Hotel Click Clack

Due to the difficulties posed in cloning the XI gene in S. cerevisiae, several advances have been made in the ‘conservative’ approach to the two-step isomerisation process. Among the various XR and XDH genes tested, the XYL1 (Verduyn et al. 1985) and XYL2 (Kötter et al. 1990) genes from P. stipitis were shown to be active in S. cerevisiae (Kötter et al. 1990; Kötter and Ciriacy 1993; Tantirungkij et al. 1993; Walfridsson et al. 1997). Although many reports exist for the expression of P. stipitis XR in S. cerevisiae, Govinden et al. (2001) expressed XR from C. shehatae in the yeast and showed improved activity to the results with P. stipitis. It has been suggested that expressing both XYL1 and XYL2 from C. shehatae in S. cerevisiae should be explored, but no reports have yet been published. The major disadvantage of the two-step isomerisation process was found to be the apparent redox cofactor imbalance, especially under anaerobic conditions. Almost all known XRs have been shown to have a preference for NADPH as cofactor (Bruinenberg et al. 1983), with the exception of some (Verduyn et al. 1985; Rizzi et al. 1988; Neuhauser et al. 1997; Govinden et al. 2001) that have dual cofactor dependence (NAD(P)H). Even among these, the NADH dependence was lower than that for NADPH and was displayed only under certain conditions. The Km of XR for xylose differed between the two cofactors. According to Kostrzynska et al. (1998), the Km for NADPH was 11 µM, while the Km for NADH was 28 µM. These values were similar to the values reported earlier – 9 µM and 21 µM respectively – by Verduyn et al. (1985). Thus the kinetics of this enzyme favoured NADPH utilisation. This contrasted with the cofactor requirement of the XDH enzyme in the next step of xylose utilisation. This enzyme was almost exclusively NAD+ dependent (Kötter et al. 1990) and hence released NADH. Cloning and expressing these two genes, XYL1 and XYL2, in S. cerevisiae, where ethanol production under fermentative conditions was redox neutral, created an imbalance in redox potential. This usually resulted in the exclusive production of xylitol from xylose or, in some cases, in the production of more glycerol because the cell adapted itself to correct the redox imbalance. This scenario was different from that of P. stipitis, where xylose was converted to ethanol and xylitol under

anaerobic conditions (Ligthelm et al. 1988), but produced no xylitol under oxygen-limited conditions. This led to the suggestion that P. stipitis maintained its redox balance through other mechanisms, which was proven by the studies of Jeppsson et al. (1995) and Hallborn (1995). They found two redox sinks that were thought to prevent xylitol formation in P. stipitis. They were: (i) an alternative cyanide-insensitive oxidase believed to regenerate the NAD+ required for xylitol oxidation; and (ii) a D-arabinitol dehydrogenase that may act as redox balancer by reducing D-ribulose to D-arabinitol. These activities were not present in native S. cerevisiae strains and therefore could explain the xylitol produced during xylose growth in S. cerevisiae and not P. stipitis. On the other hand, it was also suggested that P. stipitis produced both ethanol and xylitol under anaerobic conditions due to the dual cofactor dependency of the XR (Prior et al. 1989; Dellweg et al. 1990; Skoog and Hahn-Hägerdal 1990; Kötter and Ciriacy 1993). It is intriguing that the same enzyme when present in P. stipitis showed dual cofactor dependency but, when expressed in S. cerevisiae, was almost exclusively NADPH dependent, as observed from the exclusive xylitol production (Skoog and Hahn- Hägerdal 1990; Hallborn et al. 1991). However, Ligthelm et al. (1988) showed that very little xylose carbon was channelled through the oxidative PPP in P. stipitis. Bruinenberg et al. (1984) showed the importance of NADH as well as that of the redox couple (Bruinenberg et al. 1983) in xylose fermentation in yeasts by using a C. utilis model.

It was proposed and shown that the expression levels and ratio of the two enzymes – XR and XDH – were very important for better xylose utilisation and conversion. Walfridsson et al. (1997) studied the effect of differentially expressed XYL1 and XYL2 genes on xylose fermentation in recombinant yeast by cloning the two genes under two different promoters, phosphoglycerate kinase (PGK1) and alcohol dehydrogenase (ADH1), under different orientations in the plasmid. Analysing the resultant XR and XDH activities in the various recombinant strains led them to conclude that a ratio of 0.06 between XR and XDH was crucial for better xylose fermentation to yield products other than xylitol.

