Exploring Fungal Polyketide C-Methylation through Combinatorial Domain Swaps

Abstract

Polyketide C-methylation occurs during a programmed sequence of dozens of reactions carried out by multidomain polyketide synthases (PKSs). Fungal PKSs perform these reactions iteratively, where a domain may be exposed to and act upon multiple enzyme-tethered intermediates during biosynthesis. We surveyed a collection of C-methyltransferase (CMeT) domains from nonreducing fungal PKSs to gain insight into how different methylation patterns are installed. Our in vitro results show that control of methylation resides primarily with the CMeT, and CMeTs can intercept and methylate intermediates from noncognate nonreducing PKS domains. Furthermore, the methylation pattern is likely imposed by a competition between methylation or ketosynthase-catalyzed extension for each intermediate. Understanding site-specific polyketide C-methylation may facilitate targeted C-C bond formation in engineered biosynthetic pathways.

Figure 1:. Fungal NR-PKSs selected for domain deconstruction and combinatorial swaps.

Group VI and VII NR-PKSs share a common domain architecture (top), with Group VI terminating in a TE domain to release the polyketide as the carboxylic acid and Group VII terminating in a R domain to give an aldehyde. DtbA and PkbA contain tandem ACP domains. (SAT: starter unit-ACP transacylase, KS: ketosynthase, MAT: malonyl-CoA:ACP transacylase, PT: product template, ACP: acyl carrier protein, CMeT: C-methyltransferase, R: reductase, TE: thioesterase).

Figure 2:. Reconstitution of the “minimal methylating PKS” for.

A) PksCT minimal reconstitution with non-cognate ACPs. B) PkeA minimal reconstitution with non-cognate ACPs. C) Addition of non-cognate CMeTs to minimal PksCT D) Addition of non-cognate CMeTs to minimal PkeA E) PksCT and F) PkeA SAT-KS-MAT were combined with paired ACP and CMeT. Traces are vertically offset for clarity. See SI Figure 1 and Table 3 for UV/Vis and mass data.

Figure 3:. Synthetic standards of potential methylation products.

Triacetic acid lactone (1) and variously methylated derivatives 3–5 were obtained by published procedures^, or from commercial sources. These were further elaborated to tetraketides 6, 7, 10, 12–15 as detailed in the Supplementary Information.

Figure 4:. Increasing relative amounts of CMeT results in hypermethylation of pentaketide intermediates.

A) PksCT SAT-KS-MAT and ACP were incubated with increasing concentrations of PksCT CMeT. Traces are vertically stacked and the peak for 1 is truncated for clarity. B) Control of C-methylation programming is proposed to rely on the rates of extension and methylation for each acyl-ACP intermediate produced.