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.
Department of Chemical Engineering, University of Ulsan, Ulsan, 680-749, Republic of Korea.
Irisappan
I
Ganesh
Sambandam
S
Ravikumar
Ik-Keun
IK
Yoo
Soon Ho
SH
Hong
D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D015374:Biosensing Techniques; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D005798:Genes, Bacterial; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008293:Malates / Q000737:chemistry*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D010578:Petroleum; D010766:Phosphorylation; D018272:Porins / Q000378:metabolism; D025341:Principal Component Analysis; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D011494:Protein Kinases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; C489398:MalK protein, E coli; D008293:Malates; D009097:Multienzyme Complexes; C113724:OmpC protein; D010578:Petroleum; D018272:Porins; D049452:Green Fluorescent Proteins; C030298:malic acid; D011494:Protein Kinases; C469359:DcuS protein, E coli; C057843:envZ protein, E coli