Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

β-Carotene inhibition of aflatoxin biosynthesis amongAspergillus flavus genotypes from Illinois corn

Thirty-nineAspergillus flavus genotypes (DNA fingerprinting) isolated from corn grown in a field near Kilbourne, Illinois were evaluated for their sensitivity to β-carotene (50 μg/ml) inhibition of aflatoxin B₁ biosynthesis. Inhibition of aflatoxin was greater than 90% for 28 of the genotypes and >70% for 38 of the 39 genotypes. FiveA. flavus strains (4 fingerprint groups) isolated from molded raw peanuts, NRRL 3239, NRRL 3357, NRRL 6514, NRRL 6515 and NRRL 13135, produced greater quantities of aflatoxin than all 39 genotypes isolated from corn, and were less sensitive to β-carotene inhibition.Aspergillus flavus NRRL 3357 is commonly used as inoculum in variety trials for aflatoxin resistance. Isolate identity and sensitivity to potential inhibitors in corn can be critical in assessing corn resistance to aflatoxin.     

First report of an atypical new <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> isolates with nucleotide insertion in <Emphasis Type="Italic">aflR</Emphasis> gene resembling to <Emphasis Type="Italic">A. sojae</Emphasis>

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B₁, B₂, G₁, and G₂ and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153–1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.     

Enhanced gene targeting frequency in ku70 and ku80 disruption mutants of Aspergillus sojae and Aspergillus oryzae

In the koji molds Aspergillus sojae and Aspergillus oryzae, exogenous DNA is integrated in the genome, in most cases irrespective of the sequence homology, suggesting that DNA integration occurs predominantly through a nonhomologous end joining pathway where two ku genes, namely, ku70 and ku80, play a key role. To determine the effect of ku gene disruption on the gene targeting frequency, we constructed ku70-, ku80-, and ku70–ku80-disrupted strains of A. sojae and A. oryzae. The gene targeting frequency of the tannase gene in ku70 and ku80 strains of both Aspergillus species was markedly enhanced as compared with that of the parental strains. The gene targeting frequency of the aflR and ku80 genes was also enhanced in an A. sojae ku70 background. Therefore, the koji mold strains with ku-disrupted genes will be excellent tools as hosts for efficient gene targeting.     

Population structure of Magnaporthe oryzae isolates from green foxtail in Japan examined by DNA fingerprint analysis

The population structure of Magnaporthe oryzae from green foxtail (Setaria viridis) in Japan was examined by DNA fingerprint analyses using the transposable elements MGR586 and MAGGY as probes. Fifteen M. oryzae isolates from green foxtail were collected from 11 Japanese prefectures so that a macrogeographic population of this pathogen is represented. All the 15 isolates were sorted into distinct haplotypes by DNA fingerprint analyses with both probes. Furthermore, similarities between the DNA fingerprint profiles of the 15 isolates were exclusively low; i.e., if lineages are arbitrarily established based on greater than 70% similarities in isolates, the 15 isolates could be categorized into 13 distinct lineages by DNA fingerprinting with both probes. We also examined the MGR586 DNA fingerprint variations of this pathogen in 9 microgeographic populations each of which contained 20 to 24 isolates collected from a 1 m² or 50 m² area. In all the 9 populations, more than 2 haplotypes, which shared less than 70% similarities, were identified in the DNA fingerprint profiles. These results suggested that M. oryzae isolates from the green foxtail in Japan possessed a complex lineage structure, even at the microgeographic scale.     

A rapid identification method for aflatoxin-producing strains ofAspergillus flavus andA. parasiticus by ammonia vapor

The colony reverse of aflatoxin (AF)-producing strains ofAspergillus flavus andA. parasiticus turned pink when their cultures were exposed to ammonia vapor. The color change was visible for colonies grown on media suitable for AF production such as potato dextrose, coconut, and yeast extract sucrose agars after 2 d incubation at 25°C. Of the 120 strains ofA. flavus, A. parasiticus, and two related species inA. flavus group:A. oryzae andA. sojae tested in this study, only the AF-producing strains ofA. flavus andA. parasiticus showed the pink pigmentation. The color change occurred immediately after the colony was contacted with ammonia vapor. This method was useful for rapid screening the AF-producing strains ofA. flavus andA. parasiticus.     

Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold,Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239

We conducted genome sequencing of the filamentous fungus Aspergillus sojae NBRC4239 isolated from the koji used to prepare Japanese soy sauce. We used the 454 pyrosequencing technology and investigated the genome with respect to enzymes and secondary metabolites in comparison with other Aspergilli sequenced. Assembly of 454 reads generated a non-redundant sequence of 39.5-Mb possessing 13 033 putative genes and 65 scaffolds composed of 557 contigs. Of the 2847 open reading frames with Pfam domain scores of >150 found in A. sojae NBRC4239, 81.7% had a high degree of similarity with the genes of A. oryzae. Comparative analysis identified serine carboxypeptidase and aspartic protease genes unique to A. sojae NBRC4239. While A. oryzae possessed three copies of α-amyalse gene, A. sojae NBRC4239 possessed only a single copy. Comparison of 56 gene clusters for secondary metabolites between A. sojae NBRC4239 and A. oryzae revealed that 24 clusters were conserved, whereas 32 clusters differed between them that included a deletion of 18 508 bp containing mfs1, mao1, dmaT, and pks-nrps for the cyclopiazonic acid (CPA) biosynthesis, explaining the no productivity of CPA in A. sojae. The A. sojae NBRC4239 genome data will be useful to characterize functional features of the koji moulds used in Japanese industries.     

Effects of Microorganisms on the Peroxidation of Lipid and Fatty Acid Composition of Fermented Fish Meal

Fermentation of waste fish treated with Aspergillus oryzae, Aspergillus sojae K, and Saccharomyces cerevisiae IFO 2114 were studied independently and combined. Three microorganisms decreased the POV, MDA, and COY of fish meal at different rates. The optimum conditions for fermentation with the combination of three microorganisms was found at 30°C for 20–hr fermentation. Almost no difference was observed in the chemical composition or amino acid spectra of the protein hydrolysates of the fermented and nonfermented fish meal. On the quantity of water-soluble amino acids, the highest increase was in glutamate among others, but histidine was decreased by the combination of the three microorganisms. With A. oryzae, the highest increase was in phenylalanine and with A. sojae K in threonine. A great change was observed in some fatty acids content. Myristic acid (14:0) was decreased while the highest increase occurred in the linoleic acid (18: 2) content by fermentation with the combination of three microorganisms. The same phenomenum was observed with A. oryzae and A. sojae K. S. cerevisiae has a weaker effect than A. oryzae and A. sojae K in fermentation of waste fish.     

Improvement of Heterologous Protein Production in Aspergillus oryzae by RNA Interference with α-Amylase Genes

Aspergillus oryzae RIB40 has three α-amylase genes (amyA, amyB, and amyC), and secretes α-amylase abundantly. However, large amounts of endogenous secretory proteins such as α-amylase can compete with heterologous protein in the secretory pathway and decrease its production yields. In this study, we examined the effects of suppression of α-amylase on heterologous protein production in A. oryzae, using the bovine chymosin (CHY) as a reporter heterologous protein. The three α-amylase genes in A. oryzae have nearly identical DNA sequences from those promoters to the coding regions. Hence we performed silencing of α-amylase genes by RNA interference (RNAi) in the A. oryzae CHY producing strain. The silenced strains exhibited a reduction in α-amylase activity and an increase in CHY production in the culture medium. This result suggests that suppression of α-amylase is effective in heterologous protein production in A. oryzae.     

