A tripartite carbohydrate-binding module to functionalize cellulose nanocrystals.

The development of protein and microorganism engineering have led to rising expectations of biotechnology in the design of emerging biomaterials, putatively of high interest to reduce our dependence on fossil carbon resources. In this way, cellulose, a renewable carbon based polysaccharide and derived products, displays unique properties used in many industrial applications. Although the functionalization of cellulose is common, it is however limited in terms of number and type of functions. In this work, a Carbohydrate-Binding Module (CBM) was used as a central core to provide a versatile strategy to bring a large diversity of functions to cellulose surfaces. CBM3a from Clostridium thermocellum, which has a high affinity for crystalline cellulose, was flanked through linkers with a streptavidin domain and an azide group introduced through a non-canonical amino acid. Each of these two extra domains was effectively produced and functionalized with a variety of biological and chemical molecules. Structural properties of the resulting tripartite chimeric protein were investigated using molecular modelling approaches, and its potential for the multi-functionalization of cellulose was confirmed experimentally. As a proof of concept, we show that cellulose can be labelled with a fluorescent version of the tripartite protein grafted to magnetic beads and captured using a magnet.

Functional Analysis of Deoxyhexose Sugar Utilization in Escherichia coli Reveals Fermentative Metabolism under Aerobic Conditions.

l-Rhamnose and l-fucose are the two main 6-deoxyhexoses Escherichia coli can use as carbon and energy sources. Deoxyhexose metabolism leads to the formation of lactaldehyde, whose fate depends on oxygen availability. Under anaerobic conditions, lactaldehyde is reduced to 1,2-propanediol, whereas under aerobic conditions, it should be oxidized into lactate and then channeled into the central metabolism. However, although this all-or-nothing view is accepted in the literature, it seems overly simplistic since propanediol is also reported to be present in the culture medium during aerobic growth on l-fucose. To clarify the functioning of 6-deoxyhexose sugar metabolism, a quantitative metabolic analysis was performed to determine extra- and intracellular fluxes in E. coli K-12 MG1655 (a laboratory strain) and in E. coli Nissle 1917 (a human commensal strain) during anaerobic and aerobic growth on l-rhamnose and l-fucose. As expected, lactaldehyde is fully reduced to 1,2-propanediol under anoxic conditions, allowing complete reoxidation of the NADH produced by glyceraldehyde-3-phosphate-dehydrogenase. We also found that net ATP synthesis is ensured by acetate production. More surprisingly, lactaldehyde is also primarily reduced into 1,2-propanediol under aerobic conditions. For growth on l-fucose, 13C-metabolic flux analysis revealed a large excess of available energy, highlighting the need to better characterize ATP utilization processes. The probiotic E. coli Nissle 1917 strain exhibits similar metabolic traits, indicating that they are not the result of the K-12 strain's prolonged laboratory use. IMPORTANCE E. coli's ability to survive in, grow in, and colonize the gastrointestinal tract stems from its use of partially digested food and hydrolyzed glycosylated proteins (mucins) from the intestinal mucus layer as substrates. These include l-fucose and l-rhamnose, two 6-deoxyhexose sugars, whose catabolic pathways have been established by genetic and biochemical studies. However, the functioning of these pathways has only partially been elucidated. Our quantitative metabolic analysis provides a comprehensive picture of 6-deoxyhexose sugar metabolism in E. coli under anaerobic and aerobic conditions. We found that 1,2-propanediol is a major by-product under both conditions, revealing the key role of fermentative pathways in 6-deoxyhexose sugar metabolism. This metabolic trait is shared by both E. coli strains studied here, a laboratory strain and a probiotic strain. Our findings add to our understanding of E. coli's metabolism and of its functioning in the bacterium's natural environment.