Exploiting the Biosynthetic Potency of Taxol from Fungal Endophytes of Conifers Plants; Genome Mining and Metabolic Manipulation

Endophytic fungi have been considered as a repertoire for bioactive secondary metabolites with potential application in medicine, agriculture and food industry. The biosynthetic pathways by fungal endophytes raise the argument of acquisition of these machineries of such complex metabolites from the plant host. Diterpenoids “Taxol” is the most effective anticancer drug with highest annual sale, since its discovery in 1970 from the Pacific yew tree, Taxus brevifolia. However, the lower yield of Taxol from this natural source (bark of T. brevifolia), availability and vulnerability of this plant to unpredicted fluctuation with the ecological and environmental conditions are the challenges. Endophytic fungi from Taxus spp. opened a new avenue for industrial Taxol production due to their fast growth, cost effectiveness, independence on climatic changes, feasibility of genetic manipulation. However, the anticipation of endophytic fungi for industrial Taxol production has been challenged by the loss of its productivity, due to the metabolic reprograming of cells, downregulating the expression of its encoding genes with subculturing and storage. Thus, the objectives of this review were to (1) Nominate the endophytic fungal isolates with the Taxol producing potency from Taxaceae and Podocarpaceae; (2) Emphasize the different approaches such as molecular manipulation, cultural optimization, co-cultivation for enhancing the Taxol productivities; (3) Accentuate the genome mining of the rate-limiting enzymes for rapid screening the Taxol biosynthetic machinery; (4) Triggering the silenced rate-limiting genes and transcriptional factors to activates the biosynthetic gene cluster of Taxol.

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Molecular Identification of Endophytic Fungi and Their Pathogenicity Evaluation Against Dendrobium nobile and Dendrobium officinale

International Journal of Molecular Sciences ◽

10.3390/ijms21010316 ◽

2020 ◽

Vol 21 (1) ◽

pp. 316 ◽

Cited By ~ 7

Author(s):

Surendra Sarsaiya ◽

Archana Jain ◽

Qi Jia ◽

Xiaokuan Fan ◽

Fuxing Shu ◽

...

Keyword(s):

Molecular Identification ◽

Endophytic Fungi ◽

Fungal Endophytes ◽

Dendrobium Officinale ◽

Plant Host ◽

Efficient System ◽

Dendrobium Nobile ◽

Living Tissues ◽

Pathogenic Effects

Dendrobium are tropical orchid plants that host diverse endophytic fungi. The role of these fungi is not currently well understood in Dendrobium plants. We morphologically and molecularly identified these fungal endophytes, and created an efficient system for evaluating the pathogenicity and symptoms of endophytic fungi on Dendrobium nobile and Dendrobium officinale though in vitro co-culturing. ReThe colony morphological traits of Dendrobium myco-endophytes (DMEs) were recorded for their identification. Molecular identification revealed the presence of Colletotrichum tropicicola, Fusarium keratoplasticum, Fusarium oxysporum, Fusarium solani, and Trichoderma longibrachiatum. The pathogenicity results revealed that T. longibrachiatum produced the least pathogenic effects against D. nobile protocorms. In seedlings, T. longibrachiatum showed the least pathogenic effects against D. officinale seedlings after seven days. C. tropicicola produced highly pathogenic effects against both Dendrobium seedlings. The results of histological examination of infected tissues revealed that F. keratoplasticum and T. longibrachiatum fulfill Koch’s postulates for the existence of endophytes inside the living tissues. The DMEs are cross-transmitted inside the host plant cells, playing an important role in plant host development, resistance, and alkaloids stimulation.

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Isolation and Characterization of Fungal Endophytes Isolated from Medicinal Plant Ephedra pachyclada as Plant Growth-Promoting

Biomolecules ◽

10.3390/biom11020140 ◽

2021 ◽

Vol 11 (2) ◽

pp. 140 ◽

Cited By ~ 2

Author(s):

Ahmed Mohamed Aly Khalil ◽

Saad El-Din Hassan ◽

Sultan M. Alsharif ◽

Ahmed M. Eid ◽

Emad El-Din Ewais ◽

...

