Cell Reports Physical Science

Membrane models of scaffolded discoidal lipid bilayers called nanodiscs have proven to be a valuable tool for the study of membrane proteins in a native environment. DNA-scaffolded membrane model has emerged as an alternative tool for membrane protein studies. Taking advantage of the designability of DNA nanostructure, we created a double-decker double-stranded DNA ring (DDring) to self-assemble DNA-based nanodiscs (DNA-ND). The DDring is 17 nm wide and 4 nm high, and equipped with 28 alkyl chains on the inside that can interact with each hydrophobic leaflet of the lipid bilayer. We further demonstrate the functionality of DNA-ND membrane model with the assembly of membrane proteins. DDrings are suited to neutral or cationic charged phospholipids and detergents. This study provides more insights into the potential use of DNA- assisted nanodiscs for membrane protein characterization.

Combatting biofilm-associated methicillin-resistant Staphylococcus aureus (MRSA) infections remains a formidable challenge, largely due to bacterias heightened resistance to antibiotics and evasion of the hosts immune responses. Inhibiting biofilm formation or promoting biofilm dissolution is believed to disrupt the protective matrices and expose bacterial cells to immune clearance or antimicrobial treatments. Cross- helical amyloid fibrils of phenol-soluble modulins are the key structural components of the MRSA biofilm. In this study, we present a novel retro-inverso peptide that successfully inhibits cross- amyloid formation, induces disassembly of pre-formed fibrils of the biofilm-forming modulin peptide, and efficiently disperses MRSA biofilm biomass in a dose-dependent manner. The biophysical studies of the interaction of the designed peptide and modulins have suggested a mechanism for the biofilm disruption. The findings highlight the potential of the proteolytically resistant mirror-image peptide as a promising therapeutic agent for the treatment of biofilm-associated MRSA infections.

Living organisms achieve adaptive actuation through the seamless integration of neural motor control circuitry and proprioceptive feedback. While biohybrid robotics aims to replicate these capabilities by merging engineered muscle with synthetic scaffolds, the field remains limited by interfaces that lack the efficiency and closed-loop regulation of natural neuromuscular systems. Here, we introduce a biohybrid muscle actuator system featuring a bioelectronic interface based on soft poly(3,4-ethylenedioxythiophene) (PEDOT) fibers for stimulation and sensing. These fibers conformally couple to muscle tissues, eliciting robust contractions at voltages as low as 1 V--requiring ultra-low power (0.376 {+/-} 0.034 mW) and preserving long-term tissue viability. By leveraging the independent addressability of these fibers, we demonstrate selective actuation of individual muscle units to achieve precise spatiotemporal control of a two-muscle-powered walking biohybrid robot, reaching a locomotion speed of 5.43 {+/-} 0.79 mm/min. When configured as strain sensors, the fibers exhibit a high gauge factor of 155.45 {+/-} 6.59 and resolve contractile displacements within tens of micrometers. We demonstrate that this sensing modality can be integrated into a closed-loop controller to autonomously modulate stimulation based on real-time feedback, significantly mitigating muscle fatigue (p = 0.038) during continuous operation. This work establishes a versatile platform for efficient actuation and intrinsic feedback sensing, providing a blueprint for efficient, autonomous, and adaptive biohybrid machines. SummarySoft conductive fibers enable a bioelectronic interface for low-power actuation and closed-loop control in biohybrid robots.

Monoterpenes are a diverse class of natural products with broad industrial and pharmaceutical applications. While there is great interest in transitioning their production from chemical synthesis and natural source extraction to yeast bioprocesses, this approach remains limited by the dual functionality of the endogenous farnesyl diphosphate synthase Erg20p, which produces the monoterpene precursor geranyl diphosphate (GPP) but favors its subsequent conversion to farnesyl diphosphate (FPP). To address this limitation, we recruited Erg20p and monoterpene synthases into synthetic membraneless organelles, improving production. In doing so, we found that short C-terminal peptide fusions used for recruitment also significantly enhance GPP synthase activity relative to FPP synthase activity. The combined effects of metabolic spatial organization and GPP synthase activity enhancement significantly boost production of different monoterpenes, including geraniol titers exceeding 4 g/L. The strategies presented here can be readily integrated with other traditional metabolic engineering approaches to build yeast strains with high levels of monoterpene production.

