Construction Of A Ligd Disruptant For Efficient Gene Targeting In White Koji Mold, Aspergillus Kawachii

Several species of the filamentous fungal genus Aspergillus have traditionally been used as koji molds for brewing alcoholic beverages in Japan. Koji is rice or barley that has been polished, steamed, and covered with the hyphal growth of a fungus, whose secreted enzymes convert the starch present in the grains to glucose (Akiyama, 2010). Black koji mold, A. luchuensis, and its albino mutant, white koji mold, A. kawachii, have been used for making the Japanese distilled spirit, shochu. Since shochu is mainly produced in Kyushu, Japan, where the climate is relatively warm, citric acid-producing A. kawachii and A. luchuensis were selected to make shochu to prevent undesirable contamination of bacteria. Although these two species of koji mold are phylogenetically close to A. niger, which is used industrially to produce citric acid, they are distinctly separated from A. niger (Yamada et al., 2011). The mechanism for hyperproduction of citric acid by A. kawachii but not by sake-making A. oryzae remains to be elucidated. Although the genome information about A. kawachii IFO(NBRC)4308 has been determined (Futagami et al., 2011), genetic studies for A. kawachii are very limited due to non availability of auxotroph. In general, it is hard to accomplish the gene targeting using a homologous recombination system in filamentous fungi. This is associated with the high ability of the nonhomologous end joining (NHEJ) system in filamentous fungi. Ninomiya et al. first demonstrated that inactivation of NHEJ highly enhances the homologous recombination in Neurospora crassa (Ninomiya et al., 2004). After this, the disruptants of the genes, kuA, kuB, or ligD, that are involved in NHEJ, from various filamentous fungi including A. nidulans (Nayak et al., 2006), A. oryzae, A. sojae (Takahashi et al., 2006), A. niger (Meyer et al., 2007), and A. luchuensis (Takahashi et al., 2011) were constructed. They showed highly efficient homologous recombination. In the present paper, we constructed A. kawachii strains with the ability of highly efficient homologous recombination and with the auxotrophy to accomplish the genetic study of A. kawachii. Since we have determined genome sequence of A. kawachii IFO4308 (Futagami et al., 2011), the sequence information about the strain IFO4308 was used for design of PCR primers. A DNA cassette for disruption of the ligD was amplified by PCR using a plasmid pUC∆ligD (Takahashi et al., 2011) as a template. The DNA cassettes for disruption of pyrG and argB, and for replacement of ptrA by hph, were done J. Gen. Appl. Microbiol., 59, 257‒260 (2013)