Molecular techniques employed in CTG(Ser1) and CTG(Ala) D-xylose metabolizing yeast clades for strain design and industrial applications

Abstract: 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.