Engineered Fluorine Metabolism and Fluoropolymer Production in Living Cells

Abstract

Fluorine has become an important element for the design of synthetic molecules for use in medicine, agriculture, and materials. Despite the many advantages provided by fluorine for tuning key molecular properties, it is rarely found in natural metabolism. We seek to expand the molecular space available for discovery through the development of new biosynthetic strategies that cross synthetic with natural compounds. Towards this goal, we engineered a microbial host for organofluorine metabolism and show that we can achieve the production of the fluorinated diketide 2-fluoro-3-hydroxybutyrate at approximately 50 % yield. This fluorinated diketide can be used as a monomer in vivo to produce fluorinated poly(hydroxyalkanoate) (PHA) bioplastics with fluorine substitutions ranging from around 5-15 %. This system provides a platform to produce mm flux through the key fluoromalonyl coenzyme A (CoA) building block, thereby offering the potential to generate a broad range of fluorinated small-molecule targets in living cells.

Keywords: biocatalysis; biosynthesis; fluorine; organofluorine compounds; polymers.

Figure 1

In vivo production of fluorinated biopolymers in engineered E. coli. (A) Pathway for the production of FHB and fluorinated PHA synthesis from fluoromalonate. (B) Plasmid design for the synthetic organofluorine pathways.

Figure 2

Organofluorine levels observed from culture of pathway variants. Cultures were incubated for 48 h at 30°C with 5 mM fluoromalonate (no malonate was added). Strains containing pPOL2 with pFMAL plasmids bearing a transporter (pFMAL2, MadLM; pFMAL3, MatC; pFMAL4, MdcF) were induced at different cell densities (OD₆₀₀ = 0.3 or 0.8). Data are mean ± SD (n = 3) except for the no cell condition where n = 1.

Figure 3

In vivo production of poly(2-fluoro-3-hydroxybutyrate-co-3-hydroxybutyrate) by engineered E. coli. (A) Monomer content of polymer and growth medium for full pathway containing the PhaC polymerase (pPOL1, pFMAL2) in the presence of 5 mM fluoromalonate. Data are mean ± SD (n = 3). (B) Expansion of the COSY spectrum of poly(FHB-co-HB). Crosspeak between H_B and H_C between a fluorinated and non-fluorinated monomer is highlighted in red. (C) Characterization of poly(FHB-co-HB) compared to PHB. (D) ¹H-¹⁹F HMBC NMR spectrum shows numerous chemically distinct types of FHB monomers (red) as indicated by the number of fluorine crosspeaks between H_A, H_B, and H_C.