Production Of Bioactive Secondary Metabolites Research Articles

Fungal Endophytes: Microfactories of Novel Bioactive Compounds with Therapeutic Interventions; A Comprehensive Review on the Biotechnological Developments in the Field of Fungal Endophytic Biology over the Last Decade.

The seminal discovery of paclitaxel from endophytic fungus Taxomyces andreanae was a milestone in recognizing the immense potential of endophytic fungi as prolific producers of bioactive secondary metabolites of use in medicine, agriculture, and food industries. Following the discovery of paclitaxel, the research community has intensified efforts to harness endophytic fungi as putative producers of lead molecules with anticancer, anti-inflammatory, antimicrobial, antioxidant, cardio-protective, and immunomodulatory properties. Endophytic fungi have been a valuable source of bioactive compounds over the last three decades. Compounds such as taxol, podophyllotoxin, huperzine, camptothecin, and resveratrol have been effectively isolated and characterized after extraction from endophytic fungi. These findings have expanded the applications of endophytic fungi in medicine and related fields. In the present review, we systematically compile and analyze several important compounds derived from endophytic fungi, encompassing the period from 2011 to 2022. Our systematic approach focuses on elucidating the origins of endophytic fungi, exploring the structural diversity and biological activities exhibited by these compounds, and giving special emphasis to the pharmacological activities and mechanism of action of certain compounds. We highlight the tremendous potential of endophytic fungi as alternate sources of bioactive metabolites, with implications for combating major global diseases. This underscores the significant role that fungi can play in the discovery and development of novel therapeutic agents that address the challenges posed by prevalent diseases worldwide.

Anti-Insect Properties of Penicillium Secondary Metabolites.

In connection with their widespread occurrence in diverse environments and ecosystems, fungi in the genus Penicillium are commonly found in association with insects. In addition to some cases possibly implying a mutualistic relationship, this symbiotic interaction has mainly been investigated to verify the entomopathogenic potential in light of its possible exploitation in ecofriendly strategies for pest control. This perspective relies on the assumption that entomopathogenicity is often mediated by fungal products and that Penicillium species are renowned producers of bioactive secondary metabolites. Indeed, a remarkable number of new compounds have been identified and characterized from these fungi in past decades, the properties and possible applications of which in insect pest management are reviewed in this paper.

Endophytic fungi mediates production of bioactive secondary metabolites via modulation of genes involved in key metabolic pathways and their contribution in different biotechnological sector.

Endophytic fungi stimulate the production of an enormous number of bioactive metabolites in medicinal plants and affect the different steps of biosynthetic pathways of these secondary metabolites. Endophytic fungi possess a number of biosynthetic gene clusters that possess genes for various enzymes, transcription factors, etc., in their genome responsible for the production of secondary metabolites. Additionally, endophytic fungi also modulate the expression of various genes responsible for the synthesis of key enzymes involved in metabolic pathways of such as HMGR, DXR, etc. involved in the production of a large number of phenolic compounds as well as regulate the expression of genes involved in the production of alkaloids and terpenoids in different plants. This review aims to provide a comprehensive overview of gene expression related to endophytes and their impact on metabolic pathways. Additionally, this review will emphasize the studies done to isolate these secondary metabolites from endophytic fungi in large quantities and assess their bioactivity. Due to ease in synthesis of secondary metabolites and their huge application in the medical industry, these bioactive metabolites are now being extracted from strains of these endophytic fungi commercially. Apart from their application in the pharmaceutical industry, most of these metabolites extracted from endophytic fungi also possess plant growth-promoting ability, bioremediation potential, novel bio control agents, sources of anti-oxidants, etc. The review will comprehensively shed a light on the biotechnological application of these fungal metabolites at the industrial level.

Identification of Antimicrobial Metabolites from the Egyptian Soil-Derived Amycolatopsis keratiniphila Revealed by Untargeted Metabolomics and Molecular Docking.

