Functional Amyloids: The Biomaterials of Tomorrow?

Functional amyloid (FAs), particularly the bacterial proteins CsgA and FapC, have many useful properties as biomaterials: high stability, efficient, and controllable formation of a single type of amyloid, easy availability as extracellular material in bacterial biofilm and flexible engineering to introduce new properties. CsgA in particular has already demonstrated its worth in hydrogels for stable gastrointestinal colonization and regenerative tissue engineering, cell‐specific drug release, water‐purification filters, and different biosensors. It also holds promise as catalytic amyloid; existing weak and unspecific activity can undoubtedly be improved by targeted engineering and benefit from the repetitive display of active sites on a surface. Unfortunately, FapC remains largely unexplored and no application is described so far. Since FapC shares many common features with CsgA, this opens the window to its development as a functional scaffold. The multiple imperfect repeats in CsgA and FapC form a platform to introduce novel properties, e.g., in connecting linkers of variable lengths. While exploitation of this potential is still at an early stage, particularly for FapC, a thorough understanding of their molecular properties will pave the way for multifunctional fibrils which can contribute toward solving many different societal challenges, ranging from CO2 fixation to hydrolysis of plastic nanoparticles.

The subjugation of Nature has always been a driving force of human innovation.In prehistoric times, humankind domesticated animals and grew crops, facilitating a sedentary existence with a (reasonably) stable food supply.In the Industrial Revolution, metal was wrought into machines to automate processes ranging from the butchering of animals to the production of clothes.A more recent expression of this Promethean behavior was the invention of plastics, a collection of indestructible materials with a diversity of almost miraculous properties.What we have since learned, at the cost of varying degrees of environmental destruction, is that subjugation comes with long-term costs and lacks the sustainability of Nature's solutions.After all, evolution has had several billion years of practice before the dawn of humanity.In a sense, the development of cutting-edge materials has come full circle.We have now started to try to solve our problems (many of which are caused by our previous "solutions") by learning from Nature.Hopefully, we are in transit from the age of exploitation to that of coexistence.
The development of biomaterials, which are sustainable and environmentally friendly, could be the key to such a transition.In the last decades, several studies have demonstrated that the generation of these biomaterials is a close reality rather than a simple speculation.In this endeavor, several strategies have been explored, including the use of amyloid fibrils, which stands as an emerging approach.Despite the complexity of these structures, several applications and/or functionalities have already been described.The application of functional and artificial amyloids as materials building blocks has been comprehensively discussed in numerous reviews, [1][2][3] including a recent and excellent publication in 2016 from Knowles and Mezzenga. [1]Here, we summarize the new development after that and other earlier reviews.In particular, we focus on the potential applications of the bacterial functional amyloid (FA) CsgA from Escherichia coli, and to a lesser extent the FA FapC from Pseudomonas.Both proteins show high stability, controllable formation, easy availability, and flexible engineering to introduce new properties.These properties make FA excellent building blocks for more durable, plastic-like materials, and its redox-controllable assembly fibrillation is also a useful property when designing amyloid-based biomaterials.We discuss new emerging applications of amyloid-based biomaterials, such as slow-release cargo or filters, with special mention of catalytic amyloid.Moreover, as the development of cryo-electron microscopy (Cryo-EM) has permitted the acquisition of near-atom resolution structures of different fibers, we briefly assess how the understanding of FAs' structures can facilitate the design of amyloid-based biomaterials.

From Drug Delivery to Bioplastics and Smart Materials
Inspiration from Nature is evident across numerous research fields.In medicine, for example, drug delivery via biological polymer- [4,5] or protein-based [6,7] nanoparticles show great promise for both targeted and temporally controlled release of drugs.Also, hydrogels, based on both organic and biological polymers as well as peptide-based fibers, are starting to play a role in tissue engineering and regenerative medicine by providing cells with a 3D scaffold that closely mimics the extra-cellular matrix in terms of adhesion and flexibility/ elasticity. [8]he development of bio-based plastics is also under rapid development in multiple parallel tracks.12] These bioplastics have been shown to be both sustainable and biodegradable.[12] Another approach involves the production of conventional plastic precursors in microorganisms.[12] One of the newest trends in materials science involves smart materials which encompass a wide range of compounds, from titanium foams to self-healing hydrogels. [13,14]They all have one or more properties that respond to external stimuli.This has close parallels to natural materials such as bacterial biofilm (the matrix in which sedentary bacteria are embedded), connective tissues, and woody tissue whose complex composition makes them both multifunctional and highly adaptive to changes in environments.An extra advantage of bacterial biofilm is that it is a living material, maintained and replenished by the embedded bacterial cells. [15]

