Phosphatidylcholine levels regulate hyphal elongation and differentiation in the filamentous fungus Aspergillus oryzae.

Filamentous fungi are eukaryotic microorganisms that differentiate into diverse cellular forms. Recent research demonstrated that phospholipid homeostasis is crucial for the morphogenesis of filamentous fungi. However, phospholipids involved in the morphological regulation are yet to be systematically analyzed. In this study, we artificially controlled the amount of phosphatidylcholine (PC), a primary membrane lipid in many eukaryotes, in a filamentous fungus Aspergillus oryzae, by deleting the genes involved in PC synthesis or by repressing their expression. Under the condition where only a small amount of PC was synthesized, A. oryzae hardly formed aerial hyphae, the basic structures for asexual development. In contrast, hyphae were formed on the surface or in the interior of agar media (we collectively called substrate hyphae) under the same conditions. Furthermore, we demonstrated that supplying sufficient choline to the media led to the formation of aerial hyphae from the substrate hyphae. We suggested that acyl chains in PC were shorter in the substrate hyphae than in the aerial hyphae by utilizing the strain in which intracellular PC levels were controlled. Our findings suggested that the PC levels regulate hyphal elongation and differentiation processes in A. oryzae and that phospholipid composition varied depending on the hyphal types.

Regulation of gliotoxin biosynthesis and protection in Aspergillus species.

Aspergillus fumigatus causes a range of human and animal diseases collectively known as aspergillosis. A. fumigatus possesses and expresses a range of genetic determinants of virulence, which facilitate colonisation and disease progression, including the secretion of mycotoxins. Gliotoxin (GT) is the best studied A. fumigatus mycotoxin with a wide range of known toxic effects that impair human immune cell function. GT is also highly toxic to A. fumigatus and this fungus has evolved self-protection mechanisms that include (i) the GT efflux pump GliA, (ii) the GT neutralising enzyme GliT, and (iii) the negative regulation of GT biosynthesis by the bis-thiomethyltransferase GtmA. The transcription factor (TF) RglT is the main regulator of GliT and this GT protection mechanism also occurs in the non-GT producing fungus A. nidulans. However, the A. nidulans genome does not encode GtmA and GliA. This work aimed at analysing the transcriptional response to exogenous GT in A. fumigatus and A. nidulans, two distantly related Aspergillus species, and to identify additional components required for GT protection. RNA-sequencing shows a highly different transcriptional response to exogenous GT with the RglT-dependent regulon also significantly differing between A. fumigatus and A. nidulans. However, we were able to observe homologs whose expression pattern was similar in both species (43 RglT-independent and 11 RglT-dependent). Based on this approach, we identified a novel RglT-dependent methyltranferase, MtrA, involved in GT protection. Taking into consideration the occurrence of RglT-independent modulated genes, we screened an A. fumigatus deletion library of 484 transcription factors (TFs) for sensitivity to GT and identified 15 TFs important for GT self-protection. Of these, the TF KojR, which is essential for kojic acid biosynthesis in Aspergillus oryzae, was also essential for virulence and GT biosynthesis in A. fumigatus, and for GT protection in A. fumigatus, A. nidulans, and A. oryzae. KojR regulates rglT, gliT, gliJ expression and sulfur metabolism in Aspergillus species. Together, this study identified conserved components required for GT protection in Aspergillus species.

Inhibition of Aspergillus oryzae Mycelium Growth and Conidium Production by Irradiation with Light at Different Wavelengths and Intensities.

