Construction of a ligD disruptant for efficient gene targeting in white koji mold, Aspergillus kawachii.
Construction of a ligD disruptant for efficient gene targeting in white koji mold, Aspergillus kawachii.
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DOI：
10.2323/jgam.59.257
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发表时间：
2013
期刊：
影响因子：
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通讯作者：
S. Tashiro;Taiki Futagami;Shotaro Wada;Y. Kajiwara;H. Takashita;T. Omori;Toru Takahashi;O. Yamada;K. Takegawa;M. Goto
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文献类型：
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作者：
S. Tashiro;Taiki Futagami;Shotaro Wada;Y. Kajiwara;H. Takashita;T. Omori;Toru Takahashi;O. Yamada;K. Takegawa;M. Goto
在日本，丝状真菌曲霉属的几个物种传统上被用作酿造酒精饮料的曲霉菌。曲是大米或大麦，经过抛光，蒸煮，并覆盖着真菌的菌丝生长，其分泌的酶将谷物中的淀粉转化为葡萄糖（Akiyama，2010）。黑曲霉A. luchuensis及其白化病突变株白色曲霉A.川内酒被用来酿造日本的蒸馏酒--烧酒。由于烧酒主要产于气候相对温暖的日本九州，因此产柠檬酸的A。kawachii和A.选择luchuensis来制作烧酒以防止不希望的细菌污染。虽然这两种曲霉菌在遗传学上与A.尼日尔，工业上用于生产柠檬酸，它们与A.尼日尔（Yamada等人，2011年）。对A. kawachii而不是酿酒A.这一点有待澄清。虽然A. kawachii IFO（NBRC）4308已确定（Futagami等人，2011），A. Kawachii由于营养缺陷型的不可用而非常有限。一般来说，在丝状真菌中使用同源重组系统很难实现基因打靶。这与丝状真菌中的非同源末端连接（NHEJ）系统的高能力有关。二宫等人首先证明NHEJ的失活高度增强粗糙脉孢菌中的同源重组（二宫等人，2004年）。在此之后，从包括A.构巢菌（Nayak等人，2006），A.米A. sojae（Takahashi等人，2006），A.尼日尔（Meyer等人，2007）和A. luchuensis（Takahashi等，2011年）建成。它们表现出高效的同源重组。在本文中，我们构造了A.利用具有高效同源重组能力和营养缺陷型的kawachii菌株完成A.川内。由于我们已经测定了A. kawachii IFO 4308（Futagami等人，2011），将关于菌株IFO 4308的序列信息用于PCR引物的设计。通过PCR使用质粒pUC pigD（Takahashi等人，2011年，作为模板。用于破坏pyrG和argB以及用于用hph替换ptrA的DNA盒由J. Gen. Appl. Microbiol.，59，257 - 260（2013）
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)