Chemical Elicitors-Induced Variation in Cellular Biomass, Biosynthesis of Secondary Cell Products, and Antioxidant System in Callus Cultures of Fagonia indica.

Fagonia indica is a rich source of pharmacologically active compounds. The variation in the metabolites of interest is one of the major issues in wild plants due to different environmental factors. The addition of chemical elicitors is one of the effective strategies to trigger the biosynthetic pathways for the release of a higher quantity of bioactive compounds. Therefore, this study was designed to investigate the effects of chemical elicitors, aluminum chloride (AlCl3) and cadmium chloride (CdCl2), on the biosynthesis of secondary metabolites, biomass, and the antioxidant system in callus cultures of F. indica. Among various treatments applied, AlCl3 (0.1 mM concentration) improved the highest in biomass accumulation (fresh weight (FW): 404.72 g/L) as compared to the control (FW: 269.85 g/L). The exposure of cultures to AlCl3 (0.01 mM) enhanced the accumulation of secondary metabolites, and the total phenolic contents (TPCs: 7.74 mg/g DW) and total flavonoid contents (TFCs: 1.07 mg/g DW) were higher than those of cultures exposed to CdCl2 (0.01 mM) with content levels (TPC: 5.60 and TFC: 0.97 mg/g) as compared to the control (TPC: 4.16 and TFC: 0.42 mg/g DW). Likewise, AlCl3 and CdCl2 also promoted the free radical scavenging activity (FRSA; 89.4% and 90%, respectively) at a concentration of 0.01 mM, as compared to the control (65.48%). For instance, the quantification of metabolites via high-performance liquid chromatography (HPLC) revealed an optimum production of myricetin (1.20 mg/g), apigenin (0.83 mg/g), isorhamnetin (0.70 mg/g), and kaempferol (0.64 mg/g). Cultures grown in the presence of AlCl3 triggered higher quantities of secondary metabolites than those grown in the presence of CdCl2 (0.79, 0.74, 0.57, and 0.67 mg/g). Moreover, AlCl3 at 0.1 mM enhanced the biosynthesis of superoxide dismutase (SOD: 0.08 nM/min/mg-FW) and peroxidase enzymes (POD: 2.37 nM/min/mg-FW), while CdCl2 resulted in an SOD activity up to 0.06 nM/min/mg-FW and POD: 2.72 nM/min/mg-FW. From these results, it is clear that AlCl3 is a better elicitor in terms of a higher and uniform productivity of biomass, secondary cell products, and antioxidant enzymes compared to CdCl2 and the control. It is possible to scale the current strategy to a bioreactor for a higher productivity of metabolites of interest for various pharmaceutical industries.

Identification and metabolomic analysis of chemical elicitors for tacrolimus accumulation in Streptomyces tsukubaensis.

Tacrolimus is a widely used immunosuppressive agent in the treatment of various clinical diseases. However, the low fermentation yield seriously limits its further application. To stimulate tacrolimus synthesis, nine chemical elicitors of five groups were evaluated for their effects on tacrolimus accumulation in S. tsukubaensis. The results showed that sodium butyrate (SB), dimethylsulfoxide (DMSO), and LaCl3 could increase tacrolimus accumulation by more than 30%. Cumulative effects of different chemical elicitors exhibited that the highest tacrolimus yield was improved by 64.7% (303.60 mg/L) in DMSO and La treatment, compared to the control. To decipher possible response mechanism, a weighted correlation network analysis (WGCNA) based on metabolomics was employed and datasets showed 13 distinct metabolic modules and 16 hub metabolites were possibly related to the stimulatory roles of DMSO, La, SB, and their combination treatments. The pathway analysis further exhibited that central carbon metabolism, amino acid metabolism, and fatty acid metabolism showed significant differences in the above chemical elicitor treatments. Thereinto, the carboxylation of propionyl-CoA from isoleucine and methionine degradation was first confirmed to be the main source of methylmalonyl-CoA by RT-PCR analysis in DMSO and La treatment. By further strengthening of the supply of methylmalonyl-CoA precursor in DMSO and La treatment, the final tacrolimus yield could reach to 372.12 mg/L, 2.02-fold higher than the control. To our knowledge, this is the first study to unveil the potential mechanism of different chemical elicitor stresses in S. tsukubaensis based on metabolomics, and the established information provide valuable guidance for further improving tacrolimus production.

