Bacterial Species in Engineered Living Materials: Strategies and Future Directions

ABSTRACT

In recent years, there has been a notable increase interest in engineered living materials (ELMs) owing to their considerable potential. One key area of research within this field is the utilisation of various species of bacteria to create innovative living materials. In order to accelerate the advancement of bacterial‐based living materials, a systematic summary of bacterial species and their design strategies is essential. Yet, up to this point, no applicable reviews have been documented. This review offers a concise introduction to living materials derived from bacteria, delves into the strategies and applications of each bacterial species in this realm, and provides perspectives and future outlooks in this field.

Bacterial ELMs can be derived from two sources: natural biofilms and hybrid living materials. In natural biofilms, the production of ELMs involves the genetic engineering of bacteria to manipulate the biofilm matrix components for desired material properties. On the other hand, hybrid living materials refer to the process of encapsulating engineered bacterial cells with distinct matrix polymers.

1. Introduction

The development of engineered living materials (ELMs) is driven by the need for new materials capable of performing complex functions and adapting to dynamic conditions. ELMs are typically composed of matrices that provide structural support and a nurturing environment for living cells to grow and function. These advanced materials are engineered to interact with their surroundings, sense and respond to various stimuli, and execute specific tasks. One of the earliest documented examples of such living functional materials was developed in 2012 by Wendy J. Stark's group, who encapsulated Penicillium chrysogenum cells within an agar matrix to achieve sustained release of penicillin (Gerber et al. 2012). This work demonstrated the potential of integrating living organisms into materials for controlled substance delivery. Building on this foundation, Timothy K. Lu's team in 2014 made significant progress by applying synthetic biology techniques to engineer E. coli biofilms (Chen et al. 2014). Their research resulted in living materials that were not only responsive and tunable but also exhibited multiscale patterning capabilities. These advancements highlight critical milestone in ELMs' evolution, emphasising the role of genetic modification in enabling cells to perform specialised functions. The ubiquitous presence of bacteria in diverse natural environments underscores the suitability of bacterial biofilms as a readily available and renewable resource for ELMs creation and advancement. Biofilms are intricate communities of bacteria that adhere to surfaces and secrete an extracellular matrix composed of polysaccharides, proteins, and DNA (Eick 2021; Tursi and Tükel 2018). The building of the “house” by bacteria itself can protect bacteria from adverse environmental factors and immune responses. Furthermore, biofilms contain water channels, which help the distribution of nutrition and molecules (Wilking et al. 2013). Three‐dimensional (3D) structures of biofilms in nature can become an Eden for bacteria in a bad environment, but they can be used as a platform for ELM design. Biofilm engineering for new living material refers to the harnessing of the biofilm formation process of bacteria. For instance, species of Comamonas are characterised as gram‐negative and aerobic bacteria that are ubiquitous in the environment. Through the continuous expression of the YedQ protein, a biofilm of Comamonas testosteroni has been genetically engineered, and the engineered biofilm has a higher degradation efficiency of 3‐chloroaniline than that of the wild type (Wu et al. 2015). Curli fibre is a major component in E. coli biofilms, and decorating the curli fibre with an α‐amylase allows the generated biofilm to maintain the activity of amylase to the substrate 4‐Nitrophenyl‐α‐D‐maltopentaoside under denaturing environmental conditions (Botyanszki et al. 2015). The genus of Komagataeibacter forms dense bacterial cellulose (BC) sheets when cultured in static condition (Azeredo et al. 2019). When co‐cultured with Saccharomyces cerevisiae yeast, the modified yeast strains release cellulase into the BC of Komagataeibacter rhaeticus, which, in turn, reduces the stiffness of the BC materials (Gilbert et al. 2021).

Over the last decade, ELMs have infiltrated numerous aspects of our daily existence, showcasing their versatility (Rivera‐Tarazona et al. 2021). Within the ELM domain, classifications include archaeal, eukaryotic, bacterial, synthetic, and cross‐domain ELMs (Lantada et al. 2022). Although substantial research has focused on archaeal, eukaryotic, synthetic, and cross‐domain ELMs (Lantada et al. 2022; Huber et al. 2022), this review narrows its scope to exclusively cover bacterial ELMs, an area that has garnered significant interest. Bacterial ELMs can be categorised into two main types: those derived from natural biofilms and those from hybrid living materials. Hybrid living materials refer to a class of ELMs formed by encapsulating cells within an inorganic or organic matrix, thereby combining biological components with synthetic elements to create innovative functional materials (An et al. 2023). Over the past decade, significant advancements have been made in developing strategies for manipulating bacterial biofilms to fabricate biofilm‐based living materials. These approaches leverage the inherent structure and functional properties of biofilms to create materials that can perform specific tasks. Meanwhile, parallel progress has been observed in the engineering of hybrid living materials, where researchers have focused on integrating live cells with non‐biological matrices to produce composites with tailored functionalities. Despite these advances, the field of biofilm and hybrid material engineering is still in its infancy, presenting ample opportunities for growth and innovation. In this review, we first discuss the bacterial species used in natural biofilm‐based living materials and bacterial species used in hybrid living materials and then provide perspectives on the bacterial species used in ELMs.

2. Bacterial Species Used in Natural Biofilm‐Based Living Materials

To date, many different living materials have been generated based on the natural biofilm formation of E. coli , Bacillus subtilis , Komagataeibacter rhaeticus, Geobacter sulfurreducens , Corynebacterium glutamicum , and Caulobacter crescentus. Figure 1 illustrates the scheme of creating diverse ELMs through natural biofilm engineering.

FIGURE 1.

Bacterial ELMs derived from natural biofilms. (A) Schematic illustration of the strategies for creating diverse types of ELMs by leveraging natural bacterial biofilms. These ELMs are developed through: The secretion and assembly of csgA‐F and TasA‐F fusion amyloid proteins by E. coli and B. subtilis respectively, where “F” represents various functional protein domains or peptides; the secretion and assembly of cellulose by K. rhaeticus; the assembly of Spa2 fusion proteins within pili structure of C. glutamicum ; the expression OmcS protein on the outer membrane of G. sulfurreducens ; and the surface display of engineered RsaA proteins on C. crescentus cell surface. (B) Representative examples of engineered biofilms: (i) B. subtilis biofilm with MHETase activity. Reprinted with permission from Ref (Huang et al. 2019). Copyright2018 The Author(s), under exclusive licence to Springer Nature America Inc. (ii) E. coli biofilms designed to disinfect virus from river water. Reprinted with permission from Ref (Pu et al. 2020). The Authors 2020. This article is published with open access at Wiley‐VCH Verlag. (iii) E. coli biofilms acting as biosensor. Reprinted with permission from Ref (Moser et al. 2019). Copyright2019 WILEY‐VCH Verlag GmbH&Co. KGaA, Weinheim.

