Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures

The effect of addition of autoclaved and filter-sterilized culture filtrate of Piriformospora indica (a root endophytic fungus) to the growing Linum album hairy root cultures on growth and lignan production was investigated. The addition resulted in a significant enhancement in lignan production and growth. The podophyllotoxin and 6-methoxypodophyllotoxin (the lignans) concentrations were maximally improved by 3.8 times (233.8 mg/L) and 4.4 times (131.9 mg/L) in comparison to control cultures, respectively, upon addition of 3.0% (v/v) filter-sterilized culture filtrate of P. indica to the hairy root cultures of L. album for exposure time of 48 h. This increase in the lignan content also coincided with the increase in phenylalanine ammonia lyase activity, which was 3.1-fold (371.4 μkat/kg protein) higher compared to control cultures under the same conditions. The maximal increase in hairy root biomass was, however, obtained under different conditions; it was enhanced by 1.4 times (21.8 g/L) in comparison to control cultures, when 2% (v/v) filter-sterilized culture filtrate was in contact with L. album cultures for 96 h.     

Influence of hairy root ecotypes on production of tropane alkaloids in Brugmansia candida

Hyoscyamine and scopolamine are tropane alkaloids widely applied in medicine. Differences in alkaloid production and growth kinetics have been observed in Argentinian and Colombian ecotypes of Brugmansia candida hairy roots. The aim of this work was to analyze the production of key intermediates in tropane alkaloid synthesis in both ecotypes to determine differences in the biosynthetic pathway. Additionally, rolC gene expression was analyzed to determine its correlation with hairy root growth. The results showed a higher accumulation of polyamines in Colombian hairy roots, suggesting that there may be a rate-limiting enzyme in the last steps of hyoscyamine biosynthesis. Additionally, rolC gene expression was correlated with an improvement in hairy root growth, which supports the function of rol genes as growth modulators and suggests that metabolic engineering approaches involving rolC manipulation may be useful for the development of more efficient B. candida hairy root cultures for biotechnological applications.     

High-yield production of tropane alkaloids by hairy-root cultures of aDatura candida hybrid

Hairy root cultures were obtained following inoculation of the stems of sterile plantlets of aDatura candida hybrid withAgrobacterium rhizogenes. The scopolamine and hyoscyamine content was quantified by HPLC and compared with the non-transformed plants. The alkaloid yield (0.68% dry weight) obtained with the hairy roots was 1.6 and 2.6 times the amount found in the aerial parts and in the roots of the parent plants, respectively. Only a small proportion of alkaloids was released into the growth medium. Scopclamine was the principal alkaloid and the scopolamine/hyoscyamine ratio of ca. 5:1 makes these hairy roct cultures worthy of consideration as a source of scopolamine.     

Production of rhoifolin and tiliroside from callus cultures of Chorisia chodatii and Chorisia speciosa

The current work aims to stimulate the production of rhoifolin and tiliroside as two valuable phytochemicals from Chorisia chodatii Hassl. and Chorisia speciosa A. St.-Hil. callus cultures. A comparison between three explants from the in vitro germinated seedlings of both species for callus induction and accumulation of both flavonoids was carried out. Highly efficient calluses were induced from the leaves, stems and roots of C. chodatii seedlings on Gamborg’s B5 (B5) and Murashige and Skoog (MS) media containing 2.0 mg/l β-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenin (BA) or kinetin (Kn), while those of C. speciosa seedlings efficiently produced calluses on both media supplemented with 0.5 or 1.0 mg/l NAA and 0.5 mg/l BA. Besides, the highest contents of rhoifolin (1.927 mg/g DW) and tiliroside (1.776 mg/g DW) from C. speciosa cultures were obtained from the calluses of seedlings’ roots and stems maintained on B5 medium containing 1.0 mg/l NAA and 0.5 mg/l BA, respectively. On the other hand, the maximum rhoifolin content (0.555 mg/g DW) from C. chodatii cultures was obtained from the calluses of seedlings’ stems grown on B5 medium supplemented with 2.0 mg/l NAA and 0.5 mg/l BA, whereas the highest tiliroside content (0.547 mg/g DW) was provided by the root explants on B5 medium containing 2.0 mg/l NAA and 0.5 mg/l Kn. Both flavonoids were bioaccumulated in greater amounts than the wild and cultivated intact plants, which provides a promising tool for their future commercial production under a controlled environment, independent of climate and soil conditions.     

