Mucor circinelloides: a model organism for oleaginous fungi and its potential applications in bioactive lipid production.

Microbial oils have gained massive attention because of their significant role in industrial applications. Currently plants and animals are the chief sources of medically and nutritionally important fatty acids. However, the ever-increasing global demand for polyunsaturated fatty acids (PUFAs) cannot be met by the existing sources. Therefore microbes, especially fungi, represent an important alternative source of microbial oils being investigated. Mucor circinelloides-an oleaginous filamentous fungus, came to the forefront because of its high efficiency in synthesizing and accumulating lipids, like γ-linolenic acid (GLA) in high quantity. Recently, mycelium of M. circinelloides has acquired substantial attraction towards it as it has been suggested as a convenient raw material source for the generation of biodiesel via lipid transformation. Although M. circinelloides accumulates lipids naturally, metabolic engineering is found to be important for substantial increase in their yields. Both modifications of existing pathways and re-formation of biosynthetic pathways in M. circinelloides have shown the potential to improve lipid levels. In this review, recent advances in various important metabolic aspects of M. circinelloides have been discussed. Furthermore, the potential applications of M. circinelloides in the fields of antioxidants, nutraceuticals, bioremediation, ethanol production, and carotenoids like beta carotene and astaxanthin having significant nutritional value are also deliberated.

Antibacterial and cytotoxic potency of thermophilic Streptomyces werraensis MI-S.24-3 isolated from an Egyptian extreme environment.

The need for novel and active antibiotics specially from actinomycetes is essential due to new and drug-resistant pathogens. In this study, 87 actinomycetes were isolated, and 18 strains among them characterized as thermophilic actinomycetes. Further fractionation and preliminary antibacterial activities indicated that one strain, coded as MI-S.24-3, showed good antibacterial activity. Based on the phenotypic, genomic, phylogenetic, and biochemical analyses, MI-S.24-3 was identified as Streptomyces werraensis. Results demonstrated that the ethyl acetate active fraction showed maximum antibacterial activity against Staphylococcus aureus and Escherichia coli with MIC (12.7 ± 0.1 and 18.3 ± 0.2 mg/mL), and MBC (96.5 ± 1.4 and 91.5 ± 0.7 mg/mL), respectively, with determination of time kill kinetics assay. The active fraction showed moderate-to-weak cytotoxic effects against human lung carcinoma (A549 cells), breast cancer cell line (MCF-7), and human cervical carcinoma (HELA cells) with a IC50 of (23.8 ± 1.2, 54 ± 1.8, 96.4 ± 3.2 µg/mL, respectively). Active components were characterised by different chemically volatile, ester, and lactone compounds, determined by GC-MS coupled with daughter ions of (GC-MS/MS). Notably, erucic acid and reynosin identified compounds are rare metabolites produced by Streptomyces werraensis. Our findings demonstrated that the MI-S.24-3 strain could be a potential source for active compounds of biomedical and pharmaceutical interest.

Biodegradation of poly (vinyl alcohol) by an orychophragmus rhizosphere-associated fungus Penicillium brevicompactum OVR-5, and its proposed PVA biodegradation pathway.

In recent years, the utilisation of endophytes has emerged as a promising biological treatment technology for the degradation of plastic wastes such as biodegradation of synthetic plastics. This study, therefore, aimed to explore and extensively screen endophytic fungi (from selected plants) for efficient in vitro polyvinyl alcohol (PVA) biodegradation. In total, 76 endophytic fungi were isolated and cultivated on a PVA screening agar medium. Among these fungi, 10 isolates showed potential and were subsequently identified based on phenotypical characteristics, ITS ribosomal gene sequences, and phylogenetic analyses. Four strains exhibited a maximum level of PVA-degradation in the liquid medium when cultivated for 10 days at 28 °C and 150 rpm. These strains showed varied PVA removal rates of 81% (Penicillium brevicompactum OVR-5), 67% (Talaromyces verruculosus PRL-2), 52% (P. polonicum BJL-9), and 41% (Aspergillus tubingensis BJR-6) respectively. The most promising PVA biodegradation isolate 'OVR-5', with an optimal pH at 7.0 and optimal temperature at 30 °C, produced lipase, manganese peroxidase, and laccase enzymes. Based on analyses of its metabolic intermediates, as identified with GC-MS, we proposed the potential PVA degradation pathway of OVR-5. Biodegradation results were confirmed through scanning electron microscopy and Fourier transform infrared spectroscopy. This study provides the first report on an endophytic P. brevicompactum strain (associated with Orychophragmus violaceus) that has a great ability for PVA degradation providing more insight on potential fungus-based applications in plastic waste degradation.

