TY - JOUR
T1 - Engineered fumarate sensing Escherichia coli based on novel chimeric two-component system
AU - Ganesh, Irisappan
AU - Ravikumar, Sambandam
AU - Lee, Seung Hwan
AU - Park, Si Jae
AU - Hong, Soon Ho
N1 - Funding Information:
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology ( 2011-0022392 ).
PY - 2013/12
Y1 - 2013/12
N2 - 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.
AB - 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.
KW - Chimeric TCS
KW - DcuS/DcuR two-component system (TCS)
KW - DcuS/EnvZ (DcuSZ) TCS
KW - Escherichia coli
KW - Fumarate
UR - http://www.scopus.com/inward/record.url?scp=84888822637&partnerID=8YFLogxK
U2 - 10.1016/j.jbiotec.2013.09.003
DO - 10.1016/j.jbiotec.2013.09.003
M3 - Article
C2 - 24056083
AN - SCOPUS:84888822637
SN - 0168-1656
VL - 168
SP - 560
EP - 566
JO - Journal of Biotechnology
JF - Journal of Biotechnology
IS - 4
ER -