Precise regulation of gene expression is crucial for the construction of high-performance cell factories,and non-nutrient source-based small molecule inducible system has a wide range of application potential in the regulation of gene expression.In this study,a Tet-on inducible system based on doxycycline response was developed in Komagataella phaffii and every elements of the system were further optimized.A series of inducible promoters that can respond to doxycycline were eventually obtained by screening appropriate core promoter sequences,optimizing the promoters that regulate rtTA expression,optimizing the transcriptional activation domains,and creating rtTA mutants.Among them,the Tet-on inducible system based on rtTA-V10 had a low background level leakage(fluorescence value was 1.05 times that of wild-type strains),and a dynamic range of fluorescence intensity reached 52.76 times after the addition of doxycycline.The rtTA-VPR-based Tet-on inducible system showed good expression intensity under various carbon source conditions,reaching 111.16％ of PAOX1 under methanol condition,and 110.65％,106.05％,112.24％ of PGCW14 under the glycerol,ethanol and glucose condition,respectively.Precise gene regulation can be achieved by adding different concentrations of inducers,which provides a powerful genetic manipulation tool for precise gene expression regulation in the fields of Komagataella phaffii metabolic engineering and synthetic biology.
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