Engineered Biosynthesis of Plant Polyketides:  Chain Length Control in an Octaketide-Producing Plant Type III Polyketide Synthase

Abstract

The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) produces a variety of plant secondary metabolites with remarkable structural diversity and biological activities (e.g., chalcones, stilbenes, benzophenones, acrydones, phloroglucinols, resorcinols, pyrones, and chromones). Here we describe an octaketide-producing novel plant-specific type III PKS from aloe (Aloe arborescens) sharing 50-60% amino acid sequence identity with other plant CHS-superfamily enzymes. A recombinant enzyme expressed in Escherichia coli catalyzed seven successive decarboxylative condensations of malonyl-CoA to yield aromatic octaketides SEK4 and SEK4b, the longest polyketides known to be synthesized by the structurally simple type III PKS. Surprisingly, site-directed mutagenesis revealed that a single residue Gly207 (corresponding to the CHS's active site Thr197) determines the polyketide chain length and product specificity. Small-to-large substitutions (G207A, G207T, G207M, G207L, G207F, and G207W) resulted in loss of the octaketide-forming activity and concomitant formation of shorter chain length polyketides (from triketide to heptaketide) including a pentaketide chromone, 2,7-dihydroxy-5-methylchromone, and a hexaketide pyrone, 6-(2,4-dihydroxy-6-methylphenyl)-4-hydroxy-2-pyrone, depending on the size of the side chain. Notably, the functional diversity of the type III PKS was shown to evolve from simple steric modulation of the chemically inert single residue lining the active-site cavity accompanied by conservation of the Cys-His-Asn catalytic triad. This provided novel strategies for the engineered biosynthesis of pharmaceutically important plant polyketides.

Type III polyketide synthases in natural product biosynthesis

“…A recent work revealed that incubation of the recombinant wildtype OKS and malonyl-CoA yielded SEK4 and SEK4b through sequential condensation of eight molecules of malonyl-CoA ( Fig. 2A) (13). These two compounds have been reported to be the spontaneously cyclized products of the nascent octaketide chain synthesized by some type II minimal PKSs such as the actinorhodin minimal PKS (14).…”

“…An OKS mutant in which Gly 207 was substituted with bulky Met as in PCS completely lost the octaketide-forming activity. However, the mutant PKS efficiently produced an unnatural pentaketide, 2,7-dihydroxy-5-methylchromone that is a regioisomer of 5,7-dihydroxy-2-methylchromone produced by PCS (13). Similarly, the PCS M207G mutant produced the octaketides SEK4/SEK4b instead of the pentaketide chromone (16).…”

LAP6/POLYKETIDE SYNTHASE AandLAP5/POLYKETIDE SYNTHASE BEncode Hydroxyalkyl α-Pyrone Synthases Required for Pollen Development and Sporopollenin Biosynthesis inArabidopsis thaliana

“…The best-studied type III PKS is CHS, which carries out the first committed step in flavonoid biosynthesis by catalyzing the sequential decarboxylative addition of three acetate units from malonyl-CoA to a p-coumaryl-CoA starter molecule derived from the general phenylpropanoid pathway. Moreover, other members of the type III PKS superfamily in plants use linear acyl-CoAs of varying length (C2 to C20) as starter substrates and give rise to a large variety of metabolites (Austin and Noel, 2003;Abe et al, 2004Abe et al, , 2005Mizuuchi et al, 2008;Flores-Sanchez and Verpoorte, 2009). …”

Synthesis of unnatural alkaloid scaffolds by exploiting plant polyketide synthase

“…In contrast, the regular CHS did not accept the bulky substrate. Further, octaketide synthase (OKS) from Aloe arborescens (12), which normally catalyzes iterative condensations of eight molecules of malonyl-CoA to produce the aromatic octaketides SEK4 and SEK4b, only afforded a tetraketide pyrone as a minor product by condensation of 2-carbamoylbenzoyl-CoA with three molecules of malonyl-CoA (Fig. S3).…”

“…Interestingly, type III PKSs exhibit remarkable substrate tolerance, accepting a series of nonphysiological substrate analogues to produce chemically and structurally distinct unnatural novel polyketides (4)(5)(6)(7)(8)(9)(10). Furthermore, recent structure-based engineering of functionally divergent type III PKSs has significantly expanded the catalytic repertoire of the enzymes and the product diversity (11)(12)(13)(14)(15)(16)(17). Due to their remarkable substrate promiscuity and catalytic potential, the structurally and mechanistically simple type III PKS enzymes represent an excellent platform for the further development of unnatural novel biocatalysts with unprecedented catalytic functions (18).…”