Silkworm pupae protein co-degradation by magnetic nanoparticles immobilized proteinase K and Mucor circinelloides aspartic protease for further utilization of sericulture by-products.

Silkworm pupae, by-product of sericulture industry, is massively discarded. The degradation rate of silkworm pupae protein is critical to further employment, which reduces the impact of waste on the environment. Herein, magnetic Janus mesoporous silica nanoparticles immobilized proteinase K mutant T206M and Mucor circinelloides aspartic protease were employed in the co-degradation. The thermostability of T206M improved by enhancing structural rigidity (t1/2 by 30 min and T50 by 5 °C), prompting the degradation efficiency. At 65 °C and pH 7, degradation rate reached the highest of 61.7%, which improved by 26% compared with single free protease degradation. Besides, the immobilized protease is easy to separate and reuse, which maintains 50% activity after 10 recycles. Therefore, immobilized protease co-degradation was first applied to the development and utilization of silkworm pupae resulting in the release of promising antioxidant properties and reduces the environmental impact by utilizing a natural and renewable resource.

Production of polyunsaturated fatty acids in pork backfat fermented by Mucor circinelloides.

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.

Enzyme Activity of Culturable Fungi and Bacteria Isolated from Traditional Agarwood Fermentation Basin Indicate Temporally Significant Lignocellulosic and Lipid Substrate Modulations.

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. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01257-y.

Lipid accumulation and SNF1 transcriptional analysis of Mucor circinelloides on xylose under nitrogen limitation.

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-ß, 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.

First Report of Mucor Rot Caused by Mucor circinelloides on Gastrodia elata in China.

Gastrodia elata Blume is used as a traditional Chinese herbal medicine and is widely planted throughout China (Zhu et al. 2019). From September to October 2022, G. elata tuberous rot occurred in 47 households in Yiliang County, Zhaotong City (27°39' N, 104°16' E), Yunnan Province, China, within a major G. elata production area covering 776 ha, with an incidence rate of 10 to 15%. Symptoms manifested as black and brown lesions on the tuber surface, which were concave, soft, foul-smelling, and surrounded by grayish-brown tissue . Three infected G. elata were randomly collected from each household, placed in transparent plastic bags, and pathogen isolation was conducted in a laboratory. Infected G. elata tubers were surface-sterilized with 0.5% NaOCl for 2 min, rinsed five times with sterile water, and dried. Symptomatic tissues from the margin between necrotic and healthy tissues were cut into 5 × 5 mm pieces, placed onto potato dextrose agar (PDA), and incubated at 28 ºC in the dark for 3 days. Hyphal tips of fungi growing from the samples were transferred onto new PDA plates and incubated until they produced conidia. Five fungal isolates (Charliezhao 417, 418, 419, 420, and 421) with the same morphological characteristics were obtained from the samples. Colonies tended to be yellow or light grey, and produced sporangiospores that were sub-globose, ellipsoid, or irregular, measuring 4.0 to 9.7 × 2.5 to 4.6 µm (n = 50). The morphological characteristics of the isolates resembled Mucor circinelloides (Wagner et al. 2020). Genomic DNA of two representative isolates (Charliezhao 417 and 418) was extracted using the DN14 cetyltrimethylammonium bromide rapid plant genome extraction kit (Aidlab Biotechnologies Co., Ltd, Beijing). The ITS and RPB1 genes were amplified by polymerase chain reaction using the primers ITS1/ITS4 (White et al. 1990) and Af/Cf (Matheny et al. 2002), respectively. All sequences were deposited in GenBank (accession no. OQ612709, OR028949 for ITS, OQ621439, OR033135 for RPB1). A BLASTN homology search with the ITS nucleotide sequences showed that they had 99.5 to 99.2% identity with M. circinelloides isolate KR056083 (603/606 bp) and isolate KJ588204 (617/622 bp), respectively; and the RPB1 sequences had 99.89% to 99.75% identity to isolates KJ588206 (874/875 bp) and isolate KJ588206 (803/805 bp), respectively. To complete Koch's postulates, five mature, healthy G. elata tubers were surface disinfected with 1% NaClO solution for 1 min, rinsed with sterile water, and dried at 25℃ for 30 min. A conidial suspension (106 spores/ml) collected from two isolates (Charliezhao 417 and 418) was sprayed onto G. elata tubers, and the control treated with distilled water. All G. elata tubers were incubated at 25 ºC with 80% relative humidity. The experiment had five replicates. After 7 days of incubation, there were obvious brown spots on inoculated tubers; no symptoms were observed on the controls. The pathogen was re-isolated from all inoculated G. elata tubers and confirmed as M. circinelloides by morphological and molecular analyses, completing Koch's postulates. This is the first report of M. circinelloides causing G. elata mucor rot in China. The tubers of G. elate are often employed in the treatment of headaches, convulsions and neurodegenerative disorders (Manavalan et al. 2012). Thus, the declining yield of G. elate due to persistent obstacles related to continuous cropping and diseases poses a potential threat to the development of the G. elate industry.