Deciphering the origin of Aspergillus flavus NRRL21882, the active biocontrol agent of Afla-Guard®

Single nucleotide polymorphisms (SNPs) of genome sequences of eight Aspergillus flavus and seven Aspergillus oryzae strains were extracted with Mauve, a multiple-genome alignment programme. A phylogenetic analysis with sequences comprised of concatenated total SNPs by the unweighted pair group method with arithmetic mean (UPGMA) of MAFFT adequately separated them into three groups, A. flavus S-morphotype, A. flavus L-morphotype and A. oryzae. Divergence time inferred for A. flavus NRRL21882, the active agent of the biocontrol product Afla-Guard^®, and S-morphotype was about 5·1 mya. Another biocontrol strain, A. flavus AF36, diverged from aflatoxigenic L-morphotype about 2·6–3·0 mya. Despite the close relatedness of A. oryzae to A. flavus, A. oryzae strains likely evolved from aflatoxigenic Aspergillus aflatoxiformans (=A. parvisclerotigenus). A survey of A. flavus populations implies that prior Afla-Guard^® applications are associated with prevalence of NRRL21882-type isolates in Mississippi fields. In addition, a few NRRL21882 relatives were identified. A. flavus Og0222, a biocontrol ingredient of Aflasafe™, was verified as a NRRL21882-type strain, having identical sequence breakpoints that led to deletion of aflatoxin and cyclopiazonic acid gene clusters. A similar UPGMA analysis suggests that the occurrence of NRRL21882-type strains is a more recent event.     

Production of the plant polyketide curcumin in Aspergillus oryzae: strengthening malonyl-CoA supply for yield improvement

The filamentous fungus Aspergillus oryzae was recently used as a heterologous host for fungal secondary metabolite production. Here, we aimed to produce the plant polyketide curcumin in A. oryzae. Curcumin is synthesized from feruloyl-coenzyme A (CoA) and malonyl-CoA by curcuminoid synthase (CUS). A. oryzae expressing CUS produced curcumin (64 μg/plate) on an agar medium containing feruloyl-N-acetylcysteamine (a feruloyl-CoA analog). To increase curcumin yield, we attempted to strengthen the supply of malonyl-CoA using two approaches: enhancement of the reaction catalyzed by acetyl-CoA carboxylase (ACC), which produces malonyl-CoA from acetyl-CoA, and inactivation of the acetyl-CoA-consuming sterol biosynthesis pathway. Finally, we succeeded in increasing curcumin yield sixfold by the double disruption of snfA and SCAP; SnfA is a homolog of SNF1, which inhibits ACC activity by phosphorylation in Saccharomyces cerevisiae and SCAP is positively related to sterol biosynthesis in Aspergillus terreus. This study provided useful information for heterologous polyketide production in A. oryzae.     

Conidium Germination Rate in Wild and Domesticated Yellow-Green Aspergilli

Conidia of domesticated yellow-green aspergilli from strains of Aspergillus oryzae (Ahlburg) Cohn and Aspergillus sojae Sakaguchi and Yamada ex Murakami, used in the preparation of koji inoculum, germinate approximately 3 h sooner than conidia produced by related wild species, Aspergillus flavus Link ex Fr. and Aspergillus parasiticus Speare. There was no consistent relationship between average conidium size and estimated 50% germination time. Germination trials were conducted on Czapek agar at 28°C. A hypothesis is offered that, in the propagation of koji inoculum, selection has favored those individuals capable of rapid conidium germination and germ tube extension, attributes that enable them to gain the available substrate during intraspecific competition.     

Improvement of Aspergillus oryzae for hyperproduction of endoglucanase: expression cloning of cmc-1 gene of Aspergillus aculeatus

FI-Carboxymethylcellulase (cmc1; family 12) is one of the endoglucanases of Aspergillus aculeatus and consists of single polypeptide chain of 221 amino acids. The cmc1 gene was expressed in Aspergillus oryzae niaD300 (niaD⁻) under promoter 8142. The plasmid pCMG14 carrying the cmc1 gene at PstI site was used as a source of the gene (920 bp) and Aspergillus oryzae was successfully transformed by the plasmid pNAN-cmc1 (harboring cmc1 gene). The plasmid was integrated in Aspergillus oryzae niaD300 genome at niaD locus and the transformed fungus constitutively produced very high amounts of endoglucanases when grown on glucose, maltose, soluble starch and wheat bran.     

