Biology subjects

Rubio, L. M.

Publications and source records attributed to Rubio, L. M..

8 recordsLinked to original sources

bioRxiv2026

Natural variation in NifU and NifS enhances chloroplasts compatibility for nitrogenase engineering

Reconstitution of functional nitrogenase in plants requires the coordinated expression of the [Fe-S] cluster assembly proteins NifU and NifS. However, the extent to which these proteins interact with endogenous Fe-S metabolism and affect plant physiology remains unclear. Here, we compared NifU and NifS homologs from diverse diazotrophs to identify variants compatible with the plant chloroplast environment. Selected variants of Azotobacter vinelandii, Fischerella thermalis, and Marinobacter lutimaris were characterized by transient expression in Nicotiana benthamiana and stable transformation in rice. Plant-produced NifU was largely devoid of [Fe-S] clusters when isolated but retained strong capacity for in vitro [Fe-S] cluster reconstitution and apo-NifH activation in a Ft > Av >Ml gradient, indicating correct folding and function but limited cluster loading or stability in vivo. NifU and NifS expression in transgenic rice resulted in variant-dependent proteome and phenotype effects, with A. vinelandii-expressing lines exhibiting severe defects, F. thermalis lines showing intermediate phenotype, and M. lutimaris lines being indistinguishable from wild type. These results reveal a trade-off between the biochemical activity of NifU and NifS and their compatibility with host metabolism, which must be considered for successful nitrogenase engineering in plants. HighlightNifU/NifS homolog selection determines trade-offs between [Fe-S] cluster assembly activity and plant compatibility, identifying variants that minimize physiological disruption while supporting nitrogenase cofactor assembly in chloroplasts.

plant biology↗

bioRxiv2026

Metabolic commitment and nitrogen control of diazotrophy in the diazoplast-containing diatom Epithemia adnata

Earths nitrogen cycle is central to sustaining ecosystem productivity and global biogeochemical balance. Although biological N2-fixation is well characterized in prokaryotes and plant symbioses, in other eukaryotic lineages it remains poorly understood. Diatoms of the family Rhopalodiacea harbor diazoplasts, endosymbiotic spheroid bodies specialized for N2-fixation. This makes these diatoms genuine N2-fixing eukaryotes that represent a unique model for organelle evolution, parallel but distinct from haptophyte nitroplasts. Here, we report the isolation and stable cultivation of an Epithemia adnata strain, the sequencing of its diazoplast genome and its proteomic profile when growing diazotrophically in the light or darkness, or upon exposure to ammonium. Our analyses reveal that ammonium induced broad down-regulation of diazoplast proteins, particularly those linked to N2-fixation, ATP synthesis, and central carbon metabolism underscoring a general regulatory commitment toward diazotrophic metabolism tightly coupled to host carbon and nitrogen status. The pentose phosphate pathway and ferredoxin-NADP oxidoreductase appear as likely source of reductant to nitrogenase. A striking enrichment of chaperones, peroxiredoxins, bacterioferritin-like proteins, and DpsA might stabilize nitrogenase and buffer against oxidative stress during light-driven diazotrophy. Importantly, we identified a plasmid-encoded GlpF as a putative glycerol transporter, pointing to glycerol-mediated host-symbiont metabolic integration in the extant symbiosis and possibly a crucial innovation during the early evolutionary stages of its establishment. Thus, diazoplast activity is not autonomous but requires integration with host carbon and nitrogen status, establishing glycerol transport, reductant supply, stress mitigation, and nutrient-responsive regulation as pivotal mechanisms of nitrogenase activity and host integration. These findings have broad implications for biogeochemical cycling, organellogenesis, and synthetic biology strategies aimed at engineering N2-fixation in crop plants. SignificanceN2-fixing eukaryotes are increasingly recognized as abundant algae containing bacterial-derived diazotrophic endosymbionts, representing an underappreciated component of global N cycling. Diazoplasts in rhopalodiacean diatoms represent a compelling example of such endosymbionts specialized for N2-fixation. By combining genomic sequencing and proteomic analysis, we demonstrate their metabolic specialization, host integration, and regulatory commitment to diazotrophy. These findings reinforce the emerging view that diazoplasts function as organelle-like entities dedicated to N2-fixation, dependent on host-supplied carbon while contributing fixed N in return. Beyond giving evolutionary insights into organellogenesis, this work establishes a framework for translational applications, such as engineering N2-fixation into agricultural plants. Such advances could reduce reliance on synthetic fertilizers, influence biogeochemical cycles, and promote sustainable food production.

