  D-xylose is the second most abundant monosaccharide found in lignocellulose
and is of biotechnological importance for producing second-generation ethanol
and other high-value chemical compounds. D-xylose conversion to ethanol is
promoted by microbial fermentation, mainly by bacteria, yeasts, or filamentous
fungi. Considering yeasts, species belonging to the CTG(Ser1) or CTG(Ala) clade
display a remarkable ability to ferment D-xylose to ethanol and other
compounds; however, these yeasts are not employed on an industrial scale due to
the poor fermentative performance compared to conventional yeasts, like
Saccharomyces cerevisiae, and also due to the lack of a molecular toolbox for
development of new strains tailored to fermentation stress tolerance and
performance. Thus, the purpose of this review is to evaluate the major
molecular tools (e.g., transformation markers and techniques, vectors,
regulatory sequences, and gene editing techniques) available for the most
studied yeasts of CTG(Ser1) clade, like Scheffersomyces, Spathaspora, Candida
and Yamadazyma species, and the CTG(Ala) clade representative Pachysolen
tannophilus. Furthermore, we synthesized the current state-of-the-art molecular
developments and perspectives for D-xylose fermenting yeast strain design.