2.1. E. coli

E. coli serves as a model organism and is widely present in laboratories around the world. Its resilience, versatility, broad metabolic capabilities, and ease of cultivation have made E. coli an extensively studied and well‐understood bacterium. Additionally, the E. coli genome is relatively simple, allowing scientists to easily perform genetic modifications. While certain strains of E. coli can be pathogenic, the strains commonly used in laboratories are typically harmless and are considered generally regarded as safe (GRAS) microorganisms. These characteristics have made E. coli the most extensively utilised organism for the construction and study of ELMs. E. coli can form biofilm virtually everywhere, and the biofilm matrix components include proteins, cellulose, and extracellular DNA (Tursi and Tükel 2018). Compared to cellulose and extracellular DNA, proteins are highly programmable. Amyloids, a type of protein polymer, are crucial for the structural integrity of biofilms. They were originally recognised as virulence factors in E. coli (Olsén et al. 1989). In Enterobacteria, csgBAC and csgDEFG operons are responsible for the synthesis of curli amyloid fibres (Bhoite et al. 2019). csgD functions as a transcriptional regulator that promotes the transcription of the csgBAC operon. The proteins csgE, csgF, and csgG collectively form the secretion system responsible for transporting both csgB and csgA proteins to the bacterial outer membrane. The csgA protein, which serves as the building block for amyloid fibres, is exported by the secretion apparatus in an unfolded state. When csgA is secreted into the extracellular space, it undergoes self‐assembly to form nanofibers, a process assisted by the csgB protein (Hammer et al. 2012).

Based on these findings, subsequent research in the field of ELMs gradually revolved around this system. To harness the unique characteristics of the curli system in E. coli, various strains have been selectively engineered or chosen for distinct applications. In 2014, researchers explored the development of strong underwater adhesion materials by utilising E. coli NEB C3016, a modified version of the E. coli BL21 strain. The study aimed to investigate how the combination of two different adhesion systems within this bacterial strain could lead to the creation of powerful adhesives designed to work effectively in water. Since the underwater attachment function of mussel foot proteins (Mfps) in Mytilus galloprovincialis depends on the presence of abundant 3, 4‐dihydroxyphenylalanine, these researchers fused the Mfps domains to the csgA protein at the C‐terminal; after expression and purification, the researchers reported that the fusion proteins exhibit strong and multifunctional underwater adhesive properties (Zhong et al. 2014). Researchers using E. coli DE3 (BL21) have found that adding fusion domains does not interfere with the standard β‐sheet structures, but they have shown that it does impact the rate of assembly, appearance, and rigidity of the resulting fibrils (Cui et al. 2019). Therefore, the robustness of this system needs more consideration before conducting additional experiments.

In another study, researchers employed a genetically modified bacterial strain, E. coli PHL628‐∆csgA, derived from the E. coli K‐12 MG1655. The E. coli PHL628 possesses an ompR234 mutation where the original ompR regulatory protein undergoes a single amino acid substitution; specifically, leucine (L) at position 43 is replaced by an arginine (R) residue. This particular alteration results in the overexpression of curli (Prigent‐Combaret et al. 2000; Vidal et al. 1998). In order to further engineer this E. coli strain, the native csgA gene was deleted from E. coli PHL628, resulting in E. coli PHL628‐∆csgA. This deletion allows for adverse peptides to be fused to csgA by constructing the corresponding plasmids in vitro. After plasmid transformation, transformants with new functions can be generated. Studies have shown that a spontaneous covalent bond forms between SpyTag and SpyCatcher (Zakeri et al. 2012; Reddington and Howarth 2015). When SpyTag is combined with csgA through fusion, it is subsequently expressed and assembled by E. coli PHL628‐∆csgA, and the resulting fibres exhibit close resemblance to the native curli fibres. Upon further fusing SpyCatcher to an amylase, the immobilisation reaction was found to be robust. Additionally, the amylase‐modified biofilms maintained their functionality even when subjected to diverse pH levels. This system is notable as it introduces a new, versatile approach for immobilising enzymes at specific sites (Botyanszki et al. 2015). A further extension of this study has explored the ability of the biofilm‐based material to immobilise more enzymes by using the E. coli PHL628‐∆csgA. To accomplish this objective, the researchers designed and tested a series of conjugation pairs. In each instance, one part of the conjugation pair was connected to the C‐terminal region of csgA, whereas the other part was attached to the N‐terminal region of enzymes. They reported that the resulting material exhibited outstanding properties, including the targeted purification and immediate immobilisation of multiple enzymes straight from unrefined cell extracts (Nussbaumer et al. 2017).

In 2020, E. coli MG1655PRO∆csgAompR234 was utilised where the gene csgA had been knocked out, featuring constitutive substantial expression of both tetR and lacI proteins from a PRO cassette, as well as harbouring an ompR 234 mutation. This E. coli strain was employed to purify virus‐contaminated river water (Pu et al. 2020). Given that C5 is a well‐characterised peptide with known affinity for influenza viruses, it has been demonstrated to specifically interact with the haemagglutinin membrane protein of the influenza virus (Matsubara et al. 2010). In this research, C5 was concatenated with the csgA protein, forming a fusion protein. The resulting materials effectively facilitated the direct extraction of virus particles from liquid mediums. Of note, the disinfection efficiency achieved levels undetectable by the qPCR assay's sensitivity threshold. Due to concern that the endogenous csgB gene might have an impact on the expression of the fusion proteins, a further deletion of the csgB gene in E. coli MG1655PRO∆csgAompR234 results in the generation of E. coli MG1655PRO∆csgBAompR234, which is utilised for the production of conductive protein nanofibers. Because aromatic amino acid contents contribute to the electrical conduction of the PilA protein of Geobacter sulfurreducens (Reardon and Mueller 2013; Tan et al. 2016). By mimicking the motif present in the PilA protein of Geobacter sulfurreducens , a group of researchers has effectively incorporated conductive peptide motifs, consisting of different tyrosine and tryptophan residue combinations, onto the C‐terminal region in csgA. This novel coupling has empowered the once non‐conductive E. coli biofilm to gain conductivity (Kalyoncu et al. 2017).

