Elucidating the mechanism of fluorinated extender unit loading for improved production of fluorine-containing polyketides

Abstract

Polyketides are a large family of bioactive natural products synthesized by polyketide synthase (PKS) enzyme complexes predominantly from acetate and propionate. Given the structural diversity of compounds produced using these two simple building blocks, there has been longstanding interest in engineering the incorporation of alternative extender units. We have been investigating the mechanism of fluorinated monomer insertion by three of the six different modules of the PKS involved in erythromycin biosynthesis (6-deoxyerythronolide B synthase, DEBS) to begin understanding the contribution of different steps, such as enzyme acylation, transacylation, C-C bond formation, and chain transfer, to the overall selectivity and efficiency of this process. In these studies, we observe that inactivation of a cis-acyltransferase (AT) domain to circumvent its native extender unit preference leads concurrently to a change of mechanism in which chain extension with fluorine-substituted extender units switches largely to an acyl carrier protein (ACP)-independent mode. This result suggests that the covalent linkage between the growing polyketide chain and the enzyme is lost in these cases, which would limit efficient chain elongation after insertion of a fluorinated monomer. However, use of a standalone trans-acting AT to complement modules with catalytically deficient AT domains leads to enzyme acylation with the fluoromalonyl-CoA extender unit. Formation of the canonical ACP-linked intermediate with fluoromalonyl-CoA allows insertion of fluorinated extender units at 43% of the yield of the wild-type system while also amplifying product yield in single chain-extension experiments and enabling multiple chain extensions to form multiply fluorinated products.polyketide synthase | fluorine | synthetic biology | natural products

Polyketide synthases as a platform for chemical product design

“…AT domains primarily select for either malonyl‐CoA or methylmalonyl‐CoA extender units . However, less utilized malonyl‐CoA derivatives with long‐chain alkyl groups, heteroatoms, halogens, and aromatic groups have been reported . The ability to predict the acyl‐CoA extender unit from an AT sequence‐structural analysis has made the AT one of the most well studied domains within the traditional Type I PKS …”

Facile Bioinspired Preparation of Fluorinase@Fluoridated Hydroxyapatite Nanoflowers for the Biosynthesis of 5′-Fluorodeoxy Adenosine

“…Therefore, approximately only 2% of fluorinated drugs were on the market in 1970, and the current number has grown to approximately 25% [2]. To date, most organofluorine compounds were synthesized using chemical methods [3][4][5], and the biosynthesis of organofluorine compounds has only been used to investigate the catalysis of the corresponding fluorinase [6][7][8]. In chemical methods, fluorination is mainly carried out in nucleophilic, electrophilic, radical, and transition metal-catalyzed fluoroalkylation reactions [9].…”

Engineered Fluorine Metabolism and Fluoropolymer Production in Living Cells

“…[8] While it was possible to observe turnover in vivo , the flux was quite low (μM) and we sought to establish this chemistry in living cells in order to take full advantage of the efficiency and scalability of biosynthesis. Since polyketide synthase (PKS) enzymes are typically limited by poor solubility in heterologous hosts, we addressed this problem by designing a high flux organofluorine biosynthetic pathway producing the 2-fluoro-3 R -hydroxybutyrate (FHB) diketide through the chain extension of acetyl-CoA with the fluoromalonyl coenzyme A (CoA) extender unit using the acetoacetyl-CoA synthase (NphT7) [9],[10] and an acetoacetyl-CoA reductase (PhaB) (Figure 1A).…”

“…[13] Plasmid pFMAL1 was designed to encode enzymes for generation of fluoromalonyl-CoA and contains a malonate:CoA ligase (MatB) from Rhodopseudomonas palustris, [14] which showed improved kinetic properties with respect to fluoromalonate activation compared to a previously used homolog (Figure 1B, Table S1 and Figure S1). [8a] A second plasmid, pPOL2, encoding NphT7 and PhaB was also constructed (Figure 1B). We assayed the ability of E. coli BL21(de3) cells harboring both pFMAL1 and pPOL2 to convert fed fluoromalonate (5 mM) to other fluorinated species by 19 F NMR analysis of culture supernatant.…”

“…The incorporation ratio approximately reflects the relative activity of NphT7 on the two substrates (5-fold difference in k cat / K M ). [8a] To further confirm the presence of fluorinated monomers within the polymer, poly(FHB- co -HB) was isolated directly from cells and analyzed by 1 H, 19 F and 13 C NMR, which all contain peaks corresponding to fluorinated monomers with characteristically large J H-F and J C-F coupling constants (Figure S11). The COSY spectrum also contains a crosspeak arising from fluorinated monomers adjacent to their nonfluorinated congeners, indicating the fluorinated monomers are integrated within the polymer (Figure 3B, Figure S12).…”