A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

Effect of Different Carbons on Lipid Production and SNF1 Transcription in Mucor Circinelloides.

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-ß 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. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01070-z.

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.

Novel 2-aryl-4-aryloxyquinoline-based fungistatics for Mucor circinelloides. Biological evaluation of activity, QSAR and docking study.

Zygomycetes are ubiquitous saprophytes in natural environments which transform organic matter. Some zygomycetes of gender Mucor have attracted interest in health sector. Due to its ability as opportunistic microorganisms infecting immuno-compromised people and to the few available pharmacological treatments, the mucormycosis is receiving worldwide attention. Concerning to the pharmacological treatments, some triazole-based compounds such as fluconazole are extensively used. Nevertheless, we focused in the quinolines since they are broadly used models for the design and development of new synthetic antifungal agents. In this study, the fungistatic activity on M. circinelloides of various 2-aryl-4-aryloxyquinoline-based compounds was discovered, and in some cases, it resulted better than reference compound fluconazole. These quinoline derivatives were synthesized via the Csp2-O bond formation using diaryliodonium(III) salts chemistry. A QSAR study was carried out to quantitatively correlate the chemical structure of the tested compounds with their biological activity. Also, a docking study to identify a plausible action target of our more active quinolines was carried out. The results highlighted an increased activity with the fluorine- and nitro-containing derivatives. In light of the few mucormycosis pharmacological treatments, herein we present some non-described molecules with excellent in vitro activities and potential use in the mucormycosis treatment.

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.

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.

Role of Snf-ß in lipid accumulation in the high lipid-producing fungus Mucor circinelloides WJ11.

BACKGROUND: Mucor circinelloides WJ11 is a high-lipid producing strain and an excellent producer of γ-linolenic acid (GLA) which is crucial for human health. We have previously identified genes that encode for AMP-activated protein kinase (AMPK) complex in M. circinelloides which is an important regulator for lipid accumulation. Comparative transcriptional analysis between the high and low lipid-producing strains of M. circinelloides showed a direct correlation in the transcriptional level of AMPK genes with lipid metabolism. Thus, the role of Snf-ß, which encodes for ß subunit of AMPK complex, in lipid accumulation of the WJ11 strain was evaluated in the present study. RESULTS: The results showed that lipid content of cell dry weight in Snf-ß knockout strain was increased by 32 % (from 19 to 25 %). However, in Snf-ß overexpressing strain, lipid content of cell dry weight was decreased about 25 % (from 19 to 14.2 %) compared to the control strain. Total fatty acid analysis revealed that the expression of the Snf-ß gene did not significantly affect the fatty acid composition of the strains. However, GLA content in biomass was increased from 2.5 % in control strain to 3.3 % in Snf-ß knockout strain due to increased lipid accumulation and decreased to 1.83 % in Snf-ß overexpressing strain. AMPK is known to inactivate acetyl-CoA carboxylase (ACC) which catalyzes the rate-limiting step in lipid synthesis. Snf-ß manipulation also altered the expression level of the ACC1 gene which may indicate that Snf-ß control lipid metabolism by regulating ACC1 gene. CONCLUSIONS: Our results suggested that Snf-ß gene plays an important role in regulating lipid accumulation in M. circinelloides WJ11. Moreover, it will be interesting to evaluate the potential of other key subunits of AMPK related to lipid metabolism. Better insight can show us the way to manipulate these subunits effectively for upscaling the lipid production. Up to our knowledge, it is the first study to investigate the role of Snf-ß in lipid accumulation in M. circinelloides.