Aspergillus oryzae is a safe filamentous fungus widely used in the food, medicine, and feed industries, but there is currently not enough research on the light response of A. oryzae. In this study, 12 different light conditions were set and A. oryzae GDMCC 3.31 was continuously irradiated for 72 h to investigate the effect of light on mycelial growth and conidium production. Specifically, each light condition was the combination of one light wavelength (475, 520, or 630 nm) and one light intensity (20, 40, 60, or 80 µmol photon m-2 s-1). The results show that mycelium growth was inhibited significantly by green light (wavelength of 520 nm and intensities of 20 and 60 µmol photon m-2 s-1) and blue light (wavelength of 475 nm and intensity of 80 µmol photon m-2 s-1). The production of conidia was suppressed only by blue light (wavelength of 475 nm and intensities of 40, 60, and 80 µmol photon m-2 s-1), and those levels of inhibition increased when the intensity of blue light increased. When the strain was irradiated by blue light (80 µmol photon m-2 s-1), the number of conidia was 57.4% less than that of the darkness group. However, within our set range of light intensities, A. oryzae GDMCC 3.31 was insensitive to red light (wavelength of 630 nm) in terms of mycelium growth and conidium production. Moreover, interaction effects between light wavelength and intensity were found to exist in terms of colony diameter and the number of conidia. This research investigated the light response of A. oryzae, which may provide a new method to regulate mixed strains in fermented foods by light. IMPORTANCE Studies on the monochromatic light response of Aspergillus nidulans and Neurospora crassa have gone deep into the molecular mechanism. However, research methods for the light response of A. oryzae remain in the use of white light sources. In this study, we first demonstrated that A. oryzae GDMCC 3.31 was sensitive to light wavelength and intensity. We have observed that blue light inhibited its growth and sporulation and the inhibitory effect increased with intensity. This research not only adds new content to the study of the photoreaction of Aspergillus but also brings new possibilities for the use of light to regulate mixed strains and ultimately improve the flavor quality of fermented foods.

Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae.

Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.

Optimization and scale-up of α-amylase production by Aspergillus oryzae using solid-state fermentation of edible oil cakes.

BACKGROUND: Amylases produced by fungi during solid-state fermentation are the most widely used commercial enzymes to meet the ever-increasing demands of the global enzyme market. The use of low-cost substrates to curtail the production cost and reuse solid wastes are seen as viable options for the commercial production of many enzymes. Applications of α-amylases in food, feed, and industrial sectors have increased over the years. Additionally, the demand for processed and ready-to-eat food has increased because of the rapid growth of food-processing industries in developing economies. These factors significantly contribute to the global enzyme market. It is estimated that by the end of 2024, the global α-amylase market would reach USD 320.1 million (Grand View Research Inc., 2016). We produced α-amylase using Aspergillus oryzae and low-cost substrates obtained from edible oil cake, such as groundnut oil cake (GOC), coconut oil cake (COC), sesame oil cake (SOC) by solid-state fermentation. We cultivated the fungus using these nutrient-rich substrates to produce the enzyme. The enzyme was extracted, partially purified, and tested for pH and temperature stability. The effect of pH, incubation period and temperature on α-amylase production using A. oryzae was optimized. Box-Behnken design (BBD) of response surface methodology (RSM) was used to optimize and determine the effects of all process parameters on α-amylase production. The overall cost economics of α-amylase production using a pilot-scale fermenter was also studied. RESULTS: The substrate optimization for α-amylase production by the Box-Behnken design of RSM showed GOC as the most suitable substrate for A. oryzae, as evident from its maximum α-amylase production of 9868.12 U/gds. Further optimization of process parameters showed that the initial moisture content of 64%, pH of 4.5, incubation period of 108 h, and temperature of 32.5 °C are optimum conditions for α-amylase production. The production increased by 11.4% (10,994.74 U/gds) by up-scaling and using optimized conditions in a pilot-scale fermenter. The partially purified α-amylase exhibited maximum stability at a pH of 6.0 and a temperature of 55 °C. The overall cost economic studies showed that the partially purified α-amylase could be produced at the rate of Rs. 622/L. CONCLUSIONS: The process parameters for enhanced α-amylase secretion were analyzed using 3D contour plots by RSM, which showed that contour lines were more oriented toward incubation temperature and pH, having a significant effect (p < 0.05) on the α-amylase activity. The optimized parameters were subsequently employed in a 600 L-pilot-scale fermenter for the α-amylase production. The substrates were rich in nutrients, and supplementation of nutrients was not required. Thus, we have suggested an economically viable process of α-amylase production using a pilot-scale fermenter.

Exploring differential traits of lipid-producing stages of the wild type and morphologically engineered strain of Aspergillus oryzae by comparative kinetic modeling.