Self-assembled peptide-based nanostructures have diverse applications in the pharmaceutical and materials fields, but accurately predicting their self-assembly behavior without time-intensive organic synthesis and characterization remains a significant challenge. Here, we assess the effectiveness of AlphaFold3 (AF3), a deep learning model for protein structure prediction, in modeling peptide-based nanostructures and the interactions driving supramolecular self-assembly. We designed amphiphilic peptides composed of alternating hydrophobic residues (valine, leucine, isoleucine, phenylalanine) and hydrophilic residues (glutamic acid), varying both sequence length and residue order. Using AF3s multimer mode, we modeled assemblies with copy numbers ranging from 10 to 1000, generating diverse morphologies such as micelles and nanotubes. We qualitatively analyzed hydrophobic regions, secondary structures, and intermolecular interactions, while also calculating radii of gyration, packing scores, and aspect ratios using PyRosetta. Our results indicate that AF3 predicts morphologies consistent with hydrophobic driving forces and steric constraints. Increased hydrophobicity correlates with smaller radii of gyration, while higher copy numbers correspond to smaller aspect ratios (more compact structures). Longer hydrophobic segments lead to disordered structures, whereas longer hydrophilic segments promote organization. While AF3 captures systemic trends consistent with biophysical principles, comparisons to literature reveal discrepancies driven by charge effects and secondary structure bias, including an overemphasis on helical propensity (e.g., alanine-rich sequences) and sensitivity to terminal charge repulsion. Additionally, since AF3 is predisposed to predict a single assembled entity rather than higher-order assemblies such as multiple micelles or fibers, finding the optimal copy number for the best prediction requires system-specific iteration. These limitations highlight the need for complementary approaches with controlled chemical potential and environmental conditions, though qualitative agreement with experimental trends in morphology and compactness supports AF3s utility for initial structure generation. Our findings highlight AF3s potential as a user-friendly design tool for structure generation in peptide design, aiding the efficient development of functional self-assembled peptide nanomaterials.

Sea water desalination is a critical solution to global water scarcity, primarily relying on membrane-based technologies and reverse osmosis. Artificial water channels (AWCs) offer a promising solution for next-generation desalination membranes. Very promising channel building blocks for AWC is oligourea foldamers repeats. These biomimetic molecules, composed of unnatural amino acids, self-assemble into protein-like superhelical channels with water-filled pores, making them ideal candidates for selective water transport. In silico studies provide essential support to experimental efforts in designing novel oligoureas. However, such studies require a dedicated force field for now unavailable for these molecules. In this work, we developed a tailored force field for oligoureas by adapting parameters from two established protein force field families: CHARMM (CHARMM36m) and Amber (GAFF2), using their structural similarities to natural amino acids. Our objective was to identify a force field that reliably preserves the structural integrity of oligourea foldamers. We evaluated two distinct oligoureas using molecular dynamics (MD) simulations across increasing system sizes. By comparing simulation results with experimental data, we assessed key structural features, including folding patterns, stability, and pore shapes. Our findings demonstrate that the CHARMM-based force field consistently reproduces experimental observations for both foldamers, outperforming the Amber-based alternative. This newly developed CHARMM-based force field paves the way for further exploration of oligoureas, enabling deeper insights into their stability and efficiency in sea water desalination.

Biohybrid robots combining compliant synthetic support structures with biological actuators could enable future applications ranging from precision microsurgery to unmanned exploration. Machines actuated by living skeletal muscles are capable of adaptive behaviors, such as sensing and responding to environmental stimuli in real-time, offering functional advantages over non-biological actuators. However, typical skeletal muscle-powered biohybrid robots depend on 3D tissues which require large cell volumes and offer limited control of muscle fiber alignment, thus reducing efficiency of force generation and transduction. Here, we present a locomotive biohybrid robot powered by 2D monolayers, or thin films, of precisely aligned skeletal muscle fibers on a micropatterned hydrogel skeleton. We demonstrate how varying skeleton design parameters, ranging from material stiffness to microscale topology, impacts muscle fiber alignment and resultant actuation strains, generating forces 10X higher than previous 2D skeletal muscle actuators, improving untethered actuation longevity by [~]4500X from < 10 minutes to > 30 days, and increasing efficiency of muscle force output (force per unit volume of muscle) by 20X as compared to 3D muscles. Utilizing our optimized design for skeletal muscle thin films, we create a multi-limbed robot composed of independent muscle-powered fins capable of on/off control and frequency-dependent speed control. With these control inputs, we achieve steered multi-directional locomotion at speeds up to 4 body lengths per minute in straight movement and 1200 degrees per minute in rotational movement, highlighting potential for such actuators to be transformed into long-lasting functional soft robots.

