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.     

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.     

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%。     

Enhanced sunflower oil utilization and gamma-linolenic acid production by Mucor circinelloides f.circinelloides CBS 108.16 in the presence of acetate

World Journal of Microbiology and Biotechnology -     

Characterization of the Mucor circinelloides life cycle by on-line image analysis

AIMS: The life cycle of the dimorphic fungus Mucor circinelloides was studied in a temperature-controlled flow-through cell, which constitutes an ideal tool when following the development of individual cells, with a view to understanding the growth and differentiation processes occurring in and between the different morphological forms of the organism. METHODS AND RESULTS: Mycelial growth and the transformation of hyphae into chains of arthrospores were characterized by image analysis techniques and described quantitatively. The influence of the nature (glucose and xylose) and concentration of the carbon source on specific growth rate and hyphal growth unit length were studied. The organism branched more profusely on xylose than on glucose while the specific growth rates determined were rather similar. Methods were developed to study the yeast-like growth phase of M. circinelloides in the flow-through cell, and combined with fluorescent microscopy which allowed new insights to bud formation. Additionally, numbers and distribution of nuclei in arthrospores, hyphae and yeasts were studied. CONCLUSIONS: The results give essential information on the morphological development of the organism. SIGNIFICANCE AND IMPACT OF STUDY: Development of any industrial process utilizing this organism will be dependent on the information obtained here for effective process optimization.     

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。     

Purification and characterization of a glutathione S-transferase from the fungus Cunninghamella elegans

Cunninghamella elegans grown on Sabouraud dextrose broth had glutathione S-transferase (GST) activity. The enzyme was purified 172-fold from the cytosolic fraction (120000 x g) of the extract from a culture of C. elegans, using Q-Sepharose ion exchange chromatography and glutathione affinity chromatography. The GST showed activity against 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, 4-nitrobenzyl chloride, and ethacrynic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel filtration chromatography revealed that the native enzyme was homodimeric with a subunit of M(r) 27000. Comparison by Western blot analysis implied that this fungal GST had no relationship with mammalian alpha-, mu-, and pi-class GSTs, although it showed a small degree of cross-reactivity with a theta-class GST. The N-terminal amino acid sequence of the purified enzyme showed no significant homology with other known GSTs.     

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.     

Potential use of Mucor circinelloides for the biological control of certain helminths affecting livestock reared in a care farm

A preliminary study to evaluate the possibilities of biological control procedures against parasites affecting livestock reared in a care farm has been conducted. Adults with mental disabilities were involved in spreading the spores of the filamentous fungus Mucor circinelloides directly onto the faeces, or as a food additive. In the first assay, the spores were sprayed directly onto the faeces of piglets and calves parasitised by roundworms (Ascaris suum) and liver flukes (Fasciola hepatica), respectively. In the second assay, the spores were mixed with on-farm mash feed. Participation of the adults in the experiments was fully satisfactory. In the manure sprayed Mucor spores, the viability of eggs of roundworms and flukes reduced by 53% and 74%, respectively. Significant reductions of viability of eggs of Ascaris (60%) and Fasciola (67%) in the faeces of piglets and calves given mash feed-added Mucor spores were achieved, which demonstrates their ability to survive in the digestive tract of the animals. It is concluded that biological control of parasites could be helpful to decrease the risk of infection in animals reared by intellectually disabled adults in a care farm, and it could motivate them to accomplish new tasks.     

昆虫谷胱甘肽S-转移酶的基因结构及其表达调控

谷胱甘肽S-转移酶（glutathione S-transferases, GSTs）属于一个超家族,目前已从20多种昆虫中克隆得到了近百个GSTs基因序列。这些基因分属于至少3个类别,Ⅰ（Delta）类,Ⅱ类和Ⅲ（Epsilon）类,其中Ⅰ类和Ⅲ类是昆虫特异性的类别。昆虫Ⅰ类GSTs基因通常由多基因家族编码,基因多态性在不同昆虫种类中差异很大。Ⅱ类基因的种类较少,基因的结构较简单,通常是单拷贝基因。Ⅲ类基因是最近才鉴定出来的新类别,目前仅在黑腹果蝇和冈比亚按蚊中明确了其在染色体上的定位。基因簇、可变剪接和基因融合等机制是导致昆虫GSTs基因多态性的主要原因。在抗性昆虫种群中,GSTs表达量的增加有mRNA水平的提高和基因扩增两种机制,但后一种机制的报道很少。GSTs活性的增加是由于属于一类或多类的多个同工酶的增量调控,也有少数是由于单个同工酶的增量调控。GSTs的表达受反式调控元件和顺式调控元件的调控。目前仅有少数含有调节基因的染色体大致位点和可能的调控元件得到鉴定。     

Use of thiopropyl Sepharose for the synthesis of an adsorbent for the affinity chromatography of glutathione S-transferase

Thiopropyl Sepharose 6B in the 2-thiopyridyl-activated form was used for the reversible immobilisation of reduced glutathione (GSH). The resulting affinity matrix was successfully tested as a sorbent for the partial purification of glutathione S-transferase (GST) from pig kidney. The specific elution of the enzyme was performed with 10 mM GSH in Tris-HCl buffer (pH 7.8), non-specific elution with 20 mM dithiotreitol (DTT) in the same buffer.     

