The major requirement for S. cerevisiae as CBP yeast would be sufficient ex­pression and production of extracellular saccharolytic enzymes [1]. In the context of creating such a CBP yeast, the first question researchers would like to answer is, “How much saccharolytic enzyme, particularly cellulase expres­sion, is enough to enable CBP conversion of plant material to ethanol, and is that amount feasible in S. cerevisiae?” The obvious follow-up question is, “How do we accomplish those levels of expression?” Recent analyses [9,44] have approached the first question from a kinetic standpoint, balancing the demand for soluble products of cellulose hydrolysis (glucose) by cells with the production of those products by cellulase systems. Demand is a simple func­tion of the growth rate and the cell yield: ^/YX/S = g glucose/gcells/h, while supply is just the cell-specific cellulase activity: g glucose/g cells/h. These re­lationships can be used to calculate a number of useful quantities, including the percentage of total cell protein that needs to be cellulase to achieve a par­ticular growth rate on a cellulosic substrate. The relative levels of individual cellulase component expression can be calculated based on the known ratios of those components in native systems.

In the last two decades there have been several reports on the expression of cellulases in S. cerevisiae. Table 2 summarizes some of the results found to date. Most reports regarding the expression of cellulases and hemicellulases in yeast employed strong glycolytic (or other constitutively expressed) pro­moters to drive expression of the heterologous gene(s). Although the choice of promoter and leader sequences will undoubtedly have a great influence on expression levels attained, there are not enough data in the literature to suggest any general trends as to what are the best promoter and leader sequences to use when expressing cellulases and hemicellulases. Several re­searchers have sought to produce cellulases in an organism that would not yield interfering activities so as to gain insight into the mechanism of the original cellulolytic enzyme [99], whereas others have sought to enable the yeast to hydrolyze nonnative cellulolytic substrates [43,59,78,102]. Although most of the cellulases that have been successfully produced in S. cerevisiae were of fungal origin, there are reports of successful bacterial cellulase pro­duction [76,82].

Full enzymatic hydrolysis of crystalline cellulose requires three major types of enzymatic activity: (1) endoglucanases (1,4-P-D-glucan 4-glucano — hydrolases; EC 3.2.1.4); (2) exoglucanases, including D-cellodextrinases (1,4-P-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (1,4-P-D-glucan cellobiohydrolases; EC 3.2.1.91); and (3) P-glucosidases (в-glucoside glucohydrolases; EC 3.2.1.21) (Fig. 2a). Cellobiohydrolase (CBH) enzymes are key components for fungal cellulase systems, and their func­tional secretion is critical for allowing CBP. For example, CBHs make up ~ 80% of the total mass for the T. reesei system, and CBH1 plays a particu­larly important role, making up 60% of the total mass [103]. CBHs have been successfully produced and secreted by S. cerevisiae and were tested for ac­tivity on a variety of substrates ranging from small synthetic molecules to amorphous and crystalline forms of cellulose (Table 1). Some reports have shown decreased specific activity on certain substrates, presumably as a re­sult of hyperglycosylation [47, 48]. However, in a recent study it was shown that the specific activity of a glycosylated heterologous CBH1 did not dif­fer significantly from that of the native enzyme produced by T. reesei [49].

U = micromole substrate released/min, NR = not reported; italics indicate calculation based on assumptions (0.45 g DCW/g glucose, 0.45 g protein/g DCW, 1.3 x 107 cells/mg DCW, 1 OD(600) = 0.57 g DCW/L).