The microbial extracellular matrix (ECM) is a complex network of self-secreted biopolymers uniting the cells in biofilms, providing them with structural integrity, and contributing to their elevated resistance to antibiotic treatments. Recently, there is a growing realization that a regulated, bidirectional cross-talk of bacteria and ECM confers biofilms with tissue-like traits, however, the mechanisms of spatio-temporal self-organisation of ECM and its regulation are still poorly understood. In the model organism for biofilm formation Bacillus subtilis, TasA is the major protein component of the extracellular matrix. We recently showed that TasA, isolated in the form of stable and structured globules, assembles into elongated and ordered fibers via a donor-strand complementation mechanism. In this study, we discovered that in the presence of zinc metal ions, TasA is able to form hydrogels with > 97% water content. Electron- and atomic force-microscopies as well as small angle X-ray scattering measurements show that cross-linking with zinc ions induces a transition in TasA morphology from one-dimensional fibers to two-dimensional sheets. Electron paramagnetic resonance measurements then show that such a significant morphological shift is associated with molecular changes in the coordination environment of zinc ions, which lead to structural changes at the protein level. When assembling into macroscopic networks, TasA-Zn metallogels exhibit viscoelastic properties and a fast recovery following an excessive strain. These metallogels represent a novel class of bacterially-derived ECMs that form easily at room temperature without covalent crosslinking, and may be used as a natural matrix-mimics in biofilm models for infection studies.

Genetic engineering experiments and therapies are constrained by the size of DNA integrations into human cells genomes. Existing AAV, lentiviral, and non-viral methods rapidly decrease in integration efficiency beyond [~]5kb of sequence. Through systematic evaluation of non-viral DNA template formats, we identified circular ssDNA and dsDNA as capable of mediating >5kb integrations. Large circular DNA delivery efficiency and its impacts on cell viability and payload expression could be significantly improved with small DNA "helper" plasmids, mRNA-encoded nucleases, and sequence design optimizations. Collectively, these modifications enabled ultra-large--up to 10 kb DNA--integrations at >20% efficiency in primary human T cells at the TRAC locus and at >60% efficiency in human iPSCs at the AAVS1 locus. Finally, we demonstrate that GMP clinical-manufactured T cells with ultra-large integrations are functional in vitro and in vivo. Overall, we identified optimal template architectures, delivery modes, and sequence design rules for ultra-large DNA integrations in both research and clinical settings to accelerate basic genetic research and next-generation cellular therapies.

Bacterial microcompartments (BMCs) are a diverse and widespread class of protein-based organelle consisting of a semi-permeable protein shell encapsulating an enzymatic core. Along with their native assembly pathway, isolated BMC shell proteins have been shown to assemble into alternative superstructures such as flat sheets and nanotubes. The self-assembly and modularity of BMC shell proteins make them of great interest as modular platforms for applications involving scaffolding, immobilization and compartmentalization. While the assembly of BMC shell proteins into higher-order structures has been well-studied, the design of controllable and modular cargo loading is underdeveloped in comparison. Recently, we reported the pH-controlled assembly of CcmK2 - the major hexameric shell protein of the {beta}-carboxysome BMC - into monodisperse mesh-like microscale particles. Here, we develop a suite of encapsulation strategies for stochastic or targeted loading of various cargos, as well as the direct conjugation of cargo to CcmK2 particles. Our systematic analysis demonstrates that cargo loading and particle assembly can be modulated by the choice of recruitment strategy and the order of cargo introduction. Our findings also reveal a cooperative cargo loading mechanism during assembly that influences particle sizing and apparent morphology. Our study serves as a blueprint for the rational design of tunable cargo loading into engineered BMC-derived microcompartment systems for diverse biotechnological applications.

