Whole-Genome De Novo Sequencing of the Lignin-Degrading Wood Rot Fungus Phanerochaete chrysosporium (ATCC 20696).

Phanerochaete chrysosporium (ATCC 20696) has a catabolic ability to degrade lignin. Here, we report whole-genome sequencing used to identify genes related to lignin modification. We determined the 39-Mb draft genome sequence of this fungus, comprising 13,560 predicted gene models. Gene annotation provided crucial information about the location and function of protein-encoding genes.

Phanerochaete chrysosporium Multienzyme Catabolic System for in Vivo Modification of Synthetic Lignin to Succinic Acid.

Whole cells of the basidiomycete fungus Phanerochaete chrysosporium (ATCC 20696) were applied to induce the biomodification of lignin in an in vivo system. Our results indicated that P. chrysosporium has a catabolic system that induces characteristic biomodifications of synthetic lignin through a series of redox reactions, leading not only to the degradation of lignin but also to its polymerization. The reducing agents ascorbic acid and α-tocopherol were used to stabilize the free radicals generated from the ligninolytic process. The application of P. chrysosporium in combination with reducing agents produced aromatic compounds and succinic acid as well as degraded lignin polymers. P. chrysosporium selectively catalyzed the conversion of lignin to succinic acid, which has an economic value. A transcriptomic analysis of P. chrysosporium suggested that the bond cleavage of synthetic lignin was caused by numerous enzymes, including extracellular enzymes such as lignin peroxidase and manganese peroxidase, and that the aromatic compounds released were metabolized in both the short-cut and classical tricarboxylic acid cycles of P. chrysosporium. In conclusion, P. chrysosporium is suitable as a biocatalyst for lignin degradation to produce a value-added product.

Whole-genome de novo sequencing of wood rot fungus Fomitopsis palustris (ATCC62978) with both a cellulolytic and ligninolytic enzyme system.

Fomitopsis palustris is a model brown rot fungus causing destructive wood decay based on the cellulase system. Endoglucanase secreted by F. palustris hydrolyzes cellulose in both the crystalline and amorphous form. In this study, whole-genome sequencing was conducted to identify genes related to F. palustris cellulose degradation and their functions. We determined the 43-Mb complete draft genome of F. palustris (ATCC 62978), comprising 14,592 predicted gene models. Gene annotation provided crucial information about the location and function of protein-encoding genes. Three types of endoglucanases were expressed: endo-1,3-beta-glucanase, endo-1,4-beta-d-glucanase, and endoglucanase. In addition, various ligninolytic enzymes such as laccase, aromatic compound dioxygenase, and aryl alcohol dehydrogenase were expressed in F. palustris (ATCC 62978). Colony polymerase chain reaction (PCR) indicated that the endo-1,4-beta-d-glucanase gene comprises 732bp. Optimization of the expression conditions of endoglucanase by real-time PCR revealed that endoglucanase was highly expressed after 7days in all conditions, which was secreted during the secondary metabolism. Studies for large-scale cellulase production from this fungus and investigation of its ligninolytic system will promote its extensive use in various applications. The genomic information determined herein provides a basis for molecular genetics studies to understand the genome functions of F. palustris (ATCC 62978).

Levulinic acid production by two-step acid-catalyzed treatment of Quercus mongolica using dilute sulfuric acid.

The objectives of this research were to produce a levulinic acid by two-step acid-catalyzed treatment of Quercus mongolica and to investigate the effect of treatment parameter (reaction temperature range: 100-230°C; sulfuric acid (SA) concentration range: 0-2%) on the levulinic acid yield. After 1st step acid-catalyzed treatment, most of the hemicellulosic C5 sugars (15.6gg/100gbiomass) were released into the liquid hydrolysate at the reaction temperature of 150°C in 1% SA; the solid fraction, which contained 53.5% of the C6 sugars, was resistant to further loss of C6 sugars. Subsequently, 2nd step acid-catalyzed treatment of the solid fractions was performed under more severe conditions. Finally, 16.5g/100g biomass of levulinic acid was produced at the reaction temperature of 200°C in 2% SA, corresponding to a higher conversion rate than during single-step treatment.

Degradation and polymerization of monolignols by Abortiporus biennis, and induction of its degradation with a reducing agent.