Bovine mastitis: An appraisal of its alternative herbal cure.

Bovine mastitis is globally recognized as the most common and costly disease affecting dairy herds. The disease causes huge financial losses to dairy industries by reduced yield and milk quality, deaths and culling of affected cows and also by associated treatment costs. The disease occurs due to invasion of the mammary glands by pathogenic bacteria followed by their multiplication in the milk producing tissues. The most common treatment method available against bovine mastitis is the intra-mammary infusion of antibiotics. However, their use is associated with the problem of antimicrobial resistance. This scenario has made search for alternative treatment approaches necessary. Medicinal plants with their well-established history are an excellent natural product resource used as an alternative therapy. Antibacterial agents from plants can act as important sources of novel antibiotics, efflux pump inhibitors, compounds that target bacterial virulence or can be used in combination with existing drugs. The plants form an essential component of ethno-veterinary medicine used in the treatment of different diseases like bovine mastitis. This review article attempts to provide an overview of the different medicinal plants used in the treatment of bovine mastitis. Antimicrobial studies of these plant species and some of their isolated constituents have been reviewed in detail. It highlights the logic and precedence behind mining this important natural product resource. Our own research findings in this direction and future scope of research are also discussed briefly.

Antimicrobial investigation of selected soil actinomycetes isolated from unexplored regions of Kashmir Himalayas, India.

The aim of the present study was to isolate and evaluate the antimicrobial potential of soil actinomycetes of Kashmir Himalayas. The secondary metabolites of actinomycetes are the prominent source of antibiotics. A total of 121 morphologically different actinomycete strains were isolated and screened for antimicrobial activity against various human pathogens. The ethyl acetate extract of fermented broth an actinomycete strain, identified as Streptomyces pratensis exhibited significant antimicrobial activity against Staphylococcus aureus ATCC 29213 with MIC 0.25 µg/ml and Mycobacterium tuberculosis Strain H37Rv with MIC 0.062 µg/ml. The strain S. pratensis IIIM06 was grown on large scale and their broth was extracted with ethyl acetate. The extract was subjected to various chromatography techniques which led to the isolation of four compounds whose structures were established as actinomycin C1, actinomycin C2, actinomycin C3 and actiphenol on the basis of spectral data analysis. Actinomycin C1, C2 and C3 exhibited potent antimicrobial activity against S. aureus as well as M. tuberculosis. The isolated indigenous actinomycetes exhibited good antibacterial activity and the study reveals that IIIM06 is a promising strain and could be of great potential for industrial applications.

Chemoprofile and functional diversity of fungal and bacterial endophytes and role of ecofactors - A review.

Endophytes represent a hidden world within plants. Almost all plants that are studied harbor one or more endophytes, which help their host to survive against pathogens and changing adverse environmental conditions. Fungal and bacterial endophytes with distinct ecological niches show important biological activities and ecological functions. Their unique physiological and biochemical characteristics lead to the production of niche specific secondary metabolites that may have pharmacological potential. Identification of specific secondary metabolites in adverse environment can also help us in understanding mechanisms of host tolerance against stress condition such as biological invasions, salt, drought, temperature. These metabolites include micro as well as macromolecules, which they produce through least studied yet surprising mechanisms like xenohormesis, toxin-antitoxin system, quorum sensing. Therefore, future studies should focus on unfolding all the underlying molecular mechanisms as well as the impact of physical and biochemical environment of a specific host over endophytic function and metabolite elicitation. Need of the hour is to reshape the focus of research over endophytes and scientifically drive their ecological role toward prospective pharmacological as well as eco-friendly biological applications. This may help to manage these endophytes especially from untapped ecoregions as a useful undying biological tool to meet the present challenges as well as lay a strong and logical basis for any impending challenges.

