Research

Designer cellulosomes

We originally proposed a new concept for the construction of designer cellulosomes, comprising recombinant chimaeric scaffoldin constructs and selected dockerin-containing enzyme hybrids. This arrangement serves to promote the synergistic action among enzyme components. In developing this objective, the CBMs of the native free enzymes are replaced with a dockerin (Figure 1), and chimaeric scaffoldins are designed to include multiple copies of cohesins of different specificities (Figure 2). This enables precise incorporation of complementary dockerin-containing components into the complex (Figure 3).

BioFuels

Designer cellulosomes as a platform for processing biomass-to-biofuels

History of the Cellulosome

In the early 1980s, Raffi Lamed and I met at Tel Aviv University and commenced our work that led to the discovery of the cellulosome concept. Raffi had just completed postdoctoral tenures in the States and I was at the tail end of a postdoc at Tel Aviv University. Raffi approached me at the time with a description of how Clostridium thermocellum, an anaerobic thermophilic cellulolytic bacterium, bound very strongly to the cellulose substrate before it commences its degradation. We decided to study this phenomenon together.

Non-cellulosomal cohesin and dockerin-like modules

The cohesin-dockerin interaction was originally discovered as the decisive modular pair that dictates the assembly of the various enzymatic subunits into the cellulosome complex. Until recently, the presence of cohesins and dockerins within a bacterial proteome was considered a definitive “signature” of a cellulosome-producing bacterium.

The avidin-biotin system

The major direction of my early work in this field was devoted to the study of the avidin-biotin complex and its general use as a tool in the biological sciences. The main reason for interest in this system is two-fold: (a) The interaction between avidin (or streptavidin) and biotin exhibits the highest known affinity (Ka ~ 10¹⁵ M^-1) between a ligand and a protein (1), and (b) the avidin-biotin system has become a universal tool in many biotechnological applications (Figure 1). We were the first to biotinylate various macromolecules, such as antibodies and lectins. We also designed and synthesized a variety of additional group-specific biotinylating reagents and developed many new applications of the avidin-biotin system (Figure 2).

Cellulosomal human gut bacteria

Very recently, cellulosomes were revealed in the human gut, due to the sequencing of metagenomic samples and isolation and characterization of novel human gut bacterial species that grow on complex polysaccharide substrates.

BioFuels

During the past several years, there has been increasing interest in the development of biofuels derived from environmentally friendly lignocellulosic biomass. The plant cell wall is composed mainly of cellulose, hemicellulose and lignin and is the most abundant carbon source on Earth. Therefore is a promising material for production of biofuels. One leading approach is to use consolidated bioprocessing (CBP) microorganisms that are capable of directly converting lignocellulose into valuable end products (e.g., ethanol).

3D structures of cellulosomal modules

Crystal structures of cellulosomal modules

In order to understand the nature of the intermodular contacts formed between the cellulosomal subunits, or how polycellulosome assembly occurs, we employed crystallographic techniques. In early collaborative studies with Linda Shimon and Felix Frolow, we succeeded in determining the 3D crystal structure of a type-I cohesin. More recently, we successfully cloned, expressed, purified, crystallized and solved several novel structures of type-II and type-III cohesin modules from different cellulosomal species. A gallery of selected crystals grown in our lab is shown in Figure 1.

Cohesin-dockerin Interactions

The cohesin-dockerin pair represents a high-affinity (Kd 10-9-10-12 M) protein-protein interaction. This interaction tends to be nonspecific within a given species but specific between species. Thus, a scaffoldin-borne cohesin of one species would be expected to recognize and bind to most or all of the enzyme-borne dockerins from the same species, but not with the dockerins from a different species

Cellulosome Systems

Bacterial cellulosomal systems can be categorized into two major types: simple cellulosome systems contain a single scaffoldin and complex cellulosome systems exhibit multiple types of interacting scaffoldins.

Enzymes

The chemical and structural intricacy of plant cell wall polysaccharides is matched by the diversity and complexity of the enzymes that degrade them. Cellulases and hemicellulases are family members of the broad group of glycoside hydrolases, which catalyze the hydrolysis of oligosaccharides and polysaccharides in general. These enzymes are composed of a series of separate modules. This fact explains the very large size of some of these enzymes and gives us some insight into their complex mode of action. Each module or domain comprises a consecutive portion of the polypeptide chain and forms an independently folding, structurally and functionally distinct unit. Each enzyme contains at least one catalytic module, which catalyzes the actual hydrolysis of the glycosidic bond and provides the basis for classification of the simple enzymes. Other accessory or "helper" domains assist or modify the primary hydrolytic action of the enzyme, thus modulating the overall properties of the enzyme. Some of the different themes illustrating the modular compositions of the cellulases and related enzymes are illustrated in Figure 1.