Unlocking the biosynthetic potential of Penicillium roqueforti for hyperproduction of the immunosuppressant mycophenolic acid: Gamma radiation mutagenesis and response surface optimization of fermentation medium.

Based on the broad clinical utility of the immunosuppressant mycophenolic acid (MPA), this article aims to intensify the biosynthetic potential of Penicillium roqueforti for more effective hyperproduction of the drug. Several mutants were generated from irradiation mutagenesis and screened. Two strains (GM1013 and GM1093) presented an elevated MPA productivity with significant yield constancy over 10 subsequent generations. By investigating the effect of some phosphorous sources and mineral salts on MPA production by the two mutants, KH2 PO4 and FeSO4 ·7H2 O were most preferred by the two mutants for higher MPA production rates. Statistics-dependent experimental designs were also employed for optimizing medium components for maximum MPA production. Medium components were primarily screened using the Plackett-Burman model to demonstrate the most important components that most significantly affect MPA production. The concentrations of these significant components were then optimized through a central composite rotatable model. In conclusion, gamma-radiation mutation and response surface optimization resulted in a promising MPA productivity by P. roqueforti GM1013. To our knowledge, the MPA-yield achieved in this study (2933.32 mg L-1 ) is the highest reported by academic laboratories from P. roqueforti cultures, which could be of economic value for a prospective large industrialized application.

Irradiation impact on biological activities of Anthraquinone pigment produced from Talaromyces purpureogenus and its evaluation, characterization and application in beef burger as natural preservative.

BACKGROUND: The demand for natural coloring and preservative agents in food industry is increasing day by day as a result of awareness of the negative health effects of synthetic color preservatives. Consumers want foods with less processing, a longer shelf life, and clear labels that list only natural ingredients and food additives with familiar names that promote good health. In order to meet consumer demands and regain consumers' confidence in the safety of food products, the food industry was compelled to search for natural alternatives with strong antibacterial and antioxidant properties. Therefore, the objective of this study was to produce a microbial pigment that not only serve as food coloring agents but also provide health advantages owing to their bioactivities. Additionally, the potential use of anthraquinone pigment (AQP) as a natural food preservative compared to gamma irradiation was also examined to extend the shelf life of the beef burger and improve its hygienic quality. RESULTS: This study used Talaromyces purpureogenus AUMC2603 to produce the red natural pigment, which was identified as an anthraquinone pigment (AQP). According to the results, gamma (γ) radiation had no significant effect on AQP's antibacterial properties. However, it has a negative, considerable effect on antioxidant activity, where a large dose of γ-ray may change the antioxidant components and lessen the AQP's capacity to scavenge free radicals. Additionally, the γ ray-treated AQP had a strong cytotoxic activity in relation to a high γ-ray dose. As a result, it is suggested that AQP-containing foods should not be irradiated. The extracted AQP was applied as a food additive to improve the quality and increase the shelf life of beef burgers. Significant antibacterial and antioxidant action has been shown at 2% (w/v) AQP. The findings demonstrated that the treatment of beef burger with AQP decreased the initial total bacterial count and psychrophilic bacteria and extended the shelf-life of beef burger in comparison to the control (beef burger with no addition of AQP, butylated hydroxytoluene (BHT) or gamma radiation treatment). On the other hand, there was no substantial difference in the overall amount of mold and yeast or coliform at zero time. According to sensory characteristics, beef burgers had a shelf life of 6 days for controls and 9, 12, and 15 days for AQP-treated samples at 0.5, 1 and 2%, respectively, compared to γ- irradiated samples, 9 and 21 days, at 3 and 5 Kilo Gray (KGy), respectively. CONCLUSIONS: This research provides a natural red pigment from Talaromyces purpureogenus with potent biological activities as antimicrobials and antioxidants to be applied as coloring, additive, and preservative agent in the food industry. Also, the tested pigment offers a powerful alternative to gamma irradiation for extending the shelf life of food products.

Optimization, purification, and structure elucidation of anthraquinone pigment derivative from Talaromyces purpureogenus as a novel promising antioxidant, anticancer, and kidney radio-imaging agent.