In vitro cultures of Silybum marianum and silymarin accumulation

In this study, a protocol for initiation of callus and shoot cultures from leaves and shoot tips explants of different silybium genotypes collected from different locations in Egypt was established. Callus cultures were initiated from leaves explants and exposed to different concentrations of the precursor (coniferyl alcohol). Shoot cultures were initiated from shoot tips explants. Moreover, the produced plants of the different Silybium shoots as well as intact plants were subjected to protein screening using SDS–PAGE analysis.Results obtained revealed that the optimum medium for growth and maintenance of friable callus was MS medium supplemented with 0.25 mg L⁻¹ 2,4-Dichlorophenoxy acetic acid (2,4-D) + 0.25 mg L⁻¹ Kinetin (Kin). The best medium for proliferation of high number of shoots was MS-medium with 0.25 mg L⁻¹ each of Benzyl Adinine (BA) and Naphthalene Acetic Acid (NAA). Coniferyl alcohol in concentration of 30 μM caused an increase in accumulation of silymarin contents in most callus cultures. SDS–PAGE of different Silybium shoots revealed that the protein profiles of 100% of in vitro produced plantlets similar to their control.     

Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.)

In this study, the effects of ploidy level and culture medium were studied on the production of tropane alkaloids. We have successfully produced stable tetraploid hairy root lines of Hyoscyamus muticus and their ploidy stability was confirmed 30?months after transformation. Tetraploidy affected the growth rate and alkaloid accumulation in plants and transformed root cultures of Egyptian henbane. Although tetraploid plants could produce 200% higher scopolamine than their diploid counterparts, this result was not observed for corresponding induced hairy root cultures. Culture conditions did not only play an important role for biomass production, but also significantly affected tropane alkaloid accumulation in hairy root cultures. In spite of its lower biomass production, tetraploid clone could produce more scopolamine than the diploid counterpart under similar growth conditions. The highest yields of scopolamine (13.87?mg?l^?1) and hyoscyamine (107.7?mg 1^?1) were obtained when diploid clones were grown on medium consisting of either Murashige and Skoog with 60?g/l sucrose or Gamborg??s B5 with 40?g/l sucrose, respectively. Although the hyoscyamine is the main alkaloid in the H. muticus plants, manipulation of ploidy level and culture conditions successfully changed the scopolamine/hyoscyamine ratio towards scopolamine. The fact that hyoscyamine is converted to scopolamine is very important due to the higher market value of scopolamine.     

Improvement in natamycin production by Streptomyces natalensis with the addition of short-chain carboxylic acids

Natamycin is an important tetraene (polyene) antibiotic produced in submerged culture by different strains of Streptomyces sp. In the present work, the effects of the addition of short-chain carboxylic acids (acetic, propionic and butyric) on cell growth and the kinetics of natamycin production were investigated during submerged cultivation of Streptomyces natalensis. The addition of acetic and propionic acids showed stimulatory effects on natamycin production when added to the fermentation medium at concentrations below 2 g L^?1 at the beginning of cultivation. In addition, when acetic and propionic acids were added in a mixture (7:1) at a total concentration of 2 g L^?1, antibiotic production increased significantly, reaching 3.0 g L^?1 (approximately 223% and 250% increases in volumetric and specific antibiotic production, respectively, compared with the control culture). Moreover, the addition of carboxylic acids not only increased the antibiotic yield but also decreased the production time from 96 h to only 84 h in shake-flask cultures. A further enhancement in natamycin production was achieved by cultivation in a 2-L stirred-tank bioreactor under controlled pH conditions. The maximum volumetric production of 3.98 g L^?1 was achieved after 84 h in carboxylic acid-supplemented culture (acetate and propionate in a ratio of 7:1).     

