Physiological properties and fatty acid composition in Mucor circinelloides f. circinelloides

Sporangiospores of Mucor circinelloides f. circinelloides CBS108.16 could germinate and grow on a wide variety of carbon sources in synthetic liquid media. Growth was supported by aldoses which have the same configuration at carbon atom number two as glucose. Di- and trisaccharides consisting of D-glucopyranosyl moieties were assimilated, while polysaccharides like inuline and starch were also utilised. Various alcohols and organic acids could be assimilated, while the phenolic compounds tested could not support aerobic growth. The fungus was able to ferment carbohydrates consisting of D-glucopyranosyl moieties, grow in the absence of vitamins and in the presence of cycloheximide. It also liquefied gelatin and produced lipases and cellulolytic enzymes. It was found that the highest percentages polyunsaturated fatty acids were produced when acetic acid, glucose, mannitol, soluble starch or trehalose was used as carbon source. The absence of vitamins in the medium lowered the percentage of these fatty acids.     

Electrofusion of Mucor circinelloides protoplasts

Summary Electrical parameters were optimized for induction of the fusion of protoplasts formed from two auxotrophic (leu^–, ade^–) Mucor circinelloides strains. These proved to be a pulse voltage of 66 V, a pulse duration of 66 us and six pulses at intervals of 1.0 s. Nutritionally complementing colonies were obtained at an average frequency up to 12.4%, which is substantially higher than achieved earlier with the PEG-Ca²⁺ method.     

Agrobacterium tumefaciens-mediated transformation ofMucor circinelloides

The Agrobacterium tumefaciens-mediated transformation of the zygomycetous fungus Mucor circinelloides is described. A method was also developed for the hygromycin B-based selection of Mucor transformants. Transformation with the hygromycin B phosphotransferase gene of Escherichia coli controlled by the heterologous Aspergillus nidulans trpC promoter resulted in hygromycin B-resistant clones. The presence of the hygromycin resistance gene in the genome of the transformants was verified by polymerase chain reaction and Southern hybridization: the latter analyses revealed integrations in the host genome at different sites in different transformants. The stability of transformants remained questionable during the latter analyses.     

Carotenoid Mutants of Mucor circinelloides

The wild-type of the filamentous fungus Mucor circinelloides accumulates the yellow pigment β-carotene. At a continuous blue-light fluence rate of 0.1 W/m² the β-carotene content increases about eight fold over the dark controls. Among the mutants isolated after exposure of spores to either N-methyl-N'-nitro-N-nitrosoguanidine or ICR-170, a red mutant accumulating lycopene, white mutants accumulating phytoene and white mutants without carotenoids were found. The biosynthesis of carotenoids in M. circinelloides shows similarities with that of the fungus Phycomyces blakesleeanus such as the presence of mutants in the same structural genes and the induction by light of the pathway. However, negative end-product regulation by β-carotene on the biosynthetic pathway, as in Phycomyces, is absent in M. circinelloides. In contrast to Phycomyces carB and carR mutants, carotenoids in corresponding mutants of M. circinelloides are photoinduced.     

Mucor circinelloides a destructive hyperparasite of Rhizopus nigricans

In vitro studies on hyphal interactions between phyllosphere and phylloplane fungi revealed Mucor circinelloides to be a destructive hyperparasite of Rhizopus nigricans. The mycoparasite penetrated the host hypha and formed sporangiophores and sporangia on different parts of Rhizopus nigricans.     

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.     

卷枝毛霉孢子原生质体的制备与再生

为了构建高产γ-亚麻酸的卷枝毛霉稳定遗传转化体系,利用酶解法对卷枝毛霉(Mucor circinelloides sp.)EIM-10的孢子进行原生质体制备。研究酶液组成、渗透压稳定剂、酶解温度、酶解时间等对卷枝毛霉孢子原生质体形成和再生的影响,建立了制备卷枝毛霉孢子原生质体的最适条件:1%纤维素酶和2%溶壁酶为酶解体系,0.5mol/L NaCl作为渗透压稳定剂,酶解温度32℃,酶解时间2.5 h,再生培养基为0.5 mol/L NaCl高渗培养基。用双层平板培养法进行原生质体再生,在此条件下原生质体的形成量为1.2×106个/mL,再生率为70.5%。     

Acetate-enhanced polymerized triacylglycerol utilization by Mucor circinelloides

We found that laboratory-prepared sunflower oil waste containing polymerized triacylglycerols (PTAGs) still within South African regulatory levels (i.e. 1, 5, 10 and 15% w/w) is effectively degraded by the fungus Mucor circinelloidesin the presence of acetate. Poor utilization was experienced in its absence. These results suggest that fungi could be used in oil waste removal, while at the same time biomass, rich in essential fatty acids such as linoleic and gamma-linolenic acid is produced.     

