Accelerating the design of pili-enabled living materials using an integrative technological workflow.

Bacteria can be programmed to create engineered living materials (ELMs) with self-healing and evolvable functionalities. However, further development of ELMs is greatly hampered by the lack of engineerable nonpathogenic chassis and corresponding programmable endogenous biopolymers. Here, we describe a technological workflow for facilitating ELMs design by rationally integrating bioinformatics, structural biology and synthetic biology technologies. We first develop bioinformatics software, termed Bacteria Biopolymer Sniffer (BBSniffer), that allows fast mining of biopolymers and biopolymer-producing bacteria of interest. As a proof-of-principle study, using existing pathogenic pilus as input, we identify the covalently linked pili (CLP) biosynthetic gene cluster in the industrial workhorse Corynebacterium glutamicum. Genetic manipulation and structural characterization reveal the molecular mechanism of the CLP assembly, ultimately enabling a type of programmable pili for ELM design. Finally, engineering of the CLP-enabled living materials transforms cellulosic biomass into lycopene by coupling the extracellular and intracellular bioconversion ability.

Living materials fabricated via gradient mineralization of light-inducible biofilms.

Living organisms have evolved sophisticated cell-mediated biomineralization mechanisms to build structurally ordered, environmentally adaptive composite materials. Despite advances in biomimetic mineralization research, it remains difficult to produce mineralized composites that integrate the structural features and 'living' attributes of their natural counterparts. Here, inspired by natural graded materials, we developed living patterned and gradient composites by coupling light-inducible bacterial biofilm formation with biomimetic hydroxyapatite (HA) mineralization. We showed that both the location and the degree of mineralization could be regulated by tailoring functional biofilm growth with spatial and biomass density control. The cells in the composites remained viable and could sense and respond to environmental signals. Additionally, the composites exhibited a maximum 15-fold increase in Young's modulus after mineralization and could be applied to repair damage in a spatially controlled manner. Beyond insights into the mechanism of formation of natural graded composites, our study provides a viable means of fabricating living composites with dynamic responsiveness and environmental adaptability.

Programmable and printable Bacillus subtilis biofilms as engineered living materials.

Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

Patterned Amyloid Materials Integrating Robustness and Genetically Programmable Functionality.

The precise manipulation, localization, and assembly of biological and bioinspired molecules into organized structures have greatly promoted material science and bionanotechnology. Further technological innovation calls for new patternable soft materials with the long-sought qualities of environmental tolerance and functional flexibility. Here, we report a patterned amyloid material (PAM) platform for producing hierarchically ordered structures that integrate these material attributes. This platform, combining soft lithography with generic amyloid monomer inks (consisting of genetically engineered biofilm proteins dissolved in hexafluoroisopropanol), along with methanol-assisted curing, enables the spatially controlled deposition and in situ reassembly of amyloid monomers. The resulting patterned structures exhibit spectacular chemical and thermal stability and mechanical robustness under harsh conditions. The PAMs can be programmed for a vast array of multilevel functionalities, including anchoring nanoparticles, enabling diverse fluorescent protein arrays, and serving as self-supporting porous sheets for cellular growth. This PAM platform will not only drive innovation in biomanufacturing but also broaden the applications of patterned soft architectures in optics, electronics, biocatalysis, analytical regents, cell engineering, medicine, and other areas.

Diatom-inspired multiscale mineralization of patterned protein-polysaccharide complex structures.

Marine diatoms construct their hierarchically ordered, three-dimensional (3D) external structures called frustules through precise biomineralization processes. Recapitulating the remarkable architectures and functions of diatom frustules in artificial materials is a major challenge that has important technological implications for hierarchically ordered composites. Here, we report the construction of highly ordered, mineralized composites based on fabrication of complex self-supporting porous structures-made of genetically engineered amyloid fusion proteins and the natural polysaccharide chitin-and performing in situ multiscale protein-mediated mineralization with diverse inorganic materials, including SiO2, TiO2 and Ga2O3. Subsequently, using sugar cubes as templates, we demonstrate that 3D fabricated porous structures can become colonized by engineered bacteria and can be functionalized with highly photoreactive minerals, thereby enabling co-localization of the photocatalytic units with a bacteria-based hydrogenase reaction for a successful semi-solid artificial photosynthesis system for hydrogen evolution. Our study thus highlights the power of coupling genetically engineered proteins and polysaccharides with biofabrication techniques to generate hierarchically organized mineralized porous structures inspired by nature.