Keyword(s):

Medicinal Plants ◽

Plant Growth ◽

Aspergillus Flavus ◽

Endophytic Fungi ◽

Fungal Endophytes ◽

Penicillium Crustosum ◽

Plant Growth Promoting ◽

Penicillium Commune ◽

Maize Plants ◽

Growth Promoting

Endophytic fungi are widely present in internal plant tissues and provide different benefits to their host. Medicinal plants have unexplored diversity of functional fungal association; therefore, this study aimed to isolate endophytic fungi associated with leaves of medicinal plants Ephedra pachyclada and evaluate their plant growth-promoting properties. Fifteen isolated fungal endophytes belonging to Ascomycota, with three different genera, Penicillium, Alternaria, and Aspergillus, were obtained from healthy leaves of E. pachyclada. These fungal endophytes have varied antimicrobial activity against human pathogenic microbes and produce ammonia and indole acetic acid (IAA), in addition to their enzymatic activity. The results showed that Penicillium commune EP-5 had a maximum IAA productivity of 192.1 ± 4.04 µg mL−1 in the presence of 5 µg mL−1 tryptophan. The fungal isolates of Penicillium crustosum EP-2, Penicillium chrysogenum EP-3, and Aspergillus flavus EP-14 exhibited variable efficiency for solubilizing phosphate salts. Five representative fungal endophytes of Penicillium crustosum EP-2, Penicillium commune EP-5, Penicillium caseifulvum EP-11, Alternaria tenuissima EP-13, and Aspergillus flavus EP-14 and their consortium were selected and applied as bioinoculant to maize plants. The results showed that Penicillium commune EP-5 increased root lengths from 15.8 ± 0.8 to 22.1 ± 0.6. Moreover, the vegetative growth features of inoculated maize plants improved more than the uninoculated ones.

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Environmental RNAi pathways in the two-spotted spider mite

BMC Genomics ◽

10.1186/s12864-020-07322-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Mosharrof Mondal ◽

Jacob Peter ◽

Obrie Scarbrough ◽

Alex Flynt

Keyword(s):

Gene Expression ◽

Small Rna ◽

Spider Mite ◽

Genetic Manipulation ◽

Model Organisms ◽

Rnai Pathway ◽

Plant Host ◽

Rnai Technology ◽

Two Spotted Spider Mite ◽

Multicellular Organisms

Abstract Background RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design. Results Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented. Conclusions Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.

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Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The ISME Journal ◽

10.1038/s41396-021-01052-3 ◽

2021 ◽

Author(s):

Valentin Waschulin ◽

Chiara Borsetto ◽

Robert James ◽

Kevin K. Newsham ◽

Stefano Donadio ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Full Length ◽

Metagenomic Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Rich Diversity ◽

Long Read ◽

The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

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The Influence of the Grapevine Bacterial and Fungal Endophytes on Biomass Accumulation and Stilbene Production by the In Vitro Cultivated Cells of Vitis amurensis Rupr.

Plants ◽

10.3390/plants10071276 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1276

Author(s):

Olga A. Aleynova ◽

Andrey R. Suprun ◽

Nikolay N. Nityagovsky ◽

Alexandra S. Dubrovina ◽

Konstantin V. Kiselev

Keyword(s):

