Engineered biosynthesis of plant polyketides: structure-based and precursor-directed approach

Area of Science:

• Biochemistry
• Molecular Biology
• Plant Science

Background:

• Novel plant-specific type III polyketide synthases (PKSs), pentaketide chromone synthase (PCS) and octaketide synthase (OKS), were identified in Aloe arborescens.
• These PKSs exhibit high sequence identity to chalcone synthase (CHS) superfamily members but possess unique active-site residues (Met207 in PCS, Gly207 in OKS) replacing the conserved Thr197 in CHS.
• The active-site residue variation is hypothesized to control polyketide chain length and product specificity.

Purpose of the Study:

• To investigate the role of specific active-site residues in controlling polyketide chain length and product specificity in PCS and OKS.
• To engineer PCS and OKS enzymes through site-directed mutagenesis to produce novel polyketides.
• To explore the potential of structure-based and precursor-directed approaches for generating diverse unnatural polyketides.

Main Methods:

• Expression of recombinant PCS and OKS in Escherichia coli.
• Site-directed mutagenesis of PCS and OKS active-site residues.
• X-ray crystallography to analyze enzyme structures.
• Biochemical assays to determine polyketide products and chain lengths.
• Use of various starter substrates (malonyl-CoA, p-coumaroyl-CoA, phenylacetyl-CoA, benzoyl-CoA).

Main Results:

• Recombinant PCS produced pentaketides, while OKS produced octaketides (SEK4 and SEK4b).
• Mutagenesis studies confirmed that single active-site residue changes (Met207 in PCS, Gly207 in OKS) dictate polyketide chain length.
• Engineered PCS and OKS mutants produced novel unnatural polyketides, including nonaketides, decaketides, C19 stilbenes, and C21 chalcones, with altered substrate specificities.

Conclusions:

• The steric bulk of the active-site residue is a critical determinant of polyketide chain length and product specificity in type III PKSs.
• Structure-guided engineering of PCS and OKS enables the production of a wide array of novel and structurally diverse unnatural polyketides.
• The combination of structure-based and precursor-directed strategies offers a powerful approach for expanding the repertoire of plant polyketides.