Design of temperature-responsive biofilms for the precise and reversible control of engineered living biomaterials

生物材料将某些机械性能与生命系统的多功能性结合在一起，在应对我们时代的生态和技术挑战方面正变得越来越重要。这种“工程生物材料”(ELM)的一个有希望的起点是细菌生物膜，如大肠杆菌的“卷曲”系统。这种生物膜可以可持续地生产和加工成宏观材料，如水凝胶或生物塑料。重要的是，卷曲材料表现出高度的机械稳定性和耐恶劣化学条件。Curli本身由纤维组成，这些纤维是由大肠杆菌分泌的蛋白质CsgA聚合形成的，然后由其结合伙伴CsgB固定在细胞表面。虽然生物膜的形成一般是遗传诱导的，但现有的卷曲生物膜形成后，由于纤维的高度稳定性，其力学性能几乎不会改变。因此，迄今为止，生物膜对外界刺激的机械适应是不可能的。该项目旨在开发可逆控制基于卷曲的榆树的机械稳定性的分子工具。使用现代蛋白质工程方法，我将产生人工的、对温度敏感的CsgA变体。然后，我将使用这些来控制卷曲纤维的生物物理性质，以响应温度的变化。在项目的第一部分，我将产生由CsgA和各种受体结构域组成的融合蛋白，这些融合蛋白根据温度同源二聚，从而在单个纤维之间形成稳定的、可逆的交叉连接。这使得有可能具体地控制所得到的榆树的流变性。在第二步中，我将通过将CsgA点突变文库与结构域插入方法相结合，最终产生仅在低温(&lt；25℃)下齐聚的合成CsgA变体。我将在随后的项目中与合作伙伴一起使用这些温度敏感、可逆和自适应的卷曲生物膜，用于活体结构的3D生物打印和益生菌的程序化释放。通过将蛋白质工程应用于卷曲生物膜，该研究项目将开启新一代生物材料的大门。

Biomaterials that combine certain mechanical properties with the versatility of living systems, are becoming increasingly important in response to the ecological and technological challenges of our time. A promising starting point for such "engineered living materials" (ELMs) are bacterial biofilms, such as the "curli" system of Escherichia coli. Such biofilms can be sustainably produced and processed into macroscopic materials such as hydrogels or bioplastic. Importantly, curli materials exhibit high mechanical stability and resistance to harsh chemical conditions. Curli themselves consist of fibrils that are formed by polymerization of the protein CsgA secreted by E. coli and are then anchored to the cell surface by its binding partner CsgB. Although the formation of biofilms is in general genetically inducible, the mechanical properties of existing curli biofilms can hardly be changed after their formation due to the high stability of the fibrils. Thus, the mechanical adaptation of the biofilm to external stimuli has thus far not been possible. This project aims to develop molecular tools for reversible control of the mechanical stability of curli-based ELMs. Using modern protein engineering methods, I will generate artificial, temperature-sensitive CsgA variants. I will then use these to control the biophysical properties of curli fibrils in response to temperature changes. In the first part of the project, I will generate fusion proteins consisting of CsgA and various receptor domains, which homo-dimerize depending on the temperature and can thus form stabilizing, reversible cross-connections between individual fibrils. This makes it possible to specifically control the rheological properties of the resulting ELMs. In a second step, I will finally produce synthetic CsgA variants that only oligomerize at low temperatures (<25°C) by combining CsgA point mutation libraries with domain insertion methods. I will use these temperature-sensitive, reversible and adaptive curli biofilms in subsequent projects together with collaboration partners for 3D bioprinting of living structures and for the programmed release of probiotic bacteria. By applying protein engineering to curli biofilms, this research project will unlock a new generation of biomaterials.