Actinomycetes are prolific producers of bioactive secondary metabolites. The prevalence of multidrug-resistant (MDR) pathogens has prompted us to search for potential natural antimicrobial agents. Herein, we report the isolation of rare actinobacteria from Egyptian soil. The strain was identified as Amycolatopsis keratiniphila DPA04 using 16S rRNA gene sequencing. Cultivation profiling, followed by chemical and antimicrobial evaluation of crude extracts, revealed the activity of DPA04 ISP-2 and M1 culture extracts against Gram-positive bacteria. Minimum inhibitory concentrations (MIC) values ranged from 19.5 to 39 µg/mL. Chemical analysis of the crude extracts using ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF) led to the identification of 45 metabolites of different chemical classes. In addition, ECO-0501 was identified in the cultures with significant antimicrobial activity. Multidrug resistance in Staphylococcus aureus is reported to be related to the multidrug efflux pump (MATE). ECO-0501 and its related metabolites were subjected to molecular docking studies against the MATE receptor as a proposed mechanism of action. ECO-0501 and its derivatives (AK_1 and N-demethyl ECO-0501) had better binding scores (-12.93, -12.24, and -11.92 kcal/mol) than the co-crystallized 4HY inhibitor (-8.99 kcal/mol) making them promising candidates as MATE inhibitors. Finally, our work established that natural products from this strain could be useful therapeutic tools for controlling infectious diseases.

Genome Mining and Screening for Secondary Metabolite Production in the Endophytic Fungus Dactylonectria alcacerensis CT-6.

Endophytic fungi are a treasure trove of natural products with great chemical diversity that is largely unexploited. As an alternative to the traditional bioactivity-guided screening approach, the genome-mining-based approach provides a new methodology for obtaining novel natural products from endophytes. In our study, the whole genome of an endophyte, Dactylonectria alcacerensis CT-6, was obtained for the first time. Genomic analysis indicated that D. alcacerensis CT-6 has one 61.8 Mb genome with a G+C content of 49.86%. Gene annotation was extensively carried out using various BLAST databases. Genome collinearity analysis revealed that D. alcacerensis CT-6 has high homology with three other strains of the Dactylonectria genus. AntiSMASH analysis displayed 45 secondary metabolite biosynthetic gene clusters (BGCs) in D. alcacerensis CT-6, and most of them were unknown and yet to be unveiled. Furthermore, only six known substances had been isolated from the fermented products of D. alcacerensis CT-6, suggesting that a great number of cryptic BGCs in D. alcacerensis CT-6 are silent and/or expressed at low levels under conventional conditions. Therefore, our study provides an important basis for further chemical study of D. alcacerensis CT-6 using the gene-mining strategy to awaken these cryptic BGCs for the production of bioactive secondary metabolites.

Comparative Genomics Analysis of Ganoderma Orthologs Involved in Plant-Pathogenesis

Ganoderma species are producers of bioactive secondary metabolites and lignin degraders. A few Ganoderma species are known to be plant pathogens that attack economically important trees. In this study, comparative genomics analysis was conducted on the proteome of ten Ganoderma species/strains, focusing on the proteins that have been reported to be involved in plant-pathogenesis in other fungi. Fungal trophic lifestyle prediction of these Ganoderma species/strains supported that G. boninense (a potent pathogen to oil palm) is a hemibiotrophic fungus while the other Ganoderma species/strains analyzed were predicted to be saprophytes or a symbiont based on their Carbohydrate-Active Enzyme (CAZyme) contents. Although these Ganoderma species/strains were demonstrated to share many CAZymes and secondary metabolite core gene clusters, individual species may produce unique CAZymes and secondary metabolite core genes that determine their lifestyles, host-specificity, and potential as a producer of bioactive secondary metabolites. Ortholog groups that are related to fungal virulence from seven Ganoderma species/strains including those involved in lignin degradation, mycotoxin, siderophore and ergosterol biosynthesis, and virulence were summarized. Potential effectors were predicted from the proteome of these Ganoderma species/strains, and putative effectors that were being expressed in G. boninense in oil palm roots but not found in other species were identified. The findings provide a useful resource to further analyze plant-pathogenesis and wood degradation activities of these Ganoderma species.

