The Identification of a Target Gene of the Transcription Factor KojR and Elucidation of Its Role in Carbon Metabolism for Kojic Acid Biosynthesis in Aspergillus oryzae.

DNA-binding transcription factors are broadly characterized as proteins that bind to specific sequences within genomic DNA and modulate the expression of downstream genes. This study focused on KojR, a transcription factor involved in the metabolism of kojic acid, which is an organic acid synthesized in Aspergillus oryzae and is known for its tyrosinase-inhibitory properties. However, the regulatory mechanism underlying KojR-mediated kojic acid synthesis remains unclear. Hence, we aimed to obtain a comprehensive identification of KojR-associated genes using genomic systematic evolution of ligands by exponential enrichment with high-throughput DNA sequencing (gSELEX-Seq) and RNA-Seq. During the genome-wide exploration of KojR-binding sites via gSELEX-Seq and identification of KojR-dependent differentially expressed genes (DEGs) using RNA-Seq, we confirmed that KojR preferentially binds to 5'-CGGCTAATGCGG-3', and KojR directly regulates kojT, as was previously reported. We also observed that kojA expression, which may be controlled by KojR, was significantly reduced in a ΔkojR strain. Notably, no binding of KojR to the kojA promoter region was detected. Furthermore, certain KojR-dependent DEGs identified in the present study were associated with enzymes implicated in the carbon metabolic pathway of A. oryzae. This strongly indicates that KojR plays a central role in carbon metabolism in A. oryzae.

Identification and characterization of methoxy- and dimethoxyhydroquinone 1,2-dioxygenase from Phanerochaete chrysosporium.

White-rot fungi, such as Phanerochaete chrysosporium, are the most efficient degraders of lignin, a major component of plant biomass. Enzymes produced by these fungi, such as lignin peroxidases and manganese peroxidases, break down lignin polymers into various aromatic compounds based on guaiacyl, syringyl, and hydroxyphenyl units. These intermediates are further degraded, and the aromatic ring is cleaved by 1,2,4-trihydroxybenzene dioxygenases. This study aimed to characterize homogentisate dioxygenase (HGD)-like proteins from P. chrysosporium that are strongly induced by the G-unit fragment of vanillin. We overexpressed two homologous recombinant HGDs, PcHGD1 and PcHGD2, in Escherichia coli. Both PcHGD1 and PcHGD2 catalyzed the ring cleavage in methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ). The two enzymes had the highest catalytic efficiency (kcat/Km) for MHQ, and therefore, we named PcHGD1 and PcHGD2 as MHQ dioxygenases 1 and 2 (PcMHQD1 and PcMHQD2), respectively, from P. chrysosporium. This is the first study to identify and characterize MHQ and DMHQ dioxygenase activities in members of the HGD superfamily. These findings highlight the unique and broad substrate spectra of PcHGDs, rendering them attractive candidates for biotechnological applications.IMPORTANCEThis study aimed to elucidate the properties of enzymes responsible for degrading lignin, a dominant natural polymer in terrestrial lignocellulosic biomass. We focused on two homogentisate dioxygenase (HGD) homologs from the white-rot fungus, P. chrysosporium, and investigated their roles in the degradation of lignin-derived aromatic compounds. In the P. chrysosporium genome database, PcMHQD1 and PcMHQD2 were annotated as HGDs that could cleave the aromatic rings of methoxyhydroquinone (MHQ) and dimethoxyhydroquinone (DMHQ) with a preference for MHQ. These findings suggest that MHQD1 and/or MHQD2 play important roles in the degradation of lignin-derived aromatic compounds by P. chrysosporium. The preference of PcMHQDs for MHQ and DMHQ not only highlights their potential for biotechnological applications but also underscores their critical role in understanding lignin degradation by a representative of white-rot fungus, P. chrysosporium.

Pathway crosstalk between the central metabolic and heme biosynthetic pathways in Phanerochaete chrysosporium.

