Homologs of Aflatoxin Biosynthesis Genes and Sequence of aflR in Aspergillus oryzae and Aspergillus sojae

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis in Aspergillus parasiticus and A. flavus was shown for A. oryzae and A. sojae. Homologs of the regulatory gene aflR were cloned and sequenced from A. oryzae and A. sojae.     

Chromosomal Location Plays a Role in Regulation of Aflatoxin Gene Expression in Aspergillus parasiticus

The nor-1 gene in the filamentous fungus Aspergillus parasiticus encodes a ketoreductase involved in aflatoxin biosynthesis. To study environmental influences on nor-1 expression, we generated plasmid pAPGUSNNB containing a nor-1 promoter-β-glucuronidase (GUS) (encoded by uidA) reporter fusion with niaD (encodes nitrate reductase) as a selectable marker. niaD transformants of A. parasiticus strain NR-1 (niaD) carried pAPGUSNNB integrated predominantly at the nor-1 or niaD locus. Expression of the native nor-1 and nor-1::GUS reporter was compared in transformants grown under aflatoxin-inducing conditions by Northern and Western analyses and by qualitative and quantitative GUS activity assays. The timing and level of nor-1 promoter function with pAPGUSNNB integrated at nor-1 was similar to that observed for the native nor-1 gene. In contrast, nor-1 promoter activity in pAPGUSNNB and a second nor-1::GUS reporter construct, pBNG3.0, was not detectable when integration occurred at niaD. Because niaD-dependent regulation could account for the absence of expression at niaD, a third chromosomal location was analyzed using pAPGUSNP, which contained nor-1::GUS plus pyrG (encodes OMP decarboxylase) as a selectable marker. GUS expression was detectable only when pAPGUSNP integrated at nor-1 and was not detectable at pyrG, even under growth conditions that required pyrG expression. nor-1::GUS is regulated similarly to the native nor-1 gene when it is integrated at its homologous site within the aflatoxin gene cluster but is not expressed at native nor-1 levels at two locations outside of the aflatoxin gene cluster. We conclude that the GUS reporter system can be used effectively to measure nor-1 promoter activity and that nor-1 is subject to position-dependent regulation in the A. parasiticus chromosome.     

黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生相关联

为探讨黄曲霉菌aflR基因启动子序列变异与黄曲霉毒素产生的关系,收集黄曲霉菌、米曲霉菌和寄生曲霉菌若干株。在有利于黄曲霉毒素产生的条件下培养后,提取各菌株的总RNA,RT-PCR法检测aflR基因的mRNA表达水平;并应用ELISA法检测各菌株产生黄曲霉毒素B1的情况。提取各菌株的基因组DNA,PCR扩增aflR基因启动子序列并测序。应用基因分析软件将不产毒素的黄曲霉菌与产毒黄曲霉菌的aflR基因启动子序列进行比较,找出不产毒菌株aflR基因启动子序列的变异位点。ELISA法和RT-PCR法结果表明,产毒的黄曲霉菌菌株均有明显的aflR基因转录,而在2株不产毒的黄曲霉菌菌株中,一株aflR基因无转录,另一株仅有较低水平的转录。序列比较结果表明,不产毒黄曲霉菌菌株的aflR基因启动子序列存在如下共同变异位点:-90、-236、-253、-262、-282位。米曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均为阴性,并且其aflR基因启动子序列中存在与上述不产毒黄曲霉菌菌株相同的变异位点。寄生曲霉菌产生黄曲霉毒素B1和aflR基因转录的检测均呈阳性,并且其aflR基因启动子序列的上述5个位点与产毒黄曲霉菌完全一致。在不产毒素的黄曲霉菌aflR基因启动子序列中发现了5个共同变异位点,实验结果提示这些变异位点可能与黄曲霉毒素的产生有关。     

Gene cloning, purification, and characterization of a novel peptidoglutaminase-asparaginase from Aspergillus sojae

Glutaminase is an enzyme that catalyzes the hydrolysis of l-glutamine to l-glutamate, and it plays an important role in the production of fermented foods by enhancing the umami taste. By using the genome sequence and expressed sequence tag data available for Aspergillus oryzae RIB40, we cloned a novel glutaminase gene (AsgahA) from Aspergillus sojae, which was similar to a previously described gene encoding a salt-tolerant, thermostable glutaminase of Cryptococcus nodaensis (CnGahA). The structural gene was 1,929 bp in length without introns and encoded a glutaminase, AsGahA, which shared 36% identity with CnGahA. The introduction of multiple copies of AsgahA into A. oryzae RIB40 resulted in the overexpression of glutaminase activity. AsGahA was subsequently purified from the overexpressing transformant and characterized. While AsGahA was located at the cell surface in submerged culture, it was secreted extracellularly in solid-state culture. The molecular mass of AsGahA was estimated to be 67 kDa and 135 kDa by SDS-PAGE and gel filtration chromatography, respectively, indicating that the native form of AsGahA was a dimer. The optimal pH of the enzyme was 9.5, and its optimal temperature was 50°C in sodium phosphate buffer (pH 7.0). Analysis of substrate specificity revealed that AsGahA deamidated not only free l-glutamine and l-asparagine but also C-terminal glutaminyl or asparaginyl residues in peptides. Collectively, our results indicate that AsGahA is a novel peptidoglutaminase-asparaginase. Moreover, this is the first report to describe the gene cloning and purification of a peptidoglutaminase-asparaginase.     

