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Background <p>Bacterial nanocellulose (BC), produced by <i>Komagataeibacter</i> 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 <i>K. rhaeticus</i> iGEM, a model bacterium for BC-based bioELMs, enabling a synthetic biology framework for single-chassis based biomaterial functionalization.</p> Results <p>Genome-wide analysis confirmed the presence of a complete Sec translocation machinery in <i>K. rhaeticus</i>. 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.</p> Conclusions <p>This study benchmarks signal peptide-dependent protein translocation in <i>K. rhaeticus</i> 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 <i>K. rhaeticus</i> as a single-chassis platform towards the production of functionalized bioELMs.</p>

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A signal peptide-guided approach towards in situ functionalization of bacterial nanocellulose in Komagataeibacter rhaeticus

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月17日

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  • Jenni Vannas,
  • Amritpal Singh,
  • Bibi Hannikainen,
  • Michelle Alexandrino de Assis,
  • Rodrigo Ledesma-Amaro,
  • Tom Ellis,
  • Rahul Mangayil

摘要

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.