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β-carotene is one of the most diverse and effective antioxidants in natural fat-soluble pigments in the food, pharmaceutical, and cosmetic industries. Much attention has been attracted in recent years, due to its potential antibacterial properties and anticancer agents. Among them, microbial synthesis can be expected to serve as the everincreasingly important source of β-carotene, due to the lower cost, higher safety, and easy large-scale production, compared with animal and plant extraction and industrial synthesis. Light stimulation of carotenoid synthesis is also the earliest and most studied regulatory mechanism in fungi. The genome of Mucor circinelloides can be better used in the β-carotene synthesis mechanism, due to the efficient genetic transformation, gene replacement, and silencing, as well as the ability to express foreign genes. This study aims to clarify the molecular mechanism in the specific metabolic process of β-carotene synthesis, where the microorganisms were stimulated by light. A systematic transcriptomic analysis was then made using high-performance liquid chromatography and Illumina Hiseq 2000. The gene changes and functional annotation was determined in β-carotene accumulation and metabolic pathway of Mucor circinelloides WJ11 after light stimulation, respectively. The fermentation results showed that the cell dry weight was slightly higher under light exposure than under dark conditions, the maximum of which was up to 14.8 g/L under light exposure for 1 440 min. There was a high rate of glucose consumption. The concentration of residual glucose was about 7.0 g/L in the medium at the late fermentation stage. In addition, the lipid content significantly increased, reaching 35.3% of the dry weight of cells. β-carotene was at the lower level under the two cultures before light. There was no difference in production. Mucor circinelloides WJ11 accumulated the maximum pigment amount under 1 440 min of continuous light. The pigment amounts under light and dark conditions were 458.3 and 218.7 μg/g, respectively, and then the β-carotene production slowly decreased. The samples were collected at different times under light and dark conditions for the latter transcriptomic analysis. The annotation of the Kyoto encyclopedia in the genes and genomes (KEGG) database showed that a maximum of 5 598 differential genes were annotated to obtain 119 metabolic pathways. The significantly enriched metabolic pathway "biosynthesis of secondary metabolites" was closely related to β-carotene accumulation in Mucor circinelloides. In addition, accumulated pyruvate by the glycolytic pathway after 120 min of light exposure was converted into acetaldehyde in the cytoplasm, and then activated the pyruvate-acetaldehyde-acetic acid cycle, thus supplying more precursor acetyl-CoA for pigment synthesis. Furthermore, the expression of mevalonate pathway genes was enhanced significantly, leading to a sharp increase in β-carotene synthesis. The lipid accumulation and β-carotene synthesis were stabilized after 1 440 min of light exposure. There was an accumulation of acetyl-CoA to affect the higher activity of acetyl-CoA acetyltransferase (ACAT). However, the activity of the mevalonate pathway decreased instead, and β-carotene accumulation decreased as well. Meantime, the decreased expression of acyl-CoA synthetase (ACS) and ATP: Citrate lyase (ACL) allowed for more carbon sources to flux into the TCA cycle. The transcriptional analysis of differential genes showed that light stimulation increased the utilization rate of glucose, in order to rapidly metabolize into the energy for growth, such as fermentation. The transcription levels of three White collar-1 (WC-1) were significantly down-regulated after 120 min of light exposure, indicating the key regulatory proteins for β-carotene synthesis. The findings can provide theoretical support for the molecular mechanism and genetic modification of β-carotene synthesis. [ABSTRACT FROM AUTHOR]

Background: Chitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes. Results: The study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample). Conclusions: The research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers. [ABSTRACT FROM AUTHOR]

The current investigation involved the isolation of 13 endophytic fungi from Taxus sp. collected in Himachal Pradesh, India. Among these, isolate PAT-3 produced 195.13 mg/L of Taxol in reformative medium broth using microbial fermentation as an alternative source. The PAT-3 isolate was characterized as Mucor circinelloides through morphologic and molecular techniques. The PAT-3 isolate was the only one to exhibit positive results for the Taxol biosynthesis-related genes 10-deacetylbaccatin-III-10-O-acetyltransferase (dbat), Baccatin-III, 3: amino, 3 phenylpropanol transferase (bapt), and taxadienol-acetyltransferase (tat). Furthermore, human breast cancer (MCF-7) and human melanoma cancer (SKMEL-28) cell lines demonstrated the cytotoxicity of Taxol extracted from isolate PAT-3, with IC50 values of 80.32 µg/mL and 77.21 µg/mL, respectively. To our knowledge, this is the first study that demonstrates the ability of the endophytic fungus M. circinelloides from Taxus sp. in the northern Himalayan region to produce paclitaxel. The study's findings show that Mucor circinelloides is an excellent alternative source of Taxol, and they may pave the way for the production of Taxol at the industrial level in future. [ABSTRACT FROM AUTHOR]

Abstract Background Chitosan oligosaccharides (COS) have great potential for applications in several fields, including agriculture, food industry or medicine. Nevertheless, the large-scale use of COS requires the development of cost-effective technologies for their production. The main objective of our investigation was to develop an effective method of enzymatic degradation of chitosan in a column reactor using Mucor circinelloides IBT-83 cells, immobilized in a polyurethane foam (PUF). These cells serve as a source of chitosanolytic enzymes. Results The study revealed that the process of freeze-drying of immobilized mycelium increases the stability of the associated enzymes during chitosan hydrolysis. The use of stabilized preparations as an active reactor bed enables the production of COS at a constant level for 16 reactor cycles (384 h in total), i.e. 216 h longer compared to non-stabilized mycelium. In the hydrolysate, oligomers ranging in structure from dimer to hexamer as well as D-glucosamine were detected. The potential application of the obtained product in agriculture has been verified. The results of phytotests have demonstrated that the introduction of COS into the soil at a concentration of 0.01 or 0.05% w/w resulted in an increase in the growth of Lepidium sativum stem and root, respectively (extensions by 38 and 44% compared to the control sample). Conclusions The research has verified that the PUF-immobilized M. circinelloides IBT-83 mycelium, which has been stabilized through freeze-drying, is a promising biocatalyst for the environmentally friendly and efficient generation of COS. This biocatalyst has the potential to be used in fertilizers.

Abstract Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry.