Scaffolded DNA origami has become a valuable nanoscale tool for applications in biomedical and physical sciences. Critical to leveraging the modular and programmable properties of DNA origami nanodevices is access to the scaffold strand, a long single-stranded DNA (ssDNA) of precise length and sequence, which is folded into a compact shape via piecewise base-pairing with many staple strands, short ssDNA oligonucleotides. Current methods to produce and manipulate long ssDNA scaffolds can be costly, time-consuming, and cumbersome. In contrast, methods to produce and manipulate the sequence of double-stranded DNA (dsDNA) are efficient and scalable. Here, we present a method for the rapid isolation of target ssDNA sequences from a variety of dsDNA sources using oligonucleotides as blocking strands that bind continuously to the undesired strand, thereby releasing the target scaffold strand. We report successful ssDNA isolation from linear and supercoiled dsDNAs of various sequences and lengths, ranging from 769 to 15,101 nucleotides. In addition to isolating ssDNA, we demonstrated this approach enables folding of DNA origami directly from dsDNA templates using both blocking and staple strands in a single-pot thermally controlled reaction. Furthermore, we explore multi-scaffold and gene-encoding DNA origami structures, expanding the framework for application-based designs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/709872v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1cc75dcorg.highwire.dtl.DTLVardef@4df8e2org.highwire.dtl.DTLVardef@10ed113org.highwire.dtl.DTLVardef@1c05bdd_HPS_FORMAT_FIGEXP M_FIG C_FIG

The mass-rearing of black soldier fly (Hermetia illucens) larvae (BSFL) is a promising solution for converting organic waste into high-quality insect protein, but preventing larval escape from open-top rearing containers remains a major management challenge. Conventional escape-control methods are often unreliable or impractical. To address this, we developed and evaluated a novel physical barrier, the anti-climbing tape, featuring regularly arranged macroscale protrusions designed to disrupt larval locomotion on vertical surfaces. We conducted a series of experiments to examine the design parameters of the anti-climbing tapes, including the gap size between protrusions and the number of protrusion rows. Our results demonstrate that the anti-climbing tape prevents escape via a dual mechanism: (1) physical obstruction, in which gaps narrower than the larval body width block larvae from passing through, and (2) adhesion reduction, in which the elevated protrusion array decreases the effective contact area for wet adhesion while increasing gravitational torque acting on the larval body. The effectiveness of these mechanisms was dependent on larval size. A design featuring 0.5-mm gaps and a 15-row protrusion array completely prevented the escape of later-instar larvae (>10 mm) in a 20-day large-scale trial, whereas the method was less effective for smaller larvae. In conclusion, the anti-climbing tape provides a robust and chemical-free approach to BSFL escape in mass rearing. To ensure reliable performance, its design parameters, both gap size and array width must be optimised to suppress the mechanical and adhesive components of larval climbing according to the target larval size. Conflict of interestS. Jang and M. Shimoda are inventors on a Japanese patent application (No. 2022-172252, filed November 27, 2022) related to the method described in this manuscript. FundingThis study was supported by Korea-Japan Joint Government Scholarship Program for the Students in Science and Engineering Departments, the Korean Scholarship Foundation, and the University of Tokyo Foundations Support Fund for International Students.

Monoterpene indole alkaloids (MIAs) are a major class of plant natural products with important pharmaceutical activities, yet the biosynthetic pathway to their universal precursor, strictosidine, has been fully elucidated in only Catharanthus roseus. In kratom (Mitragyna speciosa), only the first and last steps of strictosidine biosynthesis were previously known. Here, we applied multiplex pathway engineering in yeast to accelerate the discovery, reconstruction, and optimization of the kratom strictosidine pathway. Iterative multiplex integration and screening identified 13 functional kratom genes and enabled rapid validation of functional pathway modules, thereby completing the kratom strictosidine pathway from geranyl pyrophosphate and tryptophan. We also identified a vacuolar secologanin transporter, MsNPF2.6, which increased strictosidine production by 62% in yeast. Pathway optimization through the incorporation of nepetalactol-producing enzymes from other plants further supported strictosidine production in yeast from fed geraniol and tryptophan. These results establish the strictosidine pathway in kratom and highlight multiplex engineering as a powerful platform for rapid plant pathway discovery and optimization.

Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR-Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.

