Combinatorial pathway balancing provides biosynthetic access to 2-fluoro-cis,cis-muconate in engineered Pseudomonas putida

The wealth of bio-based building blocks produced by engineered microorganisms seldom include halogen atoms. Muconate is a platform chemical with a number of industrial applications that could be broadened by introducing fluorine atoms to tune its physicochemical properties. The soil bacterium Pseudomonas putida naturally assimilates benzoate via the ortho-cleavage pathway with cis,cis-muconate as intermediate. Here, we harnessed the native enzymatic machinery (encoded within the ben and cat gene clusters) to provide catalytic access to 2-fluoro-cis,cis-muconate (2-FMA) from fluorinated benzoates. The reactions in this pathway are highly imbalanced, leading to accumulation of toxic intermediates and limited substrate conversion. By disentangling regulatory patterns of ben and cat in response to fluorinated effectors, metabolic activities were adjusted to favor 2-FMA biosynthesis. After implementing this combinatorial approach, engineered P. putida converted 3-fluorobenzoate to 2-FMA at the maximum theoretical yield. Hence, this study illustrates how synthetic biology can expand the diversity of nature's biochemical catalysis. • Pseudomonas putida processes fluorinated benzoates via the ortho-cleavage pathway • Imbalanced utilization of fluorinated substrates by P. putida results in toxic effects • Pathway modules show divergent transcriptional responses to fluorinated metabolites • Balancing enzyme activities enables efficient 2-fluoro-cis,cis-muconate production Even after decades of research, the chemical nature and structural diversity of building blocks obtained by biocatalysis is restricted to a handful of compounds. Moreover, the potential of bio-based addition of halogen atoms is yet to be fully exploited. Here, 2-fluoro-cis,cis-muconate (2-FMA) was targeted as a new-to-industry chemical. 2-FMA acts as precursor of complex organic structures by its two terminal carboxyl groups, providing access to novel fluorinated polymers and bioactives for medicine and agriculture. Virtually impossible to produce via traditional chemical synthesis, 2-FMA was obtained by leveraging the rich metabolism of the platform bacterium Pseudomonas putida. Efficient conversion of 3-fluorobenzoate, a low-cost haloarene, into 2-FMA is only possible through finely orchestrated biochemistry. Such balance was achieved by combinatorial substitution of native regulatory elements—a strategy that can be also implemented for biosynthesis of other value-added molecules. Biocatalysis can provide access to value-added fluorinated compounds that are difficult to synthesize chemically. In this article, Wirth and Nikel describe a strategy for biological production of 2-fluoro-cis,cis-muconate, a fluorinated derivative of muconate (classified as a top-50 platform molecule). Pseudomonas strains were reprogrammed through a rational metabolic engineering approach applied to native pathways for biodegradation of aromatic xenobiotics. Following this blueprint, the catalytic potential of microbial biochemical pathways can be harnessed toward expanding the spectrum of fluorinated building blocks available for industrial applications.