Strains Of Mucor Circinelloides Research Articles

Lipid production from corn straw by cellobiohydrolase and delta-6 desaturase engineered Mucor circinelloides strains under solid state fermentation

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.

Safety evaluation of an extension of use of the food enzyme triacylglycerol lipase from the non-genetically modified Mucor circinelloides strain AE-LMH.

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.

Lipid Accumulation by Snf-β Engineered Mucor circinelloides Strains on Glucose and Xylose.

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 24h 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.

Effect of Adding Cellulose on Lipid Accumulation in Cellobiohydrolase and Delta-6 Desaturase Engineered Mucor circinelloides Strains

Effect of Adding Cellulose on Lipid Accumulation in Cellobiohydrolase and Delta-6 Desaturase Engineered Mucor circinelloides Strains

Safety evaluation of the food enzyme triacylglycerol lipase from the non-genetically modified Mucor circinelloides strain AE-LMH.

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. The food enzyme is considered free from viable cells of the production organism. The food enzyme is intended to be used in baking processes, egg processing and the manufacture of enzyme-modified dairy ingredients (EMDI). Dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 0.242mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panelidentified a no observed adverse effect level of 784mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 3,240. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panelconsidered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood of such reactions is low. Based on the data provided, the Panelconcluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Production of surface‐active sophorolipid biosurfactant and crude oil degradability by novel Rhodotorula mucilaginosa strain SKF2

AbstractBiosurfactants are produced by important types of microorganisms such as bacteria, yeast, and filamentous fungi and have been used in a variety of industries. Among the 15 crude oil‐degrading fungi, the two molds and one yeast were identified by 18S rDNA sequences as Mucor circinelloides strain SKMC, Fusarium fujikuroi strain DB2, and Rhodotorula mucilaginosa strain SKF2. These strains were isolated from crude oil–contaminated soil, diesel oil–contaminated soil, and activated sludge in the Oil Refinery Plant in Isfahan, Iran, respectively. The yeast strain was identified as a novel crude oil–degrading and biosurfactant‐producing fungi in the presence of (1% v/v) Iranian light crude oil in the minimal salt medium (MSM). The highest amount of the dry weight of produced biosurfactant was measured at 6.2 g L−1. Chemical nature of produced biosurfactant was determined as a surface‐active sophorolipid biosurfactant compound by thin‐layer chromatography, Fourier transform infra‐red spectroscopy, and gas chromatography–mass spectrometry (GC–MS) analysis. The residual hydrocarbons in the MSM were analyzed by GC–MS, and it was shown that octadecane and docosane were eliminated by this novel strain completely.

Lipid Accumulation by Xylose Metabolism Engineered Mucor circinelloides Strains on Corn Straw Hydrolysate.

Previously, we presented a novel approach for increasing the consumption of xylose and the lipid yield by overexpressing the genes coding for xylose isomerase (XI) and xylulokinase (XK) in Mucor circinelloides. In the present study, an in-depth analysis of lipid accumulation by xylose metabolism engineered M. circinelloides strains (namely Mc-XI and Mc-XK) using corn straw hydrolysate was to be explored. The results showed that the fatty acid contents of the engineered M. circinelloides strains were, respectively, increased by 19.8% (in Mc-XI) and 22.3% (in Mc-XK) when compared with the control strain, even though a slightly decreased biomass in these engineered strains was detected. Moreover, the xylose uptake rates of engineered strains in the corn straw hydrolysate were improved significantly by 71.5% (in Mc-XI) and 68.8% (in Mc-XK), respectively, when compared with the control strain. Maybe the increased utilization of xylose led to an increase in lipid synthesis. When the recombinant M. circinelloides strains were cultured in corn straw hydrolysate medium with the carbon-to-nitrogen ratio (C/N ratio) of 50 and initial pH of 6.0, at 30 °C and 500 rpm for 144 h, a total biomass of 12.6-12.9 g/L with a lipid content of 17.2-17.7% (corresponding to a lipid yield of 2.17-2.28 g/L) was achieved. Our study provides a foundation for the further application of the engineered M. circinelloides strains to produce lipid from lignocelluloses.

Exploring Simplified Methods for Insect Chitin Extraction and Application as a Potential Alternative Bioethanol Resource.