Although biohybrid robots offer the potential for soft, adaptive actuation by harnessing living muscle, practical operation in cell culture environments is often limited by the requirement of immersed leads or cumbersome stimulation equipment. Here, we present a thin, miniaturized, wireless bioelectronic stimulator that can electrically drive biohybrid robots while maintaining stability in aqueous cell culture media. Built on a 50-{micro}m liquid crystal polymer (LCP) substrate, the device integrates a planar receiving coil, interconnects, a diode-based rectifier, and a tank capacitor. This enables the device to convert an approximately 4.9-MHz radio-frequency (RF) input into pulsed direct current (DC), which is delivered through integrated stimulation electrodes. The stimulator has a footprint of [~]23 mm2 and a total thickness and mass of [~]100 {micro}m and [~]7 mg, respectively. We integrated the stimulator with a nanopatterned carbon nanotube (CNT)/gelatin hydrogel fin seeded with human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to generate propulsion through fin flapping. By optimizing the thickness of the polydimethylsiloxane (PDMS) encapsulation layer, the density was tuned, and the robot remained freely floating and retained shape integrity during operation. This produced autonomous forward locomotion of [~]70 {micro}m/s. The stimulator generated distance-dependent output voltage pulses of [~]2-6 V and reliably synchronized fin flapping rates of up to 2 Hz without an observable loss of cell attachment or sarcomeric organization. Together, these results establish a compact, media-compatible, wireless, bioelectronic interface suitable for closed-system biohybrid robotics.

Bacteroides thetaiotaomicron (Bt), a dominant bacterial species in the human gut, is a promising chassis for engineering in situ therapeutic delivery systems. Previously, we developed a secretion toolkit composed of endogenous lipoprotein signal peptides (SPs) and full-length secretory proteins from Bt. However, due to variations in length, structure, and amino-acid sequence, these SPs exhibited inconsistent secretion efficiencies across different cargo proteins. Because the activity of individual SP-cargo pairs is not readily predictable, screening and optimization is often required to achieve target secretion titers. To enhance the utility of our toolbox, we studied the impact of different SP sequence components on protein secretion, then applied this knowledge to develop a standardized toolkit to and enable predictable and tunable protein secretion across diverse SP-cargo pairs. To achieve this, we first identified the lipoprotein export sequence (LES) as the key determinant of efficient secretion of heterologous proteins by lipoprotein SPs. We next performed mutagenesis on the LES region of a representative lipoprotein SP to generate a pool of mutants featuring a standardized SP backbone with diversified LES regions. Screening and characterization of this mutant pool revealed a charge-dependent regulation of both secretion and surface display of heterologous cargo proteins. From these findings, we established a toolkit with improved tunability, enhanced predictability, and surface display capabilities that minimizes the need for iterative screening when developing protein secreting gene circuits for Bt and other Bacteroides species. By enhancing both the flexibility and control of therapeutic protein output, these results expand the potential of engineered living therapeutic applications, particularly those requiring tunable dosing or surface presentation of proteins.

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

The CRISPR-Cas9 system has revolutionized genome engineering, yet its full therapeutic potential remains constrained by challenges in precisely modulating its activity and specificity. Here we report a fully computational end-to-end pipeline for the de novo design of a single-domain VHH nanobody (NbSpCas9-v1) targeting a structurally conserved, non-catalytic epitope at the PAM-interacting (PI) and RuvC-III interface of Streptococcus pyogenes Cas9 (SpCas9; PDB: 4UN3). Nanobody sequences were generated using BoltzGen, a generative diffusion binder design framework, and co-folded with SpCas9 using Boltz-2 to evaluate structural confidence and binding affinity. The top-ranked model (SpCas9_4UN3_Bivalent_Hub_v1) achieved a complex pLDDT of 0.8406, an aggregate score of 0.8016, and an ipTM of >0.8, indicating high confidence in the nanobody-antigen interface. The designed 1,616-residue quaternary complex (SpCas9 + sgRNA + DNA + nanobody) was subjected to 10 ns of all-atom molecular dynamics (MD) simulation using the AMBER14SB force field within the GROMACS/OpenMM framework. The complex stabilized at RMSD [~]6 [A] with a radius of gyration of 39-44 [A], confirming thermodynamic stability under physiological conditions (310 K, 0.15 M NaCl). A conserved 96.3 [A] inter-molecular distance between the nanobody centroid and the HNH catalytic residue H840 establishes NbSpCas9-v1 as a distal, non-inhibitory binder -- ideally suited for a Bivalent Hub architecture recruiting secondary effectors to the Cas9 ribonucleoprotein (RNP). The nanobody-Cas9 interface is stabilized by 8 hydrogen bonds, 4 salt bridges, and [~]1,850 [A]2 of buried solvent-accessible surface area. These results provide a rigorous structural and dynamic foundation for experimental validation of VHH-based CRISPR-Cas9 enhancers and modulators. GRAPHICAL ABSTRACTThe computational workflow proceeds from SpCas9 crystal structure acquisition (PDB: 4UN3) through BoltzGen nanobody design, Boltz-2 structural co-folding, 10 ns explicit-solvent MD validation, and Bivalent Hub functional characterization. The PyMOL rendering below shows the full quaternary complex at atomistic resolution.

