Genome-Scale Model of Rhizopus microsporus: Metabolic integration of a fungal holobiont with its bacterial and viral endosymbionts.

Rhizopus microsporus often lives in association with bacterial and viral symbionts that alter its biology. This fungal model represents an example of the complex interactions established among diverse organisms in functional holobionts. We constructed a Genome-Scale Model (GSM) of the fungal-bacterial-viral holobiont (iHol). We employed a constraint-based method to calculate the metabolic fluxes to decipher the metabolic interactions of the symbionts with their host. Our computational analyses of iHol simulate the holobiont's growth and the production of the toxin rhizoxin. Analyses of the calculated fluxes between R. microsporus in symbiotic (iHol) versus asymbiotic conditions suggest that changes in the lipid and nucleotide metabolism of the host are necessary for the functionality of the holobiont. Glycerol plays a pivotal role in the fungal-bacterial metabolic interaction, as its production does not compromise fungal growth, and Mycetohabitans bacteria can efficiently consume it. Narnavirus RmNV-20S and RmNV-23S affected the nucleotide metabolism without impacting the fungal-bacterial symbiosis. Our analyses highlighted the metabolic stability of Mycetohabitans throughout its co-evolution with the fungal host. We also predicted changes in reactions of the bacterial metabolism required for the active production of rhizoxin. This iHol is the first GSM of a fungal holobiont.

Rhizopus stolonifer biomass catalytic transesterification capability: optimization of cultivation conditions.

BACKGROUND: Using fungal biomass for biocatalysis is a potential solution for the expensive cost of the use o enzymes. Production of fungal biomass with effective activity requires optimizing the cultivation conditions. RESULTS: Rhizopus stolonifer biomass was optimized for transesterification and hydrolysis of waste frying oil (WFO). Growth and biomass lipolytic activities of R. stolonifer improved under shaking conditions compared to static conditions, and 200 rpm was optimum. As biomass lipase and transesterification activities inducer, olive oil was superior to soybean, rapeseed, and waste frying oils. Biomass produced in culture media containing fishmeal as an N-source feedstock had higher lipolytic capabilities than corn-steep liquor and urea. Plackett Burman screening of 9 factors showed that pH (5-9), fishmeal (0.25-1.7%, w/v), and KH2PO4 (0.1-0.9%, w/v) were significant factors with the highest main effect estimates 11.46, 10.42, 14.90, respectively. These factors were selected for response surface methodology (RSM) optimization using central composite design (CCD). CCD models for growth, biomass lipase activity, and transesterification capability were significant. The optimum conditions for growth and lipid modification catalytic activities were pH 7.4, fishmeal (2.62%, w/v), and KH2PO4 (2.99%, w/v). CONCLUSION: Optimized culture conditions improved the whole cell transesterification capability of Rhizopus stolonifer biomass in terms of fatty acid methyl ester (FAME) concentration by 67.65% to a final FAME concentration of 85.5%, w/w.

Alteration of Chain-Length Selectivity and Thermostability of Rhizopus oryzae Lipase via Virtual Saturation Mutagenesis Coupled with Disulfide Bond Design.

Rhizopus oryzae lipase (ROL) is one of the most important enzymes used in the food, biofuel, and pharmaceutical industries. However, the highly demanding conditions of industrial processes can reduce its stability and activity. To seek a feasible method to improve both the catalytic activity and the thermostability of this lipase, first, the structure of ROL was divided into catalytic and noncatalytic regions by identifying critical amino acids in the crevice-like binding pocket. Second, a mutant screening library aimed at improvement of ROL catalytic performance by virtual saturation mutagenesis of residues in the catalytic region was constructed based on Rosetta's Cartesian_ddg protocol. A double mutant, E265V/S267W (with an E-to-V change at residue 265 and an S-to-W change at residue 267), with markedly improved catalytic activity toward diverse chain-length fatty acid esters was identified. Then, computational design of disulfide bonds was conducted for the noncatalytic amino acids of E265V/S267W, and two potential disulfide bonds, S61C-S115C and E190C-E238C, were identified as candidates. Experimental data validated that the variant E265V/S267W/S61C-S115C/E190C-E238C had superior stability, with an increase of 8.5°C in the melting temperature and a half-life of 31.7 min at 60°C, 4.2-fold longer than that of the wild-type enzyme. Moreover, the variant improved the lipase activity toward five 4-nitrophenyl esters by 1.5 to 3.8 times, exhibiting a potential to modify the catalytic efficiency. IMPORTANCE Rhizopus oryzae lipase (ROL) is very attractive in biotechnology and industry as a safe and environmentally friendly biocatalyst. Functional expression of ROL in Escherichia coli facilitates effective high-throughput screening for positive variants. This work highlights a method to improve both selectivity and thermostability based on a combination of virtual saturation mutagenesis in the substrate pocket and disulfide bond prediction in the noncatalytic region. Using the method, ROL thermostability and activity to diverse 4-nitrophenyl esters could be substantially improved. The strategy of rational introduction of multiple mutations in different functional domains of the enzyme is a great prospect in the modification of biocatalysts.

Nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry metabolomics studies on non-organic soybeans versus organic soybeans (Glycine max), and their fermentation by Rhizopus oligosporus.

BACKGROUND: Soybeans (Glycine max) are high in proteins and isoflavones, which offer many health benefits. It has been suggested that the fermentation process enhances the nutrients in the soybeans. Organic foods are perceived as better than non-organic foods in terms of health benefits, yet little is known about the difference in the phytochemical content that distinguishes the quality of organic soybeans from non-organic soybeans. This study investigated the chemical profiles of non-organic (G, T, U, UB) and organic (C, COF, A, R, B, Z) soybeans (G. max [L.] Merr.) and their metabolite changes after fermentation with Rhizopus oligosporus. RESULTS: A clear separation was only observed between non-organic G and organic Z, which were then selected for further investigation in the fermentation of soybeans (GF and ZF). All four groups (G, Z, GF, ZF) were analyzed using nuclear magnetic resonance (NMR) spectroscopy along with liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this way a total of 41 and 47 metabolites were identified respectively, with 12 in common. A clear variation (|log1.5 FC| > 2 and P < 0.05) was observed between Z and ZF: most of the sugars and isoflavone glycosides were found only in Z, while more amino acids and organic acids were found in ZF. An additional four metabolites clustered as C-glycosylflavonoids were discovered from MS/MS-based molecular networking. CONCLUSION: Chemical profiles of non-organic and organic soybeans exhibited no significant difference. However, the metabolite profile of the unfermented soybeans, which were higher in sugars, shifted to higher amino acid and organic acid content after fermentation, thereby potentially enhancing their nutritional value. © 2022 Society of Chemical Industry.

Purification, Characterization, and Application of Endoglucanase from Rhizopus oryzae as Antibiofilm Agent.

The enzyme endoglucanase is responsible for the depolymerization of cellulose. This study focuses on characterization and purification of endoglucanase from Rhizopus oryzae MTCC 9642 through a simple size exclusion method and its effective application as an antibiofilm agent. Extracellular ß-1,4-endoglucanase, an enzyme that catalyzes the hydrolysis of carboxymethyl cellulose, was found to be synthesized by Rhizopus oryzae MTCC 9642. The enzyme was purified up to homogeneity simply by size exclusion process through ultrafiltration and gel chromatography. The molecular weight of purified enzyme protein was estimated to be 39.8 kDa and it showed the highest substrate affinity towards carboxymethyl-cellulose with Km and Vmax values of 0.833 mg ml-1 and of 0.33 mmol glucose min-1 mg-1protein, respectively. The purified enzyme exhibited optimal activity at pH 6 with a broad stability range of pH 3-8. The most preferred temperature was 35 °C and 50% of activity could be retained after the thermal exposure at 40 °C for 25 min. The purified enzyme protein was inactivated by Cu2+, while the activity could be enhanced by the addition of exogenous thiols. Since biofilm is a challenge for health sector, with the aim of eradicating the biofilm, the purified endoglucanase was used to remove biofilm produced by two nosocomial bacteria. As predicted by in silico molecular docking interaction, the purified enzyme could effectively degrade biofilm architecture of bacterial strains S. aureus and P. aeruginosa by 76.52 ± 6.52% and 61.67 ± 8.76%, respectively. The properties of purified enzyme protein, as elucidated by in vitro and in silico characterization, may be favourable for its commercial applications as a potent antibiofilm agent.

