Jun-ichi Maruyama

Promotion of efficient Saccharification of crystalline cellulose by Aspergillus fumigatus Swo1.

ABSTRACT Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.

Multiple gene disruptions by marker recycling with highly efficient gene-targeting background (ΔligD) in Aspergillus oryzae

Previously we reported that double disruption of the proteinase genes (tppA and pepE) improved heterologous protein production by Aspergillus oryzae (Jin et al. Appl Microbiol Biotechnol 76:1059–1068, 2007). Since A. oryzae has 134 protease genes, the number of auxotrophy in a single host is limited for multiple disruptions of many protease genes. In order to rapidly perform multiple gene disruptions in A. oryzae, we generated the marker recycling system in highly efficient gene-targeting background. A. oryzaeligD gene homologous to Neurospora crassamus-53 gene involved in nonhomologous chromosomal integration was disrupted, followed by disruption of the pyrG gene for uridine/uracil auxotroph. We further performed successive rounds of gene disruption (tppA and pepE) by the pyrG marker with high gene-targeting efficiency allowed by the ΔligD background. After each disruption process the pyrG marker was excised by the direct repeats consisting of ~300 bp upstream flanking region of the target gene, resulting in no residual ectopic/foreign DNA fragments in the genome. Consequently, we succeeded to breed the double proteinase gene disruptant (ΔtppA ΔpepE) applicable to further sequential gene disruptions in A. oryzae.

Stress-Activated MAP Kinase Cascades in Cellular Senescence

In response to progressive telomere shortening in successive cell divisions, normal somatic cells withdraw from the cell cycle and exhibit irreversible growth arrest. This state, called cellular senescence, is induced not only by telomere shortening but also by various physico-chemical stressors that induce DNA damage and chromatin disruption as well as by strong mitogenic signals. Because senescent cells never re-enter the cell cycle, cellular senescence appears to prevent malignant transformation of damaged cells and thus contributes to tumor suppression. On the other hand, excess accumulation of senescent cells attenuates the integrity and normal function of tissues, leading to age-related diseases. In addition to the well-established roles of p53 and pRB in cellular senescence, recent evidence suggests that stress-activated mitogen-activated protein kinase (MAPK) cascades that converge on c-Jun N-terminal kinases (JNKs) and p38 MAPKs also play important roles in the regulation of cellular senescence. In this review, we focus on signaling that regulates stress-induced cellular senescence, with special focus on the JNK and p38 MAPK cascades.

Development of a Versatile Expression Plasmid Construction System for Aspergillus oryzae and Its Application to Visualization of Mitochondria

We report here a development of the MultiSite GatewayTM-based versatile plasmid construction system applicable for the rapid and efficient preparation of Aspergillus oryzae expression plasmids. This system allows the simultaneous connection of the three DNA fragments inserted in entry clones along with a destination vector in a defined order and orientation. We prepared a variety of entry clones and destination vectors containing promoters, genes encoding carrier-proteins and fusion tags, and selectable markers, which makes it possible to generate 80 expression plasmids for each target protein. Using this system, plasmids for expression of the EGFP fused with the mitochondrial-targeting signal of citrate synthase (AoCit1) were generated. Tubular structures of mitochondria were visualized in the transformants expressing the AoCit1-EGFP fusion protein. This plasmid construction system allows us to prepare a large number of expression plasmids without laborious DNA manipulations, which would facilitate molecular biological studies on A. oryzae.

Analysis of Expressed Sequence Tags from the Fungus Aspergillus oryzae Cultured Under Different Conditions

Abstract We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.

Rhomboid protease PARL mediates the mitochondrial membrane potential loss-induced cleavage of PGAM5.

Background: Mitochondrial rhomboid protease PARL mediates the cleavage of PINK1 in healthy mitochondria. Results: PARL mediates the cleavage of PGAM5 in damaged mitochondria. Conclusion: PARL regulates differential cleavage of PINK1 and PGAM5 depending on the health status of mitochondria. Significance: This is the first implication of stress-dependent regulation of PARL-mediated RIP. Regulated intramembrane proteolysis is a widely conserved mechanism for controlling diverse biological processes. Considering that proteolysis is irreversible, it must be precisely regulated in a context-dependent manner. Here, we show that phosphoglycerate mutase 5 (PGAM5), a mitochondrial Ser/Thr protein phosphatase, is cleaved in its N-terminal transmembrane domain in response to mitochondrial membrane potential (ΔΨm) loss. This ΔΨm loss-dependent cleavage of PGAM5 was mediated by presenilin-associated rhomboid-like (PARL). PARL is a mitochondrial resident rhomboid serine protease and has recently been reported to mediate the cleavage of PINK1, a mitochondrial Ser/Thr protein kinase, in healthy mitochondria with intact ΔΨm. Intriguingly, we found that PARL dissociated from PINK1 and reciprocally associated with PGAM5 in response to ΔΨm loss. These results suggest that PARL mediates differential cleavage of PINK1 and PGAM5 depending on the health status of mitochondria. Our data provide a prototypical example of stress-dependent regulation of PARL-mediated regulated intramembrane proteolysis.

Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins

Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

Occurrence of tetrodotoxin in the gastropod mollusk Tutufa lissostoma (frog shell)

Paralytic toxicity was detected in gastropod mollusk Tutufa lissostoma (frog shell) specimens collected from Suruga Bay, Shizuoka Prefecture, Japan. Seventeen of the 22 digestive glands removed were toxic; the highest toxicity, expressed as tetrodotoxin (TTX), being as high as 700 mouse units (MU) per gram. Attempts were made to isolate the toxin from pooled digestive glands by activated charcoal treatment and column chromatography on Amberlite IRC-50, CM-Sephadex C-25 and Bio-Rex 70. The toxin showed a specific toxicity (as TTX) of 4200 MU/mg. It exhibited the same thin-layer chromatographic and electrophoretic behaviors and 1H-NMR spectrum as TTX. The toxin gave the same pattern as the TTX standard when alkali-hydrolyzed and analyzed by GC--MS, indicating that it contains the quinazoline skeleton specific to TTX. From these results the frog shell toxin was identified as TTX.

Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae

In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with α-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi.

Extracellular Production of Neoculin, a Sweet-Tasting Heterodimeric Protein with Taste-Modifying Activity, by Aspergillus oryzae

ABSTRACT Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, α-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.