Development of precise genome editing and multi-copy integration tools in Hansenula polymorpha DL-1

<i>Hansenula polymorpha</i> DL-1 is a thermotolerant yeast capable of utilizing multiple renewable carbon sources, making it a promising microbial cell factory for sustainable manufacturing. However, advanced metabolic engineering efforts have been constrained by its strong non-homologous end joining (NHEJ) mechanism and limited choice of suitable genetic tools. This study presents an optimized synthetic biology toolkit to address these limitations. A high-efficiency CRISPR-Cas9-based genome editing system was established, achieving an editing efficiency of 97.2 %. To further enhance homologous recombination (HR), the NHEJ pathway was partially suppressed by knocking out <i>KU80</i> and overexpressing HR-related genes from <i>Saccharomyces cerevisiae</i>. This increased HR rates to 88.9 %. In addition, 36 neutral sites were identified for stable single-copy gene integration without disrupting native gene expression cassettes. Finally, multi-copy integration tools were developed by targeting rDNA and Ty elements, leading to a ∼60-fold increase in β-carotene production compared with single-copy integrants. Furthermore, squalene titers were increased from 0.1 mg/L in the wild-type strain to 187.2 mg/L through iterative multi-copy integration. These advances significantly expand the genetic tractability of <i>H. polymorpha</i> DL-1, underscoring its potential as a versatile platform for efficient and sustainable production of value-added compounds.