Efficient gene disruption in the koji-mold Aspergillus sojae using a novel variation of the positive-negative method

Molecular Genetics and Genomics : MGG
T TakahashiKeietsu Abe

Abstract

When no phenotypic screen is available, gene disruption in the koji-mold Aspergillus sojae is a time-consuming process, owing to the low frequency of homologous recombination. To achieve efficient gene disruption in the koji-mold, we developed a novel positive-negative selection method to enrich for homologous recombinants. The pyrG gene from A. sojae was used as a positive selection marker for transformants, and the oliC31 gene of A. nidulans, which codes for a mutant form of subunit 9 of the F1FO-ATPase, was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. The positive-negative selection markers, in combination with a pyrG deletion strain as a host, enabled enrichment for homologous recombinants, and disruption of the genes niaD, areA and tannase was successfully demonstrated. In order to examine whether the positive-negative selection technique is effective for targeting any locus, even in the absence of information on gene function or phenotype, we attempted to disrupt the aflR gene of A. sojae, which codes for a putative transcription factor for the aflatoxin biosynthetic pathway, using the method. Despite the fact that this gene is not transcribed in...Continue Reading

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Citations

Feb 14, 2006·Molecular Genetics and Genomics : MGG·Tadashi TakahashiYasuji Koyama
Oct 27, 2009·Molecular Genetics and Genomics : MGG·Feng Jie JinYasuji Koyama
Dec 31, 2011·Molecular Genetics and Genomics : MGG·Seiichi HaraYasuji Koyama
Feb 10, 2006·Cell Research·Richard J WeldHayley J Ridgway
Oct 28, 2008·Applied and Environmental Microbiology·Tadashi TakahashiYasuji Koyama
May 23, 2012·Applied and Environmental Microbiology·Kotaro ItoYasuji Koyama
Jul 3, 2007·Eukaryotic Cell·Tomonori FujiokaKeietsu Abe
Jan 24, 2006·Bioscience, Biotechnology, and Biochemistry·Tadashi TakahashiYasuji Koyama
Sep 23, 2009·Fungal Genetics and Biology : FG & B·Kazutoshi SakamotoKatsuya Gomi
Oct 9, 2008·Biotechnology Advances·David Lubertozzi, Jay D Keasling
Feb 3, 2007·Journal of Bioscience and Bioengineering·Satoshi SuzukiYutaka Kashiwagi
Mar 15, 2013·Fungal Genetics and Biology : FG & B·Zsuzsanna KovácsTünde Pusztahelyi
Apr 26, 2016·Bioscience, Biotechnology, and Biochemistry·Motoaki Sano
May 20, 2014·Applied and Environmental Microbiology·Tadashi TakahashiTetsuya Oguma
Nov 24, 2018·Bioscience, Biotechnology, and Biochemistry·Yasutomo ShinoharaYasuji Koyama
Mar 22, 2014·The Journal of General and Applied Microbiology·Feng Jie JinYasuji Koyama
Apr 25, 2019·Applied Microbiology and Biotechnology·Ying DingXiao-Jun Ji
Dec 11, 2020·The Journal of General and Applied Microbiology·Yasuko ArakiSeiichi Hara

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