Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for β-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of β-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.