Novel promoter of Pseudozyma antarctica exhibits stable glucose-mediated activation and copper-mediated suppression of flanking genes

Pseudozyma antarctica is a leaf-colonizing basidiomycetous yeast that produces a plastic-degrading enzyme (PaE) using xylose as an inducer and carbon source. Notably, glucose, a readily available carbon source, has not been utilized for PaE production. Herein, we identified two promoters of P. antarctica for stable induction of PaE gene transcription at high levels in commonly used yeast media with glucose, along with assessing the effect of copper supplementation. Through microarray analysis, the top five genes with high expression in glucose cultures were identified and their promoter activities were evaluated by reporter assay. The first and second genes were located 903 nucleotides apart on the P. antarctica chromosome and shared reciprocal inverted sequences as promoters, namely PaFRE1 and PaCTR1, respectively, owing to their weak similarity to FRE and CTR in Saccharomyces cerevisiae genome, which regulate the copper uptake. When P. antarctica was grown in three common media using glucose as a carbon source, both PaFRE1 and PaCTR1 induced high and stable expression and produced more heterologous artificial luciferase and endogenous PaE than that by the highly expressed actin promoter. Additionally, the activities of PaFRE1 and PaCTR1 promoters were suppressed by copper addition. Altogether, this study shows that these P. antarctica promoters can be used to produce various proteins such as PaE.