Cell-Free Systems to Mimic and Expand Metabolism.

Cell-free synthetic biology incorporates purified components and/or crude cell extracts to carry out metabolic and genetic programs. While protein synthesis has historically been the primary focus, more metabolism researchers are now turning toward cell-free systems either to prototype pathways for cellular implementation or to design new-to-nature reaction networks that incorporate environmentally relevant substrates or new energy sources. The ability to design, build, and test enzyme combinations has accelerated efforts to understand metabolic bottlenecks and engineer high-yielding pathways.

Allosteric regulation of pyruvate kinase enables efficient and robust gluconeogenesis by preventing metabolic conflicts and carbon overflow.

Gluconeogenesis, the reciprocal pathway of glycolysis, is an energy-consuming process that generates glycolytic intermediates from non-carbohydrate sources. In this study, we demonstrate that robust and efficient gluconeogenesis in bacteria relies on the allosteric inactivation of pyruvate kinase, the enzyme responsible for the irreversible final step of glycolysis. Using the model bacterium as an example, we discovered that pyruvate kinase activity is inhibited during gluconeogenesis via its extra C-terminal domain (ECTD), which is essential for autoinhibition and metabolic regulation.

Engineering the future of medicine: Natural products, synthetic biology and artificial intelligence for next-generation therapeutics.

The eXchange Unit between Thiolation domains approach and artificial intelligence (AI)-driven tools like Synthetic Intelligence are transforming nonribosomal peptide synthetase and polyketide synthase engineering, enabling the creation of novel bioactive compounds that address critical challenges like antibiotic resistance and cancer. These innovations expand chemical space and optimize biosynthetic pathways, offering precise and scalable therapeutic solutions. Collaboration across synthetic biology, AI, and clinical research is essential to translating these breakthroughs into next-generation treatments and revolutionizing drug discovery and patient care.

From Isolation to Application: Utilising Phage-Antibiotic Synergy in Murine Bacteremia Model to Combat Multidrug-Resistant Enterococcus faecalis.

Enterococcus species, natural inhabitants of the human gut, have become major causes of life-threatening bloodstream infections (BSIs) and the third most frequent cause of hospital-acquired bacteremia. The rise of high-level gentamicin resistance (HLGR) in enterococcal isolates complicates treatment and revives bacteriophage therapy. This study isolated and identified forty E.

Proton Tunneling Allows a Proton-Coupled Electron Transfer Process in the Cancer Cell.

Proton-coupled electron transfer (PCET) is a fundamental redox process and has clear advantages in selectively activating challenging C-H bonds in many biological processes. Intrigued by this activation process, we aimed to develop a facile PCET process in cancer cells by modulating proton tunneling. This approach should lead to the design of an alternative photodynamic therapy (PDT) that depletes the mitochondrial electron transport chain (ETC), the key redox regulator in cancer cells under hypoxia.

Light-Controlled Secretion and Injection of Proteins into Eukaryotic Cells by Optogenetic Control of the Bacterial Type III Secretion System.

Gram-negative bacteria can use the type III secretion system (T3SS) to inject effector proteins into eukaryotic target cells. In this chapter, we describe the application of a light-controlled T3SS, based on the targeted sequestration of an essential dynamic T3SS component with the help of optogenetic interaction switches. This method enables to control the secretion or injection into eukaryotic cells for a wide range of protein cargos with high temporal and spatial precision.

Impact of absolute values and changes in meteorological and air quality conditions on community-acquired pneumonia in Germany.

Community-acquired pneumonia (CAP) is a major global health concern as it is a leading cause of morbidity, mortality and economic burden to the health care systems. In Germany, more than 15,000 people die every year from CAP. Climate change is altering weather patterns, and it may influence the probability and severity of CAP.

The Dual Roles of Lamin A/C in Macrophage Mechanotransduction.

Cellular mechanotransduction is a complex physiological process that integrates alterations in the external environment with cellular behaviours. In recent years, the role of the nucleus in mechanotransduction has gathered increased attention. Our research investigated the involvement of lamin A/C, a component of the nuclear envelope, in the mechanotransduction of macrophages under compressive force.

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme.

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs.

- & : plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly.

Golden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation.

New-to-nature CO-dependent acetyl-CoA assimilation enabled by an engineered B-dependent acyl-CoA mutase.

Acetyl-CoA is a key metabolic intermediate and the product of various natural and synthetic one-carbon (C1) assimilation pathways. While an efficient conversion of acetyl-CoA into other central metabolites, such as pyruvate, is imperative for high biomass yields, available aerobic pathways typically release previously fixed carbon in the form of CO. To overcome this loss of carbon, we develop a new-to-nature pathway, the Lcm module, in this study.

Layered entrenchment maintains essentiality in the evolution of Form I Rubisco complexes.

