Engineering *Bacillus subtilis* for the formation of a durable living biocomposite material

The study addresses the challenge of creating resilient, truly living engineered living materials (ELMs) where cells actively fabricate and organize the material. Existing ELMs often embed cells physically within external matrices, limiting autonomous self-fabrication, regeneration, and environmental resilience. The authors propose using Bacillus subtilis, a spore-forming, GRAS bacterium with strong secretion capabilities and long-term viability, to secrete self-assembling protein scaffolds that cross-link cells and biomineralize silica, forming a durable, regenerable biocomposite. The central hypothesis is that engineering B. subtilis to secrete 2D EutM-based scaffolds, display SpyTags on flagella for covalent attachment to SpyCatcher-bearing scaffolds, and incorporate biomineralization peptides will yield a self-fabricating, mechanically strengthened silica-based ELM capable of regeneration and modular functional augmentation.

Prior ELMs have leveraged engineered Escherichia coli curli fibers to produce extracellular matrices and embed functions, used secreted bacterial cellulose to embed microbes, or elastin-like polypeptides interfacing with S-layers. While effective, these systems often rely on amyloid fibers or specific chassis with limited resilience. Protein-based, secreted matrices can offer programmability and control. The authors build on bacterial microcompartment shell proteins (EutM) as robust, engineerable 2D scaffolds tolerant of N- and C-terminal fusions, including SpyCatcher for covalent attachment via SpyTag/SpyCatcher chemistry. They also note natural silica biomineralization mechanisms and peptides (e.g., R5, CotB1p, SB7, Synthetic) that can nucleate silica from orthosilicic acid, motivating peptide-scaffold fusions to mineralize silica. Limitations in current ELM diversity, autonomous self-fabrication, and long-term viability motivate moving to spore-forming Bacillus.

- Growth condition optimization: Evaluated B. subtilis 168 growth in LB and Spizizen Minimal Medium (SMM), at 20 and 30 °C, monitoring OD600, pH, flagella, and sporulation over 4 days. SMM at 20 °C maintained pH ~7.0 optimal for silica polymerization for up to 48–96 h and supported flagellation without sporulation. - Scaffold secretion engineering: Constructed EutM-SpyCatcher (EutM-SpyC) with N-terminal Bacillus signal peptides (SacB, XynA, YngK, CelA, LipA) to assess secretion. Cultured recombinant strains (SMM, 20 °C, 48 h); analyzed fractions by SDS-PAGE: culture supernatant (TCA-concentrated), urea-solubilized scaffolds from pellets (4 M urea), and lysed pellet. SacB gave markedly higher apparent secretion and scaffold recovery; other signals yielded minimal secretion. LC-MS showed uncleaved SacB remained on secreted EutM, likely due to rapid folding post-Sec translocation. - Durable-cell chassis engineering: Created B. subtilis ΔlytC to retain endospores within mother cell (prevent release) and ΔflhG to cluster flagella at poles (reduced peritrichous density), generating ΔlytC ΔflhG. Phenotypes confirmed (spore retention, clustered polar flagella), with secretion comparable to WT. Growth and pH tracked; no sporulation through 48 h under standard conditions. - Flagella SpyTag display: Engineered Hag flagellin with T209C for fluorescent maleimide labeling and inserted SpyTag at nine positions in D2/D3 region; several insertions were lethal. Functional display assessed by adding purified tdTomato-SpyCatcher during growth and visualizing flagella attachment. Position 588 (HagT209C::SpyTag588) showed best SpyCatcher binding albeit with shorter flagella. In situ covalent linkage verified by co-culturing HagT209C::SpyTag588 strain with EutM-SpyC secreting strain (SMM, 20 °C, 96 h), isolating sheared flagella, detecting a ~55 kDa EutM-SpyC–Hag complex by SDS-PAGE. - Biomineralization peptide screening: Expressed and purified from E. coli His-EutM fused to R5, CotB1p (CotB), SB7, and Synthetic peptides. Quantified silica precipitation using molybdate blue assay at 100 mM silica; visualized by SEM/TEM. CotB fusion yielded the highest silica precipitation efficiency; His-EutM alone also precipitated silica. - Bacillus secretion of biomineralizing scaffolds: Replaced SpyCatcher with CotB in Bacillus expression vector (pCT-EutMCotB, with SacB). Confirmed secretion (lower than SpyC due to cationic fusion). Cultures incubated with varying silica concentrations to determine gelation thresholds. - Consolidated ELM construct: Co-expression plasmid(s) for SacB-His-EutM-SpyC and SacB-His-EutM-CotB under independent cumate-inducible promoters, plus plasmid-borne hagT209C::SpyTag588 under native promoter in ΔlytC ΔflhG. Verified secretion levels by SDS-PAGE; attempted flagella imaging showed secreted scaffolds labeled by cysteine-reactive dye indicating extracellular scaffold abundance. - Biocomposite fabrication: Induced and uninduced cultures incubated with 100 mM hydrolyzed TEOS-derived silica in 6-well plates (20 °C, 1 h) to assess aggregate formation. Prepared cylindrical gel plugs in syringe molds with 200 mM silica, cured at 20–25 °C up to 5 h for rheology. - Mechanical testing: Performed frequency sweep rheology on 15 mm diameter plugs (gap 4.5 mm, 1% strain) to measure storage (G′) and loss (G″) moduli; compared scaffold-secreting and control cultures. - Scaffold localization: Labeled induced/uninduced cultures with His-SpyTag-eGFP or His-eGFP and imaged fluorescence for scaffold localization. Processed silica gel blocks for TEM thin sections to visualize cell distribution and assess staining of scaffolded regions. - Regeneration: Used ~5 mm3 pieces from 24 h-cured (200 mM silica) plugs to inoculate fresh cultures; assessed secretion and re-fabrication. Also tested 2-week-cured plugs and sequenced plasmids from regrown colonies for stability. - Functional augmentation via co-culture: Constructed pCT-Purple-HagT209C::SpyT588 (purple chromoprotein) and co-cultured 1:1 with the scaffold-secreting strain; compared to control co-culture lacking scaffolds. Fabricated purple silica blocks (200 mM silica, 24 h cure).