Comparative profilings of cell growth and lipid production in the morphologically engineered strain (Δags1) and the wild type (WT) of Aspergillus oryzae BCC7051 were implemented. Using various nitrogen sources, a discrimination in cell morphology between the two strains was found, of which the Δags1 culture exhibited mycelial growth as small pellets in contrast to the WT. Of them, sodium nitrate and potassium nitrate were optimal for lipid production of the WT and Δags1 strains, respectively, which the highest lipid concentrations of 7.2 and 7.9 g L-1 were obtained in the respective cultures. The mathematical models of the growth kinetics and lipid phenotypes of both fungal strains were developed, enabling to distinguish three lipid-producing stages, including low lipid-producing, lipid accumulation, and lipid turnover stages. The model validation showed good performances in all nitrogen sources tested for the WT, but only NaNO3 and mixed yeast extract/NH4Cl were fitted well for the Δags1. The difference in the period of lipid-producing stages between the WT and Δags1 indicated the metabolic alterations of A. oryzae by the defect of a gene involved in the cell wall biosynthesis, which exhibited benefits for bioprocessing practices in addition to the high productivities of biomass and lipid. These findings would further permit the manipulation in the metabolic hub of the fungal production platform for other industrial purposes.

Optimization of media composition and growth conditions for production of milk-clotting protease (MCP) from Aspergillus oryzae DRDFS13 under solid-state fermentation.

This study reports the optimization of milk-clotting protease production from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) in both one-variable-at-a-time and response surface methodology (RSM). The production and optimization of milk-clotting protease obtained from Aspergillus oryzae DRDFS13 under solid-state fermentation (SSF) using different agro-industrial wastes as solid substrates were studied. The agro-industrial wastes used included wheat bran, rice bran, pea bran, and grass pea bran. The chemical composition of the best solid substrate was tested using standard methods. Others cultivation parameters were studied, and the results showed that the optimum fermentation medium composed of wheat bran, casein (1% w/w), and glucose (0.5% w/w) and the conditions for maximum milk-clotting protease production were at the moisture content of 55.0%, inoculum of 0.5*106 spores/mL, incubation temperature of 30 °C, pH of 6.0, and fermentation time of 5 days. The highest milk-clotting activity was obtained from the crude enzyme extracted using 0.1 M NaCl and partial purification of the crude enzyme using chilled acetone, and 80% (NH4)2SO4 increased the ratio of MCA/PA from 0.56 to 1.30 and 0.65, respectively. Moreover, the highest MCA (137.58 U/mL) was obtained at a casein concentration of 0.5%, pH 4.0, and 25 °C, using RSM. Thus, results from the present study showed that the optimization of milk-clotting protease production from A. oryzae DRDFS 13 under SSF by both one-variable-at-a-time and RSM significantly increased the milk-clotting activity. This is the first report from a fungus in the Ethiopian setting and a modest contribution to highlight the potential of harnessing microbial protease enzymes for industrial applications.

Identification and characterization of a DevR-interacting protein in Aspergillus oryzae.

The basic helix-loop-helix (bHLH) proteins belong to a superfamily of transcription factors. Recent research has shown that the bHLH transcription factor DevR is involved in both sexual and asexual development as well as conidial melanin production in Aspergillus species. Our previous research also found that DevR significantly influences polysaccharide metabolism in Aspergillus oryzae. In this study, to further explore the function of DevR, its interaction proteins were screened by a yeast two-hybrid assay. An A. oryzae cDNA library was transformed into the Y187 strain by using the SMART technique and the homologous recombination method, and then hybridized with a constructed DevR bait plasmid introducing strain to obtain positive clones. Through sequencing analysis, the potential interaction proteins of DevR were determined. Among them, an AO090701000363 gene-encoding protein (named DipA), which was predicted to be a basic leucine zipper (bZIP) transcription factor, was a possible candidate. Phenotypic analysis indicated that overexpression of the AodipA may significantly suppress growth of the strain. Additionally, although no obvious change in the growth rate was found, the deletion of AodipA resulted in thicker hyphae morphology relative to the control. Comparative proteomic analysis further indicated that DipA was potentially involved in the regulation of cell wall integrity, carbon utilization, acetate catabolic process and other biological processes. Partial similarity of the phenotype to that of DevR suggested a correlation between them and implied that the DipA has a function partially similar to that of DevR.