This study aims to investigate the bioproduction and prospective biological applications of a natural red pigment from Talaromyces purpureogenus AUMC2603. Maximum pigment yield was achieved by a numerical optimization at pH 6, temperature 25 °C, and an 18-day incubation period on Yeast Malt Broth (YMB) media. The crude pigment was separated and purified into two pigment fractions via solid-phase extraction and then characterized as anthraquinone (dominant) and herquinone by LC/MS and 1HNMR analysis. The crude pigment extract and the two separated fractions displayed a potential antioxidant activity. Additionally, they showed a powerful anticancer activity towards cancer cell lines, MCF-7, HepG-2, and HCT116 with less cytotoxicity on normal cell lines, MCF12F and BJ-1T. The radioiodination efficiency of the radiosynthesized 99mTc-anthraquinone pigment complex was also investigated and optimized, obtaining a radiochemical yield of 92.70 % ± 0.89 %. An in vivo biodistribution study of the 99mTc-anthraquinone pigment complex demonstrated a high kidney uptake of 34 % injected dose per gram of organ tissue 60 min after intravenous injection, and the complex retention remained high up to 120 min. The current study is the first bioassay report on the efficacy of a purified anthraquinone from T. purpureogenus as a potent agent for kidney radio-imaging that could be applied in kidney cancer diagnosis.

Biosorption optimization of lead(II) and cadmium(II) ions by two novel nanosilica-immobilized fungal mutants.

AIMS: This study aims at immobilization of fungal mutants on nanosilica (NSi)-carriers for designing efficient biosorbents as a significant new technology for decontamination practices and maximizing their heavy metal (HM) sorption proficiency through the experimental design methodology. MATERIALS AND RESULTS: Endophytic fungal mutant strains, Chaetomium globosum El26 mutant and Alternaria alternata S5 mutant were heat inactivated and then immobilized, each separately, on NSi carriers to formulate two separated nano-biosorbents. The formulated NSi-Chaetomium globosum El26 mutant (NSi-Chae El26 m) was investigated for Pb+2 uptake while, the formulated NSi-Alternaria alternata S5 mutant (NSi-Alt S5 m) was investigated for Cd+2 uptake, each through a batch equilibrium protocol. Before and after the metal sorption process, the designed nano-biosorbents were characterized via scanning electron microscopy, energy-dispersive X-ray spectroscopy, and Fourier transform infrared analysis. Sorption pH, contact time, sorbent concentration, and initial HM concentration were statistically optimized using a Box-Behnken design. Results showed that NSi-Chae El26 m was efficient in Pb+2 uptake with maximum biosorption capacities of 199.0, while NSi-Alt S5 m was efficient in Cd+2 uptake with maximum biosorption capacities of 162.0 mg g-1 . Moreover, the equilibrium data indicated that the adsorption of Pb+2 and Cd+2 by the tested nano-biosorbents fitted to the Freundlich isotherm. CONCLUSIONS: The formulated nano-biosorbents resulted in higher HM biosorption of metal ions from aqueous solution than that obtained by the free fungal biomass. The biosorption statistical modelling described the interactions between the tested sorption parameters and predicted the optimum values for maximum HM biosorption capacity by the two designed nano-biosorbents. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings verify that members of the endophytic fungal genera Alternaria and Chaetomium are suitable to produce nano-biosorbents for decontamination practices after treatment by gamma mutagenesis, heat inactivation, and NSi immobilization. Moreover, statistical optimization can assist to evaluate the optimal conditions to produce such bioremediation material.

Novel fabrication of SiO2/Ag nanocomposite by gamma irradiated Fusarium oxysporum to combat Ralstonia solanacearum.

The bacterial wilt is a global destructive plant disease that initiated by the phytopathogenic Ralstonia solanacearum. This study display a novel biofabrication of silica/silver nanocomposite using Fusarium oxysporum-fermented rice husk (RH) under solid state fermentation (SSF). The biofabricated nanocomposite was characterized by XRD, UV-Vis. spectroscopy, DLS, SEM, EDX elemental mapping, and TEM analyses as well as investigated for anti-R. solanacearum activity. Response surface methodology was also processed for optimizing the biofabrication process and improving the anti-bacterial activity of the fabricated nanocomposite. Maximum suppression zone of 29.5 mm against R. solanacearum was reached at optimum RH content of 6.0 g, AgNO3 concentration of 2.50 mM, reaction pH of 6.3, and reaction time of 2 days. The anti-R. solanacearum activity of the fabricated nanocomposite was further improved by exposing the F. oxysporum strain to a gamma irradiation dose of 200 Gy. In conclusion, RH recycling under SSF by F. oxysporum could provide an innovative, facile, non-expensive, and green approach for fabricating SiO2/Ag nanocomposite that could be applied efficiently as an eco-friendly antibacterial agent to combat R. solanacearum in agricultural applications. Moreover, the developed method could serve as a significant platform for the designing of new nanostructures for broad applications.

New and potent production platform of the acetylcholinesterase inhibitor huperzine A by gamma-irradiated Alternaria brassicae under solid-state fermentation.