plant biology↗

bioRxiv2025

Azotobacter vinelandii glutaredoxin D delivers the core cluster to nitrogenase cofactor scaffold protein NifU

The scaffold protein NifU plays a central role in assembling the precursor [Fe4S4] clusters required for nitrogenase to function. The synthesis of these precursors depends on a catalytic [Fe2S2] group within NifU core ferredoxin domain. Here, we show that the monothiol glutaredoxin GrxD delivers this cluster to the NifU scaffold protein. Consistently, grxD mutants have reduced nitrogenase activity, the result of altered iron allocation to this enzyme. Biochemical assays show that GrxD unidirectionally transfers [Fe2S2] to NifU through protein-protein interaction. This allows GrxD to restore apo-NifU functionality, enabling proper [Fe4S4] synthesis, and NifH activation. These findings are crucial to understand how iron is allocated to nitrogenase for biological nitrogen fixation.

biochemistry↗

bioRxiv2025

A high-throughput heterologous expression platform for plant synthetic biology based on Arabidopsis suspension cells

Efficient heterologous expression platforms are essential for plant synthetic biology, particularly for engineering complex multigene pathways. Here, we establish a high-throughput system for both transient and stable transformation of Arabidopsis thaliana suspension cells using plant cell pack infiltration. This method requires no specialized equipment or consumables and is compatible with several cell lines. It enables rapid generation of 100 g of transgenic cells within two weeks and allows expression of at least 6 stacked genes from a single construct. We characterized constitutive promoters for gene expression in Arabidopsis cells and validated plastid targeting peptides. A library of NifB homologs was screened for expression and solubility and several archaeal variants suitable for plant expression were identified. We further engineered stable cell lines expressing up to six genes, encoding the NifB module components NifU, NifS, FdxN, and NifB, demonstrating that the newly developed platform integrates into an established workflow for nitrogenase engineering. The platform accelerates design-build-test cycles and facilitates the production of delicate proteins that require large amounts of transgenic biomass. It thus represents a versatile and scalable tool for advancing synthetic biology and for tackling major biotechnological challenges, such as biological nitrogen fixation. HighlightWe developed a fast and scalable expression platform in Arabidopsis suspension cells, enabling transient and stable multigene expression for applications in plant synthetic biology such as nitrogenase engineering.

plant biology↗

bioRxiv2025

Molecular sorting of nitrogenase catalytic cofactors

The free-living diazotroph Azotobacter vinelandii produces three genetically distinct but functionally and mechanistically similar nitrogenase isozymes, designated as Mo-dependent, V-dependent, and Fe-only. They respectively harbor nearly identical catalytic cofactors that are distinguished by a heterometal site occupied by Mo (FeMo-cofactor), V (FeV-cofactor), or Fe (FeFe-cofactor). Completion of FeMo-cofactor and FeV-cofactor formation occurs on molecular scaffolds prior to delivery to their catalytic partners. In contrast, completion of FeFe-cofactor assembly occurs directly within its cognate catalytic partner. Because hybrid nitrogenase species that contain the incorrect cofactor type cannot reduce N2 to support diazotrophic growth there must be a way to prevent misincorporation of an incorrect cofactor when different nitrogenase isozyme systems are produced at the same time. Here, we show that fidelity of the Fe-only nitrogenase is preserved by blocking the misincorporation of either FeMo-cofactor or FeV-cofactor during its maturation. This protection is accomplished by a two-domain protein, designated AnfO. It is shown that the N-terminal domain of AnfO binds to an immature form of the Fe-only nitrogenase and the C-terminal domain, tethered to the N-terminal domain by a flexible linker, has the capacity to capture FeMo- and FeV-cofactor. AnfO does not prevent the normal activation of Fe-only nitrogenase because completion of FeFe-cofactor assembly occurs within its catalytic partner and, therefore, is never available for capture by AnfO. These results support a post-translational mechanism involving the molecular sorting of structurally similar metallocofactors that involve both protein-protein interactions and metallocofactor binding while exploiting differential pathways for nitrogenase associated catalytic cofactor assembly.

