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Lignin is a recalcitrant macromolecule formed by three alcohols (monolignols) predominantly connected by β-aryl ether linkages and is one of the most abundant organic macromolecules in the biosphere. However, the role played by environmental bacteria in lignin degradation is still not entirely understood. In this study, we identified an environmental Klebsiella strain isolated from sediment collected from an altitudinal region in a unique Brazilian biome called Caatinga. This organism can also grow in the presence of kraft lignin as a sole source of carbon and aromatic compounds. We performed whole-genome sequencing and conducted an extensive genome-based metabolic reconstruction to reveal the potential mechanisms used by the bacterium Klebsiella variicola P1CD1 for lignin utilization as a carbon source. We identified 262 genes associated with lignin-modifying enzymes (LMEs) and lignin-degrading auxiliary enzymes (LDAs) required for lignin and aromatic compound degradation. The presence of one DyP (Dye-decolorizing Peroxidase) gene suggests the ability of P1CD1 strain to access phenolic and nonphenolic structures of lignin molecules, resulting in the production of catechol and protocatechuate (via vanillin or syringate) along the peripheral pathways of lignin degradation. K. variicola P1CD1 uses aldehyde-alcohol dehydrogenase to perform direct conversion of vanillin to protocatechol. The upper funneling pathways are linked to the central pathways of the protocatechuate/catechol catabolic branches via β-ketoadipate pathways, connecting the more abundant catabolized aromatic compounds with essential cellular functions, such as energy cellular and biomass production (i.e., via acetyl-CoA formation). The combination of phenotypic and genomic approaches revealed the potential dissimilatory and assimilatory ability of K. variicola P1CD1 to perform base-catalyzed lignin degradation, acting on high- and low-molecular-weight lignin fragments. These findings will be relevant for developing metabolic models to predict the ligninolytic mechanism used by environmental bacteria and shedding light on the flux of carbon in the soil., Competing Interests: The authors have declared that no competing interests exist.

Overcoming recalcitrance of lignin has motivated bioprospecting of high-yielding enzymes from environmental ligninolytic microorganisms associated with lignocellulose degrading-systems. Here, we performed isolation of 21 ligninolytic strains belonging to the genus Klebsiella spp., driven by the presence of lignin in the media. The fastest-growing strains (FP10-5.23, FP10-5.22 and P3TM1) reached the stationary phase in approximately 24 h, in the media containing lignin as the main carbon source. The strains showed biochemical evidence of ligninolytic potential in liquid- and solid media-converting dyes, which the molecular structures are similar to lignin fragments. In liquid medium, higher levels of dye decolorization was observed for P3TM.1 in the presence of methylene blue, reaching 98% decolorization in 48 h. The highest index values (1.25) were found for isolates P3TM.1 and FP10-5.23, in the presence of toluidine blue. The genomic analysis revealed the presence of more than 20 genes associated with known prokaryotic lignin-degrading systems. Identification of peroxidases (lignin peroxidase-LiP, dye-decolorizing peroxidase-DyP, manganese peroxidase-MnP) and auxiliary activities (AA2, AA3, AA6 and AA10 families) among the genetic repertoire suggest the ability to produce extracellular enzymes able to attack phenolic and non-phenolic lignin structures. Our results suggest that the Klebsiella spp. associated with fresh water and soil may play important role in the cycling of recalcitrant molecules in the Caatinga (desert-like Brazilian biome), and represent a potential source of lignin-degrading enzymes with biotechnological applications.