Extending the "One Strain Many Compounds" (OSMAC) Principle to Marine Microorganisms.

Genomic data often highlights an inconsistency between the number of gene clusters identified using bioinformatic approaches as potentially producing secondary metabolites and the actual number of chemically characterized secondary metabolites produced by any given microorganism. Such gene clusters are generally considered as "silent", meaning that they are not expressed under laboratory conditions. Triggering expression of these "silent" clusters could result in unlocking the chemical diversity they control, allowing the discovery of novel molecules of both medical and biotechnological interest. Therefore, both genetic and cultivation-based techniques have been developed aimed at stimulating expression of these "silent" genes. The principles behind the cultivation based approaches have been conceptualized in the "one strain many compounds" (OSMAC) framework, which underlines how a single strain can produce different molecules when grown under different environmental conditions. Parameters such as, nutrient content, temperature, and rate of aeration can be easily changed, altering the global physiology of a microbial strain and in turn significantly affecting its secondary metabolism. As a direct extension of such approaches, co-cultivation strategies and the addition of chemical elicitors have also been used as cues to activate "silent" clusters. In this review, we aim to provide a focused and comprehensive overview of these strategies as they pertain to marine microbes. Moreover, we underline how changes in some parameters which have provided important results in terrestrial microbes, but which have rarely been considered in marine microorganisms, may represent additional strategies to awaken "silent" gene clusters in marine microbes. Unfortunately, the empirical nature of the OSMAC approach forces scientists to perform extensive laboratory experiments. Nevertheless, we believe that some computation and experimental based techniques which are used in other disciplines, and which we discuss; could be effectively employed to help streamline the OSMAC based approaches. We believe that natural products discovery in marine microorganisms would be greatly aided through the integration of basic microbiological approaches, computational methods, and technological innovations, thereby helping unearth much of the as yet untapped potential of these microorganisms.

The Commonly Used Bactericide Bismerthiazol Promotes Rice Defenses against Herbivores.

Chemical elicitors that enhance plant resistance to pathogens have been extensively studied, however, chemical elicitors that induce plant defenses against insect pests have received little attention. Here, we found that the exogenous application of a commonly used bactericide, bismerthiazol, on rice induced the biosynthesis of constitutive and/or elicited jasmonic acid (JA), jasmonoyl-isoleucine conjugate (JA-Ile), ethylene and H2O2 but not salicylic acid. These activated signaling pathways altered the volatile profile of rice plants. White-backed planthopper (WBPH, Sogatella furcifera) nymphs and gravid females showed a preference for feeding and/or oviposition on control plants: survival rates were better and more eggs were laid than on bismerthiazol-treated plants. Moreover, bismerthiazol treatment also increased both the parasitism rate of WBPH eggs laid on plants in the field by Anagrus nilaparvatae, and also the resistance of rice to the brown planthopper (BPH) Nilaparvata lugens and the striped stem borer (SSB) Chilo suppressalis. These findings suggest that the bactericide bismerthiazol can induce the direct and/or indirect resistance of rice to multiple insect pests, and so can be used as a broad-spectrum chemical elicitor.

Finding new elicitors that induce resistance in rice to the white-backed planthopper Sogatella furcifera.

Herein we report a new way to identify chemical elicitors that induce resistance in rice to herbivores. Using this method, by quantifying the induction of chemicals for GUS activity in a specific screening system that we established previously, 5 candidate elicitors were selected from the 29 designed and synthesized phenoxyalkanoic acid derivatives. Bioassays confirmed that these candidate elicitors could induce plant defense and then repel feeding of white-backed planthopper Sogatella furcifera.

Chemical elicitation as an avenue for discovery of bioactive compounds from fungal endophytes.

