RESUMEN
BACKGROUND: With the development of engineering the microbial cell factories, biosensors have been used widely for regulation of cellular metabolism and high-throughput screening. However, most of the biosensors constructed in Saccharomyces cerevisiae are designed for transcriptional activation. Very few studies have dedicated to the development of genetic circuit for repressive regulation, which is also indispensable for the dynamic control of metabolism. RESULTS: In this study, through transcriptional deactivation design, we developed transcription-factor-based biosensors to allow repressive regulation in response to ligand. Using a malonyl-CoA sensing system as an example, the biosensor was constructed and systematically engineered to optimize the dynamic range by comparing transcriptional activity of the activators, evaluating the positions and numbers of the operators in the promoter and comparing the effects of different promoters. A biosensor with 82% repression ratio was obtained. Based on this design principle, another two biosensors, which sense acyl-CoA or xylose and downregulate gene expression, were also successfully constructed. CONCLUSIONS: Our work systematically optimized the biosensors for repressive regulation in yeast for the first time. It provided useful framework to construct similar biosensors. Combining the widely reported biosensors for transcriptional activation with the biosensors developed here, it is now possible to construct biosensors with opposing transcriptional activities in yeast.
Asunto(s)
Técnicas Biosensibles , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación Fúngica de la Expresión Génica , Ingeniería Genética , Malonil Coenzima A/metabolismo , Ingeniería Metabólica , Regiones Promotoras Genéticas , Dominios Proteicos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Activación Transcripcional/genética , Xilosa/genética , Xilosa/metabolismoRESUMEN
Fine-tuning of the expression of genes is crucial for cell factory construction. Promoters are the most important tools to control gene expression. However, native promoters are often limited by their transcriptional ability. In this study, we sought to overcome the limitations of native promoters in Saccharomyces cerevisiae through the construction of hybrid promoter libraries for both constitutive promoters and promoters induced by diauxic shift. A series of hybrid constitutive promoters were constructed by combing the upstream activation sequences and changing the core promoter elements. The transcriptional capacity of the strongest promoter was 2-fold higher than that of the yeast native TEF1 promoter. Aside from the constitutive promoters, hybrid promoters that were induced in the post-diauxic phase were also constructed. These promoters had low transcriptional ability during growth on glucose and automatically activated upon growth with a diauxic shift. The strength of these promoters was also increased by replacing the core promoter with strong core promoters. Our study provides a series of constitutive and diauxic shift-induced promoters with a broad range of transcriptional capacity and will facilitate synthetic biology and metabolic engineering application.