Biology subjects

de Pins, B.

Publications and source records attributed to de Pins, B..

12 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↗

bioRxiv2026

Global carbon fixation in Earth's subsurface biosphere

The subsurface of our planet hosts 15% of Earths biomass and plays a key role in mediating the exchange of volatiles and elements between deep, long-residence-time geological reservoirs and rapidly cycling surface environments, influencing planetary climate and habitability. While a significant fraction of subsurface microorganisms rely on surface-derived organic carbon, an unknown portion is sustained through chemolithotrophic carbon fixation. Despite its importance, the global diversity and distribution of microbial carbon fixation pathways in the subsurface, and the environmental drivers shaping them, remain poorly constrained. Here we systematically characterise carbon fixation pathways for 412 subsurface metagenomes, including 242 new metagenomes, and compare them to surface-derived datasets. We find that subsurface environments span a broader physicochemical space than surface systems and support a higher abundance and diversity of carbon fixation strategies. Using colocated geochemical data spanning >50 variables, we show that the reductive tricarboxylic acid cycle and the reductive acetyl-CoA pathway are enriched in reducing, geochemically evolved fluids. We use the metagenomic results together with previously published carbon fixation rates in the subsurface to derive a global continental subsurface carbon fixation rate of [~]2.65 Pg C yr-1 (range: 0.31-2.99). This represents [~]2% of terrestrial photosynthetic primary production, and is an order of magnitude higher than geological fluxes between the surface and the subsurface. These results identify the subsurface as a reservoir of autotrophic strategies organized along geochemical gradients, contributing substantially to the global carbon cycle. One Sentence SummaryThe subsurface is a widespread, environmentally and functionally diverse reservoir of autotrophic carbon fixation pathways that can contribute substantially to the global carbon cycle, fixing [~]2.65 Pg C yr-1 in continental settings alone.

microbiology↗

bioRxiv2025

Rubisco is slow across the tree of life

Rubisco is the main gateway through which inorganic carbon enters the biosphere, catalyzing the vast majority of carbon fixation on Earth. This pivotal enzyme has long been observed to be kinetically constrained. Yet, this impression is based on kinetic measurements heavily focused on eukaryotic rubiscos, a rather conserved group of low genetic diversity. Moreover, the fastest rubiscos that we know of so far were found among the sparsely sampled prokaryotes. Could there be yet faster rubiscos among the uncharted regions of rubiscos phylogenetic diversity? Here, we perform a characterization of more than 250 rubiscos from a wide range of bacteria and archaea, thereby doubling the coverage of the diversity of this key enzyme. We assess the distribution of the carboxylation rates at saturating levels of CO2, and establish that rubisco is a relatively slow enzyme across the tree of life, never exceeding {approx}30 reactions per second at 30{degrees}C. We show that relatively faster subclades share similar evolutionary contexts, involving micro-oxygenic environments or a CO2 concentrating mechanism. Leveraging a simple machine learning model trained on this dataset, we predict the carboxylation rate for all {approx}68,000 sequenced rubisco variants found in nature to date. This study provides the largest and most diverse dataset of natural variants for an enzyme and their associated rates, establishing a solid benchmark for future efforts to predict catalytic rates from sequence data. SignificanceDiscovering a fast carboxylating rubisco has been a long-standing challenge in the scientific community, given its potential impact on sustainable food and fuel production. Yet, only a small fraction of rubiscos natural diversity has been kinetically characterized. Here, we present a large-scale kinetic survey covering the entire spectrum of rubiscos diversity found in nature. Focusing on genetic clusters with above-average rates, we show that rubiscos catalytic rate does not exceed {approx}30 reactions per second at 30{degrees}C. Supported by a machine-learning predictive model, we extend this finding to all sequenced natural variants. Our study provides the most comprehensive kinetic dataset for a single enzyme to date.

systems biology↗

bioRxiv2025

Trace metals availability controls terminal electron acceptor utilization in Escherichia coli

Trace metals play an essential role in the metabolism of all living organisms and many metal-containing enzymes contribute to key physiological and ecological processes such as aerobic and anaerobic respiration, photosynthesis, carbon and nitrogen fixation. Despite this, trace metals potential to control microbial functional diversity and metabolic shifts is unknown. Here we demonstrate that the availability of trace metals controls electron acceptors utilization in Escherichia coli. Physiological and proteomic data show that trace metals depleted cultures have significantly reduced growth, start fermentation and increase energy expenditure for metal homeostasis even when more energetically favourable electron acceptors are present. Overall these results suggest how evolutionary and competitive pressures arising from changes in biological trace metals availability in deep time have contributed to shaping evolution and competition.

