The basic premise of the native ethanologen CBP approach is to start with a robust ethanologen, and engineer in it the ability to produce and secrete a suite of cellulolytic enzymes. This approach generally takes advantage of already having an excellent ethanol production system with ethanol tolerance mechanisms in place, and either natural or engineered abilities to utilize the major biomass-derived sugars including xylose and arabinose. With the metabolic foundation intact, the strain development effort can focus solely on producing large amounts of cellulases and directing them to the extracellular space. However, these related processes represent distinct chal­lenges in and of themselves, and can indeed be quite complex. While most attempts at pursuing this route have expressed one to several separate cellulases, a native cellulose-degrading organism will generally produce dozens of distinct enzymes in order to achieve this task. For example, T. reesei a cellulolytic fungi with the pre­miere cellulose-degrading enzyme, CBHI [32], expresses 10 cellulose-degrading and 16 hemicellulose-degrading enzymes [12]. Optimizing the functional expres­sion of many enzymes can be complicated as it includes transcription, translation, peptide folding, and ensuring protein stability. Further complicating the case is that many enzymes, particularly of fungal origin, need post-translational modifications including glycosylation to achieve maximal activity or stability [33-35]. In addi­tion, the enzymes must be targeted either directly or indirectly to be secreted extra­cellularly. Of course, the advantage that recombinant CBP organisms have over cellulolytic organisms in nature is that they are fed a partially deconstructed sub­strate due to thermochemical pretreatment, making the task of cellulose and hemi- cellulose depolymerization less daunting, and requiring fewer enzymes. At an absolute minimum, an endoglucanase, an exoglucanase (cellobiohydrolase) and a b-glucosidase will be required to fully depolymerize cellulose [36], and very likely additional accessory enzymes will be required, making the expression of sufficient enzymes a challenging task.

There are numerous ethanologens that have been considered as candidates for conversion to a CBP organism including Escherichia coli [7, 37, 38], Klebsiella oxytoca [7, 39-41], Z. mobilis [42-47], and several yeasts including S. cerevisiae [6, 9, 48-50]. Given their strong representation in the cellulosic biofuels research landscape, we will discuss S. cerevisiae and Z. mobilis in more detail below.