Simple SummaryThe studies on chitin utilization as a source for bioethanol production are still very few. The present study explores some simple methods for insect chitin extraction and application in bioethanol production. Using insect chitin in bioethanol production, may help decreasing the dependence on energy crops as a carbon source for bioethanol. Fungal strains of Mucor circinelloides were reported previously to bio-convert chitin directly to ethanol in submerged fermentation systems. In our study, we explored the bioconversion of insect chitin to bioethanol using two different strains of Mucor circinelloides in submerged fermentation systems. An insect-isolated M. circinelloides strain was found to bio-convert the extracted chitin directly to ethanol in submerged fermentation system. The source of strain isolation and the pH of the production medium were showed to influence the chitin bioconversion directly to bioethanol. All fermentation processes can be conducted easily, using the whole growing microorganism instead of using purified enzymes. These results highlight the insect biomass as a potential new, cheap and renewable source for bioethanol production simply, using a potent insect-isolated M. circinelloides strain.Chitin, the second most plentiful biopolymer in nature, is a major component of insect cuticle. In searching for alternative resources for fossil fuels, some fungal strains of Mucor circinelloides from an insect-source were found to produce bioethanol directly using insect chitin as a substrate. Herein, simplified methods for insect chitin extraction and application as a substrate in submerged fermentation for bioethanol production were explored. Chitin of the American cockroach (Periplaneta americana (L.)) was isolated by refluxing the cockroaches dried exoskeletons with 4% NaOH. The purity of the extracted chitin was assessed to be high when the physicochemical properties of the extracted chitin matched these of commercially available crab and shrimp samples. The extracted chitin was employed as a substrate in submerged fermentation using two strains of M. circinelloides. One of these, strains M. circinelloides 6017 showed immense potential for bioethanol production directly. It could to bio-transform 15 g/L of colloidal chitin directly to 11.22 ± 0.312 g/L of bioethanol (74% of the initial chitin mass) after 6 days of incubation. These results confirm the possibility of using insect biomass as a potential alternative resource for bioethanol production in a simple manner thus contributing to the creation of an alternate energy source.

Biodegradation of crude oil by a halotolerant and biosurfactant producing strain of Mucor circinelloides in different microcosm conditions

ABSTRACT Most petroleum-polluted soils have extreme conditions such as high salinity. The purpose of this study was to evaluate the effect of salinity in various concentrations of NaCl on oil hydrocarbons degradation and biosurfactant production by Mucor circinelloides. Also, the effect of biosurfactant production by the fungal strain on crude oil removal was studied. Finally, the oil biodegradation process by M. circinelloides was investigated in microcosm conditions. The fungi could grow in the presence of up to 200 g L−1 NaCl and was able to remove 87.5% and 49.8% of 10 g L−1 crude oil in MSM at 0 and 50 g L−1 NaCl, respectively. It could also degrade 87% of aliphatic hydrocarbons in 25 g L−1 NaCl. M. circinelloides was able to produce biosurfactant, and reduce the surface tension to 27.6 mN m−1 and 32.9 mN m−1 in NaCl concentrations of 0 and 25 g L−1, respectively. The best performance of M. circinelloides in microcosm was achieved as 56.2% in soil with 10 g kg−1 crude oil, C/N/P ratio of 100:10:1, 25 g kg−1 NaCl and 20 g kg−1 wheat bran over the period of 105 days. In conclusion, here a potent strain was introduced and characterised for the biodegradation of crude oil in non-saline and saline environments. It is able to use crude oil as a sole and supplementary source of carbon and also produce biosurfactant so as to facilitate crude oil degradation.

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

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. 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 36days, 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 β-folding in the JCC protein, which may improve the digestibility when the detoxified JCC is used as a feedstuff. 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. 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.

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

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. 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. Two genes for α-subunit, one for β-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. 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-β, Snf-γ1, Snf-γ4, Snf-γ5 subunits were shown to be more pronounced and could potentially be further explored in future studies.

Comparative Analysis of β-Carotene Production by Mucor circinelloides Strains CBS 277.49 and WJ11 under Light and Dark Conditions.

Carotenoids are natural potent antioxidants and free radical scavengers which are able to modulate the pathogenesis of some cancers and heart diseases in human, indicating their importance in being provided through the diet. Mucor circinelloides accumulates β-carotene as the main carotenoid compound and has been used as a model organism in carotenogenic studies. In the present study, the potential of two M. circinelloides strains to accumulate β-carotene was investigated under light and dark conditions. The results, which were quantitated by HPLC, showed that CBS 277.49 accumulated higher pigment in comparison to WJ11 under both conditions. Continuous illumination triggered the pigment accumulation up to 2.7-fold in strain CBS 277.49 and 2.2-fold in strain WJ11 in comparison to dark. The mRNA analysis of the four key genes involved in isoprenoid pathway by RT-qPCR showed higher transcriptional levels in CBS 277.49 as compared to WJ11, indicating that the pigment production metabolic machinery is more active in CBS 277.49 strain. A new scope for further research was established by this work for improved β-carotene production in the high producing strain CBS 277.49.

