Estimation of hyphal tensile strength in production-scale Aspergillus oryzae fungal fermentations.

Fragmentation of filamentous fungal hyphae depends on two phenomena: hydrodynamic stresses, which lead to hyphal breakage, and hyphal tensile strength, which resists breakage. The goal of this study was to use turbulent hydrodynamic theory to develop a correlation that allows experimental data of morphology and hydrodynamics to be used to estimate relative (pseudo) tensile strength (sigma(pseudo)) of filamentous fungi. Fed-batch fermentations were conducted with a recombinant strain of Aspergillus oryzae in 80 m(3) fermentors, and measurements were made of both morphological (equivalent hyphal length, L) and hydrodynamic variables (specific power input, epsilon; kinematic viscosity, v). We found that v increased over 100-fold during these fermentations and, hence, Kolmogorov microscale (lambda) also changed significantly with time. In the impeller discharge zone, where hyphal fragmentation is thought to actually take place, lambda was calculated to be 700-3500 microm, which is large compared to the size of typical fungal hyphae (100-300 microm). This result implies that eddies in the viscous subrange are responsible for fragmentation. Applying turbulent theory for this subrange, it was possible to calculate sigma(pseudo)from morphological and hydrodynamic measurements. Pseudo tensile strength was not constant but increased to a maximum during the first half and then decreased during the second half of each fermentation, presumably due to differences in physiological state. When a literature correlation for hyphal fragmentation rate (k(frag)) was modified by adding a term to account for viscosity and tensile strength, the result was better qualitative agreement with morphological data. Taken together, these results imply hyphal tensile strength can change significantly over the course of large-scale, fed-batch fungal fermentations and that existing fragmentation and morphology models may be improved if they accounted for variations in hyphal tensile strength with time.     

Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation

Industrial enzymes are often produced by filamentous fungi in fed-batch cultivations. During cultivation, the different morphological forms displayed by the fungi have an impact on the overall production. The morphology of a recombinant lipase producing Aspergillus oryzae strain was investigated during fed-batch cultivations. During the exponential batch phase of the fed-batch cultivations, the average hyphal length increased as did the number of tips per hyphal element. Most striking was the finding that the diameter of the hyphal elements increased with an average factor of 1.5 during the batch phase from 2.8–2.9 up to 4.0–4.4 μm. The diameter of the hyphal elements remained constant, around 4 μm, after the feed was started. However, the diameter of the immediate hyphal tip, where the enzyme secretion is thought to take place, increased dramatically with up to a factor 2.5 during the feeding period. The expression of the recombinant lipase was induced by the feeding with maltose, and an increase in lipase activity was seen in parallel to a swelling of the tips. The results indicate that the two events are linked as a return to normal growth was observed upon cessation of production due to oxygen limitations.     

Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

Structural and functional analysis of the amdR regulatory gene of Aspergillus oryzae.

We have isolated the Aspergillus oryzae homologue of the amdR regulatory gene of Aspergillus nidulans by cross hybridization. Sequence analysis and functional studies have shown that the amdR genes are highly conserved and functionally interchangeable between the two species. The homology between the two genes extends throughout most of the coding sequences, including sequences encoding the DNA-binding domain and putative activation domains. Two regions of nonconserved sequence were also identified. Studies using various amdS::lacZ fusion constructs indicate that the A. oryzae gene product binds similar sequences and responds to inducer in a similar manner to the A. nidulans protein. Inactivation of the A. oryzae gene results in the inability to grow on gamma-amino-butyric acid (GABA) as a carbon and/or nitrogen source indicating that GABA utilization is amdR-dependent in A. oryzae as it is in A. nidulans.     

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

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

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.     

Transformation of Aspergillus aculeatus using the drug resistance gene of Aspergillus oryzae and the pyrG gene of Aspergillus nidulans

Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5'-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.     

Genetic analysis of growth inhibition of yeast cells caused by expression of Aspergillus oryzae RNase T1

Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.     