Virus Disinfection from Environmental Water Sources Using Living Engineered Biofilm Materials.

Waterborne viruses frequently cause disease outbreaks and existing strategies to remove such viral pathogens often involve harsh or energy-consuming water treatment processes. Here, a simple, efficient, and environmentally friendly approach is reported to achieve highly selective disinfection of specific viruses with living engineered biofilm materials. As a proof-of-concept, Escherichia coli biofilm matrix protein CsgA was initially genetically fused with the influenza-virus-binding peptide (C5). The resultant engineered living biofilms could correspondingly capture virus particles directly from aqueous solutions, disinfecting samples to a level below the limit-of-detection for a qPCR-based detection assay. By exploiting the surface-adherence properties of biofilms, it is further shown that polypropylene filler materials colonized by the CsgA-C5 biofilms can be utilized to disinfect river water samples with influenza titers as high as 1 × 107 PFU L-1. Additionally, a suicide gene circuit is designed and applied in the engineered strain that strictly limits the growth of bacterial, therefore providing a viable route to reduce potential risks confronted with the use of genetically modified organisms. The study thus illustrates that engineered biofilms can be harvested for the disinfection of pathogens from environmental water samples in a controlled manner and highlights the unique biology-only properties of living substances for material applications.

Immobilization of functional nano-objects in living engineered bacterial biofilms for catalytic applications.

Nanoscale objects feature very large surface-area-to-volume ratios and are now understood as powerful tools for catalysis, but their nature as nanomaterials brings challenges including toxicity and nanomaterial pollution. Immobilization is considered a feasible strategy for addressing these limitations. Here, as a proof-of-concept for the immobilization of nanoscale catalysts in the extracellular matrix of bacterial biofilms, we genetically engineered amyloid monomers of the Escherichia coli curli nanofiber system that are secreted and can self-assemble and anchor nano-objects in a spatially precise manner. We demonstrated three scalable, tunable and reusable catalysis systems: biofilm-anchored gold nanoparticles to reduce nitro aromatic compounds such as the pollutant p-nitrophenol, biofilm-anchored hybrid Cd0.9Zn0.1S quantum dots and gold nanoparticles to degrade organic dyes and biofilm-anchored CdSeS@ZnS quantum dots in a semi-artificial photosynthesis system for hydrogen production. Our work demonstrates how the ability of biofilms to grow in scalable and complex spatial arrangements can be exploited for catalytic applications and clearly illustrates the design utility of segregating high-energy nano-objects from injury-prone cellular components by engineering anchoring points in an extracellular matrix.

Directing curli polymerization with DNA origami nucleators.

The physiological or pathological formation of fibrils often relies on molecular-scale nucleators that finely control the kinetics and structural features. However, mechanistic understanding of how protein nucleators mediate fibril formation in cells remains elusive. Here, we develop a CsgB-decorated DNA origami (CB-origami) to mimic protein nucleators in Escherichia coli biofilm that direct curli polymerization. We show that CB-origami directs curli subunit CsgA monomers to form oligomers and then accelerates fibril formation by increasing the proliferation rate of primary pathways. Fibrils grow either out from (departure mode) or towards the nucleators (arrival mode), implying two distinct roles of CsgB: as nucleation sites and as trap sites to capture growing nanofibrils in vicinity. Curli polymerization follows typical stop-and-go dynamics but exhibits a higher instantaneous elongation rate compared with independent fibril growth. This origami nucleator thus provides an in vitro platform for mechanistically probing molecular nucleation and controlling directional fibril polymerization for bionanotechnology.

Modular genetic design of multi-domain functional amyloids: insights into self-assembly and functional properties.

Engineering functional amyloids through a modular genetic strategy represents new opportunities for creating multifunctional molecular materials with tailored structures and performance. Despite important advances, how fusion modules affect the self-assembly and functional properties of amyloids remains elusive. Here, using Escherichia coli curli as a model system, we systematically studied the effect of flanking domains on the structures, assembly kinetics and functions of amyloids. The designed amyloids were composed of E. coli biofilm protein CsgA (as amyloidogenic cores) and one or two flanking domains, consisting of chitin-binding domains (CBDs) from Bacillus circulans chitinase, and/or mussel foot proteins (Mfps). Incorporation of fusion domains did not disrupt the typical ß-sheet structures, but indeed affected assembly rate, morphology, and stiffness of resultant fibrils. Consequently, the CsgA-fusion fibrils, particularly those containing three domains, were much shorter than the CsgA-only fibrils. Furthermore, the stiffness of the resultant fibrils was heavily affected by the structural feature of fusion domains, with ß-sheet-containing domains tending to increase the Young's modulus while random coil domains decreasing the Young's modulus. In addition, fibrils containing CBD domains showed higher chitin-binding activity compared to their CBD-free counterparts. The CBD-CsgA-Mfp3 construct exhibited significantly lower binding activity than Mfp5-CsgA-CBD due to inappropriate folding of the CBD domain in the former construct, in agreement with results based upon molecular dynamics modeling. Our study provides new insights into the assembly and functional properties of designer amyloid proteins with increasing complex domain structures and lays the foundation for the future design of functional amyloid-based structures and molecular materials.