Bioprospecting of whole-cell biocatalysts for cholesterol biotransformation.

Microorganisms were isolated from industrial wool scouring effluents and from the soil adjacent to the wastewater treatment lagoon, both sterols-rich environments, in order to search for novel biocatalysts able to transform cholesterol. The isolates were identified on the basis of morphological and biochemical characteristics and phylogenetic analysis. Furthermore, a rapid and accurate bacteria identification by matrix assisted laser desorption/ionization-time-of-flight mass spectrometry was carried out. Bacteria and fungi including representatives of the genera Fusarium, Talaromyces, Trichoderma, Mucor, Aspergillus, Citrobacter, Proteus, Klebsiella, Exiguobacterium, Acinetobacter, Tsukamurella, Bacillus, and Streptomyces were found and evaluated for their ability to biotransform cholesterol by whole-cell treatment system. The results show that a Trichoderma koningiopsis strain, as well as two strains of Mucor circinelloides were able to transform cholesterol into value-added products. The major products were characterized as 7β-hydroxycholesterol, 4-cholesten-3-one, 5α,6α-epoxycholestan-3β-ol and 5β,6β-epoxycholestan-3β-ol. To the best of our knowledge, the present study is the first report of cholesterol biotransformation by representatives of Trichoderma and Mucor genera.

Generation of A Mucor circinelloides Reporter Strain-A Promising New Tool to Study Antifungal Drug Efficacy and Mucormycosis.

Invasive fungal infections caused by Mucorales (mucormycosis) have increased worldwide. These life-threatening infections affect mainly, but not exclusively, immunocompromised patients, and are characterized by rapid progression, severe tissue damage and an unacceptably high rate of mortality. Still, little is known about this disease and its successful therapy. New tools to understand mucormycosis and a screening method for novel antimycotics are required. Bioluminescent imaging is a powerful tool for in vitro and in vivo approaches. Hence, the objective of this work was to generate and functionally analyze bioluminescent reporter strains of Mucor circinelloides, one mucormycosis-causing pathogen. Reporter strains were constructed by targeted integration of the firefly luciferase gene under control of the M. circinelloides promoter Pzrt1. The luciferase gene was sufficiently expressed, and light emission was detected under several conditions. Phenotypic characteristics, virulence potential and antifungal susceptibility were indifferent to the wild-type strains. Light intensity was dependent on growth conditions and biomass, being suitable to determine antifungal efficacy in vitro. This work describes for the first time the generation of reporter strains in a basal fungus that will allow real-time, non-invasive infection monitoring in insect and murine models, and the testing of antifungal efficacy by means other than survival.

Aislamiento, identificación y evaluación de la actividad antimicrobiana de metabolitos producidos por Mucor circinelloides (Cepa Nativa SPG 321)

Objetivo. Identificar y evaluar la capacidad antimicrobiana de los metabolitos aislados a partir delproceso de fermentación en una cepa de Mucor circinelloides. Método. En el presente estudio setrabajó la cepa nativa de Mucor circinelloides SPG 321 suministrada por el Grupo de Investigaciónde Biotrasformación (GIBUJ), con el fin de evaluar su actividad antimicrobiana. La cinética decrecimiento determinó que la ideofase culminó a la hora 264, hora determinada como la finalizaciónde la fermentación. Se separó el micelio del caldo y posteriormente se fraccionaron los extractos concromatografías de capa fina y columna en sílica gel, eluidas con diferentes relaciones de solventes.Resultados. Los resultados arrojados por la técnica de gases acoplado a masas CG-EM confirmanla importancia de Mucor circinelloides en la producción de ácidos grasos insaturados. A partir delmicelio se obtuvo un esterol, compuesto M. cB3. La fracción CHCl3 en biomasa mostró actividadinhibitoria para los microorganismos Gram positivos.

