<i>De Novo</i> Synthesis of Reticuline and Taxifolin Using Re-engineered Homologous Recombination in <i>Yarrowia lipolytica</i>

<i>Yarrowia lipolytica</i> has been widely engineered as a eukaryotic cell factory to produce various important compounds. However, the difficulty of gene editing and the lack of efficient neutral sites make rewiring of <i>Y. lipolytica</i> metabolism challenging. Herein, a Cas9 system was established to redesign the <i>Y. lipolytica</i> homologous recombination system, which caused a more than 56-fold increase in the HR efficiency. The fusion expression of the hBrex27 sequence in the C-terminus of Cas9 recruited more Rad51 protein, and the engineered Cas9 decreased NHEJ, achieving 85% single-gene positive efficiency and 25% multigene editing efficiency. With this system, neutral sites on different chromosomes were characterized, and a deep learning model was developed for gRNA activity prediction, thus providing the corresponding integration efficiency and expression intensity. Subsequently, the tool and platform strains were validated by applying them for the <i>de novo</i> synthesis of (<i>S</i>)-reticuline and (2<i>S</i>)-taxifolin. The developed platform strains and tools helped transform <i>Y. lipolytica</i> into an easy-to-operate model cell factory, similar to <i>Saccharomyces cerevisiae</i>.