Engineering a non-model yeast Rhodotorula mucilaginosa for terpenoids synthesis.

Terpenoids have tremendous biological activities and are widely employed in food, healthcare and pharmaceutical industries. Using synthetic biology to product terpenoids from microbial cell factories presents a promising alternative route compared to conventional methods such as chemical synthesis or phytoextraction. The red yeast Rhodotorula mucilaginosa has been widely studied due to its natural production capacity of carotenoid and lipids, indicating a strong endogenous isoprene pathway with readily available metabolic intermediates. This study constructed several engineered strains of R. mucilaginosa with the aim of producing different terpenoids. Monoterpene α-terpineol was produced by expressing the α-terpineol synthase from Vitis vinifera. The titer of α-terpineol was further enhanced to 0.39 mg/L by overexpressing the endogenous rate-limiting gene of the MVA pathway. Overexpression of α-farnesene synthase from Malus domestica, in combination with MVA pathway rate-limiting gene resulted in significant increase in α-farnesene production, reaching a titer of 822 mg/L. The carotenoid degradation product ß-ionone was produced at a titer of 0.87 mg/L by expressing the ß-ionone synthase from Petunia hybrida. This study demonstrates the potential of R. mucilaginosa as a platform host for the direct biosynthesis of various terpenoids and provides insights for further development of such platforms.

Microbes: A Hidden Treasure of Polyunsaturated Fatty Acids.

Microbes have gained a lot of attention for their potential in producing polyunsaturated fatty acids (PUFAs). PUFAs are gaining scientific interest due to their important health-promoting effects on higher organisms including humans. The current sources of PUFAs (animal and plant) have associated limitations that have led to increased interest in microbial PUFAs as most reliable alternative source. The focus is on increasing the product value of existing oleaginous microbes or discovering new microbes by implementing new biotechnological strategies in order to compete with other sources. The multidisciplinary approaches, including metabolic engineering, high-throughput screening, tapping new microbial sources, genome-mining as well as co-culturing and elicitation for the production of PUFAs, have been considered and discussed in this review. The usage of agro-industrial wastes as alternative low-cost substrates in fermentation for high-value single-cell oil production has also been discussed. Multidisciplinary approaches combined with new technologies may help to uncover new microbial PUFA sources that may have nutraceutical and biotechnological importance.

Investigating the Effect of Alcohol Dehydrogenase Gene Knockout on Lipid Accumulation in Mucor circinelloides WJ11.

Mucor circinelloides is an oleaginous, dimorphic zygomycete fungus species that produces appreciable levels of ethanol when grown under aerobic conditions in the presence of high glucose, indicating the fungus is a Crabtree-positive microorganism. Engineering efforts to redirect carbon flux from ethanol to lipid biosynthesis may shed light on the critical role of ethanol biosynthesis during aerobic fermentation in M. circinelloides. Therefore, in this study, the alcohol dehydrogenase gene (ADH1) of M. circinelloides WJ11 was deleted, and its effects on growth, lipid production, and fatty acid content were analyzed. Our results showed that knocking out of adh1∆ reduced the ethanol concentration by 85-90% in fermented broth, indicating that this gene is the major source of ethanol production. Parallel to these findings, the lipid and fatty acid content of the mutant was decreased, while less change in the growth of WJ11 was observed. Furthermore, a fermentation study showed the lipid and fatty acid content was restored in the mutant strain when the fermentation media was supplemented with 0.5% external ethanol, indicating the importance of alcohol dehydrogenase and its product on growth and lipid biosynthesis in M. circinelloides. To our knowledge, this is the first study to show a link between alcohol dehydrogenase and lipid production in M. circinelloides.

