Bioprocessing of Epothilone B from Aspergillus fumigatus under solid state fermentation: Antiproliferative activity, tubulin polymerization and cell cycle analysis.

Epothilone derivatives have been recognized as one of the most powerful anticancer drugs towards solid tumors, for their unique affinity to bind with ß-tubulin microtubule arrays, stabilizing their disassembly, causing cell death. Sornagium cellulosum is the main source for Epothilone, however, the fermentation bioprocessing of this myxobacteria is the main challenge for commercial production of Epothilone. The metabolic biosynthetic potency of epothilone by Aspergillus fumigatus, an endophyte of Catharanthus roseus, raises the hope for commercial epothilone production, for their fast growth rate and feasibility of manipulating their secondary metabolites. Thus, nutritional optimization of A. fumigatus for maximizing their epothilone productivity under solid state fermentation process is the objective. The highest yield of epothilone was obtained by growing A. fumigatus on orange peels under solid state fermentation (2.2 µg/g), bioprocessed by the Plackett-Burman design. The chemical structure of the extracted epothilone was resolved from the HPLC and LC-MS/MS analysis, with molecular mass 507.2 m/z and identical molecular fragmentation pattern of epothilone B of S. cellulosum. The purified A. fumigatus epothilone had a significant activity towards HepG2 (IC50 0.98 µg/ml), Pancl (IC50 1.5 µg/ml), MCF7 (IC50 3.7 µg/ml) and WI38 (IC50 4.6 µg/ml), as well as a strong anti-tubulin polymerization activity (IC50 0.52 µg/ml) compared to Paclitaxel (2.0 µg/ml). The effect of A. fumigatus epothilone on the immigration ability of HepG2 cells was assessed, as revealed from the wound closure of the monolayer cells that was estimated by ~ 63.7 and 72.5%, in response to the sample and doxorubicin, respectively, compared to negative control. From the Annexin V-PI flow cytometry results, a significant shift of the normal cells to the apoptosis was observed in response to A. fumigatus epothilone by ~ 20 folds compared to control cells, with the highest growth arrest of the HepG2 cells at the G0-G1 stage.

Antibacterial activity of bioactive compounds extracted from red kidney bean (Phaseolus vulgaris L.) seeds against multidrug-resistant Enterobacterales.

In the present study, biologically active compounds such as phenolic-rich extract (PRE), 7S globulin (vicilin), and 11S globulin (legumin) from red kidney bean (Phaseolus vulgaris L.) seeds were extracted and evaluated as antibacterial agents against multidrug-resistant (MDR) Enterobacterales isolated from both animal and human sources. The overall occurrence rate of Enterobacterales was 43.6%, which significantly differed between animal (38.75%) and human (56.67%) sources. Antimicrobial susceptibility testing revealed that Enterobacterales isolates exhibited full resistance (100%) to amoxicillin-clavulanic acid, followed by ampicillin (75.44%), erythromycin (71.93%), cefoxitin (70.18%), amoxicillin (66.66%), ceftriaxone (64.91%), and trimethoprim/sulfamethoxazole (56.14%). Worthy of note, 97.92% of Enterobacterales isolates were MDR. The total phenolic contents (TPC; 53 ± 2 mg GAE g-1) and total flavonoid contents (TFC; 26 ± 1 mg QE g-1) were recorded. The major phenolic and flavonoid components were catechol (17.63 µg/mL) and hesperidin (11.37 µg/mL), respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect the 7S and 11S globulin's molecular mass. The data revealed that red kidney bean protein isolate (KPI) includes two major portions: 7S and 11S globulins. The bioactive compounds of Phaseolus vulgaris were investigated for their antibacterial activities against Enterobacterales for the first time. The protein component (MIC = 0.125 - 2 µg/mL; 53.85%) and its 7S and 11S globulin subunits (MIC = 0.5 - 2 µg/mL; 30.77% each) were the most potent extracts, whereas the methanolic extract was the least effective one (MIC = 2 µg/mL; 15.38%). The results displayed the potential of protein bioactive compounds as a hopeful candidate for enhancing future medication plans for the treatment of Enterobacterales originating from animal and human sources.

Triggering the biosynthetic machinery of Taxol by Aspergillus flavipes via cocultivation with Bacillus subtilis: proteomic analyses emphasize the chromatin remodeling upon fungal-bacterial interaction.

