Mucor thermorhizoides-A New Species from Post-mining Site in Sudety Mountains (Poland).

Mucor representatives are mostly rapidly growing cosmopolitan soil saprotrophs of early diverged Mucoromycotina subphylum. Although this is the most speciose genus within the group, some lineages are still understudied. In this study, new species of Mucor was isolated from the post-mining area in southwestern Poland, where soil chemical composition analysis revealed high concentration of hydrocarbons and heavy metals. Phylogenetic analysis based on multigene phylogeny showed that the new isolate clusters distinctly from other Mucor species as a sister group to Mucor microsporus. New species Mucor thermorhizoides Abramczyk (Mucorales, Mucoromycota) is characterized by the extensive rhizoid production in elevated temperatures and formation of two layers of sporangiophores. It also significantly differs from M. microsporus in the shape of spores and the size of sporangia. M. thermorhizoides was shown to be able to grow in oligotrophic conditions at low temperatures. Together with M. microsporus they represent understudied and highly variable lineage of the Mucor genus.

Features and evaluation of mucormycosis in COVID-19 patients from two referral hospitals in Iran.

OBJECTIVES: The present study aimed to assess the features, clinical characteristics, and species diversity among patients admitted to referral Hospitals for SARS-CoV-2 pneumonia and mucormycosis in Tehran, Iran, and the relationship between seasonal and species diversity was considered. METHODS: Confirmed COVID-19 patients with a positive reverse-transcriptase real-time (rRT-PCR) test for SARS-CoV2 were primarily included based on clinically suspected mucormycosis infection and confirmed by histopathology and mycology examination of biopsy specimens. The PCR technique was performed by the amplification of the high-affinity iron permease 1 (FTR1) gene for identification and discrimination between Rhizopus arrhizus and non- Rhizopus arrhizus isolates. In contrast, species identification of non-Rhizopus arrhizus was performed by sequencing of ITS rDNA region. RESULTS: Rhino-sino-orbital mucormycosis was identified in the majority of cases (n = 33), with 66 % and 34 % of the cases involving male and female patients, respectively. Rhizopus arrhizus was found to be the most prevalent (84.6 %), followed by Mucor circinelloides (7.6 %). Rhizopus arrhizus was the most prevalent species and present in all the seasons; however, Mucor circinelloides was only present in the autumn. The overall mortality of the total population was 24.6 % (16/ 65); the mortality rates occurring in patients diagnosed with rhino-sino-orbital infection and rhino-sinusal form were 21.4 % and 25 %, respectively. CONCLUSION: CAM can be a serious complication of severe COVID-19, especially in patients with uncontrolled diabetes. It is important to monitor the epidemiology of mucormycosis to raise awareness of the disease and improve diagnosis, treatment and prognosis, particularly in the setting of pandemic.

Analyzing the safety of the parasiticide fungus Mucor circinelloides: first insights on its virulence profile and interactions with the avian gut microbial community.