Evaluation of Different Standard Amino Acids to Enhance the Biomass, Lipid, Fatty Acid, and γ-Linolenic Acid Production in Rhizomucor pusillus and Mucor circinelloides.

In this study, 18 standard amino acids were tested as a single nitrogen source on biomass, total lipid, total fatty acid (TFA) production, and yield of γ-linolenic acid (GLA) in Rhizomucor pusillus AUMC 11616.A and Mucor circinelloides AUMC 6696.A isolated from unusual habitats. Grown for 4 days at 28°C, shaking at 150 rpm, the maximum fungal biomass for AUMC 6696.A was 14.6 ± 0.2 g/L with arginine and 13.68 ± 0.1 g/L with asparagine, when these amino acids were used as single nitrogen sources, while AUMC 11616.A maximum biomass was 10.73 ± 0.8 g/L with glycine and 9.44 ± 0.6 g/L with valine. These were significantly higher than the ammonium nitrate control (p < 0.05). The highest levels of TFA were achieved with glycine for AUMC 11616.A, 26.2 ± 0.8% w/w of cell dry weight, and glutamic acid for AUMC 6696.A, 23.1 ± 1.3%. The highest GLA yield was seen with proline for AUMC 11616.A, 13.4 ± 0.6% w/w of TFA, and tryptophan for AUMC 6696.A, 12.8 ± 0.3%, which were 38% and 25% higher than the ammonium tartrate control. The effects of environmental factors such as temperature, pH, fermentation time, and agitation speed on biomass, total lipids, TFA, and GLA concentration of the target strains have also been investigated. Our results demonstrated that nitrogen assimilation through amino acid metabolism, as well as the use of glucose as a carbon source and abiotic factors, are integral to increasing the oleaginicity of tested strains. Few studies have addressed the role of amino acids in fermentation media, and this study sheds light on R. pusillus and M. circinelloides as promising candidates for the potential applications of amino acids as nitrogen sources in the production of lipids.

Role of Cytosolic Malic Enzyme in Oleaginicity of High-Lipid-Producing Fungal Strain Mucor circinelloides WJ11.

Mucor circinelloides, an oleaginous filamentous fungus, is gaining popularity due to its ability to synthesize significant amounts of lipids containing γ-linolenic acid (GLA) that have important health benefits. Malic enzyme (ME), which serves as the main source of NADPH in some fungi, has been found to regulate lipid accumulation in oleaginous fungi. In the present study, the role of two cytosolic ME genes, cmalA and cmalB, in the lipid accumulation of the M. circinelloides high-lipid-producing strain WJ11, was evaluated. Strains overexpressing cmalA and cmalB showed a 9.8- and 6.4-fold rise in specific ME activity, respectively, and an elevation of the lipid content by 23.2% and 5.8%, respectively, suggesting that these genes are involved in lipid biosynthesis. Due to increased lipid accumulation, overall GLA content in biomass was observed to be elevated by 11.42% and 16.85% in cmalA and cmalB overexpressing strains, respectively. Our study gives an important insight into different studies exploring the role of the cmalA gene, while we have for the first time investigated the role of the cmalB gene in the M. circinelloides WJ11 strain.

Simultaneous overexpression of ∆6-, ∆12- and ∆9-desaturases enhanced the production of γ-linolenic acid in Mucor circinelloides WJ11.

