Production Of Novel Metabolites Research Articles

A Close View of the Production of Bioactive Fungal Metabolites Mediated by Chromatin Modifiers.

Secondary metabolites produced by fungi are well known for their biological properties, which play important roles in medicine. These metabolites aid in managing infections and treating chronic illnesses, thereby contributing substantially to human health improvement. Despite this extensive knowledge, the vast biodiversity and biosynthetic potential of fungi is still largely unexplored, highlighting the need for further research in natural products. In this review, several secondary metabolites of fungal origin are described, emphasizing novel structures and skeletons. The detection and characterization of these metabolites have been significantly facilitated by advancements in analytical systems, particularly modern hyphenated liquid chromatography/mass spectrometry. These improvements have primarily enhanced sensitivity, resolution, and analysis flow velocity. Since the in vitro production of novel metabolites is often lower than the re-isolation of known metabolites, understanding chromatin-based alterations in fungal gene expression can elucidate potential pathways for discovering new metabolites. Several protocols for inducing metabolite production from different strains are discussed, demonstrating the need for uniformity in experimental procedures to achieve consistent biosynthetic activation.

Biologically active secondary metabolites from white-rot fungi.

In recent years, there has been a considerable rise in the production of novel metabolites derived from fungi compared to the ones originating from bacteria. These organic substances are utilized in various sectors such as farming, healthcare, and pharmaceutical. Since all dividing living cells contain primary metabolites, secondary metabolites are synthesized by utilizing intermediate compounds or by-products generated from the primary metabolic pathways. Secondary metabolites are not critical for the growth and development of an organism; however, they exhibit a variety of distinct biological characteristics. White-rot fungi are the only microorganisms able to decompose all wood components. Hence, they play an important role in both the carbon and nitrogen cycles by decomposing non-living organic substrates. They are ubiquitous in nature, particularly in hardwood (e.g., birch and aspen) forests. White-rot fungi, besides ligninolytic enzymes, produce different bioactive substances during their secondary metabolism including some compounds with antimicrobial and anticancer properties. Such properties could be of potential interest for the pharmaceutical industries. Considering the importance of the untapped biologically active secondary metabolites from white-rot fungi, the present paper reviews the secondary metabolites produced by white-rot fungi with different interesting bioactivities.

Investigation of the impact of bacterial microencapsulation on natural product discovery

Developing new effective drugs to treat antibiotic resistant infections and cancer is urgently needed to reduce mortality due to these devastating diseases. Despite the promising potential of environmental microorganisms to produce such drugs, researchers are currently facing the problem of rediscovering known compounds. To address this challenge, the impact of microencapsulation on natural compound production was explored using, Kitasatospora cystarginea NRRL B-16505 which is known to produce cystargamide and the 20S proteasome inhibitory cystargolide. The current work postulates that the stress of microencapsulation process may induce secondary metabolism, potentially leading to the production of novel metabolites. Different microencapsulation techniques, including microfluidics, co-axial air flow printing, and emulsification, were compared in terms of bead size, cell viability, metabolite profile, and metabolite yields. This study has shown promising results that may be leading to the discovery of new bioactive compounds as well as activating the silent pathways for compound production. Nutrition deficiency, reduced Oxygen, presence of salt, heat shock, bead uniformity, and shear stress during microencapsulation are potential reasons for the production of these putatively novel chemicals.

Bioprospecting of new Antarctic seaweed selective antitumor molecules: chemical characterization and in vitro analysis

