Significance of two intracellular triacylglycerol lipases of Aspergillus oryzae in lipid mobilization: A perspective in industrial implication for microbial lipid production.

Microbial lipid production of oleaginous strains involves in a complex cellular metabolism controlling lipid biosynthesis, accumulation and degradation. Particular storage lipid, triacylglycerol (TAG), contributes to dynamic traits of intracellular lipids and cell growth. To explore a basis of TAG degradation in the oleaginous strain of Aspergillus oryzae, the functional role of two intracellular triacylglycerol lipases, AoTgla and AoTglb, were investigated by targeted gene disruption using CRISPR/Cas9 system. Comparative lipid profiling of different cultivation stages between the control, single and double disruptant strains (ΔAotgla, ΔAotglb and ΔAotglaΔAotglb strains) showed that the inactivation of either AoTgla or AoTglb led to the increase of total lipid contents, particularly in the TAG fraction. Moreover, the prolonged lipid-accumulating stage of all disruptant strains was obtained as indicated by a reduction in specific rate of lipid turnover, in which a holding capacity in maximal lipid and TAG levels was achieved. The involvement of AoTgls in spore production of A. oryzae was also discovered. In addition to the significance in lipid physiology of the oleaginous fungi, this study provides an impact on industrial practice by overcoming the limitation in short lipid-accumulating stage of the fungal strain, which facilitate the cell harvesting step at the maximum lipid production yield.

Relationship between pellet formation by Aspergillus oryzae strain KB and the production of ß-fructofuranosidase with high transfructosylation activity.

Aspergillus oryzae KB produces two ß-fructofuranosidases (F1 and F2). F1 has high transfructosylation activity (Ut) to produce fructooligosaccharides. F2 has high hydrolysis activity (Uh), releasing glucose and fructose. It is desirable to selectively produce F1, which can be used for production of fructooligosaccharides. Here, the relationship between filamentous pellet size and selective production of F1 in liquid culture was investigated. Our finding revealed that: (i) The mean particle size of pellets (5.88 ± 1.36 mm) was larger, and the ratio of Ut to Uh was improved (Ut/Uh = 5.0) in 10% sucrose medium compared with 1% sucrose medium (pellet size = 2.60 ± 0.37 mm; Ut/Uh = 0.96). (ii) The final culture pH of the 1% sucrose medium was 8.7; on controlling the pH of 1% sucrose medium at 5.0, increased pellet size (9.69 ± 2.01 mm) and Ut/Uh (7.8) were observed. (iii) When 3% glycerin was used as carbon source, the pellet size decreased to 1.09 ± 0.33 mm and Ut/Uh was 0.57. (iv) In medium containing 1% sucrose, the pellet size was dependent on the number of spores used in the culture inoculum, but, in these experiments, Ut/Uh was almost constant (1.05 ± 0.08). Collectively, the data show that the value of Ut/Uh is proportional to the pellet size when liquid culture of A. oryzae strain KB is performed in some conditions (such as in the presence of high sucrose concentration, low pH, or added Tween surfactant), but in other conditions Ut/Uh is independent of pellet size.

Purification and characterization of glucoamylase of Aspergillus oryzae from Luzhou-flavour Daqu.

OBJECTIVE: To obtain novel glucoamylase from Daqu microbe. RESULTS: A dominant strain known as LZ2 with high activity of hydrolyzing starch was isolated from Luzhou Daqu, a Chinese traditional fermentation starter. The LZ2 was identified as Aspergillus oryzae by 18S rDNA sequence analysis. Glucoamylase from LZ2, named as GA-LZ2, was purified to homogeneity and showed a single band with expected molecular mass of 60 kD. The GA-LZ2 effectively degraded amylose, rice starch and wheat starch. Optimal temperature and pH value of enzyme were 60 °C and pH 4.0 respectively. The GA-LZ2 displayed significant thermal stability and pH stability at moderate temperature and low pH. Intriguingly, the thermostability was enhanced in the presence of starch. In addition, GA-LZ2 exhibited insensitivity to glucose, independence of metal ions and tolerance to organic solvents. The GA-LZ2 retained complete activity in the presence of 100 mM glucose and 5% ethanol and methanol. CONCLUSION: Glucoamylase GA-LZ2 displayed broad substrate specificity, strong stability and tolerance, suggesting that GA-LZ2 carry potential for industrial application in bioethanol production.

A comparative genomics study of 23 Aspergillus species from section Flavi.

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

Evolution of Aspergillus oryzae before and after domestication inferred by large-scale comparative genomic analysis.

