Metabolic engineering of Yarrowia lipolytica with massive gene assembly and genomic integration

Synthetic biology has developed varied strategies of one-pot multigene assembly and genomic integration to promote metabolic engineering of chassis. However, such strategies for engineering oleaginous yeast Yarrowia lipolytica is lacking, given the current stage that at most 5 exogenous genes (around 13 kb) can be assembled and integrated in the genome at once. Here, we develop a strategy of massive gene assembly and integration in Y. lipolytica genome. As a proof of concept, dozen-gene assembly (around 30 kb) and integration are achieved stably and reproducibly, and a Y.lipolytica chassis containing a total of 35 exogenous genes (sum of about 93.5 kb) is constructed. The introductions ofmassive genes exhibit modulation of natural product synthesis to different extents, significantly expands the capability of Y.lipolytica metabolic engineering.