Genetic mining of the “dark matter” in fungal natural products

<正>Filamentous fungi are one of the major sources of natural products (NPs; which are also termed as secondary metabolites (SMs)) with diverse biological activities, which have been widely used in agriculture, industry, and pharmaceuticals. Fungi are the second largest species in nature, and their biodiversity implies genomic diversity, which, in turn,predicts the structural diversity of metabolites. In general,     

The effect of biologically active substances from coniferous plants on the <Emphasis Type="Italic">L</Emphasis>-phenylalanine ammonia lyase and peroxidase activities in wheat leaves

The effect of the preparations produced from needles and wood of various coniferous species on the activities of L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and peroxidase (PO; EC 1.11.1.7), the enzymes involved in the development of plant defense response, was studied. It was demonstrated that treatment of brend wheat (Triticum aestivum L.) primary leaves with biological preparations produced from coniferous plants caused a transient increase in PAL and PO activities. The induction of these enzyme activities depends on the concentration of preparations and plant immune status. The results obtained suggest that coniferous metabolites are of interest as a source of plant extracts with the elicitor effect, increasing the resistance of plants to phytopathogens and adverse environmental factors. Original Russian Text ￠ E.V. Evtushenko, V.A. Saprykin, M.Yu. Galitsyn, V.M. Chekurov, 2008, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2008, Vol. 44, No. 1, pp. 123–128.     

Effects of low salinity on antioxidant enzymes activities in kidney and muscle of juvenile silver pomfret Pampus argenteus

Silver pomfret (Pampus argenteus) is an important fish species in China, whose fatality is strongly affected by marine salinity. To better understand the tolerance of sliver pomfret to varying environmental salinity at its early developmental stages, the influence of salinity on the activities of five antioxidant enzymes – superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST), and glutathione reductase (GR) – in the kidney and muscle of juvenile silver pomfret was investigated in this study. Divided groups A, B, C, D at random, the juvenile silver pomfrets were then treated with different salinity levels of 25, 20, 15, and 10 with time periods of 24, 48, 96, and 120 h respectively. Generally speaking, the results show that the renal and muscular SOD as well as the renal CAT activities first rise then drop (P < 0.05), while the muscular CAT activity increases with the elapse of treatment time (P < 0.05) and peaked at 96 h, then decreased at 120 h, while the muscular CAT activity in group D increased consistently and peaked at 120 h. The renal GPX activity decreases significantly (P < 0.05) as the salinity drops and treatment time prolongs with its lowest level occurring at 96 h in group D, while the muscular GPX activity increases first then decrease (P < 0.05); moreover the renal and muscular GPX activities vary reversely in compensation. On a whole, the renal and muscular GST and GR activities first rise then drop (P < 0.05) with the drop of salinity and elapse of time. To sum up, the results acquired from study indicate that the salinity decrease could effectively stimulate and enhance the antioxidant enzyme activity in the kidney and muscle of the juvenile silver pomfrets, thus effectively eliminating the excessive reactive oxygen species (ROS) and minimizing the body damage. Characterized by certain sequentiality and tissue specificity, the activation of the antioxidant enzymes could also be inhibited when the salinity varies beyond the tolerance range of the body.     

Effects of low salinity on antioxidant enzymes activities in kidney and muscle of juvenile silver pomfret Pampus argenteus

