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

• Published in: Journal of biotechnology Vol. 168; no. 4; pp. 560 - 566
• Main Authors: Ganesh, Irisappan, Ravikumar, Sambandam, Lee, Seung Hwan, Park, Si Jae, Hong, Soon Ho
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2013
• Subjects: active sites; Aerobiosis; Bacterial Proteins - genetics; Biosensing Techniques - methods; Biotechnology; Chimeric TCS; chimerism; DcuS/DcuR two-component system (TCS); DcuS/EnvZ (DcuSZ) TCS; Dicarboxylic Acid Transporters - genetics; Dicarboxylic Acid Transporters - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fumarate; Fumarates - isolation & purification; Fumarates - metabolism; Gene expression; Gene Expression Regulation, Bacterial; Genes; histidine kinase; Kinases; Metabolic Engineering; Microorganisms; Phosphorylation; Porins - genetics; principal component analysis; Promoter Regions, Genetic; Screening; Screens; Signal Transduction - genetics; Trans-Activators - genetics; DcuS/DcuR two-component system (TCS); Chimeric TCS; DcuS/EnvZ (DcuSZ) TCS; Fumarate; Escherichia coli
• ISSN: 0168-1656; 1873-4863; 1873-4863
• DOI: 10.1016/j.jbiotec.2013.09.003

•The expression of the gfp gene regulated by DcuS/DcuR two-component system (TCS) was examined for the detection of fumarate in the medium.•Chimeric DcuS/EnvZ (DcuSZ) TCS was further developed for the expression of the gfp gene responding to the existence of fumarate in the medium.•Escherichia coli strain could be engineered to specifically detect fumarate based on DcuS/EnvZ (DcuSZ) TCS. 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.