Class VI G protein-coupled receptors in Aspergillus oryzae regulate sclerotia formation through GTPase-activating activity.

G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane receptors in eukaryotes that sense and transduce extracellular signals into cells. In Aspergillus oryzae, 16 canonical GPCR genes are identified and classified into nine classes based on the sequence similarity and proposed functions. Class VI GPCRs (AoGprK-1, AoGprK-2, and AoGprR in A. oryzae), unlike other GPCRs, feature a unique hybrid structure containing both the seven transmembrane (7-TM) and regulator of G-protein signaling (RGS) domains, which is not found in animal GPCRs. We report here that the mutants with double or triple deletion of class VI GPCR genes produced significantly increased number of sclerotia compared to the control strain when grown on agar plates. Interestingly, complementation analysis demonstrated that the expression of the RGS domain without the 7-TM domain is sufficient to restore the phenotype. In line with this, among the three Gα subunits in A. oryzae, AoGpaA, AoGpaB, and AoGanA, forced expression of GTPase-deficient mutants of either AoGpaA or AoGpaB caused an increase in the number of sclerotia formed, suggesting that RGS domains of class VI GPCRs are the negative regulators of these two GTPases. Finally, we measured the expression of velvet complex genes and sclerotia formation-related genes and found that the expression of velB was significantly increased in the multiple gene deletion mutants. Taken together, these results demonstrate that class VI GPCRs negatively regulate sclerotia formation through their GTPase-activating activity in the RGS domains. KEY POINTS: • Class VI GPCRs in A. oryzae regulate sclerotia formation in A. oryzae • RGS function of class VI GPCRs is responsible for regulation of sclerotia formation • Loss of class VI GPCRs resulted in increased expression of sclerotia-related genes.

Autophagy deficiency boosts the production of kojic acid in the filamentous fungus Aspergillus oryzae.

We found that the expression of genes involved in kojic acid (KA) biosynthesis, kojA, kojR, and kojT, was highly elevated in the Aspergillus oryzae autophagy-deficient mutants. In agreement, KA production was much increased in these mutants. Nuclear translocation of KojR, a transcription factor, was observed in the autophagy mutants before they were starved, explaining why KA production was boosted.

In vitro and in vivo characterization of genes involved in mannan degradation in Neurospora crassa.

To better understand the roles of genes involved in mannan degradation in filamentous fungi, in this study we searched, identified, and characterized one putative GH5 endo-ß-mannanase (GH5-7) and two putative GH2 mannan-degrading enzymes (GH2-1 and GH2-4) in Neurospora crassa. Real-time RT-PCR analyses showed that the expression levels of these genes were significantly up-regulated when the cells were grown in mannan-containing media where the induction level of gh5-7 was the highest. All three proteins were heterologously expressed and purified. GH5-7 displayed a substrate preference toward galactomannan by showing 10-times higher catalytic efficiency than to linear ß-mannan. In contrast, GH2-1 preferred short manno-oligosaccharides or ß-mannan as substrates. Compared to the wild type strain, the growth of Δgh5-7 and Δgh5-7Δgh2-4 mutants, but not Δgh2-1, Δgh2-4, and Δgh2-1Δgh2-4 mutants, was poor in the cultures containing glucomannan or galactomannan as the sole carbon source, suggesting that GH5-7 plays a critical role in the utilization of heteromannans in vivo. On the other hand, all the mutants showed significantly slow growth when grown in the medium containing linear ß-mannan. Collectively, these results indicate that N. crassa can utilize glucomannan and galactomannan without GH2-1 and GH2-4, but efficient degradation of ß-mannan requires a concerted action of three enzymes, GH5-7, GH2-1, and GH2-4.

Distinct enzymatic and cellular characteristics of two phospholipases A1 in Aspergillus oryzae.