The present study investigated the molecular phylogeny, antimicrobial and cytotoxic activities of fungal endophytes obtained from the A*STAR Natural Organism Library (NOL) and previously isolated from Sungei Buloh Wetland Reserve, Singapore. Phylogenetic analysis based on ITS2 gene suggests that these isolates belong to 46 morphotypes and are affiliated to 23 different taxa in 17 genera of the Ascomycota phylum. Colletotrichum was the most dominant fungal genus accounting for 37% of all the isolates, followed by Diaporthe (13%), Phyllosticta (10.9%) and Diplodia (8.7%). Chemical elicitation using 5-azacytidine, a DNA methyltransferase inhibitor and suberoylanilide hydroxamic acid, a histone deacetylase inhibitor resulted in an increase in the number of active strains. Bioassay-guided isolation and structural elucidation yielded pestahivin and two new analogues from Bartalinia sp. F9447. Pestahivin and its related analogues did not exhibit antibacterial activity against Staphylococcus aureus but displayed strong antifungal activities against Candida albicans and Aspergillus brasiliensis, with IC50 values ranging from 0.46 ± 0.06 to 144 ± 18 µM. Pestahivin and its two analogues furthermore exhibited cytotoxic activity against A549 and MIA PACA-2 cancer cell lines with IC50 values in the range of 0.65 ± 0.12 to 42 ± 5.2 µM. The finding from this study reinforces that chemical epigenetic induction is a promising approach for the discovery of bioactive fungal secondary metabolites encoded by cryptic gene clusters.

Integrated Metabolomic and Network Analysis to Explore the Potential Mechanism of Three Chemical Elicitors in Rapamycin Overproduction.

Rapamycin is a polyketide macrocyclic antibiotic with exceptional pharmacological potential. To explore the potential mechanism of rapamycin overproduction, the intracellular metabolic differences of three chemical elicitor treatments were first investigated by combining them with dynamic metabolomics and network analysis. The metabolic response characteristics of each chemical elicitor treatment were identified by a weighted gene co-expression network analysis (WGCNA) model. According to the analysis of the identified metabolic modules, the changes in the cell membrane permeability might play a key role in rapamycin overproduction for dimethyl sulfoxide (DMSO) treatment. The enhancement of the starter unit of 4,5-dihydroxycyclohex-1-ene carboxylic acid (DHCHC) and the nicotinamide adenine dinucleotide phosphate (NADPH) availability were the main functions in the LaCl3 treatment. However, for sodium butyrate (SB), the improvement of the methylmalonyl-CoA and NADPH availability was a potential reason for the rapamycin overproduction. Further, the responsive metabolic pathways after chemical elicitor treatments were selected to predict the potential key limiting steps in rapamycin accumulation using a genome-scale metabolic network model (GSMM). Based on the prediction results, the targets within the reinforcement of the DHCHC and NADPH supply were selected to verify their effects on rapamycin production. The highest rapamycin yield improved 1.62 fold in the HT-aroA/zwf2 strain compared to the control.

Production and Secretion of Isowighteone in Hairy Root Cultures of Pigeon Pea (Cajanus cajan) Co-Treated with Multiple Elicitors.

Isowighteone (3'-isoprenyl genistein) is a prenylated stilbenoid derivative that exhibits neuroprotective, antibacterial, and anti-inflammatory properties. To establish a bioproduction system for this bioactive compound, hairy root cultures of pigeon pea (Cajanus cajan (L.) Millsp.) were developed via Agrobacterium rhizogenes-mediated transformation. The cultures were co-treated with methyl jasmonate, cyclodextrin, hydrogen peroxide, and magnesium chloride to enhance the production of isowighteone. The amount of isowighteone that accumulated in the culture medium upon elicitation varied with the period of elicitation. Isowighteone was purified from extracts of the culture medium by semi-preparative HPLC, and its identity was confirmed by tandem mass spectrometry. After 144 h of elicitation in 12-day-old hairy root culture, the total yield of isowighteone was 8058.618 ± 445.78 µg/g DW, of which approximately 96% was found in the culture medium. The yield of isowighteone in the elicited hairy root culture was approximately 277-fold higher than in the non-elicited root culture. The difference between the phenotypes of the elicited and non-elicited pigeon pea hairy roots was studied using scanning electron microscopy. The non-elicited hairy roots had uniform surfaces whereas the elicited roots had non-uniform shapes. Pigeon pea hairy roots provide a sustainable platform for producing and studying the biosynthesis of isowighteone.

