The recombinant cellulolytic strategy involves engineering non-cellulolytic organisms that exhibit high product yields by producing a heterologous cellulase system enabling cellulose utilization. Early research advances have been reviewed previously (15). The yeast, S. cere — visiae, is a promising host organism for this strategy because of its high ethanol productivity at high yields, high osmo — and ethanol-tolerance, natural robustness in industrial processes, ease of genetic manipulation, and generally regarded as safe status due to its long association with the food and beverage industries. Cellulases from bacterial and fungal sources have been transferred to S. cerevisiae, enabling the hydrolysis of cellulosic derivatives (15), or growth on cellobiose (63, 64).

Three recombinant enzymes — Trichoderma reesei endoglucanase II, T. reesei cellobiohy — drolase II, as well as Aspergillus aculeatus p — glucosidase cellulase — have been co-expressed in S. cerevisiae via individual fusion proteins with the C-terminal-half region of a-agglutinin (65). However, this recombinant strain cannot grow on cellulose using these recombinant cellulases, possibly because of poor recombinant cellulase expression or low enzyme activity or both.

van Zyl and coworkers (66) were the first to produce recombinant S. cerevisiae that can grow on pure insoluble cellulose by expressing two recombinant cellulases — the T. reesei endoglucanase (EG I) and the S. fibuligera p-glucosidase (BGL 1). The resulting strain was able to grow on phosphoric acid swollen cellulose (PASC) through simultaneous production of sufficient extracellular endoglucanase and p-glucosidase activity. Anaerobic growth was observed on the medium containing 10 g/L PASC as sole carbohydrate source with concomi­tant ethanol production of up to 1.0 g/L. Since crystalline cellulose hydrolysis requires three types of cellulases (endoglucanase, cellobiohydrolase, and p-glucosidase) to work together (24, 25), it is still a challenge to develop recombinant cellulolytic microorganisms that can express high levels of these cellulases to support cell growth on crystalline cellulose.

To achieve the self-supporting growth based on recombinant cellulases, it is appropriate to estimate the feasibility of cellulase expression levels. On the basis of the sufficiency of ex­pression of growth-enabling heterologous enzymes, the level of enzyme expression required to achieve a specified growth rate maybe calculated as a function of enzyme-specific activity (63). For growth enabled by cellulases with specific activities in the range available, required expression levels are well within the range reported in the literature (1-10% of cellular pro­tein) (67, 68). Protein expression at this level has been reported in both S. cerevisiae (67) and E. coli (69), although not to date for active cellulases. On the other hand, nature has created a diversity of cellulolytic microorganisms. With time, we anticipate that recombi­nant cellulolytic microorganisms with activity on crystalline cellulose will be created in the laboratory.