Improved metabolites of pharmaceutical ingredient grade Ginkgo biloba and the correlated proteomics analysis

Ginkgo biloba is an attractive and traditional medicinal plant, and has been widely used as a phytomedicine in the prevention and treatment of cardiovascular and cerebrovascular diseases. Flavonoids and terpene lactones are the major bioactive components of Ginkgo, whereas the ginkgolic acids (GAs) with strong allergenic properties are strictly controlled. In this study, we tested the content of flavonoids and GAs under ultraviolet‐B (UV‐B) treatment and performed comparative proteomic analyses to determine the differential proteins that occur upon UV‐B radiation. That might play a crucial role in producing flavonoids and GAs. Our phytochemical analyses demonstrated that UV‐B irradiation significantly increased the content of active flavonoids, and decreased the content of toxic GAs. We conducted comparative proteomic analysis of both whole leaf and chloroplasts proteins. In total, 27 differential proteins in the whole leaf and 43 differential proteins in the chloroplast were positively identified and functionally annotated. The proteomic data suggested that enhanced UV‐B radiation exposure activated antioxidants and stress‐responsive proteins as well as reduced the rate of photosynthesis. We demonstrate that UV‐B irradiation pharmaceutically improved the metabolic ingredients of Ginkgo, particularly in terms of reducing GAs. With high UV absorption properties, and antioxidant activities, the flavonoids were likely highly induced as protective molecules following UV‐B irradiation.     

Underestimated biodiversity as a major explanation for the perceived rich secondary metabolite capacity of the cyanobacterial genus Lyngbya

Marine cyanobacteria are prolific producers of bioactive secondary metabolites responsible for harmful algal blooms as well as rich sources of promising biomedical lead compounds. The current study focused on obtaining a clearer understanding of the remarkable chemical richness of the cyanobacterial genus Lyngbya. Specimens of Lyngbya from various environmental habitats around Curaçao were analysed for their capacity to produce secondary metabolites by genetic screening of their biosynthetic pathways. The presence of biosynthetic pathways was compared with the production of corresponding metabolites by LC‐ESI‐MS² and MALDI‐TOF‐MS. The comparison of biosynthetic capacity and actual metabolite production revealed no evidence of genetic silencing in response to environmental conditions. On a cellular level, the metabolic origin of the detected metabolites was pinpointed to the cyanobacteria, rather than the sheath‐associated heterotrophic bacteria, by MALDI‐TOF‐MS and multiple displacement amplification of single cells. Finally, the traditional morphology‐based taxonomic identifications of these Lyngbya populations were combined with their phylogenetic relationships. As a result, polyphyly of morphologically similar cyanobacteria was identified as the major explanation for the perceived chemical richness of the genus Lyngbya, a result which further underscores the need to revise the taxonomy of this group of biomedically important cyanobacteria.     

Secondary Metabolites,Glandular Trichomes and Biological Activity of Sideritis montana L. subsp. montana from Central Italy

Sideritis montana subsp. montana is a small annual herb occurring in countries bordering the Mediterranean and Balkan regions. The secondary metabolism of this plant has not been fully explored so far. The aim of the present study was to understand the complex mixture of secondary metabolites and the type of secretory structures. The polar constituents were isolated by column chromatography from the ethanolic extract, and their structure was elucidated by NMR and MS. The essential oil was isolated by hydrodistillation and analysed by GC/MS. The plant indumentum was studied by light and scanning electron microscopy. To complete the work, the essential oil antioxidant activity and cytotoxicity on tumor cells were evaluated by DPPH, ABTS, FRAP, and MTT methods. Four different classes of secondary metabolites were isolated, namely flavonoids, caffeoylquinic derivatives, glycosidic hydroquinones and iridoids. The essential oil was mainly characterized by sesquiterpenene hydrocarbons. Peltate and long‐capitate hairs were the main sites where terpenes and polar constituents are produced. The secondary metabolites found in S. montana subsp. montana are of chemotaxonomic interest, some of them being typical of the genus Sideritis. The trichomes types observed partially differ from those described in other members of the genus Sideritis. The essential oil showed noteworthy inhibition on tumor cells.     

Identification of Genes Involved in Flavonoid Biosynthesis in Sophora japonica Through Transcriptome Sequencing

Sophora japonica is a traditional Chinese medicinal ingredient that is widely used in the medicine, food, and industrial dye industries. Since flavonoids are the main components of S. japonica, studying the flavonoid composition and content of this plant is important. This study aimed to identify molecules involved in the flavonoid biosynthetic pathways in S. japonica. Deep sequencing was performed, and 85,877,352 clean reads were filtered from 86,095,152 raw reads. The clean reads were spliced to obtain 111,382 unigenes, which were then annotated with NR, GO, KEGG, eggNOG. Differential expression analysis and NR function prediction revealed 18 differentially expressed unigenes associated with 13 enzymes in flavonoid biosynthetic pathways. Our results reveal new insights on secondary metabolite biosynthesis‐related genes in S. japonica and enhance the potential applications of S. japonica in genetic engineering.     