Cell Culture ◽

Cell Suspension ◽

Endophytic Fungi ◽

Endophytic Bacteria ◽

Fungal Endophytes ◽

Biomass Accumulation ◽

Vitis Amurensis ◽

Grape Cell ◽

Stilbene Biosynthesis

Plant endophytes are known to alter the profile of secondary metabolites in plant hosts. In this study, we identified the main bacterial and fungal representatives of the wild grape Vitis amurensis Rupr. microbiome and investigated a cocultivation effect of the 14 endophytes and the V. amurensis cell suspension on biomass accumulation and stilbene biosynthesis. The cocultivation of the V. amurensis cell culture with the bacteria Agrobacterium sp., Bacillus sp., and Curtobacterium sp. for 2 weeks did not significantly affect the accumulation of cell culture fresh biomass. However, it was significantly inhibited by the bacteria Erwinia sp., Pantoea sp., Pseudomonas sp., and Xanthomonas sp. and fungi Alternaria sp., Biscogniauxia sp., Cladosporium sp., Didymella sp. 2, and Fusarium sp. Cocultivation of the grapevine cell suspension with the fungi Didymella sp. 1 and Trichoderma sp. resulted in cell death. The addition of endophytic bacteria increased the total stilbene content by 2.2–5.3 times, while the addition of endophytic fungi was more effective in inducing stilbene accumulation by 2.6–16.3 times. The highest content of stilbenes in the grapevine cells cocultured with endophytic fungi was 13.63 and 13.76 mg/g of the cell dry weight (DW) after cultivation with Biscogniauxia sp. and Didymella sp. 2, respectively. The highest content of stilbenes in the grapevine cells cocultured with endophytic bacteria was 4.49 mg/g DW after cultivation with Xanthomonas sp. The increase in stilbene production was due to a significant activation of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) gene expression. We also analyzed the sensitivity of the selected endophytes to eight antibiotics, fluconazole, and trans-resveratrol. The endophytic bacteria were sensitive to gentamicin and kanamycin, while all selected fungal strains were resistant to fluconazole with the exception of Cladosporium sp. All endophytes were tolerant of trans-resveratrol. This study showed that grape endophytes stimulate the production of stilbenes in grape cell suspension, which could further contribute to the generation of a new stimulator of stilbene biosynthesis in grapevine or grape cell cultures.

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Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

Marine Drugs ◽

10.3390/md19020108 ◽

2021 ◽

Vol 19 (2) ◽

pp. 108

Author(s):

Xiyan Wang ◽

Thomas Isbrandt ◽

Mikael Lenz Strube ◽

Sara Skøtt Paulsen ◽

Maike Wennekers Nielsen ◽

...

Keyword(s):

Secondary Metabolite ◽

Genetic Manipulation ◽

Genome Mining ◽

Hydrolytic Enzymes ◽

Metabolite Profile ◽

Chitinolytic Enzyme ◽

Chitin Degradation ◽

Metabolite Profiles ◽

Bioactive Potential ◽

Pseudoalteromonas Rubra

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

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Unexpected genomic, biosynthetic and species diversity of Streptomyces bacteria from bats in Arizona and New Mexico, USA

BMC Genomics ◽

10.1186/s12864-021-07546-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Cooper J. Park ◽

Nicole A. Caimi ◽

Debbie C. Buecher ◽

Ernest W. Valdez ◽

Diana E. Northup ◽

...

Keyword(s):

New Mexico ◽

Genetic Manipulation ◽

Distinct Species ◽

Biosynthetic Gene Cluster ◽

Genomic Variation ◽

Species Variation ◽

Nucleotide Polymorphisms ◽

Peptide Synthetases ◽

Genome Wide ◽

Diversity Estimates

Abstract Background Antibiotic-producing Streptomyces bacteria are ubiquitous in nature, yet most studies of its diversity have focused on free-living strains inhabiting diverse soil environments and those in symbiotic relationship with invertebrates. Results We studied the draft genomes of 73 Streptomyces isolates sampled from the skin (wing and tail membranes) and fur surfaces of bats collected in Arizona and New Mexico. We uncovered large genomic variation and biosynthetic potential, even among closely related strains. The isolates, which were initially identified as three distinct species based on sequence variation in the 16S rRNA locus, could be distinguished as 41 different species based on genome-wide average nucleotide identity. Of the 32 biosynthetic gene cluster (BGC) classes detected, non-ribosomal peptide synthetases, siderophores, and terpenes were present in all genomes. On average, Streptomyces genomes carried 14 distinct classes of BGCs (range = 9–20). Results also revealed large inter- and intra-species variation in gene content (single nucleotide polymorphisms, accessory genes and singletons) and BGCs, further contributing to the overall genetic diversity present in bat-associated Streptomyces. Finally, we show that genome-wide recombination has partly contributed to the large genomic variation among strains of the same species. Conclusions Our study provides an initial genomic assessment of bat-associated Streptomyces that will be critical to prioritizing those strains with the greatest ability to produce novel antibiotics. It also highlights the need to recognize within-species variation as an important factor in genetic manipulation studies, diversity estimates and drug discovery efforts in Streptomyces.