Parasiticide fungi are considered an accurate, sustainable, and safe solution for the biocontrol of animal gastrointestinal (GI) parasites. This research provides an initial characterization of the virulence of the native parasiticide fungus Mucor circinelloides (FMV-FR1) and an assessment of its impact on birds' gut microbes. The genome of this fungus was sequenced to identify the genes coding for virulence factors. Also, this fungus was checked for the phenotypic expression of proteinase, lecithinase, DNase, gelatinase, hemolysin, and biofilm production. Finally, an in vivo trial was developed based on feeding M. circinelloides spores to laying hens and peacocks three times a week. Bird feces were collected for 3 months, with total genomic DNA being extracted and subjected to long-read 16S and 25S-28S sequencing. Genes coding for an iron permease (FTR1), iron receptors (FOB1 and FOB2), ADP-ribosylation factors (ARFs) (ARF2 and ARF6), and a GTPase (CDC42) were identified in this M. circinelloides genome. Also, this fungus was positive only for lecithinase activity. The field trial revealed a fecal microbiome dominated by Firmicutes and Proteobacteria in laying hens, and Firmicutes and Bacteroidetes in peacocks, whereas the fecal mycobiome of both bird species was mainly composed of Ascomycetes and Basidiomycetes fungi. Bacterial and fungal alpha-diversities did not differ between sampling time points after M. circinelloides administrations (P = 0.62 and P = 0.15, respectively). Although findings from this research suggest the lack of virulence of this M. circinelloides parasiticide isolate, more complementary in vitro and in vivo research is needed to conclude about the safety of its administration to birds, aiming at controlling their GI parasites.IMPORTANCEA previous study revealed that the native Mucor circinelloides isolate (FMV-FR1) can develop parasiticide activity toward coccidia oocysts, one of the most pathogenic GI parasites in birds. However, ensuring its safety for birds is of utmost importance, namely by studying its virulence profile and potential effect on commensal gut microbes. This initial study revealed that although this M. circinelloides isolate had genes coding for four types of virulence factors-iron permease, iron receptors, ADP-ribosylation factors, and GTPase-and only expressed phenotypically the enzyme lecithinase, the administration of its spores to laying hens and peacocks did not interfere with the abundances and diversities of their gut commensal bacteria and fungi. Although overall results suggest the lack of virulence of this M. circinelloides isolate, more complementary research is needed to conclude about the safety of its administration to birds in the scope of parasite biocontrol programs.

The role of areA in lipid accumulation in high lipid-producing fungus Mucor circinelloides WJ11.

In the oleaginous fungus Mucor circinelloides, lipid accumulation is regulated by nitrogen metabolism, which is regulated by the areA gene, a member of the GATA zinc finger transporter family and a major regulator for nitrogen metabolism. However, the role of areA in lipid accumulation in this fungus has not been reported. In order to explore the regulatory effect of areA gene on nitrogen metabolism and lipid accumulation in M. circinelloides, we constructed areA gene knockout and overexpression strains. Then, the recombinant strains were cultured and their biochemical indexes were measured. Simultaneously, transcriptomic studies on the recombinant strains were conducted to infer the regulatory mechanism of areA. The results showed that the areA knockout strain accumulated more lipid, which is 42 % higher than the control. While the areA overexpressing strain obtained the higher biomass accumulation (23 g/L) and used up the nitrogen source in the medium earlier than the control strain and knockout strain. Transcriptome data analysis showed that nr and nit-6 genes related to nitrogen metabolism were up-regulated. And the expression levels of key genes acc and aclY were higher in the areA knockout strain than others, which was positively correlated with the increased lipid accumulation. In addition, in knockout strains, protein catabolism tended to provide substrates for the lipid production, and the expression levels of the related genes were also higher than others. These results indicated that the areA gene not only controls the transcription level of genes related to nitrogen metabolism but also affects lipid accumulation.

Role of AMP Deaminase in Mucor circinelloides: Implications for Nitrogen Utilization and Lipid Biosynthesis.

Lipid accumulation in oleaginous organisms is initiated by AMP deaminase (AMPD) after nitrogen depletion because it mediates the concentration of intracellular adenosine monophosphate (AMP). However, the role of AMPD in lipogenesis in the oleaginous fungus Mucor circinelloides is largely unknown. Therefore, we identified the genes (ampd1 and ampd2) encoding AMPD and investigated the role of AMPD in lipid synthesis in this fungus by overexpressing and deleting ampd genes. Deletion of ampd1 and ampd2 caused 21 and 28% increments in lipid contents under N-limited conditions, respectively. These increases were correlated with the activation of enzymes involved in lipogenesis and the alteration of energy balance. Unexpectedly, overexpression of ampd genes affected nitrogen consumption in both N-limited and N-excess media, which resulted in an increase in cell growth and lipid accumulation compared with the control strain when nitrogen was available. Furthermore, the increased lipid accumulation in the ampd-overexpressing mutants in N-excess media was accompanied by enhanced activities of lipid biosynthetic enzymes. These data suggested that nitrogen metabolism and energy metabolism are affected by AMPD, and overexpression of ampd genes induced lipid accumulation under nitrogen-rich conditions by mimicking the nitrogen limitation response. This highlights an intriguing function of AMPD in M. circinelloides.

