Evolution of an alkane-inducible biosensor for increased responsiveness to short-chain alkanes.
Synthetic alkane-inducible biosensors have applications as detectors for environmental hydrocarbon contamination and as novel inducible expression systems with low-cost inducers. Here, we have assembled and evolved an alkane-responsive biosensor with a fluorescence output signal in Escherichia coli by utilizing regulatory machinery from Pseudomonas putida's alkane metabolism. Within our system, the transcriptional regulator, AlkSp, is activated by the presence of alkanes and binds to the P(alkB) promoter, stimulating transcription of a Green Fluorescent Protein reporter. Through two successive rounds of directed evolution via error prone PCR and fluorescence activated cell sorting, we isolated alkS mutants enabling up to a 5 fold increase in fluorescence output signal in response to short-chain alkanes such as hexane and pentane. Further characterization of selected mutants demonstrated altered responsiveness to a wide range of linear alkanes (pentane to dodecane). Sequence analysis highlighted the S470T mutation as a likely candidate responsible for increased effectiveness of the AlkS protein for short-chain alkanes. This work represents the first evolution of a synthetic biosensor system for alkanes.
Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, United States.
Ben
B
Reed
John
J
Blazeck
Hal
H
Alper
D000473:Alkanes / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D019943:Amino Acid Substitution; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D020125:Mutation, Missense; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics
D000473:Alkanes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins