Giving Back Lost Enzymes Slows a Brown-Rot Fungus Down

Wood-decay fungi split into two evolutionary camps, and a new genetic experiment shows what happens when the camps are forced to share tools. White-rot fungi deploy a suite of lignin-modifying enzymes to dismantle the tough aromatic polymer that shields cellulose in plant cell walls. Brown-rot fungi, which descended from white-rot ancestors, largely abandoned those enzymes and instead rely on a small-molecule-driven Fenton reaction that floods wood with reactive oxygen species. Researchers have now engineered the brown-rot fungus Gloeophyllum trabeum to produce lignin-modifying enzymes from the white-rot fungus Trametes versicolor, and the outcome is a striking trade-off: the engineered hybrids regained part of their lost capacity to degrade lignin, but their overall breakdown of lignocellulose slowed down.

The study, published in Fungal Biology and Biotechnology, set out to test a long-standing evolutionary puzzle. Brown-rot fungi lost not only the genes encoding lignin-modifying enzymes but also more than 30 percent of the genes encoding carbohydrate-active enzymes relative to white-rot fungi, according to genomic comparisons cited by the authors. That streamlining apparently pays off in the wild, where brown-rot fungi deconstruct wood rapidly and selectively harvest the embedded polysaccharides while only partially modifying lignin. Why a lineage would discard such powerful enzymatic machinery has remained unclear, and the new work offers a direct experimental answer: reintroducing the enzymes disrupts a decay system that had been fine-tuned to run without them.

To build the hybrid fungi, the team first mined transcriptomic data from Trametes versicolor and Pleurotus ostreatus decaying aspen wood wafers, using a publicly available dataset to identify which lignin-modifying enzyme genes are most active and at which decay stages. Class II peroxidase genes in both white-rot species were mostly switched on at later decay stages, consistent with lignin oxidation ramping up as decay progresses, while laccase genes peaked early. From this analysis the researchers selected two manganese peroxidase genes, MnP1 and MnP2, a lignin peroxidase gene, LiP2, and a laccase gene, LCC3, from T. versicolor, choosing representatives with the highest transcriptional abundance and clear stage-specific regulation.

Each gene was then transformed individually into G. trabeum protoplasts together with a hygromycin resistance marker, using expression cassettes driven by promoter and terminator sequences from another brown-rot fungus, Rhodonia placenta. After screening dozens of transformants per genotype and confirming gene insertion by PCR, the team selected three representative strains for each enzyme and verified transcription by quantitative RT-PCR. Enzyme activity assays confirmed functional production of MnP1, MnP2, and LCC3, although activity levels varied among transformants of the same genotype. Notably, no clear lignin peroxidase activity was detected in the LiP2 transformants, so the functional conclusions rest mainly on the manganese peroxidases and the laccase.

Growth measurements revealed the first signs of incompatibility. On nutrient-rich agar or minimal medium with glucose, the engineered strains extended their hyphal fronts at normal speeds, indicating that polarized mycelial growth was unaffected when readily available nutrients were present. Total biomass accumulation, however, dropped substantially in the mutants, particularly those expressing MnP2 and LCC3, a pattern the authors attribute to the metabolic burden of synthesizing large amounts of recombinant protein. When the fungi were grown on complex lignocellulosic substrates such as corn stover and spruce, the MnP2 and LCC3 mutants showed clear radial growth defects relative to their performance on rich medium, suggesting that the cost of enzyme production compounds on substrates that already demand heavy secretory investment.

The central degradation experiment used corn stover in solid-state culture for 30 days. Because soluble extractives make up 25 to 30 percent of corn stover, the researchers measured the net mass change of the insoluble lignocellulose fraction after hot-water extraction, rather than whole-substrate mass loss, to gauge true lignocellulose-solubilizing capacity. The wild-type strain solubilized 36.8 plus or minus 1.2 percent of the insoluble lignocellulose in 30 days. The LME mutants fell short of that benchmark, with the reduction especially pronounced in MnP1 and LCC3 strains. All three LCC3 mutants degraded the substrate more slowly than the wild type, and the decrease was statistically significant in all three MnP1 strains even though those mutants showed no growth defects, pointing to interference with the decay machinery itself rather than a simple growth penalty.