Chemical Elicitors Induce Rare Bioactive Secondary Metabolites in Deep-Sea Bacteria under Laboratory Conditions.

Bacterial genome sequencing has revealed a vast number of novel biosynthetic gene clusters (BGC) with potential to produce bioactive natural products. However, the biosynthesis of secondary metabolites by bacteria is often silenced under laboratory conditions, limiting the controlled expression of natural products. Here we describe an integrated methodology for the construction and screening of an elicited and pre-fractionated library of marine bacteria. In this pilot study, chemical elicitors were evaluated to mimic the natural environment and to induce the expression of cryptic BGCs in deep-sea bacteria. By integrating high-resolution untargeted metabolomics with cheminformatics analyses, it was possible to visualize, mine, identify and map the chemical and biological space of the elicited bacterial metabolites. The results show that elicited bacterial metabolites correspond to ~45% of the compounds produced under laboratory conditions. In addition, the elicited chemical space is novel (~70% of the elicited compounds) or concentrated in the chemical space of drugs. Fractionation of the crude extracts further evidenced minor compounds (~90% of the collection) and the detection of biological activity. This pilot work pinpoints strategies for constructing and evaluating chemically diverse bacterial natural product libraries towards the identification of novel bacterial metabolites in natural product-based drug discovery pipelines.

Silver Can Induce Oxidative Stress in Parallel to Other Chemical Elicitors to Modulate the Ripening of Chili Cultivars.

Two chili cultivars, i.e., cv. Bullet and cv. Tejaswini, were evaluated on postharvest related ripening characteristics with varying durations under hydrogen peroxide, putrescine and silver treatments. The reducing sugar was inversely related to the maximum values at 7 days of ripening. Silver and putrescine were the most regulatory in terms of changing of the total carbohydrate content as compared to hydrolysis of the total reducing sugar. Regarding pectin methylesterase activity, both chilies were consistent, regardless of the number of days of incubation. Still, putrescine and silver were significant contributors to variations in cv. Bullet and cv. Tejaswani. For the pigment content, lycopene and chlorophyll increased in a linear manner, although these treatments significantly varied over time. Hydrogen peroxide and putrescine were responsible for the maximum accumulation of lycopene for both the cultivars, whereas, only cv. Tejaswani displayed maximum carotenoid for putrescine. Silver for both chili varieties was the most inhibitory for lycopene and carotenoid content. Superoxide had a good impact on the accumulation of lipid peroxides, irrespective of the chili variety. The maximum accumulation of lipid peroxide was recorded at seven days of treatment. Phenolics and flavonoids were in decreasing order for both the chili varieties, progressing through the days of the study period in a similar manner. Silver was the main contributor to variations in the phenolics and flavonoid contents in cv. Tejaswani. The solubilization of total carbohydrate into reducing sugar was in an inverse relationship, with the maximum values being reached at 7 days of ripening.

Recent advances in inducing endophytic fungal specialized metabolites using small molecule elicitors including epigenetic modifiers.

Today when the quest of new lead molecules to supply the development pipeline is driving the course of drug discovery, endophytic fungi with their outstanding biosynthetic potential seem to be highly promising avenues for natural product scientists. However, challenges such as the production of inadequate quantities of compounds, the attenuation or loss of ability of endophytes to produce the compound of interest when grown in culture and the inability of fungal endophytes to express their full biosynthetic potential in laboratory conditions have been the major constraints. These have led to the application of small chemical elicitors that induce epigenetic changes in fungi to activate their silent gene clusters optimizing the amount of metabolites of interest or inducing the synthesis of hitherto undescribed compounds. In this respect small molecular weight compounds which are known to function as inhibitors of histone deacetylase (HDAC), DNA methyltransferase (DNMT) and proteasome have proven their efficacy in enhancing or inducing the production of specialized metabolites by fungi. Moreover, organic solvents, metals and plants extracts are also acknowledged for their ability to cause shifts in fungal metabolism. We highlight the successful studies from the past two decades reporting the ability of structurally diverse small molecular weight compounds to elicit the production of previously undescribed metabolites from endophytic fungi grown in culture. This mini review argues in favor of chemical elicitation as an effective strategy to optimize the production of fungal metabolites and invigorate the pipeline of drug discovery with new chemical entities.