Previously, we constructed engineered M. circinelloides strains that can not only utilize cellulose, but also increase the yield of γ-linolenic acid (GLA). In the present study, an in-depth analysis of lipid accumulation by engineered M. circinelloides strains using corn straw was to be explored. When a two-stage temperature control strategy was adopted with adding 1.5% cellulase and 15% inoculum, the engineered strains led to increases in the lipid yield (up to 1.56 g per 100 g dry medium) and GLA yield (up to 274 mg per 100 g dry medium) of 1.8- and 2.3-fold, respectively, compared with the control strain. This study proved the engineered M. circinelloides strains, especially for Mc-C2PD6, possess advantages in using corn straw to produce GLA. This work provided a reference for transformation from agricultural cellulosic waste to functional lipid in one step, which might play a positive role in promoting the sustainable development of biological industry. [ABSTRACT FROM AUTHOR]

Immuno-compromised COVID-19 patients being treated with glucocorticoids like Dexamethasone for prolonged periods had led to the surge of mucormycosis. Mucor spores proliferate and spread faster in COVID afflicted persons. In India, more than 51,775 cases and 4300 deaths due to COVID-associated mucormycosis were reported till date. The Government of J&K declared an epidemic of COVID-19 associated mucormycosis under Sect. 2 of the Epidemic Disease Act, 1897. In the backdrop of current COVID scenario, it becomes imperative to study the etiological factors, occurrence of causative agents and aerosolisation of the Mucor spores from the environmental samples leading to mucormycosis. The present study was undertaken to check the presence of Mucor spores in the environment so as to find the reinforcing sources of COVID associated mucormycosis. We isolated three strains of Mucor species: Mucor circinelloides and two Mucor hiemalis species, from environmental samples (air, soil and water), all having a positive relevance to mucormycosis. This is the first study on the presence of mucormycosis causing Mucor species in Kashmir environment. In this study, some pathogenicity marker tests were performed to detect the virulence of these fungal strains such as Anti-fungal susceptibility test (AST), biofilm formation and heat stress test. The in vitro antifungal susceptibility of these species showed the major zone of inhibition by Amphotericin B (Amph) and Nystatin (NS), whereas, Fluconazole (FLC) showed no activity. Biofilm was formed by both Mucor species which was confirmed by crystal violet assay (CV) with positive absorbance readings in microplate reader. Under heat stress, pathogenicity marker test was performed in which both species formed considerable aseptate hyphal growth and small scattered spores. During SEM (Scanning Electron Microscopy) analysis, both the species of Mucor were observed with intact sporangia producing several sporangiospores, whose size dimorphism is linked to its virulence. Thus, augmented knowledge of the epidemiology and clinical presentations of these Mucor species may prevent environmental exposure of immune-compromised patients post-COVID and improve early diagnosis and treatment. [ABSTRACT FROM AUTHOR]

The replacement of expensive components in microbial growth media with pretreated lignocellulosic waste component to increase the product spectrum and add value to the bioproducts has been encouraged to achieve sustainable and feasible utilization of waste biomass as per the biorefinery approach. This study demonstrates an integrated biorefinery approach towards utilization of sugarcane bagasse and biomass of Mucor circinelloides ZSKP. A maximum reducing sugar recovery of 80.67 g/l was achieved after combining pretreatment with saccharification. A low temperature, glycerol, and ammonium phosphate pretreatment method was established, where glycerol pretreatment conditions were reduced from 250 to 150 °C and from 120 to 45 min. The ammonium phosphate-containing hydrolysate yielded 12.89 g/l of fungal biomass after fermentation to add to 20.8 g lignin from the delignification step. The biomass production was further improved to 17.69 g/l after supplementation with corn steep solids and mineral salts. The fermentation process also yielded 2.36 g/l chitosan and 4.9 g/l of lipids after extraction from the oleaginous fungus. The lignin infused glycerol plasticized chitosan biocomposite plastic had a 100% improvement in thermogravimetric properties with almost 50% more energy needed to increase the temperature of the material when compared to glycerol only plasticized biocomposite. The fungal chitosan showed antimicrobial properties and was effective as a preservative spray for fresh tomatoes and apples extending their shelf life to at least 14 and 18 days, respectively. This study therefore demonstrated that a novel two-step pretreatment process could be environmentally beneficial and yielded multiple products for biotechnological applications. [ABSTRACT FROM AUTHOR]

Agarwood oil is one of the costliest essential oils used in perfumery, medicine and aroma. Production of the oil traditionally involves a soaking/fermentation step. Studies have indicated a definite role of the diverse microorganisms growing during the open soaking step, and in the emergent aroma of the essential oil. However, the temporal nature of fermentation and a key functional aspect i.e., the enzymatic properties of the microbes from the fermentation basin have not been studied yet. A total of 20 bacteria and 14 fungi isolated from fermentation basins located in Assam, India, at different soaking periods classified as early (0–20 days), medium (20–40 days) and late (40–60 days) clearly pointed towards an early fungal domination followed by succession of bacteria. The physico-chemical transformations of the wood are controlled by enzymatic properties (cellulase, xylanase, amylase and lipase) of the isolates. The results indicated a strong lignocellulosic substrate modulation potential in the four isolates, viz- Purpureocillium lilacinum (0.354 mg/mL), Mucor circinelloides (0.331 mg/mL), Penicillium citrinum (0.324 mg/mL) and Bacillus megaterium (0.152 mg/mL). The highest culturable abundance (CFU/mL) was found in M. circinelloides (2 × 109) among fungi and B. megaterium (4.5 × 109) among bacteria. The highest cellulase activity was shown by P. lilacinum (0.354 mg/mL) while xylanase and lipase by M. circinelloides (0.873 and 0.128 mg/mL). An interesting revelation was that a substantial proportion of the isolates (70% bacteria and 78% fungi) were positive for lipase activity. This is the first report on the "culturable microbiome" of the agarwood fermentation basin from a temporal and functional bioactivity perspective. [ABSTRACT FROM AUTHOR]

Abstract The biological control of gastrointestinal (GI) parasites using predatory fungi has been recently proposed as an accurate and sustainable approach in birds. The current study aimed to assess for the first time the efficacy of using the native ovicidal fungus Mucor circinelloides (FMV-FR1) in reducing coccidia parasitism in peacocks. For this purpose, an in vivo trial was designed in the resident peacock collection (n = 58 birds) of the São Jorge Castle, at Lisbon, Portugal. These animals presented an initial severe infection by coccidia of the genus Eimeria (20106 ± 8034 oocysts per gram of feces, OPG), and thus received commercial feed enriched with a M. circinelloides suspension (1.01 × 108 spores/kg feed), thrice-weekly. Fresh feces were collected every 15 days to calculate the coccidia shedding, using the Mini-FLOTAC technique. The same bird flock served simultaneously as control (t0 days) and test groups (t15–t90 days). The average Eimeria sp. shedding in peacocks decreased up to 92% following fungal administrations, with significant reduction efficacies of 78% (p = 0.004) and 92% (p = 0.012) after 45 and 60 days, respectively. Results from this study suggest that the administration of M. circinelloides spores to birds is an accurate solution to reduce their coccidia parasitism.