Comparison of Alkaline Proteinase from Hyperproductive Mutants and Parent Strain of Aspergillus sojae

Physico-chemical properties of alkaline proteinase from the parent strain were compared with those from hyperproductive mutants of Aspergillus sojae. All the results on behavior of enzyme protein to ion exchange resin and celluloses, gel filtration, ultracentrifugal sedimentation, disc electrophoresis and isoelectrofocusing on polyacrylamide gel column, specific activity, substrate specificity, and kinetic constants provided evidence in favor of the conclusion that the parent and mutant strains produced the chemically identical enzymes and that superactivity of alkaline proteinase in culture extracts or filtrates of mutant strains was not attributed to alteration of catalytic property of the enzyme, but to hyperproduction of the identical enzyme resulting from the genetic change in the regulatory mechanism of enzyme synthesis.     

Metabolism of l-Tyrosine in Aspergillus sojae

Circadian rhythmicity was investigated in isolated small intestine and mucosal epithelial cells from rats on restricted-feeding regimen (food available from 17:00 to 23:00 every day). In the isolated intestine, daily rhythms synchronized to meal-timing were found in the activity patterns of l-leucine, l-lysine and d-glucose transport, and mucosal γ-glutamyltransferase and sucrase, and in the rates of lactate formation from glucose; the nadirs occurred at 12:00 and the peaks at 23:00. These same patterns were also noted with the mucosal epithelial cells prepared at distinct times of day from rats on meal-feeding regimen. The fasted rat intestine responded to refeeding with prompt increase in transport activity, i.e., out of phase with the original rhythm. Intraperitoneal administration of cyloheximide suppressed the daily rise in leucine transport activity, indicating that the transport rhythm was entrained by or closely associated with the rhythmic fluctuation in protein synthesis in the epithelial cells, which in turns is cued by the feeding schedule. The kinetic parameters estimated for leucine transport, the apparent affinity constant for transport and the maximal transport rate, were significantly higher at high activity periods. It is suggested that the rhythmic increase in transport activity is not only associated with membrane hyperpolarization but may be mediated by the emergence of a high capacity transport system.     

Metabolism of Phenylalanine in Aspergillus sojae

It was found that a new compound of phenylalanine metabolites (2-hydroxy-3-phenylpropenoic acid) and phenylacetic acid were formed in the cultured Czapek medium containing phenylalanine by Aspergillus sojae. 2-Hydroxy-3-phenylpropenoic acid (HPPA) was formed from phenylalanine (d- and l-form) via phenyllactic acid (d- and l-form), and degraded to benzoic acid, p-hydroxybenzoic acid, protocatechuic acid, and catechol in this order.

On the other hand, phenylacetic acid was formed from phenylpyruvic acid, and converted to homogentisic acid via o-hydroxyphenylacetic acid. From these results, a metabolic pathway of phenylalanine in Asp. sojae was proposed.     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

An Aminopeptidase of Aspergillus sojae

An aminopeptidase was purified from Aspergillus sojae X–816. The molecular weight of the enzyme was estimated to be 220,000. The isoelectric point was at pH 5.3. The optimum pH for l-leucylglycylglycine was 7.5. The enzyme was stable up to 37°C against temperature treatment for 15 min. Some chelating agents inhibited the enzyme activity. The Km value for l-leucylglycylglycine at pH 7.5 and 37°C was 45 mm. The Km value for l-leucyl-β-naphthylamide at pH 7.0 and 37°C was 2.2 mm.     

Aflatoxin production by Aspergillus parasiticus in a competitive environment

Aspergillus parasiticus NRRL 2999 was grown in the presence of Rhizopus nigricans, Saccharomyces cerevisiae, Acetobacter aceti, or Brevibacterium linens and aflatoxin concentration was determined after 3,5,7, and 10 days of incubation at 28C. R. nigricans and S. cerevisiae inhibited growth and aflatoxin production by A. parasiticus. B. linens caused slight inhibition and A. aceti stimulated growth and aflatoxin production by A. parasiticus.     

Cloning of a sugar utilization gene cluster in Aspergillus parasiticus

At one end of the 70 kb aflatoxin biosynthetic pathway gene cluster in Aspergillus parasiticus and Aspergillus flavus reported earlier, we have cloned a group of four genes that constitute a well-defined gene cluster related to sugar utilization in A. parasiticus: (1) sugR, (2) hxtA, (3) glcA and (4) nadA. No similar well-defined sugar gene cluster has been reported so far in any other related Aspergillus species such as A. flavus, A. nidulans, A. sojae, A. niger, A. oryzae and A. fumigatus. The expression of the hxtA gene, encoding a hexose transporter protein, was found to be concurrent with the aflatoxin pathway cluster genes, in aflatoxin-conducive medium. This is significant since a close linkage between the two gene clusters could potentially explain the induction of aflatoxin biosynthesis by simple sugars such as glucose or sucrose.     

寄生曲霉脂肪酶的全基因合成、原核表达及其结构分析

根据寄生曲霉脂肪酶基因序列(GenBank登录号:AF404488)及大肠杆菌密码子的偏爱性,应用重叠延伸PCR技术,人工合成了寄生曲霉脂肪酶基因lipAP.将基因克隆到pFL-B13cl载体上获得重组质粒pFL-B13cl/Lip AP,并在大肠杆菌BL21(DE3)中进行优化表达.融合蛋白Sumo-LipAP在0.1 mmol/L IPTG、37℃条件下诱导6 h表达量最高.SDS-PAGE分析显示融合蛋白His-Sumo-LipAP以包涵体形式表达,大小约为53 kD.复性后透析处理,大部分融合蛋白转化为正确构象且具有催化活性,经一步疏水层析其纯度可达98％.生物信息学工具预测和分析发现,LipAP空间结构保守,具有催化三联体(Ser173-Asp226-His288)、活性部位盖子(S111YSIRNWVTDAT122)及保守五肽(GHSLG)3个功能域.