Orozco-Hidalgo Maria Teresa, Charrier Marimikel, Tjahjono Nicholas, Tesoriero Robert F, Li Dong, Molinari Sara, Ryan Kathleen R, Ashby Paul D, Rad Behzad, Ajo-Franklin Caroline M
Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, USA.
Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, California, USA
mSystems. 2021 Mar 23;6(2):e00903-20. doi: 10.1128/mSystems.00903-20.
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 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 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 and demonstrates the autonomous secretion and assembly of programmable extracellular protein matrices, offering a path forward toward the formation of cohesive engineered living materials. 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.
细菌细胞外基质自主形成,产生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,我们设计了一个双菌株细菌系统,该系统能自我合成并构建蛋白质合成细胞外基质。我们对其进行工程改造,使其分泌一种由与带正电荷的SpyCatcher [SC]融合的类弹性蛋白多肽(ELP)水凝胶组成的细胞外基质蛋白。这种生物聚合物的分泌水平为60毫克/升,这是天然I型分泌装置前所未有的生物材料分泌水平。ELP结构域被替换为ELP的可交联变体或类弹性蛋白多肽,表明该系统具有灵活性。SC-ELP基质蛋白通过其工程化的表面层与展示高密度SpyTag(ST)肽阵列的菌株的细胞表面特异性共价结合。我们的工作为大肠杆菌中的I型分泌制定了蛋白质设计指南,并展示了可编程细胞外蛋白质基质的自主分泌和组装,为形成有凝聚力的工程活材料提供了一条前进的道路。工程活材料(ELM)旨在模仿自然系统的特性,为传统材料带来自我修复、合成、自主组装和响应性等优点。先前的研究表明,复制细菌细胞外基质(ECM)以模仿生物膜具有潜力。然而,这些努力需要耗能的加工过程或可调性有限。我们提出了一种细菌合成系统,该系统可操纵ECM的蛋白质含量,实现可编程的相互作用和自主材料形成。为了实现这一目标,我们设计了一个双菌株系统来分泌合成细胞外蛋白质基质(sEPM)。这项工作是朝着理解工程活细胞自主构建ELM所需参数迈出的一步。