Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials.
Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials.
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工程高产生物聚合物的分泌为生物材料创造了细胞外蛋白质基质。
DOI：
10.1128/msystems.00903-20
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发表时间：
2021-03-23
期刊：
影响因子：
6.4
通讯作者：
Ajo-Franklin CM
中科院分区：
文献类型：
--
作者：
Orozco-Hidalgo MT;Charrier M;Tjahjono N;Tesoriero RF Jr;Li D;Molinari S;Ryan KR;Ashby PD;Rad B;Ajo-Franklin CM
细菌细胞外基质自主形成，产生复杂的材料性质和多细胞行为。合成基质类似物可以复制这些功能，但需要外源添加材料或具有有限的可编程性。在这里，我们设计了一个双菌株的细菌系统，自我合成和结构的蛋白质合成细胞外基质。我们设计了新月柄杆菌，使其分泌一种由弹性蛋白样多肽（ELP）水凝胶与增压SpyCatcher [SC（−）]融合组成的细胞外基质蛋白。该生物聚合物以60 mg/L的水平分泌，这是天然I型分泌装置前所未有的生物材料分泌水平。ELP结构域与ELP的交叉突变体或节枝弹性蛋白样多肽交换，表明该系统是灵活的。SC（-）-ELP基质蛋白特异性地共价结合到C. crescentus菌株，其通过其工程化表面层展示SpyTag（ST）肽的高密度阵列。我们的工作为C中的I型分泌开发了蛋白质设计指南。crescentus，并展示了自主分泌和可编程的细胞外蛋白质基质的组装，提供了一条通往形成有凝聚力的工程生物材料的道路。工程生命材料（ELM）旨在模仿自然发生系统的特性，带来自我修复、合成、自主组装和响应传统材料的好处。先前的研究已经显示了复制细菌细胞外基质（ECM）以模拟生物膜的潜力。然而，这些努力需要能量密集型处理或具有有限的可调谐性。我们提出了一个细菌合成的系统，操纵ECM的蛋白质含量，允许可编程的相互作用和自主材料的形成。为了实现这一点，我们设计了一个双菌株系统分泌合成的细胞外蛋白基质（saponin）。这项工作是朝着理解工程活细胞自主构建ELM的必要参数迈出的一步。
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.