A Comprehensive in vitro and in silico Assessment on Inhibition of CYP51B and Ergosterol Biosynthesis by Eugenol in Rhizopus oryzae.

Mucormycosis, also known as Zygomycosis, is a disease caused by invasive fungi, predominantly Rhizopus species belonging to the Order of Mucorales. Seeing from the chemistry perspective, heterocyclic compounds with an "azole" moiety are widely employed as antifungal agent for minimising the effect of mucormycosis as a prescribed treatment. These azoles serve as non-competitive inhibitors of fungal CYP51B by predominantly binding to its heme moiety, rendering its inhibition. However, long-term usage and abuse of azoles as antifungal medicines has resulted in drug resistance among certain fungal pathogens. Hence, there is an unmet need to find alternative therapeutic compounds. In present study, we used various in vitro tests to investigate the antifungal activity of eugenol against R. oryzae/R. arrhizus, including ergosterol quantification to test inhibition of ergosterol production mediated antifungal action. The minimum inhibitory concentration (MIC) value obtained for eugenol was 512 µg/ml with reduced ergosterol concentration of 77.11 ± 3.25% at MIC/2 concentration. Further, the molecular interactions of eugenol with fungal CYP51B were meticulously studied making use of proteomics in silico study including molecular docking and molecular dynamics simulations that showed eugenol to be strongly interacting with heme in an identical fashion to that shown by azole drugs (in this case, clotrimazole was evaluated). This is the first of a kind study showing the simulation study of eugenol with CYP51B of fungi. This inhibition results in ergosterol synthesis and is also studied and compared with keeping clotrimazole as a reference.

Production of Primary Metabolites by Rhizopus stolonifer, Causal Agent of Almond Hull Rot Disease.

Species in the fungal genus Rhizopus are able to convert simple sugars into primary metabolites such as fumaric acid, lactic acid, citric acid, and, to a lesser extent, malic acid in the presence of specific carbon and nitrogen sources. This ability has been linked to plant pathogenicity. Rhizopus stolonifer causes hull rot disease in almonds, symptoms of which have been previously associated with the fungus's production of fumaric acid. Six isolates of R. stolonifer taken from infected almond hulls were grown in artificial media amended with one of four carbon sources (glucose, fructose, sucrose, and xylose) and two nitrogen sources (asparagine and ammonium sulphate) chosen based on almond hull composition and used in industry. Proton nuclear magnetic resonance (1H NMR)-based metabolomics identified that R. stolonifer could metabolise glucose, fructose, sucrose, and to a lesser extent xylose, and both nitrogen sources, to produce three metabolites, i.e., fumaric acid, lactic acid, and ethanol, under in vitro conditions. Sugar metabolisation and acid production were significantly influenced by sugar source and isolates, with five isolates depleting glucose most rapidly, followed by fructose, sucrose, and then xylose. The maximum amounts of metabolites were produced when glucose was the carbon source, with fumaric acid produced in higher amounts than lactic acid. Isolate 19A-0069, however, preferred sucrose as the carbon source, and Isolate 19A-0030 produced higher amounts of lactic acid than fumaric acid. This is the first report, to our knowledge, of R. stolonifer producing lactic acid in preference to fumaric acid. Additionally, R. stolonifer isolate 19-0030 was inoculated into Nonpareil almond fruit on trees grown under high- and low-nitrogen and water treatments, and hull compositions of infected and uninfected fruit were analysed using 1H NMR-based metabolomics. Glucose and asparagine content of uninfected hulls was influenced by the nitrogen and water treatments provided to the trees, being higher in the high-nitrogen and water treatments. In infected hulls, glucose and fructose were significantly reduced but not sucrose or xylose. Large amounts of both fumaric and lactic acid were produced, particularly under high-nitrogen treatments. Moreover, almond shoots placed in dilute solutions of fumaric acid or lactic acid developed leaf symptoms very similar to the 'strike' symptoms seen in hull rot disease in the field, suggesting both acids are involved in causing disease.

