Irisappan Ganesh

Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system

DcuS/DcuR two component system (TCS) was firstly employed for the expression of the gfp gene under the dcuB gene promoter in aerobic condition to develop high throughput screening system able to screen microorganisms producing high amount of fumarate. However, the DcuS/DcuR TCS could not produce a signal strong enough to mediate the expression of the gfp gene responding fumarate concentration. Thus, DcuS/DucR TCS was engineered by recruiting the EnvZ/OmpR system, the most-studied TCS in E. coli. A chimeric DcuS/EnvZ (DcuSZ) TCS was constructed by fusing the sensor histidine kinase of DcuS with the cytoplasmic catalytic domain of EnvZ, in which the expression of the gfp gene or the ompC gene was mediated by the ompC gene promoter through the cognate response regulator, OmpR. The output signals produced by the chimeric DcuSZ TCS were enough to detect fumarate concentration quantatively, in which the expressions of the gfp gene and the ompC gene were proportional to the fumarate concentration in the medium. Moreover, principal component analysis of C4-dicarboxylates showed that DcuSZ chimera was highly specific to fumarate but could also respond to other C4-dicarboxylates, which strongly suggests that TCS-based high throughput screening system able to screen microorganisms producing target chemicals can be developed.

Metabolically engineered Escherichia coli as a tool for the production of bioenergy and biochemicals from glycerol

Currently, a variety of feedstock is utilized by metabolically engineered bacteria for the production of bioenergy and biochemicals. Recent studies have shown that glycerol can be used as an alternative feedstock for glucose, considering its higher availability, lower price, and high degree of reduction. Hence, this review focuses on recent developments in the bioconversion of glycerol to bioenergy (ethanol and hydrogen) and biochemicals (1,3-propanediol, 1,2-propanediol, 3-hydroxypropionic acid, succinic acid, lactic acid, polyhydroxyalkanoates and Lphenyl alanine) using metabolically engineered Escherichia coli.

Construction of malate-sensing Escherichia coli by introduction of a novel chimeric two-component system

In an attempt to develop a high-throughput screening system for screening microorganisms which produce high amounts of malate, a MalKZ chimeric HK-based biosensor was constructed. Considering the sequence similarity among Escherichia coli (E. coli) MalK with Bacillus subtilis MalK and E. coli DcuS, the putative sensor domain of MalK was fused with the catalytic domain of EnvZ. The chimeric MalK/EnvZ TCS induced the ompC promoter through the cognate response regulator, OmpR, in response to extracellular malate. Real-time quantitative PCR and GFP fluorescence studies showed increased ompC gene expression and GFP fluorescence as malate concentration increased. By using this strategy, various chimeric TCS-based bacteria biosensors can be constructed, which may be used for the development of biochemical-producing recombinant microorganisms.

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.

Expression characteristics of the maeA and maeB genes by extracellular malate and pyruvate in Escherichia coli

The malate-pyruvate conversion pathway is catalyzed by two malic enzyme isomers, MaeA and MaeB. qRT-PCR was carried out under malate and pyruvate supplemented conditions to understand the dynamics of maeA and maeB gene expression. maeA expression was elevated by malate, and maeB expression was inhibited by levels of both malate and pyruvate higher than 0.1 mM. Green fluorescent protein (GFP) reporter plasmids were also constructed by integration of the maeA/maeB promoter with the gfp gene. We showed that maeA driven GFP expression was positively and negatively correlated with extracellular malate and pyruvate induction. In contrast, no significant changes in maeB driven GFP expression were observed under both malate and pyruvate supplemented conditions.

Construction of methanol sensing Escherichia coli by the introduction of Paracoccus denitrificans MxaY based chimeric two-component system.

Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.

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.