Antimicrobial and antioxidant potentials of crude extracts of culturally dissimilar endophytic fungi

Endophytes from an unexplored plant such as Spatterdock (SD) Nuphar lutea variegata could be a dependable producer of novel secondary metabolites active against drug resistance organisms. This study explored the diversity of fungal endophytes from mid-rib (MR) and leaf blade (LB) of Nuphar lutea variegata as well as the antimicrobial and antioxidant potentials of their secondary metabolites. Morphological features observed showed four species were isolated. A broad-spectrum antimicrobial activity observed to be concentration-dependent was observed for all the fungal crude extracts 1 mg/mL. Crude extract of SD-LB3 showed the best antibacterial activities against S. aureus and P. aeruginosa with a minimum inhibitory concentration of 0.5 and 0.25 µg/mL respectively. The antioxidant capacities of the fungal crude extracts showed a better neutralizing effect of free radicals compared to the standard Quercitin. Effective inhibition of free radicals was observed for all the fungal extracts from 20 to 100 mg/mL (96.7 – 99.3 %). The highest antioxidant activity was shown by the extracts of fungi isolated from the leaf blade with an average of 98 to 99.2 % at the tested concentrations of 20 to 100 mg/mL. Our study provides evidence of diverse endophytic fungi associated with Nuphar lutea variegata, and the antibacterial and antioxidant activities observed confirm their capacities as producers of bioactive secondary metabolites. This study is the first to isolate endophytic fungi form Nuphar lutea variegata and screen the antimicrobial and antioxidant activities of their secondary metabolites.

Regulation of Multidrug Efflux Pumps by TetR Family Transcriptional Repressor Negatively Affects Secondary Metabolism in Streptomyces coelicolor A3(2).

Streptomyces spp. are well-known producers of bioactive secondary metabolites (SMs) that serve as pharmaceutical agents. In addition to their ability to produce SMs, Streptomyces spp. have evolved diverse membrane transport systems to protect cells against antibiotics produced by itself or other microorganisms. We previously screened mutants of Streptomyces coelicolor that show a phenotype of reduced undecylprodigiosin (RED) production in a combined-culture with Tsukamurella pulmonis. Here, we identified a point mutation, which reduced RED production, by performing genome resequencing and genetic complementation. We found that inactivation of the sco1718 gene encoding the TetR family transcriptional regulator (TFR) produced a deficient phenotype for several SMs in Streptomyces coelicolor A3(2). In the genome of S. coelicolor A3(2), two other sets of TFR and two-component ATP-binding cassette (ABC) transporter genes (sco4358-4360 and sco5384-5382) were found which had similar effects on the phenotype for both secondary metabolism and antibiotic resistance. An electrophoretic mobility shift assay and quantitative reverse transcription-PCR experiments demonstrated that TFRs repressed the expression of each adjacent two-component ABC transporter genes by binding to the operator sequence. Notably, the Δsco1718 mutant showed increased resistance to several antibiotics of other actinomycete origin. Our results imply the switching of cell metabolism to direct offense (antibiotic production) or defense (efflux pump activation) using costly and limited quantities of cell energy sources (e.g., ATP) in the soil ecosystem. IMPORTANCE The bacterial metabolic potential to synthesize diverse secondary metabolites in the environment has been revealed by recent (meta)genomics of both unculturable and culturable bacteria. These studies imply that bacteria are continuously exposed to harmful chemical compounds in the environment. Streptomyces spp. contain antibiotic efflux pumps and SM biosynthetic gene clusters. However, the mechanism by which soil bacteria, including Streptomyces, survive against toxic compounds in the environment remains unclear. Here, we identified three sets of TFR-ABC transporter genes in Streptomyces coelicolor A3(2). We found that each TFR controlled the expression of respective ABC transporter, and the expression of all ABC transporters negatively impacted SM production and increased antibiotic resistance. Notably, bioinformatic analysis indicated that these TFR-ABC transporter gene sets are highly conserved and widely distributed in the genome of Streptomyces species, indicating the importance of systematic regulation that directs antibiotic production and xenobiotic excretion.

