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.
Department of Chemical Engineering, University of Ulsan, 93 Daehakro, Nam-gu, Ulsan 680-749, Republic of Korea.
Irisappan
I
Ganesh
Sambandam
S
Ravikumar
Seung Hwan
SH
Lee
Si Jae
SJ
Park
Soon Ho
SH
Hong
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000235:genetics; D015374:Biosensing Techniques / Q000379:methods*; D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005650:Fumarates / Q000302:isolation & purification* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D060847:Metabolic Engineering; D010766:Phosphorylation; D018272:Porins / Q000235:genetics; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics
D001426:Bacterial Proteins; D029382:Dicarboxylic Acid Transporters; D005650:Fumarates; C113724:OmpC protein; D018272:Porins; D015534:Trans-Activators; C112177:osmolarity response regulator proteins
Chimeric TCS; DcuS/DcuR two-component system (TCS); DcuS/EnvZ (DcuSZ) TCS; Escherichia coli; Fumarate