The biological control of gastrointestinal (GI) parasites using predatory fungi has been recently proposed as an accurate and sustainable approach in birds. The current study aimed to assess for the first time the efficacy of using the native ovicidal fungus Mucor circinelloides (FMV-FR1) in reducing coccidia parasitism in peacocks. For this purpose, an in vivo trial was designed in the resident peacock collection (n = 58 birds) of the São Jorge Castle, at Lisbon, Portugal. These animals presented an initial severe infection by coccidia of the genus Eimeria (20106 ± 8034 oocysts per gram of feces, OPG), and thus received commercial feed enriched with a M. circinelloides suspension (1.01 × 108 spores/kg feed), thrice-weekly. Fresh feces were collected every 15 days to calculate the coccidia shedding, using the Mini-FLOTAC technique. The same bird flock served simultaneously as control (t0 days) and test groups (t15–t90 days). The average Eimeria sp. shedding in peacocks decreased up to 92% following fungal administrations, with significant reduction efficacies of 78% (p = 0.004) and 92% (p = 0.012) after 45 and 60 days, respectively. Results from this study suggest that the administration of M. circinelloides spores to birds is an accurate solution to reduce their coccidia parasitism. [ABSTRACT FROM AUTHOR]

Simple Summary: The lipid yield of Mucor circinelloides WJ11 has been much studied for industrial production improvement. In this study, the carRP gene responsible for the production of carotenoids was knocked out, which resulted in the interruption of the carotenoid production pathway and a simultaneous impact on lipid production. Further, an integrative transcriptome and genome-scale metabolic model-driven analysis was conducted, which provides insights into the coordinated relationship between carotenoid and fatty acid biosynthesis in M. circinelloides. The findings can be used to design efficient M. circinelloides cell factories. Mucor circinelloides WJ11 is a lipid-producing strain with industrial potential. A holistic approach using gene manipulation and bioprocessing development has improved lipid production and the strain's economic viability. However, the systematic regulation of lipid accumulation and carotenoid biosynthesis in M. circinelloides remains unknown. To dissect the metabolic mechanism underlying lipid and carotenoid biosynthesis, transcriptome analysis and reporter metabolites identification were implemented between the wild-type (WJ11) and ΔcarRP WJ11 strains of M. circinelloides. As a result, transcriptome analysis revealed 10,287 expressed genes, with 657 differentially expressed genes (DEGs) primarily involved in amino acid, carbohydrate, and energy metabolism. Integration with a genome-scale metabolic model (GSMM) identified reporter metabolites in the ΔcarRP WJ11 strain, highlighting metabolic pathways crucial for amino acid, energy, and nitrogen metabolism. Notably, the downregulation of genes associated with carotenoid biosynthesis and acetyl-CoA generation suggests a coordinated relationship between the carotenoid and fatty acid biosynthesis pathways. Despite disruptions in the carotenoid pathway, lipid production remains stagnant due to reduced acetyl-CoA availability, emphasizing the intricate metabolic interplay. These findings provide insights into the coordinated relationship between carotenoid and fatty acid biosynthesis in M. circinelloides that are valuable in applied research to design optimized strains for producing desired bioproducts through emerging technology. [ABSTRACT FROM AUTHOR]

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the non‐genetically modified Mucor circinelloides strain AE‐LMH by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. The dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.845 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (784 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 928. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use. [ABSTRACT FROM AUTHOR]

A culture-dependent method was used to isolate fungal endophytes from the leaves, stems, and roots of the invasive plant Solanum rostratum Dunal. growing in Xinjiang Province, China. All isolates were identified according to ITS (internal transcribed spacer) region of ribosomal DNA sequences and analyzed by Nucleotide BLAST according to NCBI GenBank and Mycobank database. Altogether 176 endophytic fungal isolates corresponding to 44 OTUs were identified, which were classified into 12 genera, with Penicillium (59.66%) and Aspergillus (23.29%) being the highly dominant genera. Ten endophytic isolates (OTU1, OTU15, OTU16, OTU21, OTU23, OTU25, OTU26, OTU30, OTU37 and OTU44) were identified as potential new species. [ABSTRACT FROM AUTHOR]

Premyrsinane-type diterpenoids have been considered to originate from the cyclization of a suitable 5,6- or 6,17-epoxylathyrane precursor. Their biological activities have not been sufficiently explored to date, so the development of synthetic or microbial approaches for the preparation of new derivatives would be desirable. Epoxyboetirane A (4) is an 6,17-epoxylathyrane isolated from Euphorbia boetica in a large enough amount to be used in semi-synthesis. Transannular cyclization of 4 mediated by Cp2TiIIICl afforded premyrsinane 5 in good yield as an only diasteroisomer. To enhance the structural diversity of premyrsinanes so their potential use in neurodegenerative disorders could be explored, compound 5 was biotransformed by Mucor circinelloides NRRL3631 to give rise to hydroxylated derivatives at non-activated carbons (6–7), all of which were reported here for the first time. The structures and absolute configurations of all compounds were determined through extensive NMR and HRESIMS spectroscopic studies. [ABSTRACT FROM AUTHOR]

Definition: In recent decades, important modifications have been introduced in zoos in order to guarantee the welfare of captive wild animals. Thus, many of these species are housed in enclosures with access to vegetation, where they can enjoy habitats close to those in their natural surroundings, interact with the environment, etc. These habitats present beneficial conditions for some species of parasites to survive and spread. This is a very similar problem to that affecting livestock, and the same solution, based on deworming, is currently being applied. However, the free-living stages of certain parasites that develop in the soil are responsible for high rates of ground contamination throughout the year, so that animals become infected soon after successful deworming, resulting in chemical parasiticides being frequently administered. Preventive measures are seldom considered, which worsens the situation. This entry summarizes the usefulness of the dissemination of certain saprophytic filamentous fungi with proven antagonism against some of the parasites. [ABSTRACT FROM AUTHOR]

ABSTRACT Dimorphism is known among the etiologic agents of endemic mycoses as well as in filamentous Mucorales. Under appropriate thermal conditions, mononuclear yeast forms alternate with multi-nucleate hyphae. Here, we describe a dimorphic mucoralean fungus obtained from the sputum of a patient with Burkitt lymphoma and ongoing graft-versus-host reactions. The fungus is described as Mucor germinans sp. nov. Laboratory studies were performed to simulate temperature-dependent dimorphism, with two environmental strains Mucor circinelloides and Mucor kunryangriensis as controls. Both strains could be induced to form multinucleate arthrospores and subsequent yeast-like cells in vitro. Multilateral yeast cells emerge in all three Mucor species at elevated temperatures. This morphological transformation appears to occur at body temperature since the yeast-like cells were observed in the lungs of our immunocompromised patient. The microscopic appearance of the yeast-like cells in the clinical samples is easily confused with that of Paracoccidioides. The ecological role of yeast forms in Mucorales is discussed.IMPORTANCEMucormycosis is a devastating disease with high morbidity and mortality in susceptible patients. Accurate diagnosis is required for timely clinical management since antifungal susceptibility differs between species. Irregular hyphal elements are usually taken as the hallmark of mucormycosis, but here, we show that some species may also produce yeast-like cells, potentially being mistaken for Candida or Paracoccidioides. We demonstrate that the dimorphic transition is common in Mucor species and can be driven by many factors. The multi-nucleate yeast-like cells provide an effective parameter to distinguish mucoralean infections from similar yeast-like species in clinical samples.

In recent decades, important modifications have been introduced in zoos in order to guarantee the welfare of captive wild animals. Thus, many of these species are housed in enclosures with access to vegetation, where they can enjoy habitats close to those in their natural surroundings, interact with the environment, etc. These habitats present beneficial conditions for some species of parasites to survive and spread. This is a very similar problem to that affecting livestock, and the same solution, based on deworming, is currently being applied. However, the free-living stages of certain parasites that develop in the soil are responsible for high rates of ground contamination throughout the year, so that animals become infected soon after successful deworming, resulting in chemical parasiticides being frequently administered. Preventive measures are seldom considered, which worsens the situation. This entry summarizes the usefulness of the dissemination of certain saprophytic filamentous fungi with proven antagonism against some of the parasites.

The exploitation of food industry wastes and their conversion into value-added products present a promising and continuously growing field, given the diversity of elaborated wastes. The current work aimed to utilize sweet cheese whey as a growth medium for acid protease production by a local fungus strain. The biochemical and physicochemical properties of the cheese whey, such as pH, conductivity, chemical oxygen demand, biological oxygen demand (BOD 5 ), total nitrogen and protein contents, and mineral salts, were assessed using various analytical methods. The effect of certain parameters on acid protease production by Mucor circinelloides MG603064.1 through the SmF process was investigated using the conventional design method “One factor at a time”. Subsequent to characterization, the crude extract was used in a trial to create fromage frais , compared to the commercial rennin CHY-MAX ® Powder Extra. Cheese whey characterization revealed its richness in total nitrogen (1.044±0.044 g/l), protein content (6.52±0.04 g/l), and principal mineral salts: calcium (1.637±0.037 g/l), phosphorus (1.173±0.023 g/l), and chloride (1.66±0.09 g/l). The optimal values of the SmF process for acid protease production, such as the inoculum size, beef extract, and KH 2 PO 4 supplements, the initial pH of cheese whey, and incubation temperature were, respectively, 11% (v/v), 0.4% (w/v), 0.5% (w/v), 5.5, and 30̊C. Under these conditions, the lowest milk-clotting time of 290 s was achieved, representing an 18.41-fold increase com¬pared to the initial step using the unoptimized medium. The enzyme showed maximum milk-clotting activity at pH 5, a temperature of 60̊C, and in the presence of 0.025 M of CaCl 2 . The enzyme activity also significantly im¬proved with sonication (35 kHz) for 10 min. The crude extract of M. circinelloides ensured the production of fresh cheese samples with characteristics roughly similar to those obtained by the control (CHY-MAX ® rennin). The acid protease of M. circinelloides could successfully substitute the conventional rennin in the manufacture of fresh cheese.

Pork backfat (PB) contains excessive saturated fatty acids (SFAs), but lacks polyunsaturated fatty acids (PUFAs). Excessive SFAs can be used as a substrate for the growth of certain microorganisms that convert them into PUFAs and monounsaturated fatty acids (MUFAs), and the added value of PB can be enhanced. In this study, Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189 were co-cultured for conversion of PB into fermented pork backfat (FPB) with high level of PUFAs. Our results showed that the content of γ-linolenic acid (GLA) and linoleic acid (LA) in the surface of FPB reached 9.04 ± 0.14 mg/g and 107.31 ± 5.16 mg/g for 7-day fermentation, respectively. To convert the internal SFAs of PB, ultrasound combined with papain was used to promote the penetrative growth of M. circinelloides into the internal PB, and the GLA level in the third layer of fat reached 2.58 ± 0.31 mg/g FPB. The internal growth of M. circinelloides in PB was promoted by adjusting the oxygen rate and ventilation rate through the wind velocity sensor. When the oxygen rate is 2 m/s and the ventilation rate is 18 m3/h, the GLA level in the third layer of fat reached 4.13 ± 1.01 mg/g FPB. To further improve the level of PUFAs in PB, FPB was produced by M. circinelloides at 18 °C. The GLA content on the surface of FPB reached 15.73 ± 1.13 mg/g FPB, and the GLA yield in the second and third layers of fat reached 8.68 ± 1.77 mg/g FPB and 6.13 ± 1.28 mg/g FPB, the LA yield in the second and third layers of fat reached 105.45 ± 5.01 mg/g FPB and 98.46 ± 4.14 mg/g FPB, respectively. These results suggested that excessive SFAs in PB can be converted into PUFAs and provided a new technique for improving PUFAs in FPB. Key points: • This article achieved the conversion of PUFAs in pork backfat by Mucor circinelloides CBS 277.49 and Lactiplantacillus plantarum CGMCC 24189. • This article solved the internal growth of M. circinelloides CBS277.49 in pork backfat by ultrasound combined with papain. • This article proposed an innovative of promoting the internal growth of M. circinelloides and increasing the PUFAs production by oxygen ventilation in pork backfat. [ABSTRACT FROM AUTHOR]

Fatty acid elongases play crucial roles in synthesizing long-chain polyunsaturated fatty acids. Identifying more efficient elongases is essential for enhancing oleaginous microorganisms to produce high yields of target products. We characterized three elongases that were identified with distinct specificities: McELO from Mucor circinelloides, PrELO from Phytophthora ramorum, and PsELO from Phytophthora sojae. Heterologous expression in Saccharomyces cerevisiae showed that McELO preferentially elongates C16 to C18 fatty acids, PrELO targets Δ6 polyunsaturated fatty acids, and PsELO uses long chain saturated fatty acids as substrates. McELO and PrELO exhibited more homology, potentially enabling fatty acid composition remodeling and enhanced LC-PUFAs production in oleaginous microorganisms. Site-directed mutagenesis of conserved amino acids across elongase types identified residues essential for activity, supported by molecular docking. Alanine substitution of conserved polar residues led to enzyme inactivation, underscoring their importance in the condensation reaction. Our findings offer promising elongase candidates for polyunsaturated fatty acid production, contributing to the bioindustry's sustainable development. [ABSTRACT FROM AUTHOR]

Mucor circinelloides lipase Lip10 is a dual-functional enzyme with lipolytic activity and phospholipid:diacylglycerol acyltransferase (PDAT) activity in aqueous phase. However, the catalytic properties of Lip10 and its structural characteristics remain unknown. The present study systematically investigated its PDAT activity and lipolytic activity using six phospholipid substrates, 18:0 triacylglycerol and 16:0 1,2-diacylglycerol, predict the 3D structure of Lip10 using AlphaFold2, and evaluated residues' flexibility through molecular dynamics analysis. Lip10 exhibited a preference for phosphorylethanolamine (PE) with a long-chain fatty acyl group and showed the lipolytic activity to 18:0 triacylglycerol and 16:0 1.2-diacylglycerol. 20 mM Mg2+ and Fe2+ can significantly increase the PDAT activity of Lip10 by 50–80%. Molecular docking analysis suggest that the lower binding energy for 18:0 PE with Lip10 contributes to the higher structural stability of the complex of 18:0 PE and Lip10. Mutations of Phe565Asp and Glu318Met increase PDAT activity of Lip10 by 3.6-fold and 2.7-fold, while the TAG content produced by mutants Lip10Phe103Pro, Lip10Ile487Lys and Lip10Phe570Asp decrease by 33%− 43%. The present study demonstrated structural properties of the dual-functional lipase Lip10 and provide a theoretical basis to study its roles in the lipid metabolism of M. circinelloides in the future. [Display omitted] • Lip10 is a dual-functional enzyme with lipolytic activity and PDAT activity in aqueous phase. • Lip10 exhibited stronger PDAT activity when 18:0 PE was used as the substrate. • Lip10 showed the lipolytic activity to 18:0 triacylglycerol and 16:0 1.2-diacylglycerol. • Mutations of Phe565Asp and Glu318Met increase PDAT activity of Lip10 by 3.6-fold and 2.7-fold. • TAG produced by mutants Lip10Phe103Pro, Lip10Ile487Lys and Lip10Phe570Asp decrease by 33%− 43%. [ABSTRACT FROM AUTHOR]

Chitooligosaccharides (COS) have numerous biological activities, including antibacterial, antifungal, antiviral, anticancer and antioxidant. Unlike high-molecular-weight chitosan, COS have a number of advantages in large-scale commercial applications due to their water solubility and low viscosity. COS are used in the food, agricultural, medical and pharmaceutical industries. The present study demonstrated that intracellular, partially purified enzymes from Mucor circinelloides IBT-83 can hydrolyse chitosan and effectively catalyse the synthesis of a heterodimer composed of D-glucosamine and N-acetyl-D-glucosamine (carried out at a 2.5-fold molar excess of N-acetyl-D-glucosamine relative to D-glucosamine in an acetate buffer [pH 5.5] containing 20% ammonium sulfate). The obtained product was analysed using high-performance liquid chromatography. [ABSTRACT FROM AUTHOR]

Control of infection by gastrointestinal nematodes remains a big problem in ruminants under continuous grazing. For the purpose of decreasing the risk of infection by Trichuris sp. in captive bison (Bison bison) always maintained in the same plot, dried gelatins having ≥106 chlamydospores of both Mucor circinelloides and Duddingtonia flagrans were given to them for one week, and at the end, fecal samples (FF) collected each week for four weeks were analyzed immediately. Feces taken one week prior to gelatin administration served as controls (CF). Eggs of Trichuris sp. were sorted into non-viable and viable, then classified into viable undeveloped (VU), viable with cellular development (VCD), or viable infective (VI). Ovistatic and ovicidal effects were determined throughout the study. In FF, viability of Trichuris eggs decreased between 9% (first week) and 57% (fourth week), egg development was delayed during the first two weeks, and VI percentages were significantly lower than in CF (p = 0.001). It is concluded that the preparation of gelatins with chlamydospores of parasiticidal fungi and their subsequent dehydration offer an edible formulation that is ready to use, stress-free to supply, and easy to store, as well as being well-accepted by ruminants and highly efficient to reduce the risk of Trichuris sp. infection among animals under continuous grazing regimes. [ABSTRACT FROM AUTHOR]

Sucrose non-fermenting 1 (SNF1) protein kinase plays the regulatory roles in the utilization of selective carbon sources and lipid metabolism. Previously, the role of β subunit of SNF1 in lipid accumulation was evaluated by overexpression and knockout of Snf-β in oleaginous fungus M. circinelloides. In the present study, the growth and lipid accumulation of Snf-β overexpression and knockout strains were further analyzed and compared with glucose or xylose as a single or mixed carbon sources. The results showed that the lipid contents in Snf-β knockout strain improved by 23.2% (for glucose), 28.4% (for xylose), and 30.5% (for mixed glucose and xylose) compared with that of the control strain, respectively. The deletion of Snf-β subunit also altered the transcriptional level of acetyl-CoA carboxylase (ACC). The highest transcriptional levels of ACC1 in Snf-β knockout strain at 24 h were increased by 2.4-fold (for glucose), 2.8-fold (for xylose), and 3.1-fold (for mixed glucose and xylose) compared with that of the control strain, respectively. Our results indicated that Snf-β subunit enhanced lipid accumulation through the regulation of ACC1 in response to xylose or mixed sugars of glucose and xylose more significantly than that of response to glucose. This is the first study to explore the effect of Snf-β subunit of M. circinelloides in regulating lipid accumulation responding to different carbon nutrient signals of glucose and xylose. This study provides a foundation for the future application of the Snf-β engineered strains in lipid production from lignocellulose. [ABSTRACT FROM AUTHOR]

Abstract The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the non‐genetically modified Mucor circinelloides strain AE‐LMH by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. The dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.845 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (784 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 928. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

The exploitation of food industry wastes and their conversion into value-added products present a promising and continuously growing field, given the diversity of elaborated wastes. The current work aimed to utilize sweet cheese whey as a growth medium for acid protease production by a local fungus strain. The biochemical and physicochemical properties of the cheese whey, such as pH, conductivity, chemical oxygen demand, biological oxygen demand (BOD5), total nitrogen and protein contents, and mineral salts, were assessed using various analytical methods. The effect of certain parameters on acid protease production by Mucor circinelloides MG603064.1 through the SmF process was investigated using the conventional design method “One factor at a time”. Subsequent to characterization, the crude extract was used in a trial to create fromage frais, compared to the commercial rennin CHY-MAX® Powder Extra. Cheese whey characterization revealed its richness in total nitrogen (1.044±0.044 g/l), protein content (6.52±0.04 g/l), and principal mineral salts: calcium (1.637±0.037 g/l), phosphorus (1.173±0.023 g/l), and chloride (1.66±0.09 g/l). The optimal values of the SmF process for acid protease production, such as the inoculum size, beef extract, and KH2PO4 supplements, the initial pH of cheese whey, and incubation temperature were, respectively, 11% (v/v), 0.4% (w/v), 0.5% (w/v), 5.5, and 30̊C. Under these conditions, the lowest milk-clotting time of 290 s was achieved, representing an 18.41-fold increase com¬pared to the initial step using the unoptimized medium. The enzyme showed maximum milk-clotting activity at pH 5, a temperature of 60̊C, and in the presence of 0.025 M of CaCl2. The enzyme activity also significantly im¬proved with sonication (35 kHz) for 10 min. The crude extract of M. circinelloides ensured the production of fresh cheese samples with characteristics roughly similar to those obtained by the control (CHY-MAX® rennin). The acid protease of M. circinelloides could successfully substitute the conventional rennin in the manufacture of fresh cheese. [ABSTRACT FROM AUTHOR]

