Biology subjects

Linder, M. B.

Publications and source records attributed to Linder, M. B..

8 recordsLinked to original sources

bioRxiv2026

Engineering the bacterial cellulose-forming surface as a programmable protein recruitment interface

Synthetic biology has advanced microorganisms to be programmed as production hosts, but its application to bacteria that inherently assemble extracellular materials remains limited. Komagataeibacter spp., natively synthesizes cellulose at the bacterial cell surface, creating a material-forming interface that has not been used as a programmable recruitment platform. Here we establish cell-surface display in Komagataeibacter intermedius and show that this interface can recruit defined proteins, making functionalization part of cellulose formation. By engineering LppOmpA, we displayed a fluorescent protein and genetically encoded capture modules (SpyTag and SilkTag) to selectively capture catcher-fused protein cargos onto K. intermedius cell surface. Recruitment of silk-derived structural protein before cellulose production generated silk-associated fibrous structures within the pellicles, with retained cargo signal after washing. The resulting biocomposite showed reorganized fibre-network morphology, increased surface hydrophobicity, mesoscale ordering, and improved wet-state compressive strength. Wild-type cells exposed to same conditions did not reproduce these changes, demonstrating that material properties arise from surface-directed recruitment rather than protein exposure alone. This work demonstrates the material-forming bacterial surface as a programmable engineering interface for organizing extracellular proteins, providing a general strategy for engineering living materials.

bioengineering↗

bioRxiv2026

Non-ribosomal Peptides as Structural Determinants of Fungal Hydrophobicity

Fungal surfaces must remain hydrophobic to enable growth, dispersal, and survival under fluctuating environmental conditions, yet the molecular basis of this property remains incompletely understood. Here, we identify fungisporins, fusahexins, and related cyclic non-ribosomal peptides (NRPs) as members of a conserved functional class of fungal metabolites, termed WAter Repellent Peptides (WARPs), that are required for fungal surface hydrophobicity. Across filamentous fungi, WARPs vary substantially in sequence and length but share conserved structural features, including cyclization, hydrophobic amino acid composition, and alternating D- and L-configurations, consistent with a flexible amphiphilic scaffold. Loss of WARP-producing non-ribosomal peptide synthetases results in rapid collapse of aerial hyphae upon water exposure, demonstrating that these peptides are required for maintenance of hydrophobic aerial structures. Using phage-display-derived antibodies, we localize WARPs to the hyphal surface, supporting their role as surface-associated structural components. Together, these findings identify a conserved NRPS-encoded peptide system that contributes to fungal hydrophobicity and establish WARPs as a broadly distributed class of surface-associated metabolites with structural function in filamentous fungi.

microbiology↗

bioRxiv2024

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation (LLPS) occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. We find that liquid spidroin condensates absorb visible light, which enables us to control sol-gel transitions of individual droplets through laser pulses. Fluorescence microscopy reveals that laser-induced gelation significantly alters the interactions between droplet proteins and small molecules, which allows us to load single droplets with an anticancer drug. In summary, our findings demonstrate direct control of phase transitions in individual condensates opening new avenues for functional and structural characterization. SYNOPSIS TOCThe liquid-to-solid transitions of phase-separated protein condensates are challenging to control. Leppert et al. show that condensates of engineered mini-spidroins gelate at slightly elevated temperatures. Using high-energy laser pulses at wavelengths that are absorbed by the droplets, the authors induce sol-gel transitions in single droplets. These gelated droplets are chemically stable and exhibit an increased ability to sequester drug molecules. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/584573v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@a7186corg.highwire.dtl.DTLVardef@345bd0org.highwire.dtl.DTLVardef@177c94forg.highwire.dtl.DTLVardef@1416456_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Repurposing host-guest chemistry to sequester virulence and eradicate biofilms in multidrug resistant Pseudomonas aeruginosa and Acinetobacter baumannii

The limited diversity in targets of available antibiotic therapies has put tremendous pressure on the treatment of bacterial pathogens, where numerous resistance mechanisms that counteract their function are becoming increasingly prevalent. Here, we utilize an unconventional anti-virulence screen of host-guest interacting macrocycles, and identify a water-soluble synthetic macrocycle, Pillar[5]arene, that is non-bactericidal/bacteriostatic and has a mechanism of action that involves binding to both homoserine lactones and lipopolysaccharides, key virulence factors in Gram-negative pathogens. Pillar[5]arene is active against Top Priority carbapenem- and third/fourth-generation cephalosporin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, suppressing toxins and biofilms and increasing the penetration and efficacy of standard-of-care antibiotics in combined administrations. The binding of homoserine lactones and lipopolysaccharides also sequesters their direct effects as toxins on eukatryotic membranes, neutralizing key tools that promote bacterial colonization and impede immune defenses, both in vitro and in vivo. Pillar[5]arene evades both existing antibiotic resistance mechanisms, as well as the build-up of rapid tolerance/resistance. The versatility of macrocyclic host-guest chemistry provides ample strategies for tailored targeting of virulence in a wide range of Gram-negative infectious diseases.

biochemistry↗

bioRxiv2022

Engineering a minimal SilkCatcher/Tag pair compatible with SpyCatcher/Tag pair for the production of native-sized spider silk

