Using an AraC-based three-hybrid system to detect biocatalysts in vivo.
Recent methods to create large libraries of proteins have greatly advanced the discovery of proteins with novel functions. However, one limitation in the discovery of new biocatalysts is the screening or selection methods employed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid system), which allows the construction of an easily screened or selected phenotype for, in theory, any type of enzymatic reaction. The method couples the in vivo concentration of an enzyme's substrate to changes in the transcriptional level of a reporter operon. Using the arabinose operon activator AraC, we constructed a system capable of detecting the fungal enzyme scytalone dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefulness in library screening.
Firestine
S M
SM
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA.
Salinas
F
F
Nixon
A E
AE
Baker
S J
SJ
Benkovic
S J
SJ
D051777:AraC Transcription Factor; D001426:Bacterial Proteins; D001578:Benzopyrans / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular / Q000379:methods*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification; D017930:Genes, Reporter; D006836:Hydro-Lyases / Q000235:genetics* / Q000302:isolation & purification; D015336:Molecular Probe Techniques; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D012641:Selection, Genetic; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
C111394:2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one; D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D001426:Bacterial Proteins; D001578:Benzopyrans; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D006836:Hydro-Lyases; C082741:scytalone dehydratase