Bio-detoxification of Jatropha curcas L. cake by a soil-borne Mucor circinelloides strain using a zebrafish survival model and solid-state fermentation.

AIMS: The aims of the study were to (i) improve the evaluation criteria of detoxifying Jatropha curcas L. cake (JCC), (ii) isolate and characterize a JCC tolerant strain, (iii) explore its JCC detoxifying potential. METHODS AND RESULTS: The zebrafish was employed as a survival model to screen the strains capable of detoxifying JCC. A strain identified as Mucor circinelloides SCYA25, which is highly capable of degrading all toxic components, was isolated from soil. Different solid-state fermentation parameters were optimized by response surface methodology. The optimal values for inoculation amount, moisture content, temperature, and time were found to be 18% (1·8 × 106 spores g-1 cake), 66%, 26, and 36 days, respectively, to achieve maximum detoxification of the JCC (92%). Under optimal fermentation conditions, the protein content of JCC was increased, while the concentrations of ether extract, crude fiber, toxins, and anti-nutritional substances were all degraded considerably (P < 0·05). Scanning electron microscopy and Fourier transform infrared spectrometer analysis revealed that the fermentation process could disrupt the surface structure and improve the ratio of α-helix to ß-folding in the JCC protein, which may improve the digestibility when the detoxified JCC is used as a feedstuff. CONCLUSIONS: Our results indicate that M. circinelloides SCYA25 is able to detoxify JCC and improve its nutritional profile, which is beneficial to the safe utilization of JCC as a protein feedstuff. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly identified M. circinelloides SCYA25 detoxified JCC in a safe manner to provide a potential alternative to soybean meal for the feed industry. These results also provide a new perspective and method for the toxicity evaluation and utilization of JCC and similar toxic agricultural by-products.

Improved glucose and xylose co-utilization by overexpression of xylose isomerase and/or xylulokinase genes in oleaginous fungus Mucor circinelloides.

Most of the oleaginous microorganisms cannot assimilate xylose in the presence of glucose, which is the major bottleneck in the bioconversion of lignocellulose to biodiesel. Our present study revealed that overexpression of xylose isomerase (XI) gene xylA or xylulokinase (XK) gene xks1 increased the xylose consumption by 25 to 37% and enhanced the lipid content by 8 to 28% during co-fermentation of glucose and xylose. In xylA overexpressing strain Mc-XI, the activity of XI was 1.8-fold higher and the mRNA level of xylA at 24 h and 48 h was 11- and 13-fold higher than that of the control, respectively. In xks1 overexpressing strain Mc-XK, the mRNA level of xks1 was 4- to 11-fold of that of the control strain and the highest XK activity of 950 nmol min-1 mg-1 at 72 h which was 2-fold higher than that of the control. Additionally, expression of a translational fusion of xylA and xks1 further enhanced the xylose utilization rate by 45%. Our results indicated that overexpression of xylA and/or xks1 is a promising strategy to improve the xylose and glucose co-utilization, alleviate the glucose repression, and produce lipid from lignocellulosic biomass in the oleaginous fungus M. circinelloides. KEY POINTS: • Overexpressing xylA or xks1 increased the xylose consumption and the lipid content. • The xylose isomerase activity and the xylA mRNA level were enhanced in strain Mc-XI. • Co-expression of xylA and xks1 further enhanced the xylose utilization rate by 45%.

Annotation of AMP-activated protein kinase genes and its comparative transcriptional analysis between high and low lipid producing strains of Mucor circinelloides.