Silver pomfret (Pampus argenteus) is an important fish species in China, whose fatality is strongly affected by marine salinity. To better understand the tolerance of sliver pomfret to varying environmental salinity at its early developmental stages, the influence of salinity on the activities of five antioxidant enzymes – superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferase (GST), and glutathione reductase (GR) – in the kidney and muscle of juvenile silver pomfret was investigated in this study. Divided groups A, B, C, D at random, the juvenile silver pomfrets were then treated with different salinity levels of 25, 20, 15, and 10 with time periods of 24, 48, 96, and 120 h respectively. Generally speaking, the results show that the renal and muscular SOD as well as the renal CAT activities first rise then drop (P < 0.05), while the muscular CAT activity increases with the elapse of treatment time (P < 0.05) and peaked at 96 h, then decreased at 120 h, while the muscular CAT activity in group D increased consistently and peaked at 120 h. The renal GPX activity decreases significantly (P < 0.05) as the salinity drops and treatment time prolongs with its lowest level occurring at 96 h in group D, while the muscular GPX activity increases first then decrease (P < 0.05); moreover the renal and muscular GPX activities vary reversely in compensation. On a whole, the renal and muscular GST and GR activities first rise then drop (P < 0.05) with the drop of salinity and elapse of time. To sum up, the results acquired from study indicate that the salinity decrease could effectively stimulate and enhance the antioxidant enzyme activity in the kidney and muscle of the juvenile silver pomfrets, thus effectively eliminating the excessive reactive oxygen species (ROS) and minimizing the body damage. Characterized by certain sequentiality and tissue specificity, the activation of the antioxidant enzymes could also be inhibited when the salinity varies beyond the tolerance range of the body.     

Effect of Microwave and He-Ne Laser on Enzyme Activity and Biophoton Emission of Isatis indigotica Fort

The seed embryos of lsatis indigotica Fort were exposed to He-Ne laser (5.23 mW/mm^2, radiated for 5 min) and microwave ( 1.26 mW/mm^2, radiated for 8 s) irradiation to determine the effects of microwave and He-Ne laser pretreatment on enzyme activities, and biophoton emission of cotyledon. Then: (i) changes in the activities of enzymes in L indigotica cotyledon (such as amylase, transaminase, and proteinase) were measured to investigate the effects of He-Ne laser and microwave pretreatment; and (ii) biophoton emission was measured to determine the speed of cell division and metabolism. Results from these experiments indicated that: (i) the activities of amylase, transaminase, and proteinase of the cotyledon pretreated by HeNe laser and microwave were significantly increased; and (ii) the intensity of biophoton emission was enhanced significantly by He-Ne laser and microwave irradiation. These changes suggest that He-Ne laser and microwave pretreatment can improve the inner energy of seeds, lead to an enhancement of cotyledon enzymes, and speed up the metabolism of the cell, resulting in significantly increased biophoton emission. Moreover, the mechanism of action of the effects of laser and microwave radiation on the microcalorimetric parameters, enzyme activities, and biophoton emission of seeds is discussed on the basis of the results obtained.     

蝉拟青霉代谢产物清除DPPH自由基和抗真菌活性的研究

DPPH-TLC and DPPH-Microplate assays were used to determine the free radical scavenging activities of methanol extracts from the fermentation broth and the mycelia of Paecilomyces cicadae P3. The results revealed that the extracts had strong free radical scavenging activities. At the concentration of 5.0mg/mL the methanol extract from fermentation broth of P3 could decrease 74.86% of 0.4mg/mL DPPH radicals after incubating at 37℃ for 10 minutes. An anti-fungal TLC assay was applied to test antimicrobial metabolites of Paecilomyces cicadae P3. Meanwhile, an anti-Candida albicans Oxford Cup assay was made to confirm their anti-fungal activities. At the concentration of 5.0mg/mL the methanol extracts from fermentation broth and mycelia of P3 had significant anti-Candida albicans activities and the diameters of inhibition zone were 21.42mm and 11.23mm respectively.     

Effects of simulated elevated concentration of atmospheric CO2 and temperature on soil enzyme activity in the subalpine fir forest