Although the E. coli strains mentioned above have been used in several studies, there may be other elements of the two operons responsible for curli formation in the E. coli genome that may have some unknown effects on curli production. To address this issue, researchers have utilised an E. coli strain where all operons are deleted in E. coli PQN4 to conduct additional studies. New research has uncovered a significant increase in mercury pollution in the environment compared to previous estimates (agles‐Smith et al. 2016). This highlights the necessity for novel methods to clean up contaminated areas. The mer operon is a commonly observed and long‐standing bacterial operon present in plasmids and transposons, where the MerR protein controls the expression of the mer operon (Summers 1992). Once Hg²⁺ binds to MerR, a structural transformation occurs, causing the liberation of the mer operon. Recent research has also shed light on the potential protective role of curli in bacterial defence, suggesting that it may effectively safeguard bacteria within biofilms by adsorbing heavy metal (Hidalgo et al. 2010). Substituting the mer operon genes with a yellow fluorescent protein gene and curli operons, mercury‐inducible biosensors that can report and sequester mercury have been developed (Tay et al. 2017). In addition to the design for reporting and binding of mercury, this E. coli strain can also be used for abstracting various rare earth elements (REEs). This is particularly significant due to the growing need for REEs in recent decades. Lanthanide‐binding tags (LBTs) are oligopeptides that exhibit strong binding for many lanthanides (Nitz et al. 2004). LBTs have been fused to the csgA protein, resulting in the construction of a fibre with selective REEs binding activity that shows a preference for lanthanides; the recovery procedure for this fibre is simple, requiring only the use of a dilute acid wash (Tay et al. 2018). Additionally, the application of E. coli PQN4 has been broadened into the field of bioelectronics. By strategically modifying multiple amino acid residues in the core region of the csgA protein, engineered curli fibres have been developed that exhibit significantly enhanced electrical conductivity in comparison to their wild‐type counterparts. These modified curli fibres thus possess the capability for electron conduction, thereby expanding their potential applications in this cutting‐edge field (Dorval Courchesne et al. 2018). Leveraging microbial engineering to fabricate materials with adverse functions has been established by these strains. Building 3D structures in any pattern is a bottleneck in this field. A team has recently created a customisable biological substance for 3D printing of living materials. In pursuit of this objective, the researchers utilised the crosslinking of a fibrin‐inspired fusion proteins to create a bioink. The process of fibrin polymerisation is partly facilitated by noncovalent interactions occurring between an alpha‐chain domain located at the N‐terminal of one fibrin monomer and a gamma‐chain domain situated at the C‐terminal of an adjacent monomer (Pratt et al. 1997). By integrating these two domains separately at the N‐terminus and C‐terminus of csgA, csgA‐α and csgA‐β fusion proteins are generated. Introducing specially engineered E. coli PQN4 to the ink enables the fabrication of 3D‐printed living materials with specific functions, such as releasing the anticancer drug azurin and regulating their own cell growth (Duraj‐Thatte et al. 2021).

E. coli JF1∆csg is a distinct strain that has been specifically engineered for controlled expression and secretion of the csgA fusion proteins, with meticulous attention to avoiding interference from its native curli production system. In order to accomplish this, the researchers have methodically knocked out all the endogenous curli‐associated genes within the E. coli JF1∆csg genome (Moser et al. 2019). In this particular strain of E. coli , a light regulation system is employed to carefully manage the activation of the csgBAC operon via blue light. Furthermore, green light and red light are utilised to modulate the expression of csgA fusion proteins labelled with HA and His tags, respectively. The operon, encompassing the genes that govern the secretion apparatus of curli (csgE, csgF, csgG), is subjected to precise regulation by the IPTG inducer. The csgD was excluded because it encodes a regulatory protein. Thus, the E. coli JF1∆csg, equipped with this advanced light control system, can be effectively utilised for the precise patterning and immobilisation onto various surfaces such as textiles, ceramics, and plastics, demonstrating the adaptability of this method in the fields of materials science and bioengineering. To explore additional functions, this strain has also been combined with other E. coli strains to form two strain systems. By combining with the E. coli MG1655PRO∆csgAompR234, a living glue system with autonomous mechanical repair properties, has been developed (An et al. 2020). In this dual‐strain living glue system, E. coli MG1655PRO ∆csgAOmpR234 was used to produce csgA‐Mfp3s fusion proteins and constitutively produced acyl‐homo‐serine lactone (AHL), and the E. coli JF1∆csg was used to produce tyrosinase, which can enhance the adhesion ability of bacteria biofilm. Upon the addition of horse blood to induce the system, the E. coli MG1655PRO ∆csgAOmpR234 strain is triggered to produce csgA‐Mfp3s fusion proteins and continuously generates AHL. In response to the AHL signal, E. coli JF1∆csg strain is activated to synthesise tyrosinase. Through the cooperation of these two strains, the small holes in the microfluidic device can be repaired when horse blood is pumped into the system. Although both cellulose and extracellular DNA constitute crucial elements of the biofilm matrix in E. coli , there have been no reports of living materials based on these two components to date. It is noteworthy that cell‐surface lipopolysaccharides have been effectively restructured using metabolic pathway engineering. The inherent GDP‐fucose de novo pathway in E. coli has been substituted with a GDP‐fucose salvage pathway originating from Bacteroides fragilis . Many different fucose analogs can be added into the polysaccharides to decorate the cell surface (Yi et al. 2009). This strategy will be helpful to build living materials based on polysaccharides with more functions in the future.