Phospholipases A1 (PLA1s) catalyze the hydrolysis of sn-1 linkage in the glycerophospholipids, thereby releasing fatty acids and 2-acyl lysophospholipids. PLA1s are found in various organisms and tissues where they play diverse cellular functions, but their roles in filamentous fungi remain elusive. In this study we analyzed the enzymatic properties and physiological functions of two secretory PLA1s, PLA1-1 and its paralog PLA1-2, in the filamentous fungus Aspergillus oryzae. Although PLA1-1 and PLA1-2 share 49% amino acid sequence identity, they significantly differ in various aspects. While PLA1-1 displayed PLA1 activity to phosphatidylcholine and phosphatidylethanolamine, and degraded various phospholipids, PLA1-2 exhibited PLA1 activity only to phosphatidylglycerol. PLA1-1 was secreted to the culture medium, but PLA1-2 was not secreted and retained in the mycelium. Fluorescence microscopic observation of A. oryzae strains expressing EGFP-fused PLA1-1 and PLA1-2 demonstrated that they display overlapping but distinct cellular localization. A. oryzae mutants deleted for pla1-1 or pla1-2 grew normally, but the secreted phospholipase activity was significantly reduced in the Δpla1-1 strain. These data suggest that two sPLA1 enzymes are not redundant and play distinct cellular functions in A. oryzae.

A novel glucuronoyl esterase from Aspergillus fumigatus-the role of conserved Lys residue in the preference for 4-O-methyl glucuronoyl esters.

Cellulose in plant cell walls is mainly covered by hemicellulose and lignin, and thus efficient removal of these components is thought to be a key step in the optimal utilization of lignocellulose. The recently discovered carbohydrate esterase (CE) 15 family of glucuronoyl esterases (GEs) which cleave the linkages between the free carboxyl group of D-glucuronic acid in hemicellulose and the benzyl groups in lignin residues could contribute to this process. Herein, we report the identification, functional expression, and enzymatic characterization of a GE, AfGE, from the filamentous fungus Aspergillus fumigatus. AfGE was heterologously expressed in Aspergillus oryzae, and the purified enzyme displayed the ability to degrade the synthetic substrates mimicking the ester linkage between hemicellulose and lignin. AfGE is a potentially industrially applicable enzyme due to its characteristic as a thermophilic enzyme with the favorable temperature of 40-50 °C at pH 5. Molecular modeling and site-directed mutagenesis studies of AfGE demonstrated that Lys209 plays an important role in the preference for the substrates containing 4-O-methyl group in the glucopyranose ring.

Trp residue at subsite - 5 plays a critical role in the substrate binding of two protistan GH26 ß-mannanases from a termite hindgut.

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two ß-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to ß-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite - 5. The Km values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.

Yor022c protein is a phospholipase A1 that localizes to the mitochondrial matrix.

In mammals, three types of intracellular phospholipase A1 (iPLA1) enzymes have been characterized and are thought to be involved in various cellular processes such as phospholipid metabolism, organelle biogenesis, and membrane trafficking. In this study we analyzed the unique iPLA1-like protein, Yor022c, in the budding yeast Saccharomyces cerevisiae. By the mass spectrometry analysis, we demonstrate that Yor022c is actually a phospholipase displaying sn-1-specific activity toward phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid, generating 2-acyl lysophospholipids. GFP-fused Yor022c co-stained with the mitochondrial dye MitoTracker, indicating that, unlike its mammalian counterparts, it is a mitochondrial protein. Further biochemical fractionation experiment combined with protease sensitivity assay showed that Yor022c localizes to the mitochondrial matrix. Thus Yor022c is the first PLA1 putatively involved in the maintenance of sn-1 acyl chains of phospholipids in the mitochondrial inner membrane.

Mitochondria-localized phospholipase A2, AoPlaA, in Aspergillus oryzae displays phosphatidylethanolamine-specific activity and is involved in the maintenance of mitochondrial phospholipid composition.

