Cellulose, the major component of plant cell wall and the most abundant biopolymer on earth, is a source of energy for polysaccharide-degrading microorganisms, and a potential, currently unexploited, source of renewable energy for conversion into biofuels (Lynd L R, et al. (2008) Nature Biotechnol. 26:169-172; Ragauskas A J, et al. (2006) Science 311:484-489. Due to the highly ordered, insoluble, crystalline nature of the cellulose, very few microorganisms possess the necessary enzymatic system to efficiently degrade cellulosic substrates to soluble sugar (Himmel M E, et al. (2007) Science 315:804-807; Erratum: 316, 982).
Highly efficient cellulose degradation has been demonstrated by a multi-enzyme complex, termed cellulosome, which was found to be produced by several cellulolytic microorganisms. An exemplary, well characterized cellulosome system is the one produced by the anaerobic, thermophilic, cellulolytic bacterium, Clostridium thermocellum (Bayer E A, Belaich J-P, Shoham Y, & Lamed R (2004) Annu. Rev. Microbiol. 58:521-554). The cellulosome contains a non-catalytic subunit called scaffoldin that binds the insoluble substrate via a cellulose-specific carbohydrate-binding module (CBM). The scaffoldin subunit also functions as an integrator of various enzymatic subunits into the complex—it typically contains a set of subunit-binding modules, termed cohesins, that mediate specific incorporation and organization of the enzymatic subunits into the complex through interaction with a complementary binding module, termed dockerin, that is present in each enzymatic subunit. For example, the C. thermocellum scaffoldin contains a set of nine (9) cohesins, allowing the incorporation of nine dockerin-bearing subunits into the complex. In some cellulosome-producing microorganisms, the scaffoldin further contains a dockerin, whose type is different from the type of dockerin found in the enzymatic subunits, which connects the cellulosome to the microorganism cell via interaction with a matching cohesin present in cell-anchoring proteins. There is essentially no cross-specificity between cohesin-dockerin partners that mediate enzyme integration, and cohesin-dockerin partners that mediate cell anchoring, thus ensuring a reliable mechanism for cell-surface attachment and cellulosome assembly. For example, in C. thermocellum, the enzymatic subunits contain type I dockerins which interact with complementary type I cohesins of the scaffoldin. The scaffoldin contains a type-II dockerin at its C terminus that mediates the attachment of the cellulosome to the bacterial cell wall through a selective binding interaction with a set of cell-anchoring proteins that contains type II cohesins. The degree of cellulosome attachment to the cell is varied and decreases in high cell density. The assembly of the enzymes into the complex ensures their collective targeting to a specific region of the substrate thereby facilitating stronger synergism among the catalytic components (Bayer E A, Morag E, & Lamed R (1994) Trends Biotechnol. 12:378-386; Shoham Y, Lamed R, & Bayer E A (1999) Trends Microbiol. 7:275-281).
The Lego-like architecture of the Clostridium thermocellum cellulosome holds great potential for creating “designer cellulosomes”, namely, artificial assemblies comprising hybrid forms of cellulosomal components, for improved hydrolysis of cellulosic substrates (Bayer E A, Morag E, & Lamed R (1994) Trends Biotechnol. 12:378-386). To date, most of the designer cellulosome experiments try to mimic the enzymatic synergism observed for native cellulosome systems by fabricating complexes composed of an artificial chimaeric cohesin-containing scaffoldin and a set of matching dockerin-containing cellulases (Fierobe H-P, et al. (2002) J. Biol. Chem. 277:49621-49630; Fierobe H-P, et al. (2005) J. Biol. Chem. 280:16325-16334; Moraïs S, et al. (2010) mBio 1:e00285-00210).
The synergistic degradation of the different cellulosomal enzymes results in the formation of large concentrations of the major soluble disaccharide end product cellobiose. In the native environment, the cellobiose and other oligodextrins are transported directly into the cell by ABC transporter systems (Nataf Y, et al. (2009) J. Bacteriol. 191:203-209), during which they are hydrolyzed to glucose by periplasmic β-glucosidases (Strobel H J (1995) Curr. Microbiol. 31:210-214). The assimilation of oligodextrins can be accomplished by various additional microorganisms in the environment, and cellobiose is rapidly removed from the medium (Bayer E A, Morag E, & Lamed R (1994) Trends Biotechnol. 12:378-386). In the native ecosystem, cellobiose plays a regulatory role and acts as a strong inhibitor of cellulose-degrading enzymes. Near-complete inhibition of the C. thermocellum cellulosome typically occurs at a concentration of 2% cellobiose (Lamed R, Kenig R, Setter E, & Bayer E A (1985) Enzyme Microb. Technol. 7:37-41). Therefore, in a cell-free system, removal of the inhibitory cellobiose is essential for constant degradation of cellulosic substrates.
Previous studies have shown that addition of a β-glucosidase to reaction mixtures containing the C. thermocellum cellulosome can enhance the rate and degree of solubilization of crystalline cellulose by the cellulosome (Lamed R, et al. (1991) Appl. Biochem. Biotechnol. 27:173-183; Kadam et al. (1989) Biochem Biophys Res Commun 161(2):706-711), presumably by converting cellobiose to two molecules of non-inhibitory glucose. However, in the process of crystalline cellulose degradation, the cellulosome binds to the insoluble cellulosic substrate, and therefore only a fraction of the free β-glucosidase can be involved directly in digestion of cellobiose, which accumulates in the immediate environment of the substrate-attached cellulosome.
The degradation of phosphoric acid-swollen cellulose (PASC) by artificial mini-cellulosomes composed of two dockerin-bearing cellulases and a dockerin-bearing β-glucosidase attached to yeast cells has been previously examined (Tsai S L, Oh J, Singh S, Chen R, & Chen W (2009) Appl. Environ. Microbiol. 75:6087-6093).
Addition of exogenous components to the native cellulosome has been proposed in the form of a “super-cellulosome”, where exogenous enzymes are incorporated into the intact cellulosome using bi-functional crosslinking reagents (Bayer E A, Morag E, & Lamed R (1994) Trends Biotechnol. 12:378-386). However, the non-specific chemical nature of crosslinking could impair the activities of the enzymes, and is also time and resource consuming (Rao S V, Anderson K W, & Bachas L G (1998) Microchim. Acta 128:127-143).
Targeted integration of a cohesin-fused β-glucosidase into the C. thermocellum cellulosome has been described in Gefen et al. (2012) PNAS, 109(26); 10298-10303, to some of the inventors of the present invention, published after the priority date of the present application.
There still remains a need for compositions and methods for improved degradation of biomass, especially recalcitrant cellulosic biomass.
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