This study was carried out to better understand the characteristic modification mechanisms of monolignols by enzyme system of Abortiporus biennis and to induce the degradation of monolignols. Degradation and polymerization of monolignols were simultaneously induced by A. biennis. Whole cells of A. biennis degraded coniferyl alcohol to vanillin and coniferyl aldehyde, and degraded sinapyl alcohol to 2,6-dimethoxybenzene- 1,4-diol, with the production of dimers. The molecular weight of monolignols treated with A. biennis increased drastically. The activities of lignin degrading enzymes were monitored for 24 h to determine whether there was any correlation between monolignol biomodification and ligninolytic enzymes. We concluded that complex enzyme systems were involved in the degradation and polymerization of monolignols. To degrade monolignols, ascorbic acid was added to the culture medium as a reducing agent. In the presence of ascorbic acid, the molecular weight was less increased in the case of coniferyl alcohol, while that of sinapyl alcohol was similar to that of the control. Furthermore, the addition of ascorbic acid led to the production of various degraded compounds: syringaldehyde and acid compounds. Accordingly, these results demonstrated that ascorbic acid prevented the rapid polymerization of monolignols, thus stabilizing radicals generated by enzymes of A. biennis. Thereafter, A. biennis catalyzed the oxidation of stable monolignols. As a result, ascorbic acid facilitated predominantly monolignols degradation by A. biennis through the stabilization of radicals. These findings showed outstanding ability of A. biennis to modify the lignin compounds rapidly and usefully.

Transcriptomic analysis of the white rot fungus Polyporus brumalis provides insight into sesquiterpene biosynthesis.

Object of this study was to identify genes and enzymes that are involved in sesquiterpene biosynthesis in the wood rotting fungus, Polyporus brumalis. Sesquiterpenes, ß-eudesmane and ß-eudesmol, were produced by the mycelium of P. brumalis cultured in modified medium. However, theses final products were not observed when the fungus was grown in potato dextrose medium. We used next generation sequencing (NGS) to identify differentially expressed genes (DEGs) related to terpene metabolism. This approach generated 25,000 unigenes and 127 metabolic pathways that were assigned to Kyoto Encyclopedia Genes Groups (KEGG). Further analysis of samples from modified medium indicated significant upregulation of 8 unigenes involved in the mevalonate (MVA) and methylerythritol phosphate (MEP) biosynthetic pathways. These pathways generate isopentenyl pyrophosphate (IPP) and farnesyl pyrophosphate (FPP), which are precursors for the synthesis of sesquiterpenes. Furthermore, genes encoding germacrene A synthase, which facilitate the cyclization of FPP, were only differentially expressed in mycelium from fungi grown in modified medium. Our data provide a resource for studying the molecular mechanisms underpinning sesquiterpene biosynthesis and terpene metabolism.

Biotransformation of (-)-α-Pinene by Whole Cells of White Rot Fungi, Ceriporia sp. ZLY-2010 and Stereum hirsutum.

Two white rot fungi, Ceriporia sp. ZLY-2010 (CER) and Stereum hirsutum (STH) were used as biocatalysts for the biotransformation of (-)-α-pinene. After 96 hr, CER converted the bicyclic monoterpene hydrocarbon (-)-α-pinene into α-terpineol (yield, 0.05 g/L), a monocyclic monoterpene alcohol, in addition to, other minor products. Using STH, verbenone was identified as the major biotransformed product, and minor products were myrtenol, camphor, and isopinocarveol. We did not observe any inhibitory effects of substrate or transformed products on mycelial growth of the fungi. The activities of fungal manganese-dependent peroxidase and laccase were monitored for 15 days to determine the enzymatic pathways related to the biotransformation of (-)-α-pinene. We concluded that a complex of enzymes, including intra- and extracellular enzymes, were involved in terpenoid biotransformation by white rot fungi.

Biotransformation of (-)-α-pinene and geraniol to α-terpineol and p-menthane-3,8-diol by the white rot fungus, Polyporus brumalis.

In this study, the monoterpenes, α-pinene and geraniol, were biotransformed to synthesize monoterpene alcohol compounds. Polyporus brumalis which is classified as a white rot fungus was used as a biocatalyst. Consequently α-terpineol was synthesized from α-pinene by P. brumalis mycelium, after three days. Moreover, another substrate, the acyclic monoterpenoids geraniol was transformed into the cyclic compound, p-menthane-3, 8-diol (PMD). The main metabolites, i.e., α-terpineol and PMD, are known to be bioactive monoterpene alcohol compounds. This study highlights the potential of fungal biocatalysts for monoterpene transformation.