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Revealing the endophytic mycoflora in tea (Camellia sinensis) leaves in Sri Lanka: the first comprehensive study

Phytotaxa ◽

10.11646/phytotaxa.514.3.5 ◽

2021 ◽

Vol 514 (3) ◽

pp. 247-260

Author(s):

KASUN THAMBUGALA ◽

DINUSHANI DARANAGAMA ◽

SAGARIKA KANNANGARA ◽

THENUKA KODITUWAKKU

Keyword(s):

Sri Lanka ◽

Camellia Sinensis ◽

Endophytic Fungi ◽

Sequence Data ◽

Fungal Endophytes ◽

Fungal Endophyte ◽

Tea Leaves ◽

Molecular Sequence Data ◽

Molecular Sequence ◽

Rdna Sequence Data

Endophytic fungi are a diverse group of microorganisms that live asymptomatically in healthy tissues of host and they have been reported from all kinds of plant tissues such as leaves, stems, roots, flowers, and fruits. In this study, fungal endophytes associated with tea leaves (Camellia sinensis) were collected from Kandy, Kegalle, and Nuwara Eliya districts in Sri Lanka and were isolated, characterized, and identified. A total of twenty endophytic fungal isolates belonging to five genera were recovered and ITS-rDNA sequence data were used to identify them. All isolated endophytic fungal strains belong to the phylum Ascomycota and the majority of these isolates were identified as Colletotrichum species. Phyllosticta capitalensis was the most commonly found fungal endophyte in tea leaves and was recorded in all three districts where the samples were collected. This is the very first investigation on fungal endophytes associated with C. sinensis in Sri Lanka based on molecular sequence data. In addition, a comprehensive account of known endophytic fungi reported worldwide on Camellia sinensis is provided.

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Discovery of a Potential Liver Fibrosis Inhibitor from a Mushroom Endophytic Fungus by Genome Mining of a Silent Biosynthetic Gene Cluster

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.1c03639 ◽

2021 ◽

Vol 69 (38) ◽

pp. 11303-11310

Author(s):

Jin-Tao Cheng ◽

Han-Min Wang ◽

Jia-Hui Yu ◽

Chen-Fan Sun ◽

Fei Cao ◽

...

Keyword(s):

Liver Fibrosis ◽

Gene Cluster ◽

Endophytic Fungus ◽

Genome Mining ◽

Biosynthetic Gene Cluster ◽

Biosynthetic Gene

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Isolation and antimicrobial effects of endophytic fungi from Edgeworthia chrysantha

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v10i3.23575 ◽

2015 ◽

Vol 10 (3) ◽

pp. 529 ◽

Cited By ~ 4

Author(s):

Huawei Zhang ◽

Chuanfeng Ruan ◽

Xuelian Bai

Keyword(s):

Ethyl Acetate ◽

Endophytic Fungi ◽

Fermentation Broth ◽

Fungal Endophytes ◽

Antimicrobial Effect ◽

Antibacterial Activities ◽

Antimicrobial Activities ◽

Human Pathogens ◽

Antimicrobial Effects ◽

Staphyloccocus Aureus

Ten fungal strains isolated from Edgeworthia chrysantha, one of traditional medicinal plants in China, were evaluated their antimicrobial activities against three human pathogens, Escherichia coli, Staphyloccocus aureus and Candida albicans, and two phytopathogens, Rhizoctonia cerealis and Colletotrichum gloeosporioides. The results indicated that most ethyl acetate extracts of fermentation broth of these fungal endophytes had stronger antimicrobial activities than their fermentation broth. Among these endophytic strains, both fermentation broth and the ethyl acetate extract of strain D showed the strongest inhibitory effects on all pathogens. Strains 5-19 and BZ also exhibited potent antibacterial activities. However, other strains had weak or no antimicrobial effect. This was the first report on the isolation and antimicrobial effects of endophytic fungi from E. chrysantha.

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