Engineering High-Yield Biopolymer Secretion Creates an Extracellular Protein Matrix for Living Materials

Abstract

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.

Bottom-up approaches to engineered living materials: Challenges and future directions

“…First of all, protein production must be coupled with secretion so that PBMs can assemble in the extracellular space and create a matrix. Compared 12 The panel showing the seeded chain-growth polymerization was adapted with permission from Bowen et al 7 The panel showing the optimization of protein secretion is from Orozco-Hidalgo et al 13 ll Matter 4, 3095-3120, October 6, 2021 3099 with intracellular expression, exporting proteins outside of the cell is an onerous task. Protein secretion or translocation to the cell membrane is crucial for myriad processes in bacterial physiology, such as pathogenicity, membrane homeostasis, nutrition, and protection.…”

“…28 However, type I secretion systems export only partially or fully unfolded proteins. To secrete proteins with folded domains, Orozco-Hidalgo et al 13 took the innovative approach of swapping the folded domain with a mutant version that folds slowly. This boosted the yield from undetectable levels to $60 mg L À1 , the highest yield of a secreted biopolymer by a type I secretion system.…”

Synthetic biology as driver for the biologization of materials sciences

“…In a recent study, the same group engineered C. crescentus to produce a crosslinked extracellular protein matrix for living materials [ 220 ]. To this aim, C. crescentus was engineered to secrete via its type-1 secretion apparatus an extracellular matrix protein constructed from elastin-like polypeptides fused to a supercharged SpyCatcher variant.…”

“…It was shown that this secreted protein bound covalently to a SpyTag-functionalized S-layer (see paragraph above) of C. crescentus . This two-strain system to secrete a synthetic extracellular protein matrix was suggested as a step toward understanding the parameters required to engineer living cells to autonomously construct ELMs [ 220 ].…”

Engineering Bacillus subtilis for the formation of a durable living biocomposite material

“…True ELMs have been created by engineering E. coli to produce an extracellular matrix from curli fibers 8–25 . Other types of extracellular matrices for ELM fabrication were created from secreted bacterial cellulose to embed microbial cells 26, 27 or from elastin-like polypeptides to attach Caulobacter cells via their protein S-layers 28 . Among these examples, the secretion of a polypeptide-based scaffolding system or matrix offers greater control over material assembly and functionalization due to the genetic programmability of polypeptide structures and functions.…”