Yaver D S, Lamsa M, Munds R, Brown S H, Otani S, Franssen L, Johnstone J A, Brody H
Novo Nordisk Biotech, Davis, California 95616, USA.
Fungal Genet Biol. 2000 Feb;29(1):28-37. doi: 10.1006/fgbi.1999.1179.
Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Fungal Genetics and Biology 29, 28-37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzae pyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.
利用DNA标签诱变提高米曲霉中异源蛋白的产量。《真菌遗传学与生物学》29卷，第28 - 37页。限制酶介导的整合（REMI）已被用作诱变剂，在表达嗜热栖热菌脂肪酶的米曲霉菌株中构建了两个插入文库。使用含有米曲霉pyrG基因以及BamHI或EcoRI酶的线性化质粒构建REMI文库。对这些文库进行脂肪酶产量筛选，并分离出产量增加的突变体。从脂肪酶滴度增加的一个突变体（DEBY10.3）中克隆了整合事件侧翼的基因组DNA。侧翼DNA的核苷酸序列显示与构巢曲霉palB基因相似。在产脂肪酶的菌株中破坏palB基因导致脂肪酶表达增加。此外，对DEBY10.3的palB表型进行互补导致脂肪酶产量下降。这些证据表明，DEBY10.3中脂肪酶产量的增加与pyrG基因整合到palB基因产生的palB表型有关。结果还表明，用REMI进行标签诱变可用于鉴定影响异源蛋白表达的基因。
