Xylose isomerase (XI, D-xylose ketol isomerase, EC 5.3.1.5) catalyses the re­versible isomerisation of D-xylose to D-xylulose. This enzyme has been the subject of much applied research because it also catalyses the isomerisation of D-glucose and D-fructose. In this role of “glucose isomerase”, xylose iso — merase is applied on a huge scale for the production of high-fructose corn syrup and continues to be one of the most abundantly applied industrial en­zymes. The high-fructose syrup application has led to intensive screening and protein engineering studies, with increased activity and stability of XIs at el­evated temperature as a priority target [11,23]. For excellent reviews on the molecular and industrial aspects of XI, the reader is referred to a number of specialised reviews [4,11,12].

In the context of the present paper, several characteristics of XIs are note­worthy. First and foremost, and in contrast to the xylose reductase/xylitol dehydrogenase pathway, the XI reaction does not involve pyridine nucleotide cofactors. As this will entirely circumvent the cofactor regeneration chal­lenges associated with the xylose reductase/xylose dehydrogenase pathway, functional expression of a XI in S. cerevisiae has long been regarded the most promising approach to engineering S. cerevisiae for alcoholic fermentation of D-xylose [14].

XIs generally require divalent cations, but the specificity of the metal re­quirement is strongly dependent on the source of the enzyme, with many enzymes requiring Co2+, but others Mn2+ or Mg2+ [11]. Although S. cere­visiae has been demonstrated to accumulate cobalt intracellularly [18], it is not clear whether this metal is available in the cytosol or sequestered in, for example, the vacuole. Other aspects with potential relevance for yeast metabolic engineering include the high temperature optimum (60-80 °C) and the relatively high pH optimum (7.0-9.0) of many of the XIs that have been characterised [11]. As S. cerevisiae is a mesophilic micro-organism with a cytosolic pH slightly below 7, intracellular expression of heterologous struc­tural genes for XIs may not always lead to optimal activity.

Even in the pre-genomics era, it was clear that XIs are widespread among prokaryotic micro-organisms, and also occur in several plants [11]. Figure 3 shows a phylogenetic tree of XI gene sequences based on an October 2006 GenBank database search. This phylogenetic tree gives a good indication of the diversity of XI genes and the phylogenetic relationships between se­quences from related organisms. With respect to eukaryotes, the tree contains four sequenced XI sequences from the plants Hordeum vulgare, Arabidop — sis thaliana, Oryza sativa and Medicago truncatula, which cluster together (Fig. 3). The phylogenetic tree contains only one other eukaryotic XI se­quence, namely that of the anaerobic fungus Piromyces sp. E2 [28]. Inter­estingly, this eukaryotic XI sequence clusters with those of the prokaryotic phylum Bacteroidetes, which has led to the suggestion that the fungus may have acquired XI via horizontal gene transfer [28], as previously suggested for other enzymes in anaerobic fungi [20].

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