Mucor circinelloides WJ11 is a lipid-producing strain with industrial potential. A holistic approach using gene manipulation and bioprocessing development has improved lipid production and the strain’s economic viability. However, the systematic regulation of lipid accumulation and carotenoid biosynthesis in M. circinelloides remains unknown. To dissect the metabolic mechanism underlying lipid and carotenoid biosynthesis, transcriptome analysis and reporter metabolites identification were implemented between the wild-type (WJ11) and ΔcarRP WJ11 strains of M. circinelloides. As a result, transcriptome analysis revealed 10,287 expressed genes, with 657 differentially expressed genes (DEGs) primarily involved in amino acid, carbohydrate, and energy metabolism. Integration with a genome-scale metabolic model (GSMM) identified reporter metabolites in the ΔcarRP WJ11 strain, highlighting metabolic pathways crucial for amino acid, energy, and nitrogen metabolism. Notably, the downregulation of genes associated with carotenoid biosynthesis and acetyl-CoA generation suggests a coordinated relationship between the carotenoid and fatty acid biosynthesis pathways. Despite disruptions in the carotenoid pathway, lipid production remains stagnant due to reduced acetyl-CoA availability, emphasizing the intricate metabolic interplay. These findings provide insights into the coordinated relationship between carotenoid and fatty acid biosynthesis in M. circinelloides that are valuable in applied research to design optimized strains for producing desired bioproducts through emerging technology.

Eicosatetraenoic acid (ETA, 20:4, ω-3) is the desaturation product of dihomo-gamma linolenic acid (DGLA, 20:3, ω-6) catalyzed by delta-17 desaturase, which is considered as a healthy product that helps to lower risk of heart diseases. The oleaginous filamentous fungus, Mucor circinelloides, has been used for a long time as a model micro-organism for GLA production at industrial scales. However, M. circinelloides lacks the key enzymes to synthesize C20 polyunsaturated fatty acids (PUFAs). M. circinelloides could produce DGLA by overexpressing the D6E(GLELO) gene, which could be a useful tool to produce ETA due to the availability of established genetic manipulation tools. Therefore, in this study, delta-17 desaturase (PpD17 and PaD17) genes from Phytophthora parasitica and Pythium aphanidermatum, respectively, were introduced into M. circinelloides to construct an ETA-producing cell factory. Our results showed that the PaD17 and PpD17 overexpression strains' biomass increased by 25.98 and 23.34 g/L (39.98 and 25.75%), respectively, compared with the control strain. Meanwhile, the lipid contents of the recombinant strains also increased and reached up to 28.88% in Mc-PaD17 and 30.95% in Mc-PpD17, respectively, compared with the control strain (23.38% in Mc-2076). The RT-qPCR results showed that overexpression of delta-17 desaturase genes promoted the expression of cme2, fas2, and D6E, thereby contributing to lipid biosynthesis in M. circinelloides. Meanwhile, the content of ETA reached up to 1.95%, and the yield of ETA was up to 114.69 mg/L in PpD17 overexpression mutants at 96 h. This study provided the first report on the construction of an ETA-producing cell factory by heterologous overexpression of the PpD17 gene in M. circinelloides, which established a new scope for further research in the production of ETA in oleaginous fungi. [ABSTRACT FROM AUTHOR]

Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism's cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme's superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production. [ABSTRACT FROM AUTHOR]

As the Greek-style yogurt market continues to experience prosperous growth, finding the most appropriate destination for yogurt acid whey (YAW) is still a challenge for Greek yogurt manufacturers. This study provides a direct alternative treatment of YAW by leveraging the abilities of Mucor circinelloides and Mucor genevensis to raise the pH of YAW and to produce fungal biomass with a high lipid content. Aerobic cultivations of these species were conducted in YAW, both with and without the addition of lactase, at 30 °C, and 200 rpm agitation. The density, pH, biochemical oxygen demand (BOD), biomass production, lipid content, fatty acid profile, and sugar and lactic acid concentrations were regularly measured throughout the 14-day cultivations. The data showed that M. genevensis was superior at deacidifying YAW to a pH above 6.0—the legal limit for disposing of cultured dairy waste. On the other hand, M. circinelloides generated more fungal biomass, containing up to 30% w/w of lipid with high proportions of oleic acid and γ-linolenic acid. Additionally, the treatments with lactase addition showed a significant decrease in the BOD. In conclusion, our results present a viable treatment to increase the pH of YAW and decrease its BOD, meanwhile generating fungal oils that can be further transformed into biodiesel or processed into functional foods or dietary supplements. [ABSTRACT FROM AUTHOR]

Malate transporter proteins (MTPs) play a pivotal role in regulating flux in the citrate/malate/pyruvate shuttle to deliver acetyl-CoA from the mitochondria to the cytosol and thus regulate lipid biosynthesis in oleaginous fungi. Despite the recent successful exploration of the mitochondrial malate transporters in Mucor circinelloides, research with in silico analyses that include molecular docking and their dynamics, in addition to homology modelling of malate transporters, have not been reported. In this study, the physico-chemical properties and nucleotide sequence analysis of two mitochondrial MTPs (MT and SoDIT-a with Gene/protein ID scafold00018.48 and scafold00239.15, respectively), in M. circinelloides WJ11 were performed. The three-dimensional (3D) model of the mitochondrial MTPs was determined and the best-docked complex stabilities were demonstrated with molecular dynamic (MD) simulations. The activity domain was revealed to form hydrogen bonds and piling interactions with citrate and malate upon docking. Our study showed better binding affinities for the MTPs—reaching up to −3.44 and −7.27 kcal/mol with the MT and SoDIT-a proteins, respectively (compared to the target of −2.85 and −6.00 kcal/mol for citric acid-binding). MD simulations illustrated that the protein complexes demonstrated conformational stability throughout the simulation. This study was the first to elucidate the structural characteristics of mitochondrial MTPs in M. circinelloides WJ11, providing direct evidence regarding the transport mechanism of specific substrates. Furthermore, the current results support ongoing efforts to combine functional and structural data to better understand the MTPs (at the molecular and atomic levels) of an oleaginous fungus such as M. circinelloides. [ABSTRACT FROM AUTHOR]