BackgroundThe Antarctic continent exposes seaweeds to extreme environmental conditions, which may facilitate the production of novel metabolites. Algae represent a highly diverse group of little explored organisms. Thus, marine biomass has emerged as a possible source of new biologically active molecules for the treatment of diseases requiring novel therapeutic options. PurposeThe ultrasonic and Soxhlet extractions were used to evaluate chemical profiles and antitumor activities of Desmarestia anceps, Iridaea cordata, and Pyropia endiviifolia extracts using solvents of different polarity. MethodsChemical characterization of the extracts was performed using gas chromatography. The anticancer activities of the extracts were evaluated by exposing rat glioma (C6), human glioblastoma (U87), and human lung adenocarcinoma epithelial (A549) cell lines to increasing extract concentrations (10–1000 µg mL−1) for 24 and 48 h. In parallel, rat astrocyte and human lung fibroblast (MRC-5) were treated under the same conditions to represent non-transformed cell controls, and permit tumor selectivity evaluations of the extracts. ResultsAn MTT assay was used to measure cell cytotoxicity. Hexane, chloroform, and ethanol extracts reduced glioma and lung cancer cell viability. The hexane and ethanol extracts of P. endiviifolia showed the best antiproliferative effect, decreasing glioma cell viability by 40% at 10 µg mL−1, and the chloroform extract reduced lung adenocarcinoma cell viability by 50% at 46 µg mL−1 after 24 h of treatment. None of the extracts affected the viability of non-transformed cells, as was observed in tumor cells. Fatty acids, fatty alcohols, and phthalate esters were the main compounds identified in the seaweed extracts. ConclusionsAntarctic seaweeds displayed selective antitumor activity against glioma and lung cancer cells, particularly the endemic red alga P. endiviifolia. Therefore, these algae could play an important role as novel prototype molecule source for oncology drug development.

Endophytic Fungi: Key Insights, Emerging Prospects, and Challenges in Natural Product Drug Discovery.

Plant-associated endophytes define an important symbiotic association in nature and are established bio-reservoirs of plant-derived natural products. Endophytes colonize the internal tissues of a plant without causing any disease symptoms or apparent changes. Recently, there has been a growing interest in endophytes because of their beneficial effects on the production of novel metabolites of pharmacological significance. Studies have highlighted the socio-economic implications of endophytic fungi in agriculture, medicine, and the environment, with considerable success. Endophytic fungi-mediated biosynthesis of well-known metabolites includes taxol from Taxomyces andreanae, azadirachtin A and B from Eupenicillium parvum, vincristine from Fusarium oxysporum, and quinine from Phomopsis sp. The discovery of the billion-dollar anticancer drug taxol was a landmark in endophyte biology/research and established new paradigms for the metabolic potential of plant-associated endophytes. In addition, endophytic fungi have emerged as potential prolific producers of antimicrobials, antiseptics, and antibiotics of plant origin. Although extensively studied as a “production platform” of novel pharmacological metabolites, the molecular mechanisms of plant–endophyte dynamics remain less understood/explored for their efficient utilization in drug discovery. The emerging trends in endophytic fungi-mediated biosynthesis of novel bioactive metabolites, success stories of key pharmacological metabolites, strategies to overcome the existing challenges in endophyte biology, and future direction in endophytic fungi-based drug discovery forms the underlying theme of this article.

Reduced cytotoxicity of polyethyleneimine by covalent modification of antioxidant and its application to microalgal transformation

ABSTRACT The conversion of carbon dioxide into valuable chemicals is an effective strategy for combating augmented concentrations of carbon dioxide in the environment. Microalgae photosynthetically produce valuable chemicals that are used as biofuels, sources for industrial materials, medicinal leads, and food additives. Thus, improvements in microalgal technology via genetic engineering may prove to be promising for the tailored production of novel metabolites. For the transformation of microalgae, nucleic acids such as plasmid DNA (pDNA) are delivered into the cells using physical and mechanical techniques, such as electroporation, bombardment with DNA-coated microprojectiles, and vortexing with glass beads. However, owing to the electrostatic repulsion between negatively charged cell walls and nucleic acids, the delivery of nucleic acids into the microalgal cells is challenging. To solve this issue, in this study, we investigated microalgal transformation via electroporation using polyplexes with linear polyethyleneimine (LPEI) and pDNA. However, the high toxicity of LPEI decreased the transformation efficiency in Chlamydomonas reinhardtii cells. We revealed that the toxicity of LPEI was due to oxidative stress resulting from the cellular uptake of LPEI. To suppress the toxicity of LPEI, an antioxidant, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was covalently conjugated with LPEI; the conjugate was named as TEMPO-LPEI. Interestingly, with a cellular uptake tendency similar to that of LPEI, TEMPO-LPEI dramatically decreased oxidative stress and cytotoxicity. Electroporation using polyplexes of TEMPO-LPEI and pDNA enhanced the transformation efficiency, compared to those treated with bare pDNA and polyplexes of LPEI/pDNA. This result indicates that polycations conjugated with antioxidants could be useful in facilitating microalgal transformation.