Isolation, antibacterial screening, and identification of bioactive cave dwelling bacteria in Fiji.

Bacteria are well known producers of bioactive secondary metabolites, including some of the most effective antibiotics in use today. While the caves of Oceania are still largely under-explored, they form oligotrophic and extreme environments that are a promising source for identifying novel species of bacteria with biologically active compounds. By using selective media that mimicked a cave environment, and pretreatments that suppressed the growth of fast-growing bacteria, we have cultured genetically diverse bacteria from a limestone cave in Fiji. Partial 16S rRNA gene sequences from isolates were determined and compared with 16S rRNA gene sequences in EzBioCloud and SILVA data bases. Fifty-five isolates purified from culture had Actinomycete-like morphologies and these were investigated for antibacterial activity. Initial screening using a cross streak test with pathogenic bacteria indicated that 34 of the isolates had antibacterial properties. The best matches for the isolates are bacteria with potential uses in the manufacture of antibiotics and pesticides, in bioremediation of toxic waste, in biomining, in producing bioplastics, and in plant growth promotion. Nineteen bacteria were confirmed as Actinomycetes. Thirteen were from the genus Streptomyces and six from genera considered to be rare Actinomycetes from Pseudonocardia, Kocuria, Micromonospora, Nonomuraea. Ten isolates were Firmicutes from the genera Bacillus, Lysinbacillus, Psychrobacillus and Fontibacillus. Two were Proteobacteria from the genera Mesorhizobium and Cupriavidus. Our findings identify a potentially rich source of microbes for applications in biotechnologies.

Selective isolation of Arctic marine actinobacteria and a down-scaled fermentation and extraction strategy for identifying bioactive compounds.

Actinobacteria are among the most prolific producers of bioactive secondary metabolites. In order to collect Arctic marine bacteria for the discovery of new bioactive metabolites, actinobacteria were selectively isolated during a research cruise in the Greenland Sea, Norwegian Sea and the Barents Sea. In the frame of the isolation campaign, it was investigated how different sample treatments, isolation media and sample-sources, such as animals and sediments, affected the yield of actinobacterial isolates to aid further isolation campaigns. Special attention was given to sediments, where we expected spores of spore forming bacteria to enrich. Beside actinobacteria a high share of bacilli was obtained which was not desired. An experimental protocol for down-scaled cultivation and extraction was tested and compared with an established low-throughput cultivation and extraction protocol. The heat-shock method proved suitable to enrich spore-, or endospore forming bacteria such as bacilli. Finally, a group bioactive compounds could be tentatively identified using UHPLC-MS/MS analysis of the active fractions.

Endophytic microbes and their diverse beneficial aspects in various sectors: A critical insight

Endophytes are ubiquitous and grow in plant tissues without causing any harmful effects to the host. They include different groups of microorganisms such as bacteria, fungi and actinomycetes. Along with the host plants, the existing endophytes also co-evolve after a long relationship between them. Host plant-endophyte interaction is similar to that of plant growth promoting microbes as they induce the growth of the host plant and increase resilience against biotic and abiotic stresses. The interaction of plant endophytes at the molecular level and the effect of endophytes on host gene expression is a new field of study and are still rarely explored. Endophytes act as a promising resource of many invaluable bioactive secondary metabolites. Some of these bioactive compounds include alkaloids, polyphenols, sterols, xanthones, terpenoids, flavones, coumarins, polyketides, quinones, saponins, tannins, benzopyrones, dibenzofurans. These secondary metabolites are beneficial for agriculture, industrial and pharmacological purposes. As endophytes have beneficial effects in sustainable agriculture, plant disease management, pharmaceuticals, industry and environmental management in an eco-friendly way, thus improving the strategy of application of endophytes as biological agents in every aspect of our life is a very challenging field of research. Our aim in this present review is to focus on plant-endophyte interactions and their various dimensions in order to address some future possibilities for expediting the bioactive secondary metabolite production.