Aspergillus oryzae is an industrially useful species, of which various strains have been identified; however, their genetic relationships remain unclear. A. oryzae was previously thought to be asexual and unable to undergo crossbreeding. However, recent studies revealed the sexual reproduction of Aspergillus flavus, a species closely related to A. oryzae. To investigate potential sexual reproduction in A. oryzae and evolutionary history among A. oryzae and A. flavus strains, we assembled 82 draft genomes of A. oryzae strains used practically. The phylogenetic tree of concatenated genes confirmed that A. oryzae was monophyletic and nested in one of the clades of A. flavus but formed several clades with different genomic structures. Our results suggest that A. oryzae strains have undergone multiple inter-genomic recombination events between A. oryzae ancestors, although sexual recombination among domesticated species did not appear to have occurred during the domestication process, at least in the past few decades. Through inter- and intra-cladal comparative analysis, we found that evolutionary pressure induced by the domestication of A. oryzae appears to selectively cause non-synonymous and gap mutations in genes involved in fermentation characteristics, as well as intra-genomic rearrangements, with the conservation of industrially useful catalytic enzyme-encoding genes.

Discrimination of Aspergillus flavus from Aspergillus oryzae by matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry.

BACKGROUND: Aspergillus flavus is a major cause of severe non-invasive fungal infections in the Middle Eastern countries. However, it is difficult to distinguish A flavus from A oryzae. OBJECTIVES: To assess the potential of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) in discriminating between A flavus and A oryzae and compare it with ß-tubulin gene sequencing. METHODS: We used the Bruker Daltonik MALDI-TOF MS system to analyse 200 clinical and environmental A flavus isolates and one A pseudonomius and one A alliaceus (Aspergillus section Flavi) isolate a priori identified as such by sequencing of the ß-tubulin gene. RESULTS: All 200 A flavus isolates were identified at the genus level and 176 (88%) at the species levels by MALDI-TOF MS based on the spectral log-scores (≥2.0 and 1.7-1.99, respectively); among them, only 18 (10.2%) were confirmed as A flavus, whereas 35 (19.9%) were identified as A oryzae and 123 (69.9%) as A flavus/A oryzae. Aspergillus pseudonomius and A alliaceus were misidentified as A flavus and A parasiticus with log-score values of 1.39 and 1.09, respectively. CONCLUSIONS: The results indicate that the commercially available Bruker Daltonik MALDI-TOF MS score database cannot separate A flavus and A oryzae species. We also showed that establishment of an in-house library is a useful tool to discriminate closely related Aspergillus species, including A flavus and A oryzae.

First record of Aspergillus oryzae as an entomopathogenic fungus against the poultry red mite Dermanyssus gallinae.

The poultry red mite, Dermanyssus gallinae, is a blood-feeding ectoparasite that affects egg-laying hens worldwide. Strategies to control this parasite have focused in the use of entomopathogenic fungi, such as Metarhizium anisopliae. However, only a few studies have evaluated the use of Aspergillus oryzae to control D. gallinae and none of them have employed native strains. In the work presented here, a novel entomopathogenic fungus was isolated from a dead D. gallinae. The results of phylogenetic analysis showed 100% similarity between the isolated strain and those of two species, A. oryzae and Aspergillus flavus, and 99.82% similarity with A. parvisclerotigenus, which were in the same branch of the Flavi section of the genus Aspergillus. This entomopathogenic fungus was a non-aflatoxin B1 producer, as shown by the presence of aflatoxin B1 in the conidial infection suspension. Morphological features of fungus in comparison with A. oryzae and A. flavus indicated that the isolated strain belonged to A. oryzae, and was named Aspergillus sp. Dg-1. The pathogenicity of Aspergillus sp. Dg-1 on D. gallinae at different life stages was then assessed under laboratory conditions. The experiments showed that the isolated strain significantly increased the mortality rate in adult mites, up to 24.83 ± 2.25, compared to the mortality rates in the control group, which were 15.17 ± 2.75 (P ＜ 0.05). However, Aspergillus sp. Dg-1 did not have pathogenic effects on the second nymph stage of D. gallinae. Our findings demonstrate that Aspergillus sp. Dg-1 has pathogenic effects on D. gallinae in their adult stage, presenting biocontrol potential against D. gallinae.

Production and biochemical characterization of an alkaline protease from Aspergillus oryzae CH93.