A comprehensive analysis to survey heme-binding proteins produced by the white-rot fungus Phanerochaete chrysosporium was achieved using a biotinylated heme-streptavidin beads system. Mitochondrial citrate synthase (PcCS), glyceraldehyde 3-phosphate dehydrogenase (PcGAPDH), and 2-Cys thioredoxin peroxidase (mammalian HBP23 homolog) were identified as putative heme-binding proteins. Among these, PcCS and PcGAPDH were further characterized using heterologously expressed recombinant proteins. Difference spectra of PcCS titrated with hemin exhibited an increase in the Soret absorbance at 414 nm, suggesting that the axial ligand of the heme is a His residue. The activity of PcCS was strongly inhibited by hemin with Ki oxaloacetate of 8.7 µM and Ki acetyl-CoA of 5.8 µM. Since the final step of heme biosynthesis occurred at the mitochondrial inner membrane, the inhibition of PcCS by heme is thought to be a physiological event. The inhibitory mode of the heme was similar to that of CoA analogues, suggesting that heme binds to PcCS at His347 at the AcCoA-CoA binding site, which was supported by the homology model of PcCS. PcGAPDH was also inhibited by heme, with a lower concentration than that for PcCS. This might be caused by the different location of these enzymes. From the integration of these phenomena, it was concluded that metabolic regulations by heme in the central metabolic and heme synthetic pathways occurred in the mitochondria and cytosol. This novel pathway crosstalk between the central metabolic and heme biosynthetic pathways, via a heme molecule, is important in regulating the metabolic balance (heme synthesis, ATP synthesis, flux balance of the tricarboxylic acid (TCA) cycle and cellular redox balance (NADPH production) during fungal aromatic degradation. KEY POINTS: • A comprehensive survey of heme-binding proteins in P. chrysosporium was achieved. • Several heme-binding proteins including CS and GAPDH were identified. • A novel metabolic regulation by heme in the central metabolic pathways was found.

Identification of Desiccation Stress-Inducible Antioxidative and Antiglycative Ultraviolet-Absorbing Oxylipins, Saclipin A and Saclipin B, in an Edible Cyanobacterium Aphanothece sacrum.

Aphanothece sacrum, a freshwater cyanobacterium, is an edible cyanobacterial strain. We identified two compounds belonging to the oxylipin family that possess UV-absorbing abilities and accumulate in the dried sample of A. sacrum. The compounds, named saclipin A and saclipin B, exhibited strong UV-absorption properties with the absorption maxima at 316 and 319 nm, respectively, and the molar extinction coefficients of 26,454 and 30,555 M-1 cm-1, respectively. The chemical structures of saclipins A and B have been elucidated, revealing that they have an all-E and a 12Z isomeric relationship within the triene structure. The saclipins could be isomerized by photoirradiation, with the cis-form saclipin B proving to be more stable in methanol, ethanol, or acetonitrile. Under drought stress conditions, the accumulation of saclipins A and B in A. sacrum was found to be increased 20- and 10-fold, respectively. Purified saclipins from A. sacrum showed biocompatibility and valuable bioactivities. Specifically, saclipins exhibited radical scavenging activity, maintaining their activity even 40 min after the reaction began. Additionally, they demonstrated inhibitory activity against glycation of elastin and collagen, which are constituents of dermal tissue. Notably, saclipins showed higher activity than the well-known glycation inhibitor aminoguanidine against collagen glycation.

A very long chain fatty acid responsive transcription factor, MYB93, regulates lateral root development in Arabidopsis.

Lateral roots (LRs) are critical to root system architecture development in plants. Although the molecular mechanisms by which auxin regulates LR development have been extensively studied, several additional regulatory systems are hypothesized to be involved. Recently, the regulatory role of very long chain fatty acids (VLCFAs) has been shown in LR development. Our analysis showed that LTPG1 and LTPG2, transporters of VLCFAs, are specifically expressed in the developing LR primordium (LRP), while the number of LRs is reduced in the ltpg1/ltpg2 double mutant. Moreover, late LRP development was hindered when the VLCFA levels were reduced by the VLCFA synthesis enzyme mutant, kcs1-5. However, the details of the regulatory mechanisms of LR development controlled by VLCFAs remain unknown. In this study, we propose a novel method to analyze the LRP development stages with high temporal resolution using a deep neural network and identify a VLCFA-responsive transcription factor, MYB93, via transcriptome analysis of kcs1-5. MYB93 showed a carbon chain length-specific expression response following treatment of VLCFAs. Furthermore, myb93 transcriptome analysis suggested that MYB93 regulated the expression of cell wall organization genes. In addition, we also found that LTPG1 and LTPG2 are involved in LR development through the formation of root cap cuticle, which is different from transcriptional regulation by VLCFAs. Our results suggest that VLCFA is a regulator of LRP development through transcription factor-mediated regulation of gene expression and the transportation of VLCFAs is also involved in LR development through root cap cuticle formation.