Bioactive Alkaloids from the Marine-Derived Fungus Metarhizium sp. P2100.

The Metarhizium fungal species are considered the prolific producers of bioactive secondary metabolites with a variety of chemical structures. In this study, the biosynthetic potential of marine-derived fungus Metarhizium sp. P2100 to produce bioactive alkaloids was explored by using the one strain many compounds (OSMAC) strategy. From the rice solid medium (mixed with glucose peptone and yeast broth (GPY)), wheat solid medium (mixed with Czapek) and GPY liquid medium, one rare N-butenone spiroquinazoline alkaloid, N-butenonelapatin A (1), together with nine known compounds (2-10), were isolated and identified. Their structures were elucidated by analysis of the comprehensive spectroscopic data, including 1D and 2D NMR and HRESIMS, and the absolute configuration of 1 was determined by a single-crystal X-ray crystallographic experiment. N-butenonelapatin A (1) represents the first example of N-butenone spiroquinazoline with a rare α, β-unsaturated ketone side chain in the family of spiroquinazoline alkaloids. Compound 4 displayed antibacterial activity against Vibrio vulnificus MCCC E1758 with a minimum inhibitory concentration (MIC) value of 6.25 µg/mL. Compound 7 exhibited antibacterial activities against three aquatic pathogenic bacteria, including V. vulnificus MCCC E1758, V. rotiferianus MCCC E385 and V. campbellii MCCC E333 with the MIC values of 12.5, 12.5 and 6.25 μg/mL, respectively. Compounds 3 and 6 demonstrated anti-inflammatory activity against NO production induced by lipopolysaccharide (LPS) with the IC50 values of 37.08 and 37.48 μM, respectively. In addition, compound 1 showed weak inhibitory activity against the proliferation of tumor cell lines A-375 and HCT 116. These findings further demonstrated that fungi of the Metarhizium species harbor great potentials in the synthesis of a variety of bioactive alkaloids.

Natural products, including a new caboxamycin, from Streptomyces and other Actinobacteria isolated in Spain from storm clouds transported by Northern winds of Arctic origin.

Actinobacteria, mostly Streptomyces species, are the main source of natural products essential in medicine. While the majority of producer microorganisms of secondary metabolite are reported from terrestrial or marine environments, there are limited reports of their isolation from atmospheric precipitations. Clouds are considered as atmospheric oases for microorganisms and there is a recent paradigm shift whereby atmospheric-derived Actinobacteria emerge as an alternative source for drug discovery. In this context, we studied a total of 18 bioactive Actinobacteria strains, isolated by sampling nine precipitation events with prevailing Northern winds in the Cantabrian Sea coast, Northern Spain. Backward trajectories meteorological analyses indicate that air masses were originated mostly in the Arctic Ocean, and their trajectory to downwind areas involved the Atlantic Ocean and also terrestrial sources from continental Europe, and in some events from Canada, Greenland, Mauritania and Canary Islands. Taxonomic identification of the isolates, by 16S rRNA gene sequencing and phylogenetic analyses, revealed that they are members of three Actinobacteria genera. Fifteen of the isolates are Streptomyces species, thus increasing the number of bioactive species of this genus in the atmosphere to a 6.8% of the total currently validated species. In addition, two of the strains belong to the genus Micromonospora and one to genus Nocardiopsis. These findings reinforce a previous atmospheric dispersal model, extended herein to the genus Micromonospora. Production of bioactive secondary metabolites was screened in ethyl acetate extracts of the strains by LC-UV-MS and a total of 94 secondary metabolites were detected after LC/MS dereplication. Comparative analyses with natural products databases allowed the identification of 69 structurally diverse natural products with contrasted biological activities, mostly as antibiotics and antitumor agents, but also anti-inflammatory, antiviral, antiparasitic, immunosuppressant and neuroprotective among others. The molecular formulae of the 25 remaining compounds were determined by HRMS. None of these molecules had been previously reported in natural product databases indicating potentially novel metabolites. As a proof of concept, a new metabolite caboxamycin B (1) was isolated from the culture broth of Streptomyces sp. A-177 and its structure was determined by various spectrometric methods. To the best of our knowledge, this is the first novel natural product obtained from an atmospheric Streptomyces, thus pointing out precipitations as an innovative source for discovering new pharmaceutical natural products.