Exploring membrane and cytoplasm proteomic responses of Alkalimonas amylolytica N10 to different external pHs with combination strategy of de novo peptide sequencing

Identification of differentially proteomic responses to external pHs would pave an access for understanding of survival mechanisms of bacteria living at extreme pH environment. We cultured Alkalimonas amylolytica N10 (N10), a novel alkaliphilic bacterium found in Lake Chahannor, in media with three different pHs and extracted the correspondent membrane and cytoplasm proteins for proteomic analysis through 2‐DE. The differential 2‐DE spots corresponding to the altered pHs were delivered to MALDI TOF/TOF MS for protein identification. Since the genomic data of strain N10 was unavailable, we encountered a problem at low rate of protein identification with 18.1%. We employed, therefore, a combined strategy of de novo sequencing to analyze MS/MS signals generated from MALDI TOF/TOF MS. A significantly improved rate of protein identification was thus achieved at over than 70.0%. Furthermore, we extensively investigated the expression of these pH‐dependent N10 genes using Western blot and real‐time PCR. The conclusions drawn from immunoblot and mRNA measurements were mostly in agreement with the proteomic observations. We conducted the bioinformatic analysis to all the pH‐dependent N10 proteins and found that some membrane proteins participated in iron transport were differentially expressed as external pH elevated and most of differential proteins with increased or bell‐shape mode of pH‐dependence were involved in bioenergetic process and metabolism of carbohydrates, fatty acid, amino acids, and nucleotides. Our data thus provide a functional profile of the pH‐responsive proteins in alkaliphiles, leading to elucidation of alkaliphilic‐adaptive mechanism.     

Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated protein

Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully ¹³C‐labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Chitosan‐triggered immunity to Fusarium in chickpea is associated with changes in the plant extracellular matrix architecture,stomatal closure and remodeling of the plant metabolome and proteome

Pathogen‐/microbe‐associated molecular patterns (PAMPs/MAMPs) initiate complex defense responses by reorganizing the biomolecular dynamics of the host cellular machinery. The extracellular matrix (ECM) acts as a physical scaffold that prevents recognition and entry of phytopathogens, while guard cells perceive and integrate signals metabolically. Although chitosan is a known MAMP implicated in plant defense, the precise mechanism of chitosan‐triggered immunity (CTI) remains unknown. Here, we show how chitosan imparts immunity against fungal disease. Morpho‐histological examination revealed stomatal closure accompanied by reductions in stomatal conductance and transpiration rate as early responses in chitosan‐treated seedlings upon vascular fusariosis. Electron microscopy and Raman spectroscopy showed ECM fortification leading to oligosaccharide signaling, as documented by increased galactose, pectin and associated secondary metabolites. Multiomics approach using quantitative ECM proteomics and metabolomics identified 325 chitosan‐triggered immune‐responsive proteins (CTIRPs), notably novel ECM structural proteins, LYM2 and receptor‐like kinases, and 65 chitosan‐triggered immune‐responsive metabolites (CTIRMs), including sugars, sugar alcohols, fatty alcohols, organic and amino acids. Identified proteins and metabolites are linked to reactive oxygen species (ROS) production, stomatal movement, root nodule development and root architecture coupled with oligosaccharide signaling that leads to Fusarium resistance. The cumulative data demonstrate that ROS, NO and eATP govern CTI, in addition to induction of PR proteins, CAZymes and PAL activities, besides accumulation of phenolic compounds downstream of CTI. The immune‐related correlation network identified functional hubs in the CTI pathway. Altogether, these shifts led to the discovery of chitosan‐responsive networks that cause significant ECM and guard cell remodeling, and translate ECM cues into cell fate decisions during fusariosis.     

