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Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii

&NA; The non‐conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non‐homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4‐disrupted strain. To assess the roles of non‐homologous end joining (NHEJ)‐related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker‐recycling methods (yielding either a seamless gene deletion or a Cre‐loxP‐mediated gene deletion). The resulting dnl4‐ and/or ku70‐deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock‐out and gene knock‐in experiments. The Dnl4p‐defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild‐type and Ku70p‐deffective strains. The dnl4 ku70 double knock‐out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.