Basal transcription profiles of the rhamnose-inducible promoter P-LRA3 and the development of efficient P-LRA3-based systems for markerless gene deletion and a mutant library in Pichia pastoris

文献类型： 外文期刊

第一作者： Jiao, Jian

作者： Jiao, Jian;Wang, Shuai;Liang, Mingli;Zhang, Yuhong;Xu, Xinxin;Zhang, Wei;Liu, Bo

作者机构：

关键词： Pichia pastoris; Rhamnose-inducible promoter; Basal transcription; Markerless gene deletion; Mutation system

期刊名称：CURRENT GENETICS （ 影响因子：3.886； 五年影响因子：3.697 ）

ISSN： 0172-8083

年卷期： 2019 年 65 卷 3 期

页码：

收录情况： SCI

摘要： An ideal inducible promoter presents inducibility with an inducer and no basal transcription without inducer. Previous studies have shown that P-LRA3 in Pichia pastoris is a strong rhamnose-inducible promoter for driving the industrial production of recombinant proteins. However, another important profile of P-LRA3, the basal transcription, was not investigated yet. In this study, the basal transcription of P-LRA3 was assessed according to the profiles of two test strains grown in media lacking rhamnose: (1) the production of secretory -galactosidase in P. pastoris GS115/P-LRA3-LacB, in which lacB expression was regulated by P-LRA3,P- and (2) growth in P. pastoris GS115/P-LRA3-MazF, in which the expression of mazF, which encodes an intracellular toxic protein, was controlled by P-LRA3. Analyses revealed low -galactosidase production and non-obviously inhibited growth of the test strains, which suggests that there was a low basal transcription level of P-LRA3 when rhamnose was absent. Thus, P-LRA3 was an excellent candidate for genetic manipulation in P. pastoris due to its strict regulation, a strong and a low transcriptional activity with and without rhamnose, respectively. Subsequently, two systems were developed based on P-LRA3 in P. pastoris: (1) an efficient markerless gene deletion system for single or multiple genes and (2) a high efficient piggyBac transposase-mediated mutation system for investigating the functions of unknown genes, as well as for the screening of expected mutants.

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