- Optimal cultivation: SMM at 20 °C maintained pH ≈7.0 for 48–96 h and preserved flagella while minimizing sporulation; EutM-SpyC expression reduced growth somewhat, indicating metabolic burden. - Secretion signal: SacB signal peptide enabled substantial extracellular recovery of EutM-SpyC scaffolds (observed in supernatant and urea-solubilized pellet fractions); other signal peptides yielded minimal secretion. LC-MS confirmed uncleaved SacB on secreted proteins. - Engineered chassis: ΔlytC prevented spore release (endospores retained), ΔflhG produced clustered polar flagella; secretion levels and growth comparable or improved versus WT under standard conditions. - SpyTag display: Among nine Hag insertion sites, SpyTag at position 588 provided strongest functional display; other sites showed no/weak binding or were deleterious. In situ covalent attachment of EutM-SpyC to Hag::SpyTag588 confirmed by a ~55 kDa complex on SDS-PAGE after co-culture. - Biomineralization efficacy: In molybdate blue assay at 100 mM silica, His-EutM-CotB precipitated 7.44 ± 0.36 mmol SiO2 per g protein; His-EutM precipitated 1.98 ± 0.01 mmol/g. SEM/TEM showed increased surface roughness and silica nanoparticles on scaffolds, with CotB indicating surface-catalyzed biomineralization. - Bacillus-secreted EutM-CotB enhanced silica gelation: Cultures expressing EutM-CotB formed gels at lower silica concentrations than controls; with 100 mM silica, EutM-CotB cultures formed solidifying gels while controls remained liquid-soft gels. - Mechanical properties: Silica gel blocks (200 mM silica, 25 °C, 5 h) from scaffold-expressing cultures showed increased storage modulus G′ versus control: 1.23-fold (pCT-EutMCotB-EutMSpyC) and 1.37-fold when also displaying HagT209C::SpyTag588, with G″ unchanged and lower than G′. - Scaffold localization: SpyTag-eGFP labeled regions between cells in induced cultures, indicating extracellular EutM scaffold assembly with available SpyCatcher binding sites. In silica thin sections, induced samples showed unstained clear zones around cells (consistent with silicified scaffold regions interfering with staining), and SDS-PAGE of dissolved gels detected EutM bands. - Regeneration: 24 h-cured silica blocks yielded viable cells that re-expressed scaffolds and re-formed cross-linked silica materials. After 2 weeks curing, cells could regrow with plasmid retention but exhibited multiple mutations in EutM regions and lost scaffold secretion. - Functional augmentation: Co-cultures of scaffold-secreting strain with a purple chromoprotein-expressing SpyTag-display strain formed purple cross-linked silica materials; controls lacking scaffold secretion did not. A purple silica block (15 × 30 mm) was fabricated at 200 mM silica after 24 h curing.

The work demonstrates that B. subtilis can be engineered to actively fabricate a living silica biocomposite by secreting self-assembling EutM scaffolds that biomineralize silica and covalently cross-link cells via SpyTag/SpyCatcher. Using a spore-forming chassis with retained endospores (ΔlytC) ensures the living component persists as a structural element and enables regeneration. Polar clustering of flagella (ΔflhG) and optimized SpyTag insertion (position 588) enabled effective, spatially controlled cross-linking, improving material robustness. Screening biomineralization peptides identified CotB as a strong silica nucleator when fused to EutM, enhancing gelation and contributing to increased mechanical rigidity (higher G′). The system supports modular functionalization, as shown by co-culturing with a chromoprotein-expressing strain to create colored materials. Collectively, these results address the goal of a resilient, self-fabricated, and regenerable ELM platform that can be augmented with new functions by genetic programming and co-culture strategies.

This study establishes a framework for resilient engineered living materials built by B. subtilis that secrete programmable EutM-based scaffolds for cell cross-linking and silica biomineralization. Key advances include efficient secretion via SacB, a durable chassis retaining endospores, functional SpyTag display on clustered polar flagella (optimal at Hag position 588), and incorporation of a CotB biomineralization peptide to enhance silica polymerization and mechanical strength. The ELM can be regenerated from cured silica pieces and readily accepts new functions through co-cultivation with engineered strains (e.g., chromoprotein coloration). Future directions include genomic integration of expression modules to prevent plasmid instability and enable long-term regeneration, increasing biomineralization peptide density or diversity to reduce required silica concentrations and expand to other minerals, optimizing secretion of cationic fusions, and extending the approach to other resilient, spore-forming chassis for diverse functional materials.

- High silica concentrations were required for gel formation (≥100 mM for aggregation; 200 mM for solid plugs), higher than natural biosilicification environments. - Plasmid-based expression led to mutational instability over prolonged curing/storage (2 weeks), resulting in loss of scaffold secretion; genomic integration is needed for long-term stability. - EutM-CotB secretion levels were lower than EutM-SpyC, likely due to cationic fusion effects, potentially limiting biomineralization efficiency. - Growth burden from high-level scaffold expression reduced culture density under some conditions. - Scaffold visualization in silica thin sections was hindered (clear zones suggesting silicified regions that resist staining), complicating direct structural imaging of the matrix. - Uncleaved SacB on secreted EutM may influence assembly properties; effects on long-term material performance were not fully characterized.