Characteristics of cell growth and lipid accumulation of high and low lipid-producing strains of Mucor circinelloides grown on different glucose-oil mixed media

Abstract Mucor circinelloides is one of the few oleaginous fungi that produce large amounts of γ-linolenic acid (GLA). However, there are few reports regarding the lipid accumulation characteristics and assimilation preferences of M. circinelloides grown on different oils. The present study investigated the characteristics of cell growth, lipid accumulation, and lipase activity of a low lipid-producing strain M. circinelloides CBS 277.49 and a high lipid-producing strain M. circinelloides WJ11. The highest biomass of 21.2 g/L was obtained for CBS 277.49 grown on soybean oil, while the highest lipid content of 65% was obtained when WJ11 was grown on soybean phospholipids. M. circinelloides preferred to assimilate exogenous palmitic, stearic, and α-linolenic acid to synthesize intracellular triacylglycerols. Soybean phospholipids and phytosterol esters stimulated the production of intracellular lipases in both strains more efficiently than soybean oils. The maximal lipase activity of 2200 U/g protein was observed for CBS 277.49 grown on phytosterol esters, while the maximal lipase activity of 2000 U/g protein was observed for WJ11 grown on soybean phospholipids. Although the transcriptional levels of the lipase genes were sharply upregulated when strains were grown on glucose-oil mixed media, the extracellular lipase activity in the medium was still low at 4–210 U/g protein.

Dissecting metabolic behavior of lipid over-producing strain of Mucor circinelloides through genome-scale metabolic network and multi-level data integration

Lipid accumulation is an important cellular process of oleaginous microorganisms. To dissect metabolic behavior of oleaginous Zygomycetes, the lipid over-producing strain, Mucor circinelloides WJ11, was subjected for omics-scale analysis. The genome annotation was improved and used for construction of genome-scale metabolic network of WJ11 strain. Then, the quality of the metabolic network was enhanced by incorporating gene and protein expression data. In addition to the known oleaginous genes, our results showed a number of newly identified unique genes of WJ11 strain, which involved in central carbon metabolism, lipid, amino acid and nitrogen metabolisms. The systematic compilations indicated the additional metabolic routes with the involvement in supplying precursors (acetyl-CoA, NADPH and fatty acyl substrate) for fatty acid and lipid biosynthesis. Interestingly, amino acid metabolism played a substantial role in responsive mechanism of the fungal cells to nutrient imbalance circumstance through lipogenesis as the finding of reporter metabolites (l-methionine, l-glutamate, l-aspartate, l-asparagine and l-glutamine) at lipid-accumulating stage. The cooperative function of certain lipid-degrading enzymes at the particular growth stage was elucidated by integrating the metabolic networks with gene expression data. The unique feature of carotenoid biosynthetic route in WJ11 strain was also identified by protein domain analysis. Taken together, there were cross-functional metabolisms in regulating lipid biosynthesis and retaining high level of cellular lipids in the representative of lipid over-producing strains.

Outbreak of Invasive Wound Mucormycosis in a Burn Unit Due to Multiple Strains of Mucor circinelloides f. circinelloides Resolved by Whole-Genome Sequencing.

ABSTRACTMucorales 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.

A protein complex bearing an oxidase with napthalene dihydrodiol dehydrogenase activity is induced in Mucor circinelloides strain YR-1 during growth on polycyclic aromatic compounds

Fungi are organisms capable of growing in a myriad of conditions and respond to counteract environmental cues. Several locations in the world are polluted with oil and its derivatives, and some microorganisms tolerant to these compounds have been isolated. Some fungi can grow in the presence of molecules such as polycyclic aromatic hydrocarbons as sole carbon sources. In this report, we further characterized the induced enzymes with phenanthrene from Mucor circinelloides YR-1 strain, isolated from a polluted field near a petrochemical facility in México. We identified a putative oxidase that is induced when growth with phenanthrene as sole carbon source at a pH of 8.5 and is NADP+ dependent. We show that this enzyme bears naphthalene dihydrodiol dehydrogenase activity with substrate preference for the cis-naphthalene over the trans-naphthalene, with an optimal pH in the range of 8-10. Mass spectrometry analysis revealed that the induced enzyme belongs to the NADP+ oxidase family enzymes with the typical Rossmann-fold for NADP+ binding. This enzyme seems to form a high molecular weight structure (~541kDa) and with a monomer of 57kDa, suggesting that the multimer is constituted of 10 subunits. Our findings contribute to understanding of the roles that dihydrodiol dehydrogenases have in organisms exposed to toxic compounds in the environment and can regulate their expression.

Canthaxanthin production with modified Mucor circinelloides strains

Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.