Little is known about the responses of the activities of soil enzymes that are related to mass cycle to simulated climate change. Therefore, 72 intact soil columns from the primary fir (Abies faxoniana Rehder & E. H. Wilson) forest were parked in environment-controlled chambers with the CK (outside ambient CO₂ concentration and temperature), EC (elevated concentration CO₂ with (347.1 ± 22.1) μmol·mol^?1), ET (elevated temperature with (2.4 ± 0.4)°C), and ECT (elevated CO₂ concentration with (352.8 ± 27.6) μmol·mol^?1 and temperature with (2.2 ± 0.5)°C) treatments, and the activities of invertase, urease, nitrate reductase and acid phosphatase, which are related to the cycles of carbon, nitrogen and phosphorus in mineral soil (MS) and organic layer (OL) were measured simultaneously to understand the responses of these enzymes to climate change. Significant monthly variations on the activities of the studied enzymes were found in both OL and MS with the highest enzyme activities in summer, which were of ecological significance for soil nutrient availability and tree nutrition in the subalpine forest ecosystem. Different monthly patterns of enzyme activities were attributed to enzyme sources and soil layer. EC treatment had influenced slightly on the activities of the studied enzymes resulting from the higher CO₂ concentration in soil atmosphere and no indirect effect from the EC owing to a lack of trees planted on soils. ET treatment increased enzyme activities in comparison with the CK treatment because ET was beneficial to microbial growth and propagation. The increments of the enzyme activities in OL were higher than those in MS, implying that OL is more sensitive to climate change. ECT treatment sharply increased enzyme activities in comparison with the EC and CK, but there was no significant difference between ET and ECT, which was also attributed to no indirect effect by EC treatment owing to trees not planted on soils, implying that the increment of enzyme activities resulted from the temperature effect. However, further studies on indirect effect and complex effect on soil enzyme activity caused by EC, ET and ECT are needed to understand the soil enzyme responses to the climate change.     

Structural and mutational analysis of the interaction between the Middle-East respiratory syndrome coronavirus (MERS-CoV) papain-like protease and human ubiquitin

The papain-like protease(PL^pro) of Middle-East respiratory syndrome coronavirus（MERS-CoV） has proteolytic,deubiquitinating,and de ISGylating activities.The latter two are involved in the suppression of the antiviral innate immune response of the host cell.To contribute to an understanding of this process,we present here the X-ray crystal structure of a complex between MERS-CoV PL^pro and human ubiquitin（Ub） that is devoid of any covalent linkage between the two proteins.Five regions of the PL^pro bind to two areas of the Ub.The C-terminal five residues of Ub,RLRGG,are similar to the P5–P1 residues of the polyprotein substrates of the PL^pro and are responsible for the major part of the interaction between the two macromolecules.Through sitedirected mutagenesis,we demonstrate that conserved Asp165 and non-conserved Asp164 are important for the catalytic activities of MERS-CoV PL^pro.The enzyme appears not to be optimized for catalytic efficiency; thus,replacement of Phe269 by Tyr leads to increased peptidolytic and deubiquitinating activities.Ubiquitin binding by MERS-CoV PL^pro involves remarkable differences compared to the corresponding complex with SARS-CoV PL^pro.The structure and the mutational study help understand common and unique features of the deubiquitinating activity of MERS-CoV PL^pro.     

利用组织培养对秦岭百合属植物的保种繁殖研究

通过组织培养的方法对秦岭野生百合属植物进行了保种繁殖研究。实验证明,在ＭＳ＋０．５ｍｇ／ＬＮＡＡ上可诱导云南大百合、秦岭麝香百合和川百合产生较多的小鳞茎,在ＭＳ＋０．５ｍｇ／ＬＮＡＡ＋０．１ｍｇ／Ｌ６－ＢＡ上继代培养２０多次仍保持较强的活力和稳定的遗传性。而卷丹在ＭＳ＋１ｍｇ／ＬＮＡＡ上诱导产生小鳞茎,在ＭＳ＋０．５ｍｇ／ＬＮＡＡ＋０．１ｍｇ／Ｌ６－ＢＡ上继代培养较好。结果表明,通过组织培养可以建立百合属植物的种质基因库。     