Change over Time in the Risk of Death among Japanese COVID-19 Cases Caused by the Omicron Variant Depending on Prevalence of Sublineages.

To assess temporal changes to the risk of death in COVID-19 cases caused by the Omicron variant, we calculated age-standardized case fatality rates (CFR) in patients aged ≥40 years over nine diagnostic periods (3 January to 28 August 2022) in ten Japanese prefectures (14.8 million residents). Among 552,581 study subjects, we found that there were 1836 fatalities during the isolation period (up to 28 days from date of onset). The highest age-standardized CFR (0.85%, 95% confidence interval (CI):0.78-0.92) was observed in cases diagnosed in the second 4-week period (January 31 to February 27), after which it declined significantly up to the 6th 4-week period (0.23%, 95% CI: 0.13-0.33, May 23 to June 19). The CFR then increased again but remained at 0.39% in the eighth period (July 18 to August 28). The CFR in cases with the BA.2 or BA.5 sublineages in the age range 60-80 years was significantly lower than that with BA.1 infections (60 years: 0.19%, 0.02%, 0.053%, respectively; 70 years: 0.91%, 0.33%, 0.39%; ≥80 years: 3.78%, 1.96%, 1.81%, respectively). We conclude that the risk of death in Japanese COVID-19 patients infected with Omicron variants declined through February to mid-June 2022.

Biochemical characterization of hydroquinone hydroxylase from Phanerochaete chrysosporium.

The white-rot fungus Phanerochaete chrysosporium can degrade lignin polymers using extracellular, non-specific, one-electron oxidizing enzymes. This results in the formation of guaiacyl (G), syringyl (S), and hydroxyphenyl (H) units, such as vanillic acid, syringic acid, and p-hydroxybenzoic acid (p-HBA) and the corresponding aldehydes, which are further metabolized intracellularly. Therefore, the aim of this study was to identify proteins involved in the hydroxylation of H-unit fragments such as p-HBA and its decarboxylated product hydroquinone (HQ) in P. chrysosporium. A flavoprotein monooxygenase (FPMO), PcFPMO2, was identified and its activity was characterized. Recombinant PcFPMO2 with an N-terminal polyhistidine tag was produced in Escherichia coli and purified. In the presence of NADPH, PcFPMO2 used six phenolic compounds as substrates. PcFPMO2 catalyzed the hydroxylation of the H-unit fragments such as p-HBA and HQ, and the G-unit derivative methoxyhydroquinone (MHQ). The highest catalytic efficiency (kcat/Km) was observed with HQ, indicating that PcFPMO2 could be involved in HQ hydroxylation in vivo. Additionally, PcFPMO2 converted MHQ to 3-, 5-, and 6-methoxy-1,2,4-trihydroxybenzene (3-, 5-, and 6-MTHB), respectively, suggesting that PcFPMO2 might partially be involved in MHQ degradation, following aromatic ring fission, via three MTHBs. FPMOs are divided into eight groups (groups A to H). This is the first study to show MHQ hydroxylase activity of a FPMO-group A superfamily member. These findings highlight the unique substrate spectrum of PcFPMO2, making it an attractive candidate for biotechnological applications.

Biochemical Characterization of a Pectate Lyase AnPL9 from Aspergillus nidulans.

Pectinolytic enzymes have diverse industrial applications. Among these, pectate lyases act on the internal α-1,4-linkage of the pectate backbone, playing a critical role in pectin degradation. While most pectate lyases characterized thus far are of bacterial origin, fungi can also be excellent sources of pectinolytic enzymes. In this study, we performed biochemical characterization of the pectate lyase AnPL9 belonging to the polysaccharide lyase family 9 (PL9) from the filamentous fungus Aspergillus nidulans. Recombinant AnPL9 was produced using a Pichia pastoris expression system and purified. AnPL9 exhibited high activity on homogalacturonan (HG), pectin from citrus peel, pectin from apple, and the HG region in rhamnogalacturonan-I. Although digalacturonic acid and trigalacturonic acid were not degraded by AnPL9, tetragalacturonic acid was converted to 4,5-unsaturated digalacturonic acid and digalacturonic acid. These results indicate that AnPL9 degrades HG oligosaccharides with a degree of polymerization > 4. Furthermore, AnPL9 was stable within a neutral-to-alkaline pH range (pH 6.0-11.0). Our findings suggest that AnPL9 is a candidate pectate lyase for biotechnological applications in the food, paper, and textile industries. This is the first report on a fungal pectate lyase belonging to the PL9 family.

