Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

Abstract

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.

Laccase detoxification mediates the nutritional alliance between leaf-cutting ants and fungus-garden symbionts

“…Phenolic compounds are not only present in live plant tissue, but to a lesser extent also in the shed leaves and leaf litter (24) collected by most other fungus-growing ants, suggesting that polyphenol-oxidase activity was an inherent property of ant fungus farming before active leaf cutting arose. Ant-cultivated leucocoprinaceous fungi are closely related to saprophytic basidiomycetes that mostly decompose leaf litter (25), and laccase enzymes are known from the gardens of leaf-litter collecting fungus-growing termites (26), indicating the inherent need for phenol degradation when using leaf litter as fungal substrate. The genomes of saprophytic fungi normally contain multiple laccase-coding genes (27,28), so that the actual number of laccases that we found in L. gongylophorus may merely represent the enzyme spectrum of the last common ancestor of the gongylidiabearing cultivars (14,29).…”

Lignocellulose pretreatment in a fungus-cultivating termite

“…In contrast to characteristic ligninolytic peroxidases, laccase is the only enzyme with the potential to act on lignin that has been found in Termitomyces spp. (15,16,21), but its functional role in ligninolysis remains unclear and controversial (22). In addition to genetic and biochemical research approaches, several solid-state NMR studies have shown different peak intensities assigned to lignin structures between spectra of the fresh comb and mature comb (23,24).…”

“…from several Macrotermitinae termite hosts apparently lack typical ligninolytic agents, such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidase (VP), that are found in, for example, white-rot fungi (8,15,16). Research on these enzymes shows that lignin depolymerization may occur via many mechanisms: (i) LiPs and VPs are known to efficiently oxidize nonphenolic β-ether units in lignin within the wood cell wall-beyond where enzymes are permitted to travel-via single electron transfer, creating aryl cation radical intermediates and cleaving the β-ether unit between its Cα and Cβ carbons (17,18); (ii) MnPs preferentially oxidize Mn 2+ to the reactive Mn 3+ which, in its chelated form, can act as a diffusible redox mediator and attack phenolic structures in lignin (19); (iii) unsaturated fatty acids undergo peroxidation by MnP to form organoperoxyl radicals that have also been shown to be potential ligninolytic agents if able to diffuse into the wood cell wall (20).…”

Diversity of Two-Domain Laccase-Like Multicopper Oxidase Genes in Streptomyces spp.: Identification of Genes Potentially Involved in Extracellular Activities and Lignocellulose Degradation during Composting of Agricultural Waste

“…Traditional three-domain fungal and bacterial laccases have been widely studied for their importance in various biotechnological applications (13)(14)(15). A novel lineage of relatively small LMCOs-the two-domain LMCOs-has now attracted the attention of researchers (16), and two-domain LMCOs were found to be typically present in Streptomyces spp., such as Streptomyces griseus, Streptomyces ipomoeae, and Streptomyces coelicolor (17)(18)(19)(20).…”