RIKEN Review No. 42 (December, 2001): Focused on Ecomolecular Science Research

Lignin degradation and roles of white rot fungi: Studyon an efficient symbiotic system in fungus-growing

termites and its application to bioremediationMoriya Ohkuma,∗1 Yoshimasa Maeda,∗1 Toru Johjima,∗2 and Toshiaki Kudo∗1

∗1Microbiology Laboratory, RIKEN∗2JST-ICORP

White rot fungi produce extracellular phenoloxidases and can decompose lignin efficiently. Elfvingia applanatahas been successfully applied for the bioconversion of bisphenol A, suggesting the usefulness of white rot fungifor bioremediation. In order to attain real bioremediation, the recycling of ecomolecules in the ecosystem isimportant. The sophisticated symbiotic system of white rot fungi of the genera Termitomyces with fungus-growing termites is an attractive example of an efficient natural system to be studied, in which cooperationbetween termites and fungi accomplishes efficient decomposition of lignin and complete biorecycling of plantlitter.

Lignocellulose is the predominant component of woody plantand dead plant materials, and the most abundant biomass onearth. Lignin is a heterogenous and irregular arrangement ofphenylpropanoid polymer that resists chemical or enzymaticdegradation to protect cellulose. It has been thought thatlignin degradation is a rate-limiting step of carbon recycling.Microbial activity of metabolizing lignin and its componentsis one of the plausible evolutional origins for the degradingpathway of aromatic xenobiotics and/or environmental pol-lutants, such as PCB and dioxin. For applied and environ-mental science, it is important to understand the nature ofthe decomposition and biorecycling of lignocellulose by re-sponsible organisms.

Basidiomycetes, which cause white rot decay, are able to de-grade lignin in wood. These fungi are called white rot fungi.Lignin degradation by white rot fungi has been extensivelystudied, and results revealed that three kinds of extracellularphenoloxidases, namely, lignin peroxidase (LiP), manganeseperoxidase (MnP) and laccase (Lac), are responsible for ini-tiating the depolymerization of lignin.1) The expression pat-tern of these enzymes depends on the organisms: some se-crete LiP and MnP (no Lac), whereas others secrete MnPand Lac (no LiP). In addition to lignin, white rot fungi areable to degrade a variety of environmentally persistent pollu-tants, such as chlorinated aromatic compounds, heterocyclicaromatic hydrocarbons, various dyes and synthetic high poly-mers.2) Probably, this degradability of white rot fungi is dueto the strong oxidative activity and the low substrate speci-ficity of their ligninolytic enzymes. Thus, white rot fungi andtheir enzymes are thought to be useful not only in some in-dustrial processes like biopulping and biobreaching but alsoin bioremediation.

We investigated the degradation of an endocrine disruptor,bisphenol A (BPA), by a white rot fungus, Elfvingia ap-planata (Fig. 1), which is known in Japan as “kofuki-saru-no-koshikake” mushroom. Decolorization of lignin and degra-dation of aromatic rings in lignin were observed in the cul-ture (Fig. 2), indicating that E. applanata is a strong lignin-

Fig. 1. Slant culture of Elfvingia applanata.

Fig. 2. Lignin decomposition in the culture of Elfvingia applanata.

39

Table 1. Ligninolytic activity of enzymes produced by Elfvingia applanata.

Enzyme Method Rate(∆A min−1 ml−1)

Laccase 2,6-Dimethoxyphenol oxidation 0.09monitored at 469 nm

LiP Veratryl alcohol oxidation to ND

veratraldehyde monitored at 310 nmMnP Mn3+-malonate complex monitored at 0.435

270 nm

The enzyme activities in cultures were measured after four days of culture. ND, notdetected.

Fig. 3. Conversion of bisphenol A by the culture supernatant ofElfvingia applanata.

degrading white rot fungus. Under ligininolytic condition,E. applanata secreted MnP and Lac but not LiP (Table 1).The molecular cloning of the gene encoding MnP of E. ap-planata has been reported and the production of MnP wasinduced at the transcriptional level by the presence of MnII

and aromatic compounds like 2,5-xylidine.3) When the cul-ture supernatant was incubated with BPA, the conversionof BPA was observed, showing the usefulness of E. applanatafor bioremediation. The presence of either veratryl alcohol or2,6-dimethoxyphenol in the culture stimulated the conversionof BPA (Fig. 3). The addition of hydrogen peroxide (1mM) inthe reaction mixture stimulated the conversion only slightly,suggesting a sufficient endogenous supply of hydrogen perox-ide in the culture. Higher concentrations of hydrogen per-oxide inhibited the conversion. The putative pathway of theconversion of BPA by MnP was shown in Fig. 4.