Huperzine-A (HupA) is an emerging, powerful, and promising natural acetylcholinesterase inhibitor. Despite that, the achieved yields of HupA from microbial sources are still far from the industrial applications. Accordingly, this paper was conducted to valorize solid-state fermentation (SSF) as an efficient production platform of HupA. Four agro-industrial wastes, namely rice bran, potato peel, sugarcane bagasse, and wheat bran, were tested and screened as cultural substrates for the production of HupA by the endophytic Alternaria brassica under SSF. Maximum HupA production was attained on using rice bran moistened by Czapex's dox mineral broth. In the effort to increase the HupA titer, supplementation of the best moistening agent by different carbon and nitrogen sources was successfully investigated. Additionally, factors affecting HupA production under SSF including substrate concentration, moistening level, and inoculum concentration were optimized using response surface methodology. A Box-Behnken design was applied for generating a predictive model of the interactions between these factors. Under the optimum conditions of 15 g rice bran, inoculum concentration of 5 × 106 spores mL-1, and 60% moisture level, HupA concentration was intensified to 518.93 µg g-1. Besides, HupA production by the fungal strain was further enhanced using gamma-irradiation mutagenesis. The final HupA production was significantly intensified following exposure to 0.5 KGy gamma radiation to 1327 µg g-1, which represents a 12.85-fold increase. This is the first report on the successful production of the natural fungal metabolite HupA under SSF. Moreover, the achieved yield in this study using agro-industrial wastes may contribute to reducing the cost of HupA manufacture.Key points• Different agro-industrial by-products were tried as cultural substrates for the production of the acetylcholinesterase inhibitor HupA under SSF for the first time.• Factors affecting HupA production under SSF were optimized using response surface methodology.• The final HupA production was intensified following exposure to gamma radiation recording 1327 µg g-1, which represents a 12.85-fold increase.

Molecular identification and evaluation of gamma irradiation effect on modulating heavy metals tolerance in some of novel endophytic fungal strains.

Heavy metal (HM) pollution is a worldwide environmental issue. Given the urgent need to develop more powerful approaches for effective phytoremediation of HMs, isolation of novel endophytic strains from hyperaccumulator plants having potent HM tolerance is the main objective in this research. Moreover, the recovered strains were characterized and subjected to radiation mutagenesis to enhance their tolerance to HMs. Among 105 isolates, Alternaria alternata AUMC14431 was identified as the most effective Cd+2 tolerant strain having high recorded tolerance index (TI) (76.24%); in addition, the recorded minimum inhibitory concentration (MIC) was 300 ppm. Meanwhile, Chaetomium globosum AUMC14432 was identified as the most effective Pb+2 and Ni+2 tolerant strain having high recorded TI (97.46 and 93.34%, respectively); in addition, the evaluated MICs were 250 and 200 ppm, respectively. UV and gamma irradiation of the tested strains enhanced their Cd+2 and Pb+2 tolerance significantly (P ≤ 0.05). Meanwhile, irradiation had a negative impact on Ni+2 tolerance of C. globosum. The mutation incidence at the molecular level arising from exposure to irradiation was investigated. Genomic DNA of both the wild and mutated endophytic strains were isolated followed by random amplified polymorphic DNA (RAPD-PCR) analysis, using two short primers. A remarkable difference in DNA gel pattern between the wild type and mutated strains was observed. In conclusion, the novel isolated and irradiated endophytic strains, A. alternata S5 and C. globosum El26, having high efficiency in Cd+2 and Pb+2 tolerance, respectively, are considered to be prospective and powerful bioremediation candidates for potential application in microbially assisted phytoremediation.

Response surface methodology-mediated improvement of the irradiated endophytic fungal strain, Alternaria brassicae AGF041 for Huperzine A-hyperproduction.

Huperzine A (HupA) is an anti-Alzheimer's therapeutic and a dietary supplement for memory boosting that is extracted mainly from Huperziacae plants. Endophytes represent the upcoming refuge to protect the plant resource from distinction but their HupA yield is still far from commercialization. In this context, UV and gamma radiation mutagenesis of the newly isolated HupA-producing Alternaria brassicae AGF041 would be applied in this study for improving the endophytic HupA yield. Compared to non-irradiated cultures, UV (30-40 min, exposure) and γ (0·5 KGy, dose) irradiated cultures, each separately, showed a significant higher HupA yield (17·2 and 30·3%, respectively). While, application of a statistically optimized compound irradiation (0·70 KGy of γ treatment and 42·49 min of UV exposure, sequentially) via Response Surface Methodology (RSM) resulted in 53·1% production increase. Moreover, a stable selected mutant strain CM003 underwent batch cultivation using a 6·6 l bioreactor for the first time and was successful for scaling up the HupA production to 261·6 µg l-1 . Findings of this research are demonstrated to be valuable as the employed batch fermentation represents a successful starting step towards the promising endophytic HupA production at an industrial scale.