Characterization of two 1,2,4-trihydroxybenzene 1,2-dioxygenases from Phanerochaete chrysosporium.

Lignin is the most abundant aromatic compound in nature, and it plays an important role in the carbon cycle. White-rot fungi are microbes that are capable of efficiently degrading lignin. Enzymes from these fungi possess exceptional oxidative potential and have gained increasing importance for improving bioprocesses, such as the degradation of organic pollutants. The aim of this study was to identify the enzymes involved in the ring cleavage of the lignin-derived aromatic 1,2,4-trihydroxybenzene (THB) in Phanerochaete chrysosporium, a lignin-degrading basidiomycete. Two intradiol dioxygenases (IDDs), PcIDD1 and PcIDD2, were identified and produced as recombinant proteins in Escherichia coli. In the presence of O2, PcIDD1 and PcIDD2 acted on eight and two THB derivatives, respectively, as substrates. PcIDD1 and PcIDD2 catalyze the ring cleavage of lignin-derived fragments, such as 6-methoxy-1,2,4-trihydroxybenzene (6-MeOTHB) and 3-methoxy-1,2-catechol. The current study also revealed that syringic acid (SA) was converted to 5-hydroxyvanillic acid, 2,6-dimethoxyhydroquinone, and 6-MeOTHB by fungal cells, suggesting that PcIDD1 and PcIDD2 may be involved in aromatic ring fission of 6-MeOTHB for SA degradation. This is the first study to show 6-MeOTHB dioxygenase activity of an IDD superfamily member. These findings highlight the unique and broad substrate spectra of PcIDDs, rendering it an attractive candidate for biotechnological application. KEY POINTS: • Novel intradiol dioxygenases (IDD) in lignin degradation were characterized. • PcIDDs acted on lignin-derived fragments and catechol derivatives. • Dioxygenase activity on 6-MeOTHB was identified in IDD superfamily enzymes.

Enhanced Bioremediation of 4-Chlorophenol by Electrically Neutral Reactive Species Generated from Nonthermal Atmospheric-Pressure Plasma.

4-Chlorophenol (4-CP) is a chlorinated aromatic compound with broad industrial applications. It is released into the environment as an industrial byproduct and is highly resistant to biodegradation. Pseudomonas sp. in the environment and activated sludge are used for 4-CP bioremediation; however, the degradation of 4-CP takes a long time. Consequently, the toxicity of 4-CP is a major barrier to its bioremediation. In this study, we investigated the synergistic effect of electrically neutral reactive species on the bacterial bioremediation of 4-CP. Our results showed that the concentration of 4-CP decreased from 2.0 to 0.137 mM and that it was converted to 4-chlorocatechol (4-CC; 0.257 mM), 4-chlororesorcinol (0.157 mM), hydroquinone (0.155 mM), and trihydroxy chlorobenzene and their respective ring-cleaved products following irradiation of neutral reactive species. These compounds were less toxic than 4-CP, except for 4-CC, which reduced the toxicity of 4-CP to Pseudomonas putida. When the neutral reactive species-treated 4-CP fraction was added to P. putida cultured in a synthetic sewage medium for 48 h, the 4-CP concentration was reduced to 0.017 mM, whereas nontreated 4-CP (2.0 mM) was hardly degraded by P. putida. These results suggest that the biodegradation of 4-CP can be efficiently improved by combining irradiation of neutral reactive species with microbial treatment. The irradiation of neutral reactive species of environmental pollutants may additionally lead to further improvements in bioremediation processes.

Administration of Aspergillus oryzae suppresses DSS-induced colitis.

Aspergillus oryzae, a filamentous fungus, has long been used for the production of traditional Japanese foods. Here, we analyzed how A. oryzae administration affects the intestinal environment in mice. The results of 16S rRNA gene sequencing of the gut microbiota indicated that after the administration of heat-killed A. oryzae spores, the relative abundance of an anti-inflammatory Bifidobacterium pseudolongum strain became 2.0-fold greater than that of the control. Next, we examined the effect of A. oryzae spore administration on the development of colitis induced by dextran sodium sulfate in mice; we found that colitis was alleviated by not only heat-killed A. oryzae spores, but also the cell wall extracted from the spores. Our findings suggest that A. oryzae holds considerable potential for commercial application in the production of both traditional Japanese fermented foods and new foods with prebiotic functions.