In this study, Aspergillus oryzae CH93 was isolated from soil sample and examined using molecular analysis. Following culture of A. oryzae CH93 under optimal enzyme production, a 47.5kDa extracellular protease was purified using ammonium sulfate precipitation and Q-Sepharose chromatography. The optimal pH 8 and temperature of 50°C obtained for the isolated protease. Sodium dodecyl sulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), H2O2 decreased activity, while Triton X-100 and phenylmethanesulfonyl fluoride (PMSF) had no inhibitory effect on the enzyme activity; meanwhile, 2-mercaptoethanol and ethylenediaminetetraacetic acid (EDTA) declined the protease activity. Isoamyl alcohol and acetone (30%) enhanced activity whereas 2-propanol, isopropanol and dimethyl sulfoxide (DMSO) (30%) reduced protease activity. The enzyme exhibited a half-life of 100min at its optimum temperature. Among five substrates of bovine serum albumin (BSA), N-acetyl-l-tyrosine ethyl ester monohydrate (ATEE), casein, azocasein and gelatin results showed that casein is the best substrate with Vmax of 0.1411±0.004µg/min and Km of 2.432±0.266µg/ml. In conclusion, the extracted protease from A. oryzae CH93 as a fungal source possessed biochemical features which could be useful in some application usages.

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 1 kb 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.

Degradation of 3-phenoxybenzoic acid by a filamentous fungus Aspergillus oryzae M-4 strain with self-protection transformation.

A novel filamentous fungus M-4 strain was isolated from soy sauce koji and identified as Aspergillus oryzae (Collection number: CGMCC 11645) on the basis of morphological characteristics and internal transcribed spacer sequence. M-4 could degrade 80.62 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L-1) within 5 days. 3-PBA degradation occurred in accordance with first-order kinetics. The degradation metabolites of 3-PBA were identified through high-performance liquid chromatography-mass spectrometry (HPLC-MS). Relevant enzymatic activities and substrate utilization were also investigated, which indicated that M-4 could effectively degrade the intermediates of 3-PBA. Base on analysis of these metabolites, a novel biochemical pathway for the degradation of 3-PBA was proposed. There exists a mutual transformation between 3-phenoxy-benzyl alcohol and 3-PBA, which was firstly reported about the degradation of 3-PBA and may be attributed to self-protection transformation of M-4; subsequently, 3-PBA was gradually transformed into phenol, 3-hydroxy-5-phenoxy benzoic acid, protocatechuic acid and gallic acid. The safety of M-4 was evaluated via an acute toxicity test in vivo. The biodegradation ability of M-4 without toxic effects reveals that this fungus may be likely to be used for eliminating 3-PBA from contaminated environment or fermented foods.

Evaluation of commercial soy sauce koji strains of Aspergillus oryzae for γ-aminobutyric acid (GABA) production.

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production.

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 1.5 kb 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.

Purification and characterization of a thermostable hypothetical xylanase from Aspergillus oryzae HML366.

In the current study, fermentation broth of Aspergillus oryzae HML366 in sugar cane bagasse was subjected to ultrafiltration and ion exchange chromatography, and two xylanases, XynH1 and XynH2, were purified. Time-of-flight mass spectrometry coupled with SDS-PAGE analysis revealed that XynH1 is identical to the hypothetical A. oryzae RIB40 protein XP_001826985.1, with a molecular weight of 33.671 kDa. Likewise, XynH2 was identified as xylanase XynF1 with a molecular weight of 35.402 kDa. Sequence analysis indicated that XynH1 belongs to glycosyl hydrolases family 10. The specific activity of XynH1 was measured at 476.9 U/mg. Optimal xylanase activity was observed at pH 6.0, and enzyme remained active within pH 4.0-10.0 and at a temperature below 70 °C. Mg(2+), Mn(2+), Ca(2+), and K(+) enhanced the XynH1 xylanase activity to 146, 122, 114, and 108%, respectively. XynH1 hydrolyzed Birchwood xylan and Larchwood xylan effectively. The K m and V max of XynH1 values determined were 1.16 mM and 336 µmol/min/mg with Birchwood xylan as the substrate. A. oryzae HML366 xylanase XynH1 showed superior heat and pH tolerance, therefore may have significant applications in paper and biofuel industries. These studies constitute the first investigation of the xylanase activities of the hypothetical protein XP_001826985.1 form A. oryzae.

Comparison of the genomes and transcriptomes associated with the different protease secretions of Aspergillus oryzae 100-8 and 3.042.