Deletion of admB gene encoding a fungal ADAM affects cell wall construction in Aspergillus oryzae

Mammals possess a unique signaling system based on the proteolytic mechanism of a disintegrin and metalloproteinases (ADAMs) on the cell surface. We found two genes encoding ADAMs in Aspergillus oryzae and named them admA and admB. We produced admA and admB deletion strains to elucidate their biological function and clarify whether fungal ADAMs play a similar role as in mammals. The ?admA?admB and ?admB strains were sensitive to cell wall-perturbing agents, congo red, and calcofluor white. Moreover, the two strains showed significantly increased weights of total alkali-soluble fractions from the mycelial cell wall compared to the control strain. Furthermore, ?admB showed MpkA phosphorylation at lower concentration of congo red stimulation than the control strain. However, the MpkA phosphorylation level was not different between ?admB and the control strain without the stimulation. The results indicated that A. oryzae AdmB involved in the cell wall integrity without going through the MpkA pathway.     

Cloning,expression and characterization of endo‐β‐1,4‐mannanase from Aspergillus fumigatus in Aspergillus sojae and Pichia pastoris

To be utilized in biomass conversion, including ethanol production and galactosylated oligosaccharide synthesis, namely prebiotics, the gene of extracellular endo‐β‐1,4‐mannanase (EC 3.2.1.78) of Aspergillus fumigatus IMI 385708 (formerly known as Thermomyces lanuginosus IMI 158749) was expressed first in Aspergillus sojae and then in Pichia pastoris under the control of the glyceraldehyde triphosphate dehydrogenase (gpdA ) and the alcohol oxidase (AOX1 ) promoters, respectively. The highest production of mannanase (352 U mL^?1) in A. sojae was observed after 6 days of cultivation. In P. pastoris, the highest mannanase production was observed 10 h after induction with methanol (61 U mL^?1). The fold increase in mannanase production was estimated as ～12‐fold and ～2‐fold in A. sojae and P. pastoris, respectively, when compared with A. fumigatus. Both recombinant enzymes showed molecular mass of about 60 kDa and similar specific activities (～350 U mg^?1 protein). Temperature optima were at 60°C and 45°C, and maximum activity was at pH 4.5 and 5.2 for A. sojae and P. pastoris, respectively. The enzyme from P. pastoris was more stable retaining most of the activity up to 50°C, whereas the enzyme from A. sojae rapidly lost activity above 40°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

米曲霉异源蛋白表达系统研究进展及展望

米曲霉作为一种重要的工业微生物,在异源蛋白表达方面已有广泛应用,受限于被表达蛋白的修饰及分泌过程,目前实际生产使用的基因供体主要局限于其他真菌,尤其是丝状真菌。当外源基因来源于植物、昆虫和哺乳动物时,米曲霉所生产的异源蛋白产量及生物活性往往不尽如人意。本文综述了米曲霉作为宿主表达异源蛋白的研究进展,包括其现有的遗传操作手段及异源表达方面的应用及探索,重点介绍了应用过程中面临的挑战和解决策略,另外,对米曲霉表达异源蛋白的应用前景及发展方向进行了展望。     

Analysis of phenanthrene degradation by Ascomycota fungi isolated from contaminated soil from Reynosa,Mexico

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds generated mainly by anthropogenic sources. They are considered toxic to mammals, since they have carcinogenic, mutagenic and genotoxic properties, among others. Although mycoremediation is an efficient, economical and eco-friendly technique for degrading PAHs, the fungal degradation potential of the phylum Ascomycota has not been widely studied. In this work, we evaluated different fungal strains from the polluted soil of ‘La Escondida’ lagoon in Reynosa, Mexico to know their potential to degrade phenanthrene (PHE). Forty-three soil isolates with the capacity to grow in the presence of PHE (0·1% w/v) were obtained. The fungi Aspergillus oryzae MF13 and Aspergillus flavipes QCS12 had the best potential to degrade PHE. Both fungi germinated and grew at PHE concentrations of up to 5000 mg l⁻¹ and degraded 235 mg l⁻¹ of PHE in 28 days, with and without an additional carbon source. These characteristics indicate that A. oryzae MF13 and A. flavipes QCS12 could be promising organisms for the remediation of sites contaminated with PAHs and detoxification of recalcitrant xenobiotics.     

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.