Biology subjects

Lindberg, P.

Publications and source records attributed to Lindberg, P..

5 recordsLinked to original sources

bioRxiv2026

Conversion of CO2 into valuable products: Engineering the PirC-PGAM switch in cyanobacteria to direct carbon flux into desired products

BackgroundIn response to rising CO2 emissions driving global warming, there is an urgent need for a transition toward a sustainable bioeconomy. Photo-biotechnological processes based on oxygenic photosynthesis hold high potential for achieving CO2 neutrality and in this regard, cyanobacteria have emerged as promising biocatalysts. Rational metabolic engineering of cyanobacteria depends on a thorough understanding of native regulatory mechanisms governing primary metabolism, which can limit metabolic flux through specific pathways and, consequently, the formation of target products. Recent insights have identified a key regulatory node at the 2,3-bisphosphogylcerate-independent phosphoglycerate mutase (PGAM) reaction, where the metabolic flux from newly fixed carbon is redirected from the Calvin-Benson-Bassham (CBB) cycle towards lower glycolysis. This metabolic valve is controlled by the small inhibitor protein PirC, whose binding to PGAM is determined by the central signal transduction protein PII. ResultsIn this study, we exploit the PirC-PGAM interaction as a novel target for regulatory metabolic engineering in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Chassis strains with engineered control of PGAM, defined as PGAM-ON or PGAM-OFF states, were generated using two complementary approaches: tuning pgam gene expression and modulating PirC abundance to regulate PGAM activity. The effectiveness of this regulatory engineering strategy was demonstrated by redirecting carbon flux toward two representative, naturally occurring products: sucrose, produced via gluconeogenesis fueled by the Calvin-Benson-Bassham (CBB) cycle, and succinate, an intermediate of the tricarboxylic acid (TCA) cycle. Narrowing the PGAM valve resulted in a threefold increase in sucrose accumulation. In contrast, opening the PGAM valve by relieving PGAM inhibition through pirC deletion or separate pgam overexpression resulted in up to an 18-fold increase in succinate excretion. Furthermore, similar genetic configurations were applied to enhance production of a heterologous compound, isoprene, derived from pyruvate. ConclusionsThis study establishes the PGAM valve as a tunable control point for the rational re-direction of carbon flux in Synechocystis and highlights small regulatory proteins as powerful targets for metabolic engineering. Together, these findings provide proof of concept for an advanced level of molecular engineering in cyanobacteria and to fully harness their biocatalytic potential in future photosynthesis-driven biotechnological applications.

bioengineering↗

bioRxiv2024

microRNA and non-targeted proteome analysis of liquid biopsies from the distal lung collected by Particles in Exhaled Air (PExA) reveals presence from extracellular vesicles

Background. Early detection and longitudinal follow-up are essential for timely diagnosis and treatment for lung diseases. Currently, invasive methods are often required to examine the distal parts of the lungs. The growing need to explore the bio-molecular mechanisms in various lung diseases highlights the importance of non-invasive methods. The use of particles in exhaled air (PExA), a non-invasive technique for sampling of epithelial lining fluid from distal airways, is gaining attention. MicroRNAs (miRNAs) are crucial in modulating protein expression both intracellularly and intercellularly, often transported via extracellular vesicles. Dysregulated miRNAs have been linked to many pulmonary diseases, and their relative stability, especially when encapsulated in EVs, makes them promising biomarkers. Here we report for the first time multi-modal analysis of miRNAs and proteins in PExA, offering an opportunity to study the role of miRNAs in the pathophysiology of respiratory diseases in a non-invasive manner. Methods. Exhaled particles were collected from healthy subjects using the PExA 2.0 instrument utilizing the optimized PExA breathing maneuver. PExA samples collected on different types of impaction membranes were analyzed using a non-targeted mass spectrometry-based proteomics workflow optimized for single-cell detection, and a miRNAseq workflow optimized for low input starting material. Technical validation of a subset of the detected miRNAs was performed using custom-designed miRCURY LNA miRNA PCR assays. Pathway enrichment analyses for the detected proteins were performed using STRING. Results. Proteomic analysis consistently identified over 50 proteins across multiple types of impaction membranes, sample dilution series, and individuals down to a single PEx sPOT (24ng starting material). We observed a significant enrichment of proteins associated with extracellular vesicles, including blood microparticles, and secretory granules. miRNA-seq revealed 39 mature miRNAs, the majority of which have been previously reported to be detected in the airways. Some were also reported to be secreted by primary human airway epithelial cells via extracellular vesicles. miRNA-125b and the members of the let-7 family were among the most abundant miRNAs detected. Fluorometric assays showed significant RNase activity in both PExA and other lung-related samples, such as bronchoalveolar lavage fluid, suggesting that this activity originates from the airways and is independent of the sampling techniques used. The workflow for extraction and processing of the PExA collection membrane, tested with abundant synthetic miRNAs and analyzed using the miRCURY LNA miRNA PCR assay, yielded results comparable to control samples, indicating that the membrane material does not interfere with the assay. Conclusions. Using PExA, we identified several miRNAs reported to be dysregulated in pulmonary disorders. The enrichment of extracellular secretory components in the core protein list, along with the elevated RNAse activity in the respiratory tract, suggest that the detected miRNAs may be encapsulated within extracellular vesicles. These miRNAs are of particular interest due to their potential role in intercellular communication. Our findings suggest that PExA holds a potential as a non-invasive tool for studying extracellular vesicle-mediated miRNA cargo in the small airways.

physiology↗

bioRxiv2024

CORACLE (COVID-19 liteRAture CompiLEr): A platform for efficient tracking and extraction of SARS-CoV-2 and COVID-19 literature, with examples from post-COVID with respiratory involvement

