Murali kannan Maruthamuthu

Engineering Escherichia coli to sense acidic amino acids by introduction of a chimeric two-component system

In an attempt to create an acidic amino acid-sensing Escherichia coli, a chimeric sensor kinase (SK)-based biosensor was constructed using Pseudomonas putida AauS. AauS is a sensor kinase that ultimately controls expression of the aau gene through its cognate response regulator AauR, and is found only in P. putida KT2440. The AauZ chimera SK was constructed by integration of the sensing domain of AauS with the catalytic domain of EnvZ to control the expression of the ompC gene in response to acidic amino acids. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as the concentration of acidic amino acids increased. These data suggest that AauS-based recombinant E. coli can be used as a bacterial biosensor of acidic amino acids. By employing the chimeric SK strategy, various bacteria biosensors for use in the development of biochemical-producing recombinant microorganisms can be constructed.

Engineering chimeric two-component system into Escherichia coli from Paracoccus denitrificans to sense methanol

Escherichia coli does not have the methanol sensing apparatus, was engineered to sense methanol by employing chimeric two-component system (TCS) strategy. A chimeric FlhS/EnvZ (FlhSZ) chimeric histidine kinase (HK) was constructed by fusing the sensing domain of Paracoccus denitrificans FlhS with the catalytic domain of E. coli EnvZ. The constructed chimeric TCS FlhSZ/OmpR could sense methanol by the expression of ompC and gfp gene regulated by ompC promoter. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed the dynamic response of the chimeric TCS to methanol. The expression of ompC and the gfp fluorescence was maximum at 0.01 and 0.5% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric HK FlhSZ. This strategy can be employed for the construction of several chimeric TCS based bacterial biosensors for the development of biochemical producing recombinant microorganisms.

Construction of methanol sensing Escherichia coli by the introduction of novel chimeric MxcQZ/OmpR two-component system from Methylobacterium organophilum XX

Methylobacterium organophilum XX is a type II facultative methylotroph that can grow on methanol. In M. organophilum XX, the MxcQ/MxcE two-component system (TCS) is involved in methanol metabolism. EnvZ/OmpR in E. coli TCS was exploited to develop a methanol biosensor by engaging the MxcQ/MxcE TCS system. The MxcQZ/OmpR methanol sensing chimeric TCS was constructed by integrating the sensing domain of M. organophilum MxcQ with the transmitter domain of E. coli EnvZ. The response regulator of the chimeric TCS system is OmpR, which regulates the expression of the ompC and gfp. The expression of ompC was monitored by real-time quantitative PCR analysis. The expression of gfp also confirmed the expression of the ompC. The maximum expression of ompC and gfp occurred with 0.05% of methanol, and the expression started to decline with further increases in methanol concentration. This system delivers rapid detection of methanol in the environment.

Engineering a chimeric malate two-component system by introducing a positive feedback loop in Escherichia coli

Previous studies constructed a chimeric MalKZ two-component system to sense environmental malate. In this study, we used a positive feedback loop to accelerate and amplify the output signal indicating malate concentration. The positive feedback loop was constructed by cloning ompR gene, which encodes ompC and induces OmpR protein; ompC promoter was used to control the process. The transcriptional expression profile showed that the expression level of ompC gene increased about two-fold after the positive feedback loop was introduced. When GFP was used as a reporter protein, a 71% increase in fluorescence level was observed. The results indicate that the signal transduction kinetics of MalKZ can be engineered by introducing the positive feedback loop.

Modification of the dynamic nature of the chimeric fumarate two-component system in Escherichia coli via positive feedback loop

A positive feedback loop was introduced to modify the dynamic behavior of fumarate sensing DcuSZ chimera TCS. To construct the positive feedback loop, the ompR gene was cloned downstream of the ompC promoter. The ompC promoter induced the expression of OmpR, which in turn induced the expression of the ompC promoter. Through the introduction of this positive feedback loop, the transcriptional expression levels of ompC increased 2.6-fold. When GFP was used as a reporter protein, a 64% increase in fluorescence level was observed. These results suggest that sensitivity of the TCS based fumarate sensing system can be engineered through the introduction of a positive feedback loop.