Biomaterials made by living systems, while very diverse, generally share similar organization: different lineages of cells are precisely patterned within a matrix to create a hierarchically defined structure. This meticulous organization allows for their multifunctional properties, which often exceed those of traditional materials. For these reasons, there is growing interest in creating engineered living materials (ELMs) that will have capabilities similar to those of natural living materials yet with tailored functions. This review aims to highlight technologies still missing from the field of ELMs, which currently prevents more impactful applications. We briefly review the existing literature and identify challenges in designing novel protein-based and non-ribosomally synthesized matrix elements, producing and incorporating biominerals, and improving critical material properties such as lifespan, spatial patterning, and cell-cell communication. We also discuss the interplay between these challenges and the need for the development of new chassis and corresponding genetic toolboxes. By overcoming these obstacles, we come ever closer to unlocking the potential and versatility of biomaterials to create designer ELMs.Protein-based materials (PBMs) are ubiquitous in nature and have often been studied and explored for commercial applications-especially in the biomedical field.

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.

Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the composition, mechanical properties, and catalytic function of these ELMs. This work provides genetic tools, design and assembly rules, and a platform for growing ELMs with control over both matrix and cellular structure and function.

Engineered living materials (ELMs) embed living cells in a biopolymer matrix to create novel materials with tailored functions. While bottom-up assembly of macroscopic ELMs with a de novo matrix would offer the greatest control over material properties, we lack the ability to genetically encode a protein matrix that leads to collective self-organization. Here we report growth of ELMs from Caulobacter crescentus cells that display and secrete a self-interacting protein. This protein formed a de novo matrix and assembled cells into centimeter-scale ELMs. Discovery of design and assembly principles allowed us to tune the mechanical, catalytic, and morphological properties of these ELMs. This work provides novel tools, design and assembly rules, and a platform for growing ELMs with control over matrix and cellular structure and function.One-Sentence SummaryWe discovered rules to grow bacteria into macroscopic living materials with customizable composition, structure, and function.