Development of a saprophytic fungal inoculum for the biodegradation of sub-bituminous coal

The rhizospheres of the weeds Ageratum conyzoides, Axonopus compressus, Emilia coccinea, Synedrella nodiflora, Urena lobata and Sida acuta from a sub-bituminous coal mining site and a control site, without coal discards, were screened for new fungi with ability to degrade sub-bituminous coal in the laboratory. The isolates were identified by cultural and molecular methods. Seventeen out of the sixty-one fungal isolates tested could utilize sub-bituminous coal as an energy source. Upon further evaluation, only seven of these were promising candidates for coal biodegradation, and they were assayed for their biosolubilization and depolymerization activities to determine their mechanisms of coal biodegradation. Based on the accumulation of humic acid (HA), which is the marker for biosolubilization, Mucor circinelloides and Aspergillus tubingensis were the most active. On the other hand, Cunninghamella bertholletiae, Simplicillium subtropicum, Penicillium daleae and Trichoderma koningiopsis were the highest producers of fulvic acid (FA), the indicator of depolymerization. Purpureocillium lilacinum produced the lowest yields of both HA and FA compared to the other six coal-degrading candidates. The presence of laccase in Trichoderma koningiopsis, Penicillium daleae and Simplicillium subtropicum suggests a role for this enzyme in the enhancement of the coal biodegradation process. However, the inability to amplify the laccase gene in Cunninghamella bertholletiae indicates that another enzyme probably aids its coal bioconversion. The current investigation highlights the potentials of these strains in harnessing biotechnological processes of sub-bituminous coal conversion into value-added products, which could be extended to the bioremediation of coal-polluted soils. The fungi with the highest coal bioconversion capabilities belonged to Ascomycota and Zygomycota and were found in the rhizospheres of the weeds Emilia coccinea, Ageratum conyzoides and Axonopus compressus.

Duplication and overexpression of the genes encoding a beta 1,3-glucan synthase confer the intrinsic resistance to echinocandin in Mucor circinelloides

Procedures such as solid organ transplants and cancer treatments can leave many patients in an immunocompromised state resulting in an increased susceptibility to opportunistic diseases including fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Currently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling SARS-CoV-2 infections. Mucorales fungi are notorious for presenting resistance to most antifungal drugs. The absence of effective means to treat these infections results in mortality rates approaching 100% in cases of disseminated infection. One of the most effective antifungal drug classes currently available are echinocandins. Echinocandins seem to be efficacious in treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have no to little effect on Mucorales. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding for the echinocandin target protein β-(1,3)-D-glucan synthase (fksA, fksB, and fksC). Interestingly, we revealed that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes when compared to an untreated control. We further uncovered that the serine/threonine phosphatase calcineurin is responsible for the overexpression of fksA and fksB as deletion of calcineurin results in a decrease in expression of all three fks genes and a lower minimal inhibitory concentration (MIC) to micafungin. Taken together, this study demonstrates that the fks gene duplication and overexpression by calcineurin contribute to the intrinsic resistance to echinocandins in Mucor.

Antimicrobial Activities and Molecular Signature of Endophytic Fungi of Opuntia ficus-Indica Cacti and the Cactus-Like Plant Aloe vera

Endophytes are fungi that colonize the internal tissues of plants without causing immediate adverse effects. Saudi Arabia (SA) is rich in Opuntia ficus-indica cacti and the cactus-like plant Aloe vera, which grow in the southern and western areas of SA. This study aimed to isolate and identify endophytic fungi from cacti and cactus-like plants in the Jeddah, Taif, and Al Baha regions KSA and then determine their effects on pathogenic fungal and bacterial growth. The isolates were groupedAloe Vera; Opuntia Ficus-Indica; Endophytic Fungi; Antimicrobial Activities; Pathogenic Bacteria. into 16 distinct operational taxonomic units based on the sequence of the internal transcribed spacer in the rDNA gene with the primers ITS1 and ITS4. Mucor circinelloides was the endophytic fungus found most frequently, with a relative frequency of 20.43%, followed by Talaromyces funiculosus, with a relative frequency of 16.12% when isolated from Opuntia ficus-indica and Aloe vera. Nine out of sixteen endophytic fungi exhibited strong antifungal activity against all the tested pathogens. P. funiculosum, Aspergillus versicolor, Penicillium janthinellum, and Fusarium oxysporum showed vigorous antimicrobial activities against the human pathogenic bacteria Escherichia coli, Shigella sp., and Salmonella typhimurium.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of −4.87 kcal/mol to citric acid, while −3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Submerged Fermentation of Animal Fat By-Products by Oleaginous Filamentous Fungi for the Production of Unsaturated Single Cell Oil

Animal waste fats were explored as a fermentation substrate for the production of high-value unsaturated single cell oil (SCO) using oleaginous fungi, Mucor circinelloides and Mortierella alpina. Both strains showed good growth and lipid accumulation when using animal fat as a single carbon source. The biomass concentration of 16.7 ± 2.2 gDCW/L and lipid content of 54.1%wt (of dry cell weight) were obtained for Mucor circinelloides in shake flask experiments, surpassing the biomass yield achieved in batch and fed-batch fermentation. In contrast, Mortierella alpina gave the highest biomass concentration (8.3 ± 0.3 gDCW/L) and lipid content (55.8%wt) in fed-batch fermentation. Fat grown Mortierella alpina was able to produce arachidonic acid (ARA), and the highest ARA content of 23.8%wt (of total lipid weight) was in fed-batch fermentation. Gamma-linolenic acid (GLA) was produced by both fungal strains. At the end of fed-batch fermentation, the GLA yields obtained for Mucor circinelloides and Mortierella alpina were 4.51%wt and 2.77%wt (of total lipid weight), respectively. This study demonstrates the production of unsaturated SCO-rich fungal biomass from animal fat by fermentation.

