Combinatorial assembly of ferredoxin-linked modules in Escherichia coli yields a testing platform for Rnf-complexes

The Rnf complex is a membrane-bound ferredoxin(Fd):NAD(P)⁺ oxidoreductase (Fno) that couples Fd oxidation to vectorial H⁺ /Na⁺ transport across the cytoplasmic membrane. Here, we produced two putative Rnf-complexes from Clostridioides difficile (Cd-Rnf) and Clostridium ljungdahlii (Cl-Rnf) for the first time in Escherichia coli. A redox-responsive low-expression system enabled Rnf assembly in the membranes of E. coli as confirmed by in vitro activity measurements. To study the physiological effects of Rnf on the metabolism of E. coli, we assembled additional Fd-dependent enzymes by plasmid-based multigene expression: (a) an Fd-linked butyrate pathway (But) from C. difficile, (b) an [FeFe]-hydrogenase (Hyd) to modulate the redox state of Fd, and (c) heterologous ferredoxins as electron carriers. The hydrogenase efficiently modulated butyrate formation by H₂ -mediated Fd reoxidation under nitrogen. In its functionally assembled state, Rnf severely impaired cell growth. Including Hyd in the But/Rnf background, in turn, restored normal growth. Our findings suggest that Rnf mediates reverse electron flow from NADH to Fd, which requires E. coli's F-type ATPase to function in its reverse, ATP hydrolyzing direction. The reduced Fd is then reoxidized by endogenous Fd:NAD(P)H oxidoreductase (Fpr), which regenerates NADH and, thereby, initiates a futile cycle fueled by ATP hydrolysis. The introduction of hydrogenase interrupts this futile cycle under N₂ by providing an efficient NAD(P)⁺ -independent Fd reoxidation route, whereas under H₂ , Hyd outcompetes Rnf for Fd reduction. This is the first report of an Rnf complex being functionally produced and physiologically investigated in E. coli.