Page 75 - Advances in bioenergy (2016)

inhibitors. A way to exploit these intrinsic metabolic capabilities of S. cerevisiae, to improve
its fermentative performance in the presence of toxic compounds, is to adapt it to toxic
conditions by constant exposure to tolerable inhibitor concentrations in repetitive batch, fed-
batch, or continuous cultivation. A truly mechanistic understanding of the action of inhibitors is
not needed, but a well-designed selection procedure, on the other hand, is necessary to have a
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successful adaptation. Cultivation in increasing concentrations of selected individual
inhibitors or in increasing concentration of toxic hydrolysates will result in the increase in
frequency of adapted cells in the culture and accordingly in the isolation of an adapted strain.
However, the adapted properties of the strain may be inducible only under the selection
pressure imposed by the experimental conditions, as in the recent study on the adaptation of a
xylose-fermenting S. cerevisiae to acetic acid performed by Wright et al. 100

The promise of a successful coconsumption of xylose and glucose really boosted the
commercial bioethanol perspective. Kuyper et al. 101 managed to develop through a very
systematic experimental setup, an adapted S. cerevisiae xylose fermenting strain, showing
significantly improved xylose consumption rates under various conditions. More recently,
combined approaches of genetic engineering, chemical mutagenesis, and evolutionary
engineering were used to obtain high xylose-consuming rates by a recombinant S. cerevisiae

strain. 102 A significantly improved strain was developed from a parental chemically
mutagenized, when the latter was exposed to serial batch cultivations for growth in xylose
medium, first by aerobic and then under oxygen-limiting conditions using increasing xylose
concentrations for selection.

If not only improved strains, but also knowledge output, through evolutionary engineering are
desirable, the genetic alterations connected with the improved performance of the strains
developed have to be identified (Box 2.2). Inverse metabolic engineering (in later articles also
reported as reverse metabolic engineering) is the term introduced by Bailey et al. 103 to
describe biotechnological approaches which in contrast to the conventional ‘forward’
metabolic engineering cycle, begin from successful phenotypes toward the knowledge of the

factors and mechanisms that create or affect them. Genome-wide analytical approaches are
probably the key tool to a deeper understanding of them. As Oud et al. 104 recently underlined,
although whole genome sequencing applied for the reverse metabolic engineering of yeasts is
still small, the available information consistently indicates that this technique is a real game
changer. Adopting microarray and statistical two-way ANOVA analysis for analyzing
‘successful’ S. cerevisiae phenotypes, Stanley et al. 105 identified significant transcriptional
changes relevant to ethanol tolerance, revealing that improved ethanol tolerance is related to
increased mitochondrial and NADH oxidation activities, which also stimulates glycolysis.