LAUSR

With the rapid development of synthetic biology, the oleaginous yeast Yarrowia lipolytica has become an attractive microorganism for chemical production. To better optimize and reroute metabolic pathways, we have expanded the CRISPR‐based gene expression toolkit of Y. lipolytica. By sorting the integration sites associated with high expression, new neutral integration sites associated with high expression and high integration efficiency were identified. Diverse genetic components, including promoters and terminators, were also characterized to expand the expression range. We found that in addition to promoters, the newly characterized terminators exhibited large variations in gene expression. These genetic components and integration sites were then used to regulate genes involved in the lycopene biosynthesis pathway, and different levels of lycopene production were achieved. The CRISPR‐based gene expression toolkit developed in this study will facilitate the genetic engineering of Y. lipolytica.