Cohesin-dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity.
ABSTRACT The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.
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ABSTRACT: Bacterial cellulosomes are generally believed to assemble at random like those produced by Clostridium cellulolyticum. They are composed of one scaffolding protein displaying 8 homologous type I cohesins that bind to any of the type I dockerins borne by the 62 cellulosomal subunits, thus generating highly heterogeneous complexes. In the present study the heterogeneity and random assembly of the cellulosomes were evaluated using a simpler model: a miniscaffoldin containing three C. cellulolyticum cohesins and three cellulases of the same bacterium bearing the cognate dockerin (Cel5A, Cel48F and Cel9G). Surprisingly, in lieu of the anticipated randomized integration of enzymes, the assembly of the minicellulosome generated only three distinct types of complex out of the ten possible combinations, thus indicating preferential integration of enzymes upon binding to the scaffoldin. A hybrid scaffoldin that displays one cohesin from C. cellulolyticum and one from C. thermocellum thus allowing sequential integration of enzymes was exploited to further characterize this phenomenon. The initial binding of a given enzyme onto the C. thermocellum cohesin was found to influence the type of enzyme which is bound subsequently onto the C. cellulolyticum cohesin. The preferential integration appears to be related to the length of the inter-cohesin linker. The data indicate that the binding of a cellulosomal enzyme onto a cohesin has direct influence on the dockerin-bearing proteins that will subsequently interact with adjacent cohesins. Thus, despite the general lack of specificity of the cohesin/dockerin interaction within a given species and type, bacterial cellulosomes are not necessarily assembled at random. This article is protected by copyright. All rights reserved.FEBS Journal 08/2013; · 3.99 Impact Factor
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ABSTRACT: Ethylene is an industrially important compound, but more sustainable production methods are desirable. Since cellulosomes increase the ability of cellulolytic enzymes by physically linking the relevant enzymes via dockerin-cohesin interactions, in this study, we genetically engineered a chimeric cellulosome-like complex of two ethylene-generating enzymes from tomato using cohesin-dockerins from the bacteria Clostridium thermocellum and Acetivibrio cellulolyticus. This complex was transformed into Escherichia coli to analyze kinetic parameters and enzyme complex formation and into the cyanobacterium Synechococcus elongatus PCC 7942, which was then grown with and without 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. Only at minimal protein expression levels (without IPTG), the chimeric complex produced 3.7 times more ethylene in vivo than did uncomplexed enzymes. Thus, cyanobacteria can be used to sustainably generate ethylene, and the synthetic enzyme complex greatly enhanced production efficiency. Artificial synthetic enzyme complexes hold great promise for improving the production efficiency of other industrial compounds.ACS Synthetic Biology 02/2014; · 3.95 Impact Factor
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ABSTRACT: Thermophilic microorganisms are attractive candidates for conversion of lignocellulose to biofuels since they produce robust, effective, carbohydrate-degrading enzymes, and survive under harsh bioprocessing conditions that reflect their natural biotopes. However, no naturally occurring thermophile is known that can convert plant biomass into a liquid biofuel at rates, yields and titers that meet current bioprocessing and economic targets. Meeting those targets requires either metabolically engineering solventogenic thermophiles with additional biomass deconstruction enzymes, or engineering plant biomass degraders to produce a liquid biofuel. Thermostable enzymes from microorganisms isolated from diverse environments can serve as genetic reservoirs for both efforts. Because of the the sheer number of enzymes that are required to hydrolyze plant biomass to fermentable oligosaccharides, the latter strategy appears to be the preferred route and thus has received the most attention to date. Thermophilic plant biomass degraders fall into one of two categories: cellulosomal (i.e., multi-enzyme complexes) and non-cellulosomal (i.e., "free" enzyme systems). Plant biomass deconstructing thermophilic bacteria from the genera Clostridium (cellulosomal) and Caldicellulosiruptor (non-cellulosomal), which have potential as metabolic engineering platforms for producing biofuels, are compared and contrasted from a systems biology perspective. This article is protected by copyright. All rights reserved.FEMS microbiology reviews 10/2013; 38(3). · 13.81 Impact Factor