Mucor circinelloides WJ11, an oleaginous filamentous fungus, produces 36% lipid of its cell dry weight when cultured in a high C/N ratio medium, however, the yield of γ-linolenic acid (GLA) is insufficient to make it competitive with other plant sources. To increase the GLA content in M. circinelloides WJ11, this fungus was engineered by overexpression of its key genes such as Δ6-, Δ12-, and Δ9-desaturases involved in GLA production. Firstly, we tried to overexpress two Δ6-desaturase isozymes to determine which one played important role in GLA synthesis. Secondly, Δ6-and Δ12-desaturase were co-overexpressed to check whether linoleic acid (LA), the precursor for GLA synthesis, is a limiting factor or not. Moreover, we tried to explore the effects of simultaneous overexpression of Δ6-, Δ12-, and Δ9-desaturases on GLA production. Our results showed that overexpression (1 gene) of DES61 promoted higher GLA content (21% of total fatty acids) while co-overexpressing (2 genes) DES61 and DES12 and simultaneous overexpressing (3 genes) DES61, DES12, and DES91 increased the GLA production of engineered strains by 1.5 folds and 1.9 folds compared to the control strain, respectively. This study provided more insights into GLA biosynthesis in oleaginous fungi and laid a foundation for further increase in GLA production into fungus such as M. circinelloides.

Coculturing of Mucor plumbeus and Bacillus subtilis bacterium as an efficient fermentation strategy to enhance fungal lipid and gamma-linolenic acid (GLA) production.

This study aimed to improve lipid and gamma-linolenic acid (GLA) production of an oleaginous fungus, Mucor plumbeus, through coculturing with Bacillus subtilis bacteria, optimising the environmental and nutritional culture conditions, and scaling them for batch fermentation. The maximum levels of biomass, lipid, fatty acid, and GLA in a 5 L bioreactor containing cellobiose and ammonium sulfate as the optimal carbon and nitrogen sources, respectively, achieved during the coculturing processes were 14.5 ± 0.4 g/L, 41.5 ± 1.3, 24 ± 0.8, and 20 ± 0.5%, respectively. This strategy uses cellobiose in place of glucose, decreasing production costs. The nutritional and abiotic factor results suggest that the highest production efficiency is achieved at 6.5 pH, 30 °C temperature, 10% (v/v) inoculum composition, 200 rpm agitation speed, and a 5-day incubation period. Interestingly, the GLA concentration of cocultures (20.0 ± 0.5%) was twofold higher than that of monocultures (8.27 ± 0.11%). More importantly, the GC chromatograms of cocultures indicated the presence of one additional peak corresponding to decanoic acid (5.32 ± 0.20%) that is absent in monocultures, indicating activation of silent gene clusters via cocultivation with bacteria. This study is the first to show that coculturing of Mucor plumbeus with Bacillus subtilis is a promising strategy with industrialisation potential for the production of GLA-rich microbial lipids and prospective biosynthesis of new products.

Overexpression of the Mitochondrial Malic Enzyme Genes (malC and malD) Improved the Lipid Accumulation in Mucor circinelloides WJ11.

Mucor circinelloides serves as a model organism to investigate the lipid metabolism in oleaginous microorganisms. It is considered as an important producer of γ-linolenic acid (GLA) that has vital medicinal benefits. In this study, we used WJ11, a high lipid-producing strain of M. circinelloides (36% w/w lipid, cell dry weight, CDW), to examine the role in lipid accumulation of two mitochondrial malic enzyme (ME) genes malC and malD. The homologous overexpression of both malC and malD genes enhanced the total lipid content of WJ11 by 41.16 and 32.34%, respectively. In parallel, the total content of GLA was enhanced by 16.73 and 46.76% in malC and malD overexpressing strains, respectively, because of the elevation of total lipid content. The fact that GLA content was enhanced more in the strain with lower lipid content increase and vice versa, indicated that engineering of mitochondrial MEs altered the fatty acid profile. Our results reveal that mitochondrial ME plays an important role in lipid metabolism and suggest that future approaches may involve simultaneous overexpression of distinct ME genes to boost lipid accumulation even further.

Isolation and Screening of Microorganisms for the Effective Pretreatment of Lignocellulosic Agricultural Wastes.

