Abstract
BackgroundComplete hydrolysis of cellulose to glucose requires the synergistic action of three general types of glycoside hydrolases; endoglucanases, exoglucanases, and cellobiases. Cellulases that are found in Nature vary considerably in their modular diversity and architecture. They include: non-complexed enzymes with single catalytic domains, independent single peptide chains incorporating multiple catalytic modules, and complexed, scaffolded structures, such as the cellulosome. The discovery of the latter two enzyme architectures has led to a generally held hypothesis that these systems take advantage of intramolecular and intermolecular proximity synergies, respectively, to enhance cellulose degradation. We use domain engineering to exploit both of these concepts to improve cellulase activity relative to the activity of mixtures of the separate catalytic domains.ResultsWe show that engineered minicellulosomes can achieve high levels of cellulose conversion on crystalline cellulose by taking advantage of three types of synergism; (1) a complementary synergy produced by interaction of endo- and exo-cellulases, (2) an intramolecular synergy of multiple catalytic modules in a single gene product (this type of synergism being introduced for the first time to minicellulosomes targeting crystalline cellulose), and (3) an intermolecular proximity synergy from the assembly of these cellulases into larger multi-molecular structures called minicellulosomes. The binary minicellulosome constructed in this study consists of an artificial multicatalytic cellulase (CBM4-Ig-GH9-X11-X12-GH8-Doc) and one cellulase with a single catalytic domain (a modified Cel48S with the structure CBM4-Ig-GH48-Doc), connected by a non-catalytic scaffoldin protein. The high level endo-exo synergy and intramolecular synergies within the artificial multifunctional cellulase have been combined with an additional proximity-dependent synergy produced by incorporation into a minicellulosome demonstrating high conversion of crystalline cellulose (Avicel). Our minicellulosome is the first engineered enzyme system confirmed by test to be capable of both operating at temperatures as high as 60°C and converting over 60% of crystalline cellulose to fermentable sugars.ConclusionWhen compared to previously reported minicellulosomes assembled from cellulases containing only one catalytic module each, our novel minicellulosome demonstrates a method for substantial reduction in the number of peptide chains required, permitting improved heterologous expression of minicellulosomes in microbial hosts. In addition, it has been shown to be capable of substantial conversion of actual crystalline cellulose, as well as of the less-well-ordered and more easily digestible fraction of nominally crystalline cellulose.
Highlights
Complete hydrolysis of cellulose to glucose requires the synergistic action of three general types of glycoside hydrolases; endoglucanases, exoglucanases, and cellobiases
Construction of a multifunctional cellulase with high intramolecular synergy Given the current limitations of minicellulosomes constructed using only cellulosomal enzymes with one catalytic module, an artificial multifunctional cellulase intended for incorporation into new minicellulosomes was constructed by fusing a truncated version of a processive endoglucanase, C. thermocellum CbhA (CBM4Ig-GH9-X11-X12, tCbhA) and a classical endoglucanase, Cel8A (GH8-Doc) resulting in a new molecule with architecture of CBM4-Immunoglobulin-like fold (Ig)-GH9-X11-X12-GH8-Doc (Table 1 and Figure 1)
The two consecutive X1 domains were considered special “spacer” or “linker” segments between two component peptide domains [27,28]. This large protein was found to be soluble and stable when overexpressed in E. coli. The activity of this multifunctional cellulase was measured on Avicel and compared with the activity of the truncated CbhA and Cel8A both individually and in a simple mixture (Figure 2)
Summary
Complete hydrolysis of cellulose to glucose requires the synergistic action of three general types of glycoside hydrolases; endoglucanases, exoglucanases, and cellobiases. Cellulases that are found in Nature vary considerably in their modular diversity and architecture They include: non-complexed enzymes with single catalytic domains, independent single peptide chains incorporating multiple catalytic modules, and complexed, scaffolded structures, such as the cellulosome. The discovery of the latter two enzyme architectures has led to a generally held hypothesis that these systems take advantage of intramolecular and intermolecular proximity synergies, respectively, to enhance cellulose degradation. The most common is the free cellulase system, which exists mainly in cellulolytic fungi and bacteria and uses mainly intermolecular endo-, and exo-cellulase synergy [3]. Clostridium thermocellum is a well-studied producer of cellulosomes, known to be extremely large and complex self-assembling arrays of as many as 91 cellulase enzymes [10,11]
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