Aspergillus oryzae spore germination is enhanced by non-thermal atmospheric pressure plasma.

Poor and unstable culture growth following isolation presents a technical barrier to the efficient application of beneficial microorganisms in the food industry. Non-thermal atmospheric pressure plasma is an effective tool that could overcome this barrier. The objective of this study was to investigate the potential of plasma to enhance spore germination, the initial step in fungal colonization, using Aspergillus oryzae, a beneficial filamentous fungus used in the fermentation industry. Treating fungal spores in background solutions of phosphate buffered saline (PBS) and potato dextrose broth (PDB) with micro dielectric barrier discharge plasma using nitrogen gas for 2 and 5 min, respectively, significantly increased the germination percentage. Spore swelling, the first step in germination, was accelerated following plasma treatment, indicating that plasma may be involved in loosening the spore surface. Plasma treatment depolarized spore membranes, elevated intracellular Ca2+ levels, and activated mpkA, a MAP kinase, and the transcription of several germination-associated genes. Our results suggest that plasma enhances fungal spore germination by stimulating spore swelling, depolarizing the cell membrane, and activating calcium and MAPK signaling.

The Biogenetic Origin of the Biologically Active Naematolin of Hypholoma Species Involves an Unusual Sesquiterpene Synthase.

Naematolin is a biologically active sesquiterpene produced by Hypholoma species. Low titres and complex structure constrain the exploitation of this secondary metabolite. Here, we de novo sequenced the H. fasciculare genome to identify a candidate biosynthetic gene cluster for production of naematolin. Using Aspergillus oryzae as a heterologous host for gene expression, the activity of several sesquiterpene synthases were investigated, highlighting one atypical sesquiterpene synthase apparently capable of catalysing the 1,11 and subsequent 2,10 ring closures, which primes the synthesis of the distinctive structure of caryophyllene derivatives. Co-expression of the cyclase with an FAD oxidase adjacent within the gene cluster generated four oxidised caryophyllene-based sesquiterpenes: 5ß,6α,8ß-trihydroxycariolan, 5ß,8ß-dihydroxycariolan along with two previously unknown caryophyllene derivatives 2 and 3. This represents the first steps towards heterologous production of such basidiomycete-derived caryophyllene-based sesquiterpenes, opening a venue for potential novel antimicrobials via combinatorial biosynthesis.

Morphology engineering of Aspergillus oryzae for l-malate production.

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l-malate production. Specifically, correlation between A. oryzae morphology and l-malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l-malate production was 120-130 mm3 /ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l-malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l-malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.

Metabolomic study of the soybean pastes fermented by the single species Penicillium glabrum GQ1-3 and Aspergillus oryzae HGPA20.

Penicillium glabrum GQ1-3 and Aspergillus oryzae HGPA20 isolated from home-made soybean pastes were separately inoculated into soybean paste subjected to brine fermentation for 90 days. The amino acid nitrogen contents of the two fermentation systems were detected every 10 days, and both reached the maximum level at 40 days. The samples fermented for 40 days were analyzed via gas chromatography-time of flight mass spectrometry. Using univariate, multivariate and KEGG analyses, 72 differential metabolites were obtained, and 7 metabolic pathways closely related to fermentation were screened. The relative contents of 2-oxoglutarate, ornithine, glutamine, and citrulline were higher in GQ1-3, whereas those of l-homoserine, aspartic acid, and asparagine were higher in HGPA20. These findings indicate that α-ketoglutaric acid-derived amino acid synthesis is preponderant in GQ1-3, whereas oxaloacetate-derived type is predominant in HGPA20. The different pathways of amino acid synthesis lead to the distinct nutrients and umami substances in the fermented soybean pastes.

Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae.

In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae.

Fathoming Aspergillus oryzae metabolomes in formulated growth matrices.