biochemistry↗

bioRxiv2024

Dynamics of genetic circuits in Pseudomonas protegens

The engineering of genetic circuits to perform predefined computations is central to synthetic biology, enabling living cells with new functionalities applicable across various domains. However, these circuits are often specifically tailored to particular cellular hosts, with Escherichia coli being the most popular. Consequently, their intended functions may not translate well to other organisms, limiting their scope. Understanding circuit dynamics in less familiar organisms is crucial, especially for niche-specific applications requiring cellular chassis different from model organisms generally used in synthetic biology. Here, we develop a combined experimental and theoretical pipeline to evaluate the performance of NOT logic circuits, also called inverters, in the soil bacterium Pseudomonas protegens Pf-5--a host renowned for its unique environmental functions and a newcomer to genetic circuitry. Inverters were experimentally tested to characterize input-output functionality, and mathematical modelling was used to infer the dynamic principles of circuit modules. The model quantified the individual impacts of key parameters--such as translation efficiency, repressor performance, and promoter activity--on output levels, enabling predictions about inter-host circuit portability. This parameter calibration revealed unique properties of the chassis, including faster transitions between on and off circuit states compared to the synthetic biology workhorse Pseudomonas putida. These characteristics may reflect adaptations to the fluctuating conditions of the plant rhizosphere, where this bacteria thrives. As a result, our work provides DNA parts, circuits and mathematical characterizations to establish P. protegens Pf-5 as a viable chassis for environmental synthetic biology.

synthetic biology↗

bioRxiv2021

Azotobacter vinelandii scaffold protein NifU transfers iron to NifQ as part of the iron-molybdenum cofactor biosynthesis pathway for nitrogenase

Azotobacter vinelandii molybdenum-dependent nitrogenase obtains molybdenum from NifQ, a monomeric iron-sulfur molybdoprotein. This protein requires of a preexisting [Fe-S] cluster to form a [MoFe3S4] group to serve as specific donor during nitrogenase cofactor biosynthesis. Here, we show biochemical evidence for NifU being the donor of the [Fe-S] cluster. Protein-protein interaction studies using apo-NifQ and as-isolated NifU demonstrated the interaction between both proteins which is only effective when NifQ is unoccupied by its [Fe-S] cluster. The apo-NifQ iron content increased after the incubation with as-isolated NifU, reaching similar levels to holo-NifQ after the interaction between apo-NifQ and NifU with reconstituted transient [Fe4-S4] groups. These results also indicate the necessity of co-expressing NifU together with NifQ in the pathway to provide molybdenum for the biosynthesis of nitrogenase in engineered nitrogen-fixing plants.

biochemistry↗

bioRxiv2021

Deploying an in vitro gut model to assay the impact of a mannan-oligosaccharide prebiotic, Bio-Mos(R) on the Atlantic salmon (Salmo salar) gut microbiome.

Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and the associated metabolism. Bio-Mos is a commercially available pre-biotic currently used in the agri-feed industry. To assess Bio-Mos for potential use as a prebiotic growth promotor in salmonid aquaculture, we have modified an established Atlantic salmon in vitro gut model, SalmoSim, to evaluate its impact on the host microbial communities. Inoculated from biological triplicates of adult farmed salmon pyloric caeca compartments, the microbial communities were stabilised in SalmoSim followed by a twenty-day exposure to the prebiotic and in turn followed by an eight day wash out period. Dietary inclusion of MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium, Agarivorans, Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to MOS supplementation with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggests that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators. ImportanceIn this paper we report the results of the impact of prebiotic (MOS supplementation) on microbial communities within recently developed Atlantic salmon gut microbiome in vitro simulator. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production in the Atlantic salmon gut and correlates with a significant shift in microbial community composition with observed increase in lactic acid producing Carnobacterium. In conjunction with previous in vivo studies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role of in vitro gut models to augment in vivo trials of microbiome modulators.

bioengineering↗