Outlook for cellulase improvement: Screening and selection strategies

Abstract

Cellulose is the most abundant renewable natural biological resource, and the production of biobased products and bioenergy from less costly renewable lignocellulosic materials is important for the sustainable development of human beings. A reduction in cellulase production cost, an improvement in cellulase performance, and an increase in sugar yields are all vital to reduce the processing costs of biorefineries. Improvements in specific cellulase activities for non-complexed cellulase mixtures can be implemented through cellulase engineering based on rational design or directed evolution for each cellulase component enzyme, as well as on the reconstitution of cellulase components. Here, we review quantitative cellulase activity assays using soluble and insoluble substrates, and focus on their advantages and limitations. Because there are no clear relationships between cellulase activities on soluble substrates and those on insoluble substrates, soluble substrates should not be used to screen or select improved cellulases for processing relevant solid substrates, such as plant cell walls. Cellulase improvement strategies based on directed evolution using screening on soluble substrates have been only moderately successful, and have primarily targeted improvement in thermal tolerance. Heterogeneity of insoluble cellulose, unclear dynamic interactions between insoluble substrate and cellulase components, and the complex competitive and/or synergic relationship among cellulase components limit rational design and/or strategies, depending on activity screening approaches. Herein, we hypothesize that continuous culture using insoluble cellulosic substrates could be a powerful selection tool for enriching beneficial cellulase mutants from the large library displayed on the cell surface.

Introduction

Cellulose is the primary product of photosynthesis in terrestrial environments, and the most abundant renewable bioresource produced in the biosphere (∼ 100 billion dry tons/year) (Holtzapple, 1993, Jarvis, 2003, Zhang and Lynd, 2004b). Cellulose biodegradation by cellulases and cellulosomes, produced by numerous microorganisms, represents a major carbon flow from fixed carbon sinks to atmospheric CO₂ (Berner, 2003, Falkowski et al., 2000, Melillo et al., 2002), is very important in several agricultural and waste treatment processes (Angenent et al., 2004, Das and Singh, 2004, Haight, 2005, Hamer, 2003, Humphrey et al., 1977, Russell and Rychlik, 2001, Schloss et al., 2005, van Wyk, 2001), and could be widely used to produce sustainable biobased products and bioenergy to replace depleting fossil fuels (Angenent et al., 2004, Demain et al., 2005, Galbe and Zacchi, 2002, Hall et al., 1993, Hoffert et al., 2002, Kamm and Kamm, 2004, Lynd, 1996, Lynd et al., 1991, Lynd et al., 2002, Lynd et al., 1999, Mielenz, 2001, Mohanty et al., 2000, Moreira, 2005, Reddy and Yang, 2005, Wyman, 1994, Wyman, 1999, Wyman, 2003). Additionally, studies have shown that the use of biobased products and bioenergy can achieve zero net carbon dioxide emission (Demain, 2004, Demain et al., 2005, Hoffert et al., 2002, Lynd et al., 1991, Lynd et al., 1999). Development of technologies for effectively converting less costly agricultural and forestry residues to fermentable sugars offers outstanding potential to benefit the national interest through: (1) improved strategic security, (2) decreased trade deficits, (3) healthier rural economies, (4) improved environmental quality, (5) technology exports, and (6) a sustainable energy resource supply (Angenent et al., 2004, Caldeira et al., 2003, Demain et al., 2005, Hoffert et al., 1998, Hoffert et al., 2002, Kamm and Kamm, 2004, Lynd, 1996, Lynd et al., 1991, Lynd et al., 1999, Lynd et al., 2002, Moreira, 2005, Wirth et al., 2003, Wyman, 1999).

Effective conversion of recalcitrant lignocellulose to fermentable sugars requires three sequential steps: (1) size reduction, (2) pretreatment/fractionation, and (3) enzymatic hydrolysis (Wyman, 1999, Zhang and Lynd, 2004b). One of the most important and difficult technological challenges is to overcome the recalcitrance of natural lignocellulosic materials, which must be enzymatically hydrolyzed to produce fermentable sugars (Chang et al., 1981, Demain et al., 2005, Fan et al., 1982, Grethlein, 1984, Hsu, 1996, Lin et al., 1981, McMillian, 1994, Millett et al., 1976, Moreira, 2005, Mosier et al., 2005, Saddler et al., 1993, Weil et al., 1994, Wyman, 1999, Wyman et al., 2005a).