The stochasticity of Aspergillus oryzae (Trivially: the koji mold) pan-metabolomes commensurate with its ubiquitously distributed landscapes, i.e. growth matrices have been seemed uncharted since its food fermentative systems are mostly being investigated. In this review, we explicitly have discussed the likely tendencies of A. oryzae metabolomes pertaining to its growth milieu formulated with substrate matrices of varying nature, composition, texture, and associated physicochemical parameters. We envisaged typical food matrices, namely, meju, koji, and moromi as the semi-natural cultivation models toward delineating the metabolomic patterns of the koji mold, which synergistically influences the organoleptic and functional properties of the end products. Further, we highlighted how tailored conditions in sub-natural growth matrices, i.e. synthetic cultivation media blends, inducers, and growth surfaces, may influence A. oryzae metabolomes and targeted phenotypes. In general, the sequential or synchronous growth of A. oryzae on formulated matrices results in a number of metabolic tradeoffs with its immediate microenvironment influencing its adaptive and regulatory metabolomes. In broader context, evaluating the metabolic plasticity of A. oryzae relative to the tractable variables in formulated growth matrices might help approximate its growth and metabolism in the more complex natural matrices and environs. These approaches may considerably help in the design and manipulation of hybrid cultivation systems towards the efficient harnessing of commercial molds.

A new method for determining the mycelial weight of the koji-mold Aspergillus oryzae by measuring its glycosylceramide content.

At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

Rapid evaluation of γ-aminobutyric acid in foodstuffs by direct real-time mass spectrometry.

Direct analysis in real-time ionization coupled with mass spectrometry (DART-MS) was first applied for the rapid determination of gamma-aminobutyric acid (GABA) in foods. Samples of germinated barley and fermented beans containing GABA at different levels were used, and the results were compared with those obtained by ultrahigh-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (UHPLC-ESI-MS). After a series of optimization, a simple sample extraction procedure using 30% methanol aqueous solution was conducted, followed by direct determination of sample extracts without chromatographic separation or prior derivatization. The optimized DART-MS method exhibited low limits of detection (0.040 mg·kg-1) and good recovery rates (88.6%-104%). The Aspergillus oryzae-fermented black beans produced the highest amount GABA. The results for the samples slightly varied between DART-MS and UHPLC-ESI-MS. Current findings indicate that DART-MS could be a high-throughput alternative to classic UHPLC-ESI-MS.

Measuring Bacterial Glycosyl Hydrolase Activity with a Soluble Capture Probe by Mass Spectrometry.

A solution-phase enzymatic assay has been developed to track bacterial glycosyl hydrolase activity by surface-assisted MALDI-TOF mass spectrometry. Lactose was equipped with an azide-functionalized linker and was supplemented to bacterial cultures as an artificial substrate for bacterial ß-galactosidase enzyme. The azide linked glycoside probe was then covalently captured on an alkyne-functionalized indium tin oxide sample plate via a bio-orthogonal copper-catalyzed azide alkyne cycloaddition (CuAAC). The noncovalent immobilization of the alkyne capture tag via hydrophobic interactions on the ITO-sample plate allowed the analysis of the probe conjugate by surface-based mass spectrometry. The ratio of digested to nondigested lactose probe was then employed as a measure for bacterial hydrolase activity, which correlated well with bacterial growth measured by optical density. In addition, we established in a proof of concept experiment that the setup was well suited to identify antibiotic susceptibility of bacterial strains with a performance comparable to current state-of-the-art methods. While the proof of concept version is limited to the identification of a single enzyme activity, we envisage that the use of multiple substrate probes in a multiplexed version will allow the quantification of various glycosyl hydrolase activities with clinical relevance in a single experiment.

Systematic metabolic profiling and bioactivity assays for bioconversion of Aceraceae family.

