Engineering the architecture of erythritol-inducible promoters for regulated and enhanced gene expression in Yarrowia lipolytica

The non-conventional model yeast Yarrowia lipolytica is of increasing interest as a cell factory for producing recombinant proteins or biomolecules with biotechnological or pharmaceutical applications. To further develop the yeast's efficiency and construct inducible promoters, it is crucial to better understand and engineer promoter architecture. Four conserved cis-regulatory modules (CRMs) were identified via phylogenetic footprinting within the promoter regions of EYD1 and EYK1, two genes that have recently been shown to be involved in erythritol catabolism. Using CRM mutagenesis and hybrid promoter construction, we identified four upstream activation sequences (UASs) that are involved in promoter induction by erythritol. Using RedStarII fluorescence as a reporter, the strength of the promoters and the degree of erythritol-based inducibility were determined in two genetic backgrounds: the EYK1 wild type and the eyk1Δ mutant. We successfully developed inducible promoters with variable strengths, which ranged from 0.1 SFU/h to 457.5 SFU/h. Erythritol-based induction increased 2.2 to 32.3 fold in the EYK1 + wild type and 2.9 to 896.1 fold in the eyk1Δ mutant. This set of erythritol-inducible hybrid promoters could allow the modulation and fine-tuning of gene expression levels. These promoters have direct applications in protein production, metabolic engineering and synthetic biology.