YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica .

Effective metabolic engineering of microorganisms relies on balanced expression of both heterologous and endogenous genes to channel metabolic flux towards products of interest while achieving reasonable biomass buildup. To facilitate combinatorial pathway engineering and facile genetic operation, we engineered a set of modular cloning vectors compatible with BioBrick standards, called YaliBricks, to allow for rapid assembly of multigene pathways with customized genetic control elements (promoters, intronic sequences and terminators) in the oleaginous yeast Yarrowia lipolytica . We established a sensitive luciferase reporter and characterized a set of 12 native promoters to expand the oleaginous yeast genetic toolbox for transcriptional fine-tuning. We harnessed the intron alternative splicing mechanism and explored three unique gene configurations that allow us to encode genetic structural variations into metabolic function. We elucidated the role of how these genetic structural variations affect gene expression. To demonstrate the simplicity and effectiveness of streamlined genetic operations, we assembled the 12 kb five-gene violacein biosynthetic pathway in one week. We also expanded this set of vectors to accommodate self-cleavage ribozymes and efficiently deliver guide RNA (gRNA) for targeted genome-editing with a codon-optimized CRISPR-Cas9 nuclease. Taken together, the tools built in this study provide a standard procedure to streamline and accelerate metabolic pathway engineering and genetic circuits construction in Yarrowia lipolytica .