Two alternative pathways for substrate assimilation byMucor circinelloides

Transition of an oleaginous strain ofMucor circinelloides from mycelial to yeast-like form was studied in conditions favoring lipogenesis. In culture media with citric acid as the sole carbon source at concentrations of 7.5 and 10 g/L (C/N ratio of 26 and 35, respectively), the mold accumulated significant quantities of lipid. At a higher citric acid concentration liposynthesis was inhibited and the fermentation mechanism decreased the high substrate concentration in the culture media. Under these conditions yeast-like morphogenesis was observed. In the yeast-like cells, biosynthesis of linoleic and γ-linolenic acid was inhibited. In contrast, no significant changes were observed in the biosynthesis of palmitoleic acid whereas the concentration of oleic acid was increased in the storage lipids of yeast-like cells.     

Critical applications of Mucor circinelloides within a biorefinery context

The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.     

Detection of 3-nitropropionic acid and cytotoxicity inMucor circinelloides

Mucorales are regarded as the aetiological agents of Mucormycosis. Their capabilities to produce mycotoxins are not profoundly investigated, in contrast to those of the fungi from the generaPenicillium, Aspergillus, orFusarium. The aim of this study was to isolate and identify fungi of the order Mucorales and investigate mycotoxins production. Twelve samples of visibly moulded grass silage and eight samples of damaged whole crop maize silage were analysed. Malt extract agar plates were used for sub cultivation. Three fungal species of the order Mucorales were isolated from grass silage, which were identified by their macro-and micro-morphology asAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer. The cytotoxicity ofMucor circinelloides extract was analysed using the cytotoxicity test (MTT assay) and the result, showed a low cytotoxicity. Additionally extracts fromAbsidia corymbifera, Mucor circinelloides andRhizopus stolonifer were tested for mycotoxin-production using an LC/MS/MS-based multimycotoxin method. 3-nitropropionic acid was detected in the culture extract ofMucor circinelloides. Presented at the 30^th Mykotoxin Workshop Utrecht, Netherlands, April 28–30, 2008.     

Integrative transformation by homologous recombination in the zygomycete Mucor circinelloides

Summary Transformation of a Mucor circinelloides Leu^– strain with the plasmid pAD45, harbouring the wild-type allele (leuA⁺) and a chymosin gene, led to the identification of mitotically stable transformants after one to three vegetative growth cycles on non-selective medium. Southern analysis of the stable transformed strains demonstrated that the vector is integrated, as an intact molecule, into the resident Mucor leuA locus. Retransformation of Escherichia coli with genomic DNA restricted with enzymes having no or only a single recognition site within the inserted sequence did not permit isolation of plasmids or fragments carrying the leuA or chymosin gene.     

Isolation and purification of Mucor circinelloides intracellular chitosanolytic enzymes

This study aimed at isolation, purification and characterization of a chitosanase from Mucor circinelloides mycelium. The latter contains also a mycelium-bound lipase and lipids. The chitosanase and lipase were extracted from defatted M. circinelloides mycelium with a detergent and purified through a two-step procedure comprising chromatography on bacitracin–CNBr-Sepharose 4B and gel filtration on Sephadex G-100. Purification degree of the chitosanase (endo-type enzyme) and lipase was 23 and 12, respectively. These enzymes were optimally active at pH of 5.5–6.0 (chitosanase) and 7.2 (lipase in olive oil hydrolysis) and at 37 °C. Both purified enzymes were activated by Ca²⁺ and Mg²⁺ ions. The preferred substrates of chitosanase were chitosan preparations with a high degree of deacetylation. This enzyme showed no activity for colloidal chitin, Na-CMC and starch. SDS–PAGE of both purified enzymes showed two bands with molecular masses of 42 and 43 kDa. Our results suggest that M. circinelloides synthesizes an oligomeric (bifunctional) lipase which also efficiently depolymerizes chitosan.     

