Jin Feng Jie, Maruyama Jun-Ichi, Juvvadi Praveen Rao, Arioka Manabu, Kitamoto Katsuhiko
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
FEMS Microbiol Lett. 2004 Oct 1;239(1):79-85. doi: 10.1016/j.femsle.2004.08.025.
We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD-sC-). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD-sC-adeA-) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD-sC-DeltaargB adeA- or niaD-sC-DeltaargB adeB-) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD-sC-DeltaargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.
我们之前通过对双营养缺陷型宿主(niaD-sC-)进行紫外线诱变,分离出红色腺嘌呤营养缺陷型突变体,从而在米曲霉中设计了一种三重营养缺陷型宿主-载体系统。在本研究中,我们通过破坏参与精氨酸生物合成的argB基因,努力利用该系统构建一种新型的四重营养缺陷型宿主。argB基因破坏盒通过融合PCR产生,该方法只需两步PCR就能将选择标记adeA插入目标argB基因。将嵌合DNA片段转化到三重营养缺陷型菌株(niaD-sC-adeA-)中,以较高的效率(约40%)获得了argB破坏体。通过正常的菌落颜色和引入野生型argB基因使精氨酸营养缺陷型逆转来对argB破坏体进行表征。随后,对三重营养缺陷型菌株(niaD-sC-ΔargB)进行紫外线诱变,然后筛选红色菌落以检测腺嘌呤营养缺陷型,从而分离出四重营养缺陷型菌株(niaD-sC-ΔargB adeA-或niaD-sC-ΔargB adeB-)。所得结果表明,adeA基因在开发具有四重营养缺陷型的新型米曲霉宿主-载体系统中作为一种有效的选择标记,从而为从无法进行有性杂交的半知菌中培育多种营养缺陷型突变体提供了一种强大的工具。