Biology subjects

Partipilo, M.

Publications and source records attributed to Partipilo, M..

3 recordsLinked to original sources

bioRxiv2026

A microbial growth-coupled platform for in vivo interrogation of Rubisco oxygenase activity

Rubisco catalyzes the primary CO2-fixing reaction of the biosphere, yet its competing oxygenation reaction reduces net global carbon fixation and has resisted direct exploration in living cells. Here, we engineer an auxotrophic Escherichia coli strain in which 2-phosphoglycolate, the direct product of Rubisco oxygenation, becomes essential for growth, making bacterial fitness a quantitative proxy for oxygenation flux in vivo. This provides direct access to catalytic selectivity, something previously inaccessible to carboxylation-coupled assays. The platform enables screening of phylogenetically diverse Form II Rubisco and phosphoribulokinase (Prk) variants circumventing protein purification and extensive in vitro characterization. Adaptive laboratory evolution under oxygenation-selective pressure identified two mutations: Rubisco M115I genetically rebalances the in vivo carboxylation/oxygenation trade-off (resulting in 6-fold reduction in kcat,C), while Prk N216T improves overall flux without altering selectivity. This platform makes Rubiscos least-studied catalytic function selectable and evolvable in vivo, opening the carboxylation/oxygenation trade-off to systematic genetic dissection and engineering.

synthetic biology↗

bioRxiv2025

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

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

systems biology↗

bioRxiv2023

The S-component fold: A link between bacterial transporters and receptors

The processes of nutrient uptake and signal sensing are crucial for microbial survival and adaptation. Membrane-embedded proteins involved in these functions (transporters and receptors) are commonly regarded as unrelated in terms of sequence, structure, mechanism of action and evolutionary history. Here, we analyze the protein structural universe using recently developed artificial intelligence-based structure prediction tools, and find an unexpected link between prominent groups of microbial transporters and receptors. The so-called S-components of energy-coupling factor (ECF) transporters, and the membrane domains of sensor histidine kinases of the 5TMR cluster share a structural fold. The discovery of their relatedness manifests a widespread case of prokaryotic "transceptors" (related proteins with transport or receptor function), showcases how artificial intelligence-based structure predictions reveal unchartered evolutionary connections between proteins, and provides new avenues for engineering transport and signaling functions in bacteria.

biochemistry↗