In mammals, cytosolic phospholipases A2 (cPLA2s) play important physiological roles by releasing arachidonic acid, a precursor for bioactive lipid mediators, from the biological membranes. In contrast, fungal cPLA2-like proteins are much less characterized and their roles have remained elusive. AoPlaA is a cPLA2-like protein in the filamentous fungus Aspergillus oryzae which, unlike mammalian cPLA2, localizes to mitochondria. In this study, we investigated the biochemical and physiological functions of AoPlaA. Recombinant AoPlaA produced in E. coli displayed Ca2+-independent lipolytic activity. Mass spectrometry analysis demonstrated that AoPlaA displayed PLA2 activity to phosphatidylethanolamine (PE), but not to other phospholipids, and generated 1-acylated lysoPE. Catalytic site mutants of AoPlaA displayed almost no or largely reduced activity to PE. Consistent with PE-specific activity of AoPlaA, AoplaA-overexpressing strain showed decreased PE content in the mitochondrial fraction. In contrast, AoplaA-disruption strain displayed increased content of cardiolipin. AoplaA-overexpressing strain, but not its counterparts overexpressing the catalytic site mutants, exhibited retarded growth at low temperature, possibly because of the impairment of the mitochondrial function caused by excess degradation of PE. These results suggest that AoPlaA is a novel PE-specific PLA2 that plays a regulatory role in the maintenance of mitochondrial phospholipid composition.

Structural and biochemical analyses of glycoside hydrolase family 26 ß-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of ß-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

Expression, purification, refolding, and enzymatic characterization of two secretory phospholipases A2 from Neurospora crassa.

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of sn-2 linkage in the glycerophospholipid, thereby releasing fatty acid and 1-acyl lysophospholipid. Among sPLA2s from various organisms and tissues, group XIV fungal/bacterial sPLA2s are relatively less characterized compared to their mammalian counterparts. Here we report cloning, recombinant expression, refolding, and enzymatic characterization of two sPLA2s, NCU06650 and NCU09423, from the filamentous fungus Neurospora crassa. The hexahistidine-tagged putative mature region of both proteins was expressed in Escherichia coli. Inclusion bodies were solubilized using a high hydrostatic pressure refolding technique. NCU06650 was solubilized without any additives at alkaline pH, and the addition of arginine or non-detergent sulfobetain (NDSB) significantly improved the process at acidic pH. In contrast, NCU09423 was solubilized only when NDSB was added at alkaline pH. Both enzymes displayed a Ca(2+)-dependent lipolytic activity toward E. coli membrane. Mass spectrometry analysis using the synthetic phospholipids as substrates demonstrated that both enzymes preferentially cleaved the sn-2 ester linkage of substrates and generated 1-acyl lysophospholipids, demonstrating that they are bona fide PLA2.

The GH26 ß-mannanase RsMan26H from a symbiotic protist of the termite Reticulitermes speratus is an endo-processive mannobiohydrolase: heterologous expression and characterization.

Symbiotic protists in the gut of termites are prominent natural resources for enzymes involved in lignocellulose degradation. Here we report expression, purification, and biochemical characterization of a glycoside hydrolase family 26 mannanase RsMan26H from the symbiotic protist of the lower termite, Reticulitermes speratus. Biochemical analysis of RsMan26H demonstrates that this enzyme is an endo-processive mannobiohydrolase producing mannobiose from oligo- and polysaccharides, followed by a minor accumulation of oligosaccharides larger than mannobiose. To our knowledge, this is the first report describing the unique mannobiohydrolase enzyme from the eukaryotic origin.

Functional analysis of Abp1p-interacting proteins involved in endocytosis of the MCC component in Aspergillus oryzae.

We have investigated the functions of three endocytosis-related proteins in the filamentous fungus Aspergillus oryzae. Yeast two-hybrid screening using the endocytic marker protein AoAbp1 (A.oryzae homolog of Saccharomyces cerevisiae Abp1p) as a bait identified four interacting proteins named Aip (AoAbp1 interacting proteins). In mature hyphae, EGFP (enhanced green fluorescent protein) fused to Aips colocalized with AoAbp1 at the hyphal tip region and the plasma membrane, suggesting that Aips function in endocytosis. aipA is a putative AAA ATPase and its function has been dissected (Higuchi et al., 2011). aipB, the homolog of A. nidulans myoA, encodes an essential class I myosin and its conditional mutant showed a germination defect. aipC and aipD do not contain any recognizable domains except some proline-rich regions which may interact with two SH3 (Src homology 3) domains of AoAbp1. Neither aipC nor aipD disruptants showed any defects in their growth, but the aipC disruptant formed less conidia compared with the control strain. In addition, the aipC disruptant was resistant to the triazole antifungal drugs that inhibit ergosterol biosynthesis. Although no aip disruptants showed any defects in the uptake of the fluorescent dye FM4-64, the endocytosis of the arginine permease AoCan1, one of the MCC (membrane compartment of Can1p) components, was delayed in both aipC and aipD disruptants. In A. oryzae, AoCan1 localized mainly at the plasma membrane in the basal region of hyphae, suggesting that different endocytic mechanisms exist in apical and basal regions of highly polarized cells.