Plants are an important and inexhaustible source of bioactive molecules in food, medicine, agriculture, and industry. In this study, we performed systematic liquid chromatography-mass spectrometry (LC-MS)-based metabolic profiling coupled with antioxidant assays for indigenous plant family extracts. Partial least-squares discriminant analysis of LC-MS datasets for the extracts of 34 plant species belonging to the families Aceraceae, Asteraceae, and Rosaceae showed that these species were clustered according to their respective phylogenies. In particular, seven Aceraceae species were clearly demarcated with higher average antioxidant activities, rationalizing their application for bioconversion studies. On the basis of further evaluation of the interspecies variability of metabolic profiles and antioxidant activities among Aceraceae family plants, we found that Acer tataricum (TA) extracts were clearly distinguished from those of other species, with a higher relative abundance of tannin derivatives. Further, we detected a strong positive correlation between most tannin derivatives and the observed higher antioxidant activities. Following Aspergillus oryzae-mediated fermentative bioconversion of Acer plant extracts, we observed a time-correlated (0-8 days) linear increase in antioxidant phenotypes for all species, with TA having the highest activity. Temporal analysis of the MS data revealed tannin bioconversion mechanisms with a relatively higher abundance of gallic acid (m/z 169) accumulated at the end of 8 days, particularly in TA. Similarly, quercetin precursor (glycoside) metabolites were also transformed to quercetin aglycones (m/z 301) in most Acer plant extracts. The present study underscores the efficacy of fermentative bioconversion strategies aimed at enhancing the quality and availability of bioactive metabolites from plant extracts.

Comparison of volatile and non-volatile metabolites in rice wine fermented by Koji inoculated with Saccharomycopsis fibuligera and Aspergillus oryzae.

This study investigated volatile and nonvolatile metabolite profiles of makgeolli (a traditional rice wine in Korea) fermented by koji inoculated with Saccharomycopsis fibuligera and/or Aspergillus oryzae. The enzyme activities in koji were also examined to determine their effects on the formation of metabolites. The contents of all 18 amino acids detected were the highest in makgeolli fermented by S. fibuligera CN2601-09, and increased after combining with A. oryzae CN1102-08, unlike the contents of most fatty acids. On the other hand, major volatile metabolites were fusel alcohols, acetate esters, and ethyl esters. The contents of most fusel alcohols and acetate esters were the highest in makgeolli fermented by S. fibuligera CN2601-09, for which the protease activity was the highest, leading to the largest amounts of amino acods. The makgeolli samples fermented only by koji inoculated with S. fibuligera could be discriminated on PCA plots from the makgeolli samples fermented in combination with A. oryzae. In the case of nonvolatile metabolites, all amino acids and some metabolites such as xylose, 2-methylbenzoic acid, and oxalic acid contributed mainly to the characteristics of makgeolli fermented by koji inoculated with S. fibuligera and A. oryzae. These results showed that the formations of volatile and nonvolatile metabolites in makgeolli can be significantly affected by microbial strains with different enzyme activities in koji. To our knowledge, this study is the first report on the effects of S. fibuligera strains on the formation of volatile and non-volatile metabolites in rice wine, facilitating their use in brewing rice wine.

Transcriptome analysis of different growth stages of Aspergillus oryzae reveals dynamic changes of distinct classes of genes during growth.

BACKGROUND: The gene expression profile and metabolic pathways of Aspergillus oryzae underlying the anatomical and morphological differentiation across different growth stages have not been fully characterized. The rapid development of next-generation sequencing technologies provides advanced knowledge of the genomic organization of A. oryzae. RESULTS: In this study, we characterized the growth and development of A. oryzae at different growth stages, including the adaptive phase, logarithmic phase, and stationary phase. Our results revealed that A. oryzae undergoes physiological and morphological differentiation across the different stages. RNA-seq was employed to analyze the three stages of A. oryzae, which generated more than 27 million high-quality reads per sample. The analysis of differential gene expression showed more genes expressed differentially upon transition from the adaptive phase to the logarithmic and stationary phases, while relatively steady trend was observed during the transition from the logarithmic phase to the stationary phase. GO classification of the differentially expressed genes among different growth stages revealed that most of these genes were enriched for single-organism process, metabolic process, and catalytic activity. These genes were then subjected to a clustering analysis. The results showed that the cluster with the majority of genes with increased expression upon transition from the adaptive phase to the logarithmic phase, and steady expression from the logarithmic phase to the stationary phase was mainly involved in the carbohydrate and amino acid metabolism. CONCLUSION: Our results provide a foundation for identifying developmentally important genes and understanding the biological processes across various growth stages.