Functional Bacterial Amyloids: Smart Protein Self-Assembly in the Biofilm
This short and non-exhaustive list of both currently developed and hypothetical biomaterials indicates that Nature is a treasure trove for materials with useful properties.Here, we will focus on FAs, whose unique scaffolds give them particularly advantageous material properties, as summarized in Figure 1.
One of the best examples is CsgA, the major component of Curli fibrils, which in turn constitutes a major part of the E. coli biofilm, providing both structural and chemical stability to the bacterial community. [16,17][19] The E. coli cell has evolved a dedicated channel (CsgG) chaperones (CsgC and CsgE) and a nucleator protein (CsgB).All these proteins are dedicated to the safe translocation of CsgA from the intracellular medium through the periplasm to the extracellular medium, where CsgA is then efficiently turned into amyloid fibrils, either de novo by binding to the nucleator protein CsgB or by attaching to growing ends of alreadyestablished fibrils. [17]mportantly, as a native E. coli protein, expression and purification of CsgA is a straightforward process.Recombinant production in the BL21-DE3 strain, using simple one-step chromatographic purification, provides moderate yields (≈4 mg L −1 of culture).Even better, the Joshi group has developed a scalable protocol that leads to high yields (>100 mg L −1 of culture) of naturally self-assembled curli fibrils, [20] based on filtration and washing without any chromatographic step.The ease of production likewise means that even a basic laboratory with the capability to handle GMO E. coli can produce amyloid fibrils for analysis and applications.
[23] The major component of Fap fibrils is FapC, which contains a highly amyloidogenic region; [24,25] the homologue FapB could be a nucleator protein but also a fibril component since the removal of FapA switches the composition of the fibrils from mainly FapC to mainly FapB. [21]The translocation of FapC is mediated by an interplay between various components, including a porin-based membrane channel (FapF) and the water-soluble proteins FapD, and FapE. [26]igh production yields and a tightly controlled aggregation process are clear advantages of FAs when compared to other amyloids.Protocols for high-throughput screenings of pathological amyloids such as -Syn report protein yields of ≈35 mg mL −1 , [27] which is ≈3 times lower than the protocol reported by the Joshi group.Moreover, the purification of these proteins is usually complex and requires several steps and treatments to isolate and/or solubilize the protein.Furthermore, fibrillation of -Syn and other pathological amyloids is often incomplete, requires prolonged shaking or addition of seeds, [28,29] and can lead to polymorphism, i.e., different types of fibrils depending on conditions. [30,31]Amyloid formed by other types of proteins generally require harsh conditions such as elevated temperatures and/or extreme pH. [32]In contrast, the aggregation of CsgA and FapC is fast, spontaneous, efficient, occurs under physiological conditions, and usually provides only one type of fibril.FAs simply constitute a more reliable scaffold for developing biomaterials, since the formation of the fibrils is controlled, and sample variation reduced.Admittedly, amyloid derived from food waste as -lactoglobulin (-lac) outperform FAs in terms of production and also contribute to a circular economy by profiting industrial wastes.Nevertheless, this generating process precludes the rational design of the fibrils for a particular application in contrast to FAs, which can be easily engineered without significantly altering their yield nor general properties.