Distinct chemotypes of Tephrosia vogelii and implications for their use in pest control and soil enrichment

Tephrosia vogelii Hook. f. (Leguminosae) is being promoted as a pest control and soil enrichment agent for poorly-resourced small-scale farmers in southern and eastern Africa. This study examined plants being cultivated by farmers and found two chemotypes. Chemotype 1 (C1) contained rotenoids, including deguelin, rotenone, sarcolobine, tephrosin and α-toxicarol, required for pest control efficacy. Rotenoids were absent from chemotype 2 (C2), which was characterised by prenylated flavanones, including the previously unrecorded examples (2S)-5,7-dimethoxy-8-(3-hydroxy-3-methylbut-1Z-enyl)flavanone, (2S)-5,7-dimethoxy-8-(3-methylbut-1,3-dienyl)flavanone, (2S)-4′-hydroxy-5-methoxy-6″,6″-dimethylpyrano[2″,3″:7,8]flavanone, (2S)-5-methoxy-6″,6″-dimethyl-4″,5″-dihydrocyclopropa[4″,5″]furano[2″,3″:7,8]flavanone, (2S)-7-hydroxy-5-methoxy-8-prenylflavanone, and (2R,3R)-3-hydroxy-5-methoxy-6″,6″-dimethylpyrano[2″,3″:7,8]flavanone. The known compounds (2S)-5-methoxy-6″,6″-dimethylpyrano[2″,3″:7,8]flavanone (obovatin 5-methyl ether) and 5,7-dimethoxy-8-(3-hydroxy-3-methylbut-1Z-enyl)flavone (Z-tephrostachin) were also found in C2. This chemotype, although designated Tephrosia candida DC. in collections originating from the World Agroforestry Centre (ICRAF), was confirmed to be T. vogelii on the basis of morphological comparison with verified herbarium specimens and DNA sequence analysis. Sampling from 13 locations in Malawi where farmers cultivate Tephrosia species for insecticidal use indicated that almost 1 in 4 plants were T. vogelii C2, and so were unsuitable for this application. Leaf material sourced from a herbarium specimen of T. candida contained most of the flavanones found in T. vogelii C2, but no rotenoids. However, the profile of flavonol glycosides was different to that of T. vogelii C1 and C2, with 6-hydroxy-kaempferol 6-methyl ether as the predominant aglycone rather than kaempferol and quercetin. The structures of four unrecorded flavonol glycosides present in T. candida were determined using cryoprobe NMR spectroscopy and MS as the 3-O-α-rhamnopyranosyl(1 → 6)-β-galactopyranoside-7-O-α-rhamnopyranoside, 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside, 3-O-α-rhamnopyranosyl(1 → 2)[α-rhamnopyranosyl(1 → 6)]-β-galactopyranoside-7-O-α-rhamnopyranoside, and 3-O-α-rhamnopyranosyl(1 → 2)[(3-O-E-feruloyl)-α-rhamnopyranosyl(1 → 6)]-β-galactopyranosides of 6-hydroxykaempferol 6-methyl ether. Tentative structures for a further 37 flavonol glycosides of T. candida were assigned by LC–MS/MS. The correct chemotype of T. vogelii (i.e. C1) needs to be promoted for use by farmers in pest control applications.     

