Bringing up-to-date the toolkit for the catabolism of aromatic compounds in fungi: the unexpected 1,2,3,5-tetrahydroxybenzene central pathway

Saprophytic fungi are able to catabolize many plant-derived aromatics, including, for example, gallate. The catabolism of gallate in fungi is assumed to depend on the five main central pathways, i.e., of the central intermediates catechol, protocatechuate, hydroxyquinol, homogentisate, and gentisate, but a definitive demonstration is lacking. To shed light on this process, we analyzed the transcriptional reprograming of the growth of Aspergillus terreus on gallate compared with acetate as the control condition. Surprisingly, the results revealed that the five main central pathways did not exhibit significant positive regulation. Instead, an in-depth analysis identified four highly expressed and upregulated genes that are part of a conserved gene cluster found in numerous species of fungi, though not in Aspergilli. The cluster comprises a monooxygenase gene and a fumarylacetoacetate hydrolase-like gene, which are recognized as key components of catabolic pathways responsible for aromatic compound degradation. The other two genes encode proteins with no reported enzymatic activities. Through functional analyses of gene deletion mutants, the conserved short protein with no known domains could be linked to the conversion of the novel metabolite 5-hydroxydienelatone, whereas the DUF3500 gene likely encodes a ring-cleavage enzyme for 1,2,3,5-tetrahydroxybenzene. These significant findings establish the existence of a new 1,2,3,5-tetrahydroxybenzene central pathway for the catabolism of gallate and related compounds (e.g., 2,4,6-trihydroxybenzoate) in numerous fungi where this catabolic gene cluster was observed. IMPORTANCEThe lignin found in various economically significant plants, such as major grains like rice, wheat, and maize, comprises a substantial portion of syringyl units (up to 60%). As a result, the future utilization of residues from these plants in biorefineries will yield significant quantities of syringyl derivatives. However, our understanding of how fungi degrade these syringyl derivatives is to date scarce and mostly relies on unproven assumptions. Our study, demonstrates the existence of a new 1,2,3,5-tetrahydroxybenzene central intermediate for the catabolism of gallate in numerous fungi. This finding expands the toolkit of central pathways, proving that the generalized assumption that gallate catabolism depends on the previously known five main central pathways was incorrect. This research reveals a novel crucial central pathway of great ecological and biotechnological importance, not only for fungi but also potentially for bacteria.