BACKGROUND: AMP-activated protein kinase (AMPK) is an important regulator for lipid accumulation, potentially known to have an inhibitory role in lipid synthesis. It inactivates acetyl-CoA carboxylase (ACC), an important regulatory enzyme required for lipid synthesis. However, in Mucor circinelloides, AMPK and its association with lipid accumulation has not been studied yet. OBJECTIVES: To identify AMPK genes in M. circinelloides and to compare their expression levels in high and low lipid-producing strains of M. circinelloides to predict the possible roles of AMPK in lipid metabolism and to select candidate genes for further studies to enhance lipid accumulation. RESULTS: Two genes for α-subunit, one for ß-subunit and six for γ-subunit were identified and annotated. Bioinformatic analysis confirmed the presence of typical conserved domains in these genes. Furthermore, transcriptional profiling displayed marked differences in expression kinetics of subunits among the selected strains. The expression of AMPK genes decreased rapidly in WJ11, high lipid producer strain during the lipid accumulation phase while contrasting profile of expression was observed in CBS 277.49, low lipid producer strain. CONCLUSION: The present study has shown the association of AMPK genes with lipid metabolism at the transcriptional level. The involvement of Snf-α1, Snf-α2, Snf-ß, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

Biosorption capacity of Mucor circinelloides bioaugmented with Solanum nigrum L. for the cleanup of lead, cadmium and arsenic.

The biosorption and bioaugmentation performances of Mucor circinelloides were investigated under different contact time, initial metal(loid) concentration and species. The microbe-plant interaction appeared synergistic with enhancing plant growth and alleviating oxidative damages induced by lead, cadmium and arsenic. The bioaugmentation with M. circinelloides led to significant immobilization on lead, cadmium and arsenic as indicated by the decreases of metal(loid) transfer and bioavailability in plant-microbe aqueous system. Lead, cadmium and arsenic were mainly allocated on cell wall and a few parts entered into intercellular system, suggesting cell wall adsorption and intracellular bioaccumulation served as the main mechanisms of M. circinelloides. The adsorption kinetics and isotherms on lead, cadmium and arsenic were fitted well with the pseudo-second-order and Langmuir models, with the maximum adsorption capacities of 500, 15.4 and 29.4 mg·g-1 fungal biomass at pH 6.0 and 25 â„ƒ. The optimum initial concentration and contact time were 300-10-20 mg·L-1 and 2 h. This study provides a basis for M. circinelloides as a promising adsorbent and bioaugmented agent for the cleanup of soil/aqueous environment contaminated with lead, cadmium and arsenic.

Alteration of Fermentative Metabolism Enhances Mucor circinelloides Virulence.

The fungus Mucor circinelloides undergoes yeast-mold dimorphism, a developmental process associated with its capability as a human opportunistic pathogen. Dimorphism is strongly influenced by carbon metabolism, and hence the type of metabolism likely affects fungus virulence. We investigated the role of ethanol metabolism in M. circinelloides virulence. A mutant in the adh1 gene (M5 strain) exhibited higher virulence than the wild-type (R7B) and the complemented (M5/pEUKA-adh1+) strains, which were nonvirulent when tested in a mouse infection model. Cell-free culture supernatant (SS) from the M5 mutant showed increased toxic effect on nematodes compared to that from R7B and M5/pEUKA-adh1+ strains. The concentration of acetaldehyde excreted by strain M5 in the SS was higher than that from R7B, which correlated with the acute toxic effect on nematodes. Remarkably, strain M5 showed higher resistance to H2O2, resistance to phagocytosis, and invasiveness in mouse tissues and induced an enhanced systemic inflammatory response compared with R7B. The mice infected with strain M5 under disulfiram treatment exhibited only half the life expectancy of those infected with M5 alone, suggesting that acetaldehyde produced by M. circinelloides contributes to the toxic effect in mice. These results demonstrate that the failure in fermentative metabolism, in the step of the production of ethanol in M. circinelloides, contributes to its virulence, inducing a more severe tissue burden and inflammatory response in mice as a consequence of acetaldehyde overproduction.

Discovery and characterization of a stable lipase with preference toward long-chain fatty acids.