Beyond Biofilms: Bioplastics, Hydrogels, and Redox-Controlled Assembly
In contrast to globular proteins, which require harsh denaturing conditions to induce amyloid formation, or pathological amyloid, which typically forms a spectrum of different amyloids (polymorphism), CsgA and FapC spontaneously form what appears to be a single type of fibrils under a broad spectrum of pH-values and salt concentrations.They can even aggregate in the presence of molar concentrations of the denaturants urea and guanidium hydrochloride. [19,33]The capacity of these fibrils to readily selfassemble, without seeding or templating, at ambient pressure, at physiological pH and salt concentration, without the need for heating or agitation, is a major advantage in the development of any biomaterial that has to be biocompatible in, for instance, drug delivery, tissue engineering, 3D cell culturing, and living materials (Figure 1).Additionally, since both the protein and the fibrils are produced under mild conditions, without the need for traditional chemistry, the entire production process is sustainable and environmentally friendly.
Once formed, CsgA amyloid fibrils are thermodynamically and exceptionally stable, able to withstand heat and chemical denaturants at concentrations that would result in the unfolding of nearly all globular proteins. [19]This elevated stability is the product of the extensive hydrogen bond network of the crossbeta structure as well as the zipper-like orientation of hydrophobic residues forming a tightly packed hydrophobic core.[36] These are examples of protein-based materials that are incredibly durable, strong, and versatile while being 100% biobased.Amyloid fibrils are, again due to their extensive intermolecular bonding, incredibly resilient toward chemical denaturation and enzymatic degradation.These properties make FAs excellent building blocks for more durable, plastic-like materials (Figure 1).[39] One specific study described the development of a curli-based plastic produced from dehydrated Curli hydrogels that could be molded, and had self-healing properties; its material properties, such as tensile strength, yield strength, and Young's modulus, were at the same level as traditional fossil-fuel-based plastics. [35]There are emerging reports for tuning both the properties and functionality of amyloid-based bioplastic materials by hybridization or mechanochemically functionalization.
Bioplastics are only one type of biomaterial where functional bacterial amyloids have been used as the main component (Figure 1).Hydrogels, which have become an important tool for tissue engineering, regenerative medicine, and drug delivery, are another (Figure 1).Current research aims to develop hydrogels able to mimic various environments.Synthetic polymerbased hydrogels such as poly(vinyl alcohol), poly(propylene fumarate-co-ethylene glycol), and poly(ethylene oxide), distinguish themselves by reliable production, well-defined chemistry, and controllable mechanical properties.][42][43] While less well-defined compared to synthetic hydrogels, naturally derived hydrogels have macromolecular properties that more closely resemble those of tissue extracellular matrix than polymer hydrogels do.Furthermore, successful cell growth and differentiation are often correlated with proper cell adhesion and signaling, which are much easier achieved with protein and peptide-based hydrogels. [44,45]n vivo, Curli fibrils are a major component of E. coli biofilm and thus inherently form a type of hydrogel.CsgA fibrils produced in vitro likewise readily form hydrogels above a certain concentration threshold.These biofilms and hydrogels have already been utilized in several studies to make functionalized gels with increased adhesion [46] and enzymatically active biofilms. [47] particularly interesting example is living hydrogels: when CsgA is displayed on the surface of E. coli in fusion with mucin-binding proteins, they help the bacteria stably colonize the GI tract of mice.[37] These studies have likewise highlighted the malleability of these hydrogels, which are able to retain structural integrity and revert back to their hydrogel state after being mixed with cells or other reinforcing materials.[38] In addition, they can be combined with cells for regenerative tissue engineering, and injected into brain tissue cavities as a space-filling agent that simultaneously acts as a cell anchorage and differentiation medium [48] (Figure 1).To date, we are not aware of any similar studies conducted using other functional bacterial amyloid but considering that the Fap system in many aspects is similar to Curli, it should be an obvious next step to investigate if similar hydrogels can be produced with FapC amyloid fibrils.The very straightforward production of CsgA amyloid hydrogels argues for the continued development of new materials based on these building blocks.
Another compelling argument for the use of functional bacterial amyloids as biomaterials is the ability to modify them, both chemically and translationally, without disrupting their amyloidogenicity. [15]Conveniently, CsgA can be modified by translationally attaching small proteins and peptides to the Cterminal end without disrupting the ability to form amyloid fibrils. [39]This capability has been utilized in several studies to produce CsgA fibrils functionalized with enzymes, [47] adhesion proteins, [46] and fluorescent markers. [49]The use of the SpyCatcher-SpyTag system [39,50] facilitates attachment of virtually any kind of protein to CsgA amyloid fibrils and can thus help to diversify any material or hydrogel that is produced with CsgA fibrils.The same modifications are likely possible to produce using FapC as a backbone instead of CsgA.
The previously mentioned modifications functionalize the otherwise inert CsgA amyloid fibrils.However, other modifications are underway to alter the actual fibrillation of CsgA.[53][54][55] Thus, incorporation/removal of gatekeeper residues in CsgA slows down/accelerates the fibrillation rate, respectively. [56]Likewise, the incorporation of a disulfide bridge into CsgA worked as a redox-controlled gatekeeper, since CsgA only fibrillated upon adding a reductant (here tris(2carboxyethyl)phosphine) to break the disulfide bridge. [57]Clearly, the ability to control the speed or onset of fibrillation is a useful property when designing amyloid-based biomaterials.

Slow Release of Cargo
As a general trend, amyloid fibrils, whether based on small peptides, globular proteins, or FAs, intrinsically form hydrogels at elevated concentrations, most likely due to their propensity to form large, entangled networks that have a large capacity to retain water through water molecule-sidechain hydrogen bonds.This feature has led to a variety of purely amyloidbased hydrogels, [8,40,41,44,[58][59][60][61][62] some of which are biodegradable and self-healing [63][64][65] and promote neuron differentiation and proliferation. [48]An example involving a globular protein are fibrils of lysozyme, which were used to generate capsules of different sizes.The size was tightly controlled by regulating the concentration of monomeric protein and preformed aggregates used for seeding the aggregation, and by modifying the relative flow rates of the aqueous and oil phases and the channel width of the microfluidic droplet maker.The resulting capsules gradually released encapsulated proteins over time.This approach demonstrated that the capsules presented a progressive release of monomeric protein, denoting that it might be applied to therapeutic protein delivery, but also to antimicrobial, small molecules with a higher impact than their nonencapsulated counterparts. [66]Fibrils made of bovine serum album fibrils also form amyloid-based hydrogels containing pesticides; these hydrogels adhere to plant leaves and permit a more effective and localized release. [67]Also, whey proteins were induced to form amyloid structures capable of encapsulating and progressively releasing small molecules (Figure 1). [68]Another release strategy combined -lac fibrils and polysaccharides to form coacervates that prevented or decreased stomach lesions through the release of the coacervate components. [69]These approaches can be extended by creative extensions to the core of amyloidogenic proteins.The soft (i.e., a small sequence) amyloid core of Sup35 has been implemented in multiple strategies to develop cell-specific therapies.First, Sup35 was fused to the dihydrofolate reductase, which encapsulates methotrexate, and the Zdomain-bound antibody that directs the complex to a specific cell type. [70]Similarly, Sup35 fused to the Z-domain-bound to different antibodies was developed to promote the interaction of cancer cells and T lymphocytes, enhancing the immune response to the tumor. [71] 4