Compositional analysis of the residual insoluble material revealed the flip side of the trade-off. The wild-type fungus removed 37.5 percent of cellulose and 36.7 percent of hemicellulose but only 20.7 percent of Klason lignin, the classic carbohydrate-selective signature of brown rot. In the MnP1 and LCC3 mutants, the lignin-to-cellulose and lignin-to-hemicellulose ratios of the remaining residue dropped substantially, meaning lignin was degraded proportionally faster than the carbohydrates. In other words, the reintroduced enzymes partially restored ligninolytic function and shifted the decay pattern away from carbohydrate selectivity, but the total removal of every component, especially cellulose and hemicellulose, declined. MnP2 mutants behaved inconsistently: one strain solubilized 51.1 percent less lignocellulose than the wild type, another actually solubilized 13.3 percent more, and a third showed no change, with the variability likely reflecting differences in expression levels caused by random insertion sites or gene copy numbers.

Enzyme assays and redox measurements helped explain the slowdown. In the MnP1 and LCC3 mutants, the specific productivities of cellulase, xylanase, and the ferric iron-reducing agent all fell, and iron-reduction capacity at the hyphal front dropped sharply. Iron reduction is a proxy for the low-molecular-weight metabolite-mediated Fenton system, in which fungal metabolites such as hydroquinones shuttle electrons to ferric iron and oxygen to generate hydroxyl radicals. The authors propose that the reintroduced enzymes compete for these shared redox resources: peroxidases and the Fenton reaction both consume hydrogen peroxide, and the reduced quinones that fuel Fenton chemistry can be oxidized by the enzymes instead. Consistent with this, a previous cell-free study by the same group showed that adding laccase or heme-thiolate peroxidase to a quinone redox system reduced Fenton reagent production. Interestingly, the mutants also grew better than expected on media containing the aromatic toxins 1,4-benzoquinone and corn stover phenolic extractives, indicating that the enzymes conferred a detoxification benefit, possibly by polymerizing or degrading quinones, at the cost of weakening the redox shuttles that brown rot depends on.

An integrated genotype-phenotype analysis tied the threads together. Principal component analysis separated the enzyme genotypes along phenotypic axes, and Pearson correlation coefficients quantified the associations. LCC3 had the broadest impact, correlating negatively with biomass on glucose, corn stover, and spruce, with cellulase and xylanase productivity, with iron-reduction capacity, and with the removal rates of cellulose, hemicellulose, and lignin, while correlating positively with detoxification-associated growth. MnP1 also correlated negatively with lignocellulolytic traits despite leaving growth untouched, whereas MnP2 showed the weakest and most variable correlations. The authors conclude that lignin-modifying enzymes, though prevalent in white-rot fungi, are fundamentally incompatible with the carbohydrate-selective brown-rot system, and that this incompatibility may help explain why brown-rot lineages lost the genes in the first place. They also note a practical implication: combining the two decay strategies in one organism may still be possible, but likely requires temporally segregated decay chemistries and tunable, inducible control of enzyme dosage so that lignin modification and carbohydrate removal do not compete for the same redox intermediates.

Subject of Research: Reintroducing white-rot lignin-modifying enzyme genes into the brown-rot fungus Gloeophyllum trabeum restores ligninolytic activity but reduces lignocellulose degradation

Article Title: Reintroduction of lignin-modifying enzymes in the brown-rot fungus Gloeophyllum trabeum restores ligninolytic activities but diminishes lignocellulose degradation

Article References: Ganjoloo, N., Li, W., & Zhang, J. (2026). Reintroduction of lignin-modifying enzymes in the brown-rot fungus Gloeophyllum trabeum restores ligninolytic activities but diminishes lignocellulose degradation. Fungal Biology and Biotechnology, 13(1), Article 11. https://doi.org/10.1186/s40694-026-00218-y

Image Credits: AI Generated

DOI: 10.1186/s40694-026-00218-y

Keywords: brown-rot fungi, white-rot fungi, lignin-modifying enzymes, Gloeophyllum trabeum, Trametes versicolor, Fenton chemistry, lignocellulose degradation, manganese peroxidase, laccase, reactive oxygen species, genetic engineering, fungal evolution