The expression of a cofactor-independent, heterologous XI is the solution for bypassing the intrinsic redox constraints of the XR/XDH approach. Suc­cessful implementation, however, requires an in vivo activity of XI similar to that of key glycolytic enzymes such as hexokinase and phosphofructokinase. In practice, this corresponds to an activity, under physiological conditions, of 0.5-1.0 ^mol D-xylose converted per milligram soluble cell protein per minute [68]. The apparent simplicity of this objective turned out to be decep­tive. In fact, studies on the functional expression of heterologous structural genes for XI in S. cerevisiae now spans roughly two decades.

Expression in S. cerevisiae of the E. coli xylA gene (which clusters with the XI genes from other Proteobacteria, Fig. 3), resulted in no [13] or very low in vitro XI activities [59]. Sarthy et al. (1987) showed that, while the E. coli XylA protein was produced in S. cerevisiae, its specific activity was three orders of magnitude below that of XylA protein produced in E. coli [59]. Improper protein folding, sub-optimal intracellular pH, post-translational modification, inter — or intramolecular disulfide bridge formation and a lack of specific cofactors or metal ions in S. cerevisiae were mentioned as possible causes [59]. However, no single factor was identified that could explain the low activity, and attempts to increase E. coli XI expression levels in S. cere­visiae were unsuccessful [59]. Subsequently, attempts were made to express XI-encoding genes from other prokaryotic phyla. Attempts to express XI genes from Clostridium thermosulfurogenes [48], Bacillus subtilis or Actino — planes missouriensis [1], which originate from different prokaryotic phyla (Fig. 3), also failed to result in the production of an active XI enzyme in S. cerevisiae.

The first study that achieved significant activities of a heterologous XI enzyme in S. cerevisiae was based on expression of the XI gene from the thermophile Thermus thermophilus [70]. Indeed, an enzyme activity of up to 1.0 ^mol(mg protein)-1 min-1 was found in cell extracts of the engineered S. cerevisiae strain. However, this activity was assayed at the optimum tem­perature for activity of the T. thermophilus XI of 85 °C, which is not com­patible with yeast growth or survival. At 30 °C, the optimum temperature for growth of S. cerevisiae, activity was only 0.04 ^mol (mg protein)-1 min-1 [70]. Although subsequent random mutagenesis resulted in variants of the T. ther­mophilus XI with improved temperature characteristics [26,47], in vivo en­zyme activities of the T. thermophilus XI in S. cerevisiae strains remained too low to sustain rapid anaerobic growth on D-xylose ( [35], see Sect. 5).

A breakthrough came with the discovery of a XI in an unicellular eu­karyote, the anaerobic fungus Piromyces sp. E2 [28]. Expression of this Piromyces xylA gene in S. cerevisiae resulted in high enzyme activities (up to

1.1 ^mol(mg protein)-1 min-1 at 30 °C [42].

The molecular basis for the high functional expression levels obtained with the Piromyces xylA gene remains unclear. We have recently expressed the XI sequence from Bacteroides thetaiotaomicron into S. cerevisiae. This prokary­otic sequence is 83% identical and 88% similar to the Piromyces xylA gene. S. cerevisiae strains expressing this prokaryotic XI can utilise D-xylose, albeit

at a somewhat lower rate than similar strains expressing the Piromyces xylA gene (A. A. Winkler et al. unpublished). This indicates that its probable evolu­tionary history (horizontal gene transfer followed by evolutionary adaptation to a eukaryotic host) may not be the sole factor in the successful expression of the Piromyces enzyme.

In terms of GC content and codon usage, the Piromyces xylA gene appears to have favourable characteristics for expression in S. cerevisiae. At 45%, its GC content is much closer to that of S. cerevisiae (39%), than that of, for ex­ample, the T. thermophilus gene (little over 64% GC). Also the high codon bias index of the Piromyces gene for expression in S. cerevisiae (0.642 versus — 0.018 for the T. thermophilus gene) may contribute to its efficient expression. Future structure-function studies will likely identify critical factors for high- level functional expression in yeast, in the S. cerevisiae genome as well as in the sequence of heterologous XI genes. However, while of great scientific interest, innovation in D-xylose fermentation is no longer dependent on such research, as the availability of the Piromyces xylA gene has paved the way for metabolic engineering of S. cerevisiae for anaerobic fermentation of D-xylose to ethanol. Recent progress in this area will be discussed in the following paragraphs.

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