Functional analysis of the cyclopiazonic acid biosynthesis gene cluster in Aspergillus oryzae RIB 40

The cyclopiazonic acid (CPA) nonproducing strain, Aspergillus oryzae RIB 40, does not biosynthesize cyclo-acetoacetyl-L-tryptophan (cAATrp) due to a truncation in the responsible PKS-NRPS gene. We found that RIB 40 converted cAATrp to 2-oxocyclopiazonic acid, the final product of CPA biosynthesis in A. oryzae. This indicates that the CPA biosynthesis gene cluster, except for the PKS-NRPS gene, is functional in RIB 40.     

Identification of a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene required for the biosynthesis of cyclopiazonic acid in Aspergillus oryzae

Cyclopiazonic acid (CPA) is a mycotoxin produced by several strains of Penicillium and Aspergillus species. Aspergillus oryzae strains used in fermented foods do not produce CPA; however, several wild-type A. oryzae strains produce CPA. Here, we identified a novel polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) gene involved in CPA production by comparing the telomere-adjacent region of a CPA-producing strain (A. oryzae NBRC 4177) with that of a nonproducing strain (A. oryzae RIB40). NBRC 4177 has an additional 17-18-kb sequence beyond the region corresponding to the telomere repeat in RIB40 and this additional regions contains 3' region of the PKS-NRPS gene, while RIB40 has only the 5' region of the PKS-NRPS gene. Gene disruption of the PKS-NRPS gene in NBRC 4177 resulted in elimination of CPA production. Thus, the PKS-NRPS gene is required for CPA biosynthesis, and the truncation of this gene is presumed as one of the determinants of CPA nonproductivity in A. oryzae RIB40.     

Identification of a Gene Involved in the Synthesis of a Dipeptidyl Peptidase IV Inhibitor in Aspergillus oryzae

WYK-1 is a dipeptidyl peptidase IV inhibitor produced by Aspergillus oryzae strain AO-1. Because WYK-1 is an isoquinoline derivative consisting of three l-amino acids, we hypothesized that a nonribosomal peptide synthetase was involved in its biosynthesis. We identified 28 nonribosomal peptide synthetase genes in the sequenced genome of A. oryzae RIB40. These genes were also identified in AO-1. Among them, AO090001000009 (wykN) was specifically expressed under WYK-1-producing conditions in AO-1. Therefore, we constructed wykN gene disruptants of AO-1 after nonhomologous recombination was suppressed by RNA interference to promote homologous recombination. Our results demonstrated that the disruptants did not produce WYK-1. Furthermore, the expression patterns of 10 genes downstream of wykN were similar to the expression pattern of wykN under several conditions. Additionally, homology searches revealed that some of these genes were predicted to be involved in WYK-1 biosynthesis. Therefore, we propose that wykN and the 10 genes identified in this study constitute the WYK-1 biosynthetic gene cluster.     

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.     

Presence and functionality of mating type genes in the supposedly asexual filamentous fungus Aspergillus oryzae

The potential for sexual reproduction in Aspergillus oryzae was assessed by investigating the presence and functionality of MAT genes. Previous genome studies had identified a MAT1-1 gene in the reference strain RIB40. We now report the existence of a complementary MAT1-2 gene and the sequencing of an idiomorphic region from A. oryzae strain AO6. This allowed the development of a PCR diagnostic assay, which detected isolates of the MAT1-1 and MAT1-2 genotypes among 180 strains assayed, including industrial tane-koji isolates. Strains used for sake and miso production showed a near-1:1 ratio of the MAT1-1 and MAT1-2 mating types, whereas strains used for soy sauce production showed a significant bias toward the MAT1-2 mating type. MAT1-1 and MAT1-2 isogenic strains were then created by genetic manipulation of the resident idiomorph, and gene expression was compared by DNA microarray and quantitative real-time PCR (qRT-PCR) methodologies under conditions in which MAT genes were expressed. Thirty-three genes were found to be upregulated more than 10-fold in either the MAT1-1 host strain or the MAT1-2 gene replacement strain relative to each other, showing that both the MAT1-1 and MAT1-2 genes functionally regulate gene expression in A. oryzae in a mating type-dependent manner, the first such report for a supposedly asexual fungus. MAT1-1 expression specifically upregulated an α-pheromone precursor gene, but the functions of most of the genes affected were unknown. The results are consistent with a heterothallic breeding system in A. oryzae, and prospects for the discovery of a sexual cycle are discussed.     

Draft Genome Sequence of Aspergillus oryzae Strain 3.042

Aspergillus oryzae is the most important fungus for the traditional fermentation in China and is particularly important in soy sauce fermentation. We report the 36,547,279-bp draft genome sequence of A. oryzae 3.042 and compared it to the published genome sequence of A. oryzae RIB40.     

Purification, characterization, and heterologous expression in Fusarium venenatum of a novel serine carboxypeptidase from Aspergillus oryzae.

A novel serine carboxypeptidase (EC 3.4.16.1) was found in an Aspergillus oryzae fermentation broth and was purified to homogeneity. This enzyme has a molecular weight of ca. 67,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its specific activity is 21 U/mg for carbobenzoxy (Z)-Ala-Glu at pH 4.5 and 25 degrees C. It has a ratio of bimolecular constants for Z-Ala-Lys and Z-Ala-Phe of 3.75. Optimal enzyme activity occurs at pH 4 to 4.5 and 58 to 60 degrees C for Z-Ala-Ile. The N terminus of this carboxypeptidase is blocked. Internal fragments, obtained by cyanogen bromide digestion, were sequenced. PCR primers were then made based on the peptide sequence information, and the full-length gene sequence was obtained. An expression vector that contained the recombinant carboxypeptidase gene was used to transform a Fusarium venenatum host strain. The transformed strain of F. venenatum expressed an active recombinant carboxypeptidase. In F. venenatum, the recombinant carboxypeptidase produced two bands which had molecular weights greater than the molecular weight of the native carboxypeptidase from A. oryzae. Although the molecular weights of the native and recombinant enzymes differ, these enzymes have very similar kinetic parameters.     

