| Summary: | Converting lignocellulosic biomass into value-added products is one of the challenges in developing a sustainable economy. Attempts to engineer fermenting yeasts to recover plant waste are underway. Although intensive metabolic engineering has been conducted to obtain <i>Saccharomyces cerevisiae</i> strains capable of metabolising pentose sugars mainly found in hemicellulose, enzymatic hydrolysis after pretreatment is still required. <i>Blastobotrys raffinosifermentans</i>, which naturally assimilates xylose and arabinose and displays numerous glycoside hydrolases, is a good candidate for direct and efficient conversion of renewable biomass. However, a greater diversity of tools for genetic engineering is needed. Here, we report the characterisation of four new promising promoters, a new dominant marker, and two vectors for the secretion of epitope tagged proteins along with a straightforward transformation protocol. The <i>TDH3</i> promoter is a constitutive promoter stronger than <i>TEF1</i>, and whose activity is maintained at high temperature or in the presence of ethanol. The regulated promoters respond to high temperature for <i>HSP26</i>, gluconeogenic sources for <i>PCK1</i> or presence of xylose oligomers for <i>XYL1</i>. Two expression/secretion vectors were designed based on p<i>TEF1</i> and p<i>TDH3</i>, two endogenous signal peptides from an α-arabinanase and an α-glucuronidase, and two epitopes. A heterologous α-arabinoxylan hydrolase from <i>Apiotrichum siamense</i> was efficiently secreted using these two vectors.
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