Flavin Cofactor Engineering Rewires Metabolism and Modulates Stress Response in Escherichia coli

Abstract Cofactors such as flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide, coenzyme A, and S-adenosylmethionine are essential for primary metabolism. Interestingly, they share a common adenosine moiety that does not contribute directly to catalysis and is therefore amenable to engineering for synthetic biology applications. In FAD biosynthesis, the adenosine group is installed by the flavin mononucleotide adenylyltransferase (FMNAT) domain of the bifunctional FAD synthetase using FMN and ATP. This study explores the nucleotide selectivity of the Escherichia coli FMNAT domain and the physiological effects of FAD nucleotide analogues synthesized from cellular nucleotides other than ATP. First, we engineer the FAD synthetase enzyme at an active site loop to produce FAD nucleotide analogues. Chromosomal substitution of the wild-type E. coli FAD synthetase with the mutated version results in the intracellular synthesis of these noncanonical FAD nucleotide analogues and confers tolerance to aminoglycoside antibiotics. Upon investigating their potential downstream metabolic roles, we find that the intracellular production of FAD nucleotide analogues perturbs cellular metabolism and the FAD analogues may bind to a range of E. coli enzymes. These findings validate the FMNAT domain loop as a viable site for cofactor engineering, with potential for synthetic biology. Finally, the observed correlation between FAD analogue production and increased tolerance to aminoglycosides suggests the existence of unexplored metabolic mechanisms underlying antibiotic sensitivity.