Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Isolation of nuclei from Aspergillus nidulans protoplasts

Intact nuclei were isolated from the protoplasts of the filamentous fungus Aspergillus nidulans. The large amounts of protoplasts required for such nuclear preparations were produced from young mycelia grown in liquid culture. For final purification of the crude nuclear fraction, a Nycodenz density-gradient centrifugation was applied. The resulting nuclei were of good purity and morphological state, as demonstrated by fluorescence microscopy and electronmicroscopy. The weight ratio of DNA:RNA:protein was 1:3.0:10.8 in the nuclear fraction.     

Endochitinase from Aspergillus nidulans implicated in the autolysis of its cell wall

An endochitinase from centrifuged autolyzed cultures of Aspergillus nidulans has been purified 100 times. The enzyme has Mw 27,000, pI of 4.8 units, pH optimum around 5 pH units. It is unstable at temperature greater than 70 degrees C and does not have a cation requirement. It is inhibited by Hg2+, Cu2+, Ca2+ and Ag+ and it does not have muramidase activity. The enzyme depolymerizes chitin rapidly with production of high molecular weight polysaccharides, and then slowly degrades these with production of N,N'-diacetylchitobiose. The enzyme hydrolyzes N,N',N'-triacetylchitotriose with production of N,N'-diacetylchitobiose and N-acetylglucosamine and this hydrolysis is inhibited by other chitin oligomers and N-acetylglucosamine. This enzyme hydrolyzes in the same way the chitin obtained from the cell wall of Aspergillus nidulans.     

The Aspergillus nidulans carnitine carrier encoded by the acuH gene is exclusively located in the mitochondria

The location of the Aspergillus nidulans carnitine/acyl-carnitine carrier (ACUH) was studied. ACUH with a His-tag at its N-terminus was over-expressed in Escherichia coli and purified by Ni(2+) affinity chromatography. The purified protein was utilised to raise polyclonal antibodies which were characterised by Western blotting. For localisation studies A. nidulans T1 strain, that contains the acuH gene under control of the strong promoter alcA(p), was derived. Results obtained demonstrate the exclusively mitochondrial localisation of ACUH and therefore exclude the targeting of the acuH gene product to the peroxisomal membrane.     

Carnitine acetyltransferase in pea cotyledon mitochondria

Purified pea cotyledon mitochondria did not oxidise acetyl-CoA in the presence of carnitine. However, acetylcarnitine was oxidised. It was concluded that acetylcarnitine passed through the mitochondrial membrane barrier but acetyl-CoA did not. Only a sensitive radioactive assay detected carnitine acetyltransferase in intact mitochondrion or intact mitoplast preparations. When the mitochondria or mitoplasts were burst, acetyl-CoA substrate was available to the matrix carnitine acetyltransferase and a high activity of the enzyme was measured. The inner mitochondrial membrane is there-fore the membrane barrier to acetyl-CoA but acetylcarnitine is suggested to be transported through this membrane via an integral carnitine: acylcarnitine translocator. Evidence is presented to indicate that when the cotyledons from 48-h-grown peas are oxidising pyruvate, acetylcarnitine formed in the mitochondrial matrix by the action of matrix carnitine acetyltransferase may be transported to extra-mitochondrial sites via the membrane translocator.     

Transformation of Aspergillus parasiticus using autonomously replicating plasmids from Aspergillus nidulans

Abstract A genetic transformation system for the aflatoxin-producing fungus Aspergillus parasiticus using two autonomously replicating plasmids from A. nidulans (ARp1 and pDHG25) is reported. Transformation frequencies using the plasmid pDHG25 were from 5 × 10² to 2.5 × 10⁴ transformants per 10⁶ viable protoplasts and μg DNA. The stability of the plasmids in the transformants was also studied. This transformation system offers a new opportunity to clone genes related to aflatoxin production using appropriate aflatoxin-defective mutants.     

应用实时荧光PCR技术检测构巢曲霉的初步研究

目的 根据构巢曲霉（Aspergillus nidulans）3-磷酸甘油醛脱氢酶（Glyceraldehyde-3-phosphate dehydrogenase,GAPDH）基因特异位点设计并合成Taqman探针及引物,建立构巢曲霉实时荧光 PCR检测方法。方法 应用lasergene7.1软件对构巢曲霉与13种常见曲霉主要包括黑曲霉（A.niger）、烟曲霉（A.fumigatus）、杂色曲霉（A.versicolor）、土曲霉（A.terrus）、黄曲霉（A. flavus）、温特曲霉（A.wentii）、寄生曲霉（A. parasiticus）、泡盛曲霉（A.awamori）、米曲霉（A. oryzae ）、棒曲霉（A.cavatus）、赤曲霉（A.ruber ）、亮白曲霉（A.ochraceus）及赭曲霉（A.ochraceus）GAPDH基因序列比对分析,在特异位点设计引物和探针,建立构巢曲霉实时荧光 PCR检测方法,并对该方法进行特异性及敏感性分析。结果 用曲霉属22种41株不同曲霉及其他属的12株病原真菌验证实验表明,所建立的荧光PCR方法特异性强;检测灵敏度可达4.03×10－12μg/ml的模板DNA。 结论 应用实时荧光PCR技术能够有效检测构巢曲霉,该方法具有特异、灵敏、快速等特点,可在实际工作中应用。     

pH regulation of penicillin production in Aspergillus nidulans

As shown by both bioassay and high-performance liquid chromatographic (HPLC) analysis, penicillin G production by Aspergillus nidulans is subject to regulation by the pH of the growth medium. Penicillin titres were highest at alkaline pH and in strains carrying mutations in the regulatory gene pacC which mimics the effects of growth at alkaline pH. They were lowest at acid pH and in strains carrying mutations in the palA, palB, palC, palE or palF genes which mimic the effects of growth at acid pH.     

