Autodisplay of cellulases on the surface of E. coli for the concerted biotransformation of lignocellulose

Bellof M, Jose J, Brossette T, Dobler N, Maas R

Abstract

Cellulolysis The enzymatic hydrolysis of cellulose to glucose by cellulases is one of the major steps involved in the conversion of lignocellulosic biomass. Natural lignocellulosic substrates are composed of heterogeneous intertwined polysaccharide chains with varying degrees of crystallinity, hemicelluloses and pectins, embedded in lignin. Hydrolysis and utilization of cellulose is carried out mainly by microorganisms, distributed among many genera in the domain Bacteria and in the fungal groups. Cellulose cannot get into the cells, therefore cellolytic enzymes are by necessity secreted into the medium or bound to the outside surface of cellulolytic microorganisms. Autodisplay - surface display of recombinant proteins and peptides Surface display of active proteins on living cells provides several advantages for biotechnological applications. Among other display systems in bacteria and yeast, Autodisplay was developed based on the secretion mechanism of the autotransporter family of proteins and represents a very elegant way to express a recombinant protein on the surface of Escherichia coli [1]. The gene encoding the autotransporter fusion protein exhibits a particular assembly (Fig. A). The protein of interest has to be inserted between the signal peptide (SP) and the linker domain. The N-terminal signal peptide (SP) of the nascent protein chain mediates the transport of the remaining precursor protein into the periplasm (Fig. B). The signal peptidase cuts off the SP and enables the formation of the β-barrel within the outer membrane [2]. The hydrophilic pore facilitates the transport of the passenger to the bacterial surface. The Autodisplay system allows a broad variety of recombinant proteins to be translocated. Using such cells as whole-cell biocatalysts, a substrate to be processed does not need to cross a membrane barrier but has free access. Moreover, being connected to a carrier (the cell as a biological matrix), the surface displayed protein can be purified, stabilized and applied to industrial processes much more convenient than a free molecule [3]. These advantages of Autodisplay biocatalysts can be exploited for the degradation of lignocellulosic biomass [4]. Since cellulose is degraded synergistically into glucose by three types of cellulases, we developed three types of Autodisplay biocatalysts displaying different enzymes: i) endocellulase Cel5A from Bacillus subtilis which attacks regions of low crystallinity in the cellulose fiber, ii) exocellulase CelK from Clostridium thermocellum acting in a process manner on non-reducing free ends of the crystalline cellulose polysaccharide chains and iii) β-Glucosidase BglA from Clostridium thermocellum hydrolyzing cellobiose to glucose (Fig C). We obtained three active Autodisplay whole cell catalysts acting in concert to efficiently hydrolyze cellulose.