture. In fed-batch fermentation, yeast cells were cultivated, firstly, in the starting medium containing molasses with 20 g/l of total sugar, 1.68 g/l of phosphate acid, and 6 g/l of urea (pH 5.4) for 5 h, then molasses containing 350 g/l of total sugar was fed exponentially into the bioreactor to keep the ethanol level in the broth below 0.5%. After 40 h of cultivation, the ergosterol yield reached 1,707 mg/l, which was 3.1-fold of that in the batch fermentation.     

Carotenoid production by lactoso-negative yeasts co-cultivated with lactic acid bacteria in whey ultrafiltrate

Two strains were selected--the lactoso-negative yeast Rhodotorula rubra GED2 and the homofermentative Lactobacillus casei subsp. casei Ha1 for co-cultivation in cheese whey ultrafiltrate (WU) and active synthesis of carotenoids. Under conditions of intensive aeration (1.0 l/l min, 220 rpm), a temperature of 30 degrees C, WU with 55.0 g lactose/l, initial pH = 5.5, the carotenoid content in the cells reached a maximum, when the growth of the cultures had come to an end, i.e. in the stationary phase of the yeast. The maxima for dry cell accumulation (27.0 g/l) and carotenoid formation (12.1 mg/l culture medium) did not coincide on the 5th and 6th day, respectively. A peculiarity of the carotenoid-synthesizing Rh. rubra GED2 strain, co-cultivated with L. casei Ha1, was the production of carotenoids with high beta-carotene content (46.6% of total carotenoids) and 10.7% and 36.9% for torulene and torularhodin, respectively.     

In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8.Variations in total nitrogen availability in the medium in terms of different ratios of nitrate: ammonium showed that the ratio 4:1 revealed a profound effect, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium.Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate: ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).     

Optimization of carotenoid production from hyper-producing Rhodotorula glutinis mutant 32 by a factorial approach

AIMS: Optimization of carotenoid production by a mutant of Rhodotorula glutinis. METHODS: The growth and carotenoid production was optimized in shake flasks using a two-level, three-variable factorial design. RESULTS: The volumetric carotenoid production could be increased to 129 +/- 2 mg x 1(-1) in a medium containing (g x l(-1)) yeast extract 11.74, glucose 46 and threonine 18 along with other micronutrients, wherein, beta-carotene yield was 102 +/- 2 mg x l(-1), accounting for 80% of the total carotenoids. SIGNIFICANCE AND IMPACT OF THE STUDY: The medium optimization resulted in a fourfold increase in volumetric production and a twofold increase in the cellular accumulation of carotenoids. In view of such high yields, the mutant of Rhodotorula glutinis can be a potential source of beta-carotene.     

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.     

Enhanced production of recombinant proteins from plant cells by the application of osmotic stress and protein stabilization

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) was produced from transgenic Nicotiana tabacum cells. The application of osmotic stress through the addition of 90 g/l mannitol to the plant cell medium enhanced the maximum extracellular GM-CSF concentration from 76 g/l to 130 g/l (1.7-fold increase). The addition of bovine serum albumin (BSA), along with mannitol, further increased the maximum extracellular GM-CSF concentration by as much as 2.5-fold over the control. GM-CSF degradation studies in conditioned medium demonstrated that mannitol and BSA both stabilize the GM-CSF protein. The addition of gelatin together with mannitol to the plant cell medium also enhanced the maximum extracellular GM-CSF concentration and stability over time.     

Stability of beta-carotene in spray dried preparation of Rhodotorula glutinis mutant 32

AIMS: To obtain beta-carotene-rich dry cell preparation from mutant 32 of Rhodotorula glutinis and determination of its pigment stability. METHODS AND RESULTS: The mutant 32 of R. glutinis was grown in a 14 l stirred tank fermenter. Cell mass was concentrated 10-fold by cross-flow microfiltration and then spray dried. Butylated hydroxy toluene (BHT) and d-tocopherol were used as protecting agents. A two-level, three-variable, factorial optimization was performed to achieve moisture-free, non-viable and beta-carotene-rich feed additive. CONCLUSIONS: The beta-carotene and cell mass in stirred tank fermenter were found to be 54 +/- 5 mg l-1 and 12.8 +/- 2 g l-1, respectively. In the presence of BHT, 97 +/- 3% (w/w) beta-carotene was recovered for all the inlet temperatures studied. The best beta-carotene and yeast powder recoveries were obtained at 160 degrees C, 11.6% (w/v) cell mass concentration and 1 g l-1 BHT. The pigments inside dried yeast powder were stable in dark and cold condition for at least 10 weeks. The purified beta-carotene got almost totally denatured, under similar conditions of storage, within 76 h. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray dried and stable preparation of beta-carotene-rich yeast, R. glutinis can provide alternative source of beta-carotene for use in animal nutrition.     