Glycoengineering of Aspergillus nidulans to produce precursors for humanized N-glycan structures

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man₅GlcNAc₂ glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man_3-5GlcNAc₂ species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man₃GlcNAc₂ glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex_6-7HexNAc₂ structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man_3-5GlcNAc₂ species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures.     

微粒添加对棘白菌素B发酵过程的影响

阿尼芬净是一种新型的抗真菌药物,能够抑制各种致病念珠菌在活体内外的活性。棘白菌素B(Echinocandin B,ECB)是合成阿尼芬净的关键前体,其发酵单位的高低直接关系到阿尼芬净的价格及市场前景。文中考察了构巢曲霉在摇瓶发酵生产ECB的过程中,添加滑石粉、Al2O3、玻璃珠等微粒对其发酵单位的影响。发现微粒的粒径和添加浓度是菌体形态和ECB产量的关键影响因素,添加20 g/L滑石粉(d50=14.2μm)和7颗玻璃珠(d=6 mm)可使ECB发酵产量分别比对照提高33.2%和41.7%,达到1 262.9 mg/L和1 344.1mg/L。结果表明微粒的添加可以显著地改善丝状微生物发酵过程中的菌丝形态,提高其产物的发酵产量,为丝状微生物发酵过程的优化提供了一种重要手段。     

Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414–12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2‐DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans.     

Purification and characterization of laccase II of Aspergillus nidulans

Sexual development in Aspergillus nidulans is a morphogenetic differentiation process triggered by internal and environmental signals. As a first step in analyzing the developmental pathway at the molecular level, laccase II (EC 1.10.3.2), which is specifically expressed in early stages of fruitbodies, was isolated. The enzyme was purified to apparent homogeneity from a mutant strain (SMS1) in which the sexual cycle dominates and the number of cleistothecia is increased tenfold. Laccase II was enriched 560-fold to a specific activity of 892 U (mg protein)^–1. The apparent molecular mass was determined to be 80 kDa under denaturing conditions and to be 100–120 kDa under native conditions. The internal peptide sequences gained from the protein will allow the isolation of the corresponding gene as a first step in determining the key regulators of sexual development. Received: 8 January 1998 / Accepted: 14 April 1998     

构巢曲霉内切纤维素酶A10在毕赤酵母中的表达及重组蛋白质的表征

内切纤维素酶是纤维素酶高效降解纤维素的一个关键因子,已广泛应用于工业生物技术领域。对来源于腐生真菌构巢曲霉的一个内切纤维素酶进行过表达及详细的酶学性质研究,研究结果表明:该内切纤维素酶在摇瓶和发酵罐条件下都成功获得表达,发酵罐条件下的蛋白质表达量达到0.89 mg/mL;该酶的最适反应p H和温度分别为4.0和80℃,在pH 2.0–12.0之间表现出了很好的稳定性;在温度￡60℃时,该酶非常稳定,当温度370℃,酶的稳定性大大降低;Co~(2+)、Mn~(2+)和Fe~(2+)促进了CMCase活性,而Pb~(2+)、Ni~(2+)和Cu~(2+)等金属离子表现出了一定的抑制作用。因此,该构巢曲霉内切纤维素酶表现出了非常好的耐酸、耐碱以及一定的耐热性等性能,具有开发为商品酶的潜力,为深入开发构巢曲霉来源糖苷酶的应用奠定了基础。     

Solubilisation of a cell wall bound invertase in Aspergillus nidulans

Aspergillus nidulans produces an extracellular invertase when incubated in the presence of sucrose and about half of the activity produced was found to be associated with the mycelium. Sixty percent of this mycelial invertase could be solubilised by simple mechanical disruption. Among the agents tested for solubilisation of invertase, proteinase K and dithiothreitol were the most effective.     

Long-Distance Movement of Aspergillus nidulans Early Endosomes on Microtubule Tracks

In fungal hyphal cells, intracellular membrane trafficking is constrained by the relatively long intracellular distances and the mode of growth, exclusively by apical extension. Endocytosis plays a key role in hyphal tip growth, which involves the coupling of secretory membrane delivery to the apical region with subapical compensatory endocytosis. However, the identity, dynamics and function of filamentous fungal endosomal compartments remain largely unexplored. Aspergillus nidulans RabA^Rab5 localizes to a population of endosomes that show long range bidirectional movement on microtubule (MT) tracks and are labelled with FM4-64 shortly after dye internalization. RabA^Rab5 membranes do not overlap with largely static mature endosomes/vacuoles. Impaired delivery of dynein to the MT plus ends or downregulation of cytoplasmic dynein using the dynein heavy chain nudA1^ts mutation results in accumulation of RabA^Rab5 endosomal membranes in an abnormal NudA1 compartment at the tip, strongly supporting the existence in A. nidulans hyphal tips of a dynein loading region. We show that the SynA synaptobrevin endocytic recycling cargo traffics through this region, which strongly supports the contention that polarized hyphal growth involves the association of endocytic recycling with the plus ends of MTs located at the tip, near the endocytic internalization collar.