Plant Metabolic Gene Clusters: Evolution, Organization, and Their Applications in Synthetic Biology.

Plants are a remarkable source of high-value specialized metabolites having significant physiological and ecological functions. Genes responsible for synthesizing specialized metabolites are often clustered together for a coordinated expression, which is commonly observed in bacteria and filamentous fungi. Similar to prokaryotic gene clustering, plants do have gene clusters encoding enzymes involved in the biosynthesis of specialized metabolites. More than 20 gene clusters involved in the biosynthesis of diverse metabolites have been identified across the plant kingdom. Recent studies demonstrate that gene clusters are evolved through gene duplications and neofunctionalization of primary metabolic pathway genes. Often, these clusters are tightly regulated at nucleosome level. The prevalence of gene clusters related to specialized metabolites offers an attractive possibility of an untapped source of highly useful biomolecules. Accordingly, the identification and functional characterization of novel biosynthetic pathways in plants need to be worked out. In this review, we summarize insights into the evolution of gene clusters and discuss the organization and importance of specific gene clusters in the biosynthesis of specialized metabolites. Regulatory mechanisms which operate in some of the important gene clusters have also been briefly described. Finally, we highlight the importance of gene clusters to develop future metabolic engineering or synthetic biology strategies for the heterologous production of novel metabolites.

The symbioses of endophytic fungi shaped the metabolic profiles in grape leaves of different varieties.

Endophytic fungi produce many novel bioactive metabolites that are directly used as drugs or that function as the precursor structures of other chemicals. The metabolic shaping of endophytes on grape cells was reported previously. However, there are no reports on the interactions and metabolic impact of endophyte symbiosis on in vitro vine leaves, which may be examined under well-controlled conditions that are more representative of the natural situation of endophytes within grapevines. The present study used an in vitro leaf method to establish endophyte symbiosis of grapevines and analyze the effects on the metabolic profiles of grape leaves from two different cultivars, ‘Rose honey’ (RH) and ‘Cabernet sauvignon’ (CS). The effects of endophytic fungi on the metabolic profiles of grape leaves exhibited host selectivity and fungal strain specificity. Most of the endophytic fungal strains introduced novel metabolites into the two varieties of grape leaves according to the contents of the detected metabolites and composition of metabolites. Strains RH49 and MDR36, with high or moderate symbiosis rates, triggered an increased response in terms of the detected metabolites, and the strains MDR1 and MDR33 suppressed the detected metabolites in CS and RH leaves despite having strong or moderate symbiosis ability. However, the strain RH12 significantly induced the production of novel metabolites in RH leaves due to its high symbiosis ability and suppression of metabolites in CS leaves.

Neurobehavioral effects of cyanobacterial biomass field extracts on zebrafish embryos and potential role of retinoids

