Redox cofactors imbalance

The CO-factor imbalance generated by the first two steps in xylose metabolism could be entirely circumvented if the conversion of xylose to xylulose were to be catalyzed by the prokaryotic enzyme xylose isomerase (XI, Fig. 1). o-Xylose (glucose) isomerase EC 5.3.1.5 catalyses the reversible isomerization of o-xylose and D-glucose to D-xylulose and D-fructose, respectively. XI does not require redox cofactors and cannot generate cofactor imbalance during anaerobic xylose utilization. 

Recombinant Saccharomyees cerevisiae, able to ferment the pentoses D-xylose and L-arabinose, was modified for improved fermentation rates and yields. Pentose fermentation is relevant when low cost raw materials such as plant hydrolysates are fermented to ethanol. The two most widespread pentose sugars in our biosphere are D-xylose and L-arabinose. S. cerevisiae is unable to ferment pentoses but has been engineered to do so however rates and yields are low. The imbalance of redox cofactors (excess NADP and NADH are produced) is considered a major limiting factor. For the L-arabinose fermentation we identified an NADH-dependent L-xylulose reductase replacing the previously known NADPH-dependent enzyme. For D-xylose fermentation we introduced an NADP-dependent glyceraldehyde 3-phospate dehydrogenase to regenerate NADPH. 

Many redox enzymes used in BFCs require cofactors for catalysis. For example, yeast alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde, with the concomitant reduction of NAD" to NADH. Most commonly, cofactor specificity has been engineered to increase the activity of NADP(H)-dependent enzymes with NAD(H) or to correct a cofactor imbalance in a metabolic process. As the NAD(H) cofactor is more stable and less expensive than NADP(H), this generally improves the economics of a process [42—44]. 

The pentoses, such as xylose (Xyl), that result from the hydrolysis of lignocel-lulose (see above) resist fermentation by Saccharomyces, because it lacks an efficient mechanism to convert Xyl into xylulose (Xlu). The isomerization redox interconversion pathway of Xyl and Xlu, via xylitol, that is native to Saccharomyces, is inefficient due to a cofactor incompatibility (see Fig. 8.5) and results in a redox imbalance and the accumulation of xylitol [24]. Many bacteria, in contrast,... 

In fungi, xylose is reduced to xylitol by NADH- or NADPH-dependent xylose reductase (XR) and thereafter is oxidized to xylulose by NAD -dependent xylitol dehydrogenase (XDH). The xylulose is phosphorylated, channeled into the pentose phosphate pathway [3]. XR of most fungi, including most yeasts, prefers NADPH to NADH. Because of the cofactor preference of XR (NADPH) and XDH (NAD, redox imbalance occurs under anaerobic condition [4]. Therefore, the oxygen-limited rather than anaerobic condition is ideal for bioconversion of xylose to ethanol, so that the accumulated reduced cofactor can be oxidized to reach redox balance. A critical level of oxygen should exist for the highest ethanol yield and productivity. 

Fig. 7 Hexose and pentose pathways for Eukarya and Bacteria. Mannose, glucose, and galactose are quickly phosphorylated after uptake in the cell. Pentoses are assimilated by yeast (solid lines) through an oxidoreductase pathway, whose bottleneck to xylose assimilation is the imbalance redox generated by xylose reductase (XR) and xylitol dehydrogenase (XDH) distinct cofactor preference. The same does not occur in bacteria, once single step xylose assimilation is done by enzyme xylose isomerase (XI). Blue dotted lines represent pentose assimilation pathways for Bacteria...

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