Control of gene expression from the SUC2 promoter of Saccharomyces cerevisiae with the aid of a glucose analyser

Summary A Saccharomyces cerevisiae strain harbouring the recombinant plasmid pSMF38TMA was cultured in a jar fermentor under the control of glucose concentration. In the recombinant plasmid, the mouse -amylase gene was fused to the S. cerevisiae SUC2 promoter. When glucose concentration in the medium was controlled at 10 g/l, the gene expression was completely repressed. On the other hand, the -amylase was produced and secreted in the medium at a very high level, around 200 mg/l as evaluated from the specific activity of commercially available human salivary amylase, when the glucose was kept at 0.15 g/l. This amount was almost 20-fold that obtained at 10 g/l glucose. The specific growth rate of the yeast in this culture was almost 60% of that attained with 10 g/l glucose. To obtain higher cell growth and productivity, the yeast was at first cultured at 2 g/l glucose and the concentration was then lowered to 0.15 g/l. By this control of the glucose concentration, on-off regulation of gene expression from the SUC promoter could be attained.     

Isolation and distribution of oligotrophic marine bacteria.

A useful plate culture method for isolating oligotrophic bacteria found in the low-nutrient environment of the open sea has been developed. The method uses a glass-fiber filter substitute for agar. Nutritional requirements of oligotrophic bacteria consisted of a dilute mutrient solution containing 16.8 mg C/l total organic carbon aseptically added to the sterilized filter. Distribution of bacteria in oceanic and neritic seawater was determined using the membrane filter method. In the case of seawater containing less than 0.5 mg/l dissolved carbohydrates, plate counts of oligotrophic bacteria were found to be several- to 100-fold greater than the heterotrophic bacterial counts enumerated by standard methods routinely used for enumeration. However, in seawater containing approximately over 0.5 mg/l dissolved carbohydrates, heterotrophic bacterial counts were 10-fold greater than oligotrophic bacterial counts.     

Effects of Rapeseed Oil on Activity of Methylmalonyl-CoA Carboxyltransferase in Culture of Streptomyces fradiae

To investigate why more tylosin was produced when Streptomyces fradiae T1558 was cultured in a rapeseed oil medium than in a glucose or starch medium, we measured the activity of methylmalonyl-CoA carboxyltransferase (EC 2.1.3.1) and intracellular propionic acid. The activity of the enzyme, which catalyzes the formation of the precursor of tylosin, protylonolide, was 0.19 U/mg protein in 5 days of culture in rapeseed oil medium, which was 2.5- and 1.3-fold that with the glucose or starch medium, respectively. The intracellular propionic acid concentration was 1.2 g/g of dry weight, which was 4.3- and 2.1-fold that with the glucose or starch medium, respectively. The addition of propionic acid increased tylosin production in batch culture: when 0.2 g/l (final concentration) propionic acid was added to the glucose medium, 3.8 g/l tylosin was produced in 10 days of culture, 4.7-fold the amount without propionic acid. These findings suggest that in glucose medium, intracellular propionic acid is a limiting factor because of the low activity of methylmalonyl-CoA carboxyltransferase of the tylosin biosynthesis pathway.     

Rosmarinic acid production by Lavandula vera MM cell suspension culture: nitrogen effect

The growth of Lavandula vera MM cell suspension and the biosynthesis of rosmarinic acid (RA) were followed during its cultivation in Linsmayer–Skoog media, containing different concentrations of ammonium and nitrate ions. The results showed that cultivation in a medium with 0.09g ammonium ions/l (1/4 of standard medium) ensured intensive growth (16g dry biomass/l) and enhanced biosynthesis of RA (15mg/g dry biomass). Cultivation of L. vera MM cell culture in a medium with 1.2-fold concentration of nitrate ions led to accumulation of 11mg RA/g dry biomass which was twice as much as in the standard Linsmayer–Skoog medium.     

Enhanced biomass and gamma-linolenic acid production of mutant strain Arthrospira platensis

A mutant of Arthrospira platensis PCC 9108, strain M9108, obtained by mutagenesis with UV treatment, was able to mixotrophically grow in an SOT medium containing 40 g of glucose/l. The biomass and specific growth rate of strain M9108 (4.10 g/l and 0.70/d) were 1.9-fold and 1.4-fold higher, respectively, than those of the wild type (2.21 g/l and 0.58/d) under mixotrophic culture condition. In addition, when compared with the wild type, the content of gamma- linolenic acid (GLA) in the mutant was increased when glucose concentration was increased. Compared with the wild type, the GLA content of the mutant was 2-fold higher in autotrophic culture and about 3-fold higher in mixotrophic culture. Thus, the mutant appears to possess more efficient facility to assimilate and metabolize glucose and to produce more GLA than its wild-type strain.     

Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.)