Artificial AmyR::XlnR transcription factor induces α-amylase production in response to non-edible xylan-containing hemicellulosic biomass.

Filamentous fungi belonging to the Aspergillus genus are one of the most favored microorganisms for industrial enzyme production because they can secrete large amounts of proteins into the culture medium. α-Amylase, an enzyme produced by Aspergillus species, is important for food and industrial applications. The production of α-amylase is induced by starch, mainly obtained from the edible biomass; however, the increasing demand for foods is limiting the application of the latter. Therefore, it is expected that using the non-edible biomass, such as rice straw, could improve the competition for industrial application starch containing resources. The transcription factor AmyR activates the transcription of amylolytic enzyme genes, while the transcription factor XlnR activates the transcription of xylanolytic enzyme genes in response to xylose. In this study, we aimed to construct an artificial AmyR::XlnR transcription factor (AXTF) by replacing the DNA-binding domain (1-159 amino acids) of XlnR with that (1-68 aa) of AmyR, which is capable of inducing amylolytic enzyme production in response to xylan-containing hemicellulosic biomass. The chimeric transcription factor AXTF was constructed and expressed using the gapA promoter in the amyR-deficient mutant strain SA1. When the AXTF strain was cultured in the minimal medium containing xylose as the carbon source, the amyB, amyF, agdB, and agdE transcription levels were 41.1-, 11.3-, 37.9-, and 23.7-fold higher, respectively, than those of the wild-type strain. The α-amylase and α-glucosidase activities in the culture supernatant of the AXTF strain grown with xylose for 48 h were 696.6 and 536.1 U/mL, respectively, while these activities were not detected in the culture supernatant of the wild-type and SA1 strains. When rice straw hydrolysate was used as a carbon source, the α-amylase and α-glucosidase activities were 590.2 and 362.7 U/mL, respectively. Thus, we successfully generated an Aspergillus nidulans strain showing amylolytic enzyme production in response to non-edible xylan-containing hemicellulosic biomass by transforming it with the chimeric transcription factor AXTF. Furthermore, the use of genes encoding engineered transcription factors is advantageous because introducing such genes into an industrial Aspergillus strain has similar simultaneous effects on multiple amylase genes controlled by AmyR.

Characterization of FsXEG12A from the cellulose-degrading ectosymbiotic fungus Fusarium spp. strain EI cultured by the ambrosia beetle.

Despite the threat of Fusarium dieback posed due to ambrosia fungi cultured by ambrosia beetles such as Euwallacea spp., the wood-degradation mechanisms utilized by ambrosia fungi are not fully understood. In this study, we analyzed the 16S rRNA and 18S rRNA genes of the microbial community from the Ficus tree tunnel excavated by Euwallacea interjectus and isolated the cellulose-degrading fungus, Fusarium spp. strain EI, by enrichment culture with carboxymethyl cellulose as the sole carbon source. The cellulolytic enzyme secreted by the fungus was identified and expressed in Pichia pastoris, and its enzymatic properties were characterized. The cellulolytic enzyme, termed FsXEG12A, could hydrolyze carboxymethyl cellulose, microcrystalline cellulose, xyloglucan, lichenan, and glucomannan, indicating that the broad substrate specificity of FsXEG12A could be beneficial for degrading complex wood components such as cellulose, xyloglucan, and galactoglucomannan in angiosperms. Inhibition of FsXEG12A function is, thus, an effective target for Fusarium dieback caused by Euwallacea spp.

Oxygen radical based on non-thermal atmospheric pressure plasma alleviates lignin-derived phenolic toxicity in yeast.

