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 elastin-like polypeptide (ELP) hydrogel fused to Supercharged SpyCatcher (SC(-)). This biopolymer was secreted at levels of 60 mg/L, an unprecedented level of biopolymer secretion by a gram-negative bacterium. The ELP domain was swapped with either a crosslinkable variant of ELP or 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 peptides via its engineered Surface-layer. Our work develops protein design rules for Type I secretion in C. crescentus, and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward towards 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 towards understanding the necessary parameters to engineering living cells to autonomously construct ELMs. Keywords: Caulobacter crescentus/ engineered living materials/ protein hydrogels/ surface-layer protein/ type I secretion

中文翻译：

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工程化高产生物聚合物的分泌可为生物材料创造细胞外蛋白质基质

细菌细胞外基质自动形成，从而导致复杂的材料特性和多细胞行为。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可编程性有限。在这里，我们设计了一个两株细菌的系统，可以自我合成和构建蛋白质的合成细胞外基质。我们工程化新月形杆菌以分泌由弹性蛋白样多肽（ELP）水凝胶组成的细胞外基质蛋白，该蛋白与Supercharged SpyCatcher（SC（-））融合在一起。这种生物聚合物的分泌水平为60 mg / L，这是革兰氏阴性细菌前所未有的生物聚合物分泌水平。ELP结构域与ELP的可交联变异体或resilin样多肽交换，证明该系统具有灵活性。SC（-）-ELP基质蛋白与C.crescentus菌株的细胞表面特异性和共价结合，该菌株通过其工程化的表面层展示了SpyTag肽的高密度阵列。我们的工作为C. crescentus的I型分泌制定了蛋白质设计规则，并演示了可编程细胞外蛋白质基质的自主分泌和组装，为形成具有凝聚力的工程化生活材料提供了一条途径。重要信息：活体工程材料（ELM）旨在模仿天然系统的特性，带来自我修复，合成，自主组装以及对传统材料反应灵敏的好处。先前的研究表明复制细菌细胞外基质（ECM）来模仿生物膜的潜力。然而，这些工作需要耗能的过程或可调性有限。我们提出了一种细菌合成的系统，该系统可操纵ECM的蛋白质含量，从而实现可编程的相互作用和自主的物质形成。为了实现这一目标，我们设计了一种两株菌株的系统来分泌合成的细胞外蛋白基质（sEPM）。这项工作是朝着理解工程细胞以自主构建ELM的必要参数迈出的一步。关键词：新月形杆菌/生物工程材料/蛋白质水凝胶/表层蛋白质/ I型分泌 我们设计了一种两株系统来分泌合成的细胞外蛋白基质（sEPM）。这项工作是朝着理解工程细胞以自主构建ELM的必要参数迈出的一步。关键词：新月形杆菌/生物工程材料/蛋白质水凝胶/表层蛋白质/ I型分泌 我们设计了一种两株系统来分泌合成的细胞外蛋白基质（sEPM）。这项工作是朝着理解工程细胞以自主构建ELM的必要参数迈出的一步。关键词：新月形杆菌/生物工程材料/蛋白质水凝胶/表层蛋白质/ I型分泌