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 emp loyed to find enzymes from these libraries. We have developed a potentially general method termed QUEST (QUerying for EnzymeS using the Three-hybrid s ystem), which allows the construction of an easily screened or selected phe notype for, in theory, any type of enzymatic reaction. The method couples t he in vivo concentration of an enzyme's substrate to changes in the transcr iptional level of a reporter operon. Using the arabinose operon activator A raC, we constructed a system capable of detecting the fungal enzyme scytalo ne dehydratase (SD) in bacteria, and demonstrated its sensitivity and usefu lness in library screening.