Sucrose non-fermenting 1 (SNF1) plays a crucial role in utilizing non-glucose carbon sources and regulating lipid metabolism. However, the mechanism by which SNF1 regulates lipid accumulation in oleaginous filamentous fungi in response to nutrient signals remains unclear. In the present study, by analysing the growth and lipid accumulation of M. circinelloides on xylose under nitrogen limitation, combined with the transcriptional changes of each subunit of SNF1, the regulation of SNF1 between nutrient signal and lipid accumulation was explored. The results showed that with the increase of carbon/nitrogen (C/N) ratio, the xylose consumption and cell growth of M. circinelloides decreased, and the lipid accumulation increased gradually. The optimal C/N ratio was 160:1, and the maximum lipid yield was 4.1 g/L. Two subunits of SNF1, Snf-α1 and Snf-β, are related to the regulation of lipid metabolism in response to nutrient signals from different external nitrogen sources. This is the first report on the transcriptional analysis of SNF1 subunits on xylose metabolism under nitrogen limitation. This study provides a basis for further understanding of lipid synthesis mechanism on xylose in oleaginous fungi, and it also lays a foundation for the genetic engineering of high-lipid strain. [ABSTRACT FROM AUTHOR]

Sucrose non-fermenting 1 (SNF1) protein kinase plays an important role in the utilization of selective carbon sources and regulation of lipid metabolism. In order to further explore the function of SNF1 in regulating lipid accumulation by responding nutritional signals from non-glucose carbon sources, in the present study, the lipid production and SNF1 transcriptional levels of Mucor circinelloides were analyzed and compared on different carbon sources. The results indicated that M. circinelloides could effectively utilize some secondary metabolic carbon sources of monosaccharides and disaccharides for growth and lipids production, such as fructose, maltose and galactose. Snf-β subunit was associated with the regulation of lipid metabolism in response to nutritional signals from different carbon sources. This is the first report on the transcriptional analysis of SNF1 subunits on different carbons metabolism in oleaginous filamentous fungi. This research has suggested that genetic engineering of SNF1 subunits will alter the lipid production of M. circinelloides from alternative carbon sources. [ABSTRACT FROM AUTHOR]

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Fatty acid elongases play crucial roles in synthesizing long-chain polyunsaturated fatty acids. Identifying more efficient elongases is essential for enhancing oleaginous microorganisms to produce high yields of target products. We characterized three elongases that were identified with distinct specificities: McELO from Mucor circinelloides, PrELO from Phytophthora ramorum, and PsELO from Phytophthora sojae. Heterologous expression in Saccharomyces cerevisiae showed that McELO preferentially elongates C16 to C18 fatty acids, PrELO targets Δ6 polyunsaturated fatty acids, and PsELO uses long chain saturated fatty acids as substrates. McELO and PrELO exhibited more homology, potentially enabling fatty acid composition remodeling and enhanced LC-PUFAs production in oleaginous microorganisms. Site-directed mutagenesis of conserved amino acids across elongase types identified residues essential for activity, supported by molecular docking. Alanine substitution of conserved polar residues led to enzyme inactivation, underscoring their importance in the condensation reaction. Our findings offer promising elongase candidates for polyunsaturated fatty acid production, contributing to the bioindustry’s sustainable development.

Control of infection by gastrointestinal nematodes remains a big problem in ruminants under continuous grazing. For the purpose of decreasing the risk of infection by Trichuris sp. in captive bison (Bison bison) always maintained in the same plot, dried gelatins having ≥106 chlamydospores of both Mucor circinelloides and Duddingtonia flagrans were given to them for one week, and at the end, fecal samples (FF) collected each week for four weeks were analyzed immediately. Feces taken one week prior to gelatin administration served as controls (CF). Eggs of Trichuris sp. were sorted into non-viable and viable, then classified into viable undeveloped (VU), viable with cellular development (VCD), or viable infective (VI). Ovistatic and ovicidal effects were determined throughout the study. In FF, viability of Trichuris eggs decreased between 9% (first week) and 57% (fourth week), egg development was delayed during the first two weeks, and VI percentages were significantly lower than in CF (p = 0.001). It is concluded that the preparation of gelatins with chlamydospores of parasiticidal fungi and their subsequent dehydration offer an edible formulation that is ready to use, stress-free to supply, and easy to store, as well as being well-accepted by ruminants and highly efficient to reduce the risk of Trichuris sp. infection among animals under continuous grazing regimes.

The fourth generation of bioethanol production is on a lookout for non-lignocellulosic biomass waste. One such candidate is chitin, the second most abundant biopolymer on earth. However, the crystalline nature of chitin hinders its application potential for bioethanol production. This limitation can be circumvented by hydrolysing this polymer into oligomers using chitinases. We used this hypothesis and isolated a Bacillus haynesii, a marine bacterium that utilizes colloidal chitin as a substrate and produces chitin oligosaccharides. Further, we utilized Mucor circinelloides to produce bioethanol using the chitin oligosaccharides in the shake flask. We investigated the effect of inoculum age, filling volume, different substrates, and substrate concentration on bioethanol production using Mucor circinelloides from Bacillus haynesii-produced chitin oligosaccharides. Bacillus haynesii demonstrated a maximum chitinase activity of 3.08 U/mL with specific activity of 96 U/mg at the 90th h. Chitin oligosaccharides produced by Bacillus haynesii were confirmed using mass spectrometry. Bioethanol concentration was determined using dichromate oxidation assay as well as gas chromatography. The research resulted in 7.4 g/L of ethanol from 30 g/L of chitin oligosaccharides, with a maximum ethanol yield of 0.25 g of ethanol/g substrate at the 55th h with 48 h inoculum in 80 mL of fermentation medium. Results suggest that chitin oligosaccharides from Bacillus haynesii are an effective and renewable substrate for bioethanol production. [ABSTRACT FROM AUTHOR]