2.2. B. subtilis

Another popular model organism, B. subtilis , has been extensively harnessed for enzyme, chemical, and antimicrobial material production (Su et al. 2021; Su et al. 2020). As a model organism, numerous genetic manipulation techniques have emerged over the past decades (Liu et al. 2019). Moreover, under very harsh conditions, it forms spores (Errington 2003), which, along with its ability to develop biofilms on diverse surfaces (Arnaouteli et al. 2021; Arkatkar et al. 2010), have inspired the development of B. subtilis ‐based ELMs. The biofilm matrix of B. subtilis has been reported to include DNA, polysaccharides, and proteins. TasA is an amyloid protein distinct from curli types and is one of the main components of the B. subtilis biofilm matrix. Studies have shown that it is essential for maintaining the structural integrity of the biofilms (Romero et al. 2010). The tapA‐sipW‐tasA operon directs the synthesis of TasA (Winkelman et al. 2013). With an understanding of the role of the TasA protein in B. subtilis biofilms and how it is synthesised by the tapA‐sipW‐tasA operon, researchers have begun exploring the fusion of TasA with other functional proteins through genetic engineering to create living materials with new properties. By fusing TasA with mCherry protein and MHETase, the resulting fusion proteins produced by B. subtilis 2569∆tasA∆sinR∆eps can equip living materials with additional capabilities, such as red fluorescence and MHETase enzymatic activity (Huang et al. 2019). In another study, a strain B. subtilis 1935∆eps∆bslA was used to fabricate living materials for bioremediation. By fusing the TasA protein with metallothionein, the engineered biofilm was shown to have the ability to remove Pb²⁺, Hg²⁺, and Cu²⁺ ions from water (Zhu et al. 2024). While progress has been made in harnessing the TasA protein from B. subtilis for the construction of living functional materials, a literature review indicates that these are among the few instances reporting the utilisation of the TasA protein specifically for the production of such materials. However, there is an emerging perspective suggesting that exploring non‐amyloid proteins for the constitution of the extracellular matrix in B. subtilis could present a strategic option for the design of next‐generation ELMs. Utilising non‐amyloid proteins might unlock new possibilities by virtue of their distinct properties and functions, which could enhance the versatility, robustness, and tunability of these materials. The EutM protein is a type of a bacterial microcompartment shell protein from Salmonella enterica (Schmidt‐Dannert et al. 2018). This protein offers great flexibility for engineering, readily accepting N‐ and C‐terminal fusions without loss of function (Zhang et al. 2018; Zhang et al. 2019). A recent study has genetically modified B. subtilis to display SpyTags on flagella for the purposes of cellular attachment and linkage with EutM‐SpyCatcher scaffold building blocks to form the protein matrix of ELMs (Kang et al. 2021). Because CotB1p is a silica biomineralisation peptide, after the expression of EutM‐SpyCatcher, EutM‐CotB1p fusion proteins, and the display of SpyTags on flagella, and upon incubation with silica, a biocomposite of ELMs with improved mechanical properties was developed.

2.3. K. rhaeticus

K. rhaeticus is also regarded as a model organism for studying BC production. Cellulose, a major biopolymer produced by plants, is an important component of the biofilm matrix in many bacterial species (Lamas et al. 2016; Da Re and Ghigo 2006; Matthysse et al. 2005). The main distinction between BC and plant cellulose is that BC is an ultrapure nanocellulose that does not contain polymers like pectin and lignin. Moreover, BC has strong, flexible, and hydrophilic characteristics (Iqbal et al. 2014), allowing many potential applications for BC (Tang et al. 2022). Bacteria belonging to the Acetobacteraceae family, such as Acetobacter and Komagataeibacter, are famous for their cellulose production (Gupte et al. 2021; Avcioglu 2022). Biofilm formation, a common trait among many species in this family, is predominantly comprised of cellulose as the primary biofilm matrix. (Jacek et al. 2019; Subbiahdoss et al. 2022; Fontana et al. 1990) K. rhaeticus belongs to the genus Komagataeibacter and is generally considered a GRAS organism. The cellulose produced by this bacterium can self‐organise into ordered structures without the need for an external template, which is particularly useful for constructing materials with complex shapes and functions. Moreover, this bacterium can grow under static culture conditions, eliminating the need for complex agitation equipment. These characteristics have motivated researchers to explore the development of ELMs based on K. rhaeticus. Currently, living functional materials are created by harnessing the extracellular matrix component cellulose produced by K. rhaeticus iGEM. Under static conditions, K. rhaeticus iGEM cultured flat pellicles, consisting of a substantial amount of thick BC. Researchers have adapted an AHL‐based cell‐to‐cell communication system, originally designed for E. coli , to enable the production of sender and receiver pellicles (Walker et al. 2019). The sender pellicles produce the AHL synthase LuxI, while the receiver pellicles function as a receiver and express the transcriptional activator LuxR. Upon binding with AHL produced by sender pellicles, LuxR gets activated and subsequently boosts the activity of the Plux promoter. This activation event leads to the upregulation of a red fluorescent protein in the receiver pellicles. When cultured under shaking conditions, K. rhaeticus iGEM can form millimetre‐scale rounded BC spheroids. These BC spheroids equipped with the AHL‐based cell‐to‐cell communication system produce sender spheroids and receiver spheroids. Sender spheroids and receiver spheroids can function as interactive entities capable of interacting and responding to each other. Furthermore, these spheroids can also be used to regenerate damaged BC materials, further expanding the scope of this innovative technology in the living materials field (Caro‐Astorga et al. 2021). Alteration of BC properties will increase the functionality of BC‐based ELMs. The modification of BC characteristics has been achieved by the introduction of another polymer at the genetic level. For instance, the crdS gene is a curdlan synthase gene, and the introduction of the gene crdS into G. xylinus AY201 leads to the intracellular polymerisation of uridine diphosphate glucose, allowing the secretion of cellulose along with curdlan (Fang et al. 2015). Comparison of the resulting polysaccharides to normal BC pellicles has indicated that the nanofiber structure in the pellicle remains intact, and the water permeability of the bio‐nanohybrids is minimal. Another method is the addition of N‐acetyl‐glucosamine to cellulose fibres, which not only renders BC vulnerable to lysozyme but also undermines its highly ordered structure (Yadav et al. 2010). Even though the above two studies have modified BC material properties, the limited availability of genetic tools and expertise for promoting the secretion of recombinant proteins from bacteria generating BC severely impedes their real‐world implementation. To introduce more functions to cellulose‐based living materials, an ELMs system inspired by kombucha tea has been developed. The drink known as kombucha is the result of fermentation brought about by a mixed community of bacteria and yeast often referred to as a symbiotic culture (Jayabalan et al. 2010; Villarreal‐Soto et al. 2018). One research group has employed bacterial co‐culture with Saccharomyces cerevisiae yeast to secrete TEM1 β‐lactamase into K. rhaeticus cellulose, creating catalytic materials that can be cultured and preserved at room temperature (Gilbert et al. 2021).

