Abstract
Laccases are present as isozymes in white-rot fungi, yet their evolutionary history and functional role in lignin degradation remain controversial. Trametes hirsuta, a ubiquitous fungus in forest ecosystems, can completely break down cellulose, hemicellulose, and lignin in wood. Based on bioinformatic and biochemical characterization, we have shown that five laccase isozyme genes (lacA-E) in Trametes hirsuta AH28-2 were derived from a single ancestral laccase gene, lacF, with lacA and lacB originating from disparate evolutionary branches. The syringyl-type (S-type) lignin model compounds significantly induced the expression of lacA-lacE at both transcriptional and expression levels. Furthermore, in vitro and in vivo analyses demonstrated that the later-emerging laccases, LacA and LacB, primarily contribute to oxidizing S-type lignin present in hardwood, whereas laccase LacF plays a primary role in oxidizing guaiacyl-type (G-type) lignin found in softwood. Finally, evolutionary analysis of ancestral laccases from Agaricomycetes fungi also revealed a shift from better oxidation of G-type lignin in softwood by earlier ancestral laccases to better oxidation of S-type lignin in hardwood by later ancestral laccases. These findings indicate that laccase evolution in Agaricomycetes fungi is consistent with lignin synthesis. We have demonstrated the direct involvement of laccases at different evolutionary stages in preferentially oxidizing different types of lignin.IMPORTANCELaccases in white-rot fungi always exist in the form of isozymes. However, the evolutionary history and functional diversification of laccase isozymes remain controversial. Our study demonstrates that the six laccase isozymes in Trametes hirsuta AH28-2 belong to three distinct evolutionary branches. Among them, LacF represents an earlier-emerging branch and primarily contributes to oxidizing the G-type units of gymnosperm lignin. In contrast, LacA and LacB, which are later-emerging, primarily contribute to oxidizing the S-type units in angiosperm lignin. Interestingly, ancestral laccases reconstructed at different evolutionary nodes also display distinct lignin oxidation preferences. This suggests that the evolution of laccases in Agaricomycetes fungi is closely linked to the emergence of S-type lignin units in angiosperms. These findings reveal the co-evolutionary relationship between lignin structural changes and fungal laccase diversification, providing new insights into the evolutionary mechanisms and biological functions of laccase isozymes.