Involvement of extracellular and intracellular enzymes of Ceriporia sp. ZLY-2010 for biodegradation of polychlorinated biphenyls (PCBs).

This study examined the interrelation between the biodegradation of polychlorinated biphenyls (PCBs) by Ceriporia sp. ZLY-2010 and its fungal enzyme systems. The degradation rates of Aroclor 1254 and 1260 were 29.01% on day 5 and 36.80% on day 10, respectively. MnP (Manganese dependent peroxidase) and laccase activities showed the greatest increases in the samples containing Aroclors, indicating that extracellular enzymes of Ceriporia sp. ZLY-2010 were affected by the addition of Aroclors. However, the relationship between the biodegradation rate and extracellular enzymes might be obscured by the complexity of the biodegradation process. Cytochrome P450 monooxygenase was inhibited and the biodegradation rate of the Aroclor decreased by adding the inhibitor 1-aminobenzotriazole. Two-dimensional gel electrophoresis showed that intracellular enzymes play a significant role in the biodegradation of Aroclor. Complex extracellular and intracellular enzyme systems in Ceriporia sp. ZLY-2010 play an important role in degrading PCBs. Physiological changes of Ceriporia sp. ZLY-2010 caused by PCBs appeared to affect biodegradation of PCBs. However, it is necessary to further study the unidentified enzymes related to the biodegradation of Aroclor.

Analysis of the effects of essential oils on airborne bacteria in a customized bio-clean room.

Essential oils have a sedative effect on stress, and are also known to have antibiotic and anti-carcinogenic effects. These compounds have long been used as natural microbial agents, and have recently been added to a number of pharmaceutical, food and cosmetic products. Controlling the exposure to allergens and pathogens are important factors for the treatment of allergy, and potentially reducing the risk of sensitization and infection. Low humidity, at levels under 35%, may affect human comfort and health during the winter. Patients and other individuals require optimal humidification to maintain a moisturized respiratory tract necessary for protecting against bacterial infection. We designed an analytical system to examine the effects of aromatherapeutic essential oils on airborne bacteria. The antibacterial activities of essential oils were assayed using agar plate air-sampling methods. A bacterial suspension was sprayed into a bio-clean room through the upper holes using a spray gun. Free-floating airborne bacteria were collected from the bio-clean room (blank) in blood agar plates for 10 sec using an air sampler. Three different concentrations of essential oils (0.0005, 0.005 and 0.05 ppm) were then sprayed into the bio-clean room for 5 min. Free-floating airborne bacteria were collected every 10 min for 10 sec each. Treatment with 0.0005 ppm essential oils inhibited the growth of colonies; this effect appeared to persist after 60 min. Decreased bacterial colony growth was more apparent in the presence of 0.005 ppm and 0.05 ppm essential oils than 0.0005 ppm. These effects were observed after 60 min compared to the control (distilled water). These results indicate that essential oils are able to inhibit the growth of airborne bacteria.

Biodegradation of PCB congeners by white rot fungus, Ceriporia sp. ZLY-2010, and analysis of metabolites.

Polychlorinated biphenyls (PCBs) are difficult to degrade due to poor solubility, toxicity, and thermal stability. In the present study, the feasibility of PCB congener biodegradation by Ceriporia sp. ZLY-2010 was evaluated. The biodegradation rates of four PCB congeners, 4,4'-dichlorobiphenyl, 2,3',4',5-tetrachlorobiphenyl, 2,2',4,5,5'-pentachlorobiphenyl, and 2,2',4,4',5,5'-hexachlorobiphenyl were evaluated. The degradation rate of 4,4'-dichlorobiphenyl was 34.03% on incubation day 13, while that of 2,2',4,4',5,5'-hexachlorobiphenyl reached 40.05% on incubation day 17. Therefore, Ceriporia sp. ZLY-2010 was degrading the higher PCB congeners more efficiently. PCB congener degradation products were extracted using acetone and ethyl acetate. No 2,2',4,5,5'-pentachlorobiphenyl metabolites were detected in Ceriporia sp. ZLY-2010 culture, whereas 2,2',4,4',5,5'-hexachlorobiphenyl appeared to degrade to benzoic acid. However, intermediates of 2,2',4,4',5,5'-hexachlorobiphenyl were not detected during degradation. Therefore, additional studies should be performed to explore the mechanisms of PCB degradation. Our results indicate that Ceriporia sp. ZLY-2010 is able to degrade highly chlorinated biphenyls and has potential for use in PCB biodegradation and bioremediation.