Oryzae RIB40 Research Articles

Locust Pathogen Aspergillus oryzae XJ1 Is Different from Aspergillus oryzae and Aspergillus flavus Based on Genomics Comparisons

Fungi play an increasingly important role in the biological control of insect pests. Aspergillus oryzae XJ1 is highly virulent to locust adults and nymphs, which are a destructive economic pest worldwide. Because of its host association with locusts, which is unique in Aspergillus, in this study, we examined the genetic relationships of A. oryzae XJ1 within Aspergillus. We sequenced the genome of A. oryzae XJ1 and compared it with the genomes of other Aspergillus species. The complete genome of A. oryzae XJ1 is 37.9 Mb, comprising 11,720 putative genes, assembled into eight chromosomes. The genome size is similar to that of other A. oryzae strains. Phylogenomic analysis indicated that A. oryzae XJ1 was most closely related to A. flavus NRRL3357, not A. oryzae RIB40. Core/pan-genome analysis of A. oryzae XJ1 and other Aspergillus species revealed that A. oryzae XJ1 had 704 strain-specific genes, whereas A. flavus NRRL3357, A. oryzae KDG 21, and A. parasiticus NRRL 2999 had 646, 955, and 779 unique genes, respectively. The A. oryzae XJ1 genome showed structural differences compared with the genomes of A. oryzae RIB40 and A. flavus NRRL3357 in genomic synteny analysis. These results indicate that A. oryzae XJ1 is genetically distinct at the genome level from other Aspergillus species, including A. oryzae and A. flavus, and may be as a distinct species. This will provide new insight into the classification of Aspergillus based on genomics.

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Isolation of Aspergillus oryzae mutants producing low levels of 2,4,6-trichloroanisole.

Musty or moldy off-odor in sake severely reduces its quality. Such off-odor is caused by 2,4,6-trichloroanisole (TCA), a compound that is produced by Aspergillus oryzae during sake production by O-methylating the precursor 2,4,6-trichlorophenol (TCP). TCP suppresses the growth of fungi, including A. oryzae, although TCA does not. Therefore, strains that are unable to convert TCP to TCA should be sensitive to TCP in the medium. Nevertheless, A. oryzae with a disrupted O-methyltransferase gene (ΔomtT) grew in a medium containing TCP. In agar medium, we observed no growth difference between the ΔomtT strain and a non-disrupted transformant; however, a significant growth delay was observed with the ΔomtT strain grown in liquid medium containing 0.5 µg/mL of TCP. This strain was more sensitive to low concentrations of TCP, suggesting that omtT contributes to the conversion (detoxification) of TCP in liquid culture. We generated A. oryzae RIB 40 mutants by ultraviolet irradiation and then cultured them in liquid medium containing TCP to obtain strains that did not produce moldy odor. The slow-growing strains were cultured in agar plates and then used to make koji with added TCP. We obtained three strains with lower TCA-producing ability and with sufficient hydrolase activities for sake brewing.

Lessons Learned: the Importance of Biological Curation.

Proper species identification of sequenced fungal isolates or strains is imperative for the interpretation of genomics data. Through this letter (1), and through another recently published research note (2), it has come to our attention that one of the isolates presented in our previous publication (3) was misidentified. The presumptive Aspergillus parasiticus isolate NRRL 2999 has been shown to be an Aspergillus flavus isolate, a clonal derivative of A. flavus NRRL 3357 (2). In our recent publication presenting two new reference genomes for A. flavus (4), we performed a phylogenomics analysis of several publicly available Aspergillus assemblies and noted the sequence similarity between A. flavus NRRL 3357 and A. parasiticus NRRL 2999. We also noted a close relationship between A. flavus WRRL 1519 and A. oryzae RIB40 and that A. oryzae RIB40 grouped among A. flavus isolates, not as an outgroup. Based on these analyses, we concluded that the A. flavus species is polyphyletic. The revelation that NRRL 2999 is indeed an A. flavus isolate clonal to NRRL 3357 (2) does point toward A. flavus as a distinct species from A. parasiticus but does not change the conclusion of the potential polyphyletic nature of A. flavus based on the position of A. oryzae in the analysis (4). All additional conclusions were based on phylogenetic relationships defined within the scope of the manuscript (4) and were therefore independent of species labels.