Jeppsson et al. (2006) expressed a mutated P. stipitis XR-encoding XYL1 gene (Kostrzynska et al. 1998) that showed altered Km in a recombinant S. cerevisiae strain at two different levels together with the native P. stipitis XYL2 gene and the overexpressed endogenous XKS1 gene. The resultant strain displayed a decrease in xylitol yield, accompanied by enhanced ethanol yields. Flux analysis showed that the mutated XR utilised a larger fraction of NADH for xylose reduction. Petschacher et al. (2005) reported the kinetics of a similar mutant XR in Candida tenuis. The mutated XR (K270M) had an increased catalytic constant (kcat) with both NADH and NADPH as cofactors, with the fold change being significantly higher for NADPH (2.8) than for NADH (1.7). This result suggested that, apart from reducing the in vitro affinity for NADPH, the mutation may also have enhanced the catalytic rate of the similarly mutated P. stipitis XR. However, the fermentation properties of this enzyme have not yet been described. Metzger and Hollenberg (1995) had earlier reported on direct mutagenesis on the coenzyme binding sites of P. stipitis XDH enzyme, with the

resultant mutant showing decreased NAD+ affinity. They also introduced the proposed NADP-recognition sequence (GSRPVC) of alcohol dehydrogenase from Thermoanaerobium brockii into the XDH sequence. This increased the NADP- dependent activity but, of greater significance, was the reduction in NAD+ affinity. Woodyer et al. (2003) reported on relaxing the nicotinamide specificity of phosphite dehydrogenase, while Watanabe et al. (2005) performed multiple site-directed mutageneses on amino acids from the coenzyme-binding domain of XDH. However, the effect of expressing the mutant XDH in S. cerevisiae has not yet been reported.

2.5.4 Xylulokinase

Xylulokinase (XK), as the name suggests, phosphorylates xylulose so that xylulose-5- phosphate can enter the PPP. This phosphorylation step consumes energy in the form of ATP. The apparent growth of S. cerevisiae on D-xylulose is made possible by this

enzyme. However, its growth on this substrate is so slow that its improved growth on D-

xylulose was used as a screen to identify a xylulokinase gene (Ho and Chang 1989; Deng and Ho 1990). Ho and Tsao (1995) published the sequence of this gene first, although Rodriguez-Pena et al. (1998) reported an almost identical sequence of the open reading frame YGR194c in the S. cerevisiae genome and this was renamed XKS1. Various views have been aired about the importance of XKS1 in fermentation (Ho et al. 1998; Eliasson et al. 2000; Johansson et al. 2001; Toivari et al. 2001; Jin et al. 2003). While most of the reports suggested that XKS1 should be overexpressed so that more xylulose could go through the PPP, other reports (Rodriguez-Pena et al. 1998; Jin et al. 2003) argued that, because of the high ATP requirement, an uncontrolled overexpression of this gene might lead to energy depletion, resulting in cessation of fermentation and cell toxicity. The authors suggested such a condition would be similar to the substrate-accelerated cell death observed with an S. cerevisiae TPS1 mutant during glucose metabolism. Thus, it seemed that an optimal/moderated level of XKS1 expression was needed (Jin et al. 2003) for better xylose fermentation, although several strains that have overexpressed XKS1 have shown better performance through modification of other steps in xylose metabolism (Jeppsson et al. 2003b; Karhumaa et al. 2005). Richard et al. (2000) explored the kinetics of this enzyme in S. cerevisiae and reported that the overexpression of xylulokinase did not increase the growth rate of the cell on xylulose to a level comparable to that of glucose, which meant that other factors such as activities of other enzymes may be limiting, e.g. transaldolase, because sedoheptulose-7-phosphate has been shown to accumulate in D-xylulose fermentation

(Senac and Hahn-Hägerdal 1990). These authors also concluded that the activities of the xylulokinase from S. cerevisiae resembled those of P. stipitis (Jin et al. 2002). Thus, the expression level of xylulokinase was very crucial in xylose and xylulose fermentation, although this might in turn be shown to be dependent on other factors such as downstream pathway enzymes. Eliasson et al. (2001) proposed a kinetic model, which implied that, under simplified simulation conditions, a 1:≥10:≥4 relationship of the XR/XDH/XK ratio was optimal in minimising xylitol formation during

xylose utilisation in yeast. Overexpression of XK was found to be necessary for ethanol formation from xylose. Xylitol formation decreased with a decreasing XR/XDH ratio, while ethanol formation increased. A recombinant S. cerevisiae strain (TMB3004) with a XR/XDH/XK ratio corresponding to the theoretical optimal ratio fermented xylose to ethanol efficiently (Eliasson et al. 2001).