Evaluation of antioxidant, antimicrobial and antitumor compounds production potential of Streptomyces antioxidans PU-12M

Streptomycetes are prolific producers of biologically active secondary metabolites with great structural and functional diversity, which are widely used as pharmaceutical products. The strain Streptomyces sp. PU-12M was investigated for its potential to produce antioxidant, antimicrobial and antitumor compounds. For this purpose, the laboratory scale fermentation (10 liters working volume) of the strain was performed followed by cell separation, solvent extraction and partial purification of biologically active compounds by silica gel fractionation column chromatography and preparative TLC. The antioxidant activity was determined by DPPH radical scavenging assay, antimicrobial activity by agar well diffusion assay while in vitro antitumor activity was determined by MTT and SRB assays. Altogether 24 partially purified compounds or fractions were retrieved from the extract of strain Streptomyces Sp. PU-12M and were subsequently investigated for biological activity. Among them, three fractions were found to have potent antioxidant activity. The crude extracts and the partially purified fractions were found to have promising antimicrobial activity with MIC up to 3.2mg/ml. The partially purified fractions exhibited varied cytotoxicity ranging from 40% to 100% mortality against brine shrimp (Artemia salina) larvae and 22% to 67% growth inhibition of HCT 116 colorectal cancer cell lines. The study reveals that Streptomyces antioxidans PU-12M is a proliferent producer of bioactive secondary metabolites and can be exploited further to produce the commercially useful bioactive molecules.

Overview on Strategies and Assays for Antibiotic Discovery.

The increase in antibiotic resistance poses a major threat to global health. Actinomycetes, the Gram-positive bacteria of the order Actinomycetales, are fertile producers of bioactive secondary metabolites, including antibiotics. Nearly two-thirds of antibiotics that are used for the treatment of bacterial infections were originally isolated from actinomycetes strains belonging to the genus Streptomyces. This emphasizes the importance of actinomycetes in antibiotic discovery. However, the identification of a new antimicrobial compound and the exploration of its mode of action are very challenging tasks. Therefore, different approaches that enable the “detection” of an antibiotic and the characterization of the mechanisms leading to the biological activity are indispensable. Beyond bioinformatics tools facilitating the identification of biosynthetic gene clusters (BGCs), whole cell-screenings—in which cells are exposed to actinomycete-derived compounds—are a common strategy applied at the very early stage in antibiotic drug development. More recently, target-based approaches have been established. In this case, the drug candidates were tested for interactions with usually validated targets. This review focuses on the bioactivity-based screening methods and provides the readers with an overview on the most relevant assays for the identification of antibiotic activity and investigation of mechanisms of action. Moreover, the article includes examples of the successful application of these methods and suggestions for improvement.