DIFFERENTIAL PROTEOME ANALYSIS OF THE MALE AND FEMALE ANTENNAE FROM Holotrichia parallela

To understand the olfactory mechanisms of Holotrichia parallela antennae in detecting volatile compounds in the environment, protein profiles of H. parallela antennae were analyzed using two‐dimensional electrophoresis followed by mass spectrometry and bioinformatics analyses. Approximately 1,100 protein spots in silver staining gel were detected. Quantitative image analysis revealed that in total 47 protein spots showed significant changes in different genders of adult antennae. Thirty‐five differentially expressed proteins were identified by Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI‐TOF/TOF) tandem mass spectrometer, among which 65.7% are involved in carbohydrate and energy metabolism, antioxidant system, transport, and amino acid/nucleotide metabolism. Some proteins identified here have not been reported previously in insect antennae. Identified male‐biased proteins included odorant‐binding protein 4, pheromone‐binding protein‐related protein 2, odorant‐binding protein 14, prophenoloxidase‐I, acyl‐CoA dehydrogenase, aldo‐keto reductase‐like, carbamoyl phosphate synthetase, etc. whereas some proteins are female biased, such as antennae‐rich cytochrome P450, aldehyde dehydrogenase, and putative glutamine synthetase. Alterations in the levels of some proteins were further confirmed by real time polymerase chain reaction (RT‐PCR). The proteomic resources displayed here are valuable for the discovery of proteins from H. parallela antennae.     

Proteomic analysis reveals responsive proteins of Vibrio parahaemolyticus on exposure to cationic antimicrobial peptides

Aims: To investigate whether Vibrio parahaemolyticus can sense and directly respond to the presence of cationic antimicrobial peptides (AMPs). Methods and Results: We performed proteomic methodologies to investigate the responsive proteins of V. parahaemolyticus on exposure to AMP Q6. Differential expression patterns of the outer membrane, inner membrane and cytoplasmic proteins (OMPs, IMPs and CPs) from the bacteria with or without Q6 treatment were obtained using two‐dimensional gel electrophoresis (2‐DE). Three OMPs (maltoporin, flagellin and OmpV), two IMPs (ATP synthase F1, alpha subunit; and OmpV) and three CPs (pyruvate dehydrogenase subunit E1, glyceraldehyde‐3‐phosphate dehydrogenase and inositol‐5‐monophosphate dehydrogenase) were identified using LC‐ESI‐Q‐TOF MS/MS and Mascot program. Real‐time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. Conclusions: Our results suggested that V. parahaemolyticus may directly respond to AMPs through the upregulation of the efflux channel, increased yield of energy, effective repair of damaged membranes and downregulation of carbohydrate and nucleotide metabolism for energy preservation. Significance and Impact of the Study: AMP‐responsive proteins identified in the current study could serve as attractive targets for developing more effective antimicrobial agents.     

Allocation of carbon to growth and secondary metabolites in birch seedlings under UV-B radiation and CO2 exposure

In plants, the allocation of carbon to secondary metabolites has been shown to be determined by both the availability of resources (e.g., CO₂ concentration) and by specific stress factors (e.g., ultraviolet [UV]‐radiation). It has been suggested that, in combination, CO₂ and UV‐B radiation may differentially affect plant growth and morphogenic parameters, and elevated CO₂ may ameliorate the effects of UV‐B radiation. In the present work, the effects of increased atmospheric CO₂ concentration and UV‐B radiation on growth and the accumulation of different types of secondary metabolites were studied in silver birch (Betula pendula Roth). Seedlings were exposed to 350 and 700 μmol mol^?1 of CO₂ in a greenhouse. At both CO₂ levels, additional UV‐B was either present (8.16 kJ m^?2 day^?1 of biologically effective UV‐B irradiance) or absent. The time course of accumulation of individual secondary compounds and the shifts in allocation of carbon between biomass and the secondary metabolites (phenolic acids, flavonoids, condensed tannins) were studied during a 1‐month‐long exposure. Additionally, the activities of enzymes ( l ‐phenylalanine ammonia‐lyase [PAL], EC 4.3.1.5; peroxidase, EC 1.11.1.7; polyphenol oxidase, EC 1.10.3.1) were determined for leaves. UV‐B radiation significantly increased biomass, PAL activity, and the accumulation of phenolic acids and flavonoids in seedlings. Elevated CO₂ concentration increased the activities of all the enzymes studied and the accumulation of condensed tannins in leaves, especially with UV‐B radiation. Because the observed UV‐B induction of flavonoids was smaller under a high CO₂ concentration, it was suggested that the excess of carbon in the atmosphere may moderate the effect of UV‐B by increasing the metabolic activity of leaves (high enzyme activities) and by changing the allocation of internal carbon between different primary and secondary metabolites in the plant. Our results demonstrate the significant increase in the allocation of carbon to secondary metabolites without any large change in growth due to the elevation of CO₂ concentration and UV‐B radiation. There also was a stronger impact of CO₂ than UV‐B on the phenolic metabolism of birch seedlings.     

Matrix‐free UV‐laser desorption/ionization (LDI) mass spectrometric imaging at the single‐cell level: distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species

The present paper describes matrix‐free laser desorption/ionisation mass spectrometric imaging (LDI‐MSI) of highly localized UV‐absorbing secondary metabolites in plant tissues at single‐cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 μm, was much higher than that achieved by commonly used matrix‐assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI‐TOF/MS analysis of cryo‐sectioned, laser‐microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI‐TOF/MS or LDI‐MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI‐MSI for other commonly occurring compounds such as flavonoids. The organ‐specific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level.     

