Biology subjects

Krüger, A.

Publications and source records attributed to Krüger, A..

6 recordsLinked to original sources

bioRxiv2025

Genome-wide analysis of DtxR and HrrA regulons reveals novel targets and a high level of interconnectivity between iron and heme regulatory networks in Corynebacterium glutamicum

Iron is an essential trace element required by nearly all organisms as a cofactor in enzymes, regulatory proteins, and cytochromes of the respiratory chain. Maintaining iron homeostasis is crucial, since elevated levels cause oxidative stress through the formation of reactive oxygen species. In Corynebacterium glutamicum, iron and heme homeostasis are tightly interconnected and controlled by the global regulators DtxR and HrrA. While DtxR senses intracellular Fe2+, the two-component system HrrSA is activated by heme, functioning as a global regulator of heme homeostasis. This study provides the first genome-wide analysis of DtxR and HrrA binding dynamics under varying iron and heme conditions using chromatin affinity purification sequencing (ChAP-seq). Our conditional ChAP- Seq approach revealed 25 novel DtxR targets and 210 previously unrecognized HrrA targets. Among these, metH, encoding homocysteine methyltransferase, and xerC, encoding a tyrosine recombinase, were bound by DtxR exclusively under heme conditions, underscoring condition-dependent variation in DtxR binding. Activation of metH by DtxR links iron metabolism to methionine synthesis, potentially relevant for the mitigation of oxidative stress. Beyond novel targets, this study highlights the interconnected nature of the DtxR and HrrA regulons, identifying 16 shared targets with in some cases overlapping operator sequences. Strikingly, we provide several examples for weak ChAP-Seq peaks, often disregarded in global approaches, that feature a significant impact of the regulator on differential gene expression. These findings emphasize the importance of genome-wide profiling under different conditions to uncover novel targets and shed light on the complexity and dynamic nature of bacterial regulatory networks. ImportanceThe trace element iron is essential for life, but elevated levels can rapidly cause cellular damage through oxidative stress. Bacteria, like Corynebacterium glutamicum, tightly regulate iron and heme homeostasis via the global regulators DtxR and HrrA. This study provides the first analysis of the genome-wide binding patterns of these two regulators demonstrating significant differences in binding dependent on the tested iron regimes. Overall, we identified 25 new DtxR targets and 210 previously unknown HrrA targets, including genes with crucial roles in central metabolism and DNA repair. Notably, DtxR was shown to link iron metabolism to methionine synthesis, which might be important to protect the cell from oxidative stress. Our findings highlight the interconnected nature of DtxR and HrrA networks and underscore the value of condition-specific analysis to deepen the understanding of how bacteria adapt to environmental changes.

microbiology↗

bioRxiv2024

Scalable cell-free production of active T7 RNA polymerase

The SARS-CoV-2 pandemic highlighted the urgent need for biomanufacturing paradigms that are robust and fast. Here, we demonstrate the rapid process development and scalable cell-free production of T7 RNA polymerase, a critical component in mRNA vaccine synthesis. We carry out a one-liter cell-free gene expression (CFE) reaction that achieves over 80% purity, low endotoxin levels, and enhanced activity relative to commercial T7 RNA polymerase. To achieve this demonstration, we implement rolling circle amplification to circumvent difficulties in DNA template generation, and tune cell-free reaction conditions, such as temperature, additives, purification tags and agitation to boost yields. We achieve production of a similar quality and titer of T7 RNA polymerase over more than 4 orders of magnitude reaction volume. This proof of principle positions CFE as a viable solution for decentralized biotherapeutic manufacturing, enhancing preparedness for future public health crises or emergent threats.

synthetic biology↗

bioRxiv2023

Network-based prioritisation and validation of novel regulators of vascular smooth muscle cell proliferation in disease

