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 Lpp’OmpA, 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.
Shuang Zhang, Chengji Yang, Ruxia Fan et al.· bioRxiv· 0 citations
BACKGROUND
Bacterial nanocellulose (BC), produced by Komagataeibacter species, is an ideal scaffold for biological Engineered Living Materials (bioELMs) research. Current BC functionalization strategies often rely on secondary microbial hosts or post-production enzyme immobilization, limiting the scalability and modularity required for programmable bioELMs. Establishing a single-chassis system capable of simultaneous biopolymer synthesis and in situ functionalization remains a primary objective in bioELM research. This study addresses the need by benchmarking signal peptide-mediated protein translocation in K. rhaeticus iGEM, a model bacterium for BC-based bioELMs, enabling a synthetic biology framework for single-chassis based biomaterial functionalization.
RESULTS
Genome-wide analysis confirmed the presence of a complete Sec translocation machinery in K. rhaeticus. Through liquid chromatography-tandem mass spectrometry and SignalP 5.0 prediction, native signal peptides were identified and evaluated alongside previously characterized heterologous signal peptides using β-lactamase and mScarlet as cargo proteins. Protein translocation was found to depend on signal peptide identity, cargo type, and expression mode. Fluorescence imaging revealed cytoplasmic, polar, and peripheral localization patterns, confirming functional engagement with the native translocation machinery. A key limitation identified was the retention of recombinant proteins within the periplasm, restricting extracellular availability. Despite this, signal peptide-mediated translocation enabled the incorporation of enzymatic activity into BC during biosynthesis. A post-growth osmotic shock-release strategy increased measurable enzymatic activity by 30%, demonstrating a practical route to overcome this physiological bottleneck while maintaining the biomaterial production capacity.
CONCLUSIONS
This study benchmarks signal peptide-dependent protein translocation in K. rhaeticus and identifies periplasmic retention as a key constraint for extracellular protein release. By linking protein translocation to in situ BC functionalization, this work establishes a synthetic biology framework that supports the development of K. rhaeticus as a single-chassis platform towards the production of functionalized bioELMs.
Jenni Vannas, Amritpal Singh, Bibi Hannikainen et al.· Journal of Biological Engine...· 0 citations