Rhizopus oryzae fermentation wastewater nutrient removal coupling protein fodder production by employing Chlorella pyrenoidosa.

The scenario was to investigate feasibilities of employing Chlorella pyrenoidosa for Rhizopus oryzae fermentation wastewater nutrient removal coupling protein fodder production. Results stated that TN, TP, NH3-N, COD, BOD removal reached 99.79%, 94.70%, 98.80%, 97.60%, 99.60% to acquire discharge permit under fed-batch manipulation, whilst the peaked protein yield (19.94 g/L) was 6.04-fold more than batch manipulation. Rhizopus oryzae fermentation wastewater feeding C. pyrenoidosa was praised as high-quality protein not only with 26.78% essential amino acids and essential amino acids/nonessential amino acids value of 0.84 but also pathogenic bacteria and heavy metal loads complying with fodder standards. In vitro digestibility of dry matter, protein, lipid, and starch achieving 80.07%, 92.13%, 95.93%, 91.9% and bioavailability of polypeptides, triglycerides, free fatty acids, and oligosaccharides displaying 98.67%, 87.12%, 93.86%, 30.21%, which were roughly-equivalent to corn/soybean fodder. The findings formed exemplifications in utilizing other microalgal systems for wastewater nutrient removal coupling chemicals production.

Energy metabolism as the target of 3-phenyllactic acid against Rhizopus oryzae.

3-phenyllactic acid (PLA) has broad anti-fungal activity, however, target sites of PLA on fungal cells and its anti-fungal mechanism of action have been poorly studied. In this study, we explored the inhibition mechanism of Rhizopus oryzae (R. oryzae) on rotten lily bulbs by PLA. The minimum inhibitory concentration value of PLA against R. oryzae was 8 mg/mL. We observed the ultrastructure of R. oryzae by scanning electron microscopy and transmission electron microscopy which indicated that PLA did not damage the cell membrane, but destroyed the mitochondria and other organelles. Tandem mass tag proteomes showed that PLA significantly down-regulated (P < 0.05) the expression of hexokinase (HK), phosphofructokinase (PFK), a-ketoglutarate dehydrogenase (a-KGDH), adenylate kinase (ADK1), Cytochrome C oxidase and NADH dehydrogenase, and up-regulated (P < 0.05) the expression of mitochondrial ADP/ATP carrier proteins (AAC) and subunit IV (CCIO IV) in glycolysis, tricarboxylic acid cycle or oxidative phosphorylation metabolism. Following these findings, down-regulated HK and a-KGDH activity of aforementioned pathways was shown by enzyme activity assay, and regulated gene expression of ADK1, AAC, CCIO IV and NADH dehydrogenase was further confirmed by real-time quantitative PCR. Central carbon metabolomics showed that citric acid, cis-Aconitic acid, isocitric acid, alpha-Ketoglutaric acid, succinate, fumarate and malic acid of the tricarboxylic acid cycle metabolites were significantly down-regulated (P < 0.05), and ATP production by oxidative phosphorylation was also significantly reduced (P = 0.02), resulting in insufficient energy production. Thus, ROS levels increased by 141% of the control values and cytochrome C was released, resulting in gradual cell apoptosis. All data indicated that energy metabolism was the target of PLA against R. oryzae. This was the first study to show that energy metabolism could be the target of PLA against R. oryzae, which could provide a theoretical basis to study the mechanism of fungal inhibition.

Improved synthesis of dietary triglycerides by using lipase supported on clay carriers.

