Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58

Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.     

Alkaloid production by plant cell cultures of Holarrhena antidysenterica: II. Effect of precursor feeding and cultivation in stirred tank bioreactor

Precursor feeding strategy for increasing the yield of conessine, a steroidal alkaloid of Holarrhena antidysenterica, was established in cell suspension culture. A total of 50 mg/L added cholesterol was converted into 43 mg/L of alkaloid, 90% of which constituted the conessine. By applying the precursor feeding policy to the cell suspension culture in modified Murashige and Skoog (MS) medium, a total of 143 mg/L of alkaloid was produced in 8 days. In this way the alkaloid content of the cells was increased more than six times compared to that obtained in the standard MS medium. The steps leading to biotransformation of cholesterol into alkaloids were unaffected by phosphate. The shake flask data were successfully transferred to a bench scale 6-L stirred tank bioreactor in which the specific biosynthetic rate of alkaloid production was 110 mg/100 g dry cell weight per day, about 160 times higher than that of whole plant.     

Characteristics of growth and tropane alkaloid production in Hyoscyamus albus hairy roots transformed with Agrobacterium rhizogenes A4

Summary Hairy root culture of Hyoscyamus albus was established by transformation with Agrobacterium rhizogenes strain A4. The growth and production of five tropane alkaloids were investigated under various culture conditions. Among the four basal culture media tested, Woody Plant medium was the best for growth of the hairy roots, but a high amount of tropane alkaloids was obtained with Gamborg's B5 medium. Sucrose concentration in B5 medium had little effect on the growth, while 3% sucrose was suitable for the alkaloid production. Addition of KNO₃ to Woody Plant medium affected the growth, whereas the alkaloid content was not markedly improved. Supplement of some metal ions to B5 medium stimulated the alkaloid production. In particular, Cu²⁺ remarkably enhanced both the growth and the alkaloid yield. The hairy roots cultured under 16 h/day light survived for more than 32 days compared with those cultured in the dark.Abbreviations EDTA ethylenediaminetetraacetic acid - HPLC high performance liquid chromatography - MeOH methanol - MS medium Murashige and Skoog medium - WP medium McCown's Woody Plant medium - B5 medium Gamborg B5 medium - wt weight     

Alkaloid production by plant cell suspension cultures of Holarrhena antidysenterica: I. Effect of major nutrients

The effect of major nutrients on growth and alkaloid production by plant cell culture of Holarrhena antidysenterica was studied with a view to increasing the yield of the alkaloid conessine, a therapeutic drug used for treatment of dysentery and helminthic disorders. The studies resulted in development of a modified Murashige and Skoog (MS) medium that contained 60 mM total nitrogen with a NH(4) (+)-to-NO(3) (-) ratio of 5:1, 0.25 mM phosphate, and 40 g/L sucrose. The growth regulators 2,4-dichlorophenoxy acetic acid (2,4-D) and kinetin (Kn) were also found to affect the synthesis of alkaloid. Using an optimal level of inoculum (3 g/L), the modified medium resulted in alkaloid synthesis of 0.66 g/100 g dry cell weight, which represented a 4.25-fold increase over that obtained in standard MS medium.     

The Effect of Nutrient Medium Composition on the Growth Cycle of Catharanthus roseus G. Don Cells Grown in Batch Culture

Growth of C. roseus cell suspension culture was defined in termsof dry weight, cell number, mitotic index, and packed cell volume.Removal of major nutrients from the medium was monitored asa function of culture growth. Phosphate and sucrose were theonly macronutrients completely exhausted. Utilization of thesetwo nutrients occurred parallel with increments in dry weightand cell number. Increasing the nutrient medium levels of sucroseand phosphate prolonged growth of this culture; lag and exponentialphases were extended; cell number and dry weight yield weredoubled. Dry weight assimilation was enhanced by increasingthe nutrient medium level of sucrose, whereas increments incell number were related to phosphate level. Two alkaloid fractions(fractions 1 and 2) were identified in this cell line. Fraction2 alkaloid level declined as the nutrient medium supply of nitrogenwas depleted.     