Attenuating the Taxol biosynthesis by fungi with storage and subculturing is the major challenge that limits their further industrial applications. Aspergillus flavipes has been reported as a potent Taxol producer, with plausible increasing to its Taxol yield upon coculturing with the microbiome of Podocarpus gracilior (El-Sayed et al., Process Biochemistry 76:55-67, 2019a; Scientific Reports 9, 2019b; Enzyme and Microbial Technology 131, 2019c); however, the identity of these microbial inducers remains ambiguous. Thus, this study was to assess the potency of individual microbes to trigger the Taxol biosynthesis by A. flavipes and to unravel the differentially expressed protein in response to bacterial interaction. Among the 25 bacterial endophytes of P. gracilior, Bacillus subtilis was the potent isolate enhancing the Taxol yield of A. flavipes by ~1.6-fold. Strikingly, this bacterial elicitor displayed a reliable inhibition to the growth of A. flavipes, so the released antifungal compound by B. subtilis could be the same signals for triggering the expression of A. flavipes Taxol synthesis. The highest Taxol yield by A. flavipes was obtained with the viable cells of B. subtilis, ensuring the pivotality of physical intimate bacterial-fungal interaction. Differential proteome of the cocultures A. flavipes and B. subtilis as well as the axenic A. flavipes was conducted by LC-MS/MS. From the total of 106 identified proteins, 50 proteins were significantly expressed, 47 were upregulated ones, and 59 were downregulated ones for the cocultures normalizing to the axenic one. From the Gene Ontology (GO) and KEGG enrichment analyses, the cellular process, primary metabolic process, and nitrogen compound metabolic process were significantly changed in the coculture normalizing to monoculture of A. flavipes. The molecular function terms (histones H2B, H2A, peptidyl-prolyl cis-trans isomerase, and nucleoside-diphosphate kinase (NDPK)) were the highly significantly expressed proteins of A. flavipes in response to B. subtilis, with strong correlation to triggering of Taxol biosynthesis. The intimate interaction of A. flavipes with B. subtilis strongly modulates the Taxol biosynthetic machinery of A. flavipes by modulating the chromatin remodeling.

Production and bioprocess optimization of antitumor Epothilone B analogue from Aspergillus fumigatus, endophyte of Catharanthus roseus, with response surface methodology.

Epothilones are secondary metabolites produced by Sorangium cellulosum with powerful antiproliferative activity against tumor cells by stabilizing their microtubule arrays, arresting their cellular division at G2-M phase. Unfortunately, the lower yield of epothilone is the challenge for its higher accessibility, thus, searching for alternative sources with promising epothilone producing potency is the prospective. Endophytic fungi are the potential repertoire for bioactive metabolites, thus exploring the epothilone producing potency of endophytic fungi of medicinal plants was objective. Thirty-two fungal isolates were recovered from the tested medicinal plants and their potency to produced epothilone have been assessed using the TLC, HPLC and molecular markers epoA, epoC and epoK. Aspergillus fumigatus EFBL, an endophyte of Catharanthus roseus, was the potent epothilone producer (21.5 µg/g biomass) as revealed from the chromatographic analyses and PCR of molecular markers. The chemical identity of extracted epothilone was verified from the HPLC, NMR, FTIR and LC-MS analyses as epothilone B analogue. The putative epoA gene from A. fumigatus was amplified using RT-PCR with the conservative corresponding primers to the active-sites of S. cellulosum. The amplicons of epoA was 517 bp displayed 98 % similarity with A. fumigatus PKS-NRPS domains, and 40 % similarity with epoA of S. cellulosum. From the in silico analyses, Val506, Ala605 and Ser630 are the conservative amino acids of epoA protein of A. fumigatus and S. cellulosum. Epothilone B from A. fumigatus displayed a strong antiproliferative activity against HepG-2, MCF-7 and LS174 T as revealed from the IC50 values 6.4, 8.7 and 10.21 µM, respectively. The productivity of epothilone B from A. fumigatus was optimized by surface response methodology with Plackett-Burman and Faced Centered Central Composite. With the Plackett-Burman design, the yield of epothilone (54.4-60.1 µg/g biomass) by A. fumigatus was increased by about 2.8-3.0 folds comparing to non-optimized cultures (21.5 µg/ g biomass). From the FCCD design, sucrose, tryptone and incubation time being the highest significant variables medium components affecting the epothilone yield of A. fumigatus. This is the first report exploring the feasibility of endophytic fungi for epothilone producing potency, that could be a novel platform for industrial production of epothilone.

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.

Aspergillus nidulans thermostable arginine deiminase-Dextran conjugates with enhanced molecular stability, proteolytic resistance, pharmacokinetic properties and anticancer activity.