BACKGROUND: In the search for healthier and more functional foods, dietary triglycerides (TAGs) have played a prominent role in the food industry. The objective of this work was to evaluate new clay supports to immobilize lipase from Rhizopus oryzae and use it in the synthesis of TAGs. MAIN METHODS AND MAJOR RESULTS: The immobilization of lipase by physical adsorption was carried out, determining the hydrolytic activity, esterification, immobilization yield, thermal stability, and kinetic and thermodynamic parameters. Afterwards, acidolysis was carried out and the product characterized as to composition, acidity index (AI), peroxide index (PI), and analysis of the sn-2 position. The mean hydrolytic activity of free lipase was 23,568.43 ± 559.20 U g-1 and for lipase immobilized in acidified kaolin and diatomite, 1409.04 ± 51.07 and 1235.67 ± 9.56 U g-1 ; immobilization yield of 23.78% and 20.21% and esterification activity of 1827.99 and 1722.66 U g-1 , respectively, maintaining more than 60% of the relative activity after 8 h of incubation at different temperatures. The reaction using the derivative immobilized in acidified diatomite resulted in the degree of incorporation (DI) 41.53% ± 3.99%. CONCLUSIONS AND IMPLICATIONS: From the results, the potential of diatomite and kaolin as support in the immobilization of lipase for the synthesis of dietary TAGs was verified.

Propeptide in Rhizopus chinensis Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design.

Most fungal lipases contain a propeptide, which is very important for their function and substrate selectivity. In the present study, Rhizopus chinensis lipase (RCL) was used as a research model to explore the mechanism of the propeptide of the lipase. Conventional molecular dynamics (MD) and metadynamics simulations were used to explore the mechanism by which the propeptide affects the activity of the lipase, which was subsequently verified by mutation experiments. MD simulations indicated that the propeptide had an inhibitory effect on the lid movement of RCL and found a key region (Val5-Thr10) on the propeptide. Subsequently, site-directed mutations were created in this region. The mutations enhanced the lipase catalytic efficiency to 700% and showed the potential for the propeptide to shift the substrate specificity of RCL. The specificity and activity of RCL mutants also had similar trends to wild-type RCL toward triglycerides with varying chain lengths. The mutual corroboration of simulation and site-directed mutagenesis results revealed the vital role of the key propeptide region in the catalytic activity and substrate specificity of the lipase.

Investigation of presence of endofungal bacteria in Rhizopus spp. isolated from the different food samples.

Rhizopus species are opportunistic pathogens and cause infections which lead to deaths in individuals with the weakened immune system. Some strains of Rhizopus species have been detected to have a symbiotic relationship with bacteria. The toxicity of the Rhizopus species is important. Because strains harbouring endofungal bacteria are able to produce secondary metabolites and if endofungal bacteria are released from mycelium, serious problems can occur. We aimed to investigate the presence of endofungal bacteria in Rhizopus species isolated from food samples. Rhizopus species were isolated from different food samples. The presence of endofungal bacteria in the Rhizopus isolates was investigated. Rhizopus strains containing the endofungal bacteria were identified through phenotypic and genotypic methods. Universal primers amplifying bacterial 16S rRNA region were used to amplify 1.2-1.5-kb fragment from fungal metagenomic DNA. Sequence analysis of PCR products amplified from fungal metagenomic DNA was made. Fluorescence microscopy and scanning electron microscopy were used to visualize the presence of endofungal bacteria in fungal hyphae. According to our results, the Rhizopus strains is associated with Serratia marcescens, Pseudomonas fluorescens and Klebsiella pneumoniae. Until now there is no evidence that Pseudomonas fluorescens and Klebsiella pneumoniae were identified as endofungal. These species are opportunistic pathogen dangerous for humans. It is important for humans not only the presence of the fungi but also the presence of the endofungal bacteria in foods. Our work is important because it draws attention to the presence of endofungal bacteria in foods. Because there is danger releasing of a bacterium from the mycelium, it is likely to face sepsis or serious problems.

A metabolomics approach to evaluate post-fermentation enhancement of daidzein and genistein in a green okara extract.