The effect of bacterial glutathione S-transferase on morpholine degradation

Glutathione S-transferases (GSTs) constitute a large family of enzymes that catalyze the addition of glutathione to endogenous, or xenobiotic, often toxic electrophilic compounds. The effect of this enzyme in facilitating polychlorinated biphenyls degradation has been studied previously. Here the effects of induced cell-free extracts of Acinetobacter calcoaceticus and Pseudomonas aeruginosa (grown on hexadecane), and E. coli BL21 (induced with pGEX-2T plasmid on isothiopropylgalactoside) were recruited to facilitate morpholine degradation by Mycobacterium and were compared with non-induced strains. The results showed that all induced strains had significantly more GST activity compared to non-induced ones, and the strain with most GST activity, A. calcoaceticus BS, removed morpholine faster. Eukaryotic GST gene expressed in E. coli BL21 also could facilitate morpholine degradation by Mycobacterium, The same experiments performed with cell-free extracts of non-induced cells did not show any significant effects on morpholine removal. These results showed that there is a correlation between GST activity and acceleration of morpholine degradation.     

Characterization of the complex of glutathione S-transferase pi and 1-cysteine peroxiredoxin

Glutathione S-transferase pi has been shown to reactivate 1-cysteine peroxiredoxin (1-Cys Prx) by formation of a complex [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360-372]. A model of the complex was proposed based on the crystal structures of the two enzymes. We have now characterized the complex of GST pi/1-Cys Prx by determining the M_w of the complex, by measuring the catalytic activity of the GST pi monomer, and by identifying the interaction sites between GST pi and 1-Cys Prx. The M_w of the purified GST pi/1-Cys Prx complex is 50,200 at pH 8.0 in the presence of 2.5 mM glutathione, as measured by light scattering, providing direct evidence that the active complex is a heterodimer composed of equimolar amounts of the two proteins. In the presence of 4 M KBr, GST pi is dissociated to monomer and retains catalytic activity, but the K_m value for GSH is increased substantially. To identify the peptides of GST pi that interact with 1-Cys Prx, GST pi was digested with V8 protease and the peptides were purified. The binding by 1-Cys Prx of each of four pure GST pi peptides (residues 41-85, 115-124, 131-163, and 164-197) was investigated by protein fluorescence titration. An apparent stoichiometry of 1 mol/subunit 1-Cys Prx was measured for each peptide and the formation of the heterodimer is decreased when these peptides are included in the incubation mixture. These results support our proposed model of the heterodimer.     

Characterization of the safener-induced glutathione S-transferase isoform II from maize

The safener-induced maize (Zea mays L.) glutathione S-transferase, GST II (EC 2.5.1.18) and another predominant isoform, GST I, were purified from extracts of maize roots treated with the safeners R-25788 (N,N-diallyl-2-dichloroacetamide) or R-29148 (3-dichloroace-tyl-2,2,5-trimethyl-1,3-oxazolidone). The isoforms GST I and GST II are respectively a homodimer of 29-kDa (GST-29) subunits and a heterodimer of 29 and 27-kDa (GST-27) subunits, while GST I is twice as active with 1-chloro-2,4-dinitrobenzene as GST II, GST II is about seven times more active against the herbicide, alachlor. Western blotting using antisera raised against GST-29 and GST-27 showed that GST-29 is present throughout the maize plant prior to safener treatment. In contrast, GST-27 is only present in roots of untreated plants but is induced in all the major aerial organs of maize after root-drenching with safener. The amino-acid sequences of proteolytic fragments of GST-27 show that it is related to GST-29 and identical to the 27-kDa subunit of GST IV.Abbreviations CDNB 1-chloro-2,4-dinitrobenzene - DEAE di-ethylaminoethyl - FPLC fast protein liquid chromatography - GSH reduced glutathione - GST glutathione S-transferase - GST-26 26-kDa subunit of maize GST - GST-27 27-kDa subunit of maize GST - GST-29 29-kDa subunit of maize GST - R-25788 safener N,N-diallyl-2-dichloroacetamide - R-29148 safener 3-dichloroacetyl-2,2,5-trimethyl-1,3-oxazolidone - RPLC reverse phase liquid chromatography We are grateful to M-M. Lay, ZENECA AG Products (formerly ICI Americas), Richmond, Calif., USA for providing [¹⁴C] R-25788. ZENECA Seeds in the UK is part of ZENECA Limited.     

Black shank resistant tobacco by silencing of glutathione S-transferase

A glutathione S-transferase gene was amplified from cDNA of Nicotiana tabacum roots infected with Phytophthora parasitica var. nicotianae. The gene was cloned in sense and anti-sense orientation to an RNAi vector for induced gene silencing, and reduced expression of the gene was detected by RT-PCR. A statistically significant increase in resistance of N. tabacum to infection following gene silencing was found for glutathione S-transferase-silenced plants compared with control plants. Some defense genes were up-regulated in glutathione S-transferase-silenced plants during the interaction with the pathogen. This is the first evidence of the role of glutathione S-transferase as negative regulator of defense response.     

The effect of some antineoplastic agents on glutathione S-transferase from human erythrocytes

Glutathione S-transferase was purified from human erythrocytes and effects of some antineoplastic agents were investigated on the enzyme activity. The purification procedure was composed of Glutathione-Agarose affinity chromatography after preparation of erythrocytes hemolysate. Using this procedure, the enzyme, having the specific activity of 16.00 EU/mg proteins, was purified 1143-fold with a yield of 80%. The purified enzyme showed a single band on the SDS-PAGE. The effects of paclitaxel, cyclophosphamide, and gemcitabine, are antineoplastic agents, were examined on the in vitro enzyme activity of glutathione S-transferase and were determined to be inhibitors for the enzyme. IC₅₀ values were 0.23 mM for paclitaxel, 5.57 mm for cyclophosphamide, and 6.35 mM for gemcitabine. These constants were 0.182 ± 0.028 mM and 0.162 ± 0.062 mM for paclitaxel, 6.97 ± 0.49 mM and 10.50 ± 5.43 mM for cyclophosphamide, and 6.71 mM and 7.93 mM for gemcitabine, with GSH and CDNB substrates, respectively. Inhibition types of all inhibitors were noncompetitive.