Cyanobacteria are known for their ability to produce and release mixtures of up to thousands of compounds into the environment. Recently, the production of novel metabolites, retinoids, was reported for some cyanobacterial species along with teratogenic effects of samples containing these compounds. Retinoids are natural endogenous substances derived from vitamin A that play a crucial role in early vertebrate development. Disruption of retinoid signalling- especially during the early development of the nervous system- might lead to major malfunctions and malformations. In this study, the toxicity of cyanobacterial biomass samples from the field containing retinoids was characterized by in vivo and in vitro bioassays with a focus on the potential hazards towards nervous system development and function. Additionally, in order to identify the compounds responsible for the observed in vitro and in vivo effects the complex cyanobacterial extracts were fractionated (C18 column, water-methanol gradient) and the twelve obtained fractions were tested in bioassays. In all bioassays, all-trans retinoic acid (ATRA) was tested along with the environmental samples as a positive control. Retinoid-like activity (mediated via the retinoic acid receptor, RAR) was measured in the transgenic cell line p19/A15. The in vitro assay showed retinoid-like activity by specific interaction with RAR for the biomass samples. Neurotoxic effects of selected samples were studied on zebrafish (Danio rerio) embryos using the light/dark transition test (Viewpoint, ZebraLab system) with 120 hpf larvae. In the behavioural assay, the cyanobacterial extracts caused significant hyperactivity in zebrafish at 120 hpf after acute exposure (3 h prior to the measurement) at concentrations below the teratogenicity LOEC (0.2 g dw L−1). Similar effect was observed after exposure to fractions of the extracts with detected retinoid-like activity and additive effect was observed after combining the fractions. However, the effect on behaviour was not observed after exposure to ATRA only. To provide additional insight into the behavioural effects and describe the underlying mechanism gene expression of selected biomarkers was measured. We evaluated an array of 28 genes related to general toxicity, neurodevelopment, retinoid and thyroid signalling. We detected several affected genes, most notably, the Cyp26 enzymes that control endogenous ATRA concentration, which documents an effect on retinoid signalling.

Diversity of Bacterial Biosynthetic Genes in Maritime Antarctica.

Bacterial natural products (NPs) are still a major source of new drug leads. Polyketides (PKs) and non-ribosomal peptides (NRP) are two pharmaceutically important families of NPs and recent studies have revealed Antarctica to harbor endemic polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, likely to be involved in the production of novel metabolites. Despite this, the diversity of secondary metabolites genes in Antarctica is still poorly explored. In this study, a computational bioprospection approach was employed to study the diversity and identity of PKS and NRPS genes to one of the most biodiverse areas in maritime Antarctica—Maxwell Bay. Amplicon sequencing of soil samples targeting ketosynthase (KS) and adenylation (AD) domains of PKS and NRPS genes, respectively, revealed abundant and unexplored chemical diversity in this peninsula. About 20% of AD domain sequences were only distantly related to characterized biosynthetic genes. Several PKS and NRPS genes were found to be closely associated to recently described metabolites including those from uncultured and candidate phyla. The combination of new approaches in computational biology and new culture-dependent and -independent strategies is thus critical for the recovery of the potential novel chemistry encoded in Antarctica microorganisms.

Biotechnological vistas in medicinal plants-A way forward

The major challenges in the field of medicinal plants are conservation of rare, endangered and threatened (RET) species and enhancement of secondary metabolites production by using different technologies right from conventional methods to advanced biotechnological methods in order to produce quality drug so as to meet the current demands of pharmaceutical industries. In this review paper, status of various biotechnological methods viz., cryopreservation, propagation of medicinal plant species, metabolic engineering techniques for identification of key enzymes in pathways of bioactive compounds, transgenic medicinal plants for consistent production of novel metabolites are discussed. Further, functional genomic techniques viz., DNA microarrays for simultaneous analysis of multiple genes, identification of putative micro RNAs, phytoregulation of secondary metabolites via RNA interference and transcriptomics in important medicinal plants are also reviewed.

A novel metabolite (1,3-benzenediol, 5-hexyl) production by Exophiala spinifera strain FM through dibenzothiophene desulfurization.

