A High-Throughput Screen
for the Engineered Production
of β-Lactam Antibiotics
- Publication date
- Publisher
Abstract
High-throughput screens and selections have had profound
impact
on our ability to engineer proteins possessing new, desired properties.
These methods are especially useful when applied to the modification
of existing enzymes to create natural and unnatural products. In an
advance upon existing methods we developed a high-throughput, genetically
regulated screen for the <i>in vivo</i> production of β-lactam
antibiotics using a green fluorescent protein (gfp) reporter. This
assay proved reliable and sensitive and presents a dynamic range under
which a wide array of β-lactam architectural subclasses can
be detected. Moreover, the graded response elicited in this assay
can be used to rank mutant activity. The utility of this development
was demonstrated <i>in vivo</i> and then applied to the
first experimental investigation of a putative catalytic residue in
carbapenem synthase (CarC). Information gained about the mutability
of this residue defines one parameter for enzymatic activity and sets
boundaries for future mechanistic and engineering efforts