Insects body barriers rely on specialized extracellular matrices that protect against harmful environmental influences. The outer barrier is the cuticle, which is composed of chitin, cuticle proteins and lipids. The peritrophic matrix (PM) serves as an inner barrier lining the midgut epithelium. It is composed of chitin fibers that are organized by PM proteins. While cuticle and PM proteins have received considerable attention in the past, supramolecular organization and physicochemical properties of the chitin component - particularly of the PM - remain poorly understood. Here, we combine synchrotron-based X-ray diffraction data from the PMs of lepidopteran and coleopteran insects with RNA interference (RNAi), mass spectrometric and histochemical analyses of the PM from Tribolium castaneum to determine chitins allomorphic state and degree of acetylation. The chitin of the PM exhibits signatures characteristic of dihydrate {beta}-chitin along the entire midgut. In contrast, the cuticle is made of tightly packed -chitin nanofibrils. Mass spectrometry revealed that the PMs chitin is highly acetylated (>95%). RNAi silencing of gut-specific genes encoding chitin deacetylasesTcCDA6-9 further increases the degree of acetylation. Histochemical analyses staining chitin with different degrees of acetylation confirm the predominance of highly acetylated chitin in the PM. Notably, the larval cuticle has a layered organization with deacetylated chitin present in exo- and highly acetylated chitin in endocuticles. Depletion of both TcCDA1 or TcCDA2 impairs chitin deacetylation, which indicates that both proteins cooperate in their activity in the integument. These results establish fundamental principles of polysaccharide-based extracellular matrices, with broad implications for insect biology.

Despite its industrial importance, microbial L-lysine production has largely been confined to classical producer strains, leaving the fast-growing, non-pathogenic marine microorganism V. natriegens largely untapped as an unconventional biosynthetic platform. In this work, we established an L-lysine-overproducing V. natriegens DSM759 strain through a step-wise, systematic rational engineering strategy targeting the native biosynthetic pathway. Guided by our prior systems-level analysis of the strains genetic and regulatory architecture, we identified key metabolic bottlenecks and implemented knowledge-driven interventions to relieve pathway constraints. Central to production was alleviation of feedback inhibition in the native key regulatory enzymes, aspartate kinase (AK, lysC) and dihydrodipicolinate synthase (DHDPS, dapA). Site-directed amino-acid substitutions, replicating established E. coli feedback-resistance mechanisms, were introduced into conserved regions of the V. natriegens DSM759 enzymes, producing L-lysine-insensitive variants with kinetic parameters comparable to that of corresponding wild type enzymes. Among the tested configurations, the strain co-expressing Vn.lysC2 and Vn.dapA1:E84T reached the highest L-lysine titer (9.0{+/-}0.6 mM) and yield (0.11{+/-}0.01 molLys molGlc-1), whereas overexpression of additional L-lysine pathway genes provided no further benefit. Leveraging the hosts metabolic versatility, L-lysine synthesis was also demonstrated from the chitin-derived amino-sugar N-acetylglucosamine (0.09{+/-}0.00 molLys molGlcNAc-1), highlighting the potential to valorize chitin-rich waste streams from the seafood industry. This work establishes a minimal, rational strategy for L-lysine biosynthesis in V. natriegens DSM759 and positions it as a promising platform for sustainable amino acid production.