Sulfur dioxide which is released from petroleum oil combustion causes pollution over the atmosphere and the soil. Biodesulfurization can be used as a complementary method of hydrodesulfurization, the common method of petroleum desulfurization in refineries. Many studies have been carried out to develop biological desulfurization of dibenzothiophene (DBT) with bacterial biocatalysts. However, fungi are capable to metabolize a wide range of aromatic hydrocarbons through cytochrome P450 and their extracellular enzymes. The aim of the present work was isolation and identification of fungi biocatalysts capable for DBT utilization as sulfur source and production of novel metabolites. DBT consumption and the related produced metabolites were analyzed by HPLC and GC-MS respectively. One of the isolated fungi that could utilize DBT as sole sulfur source was identified by both traditional and molecular experiments and registered in NCBI as Exophiala spinifera FM strain (accession no. KC952672). This strain could desulfurize 99% of DBT (0.3mM) as sulfur source by co-metabolism reaction with other carbon sources through the same pathway as 4S and produced 2-hydroxy biphenyl (2-HBP) during 7days of incubation at 30°C and 180rpm shaking. However, the isolate was able to transform 2-HBP to 1,3-benzenediol, 5-hexyl. While biphenyl compounds are toxic to leaving cells, biotransformation of them can reduce their toxicity and the fungi will be more tolerant to the final product. These data are the first report about the desulfurization of DBT comparable to 4S-pathway and production of innovative metabolite by E. spinifera FM strain.

De Novo Production of Metabolites by Fungal Co-culture of Trichophyton rubrum and Bionectria ochroleuca

The co-cultivation of fungi has recently been described as a promising strategy to induce the production of novel metabolites through possible gene activation. A large screening of fungal co-cultures in solid media has identified an unusual long-distance growth inhibition between Trichophyton rubrum and Bionectria ochroleuca. To study metabolite induction in this particular fungal interaction, differential LC-MS-based metabolomics was performed on pure strain cultures and on their co-cultures. The comparison of the resulting fingerprints highlighted five de novo induced compounds, which were purified using software-oriented semipreparative HPLC-MS. One metabolite was successfully identified as 4″-hydroxysulfoxy-2,2″-dimethylthielavin P (a substituted trimer of 3,5-dimethylorsellinic acid). The nonsulfated form, as well as three other related compounds, were found in the pure strain culture of B. ochroleuca.

Classical and epigenetic approaches to metabolite diversification in filamentous fungi

Studies on fungal metabolites have produced an overwhelming expectation concerning the production of novel bioactive compounds for pharmaceutical applications. The adding of various biosynthetic precursors and the changing of nutritional components in the fermentation medium can change biosynthesis pathways, also leading to the production of novel metabolites. In addition, several growing conditions can be classically manipulated to modify fungal metabolite profiles. Recently, modern genome sequence tools have shown that not all gene clusters are regularly expressed in conventional growing conditions, thus expanding the possibilities of modulating the chemical metabolite profiles produced by filamentous fungi. This review discusses and exemplifies classical and epigenetic tools successfully applied to diversify metabolite production and to produce fungal metabolites from silent metabolic pathways.

Studies on Microbial Transformation of Meloxicam by Fungi

Screening-scale studies were performed with 26 fungal cultures for their ability to transform the anti-inflammatory drug meloxicam. Among the different fungi screened, a filamentous fungus, Cunninghamella blakesleeana NCIM 687, transformed meloxicam to three metabolites in significant quantities. The transformation of meloxicam was confirmed by high-performance liquid chromatography (HPLC). Based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) data, two metabolites were predicted to be 5-hydroxymethyl meloxicam and 5-carboxy meloxicam, the major mammalian metabolites reported previously. A new metabolite was produced, which is not detected in mammalian systems. Glucose medium, pH of 6.0, temperature of 27 degrees , 5-day incubation period, dimethylformamide as solvent, and glucose concentration of 2.0%were found to be suitable for maximum transformation of meloxicam when studied separately. It is concluded that C. blakesleeana can be employed for biotransformation of drugs for production of novel metabolites.