2.4. Geobacter sulfurreducens

Besides utilising these model organisms for developing living materials, there has been a growing interest in engineering living materials derived from non‐model microorganisms, such as G. sulfurreducens. This Gram‐negative, electroactive bacterium not only survives but also remains metabolically active in low‐oxygen environments (Engel et al. 2020). Moreover, its metabolism is highly flexible, enabling it to utilise a variety of organic compounds, including acetate and lactate, as energy sources (Speers and Reguera 2012). Additionally, in recent years, the development of synthetic biology has led to the creation of genetic tools for manipulating bacteria in the Geobacter genus, making genomic editing of G. sulfurreducens more straightforward (Liu, Min, et al. 2023). The combination of these diverse biological characteristics makes G. sulfurreducens an ideal candidate for the development of ELMs. Currently, the development of ELMs using G. sulfurreducens is based on its ability to form biofilms. The typical features of G. sulfurreducens biofilms include a matrix made of polysaccharides, which contains pili and cytochromes. This structural composition of biofilms not only confers excellent electrical conductivity but also enhances hydrophilicity. Utilising this hydrophilic property of the biofilms, one research group induced electricity generation through water evaporation (Hu et al. 2022). Specifically, the process begins with forming a G. sulfurreducens biofilms on a glass slide. Once the biofilm has been established, a bottom copper electrode is fixed onto the biofilm, followed by the placement of a top copper electrode above it. This setup completes the fabrication of a power‐generating device. When one end of the G. sulfurreducens biofilm formed on glass slides is immersed in water while ensuring that the bottom copper electrode remains above the water level, the device can generate power as water continuously evaporates. Under these conditions, the maximum power density achieved by this setup can reach approximately 685.12 μW/cm². OmcS protein is one of the many cytochromes produced by G. sulfurreducens. It is not only essential for interspecies electron transfer (Yalcin and Malvankar 2020), but research has also shown that overexpressing the OmcS protein in the cyanobacterium Synechococcus elongatus PCC 7942 can significantly enhance its ability to generate photo‐current (Sekar et al. 2016). A recent research group hypothesised that OmcS may have the potential to function as a photoconductor. To test this hypothesis, they selected G. sulfurreducens CL‐1, which is capable of overexpressing the OmcS protein, for their experiments. After this strain formed a biofilm, upon photoexcitation, the living biofilm demonstrated the ability of photoconductivity, and the conductance of the biofilm followed Ohm's law (Neu et al. 2022).

2.5. Corynebacterium glutamicum

Following G. sulfurreducens, another non‐model organism that has garnered attention is Corynebacterium glutamicum. C. glutamicum is a Gram‐positive bacterium renowned for its pivotal role in amino acid production. This microorganism is a GRAS organism and features a flexible metabolism, enabling it to grow on various carbon sources (Becker et al. 2005). Moreover, C. glutamicum exhibits excellent compatibility with a wide range of genetic tools and methods, such as plasmid construction, gene editing (e.g., CRISPR/Cas9), and homologous recombination (Zha et al. 2023). These attributes make it particularly convenient for scientists to engineer C. glutamicum to achieve specific functions. Additionally, the cell‐surface protein structures known as pili in C. glutamicum are composed of Spa1, Spa2, and Spa3 proteins. These pili components offer unique opportunities for constructing ELMs. Specifically, one research group developed ELMs by forming fusion proteins with Spa2, the major component of C. glutamicum pili. By fusing Spa2 with enzymes like TrEgl and SdBgl, two enzymes that collaborate to degrade cellulose into glucose, and coassembling them in pili on the cell surface, it becomes possible to generate ELMs that efficiently convert cellulose into glucose (Huang et al. 2024).

2.6. Caulobacter crescentus

Expanding the range of non‐model organisms used in ELMs research, C. crescentus presents distinctive features. This Gram‐negative bacterium is well‐known for its extensive use in cell cycle research. It exhibits high sensitivity to environmental changes and can survive in nutrient‐poor conditions, such as those found in rivers and streams (Govers and Jacobs‐Wagner 2020). Additionally, researchers have developed a variety of tools and techniques to engineer its genome (Guzzo et al. 2020). These characteristics make it very suitable as a cellular component in ELMs. Because it possesses a Surface‐layer protein RsaA with a well‐characterised atomic structure, using it as a basis for constructing ELMs would be an excellent choice. One group initially fused the RsaA protein with SpyTag peptides for expression on the surface of CB15N∆sapA cells. Since SpyCatcher can form an isopeptide bond with SpyTag peptides, this allows for their conjugation. Subsequently, the team linked SpyCatcher to elastin‐like polypeptides and quantum dots separately, then incubated these complexes with cells expressing the RsaA‐SpyTag fusion proteins. As a result, soft materials and hard materials were formed on the cell surfaces, respectively (Charrier et al. 2019). In a follow‐up study, this group replaced residues 251–689 of the native RsaA with an elastin‐like polypeptide and a SpyTag. As a result, they were able to successfully display this bottom‐up de novo (BUD) protein on the cell surface. The matrix formed by the BUD protein can encapsulate cells to form centimetre‐scale ELMs. Notably, by fusing the oxidoreductase PQQ‐glucose dehydrogenase with SpyCatcher and subsequently incubating this fusion protein with ELMs formed based on the BUD protein, the resulting materials exhibit PQQ‐glucose dehydrogenase activity (Molinari et al. 2022).

3. Bacterial Species Used in Hybrid Living Materials

Not limited to biofilm engineering for constructing ELMs, the development of these materials can also involve encapsulating bacteria in artificially formed extracellular matrices. This approach enables the creation of hybrid living materials with a wide range of functions. Figure 2 illustrates the scheme of creating diverse hybrid living materials that combine engineered bacteria with different polymers. The bacterial species used in hybrid living materials include E. coli , Shewanella oneidensis, B. subtilis, Lactococcus lactis, Methylobacterium radiotolerans, Azospirillum brasilense , Bradyrhizobium sp., Synechococcus elongatus , Pseudomonas aeruginosa , Lactobacillus casei , Lactobacillus plantarum , Lactobacillus acidophilus , Lactobacillus gasseri , Bifidobacterium adolescentis and Lactobacillus bulgaricus.

FIGURE 2.