Sekundärstoffe – die Geheimwaffen der Pflanzen

Secondary metabolites Already 400 million years ago when land plants evolved, they probably produced secondary metabolites as means of defence against herbivores, microbes and competing plants. Secondary metabolites usually are bioactive agents, which can interfere with molecular targets in animals and microbes. Therefore, many plants and substances isolated from them can serve as valuable drugs in medicine and pharmacy. Some secondary metabolites also serve as signal compounds to attract pollinating animals and seed‐dispersing animals, but also for UV protection, as antioxidants or mobile nitrogen stores. Biology and evolution but also physiological and genetic bases of secondary metabolism are discussed in this overview.     

Heterologous expression system in <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for fungal biosynthetic gene clusters of secondary metabolites

Fungal secondary metabolites have been considered promising resources in the search for novel bioactive compounds. Given the high potential of fungi as genetic resources, it is essential to find an efficient way to link biosynthetic genes to the product in a heterologous system, because many genes for the secondary metabolite in the original strain are silent under standard laboratory conditions. In a previous study, we constructed a heterologous expression system for a biosynthetic gene cluster using Aspergillus oryzae as the host. To make the host more versatile for the expression of secondary metabolism genes, the expression levels of a global regulator, laeA, were increased by placing the A. oryzae laeA gene under the control of the constitutive active pgk promoter. In the A. oryzae overexpressing laeA, two clusters of heterologous biosynthetic genes [the monacolin K (MK) gene cluster from Monascus pilosus and the terrequinone A (TQ) gene cluster from Aspergillus nidulans] were successfully overexpressed, resulting in the production of the corresponding metabolite, MK or TQ. The successful production of secondary metabolites belonging to different structural groups, namely MK as a polyketide and TQ as a hybrid of amino acid and isoprenoid, indicated that the laeA-enriched A. oryzae was a versatile host for the heterologous expression of the biosynthetic gene cluster.     

A radiochemical technique with potential for revealing novel fungal metabolites according to expression of specific biosynthetic activities

Fungal secondary metabolites are mostly derived from a few key intermediates in primary metabolism, such as acetate and some amino acids. Classical screens for novel fungal compounds of possible industrial interest have used chromatographic displays of extract components, as was the practice for plant natural products, followed by structural determination and pharmacological study. In contrast, modern robotic screens usually focus initially on specific bioassay applied to fermentation products and crude extracts. Both strategies are expensive in terms of human resources and/or in sophisticated equipment. A relatively inexpensive technique, exploiting biosynthetic principles through use of ¹⁴C-labelled probes to recognise particular structural features by autoradiography of tlc plates is described and discussed. Application to 80 isolates of filamentous fungi from Caribbean and European/Mediterranean environments enabled recognition of arrays of metabolites according to their biosynthetic origin, showing the potential of the technique in novel product discovery in unsophisticated laboratory facilities, as exemplified by reference to subsequent discovery of novel metabolites produced by Amorosia littoralis.     

Comparative transcriptome mining for terpenoid biosynthetic pathway genes in wild and cultivated species of Plantago

Protoplasma - Plantagos are important economical and medicinal plants that possess several bioactive secondary metabolites, such as phenolics, iridoids, triterpenes, and alkaloids. Triterpenoids...     

A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: Identification of biosynthetic pathways of the major terpenoid‐related compounds and possible transport proteins

Female flowers of hop (Humulus lupulus) are an essential source of terpenoid‐related compounds, which are mainly used as flavoring in the beer brewing process. The compounds involved are bitter acids, terpenophenolics, as well as mono‐ and sesquiterpenoids. In this work, we analyzed the proteome of purified glandular trichomes (lupulin glands) from female flowers, which produce and accumulate these compounds. An extensive 2D‐LC‐MS/MS analysis identified 1015 proteins. Of these, most correspond to housekeeping and primary metabolism‐related proteins, albeit predominantly including amino acid and lipid metabolism, which feeds the specialized (secondary) metabolism. Indeed, 75 proteins belong to the specialized metabolism. No less than 40 enzymes are involved in the synthesis of terpenoid‐derived compounds and 21 are predicted transporters, some of which might be involved in the transport of specialized metabolites. We discuss the possible routes involved in the intra‐ and intercellular translocation of terpenoids and their precursors. This comprehensive proteomic map of the glandular trichomes of hop female flowers represents a valuable resource to improve our knowledge on the function of glandular trichomes.