Artificial spider silk is an attractive material for many technical applications since it is a biobased fiber that can be produced under ambient conditions but still outcompetes synthetic fibers (e.g., Kevlar) in terms of toughness. Industrial use of this material requires bulk‐scale production of recombinant spider silk proteins in heterologous host and replication of the pristine fiber's mechanical properties. High molecular weight spider silk proteins can be spun into fibers with impressive mechanical properties, but the production levels are too low to allow commercialization of the material. Small spider silk proteins, on the other hand, can be produced at yields that are compatible with industrial use, but the mechanical properties of such fibers need to be improved. Here, the literature on wet‐spinning of artificial spider silk fibers is summarized and analyzed with a focus on mechanical performance. Furthermore, several strategies for how to improve the properties of such fibers, including optimized protein composition, smarter spinning setups, innovative protein engineering, chemical and physical crosslinking as well as the incorporation of nanomaterials in composite fibers, are outlined and discussed.

Effective treatment of tuberculosis is frequently hindered by the emerging antimicrobial resistance of Mycobacterium tuberculosis. The present study evaluates monocyclic β-lactam compounds targeting the mycobacterial cell wall remodeling. Novel N-thio-β-lactams were designed, synthesized, and characterized on the L,D-transpeptidase-2, a validated target in M. tuberculosis. The candidates were evaluated in biochemical assays identifying five compounds presenting target-specific kinetic constants equal or superior to meropenem, a carbapenem currently considered for tuberculosis therapy. Mass spectrometry in line with the crystal structures of five target-ligand complexes revealed that the N-thio-β-lactams act via an unconventional mode of adduct formation, transferring the thio-residues from the lactam ring to the active-site cysteine of LdtMt2. The resulting stable adducts lead to a long-term inactivation of the target protein. Finally, the candidates were evaluated in vitro against a drug-susceptible and multidrug-resistant clinical isolates of M. tuberculosis, confirming the antimycobacterial effect of these novel compounds.

Chemical reactions can be performed to covalently modify specific residues in proteins. When applied to native enzymes, these chemical modifications can greatly expand the available set of building blocks for the development of biocatalysts. Nucleophilic canonical amino acid sidechains are the most readily accessible targets for such endeavors. A rich history of attempts to design enhanced or novel enzymes, from various protein scaffolds, has paved the way for a rapidly developing field with growing scientific, industrial, and biomedical applications. A major challenge is to devise reactions that are compatible with native proteins and can selectively modify specific residues. Cysteine, lysine, N-terminus, and carboxylate residues comprise the most widespread naturally occurring targets for enzyme modifications. In this review, chemical methods for selective modification of enzymes will be discussed, alongside with examples of reported applications. We aim to highlight the potential of such strategies to enhance enzyme function and create novel semisynthetic biocatalysts, as well as provide a perspective in a fast-evolving topic.

Spider silk is an extraordinary natural material that combines strength, extensibility, and toughness in a lightweight, protein-based fiber. While the recombinant production of spidroins has advanced, the creation of silk fibers with additional intrinsic functionalities, such as color, remains a major challenge. Here, we report the rational design, expression, and spinning of intrinsically red-colored artificial silk fibers. A mini-spidroin variant was engineered as a fusion with the red fluorescent protein mCherry, expressed at high yields (20 g/L) in E. coli fed-batch fermentations, and purified under native conditions. Although the presence of the mCherry globular domain reduced spinnability when used alone, blending with wild-type mini-spidroins enabled continuous wet spinning into robust, fluorescent fibers. Our biomimetic spinning approach preserved the correct folding of the mCherry domain within the fiber, resulting in stable red coloration and fluorescence, while maintaining mechanical properties comparable to those of other recombinant silks. This work establishes a scalable, sustainable strategy for fabricating colored bio-based fibers, opening avenues for environmentally friendly textiles and functional biomaterials that may reduce reliance on synthetic and chemically dyed fibers.

The development of bioactive protein-based materials hinges on scalable expression systems and robust and mild processing methods. Here we report the successful bacterial overexpression and purification of a fusion protein comprising the mini-spidroin (A ₃ I) ₃ -A ₁₄ and the Immunoglobulin G (IgG)-degrading enzyme IdeS. High-density fed-batch bioreactor cultivation resulted in expression levels of 7.2 g/L, demonstrating the scalability of the system. Structural and functional characterization confirmed that the IdeS moeity was folded and retained enzymatic activity. Importantly, the high solubility of the fusion protein ( > 200 mg/mL), combined with the mini-spidroin’s ability to assemble into gels and fibers, allowed us to develop self-assembled hydrogels and wet-spun fibers that maintained enzymatic function. Notably, enzymatic activity was preserved in fibers stored for prolonged times under both wet and dry conditions. These findings illustrate the potential of spidroin fusion proteins as a modular platform for producing robust bioactive materials in a scalable process, offering new avenues for biomedical applications.

Spider silk formation involves tightly regulated protein assembly influenced by pH and the presence of ions. Kosmotropic salts induce phase separation of spidroins; however, their exact role in assembly is not clear. Here, we investigate how sodium and potassium phosphate affect spidroin interactions via the single-molecule method of mass photometry. We observed that spidroin oligomerization occurs at low nanomolar protein concentrations. Potassium ions were found to stabilize a compact conformation of individual spidroins and slow down pH-induced β-sheet aggregation, consistent with its more kosmotropic nature. Microfluidic MP showed that pre-assembly of the protein through salt-induced phase separation reduced the number and size of oligomeric intermediates that form upon acidification. Together, the findings suggest that spidroins have an inherent ability to self-assemble, blurring the line between one- and two-phase status. Subtle differences in ion composition are sufficient to change spidroin stability and assembly, potentially contributing to silk spinning in vivo by balancing storage stability with rapid fiber formation.