In order to attain real bioremediation, the degradation prod-ucts should be effectively mineralized and reutilized or re-cycled in the ecosystem. As shown in the case of E. ap-planata, white rot fungi and the enzymes produced by themare useful as primary agents in bioremediation. It is consid-ered beneficial to learn from the most efficient natural sys-tems in which white rot fungi are involved in recycling lig-

Fig. 4. Proposed pathway for the conversion of bisphenol A by Elfvin-gia applanata MnP.

nocellulose. A symbiotic relationship between termites andfungi is such an attractive example of an efficient natural sys-tem. Termites (Isoptera) are extremely abundant in tropicalterrestrial ecosystems, and play important roles in biorecy-cling of plant litter. The so-called fungus-growing termitesbelong to an evolutionary related group of termites (Ter-mitidae, Macrotermitinae) and are abundant in Asian andAfrican tropics.4) They consume more than 90% of dry woodin some arid tropical areas and directly mineralize up to 20%

40

of the net primary production in wetter savannas. This groupof termites has an interesting symbiotic relationship with ba-sidiomycete fungi of the genus Termitomyces, such that theycultivate the symbiotic fungi within their nests. In sometypes of fungus-growing termites, the “termite mushroom”,the fruiting body of Termitomyces, blooms seasonally fromtermite nests (Fig. 5). The termite mushroom is unique innature, growing from only the termite nests.

There have been several suggestions for the roles of the sym-biotic fungi in termite nutrition: (1) decomposition of lignin;(2) supply of cellulase and xylanase to work synergisticallywith the enzymes produced by the termite; and (3) concentra-

Fig. 5. Mushroom blooming fungus comb of termite. The photo-graph is courtesy of Drs. Moriya and Inoue.

Fig. 6. Symbiotic relationship between fungus-growing termites and Termitomyces fungi.

tion of nutrients such as nitrogen for the termite. Many worksare involved in studies of the second suggestion, also knownas the “acquired enzyme hypothesis”,5) but some researchersquestioned a part of this hypothesis. Since an endogenouscellulase, an enzyme produced by the termite, has recentlybeen recognized in wood- and litter-feeding termites,6) it isdifficult to make generalizations on the significance of the“acquired” fungal cellulase in cellulose digestion in fungus-growing termites.

The sophisticated and well-coordinated cooperation betweenthe termites and the fungi enables efficient utilization of lig-nocellulose (Fig. 6).4) The so-called old workers forage outsidethe nest and collect plant litter. In the nest, young workersmasticate and ingest the collected plant litter which passesrapidly through the termite gut without digestion. The re-sulting fecal pellets (primary feces) are pressed together toform a sponge-like structure (called fungus comb). The sym-biotic fungi grow on the comb-like matrices of the funguscomb. They form mycelia and white round and asexual coni-dial structures called fungus nodules. It has been reportedthat the lignin content progressively decreased as the fun-gus comb matured.7) It has also been shown that in vitrodigestibility of cellulose in a matured fungus comb was ap-proximately 3-fold higher than that in a newly formed one.The fungus nodules are usually consumed by young workers,whereas the old senescent combs are consumed by old workersto produce final feces. However, final feces are rarely found inthe nest of fungus-growing termites, suggesting the highly ef-ficient decomposition and the complete biorecycling of plantlitter. These observations support the finding that symbioticfungi have the ability to degrade lignin, which makes cellu-lose more easily degraded by the cellulase produced by thetermite.

We are now studying the extracellular phenoloxidases pro-duced by a symbiotic fungus, Termitomyces albuminosus.Two genes encoding MnP (tam1 and 2 ) were identified, bothof which have essential amino acid residues for peroxidase ac-tivity and MnII ion binding whereas the residue for veratrylalcohol oxidation was not observed. These features suggest

41

that both genes encode typical MnP. The mRNA level of thetam2 was higher than that of tam1, and interestingly, theexpression of tam2 was not affected by the presence of MnII

in the culture medium in spite of the induction of MnP byMnII in general. Moreover, we have found a novel peroxidase(designated as TAP) in the culture supernatant. TAP wasable to oxidize phenolic compounds in the presence of hydro-gen peroxide. However, MnII was not required in the reactionand veratryl alcohol was not oxidized. TAP has some inter-esting characteristics with respect to the optimum pH andthe optimum concentration of hydrogen peroxide, and we arenow characterizing them in detail.

We have cultivated a number of the symbiotic fungi from thefungus nodules of various termite species. Molecular phylo-genetic analysis of the cultivated fungi is in progress. Identi-fication of fungal species in the combs without cultivation isprobably advantageous to avoid some biases introduced dur-ing their cultivation. These phylogenetic analyses are ben-eficial for understanding the selection and evolution of theassociations of symbiotic fungi with fungus-growing termites.

Moreover, we are now studying the lignin-degrading enzymesof the cultivates, particularly laccase, because laccase is themajor enzyme exhibiting ligninolytic activity in the funguscombs. The characterization of ligninolytic activity of thesymbiotic fungi is important for understanding the nature ofthis symbiotic relationship, which is most successful in biore-cycling the naturally occurring ecomolecule, lignocellulose.

References1) T. K. Kirk and R. L. Farrell: Annu. Rev. Microbiol. 41, 465

(1987).2) J. A. Bumpus et al.: Science 228, 1434 (1985).3) Y. Maeda et al.: Biotech. Lett. 23, 103 (2001).4) C. Rouland: in Termites: Evolution, Sociality, Symbioses,

Ecology, edited by T. Abe, D. E. Bignell, and M. Higashi(Kluwer Academic Publishers, Dordrecht, 2000), p. 289.

5) M. M. Martin and J. S. Martin: Science 199, 1453 (1978).6) H. Watanabe et al.: Nature 394, 330 (1998).7) F. Hyodo et al.: Soil Biol. Biochem. 32, 653 (2000).

42