Multimodular fused acetyl–feruloyl esterases from soil and gut Bacteroidetes improve xylanase depolymerization of recalcitrant biomass

Abstract

Background: Plant biomass is an abundant and renewable carbon source that is recalcitrant towards both chemical and biochemical degradation. Xylan is the second most abundant polysaccharide in biomass after cellulose, and it possesses a variety of carbohydrate substitutions and non-carbohydrate decorations which can impede enzymatic degradation by glycoside hydrolases. Carbohydrate esterases are able to cleave the ester-linked decorations and thereby improve the accessibility of the xylan backbone to glycoside hydrolases, thus improving the degradation process. Enzymes comprising multiple catalytic glycoside hydrolase domains on the same polypeptide have previously been shown to exhibit intramolecular synergism during degradation of biomass. Similarly, natively fused carbohydrate esterase domains are encoded by certain bacteria, but whether these enzymes can result in similar synergistic boosts in biomass degradation has not previously been evaluated. Results: Two carbohydrate esterases with similar architectures, each comprising two distinct physically linked catalytic domains from families 1 (CE1) and 6 (CE6), were selected from xylan-targeting polysaccharide utilization loci (PULs) encoded by the Bacteroidetes species Bacteroides ovatus and Flavobacterium johnsoniae. The full-length enzymes as well as the individual catalytic domains showed activity on a range of synthetic model substrates, corn cob biomass, and Japanese beechwood biomass, with predominant acetyl esterase activity for the N-terminal CE6 domains and feruloyl esterase activity for the C-terminal CE1 domains. Moreover, several of the enzyme constructs were able to substantially boost the performance of a commercially available xylanase on corn cob biomass (close to twofold) and Japanese beechwood biomass (up to 20-fold). Interestingly, a significant improvement in xylanase biomass degradation was observed following addition of the full-length multidomain enzyme from B. ovatus versus the addition of its two separated single domains, indicating an intramolecular synergy between the esterase domains. Despite high sequence similarities between the esterase domains from B. ovatus and F. johnsoniae, their addition to the xylanolytic reaction led to different degradation patterns. Conclusion: We demonstrated that multidomain carbohydrate esterases, targeting the non-carbohydrate decorations on different xylan polysaccharides, can considerably facilitate glycoside hydrolase-mediated hydrolysis of xylan

A polysaccharide utilization locus from the gut bacterium Dysgonomonas mossii encodes functionally distinct carbohydrate esterases

“…The overall architecture of the D. mossii PUL17 however appears unique, and no similar PULs can be found currently in the PULDB (14). Our recent biochemical characterization of the BoXylLencoded multicatalytic BoCE6-CE1 enzyme showed how it could greatly boost the GAX degradation ability of a commercial xylanase through its combined acetyl-and feruloyl esterase activities (32). Interestingly, DmCE1B in the D. mossii PUL 17 comprises two individual CE1 catalytic domains separated by a small CBM48 domain (Fig.…”

“…1B). While numerous putative CE6-CE1 fusions can be found in the PULDB, only two have been characterized (32). As mentioned, no CE1-CE1 fusion enzymes have previously been characterized, though such enzymes can be found in various Dysgonomonas species and other genera from both gastrointestinal and environmental habitats including Chryseobacterium, Dyadobacter, and Chitinophaga (14).…”

“…In the densely colonized gut, there is fierce competition for nutrients, and it is likely that possession of efficient CEs that enable greater access to cell wall polysaccharides can give microorganisms a competitive advantage. In a recent study, we demonstrated that in two unique PULs from the gut bacterium Bacteroides ovatus and the aerobic soil bacterium Flavobacterium johnsoniae, CE1 and CE6 enzymes are found in "multicatalytic" enzymes, i.e., enzymes comprised of multiple catalytic domains on the same polypeptide (32). The combination of complementary enzymatic activities into single proteins with strongly enhanced activity has previously been shown mainly for enzyme activities targeting the main polysaccharide chain (20,33), but research on multicatalytic enzymes is often impeded by their large size and resulting difficulties in protein production (34).…”

“…Why a simultaneous attack by fused enzymes is desirable is not clear. However, we recently showed that CE action may also be enhanced by a multicatalytic protein architecture when aiding endo-acting GH enzymes (32).…”

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

“…synergistic effects of having both acting in close physical proximity on the same biomass). It might however be possible that a physical connection between the catalytic domains is required to see boosting effects, as has been demonstrated recently for enzymes comprising fused carbohydrate esterase domains, where the added efficiency displayed by full-length enzymes was suggested to stem from the simultaneous action of both domains on their different respective substrates in close proximity [70]. Also, in a previous study investigating GEmediated boosting of biomass hydrolysis, only a small effect on xylose release was shown, where out of the three tested enzymes, xylose release was only increased by approximately one-third for the best-performing enzyme and for the least performing GE no significant increased xylose release was observed [32].…”