BackgroundDuring the COVID-19 pandemic there emerged a need to efficiently monitor and process large volumes of scientific literature on the subject. Currently, as the pandemic is winding down, the clinicians encountered a novel syndrome - Post-acute Sequelae of COVID- 19 (PASC) - that affects over 10% of those who contract SARS-CoV-2 and presents a significant and growing challenge in the medical field. The continuous influx of new research publications underscores a critical need for efficient tools for navigating the literature. ObjectivesWe aimed to develop an application which will allow monitoring and categorizing COVID-19-related literature through building publication networks and medical subject headings (MeSH) maps to be able to quickly identify key publications and publication networks. MethodsWe introduce CORACLE (COVID-19 liteRAture CompiLEr), an innovative web application designed for the analysis of COVID-19-related scientific articles and the identification of research trends. CORACLE features three primary interfaces: The "Search" interface, which displays research trends and citation links; the "Citation Map" interface, allowing users to create tailored citation networks from PubMed Identifiers (PMIDs) to uncover common references among selected articles; and the "MeSH" interface, highlighting current MeSH trends and associations between MeSH terms. ResultsOur web application, CORACLE, leverages regularly updated PubMed data to aggregate and categorize the extensive literature on COVID-19 and PASC, aiding in the identification of relevant research publication hubs. Using lung function in PASC patients as a search example, we demonstrate how to identify and visualize the interactions between the relevant publications. ConclusionCORACLE proves to be an effective tool for the extraction and analysis of literature. Its functionalities, including the MeSH trends and customizable citation mapping, facilitate the discovery of relevant information and emerging trends in COVID-19 and PASC research.

bioinformatics↗

bioRxiv2022

L-Aspartate oxidase provides new insights into fumarate reduction in anaerobic darkness in Synechocystis sp. PCC6803

Cyanobacteria are promising microbial hosts for production of various industrially relevant compounds, such as succinate, a central metabolite of the tricarboxylic acid cycle (TCA). Cyanobacteria have been engineered to produce succinate during photoautotrophic growth, and are also able to secrete it during anoxic fermentation conditions. It has been assumed that under anoxic darkness, succinate can be formed by reduction of fumarate catalyzed by the succinate dehydrogenase complex (SDH), however, no characterization of SDH regarding this activity has been performed. In this study, we address this issue by generating strains of the unicellular cyanobacterium Synechocystis PCC 6803 (Synechocystis) deficient in one or several subunits of SDH, and investigating succinate accumulation in these strains during dark anaerobic fermentation. The results showed higher succinate accumulation in SDH deletion strains than in the wild type, indicating a succinate dehydrogenase activity of SDH rather than fumarate reduction under these conditions. We further explored the possibility of another potential route for succinate formation from fumarate via L-aspartate oxidase (Laspo). The gene encoding Laspo in Synechocystis could not be inactivated, indicating an essential function for this enzyme. Using purified SynLaspo, we could demonstrate in vitro that in addition to L-aspartate oxidation the enzyme exhibits an L-aspartate-fumarate oxidoreductase activity. We therefore suggest that reduction of fumarate to succinate during anoxic darkness can be a byproduct of the Laspo reaction, which is the first step in biosynthesis of NAD cofactors. This work contributes to the understanding of cyanobacterial TCA cycle for future engineering and sustainable production of dicarboxylic acids.

biochemistry↗

bioRxiv2019

High density cultivation for efficient sesquiterpenoid biosynthesis in Synechocystis sp. PCC 6803

Cyanobacteria and microalgae are attractive phototrophic host systems for climate-friendly production of fuels and other high-value chemicals. The biosynthesis of an increasing diversity of industrially relevant compounds such as terpenoids has been demonstrated in recent years. To develop economically feasible and sustainable process designs, major challenges still remain regarding intracellular carbon partitioning, specific metabolic pathway activities and efficient cultivation strategies. Here, we present a technical study on comparative characteristics of sesquiterpene and sesquiterpene alcohol accumulation in engineered strains of Synechocystis sp. PCC 6803 (substrain GT-U) under different growth conditions and cell densities. This study particularly focuses on the basic applicability of a commercial High Density Cultivation platform in the presence of a dodecane overlay, which serves as a standard in-situ extractant and sink for various hydrophobic biochemicals. Significantly, the presented data demonstrate high volumetric productivities of (E)--bisabolene under high-density conditions that are more than two orders of magnitude higher than previously reported for cyanobacteria. Operating in a two-step semi-batch mode over a period of eight days, average final volumetric titers of 179.4 {+/-} 20.7 mg * L-1 were detected. Likewise, the sesquiterpene alcohols (-)-patchoulol and (-)--bisabolol accumulated to many times higher levels in high density cultivation than under standard batch conditions, with final titers of 17.3 {+/-} 1.85 mg * L-1 and 96.3 {+/-} 2.2 mg * L-1, respectively. In contrast, specific product accumulation (mg * L-1 * OD750-1) was compromised particularly for bisabolene in the high density system during phases of high biomass accumulation rates. Volumetric productivities were high during linear growth at high densities, distinctly outperforming standard batch systems. While the presented data highlight the benefits of high-density strategies for highly efficient phototrophic terpenoid production, they further point at the presence of major metabolic bottlenecks for engineered terpenoid biosynthesis and the requirement for systematic and/or targeted strategies to sustainably redirect inherent carbon fluxes in cyanobacteria. Together, our data provide additional insights into growth- and density-related effects on the efficiency of product accumulation, introducing low-scale High Density Cultivation as a rapid and efficient platform for screening of heterologous terpenoid production in cyanobacteria.

bioengineering↗