Homolog separation, a necessity for the proper identification of fungal metabolites

Monitoring of fungal extracts for the production of novel metabolites, using a modular analytical system combining HPLC with UV–MS–ELS detection, identified culture LL-W1278 as a fungus producing new biopolymers. Only a non-routine HPLC analysis of a culture extract revealed that the standard water–acetonitrile elution method did not separate all members of the metabolite complex. Fine-tuning the eluting solvents established that it was essential to include acid with the water–methanol system to separate the new materials. The routinely used water–acetonitrile system, with or without acid, was incapable of separating all homologues. With the modified method the new homologues W1278-Ax, Bx, and Cx were separated. LC/MS analysis indicated that these compounds had molecular weights of 706, 900, and 1094, respectively, 44 mass units lower than their three major homologues, W1278-A, B, and C, identified previously. UV and NMR data as well as mass fragmentation patterns established unambiguously that the new compounds lacked a carboxyl group at the terminal resorcinol unit of the biopolymer, consisting of several catenated hydroxymellein residues. A time study concerning the stability of these fungal metabolites showed a slow, but complete degradation of the primary metabolites over several months when kept as a DMSO solution.

Bacterial type III polyketide synthases: phylogenetic analysis and potential for the production of novel secondary metabolites by heterologous expression in pseudomonads

Type III polyketide synthases (PKS) were regarded as typical for plant secondary metabolism before they were found in microorganisms recently. Due to microbial genome sequencing efforts, more and more type III PKS are found, most of which of unknown function. In this manuscript, we report a comprehensive analysis of the phylogeny of bacterial type III PKS and report the expression of a type III PKS from the myxobacterium Sorangium cellulosum in pseudomonads. There is no precedent of a secondary metabolite that might be biosynthetically correlated to a type III PKS from any myxobacterium. Additionally, an inactivation mutant of the S. cellulosum gene shows no physiological difference compared to the wild-type strain which is why these type III PKS are assumed to be "silent" under the laboratory conditions administered. One type III PKS (SoceCHS1) was expressed in different Pseudomonas sp. after the heterologous expression in Escherichia coli failed. Cultures of recombinant Pseudomonas sp. harbouring SoceCHS1 turned red upon incubation and the diffusible pigment formed was identified as 2,5,7-trihydroxy-1,4-naphthoquinone, the autooxidation product of 1,3,6,8-tetrahydroxynaphthalene. The successful heterologous production of a secondary metabolite using a gene not expressed under administered laboratory conditions provides evidence for the usefulness of our approach to activate such secondary metabolite genes for the production of novel metabolites.

Therapeutic-antagonists of oestrogens can be produced for cancer and other therapies using cytochromes P450 (CYP)

Oestrogens such as 17beta-oestradiol initiates nuclear-gene transcription in gender-specified tissues such as the ovaries and mammaries; and unfortunately too in cancer cells derived from target tissues. Consequently, there has been the development of novel agents for particular cancer therapies that are antagonists of oestrogens for oestrogen-receptor (ER) binding and of drugs with ER-specific interference RNA (RNAi) abilities. Therapeutic-antagonists of oestrogens will be re-designed and biosynthesised and deployed to circumvent the gene DNA-transcription abilities of oestrogens and mimics: and their metabolites in oestrogen-target tissues (see above). Furthermore, opportunities will emerge for adjunct-chemotherapy of particular tissue cancers: and in the prevention of recurrence outcomes. Cytochromes P450 can play an important part in these developments especially for the production of novel metabolites of oestrogens as therapeutic-antagonists of oestrogen-stimulated cancers.

Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolor.

The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their annotated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites.

Approaches to improve stress tolerance using molecular genetics

ABSTRACTPlant productivity is greatly affected by environmental stress factors. In this review, we give an overview of molecular approaches that have been taken to study stress tolerance; in particular, we consider drought, salt and cold stress. Strategies and perspectives in using molecular biology to improve stress tolerance are outlined describing specific examples. Osmotic stress is associated with the synthesis of novel polypeptides and/or osmolytes. The spectrum and action of these different metabolites are summarized. A number of stress‐related genes have been characterized and several representatives are discussed. After transforming plants with these genes, the effect of the encoded proteins on altered stress behaviour is examined. If genes with enzymatic functions were chosen for these experiments, complete pathways may be altered and this can implement the production of novel metabolites conferring stress tolerance.