Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis

Abstract

Polyketides are biosynthesized through consecutive decarboxylative Claisen condensations between a carboxylic acid and differently substituted malonic acid thioesters, both tethered to the giant polyketide synthase enzymes. Individual malonic acid derivatives are typically required to be activated as coenzyme A-thioesters prior to their enzyme-catalyzed transfer onto the polyketide synthase. Control over the selection of malonic acid building blocks promises great potential for the experimental alteration of polyketide structure and bioactivity. One requirement for this endeavor is the supplementation of the bacterial polyketide fermentation system with tailored synthetic thioester-activated malonates. The membrane permeable N-acetylcysteamine has been proposed as a coenzyme A-mimic for this purpose. Here, the incorporation efficiency into different polyketides of N-acetylcysteamine activated methylmalonate is studied and quantified, showing a surprisingly high and transferable activity of these polyketide synthase substrate analogues in vivo.

Keywords: biosynthesis; coenzyme A; malonic acid; polyketide; polyketide synthase.

Figure 1

The most intensively studied PKS, deoxyerythronolide B synthase (DEBS), which catalyzes the key steps in the biosynthesis of the antibiotic erythromycin. DEBS catalyzes the extension of a propionate starter unit with six equivalents of methylmalonyl-coenzyme A (MM-CoA). After six rounds of decarboxylative Claisen condensations and varying degrees of reduction of the initially formed β-keto thioesters, the polyketide core of erythromycin is released from the enzyme via a terminal esterase [–8]. Abbreviations: AT: acyltransferase, ACP: acyl carrier protein, KS: ketosynthase, KR: ketoreductase, DH: dehydratase, ER: enoylreductase, TE: thioesterase.

Scheme 1

Synthesis of SNAC-activated D₃-methylmalonate. a: 2.1 equiv [(CH₃)₂CH]₂NLi, 1 equiv CD₃I, abs. THF, −78 °C → rt, Ar, 18 h, 37%. b: 1.3 equiv N,N'-carbonyldiimidazole, 0.3 equiv DMAP, 1.5 equiv N-acetylcysteamine, abs. THF, 0 °C → rt, Ar, 18 h, 82%; c: 2.5 equiv TiCl₄, CH₂Cl₂, Ar, 0 °C → rt, 6 h, then aq NaHCO₃ (pH 8.0), quant. (by TLC and ¹H NMR).

Figure 2

Structures of erythromycin (left) and rapamycin (right). In this experiment both compounds were labeled in their methyl side chains with deuterium.

Figure 3

Relative incorporation of the D₃-label into erythromycin (A) and rapamycin (B), depending on the fed concentration of rac-4. Color coding: green: no incorporation; blue: single; red: double; magenta: triple incorporation as detected by LC/ESI–MS.

Figure 4

ESI–MS spectra of feeding experiments with an erythromycin-producing culture of S. erythraea. The mass spectra show the change in the isotope ratio of the detected erythromycin, depending on the concentration of 4 in the fermentation medium. (A) control, 0 mM 4; (B) 1.25 mM 4; (C) 2.5 mM 4; (D) 5 mM 4; (E) 10 mM 4; (F) 15 mM 4; (G) 30 mM 4. The data are quantified by subtraction of the intensity of the mass for all relevant ions (beginning from 734.3 in steps of three, corresponding to the D₃-label) of the control from the experiments. An analogous figure (Figure S9) for the feeding studies with a rapamycin-producing culture of S. hygroscopicus can be found in Supporting Information File 1.

Scheme 2

Incorporation of a propargylated malonic acid derivative into erythromycin through an active-site mutation in the acyltransferase in DEBS module 6 (DEBS AT6*) [39].