The non-conventional yeast Yarrowia lipolytica is gaining interest in biotechnology as host for production of proteins, lipids and other biomolecules thanks to its capacity to achieve high biomasses, and to express high titers of proteins and lipids. Site-specific genome editing is however limited in this yeast. This was much improved by the recent development of a CRISPR-Cas9 genome editing protocol for Y. lipolytica based on a tRNA-sgRNA fusion. Nonetheless, in the latter protocol, Cas9 is under the control of a synthetic hybrid promoter, pUAS1B8-TEF(136), that although reported as strong, yet its tandem repeats are associated with in vivo and in vitro inconveniences like polymerase slippage, random genetic rearrangements and cloning difficulties. Here we report an optimized TEF promoter for Cas9 expression, pTEF(366)-Kozak, which is a rationally 3’-truncated version of the 5’-truncated pTEF(406) promoter of Y. lipolytica fused to a synthetic Kozak sequence. Comparison of the promoters’ strength using hrGFP reporters and RT-qPCR revealed that the 3’truncated synthetic pTEF(366)-Kozak maintained the same expression level as the pTEF(406), yet both are associated with at least 5-folds higher expression than the synthetic pUAS1B8-TEF(136). Moreover, Cas9 under pTEF(366)-Kozak resulted in higher genome editing efficiencies by 40% compared to pUAS1B8-TEF(136). Phenotypic losses resulting from Cas9-mediated gene deletions revealed no significant differences among tested promoters as phenotypic losses were already at their maxima. This is the first study conducting rational 3’-truncation in TEF promoter based on in silico predictions of its structure, which can be extended for the engineering of other yeast promoters to generate small-sized synthetic biology parts for convenient engineering of biological systems.

co-author: Benjamin Ouellet