Lysophosphatidylcholine potentiates BDNF-induced TrkB phosphorylation and downstream signals in cerebellar granule neurons.

We found that brain-derived neurotrophic factor (BDNF)-induced phosphorylation of mitogen-activated protein kinase (MAPK) and Akt in cerebellar granule neurons was specifically potentiated by LPC. LPC also augmented the BDNF-induced phosphorylation of TrkB, the receptor for BDNF. In TrkB-transfected CHO-K1 cells, LPC potentiated BDNF-induced MAPK phosphorylation. These results suggest that LPC plays a role in BDNF-TrkB signaling by regulating the activation of TrkB.

Glucuronoyl esterase facilitates biomass degradation in Neurospora crassa by upregulating the expression of plant biomass-degrading enzymes.

Glucuronoyl esterase (GE) is a promising agent for the delignification of plant biomass since it has been shown to cleave the linkage between xylan and lignin in vitro. In this study, we demonstrate that NcGE, a GE from Neurospora crassa, stimulates plant biomass degradation. In vitro, NcGE synergistically increased the release of reducing sugars from plant biomass when added together with cellulase or xylanase. In vivo, overexpression of NcGE in N. crassa resulted in an increase in xylanolytic activity. Consistently, elevated transcription of genes encoding the major plant biomass degrading-enzymes (PBDEs) was observed in the NcGE overexpression strain. Increased xylanolytic activity and transcription of PDBE genes were largely abolished when the transcription factors clr-1, clr-2, or xlr-1 were deleted. Interestingly, the expression of some PBDE genes was increased when the hydrolysate of plant biomass by NcGE was added to the culture medium. We propose that NcGE boosts the production of PBDEs through the activation of key transcription factors, which is presumably caused by NcGE-mediated generation of hypothetical inducer(s) from plant biomass.

Heterologous expression in Pichia pastoris and characterization of an endogenous thermostable and high-glucose-tolerant ß-glucosidase from the termite Nasutitermes takasagoensis.

Termites are well-known cellulose decomposers and can give researchers insights into how to utilize lignocellulosic biomass in the actual scenario of energy consumption. In this work, an endogenous ß-glucosidase from the midgut of the higher termite Nasutitermes takasagoensis was purified to homogeneity by Ni(2+) affinity chromatography and its properties were characterized. This ß-glucosidase (G1mgNtBG1), which belongs to glycoside hydrolase family 1, is a homotrimer in its native form, with a molecular mass of 169.5 kDa, as demonstrated by gel filtration chromatography. The enzyme displayed maximum activity at pH 5.5 and had broad substrate specificities toward several saccharides, including cellobiose. G1mgNtBG1 showed a relatively high temperature optimum of 65°C and one of the highest levels of glucose tolerance among several ß-glucosidases already characterized, with a K(i) of 600 mM glucose. To examine the applicability of G1mgNtBG1 in biomass conversion, we compared the thermostability and glucose tolerance of G1mgNtBG1 with those of Novozym 188. We found that G1mgNtBG1 was more thermostable after 5 h of incubation at 60°C and more resistant to glucose inhibition than Novozym 188. Furthermore, our result suggests that G1mgNtBG1 acts synergistically with Celluclast 1.5 L in releasing reducing sugars from Avicel. Thus, G1mgNtBG1 seems to be a potential candidate for use as a supplement in the hydrolysis of biomass.

Expression and one-step purification of recombinant proteins using an alternative episomal vector for the expression of N-tagged heterologous proteins in Pichia pastoris.