Effect of 5-fluoro-2′-deoxyuridine on toxin production and cell cycle regulation in marine dinoflagellate,Alexandrium tamarense

The effect of metabolic inhibitor, 5-fluoro-2′-deoxyuridine (FUdR) on toxin production and the cell cycle of marine dinoflagellate, Alexandrium tamarense, was investigated. Compared to untreated cells, FUdR at 3 μM (p < 0.05) to 300 μM (p < 0.01) inhibited the cell proliferation and toxin production in a dose-dependent manner for A. tamarense cultured in modified T₁ medium. FUdR at 203 μM resulted in cell cycle arrest at the S phase at day 4 and toxigenesis was inhibited after day 2. The toxin profiles of the FUdR-treated cultures were similar to those of the control culture. These results suggest that FUdR inhibits saxitoxin (STX) biosynthesis in the early stage of the pathway. This report is the first to demonstrate the inhibition of toxin production in A. tamarense by a nucleoside analog.     

Direct bioconversion of d-xylose to 1,2,4-butanetriol in an engineered Escherichia coli

The compound 1,2,4-butanetriol (BT) is a valuable chemical used in the production of plasticizers, polymers, cationic lipids and other medical applications, and is conventionally produced via hydrogenation of malate. In this report, BT is biosynthesized by an engineered Escherichia coli from d-xylose. The pathway: d-xylose → d-xylonate → 2-keto-3-deoxy-d-xylonate → 3,4-dihydroxybutanal → BT, was constructed in E. coli by recruiting a xylose dehydrogenase and a keto acid decarboxylase from Caulobacter crescentus and Pseudomonas putida, respectively. Authentic BT was detected from cultures of the engineered strain. Further improvement on the strain was performed by blocking the native d-xylose and d-xylonate metabolic pathways which involves disruption of xylAB, yjhH and yagE genes in the host chromosome. The final construct produced 0.88 g L⁻¹ BT from 10 g L⁻¹ d-xylose with a molar yield of 12.82%. By far, this is the first report on the direct production of BT from d-xylose by a single microbial host. This may serve as a starting point for further metabolic engineering works to increase the titer of BT toward industrial scale viability.     

Alkaloids from the roots of Stemona javanica (Kunth) Engl. (Stemonaceae) and their anti-malarial,acetylcholinesterase inhibitory and cytotoxic activities

Two new protostemonine-type alkaloids, javastemonine A and B (3 and 4) have been isolated from the root extracts of Stemona javanica together with four known Stemona alkaloids, 13-demethoxy-11(S*),12(R*)-dihydroprotostemonine (1), isoprotostemonine (2), protostemonine and isomaistemonine. The structures and relative configurations of the new alkaloids were determined by spectroscopic analysis. The alkaloids 1 and 2 and protostemonine showed moderated antiplasmodial activities against the Plasmodium falciparum strains, TM4 (IC₅₀ values of 17.7 ± 3.7, 16.8 ± 5.4, 16.0 ± 4.2 μg/mL, respectively) and K1 (IC₅₀ values of 16.8 ± 3.1, 14.1 ± 3.7, 11.9 ± 3.3 μg/mL, respectively). These compounds showed no significant cytotoxicities against KB or Vero cells or acetylcholinesterase inhibitory activities.     

Pollination biology of Eriocaulon parkeri in Connecticut

Eriocaulon parkeri B. L. Robinson is a monoecious, pioneer species of tidal mudflats that displays characteristics that suggest outcrossing as a preferred breeding system. Analyses of breeding system dynamics, fruit set, and pollen and seed viabilities were undertaken in Connecticut and Wisconsin to test the hypothesis of entomophyly and outcrossing as a preferred breeding strategy. Potential pollinators included syrphid and long-legged flies. Seed viability was estimated at 94 ± 16% (n = 133); pollen viability at 88 ± 13%. Pollen production averaged ∼500 grains per flower. Pollen–ovule ratios within inflorescences averaged 196:1, suggesting facultative autogamy. Seed set in natural populations averaged 74%, not significantly different between early and late season plants or between greenhouse controls and hand pollinations. Emasculated plants produced small amounts of seed under controlled greenhouse conditions. Pollen tube growth through the styles of E. aquaticum, a related species, was much more prevalent than that of E. parkeri. Results suggest that E. parkeri relies heavily on geitonogamy for seed production. Some seed may be produced by agamospermy.     