BACKGROUND: Vanillin is the main byproduct of alkaline-pretreated lignocellulosic biomass during the process of fermentable-sugar production and a potent inhibitor of ethanol production by yeast. Yeast cells are usually exposed to vanillin during the industrial production of bioethanol from lignocellulosic biomass. Therefore, vanillin toxicity represents a major barrier to reducing the cost of bioethanol production. RESULTS: In this study, we analysed the effects of oxygen-radical treatment on vanillin molecules. Our results showed that vanillin was converted to vanillic acid, protocatechuic aldehyde, protocatechuic acid, methoxyhydroquinone, 3,4-dihydroxy-5-methoxybenzaldehyde, trihydroxy-5-methoxybenzene, and their respective ring-cleaved products, which displayed decreased toxicity relative to vanillin and resulted in reduced vanillin-specific toxicity to yeast during ethanol fermentation. Additionally, after a 16-h incubation, the ethanol concentration in oxygen-radical-treated vanillin solution was 7.0-fold greater than that from non-treated solution, with similar results observed using alkaline-pretreated rice straw slurry with oxygen-radical treatment. CONCLUSIONS: This study analysed the effects of oxygen-radical treatment on vanillin molecules in the alkaline-pretreated rice straw slurry, thereby finding that this treatment converted vanillin to its derivatives, resulting in reduced vanillin toxicity to yeast during ethanol fermentation. These findings suggest that a combination of chemical and oxygen-radical treatment improved ethanol production using yeast cells, and that oxygen-radical treatment of plant biomass offers great promise for further improvements in bioethanol-production processes.

Ability of Saccharomyces cerevisiae MC87-46 to assimilate isomaltose and its effects on sake taste.

Recently, wild strains of Saccharomyces cerevisiae isolated from a variety of natural resources have been used to make bread, beer, wine, and sake. In the current study, we isolated wild S. cerevisiae MC strain from the carnation (Dianthus caryophyllus L) flower and produced sake using its cerulenin-resistant mutant strain MC87-46. Then, we characterized the components, including ethanol, amino acids, organic acids, and sugars, in the fermented sake. Sake brewed with MC87-46 is sweet owing to the high content of isomaltose, which was at a concentration of 44.3 mM. The low sake meter value of -19.6 is most likely due to this high isomaltose concentration. The genomic DNA of MC87-46 encodes for isomaltases IMA1, IMA2, IMA3, IMA4 and IMA5, as well as the isomaltose transporter gene, AGT1. However, these genes were not induced in MC87-46 by isomaltose, and the strain did not possess isomaltase activity. These results show that MC87-46 cannot utilize isomaltose, resulting in its accumulation in the fermented sake. Isomaltose concentrations in sake brewed with MC87-46 were 24.6-fold more than in commercial sake. These findings suggest that MC87-46 may be useful for commercial application in Japanese sake production because of its unique flavour and nutrient profile.

Identification and characterization of a thermostable pectate lyase from Aspergillus luchuensis var. saitoi.

Pectinolytic enzymes are used in diverse industrial applications. We sought to isolate a pectate lyase from Aspergillus luchuensis var. saitoi, a filamentous fungus used in traditional food and beverage preparation in Japan. The identified enzyme, named AsPelA, is orthologous to PelA from A. luchuensis mut. kawachii (AkPelA); the enzymes exhibit 99% amino acid sequence identity, with Ile140 and Val197 of AsPelA being replaced by Val and Asp in AkPelA, respectively. AsPelA activity decreased to 71%, 61%, and 46% of maximal activity after 60-min incubation at 60 °C, 70 °C, and 80 °C, whereas AkPelA activity dropped to 16%, 10%, and 8.5%, respectively, indicating that AsPelA is more thermostable than AkPelA. Furthermore, AsPelA was stable within a neutral-to-alkaline pH range, as well as in the presence of organic solvents, detergents, and metal ions. Our findings suggest that AsPelA represents a candidate pectate lyase for applications in food, paper, and textile industries.

Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium.

The activity of a self-sufficient cytochrome P450 enzyme, CYP505D6, from the lignin-degrading basidiomycete Phanerochaete chrysosporium was characterized. Recombinant CYP505D6 was produced in Escherichia coli and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as the substrates. Hydroxylation occurred at the ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at the ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active-site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid were analyzed. Compared with wild-type CYP505D6, its V51Y variant generated few products hydroxylated at the ω-4 to ω-6 positions. The products generated by wild-type CYP102A1 were hydroxylated at the ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regiospecificity of fatty acid hydroxylation, at least that at the ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry.IMPORTANCEPhanerochaete chrysosporium is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in this study, P. chrysosporium CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at the ω-1 to ω-7 or ω-1 to ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than those of the known fused proteins CYP102A1 and CYP505A1. The substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

NAD+/NADH homeostasis affects metabolic adaptation to hypoxia and secondary metabolite production in filamentous fungi.