Lignocellulosic waste is the most abundant biorenewable biomass on earth, and its hydrolysis releases highly valued reducing sugars. However, the presence of lignin in the biopolymeric structure makes it highly resistant to solubilization thereby hindering the hydrolysis of cellulose and hemicellulose. Microorganisms are known for their potential complex enzymes that play a dominant role in lignocellulose conversion. Therefore, the current study was designed to isolate and screen potential microorganisms for their selective delignification ability for the pretreatment of lignocellulosic biomass. An extensive isolation and screening procedure yielded 36 desired isolates (22 bacteria, 7 basidiomycete fungi, and 7 filamentous fungi). Submerged cultivation of these desired microorganisms revealed 4 bacteria and 10 fungi with potent lignocellulolytic enzyme activities. The potent isolates were identified as Pleurotus, Trichoderma, Talaromyces, Bacillus, and Chryseobacterium spp. confirmed by morphological and molecular identification. The efficiency of these strains was determined through enzyme activities, and the degraded substrates were analyzed through scanning electron microscopy (SEM) and X-ray diffraction (XRD). Among all isolated microbes, Pleurotus spp. were found to have high laccase activity. The cellulose-decomposing and selective delignification strains were subjected to solid-state fermentation (SSF). SSF of field waste corn stalks as a single-carbon source provides Pleurotus spp. better condition for the secretion of ligninolytic enzymes. These isolated ligninolytic enzymes producing microorganisms may be used for the effective pretreatment of lignocellulosic agricultural wastes for the production of high value-added natural products by fermentation.

Genomic Studies of White-Rot Fungus Cerrena unicolor SP02 Provide Insights into Food Safety Value-Added Utilization of Non-Food Lignocellulosic Biomass.

Cerrena unicolor is an ecologically and biotechnologically important wood-degrading basidiomycete with high lignocellulose degrading ability. Biological and genetic investigations are limited in the Cerrena genus and, thus, hinder genetic modification and commercial use. The aim of the present study was to provide a global understanding through genomic and experimental research about lignocellulosic biomass utilization by Cerrena unicolor. In this study, we reported the genome sequence of C. unicolor SP02 by using the Illumina and PacBio 20 platforms to obtain trustworthy assembly and annotation. This is the combinational 2nd and 3rd genome sequencing and assembly of C. unicolor species. The generated genome was 42.79 Mb in size with an N50 contig size of 2.48 Mb, a G + C content of 47.43%, and encoding of 12,277 predicted genes. The genes encoding various lignocellulolytic enzymes including laccase, lignin peroxidase, manganese peroxidase, cytochromes P450, cellulase, xylanase, α-amylase, and pectinase involved in the degradation of lignin, cellulose, xylan, starch, pectin, and chitin that showed the C. unicolor SP02 potentially have a wide range of applications in lignocellulosic biomass conversion. Genome-scale metabolic analysis opened up a valuable resource for a better understanding of carbohydrate-active enzymes (CAZymes) and oxidoreductases that provide insights into the genetic basis and molecular mechanisms for lignocellulosic degradation. The C. unicolor SP02 model can be used for the development of efficient microbial cell factories in lignocellulosic industries. The understanding of the genetic material of C. unicolor SP02 coding for the lignocellulolytic enzymes will significantly benefit us in genetic manipulation, site-directed mutagenesis, and industrial biotechnology.

Recent Molecular Tools for the Genetic Manipulation of Highly Industrially Important Mucoromycota Fungi.