New immunomodulatory steroidal alkaloids from Sarcococa saligna

Two new steroidal alkaloids, (20 S)-(bennzamido)-3β-(N,N-dimethyamino)-pregnane (1), and (20 S)-(bennzamido)-pregnane-3-one- (2), and two known steroidal alkaloids, pachysanaximine A (3) and 3β, 20α-diacetamido-5α-pregnane (4) were isolated from the whole plant of Sarcococca saligna. The structures of these compounds were identified with the help of spectroscopic techniques while spectra for known compounds were compared with spectra reported in literature. The immunomodulatory potential of the new compounds were found to be significant and dose dependent. Compound 1 showed inhibition of T cells proliferation at 10 μg/mL (95%), and inhibition of IL-2 production with an IC50 = 1.6 μg/mL.     

The effect of dissolved oxygen concentrations on (1 → 3)- and (1 → 6)-β-glucanase production by Acremonium sp. IMI 383068 in batch culture

The influence of dissolved oxygen tension (DOT) on the production of extracellular (1 → 3)- and (1 → 6)-β-glucanases by the fungus Acremonium sp. IMI 383068 was investigated in batch culture. The controlled DOT levels at which cultures were grown affected the measured (1 → 3)-β-glucanase specific activities, but these effects were less marked on the measured (1 → 6)-β-glucanase specific activities. There appeared to be no direct link between the branching frequency of the fungus as reflected by its hyphal growth unit, and changing DOT levels. Whether pustulan or scleroglucan were used as the sole carbon source also affected production of these enzymes and their response to varying DOT. The measured (1 → 3)-β-glucanase specific activities were generally higher with scleroglucan, mainly because of the production of an extra active (1 → 3)-β-glucanase whereas pustulan grown cells produced a corresponding inactive protein with an identical electrophoretic mobility by SDS-PAGE and N-terminal amino acid sequence. With pustulan grown cells, DOT levels had comparatively little influence on the optimal measured specific activities of the (1 → 3)-β-glucanases, while with scleroglucan, they increased as DOT increased.     

Accumulation of free amino acids during exposure to drought in three springtail species

Springtails are closely related to insects, but they differ from these with respect to water balance, in particular because springtails are small and have high integumental permeability to water. Here we report a series of experiments addressing the dynamics of osmoregulation, water content and accumulation of free amino acids (FAAs) in three springtail species during exposure to a gradually increasing environmental desiccation simulating conditions in drought exposed soil. Folsomia candida and Protaphorura fimata (both living in the deeper soil layers; euedaphic species) were active throughout the 3 week exposure, with the developing drought regime ending at −3.56 MPa (the soil water activity at the permanent wilting point of plants is −1.5 MPa) and remained hyperosmotic (having an body fluid osmolality higher than the corresponding environment) to their surrounding air. Sinella curviseta (living in upper soil/litter layers; hemiedaphic species) also survived this exposure, but remained hypoosmotic throughout (i.e. with lower osmolality than the environment). The body content of most FAAs increased in response to drought in all three species. Alanine, proline and arginine were the most significantly upregulated FAAs. By combining our results with data in the literature, we could account for 82% of the observed osmolality at −3.56 MPa in F. candida and 92% in P. fimata. The osmolality of S. curviseta was only slightly increased under drought, but here FAAs were considerably more important as osmolytes than in the two other species. We propose that FAAs probably have general importance in drought tolerance of springtails.     