Induced Tomato Resistance Against Bemisia tabaci Triggered by Salicylic Acid, ß-Aminobutyric Acid, and Trichoderma.

Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) biotype B is a key pest of Solanum lycopersicum L. (Solanaceae) throughout the world. In this study, we examined the induction of resistance on tomato plants treated with SA, BABA, and Trichoderma either individually or in combination against B. tabaci biotype B through the assessment of some biological and behavioral aspects of this insect pest. Also, to understand the mode of action of these inducers, we correlated and analyzed the biochemical basis of plant resistance, by measuring levels of polyphenol oxidase (PPO), peroxidase (POD), phenylalanine ammonia lyase (PAL), and phenolic content in leaves of treated tomato plants. The longest development time of whitefly immature stages was recorded for plants treated with root ß-aminobutyric acid application (RBABA) + root Trichoderma application (RT), root salicylic acid application (RSA) + RT, and RT. In a free-choice assay, B. tabaci adults showed a significantly lower preference for settling and oviposition in RBABA + RT, RSA + RT, and RT in comparison with control. In a no-choice assay, B. tabaci females laid significantly fewer eggs on treatments than those in control, with better results observed in RBABA + RT. Plants responded to different treatments and showed higher induction of PPO, POD, and PAL activities, besides the higher accumulation of phenols in RBABA + RT, RSA + RT, and RT treatments. These results suggest that RBABA + RT, RSA + RT, and RT could be utilized for the induction of effective plant defense against B. tabaci.

Exploring the Antibacterial Activity of Pestalotiopsis spp. under Different Culture Conditions and Their Chemical Diversity Using LC-ESI-Q-TOF-MS.

As a result of the capability of fungi to respond to culture conditions, we aimed to explore and compare the antibacterial activity and chemical diversity of two endophytic fungi isolated from Hyptis dilatata and cultured under different conditions by the addition of chemical elicitors, changes in the pH, and different incubation temperatures. Seventeen extracts were obtained from both Pestalotiopsis mangiferae (man-1 to man-17) and Pestalotiopsis microspora (mic-1 to mic-17) and were tested against a panel of pathogenic bacteria. Seven extracts from P. mangiferae and four extracts from P. microspora showed antibacterial activity; while some of these extracts displayed a high-level of selectivity and a broad-spectrum of activity, Pseudomonas aeruginosa was the most inhibited microorganism and was selected to determine the minimal inhibitory concentration (MIC). The MIC was determined for extracts man-6 (0.11 µg/mL) and mic-9 (0.56 µg/mL). Three active extracts obtained from P. mangiferae were analyzed by Liquid Chromatography-Electrospray Ionization-Quadrupole-Time of Flight-Mass Spectrometry (LC-ESI-Q-TOF-MS) to explore the chemical diversity and the variations in the composition. This allows us to propose structures for some of the determined molecular formulas, including the previously reported mangiferaelactone (1), an antibacterial compound.

Diversity-Oriented Synthesis of Natural-Product-like Libraries Containing a 3-Methylbenzofuran Moiety for the Discovery of New Chemical Elicitors.

Natural products are a major source of biological molecules. The 3-methylfuran scaffold is found in a variety of plant secondary metabolite chemical elicitors that confer host-plant resistance against insect pests. Herein, the diversity-oriented synthesis of a natural-product-like library is reported, in which the 3-methylfuran core is fused in an angular attachment to six common natural product scaffolds-coumarin, chalcone, flavone, flavonol, isoflavone and isoquinolinone. The structural diversity of this library is assessed computationally using cheminformatic analysis. Phenotypic high-throughput screening of ß-glucuronidase activity uncovers several hits. Further in vivo screening confirms that these hits can induce resistance in rice to nymphs of the brown planthopper Nilaparvata lugens. This work validates the combination of diversity-oriented synthesis and high-throughput screening of ß-glucuronidase activity as a strategy for discovering new chemical elicitors.