microbiology↗

bioRxiv2024

PYK2 in the dorsal striatum of Huntington disease R6/2 mouse model

Huntingtons disease (HD) is a devastating disease due to autosomal dominant mutation in the HTT gene. Its pathophysiology involves multiple molecular alterations including transcriptional defects. We previously showed that in HD patients and mouse model, the protein levels of the non-receptor tyrosine kinase PYK2 were decreased in the hippocampus and that viral expression of PYK2 improved the hippocampal phenotype. Here, we investigated the possible contribution of PYK2 in the striatum, a major brain region altered in HD. PYK2 mRNA levels were decreased in the striatum and hippocampus of R6/2 mice, a severe HD model. PYK2 protein levels were also decreased in the dorsal striatum of R6/2 mice and in the putamen of human patients. PYK2 knockout by itself did not result in motor symptoms observed in HD mouse models. Yet, we examined whether PYK2 deficiency participated in the R6/2 mice phenotype by expressing PYK2 in the dorsal striatum using AAV vectors. With an AAV1/Camk2a promoter, we did not observe significant improvement of body weight, clasping, motor activity and coordination (rotarod) alterations observed in R6/2 mice. With an AAV9/SYN1 promoter we found an improvement of body weight loss and a tendency to better rotarod performance. DARPP-32 immunofluorescence was increased following AAV9/SYN1-PYK2 injection compared to AAV9/SYN1-GFP, suggesting a possible partial beneficial effect on striatal projection neurons. We conclude that PYK2 mRNA and protein levels are decreased in the striatum as in hippocampus of HD patients and mouse models. However, in contrast to hippocampus, striatal viral expression of PYK2 has only a slight effect on the R6/2 model striatal motor phenotype. HighlightsO_LIHuntingtons disease is a lethal genetic disease altering striatum, cortex, and hippocampus C_LIO_LIRestoring PYK2 levels in hippocampus improved hippocampal phenotype of a Huntington mouse model C_LIO_LIWe show that PYK2 levels are decreased in the striatum of R6/2 mice and human patients C_LIO_LIViral expression of PYK2 in the striatum has only a small effect on R6/2 mouse model motor phenotype but improves weight loss C_LI

neuroscience↗

bioRxiv2023

Mapping the biochemical landscape of rubisco

Rubisco is the primary CO2 fixing enzyme of the biosphere yet has slow kinetics. The roles of evolution and chemical mechanism in constraining the sequence landscape of rubisco remain debated. In order to map sequence to function, we developed a massively parallel assay for rubisco using an engineered E. coli where enzyme function is coupled to growth. By assaying >99% of single amino acid mutants across CO2 concentrations, we inferred enzyme velocity and CO2 affinity for thousands of substitutions. We identified many highly conserved positions that tolerate mutation and rare mutations that improve CO2 affinity. These data suggest that non-trivial kinetic improvements are readily accessible and provide a comprehensive sequence-to-function mapping for enzyme engineering efforts.

biochemistry↗

bioRxiv2023

Systematic exploration of prokaryotic form I rubisco maximal carboxylation rates

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco - the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubiscos carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median 5-fold higher rate. In contrast to prior studies, we find that rubiscos originating from -cyanobacteria exhibit the highest carboxylation rates among form I enzymes ({approx}10 s-1 median versus <7 s-1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature.

systems biology↗

bioRxiv2023

Autotrophic growth of E. coli is achieved by a small number of genetic changes

Synthetic autotrophy is a promising avenue to sustainable bioproduction from CO2. Here, we use iterative laboratory evolution to generate several distinct autotrophic strains. Utilising this genetic diversity, we identify that just three mutations are sufficient for E. coli to grow autotrophically, when introduced alongside non-native energy (formate dehydrogenase) and carbon-fixing (RuBisCO, phosphoribulokinase, carbonic anhydrase) modules. The mutated genes are involved in glycolysis (pgi), central-carbon regulation (crp), and RNA transcription (rpoB). The pgi mutation reduces the enzymes activity, thereby stabilising the carbon-fixing cycle by capping a major branching flux. For the other two mutations, we observe down-regulation of several metabolic pathways and increased expression of native genes associated with the carbon-fixing module (rpiB) and the energy module (fdoGH), as well as an increased ratio of NADH/NAD+ - the cycles electron-donor. This study demonstrates the malleability of metabolism and its capacity to switch trophic modes using only a small number of genetic changes and could facilitate transforming other heterotrophic organisms into autotrophs.

synthetic biology↗

bioRxiv2023

Operant training for highly palatable food alters translating mRNA in nucleus accumbens D2 neurons and reveals a modulatory role of Neurochondrin