Regulation of product synthesis in cell cultures of Catharanthus roseus. V. Long-term maintenance of cells on a production medium

Three unselected cell lines of C. roseus maintained on a growth-associated alkaloid production medium were studied over a period of 2 to 5.5 years for the stability of alkaloid production (serpentine and ajmalicine). Large fluctuations in the total alkaloid content of 20-day-old cells were found for all three cell lines at each subculture over a two-year period. Growth rates increased during prolonged subculture and one cell line became unproductive after five years culture. By selection of small autofluorescent aggregates, high alkaloid production was restored in this cell line, while the parent line was found to be unresponsive to alkaloid induction treatments. The instability in both alkaloid production and spectrum and the loss of alkaloid productivity are discussed in relation to the selection pressures present during long-term maintenance of cell suspension cultures.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - nHS n-heptane sulphonate     

Inducer effect of Tween 20 permeabilization treatment used for release of stored tropane alkaloids inDatura innoxia Mill. hairy root cultures

Summary The effects of Tween 20 as permeabilizing agent on tropane alkaloids fromDatura innoxia Mill. hairy root cultures have been studied. The kinetics of the alkaloid release is detailed and shows three different stages: an initial rapid increase of the alkaloid level within the roots and in the culture medium, followed by a slower but higher increase of the alkaloid concentration in the medium. During this phase, the alkaloid concentration within the roots returned to a lower value. Finally, after a longer time, the quantity of hyoscyamine in the medium decreased significantly with a variable rate. According to the total alkaloid content per flask determinations under different conditions, it is clearly demonstrated that Tween treatment permeabilized the roots, but also acted as an inducer.     

Some Accounts on Culture Conditions of Callus Tissues of Solanum laciniatum Ait. for Producing Solasodine

Production of solasodine in callus cultures of Solanum laciniatum Ait. was examined under several culture conditions. The steroidal alkaloid was produced more actively in rapidly proliferating callus tissues cultured on PN medium. The alkaloid concentration in the tissue was about 0.05% (dry weight basis) during the first 5 weeks’ culture. The highest accumulation of the alkaloid per culture was obtained with 2,4-d concentration in the medium at 1~2 ppm. It is noteworthy that the alkaloid production was not inhibited by such high concentration of 2,4-d as up to 10 ppm in the medium. Supplementation of kinetin slightly increased the alkaloid production.     

The development of a single-stage growth and indole alkaloid production medium for Catharanthus roseus (L.) G. Don suspension cultures

By modification of a standard Murashige and Skoog plant tissue culture maintenance medium, a system has been developed for Catharanthus roseus cell suspension cultures in which both growth and indole alkaloid accumulation can occur in a single-stage culture of 14–21 days. Precise optimization of the medium depends upon the cell line under investigation, but the inclusion of lactose as the carbohydrate source, and NAA and kinetin as growth regulators, will generally increase yields of the indole alkaloid catharanthine. Treatment of cells growing in this optimized medium with agents that stimulate the accumulation of secondary metabolites both increases the yield of catharanthine and reduces the time required for production. We believe that this process could be useful for the commercial production of plant secondary metabolites.     

Accumulation of beta-Carboline Alkaloids and Serotonin by Cell Cultures of Peganum harmala L: I. CORRELATION BETWEEN PLANTS AND CELL CULTURES AND INFLUENCE OF MEDIUM CONSTITUENTS

A number of cell cultures of Peganum harmala were initiated to check for a correlation between the harman alkaloid content of seedlings and cell lines derived therefrom. Despite a poor correlation between callus or suspension culture lines and parent plants, the mean alkaloid contents of strains derived from seedlings with higher alkaloid yields were nevertheless higher than the mean contents of strains derived from low yield plants. Generally, alkaloid accumulation decreased with the numbers of transfers. By permanent visual selection for fluorescent areas of the calluses, however, a mean content of 0.1% harman alkaloids and 0.1% serotonin could be maintained, which was 10 times higher than in unselected callus cultures.     

