Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays

Aspergillus species included in section Nigri are common in plant products and processed food, such as grapes, cereals, coffee and derivatives, particularly in warm and tropical climates. Two of these species, A. carbonarius and A. niger, are known to produce ochratoxin A (OTA), a potent nephrotoxin and carcinogenic to human (group 2B). Recognition of the several species of this section is difficult and requires considerable expertise using conventional methods based on morphological features. In this work we describe rapid, sensitive and robust assays based on the PCR technique to discriminate the main species included in section Nigri: A. japonicus, A. heteromorphus, A. ellipticus and the two morphologically indistinguishable species of the A. niger aggregate: A. niger and A. tubingensis. The species-specific primers have been designed on the basis of ITS (internal transcribed spacers of rDNA units) sequence comparisons obtained from several Aspergillus strains and have been tested in a number of strains from different origins and hosts. These PCR assays, based on multi-copy sequences, are highly sensitive and specific and represent a good tool for an early detection of OTA-producing Aspergillus species in order to prevent OTA from entering the food chain.     

产赭曲霉毒素A黑曲霉的PCR法检测

【目的】快速检测产赭曲霉毒素A(OTA)的黑曲霉。【方法】根据黑曲霉(Aspergillus niger)CBS513.88中An15g07920基因编码聚酮合酶的酰基转移酶(AT)域设计引物,建立针对产OTA黑曲霉的聚合酶链式反应(PCR)检测方法。【结果】对72株曲霉属菌株(黑曲霉、炭黑曲霉、赭曲霉、佩特曲霉、寄生曲霉和塔宾曲霉)进行检测,发现产OTA的黑曲霉能够扩增出特异性条带,而产OTA的其它菌株不能扩增出条带;检测出3株假阳性的产OTA黑曲霉,实时定量PCR分析此3株菌中An15g07920的同源基因表达情况,发现在产毒条件下可正常表达,排除了因基因无法表达导致假阳性的可能。本方法的检测灵敏度为25 pg的DNA含量,在污染所试农产品孢子浓度大于4.0×10~4–4.0×10~5个/g时可有效检测出产毒菌株。【结论】本方法虽会产生4%的假阳性结果,但是仍可作为产毒黑曲霉有效的快速检测方法,并在农产品污染产毒黑曲霉时进行有效预警。     

黑曲霉发酵过程中菌体形态的分析方法建立及应用

丝状真菌(Filamentous fungi)的发酵生产通常具有较高的工业应用价值,但其菌体形态是一个区别于其他非丝状菌的一个重要发酵指标。针对目前形态分析的瓶颈,本研究使用琼脂糖凝胶对黑曲霉菌形进行固定,利用平板实现菌球样本的大量制备,并结合图形处理软件自建自动化处理程序,实现了大量准确可靠的菌体形态参数的获得,大大增加了形态数据处理通量及准确度。应用该方法于黑曲霉发酵生产糖化酶过程中不同供氧水平及剪切水平下菌体形态的研究,通过大量形态数据定量阐明了黑曲霉在不同剪切水平下的分区域形态分布特性,为进一步工业过程的形态优化提供了重要的研究方法。     

烟曲霉菌热稳定性植酸酶在黑曲霉菌中的分泌性表达

在先前克隆获得烟曲霉菌植酸酶phyA基因并构建了重组质粒的基础上,将该质粒转化黑曲霉菌pyrG基因缺陷株M54;同时制备植酸酶多克隆抗体用于植酸酶的免疫学检测。SDS-PAGE和western-blot结果表明,phyA在黑曲霉菌中获得分泌性表达。表达产物活性测定结果显示,重组植酸酶的表达量为597.6 IU/mL。在90℃加热10 min和100℃加热20 min后,重组植酸酶残余酶活分别为74%和70%,具有较好的热稳定性。实现了烟曲霉菌植酸酶在黑曲霉菌中的分泌性表达,表达产物具较高的生物活性和耐热性。     

Transformation of Aspergillus alliaceus using the Aspergillus niger niaD gene encoding nitrate reductase

Abstract A heterologous transformation system for Aspergillus alliaceus based on the Aspergillus niger nitrate reductase structural gene ( niaD ) has been developed. Two mutants of A. alliaceus (M3 and M17), each carrying an niaD mutation were isolated by screening UV-irradiated cells for the inability to grow on nitrate as sole nitrogen source. Using plasmid pSTA 10, transformation frequencies of 4 and 200 per μg DNA respectively were obtained for these two strains. All the niaD ⁺ transformants tested were mitotically stable. Southern hybridisation analyses showed that the vector DNA sequences were present.     

