Engineering accessory esterases into cellulosomes improves lignocellulosic biomass utilization in Ruminiclostridium cellulolyticum

Hydroxycinnamic acids, including ferulic acid (FA) and p -coumaric acid ( p CA), contribute to grass cell wall recalcitrance by decorating arabinoxylans and lignin and participating in cross-links between hemicellulose chains and within lignin–hemicellulose networks. In Ruminiclostridium cellulolyticum , the cellulosomal SGNH hydrolases SGNHh 1060 and SGNHh 2017 were recently identified as cinnamoyl esterases active on hydroxycinnamic acid (HCA) derivatives, whereas the intracellular esterase XuaH was involved in the processing of imported arabinoxylan-derived oligosaccharides. Here, we investigated whether increasing cinnamoyl esterase content in bacterial cellulosomes with these enzymes could improve the deconstruction of complex grass lignocellulose. We engineered R. cellulolyticum strains to overproduce SGNHh 1060 , SGNHh 2017 , or a secreted dockerin-grafted chimeric form of XuaH, named XuaHd. The corresponding purified complexes, referred to as 1060-, 2017-, and XuaHd-cellulosomes, were analyzed by SDS–PAGE, western blotting, and proteomics. These analyses confirmed secretion and incorporation of the overproduced proteins into corresponding cellulosomes, while showing that enrichment remodeled the complexes’ composition. Engineered cellulosomes displayed cinnamoyl esterase activities consistent with the substrate specificities of the newly incorporated enzymes on methyl-hydroxycinnamate model substrates. However, on crystalline cellulose, they released fewer cellodextrins than control cellulosomes, indicating a trade-off between esterase enrichment and hydrolytic CAZyme content. In addition, engineered complexes released more FA and p CA than control complexes on organosolv wheat straw lignin. On natural substrates, the effects were substrate-dependent: no difference in soluble sugars release was observed on wheat bran, whereas SGNHh 2017 or XuaHd enrichment impaired deconstruction-related activity on wheat straw. In contrast, in vivo, SGNHh 1060 and SGNHh 2017 overproduction improved growth on both complex lignocellulosic substrates, whereas XuaHd conferred no clear physiological advantage. Increasing cinnamoyl esterase content in R. cellulolyticum cellulosomes creates a functional trade-off: it can impair cellulose hydrolysis by altering cellulosome composition, while improving bacterial utilization of complex lignified biomass in a substrate-dependent manner. SGNHh 1060 and SGNHh 2017 therefore emerge as potential cellulosomal accessories for loosening hydroxycinnamate-mediated constraints in grass cell walls. More broadly, successful cellulosome engineering requires not only the addition of catalytic functions, but also preservation of multienzyme complex composition balance and alignment between enzyme activity, substrate architecture, and in vivo accessibility.