申请人:DANISCO A/S
公开号:EP1514936A2
公开(公告)日:2005-03-16
The subject invention lies in the field of microorganism mutation and selection of the mutants. In particular, the invention is directed at obtaining metabolic mutants in a simple, direct and specific manner. In a preferred embodiment it is also possible to obtain desired mutants not comprising recombinant DNA, thereby facilitating incorporation thereof in products for human consumption or application, due to shorter legislative procedures. The method according to the invention involves random mutation and specific selection of the desired metabolic mutant. A nucleic acid cassette comprising a nucleic acid sequence encoding a bidirectional marker, said nucleic acid cassette further comprising a basic transcriptional unit operatively linked to the nucleic acid sequence encoding the bidirectional marker and said nucleic acid cassette further comprising an inducible enhancer or activator sequence linked to the basic transcription unit in such a manner that upon induction of the enhancer or activator sequence the bidirectional marker encoding nucleic acid sequence is expressed, said inducible enhancer or activator sequence being derived from a gene associated with activity of part of metabolism, said inducible enhancer.or activator sequence being derived from a gene associated with metabolism is claimed as is application thereof in a selection method for mutants. In addition a regulator gene xlnR encoding an activating regulator of an inducible enhancer or activator sequence and application of said gene and/or its expression product in overexpression of homologous or heterologous protein or peptide is described. Knockout mutants wherein said gene is absent or inactivated and mutants with increased or decreased DNA binding capacity are also claimed.
本发明涉及微生物突变和突变体选择领域。特别是,本发明旨在以简单、直接和特异的方式获得代谢突变体。在一个优选的实施方案中,还可以获得不包含重组 DNA 的所需突变体,从而由于缩短了立法程序,便于将其加入供人类消费或应用的产品中。本发明的方法包括随机突变和特异性选择所需的代谢突变体。一种核酸盒,包括编码双向标记的核酸序列,所述核酸盒进一步包括与编码双向标记的核酸序列可操作地连接的基本转录单元,所述核酸盒进一步包括与基本转录单元连接的可诱导的增强子或激活子序列,其连接方式为当增强子或激活子序列被诱导时,编码双向标记的核酸序列被表达、所述可诱导的增强子或激活子序列来自与部分代谢活动有关的基因,所述可诱导的增强子。或激活剂序列来源于与新陈代谢有关的基因,要求将其应用于突变体的选择方法中。此外,还描述了一种编码可诱导增强子或激活子序列的激活调节因子的调节基因 xlnR,以及所述基因和/或其表达产物在过表达同源或异源蛋白质或肽中的应用。此外,还提出了上述基因缺失或失活的基因敲除突变体,以及 DNA 结合能力增强或减弱的突变体。