New metal-binding ethyldiamino- and dicarboxy-products from Aspergillus niger industrial wastes

The metal-binding ability of Aspergillus niger mycelial waste was improved by chemical modification. The latter was performed by introducing additional carboxy groups using oxidation methods or the introduction of the ethyldiamino group first by chlorination of A. niger using mesyl chloride and subsequent reaction of the product with ethylene diamine. Metal binding abilities of the products for Cd²⁺, Co²⁺, Ni²⁺ and Zn²⁺ were determined according to the Langmuir model, whereby pK _D ^* -values of 3.88 up to 5.02 were revealed. Maximum capacities for the metals were found to be in the range 172 to 1064 mmol/kg.     

米曲霉和黑曲霉营养缺陷型的分离及原生质体的制备

米曲霉(Aspergillus oryzae)3042是目前国内酱油生产中广泛使用的菌种,而黑曲霉(Aspergillus niger)3350则是制醋业中广泛使用的菌种。前者具有较高的蛋白酶活性而后者具有较高的淀粉酶活性。在酱油生产中,为了提高原料利用率,改善酱油风味,希望获得一株既有较高的蛋白酶活性同时又具有较高淀粉酶活性的杂交菌株作为     

Kinetic studies on the aggregation of Aspergillus niger conidia

Morphology has a crucial effect on productivity and the supply of substrate for cultures of filamentous fungi. However, cultivation parameters leading to the desired morphology are often chosen empirically as the mechanisms governing the processes involved are usually unknown. For coagulating microorganisms like Aspergillus niger the morphological development is considered to start with the aggregation of conidia right after inoculation. To elucidate the mechanism of this process, kinetic studies were carried out using an in-line particle size analyzer. Based on the data obtained from these experiments a model for conidial aggregation is proposed in this article. It consists of two separate aggregation steps. The first one takes place immediately after inoculation, but only leads to a small decrease of total particle concentration. Most suspended conidia aggregate after a second aggregation step triggered by germination and hyphal growth. Aggregation velocity of this second phase is linearly dependent on the particle growth rate.     

Mitochondrial location of pyruvate carboxylase in Aspergillus niger

Pyruvate carboxylase has been found in the mitochondrial fraction of two strains of Aspergillus niger along with the marker enzymes of citrate synthase and cytochrome c oxidase. The location of pyruvate carboxylase in A. nidulans was, however, confirmed to be in the cytosolic fraction. The enzyme from the former sources was dependent upon the presence of acetyl-CoA for full activity; the enzyme from A. nidulans was unaffected by the presence or absence of acetyl-CoA.     

蓝光诱导促进黑曲霉产糖化酶的研究

考察了蓝光对黑曲霉产糖化酶的影响并采用扫描电镜观察蓝光下黑曲霉形态发育过程,结果表明,与黑暗对照组相比,蓝光处理使菌丝粗壮,孢囊增大,分生孢子发育提前,黑曲霉糖化酶活力增加,孢子发育和产糖化酶的进程有一定的对应性。黑曲霉在黑暗下生长至36h时,经蓝光诱导糖化酶产量提高更为明显,提示了黑曲霉存在一个对蓝光反应产生最适光感应的发育阶段,对于光调节黑曲霉产糖化酶来说,蓝光诱导的光强由弱到强,比持续蓝光培养或采用较高光强诱导效果更好,表明黑曲霉产糖化酶存在一种光适应机制,能够感应和适应光强度变化,调节其自身代谢。从抑制性扣除杂交实验和蓝光光强变化对差异基因表达的分析来看,糖化酶基因以及呼吸链中部分氧化还原酶基因在蓝光诱导下表达皆有增强,蓝光信号转导影响了核基因编码的线粒体呼吸链相关酶基因表达水平,交替氧化酶可能参与了蓝光信号途径,影响了黑曲霉产糖化酶和孢子发育。研究结果可为在现有水平上应用蓝光调节提高糖化酶产量找到新的技术突破口和提供新思路。     

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding beta-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.     

Physical mapping of the mitochondrial DNA from Aspergillus niger

Abstract The mitochondrial DNA was isolated from Aspergillus niger WU-2223L, a citric acid-production strain, and characterized by restriction-endonuclease mapping. Cloned fragments which covered the total range of the mitochondrial DNA were assembled and utilized to construct the restriction-endonuclease map for nine restriction enzymes. This map showed that the mitochondrial DNA was a circular molecule of 32.6 kb.     