Three-dimensional image analysis of plugging at the septal pore by Woronin body during hypotonic shock inducing hyphal tip bursting in the filamentous fungus Aspergillus oryzae

We observed that the filamentous fungus, Aspergillus oryzae, grown on agar media burst out cytoplasmic constituents from the hyphal tip soon after flooding with water. Woronin body is a specialized organelle known to plug the septal pore adjacent to the lysed compartment to prevent extensive loss of cytoplasm. A. oryzae Aohex1 gene homologous to Neurospora crassa HEX1 gene encoding a major protein in Woronin body was expressed as a fusion with DsRed2, resulting in visualization of Woronin body. Confocal microscopy and three-dimensional reconstruction of images visualized the septal pore as a dark region surrounded by green fluorescence of EGFP-fused secretory protein, RNase T1, on the septum. Dual fluorescent labeling revealed the plugging of the septal pores adjacent to the lysed apical compartments by Woronin bodies during hypotonic shock. Disruption of Aohex1 gene caused disappearance of Woronin bodies and the defect to prevent extensive loss of cytoplasm during hypotonic shock.     

Nucleotide sequence and expression of the glucoamylase-encoding gene (glaA) from Aspergillus oryzae.

The glucoamylase-encoding gene (glaA) from Aspergillus oryzae was cloned using its cDNA as a probe, which had been isolated previously. From comparison of nucleotide (nt) sequences of genomic clones with its cDNA, the glaA gene was found to contain four short putative introns, 45-56 nt in length. The A. oryzae glaA gene shared 62% homology at the nt level with the A. niger glaA gene with the four introns located at the same position. The 5'-flanking region contained a TATA box at nt-72 from the start codon, and two putative CAAT sequences at nt-87 and -331. Genomic Southern analysis and physical mapping showed that the glaA gene is located on the smallest chromosome (3.4 Mb) of six separated bands of chromosomes. Clones containing the glaA gene, when re-introduced intro A. oryzae, resulted in a three- to eightfold increase in glucoamylase activity.     

Establishment of a new method to quantitatively evaluate hyphal fusion ability in Aspergillus oryzae

Hyphal fusion is involved in the formation of an interconnected colony in filamentous fungi, and it is the first process in sexual/parasexual reproduction. However, it was difficult to evaluate hyphal fusion efficiency due to the low frequency in Aspergillus oryzae in spite of its industrial significance. Here, we established a method to quantitatively evaluate the hyphal fusion ability of A. oryzae with mixed culture of two different auxotrophic strains, where the ratio of heterokaryotic conidia growing without the auxotrophic requirements reflects the hyphal fusion efficiency. By employing this method, it was demonstrated that AoSO and AoFus3 are required for hyphal fusion, and that hyphal fusion efficiency of A. oryzae was increased by depleting nitrogen source, including large amounts of carbon source, and adjusting pH to 7.0.     

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

ABSTRACT

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.     

Induction of contour sensing in Aspergillus niger by stress and its relevance to fungal growth mechanics and hyphal tip structure

Thigmotropism (contour sensing) has been assigned an important role in both plant and human fungal pathogens. However, outside these systems, our knowledge of the function of thigmotropism in fungal growth control is relatively poor. Furthermore, the effects of environmental stress on thigmotropic responses have received scant attention. To try to elucidate some of the mechanisms behind hyphal contour sensing in response to nutrient-poor environments, we have used micro-engineered substrates and several imaging techniques to investigate the thigmotropic reactions of the ubiquitous fungus Aspergillus niger. This organism not appear to demonstrate thigmotropic growth under normal conditions. Our results show that A. niger undergoes significant morphological changes during growth on solid substrates and demonstrate that the intensity of contour sensing varies depending on the area of the hyphal tip which initiates contact with the substrate. We propose that growth under nutrient-limited conditions triggers several factors that combine to increase thigmotropic sensitivity while conversely creating a 60 degrees arc at the hyphal tip which is blind to topographical variations. This has important consequences for our general understanding of the hyphal mode of growth in fungi as well as more specific aspects of hyphal tip development under stress.     

Efficient expression of a Phanerochaete chrysosporium manganese peroxidase gene in Aspergillus oryzae.

A manganese peroxidase gene (mnp1) from Phanerochaete chrysosporium was efficiently expressed in Aspergillus oryzae. Expression was achieved by fusing the mature cDNA of mnp1 with the A. oryzae Taka amylase promoter and secretion signal. The 3' untranslated region of the glucoamylase gene of Aspergillus awamori provided the terminator. The recombinant protein (rMnP) was secreted in an active form, permitting rapid detection and purification. Physical and kinetic properties of rMnP were similar to those of the native protein. The A. oryzae expression system is well suited for both mechanistic and site-directed mutagenesis studies.