Fungicolous Mucor on mushrooms: One novel species and six host records from southwest China and northern Thailand.

Mucor species are a group of common soil-borne fungi, known to cause infections on humans and animals, interfere in food production, and act as useful agents in biotechnological applications. This study reports one new Mucor species, M. yunnanensis, which was found to be fungicolous on an Armillaria sp. from southwest China. Further, M. circinelloides on Phlebopus sp., M. hiemalis on Ramaria sp. and Boletus sp., M. irregularis on Pleurotus sp., M. nederlandicus on Russula sp., and M. yunnanensis on Boletus sp. are reported as new host records. Mucor yunnanensis and M. hiemalis have been collected from Yunnan Province in China, whereas M. circinelloides, M. irregularis, and M. nederlandicus have been collected from Chiang Mai and Chiang Rai Provinces in Thailand. All the Mucor taxa reported herein were identified based on both morphology and phylogenetic analyses of a combined nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and partial nuc 28S rDNA (28S) sequence matrix. Comprehensive descriptions, illustrations, and a phylogenetic tree are provided for all the taxa reported in the study to show the placements of taxa, and the new taxon is compared with its sister taxa.

Characterization of the Sterol 24-C-Methyltransferase Genes Reveals a Network of Alternative Sterol Biosynthetic Pathways in Mucor lusitanicus.

Certain members of the order Mucorales can cause a life-threatening, often-fatal systemic infection called mucormycosis. Mucormycosis has a high mortality rate, which can reach 96 to 100% depending on the underlying condition of the patient. Mucorales species are intrinsically resistant to most antifungal agents, such as most of the azoles, which makes mucormycosis treatment challenging. The main target of azoles is the lanosterol 14α-demethylase (Erg11), which is responsible for an essential step in the biosynthesis of ergosterol, the main sterol component of the fungal membrane. Mutations in the erg11 gene can be associated with azole resistance; however, resistance can also be mediated by loss of function or mutation of other ergosterol biosynthetic enzymes, such as the sterol 24-C-methyltransferase (Erg6). The genome of Mucor lusitanicus encodes three putative erg6 genes (i.e., erg6a, erg6b, and erg6c). In this study, the role of erg6 genes in azole resistance of Mucor was analyzed by generating and analyzing knockout mutants constructed using the CRISPR-Cas9 technique. Susceptibility testing of the mutants suggested that one of the three genes, erg6b, plays a crucial role in the azole resistance of Mucor. The sterol composition of erg6b knockout mutants was significantly altered compared to that of the original strain, and it revealed the presence of at least four alternative sterol biosynthesis pathways leading to formation of ergosterol and other alternative, nontoxic sterol products. Dynamic operation of these pathways and the switching of biosynthesis from one to the other in response to azole treatment could significantly contribute to avoiding the effects of azoles by these fungi. IMPORTANCE The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis. Mucormycoses-causing fungi are resistant to most antifungal agents, including most of the azoles. For this reason, the drugs of choice to treat such infections are limited. The exploration of ergosterol biosynthesis is therefore of fundamental importance to understand the azole resistance of mucormycosis-causing fungi and to develop possible new control strategies. Characterization of sterol 24-C-methyltransferase demonstrated its role in the azole resistance and virulence of M. lusitanicus. Moreover, our experiments suggest that there are at least four alternative pathways for the biosynthesis of sterols in Mucor. Switching between pathways may contribute to the maintenance of azole resistance.

Whole-genome and comparative genome analysis of Mucor racemosus C isolated from Yongchuan Douchi.