BACKGROUNDHighly palatable food triggers behavioral alterations reminiscent of those induced by addictive drugs. These effects involve the reward system and dopamine neurons, which modulate neurons in the nucleus accumbens (NAc). The molecular mechanisms underlying the effects of highly palatable food on feeding behavior are poorly understood. METHODSWe studied the effects of 2-week operant conditioning of mice with standard or isocaloric highly palatable food. We investigated the behavioral effects and dendritic spine modifications in the NAc. We compared the translating mRNA in NAc neurons identified by the type of dopamine receptors they express, depending on the type of food and training. We tested the consequences of invalidation of an abundant downregulated gene, Ncdn (Neurochondrin). RESULTSOperant conditioning for highly palatable food increases motivation for food even in well-fed mice. In control mice, free access to regular or highly palatable food results in increased weight as compared to regular food only. Highly palatable food increases spine density in the NAc. In animals trained for highly palatable food, translating mRNAs are modified in NAc dopamine D2-receptor-expressing neurons, mostly corresponding to striatal projection neurons, but not in those expressing D1-receptors. Knock-out of Ncdn, an abundant down-regulated gene, opposes the conditioning-induced changes in satiety-sensitive feeding behavior and apparent motivation for highly palatable food, suggesting down-regulation may be a compensatory mechanism. CONCLUSIONSOur results emphasize the importance of mRNA alterations D2 striatal projection neurons in the NAc in the behavioral consequences of highly palatable food conditioning and suggest a modulatory contribution of Ncdn downregulation.

neuroscience↗

bioRxiv2021

Pyk2 in dorsal hippocampus plays a selective role in spatial memory and synaptic plasticity

Pyk2 is a Ca2+-activated non-receptor tyrosine kinase enriched in the forebrain, especially in pyramidal neurons of the hippocampus. Previous reports suggested its role in hippocampal synaptic plasticity and spatial memory but with contradictory findings possibly due to experimental conditions. Here we address this issue and show that novel object location, a simple test of spatial memory induced by a single training session, is altered in Pyk2 KO mice and that re-expression of Pyk2 in the dorsal hippocampus corrects this deficit. Bilateral targeted deletion of Pyk2 in dorsal hippocampus CA1 region also alters novel object location. Long term potentiation (LTP) in CA1 is impaired in Pyk2 KO mice using a high frequency stimulation induction protocol nut not with a theta burst protocol, explaining differences between previous reports. The same selective LTP alteration is observed in mice with Pyk2 deletion in dorsal hippocampus CA1 region. Thus, our results establish the role of Pyk2 in specific aspects of spatial memory and synaptic plasticity and show the dependence of the phenotype on the type of experiments used to reveal it. In combination with other studies they provide evidence for a selective role of non-receptor tyrosine kinases in specific aspects of hippocampal neurons synaptic plasticity.

neuroscience↗

bioRxiv2020

Translational profiling of mouse dopaminoceptive neurons reveals a role of PGE2 in dorsal striatum

Forebrain dopaminoceptive neurons play a key role in movement, action selection, motivation, and working memory. Their activity is dysregulated in addiction, Parkinsons disease and other conditions. To characterize the diverse dopamine target neuronal populations, we compare translating mRNAs in neurons of dorsal striatum and nucleus accumbens expressing D1 or D2 dopamine receptor and prefrontal cortex expressing D1 receptor. We identify D1/D2 and striatal dorso-ventral differences in the translational and splicing landscapes, which establish the characteristics of dopaminoceptive neurons. Expression differences and network analyses identify novel transcription factors with presumptive roles in these differences. Prostaglandin E2 appears as a candidate upstream regulator in the dorsal striatum, a hypothesis supported by converging functional evidence indicating its role in enhancing D2 dopamine receptor action. Our study provides powerful resources for characterizing dopamine target neurons, new information about striatal gene expression patterns, and reveals the unforeseen role of prostaglandin E2 in the dorsal striatum.

neuroscience↗

bioRxiv2019

Pyk2 in D1 receptor-expressing neurons of the nucleus accumbens modulates the acute locomotor effects of cocaine

The striatum is a critical brain region for locomotor response to cocaine. Although the D1 receptor-expressing neurons are centrally involved in mediating the locomotor effects of cocaine, the molecular pathways controlling this response are not fully understood. Here we studied the role of Pyk2, a non-receptor calcium-dependent protein-tyrosine kinase, in striatum-related functions. We discovered that cocaine injection increases Pyk2 phosphorylation in the striatum of mice in vivo. Pyk2-deficient mice displayed an altered locomotor response to acute cocaine injection. In contrast, they developed normal locomotor sensitization and cocaine-conditioned place preference. Accordingly, a cocaine-activated signaling pathway essential for these late responses, ERK phosphorylation, was not altered. Specific deletion of Pyk2 in the nucleus accumbens or in D1 neurons reproduced this phenotype, whereas deletion of Pyk2 in the dorsal striatum or in A2A receptor-expressing neurons did not. Mice lacking Pyk2 in D1-neurons also displayed lower locomotor response to the D1 receptor agonist SKF-81297 but not to an anticholinergic drug. Our results identify Pyk2 as a regulator of acute locomotor responses to psychostimulants and suggest that changes in Pyk2 expression or activation may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine action.

neuroscience↗