Sun-Hwa Ryu

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, β-eudesmane and β-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.

Abstract 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.

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).

Biomodification of Ethanol Organolsolv Lignin by Abortiporus biennis and Its Structural Change by Addition of Reducing Agent

The main goal of this study was to investigate biomodification mechanism of lignin by white rot fungus, Abortiporus biennis, and to depolymerize ethanol organosolv lignin for industrial application. In nitrogen-limited culture, A. biennis polymerized mainly lignin showing a rapid increase of molecular weight and structural changes depending on incubation days. At the initial incubation days, cleavage of ether bonds increased phenolic OH content, while the results were contrary in of the later part of the culture. Based on these results, ascorbic acid as a reducing agent was used to induce depolymerization of lignin during cultivation with white rot fungus. As a result, the degree of increase of average molecular weight of lignin was significantly declined when compared with those of the ascorbic acid free-experiment, although the molecular weight of fungus treated sample slightly increased than that of control. Furthermore, lignin derived oligomers in culture medium were depolymerized with the addition of ascorbic acid, showing that the average molecular weight was 381 Da, and phenolic OH content was 38.63%. These depolymerized lignin oligomers were considered to be applicable for industrial utilization of lignin. In conclusion, A. biennis led to the polymerization of lignin during biomodification period. The addition of ascorbic acid had a positive effect on the depolymerization and increase of phenolic OH content of lignin oligomers in medium.

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.

Changes in Activities of Lignin Degrading Enzymes and Lignin Content During Degradation of Wood Chips by Polyporus brumalis

In this study, laccase activity, rate of weight loss and degree of lignin degradation of pine wood chips were determined during the liquid and solid state incubation with Polyporus brumalis. The results showed that laccase enzyme activity at untreated wood chip was gradually decreased after 20 days, but enzyme activity with wood chip treatment showed 10 times higher than untreated ones at 60 incubation days. Rate of weight losses of pine chip and rate of lignin loss were 23.4% and 6.3% by P. brumalis during 80 incubation days. Gene expression of pblac1 from P. brumalis was 3 times increased under pine chip treatment at 40 incubation days. Consequently, laccase activity of white rot fungi, P. brumalis, was increased at incubation with wood chip and pblac1 gene was important factor of lignin degradation. Therefore, to regulate lignin degrading enzyme gene expression by using the tools of biotechnology will be able to develop superior strains and it will be useful for pretreatment of lignocellulosic biomass at bioethanol production.

Physiological and Genetic Changes by Mixing Culture of Shiitake

Attempts were made to investigate the physiological and genetic changes when two different shiitake (Lentinula edodes) strains are mixed. Mycelial growth of KFRI 180 strain and KFRI 1 strain were investigated 82 mm and 80 mm, respectively. Concerning the weight loss percentage of medium, KFRI 1 strain decreased 2.4% and KFRI 180 strain 1.6%. Plug-shaped spawn had no-problem to incubate and there were no differences among the ratios of mixture. Also, conditions of plug-shaped spawns were similar, When the isolated mycelia from plugshaped spawns was incubated again, KFRI 1 50%-KFRI 180 50% showed decreased growth of mycelia compared with other treatments. The same results were obtained from test tubes filled with sawdust. When surface of spawn bottles were observed, KFRI 1 50%-KFRI 180 50% showed spots, but other treatments were not different from KFRI 1 and KFRI 180. Test was made to confirm the strains by confrontation culture. The mixture of two strains was proved to be KFRI 1 regardless the ratios of mixture. However, by the RAPD primer analysis, when KFRI 1 was mixed with KFRI 180, KFRI 180 was stronger. Thus, the confrontation line on PDA was different from the bands analysis by primers. Attempts were made whether the fruit-bodies were made at the generating condition of spawn bottles. The results were that KFRI 1 100%, KFRI 1 90%-KFRI 180 10%, KFRI 1 80%-KFRI 180 20%, KFRI 1 50%-KFRI 180 50% treatment showed fruit-body formation. The shape of fruit-body was deformed, but the gill was made normally.