Optimization of conditions for storage and cultivation of the fungus Claviceps sp.--a producer of the ergot alkaloid agroclavine

Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain s 106 were studied. The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90-95%). Storage of the culture at -70 degrees C in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.     

Intracellular and extracellular alkaloids of Penicillium roqueforti

The alkaloid composition of mycelium and culture liquid filtrate of the fungus Penicillium roqueforti IBPM-F-141 was studied. The new metabolite--3,12-dihydroroquefortine, a derivative of roquefortine, the main component of the alkaloid fraction of this culture, has been isolated for the first time. The structure of 3,12-dihydroroquefortine was determined by chemical and physico-chemical methods. In addition to roquefortine and 3,12-dihydroroquefortine, representatives of a new alkaloid group, the clavine alkaloids, e. g. isofumigaclavine A, isofumigaclavine B and festuclavine, were also isolated and identified. The data on the content of these compounds in mycelium and culture medium are presented.     

Antioxidant activity of extracts from Thalictrum minus suspension culture

Low meadow-rue (Thalictrum minus L.) antioxidant complex was studied in cell extracts and culture medium. Its activity was expressed as total polyphenol content in ferulic acid equivalents. In these model systems (cell extracts and culture medium) the inhibition of lipid oxidation and diphenylpicrylhydrazine reduction (EC₅₀ = 12–15 μg/ml) were observed. At the phenolic compound concentration of 8–15 μg/ml, the reducing capacity of cell extracts was equivalent to 1.5 mM ascorbic acid. At the same time, berberine, a major alkaloid synthesized by the culture, manifested a low antioxidant activity. The analysis of phenolic acid composition in low meadow-rue showed that one of the main components of its antioxidant system were caffeic, gallic, chlorogenic, and ferulic acids.     

Studies on Claviceps purpurea (Fr.) Tul. parasitic on Phragmites communis Trin

Isolates from C. purpurea sclerotia occurring naturally on Phragmites communis usually sporulated vigorously on the culture medium employed, and their failure to produce alkaloid in vitro was associated with a thin white growth form. Such isolates also failed to produce sclerotia on the host plants tested. A variant having a plectenchymatic morphology in vitro and producing a thick pigmented non-sporulating growth form yielded alkaloid (up to approximately 300μg/ml mainly δ8–9 and δ9–10 lysergic acids and chanoclavine) in surface or submerged culture and developed typical ergot sclerotia (containing 0·2-0·4% alkaloid, mainly ergotoxine and ergotamine) in vivo. Improved alkaloid yield in vitro was obtained from a strain reselected after passing through a parasitic phase. Aetiological aspects of the P. communis ergot are discussed.     

雷公藤不定根两相培养初步研究

以雷公藤(Tripterygium wilfordii)不定根为材料,通过两相培养技术,研究有机溶剂邻苯二甲酸二丁酯(DBP)不同浓度及培养时间对雷公藤不定根生长及次生代谢产物含量的影响。结果显示,DBP浓度为6%、培养至第6 d时,不定根增长量达1.14 g/瓶,为对照(1.08 g/瓶)的1.06倍;DBP浓度为2%、培养至第8 d时,内酯醇含量达74.96μg/g,为对照(53.67μg/g)的1.40倍;DBP浓度为2%、培养至第2 d时,所收获的内酯醇总产量最高(0.40 mg/瓶),且为对照(0.27 mg/瓶)的1.48倍。本研究结果表明,在培养基中添加一定浓度DBP,虽然适合雷公藤内酯醇的形成,但不适合雷公藤生物碱的形成;不论添加DBP浓度大小、培养时间长短,不定根中吉碱和次碱含量及每瓶总产量均低于对照。     

Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures

Two year old, transformed root cultures of Catharanthus roseus accumulate ajmalicine and catharanthine (0.57 and 0.36 mg g^-1 DW, or 7.0 and 3.0 mg l^-1, respectively). Changes in the concentration of the medium components, as well as the addition of hydrolytic enzymes and biotic elicitors, were used as strategies to increase these alkaloid yields. Regarding the components of the medium, the results obtained, when sucrose was raised from 3 to 4.5%, are noteworthy. The nitrogen source induced differential responses in the individual alkaloid yields. No net change in the alkaloid content was observed either with changes in the concentration of vitamins or macro-and micronutrients. Though the root culture only shows a limited response to elicitors, Aspergillus treatment and the use of macerozyme increased the accumulation of ajmalicine selectively, while the addition of methyl jasmonate increased the yield of both alkaloids.Abbreviations MeJa methyl jasmonate - mU milliunits     

A study on the alkaloid production by resting cell suspensions of Catharanthus roseus in a continuous flow reactor

Summary During batch cultivation of Catharanthus roseus cell suspensions, alkaloids were found in the culture medium after growth had ceased. Resting cell suspensions with high alkaloid content were obtained by transferring the cells to a medium devoid of 2.4 D (2.4 dichlorophenoxy-acetic acid). A production system with continuous feeding was developped to study alkaloid production by these resting cell suspensions.     

Effects of Culture Conditions on Tropane Alkaloid Formation in Hyoscyamus niger Suspension Cultures

Tropane alkaloid formation was studied under various culture conditions in suspension cultures of Hyoscyamus niger L. High aeration increased cell growth and the contents of hyoscyamine and scopolamine. White’s medium inhibited cell growth but increased the hyoscyamine content. The low concentrations of nitrogen and phosphate in White’s medium were responsible for such effects. NAA promoted cell growth but inhibited hyoscyamine formation. A hyoscyamine content of 0.05% dry weight (DW) (0.14 mg in 25 ml of culture medium) was achieved in modified LS medium in which the phosphate concentration was decreased to 0.2 mm and the auxin omitted.

Effects of precursor feeding on alkaloid formation were also studied with the putative precursors of hyoscyamine. N-Methylputrescine, tropine, phenylalanine, and tropic acid increased alkaloid formation, but the promotive effect of these precursors varied from one experiment to another.     

Alkaloid production in the fungus Penicillium sizovae

The dynamics of production of agroclavine-I and epoxyagroclavine-I, the basic components of the Penicillium sizovae alkaloid fraction, was being studied. The biosynthesis of alkaloids occurs in two stages coinciding in time with the growth phases. The culture studied is shown to have a considerably higher content of extracellular alkaloids than that of the mycelium. When the culture is grown on a medium with mannitol and succinic acid, separate consumption of carbon sources is observed. The alkaloid production in P. sizovae occurs in the presence of high residual concentrations of phosphorus in the medium.     

种子培养基成分对裂殖壶菌发酵产DHA的影响

考察种子培养基中谷氨酸钠质量浓度和NaCl质量浓度（盐度）对Schizochytrium sp. HX-308菌种质量及发酵性能的影响。结果表明：40 g/L谷氨酸钠条件下培养菌种,发酵后DHA质量分数达到（53.25±0.48）%,而其油产量、油脂质量分数和DHA产量分别为（24.9±0.4）g/L、（45.2±0.6）%、（8.6±0.35）g/L; 利用不含谷氨酸钠的种子培养基发酵,其油产量、油脂质量分数和DHA产量分别为（30.1±0.8）g/L、（54.3±0.5）%、（12.1±0.5）g/L,比含40 g/L谷氨酸钠时分别提高了20.88%、20.13%和40.70%,为菌种驯化提供了新的思路和方向。此外,低盐度培养条件有利于菌种生产性能的发挥,6.25 g/L NaCl条件下发酵后油质量分数、DHA产量及DHA质量分数最高,分别为（62.1±0.8）%、（10.0±0.3）g/L、（48.97±0.42）%。