Aberrant vascular smooth muscle cell (VSMC) homeostasis and proliferation are hallmarks of vascular diseases causing heart attack and stroke. To elucidate molecular determinants governing VSMC proliferation, we reconstructed gene regulatory networks from single cell transcriptomics and epigenetic profiling. We find progressive activation of enhancers at disease-relevant loci in VSMCs that dont respond to injury and proliferation-predisposed cells. Our analysis suggests that while many transcription factors are shared, their target loci differ between VSMC states. Comparison of rewiring between VSMC subsets and in silico perturbation analysis prioritised novel regulators, including RUNX1 and the network target TIMP1. We experimentally validated that the pioneer factor RUNX1 increased VSMC responsiveness and show that TIMP1 feeds back to promote VSMC proliferation through CD74-mediated STAT3 signalling. Both RUNX1 and the TIMP1-CD74 axis were expressed in hVSMCs, at low frequency in normal arteries and increased in disease, suggesting clinical significance and potential as novel vascular disease targets.

molecular biology↗

bioRxiv2023

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

Cell-free gene expression (CFE) systems are powerful tools for transcribing and translating genes outside of a living cell. Given their diverse roles in nature, synthesis of membrane proteins is of particular interest, but their yield in CFE is substantially lower than for soluble protein. In this paper, we study factors that affect the cell-free synthesis of membrane proteins and develop a quantitative kinetic model of their production. We identify that stalling of membrane protein translation on the ribosome is a strong predictor of membrane protein synthesis and creates a negative feedback loop in which stalled peptide sequences quench ribosome activity through aggregation between the ribosome nascent chains. Synthesis can be improved by the addition of lipid membranes which incorporate protein nascent chains and, therefore, kinetically competes with aggregation. Using both quantitative modeling and experiment, we show that the balance between peptide-membrane association and peptide aggregation rates determines the total yield of synthesized membrane protein. We then demonstrate that this balance can be shifted by altering membrane composition or the protein N-terminal domain sequence. Based on these findings, we define a membrane protein expression score that can be used to rationalize the engineering of N-terminal domain sequences both of a native and computationally designed membrane proteins produced through CFE.

synthetic biology↗

bioRxiv2022

Structure-based discovery of a non-canonical prototype long-chain monoacylglycerol lipase through an endogenous catalysis intermediate complex

The identification and characterization of enzyme function is largely lacking behind the rapidly increasing availability of large numbers of sequences and associated high-resolution structures. This is often hampered by lack of knowledge on in vivo relevant substrates. Here, we present a case study of a high-resolution structure of an unusual orphan lipase in complex with an endogenous C18 monoacylglycerol ester reaction intermediate from the expression host, which is insoluble under aqueous conditions and thus not accessible for studies in solution. The data allowed its functional characterization as a prototypic long-chain monoacylglycerol lipase, which uses a minimal lid domain to position the substrate through a hydrophobic tunnel directly to the enzymes active site. Knowledge about the molecular details of the substrate binding site allowed us to modulate the enzymatic activity by adjusting protein/substrate interactions, demonstrating the potential of our findings for future biotechnology applications.

biochemistry↗

bioRxiv2019

The impact of CO2/HCO3- availability on anaplerotic flux in PDHC-deficient Corynebacterium glutamicum strains

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate yielding acetyl-CoA and CO2. The PDHC-deficient Corynebacterium glutamicum strain {Delta}aceE is therefore lacking an important decarboxylation step in central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15 hour lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates like acetate. Growth was successfully restored by deletion of ptsG encoding the glucose-specific permease of the PTS system, thereby linking the observed phenotype to the increased sensitivity of strain {Delta}aceE {Delta}pyc to glucose catabolism. In the following, strain {Delta}aceE {Delta}pyc was used to systematically study the impact of perturbations of the intracellular CO2/HCO3- pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3- levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via pentose phosphate pathway, at least partially eliminated the lag phase of strain {Delta}aceE {Delta}pyc on glucose medium. In accordance, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase accompanied with the excretion of L-valine and L-alanine. Transcriptome analysis as well as an adaptive laboratory evolution experiment of strain {Delta}aceE {Delta}pyc revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose/acetate mixtures. Altogether, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains suffering from low endogenous CO2 production rates as exemplified by PDHC-deficient strains.\n\nImportanceCO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3- pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which was additionally lacking pyruvate carboxylase ({Delta}aceE {Delta}pyc). This strain featured a >15 h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3- pool on growth on glucose-containing medium. Remarkably, all measures enhancing the CO2/HCO3- levels successfully restored growth emphasizing the strong impact of the intracellular CO2/HCO3- pool on metabolic flux especially in strains suffering from low endogenous CO2 production rates.

microbiology↗