Conserved Active Site Architecture Between Bacterial Cellulose and Chitin Synthases.

Glycosyltransferases (GTs) are a large and diverse group of enzymes responsible for catalyzing the formation of a glycosidic bond between a donor molecule, usually a monosaccharide, and a wide range of acceptor molecules, thus, playing critical roles in various essential biological processes. Chitin and cellulose synthases are two inverting processive integral membrane GTs, belonging to the type-2 family involved in the biosynthesis of chitin and cellulose, respectively. Herein, we report that bacterial cellulose and chitin synthases share an E-D-D-ED-QRW-TK active site common motif that is spatially co-localized. This motif is conserved among distant bacterial evolutionary species despite their low amino acid sequence and structural similarities between them. This theoretical framework offers a new perspective to the current view that bacterial cellulose and chitin synthases are substrate specific and that chitin and cellulose are organism specific. It lays the ground for future in vivo and in silico experimental assessment of cellulose synthase catalytic promiscuity against uridine diphosphate N-acetylglucosamine and chitin synthase against uridine diphosphate glucose, respectively.

Assembly of Synthetic Functional Cellulosomal Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome.

Heterologous display of enzymes on microbial cell surfaces is an extremely desirable approach, since it enables the engineered microbe to interact directly with the plant wall extracellular polysaccharide matrix. In recent years, attempts have been made to endow noncellulolytic microbes with genetically engineered cellulolytic capabilities for improved hydrolysis of lignocellulosic biomass and for advanced probiotics. Thus far, however, owing to the hurdles encountered in secreting and assembling large, intricate complexes on the bacterial cell wall, only free cellulases or relatively simple cellulosome assemblies have been introduced into live bacteria. Here, we employed the "adaptor scaffoldin" strategy to compensate for the low levels of protein displayed on the bacterial cell surface. That strategy mimics natural elaborated cellulosome architectures, thus exploiting the exponential features of their Lego-like combinatorics. Using this approach, we produced several bacterial consortia of Lactobacillus plantarum, a potent gut microbe which provides a very robust genetic framework for lignocellulosic degradation. We successfully engineered surface display of large, fully active self-assembling cellulosomal complexes containing an unprecedented number of catalytic subunits all produced in vivo by the cell consortia. Our results demonstrate that the enzyme stability and performance of the cellulosomal machinery, which are superior to those seen with the equivalent secreted free enzyme system, and the high cellulase-to-xylanase ratios proved beneficial for efficient degradation of wheat straw.IMPORTANCE The multiple benefits of lactic acid bacteria are well established in health and industry. Here we present an approach designed to extensively increase the cell surface display of proteins via successive assembly of interactive components. Our findings present a stepping stone toward proficient engineering of Lactobacillus plantarum, a widespread, environmentally important bacterium and potent microbiome member, for improved degradation of lignocellulosic biomass and advanced probiotics.

Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation.

Efficient conversion of cellulose into soluble sugars is a key technological bottleneck limiting efficient production of plant-derived biofuels and chemicals. In nature, the process is achieved by the action of a wide range of cellulases and associated enzymes. In aerobic microrganisms, cellulases are secreted as free enzymes. Alternatively, in certain anaerobic microbes, cellulases are assembled into large multienzymes complexes, termed "cellulosomes," which allow for efficient hydrolysis of cellulose. Recently, it has been shown that enzymes classified as lytic polysaccharide monooxygenases (LPMOs) were able to strongly enhance the activity of cellulases. However, LPMOs are exclusively found in aerobic organisms and, thus, cannot benefit from the advantages offered by the cellulosomal system. In this study, we designed several dockerin-fused LPMOs based on enzymes from the bacterium Thermobifida fusca. The resulting chimeras exhibited activity levels on microcrystalline cellulose similar to that of the wild-type enzymes. The dockerin moieties of the chimeras were demonstrated to be functional and to specifically bind to their corresponding cohesin partner. The chimeric LPMOs were able to self-assemble in designer cellulosomes alongside an endo- and an exo-cellulase also converted to the cellulosomal mode. The resulting complexes showed a 1.7-fold increase in the release of soluble sugars from cellulose, compared with the free enzymes, and a 2.6-fold enhancement compared with free cellulases without LPMO enhancement. These results highlight the feasibility of the conversion of LPMOs to the cellulosomal mode, and that these enzymes can benefit from the proximity effects generated by the cellulosome architecture.