Mucor racemosus is the predominant fungal in the zhiqu stage of the fermentation of Yongchuan Douchi (Mucor-type), which plays an important role in the fermentation process of Yongchuan Douchi. However, there is a lack of information on the genetic analysis of M. racemosus. In this study, we isolated and identified M. racemosus C (accession no JAPEHQ000000000) from Yongchuan Douchi and analyzed the physiological indicators, then genomic information of the strain to perform a comprehensive analysis of its fermentation capacity and safety. M. racemosus C had neutral protease activity up to 68.051 U/mL at 30 °C and alkaline protease activity up to 57.367 U/mL at 25 °C. In addition, comparing the genomic data with the COGs database (NCBI), it was predicted that M. racemosus C undergoes extensive amino acid metabolism, making C suitable for the production of fermented foods (e.g., Douchi, Syoyu, and sufu). Finally, we performed virulence genes and resistance genes analysis, hemolysis experiment, aflatoxins assay, antibiotic resistance assay to evaluate the safety of M. racemosus C, and the results showed that M. racemosus C was safe, non-toxin-producing and non-hemolytic.

Mucor indicus caused disseminated infection diagnosed by metagenomic next-generation sequencing in an acute myeloid leukemia patient: A case report.

Background: Mucormycosis commonly occurs in immunosuppressed patients with hematological diseases, which can be life-threatening. However, many cases are often misdiagnosed due to lack of specific clinical manifestations. Additionally, the traditional blood culture or serological testing, with a high false-negative rate, is time-consuming. Thus, precise and timely diagnosis of infections is essential for the clinical care of infected patients. Case presentation: We report a 29-year-old Chinese man with acute myeloid leukemia (AML) who developed febrile neutropenia after the first course of induction chemotherapy. He received empirical antibiotics, which did not relieve his symptoms. No pathogen was detected by traditional microbiologic assays, while Mucor indicus was identified by metagenomic next-generation sequencing (mNGS) in the blood specimen. Liposomal amphotericin B (LAmB) was used, resulting in the patient's temperature returning to normal. A few days later, abdominal computed tomography (CT) scan showed multiple liver abscesses; fluorescence staining, histopathology, and mNGS identified the causative agent-M. indicus. Posaconazole was combined with LAmB as long-term antifungal treatment. Finally, the patient received allogeneic hematopoietic stem cell transplantation successfully after controlled infection. During follow-up 1 year after transplantation, the number of liver abscesses was reduced to one and remained stable. Conclusion: This report described the first case of an AML patient diagnosed with culture-negative disseminated infections caused by M. indicus leading to rare hepatic manifestations using mNGS of peripheral blood and liver biopsy. LAmB combined with posaconazole was given in time, resulting in a favorable outcome. mNGS is a new method that assists in detecting the probable pathogen and increases the accuracy of identifying an etiology.

Role of the nonribosomal peptide synthetase siderophore enzyme (Rfs) of Mucor lusitanicus in controlling the growth of fungal phytopathogens.

Dimorphic species of Mucor, which are cosmopolitan fungi belonging to subphylum Mucoromycotina, are metabolically versatile. Some species of Mucor are sources of biotechnological products, such as biodiesel from Mucor circinelloides and expression of heterologous proteins from Mucor lusitanicus. Furthermore, Mucor lusitanicus has been described as a model for understanding mucormycosis infections. However, little is known regarding the relationship between Mucor lusitanicus and other soil inhabitants. In this study, we investigated the potential use of Mucor lusitanicus as a biocontrol agent against fungal phytopathogens, namely Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and Alternaria solani, which destroy economically important crops. Results showed that aerobic cell-free supernatants of the culture broth (SS) from Mucor lusitanicus inhibited the growth of the fungal phytopathogens in culture, soil, and tomato fruits. The SS obtained from a strain of Mucor lusitanicus carrying the deletion of rfs gene, which encodes an enzyme involved in the synthesis of siderophore rhizoferrin, had a decreased inhibitory effect against the growth of the phytopathogens. Contrarily, this inhibitory effect was more evident with the SS from an rfs-overexpressing strain compared to the wild-type. This study provides a framework for the potential biotechnological use of the molecules secreted from Mucor lusitanicus in the biocontrol of fungal phytopathogens.

cotH Genes Are Necessary for Normal Spore Formation and Virulence in Mucor lusitanicus.

Mucormycosis is an invasive fungal infection caused by certain members of the fungal order of Mucorales. The species most frequently identified as the etiological agents of mucormycosis belong to the genera Rhizopus, Lichtheimia, and Mucor. The frequency of systemic mucormycosis has been increasing, mainly because of increasing numbers of susceptible patients. Furthermore, Mucorales display intrinsic resistance to the majority of routinely used antifungal agents (e.g., echinocandins and short-tailed azoles), which limits the number of possible therapeutic options. All the above-mentioned issues urge the improvement of molecular identification methods and the discovery of new antifungal targets and strategies. Spore coat proteins (CotH) constitute a kinase family present in many pathogenic bacteria and fungi and participate in the spore formation in these organisms. Moreover, some of them can act as virulence factors being receptors of the human GRP78 protein during Rhizopus delemar-induced mucormycosis. We identified 17 cotH-like genes in the Mucor lusitanicus genome database. Successful disruption of five cotH genes in Mucor was performed using the CRISPR-Cas9 system. The CotH3 and CotH4 proteins play a role in adaptation to different temperatures as well as in developing the cell wall structure. We also show CotH4 protein is involved in spore wall formation by affecting the total chitin content and, thus, the composition of the spore wall. The role of CotH3 and CotH4 proteins in virulence was confirmed in two invertebrate models and a diabetic ketoacidosis (DKA) mouse model. IMPORTANCE Current treatment options for mucormycosis are inadequate, resulting in high mortality rates, especially among immunosuppressed patients. The development of novel therapies for mucormycosis has been hampered by lack of understanding of the pathogenetic mechanisms. The importance of the cell surface CotH proteins in the pathogenesis of Rhizopus-mediated mucormycosis has been recently described. However, the contribution of this family of proteins to the virulence of other mucoralean fungi and their functionality in vital processes remain undefined. Through the use of the CRISPR-Case9 gene disruption system, we demonstrate the importance of several of the CotH proteins to the virulence of Mucor lusitanicus by using three infection models. We also report on the importance of one of these proteins, CotH4, to spore wall formation by affecting chitin content. Therefore, our studies extend the importance of CotH proteins to Mucor and identify the mechanism by which one of the CotH proteins contributes to the development of a normal fungal cell wall, thereby indicating that this family of proteins can be targeted for future development of novel therapeutic strategies of mucormycosis.

Serine/Threonine Phosphatase Calcineurin Orchestrates the Intrinsic Resistance to Micafungin in the Human-Pathogenic Fungus Mucor circinelloides.

Procedures such as solid-organ transplants and cancer treatments can leave many patients in an immunocompromised state. This leads to their increased susceptibility to opportunistic diseases such as fungal infections. Mucormycosis infections are continually emerging and pose a serious threat to immunocompromised patients. Recently there has been a sharp increase in mucormycosis cases as a secondary infection in patients battling severe acute respiratory syndrome coronavirus 2 (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 is the echinocandins. Echinocandins seem to be efficacious in the treatment of many other fungal infections. Unfortunately, susceptibility testing has found that echinocandins have little to no effect on Mucorales fungi. In this study, we found that the model Mucorales Mucor circinelloides genome carries three copies of the genes encoding the echinocandin target protein ß-(1,3)-d-glucan synthase (fksA, fksB, and fksC). Interestingly, we found that exposing M. circinelloides to micafungin significantly increased the expression of the fksA and fksB genes, resulting in an increased accumulation of ß-(1,3)-d-glucan on the cell walls. However, this overexpression of the fks genes is not directly connected to the intrinsic resistance. Subsequent investigation discovered that the serine/threonine phosphatase calcineurin regulates the expression of fksA and fksB, and the deletion of calcineurin results in a decrease in expression of all three fks genes. Deletion of calcineurin also results in a lower minimum effective concentration (MEC) of micafungin. In addition, we found that duplication of the fks gene is also responsible for the intrinsic resistance, in which lack of either fksA or fksB led a lower MEC of micafungin. Together, these findings demonstrate that calcineurin and fks gene duplication contribute to the intrinsic resistance to micafungin we observe in M. circinelloides.

Solid-state fermentation produces greater stearidonic acid levels in genetically engineered Mucor circinelloides.