BACKGROUND: Okara is a major agri-industrial by-product of the tofu and soymilk industries. Employing food-wastes as substrates for the green production of natural functional compounds is a recent trend that addresses the dual concepts of sustainable production and a zero-waste ecosystem. RESULTS: Extracts of unfermented okara and okara fermented with Rhizopus oligosporus were obtained using ethanol as extraction solvent, coupled with ultrasound sonication for enhanced extraction. Fermented extracts yielded significantly better results for total phenolic content (TPC) and total flavonoid content (TFC) than unfermented extracts. A qualitative liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) analysis revealed a shift from glucoside forms to respective aglycone forms of the detected isoflavones, post-fermentation. Since the aglycone forms have been associated with numerous health benefits, a quantitative high-performance liquid chromatography (HPLC) analysis was performed. Fermented okara extracts had daidzein and genistein concentrations of 11.782 ± 0.325 µg mL-1 and 10.125 ± 1.028 µg mL-1 , as opposed to that of 6.7 ± 2.42 µg mL-1 and 4.55 ± 0.316 µg mL-1 in raw okara extracts, respectively. Lastly, the detected isoflavones were mapped to their metabolic pathways, to understand the biochemical reactions triggered during the fermentation process. CONCLUSION: Fermented okara may be implemented as a sustainable solution for production of natural bioactive isoflavonoids genistein and daidzein. © 2021 Society of Chemical Industry.

Antifungal activity screening for mint and thyme essential oils against Rhizopus stolonifer and their application in postharvest preservation of strawberry and peach fruits.

AIMS: With a goal to identify specific essential oils that can control postharvest Rhizopus rot on strawberry and peach fruits, we performed screening for 26 essential oils based on their antifungal activity against Rhizopus stolonifer in vitro and investigated the underlying mechanism. METHODS AND RESULTS: Mentha spicata (Ms), Mentha piperita (Mp), Thymus vulgaris CT carvacrol (Tc) and Thymus vulgaris CT thymol (Tt) exhibited strong inhibition on R. stolonifer growth in the screening. These essential oils increased plasma membrane permeability of R. stolonifer and resulted in the outflow of intercellular electrolyte, nucleic acid, protein and soluble sugar. Morphology of R. stolonifer mycelia was greatly altered by these essential oils. Hyphae treated by these essential oils exhibited high accumulation of superoxide anion and malonaldehyde. Combination of these essential oils in commercial package reduced Rhizopus rot on strawberry and peach fruits, with Mp showing the strongest efficiency. CONCLUSION: Ms, Mp, Tc and Tt essential oils inhibited R. stolonifer growth by targeting its plasma membrane and reduced Rhizopus rot on strawberry and peach fruits. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides potential applications of natural plant extract, as alternatives to tradition fungicides, to control postharvest decay on fruits and vegetables.

Fermentation with Edible Rhizopus Strains to Enhance the Bioactive Potential of Hull-Less Pumpkin Oil Cake.

Solid-state fermentation with food-grade fungal strains can be applied to enhance the bioactive parameters of agro-industrial by-products. Tempe-type fermentation can be adapted to various substrates, but the key factor is the appropriate strain selection. The aim of this study was to compare the potential of Rhizopus strains for obtaining products of improved antioxidant activity from pumpkin oil cake. For this purpose, substances reacting with the Folin-Ciocalteu reagent, with free radical scavenging potential, as well as reducing power were assessed. The effect of the fermentation on the phytate level and inositol phosphate profile in the material was also monitored. The fermentation resulted in the significant enhancement of the antioxidant potential of pumpkin oil cake in the case of all the strains tested, but the most efficient one was R. oligosporus ATCC 64063. During the course of fermentation, the level of phytate in the material decreased (the highest reduction rate was observed in the oil cake fermented with R. oryzae CBS 372.63), while peptides and fungal glucosamine were accumulated. Tempe-type fermentation can be considered as an alternative way of improving the bioactive parameters of pumpkin oil cake and, thanks to the various activities of different Rhizopus strains, it is possible to obtain products of desired parameters.

Fungal mediated biotransformation of melengestrol acetate, and T-cell proliferation inhibitory activity of biotransformed compounds.