Filamentous fungi are used to produce fermented foods, organic acids, beneficial secondary metabolites and various enzymes. During such processes, these fungi balance cellular NAD+:NADH ratios to adapt to environmental redox stimuli. Cellular NAD(H) status in fungal cells is a trigger of changes in metabolic pathways including those of glycolysis, fermentation, and the production of organic acids, amino acids and secondary metabolites. Under hypoxic conditions, high NADH:NAD+ ratios lead to the inactivation of various dehydrogenases, and the metabolic flow involving NAD+ is down-regulated compared with normoxic conditions. This review provides an overview of the metabolic mechanisms of filamentous fungi under hypoxic conditions that alter the cellular NADH:NAD+ balance. We also discuss the relationship between the intracellular redox balance (NAD/NADH ratio) and the production of beneficial secondary metabolites that arise from repressing the HDAC activity of sirtuin A via Nudix hydrolase A (NdxA)-dependent NAD+ degradation.

Characterization of pH-tolerant and thermostable GH 134 ß-1,4-mannanase SsGH134 possessing carbohydrate binding module 10 from Streptomyces sp. NRRL B-24484.

A GH 134 ß-1,4-mannanase SsGH134 from Streptomyces sp. NRRL B-24484 possesses a carbohydrate binding module (CBM) 10 and a glycoside hydrolase 134 domain at the N- and C-terminal regions, respectively. Recombinant SsGH134 expressed in Escherichia coli. SsGH134 was maximally active within a pH range of 4.0-6.5 and retained >80% of this maximum after 90 min at 30°C within a pH range of 3.0-10.0. The ß-1,4-mannanase activity of SsGH134 towards glucomannan was 30% of the maximal activity after an incubation at 100°C for 120 min, indicating that SsGH134 is pH-tolerant and thermostable ß-1,4-mannanase. SsGH134, SsGH134-ΔCBM10 (CBM10-linker-truncated SsGH134) and SsGH134-G34W (substitution of Gly34 to Trp) bound to microcrystalline cellulose, ß-mannan and chitin, regardless of the presence or absence of CBM10. These indicate that GH 134 domain strongly bind to the polysaccharides. Although deleting CBM10 increased the catalytic efficiency of the ß-1,4-mannanase, its disruption decreased the pH, solvent and detergent stability of SsGH134. These findings indicate that CBM10 inhibits the ß-1,4-mannanase activity of SsGH134, but it is involved in stabilizing its enzymatic activity within a neutral-to-alkaline pH range, and in the presence of various organic solvents and detergents. We believe that SsGH134 could be useful to a diverse range of industries.

Oxygen-radical pretreatment promotes cellulose degradation by cellulolytic enzymes.

BACKGROUND: The efficiency of cellulolytic enzymes is important in industrial biorefinery processes, including biofuel production. Chemical methods, such as alkali pretreatment, have been extensively studied and demonstrated as effective for breaking recalcitrant lignocellulose structures. However, these methods have a detrimental effect on the environment. In addition, utilization of these chemicals requires alkali- or acid-resistant equipment and a neutralization step. RESULTS: Here, a radical generator based on non-thermal atmospheric pressure plasma technology was developed and tested to determine whether oxygen-radical pretreatment enhances cellulolytic activity. Our results showed that the viscosity of carboxymethyl cellulose (CMC) solutions was reduced in a time-dependent manner by oxygen-radical pretreatment using the radical generator. Compared with non-pretreated CMC, oxygen-radical pretreatment of CMC significantly increased the production of reducing sugars in culture supernatant containing various cellulases from Phanerochaete chrysosporium. The production of reducing sugar from oxygen-radical-pretreated CMC by commercially available cellobiohydrolases I and II was 1.7- and 1.6-fold higher, respectively, than those from non-pretreated and oxygen-gas-pretreated CMC. Moreover, the amount of reducing sugar from oxygen-radical-pretreated wheat straw was 1.8-fold larger than those from non-pretreated and oxygen-gas-pretreated wheat straw. CONCLUSIONS: Oxygen-radical pretreatment of CMC and wheat straw enhanced the degradation of cellulose by reducing- and non-reducing-end cellulases in the supernatant of a culture of the white-rot fungus P. chrysosporium. These findings indicated that oxygen-radical pretreatment of plant biomass offers great promise for improvements in lignocellulose-deconstruction processes.