Emerging Paradigms for Synthetic Design of Functional Amyloids.

Many living organisms make use of diverse amyloid proteins as functional building blocks to fulfill a variety of physiological applications. This fact, along with the intrinsic self-assembly and outstanding material properties of amyloids, has prompted a significant amount of research in the synthetic design of functional amyloids to form diverse nanoarchitectures, molecular materials, and hybrid or composite materials. In particular, a new research paradigm has recently been advanced that uses synthetic biology to harness functional amyloids with cells as living materials or functional devices. Here we outline important progress in the synthetic design of functional amyloids, in the context of both non-living and living systems. We propose several important tools and underline emerging techniques and principles that might be useful in advancing the future synthetic design of functional amyloids.

Programming Cells for Dynamic Assembly of Inorganic Nano-Objects with Spatiotemporal Control.

Programming living cells to organize inorganic nano-objects (NOs) in a spatiotemporally precise fashion would advance new techniques for creating ordered ensembles of NOs and new bio-abiotic hybrid materials with emerging functionalities. Bacterial cells often grow in cellular communities called biofilms. Here, a strategy is reported for programming dynamic biofilm formation for the synchronized assembly of discrete NOs or hetero-nanostructures on diverse interfaces in a dynamic, scalable, and hierarchical fashion. By engineering Escherichia coli to sense blue light and respond by producing biofilm curli fibers, biofilm formation is spatially controlled and the patterned NOs' assembly is simultaneously achieved. Diverse and complex fluorescent quantum dot patterns with a minimum patterning resolution of 100 µm are demonstrated. By temporally controlling the sequential addition of NOs into the culture, multilayered heterostructured thin films are fabricated through autonomous layer-by-layer assembly. It is demonstrated that biologically dynamic self-assembly can be used to advance a new repertoire of nanotechnologies and materials with increasing complexity that would be otherwise challenging to produce.

Diverse Supramolecular Nanofiber Networks Assembled by Functional Low-Complexity Domains.

Self-assembling supramolecular nanofibers, common in the natural world, are of fundamental interest and technical importance to both nanotechnology and materials science. Despite important advances, synthetic nanofibers still lack the structural and functional diversity of biological molecules, and the controlled assembly of one type of molecule into a variety of fibrous structures with wide-ranging functional attributes remains challenging. Here, we harness the low-complexity (LC) sequence domain of fused in sarcoma (FUS) protein, an essential cellular nuclear protein with slow kinetics of amyloid fiber assembly, to construct random copolymer-like, multiblock, and self-sorted supramolecular fibrous networks with distinct structural features and fluorescent functionalities. We demonstrate the utilities of these networks in the templated, spatially controlled assembly of ligand-decorated gold nanoparticles, quantum dots, nanorods, DNA origami, and hybrid structures. Owing to the distinguishable nanoarchitectures of these nanofibers, this assembly is structure-dependent. By coupling a modular genetic strategy with kinetically controlled complex supramolecular self-assembly, we demonstrate that a single type of protein molecule can be used to engineer diverse one-dimensional supramolecular nanostructures with distinct functionalities.

A highly efficient Cu-catalyzed S-transfer reaction: from amine to sulfide.

A highly efficient Cu-catalyzed dual C-S bonds formation reaction, proceeding in alcohol and water under air, is reported, in which inodorous stable Na2S2O3 is used as a sulfurating reagent. This powerful strategy provides a practical and efficient approach to construct thioethers, using readily available aromatic amines and alkyl halides as starting materials. Sensitive and synthetic useful functional groups could be tolerated. Furthermore, pharmaceuticals, glucose, an amino acid, and a chiral ligand are successfully furnished by this late-stage sulfuration strategy.