Aspergillus oryzae is used to produce traditional fermented foods and beverages. A. oryzae 3.042 produces a neutral protease and an alkaline protease but rarely an acid protease, which is unfavourable to soy-sauce fermentation. A. oryzae 100-8 was obtained by N(+) ion implantation mutagenesis of A. oryzae 3.042, and the protease secretions of these two strains are different. Sequencing the genome of A. oryzae 100-8 and comparing it to the genomes of A. oryzae 100-8 and 3.042 revealed some differences, such as single nucleotide polymorphisms, nucleotide deletion or insertion. Some of these differences may reflect the ability of A. oryzae to secrete proteases. Transcriptional sequencing and analysis of the two strains during the same growth processes provided further insights into the genes and pathways involved in protease secretion.

Safety evaluation of filamentous fungi isolated from industrial doenjang koji.

A few starters have been developed and used for doenjang fermentation but often without safety evaluation. Filamentous fungi were isolated from industrial doenjang koji, and their potential for mycotoxin production was evaluated. Two fungi were isolated; one was more dominantly present (90%). Both greenish (SNU-G) and whitish (SNU-W) fungi showed 97% and 95% internal transcribed spacer sequence identities to Aspergillus oryzae/flavus, respectively. However, the SmaI digestion pattern of their genomic DNA suggested that both belong to A. oryzae. Moreover, both fungi had morphological characteristics similar to that of A. oryzae. SNU-G and SNU-W did not form sclerotia, which is a typical characteristic of A. oryzae. Therefore, both fungi were identified to be A. oryzae. In aflatoxin gene cluster analysis, both fungi had norB-cypA genes similar to that of A. oryzae. Consistent with this, aflatoxins were not detected in SNU-G and SNU-W using ammonia vapor, TLC, and HPLC analyses. Both fungi seemed to have a whole cyclopiazonic acid (CPA) gene cluster based on PCR of the maoA, dmaT, and pks-nrps genes, which are key genes for CPA biosynthesis. However, CPA was not detected in TLC and HPLC analyses. Therefore, both fungi seem to be safe to use as doenjang koji starters and may be suitable fungal candidates for further development of starters for traditional doenjang fermentation.

Improved production of protease-resistant phytase by Aspergillus oryzae and its applicability in the hydrolysis of insoluble phytates.

Among three hundred isolates of filamentous fungi, Aspergillus oryzae SBS50 secreted higher phytase activity at pH 5.0, 35 °C and 200 rpm after 96 h of fermentation. Starch and beef extract supported the highest phytase production than other carbon and nitrogen sources. A nine-fold improvement in phytase production was achieved due to optimization. Supplementation of the medium with inorganic phosphate repressed the enzyme synthesis. Among surfactants tested, Tween 80 increased fungal growth and phytase production, which further resulted in 5.4-fold enhancement in phytase production. The phytase activity was not much affected by proteases treatment. The enzyme resulted in the efficient hydrolysis of insoluble phytate complexes (metal- and protein-phytates) in a time dependent manner. Furthermore, the hydrolysis of insoluble phytates was also supported by scanning electron microscopy. The enzyme, being resistant to trypsin and pepsin, and able to hydrolyze insoluble phytates, can find an application in the animal food/feed industry for improving nutritional quality and also in combating environmental phosphorus pollution and plant growth promotion.

The proportion of non-aflatoxigenic strains of the Aspergillus flavus/oryzae complex from meju by analyses of the aflatoxin biosynthetic genes.

Strains of the Aspergillus flavus/oryzae complex are frequently isolated from meju, a fermented soybean product, that is used as the starting material for ganjang (soy sauce) and doenjang (soybean paste) production. In this study, we examined the aflatoxin producing capacity of A. flavus/oryzae strains isolated from meju. 192 strains of A. flavus/oryzae were isolated from more than 100 meju samples collected from diverse regions of Korea from 2008 to 2011, and the norB-cypA, omtA, and aflR genes in the aflatoxin biosynthesis gene cluster were analyzed. We found that 178 strains (92.7%) belonged to non-aflatoxigenic group (Type I of norB-cypA, IB-L-B-, IC-AO, or IA-L-B- of omtA, and AO type of aflR), and 14 strains (7.3%) belonged to aflatoxin-producible group (Type II of norB-cypA, IC-L-B+/B- or IC-L-B+ of omtA, and AF type of aflR). Only 7 strains (3.6%) in the aflatoxin-producible group produced aflatoxins on Czapek yeast-extract medium. The aflatoxin-producing capability of A. flavus/oryzae strains from other sources in Korea were also investigated, and 92.9% (52/56) strains from air, 93.9% (31/33) strains from rice straw, 91.7% (11/12) strains from soybean, 81.3% (13/16) strains from corn, 82% (41/50) strains from peanut, and 73.2% (41/56) strains from arable soil were included in the non-aflatoxigenic group. The proportion of non-aflatoxigenicity of meju strains was similar to that of strains from soybean, air and rice straw, all of which have an effect on the fermentation of meju. The data suggest that meju does not have a preference for non-aflatoxigenic or aflatoxin-producible strains of A. flavus/oryzae from the environment of meju. The non-aflatoxigenic meju strains are proposed to be named A. oryzae, while the meju strains that can produce aflatoxins should be referred to A. flavus in this study.

