Yeast is an efficient industrial host with a high productivity of ethanol and with well- developed genetic tools. However, yeast does not possess endogenous cellulolytic ability. Several heterologous cellulases have, therefore, been expressed in yeast for direct conversion of cellulose into ethanol. Endoglucanase genes from Bacillus spp. were successfully integrated (randomly, at approximately 44 sites) into the chromosome of yeast, resulting in the direct conversion of cellodextrin into ethanol (Cho et al. 1999).

With the advent of cell-surface display technologies, it has become possible to express artificial cellulosomes (rather than free cellulases) in yeast. Cellulosomes facilitate the assembly of different cellulolytic enzymes in close proximity, and thereby favor a proper synergy between the enzymes (Tsai et al. 2010). Surface display of endoglucanase from T. reesei and ^-glucosidase from A. aculeatus in yeast helped in the successful conversion of barley ^-glucan into ethanol with 93% of the theoretical yield and without any pretreatment (Fujita et al. 2002). Co-displaying the exoglucanase from Aspergillus spp. along with the endoglucanase and ^-glucosidase in yeast has resulted in the direct conversion of amorphous cellulose into ethanol (Fujita et al. 2004; McBride et al. 2005). Very recently, recombinant yeast has been further modified to express ^-glucosidase within the cell. A high-affinity transporter for cellobiose and cellodextrin has also been cloned into the recombinant yeast. This strain co-metabolizes xylose and cellobiose more efficiently (Ha et al. 2011).

Although several studies have demonstrated efficient ethanol production from amorphous cellulose, attempts to engineer yeast to hydrolyze crystalline cellulose have been unsuccessful because of low exoglucanase activity (la Grange et al. 2010). The exoglucanase and ^-glucosidase activities in recombinant cellulolytic yeast strains are insufficient to support growth with cellulose as a sole carbon source. Hence, a synthetic yeast consortium has been developed with four engineered yeast strains, each expressing either the scaffoldin from Clostridium spp. and Ruminococcus spp. or the three enzymes, namely, exo — and endoglucanases from Clostridium spp. and ^-glucosidase from Ruminococcus spp. (Fig 4) (Tsai et al. 2010). However, investigators have been unable to completely decipher the efficiency of the synthetic consortium, because the ratio of the different cellulases needed for a proper synergy has not been established. A cocktail 8-integration tool has been developed in yeast to predict the optimum ratio of different cellulases, but with little success (Yamada et al. 2010).

Another major problem with recombinant cellulase expression is that heterologous cellulases are made to function at a suboptimal temperature. The optimal temperature for the growth of recombinant hosts is 37°C, but cellulases are more active at temperatures

above 50°C. Therefore, the thermotolerant yeast Kluyveromyces marxianus has been engineered to display thermostable endoglucanase and ^-glucosidase on its surface. This engineered, thermostable yeast ferments ^-glucan directly to ethanol at 48°C (Yanase et al. 2010).

4.2 E. coli

The broad substrate range of E. coli, together with its ample genetic tools and its substantial fermentation capacity, renders the species to be a potential candidate for bioethanol production. E. coli, with chromosomally integrated genes encoding pyruvate decarboxylase and alcohol dehydrogenase, is an efficient ethanol producer (Ohta et al. 1991). Several attempts have been made to engineer cellulolytic ability in ethanologenic E. coli. The species also has endogenous cryptic genes for cellobiose metabolism and an endoglucanase for the hydrolysis of soluble cellulose (Park and Yun 1999; Kachroo et al. 2007; Vinuselvi and Lee 2011). Achieving a higher extracellular titer of cellulases is a bottleneck in the development of a recombinant cellulolytic E. coli for ethanol production. E. coli does not have a proper protein secretion system (Shin and Chen 2008). Because E. coli is a gram-negative bacterium, it has an outer membrane rich in peptidoglycan, which acts as a barrier for protein secretion. The extracellular protein concentration observed with E. coli is 0.0088 g/L, one hundred-fold less than that observed with native cellulolytic organisms (Qian et al. 2008; Xu et al. 2009;

Vinuselvi et al. 2011). Gram-negative bacteria possess five different protein-export pathways (Types I-V), two of which are found in E. coli (Type I and Type II).

Several attempts have been made to increase the extracellular titer of recombinant proteins in E. coli: by exploiting the Sec/TAT signal sequence (Zhou et al. 1999; Angelini et al. 2001), by fusion of recombinant proteins with extracellular proteins such as OsmY (Qian et al. 2008), or by increasing membrane permeability (Shin and Chen 2008). Cellulase secretion in E. coli has been achieved through the expression of endoglucanase, along with the out genes of Erwinia chrysanthemi, under the control of a surrogate promoter (Zhou et al. 1999). Deletion of Ipp weakens the outer membrane, allowing any proteins targeted to the periplasmic space to be secreted into the medium. Approximately 70% of the cellulases produced were secreted into the medium in an lpp knockout E. coli strain (Shin and Chen 2008). Several studies have used OsmY as a fusion partner for recombinant protein secretion in E. coli. However, this technique has not been exploited for cellulase secretion because of the large size of cellulases (Aristidou and Penttila 2000; Qian et al. 2008) (Fig 5).

The cellobiose metabolic operon from Klebsiella oxytoca has been introduced into E. coli, but the expression level of the cellobiose transporter and metabolic genes was poor, and hence could not support the growth of E. coli on cellobiose (Moniruzzaman et al. 1997). Cellulases from several species of Clostridium, Bacillus, Cellulomonas, and Ruminococcus have been expressed and characterized in E. coli (Hinchliffe 1984; Zappe et al. 1986; Fierobe et al. 1991; ReverbelLeroy et al. 1996; Lam et al. 1997; ReverbelLeroy et al. 1997; Lee et al. 2008; Li et al. 2009). Co-expression of endoglucanase from B. pumilus and ^-glucosidase from

Fervidobacterium spp. in E. coli favored growth of the recombinant strain, with soluble carboxymethyl cellulose (CMC) as the sole carbon source (Rodrigues et al 2010).