CBH = cellobiohydrolase, EG = endoglucanase, BGL = beta-glucosidase, AC = amorph­ous cellulose, BMCC = bacterial microcrystalline cellulose, BBG = barley beta-glucan, CC = crystalline cellulose, IOSC = insoluble cellooligosaccharides, C2 = cellobiose, C3 = cel — lotriose, C4 = cellotetraose, PNPC = p-nitrophenol cellobioside, PNPL = p-nitrophenol lactoside, MUC = methylumbelliferyl cellobioside, MUL = methylumbelliferyl lactoside, Xglu = 5-bromo-4-chloro-3-indolyl-|3-D-glucopyranoside

Reports of CBH production in yeast have also shown that a relatively low titer of secreted cellulase is found, although the range of reported values is quite large—0.002 to 1.5% of total cell protein. Coupled with the low spe­cific activity of CBHs, CBH expression has been identified as a limiting factor for CBP using yeast [9]. However, in a recent report the amount of CBH1 required to enable growth on crystalline cellulose was determined and was found to be, in terms of total cellular protein, within the capacity of heterol­ogous protein production in S. cerevisiae, i. e., between 1 and 10% of total cell protein [49,104-106].

Fig.2 Illustration of the complexity of cellulose and hemicellulose and the enzymes in — ► volved in their degradation. Cellulose (a) and hemicellulose structures for arabinoxylan (b), galactomannan (c) , and xyloglucan (d) depicting the different side chains present. Hexoses are distinguished from pentoses by the presence of a protruding line from the cyclic hexagon (pyranose ring), depicting the CH2OH group. Hydrolase enzymes and the bonds targeted for cleavage in the four polysaccharide structures are indicated by arrows [100,101]

Fungal and bacterial endoglucanase (EG) production in S. cerevisiae have been by and large more successful than CBH production (Table 2). This is not surprising considering that EG enzymes usually have specific activities 2 to 3 orders of magnitude higher on synthetic and amorphous cellulose substrates, such as phosphoric acid swollen cellulose (PASC) and carboxymethyl cellu­lose (CMC), in comparison to CBHs. It is thus easier to measure the presence of even small amounts of heterologous EG compared to CBHs. Although se­creted heterologous EGs were usually reported to be hyperglycosylated, this did not necessarily influence their specific activity negatively [61]. Sufficiency analysis shows that, assuming that a T. reesei system is reconstructed, even if all of the non-CBH cellulase system components were EG, it would still only need to make up ~ 0.3% of cell protein, well within the range of possibility for a S. cerevisiae secretion system. The successful expression of P-glucosidases in S. cerevisiae at sufficient levels to sustain growth on cellobiose as sole car­bon source at a rate comparable to glucose suggests that BGL expression will not be a limiting step in cellulase system reconstruction [43,44].

A number of studies have expressed multiple cellulase enzymes in at­tempts to recreate a fully cellulolytic, fermentative system [45,59,64,78,102]. Van Rensburg et al. [51] constructed a yeast capable of hydrolyzing numer­ous cellulosic substrates and growing on cellobiose, while Cho et al. [78] showed that decreased loadings of cellulase could be used for SSF experi­ments with their strain expressing a BGL enzyme and an enzyme with dual exo/endocellulase activity. Fujita et al. [59,64] reported coexpression and sur­face display of cellulases in S. cerevisiae, and a recombinant strain displaying the T. reesei endoglucanase II, cellobiohydrolase II, and the Aspergillus ac — uleatus P-glucosidase 1 was built. High cell density suspensions of this strain were able to directly convert PASC to ethanol with a yield of approximately 3 g L-1 from 10 g L-1 within 40 h [59]. Den Haan et al. [102] reported growth on and direct conversion of PASC to ethanol by a S. cerevisiae strain coex­pressing the T. reesei EG1 and the Saccharomycopsis fibuligera BGL1 (Fig. 3). Anaerobic growth (0.03 h-1) up to 0.27 gL-1 dry cell weight was observed with this strain on medium containing 10gL-1 PASC as sole carbohydrate source with concomitant ethanol production of up to 1.0 gL-1. As an ex- ocellulase activity such as CBH is required for the successful hydrolysis of crystalline cellulose, it is postulated that the addition of successful, high-level expression of CBH to this strain will enable CBP of crystalline cellulose to ethanol.

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