Engineering Novel and Improved Biocatalysts by Cell Surface Display

Abstract

Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts.

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…Typically, cell-free systems are used for cofactor-independent reactions, and often exhibit reactionrate-limited kinetics, resulting from the direct access to substrate in solution (Smith et al 2015). Cell-free enzymes have been exploited both in single (Kengen et al 1993;Kim et al 2010;Böhmer et al 2012) and multiple domain systems (Kanafusa-Shinkai et al 2013).…”

“…Regardless of the host organism, surface display systems often have three core features in common. These are: (1) a signal peptide to direct the protein of interest toward the secretory pathway; (2) an endogenous surface protein pliable to recombination (i.e., insertion, deletion, and fusion) to facilitate a stable surface anchorage of the target protein; and (3) an epitope tag to facilitate the detection of successful surface display (Smith et al 2015). In the surface display system, the amount of cellulase displayed is strictly dependent on the cell surface area, unlike cellfree systems, where there are no such limits .…”

“…enzyme systems Multifunctional enzymes-comprising the direct surface display of multiple enzymes in a non-complex form-are very highmolecular weight proteins of one or several CBMs and two or more CDs for effective substrate targeting and efficient degradation of plant cell walls, respectively (Moraïs et al 2012;Smith et al 2015). The several catalytic modules on the same polypeptide chain are assembled such that their enforced proximity account for an enhanced concerted action on substrates (Moraïs et al 2012).…”

“…In addition, the relatively simple modular structure of autodisplay systems allows the easy interaction of passenger proteins on the bacterial cell surface, thus displaying desired heterologous enzymes. The autodisplayed proteins normally expressed as monomers are capable of forming multimers upon membrane interaction after expression (Schumacher et al 2012;Smith et al 2015). Furthermore, the autodisplay secretion method makes subsequent, often costly, purification steps to recover the enzyme of interest unnecessary (Kranen et al 2014).…”

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

“…Bacteria engineered to display heterologous proteins have many potential biotechnological applications, including uses in protein engineering, biocatalysis, lignocellulose degradation, biosensing and bioremediation (Lee, Choi et al 2003, Wernerus and Stahl 2004, Schuurmann, Quehl et al 2014, Li and Tao 2015, Smith, Khera et al 2015). Surface display methods are attractive because the microbe produces and displays the protein during growth, circumventing the need for time-consuming protein purification and immobilization procedures that are typically used to create protein coated materials (Garcia-Galan, Berenguer-Murcia et al 2011, Mohamad, Marzuki et al 2015, Hyeon, Shin et al 2016).…”