Construction of the genetic switches in response to mannitol based on artificial MtlR box

Abstract

Synthetic biology has rapidly advanced from the setup of native genetic devices to the design of artificial elements able to provide organisms with highly controllable functions. In particular, genetic switches are crucial for deploying new layers of regulation into the engineered organisms. While the assembly and mutagenesis of native elements have been extensively studied, limited progress has been made in rational design of genetic switches due to a lack of understanding of the molecular mechanism by which a specific transcription factor interacts with its target gene. Here, a reliable workflow is presented for designing two categories of genetic elements, one is the switch element-MtlR box and the other is the transcriptional regulatory element- catabolite control protein A (CcpA) box. The MtlR box was designed for ON/OFF-state selection and is controlled by mannitol. The rational design of MtlR box-based molecular structures can flexibly tuned the selection of both ON and OFF states with different output switchability in response to varied kind effectors. Different types of CcpA boxes made the switches with more markedly inducer sensitivities. Ultimately, the OFF-state value was reduced by 90.69%, and the maximum change range in the presence of two boxes was 15.31-fold. This study presents a specific design of the switch, in a plug-and-play manner, which has great potential for controlling the flow of the metabolic pathway in synthetic biology.

Keywords

Genetic switch / MtlR box / Cre site / Bacillus licheniformis

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Funding

National Key Research & Development Program of China(2020YFA0907700)

the National Natural Foundation of China(32172174)

National First-Class Discipline Program of Light Industry Technology and Engineering(LITE2018-22)

the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions

the Postgraduate Research & Practice Innovation Program of Jiangsu Province(KYCX21-2027)