Evolution-Guided Biosynthesis of Terpenoid Inhibitors

Terpenoids, the largest and most structurally diverse
group of
natural products, include a striking variety of biologically active
compounds, from flavors to medicines. Despite their well-documented
biochemical versatility, the evolutionary processes that generate
new functional terpenoids are poorly understood and difficult to recapitulate
in engineered systems. This study uses a synthetic biochemical objectivea
transcriptional system that links the inhibition of protein tyrosine
phosphatase 1B (PTP1B), a human drug target, to the expression of
a gene for antibiotic resistance in Escherichia coli (E. coli)to evolve a terpene
synthase to produce enzyme inhibitors. Site saturation mutagenesis
of poorly conserved residues on γ-humulene synthase (GHS), a
promicuous enzyme, yielded mutants that improved fitness (i.e., the
antibiotic resistance of E. coli) by
reducing GHS toxicity and/or by increasing inhibitor production. Intriguingly,
a combination of two mutations enhanced the titer of a minority producta
terpene alcohol that inhibits PTP1Bby over 50-fold, and a
comparison of similar mutants enabled the identification of a site
where mutations permit efficient hydroxylation. Findings suggest that
the plasticity of terpene synthases enables an efficient sampling
of structurally distinct starting points for building new functional
molecules and provide an experimental framework for exploiting this
plasticity in activity-guided screens.