Overexpression of Shinorhizobium Meliloti Flavohemoglobinefficiently Improved Cell Growth and Fatty Acid Biosynthesis in Oleaginous Fungus Mucor Circinelloides

Oxygen availability is a limiting factor for lipid biosynthesis in eukaryotic microorganisms. Two bacterial hemoglobins from Vitreoscilla sp. (VHb) and Shinorhizobium meliloti (SHb), which could deliver the oxygen to the respiratory chain to produce more ATP, were introduced into Mucor circinelloides to alleviate oxygen limitation, thereby improving cell growth and fatty acid production. VHb and SHb genes were integrated into the M. circinelloides MU402 genome through homologous recombination, and their protein expression was verified by carbon monoxide difference spectrum (CO-difference spectrum)analysis. SHb-expressing strain showed higher biomass than VHb-expressing strain. The biomass of the SHb-expressing strain was increased by about 50% and the total fatty acid (TFA) content was as high as 15.7% of the dry cell weight which was about 40% higher than that of the control strain in flask conditions. In the fermenter, the maximum biomass and TFA content was obtained in SHb-expressing strains, with the biomass being 12.1 g/L and the TFA being 21.1% of the dry cell weight. VHb and SHb expression also affected the fatty acid composition with the proportion of polyunsaturated fatty acids being increased. Over-expression of bacterial hemoglobins, especially SHb increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb is better than VHb in improving the fatty acid production in oleaginous microorganisms.

Heterologous Expression of Two Malate Transporters From an Oleaginous Fungus Mucor circinelloides Improved the Lipid Accumulation in Mucor lusitanicus

The fungus, Mucor lusitanicus, is of great interest for microbial lipids, because of its ability to accumulate intracellular lipid using various carbon sources. The biosynthesis of fatty acid requires the reducing power NADPH, and acetyl-CoA, which is produced by the cleavage of citrate in cytosol. In this study, we employed different strategies to increase lipid accumulation in the low lipid-producing fungi via metabolic engineering technology. Hence, we constructed the engineered strain of M. lusitanicus CBS 277.49 by using malate transporter (mt) and 2-oxoglutarate: malate antiporter (sodit) from M. circinelloides WJ11. In comparison with the control strain, the lipid content of the overexpressed strains of mt and sodit genes were increased by 24.6 and 33.8%, respectively. These results showed that mt and sodit can affect the distribution of malate in mitochondria and cytosol, provide the substrates for the synthesis of citrate in the mitochondria, and accelerate the transfer of citrate from mitochondria to cytosol, which could play a significant regulatory role in fatty acid synthesis leading to lipids over accumulation.

Two Fungal Flavonoid-specific Glucosidases/rutinosidases for Rutin Hydrolysis and Rutinoside Synthesis Under Homogeneous and Heterogeneous Reaction Conditions

The glycosidases within GH5-23 cleave the glycosidic bond of β-glucosylated or rutinosylated flavonoids. Moreover, by virtue of their transglycosylation activity, glycoconjugates with glucosyl and rutinosyl moieties are accessible. Here we report the biochemical characterization and biotechnological assessment of two heterologously expressed members of GH5-23 – McGlc from Mucor circinelloides and PcGlc from Penicillium chrysogenum. Both enzymes exhibited the highest hydrolytic activities with quercetin-3-β-O-glucopyranoside, whereas lower specificity constants were determined with the rutinosides narcissin, rutin and hesperidin. High stabilities against thermal, ethanol and dimethyl sulfoxide-induced inactivation, a very limited secondary hydrolysis of the formed transglycosylation products, and no detectable product inhibition were additional features appropriate for biotechnological applications. The enzymes were compared in their efficiencies to hydrolyze rutin and to synthesize 2‑phenylethyl rutinoside under homogeneous and heterogeneous reaction conditions using high rutin concentrations of 100 and 300 mM. Highest transglycosylation efficiencies were achieved with fully dissolved rutin in reaction mixtures containing 25 % dimethyl sulfoxide. Molecular docking and multiple sequence alignments suggest that the hydrophobic environment of aromatic residues within the +1 subsite of GH5-23 glycosidases is very important for the binding of flavonoid glucosides and rutinosides.

Two fungal flavonoid-specific glucosidases/rutinosidases for rutin hydrolysis and rutinoside synthesis under homogeneous and heterogeneous reaction conditions

The glycosidases within GH5-23 cleave the glycosidic bond of β-glucosylated or rutinosylated flavonoids. Moreover, by virtue of their transglycosylation activity, glycoconjugates with glucosyl and rutinosyl moieties are accessible. Here we report the biochemical characterization and biotechnological assessment of two heterologously expressed members of GH5-23—McGlc from Mucor circinelloides and PcGlc from Penicillium chrysogenum. Both enzymes exhibited the highest hydrolytic activities with quercetin-3-β-O-glucopyranoside, whereas lower specificity constants were determined with the rutinosides narcissin, rutin and hesperidin. High stabilities against thermal, ethanol and dimethyl sulfoxide-induced inactivation, a very limited secondary hydrolysis of the formed transglycosylation products, and no detectable product inhibition were additional features appropriate for biotechnological applications. The enzymes were compared in their efficiencies to hydrolyze rutin and to synthesize 2-phenylethyl rutinoside under homogeneous and heterogeneous reaction conditions using high rutin concentrations of 100 and 300 mM. Highest transglycosylation efficiencies were achieved with fully dissolved rutin in reaction mixtures containing 25% dimethyl sulfoxide. Molecular docking and multiple sequence alignments suggest that the hydrophobic environment of aromatic residues within the + 1 subsite of GH5-23 glycosidases is very important for the binding of flavonoid glucosides and rutinosides.