Resin acids play key roles in shaping microbial communities during degradation of spruce bark.

The bark is the outermost defense of trees against microbial attack, largely thanks to toxicity and prevalence of extractive compounds. Nevertheless, bark decomposes in nature, though by which species and mechanisms remains unknown. Here, we have followed the development of microbial enrichments growing on spruce bark over six months, by monitoring both chemical changes in the material and performing community and metagenomic analyses. Carbohydrate metabolism was unexpectedly limited, and instead a key activity was metabolism of extractives. Resin acid degradation was principally linked to community diversification with specific bacteria revealed to dominate the process. Metagenome-guided isolation facilitated the recovery of the dominant enrichment strain in pure culture, which represents a new species (Pseudomonas abieticivorans sp. nov.), that can grow on resin acids as a sole carbon source. Our results illuminate key stages in degradation of an abundant renewable resource, and how defensive extractive compounds have major roles in shaping microbiomes.

Yeasts Have Evolved Divergent Enzyme Strategies To Deconstruct and Metabolize Xylan.

Together with bacteria and filamentous fungi, yeasts actively take part in the global carbon cycle. Over 100 yeast species have been shown to grow on the major plant polysaccharide xylan, which requires an arsenal of carbohydrate active enzymes. However, which enzymatic strategies yeasts use to deconstruct xylan and what specific biological roles they play in its conversion remain unclear. In fact, genome analyses reveal that many xylan-metabolizing yeasts lack expected xylanolytic enzymes. Guided by bioinformatics, we have here selected three xylan-metabolizing ascomycetous yeasts for in-depth characterization of growth behavior and xylanolytic enzymes. The savanna soil yeast Blastobotrys mokoenaii displays superior growth on xylan thanks to an efficient secreted glycoside hydrolase family 11 (GH11) xylanase; solving its crystal structure revealed a high similarity to xylanases from filamentous fungi. The termite gut-associated Scheffersomyces lignosus, in contrast grows more slowly, and its xylanase activity was found to be mainly cell surface-associated. The wood-isolated Wickerhamomyces canadensis, surprisingly, could not utilize xylan as the sole carbon source without the addition of xylooligosaccharides or exogenous xylanases or even co-culturing with B. mokoenaii, suggesting that W. canadensis relies on initial xylan hydrolysis by neighboring cells. Furthermore, our characterization of a novel W. canadensis GH5 subfamily 49 (GH5_49) xylanase represents the first demonstrated activity in this subfamily. Our collective results provide new information on the variable xylanolytic systems evolved by yeasts and their potential roles in natural carbohydrate conversion. IMPORTANCE Microbes that take part in the degradation of the polysaccharide xylan, the major hemicellulose component in plant biomass, are equipped with specialized enzyme machineries to hydrolyze the polymer into monosaccharides for further metabolism. However, despite being found in virtually every habitat, little is known of how yeasts break down and metabolize xylan and what biological role they may play in its turnover in nature. Here, we have explored the enzymatic xylan deconstruction strategies of three underexplored yeasts from diverse environments, Blastobotrys mokoenaii from soil, Scheffersomyces lignosus from insect guts, and Wickerhamomyces canadensis from trees, and we show that each species has a distinct behavior regarding xylan conversion. These findings may be of high relevance for future design and development of microbial cell factories and biorefineries utilizing renewable plant biomass.

Structural diversity and substrate preferences of three tannase enzymes encoded by the anaerobic bacterium Clostridium butyricum.

Tannins are secondary metabolites that are enriched in the bark, roots, and knots in trees and are known to hinder microbial attack. The biological degradation of water-soluble gallotannins, such as tannic acid, is initiated by tannase enzymes (EC 3.1.1.20), which are esterases able to liberate gallic acid from aromatic-sugar complexes. However, only few tannases have previously been studied in detail. Here, for the first time, we biochemically and structurally characterize three tannases from a single organism, the anaerobic bacterium Clostridium butyricum, which inhabits both soil and gut environments. The enzymes were named CbTan1-3, and we show that each one exhibits a unique substrate preference on a range of galloyl ester model substrates; CbTan1 and 3 demonstrated preference toward galloyl esters linked to glucose, while CbTan2 was more promiscuous. All enzymes were also active on oak bark extractives. Furthermore, we solved the crystal structure of CbTan2 and produced homology models for CbTan1 and 3. In each structure, the catalytic triad and gallate-binding regions in the core domain were found in very similar positions in the active site compared with other bacterial tannases, suggesting a similar mechanism of action among these enzymes, though large inserts in each enzyme showcase overall structural diversity. In conclusion, the varied structural features and substrate specificities of the C. butyricum tannases indicate that they have different biological roles and could further be used in development of new valorization strategies for renewable plant biomass.