Here we report the construction of an alternative episomal vector, pBGP3, which allows the expression of heterologous proteins with N-terminal hexahistidine and myc-epitope tags in Pichia pastoris. To test the usefulness of pBGP3, four cellulases from termites were expressed. Production was confirmed by activity assays and Western blot using anti-c-Myc antibody. Purification was performed by single-step Ni(2+)-affinity chromatography, which confirmed the efficiency of pBGP3.

Differential Biosynthesis and Roles of Two Ferrichrome-Type Siderophores, ASP2397/AS2488053 and Ferricrocin, in Acremonium persicinum.

Ferrichromes are a family of fungal siderophores with cyclic hexapeptide structures. Most fungi produce one or two ferrichrome-type siderophores. Acremonium persicinum MF-347833 produces ferrichrome-like potent Trojan horse antifungal antibiotics ASP2397 and AS2488053, the aluminum- and iron-chelating forms of AS2488059, respectively. Here, we show by gene sequencing followed by gene deletion experiments that A. persicinum MF-347833 possesses two nonribosomal peptide synthetase genes responsible for AS2488059 and ferricrocin assembly. AS2488059 was produced under iron starvation conditions and excreted into the media to serve as a defense metabolite and probably an iron courier. In contrast, ferricrocin was produced under iron-replete conditions and retained inside the cells, likely serving as an iron-sequestering molecule. Notably, the phylogenetic analyses suggest the different evolutionary origin of AS2488059 from that of conventional ferrichrome-type siderophores. Harnessing two ferrichrome-type siderophores with distinct biological properties may give A. persicinum a competitive advantage for surviving the natural environment.

Autophagy delivers misfolded secretory proteins accumulated in endoplasmic reticulum to vacuoles in the filamentous fungus Aspergillus oryzae.

Autophagy is a conserved intracellular degradation process of eukaryotic cells. In filamentous fungi, although autophagy has been reported to have multiple physiological roles, it is not clear whether autophagy is involved in the degradation of misfolded proteins. Here, we investigated the role of autophagy in the degradation of misfolded secretory proteins accumulated in endoplasmic reticulum (ER) in the filamentous fungus Aspergillus oryzae. In late-phase cultures, a disulfide bond-deleted mutant of the secretory protein α-amylase AmyB fused with mDsRed that had accumulated in the ER was subsequently delivered to vacuoles, whereas wild-type AmyB-mDsRed was predominantly located at cell walls and septa. To examine the involvement of autophagy in the delivery of mutant AmyB to vacuoles, mutant AmyB-EGFP was expressed in an A. oryzae autophagy-deficient strain (ΔAoatg8). Microscopic examination revealed that the protein delivery to vacuoles did not occur in the absence of autophagic activity, with mutant AmyB-mDsRed forming large spherical structures surrounded by ER membrane. Hence, we conclude that autophagy is responsible for the delivery of misfolded secretory proteins accumulated in the ER to vacuoles for degradation during late-growth phase in A. oryzae. This is the first study to provide evidence that autophagy plays a role in the degradation of misfolded secretory proteins in filamentous fungi.

High-throughput recombinant gene expression systems in Pichia pastoris using newly developed plasmid vectors.

We describe here the construction of Gateway-compatible vectors, pBGP1-DEST and pPICZα-DEST, for rapid and convenient preparation of expression plasmids for production of secretory proteins in Pichia pastoris. Both vectors direct the synthesis of fusion proteins consisting of the N-terminal signal and pro-sequences of Saccharomyces cerevisiae α-factor, the recognition sites for Kex2 and Ste13 processing proteases, the mature region of a foreign protein flanked by attB1- and attB2-derived sequences at N- and C-termini, respectively, and myc plus hexahistidine tags added at the extreme C-terminus. To test the usefulness of these vectors, production of endo-glucanases and xylanases from termite symbionts, as well as a fungal glucuronoyl esterase, was performed. Enzyme activities were detected in the culture supernatants, indicating that the chimeric proteins were synthesized and secreted as designed.

Heterologous expression and characterization of a glucose-stimulated ß-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae.

Neotermes koshunensis is a lower termite that secretes endogenous ß-glucosidase in the salivary glands. This ß-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-ß-D-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn(2+) and glycerol. The K(m) and V(max) values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-ß-D-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.