泡桐愈伤组织再生植株的诱导与培养

首先以MS为基本培养基从18个不同浓度的NAA和BA组合中,找出了毛泡桐（Paulownia tomentosa）、南方泡桐（Pauiownia australis）、白花泡桐（Paulownia fortunei）、兰考泡桐（Paulownia elongata）和豫杂一号泡桐(Paulownia tomentosa×P.fortunei)叶片愈伤组织诱导芽分化最适培养基分别为MS+0.3NAA+12 BA、MS+0.3NAA+12 BA、MS+0.5NAA+12 BA、MS+0.5NAA+12 BA和MS+0.7NAA+12 BA;然后,筛选出了5种泡桐芽诱导根的最适培养基（分别为1/2MS+0.1NAA、1/2MS+0.1NAA、1/2MS、1/2MS+0.3NAA和1/2MS+0.5NAA）。这些结果为利用不同种泡桐的原生质体融合培育泡桐新品种奠定了一定的基础。     

Production of benzylisoquinoline alkaloids in Saccharomyces cerevisiae

The benzylisoquinoline alkaloids (BIAs) are a diverse class of metabolites that exhibit a broad range of pharmacological activities and are synthesized through plant biosynthetic pathways comprised of complex enzyme activities and regulatory strategies. We have engineered yeast to produce the key intermediate reticuline and downstream BIA metabolites from a commercially available substrate. An enzyme tuning strategy was implemented that identified activity differences between variants from different plants and determined optimal expression levels. By synthesizing both stereoisomer forms of reticuline and integrating enzyme activities from three plant sources and humans, we demonstrated the synthesis of metabolites in the sanguinarine/berberine and morphinan branches. We also demonstrated that a human P450 enzyme exhibits a novel activity in the conversion of (R)-reticuline to the morphinan alkaloid salutaridine. Our engineered microbial hosts offer access to a rich group of BIA molecules and associated activities that will be further expanded through synthetic chemistry and biology approaches.     

Effect of α-Naphthalene- and α-Naphthoxyacetic Acid on the Activity of Certain Enzymes of Atropa belladonna cv. lutea Cells in Suspension Culture

The growth of Atropa belladonna cv. lutea Döll cells and the activities of several enzymes were studied as a function of time in suspension cultures without added auxin, with α-naphthaleneacedc acid (NAA) and with α-naphthoxyacetic acid (NOA). The growth curves were rather similar and therefore easy to compare. The enzyme activities (per mg dry weight of culture) were distinctly higher in cultures grown with NAA than in the other two treatments, in which root formation was common. In some cases the enzyme activities were lower in cultures with NOA than in the control series but usually the curves were quite parallel. Promotion of enzyme activities by NAA (aldolase, aminopeptidase, gluta-mate: oxaloacetate transaminase) was clearest in the beginning of the phase of rapid cell division (6-day-old culture). These three enzymes reach their maxima at this stage of growth in the other series as well. Ribonuclease activity increased in the stationary phase cells (13 and 19 days) with NAA but with the other treatments the level of this activity was relatively stable. Peroxidase and phosphatase did not show any characteristic maxima, glutamate: pyruvate transaminase activity was low and no amylase activity could be demonstrated. The results indicate that synthetic plant growth substances can regulate the physiological state of a tissue by causing alterations in enzyme activities, but their action is clearly specific.     

Sesquiterpene Lactones: More Than Protective Plant Compounds With High Toxicity

Sesquiterpene lactones (STLs) constitute a large group of secondary metabolites that are widely distributed in several angiosperm plant families and a few bryophytes, including liverworts. These metabolites are particularly diversified in the family Asteraceae, in which more than 5,000 compounds have been reported so far. In addition to their pharmacological importance and potential therapeutic applications, most STLs display a wide range of protective activities in plants, including acting as anti-herbivory and antimicrobial substances or inhibiting growth of competing plants. These activities are mainly related to their characteristic α,β-unsaturated structural elements, which can participate in Michael-type additions with biological nucleophiles that contain sulfhydryl groups. Supporting the protective roles of STLs, they are mainly located in glandular trichomes of aerial parts because the highly nonspecific toxicity of such compounds necessitates compartmentalization to prevent autotoxicity. However, STLs have also been reported in other aerial and underground organs, where they are assumed to exhibit other biological activities. Recent studies have suggested that these metabolites not only display protective activities due to toxicity but also play key physiological roles in mediating rhizosphere communication among plants, soil microorganisms and plant parasites. STLs have been directly implicated in plant phototropism, resulting in differential growth of a plant organ due to auxin inhibition when accumulated in response to a light stimulus. This review therefore not only highlights the protective roles of STLs in producing plants but also explores the physiological roles of these metabolites, thus providing insights for new research approaches for understanding the roles of STLs in plants and their potential future applications.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

