In nature, three dominant microbial paradigms for enzymatic deconstruction of plant cell walls have been observed
. Free enzymes, multifunctional enzymes and multi-enzyme complexes (cellulosomes) are common configurations of microbial cellulase systems.
Glycoside hydrolases have been classified thus far in 130 different families
, which commonly contain a catalytic module that cleaves the glycoside bond and (frequently) a carbohydrate-binding module (CBM) that targets the enzyme to the polysaccharide substrate, and, in many cases, additional types of ancillary modules. Cellulases include endo- and exo-acting enzymes and β-D-glucosidases, which work synergistically to hydrolyze the recalcitrant crystalline cellulose microfibrils of the plant cell wall.
Cellulosomes were discovered in 1983
[3–5] and are composed of non-catalytic scaffoldins, which contain a carbohydrate-binding module (CBM) for substrate targeting, as well as multiple cohesin modules for integrating dockerin-bearing enzymatic subunits to form a multi-component complex. The intermodular cohesin-dockerin interaction dictates the assembly of the cellulosome complex. It is believed that synergistic action is achieved by a combination of substrate-targeting and proximity effects whereby the set of cellulosomal enzymes is collectively concentrated at defined sites on the cellulosic substrate.
A third enzyme paradigm has also emerged: multifunctional enzymes are composed of two or more catalytic modules important for the degradation of plant cell walls
[6–11]. They generally contain one or several CBMs and are thus very high-molecular-weight proteins. Dockerin-bearing multifunctional enzymes may also be incorporated into cellulosomes. The presence of several catalytic modules in the same polypeptide chain would seem to indicate that their enforced proximity would account for an enhanced concerted action on cellulosic substrates. To date, four different types of multifunctional enzymes have been described: cellulase-cellulase, hemicellulase-hemicellulase, hemicellulase-cellulase and hemicellulase-carbohydrate esterase systems
. Cellulase-cellulase multifunctional enzymes observed to date present a variety of modular architectures and include combination of two catalytic modules (GH6 with GH12, GH9 with GH48, GH5 with GH12 or GH5 with GH6) with a minimum of one CBM (from families 2, 3, 5 and 10) and could contain supplementary ancillary modules such as FN3-like modules
The benefits of each strategy have been the subject of many research projects in the field, but remain as yet unclear. The comparison between free enzymatic and cellulosomal systems has been addressed in a number of recent studies
[13–23], and it appears that the cellulosomal paradigm offers advantages in deconstructing insoluble cellulosic substrates relative to free enzyme systems.
Another interesting attempt to increase enzyme synergism and compare enzyme paradigms was reported recently in the form of multifunctional enzyme conjugates
[24, 25]. These authors observed an increase in degradation of natural substrates, upon fusing two or three complementary xylan-degrading activities (xylanase, arabinofuranosidase and xylosidase) into the same polypeptide chain. Other multifunctional enzyme chimeras have been created successfully and revealed promising combined activities
Nevertheless, comparison between the three major enzyme paradigms has not been reported in the literature. In this communication, we describe the use of the well-characterized cellulolytic system of Thermobifida fusca as a model to compare the performance of the three enzyme paradigms. T. fusca possesses a limited set of only six cellulases and does not produce multifunctional enzymes. It thus represents an excellent system to prepare a variety of free and complexed cellulase systems, using the catalytic modules derived from this bacterium. In previous studies
[16, 18, 19, 21, 22], we have employed synthetic biology approaches to compare both free T. fusca enzymes and their respective engineered designer cellulosomes. One advantage of the T. fusca system is that even though its enzymes are not cellulosomal, a dockerin module can be readily grafted onto the parent enzyme by replacing the native CBM; the chimaeric enzyme can then be assembled into a complementary scaffoldin to form a designer cellulosome.
In the present study, we compared the combined synergistic action of two T. fusca cellulases: (i) in their free state (the wild-type system), (ii) as bifunctional enzymes in a single polypeptide chain (i.e., to mimic the natural multifunctional cellulase-cellulase system) and (iii) as dockerin-bearing enzymes attached to a scaffoldin in the cellulosomal mode. In a related work
, a designer cellulosome composed of two T. fusca cellulases (endoglucanase Cel5A and exoglucanase Cel48A) converted to the cellulosomal mode was demonstrated to be more efficient in degrading the crystalline cellulose substrate compared to a mixture of the respective wild-type enzymes. The same two cellulases were therefore selected to further compare their performance as bifunctional enzymes.
This report describes a set of bifunctional enzymes produced by gene fusion of catalytic modules together with one or two CBMs. The Cel48A exoglucanase and the Cel5A endoglucanase from T. fusca were combined to produce diverse geometrical arrangements, and the resulting chimeras were tested for their activity on microcrystalline cellulose. The results were compared to the parallel action of the two wild-type enzymes and the corresponding designer cellulosome bearing the converted dockerin-containing chimaeras (b-48A and f-5A).