.2. Hydrogels with Multiple Functionalities for Tissue Engineering
Additionally, several studies have shown that amyloid fibrils constitute a unique platform for tissue engineering and regeneration due to their cell-adhering capacities (Figure 1).][74][75][76][77][78] Remarkably, these strategies have provided a wide variety of tools that permit an optimal differentiation of the cells and their delivery into injured tissues.Interestingly, hydrogel characteristics and composition could be modified to adapt to the different tissues, including bones through a biomineralization process (i.e., adding hydroxyapatite to the hydrogel). [79]2D strategies have also been utilized to develop films that promote cell growth and differentiation.In particular, lysozyme amyloids were employed to form a film that increased the spreading and focal adhesion leading to adherence and differential growth of cultured epithelial and fibroblast cells. [78,80]4.3.Hydrogels for Bioremediation and Capturing CO 2 CO 2 fixation is essential for addressing climate change.In a pioneering study, the Eisenberg group demonstrated that amyloid fibers material containing alkylamine groups can reversibly bind CO 2 via carbamate formation, even in the presence of water and in a process more energy efficient than industrial methods.[81] Other examples have also emerged.Modification with aminosilane on amyloid fibrils that are derived from different food proteins enables the amyloid fibril-templated aerogels to capture CO 2 , including direct air capture.[82] Using MgO nanoparticles as solid cross-linkers, amyloid hydrogels consisting of -lac improved mechanical strength significantly compared to known amyloid hydrogels.[83] The hybrid hydrogel captures CO 2 and converts it into hydromagnesite, their excellent mechanical properties give a broader usage basin for practical applications.[83] 1. 4

.4. Filters
An alternative application is to exploit the robust material properties of amyloid as filter or membrane devices (Figure 1), currently largely involving globular proteins but also with potential for inclusion of FAs.A pioneering application combined -lac amyloid fibrils with activated carbon to form inexpensive and uniform hybrid membrane filters, a synergic combination that allowed the removal of toxic heavy metal ions and radioactive contaminants with high efficiency and with multiple cycles of use. [84]Conveniently, the amyloid fibrils facilitated recycling of metal ions into nontoxic functional states via thermal and chemical conversion. [84]-lac fibrils also bind and remove different arsenic ions [85] as well as pesticides, phenolic compounds, pharmaceuticals, dyes, and organic solvents in sustainable ways, [86] all of which are highly useful for purifying drinking water.Importantly, freeze-drying the cross-linked fibril gel [86] generated a water-stable aerogel that absorbed different organics found in the water solution.The aerogel could be recycled with no deleterious impact on the structure nor activity.-lac fibrils have also been functionalized in more elaborate ways.In one study, they were coated with nano-Pd and stabilized on a carbon paper or a titanium suboxide reactive electrochemical membrane, which conferred the capacity to remove toxic contaminants. [87]In another application, -lac fibrils were hybridized with zeolitic imidazolate framework-8 to constitute an air-in-water aerogel able to remove a large variety of heavy metals, synthetic dyes, and oily contaminants in solution. [88]Lysozyme fibrils have also proved useful, e.g. as a scaffold for generating porous membranes, functionalizing the surface of the fibrils with polydopamine.This dopamine-oxidized derivate is capable of binding ions, enabling polydopamine-coated fibrils to remove metal ions such as Pb(II) in the field of water purification. [89]These many examples of nanofibril-based biomaterial as water purification agents, masterfully summarized, [90] demonstrate their potential as affordable, efficient, and green solutions to deal with the global water crisis.

Biosensors
A fourth example is the exploitation of amyloid as unique scaffolds to develop new biosensors, particularly using fusion constructs (Figure 1).Fusion constructs of Sup35 with IgG or methylparathion hydrolase generate multifunctional and heterogenous fibrils that significantly enhanced the sensitivity of the IgG's target detection in an ELISA assay.As a proof of concept, the authors immobilized the F1 antigen on a microplate and compared the signal of commercial kits and their system. [91]The increased number of methyl-parathion hydrolase on the fibril surface compared to the soluble kit increased the sensitivity at the lowest concentrations.In another approach, the authors modified the constructs by fusing biotin instead of methyl parathion hydrolase.As a result, they generated a hybrid fibril that could be easily functionalized with different enzymatic capacities simply by incorporating streptavidin as a biotin binder. [92]These biotin-based strategies lend themselves well to generating new biosensors.As an example, biotinylated, Tyr-rich amyloid peptide fibrils bind gold nanoparticles and enzymes useful for biosensing approaches. [93]Other approaches include the modification of pathological -Syn [94] and functional CsgA fibrils [95] to perform colorimetric and fluorescent responses, respectively, to environmental changes in the pH or temperature among others. [94,95] 4

