(395d) De Novo-Engineered Living Materials from Bacteria

Naturally occurring living biomaterials, such as bones or wood, grow bottom-up from a small number of progenitor cells into macroscale structures. Engineered living materials (ELMs) are composites of living cells incorporated into a biopolymer matrix. They are inspired by naturally occurring living materials but use synthetic biology to introduce tailored non-natural properties in order to function as living sensors, therapeutics, biomanufacturing platforms, electronics, energy converters, and structural materials. While cells confer functionality to ELMs, the matrix assembles the material and defines its mechanical and physical properties by controlling the bulk material composition, structure, and function. For this reason, the ability to engineer the collective self-organization of cells through a genetically encoded synthetic matrix has been a longstanding challenge in the ELM field. Previous to my work, most macroscopic ELMs either required significant processing to be assembled or were scarcely tunable, based on natural biomaterials, such as the bacterial nanocellulose. I addressed this limitation by creating the first macroscopic de novo ELM, which grows from genetically-engineered bacteria. To achieve this goal, I engineered Caulobacter crescentus to display self-interacting proteins by replacing the central crystallization domain of the native surface layer (S-layer) monomer (RsaA) with a structural domain made of elastin-like polypeptides. In this way, the hydrophobic C-termini of the RsaA become exposed and able to self-interact. I thus created a synthetic extracellular matrix that mediates cell-cell interactions. This protein was also found to be partially secreted, forming a matrix able to hierarchically organize cells over four orders of magnitude, resulting in the growth of centimeter-scale living materials. The most remarkable and unique feature of the de novo ELMs is our ability to control their mechanical properties through genetic modifications. By altering the structural domain of the matrix protein, I tuned the mechanical properties of ELMs over a 25-fold range. I showed that this living material can be functionalized from complex enzyme mixtures, through the SpyTag-SpyCatcher technology, allowing it to perform biological catalysis. Moreover, it retained the natural ability of single C. crescentus cells to bind cadmium from contaminated solutions, showing the potential to be a much more useful tool for heavy metal removal than a suspension of single cells by virtue of being a macroscopic, solid ­material. De novo ELMs can be reshaped and used as cementing agents, forming hard hybrid materials; they can also be desiccated at room temperature and reseeded into fresh medium to form new material. This property facilitates their transport and storage. This study lays the foundation for growing ELMs with defined physical and mechanical properties, thus paving the way toward growing multifunctional, self-regenerating materials.