C ring may be dispensable for β-carboline: Design,synthesis, and bioactivities evaluation of tryptophan analog derivatives based on the biosynthesis of β-carboline alkaloids

According to our previous work and the latest research on the biosynthesis of β-carboline, and using the reverse thinking strategy, tryptophan, the biosynthesis precursor of β-carboline alkaloids, and their derivatives were synthesized, and their biological activities and structure–activity relationships were studied. This bioassay showed that these compounds exhibited good inhibitory activities against tobacco mosaic virus (TMV); especially (S)-2-amino-3-(1H-indol-3-yl)-N-octylpropanamide (4) (63.3 ± 2.1%, 67.1 ± 1.9%, 68.7 ± 1.3%, and 64.5 ± 3.1%, 500 μg/mL) exhibited the best antiviral activity both in vitro and in vivo. Compound 4 was chosen for the field trials and the acute oral toxicity test, the results showed that the compound exhibited good anti-TMV activity in the field and low acute oral toxicity. We also found that these compounds showed antifungal activities and insecticidal activities.     

Serotonin transporter protein (SERT) and P-glycoprotein (P-gp) binding activity of montanine and coccinine from three species of Haemanthus L. (Amaryllidaceae)

The alkaloid rich extracts from an acid/base extraction of bulb material of Haemanthus coccineus L., H. montanus Baker and H. sanguineus Jacq. revealed that two montanine type Amaryllidaceae alkaloids, montanine (1) and coccinine (2) were the major alkaloid constituents. Together these two alkaloids constituted 88, 91 and 98% of the total alkaloid extract from each species respectively. GC–MS analysis revealed that H. coccineus and H. sanguineus had a relative abundance of coccinine (74 and 91% respectively) to montanine (14 and 7% respectively); whereas H. montanus had 20% coccinine and 71% montanine. The three extracts and two isolated alkaloids were evaluated for binding to the serotonin transporter protein (SERT) in vitro. Affinity to SERT was highest in H. coccineus (IC₅₀ = 2.0 ± 1.1 μg/ml) followed by H. montanus (IC₅₀ = 6.8 ± 1.0 μg/ml) and H. sanguineus (IC₅₀ = 28.7 ± 1.1 μg/ml). Montanine (IC₅₀ = 121.3 ± 3.6 μM or 36.56 ± 1.14 μg/ml; K_i = 66.01 μM) was more active than coccinine (IC₅₀ = 196.3 ± 3.8 μM or 59.15 ± 1.08 μg/ml; K_i = 106.8 μM), both of which were less active than the total alkaloid extracts of each species investigated. The possible synergistic effects of two coccinine/montanine mixtures (80:20 and 20:80) were investigated, however the mixtures gave similar activities as the pure compounds and did not show any increase in activity or activity similar to the total alkaloid extracts. Thus the considerably higher activity observed in the total alkaloid extracts is not correlated to the relative proportions of coccinine and montanine in the extracts and thus are likely to be due to more potent unidentified minor constituents. Both alkaloids exhibited low binding affinity to P-glycoprotein (P-gp) as demonstrated by low inhibition of calcein-AM efflux in the MDCK-MDR1 cell line. This indicates that P-gp efflux will not be limiting for blood–brain-barrier passage of the alkaloids.     

Biochemical and structural characterization of recombinant hyoscyamine 6β-hydroxylase from Datura metel L.

Hyoscyamine 6β-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6β-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. metel (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. metel (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6β-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K_m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 μM each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of α-helicity in the secondary structure. From the fluorescence studies, Stern–Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M^?1 and 0.56 M^?1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes.     

Novel inhibitors of urease from Corydalis govaniana Wall.

A new compound govaniadine (1), along with three known tetrahydroprotoberberine-type alkaloids caseadine (2), caseamine (3), and protopine (4), were isolated from the plant Corydalis govaniana Wall. All alkaloids 1–4 exhibited a good urease enzyme inhibition with IC₅₀ ± S.E.M. values of 20.2 ± 3.6, 38.9 ± 2.8, 66.7 ± 1.2, and 54.1 ± 1.2 μM, respectively, which are comparable to the standard inhibitor, acetohydroxamic acid (IC₅₀ = 42.0 μM). None of these compounds showed inhibition against α-chymotrypsin. This is the first report of urease inhibiting tetrahydroprotoberberine-type alkaloids.