Cloning,nucleotide sequencing,and expression of the beta-galactosidase-encoding gene (lacA) from Aspergillus oryzae

lacA coding for beta-galactosidase (beta-gal) was cloned from the genomic DNA of Aspergillus oryzae RIB40. There were 9 exons in lacA and the coding region of 3,015 bp encoded a protein of 1,005 aa with a deduced molecular mass of 109,898. A. oryzae lacA was highly homologous to fungal beta-gals, with the highest aa identity of 70.7% to A. niger lacA, and also showed significant identity to acid beta-gals belonging to family 35 glycosyl hydrolases. Approximately 10 copies of lacA under control of A. oryzae glaA promoter were integrated into the chromosome of A. oryzae M-2-3. The recombinant strain expressed more than 700-fold of the beta-gal activity as compared to the wild type strain under induction by maltose.     

米曲霉(Aspergillus oryzae)RIB40中烯酮/烯酯还原酶的异源表达及性质分析

以米曲霉(Aspergillus oryzae)RIB40基因组DNA为模版,通过PCR扩增其烯酮烯酯还原酶(AspER)基因(asper)后连接到表达载体pET32a(+)上,在大肠杆菌BL21(DE3)中以可溶形式表达。通过Ni-NTA亲和色谱层析纯化后,蛋白纯度提高1.9倍,回收率为60.62%。根据分子筛凝胶层析结果推算,AspER以二聚体形式存在。性质分析表明此酶为依赖于NADPH的氧化还原酶,最适pH为7.0～8.0,最适温度为40℃。对2-环己烯酮Km和kcat值分别为(2.45±0.36)mmol/L和(4.4±0.4)×103s-1。底物谱分析发现AspER对马来酰亚胺及其衍生物有较高的活性,其中对2-甲基马来酰亚胺的转化率和e.e.值均高于99%。     

Contribution Ratios of amyA, amyB, amyC Genes to High-Level α-Amylase Expression in Aspergillus oryzae

Aspergillus oryzae strains express α-amylases abundantly, and the genome reference strain RIB40 has three α-amylase genes (amyA, amyB, and amyC). However, there is no information on the contribution ratios of individual α-amylase genes to total expression. In this study, we generated single, double, and triple disruptants of α-amylase genes by employing a strain (ΔligD) with high gene-targeting efficiency and pyrG marker recycling in A. oryzae. All the disruptants showed reduced activities of α-amylases, and the triple disruptant completely lost activity. Comparative analyses of the activities and mRNA amounts of the α-amylases suggest that the contribution of amyA to the α-amylase expression is smaller than those of amyB and amyC. The present study suggests that the ability to express a large amount of α-amylases in A. oryzae is attributed to gene duplication of genes such as amyB and amyC.     

A potato carboxypeptidase inhibitor gene provides pathogen resistance in transgenic rice

A defensive role against insect attack has been traditionally attributed to plant protease inhibitors. Here, evidence is described of the potential of a plant protease inhibitor, the potato carboxypeptidase inhibitor (PCI), to provide resistance to fungal pathogens when expressed in rice as a heterologous protein. It is shown that rice plants constitutively expressing the pci gene exhibit resistance against the economically important pathogens Magnaporthe oryzae and Fusarium verticillioides . A M. oryzae carboxypeptidase was purified by affinity chromatography and further characterized by mass spectrometry. This fungal carboxypeptidase was found to be a novel carboxypeptidase B which was fully inhibited by PCI. Overall, the results indicate that PCI exerts its antifungal activity through the inhibition of this particular fungal carboxypeptidase B. Although pci confers protection against fungal pathogens in transgenic rice, a significant cost in insect resistance is observed. Thus, the weight gain of larvae of the specialist insect Chilo suppressalis (striped stem borer) and the polyphagous insect Spodoptera littoralis (Egyptian cotton worm) fed on pci rice is significantly larger than that of insects fed on wild-type plants. Homology-based modelling revealed structural similarities between the predicted structure of the M. oryzae carboxypeptidase B and the crystal structure of insect carboxypeptidases, indicating that PCI may function not only as an inhibitor of fungal carboxypeptidases, but also as an inhibitor of insect carboxypeptidases. The potential impact of the pci gene in terms of protection against fungal and insect diseases is discussed.     

An internal region of rpoB is required for autogenous translational regulation of the beta subunit of Escherichia coli RNA polymerase.

In order to delineate the region involved in feedback regulation of the RNA polymerase beta subunit (encoded by rpoB), a collection of rpoB-lacZ translational fusions with different endpoints both upstream and downstream of the rpoB start site was assembled on lambda phage vectors. The extent of translational repression of beta was monitored by measuring beta-galactosidase levels in monolysogens of the fusions under conditions of increased intracellular concentrations of beta and beta' achieved via the induction of rpoBC expression from a multicopy plasmid. A construct containing as little as 29 bp upstream of the start of rpoB exhibited repression of beta-galactosidase activity to the same extent as a construct encoding the full upstream region. A construct which carried only 70 bp of the rpoB structural gene exhibited very little repression, while constructs which carried 126 or 221 bp of rpoB exhibited approximately the same degree of repression as a construct which carried 403 bp. These data suggest that the sequences important for feedback regulation of beta translation extend more than 70 bp into rpoB but are completely contained within a region which spans the sequences from 29 bp upstream to 126 bp downstream of the translational start site.