.6. Bioplastics
Plastics based on petrochemicals have revolutionized industry and our everyday life.However, poor recycling options have led to an enormous level of environmental plastic pollution, making it urgent to develop new and bio-based plastics as alternatives.A recent study produced sustainable and green flexible bioplastics films from amyloid fibril-biodegradable polymer blends, [34] where amyloids derived from whey proteins (mainly -lac) were dissolved in acidic water and fibrillated in presence of a plasticizer and a water-soluble polymer (polyvinyl alcohol (PVA) or methylcellulose).This led to a hybrid biofilm that was transparent, robust, flexible, and tough as well as water-resistant and thus able to form an effective barrier. [34]In another study, fibrillated hen egg white lysozyme was combined with PVA to generate bioplastic films, in which functionalization with the organic fluorophore perylene diimide strengthened the film as well as providing attractive optical properties such as emission of polarized light. [96]Another combination involves soybean protein isolate and the polysaccharide chitosan, which enhanced toughness, gas selectivity, and strength in packaging bioplastic. [97]Interestingly, further research using similar fibrils demonstrated that bacteriostatic agent can be added to amyloid fibers, resulting in a biofilm with good material properties to further extend their antibacterial and generate bioplastic for food packaging. [98]nother interesting approach for generating bioplastic is based on amyloids derived from rapeseed cake.The obtained rapeseed cake amyloids-based bioplastics rendered films that displayed improved properties when blended with polyvinyl alcohol and glycerol. [99]

Catalytic Amyloid: From Tethered Enzymes to Intrinsic Activity
The large and exposed surface of amyloid fibrils is part of a repetitive scaffold with enhanced and synergic activities.Such a large surface presents the option for binding and potentially transforming different molecules (Figure 1).FapC fibrils show a relatively unspecific but cumulatively important capacity to bind small molecules [100] which may even translate into a sponge-like ability to accumulate nutrients in vivo.This binding capacity is another attractive property for the development of new bio-inspired materials, which has been enriched by the recent emergence of fibrils with various catalytic properties, both extrinsic and intrinsic.In the extrinsic approach, a globular domain such as CspB or glucose oxidase is simply fused to the amyloid component to allow the display of active protein immobilized on the fibril surface. [101,102]or instance, the Ure2p soft amyloid core was fused to alkaline phosphatase and horseradish peroxidase, forming fibrils retaining the original amyloid core while displaying active and recyclable enzymatic surfaces. [103]However, it is also possible to generate amyloid fibrils with intrinsic catalytic activity.A pioneering work in this field developed short peptides with hydrolytic properties toward small soluble substrates. [104]hese peptides were built on a Leu-rich scaffold that promotes aggregation, while judiciously placed His residues coordinate with Zn 2+ to generate catalytic properties.Different derivates were developed to study structural and sequential dependent activity, leading to sequences and combinations that enhance hydrolytic capacity. [104]These peptides were employed in other studies to analyze the impact of fibril-stabilizing Tyr residues on catalytic activity.Besides its natural capacity to promote amyloid formation, Tyr residues are commonly found in the catalytic site of metalloenzymes, where they can coordinate different metal ions, providing a wide range of potential reactions.The obtained peptides were capable of forming amyloid fibrils that perform a Zn 2+ -dependent esterase activity. [105]More recently, another synthetic-based strategy implemented Tyr-rich synthetic peptides that formed fibrils capable of coordinating different metals such as Cu 2+ , Co 2+ , Ni 2+ , and Zn 2+ .The coordination of these metal ions within the fibril structure conferred an esterase activity as reported by standard substrates, which translates into a CO 2 fixation property through carbonic anhydrase activity. [106]ltogether, these studies demonstrated that the incorporation of residues such as Tyr or His into the amyloid fibril is easily implemented in the development of catalytic fibrils carrying out different reactions without modifying the overall aggregation

Peptide Catalysis
There have also been studies of catalytic fibrils formed by larger proteins; for example, lysozyme nanofibers have been coordinated with Au and Cu to form fibrils with peroxidase-like activity which turn out to have antibacterial properties. [107]Fibrils made by the peptide hormone glucagon also exhibit important hydrolytic activities, including lipid hydrolysis and ATP dephosphorylation. [108][111][112][113] An early study implemented the 11-residue A 25−35 fragment to generate different aggregate morphologies coordinating Au.This elucidated a conformation-dependent catalytic activity, suggesting that higher exposure of the particle surface enhanced enzymatic activity. [109]n a similar approach, A 17-21 (LVFFA) was covalently modified to generate a large variety of enzymatic activities, [110][111][112][113] while fibrils of A 16-22 (Ac-KLVFFAL-NH 2 ) showed aldolase-like activity.Interestingly, substitutions of Lys residues by Arg decreased enzymatic activity, while shorter Lys congeners such as ornithine increased retro-aldol cleavage capacity. [110]Similarly, imidazole-conjugated Im-KLVFFAL-NH 2 formed amyloid fibrils with hydrolytic activity in several substrates. [111]The loss of activity when Lys was substituted by Arg, Glu, and shorter variants, reinforced the relevance of fibril morphology in the catalytic activity.Moreover, acetylated variants confirmed the importance of imidazole in assisting in the hydrolysis reaction, which is mediated by covalent binding of the substrate to Lys. [111] Such bottom-up designed peptides often show significant substrate promiscuity; Im-KLVFFAL-NH 2 performs (at least) three different catalytic reactions. [112]Also, Ac-HLVFFAL-NH 2 fibril was reported to bind different molecules, including hemin cofactors.The resultant complex enhanced hydrolase-peroxidase cascade reactions compared to globular enzymes, such as cytochrome C. [114] 1. 5