Protein complexes composed of strictly essential subunits are abundant in nature and often arise through the gradual complexification of ancestral precursor proteins. Essentiality can arise through the accumulation of changes that are tolerated in the complex state but would be deleterious for the standalone complex components. While this theoretical framework to explain how essentiality arises has been proposed long ago, it is unclear which factors cause essentiality to persist over evolutionary timescales.

The emergence of Sox and POU transcription factors predates the origins of animal stem cells.

Stem cells are a hallmark of animal multicellularity. Sox and POU transcription factors are associated with stemness and were believed to be animal innovations, reported absent in their unicellular relatives. Here we describe unicellular Sox and POU factors.

Structural variation of types IV-A1- and IV-A3-mediated CRISPR interference.

CRISPR-Cas mediated DNA-interference typically relies on sequence-specific binding and nucleolytic degradation of foreign genetic material. Type IV-A CRISPR-Cas systems diverge from this general mechanism, using a nuclease-independent interference pathway to suppress gene expression for gene regulation and plasmid competition. To understand how the type IV-A system associated effector complex achieves this interference, we determine cryo-EM structures of two evolutionarily distinct type IV-A complexes (types IV-A1 and IV-A3) bound to cognate DNA-targets in the presence and absence of the type IV-A signature DinG effector helicase.

ZNF512B binds RBBP4 via a variant NuRD interaction motif and aggregates chromatin in a NuRD complex-independent manner.

The evolutionarily conserved histone variant H2A.Z plays a crucial role in various DNA-based processes, but the mechanisms underlying its activity are not completely understood. Recently, we identified the zinc finger (ZF) protein ZNF512B as a protein associated with H2A.

The mitochondrial citrate synthase from Tetrahymena thermophila does not form an intermediate filament.

The mitochondrial citrate synthase (mCS) purified from the ciliate Tetrahymena thermophila has been reported to form intermediate-filament-like structures during conjugation and to self-assemble into fibers when recombinantly expressed. This would represent a rare example of a tractable and recent origin of a novel cytoskeletal element. In an attempt to investigate the evolutionary emergence of this behavior, we re-investigated the ability of Tetrahymena's mCS to form filaments in vivo.

Haptoglobin buffers lipopolysaccharides to delay activation of NFκB.

It has remained yet unclear which soluble factors regulate the anti-inflammatory macrophage phenotype observed in both homeostasis and tumourigenesis. We show here that haptoglobin, a major serum protein with elusive immunoregulatory properties, binds and buffers bacterial lipopolysaccharides to attenuate activation of NFκB in macrophages. Haptoglobin binds different lipopolysaccharides with low micromolar affinities.

Adaptive laboratory evolution recruits the promiscuity of succinate semialdehyde dehydrogenase to repair different metabolic deficiencies.

Promiscuous enzymes often serve as the starting point for the evolution of novel functions. Yet, the extent to which the promiscuity of an individual enzyme can be harnessed several times independently for different purposes during evolution is poorly reported. Here, we present a case study illustrating how NAD(P)-dependent succinate semialdehyde dehydrogenase of Escherichia coli (Sad) is independently recruited through various evolutionary mechanisms for distinct metabolic demands, in particular vitamin biosynthesis and central carbon metabolism.

transcription-based biosensing of glycolate for prototyping of a complex enzyme cascade.

metabolic systems allow the reconstitution of natural and new-to-nature pathways outside of their cellular context and are of increasing interest in bottom-up synthetic biology, cell-free manufacturing, and metabolic engineering. Yet, the analysis of the activity of such networks is very often restricted by time- and cost-intensive methods. To overcome these limitations, we sought to develop an transcription (IVT)-based biosensing workflow that is compatible with the complex conditions of metabolism, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, a 27-component metabolic system that converts CO into glycolate.

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication.

Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences.

Mitochondria, Peroxisomes and Beyond-How Dual Targeting Regulates Organelle Tethering.

Eukaryotic cells feature distinct membrane-enclosed organelles such as mitochondria and peroxisomes, each playing vital roles in cellular function and organization. These organelles are linked at membrane contact sites, facilitating interorganellar molecule and ion exchange. Most contact-forming proteins identified to date are membrane proteins or membrane-associated proteins, which can form very stable contacts.

Golden Gate Cloning for the Standardized Assembly of Gene Elements with Modular Cloning in Chlamydomonas.

Modern synthetic biology requires fast and efficient cloning strategies for the assembly of new transcription units or entire pathways. Modular Cloning (MoClo) is a standardized synthetic biology workflow, which has tremendously simplified the assembly of genetic elements for transgene expression. MoClo is based on Golden Gate Assembly and allows to combine genetic elements of a library through a hierarchical syntax-driven pipeline.