Design of Molecular Biosensors for New or Improved Polyketide Synthase Activities

Metabolite-responsive gene regulators are valuable tools in
synthetic biology and can be used as molecular reporters for protein and metabolic
engineering. To develop biosensors for high-throughput screening of
biosynthesis pathways in E. coli, we
sought variants of the AraC regulatory protein that respond to small molecules
of interest.  One of our most effective
reporters from these efforts responds to the compound
4-hydroxy-6-methyl-2-pyrone, or triacetic acid
lactone (TAL), commonly identified as a triketide
derailment product during polyketide biosynthesis.  Our AraC-based TAL
reporter (AraC-TAL) enabled the in vivo directed evolution of Gerbera hybrida
2-pyrone synthase activity in E. coli
[Tang et al., J. Am. Chem. Soc. 2013, 135, 10099] and is now being used to evolve
variants of a fungal polyketide synthase (PKS) that functionally express in E. coli. 

Natural polyketides and their ?unnatural? analogues offer
great potential as therapeutics, including antibiotics, antifungals, and anticancer
agents.  Extensive research is aimed at
modifying PKS substrate and product profiles, as means to better understand PKS
biochemistry and to broaden chemical diversity. However, directed evolution of
these enzymes is limited by a general lack of product-specific, high-throughput
screening assays.  By exploiting the
promiscuity of select PKS domains, a variety of triketide
lactones possessing the 4-hydroxy-2-pyrone moiety have been synthesized [Winter
et al., Organic Letters. 2013, 15, 3775; Koryakina et
al., Org. Biomol. Chem. 2013, 11,
4449].  We probed the structural
specificity of AraC-TAL and found that this reporter responds to a variety of
4-hydroxy-2-pyrones to different extents, and not to similar compounds but lacking
these features.  Structural and mutational
analyses were then carried out to better understand how AraC-TAL and variants
thereof distinguish between select 2-pyrones.  This presentation highlights results from
these studies, as well as progress toward the design of AraC-TAL variants specific
for select TAL derivatives, which will facilitate PKS directed evolution
towards synthesis of the respective triketides and
their corresponding ?unnatural? products resulting from complete PKS
processing.