Cellulases are relatively costly enzymes, and a significant reduction in cost will be important for their commercial use in biorefineries. Cellulase-based strategies that will make the biorefinery processing more economical include: increasing commercial enzyme volumetric productivity, producing enzymes using cheaper substrates, producing enzyme preparations with greater stability for specific processes, and producing cellulases with higher specific activity on solid substrates. Recently, the biotechnology companies Genencor International and Novozymes Biotech have reported the development of technology that has reduced the cellulase cost for the cellulose-to-ethanol process from US$5.40 per gallon of ethanol to approximately 20 cents per gallon of ethanol (Moreira, 2005), in which the two main strategies were (1) an economical improvement in production of cellulase to reduce US$ per gram of enzyme by process and strain enhancement, e.g., cheaper medium from lactose to glucose and alternative inducer system and (2) an improvement in the cellulase enzyme performance to reduce grams of enzyme for achieving equivalent hydrolysis by cocktails and component improvement (Knauf and Moniruzzaman, 2004). But this claim has not yet been widely accepted because the cellulase mixture was tested only for the specific pretreated lignocellulosic substrate and cannot be applied to other pretreated lignocelluloses.

Currently, most commercial cellulases (including β-glucosidase) are produced by Trichoderma species and Aspergillus species (Cherry and Fidantsef, 2003, Esterbauer et al., 1991, Kirk et al., 2002). Cellulases are used in the textile industry for cotton softening and denim finishing; in the detergent market for color care, cleaning, and anti-deposition; in the food industry for mashing; and in the pulp and paper industries for de-inking, drainage improvement, and fiber modification (Cherry and Fidantsef, 2003, Kirk et al., 2002). The cellulase market is expected to expand dramatically when cellulases are used to hydrolyze pretreated cellulosic materials to sugars, which can be fermented to commodities such as bioethanol and biobased products on a large scale (Cherry and Fidantsef, 2003, Himmel et al., 1999, van Beilen and Li, 2002). For example, the potential cellulase market has been estimated to be as high as US$400 million per year if cellulases are used for hydrolyzing the available corn stover in the midwestern United States (van Beilen and Li, 2002). This market scenario represents an increase of ∼ 33% in the total US industrial enzyme market (Wolfson, 2005). The large market potential and the important role that cellulases play in the emerging bioenergy and bio-based products industries provide a great motivation to develop better cellulase preparations for plant cell wall cellulose hydrolysis. These improved cellulases must also have characteristics necessary for biorefineries, such as higher catalytic efficiency on insoluble cellulosic substrates, increased stability at elevated temperature and at a certain pH, and higher tolerance to end-product inhibition.

Fig. 1 shows that cellulase engineering for non-complexed cellulase systems contains three major research directions: (1) rational design for each cellulase, based on knowledge of the cellulase structure and the catalytic mechanism (Schulein, 2000, Wilson, 2004, Wither, 2001); (2) directed evolution for each cellulase, in which the improved enzymes or ones with new properties were selected or screened after random mutagenesis and/or molecular recombination (Arnold, 2001, Cherry and Fidantsef, 2003, Hibbert et al., 2005, Schmidt-Dannert and Arnold, 1999, Shoemaker et al., 2003, Tao and Cornish, 2002); and (3) the reconstitution of cellulase mixtures (cocktails) active on insoluble cellulosic substrates, yielding an improved hydrolysis rate or higher cellulose digestibility (Baker et al., 1998, Boisset et al., 2001, Himmel et al., 1999, Irwin et al., 1993, Kim et al., 1998, Sheehan and Himmel, 1999, Walker et al., 1993, Wilson and Walker, 1991, Zhang and Lynd, 2004b). With respect to engineering complexed cellulase systems (cellulosomes), the idea of chimeric constructs of cellulosomal domains/components was proposed by Bayer et al. (1994), and the reconstruction of cellulosome components is becoming another hot research area (Fierobe et al., 2001, Fierobe et al., 2002, Fierobe et al., 2005, Mingardon et al., 2005, Sabathe and Soucaille, 2003), which we do not review here.

The cornerstone of enzyme engineering is to achieve a direct correlation between the enzyme assays or screening approaches and the changes in enzyme functions in the desired application. Development of a useful, predictive cellulase assay or screening is particularly difficult because of the nature of solid heterogeneous substrates, such as plant cell walls. Available quantitative cellulase assays and screenings have been analyzed and compared herein, including their advantages and limitations. Also, successful cellulase examples using directed evolution are examined, and a possible strategy of combinatorial molecular breeding and continuous culture with solid cellulosic materials to select a cellulase with higher activity is discussed.