.2. From Peptides to Amino Acids
Even shorter versions of the core of the A 42 peptide ( 18 VFFA 21 ) have also been used as scaffolds for obtaining potent catalytic fibrils.One of these applications generated two different peptides that were capable of forming distinctive quaternary organization of the amyloid fibrils including twisted bundles and nanosheets. [115]Both structures displayed an esterase-like activity but to a different extent, which suggests a morphology-dependent catalytic activity of amyloidbased materials. [115]Another interesting approach combined the 18 VFFA 21 segment with the catalytic triads of two known proteases or an artificial mimic one.Despite some autoproteolytic effects, fibrils obtained with the three peptides digested preformed aggregates of A 40 and prevented its derived membrane damage. [116]Remarkably, A 42 fibrils were recently reported to catalyze the hydrolysis of standard substrates, but also the degradation of different neurotransmitters through hydrolysis (acetylthiocholine) and oxidation (dopamine and adrenaline) processes. [117]emarkably, the VFFA peptide is far from the shortest peptide capable of forming catalytically active superstructures.Gazit and co-workers have demonstrated that a single amino acid (Phe) can self-assemble in close coordination with Zn 2+ ions to form nanorods with exceptional thermal stability and significant hydrolytic activity, e.g., hydration of CO 2 , just like carbonic anhydrase. [118]This is probably the ultimate example of the intrinsic potential of the amyloid fold, no matter how formed, for the generation and engineering of useful properties.

Catalysis by Pathological Amyloid
The A peptide is not the only disease-associated peptide with catalytic properties.Other examples include insulin (diabetes) and -Syn (Parkinson's Disease).Insulin fibrils coordinated with thioflavin-T presented photochemical properties that permitted a light-dependent NADH regeneration in the presence of a redox mediator.Remarkably, Th-T has been traditionally used as a highly specific amyloid dye, which constitutes a great advantage of this application as it will i) avoid nonspecific interactions, while ii) validating the presence of the fibrils.Similarly, constructs of insulin fibrils fused to L-glutamate dehydrogenase and coordinated with thioflavin-T exhibited a light-dependent enzymatic activity. [119]-Syn fibrils show esterase and phosphatase activity [120] and this activity seems to change the metabolite composition of cell lysates. [121]Altogether, these results highlight the potential catalytic properties of amyloid fibrils but also suggest a role in disease.

New Insights in the Structures of FAs will Facilitate Design of Amyloid-Based Biomaterials
Although the development of advanced biomaterials derived from FAs has succeeded in some cases, [39,122] for catalytical amyloid, the detected catalytic activity was not comparable to the "real" enzymes, usually orders of magnitude less efficient. [123]igure 2. Structures of FA with conserved -solenoid architecture.A) Cryo-EM structure of CsgA protofibrils from Pontibacter korlensis [16] with 15.5 imperfect repeats (R15.5) at 7.6 Å (PDB: 8C50), with monomers alternatively colored green and cyan.B) AF2 predicted monomer model of FapC Pseudomonas sp.UK4, colored by pLDDT (blue -Very high (pLDDT > 90), cyan -Confident (90 > pLDDT > 70), yellow -Low (70 > pLDDT > 50), red -Very low (pLDDT < 50)). [17,125]ere is no doubt that more profound structural insights will contribute to rational design principles in the field, not only to enhance amyloid's catalytical activity but also to develop other functionalities such as biomaterial.

FA Structures
Amyloid was long a structural blind spot due to their incompatibility with conventional structure resolution methods such as crystallography and solution-state NMR. 16]Fortunately, determination of amyloid fibrils structure at near-atomic resolution has become possible due to the recent resolution revolution in Cryo-EM and helical reconstruction. [124]Yet it remains a challenge to determine structures of FAs fibers.The Remaut group recently combined Cryo-EM and AlphaFold2 to arrive at a structure of an expanded CsgA from Pontibacter korlensis with 15.5 imperfect repeats (R15.5) at low resolution (Figure 2A). [16]t revealed a typical -solenoid fold and a conserved cross- amyloid kernel for FA, proposing different structural classes of bacterial curli subunits.The proposed AF2 model for the selected CsgA candidate is in excellent agreement with Cryo-EM map with a resolution at 7.6 Å.While the experimental structure of FapC has not been solved, progress has been made recently using the deep learning-based structure-modeling methods tr-Rosetta and AF2. [125]This led to a predicted novel Greek key solenoid structure with five -strands per turn for Pseudomonas FapC (Figure 2B), not previously observed in FAs but also predicted by us using AlphaFold. [17]This structure is unique to Pseudomonas species and contradicts a previous proposal based on coevolutionary constraints that FapC has a CsgA-like structure. [26]t will be very exciting to get an experimental resolution on this quandary, all the more interesting given that FapC in different Pseudomonas species is found to have not just a variable number of repeats but also linkers of variable lengths between repeats. [126,127]Whether these linkers are dynamic regions extending from the amyloid surface or embedded into the amyloid structure (leading to different amyloid folds between otherwise closely related Pseudomonas species) remains to be determined.Obviously, this will impact the amyloids' range of catalytic and biomaterial properties.