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are important dietary components due to their health benefits and preventative role in cardiovascular disease. Fish-based and plant seed oils are rich in stearidonic acid (SDA; 18:4, n-3), which are readily metabolized into ω-3 PUFAs such as eicosapentaenoic acid. However, these natural sources of SDA are generally low yielding and are unlikely to meet global demands, so new sustainable microbial fermentative sources of SDA need to be identified. Expression of delta15-desaturase in the oleaginous filamentous fungus Mucor circinelloides (McD15D) has been used to construct a recombinant SDA-producing McD15D strain that produces 5·0% SDA levels using submerged fermentation conditions. Switching to solid-state fermentation conditions in the same medium with submerged fermentation resulted in this engineered strain producing significantly higher amounts of SDA. A Box-Behnken design of response surface methodology approach has been used to identify optimal glucose and ammonium tartrate concentrations and temperature levels to maximize SDA production. The use of these optimal solid-state fermentation conditions resulted in the spores and mycelium of the recombinant McD15D producing 19·5% (0·64 mg g-1 ) and 12·2% (1·52 mg g-1 ) SDA content, respectively, which represents an overall increase in SDA yield of 188·0% compared with SDA yields produced using submerged fermentation conditions.

Homologous and Heterologous Expression of Delta(12)-Desaturase in Mucor circinelloides Enhanced the Production of Linolenic Acid.

Linolenic acid (LA) is gaining more interest within the scientific community. This is because it has a potential medical role in reducing the risk of inflammation, carcinogenesis, atherosclerosis and diabetes and is a valuable nutraceutical for human health. The oleaginous fungus Mucor circinelloides produces a high lipid content (36%), including valuable polyunsaturated fatty acids (PUFAs). However, the critical step in which oleic acid (OA) is converted into LA is not efficient at supplying enough substrates for PUFA synthesis. Hence, we propose a method to increase LA production based on genetic engineering. The overexpression of the Δ12-desaturase gene from M. circinelloides and Mortierella alpina increased the LA content and improved the lipid accumulation (from 14.9% to 21.6% in the Δ12-desaturase gene of the M. circinelloides overexpressing strain (Mc-D12MC) and from 14.9% to 18.7% in the Δ12-desaturase gene of M. alpina overexpressing strain (Mc-D12MA)). Additionally, the up-regulated expression levels of these genes targeted the genes involved in NADPH production, implying that the elevated Δ12-desaturase gene may function as a critical regulator of NADPH and lipid synthesis in M. circinelloides. This study provides the first evidence to support the design of metabolic engineering related to LA and PUFA production in M. circinelloides for potential industrial applications.

Secretion of the siderophore rhizoferrin is regulated by the cAMP-PKA pathway and is involved in the virulence of Mucor lusitanicus.

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

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.

Improved γ-Linolenic Acid Production from Cellulose in Mucor circinelloides via Coexpression of Cellobiohydrolase and Delta-6 Desaturase.

The present study was aimed at facilitating the production of γ-linolenic acid (GLA) from the cellulosic substrate with the engineered oleaginous fungus Mucor circinelloides WJ11. Here, the homologous recombination technology was used to overexpress the cellobiohydrolase (CBH2) derived from Trichoderma longibrachiatum and the original delta-6 fatty acid desaturase (D6) in M. circinelloides to construct genetically engineered strains capable of effectively using cellulose to enhance GLA synthesis. When cultivated in modified K&R medium supplemented with microcrystalline cellulose, the CBH2 and D6 coexpressing strains led to increases in the biomass (up to 12.8 g/L) and lipid yield (up to 3.7 g/L) of 87% and 2.4-fold, respectively, compared to that of the control strain. Notably, when CBH2 and D6 were coexpressed in M. circinelloides, the yield of GLA reached 608 mg/L, which was a dramatic increase of 3.9-fold compared to that of the control strain. This is the first report on promoting the GLA production from the cellulosic substrate via coexpression of CBH2 and delta-6 desaturase. This work provides a theoretical basis for efficient transformation from the cellulosic substrate to functional GLA by CBH2 and D6 coexpressing strains, which might play a positive role in promoting the sustainable development of biological industry.