Mucorales is the largest and most well-studied order of the phylum Mucormycota and is known for its rapid growth rate and various industrial applications. The Mucorales fungi are a fascinating group of filamentous organisms with many uses in research and the industrial and medical fields. They are widely used biotechnological producers of various secondary metabolites and other value-added products. Certain members of Mucorales are extensively used as model organisms for genetic and molecular investigation and have extended our understanding of the metabolisms of other members of this order as well. Compared with other fungal species, our understanding of Mucoralean fungi is still in its infancy, which could be linked to their lack of effective genetic tools. However, recent advancements in molecular tools and approaches, such as the construction of recyclable markers, silencing vectors, and the CRISPR-Cas9-based gene-editing system, have helped us to modify the genomes of these model organisms. Multiple genetic modifications have been shown to generate valuable products on a large scale and helped us to understand the morphogenesis, basic biology, pathogenesis, and host-pathogen interactions of Mucoralean fungi. In this review, we discuss various conventional and modern genetic tools and approaches used for efficient gene modification in industrially important members of Mucorales.

Molecular Mechanism of Citrate Efflux by the Mitochondrial Citrate Transporter CT in Filamentous Fungus Mucor circinelloides WJ11.

The mitochondrial citrate transporter (MCT) plays an important role in citrate efflux from the mitochondria in eukaryotes, and hence provides a direct correlation between carbohydrate metabolism and lipid synthesis. Our previous studies on transporters confirmed the presence of two MCTs (TCT and CT) in oleaginous Mucor circinelloides WJ11 associated with high lipid accumulation. However, the molecular mechanism of citrate efflux from the mitochondria by MCT in M. circinelloides is still unclear. To study the citrate transport mechanism of CT, the citrate transporter gene was expressed in Escherichia coli, and its product was purified. The citrate transport activity of the protein was studied in CT reconstituted liposomes. Our results showed high efficiency of CT for [14C] citrate/citrate exchange with K m 0.01 mM at 25°C. Besides citrate, other molecules such as oxaloacetate, malate, fumarate, succinate aconitate, oxoadipate, isocitrate, and glutamate also promote citrate transport. In addition, the ct overexpression and knockout plasmids were constructed and transferred into M. circinelloides WJ11, and the mitochondria were isolated, and the transport activity was studied. Our findings showed that in the presence of 10 mM malate, the mitochondria of ct-overexpressing transformant showed 51% increase in the efflux rate of [14C] citrate, whereas the mitochondria of the ct-knockout transformant showed 18% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. This study provided the first mechanistic evidence of citrate efflux from the mitochondria by citrate transporter in oleaginous filamentous fungus M. circinelloides, which is associated with high lipid accumulation.

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.

Comparative Analysis of Different Isolated Oleaginous Mucoromycota Fungi for Their γ-Linolenic Acid and Carotenoid Production.

γ-Linolenic acid (GLA) and carotenoids have attracted much interest due to their nutraceutical and pharmaceutical importance. Mucoromycota, typical oleaginous filamentous fungi, are known for their production of valuable essential fatty acids and carotenoids. In the present study, 81 fungal strains were isolated from different Egyptian localities, out of which 11 Mucoromycota were selected for further GLA and carotenoid investigation. Comparative analysis of total lipids by GC of selected isolates showed that GLA content was the highest in Rhizomucor pusillus AUMC 11616.A, Mucor circinelloides AUMC 6696.A, and M. hiemalis AUMC 6031 that represented 0.213, 0.211, and 0.20% of CDW, respectively. Carotenoid analysis of selected isolates by spectrophotometer demonstrated that the highest yield of total carotenoids (640 µg/g) was exhibited by M. hiemalis AUMC 6031 and M. hiemalis AUMC 6695, and these isolates were found to have a similar carotenoid profile with, ß-carotene (65%), zeaxanthin (34%), astaxanthin, and canthaxanthin (5%) of total carotenoids. The total fatty acids of all tested isolates showed moderate antimicrobial activity against Staphylococcus aureus and Salmonella Typhi, and Penicillium chrysogenum. To the best of our knowledge, this is the first report on the highest yield of total lipid accumulation (51.74% CDW) by a new oleaginous fungal isolate R. pusillus AUMC 11616.A. A new scope for a further study on this strain will be established to optimize and improve its total lipids with high GLA production. So, R. pusillus AUMC 11616.A might be a potential candidate for industrial application.