Improving the homologous recombination efficiency of Yarrowia lipolytica
by grafting the heterogenous component from Saccharomyces cerevisiae
Abstract
The oleaginous non-conventional yeast Yarrowia lipolytica has enormous
potential as a microbial platform for the synthesis of various
bioproducts. However, while the model yeast Saccharomyces cerevisiae has
very high homologous recombination (HR) efficiency, non-homologous
recombination is dominant in Y. lipolytica, and foreign genes are
randomly inserted into the genome. Consequently, the low HR efficiency
greatly restricts the genetic engineering of this yeast. In this study,
RAD52, the key component of the HR machinery in S. cerevisiae, was
grafted into Y. lipolytica to improve HR efficiency. The gene ade2,
whose deletion can result in a brown colony phenotype, was used as the
reporter gene for evaluating the HR efficiency. The HR efficiency of Y.
lipolytica strains before and after integrating the ScRad52 gene was
compared using insets with homology arms of different length. The
results showed that the strategy could achieve gene targeting
efficiencies of up to 95% with a homology arm length of 1000 bp, which
was 6.5 times of the wildtype strain and 1.6 times of the traditionally
used ku70 disruption strategy. This study will facilitate the further
genetic engineering of Y. lipolytica to make it a more efficient cell
factory for the production of value-added compounds.