Bacterial ELMs derived from hybrid living materials. (A) Schematic illustration of using bacteria to create diverse hybrid living materials by encapsulating engineered bacterial cells within matrices such as agarose, alginate, Pluronic F‐127, APBA and gelatin. (B) Representative examples of hybrid living materials include: (i) B. subtilis encapsulated in APBA functioning as biosensors. Reprinted with permission from Ref (Jo and Sim 2022). Copyright2022 American Chemical Socity. (ii) S. elongatus PCC 7942 encapsulated in alginate to form a living material exhibiting laccase activity against ABTS. Reprinted with permission from Ref (Datta et al. 2023). The Authors 2023. This article is published with open access at Springer Nature. (iii) B. subtilis encapsulated in Pluronic F‐127 for treatment of C. albicans infections. Reprinted with permission from Ref (Lufton et al. 2018). Copyright2018 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

Agarose is commonly utilised as a matrix polymer due to its biocompatibility, inertness, gelling ability, and high water content, which aids in hydrating bacterial cells. To form hybrid living materials, researchers have selected it as the matrix for many bacteria species. To detect the heavy metals' presence in the environment poses a significant difficulty for European initiatives focusing on the health and security of the general public. Biosensors, as a robust alternative to physicochemical methods, can be employed for heavy metal and toxic organic substance detection. In order to form biosensors, a combination of agarose and E. coli was chosen. Specifically, the E. coli DH1 strain carrying the pBzntlux plasmid was selected because it contains a cadmium‐responsive promoter that induces bioluminescence in the presence of cadmium. Immobilising bioluminescent bacteria E. coli DH1 pBzntlux in an agarose matrix enables continuous cadmium detection (Affi et al. 2009). Another selected bacterium is Shewanella oneidensis ; it is a model organism extensively used in bioelectrochemical systems (BESs). Within BESs, the interaction between S. oneidensis and the electrode surface is pivotal in attaining high system efficiency. Favouring efficient direct electron transfer, the biofilm formation of S. oneidensis is a promising approach. Yet, naturally formed biofilms within the systems tend to be thin. To address this limitation, researchers have directly embedded S. oneidensis into a 3D artificial biofilm matrix based on agarose, offering scalability and biocompatibility suitable for industrial applications (Knoll et al. 2022). In the field of ELMs, a significant challenge is maintaining the viability of cells within materials when exposed to harsh conditions outside the laboratory. Spores, which are structures used by some bacteria for reproduction or surviving adverse conditions, exhibit tolerance to high temperature, desiccation, and radiation. Due to the ability of B. subtilis to form spores, this bacterium has also been selected for the development of hybrid living materials. Lina et al. proposed a 3D printing method that combines agarose with B. subtilis PY79 spores to construct 3D objects (González et al. 2020). Through genetic engineering, the cells of the materials can respond to vanillic stimuli and synthesise lysostaphin, which is an antibiotic that specifically kills Staphylococcus aureus without affecting B. subtilis . Additionally, the spore‐containing material remains stable at room temperature for extended periods.

The alginate exhibits a high degree of biocompatibility and low cytotoxicity, and it also displays a crosslinking behaviour when exposed to divalent cations. Given these characteristics of alginate, different bacterial species have been chosen to form hybrid living materials in combination with it. Lactococcus lactis is a Gram‐positive bacterium widely used in food fermentation processes. Due to its long‐term use in the food industry, it presents a safe choice for bioengineering applications. This bacterium is a facultative anaerobe, capable of growing under both aerobic and anaerobic conditions. It also coexists well with the human body and possesses an efficient protein secretion mechanism that can be utilised for the production and release of necessary enzymes and functional proteins into the surrounding environment. Because L. lactis produces lactic acid during metabolism from carbon sources, which can be detrimental to cell growth due to accumulation, one research group selected L. lactis NZ9020, which does not produce lactic acid due to a lactate dehydrogenase deficiency, for constructing ELMs. When L. lactis NZ9020 expressing the III7‐10 fragment of human fibronectin (FN_III7‐10), L. lactis NZ9020 expressing human bone morphogenetic protein‐2 (BMP‐2), and human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) were encapsulated together using alginate and co‐cultured in liquid medium, the L. lactis was able to trigger the differentiation of hBM‐MSCs within the material (Witte et al. 2019). The pink‐pigmented methylobacteria, Methylobacterium radiotolerans , is a Gram‐negative α‐proteobacteria of the genus Methylobacterium. This bacterial species is not only tolerant to radiation, but it can also colonise nodules and other plant tissues. The application of M. radiotolerans to jatropha has been observed to increase seed yield. However, traditional liquid and solid formulations encounter the issue of decreased bacterial viability during storage. Scott et al. found that encapsulation of this bacterium by alginate prolongs its viability during storage (Strobel et al. 2018). For bacteria of the Azospirillum genus and Bradyrhizobium sp., which are capable of assisting plants in nitrogen fixation, there is also an issue of reduced bacterial cell viability during storage. Similarly, encapsulating A. brasilense Az39 and Bradyrhizobium sp. SEMIA6144 in a matrix composed of alginate results in an improved cell survival rate and enhanced effectiveness in promoting the growth of Arachis hypogaea (Cesari et al. 2020). Additionally, given the unique ability of Cyanobacteria to grow in low‐cost media and utilise CO₂ as a carbon source, coupled with the genetic engineering capabilities available for cyanobacteria that enable modular plasmid design and gene expression, the cyanobacterium S. elongatus PCC 7942 is chosen for ELMs fabrication. By immobilising genetically engineered S. elongatus in alginate, it can be effectively used in bioremediation efforts (Datta et al. 2023). In the development of living materials for bioremediation, the CotA laccase from Bacillus subtilis was constitutively expressed in the S. elongatus PCC 7942. When embedded within an alginate hydrogel matrix and printed, this responsive biomaterial demonstrates the capability of decolorizing indigo carmine.

