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 the genetic engineering of the 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 into the genome at once. Here, we developed a strategy of massive gene assembly and integration in the Y. lipolytica genome. As a proof of concept, dozen-gene assembly (more than 30 kb) and integration were achieved stably and reproducibly, and a Y. lipolytica chassis containing a total of 35 exogenous genes (a sum of 93.5 kb) was constructed. The introduction of massive genes modulated the synthesis of lycopene, a heterologous natural product, to quite different extents. Ultimately, an optimized constructed strain containing 15 exogenous genes achieved the highest yield of 144.58 mg/g DCW and produced a lycopene titer of 2144.83 mg/L in a 5 L bioreactor. Our strategy significantly expands the capability of Y. lipolytica genetic manipulation and metabolic engineering.