OBJECTIVES: To identify novel lipases with stability and long-chain fatty acids preference by phylogenetic evolution analysis methods from database. RESULTS: Thermo-stable Candida antarctica Lipase-A (CALA) was set as a template for gene mining by PSI-BLAST. Based on phylogenetic analysis, three candidate lipases exhibiting 97%, 55%, and 35% identities with CALA, respectively, were selected for overexpression and characterization. Lipase, PhLip from Pseudozyma hubeiensis SY62 showed highest activity towards long-chain fatty acids, and showed maximum activity at pH 9.0 and 60 °C, and stability between 40 and 50 °C for 4 h and at pH 7-10 for 12 h. Enzymatic hydrolysis of Mucor circinelloides WJ11 oils by PhLip was about twofold higher than that by CALA, with respect to hydrolysis of long-chain fatty acids. Besides, fatty acids with 18 carbons, including oleic acid, linoleic acid, and linolenic acid, were preferred as substrates. CONCLUSION: The current investigation discovered a stable lipase PhLip with long-chain fatty acids preference. PhLip may be a potential candidate for producing polyunsaturated fatty acids from natural oils.

First Report of Damping-Off Caused by Mucor circinelloides on Aconitum carmichaelii in China.

Aconitum carmichaelii is a typical traditional Chinese medicinal herb that has been grown for more than one thousand years in China (Singhuber et al. 2009). Surveys for damping-off of A. carmichaelii were conducted from 2016 to 2018 in three of China's major planting areas (Shaanxi, Sichuan, and Yunnan Province). Damping-off was observed from April to June with disease incidences ranging from 5% to 11% in ten investigated fields. In the early stage of disease development, the roots were fully covered by white mycelia. When the disease was severe, seedlings were stunted, turned yellow, had withered roots, and some eventually died. One fungus was consistently isolated from the diseased roots in the planting areas of Shaanxi Province (33°7'42″N and 107°20'27″E) on PDA medium. It was identified as Mucor circinelloides, based on morphological characteristics (Schipper 1976). Three isolates from different regions were characterized by yellowish colonies composed of tall and short sporangiophores 6-11 µm in width, terminal and globose sporangia, ellipsoidal sporangiospores 5-8 µm in length and 4-5 µm in width, and obovoid columellae. The internal transcribed spacer regions (ITS) and the large subunit (28S) of ribosomal RNA gene from the representative isolate, MC180610 (GenBank accession no. MK087755, MT043749), were also amplified and sequenced using the universal primer sets, ITS1/ITS4 and NL1/NL4, respectively. BLAST analyses of the ITS sequence showed 99.38% identity with the type strain (CBS 195.68) of M. circinelloides (GenBank accession no. NR_126116), and the 28S sequence showed 99.44% identity with the strain (CBS 274.49) of M. circinelloides f. circinelloides (GenBank accession no. MH868051). The pathogenicity of M. circinelloides isolate (MC180610) to A. carmichaelii was examined by inoculating 150 ml of the hypha suspension of M. circinelloides prepared from a 10-day-old potato dextrose broth culture into the soils of healthy potted A. carmichaelii plants. Uninoculated potted A. carmichaelii plants served as controls. There were three replicates for the inoculated and control plants. All the potted A. carmichaelii plants were incubated at 25°C under 12 h light 12 h dark conditions. The symptoms that developed on M. circinelloides inoculated A. carmichaelii plants were similar to those observed in the field; control plants did not show symptoms. The pathogen was reisolated from symptomatic roots onto the PDA medium and morphologically identified as M. circinelloides. M. circinelloides has been reported as a pathogen of papaya (Carica papaya), Mandarin fruits (Citrus reticulata), and sweet potatoes (Ipomea batatas), respectively (Cruz-Lachica et al. 2018; Saito et al. 2016; Oladoye et al. 2016), but it has never been reported on A. carmichaelii before. This is the first report of damping-off caused by M. circinelloides on A. carmichaelii. This pathogen may present a threat to the production of A. carmichaelii in China.