Conserved 𝛽-Solenoid Fold of FA over Polymorphism of Pathological Amyloids
More and more amyloid structures have been determined in the last decade.Amyloid fibers associated with disease exhibit the property of polymorphism, the structures differ for the same amino acid sequence prepared at different conditions or even at the same condition, both within and between protofibrils. [128,129]f course, any mutation of the primary sequence can easily result in a different final ultrastructure of the amyloid fiber. [16]his sensitivity will cause trouble for the design of amyloid-based biomaterial, any external perturbations may lead to unintended consequences during the amyloidogenesis process.This stands in sharp contrast to FAs, AF2 predicts a conserved canonical solenoid structure for both CsgA and FapC, and changes in primary sequence have little effect on this conserved structure. [16]here is also no experimental evidence of fiber polymorphism in more than two decades of research on curli. [16]Both ex vivo isolated and in vitro formed curli have an identical -solenoid structure of the protofibrils, which strongly suggests that the CsgA sequence has an intrinsic property to form the -solenoid structure. [16]This solid monomeric building blocks of a conserved -solenoid fold makes FAs an excellent scaffold for biomaterial design.

Importance of Structural Understanding of the Cross-Seeding Mechanism
The conserved curli subunit structure could lead to the possible formation of mixed fiber and thus allow the formation of multispecies biofilm matrix, [16] encouraging the design of multispecies biomaterials.Nevertheless, we should also sound a note of caution in the use of FA as biomaterial for disease treatment due to the cross-seeding effect, for example, there is a significant increase in the deposition of alpha-synuclein amyloid in both the brain and gut after exposure of rats to bacterial amyloid Curli of E. coli. [130,131]It was observed that the fibrillar diameter of amyloid- increased after the seeding of FapC fiber fragment in vitro. [130]Experimental structure rather than the AlphaFold predicted structure should be obtained to understand the fact that FA FapC seeding can change the fibril structure of pathological amyloids. [125]Structural understanding of the cross-seeding mechanism is important to avoid the side-effect of biomaterial design.
The structures of CsgA and FapC provide the basis of the extreme physico-chemical stability of FA and should contribute to further engineering design to expand the usage of FA-based biomaterials.As experimental structure information of bacterial amyloid is still limited, further structure studies especially Cryo-EM structure may shed light on the structure-based design of FAbased biomaterials.

