Xylose reductase, the first step in the pathway of xylose catabolism in most yeast species, functions as an enzyme equally well with L-arabinose as with D-xylose, with a slightly higher affinity (lower Km) and a higher maximal rate (Vmax) for l-arabinose.59 Polyol dehydrogenases, active on xylitol, on the other hand, find either L — or D-arabinose to be a poor substrate.103 Although the XDH activity from P. stipitis was kinetically investigated in 1989, little is known about its func­tional physiology; the catalyzed reaction is reversible but activity is unlikely to be regulated by the NAD/NADH balance inside the cell.104 This yeast also con­tains a second XDH, quite distinct from the well characterized xyl2 gene product, but its role is presently undefined in either xylose catabolism or ethanol produc — tion.105 The NAD-specific XR from S. cerevisiae itself is even less well charac­terized, although the enzyme activity is induced by xylose with the wild-type organism.106

An outline of known enzyme-catalyzed metabolic relationship for pentitols and pentoses is given in figure 3.6; some of these pathways are of increasing contempo­rary interest because either they or their engineered variants could lead to the syn­thesis by whole cells (or in biotransformations with isolated enzymes) of “unnatural” or rare sugars useful for the elaboration of antibiotic or antiviral drugs — this is discussed later in chapter 8 when the Green Chemistry of the biorefinery concept for processing agricultural residues is discussed in depth.

Progress in defining the actual pathways operating in known ethanologenic yeasts was rapid after the year 2000

• gene encoding an L-xylulose reductase (forming xylitol; NADP-depen — dent) was then demonstrated in H. jecorina and overexpressed in S. cere — visiae; the l-arabinose pathway uses as its intermediates l-arabinitol, l-xylulose, xylitol, and (by the action of XDH) D-xylulose; the xylulose reductase exhibited the highest affinity to l-xylulose, but some activity was shown toward d-xylulose, d-fructose, and l-sorbose.109

• In H. jecorina, deletion of the gene for XDH did not abolish growth because ladl-encoded l-arabinitol 4-dehydrogenase compensated for this loss — however, doubly deleting the two dehydrogenase genes abolished the ability to grow on either d-xylose or xylitol.110

With this knowledge, expressing the five genes for L-arabinose catabolism in S. cere — visiae enabled growth on the pentose and, although at a low rate, ethanol production from l-arabinose under anaerobiosis.111 In the same year (2003), the genes of the shorter bacterial pathway for L-arabinose catabolism were inserted into S. cerevi — siae.112 The bacterial pathway (active in, e. g., B. subtilis and E. coli) proceeds via l-ribulose, l-ribulose 5-phosphate, and d-xylulose 5-phosphate (figure 3.6), using the enzymes L-arabinose isomerase, L-ribulokinase, and L-ribulose 5-phosphate epimerase. The coexpression of an arabinose-transporting yeast galactose permease allowed the selection on L-arabinose-containing media of an L-arabinose-utilizing yeast transformant capable of accumulating ethanol at 60% of the theoretical maxi­mum yield from L-arabinose under O2-limiting conditions.112