Iwakuma Hidekazu, Koyama Yoshiyuki, Miyachi Ayako, Nasukawa Masashi, Matsumoto Hitoshi, Yano Shuntaro, Ogihara Jun, Kasumi Takafumi
a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
b Neo-Morgan Laboratory Incorporated , Kawasaki , Japan.
Biosci Biotechnol Biochem. 2016;80(3):486-92. doi: 10.1080/09168451.2015.1104236. Epub 2015 Nov 5.
We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.
我们通过差异诱变获得了里氏木霉的一种新型葡萄糖去阻遏突变体。将含有缺乏校对活性的DNA聚合酶δ和自主复制序列AMAI的质粒导入里氏木霉ATCC66589。用5-氟乳清酸抗性评估的突变率比紫外线照射获得的突变率高约30倍。然后在含有潮霉素B的2-脱氧葡萄糖琼脂平板上筛选携带无功能DNA聚合酶δ的转化体。在含有葡萄糖的液体培养基中，突变体的对硝基苯-β-半乳糖苷水解活性比亲本高2至5倍。值得注意的是，参与葡萄糖阻遏的关键基因cre1的氨基酸序列在突变体和亲本菌株中是相同的，此外，cre1在突变体中的表达水平并未被消除。综上所述，这些结果表明，通过差异诱变产生的里氏木霉菌株是葡萄糖去阻遏变体，其在cre1以外的尚未鉴定的因子中含有突变。