Overexpression of Shinorhizobium meliloti flavohemoglobin improves 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 deliver 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. The VHb and SHb genes were integrated into the M. circinelloides MU402 genome by homologous recombination. VHb and SHb protein expression was verified by carbon monoxide difference spectrum analysis. The biomass was increased by ~ 50% in the strain expressing SHb compared with VHb. The total fatty acid (TFA) content of the strain expressing SHb reached 15.7% of the dry cell weight (~ 40% higher than that of the control strain) during flask cultivation. The biomass and TFA content were markedly increased (12.1 g/L and 21.1% dry cell weight, respectively) in strains expressing SHb than strains expressing VHb during fermenter cultivation. VHb and SHb expression also increased the proportion of polyunsaturated fatty acids. Overexpressed bacterial hemoglobins, especially SHb, increased cell growth and TFA content in M. circinelloides at low and high aeration, suggesting that SHb improves fatty acid production more effectively than VHb in oleaginous microorganisms.

Immobilization of fibrinolytic protease from Mucor subtilissimus UCP 1262 in magnetic nanoparticles.

This work reports the immobilization of a fibrinolytic protease (FP) from Mucor subtilissimus UCP 1262 on Fe3O4 magnetic nanoparticles (MNPs) produced by precipitation of FeCl3·6H2O and FeCl2·4H2O, coated with polyaniline and activated with glutaraldehyde. The FP was obtained by solid state fermentation, precipitated with 40-60% ammonium sulfate, and purified by DEAE-Sephadex A50 ion exchange chromatography. The FP immobilization procedure allowed for an enzyme retention of 52.13%. The fibrinolytic protease immobilized on magnetic nanoparticles (MNPs/FP) maintained more than 60% of activity at a temperature of 40 to 60 °C and at pH 7 to 10, when compared to the non-immobilized enzyme. MNPs and MNPs/FP did not show any cytotoxicity against HEK-293 and J774A.1 cells. MNPs/FP was not hemolytic and reduced the hemolysis induced by MNPs from 2.07% to 1.37%. Thrombus degradation by MNPs/FP demonstrated that the immobilization process guaranteed the thrombolytic activity of the enzyme. MNPs/FP showed a total degradation of the γ chain of human fibrinogen within 90 min. These results suggest that MNPs/FP may be used as an alternative strategy to treat cardiovascular diseases with a targeted release through an external magnetic field.

A thermal adaptation landscape related to virulence in Mucor irregularis transcriptional profiles.

OBJECTIVES: Our study aimed to better understand the different thermal adaptation in Mucor irregularis (M. irregularis) strains under high temperature and the involved virulence-related genes, and to offer more appropriate explanation for the diverse pathogenicity of M. irregularis in human infections. METHODS: M. irregularis isolates were incubated at 30 and 35°C for Illumina HiSeq technology (RNA-seq), as well as the virulence difference detected through Galleria mellonella infection models. We verified their transcriptional profile with RT-PCR and analysed differentially expressed genes with GO and KEGG annotations. RESULTS: All 25 isolates formed the biggest colonies at 28°C and did not grow at 37°C, while were differently inhibited at 22 and 35°C. Six selected M. irregularis displayed virulence in sync with their growth condition at high temperature. From the outcomes of RNA-seq, a total of 1559 differentially expressed genes (FC ≥ 2, FDR < 0.05) were obtained, of which 1021 genes were upregulated, and 538 genes were downregulated. Cell wall structure genes related to Ras-like and GH16 proteins, influx-efflux pumps consist of transmembrane proteins as ABC and MFS proteins, and metabolic genes as DGKɛ and Hsfs, seem to be essential in thermal adaptation and virulence of M. irregularis. CONCLUSION: We found some common genes expressed at high temperature, while some others specifically related to M. irregularis isolates with different virulence and thermal adaptation. Further research of genes involved in the pathogenic process is needed for the development of potential targeted antifungal.