A further study on chromosome minimization by protoplast fusion in Aspergillus oryzae.

Our goal in this work was to develop a method to minimize the chromosomes of Aspergillus oryzae, to arrive at a deeper understanding of essential gene functions that will help create more efficient industrially useful strains. In a previous study, we successfully constructed a highly reduced chromosome 7 using multiple large-scale chromosomal deletions (Jin et al. in Mol Genet Genomics 283:1-12, 2010). Here, we have created a further reduced chromosome A. oryzae mutant harboring a reduced chromosome 7 and a reduced chromosome 8 both of which contain a large number of non-syntenic blocks. These are the smallest A. oryzae chromosomes that have been reported. Protoplast fusion between the two distinct chromosome-reduced mutants produced a vigorous and stable fusant which was isolated. PCR and flow cytometry confirmed that two kinds of nuclei, derived from the parent strains, existed in this fusant and that the chromosome DNA per nucleus was doubled, suggesting that the fusant was a heterozygous diploid strain. By treating the cell with 1 µg/ml benomyl, cell nuclei haploidization was induced in the stable diploid strain. Array comparative genomic hybridization and pulsed-field gel electrophoresis confirmed that the reduced chromosomes 7 and 8 co-existed in the haploid fusant and that no other chromosomal modifications had occurred. This method provides a useful tool for chromosome engineering in A. oryzae to construct an industry-useful strain.

Functional analysis of the putative AAA ATPase AipA localizing at the endocytic sites in the filamentous fungus Aspergillus oryzae.

We searched for novel components involved in Aspergillus oryzae endocytosis by yeast two-hybrid (YTH) screening. Using the endocytic marker protein AoAbp1 (A. oryzae homolog of Saccharomyces cerevisiae Abp1p) as bait, a putative AAA (ATPases associated with diverse cellular activities) ATPase encoded by a gene termed aipA (AoAbp1 interacting protein) was identified. Further YTH analyses showed that the 346-370 amino-acid region of AipA interacts with the SH3 (Src homology 3) domains of AoAbp1. Moreover, AipA colocalized with AoAbp1 at the tip region, suggesting that AipA functions in endocytosis. Although aipA disruptants did not display defective growth, an aipA-overexpressing strain displayed impaired growth and wider hyphal morphology. In addition, we generated strains that overexpressed either aipA(K542A) or aipA(E596Q) , whose mutations were introduced into the AAA ATPase domain of AipA and would cause the defect of ATPase activity. In contrast to the aipA-overexpressing strain, neither aipA(K542A) - nor aipA(E596Q) -overexpressing strains showed defective growth. Moreover, only the aipA-overexpressing strain displayed a defect of FM4-64 transport to the Spitzenkörper, suggesting that AipA negatively regulates apical endocytic recycling and that the AAA ATPase domain of AipA is crucial for its function.

Screening and identification of a fungal ß-glucosidase and the enzymatic synthesis of gentiooligosaccharide.

After screening with 0.1% esculoside and 0.03% FeCl(3), we identified from rotten wood a fungal isolate HML0366 that produces high amount of ß-glucosidase. Phenotypic and rDNA internal transcribed spacer sequence analyses indicated that the isolate belongs to Aspergillus oryzae. The ß-glucosidase produced by HML0366 had an activity of 128 U/g. high performance liquid chromatography analysis also demonstrated a high transglycosylation activity of the crude enzyme. The ß-glucosidase was stable between pH 4-10 at 60 °C. A gentiobiose yield of 30.86 g/L was achieved within 72 h of the enzymatic reaction at pH 5 and 55 °C using 50% glucose as the substrate. For the first time, we report here the isolation of an A. oryzae strain producing ß-glucosidase with high hydrolytic activities. The crude enzyme has a high transglycosylation activity, which enables the enzymatic synthesis of gentiooligosaccharides.