Improving artemisinin and essential oil production from Artemisia plant through in vivo elicitation with gamma irradiation nano-selenium and chitosan coupled with bio-organic fertilizers

Artemisia plants process potential bioactive secondary metabolites such as artemisinin and essential oil. They are useful in controlling potential pests and microbes and have a therapeutic effect. The commercial production of artemisinin and essential oil is limited to regarding the worldwide demand. Urgent attempts must be undertaken to improve the production of bioactive secondary metabolites. The objectives of this experiment were to increase the production and improve the quality of bioactive secondary metabolites in order to limit the use of agrochemicals. Therefore, a field experiment was conducted during the 2018 and 2019 seasons. Treatments included three elicitations (gamma irradiation, nano-selenium, and chitosan) and three kinds of fertilizers (NPK, Moringa leaf extract, and humic acid). The experiment was conducted as a factorial with a completely randomized block. The designs and treatments were arranged in a split–split plot with three replicates. A single application of elicitors showed that chitosan > nano-selenium, chitosan > gamma irradiation, and Moringa > humic acid > NPK in plants’ artemisinin and essential oil content, while the interaction showed a significant synergistic relationship between elicitors and the fertilizers in enhancing the quantity and quality of artemisinin and essential oil of Artemisia plants. Without using any pesticides, there was no infection that appeared in Artemisia plants, this could be due to the enhancement of bioactive secondary metabolite production.

Macroalga-associated bacterial endophyte bioactive secondary metabolites twinning: Cystoseira myrica and its associated Catenococcus thiocycli QCm as a model

Marine ecosystems represent the largest biome on the earth. Until now, the relationships between the marine microbial inhabitants and the macroalgal species unclear, and the previous studies are insufficient. So, more research is required to advance our understanding of macroalgal- microbial interactions. In this study, we tried to investigate the relationship between the brown marine macroalga, Cystoseira myrica and its associated bacterial endophyte, Catenococcus thiocycli, as the first study concerning the production of bioactive secondary metabolites from a macroalgal species comparing with its associated endophytic bacteria. Secondary metabolites were extracted from alga and its bacterial endophyte with ethyl acetate and methanol. All extracts contained significant quantities of phenolics, flavonoids, tannins, and saponins. Strikingly, extracts possess antioxidant, anti-inflammatory and antimicrobial activities which were significantly correlated to phenolic and flavonoid contents.

Sorbicillinoids hyperproduction without affecting the cellulosic enzyme production in Trichoderma reesei JNTR5

BackgroundMicrobial production of bioactive secondary metabolites is challenging as most of the encoding genes are silent; and even if they are activated, the biosynthetic pathways are usually complex. Sorbicillinoids with multifunctional bioactivities are examples of these problems, which if solved can result in a more sustainable, simple supply of these important compounds to the pharmaceutical industry. As an excellent producer of cellulosic enzymes, Trichoderma reesei can secrete various sorbicillinoids.ResultsHere, we obtained a T. reesei mutant strain JNTR5 from the random mutation during overexpression of gene Tr69957 in T. reesei RUT-C30. JNTR5 exhibited a significant constitutive increase in sorbicillinoids production without affecting the cellulosic enzyme production. Confocal laser scanning microscope (CLSM) results indicated that sorbicillinoids were distributed in both mycelium and spores of JNTR5 with blue and green fluorescence. Compared with RUT-C30, JNTR5 displayed different cell morphology, reduced growth rate, and increased sporulation, but a similar biomass accumulation. Furthermore, transcriptome analysis revealed that all genes belonging to the sorbicillinoid gene cluster were upregulated, while most cellulase-encoding genes were downregulated. The cell wall integrity of JNTR5 was damaged, which might benefit the cellulase secretion and contribute to the almost unchanged cellulase and hemicellulase activity given that the damaged cell wall can enhance the secretion of the enzymes.ConclusionsFor the first time, we constructed a sorbicillinoids hyperproduction T. reesei platform with comparable cellulosic enzymes production. This outperformance of JNTR5, which is strain-specific, is proposed to be attributed to the overexpression of gene Tr69957, causing the chromosome remodeling and subsequently changing the cell morphology, structure, and the global gene expression as shown by phenotype and the transcriptome analysis of JNTR5. Overall, JNTR5 shows great potential for industrial microbial production of sorbicillinoids from cellulose and serves as an excellent model for investigating the distribution and secretion of yellow pigments in T. reesei.

Bioactive potentiality of secondary metabolites from endophytic bacteria against SARS-COV-2: An in-silico approach.

