TY - JOUR
T1 - A Biosensor Strategy for E. coli Based on Ligand-Dependent Stabilization
AU - Brandsen, Benjamin M.
AU - Mattheisen, Jordan M.
AU - Noel, Teia
AU - Fields, Stanley
N1 - Funding Information:
This work was supported by NIH grant P41GM103533 (to S.F.). B.M.B. was supported by NIH T32HG000035 and NIH F32GM122202. S.F. is an investigator of the Howard Hughes Medical Institute. We thank members of the Fields lab for helpful suggestions on the manuscript. We thank Scot Wolfe and George Church for gifts of plasmids and the E. coli strain lacking hisB, pyrF, and rpoZ.
Funding Information:
This work was supported by NIH grant P41GM103533 (to S.F.). B.M.B. was supported by NIH T32HG000035 and NIH F32GM122202. S.F. is an investigator of the Howard Hughes Medical Institute. We thank members of the Fields lab for helpful suggestions on the manuscript. We thank Scot Wolfe and George Church for gifts of plasmids and the E. coli strain lacking .
Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/9/21
Y1 - 2018/9/21
N2 - The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lacI mutation that expands ligand specificity to d-fucose generated a biosensor with improved response both to d-fucose and to IPTG. However, a mutation equivalent to the one that destabilized LacI in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.
AB - The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lacI mutation that expands ligand specificity to d-fucose generated a biosensor with improved response both to d-fucose and to IPTG. However, a mutation equivalent to the one that destabilized LacI in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.
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U2 - 10.1021/acssynbio.8b00052
DO - 10.1021/acssynbio.8b00052
M3 - Article
C2 - 30064218
AN - SCOPUS:85052315938
VL - 7
SP - 1990
EP - 1999
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
SN - 2161-5063
IS - 9
ER -