Metabolic Engineering of 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 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 genome. As a proof of concept, dozen-gene assembly (more than 30 kb) and integration were achieved stably and reproducibly, and a 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 genetic manipulation and metabolic engineering.