Five endophytic bacterial isolates were studied to identify morphologically and biochemically, according to established protocols and further confirmed by 16S rDNA Sanger sequencing, as Priestia megaterium, Staphylococcus caprae, Neobacillus drentensis, Micrococcus yunnanensis, and Sphingomonas paucimobiliz, which were then tested for phytohormone, ammonia, and hydrolytic enzyme production. Antioxidant compounds total phenolic content (TPC), and total flavonoid content (TFC) were assessed by using bacterial crude extracts obtained from 24-hour shake-flask culture. Phylogenetic tree analysis of those identified isolates shared sequence similarities with the members of Bacillus, Micrococcus, Staphylococcus, and Pseudomonas species, and after GenBank submission, accession numbers for the nucleotide sequences were found to be MW494406, MW494408, MW494401, MW494402, and MZ021340, respectively. In silico analysis was performed to identify their bioactive genes and compounds in the context of bioactive secondary metabolite production with medicinal value, where nine significant bioactive compounds according to six different types of bioactive secondary metabolites were identified, and their structures, gene associations, and protein-protein networks were analyzed by different computational tools and servers, which were reported earlier with their antimicrobial, anti-infective, antioxidant, and anti-cancer capabilities. These compounds were then docked to the 3-chymotrypsin-like protease (3CLpro) of the novel SARS-COV-2. Docking scores were then compared with 3CLpro reference inhibitor (lopinavir), and docked compounds were further subjected to ADMET and drug-likeness analyses. Ligand-protein interactions showed that two compounds (microansamycin and aureusimine) interacted favorably with coronavirus 3CLpro. Besides, in silico analysis, we also performed NMR for metabolite detection whereas three metabolites (microansamycin, aureusimine, and stenothricin) were confirmed from the 1H NMR profiles. As a consequence, the metabolites found from NMR data aligned with our in-silico analysis that carries a significant outcome of this research. Finally, Endophytic bacteria collected from medicinal plants can provide new leading bioactive compounds against target proteins of SARS-COV-2, which could be an effective approach to accelerate drug innovation and development.

Chromosome-level assembly and analysis of the Thymus genome provide insights into glandular secretory trichome formation and monoterpenoid biosynthesis in thyme

Thyme has medicinal and aromatic value because of its potent antimicrobial and antioxidant properties. However, the absence of a fully sequenced thyme genome limits functional genomic studies of Chinese native thymes. Thymus quinquecostatus Čelak., which contains large amounts of bioactive monoterpenes such as thymol and carvacrol, is an important wild medicinal and aromatic plant in China. Monoterpenoids are abundant in glandular secretory trichomes. Here, high-fidelity and chromatin conformation capture technologies were used to assemble and annotate the T. quinquecostatus genome at the chromosome level. The 13 chromosomes of T. quinquecostatus had a total length of 528.66 Mb, a contig N50 of 8.06 Mb, and a BUSCO score of 97.34%. We found that T. quinquecostatus had experienced two whole-genome duplications, with the most recent event occurring ∼4.34 million years ago. Deep analyses of the genome, in conjunction with comparative genomic, phylogenetic, transcriptomic, and metabonomic studies, uncovered many regulatory factors and genes related to monoterpenoids and glandular secretory trichome development. Genes encoding terpene synthase (TPS), cytochrome P450 monooxygenases (CYPs), short-chain dehydrogenase/reductase (SDR), R2R3-MYB, and homeodomain-leucine zipper (HD-ZIP) IV were among those present in the T. quinquecostatus genome. Notably, Tq02G002290.1 (TqTPS1) was shown to encode the terpene synthase responsible for catalyzing production of the main monoterpene product γ-terpinene from geranyl diphosphate (GPP). Our study provides significant insight into the mechanisms of glandular secretory trichome formation and monoterpenoid biosynthesis in thyme. This work will facilitate the development of molecular breeding tools to enhance the production of bioactive secondary metabolites in Lamiaceae.