The role of propeptide-mediated autoinhibition and intermolecular chaperone in the maturation of cognate catalytic domain in leucine aminopeptidase

Leucyl aminopeptidase A from Aspergillus oryzae RIB40 (AO-LapA) is an exo-acting peptidase, widely utilised in food debittering applications. AO-LapA is secreted as a zymogen by the host and requires enzymatic cleavage of the autoinhibitory propeptide to reveal its full activity. Scarcity of structural data of zymogen aminopeptidases hampers a better understanding of the details of their molecular action of autoinhibition and how this might be utilised to improve the properties of such enzymes by recombinant methods for more effective bioprocessing. To address this gap in the literature, herein we report high-resolution crystal structures of recombinantly expressed AO-LapA precursor (AO-proLapA), mature LapA (AO-mLapA) and AO-mLapA complexed with reaction product l-leucine (AO-mLapA-Leu), all purified from Pichia pastoris culture supernatant. Our structures reveal a plausible molecular mechanism of LapA catalytic domain autoinhibition by propeptide and highlights the role of intramolecular chaperone (IMC). Our data suggest an absolute requirement for IMC in the maturation of cognate catalytic domain of AO-LapA. This observation is reinforced by our expression and refolding data of catalytic domain only (AO-refLapA) from Escherichia coli inclusion bodies, revealing a limited active conformation. Our work supports the notion that known synthetic aminopeptidase inhibitors and substrates mimic key polar contacts between propeptide and corresponding catalytic domain, demonstrated in our AO-proLapA zymogen crystal structure. Furthermore, understanding the atomic details of the autoinhibitory mechanism of cognate catalytic domains by native propeptides has wider reaching implications toward synthetic production of more effective inhibitors of bimetallic aminopeptidases and other dizinc enzymes that share an analogous reaction mechanism.

English

Seafood is part of a healthful diet, but seafood consumption is not risk-free because of sea contaminations by anthropogenic activities and environmental factors. Their microbiological quality is of major concern to the food processors, consumers and public health authorities hence the need for proper assessment of the microbial quality. This study therefore characterizes pathogenic microorganisms isolated from fresh sea snail and shrimp in Ugbo nla, Igbokoda, Ondo State, Nigeria. Isolation and molecular identification of microorganisms from snail and shrimp using 16sRNA and 18sRNA gene sequence for bacteria and fungi respectively, effects of salt concentration and temperature on isolated microorganisms were carried out using standard microbiological methods. Molecular identification revealed the presence different pathogens namely; Proteus mirabilis LZPM-03, Pantoea agglomerans BJCP2, Pseudomonas aeruginosa R8-589, Klebsiella pneumoniae 2N3, Serratia marcescens, Alcaligenes faecalis ACBC1, Yersinia. pseudotubercules YPIII, Escherichia coli 2A, Lysinibacillus sphaericus C3-41, Listeria monocytogen J1776, Staphylococcus aureus and Proteus mirabilis LCX20, Fusarium proliferatum FproStRINI, Aspergillus flavus NRRL3357, Aspergillus oryzae RIB40 and Aspergillus peniciliodes. Also, 4% salt concentration at 45°C effectively inhibited the pathogens isolated from seafood after 18 h. The presence of pathogenic microorganisms in the sea foods could make them a potential health-risk when consumed without proper cooking. Salting, a traditional technique could be used to eliminate these pathogens before consumption.   Key words: Seafood, molecular identification and salt concentration. &nbsp

Transcriptome Analysis of Two Strains of <i>Aspergillus oryzae</i> with Different Responses to Light

The production process of such traditional Japanese fermented foods as miso, shoyu and sake using Aspergillus oryzae has been highly optimized over a long history. However, new technology is needed to achieve further improvements in productivity or production efficiency. Light affects the gene expression and metabolic pathways of fungi. Thus, the development of technology that takes advantage of the light response of A. oryzae is important. In a previous study, we found that A. oryzae strains RIB40 and RIB1187 showed opposing manners of growth and conidiation in response to light. To elucidate the effect of light on genome-wide gene expression for both strains, we conducted transcriptome analysis by RNA sequencing. We also identified 453 differentially expressed genes (DEGs) between both strains by bioinformatic analysis. Of these 453 DEGs, we identified a total of 67 light-responsive DEGs between RIB40 and RIB1187. We also analyzed the expression patterns of fermentation-related digestive enzyme genes.

