Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

Because of its potent antioxidant effects, lycopene has been used in various industries including, but not limited to, food, medical, and cosmetic industries. Yarrowia lipolytica, a non-conventional yeast species, is a promising chassis due to its natural mevalonate (MVA) pathway, abundant precursor acetyl coenzyme A content, and oleaginous properties. Several gene editing tools have been developed for Y. lipolytica along with engineering strategies for tetraterpenoid production. In this study, we engineered Y. lipolytica following multi-level strategies for efficient lycopene accumulation. We first evaluated the performance of the key lycopene biosynthetic genes crtE, crtB, and crtI, expressed via ribosomal DNA (rDNA) mediated multicopy random integration in the HMG1- and GGS1-overexpressing background strain. Further improvement in lycopene production was achieved by overexpressing the key genes for MVA synthesis via non-homologous end joining (NHEJ) mediated multi-round iterative transformation. Efficient strategies in the MVA and lipid synthesis pathways were combined to improve lycopene production with a yield of 430.5 mg/L. This strain produced 121 mg/g dry cell weight of lycopene in a 5-L fed-batch fermentation system. Our findings demonstrated iterative gene integration mediated by 26S rDNA and NHEJ for the efficient production of lycopene in Y. lipolytica. These strategies can be applied to induce Y. lipolytica to produce other tetraterpenoids.