FAs as New Biomaterials: What is Next?
Amyloid fibrils are an outstanding scaffold for the production of highly stable and resistant biomaterials that retain their functional applications under extreme conditions.Nonetheless, the application of pathological and synthetic amyloids must consider important concerns: i) first and foremost, the formation of toxic and transmissible species not only during the aggregation cess but also because of fibril fragmentation; ii) their tendency to form different types of amyloids depending on fibrillation conditions (polymorphism), which complicates the identification and production of the active conformation; iii) low and complex production and purification processes for an industrial scale.Here, FAs present an attractive alternative.These amyloid proteins are devoid of toxicity and transmissible capacity, and their aggregation process is extremely controlled leading to a particular conformation and properties.Furthermore, protocols that enhance their production have been described. [20]][134] We envision that the implementation of the current knowledge of new amyloid-based biomaterials based on FAs will lead to the development of innovative and improved applications in multiple fields.
FAs like FapC and CsgA display an extraordinary natural ability to form amyloid fibrils in a broad range of buffers, pH values, and salt concentrations.These fibrils readily produce hydrogels that inherently bind cells and promote differentiation, making them ideal material for tissue engineering or biosensors, and can be further manipulated to produce bioplastics.The fibril-producing cells can even be incorporated into the hydrogel and bioplastic to produce a living material that can continuously repair and maintain the material.CsgA can easily be modified with practically any protein, enzyme, or peptide tag thanks to the SpyCatcher-SpyTag system to, e.g., produce underwater amyloid adhesives fusing CsgA to mussel foot proteins, [133,135] or antimicrobial amyloid mats by attaching antimicrobial peptides. [107]All these characteristics highlight how CsgA-and by inference FapC and other bacterial FA still to be discovered [136] -could become a fundamental building block in multiple areas of materials science (Figure 1).
FAs, above all CsgA, have already been described as potential biomaterial for different fields.For instance, one of the very first applications of FAs as biomaterial focused on the generation of a living material. [137]In this strategy, cell-produced curli fibrils were employed to nucleate gold nanoparticles binding to generate nanowires.Additionally, the coexpression of different CsgA constructs facilitates the formation of patterned fibrils, opening the possibility to develop multifunctional living materials. [138]Moreover, CsgA could be combined with other proteins, such as the influenza-virus-binding peptide (C5). [139]his genetic fusion allowed to generate a functionalized fibril in living materials that capture virus particles, in a clear demonstration of disinfection potential. [139]Other fusions with CsgA consisted of the addition of a hybrid scaffolding that permitted the binding of different enzymes. [140]As a result, a multimodal E. coli surface could be generated thus prompting the development of living material with diverse application according to the grafted protein. [140]The capacity of curli fibrils to bind small particles has also been exploited to create materials with enhanced conductivity and potential applications such as biointerfaces and bioelectronics. [141]Furthermore, different environmental applications for curli fibrils have been explored.A pioneer work exploited the binding capacity of curli fibrils and generated a mercury-responsive bacteria that detects the presence of mercury and produces the extracellular self-assembling proteins that capture the contaminant. [142]These applications are not restricted to curli-based biofilms.Biofilms obtained from different bacterial strains have been used to remove a mutagenic dye from solution. [143]These living materials could be also exploited to generate hydrogels with biomedical applications.For instance, CsgA fibrils have been recently engineered to incorporate a hydroxyapatite-binding peptide, [79] leading to a hydrogel able to bind hydroxyapatite and, thus, undergo a mineralization process with obvious biomedical applications. [79]esides these applications, we strongly believe that amyloid fibrils and their engineering have a very high potential for revolutionizing the catalytic field.So far, a relatively limited variety of chemical reactions have been reproduced on the amyloid fibrils (phosphatase, oxidase, or peroxidase among others).However, a more comprehensive survey of activity toward cell lysates might uncover many different activities as demonstrated for syn. [121]Therefore, characterizing new intrinsic enzymatic activities or rational design of the sequence of FAs, i.e., incorporating known catalytic domains or triads, might potentially lead to new applications.FAs are particularly well-suited here.CsgA structures are a simple and highly conserved -solenoid with two welldefined sides which facilitate a higher surface exposure of residue sidechains than that observed in the catalytic moiety of globular enzymes and thus optimize substrate interactions.The two opposed sides could be employed to graft multiple activities or functions in the same sequence.FapC may be less easy but ultimately more rewarding to exploit.Its structure is predicted to be a Greek key motif. [17,144]As a result, many residues are hidden in the structure, but at the same time, multiple faces could be generated and thus exploited to graft diverse activities.Furthermore, the characteristic Greek key fold could be also used to generate catalytic cavities that resemble those of globular enzymes.Besides engineering different catalytic activities on the same sequence, the combination of different designed sequences may also lead to multifunctional fibrils with synergistic activity, i.e., combining all activities required for a cascade reaction, provided the positions and ratios of these sequences are carefully optimized.Although CsgA and FapC are highly conserved, they vary significantly in the number and position of the repeats between species. [16,145]his variability can increase the potential use of FAs as catalytic moieties, as the alternative sequential and structural arrangements could determine or promote different activities.Hence, developing new catalytic amyloids constitutes a unique opportunity to enhance the industrial production of multiple materials and to adapt greener manufacturing processes.As climate emergency and pollution have emerged as some of the major challenges for humanity, we strongly believe that FAs with catalytic properties can be designed for bioremediation strategies, i.e., as filters for the fixation of CO 2 and other greenhouse gas emissions, as rapidly generated mats that break down toxic waste such as fossil fuels leakages [106] or perhaps even hydrolyze plastic nanoparticles.Although the proposals here stated are speculative and unlikely to be achieved in the immediate future, the potential of FAs is still enormous.We hope that the cumulative knowledge and the use of new computational tools for protein design such as AlphaFold2, will converge to convert these visions into reality.
An important aspect to address when discussing the use of any amyloid fibril is the risk of toxicity.Amyloids have historically been associated with diseases, such as neurodegenerative disorders or more broadly amyloidosis, and there is ample evidence that protein misfolding can propagate given suitable circumstances.It is therefore important that any potential material that is developed for human contact or human consumption is thoroughly tested for any risk of causing amyloid-related complications.
In conclusion, amyloid fibrils, in particular FAs, have the potential to turn into key elements for the development of new materials.While they can be employed for a large variety of applications, we envision that amyloids would definitely stand out as state-of-the-art biosensors, filters, and catalytic scaffolds.Their unique stability and their repetitive (and exposed) surfaces likely constitute crucial factors contributing to synergic and enhanced activities by multiplying the number of binding and/or catalytic sites and outperforming less stable globular proteins in demanding conditions.FAs clearly have the potential to become a major class of biomaterials in the near future.

Figure 1 .
Figure1.Schematic summary of different applications for amyloid fibrils.Green color represents applications that have been tested in FAs as CsgA, while blue and brown correspond to other amyloids (i.e., pathological) tested for biomaterial or biomedical applications, respectively.