Production of the anticancer drug taxol by the endophytic fungus Epicoccum nigrum TXB502: enhanced production by gamma irradiation mutagenesis and immobilization technique.

Taxol, a phyto-extracted diterpenoid, is the most commercially needed drug in cancer chemotherapy. In spite of the microbial production of taxol being successful and prospective, the reported yields are still not sufficient for large-scale production. Thus, the discovery of new taxol-producing microbial strains and production enhancement methodologies such as process optimization, strain improvement, and immobilization technique are the main objectives. In this paper, a taxol-producing start strain Epicoccum nigrum TXB502 (initial yield 61.35 µg L-1) was isolated from Taxus baccata and identified by morphological and molecular tools. The optimum cultivation and nutritional conditions were assessed by testing one parameter at a time approach that resulted in 88.59% significant production increase. In addition, a stable mutant with improved productivity (40.07% yield increase in comparison with the parent strain) was successfully developed after gamma irradiation mutagenesis of the start strain. The taxol titer was further improved via testing different immobilization carriers for both spores and mycelia of this mutant. Over taxol production was achieved using alginate-immobilized mycelia with the feasibility of conducting six successive production cycles in a semi-continuous form. The final total concentration reached 8187.77 µg taxol 6 L-1 which represents approximately 22-fold increase, as compared to the initial titer of the start strain. These findings can pave the way for the prospective industrial manufacturing of taxol, as the achieved taxol production in this study is the highest reported by academic laboratories for microbial cultures. KEY POINTS: • Discovery of a new taxol-producing endophytic fungus E. nigrum TXB502 strain. • Taxol yield was successfully improved via bioprocess optimization and strain mutagenesis. • Alginate-immobilized mycelia were efficient for a semi-continuous production of taxol. • The final total concentration of taxol showed approximately 22-fold increase as compared to the initial titer.

Microbial acetylcholinesterase inhibitors for Alzheimer's therapy: recent trends on extraction, detection, irradiation-assisted production improvement and nano-structured drug delivery.

Neurodegenerative disorders especially Alzheimer's disease (AD) are significantly threatening the public health. Acetylcholinesterase (AChE) inhibitors are compounds of great interest which can be used as effective agents for the symptomatic treatment of AD. Although plants are considered the largest source for these types of inhibitors, the microbial production of AChE inhibitors represents an efficient, easily manipulated, eco-friendly, cost-effective, and alternative approach. This review highlights the recent advances on the microbial production of AChE inhibitors and summarizes all the previously reported successful studies on isolation, screening, extraction, and detecting methodologies of AChE inhibitors from the microbial fermentation, from the earliest trials to the most promising anti-AD drug, huperzine A (HupA). In addition, improvement strategies for maximizing the industrial production of AChE inhibitors by microbes will be discussed. Finally, the promising applications of nano-material-based drug delivery systems for natural AChE inhibitor (HupA) will also be summarized. KEY POINTS: • AChE inhibitors are potential therapies for Alzheimer's disease. • Microorganisms as alternate sources for prospective production of such inhibitors. • Research advances on extraction, detection, and strategies for production improvement. • Nanotechnology-based approaches for an effective drug delivery for Alzheimer's disease.

Production and enhancement of the acetylcholinesterase inhibitor, huperzine A, from an endophytic Alternaria brassicae AGF041.

Huperzine A (HupA) is a potent acetylcholinesterase (AChE) inhibitor of a great consideration as a prospective drug candidate for Alzheimer's disease treatment. Production of HupA by endophytes offers an alternative challenge to reduce the massive plant harvest needed to meet the increasing demand of HupA. In the current study, some endophytic fungal and actinobacterial isolates from the Chinese herb, Huperzia serrata, underwent liquid fermentation, alkaloid extraction, and screening for AChE inhibition and HupA production. Among these isolates, Alternaria brassicae AGF041 strain was the only positive strain for HupA production with the maximum AChE inhibition of 75.5%. Chromatographic analyses verified the identity of the produced HupA. The HupA production was efficiently maximized up to 42.89 µg/g of dry mycelia, after optimization of thirteen process parameters using multifactorial statistical approaches, Plackett-Burman and central composite designs. The statistical optimization resulted in a 40.8% increase in HupA production. This is the first report to isolate endophytic actinobacteria with anti-AChE activity from H. serrata, and to identify an endophytic fungus A. brassicae as a new promising start strain for a higher HupA yield.