Significant advances related to recombinant enzyme expression support the potential for S. cerevisiae as a CBP host. However, the challenge of integrating all the different aspects of enzymatic hydrolysis of cellulose and hemicellu — lose and subsequent fermentation of the sugars released to ethanol in a single reactor with a CBP should not be underestimated. A pertinent question of­ten asked by critics is, “Would S. cerevisiae be able to simultaneously express multiple genes, while producing and secreting the different cellulases, hemi- cellulases, and pentose utilizing enzymes required?” Several studies demon­strate coexpression of multiple genes in S. cerevisiae, for example in the case of the expression of tethered cellulolytic and xylanolytic enzymes [59,119], xylose and arabinose utilizing enzymes [40,162], as well as xylose and cel — looligosaccharide utilizing enzymes [45]. The expression and secretion of a variety of cellulases, amylases, and pectinase has also been demonstrated without adversely affecting yeast growth [51, 52].

However, the number of genes expressed is probably not as important a challenge as the need for high-level expression as well as the stress re­sponses that may accompany such high-level expression. Factors that may impose unnecessary stress on the cell are (1) sequestering of transcription factors at highly expressed promoters used for heterologous gene expression,

(2) impact of unfavorable codon bias on the translation of heterologous pro­tein (can be overcome by the use of codon-optimized synthetic genes), and

(3) improper folding of foreign proteins that can evoke the (4) unfolded pro­tein response (UPR) and consequently the endoplasmic reticulum-associated protein degradation (ERAD) response [163]. Some of these effects may be exacerbated by (5) interrupted transport of foreign proteins through the se­cretion pathway, or (6) accumulation of larger proteins at the cell wall due to low permeability [164]. The answer would thus not be simply overexpression of all the required genes to ensure a functional CBP yeast with the desirable enzymatic activities, but much more attention should also be devoted to the careful manipulation of the required enzyme activities and producing them at the right concentration to provide functionality without exerting too much unnecessary stress on the CBP yeast.

Essentially all work carried out thus far involving heterologous expres­sion of saccharolytic enzymes in yeast has involved laboratory strains. Much of this work has to be transferred to industrial strains that provide the fer­mentation capacity and robustness desired for industrial processes. Different strategies have been used for the overexpression of multiple genes in in­dustrial S. cerevisiae strains. High copy-number episomal YEp vectors, often using the two-micron autonomous replicating sequence (ARS), have been very helpful in demonstrating proof of concept in laboratory strains of S. cere­visiae [43,51,102,115]. However, these vectors are usually mitotically unsta­ble and require selection for the episomal plasmid, which often means using a defined medium that is not applicable to industrial uses [164]. The preferred route taken for industrial strains has been the use of integrative YIp vectors that facilitate direct integration of foreign expression cassettes into a target gene on the yeast genome [165,166] or recycling dominant selectable markers for multiple integration [167-170]. Although these methods provide stable expression from the yeast genome and are amendable to industrial strains, the major drawback has been low expression levels and often not delivering high enough quantities of the required gene product.

Different approaches have been pursued in an attempted to combine the advantages of overexpression from multicopy plasmids with the stability of chromosomal integration, which is also applicable to industrial strains when dominant selectable markers are used. These include the use of repetitive chromosomal DNA sequences such as rDNA [171] and 8-sequences [172]. There are approximately 140-200 copies of rDNA existing in the haploid yeast genome; however, rDNA is located in the nucleolus, which may af­fect the accessibility to RNA polymerase II transcription. Also, the size of pMIRY (multiple integration into ribosomal DNA in yeast) vectors could determine the mitotic stability of these multiple integrations [173]. The 8- sequences are the long terminal repeats of S cerevisiae retrotransposon Ty. More than 400 copies of 8-sequences can exist either Ty associated or as sole sites in the haploid yeast genome [174]. 8-Integration thus makes it possible to integrate more copies of a gene of interest into the yeast genome than the conventional integration systems [78,175]. Host strains and integrated gene size can significantly affect the transformation efficiency at 8-sequences; however, the transformation frequency can be 10- to 100-fold those ob­tained when transforming with vectors that target a single gene on the yeast genome [176].

Although the necessary tools exist for multiple and repeated integration of genes of interest into the genome of industrial strains to complement the required features for CBP (Table 1), a more strategic approach would be re­quired to design a yeast that produces the required enzyme activities, yet re­tains the competence to still perform well under industrial conditions for the economic conversion of plant biomass to ethanol. Such an approach will most probably start by building on a platform industrial yeast that cometabolises hexoses and pentoses, and subsequently finding the right combination and level of expression for saccharolytic enzymes. This approach will inevitably use reiterated metabolic engineering and flux analysis, selection and mutage­nesis strategies, and even strain breeding to allow the microorganism itself to overcome rate-limiting hurdles toward developing an efficient CBP yeast. Ex­amples of such approaches in the past have been performed to enhance xylose fermentation in laboratory and industrial strains [33,37,39,177].