The potential anticancer activity of arginine deiminase (ADI) via deimination of l-arginine into citrulline has been extensively verified against various arginine-auxotrophic tumors, however, the higher antigenicity, structural instability and in vivo proteolysis are the major challenges that limit this enzyme from further clinical implementation. Since, this clinically applied enzyme was derived from Mycobacterium spp, thus, searching for ADI from eukaryotic microbes "especially thermophilic fungi" could have a novel biochemical, conformational and catalytic properties. Aspergillus nidulans ADI was purified with 5.3 folds, with molecular subunit structure 48 kDa and entire molecular mass 120 kDa, ensuring its homotrimeric identity. The peptide fingerprinting analysis revealing the domain Glu95-Gly96-Gly97 as the conserved active site of A. nidulans ADI, with higher proximity to Mycobacterium ADI clade IV. In an endeavor to fortify the structural stability and anticancer activity of A. nidulans ADI, the enzyme was chemically modified with dextran. The optimal activity of Dextran-ADI conjugates was determined at 0.08:20 M ratio of ADI: Dextran, with an overall increase to ADI molecular subunit mass to ˜100 kDa. ADI was conjugated with dextran via the ε-amino groups interaction of surface lysine residues of ADI. The resistance of Dextran-ADI conjugate to proteolysis had been increased by 2.5 folds to proteinase K and trypsin, suggesting the shielding of >50% of ADI surface proteolytic recognition sites. The native and Dextran-ADI conjugates have the same optimum reaction temperature (37 °C), reaction pH and pH stability (7.0-8.0) with dependency on K+ ions as a cofactor. Dextran-ADI conjugates exhibited a higher thermal stability by ˜ 2 folds for all the tested temperatures, ensuring the acquired structural and catalytic stability upon dextran conjugation. Dextran conjugation slightly protect the reactive amino and thiols groups of surface amino acids of ADI from amino acids suicide inhibitors. The affinity of ADI was increased by 5.3 folds to free L-arginine with a dramatic reduction in citrullination of peptidylarginine residues upon dextran conjugation. The anticancer activity of ADI to breast (MCF-7), liver (HepG-2) and colon (HCT8, HT29, DLD1 and LS174 T) cancer cell lines was increased by 1.7 folds with dextran conjugation in vitro. Pharmacokinetically, the half-life time of ADI was increased by 1.7 folds upon dextran conjugation, in vivo. From the biochemical and hematological parameters, ADIs had no signs of toxicity to the experimental animals. In addition to the dramatic reduction of L-arginine in serum, citrulline level was increased by 2.5 folds upon dextran conjugation of ADI. This is first report exploring thermostable ADI from thermophilic A. nidulans with robust structural stability, catalytic efficiency and proteolytic resistance.

Restoring the Taxol biosynthetic machinery of Aspergillus terreus by Podocarpus gracilior Pilger microbiome, with retrieving the ribosome biogenesis proteins of WD40 superfamily.

Attenuating the Taxol yield of Aspergillus terreus with the subculturing and storage were the technical challenges that prevent this fungus to be a novel platform for industrial Taxol production. Thus, the objective of this study was to unravel the metabolic machineries of A. terreus associated with attenuation of Taxol productivity, and their restoring potency upon cocultivation with the Podocarpus gracilior microbiome. The Taxol yield of A. terreus was drastically reduced with the fungal subculturing. At the 10th subculture, the yield of Taxol was reduced by four folds (78.2 µg/l) comparing to the original culture (268 µg/l), as authenticated from silencing of molecular expression of the Taxol-rate limiting enzymes (GGPPS, TDS, DBAT and BAPT) by qPCR analyses. The visual fading of A. terreus conidial pigmentation with the subculturing, revealing the biosynthetic correlation of melanin and Taxol. The level of intracellular acetyl-CoA influx was reduced sequentially with the fungal subculturing, rationalizing the decreasing on Taxol and melanin yields. Fascinatingly, the Taxol biosynthetic machinery and cellular acetyl-CoA of A. terreus have been completely restored upon addition of 3% surface sterilized leaves of P. gracilior, suggesting the implantation of plant microbiome on re-triggering the molecular machinery of Taxol biosynthesis, their transcriptional factors, and/or increasing the influx of Acetyl-CoA. The expression of the proteins of 74.4, 68.2, 37.1 kDa were exponentially suppressed with A. terreus subculturing, and strongly restored upon addition of P. gracilior leaves, ensuring their profoundly correlation with the molecular expression of Taxol biosynthetic genes. From the proteomic analysis, the restored proteins 74.4 kDa of A. terreus upon addition of P. gracilior leaves were annotated as ribosome biogenesis proteins YTM and microtubule-assembly proteins that belong to WD40 superfamily. Thus, further ongoing studies for molecular cloning and expression of these genes with strong promotors in A. terreus, have been initiated, to construct a novel platform of metabolically stable A. terreus for sustainable Taxol production. Attenuating the Taxol yield of A. terreus with the multiple-culturing and storage might be due to the reduction on main influx of acetyl-CoA, or downregulation of ribosome biogenesis proteins that belong to WD40 protein superfamily.

