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bioRxiv · 10.1101/2025.11.04.686620

Integrated control of redox and energy metabolism by the membrane-bound and soluble transhydrogenases of Pseudomonas putida across metabolic regimes

Partipilo, M.·Favoino, G.·Puiggene, O.·Rocha, C.·Meiners, C.·Gurdo, N.·Donati, S.·Volke, D. C.·Nikel, P. I.
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Abstract

Redox homeostasis is central to microbial physiology and stress adaptation, yet the functional roles of transhydrogenases remain poorly understood beyond a few organisms. In this study, we systematically explored how Pseudomonas putida, a model soil bacterium, integrates two distinct transhydrogenases (membrane-bound PntAB and soluble SthA) into a flexible and reversible redox-balancing system that supports metabolic robustness across diverse metabolic regimes. While single deletions of either enzyme had minimal impact on the overall fitness, the double {Delta}pntAB {Delta}sthA mutant exhibited growth defects, disrupted energy charge, and redox imbalance. Unexpectedly, SthA proved essential for acetate-dependent growth, a phenotype traced to a transcriptional regulator involved in glyoxylate metabolism. Transhydrogenases also mediated tolerance to formate, a key one-carbon (C1) substrate for biotechnology, by channeling reducing equivalents released during feedstock oxidation. Synergistic activity with native formate dehydrogenases enabled redox buffering, even under stressful conditions. Functional complementation with native and engineered NAD+- or NADP+-dependent dehydrogenases validated SthA as the main sink for excess NADH. Comparative genomics linked transhydrogenase gene neighborhoods to stress and membrane processes, highlighting their evolutionary significance. These findings redefine transhydrogenases as dynamic regulators of redox metabolism, not passive cofactor shuttles. Furthermore, this work positions P. putida as a prime host for redox-intensive applications, informing design principles for C1-based metabolic engineering. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/686620v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@17c0e06org.highwire.dtl.DTLVardef@17cd094org.highwire.dtl.DTLVardef@161b5b9org.highwire.dtl.DTLVardef@1e1897e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Partipilo, M., Favoino, G., Puiggene, O., Rocha, C., Meiners, C., Gurdo, N., Donati, S., Volke, D. C., Nikel, P. I.. 2025-11-06. Integrated control of redox and energy metabolism by the membrane-bound and soluble transhydrogenases of Pseudomonas putida across metabolic regimes. https://doi.org/10.1101/2025.11.04.686620

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