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Microbiology and Biotechnology Letters

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Microbial Biotechnology (MB)  |  Synthetic Biology and Nanobiotechnology

Microbiol. Biotechnol. Lett. 2020; 48(1): 24-31

https://doi.org/10.4014/mbl.1908.08009

Received: August 20, 2019; Accepted: November 19, 2019

Engineering of Recombinant Escherichia coli towards Methanol Sensing Using Methylobacterium extroquens Two-component Systems

Vidhya Selvamani , Irisappan Ganesh , Sowon Chae , Murali kannan Maruthamuthu and Soon Ho Hong *

University of Ulsan

Five genes (mxbDM, mxcQE and mxaB) are responsible for the transcription of methanol oxidation genes in Methylobacterium strains. Among these, MxbDM and MxcQE constitute the two-component system (TCS) regulating methanol metabolism. In this study, we integrated the methanol-sensing domain of MxbD and MxcQ with the EnvZ/OmpR from Escherichia coli. The domain-swapping strategy resulted in chimeric histidine kinases (HK’s) MxbDZ and MxcQZ AM1 containing recombinant E. coli. Real-time quantitative PCR was used to monitor OmpC expression mediated by the chimeric HK and response regulator (RR) OmpR. Further, an ompC promoter based fluorescent biosensor for sensing methanol was developed. GFP fluorescence was studied both qualitatively and quantitatively in response to environmental methanol. GFP measurement also confirmed ompC expression. Maximum fluorescence was observed at 0.05% methanol and 0.01% methanol using MxbDZ and MxcQZ AM1, respectively. Thus the chimeric HK containing E. coli were found to be highly sensitive to methanol, resulting in a rapid response making them an ideal sensor.

Keywords: Methanol, chimeric two-component system, green fluorescent protein, Escherichia coli, biosensor

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