Identification of a gene cluster for biosynthesis of the sesquiterpene antibiotic, heptelidic acid, in Aspergillus oryzae.

Heptelidic acid (HA), a sesquiterpene lactone, is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recently, we found that HA was produced by Aspergillus oryzae RIB40 and acted as the growth inhibitor of the salt-tolerant lactic acid bacteria in soy sauce brewing. Although several decades have passed since the discovery of HA, the genes involved in its biosynthesis and biosynthetic pathway have not yet been fully identified. In this study, we identified the HA biosynthetic gene cluster (HA cluster) using gene disruption and expression analysis. We also revealed that two transcription regulatory genes adjacent to the HA cluster were responsible for the expression of HA biosynthetic genes in A. oryzae. Interestingly, the HA cluster contained a gene encoding GAPDH (gpdB), which showed much higher resistance to HA than the GAPDH gene (gpdA) located at the other locus, but which did not seem to act as a self-resistant gene.

Characterization and expression in Pichia pastoris of a raw starch degrading glucoamylase (GA2) derived from Aspergillus flavus NSH9

The Aspergillus flavus NSH9 gene, encoding a pH and thermostable glucoamylase with a starch binding domain (SBD), was expressed in Pichia pastoris to produce recombinant glucoamylase (rGA2). The full-length glucoamylase gene (2039 bp), and cDNA (1839 bp) encode a 612 amino acid protein most similar to glucoamylase from Aspergillus oryzae RIB40; the first 19 amino acids are presumed to be a signal peptide for secretion, and the SBD is at the C-terminal. The cDNA was successfully secreted by Pichia at 8.23 U mL-1, and the rGA2 was found to be: a 80 kDa monomer, stable from pH 3.0-9.0, with optimum catalytic activity at pH 5.0, active at temperatures up to 80°C (rGA2 retained 58% of its activity after 60 min of incubation at 70°C), and metal ions such as Na+, K+, Ca++ and Mg++ enhanced rGA2 enzyme activity. The starch degrading ability of rGA2 was also observed on raw sago starch and where prolonged incubation generated larger, deeper, holes on the starch granules, indicating rGA2 is an excellent candidate for industrial starch processing applications.

The microenvironment surrounding FAD mediates its conversion to 8-formyl-FAD in Aspergillus oryzae RIB40 formate oxidase.

Aspergillus oryzae RIB40 formate oxidase has Arg87 and Arg554 near the formyl group and O(4) atom of 8-formyl-flavin adenine dinucleotide (FAD), respectively, with Asp396 neighbouring Arg554. Herein, we probed the roles of these three residues in modification of FAD to 8-formyl-FAD. Replacement of Arg87 or Arg554 with Lys or Ala decreased and abolished the modification, respectively. Replacement of Asp396 with Ala or Asn lowered the modification rate. The observation of unusual effects of maintaining pH 7.0 on the modification in R87K, R554K and D396 variants indicates initial and subsequent processes with different pH dependencies. Comparison of the initial process at pH 4.5 and 7.0 suggests that the microenvironment around Arg87 and the protonation state of Asp396 affect the initial process in the native enzyme. Comparison of the crystal structures of native and R554 variants showed that the replacements had minimal effect on catalytic site structure. The positively charged Arg87 might contribute to the formation of an anionic quinone-methide tautomer intermediate, while the positively charged Arg554, in collaboration with the negatively charged Asp396, might stabilize this intermediate and form a hydrogen bonding network with the N(5)/O(4) region, thereby facilitating efficient FAD modification.

Survival strategy of the salt-tolerant lactic acid bacterium, Tetragenococcus halophilus, to counteract koji mold, Aspergillus oryzae, in soy sauce brewing.

In soy sauce brewing, the results of the fermentation of lactic acid greatly affect the quality of soy sauce. The soy sauce moromi produced with Aspergillus oryzae RIB40 allows the growth of Tetragenococcus halophilus NBRC 12172 but not T. halophilus D10. We isolated and identified heptelidic acid (HA), an inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), produced by A. oryzae RIB40 as the growth inhibitor of the salt-tolerant lactic acid bacteria. The growth inhibition of T. halophilus D10 by HA was suggested to be associated with the direct inhibition of GAPDH activity under high salt environment. The difference in the susceptibility to HA among various strains of T. halophilus was caused by the mutations in the gene encoding GAPDH.

