Combinatorial Pathway Balancing Provides Biosynthetic Access to 2-fluoro-,-muconate in Engineered

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 naturally assimilates benzoate via the -cleavage pathway with ,-muconate as intermediate. Here, we harnessed the native enzymatic machinery (encoded within the and gene clusters) to provide catalytic access to 2-fluoro-,-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 and in response to fluorinated effectors, metabolic activities were adjusted to favor 2-FMA biosynthesis. After implementing this combinatorial approach, engineered . 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.