Another man‐made matrix polymer, Pluronic F‐127, possesses a lower critical solution temperature, which enables it to rapidly solidify when the temperature drops below a certain threshold (Lee et al. 2010). Both B. subtilis and E. coli have been explored for forming hybrid living materials with this polymer. Microorganisms often release secondary metabolites such as antibiotics into their environment to defend against other microorganisms and maintain ecological balance. Over the past decade, there has been extensive research into using beneficial live bacteria for disease treatment. One research group chose to encapsulate B. subtilis within Pluronic F‐127 to develop a formulation aimed at treating C. albicans infections. B. subtilis was selected because it is generally recognised as a safe microorganism that can produce various antimicrobial substances. In the C. albicans infection model, the formulation containing live B. subtilis 3610, which persistently releases the antifungal agent surfactin, resulted in no inflammation in mice (Lufton et al. 2018). 3D printing technology allows scientists to precisely design and manufacture objects with complex geometries and internal structures tailored to specific needs. Materials generated using this technology have been applied in various fields, including drug delivery and tissue engineering. A recent study selected Pluronic F‐127 as one of the main components of bioink, and by utilising Aerotech's three‐axis robotic deposition stage, it was possible to print high‐resolution living materials. The incorporation of E. coli DH5αPRO, a cell type suitable for efficient plasmid transformation, enables the 3D material to produce green fluorescent protein when induced by AHL or IPTG (Liu et al. 2018).

APBA is an organic compound with a molecular formula of C8H10BNO3. It is a white crystalline powder commonly used in the organic synthesis of pharmaceuticals. The boronic acid functionality of APBA allows it to form reversible covalent bonds with specific biomolecules, making it a valuable tool in drug research. To further improve the current application of APBA in ELMs, a programmable living material has been created featuring a dynamic covalent connection between a tailored B. subtilis and APBA (Jo and Sim 2022). This breakthrough was inspired by the discovery that APBA and APBA‐based polymers have the ability to establish covalent connections with the diols present on the cell surface of B. subtilis . In this innovative system, B. subtilis PY79 underwent genetic modification to enable the production of red fluorescent protein (RFP) integrated with the N‐terminal signalling sequence of gene AmyQ (Phan et al. 2006), enabling the newly synthesised RFP to be secreted into the extracellular space. When these living materials containing the engineered B. subtilis PY79 were cultivated in suitable media, the fluorescence levels of the material and gathered liquid supernatant resulting from RFP production gradually increased, demonstrating the capability of materials to generate and release recombinant RFP. Furthermore, by engineering B. subtilis PY79 to express green fluorescent proteins in response to specific small molecules like cuminic acid and IPTG, biosensor living materials can be developed.

Gelatin, derived from collagen present in animal connective tissues, is a protein known for its capacity to form a gel upon hydration. This characteristic stems from the capability of the protein to absorb water and create a three‐dimensional network of molecules. Quorum sensing (QS) serves as a cell‐to‐cell communication mechanism utilised by numerous bacteria to synchronise their actions and control gene expression based on population density. Pseudomonas aeruginosa possesses a complex QS system, which it uses to regulate the expression of a wide array of genes through multiple signalling molecules. These genes are involved in various behaviours, including biofilm formation and antibiotic resistance. Understanding P. aeruginosa 's QS system is crucial for deciphering how pathogenic bacteria coordinate collective behaviour to adapt to environmental changes. This area of study has garnered significant interest in the medical field because it is closely related to the infection process. When Pseudomonas aeruginosa are connected by gelatin and printed using the Micro‐3D technique, the resulting materials can be utilised to investigate the QS‐mediated communication behaviour of cell aggregates as small as 500 cells (Connell et al. 2014). However, the applications of gelatin as a gel matrix are limited by its network stiffness. On the other hand, the utilisation of an alginate matrix is restricted due to its sensitivity to low pH conditions, which may lead to undesired structural collapse. Lactobacillus casei is a common lactic bacterium that can grow over a relatively wide range of pH conditions and is widely found in nature, including in the gastrointestinal tracts of humans and animals. As a probiotic, it has immune‐modulating properties and contributes to improving gut health. However, it may also experience reduced viability during storage and use. By combining alginate and gelatin, a dual‐network hydrogel system is formed, capitalising on their complementary properties. This integration enhances the viability of probiotic bacteria L. casei ATCC 393 compared to non‐encapsulated free cells (Li et al. 2009). In a similar manner, for L. casei 01, the incorporation of prebiotics, specifically starch, into the alginate matrix can improve the durability and protective capabilities of the matrix (Ta et al. 2021). Furthermore, egg lecithin, a natural emulsifier and stabiliser derived from eggs, has been explored for its effects on the survivability of alginate‐starch encapsulated bacteria. Bacteria of the genera Bifidobacteria and Lactococcus also play a promoting role in maintaining host health. Encapsulating Lactobacillus, Bifidobacterium species, and Lactococcus lactis in freeze‐dried beads containing lecithin demonstrates promising stability in terms of survival (Donthidi et al. 2010).

4. Perspectives

Over the past 10 years, the field of ELMs has grown due to the combination of techniques employed in synthetic biology and material science. The pursuit of additional materials with diverse functions similar to living organisms on the earth will continue to stimulate the development of this area. For bacterial cells, significant efforts have been dedicated to E. coli for the fabrication of ELMs. Specially, modular engineering has been used to design amyloid proteins with diverse functions. Amyloid proteins are controlled by the csg operons, and the monomeric unit of amyloid is the csgA protein, which is secreted in an unfolded conformation. When the csgA protein is fused with various protein domains or peptides, novel fusion proteins with unique functions can be generated. As a proof of concept, E. coli NEBC3016 was the first E. coli strain used to express strong and multifunctional underwater materials in vitro. The E. coli PHL628‐∆csgA has been used to obtain materials with biocatalytic function. Subsequently, E. coli PQN4, E. coli JF1∆csg, E. coli MG1655PRO∆csgA, and E. coli MG1655PRO∆csgBAompR234 were engineered to secrete and assemble fusion proteins with diverse functions (Table 1). The characteristics of these strains used in ELMs are that the csgA gene, csgA/csgB genes, or all curli genes are deleted. As csgC effectively prevents the formation of curli amyloid proteins by inhibiting primary nucleation through electrostatically guided interactions between molecules (Taylor et al. 2016). Perhaps further deletion of such a gene in the csgBA mutant strain, E. coli MG1655PRO∆csgBAompR234, will stimulate amyloid protein secretion. However, the inhibition of csgA amyloid protein formation by csgC may serve as a protective mechanism for bacteria. Therefore, it is essential to evaluate the impact of such a modification on bacterial viability and stability before proceeding with genetic manipulation. Compared to directly deleting csgC, upregulating csgE, csgF, and especially csgG might provide a more straightforward and safer approach to increase the yields of secreted curli biofilms. To date, however, no attempt at generating such an E. coli strain has been reported.

TABLE 1.

