A review of genetic engineering techniques for CTG(Ser1) and CTG(Ala) D-xylose-metabolizing yeasts employed for second-generation bioethanol production

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. Among 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 given their poor fermentative performance compared to that of conventional yeasts, such as Saccharomyces cerevisiae, and because of the lack of a molecular toolbox for the development of new strains tailored to fermentation stress tolerance and performance. Thus, the purpose of this review was to evaluate the major genetic engineering tools (e.g., transformation markers and techniques, vectors, regulatory sequences, and gene editing techniques) available for the most studied yeasts of the CTG(Ser1) clade, such as Scheffersomyces, Spathaspora, Candida, and Yamadazyma species, and the CTG(Ala) clade, representative Pachysolen tannophilus. Furthermore, we systematized state-of-the-art molecular developments and perspectives to design D-xylose-fermenting yeast strains.