短刺小克银汉霉与卷枝毛霉对昂丹司琼的转化

采用微生物转化法考察11株放线菌及11株小型丝状真菌对昂丹司琼的转化能力,通过高效液相色谱-多级质谱(HPLC-MSn)检测转化产物。7株真菌可将昂丹司琼转化为7-羟基昂丹司琼和N-去甲基昂丹司琼,与文献中报道的人体内主要代谢产物相同,其中短刺小克银汉霉AS 3.153转化能力最强,在优化的转化系统中7-羟基昂丹司琼和N-去甲基昂丹司琼的产率分别达到57.80%和15.60%。此外,3株真菌和7株放线菌将昂丹司琼转化为1-羟基昂丹司琼,其中卷枝毛霉AS 3.3421转化能力最强,在优化的转化系统中,1-羟基异构体的总产率达43.10%。表明筛选出的2模型菌株对形成昂丹司琼的类哺乳动物代谢产物具有互补能力,在确定药物代谢产物种类及制备相应的对照品中具有应用价值,可作为药物代谢体外研究的有效辅助工具。     

Molecular characterization of a G protein alpha-subunit-encoding gene from Mucor circinelloides

Genes encoding the Galpha subunit were cloned from Mucor circinelloides, a zygomycete dimorphic fungus. There are at least four genes that encode for Galpha subunits, gpa1, gpa2, gpa3, and gpa4. The genes gpa1 and gpa3 were isolated and characterized, and their predicted products showed 36%-67% identity with Galpha subunits from diverse fungi. Northern blot analysis of gpa3 showed that it is present in spores and constitutively expressed during mycelium development and during yeast-mycelium and mycelium-yeast transitions. However, during yeast cell growth, decreased levels of mRNA were observed. Sequence analysis of gpa3 cDNA revealed that Gpa3 encodes a polypeptide of 356 amino acids with a calculated molecular mass of 40.8 kDa. The deduced sequence of Gpa3 protein contains all the consensus regions of Galpha subunits of the Galpha(i/o/t) subfamily except the cysteine near the C terminus for potential ADP-ribosylation by pertussis toxin. This cDNA was expressed in Escherichia coli and purified by affinity chromatography. Based on its electrophoretic mobility in SDS-PAGE, the molecular mass of the His6-tagged Gpa3 was 45 kDa. The recombinant protein was recognized by a polyclonal antibody against a fragment of a human Galpha(i/o/t). Furthermore, the recombinant Gpa3 was ADP-ribosylated by activated cholera toxin and [32P]NAD but not by pertussis toxin. These results indicate that in M. circinelloides the Galpha subunit Gpa3 is expressed constitutively during differentiation.     

High reliability transformation of the basal fungus Mucor circinelloides by electroporation

Molecular approaches to study the biology of the zygomycete Mucor circinelloides depend mainly on the existence of a polyethylene glycol-based transformation method, which is one of the most efficient in zygomycete fungi. However, the poor reliability and low transformation rates of this method are major obstacles in the molecular study of a number of biological processes. This paper describes an easy and reliable method to transform M. circinelloides protoplasts by electroporation. A high-voltage pulse of 25 μF capacitance, 400 Ω resistance, and 4 kV/cm field strength were seen to be the optimal electrical conditions for delivering DNA into M. circinelloides protoplasts. Under these electrical conditions, successful transformations were carried out with several self-replicative plasmid and strain combinations, producing up to more than 500 transformants per μg DNA. Targeted DNA integration of a transgene (atfA gene of Acinetobacter baylyi) in a particular locus (carRP) was also achieved. This transformation method will considerably facilitate in-depth molecular genetic studies of the biology of this fungus.     

卷枝毛霉pyrG基因缺陷突变株的诱变筛选与鉴定

为制备新的遗传筛选标记用于构建高产番茄红素的工程菌株,实验运用化学诱变的手段,以N-甲基-N'-硝基-N-亚硝基胍为诱变剂,以番茄红素生产菌株卷枝毛霉MU616为出发菌株,诱变获得5株尿嘧啶缺陷型突变株,突变株在基本培养基中培养5天后仍不能生长。通过同源转化带有pyrG基因(编码乳清酸核苷-磷酸盐脱羧酶)的质粒pEPM1确定突变株Mt1、Mt4和Mt5为pyrG基因缺陷突变株。随之对pyrG突变株进行生长特性的研究和产番茄红素性能的检测,结果表明,其中突变株Mt4的生物量为(9.0±0.6)g/L,番茄红素产量在黑暗和光照条件下分别为(1 648±185)μg/g和(3 234±281)μg/g,均与出发菌株相似,适宜作为进行卷枝毛霉转化的带有遗传标记的受体菌。pyrG基因缺陷突变菌株的获得对构建高产番茄红素的卷枝毛霉工程菌具有重要的意义。