Context: Mucor circinelloides is reported to be the second among the most common causative agents of emerging mucormycosis. It is intrinsically resistant to most known antifungals. Further the use of antifungals cause side effects. Traditional knowledge system for treating various ailments is stronger in India and it also backs deriving various bioactive compounds from herbal sources, in the modern system of medicine. Therefore, two most commonly used culinary herbal materials viz., ginger and omam were studied in vitro against M. circinelloides, as an alternate to antifungal drugs. Aims: To explore the traditional herbal resources as alternate to Amphotericin B to use against M. circinelloides, a fungus causing mucormycosis. Methods and Material: Aqueous extracts of garlic and omam were prepared and tested against M. circinelloides, at different concentrations. A positive control with Amphotericin B and negative control without any supplements were also maintained. The inhibitory effect was assessed by adopting optical density (OD) measurement method in SD broth and SD Agar Well Plate using spore suspension as inoculum. Statistical Analysis Used: Paired student T test was employed using SPSS Version 1.6. Results and Conclusions: Both garlic and omam extracts were found to inhibit the M. circinelloides and their MICs were 600 and 700 μL/mL. It is comparable with the MIC of Amphotericin B, 200 μg/mL. Thus, the regular use of garlic and omam can reduce the risk of possible mucormycosis and these herbs can be explored for drug formulations against M. circinelloides. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The oleaginous fungus Mucor circinelloides has been studied for microbial oil production. Solid–state fermentation may be more suitable for lipid production than submerged fermentation due to its special filamentous structure and lower fermentation costs. M. circinelloides WJ11 under solid–state fermentation indicated that the total fatty acid content of mycelia was significantly higher than that of spores (15.0 and 10.4% in mycelia and spores after 192 h, respectively), while the biomass of the fungal mycelia was lower than that of the spores, reaching 78.2 and 86.9 mg/g, respectively. Transcriptomic studies showed that a total of 9069 genes were differentially expressed between spores and mycelia during solid–state fermentation, of which 4748 were up-regulated and 4321 were down-regulated. Among them, triglyceride-related synthases in M. circinelloides were significantly up-regulated in the mycelia. The mRNA expression level of ATP: citrate lyase was obviously increased to provide more acetyl-CoA for fatty acid synthesis in mycelia, moreover, the metabolism of leucine and isoleucine can also produce more acetyl-CoA for lipid accumulation in M. circinelloides. For NADPH supply, the expression of the pentose phosphate pathway was significantly up-regulated in mycelia, while NADP+-dependent malic enzyme was also increased by 9.5-fold under solid–state fermentation. Compared with gene expression in spores, the autophagy pathway was clearly up-regulated in mycelia to prove that autophagy was related to lipid accumulation in M. circinelloides. [ABSTRACT FROM AUTHOR]

Mucormycosis is a rare disease, which has a poor prognosis among burn patients with a reported mortality of 40–88%. We report on the survival of a 54-year-old male patient with burns of 50% of his total body surface area (TBSA) who survived despite rapid deterioration following cutaneous and probable systemic mucormycosis. A combination of antifungal treatment with extensive debridement and amputation likely resulted in the positive outcome in this case.The case highlights the importance of early diagnosis, antifungal treatment, and aggressive surgical management in burn patients with mucormycosis.

[Display omitted] • A novel nitrogen and phosphate-removing fungus was isolated. • Nitrogen was removed through assimilation and HN-AD. • Different phosphorus species deposited in cell membranes and EPS. • Removal pathway of nitrogen and phosphate were revealed. Fungi capable of simultaneous nitrogen and phosphorus removal from wastewater is rarely found. Here, a novel fungal strain (SNDM1) performing heterotrophic nitrification, aerobic denitrification, and phosphate removal was isolated and identified as Mucor circinelloides. The favorable nutrient removal conditions by the strain using glucose were C/N ratios of 25–30, salinities of 0 %–3 %, and pH of 7.5. Strain SNDM1 achieved ammonium, nitrite, nitrate, and phosphate removal rates of 5.23, 10.08, 4.88, and 0.97 mg/L/h. Nitrogen balance indicated that gaseous (18.60 %–24.55 %) and intracellular nitrogen (43.76 %–70.63 %) were primary fate of initial nitrogen. Enzyme activity revealed that ammonium removal occurred through heterotrophic nitrification and aerobic denitrification. Removed phosphorus was mainly transformed into cell membranes (56 %–64 %) and extracellular polymeric substances (20 %–26 %). Orthophosphate was the major intracellular phosphorus species, while polyphosphate and pyrophosphate existed extracellularly. These findings highlight the potential of this fungal strain for bioremediating polluted wastewater. [ABSTRACT FROM AUTHOR]

Abstract 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.

Mucorales are ubiquitous environmental molds responsible for mucormycosis in diabetic, immunocompromised, and severely burned patients. Small outbreaks of invasive wound mucormycosis (IWM) have already been reported in burn units without extensive microbiological investigations. We faced an outbreak of IWM in our center and investigated the clinical isolates with whole-genome sequencing (WGS) analysis. We analyzed M. circinelloides isolates from patients in our burn unit (BU1, Hôpital Saint-Louis, Paris, France) together with nonoutbreak isolates from Burn Unit 2 (BU2, Paris area) and from France over a 2-year period (2013 to 2015). A total of 21 isolates, including 14 isolates from six BU1 patients, were analyzed by whole-genome sequencing (WGS). Phylogenetic classification based on de novo assembly and assembly free approaches showed that the clinical isolates clustered in four highly divergent clades. Clade 1 contained at least one of the strains from the six epidemiologically linked BU1 patients. The clinical isolates were specific to each patient. Two patients were infected with more than two strains from different clades, suggesting that an environmental reservoir of clonally unrelated isolates was the source of contamination. Only two patients from BU1 shared one strain, which could correspond to direct transmission or contamination with the same environmental source. In conclusion, WGS of several isolates per patients coupled with precise epidemiological data revealed a complex situation combining potential cross-transmission between patients and multiple contaminations with a heterogeneous pool of strains from a cryptic environmental reservoir.IMPORTANCE Invasive wound mucormycosis (IWM) is a severe infection due to environmental molds belonging to the order Mucorales. Severely burned patients are particularly at risk for IWM. Here, we used whole-genome sequencing (WGS) analysis to resolve an outbreak of IWM due to Mucor circinelloides that occurred in our hospital (BU1). We sequenced 21 clinical isolates, including 14 from BU1 and 7 unrelated isolates, and compared them to the reference genome (1006PhL). This analysis revealed that the outbreak was mainly due to multiple strains that seemed patient specific, suggesting that the patients were more likely infected from a pool of diverse strains from the environment rather than from direct transmission among them. This study revealed the complexity of a Mucorales outbreak in the settings of IWM in burn patients, which has been highlighted based on WGS combined with careful sampling.

The innocuousness of the prolonged ingestion of commercial nutritional pellets containing spores of the fungal species Mucor circinelloides and Duddingtonia flagrans on grazing dairy heifers was tested. During two years, one group (G-F) received daily 2.5 kg pellets with 3 × 105 spores each fungus / kg pellet, and group G-C without them. In the third year, ruminants in both groups received spore-free feed. Evaluation of red and white blood cell parameters (RBC and WBC, respectively) was performed monthly. Alterations on the digestive activity, respiratory function and reproductive functionality were also recorded. Three heifers of G-F were necropsied in the second year of study and four in the third. Samples collected from different tissues and organs were preserved in 10% neutral buffered formalin, sections included in paraffin and stained with haematoxylin/eosin, periodic acid Schiff, Fontana–Masson and Ziehl–Neelsen. During the first two years, normal numbers of RBC and WBC were recorded in G-F, by values of RBC and haemoglobin (HB) below physiological standards in G-C. No differences between the two groups were obtained in the third year, with RBC and HB levels lower than normal (physiological). No fungal structures or lesions related to fungal administration were visualised in the tissues of G-F. It is concluded that the daily ingestion of pellets containing spores of the parasiticidal fungi M. circinelloides and D. flagrans over a long period of time is completely safe. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]