Glomerella fusaroide, and Rhizopus stolonifer were effectively able to transform the steroidal hormone melengestrol acetate (MGA) (1) into four (4) new metabolites, 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (2), 17α-acetoxy-11α-hydroxy-6-methyl-16-methylenepregna-1,4,6-triene-3,20-dione (3), 17α-acetoxy-6,7α-epoxy-6ß-methyl-16-methylenepregna-4,6-diene-3,20-dione (4), and 17α-acetoxy-11ß,15ß-dihydroxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione (5). All these compounds were structurally characterized by different spectroscopic techniques. The objective of the current study was to assess the anti-inflammatory potential of melengestrol acetate (1), and its metabolites 2-5. The metabolites and the substrate were assessed for their inhibitory effects on proliferation of T-cells in vitro. The substrate (IC50 = 2.77 ± 0.08 µM) and its metabolites 2 (IC50 = 2.78 ± 0.07 µM), 4 (IC50 = 2.74 ± 0.1 µM), and 5 (IC50 = < 2 µM) exhibited potent T- cell proliferation inhibitory activities, while compound 3 (IC50 = 29.9 ± 0.09 µM) showed a moderate activity in comparison to the standard prednisolone (IC50 = 9.73 ± 0.08 µM). All the metabolites were found to be non-toxic against 3T3 normal cell line. This study thus identifies some potent compounds active against T-cell proliferation. Their anti-inflammatory potential, therefore, deserves to be further investigated.

Rhizopus oligosporus and Lactobacillus plantarum Co-Fermentation as a Tool for Increasing the Antioxidant Potential of Grass Pea and Flaxseed Oil-Cake Tempe.

Tempe-type fermentation originating from Indonesia can enhance the antioxidant activity of plant material. However, this biological potential depends on substrates and applied microorganisms. This study aimed to determine whether co-fermentation with Rhizopus oligosporus and Lactobacillus plantarum improved antioxidant activity of tempe obtained from grass pea seeds with flaxseed oil-cake addition (up to 30%). For this purpose, substances reacting with Folin-Ciocalteu reagent and free radicals scavenging potential were measured in water-soluble fractions and dialysates from simulated in vitro digestion. Additionally, the water-soluble phenolic profile was estimated. The higher level of water-extractable compounds with antioxidant activity was determined in co-fermentation products than in fungal fermentation products. Moreover, the fermentation process with the use of L. plantarum contributed to a greater accumulation of some phenolic acids (gallic acid, protocatechuic acid) in tempe without having a negative effect on the levels of other phenolic compounds determined in fungal fermented tempe. During in vitro digestion simulating the human digestive tract, more antioxidant compounds were released from products obtained after co-fermentation than fungal fermentation. An addition of 20% flaxseed oil-cake and the application of bacterial-fungal co-fermentation, can be considered as an alternative tool to enhance the antioxidant parameters of grass pea tempe.

Molecular Dialogues between Early Divergent Fungi and Bacteria in an Antagonism versus a Mutualism.

Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.IMPORTANCE Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter.

A TAL effector-like protein of an endofungal bacterium increases the stress tolerance and alters the transcriptome of the host.

Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica-R. microsporus symbiosis.

Semi-pilot scale production of biodiesel from waste frying oil by genetically improved fungal lipases.

This paper addresses the issue of combining the usage of waste frying oil (WFO), as a feedstock, and a lipase produced in solid-state fermentation (SSF), as a biocatalyst, for semi-pilot scale production of biodiesel as fatty acid methyl esters (FAME). Two fungal mutants namely; Rhizopus stolonifer 1aNRC11 mutant F (1F) and Aspergillus tamarii NDA03a mutant G (3G) were used as a cocatalyst. The two mutants were cultivated separately by SSF in a tray bioreactor. The dried fermented solid of 1F and 3G mutants were used in a ratio of 3:1, respectively, for WFO transesterification. Optimization of several semi-pilot process stages including SSF and WFO transesterification reaction conditions resulted in 92.3% conversion of WFO to FAME. This FAME yield was obtained after 48 h using 10% cocatalyst (w/w of WFO), 10% water (w/w of WFO) and 3:1 methanol/ WFO molar ratio at 30 °C and 250 rpm. A preliminary economic evaluation of produced biodiesel price (190 $/Ton) is less than half the price of petroleum diesel in Egypt (401$/Ton) and is about 40.3% the price of biodiesel produced using a pure enzyme, which is a promising result. This strategy makes the biodiesel synthesis process greener, economical and sustainable.