Isolation, identification and in silico analysis of alpha-amylase gene of Aspergillus niger strain CSA35 obtained from cassava undergoing spoilage

In this investigation, a gene (CDF_Amyl) encoding extracellular α-amylase in Aspergillus niger strain CSA35 associated with cassava spoilage was amplified using specific primers and characterized in silico. The gene had a partial nucleotide sequence of 968 bp and encoded a protein of 222 aa residues with a molecular weight and isoelectric point of 25.13 kDa and 4.17, respectively. Its catalytic site was located in the active site domain. BLASTp analysis showed that the protein primary sequence of the α-amylase gene had 98% and 99% homologies with the α-amylase of A. niger and A. oryzae RIB40, respectively. The gene is more closely related to α-amylase genes from fungi than to bacterial, plant, or animal α-amylase genes. Restriction mapping of the gene showed it can be digested with restriction enzymes like NcoI, PstI, SmaI, and BcLI among others but not with EcoRI and EcoRV. Its protein product had a hydrophobicity score of − 0.43 but no transmembrane helix. The CDF_Amyl protein was subcellularly localized in the secretory pathway, an indication of its release into extracellular space after secretion. Also, the 3D structure of the CDF-Amyl protein was barrel-shaped with domains characteristic of α-amylases. The encoded α-amylase Vmax is 6.90 U/mg protein and Km is 6.70 mg/ml. It was concluded that the unique characteristics of the CDF_Amyl gene and its deduced protein could find applications in biotechnological, food and pharmaceutical industries where cloning and further modification of this gene would be required for product development and improvement.

Volatiles Mediated Interactions Between Aspergillus oryzae Strains Modulate Morphological Transition and Exometabolomes.

Notwithstanding its mitosporic nature, an improbable morpho-transformation state i. e., sclerotial development (SD), is vaguely known in Aspergillus oryzae. Nevertheless an intriguing phenomenon governing mold's development and stress response, the effects of exogenous factors engendering SD, especially the volatile organic compounds (VOCs) mediated interactions (VMI) pervasive in microbial niches have largely remained unexplored. Herein, we examined the effects of intra-species VMI on SD in A. oryzae RIB 40, followed by comprehensive analyses of associated growth rates, pH alterations, biochemical phenotypes, and exometabolomes. We cultivated A. oryzae RIB 40 (S1VMI: KACC 44967) opposite a non-SD partner strain, A. oryzae (S2: KCCM 60345), conditioning VMI in a specially designed “twin plate assembly.” Notably, SD in S1VMI was delayed relative to its non-conditioned control (S1) cultivated without partner strain (S2) in twin plate. Selectively evaluating A. oryzae RIB 40 (S1VMI vs. S1) for altered phenotypes concomitant to SD, we observed a marked disparity for corresponding growth rates (S1VMI < S1)7days, media pH (S1VMI > S1)7days, and biochemical characteristics viz., protease (S1VMI > S1)7days, amylase (S1VMI > nS1)3–7days, and antioxidants (S1VMI > S1)7days levels. The partial least squares—discriminant analysis (PLS-DA) of gas chromatography—time of flight—mass spectrometry (GC-TOF-MS) datasets for primary metabolites exhibited a clustered pattern (PLS1, 22.04%; PLS2, 11.36%), with 7 days incubated S1VMI extracts showed higher abundance of amino acids, sugars, and sugar alcohols with lower organic acids and fatty acids levels, relative to S1. Intriguingly, the higher amino acid and sugar alcohol levels were positively correlated with antioxidant activity, likely impeding SD in S1VMI. Further, the PLS-DA (PLS1, 18.11%; PLS2, 15.02%) based on liquid chromatography—mass spectrometry (LC-MS) datasets exhibited a notable disparity for post-SD (9–11 days) sample extracts with higher oxylipins and 13-desoxypaxilline levels in S1VMI relative to S1, intertwining Aspergillus morphogenesis and secondary metabolism. The analysis of VOCs for the 7 days incubated samples displayed considerably higher accumulation of C-8 compounds in the headspace of twin-plate experimental sets (S1VMI:S2) compared to those in non-conditioned controls (S1 and S2—without respective partner strains), potentially triggering altered morpho-transformation and concurring biochemical as well as metabolic states in molds.

Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant

Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD.

Genome mining and motif truncation of glycoside hydrolase family 5 endo-β-1,4-mannanase encoded by Aspergillus oryzae RIB40 for potential konjac flour hydrolysis or feed additive.

Two novel glycosyl hydrolase family 5 (GH5) β-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9KM. The specific enzyme activity of the purified reAoMan5A, reAoMan5B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40°C for 10min, was 8.3, 104.2 and 15.8U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant β-mannanases potential additives for use in the food and feed industries.

A biotech-systematic approach to select fungi for bioconversion of winery biomass wastes to nutrient-rich feed

Grape marc and lees are main waste biomass in wine industry, which contain substantial amount of carbohydrates and nutrients, but currently deliver considerable carbon emission through landfill in Australia. This study was aimed to develop a biotechnological systematic approach to select suitable fungi for bioconversion of the winery biomass wastes into animal feed. The biotech-systematic approach was developed through assessment of nutrient/carbon source accessibility of the fungi, understanding of their metabolic reactions in solid state fermentation, and nutritive value of the fungi-fermented grape marc. From 13 Aspergillus, Rhizopus, and Trichoderma fungal species, A. oryzae DAR 3699, A. oryzae RIB40 and T. reesei RUT C30 were determined as the best fungi due to their promising biochemical capabilities to enhance protein contents and in vitro digestibility of grape marc in mono- and co-cultivations. The mono-fungous-cultured SSF process used these selected fungi is able to convert grape marc to nutrient-rich feed by increasing the protein contents from ∼5% to 26% and the digestibility from ∼25% to over 50%. R. oligosporus 2710 with R. oryzae 6201 or T. viride 15719 showed promising protein enrichment to 18–23% and digestibility improvement by ∼50% in their co-cultivations. This preliminary bioengineering strategy will be useful for selecting microorganisms for the bioconversion of organic wastes to high valuable products. This “green cycle” bioprocess will be useful to promote the old-fashioned waste treatment technologies for the cleaner production in food processing industries.

Phylogenetic and Structural Analysis of Polyketide Synthases inAspergilli

Polyketide synthases (PKSs) of Aspergillus species are multidomain and multifunctional megaenzymes that play an important role in the synthesis of diverse polyketide compounds. Putative PKS protein sequences from Aspergillus species representing medically, agriculturally, and industrially important Aspergillus species were chosen and screened for in silico studies. Six candidate Aspergillus species, Aspergillus fumigatus Af293, Aspergillus flavus NRRL3357, Aspergillus niger CBS 513.88, Aspergillus terreus NIH2624, Aspergillus oryzae RIB40, and Aspergillus clavatus NRRL1, were selected to study the PKS phylogeny. Full-length PKS proteins and only ketosynthase (KS) domain sequence were retrieved for independent phylogenetic analysis from the aforementioned species, and phylogenetic analysis was performed with characterized fungal PKS. This resulted into grouping of Aspergilli PKSs into nonreducing (NR), partially reducing (PR), and highly reducing (HR) PKS enzymes. Eight distinct clades with unique domain arrangements were classified based on homology with functionally characterized PKS enzymes. Conserved motif signatures corresponding to each type of PKS were observed. Three proteins from Protein Data Bank corresponding to NR, PR, and HR type of PKS (XP_002384329.1, XP_753141.2, and XP_001402408.2, respectively) were selected for mapping of conserved motifs on three-dimensional structures of KS domain. Structural variations were found at the active sites on modeled NR, PR, and HR enzymes of Aspergillus. It was observed that the number of iteration cycles was dependent on the size of the cavity in the active site of the PKS enzyme correlating with a type with reducing or NR products, such as pigment, 6MSA, and lovastatin. The current study reports the grouping and classification of PKS proteins of Aspergilli for possible exploration of novel polyketides based on sequence homology; this information can be useful for selection of PKS for polyketide exploration and specific detection of Aspergilli.