Phylogenetic analysis of the UDP-glycosyltransferase multigene family of Arabidopsis thaliana

A class of UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence is found throughout the plant and animal kingdoms. Whereas mammalian enzymes use UDP-glucuronic acid, the plant enzymes typically use UDP-glucose in the transfer reactions. A diverse array of aglycones can be glucosylated by these UGTs. In plants, the aglycones include plant hormones, secondary metabolites involved in stress and defense responses, and xenobiotics such as herbicides. Glycosylation is known to regulate many properties of the aglycones such as their bioactivity, their solubility, and their transport properties within the cell and throughout the plant. As a means of providing a framework to start to understand the substrate specificities and structure-function relationships of plant UGTs, we have now applied a molecular phylogenetic analysis to the multigene family of 99 UGT sequences in Arabidopsis. We have determined the overall organization and evolutionary relationships among individual members with a surprisingly high degree of confidence. Through constructing a composite phylogenetic tree that also includes all of the additional plant UGTs with known catalytic activities, we can start to predict both the evolutionary history and substrate specificities of new sequences as they are identified. The tree already suggests that while the activities of some subgroups of the UGT family are highly conserved among different plant species, others subgroups shift substrate specificity with relative ease.     

Auxin transport at cellular level: new insights supported by mathematical modelling

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.     

Production of 8-epi-tomentosin by plant cell culture ofXanthium strumarium

This study was conducted to establish a plant cell culture system for the production of medically important secondary metabolites fromXanthium strumarium. The effects of plant growth regulators including NAA, 2,4-D, kinetin, and ABA were examined in terms of callus induction, maintenance of callus and suspension cultures. It was shown that callus was induced upon treatment with NAA while embryo was induced after treatment with 2,4-D. Callus formation was further improved by treatment with ABA and NAA. The level of callusing increased by 17–29% for the seed case, cotyledon, leaf, and hypocotyl and by 96% in the case of the root. Suspension cell lines were established using calli produced from cotyledon, hypocotyl and root and cultured at 25°C under light conditions. The cells grew up to 15 g/L with NAA 2 ppm, BA 2 ppm, and ABA 1 ppm treatment. Supernatants of suspension cultures of cell lines derived from coyledon and hypocotyl produced some distinctive secondary metabolites, one of which was identified as 8-epi-tomentosin, which belongs to the xanthanolides. The amounts of 8-epi-tomentosin produced by the cotyledon-and hypocotylderived cell lines were 13.4 mg/L and 11.0 mg/L, respectively.     

Improvement of hairy root cultures and plants by changing biosynthetic pathways leading to pharmaceutical metabolites: Strategies and applications

A plethora of bioactive plant metabolites has been explored for pharmaceutical, food chemistry and agricultural applications. The chemical synthesis of these structures is often difficult, so plants are favorably used as producers. While whole plants can serve as a source for secondary metabolites and can be also improved by metabolic engineering, more often cell or organ cultures of relevant plant species are of interest. It should be noted that only in few cases the production for commercial application in such cultures has been achieved. Their genetic manipulation is sometimes faster and the production of a specific metabolite is more reliable, because of less environmental influences. In addition, upscaling in bioreactors is nowadays possible for many of these cultures, so some are already used in industry. There are approaches to alter the profile of metabolites not only by using plant genes, but also by using bacterial genes encoding modifying enzymes. Also, strategies to cope with unwanted or even toxic compounds are available. The need for metabolic engineering of plant secondary metabolite pathways is increasing with the rising demand for (novel) compounds with new bioactive properties. Here, we give some examples of recent developments for the metabolic engineering of plants and organ cultures, which can be used in the production of metabolites with interesting properties.