Summary Suspension cultures which maintained embryogenic potency for more than 18 months were established from excised immature embryos of rice (Oryza sativa L. cv. Konansou). The cultures were subcultured every three days in N6 medium supplemented with proline (10 mM), casein hydrolysate (300 mg/l), sucrose (30 g/l) and 2,4-D (1 mg/l). The frequency of embryogenesis from the embryogenetic suspension cultures reached about 90% when cell clusters (about 1 mm in diameter) were transferred to a solid medium which consisted of N6 medium, NAA (1 mg/l), kinetin (5 mg/l), sucrose (30 g/l) and Gelrite (2 g/l). When smaller clusters of cells (approximately 200–400 m in diameter) were transferred to a liquid medium which consisted of salts of N6 medium diluted with an equal volume of water plus sucrose (45 g/l), NAA (0.01 mg/l) and 4-PU (0.1 mg/l) at a cell density of 13 clusters/ml in 2 ml of medium, somatic embryogenesis was initated at high frequency (about 50%). Morphological evidence is provided to demonstrate that the regeneration occurred via embryogenesis. This is the first report of high-frequency embryogenesis in suspension cultures of rice cells.     

Effects of high density lipoprotein containing high or low beta-carotene concentrations on progesterone production and beta-carotene uptake and depletion by bovine luteal cells

Luteal cells were isolated from mid-luteal heifer ovaries by collagenase digestion. Cells were cultured with DMEM/Ham's F12 medium in serum pre-treated plastic culture dishes for periods of up to 11 days. As beta-carotene is almost completely insoluble in all polar solvents, it was added to cultures in either dimethyl sulphoxide (DMSO), tetrahydrofuran (THF) or as high-density lipoprotein (HDL) containing high or low beta-carotene concentrations. Medium was replaced after 24 h, thereafter medium was changed every 48 h. Treatment of cells with DMSO alone or with beta-carotene (5 micromol/l) in DMSO both resulted in significant (P<0.01) stimulation of progesterone production. beta-Carotene (5 micromol/l) in THF did not alter progesterone production but 50 micromol/l beta-carotene in THF resulted in significant inhibition (P<0.02) of progesterone production on days 3 and 7. Cultures were also supplemented with bovine HDL preparations containing equal concentrations of cholesterol (25 microg/ml) but high or low beta-carotene (12.4 or 0.44 microg/mg of cholesterol). Both HDL preparations significantly stimulated progesterone production (P<0. 001) but the high beta-carotene HDL was significantly (P<0.02) more effective than the low beta-carotene HDL. However, when given together with bovine luteinizing hormone (bLH) or dibutyryl cAMP (dbcAMP), the high beta-carotene HDL stimulated progesterone production less than did the low HDL (P<0.01). Uptake and depletion of beta-carotene by luteal cells were also examined in culture. beta-Carotene supplementation increased luteal cell beta-carotene from an initial level of 373 ng per 10(6) cells to 2030 ng per 10(6) cells by day 6. In contrast, the levels in control cells decreased to 14% of starting values during the same period. Cells treated with HDL containing high beta-carotene on day 1 or days 1 and 3 were then incubated with or without bLH or dbcAMP for a further 2 days to investigate the effect of bLH and dbcAMP on depletion of beta-carotene by luteal cells. beta-Carotene depletion in the luteal cells was significantly higher (P<0.05) in LH- and dbcAMP-treated cells than in the control cells in both groups. These results indicate that the use of solvents such as DMSO or THF may have undesirable effects due to alteration of cell membrane permeability. Supplementation with bLH or dbcAMP may increase the metabolism of beta-carotene in luteal cells. bLH or dbcAMP together with high beta-carotene HDL may, when combined with the effect of increased beta-carotene metabolism, give less stimulation than with low beta-carotene HDL.     

Degradation of the herbicide atrazine by the soil mycelial fungus INBI 2-26(-)--a producer of cellobiose dehydrogenase

Nonsporulating mycelial fungi producing cellobiose dehydrogenase (CDH) and isolated from soils of South Vietnam with high residual content of dioxins are capable of growing on a solid medium in the presence of high atrazine concentrations (to 500 mg/l). At 20 and 50 mg/l atrazine, the area of fungal colonies was 1.5-1.2-fold larger, respectively, compared with control colonies of the same age, whereas development of the colonies at 500 mg/l atrazine was delayed by 5 days, compared with controls grown in the absence of atrazine. Surface cultivation of the fungus on a minimal medium with glucose as a sole source of carbon and energy decreased the initial concentration of atrazine (20 mg/l) 50 times in 40 days; in addition, no pronounced sorption of atrazine by mycelium was detected. This was paralleled by accumulation in the culture medium of extracellular CDH; atrazine increased the synthesis of this enzyme two- to threefold. Accumulation of beta-glucosidase (a mycelium-associated enzyme) and cellulases preceded the formation of CDH.