Page 77 - Advances in bioenergy (2016)

Among the strategies that have been adopted, some aim to alter the cofactor specificities of D-
xylose reductase (XR) and xylitol dehydrogenase (XDH) from P. stipitis, whereas others
adjust the redox balance through regulating the central pathways. 109 A strain developed by
chromosomal integration of the genes encoding XR, XDH, and xylulokinase (XK) fermented
whole pretreated slurry of corn stover at high WIS preferably in a fed-batch SSF process,
showing that the simultaneous coutilization of xylose was not favorable in the presence of high

amounts of glucose. 110 On the other hand, when fungal xylose isomerase (XI) genes were
functionally expressed in S. cerevisiae at high levels, the growth on xylose was very slow. As
mentioned above, a combined research approach implementing evolutionary engineering of the
constructed strain, improved significantly the fermentation rate of xylose in the presence of
glucose. 101 Promising results have recently been published describing the construction of
111
engineered yeasts, which coferment mixtures of xylose and cellobiose. In these yeast strains,
hydrolysis of cellobiose takes place inside yeast cells through the action of an intracellular β-
glucosidase following import by a high-affinity cellodextrin transporter. As shown,

intracellular hydrolysis of cellobiose minimizes glucose repression of xylose fermentation
allowing co-consumption of cellobiose and xylose.

CONCLUSION

After some decades of scientific efforts, the production of cellulosic ethanol in commercial

scale seems feasible, as demonstration and industrial scale ethanol plants pullulate year by
year in various countries. The great majority of the scientific reports on the field have so far
focused on one of three main targets: (1) the amelioration of the feedstock (plant or residue)
with regard to a readily convertible and efficient substrate, (2) the improvement of the
bioconversion process, and (3) the development of a desirable microbial factory which
efficiently carries out the bioconversion. When bioethanol feedstocks refer to residues, the
improvement is attempted through the pretreatment of the raw materials by chemical, physical,
and/or biological means, whereas the amelioration of the raw material refers to the genetic
engineering of plant cells, targeted to biofuels production, when whole plants are cultivated on
this purpose. Enzymatic hydrolysis is advanced by the discovery of a new class of oxidative
enzymes that cleave cellulose (GH61s), offering an exciting possibility for the improvement of
cellulose deconstruction. Metagenomic DNA and genetically engineered libraries should be
explored for the discovery of novel enzymes with improved cellulolytic activity on natural
substrates and increased inhibitor tolerance. The integration of the bioconversion process in a
more wide production scheme, where all biomass components are exploited and, at the same
time, different kind of products (fuels, chemicals, electricity, heat) are produced, is considered

as the most effective way to make cellulosic ethanol production profitable.

S. cerevisiae, the microorganism most widely and longer used in fermentation processes is still
a paradigm and a model for most modifications and improvements. Powerful tools as the
metabolic engineering and the inverse metabolic engineering based on evolutionary engineered
S. cerevisiae strains advance day by day. Solutions to the problem of the cofermentation of
pentoses and hexoses have been proved feasible through the combination of these techniques,