Degradable living plastics programmed by engineered spores.

Plastics are widely used materials that pose an ecological challenge because their wastes are difficult to degrade. Embedding enzymes and biomachinery within polymers could enable the biodegradation and disposal of plastics. However, enzymes rarely function under conditions suitable for polymer processing. Here, we report degradable living plastics by harnessing synthetic biology and polymer engineering. We engineered Bacillus subtilis spores harboring the gene circuit for the xylose-inducible secretory expression of Burkholderia cepacia lipase (BC-lipase). The spores that were resilient to stresses during material processing were mixed with poly(caprolactone) to produce living plastics in various formats. Spore incorporation did not compromise the physical properties of the materials. Spore recovery was triggered by eroding the plastic surface, after which the BC-lipase released by the germinated cells caused near-complete depolymerization of the polymer matrix. This study showcases a method for fabricating green plastics that can function when the spores are latent and decay when the spores are activated and sheds light on the development of materials for sustainability.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Engineering living materials by synthetic biology.

Natural biological materials are programmed by genetic information and able to self-organize, respond to environmental stimulus, and couple with inorganic matter. Inspired by the natural system and to mimic their complex and delicate fabrication process and functions, the field of engineered living materials emerges at the interface of synthetic biology and materials science. Here, we review the recent efforts and discuss the challenges and future opportunities.

B12-induced reassembly of split photoreceptor protein enables photoresponsive hydrogels with tunable mechanics.

Although the tools based on split proteins have found broad applications, ranging from controlled biological signaling to advanced molecular architectures, many of them suffer from drawbacks such as background reassembly, low thermodynamic stability, and static structural features. Here, we present a chemically inducible protein assembly method enabled by the dissection of the carboxyl-terminal domain of a B12-dependent photoreceptor, CarHC. The resulting segments reassemble efficiently upon addition of cobalamin (AdoB12, MeB12, or CNB12). Photolysis of the cofactors such as AdoB12 and MeB12 further leads to stable protein adducts harboring a bis-His-ligated B12. Split CarHC enables the creation of a series of protein hydrogels, of which the mechanics can be either photostrengthened or photoweakened, depending on the type of B12. These materials are also well suited for three dimensional cell culturing. Together, this new protein chemistry, featuring negligible background autoassembly, stable conjugation, and phototunability, has opened up opportunities for designing smart materials.

Directed assembly of genetically engineered eukaryotic cells into living functional materials via ultrahigh-affinity protein interactions.

Engineered living materials (ELMs) are gaining traction among synthetic biologists, as their emergent properties and nonequilibrium thermodynamics make them markedly different from traditional materials. However, the aspiration to directly use living cells as building blocks to create higher-order structures or materials, with no need for chemical modification, remains elusive to synthetic biologists. Here, we report a strategy that enables the assembly of engineered Saccharomyces cerevisiae into self-propagating ELMs via ultrahigh-affinity protein/protein interactions. These yeast cells have been genetically engineered to display the protein pairs SpyTag/SpyCatcher or CL7/Im7 on their surfaces, which enable their assembly into multicellular structures capable of further growth and proliferation. The assembly process can be controlled precisely via optical tweezers or microfluidics. Moreover, incorporation of functional motifs such as super uranyl-binding protein and mussel foot proteins via genetic programming rendered these materials suitable for uranium extraction from seawater and bioadhesion, respectively, pointing to their potential in chemical separation and biomedical applications.

Injectable, photoresponsive hydrogels for delivering neuroprotective proteins enabled by metal-directed protein assembly.

Axon regeneration constitutes a fundamental challenge for regenerative neurobiology, which necessitates the use of tailor-made biomaterials for controllable delivery of cells and biomolecules. An increasingly popular approach for creating these materials is to directly assemble engineered proteins into high-order structures, a process that often relies on sophisticated protein chemistry. Here, we present a simple approach for creating injectable, photoresponsive hydrogels via metal-directed assembly of His6-tagged proteins. The B12-dependent photoreceptor protein CarHC can complex with transition metal ions through an amino-terminal His6-tag, which can further undergo a sol-gel transition upon addition of AdoB12, leading to the formation of hydrogels with marked injectability and photodegradability. The inducible phase transitions further enabled facile encapsulation and release of cells and proteins. Injecting the Zn2+-coordinated gels decorated with leukemia inhibitory factor into injured mouse optic nerves led to prolonged cellular signaling and enhanced axon regeneration. This study illustrates a powerful strategy for designing injectable biomaterials.

Synthesis of Entirely Protein-Based Hydrogels by Enzymatic Oxidation Enabling Water-Resistant Bioadhesion and Stem Cell Encapsulation.

Growing complexity in modern surgery and guided tissue repair calls for new materials capable of dual functions-water-resistant adhesion to biological tissues and stem cell encapsulation/delivery. Here, we demonstrate the creation of entirely recombinant protein-based adhesive hydrogels by leveraging the sequence derived from natural adhesive molecules-mussel foot protein-3 (Mfp-3), in vitro enzymatic oxidation mediated by recombinant tyrosinase and genetically encoded SpyTag/SpyCatcher chemistry. The resulting materials exhibited varied stiffness dependent on the polymer concentration, strong water-resistant adhesion to porcine skin, and excellent compatibility with 3D stem cell culture. The presence of SpyCatcher domains within the protein networks also enabled postgelation decoration with SpyTagged proteins under mild physiological conditions. These results point to a new approach for designing genetically programmable materials that enable both water-resistant bioadhesion and stem cell encapsulation for biomedical applications.