Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials

ABSTRACT The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacter...

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Autores principales:	Maria Teresa Orozco-Hidalgo, Marimikel Charrier, Nicholas Tjahjono, Robert F. Tesoriero, Dong Li, Sara Molinari, Kathleen R. Ryan, Paul D. Ashby, Behzad Rad, Caroline M. Ajo-Franklin
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	Caulobacter crescentus engineered living material extracellular matrix protein hydrogel protein secretion surface structures Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/b74ed5c6829c463ea106d7231650ee91
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spelling	oai:doaj.org-article:b74ed5c6829c463ea106d7231650ee912021-12-02T19:22:27ZEngineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials10.1128/mSystems.00903-202379-5077https://doaj.org/article/b74ed5c6829c463ea106d7231650ee912021-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.00903-20https://doaj.org/toc/2379-5077ABSTRACT The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a resilin-like polypeptide, demonstrating this system is flexible. The SC(−)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. IMPORTANCE Engineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy-intensive processing or have limited tunability. We propose a bacterially synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.Maria Teresa Orozco-HidalgoMarimikel CharrierNicholas TjahjonoRobert F. TesorieroDong LiSara MolinariKathleen R. RyanPaul D. AshbyBehzad RadCaroline M. Ajo-FranklinAmerican Society for MicrobiologyarticleCaulobacter crescentusengineered living materialextracellular matrixprotein hydrogelprotein secretionsurface structuresMicrobiologyQR1-502ENmSystems, Vol 6, Iss 2 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Caulobacter crescentus engineered living material extracellular matrix protein hydrogel protein secretion surface structures Microbiology QR1-502
spellingShingle	Caulobacter crescentus engineered living material extracellular matrix protein hydrogel protein secretion surface structures Microbiology QR1-502 Maria Teresa Orozco-Hidalgo Marimikel Charrier Nicholas Tjahjono Robert F. Tesoriero Dong Li Sara Molinari Kathleen R. Ryan Paul D. Ashby Behzad Rad Caroline M. Ajo-Franklin Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
description	ABSTRACT The bacterial extracellular matrix forms autonomously, giving rise to complex material properties and multicellular behaviors. Synthetic matrix analogues can replicate these functions but require exogenously added material or have limited programmability. Here, we design a two-strain bacterial system that self-synthesizes and structures a synthetic extracellular matrix of proteins. We engineered Caulobacter crescentus to secrete an extracellular matrix protein composed of an elastin-like polypeptide (ELP) hydrogel fused to supercharged SpyCatcher [SC(−)]. This biopolymer was secreted at levels of 60 mg/liter, an unprecedented level of biomaterial secretion by a native type I secretion apparatus. The ELP domain was swapped with either a cross-linkable variant of ELP or a resilin-like polypeptide, demonstrating this system is flexible. The SC(−)-ELP matrix protein bound specifically and covalently to the cell surface of a C. crescentus strain that displays a high-density array of SpyTag (ST) peptides via its engineered surface layer. Our work develops protein design guidelines for type I secretion in C. crescentus and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. IMPORTANCE Engineered living materials (ELM) aim to mimic characteristics of natural occurring systems, bringing the benefits of self-healing, synthesis, autonomous assembly, and responsiveness to traditional materials. Previous research has shown the potential of replicating the bacterial extracellular matrix (ECM) to mimic biofilms. However, these efforts require energy-intensive processing or have limited tunability. We propose a bacterially synthesized system that manipulates the protein content of the ECM, allowing for programmable interactions and autonomous material formation. To achieve this, we engineered a two-strain system to secrete a synthetic extracellular protein matrix (sEPM). This work is a step toward understanding the necessary parameters to engineering living cells to autonomously construct ELMs.
format	article
author	Maria Teresa Orozco-Hidalgo Marimikel Charrier Nicholas Tjahjono Robert F. Tesoriero Dong Li Sara Molinari Kathleen R. Ryan Paul D. Ashby Behzad Rad Caroline M. Ajo-Franklin
author_facet	Maria Teresa Orozco-Hidalgo Marimikel Charrier Nicholas Tjahjono Robert F. Tesoriero Dong Li Sara Molinari Kathleen R. Ryan Paul D. Ashby Behzad Rad Caroline M. Ajo-Franklin
author_sort	Maria Teresa Orozco-Hidalgo
title	Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
title_short	Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
title_full	Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
title_fullStr	Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
title_full_unstemmed	Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials
title_sort	engineering high-yield biopolymer secretion creates an extracellular protein matrix for living materials
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/b74ed5c6829c463ea106d7231650ee91
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Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials

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