Biotransformation of dopamine to norlaudanosoline by Aspergillus niger

Norlaundanosoline is a key intermediate in the synthesis of the benzylisoquinoline alkaloids providing the upper isoquinoline portion of the morphinan skeleton. This study evaluates the feasibility of using Aspergillus niger as an in situ biotransformation system to produce norlaudanosoline from dopamine. A. niger was chosen because monoamine oxidase can be readily induced in this organism. Monoamine oxidase catalyzes the conversion of dopamine to 3,4-dihydroxyphenylacetaldehyde. In the presence of dopamine, this aldehyde will then undergo a spontaneous Picket-Spengler condensation to form norlaudanosoline. Fermentation condition to form norlaudanosoline. Fermentation conditions were optimized for the production monoamine oxidase by using a two-stage process consisting of a growth stage and an induction stage. pH control was found to be important, and at pH 4.5 dopamine accumulation in the cells was high as was the level of monoamine oxidase. With pH control at 4.5, up to 21% of the cellular dopamine was converted to norlaudanosoline. It is proposed that with further protein engineering improvements, this system may prove suitable for the in situ bio-transformation of dopamine to norlaudanosoline.     

The Aspergillus niger acuA and acuB genes correspond to the facA and facB genes in Aspergillus nidulans

Mutants in Aspergillus niger unable to grow on acetate as a sole carbon source were previously isolated by resistance to 1.2% propionate medium containing 0.1% glucose. AcuA mutants lacked acetyl-CoA synthetase (ACS) activity and acuB mutants lacked both ACS and isocitrate lyase activity. An acuA mutant was transformed to the acu+ phenotype with a clone of ACS (facA) from Aspergillus nidulans. The acuB mutant was transformed with the A. niger facB clone which has been identified by cross-hybridisation of an A. nidulans facB clone. These results confirm that acuA in A. niger is the gene for ACS and acuB is analogous to the A. nidulans facB regulatory gene.     

Isolation and characterization of six polymorphic microsatellite loci in Aspergillus niger

Certain strains of Aspergillus niger produce ochratoxin A in food and in animal feeds. Six polymorphic microsatellite markers suitable for population analysis were developed for A. niger through screening published sequences for microsatellite repeats. Polymorphism was evaluated for 28 isolates of A. niger, including toxigenic strains. Loci displayed six to 13 alleles. Investigation of cross‐species amplifications with Aspergillus carbonarius and Aspergillus japonicus showed limited success.     

黑曲霉果胶裂解酶的生物信息分析

用生物信息方法对果胶裂解酶(PNL)基因的核酸序列及其推导氨基酸序列的组成、亚细胞定位、疏水性/亲水性以及二、三级结构等进行分析.结果表明,黑曲霉的PNL为具有一定亲水性的稳定酸性分泌蛋白,具有明显的信号肤,无跨膜结构区,保守功能结构域为Pee_lyase_C.二级结构主要构成是不规则卷曲,具有以β片层结构为基础的相似三维空间结构.     

农杆菌介导凝乳酶基因转化黑曲霉载体的构建

目的:研究针对黑曲霉中高表达的糖化酶基因( glaA)位点,构建含有潮霉素抗性的农杆菌介导的牛凝乳酶基因转化黑曲霉基因置换载体,并在潮霉素基因两端设计了正向重复序列,使在后续的研究中消除抗性基因成为可能.方法:将glaA上游( Gla5)和下游(Gla3)片段作为同源臂,通过重叠延伸PCR技术连接在Cym基因两端构成GlaA5-Cym-GlaA3( CYM)片段,并在潮霉素基因下游引入与Cym基因下游方向相同的Gla3,通过中间载体获得GlaA5-Cym-GlaA3-hph-Gla3结构.结果:将上述结构克隆至在T-DNA区内只含有多克隆位点的Ti质粒载体pSZA,经过酶切鉴定,成功获得载体pSZH-CYM.     

Ultraviolet mutagenesis of the cellulase producer Aspergillus niger using protoplasts

Abstract Ultraviolet mutagenesis of Aspergillus niger strain O97 was achieved using protoplasts. Protoplasts of A. niger O97 showed the same ultraviolet killing kinetics as intact cells. After mutagenesis, several mutants were found in regenerated collonies. These mutants differ from the original strain in spore colour and cellulase-producing ability. The most active strain, designated 97V3-3 has an altered spore colour, and its carboxymethylcellose-hydrolysing, filter-paper-degradation, cotton-de-gradation and β-glucosidase activities were increased by 45.4%, 19.1%, 28.2% and 18.2%, respectively.