Biochemical characterization of peptidylarginine deiminase-like orthologs from thermotolerant Emericella dentata and Aspergillus nidulans.

Peptidylarginine deiminases (PADs) are a group of hydrolases, mediating the deimination of peptidylarginine residues into peptidyl-citrulline. Equivocal protein citrullination by PADs of fungal pathogens has a strong relation to the progression of multiple human diseases, however, the biochemical properties of fungal PADs remain ambiguous. Thus, this is the first report exploring the molecular properties of PAD from thermotolerant fungi, to imitate the human temperature. The teleomorph Emericella dentata and anamorph Aspergillus nidulans have been morphologically and molecularly identified, with observed robust growth at 37-40 °C, and strong PAD productivity. The physiological profiles of E. dentata and A. nidulans for PADs production in response to carbon, nitrogen sources, initial medium pH and incubation temperature were relatively identical, emphasizing the taxonomical proximity of these fungal isolates. PADs were purified from E. dentata and A. nidulans with apparent molecular masses 41 and 48 kDa, respectively. The peptide fingerprints of PADs from E. dentata and A. nidulans have been analyzed by MALDI-TOF/MS, displaying a higher sequence similarity to human PAD4 by 18% and 31%, respectively. The conserved peptide sequences of E. dentata and A. nidulans PADs displayed a higher similarity to human PAD than A. fumigatus PADs clade. PADs from both fungal isolates have an optimum pH and pH stability at 7.0-8.0, with putative pI 5.0-5.5, higher structural denaturation at pH 4.0-5.5 and 9.5-12 as revealed from absorbance at λ280nm. E. dentata PAD had a higher conformationally thermal stability than A. nidulans PAD as revealed from its lower Kr value. From the proteolytic mapping, the orientation of trypsinolytic recognition sites on the PADs surface from both fungal isolates was very similar. PADs from both isolates are calcium dependent, with participation of serine and cysteine residues on their catalytic sites. PADs displayed a higher affinity to deiminate the peptidylarginine residues with a feeble affinity to work as ADI. So, PADs from E. dentata and A. nidulans had a relatively similar conformational and kinetic properties. Further molecular modeling analysis are ongoing to explore the role of PADs in citrullination of human proteins in Aspergillosis, that will open a new avenue for unraveling the vague of protein-protein interaction of human A. nidulans pathogen.

Purification and immobilization of L-arginase from thermotolerant Penicillium chrysogenum KJ185377.1; with unique kinetic properties as thermostable anticancer enzyme.

L-Arginase, hydrolyzing L-arginine to L-ornithine and urea, is a powerful anticancer, L-arginine-depleting agent, against argininosuccinate synthase expressing tumors. Otherwise, the higher antigenicity and lower thermal stability of this enzyme was the main biochemical hurdles. Since, the intrinsic thermal stability of enzymes follow the physiological temperature of their producer, thus, characterization of L-arginase from thermotolerant Penicillium chrysogenum was the objective of this study. L-Arginase (Arg) was purified to its homogeneity from P. chrysogenum by 10.1-fold, with 37.0 kDa under denaturing PAGE, optimum reaction at 50 °C, pH stability (6.8-7.9), with highest molar ratio of constitutional arginine, glutamic acid, lysine and aspartic acid. The purified enzyme was PEGylated and immobilized on chitosan, with 41.9 and 22.1 % yield of immobilization. At 40 °C, the T1/2 value of free-Arg, PEG-Arg and Chit-Arg was 10.4, 15.6, 20.5 h, respectively. The free-Arg and Chit-Arg have a higher affinity to L-arginine (K m 4.8 mM), while, PEG-Arg affinity was decreased by about 3 fold (K m 15.2 mM). The inhibitory constants to the free and PEG-Arg were relatively similar towards HA and PPG. The IC50 for the free enzyme against HEPG-2 and A549 tumor cells was 0.136 and 0.165 U/ml, comparing to 0.232 and 0.496 U/ml for PEG-Arg, respectively. The in vivo T1/2 to the free Arg and PEG-Arg was 16.4 and 20.4 h, respectively as holo-enzyme. The residual L-arginine level upon using free Arg was 156.9 and 144.5 µM, after 6 and 8 h, respectively, regarding to initials at 253.6 µM, while for Peg-Arg the level of L-arginine was nil till 7 h of initial dosing. The titer of IgG was induced by 10-15 % in response to free-Arg after 28 days comparing to IgG titer for PEG-Arg.