Protein/peptide pairs, called Catcher/Tag pairs, are applied for biological isopeptide-bond mediated Click-reactions. Covalent protein ligation using Catcher/Tag pairs has turned out to be a valuable tool in biotechnology and biomedicines. It is essential to increase the current toolbox of Catcher/Tag pairs to expand the range of applications further, e.g., for multiple-fragment ligation that requires several orthogonal ligases. We report here engineering of novel Catcher/Tag pairs for protein ligation, aided by a new crystal structure of a minimal CnaB domain from Lactobacillus plantarum. We engineer several split variants, characterize in detail one of them, named SilkCatcher/Tag pair, and show that the newly engineered SilkCatcher/Tag pair is orthogonal and compatible with the widely used SpyCatcher/Tag pair. Finally, we demonstrate the use of the new SilkCatcher/Tag pair in the production of native-sized highly repetitive spider-silk-like proteins with >90% purity, which is not possible with the traditional recombinant production.

biochemistry↗

bioRxiv2022

Recombinant protein condensation inside E. coli enables the development of building blocks for bioinspired materials engineering - biomimetic spider silk protein as a case study

Recombinant expression of proteins destined to form biological materials often results in poor production yields or loss of their function due to premature aggregation. Recently, liquid-liquid phase separation has been proposed as a mechanism to control protein solubility during expression and accumulation in the cytoplasm. Here, we investigate this process in vivo during the recombinant overexpression of the spider silk-mimetic mini-spidroin NT2RepCT in Escherichia coli. The protein forms intracellular liquid-like condensates that shift to a solid-like state triggered by a decrease in their microenvironmental pH. These features are also maintained in the purified sample in vitro both in the presence of a molecular crowding agent mimicking the bacterial intracellular environment, and during a biomimetic extrusion process leading to fiber formation. Overall, we demonstrate that characterization of protein condensates inside E. coli could be used as a basis for selecting proteins for both materials applications and their fundamental structure-function studies.

synthetic biology↗

bioRxiv2019

Analysis of Finnish blue mussel (Mytilus edulis L.) shell: Biomineral ultrastructure, organic-rich interfacial matrix and mechanical behavior

Studying various marine biomineralized ultrastructures reveals the appearance of common architectural designs and building blocks in materials with fascinating mechanical properties that match perfectly to their biological tasks. Advanced mechanical properties of biological materials are attributed to evolutionary optimized molecular architectures and structural hierarchy. One example which has not yet been structurally investigated in great detail is the shell of Mytilus edulis L. (Finnish blue mussel) found in the archipelago of SW-Finland. Through a combination of various state-of-the-art techniques such as high-resolution electron microscopy imaging, Fourier-transformed infrared spectroscopy, powder X-ray diffraction, synchrotron wide-angle X-ray diffraction, nanoindentation and protein analysis, both the inorganic mineralized components as well as the organic-rich matrix were extensively characterized. We found very similar ultra-architecture across the shell of M. edulis L. as compared to the widely studied and closely related M. edulis. However, we also found interesting differences, for instance in the thickness and degree of orientation of the mineralized layers indicating dissimilar properties and related alterations in the biomineralization processes. Our results show that the shell of M. edulis L. has a gradient of mechanical properties, with the increase in the stiffness and the hardness from anterior to the posterior region of the shell. The shell is made from distinct and recognizable mineralized layers each varying in thickness and microstructural features. At posterior regions of the shell, moving from dorsal to ventral side, these layers are an oblique prismatic layer, a prismatic layer and a nacreous layer, in which the oblique prismatic layer is found to be the main and thickest mineralized layer of the shell. Probing the calcified rods in the oblique prismatic layer using high resolution SEM imaging revealed opening of channels with a diameters of 40-50 nm and lengths up to a micrometer extending through the rods. The chitin and protein have been found to be the main component of the organic-rich interfacial matrix as expected. Protein analysis showed two abundant proteins with sizes around 100 kD and 45 kD which likely not only regulates biomineralization and adhesion of the crystals but also governing the intrinsic-extrinsic toughening in the shell. Overall, this detailed analysis provides new structural insights into biomineralization of marine shells in general.

developmental biology↗

bioRxiv2019

Analyzing the weak dimerization of a cellulose binding module by sedimentation velocity experiments

Cellulose binding modules (CBMs) are found widely in different proteins that act on cellulose. Because they allow a very easy way of binding recombinant proteins to cellulose, they have become widespread in many biotechnological applications involving cellulose. One commonly used variant is the CBMCipA from Clostridium thermocellum. Here we studied the dimerization of CBMCipA, because we were interested if its solution behavior could have an impact on its use in biotechnical applications. As the principal approach, we used sedimentation velocity analytical ultracentrifugation. To enhance our understanding of the possible interactions, we used molecular dynamics simulations. By analysis of the sedimentation velocity data using a discrete model genetic algorithm we found that the CBMCipA shows a weak dimerization interaction with a dissociation constant KD of about 87 M. As the KD of CBMCipA binding to cellulose is about 0.6 M, we conclude that the dimerization is unlikely to affect cellulose binding. However, at the high concentrations used in some applications of the CMBCipA, its dimerization is likely to have an effect on its solution behavior. The work shows that analytical ultracentrifugation is a very efficient tool to analyze this type of weak interactions. Moreover, we provide here a protocol for data analysis in the program Ultrascan for determining dissociation constants by sedimentation velocity experiments.

biophysics↗