Acid protease production by Aspergillus oryzae on potato pulp powder with emphasis on glycine releasing activity: A benefit to the food industry

Potato pulp powder is a major byproduct generated in huge amounts globally from potato processing industry. The disposal of these residues causes environmental pollution. Considering the use of agro byproduct as economic and environmental benefits, biotranformation of Potato pulp powder by solid state fermentation for the production of acid protease a valuable food enzyme is explored. Screening of food grade and industrial Aspergillus oryzae strains on enriched media identified A. oryzae RIB 40 (ATCC 42149) yielding 20.5U/ml of acid protease as a potential candidate. Further, the productivity of the parent strain was improved by UV mutagenesis with a lethality of 1.8×10−4. The mutant strain (F6) produced 5.6 fold higher than the parent. The solid state fermentation of A. oryzae (F6) with potato pulp powder as a sole source and along with supplementation of nitrogen yielded 2.85±5.0 and 18.68U/g of acid protease, respectively. Statistical optimization of biovariables, namely moisture (50%), temperature (30°C) and fermentation time (120h) accomplished using a central composite rotatable experimental design, led to an 11-fold increase in protease activity (31U/g dry substrate). Model validations indicated 95% agreement between the experimental results and the predicted responses. The acid protease from A. oryzae liberated glycine (0.57±0.25U/g), which can enhance food palatability. Thus, the present study established fermenting potato pulp, with a starter culture of A. oryzae F6 strain and optimized variables can produce acid protease that has great potential for use in the food industry. This will also contribute to sustainable management in the potato industry and adds value-addition.

Aspergillus oryzae pathways that convert phenylalanine into the flavor volatile 2-phenylethanol

The filamentous fungus Aspergillus oryzae RIB40 produced 2-phenylethanol (PE) when cultured in minimum medium containing l-phenylalanine as a sole source of nitrogen. The fungus accumulated less PE in the absence of l-phenylalanine, indicating that it converted l-phenylalanine to PE. The PE production associated with fungal glucose consumption was repressed by exogenous ammonium, indicating that nitrogen-metabolite repression controls the pathway that produces PE. We identified the A. oryzae ppdA gene that is expressed at high levels in the presence of exogenous l-phenylalanine and its encoded protein was an active phenylpyruvate decarboxylase. The fungal genome encodes predicted aminotransferases of phenylalanine and PE dehydrogenases, which, together with PpdA, are likely to constitute an Erlich pathway similar to that in Saccharomyces cerevisiae that produces PE. We also identified an A. oryzae aromatic amino acid decarboxylase (AadA) that converted l-phenylalanine to phenylethylamine (PEA), and phenylalanine-inducible PEA oxidase activity in fungal cell extracts, and found that both constitute an alternative pathway through which PEA generates PE. Incubating fungal cultures with l-[2H8] phenylalanine to distinguish PE produced by these pathways, indicated that the fungus produced PE by both pathways, but to a greater extent by the Erlich pathway. Gene disruption of ppdA and aadA showed that both pathways participate in the fungal conversion of l-phenylalanine to PE.

High sequence variations in the region containing genes encoding a cellular morphogenesis protein and the repressor of sexual development help to reveal origins of Aspergillus oryzae

Aspergillus oryzae and Aspergillus flavus are closely related fungal species. The A. flavus morphotype that produces numerous small sclerotia (S strain) and aflatoxin has a unique 1.5kb deletion in the norB-cypA region of the aflatoxin gene cluster (i.e. the S genotype). Phylogenetic studies have indicated that an isolate of the nonaflatoxigenic A. flavus with the S genotype is the ancestor of A. oryzae. Genome sequence comparison between A. flavus NRRL3357, which produces large sclerotia (L strain), and S-strain A. flavus 70S identified a region (samA-rosA) that was highly variable in the two morphotypes. A third type of samA-rosA region was found in A. oryzae RIB40. The three samA-rosA types were later revealed to be commonly present in A. flavus L-strain populations. Of the 182 L-strain A. flavus field isolates examined, 46%, 15% and 39% had the samA-rosA type of NRRL3357, 70S and RIB40, respectively. The three types also were found in 18 S-strain A. flavus isolates with different proportions. For A. oryzae, however, the majority (80%) of the 16 strains examined had the RIB40 type and none had the NRRL3357 type. The results suggested that A. oryzae strains in the current culture collections were mostly derived from the samA-rosA/RIB40 lineage of the nonaflatoxigenic A. flavus with the S genotype.