Abstract
BackgroundThe 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.ResultsFragments 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.ConclusionsWe 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.
Highlights
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
Regulated expression of CipAfrags yields the surfacedisplay of scaffold proteins L. lactis HtrA NZ9000 cells were successfully transformed with either the pAW500 series or pAW300 series of vectors (Fig. 1A), resulting in strains expressing fragments of CipA (CipAfrags) alone, or as fusions with the NucA export-specific reporter, and/or the cwaM6 for anchoring of the scaffold to the cell-surface (Fig. 1B)
Growth curves of engineered L. lactis strains were used to determine if the expression and secretion of scaffold proteins resulted in growth inhibition
Summary
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. 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 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 cellulosome’s association with the surface of cells, yielding cellulose-enzymemicrobe (CEM) ternary complexes [6,7,13,14,15,16,17,18]. CEM ternary complexes benefit from the effects of microbeenzyme synergy, 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 well as optimizing the chemical environment at the substrate-microbe interface [6,7,13,14,15,16]
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