Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

Microbial Cell Factories, Sep 2010

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals.

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Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

Andrew S Wieczorek 0 Vincent JJ Martin 0 0 Department of Biology, Concordia University , Montreal, Quebec, H4B 1R6 , Canada Background: The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multiprotein cellulosome complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results: Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli b-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and b-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions: We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. - Background Macromolecular enzyme complexes catalyze an array of biochemical and metabolic processes such as the degradation of proteins [1,2] or recalcitrant polymers [3] as well as the high-yield synthesis of valuable metabolic products via substrate channeling [4]. From a biotechnological perspective, mimicking such process by incorporating catalytic modules or enzymes of interest within synthetic complexes can significantly enhance the efficiency of such bioprocesses via substrate channeling [5] and increased enzyme synergy [3]. In a cellulosome, multiple enzymes assemble into a macromolecular complex by their association with a scaffold protein for the efficient degradation of cellulose [6]. In the case of the gram-positive thermophile Clostridium thermocellum, the cellulosome is anchored to the surface of cells, resulting in one of the most efficient systems for bacterial cellulose hydrolysis [3,7]. Cellulosomal enzymes bear C-terminal type 1 dockerin (dock1) modules, which target them to a central scaffold protein (CipA) via chemically and thermally stable non-covalent interactions with one of nine cohesin (coh) modules [8]. CipA also contains a type 3a cellulose-binding module (CBM3a), allowing the different cellulases to act in synergy on the crystalline substrate, as well as a type 2 dockerin module which binds anchor proteins, ensuring the cellulosomes attachment to the cell [9,10]. Therefore, the architecture of the cellulosome establishes proximal and synergistic effects of enzymes within the complex when associated with the substrate [8,11,12]. These synergistic effects are further augmented by an extra level of synergy resulting from the cellulosomes association with the surface of cells, yielding cellulose-enzymemicrobe (CEM) ternary complexes [6,7,13-18]. CEM ternary complexes benefit from the effects of microbeenzyme synergy, ultimately limiting the escape of hydrolysis products and enzymes, increasing access to substrate hydrolysis products, minimizing the distance products must diffuse before cellular uptake occurs, concentrating enzymes at the substrate surface, protecting hydrolytic enzymes from proteases and thermal degradation, as w (...truncated)


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Andrew S Wieczorek, Vincent JJ Martin. Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis, Microbial Cell Factories, 2010, pp. 69, 9, DOI: 10.1186/1475-2859-9-69