Bacterial species used in the natural biofilm‐based living materials.

Strain name	Year	Description	Features	Advantages	Application
E. coli PHL628‐∆csgA (Botyanszki et al. 2015; Nussbaumer et al. 2017)	2015, 2017	E. coli MG1655 with a deletion of the csgA gene and an ompR234 mutation	Deletion of csgA; ompR234 mutation	Capable of fusing various peptides or proteins to curli fibres, enhanced biofilm formation	Biocatalysis
E. coli MG1655PRO ∆csgA ompR234 (Pu et al. 2020; An et al. 2020)	2020, 2020	E. coli MG1655 with constitutive high level expression of tetR and lacI from PRO cassette, with csgA knock out, and with ompR 234 mutation	Constitutive expression of TetR and LacI proteins; deletion of csgA gene; ompR234 mutation	Robust regulatory protein expression, enhanced genetic manipulation flexibility, improved biofilm formation	Bioremediation; Biosensing
E. coli MG1655PRO ∆csgBAompR234 (Kalyoncu et al. 2017)	2017	E. coli MG1655 PRO ∆csgA ompR234 with further deletion of csgB gene	Constitutive expression of TetR and LacI proteins; deletion of csgA and csgB gene; ompR234 mutation	Improved control over gene expression, elimination of potential inference from endogenous csgB, improved biofilm formation	Bioelectronics
E coli PQN4 (Tay et al. 2017)	2017	An E. coli MC4100 strain that has undergone genetic modification to eliminate the curli operon	Complete deletion of curli formation operons	Improved reproducibility, facilitate functional peptide integration	Bioremediation
E. coli JF1 ∆csg (Moser et al. 2019)	2019	Permanently delete all curli associated genes from the E. coli JF1	Native curli operons knockout	Elimination of cross‐talk	Biosensing
B. subtilis 2569 ∆tasA∆sinR ∆eps (Huang et al. 2019)	2019	B. subtilis 2569 lacks the tasA gene, as well as the sinR gene and the epsA ~ O gene	Deletion of tasA, sinR, epsA ~ O genes	Facilitates detection of functionalized TasA variants, enhanced biofilm formation, simplified biofilm matrix composition	3D printing
B. subtilis 1935 ∆eps∆bslA (Zhu et al. 2024)	2024	Knocking out epsA ~ O and bslA genes of wild‐type strain B. subtilis 1935	Deletion of epsA ~ O genes; deletion of bslA gene	Reduced background interference, simplified biofilm architecture	Biosensing, bioremediation
K. rhaeticus iGEM (Caro‐Astorga et al. 2021)	2021	BC‐producing bacterium isolated from kombucha tea	High cellulose production; high resistance to toxic chemicals	Significant chemical tolerance, development of a genetic toolkit	3D printing, patterns formation
G. sulfurreducens CL‐1 (Neu et al. 2022)	2022	A G. sulfurreducens strain that Overexpresses OmcS nanowires	Overexpression of OmcS protein	Enhanced electron transfer capability, increased photo‐current generation efficiency	Bioelectronics
C glutamicum∆spa2∆dec (Huang et al. 2024)	2024	Deletion of the spa2 and dec genes in C. glutamicum ATCC 14067	Deletion of spa2; deletion of dec	Biomass‐to‐chemical conversion, colour change marker	Biocatalysis
C. crescentus CB15N∆sapA (Charrier et al. 2019)	2018	C. crescentus CB15N with deletion of gene sapA	Deletion of sapA gene	Enhanced surface display stability, higher display efficiency	Soft materials
C. crescentus NA1000 ∆sapA::PxylmKate2 (Molinari et al. 2022)	2022	Deletion of gene sapA and insertion of the mKate2 gene, controlled by the xylose‐inducible promoter Pxyl at the same locus in C. crescentus NA1000	Deletion of sapA gene; Pxyl‐mKate2 insertion	Enhanced surface display stability, visual tracking	Biocatalysis

To extend the spectrum of bacteria employed in the engineering of amyloid proteins, the TasA protein from B. subtilis has also been incorporated. By fusing the TasA protein with various proteins or protein domains, programmable, printable, and 3D objects with diverse functions have been developed. To enable broader applications and industrial‐scale production of living materials utilising B. subtilis biofilms in the future, it is essential to enhance the biofilm formation capability of B. subtilis through genetic engineering. Studies have shown that integrating the signal peptide of the gene amyQ with red fluorescent protein and microbial transglutaminase promotes the release of both proteins in B. subtilis (Jo and Sim 2022; Mu et al. 2018). To enhance the release of TasA proteins, future efforts can be directed to explore the possibility of integrating the signal peptide of the gene amyQ with the TasA protein. Another way to enhance matrix production is to identify new strains that can produce more amyloids. Conducting PSI‐Blast searches in the NCBI RefSeq protein database utilising csg proteins from E. coli MG1655 as query sequences has successfully identified numerous curli‐containing strains within the same genus (Dueholm et al. 2012). For instance, we can identify 30 strains containing curli within the genus Salmonella, 10 strains containing curli within the genus Shigella, and 4 strains containing curli within the genus Citrobacter (Figure 3). Importantly, strains of these genera have been shown to have biofilm formation abilities. (Choong et al. 2021; Chiang et al. 2021; Zhou et al. 2021) Over the last decades, numerous alternative amyloid systems beyond curli type have also been discovered in various bacterial biofilms. Chaplins, a functional amyloid secreted by Streptomyces spp., assemble at the air‐liquid interface to form hydrophobic amyloid protein sheets (Elliot et al. 2003). The fap operon in Pseudomonas species secretes amyloid proteins that function not only in initial adhesion but also in mature biofilm formation (Dueholm et al. 2013). In a similar manner, Staphylococcus species release Bap proteins, which assemble into amyloid‐like fibres outside the cells and aid in the construction of the biofilm matrix (Taglialegna et al. 2016). However, it is worth mentioning that, among all functional amyloids distinct from curli types, only the TasA protein from B. subtilis has been recently modified for the development of ELMs. This may be attributed to the well‐known pathogenicity of Pseudomonas and Staphylococcus, as well as the difficulty in lab handling of Streptomyces (Serra et al. 2015; Shepherd et al. 2010). To address the issue of pathogenicity of Pseudomonas and Staphylococcus, future efforts should be directed to construct both attenuated strains based on living materials (Valentine et al. 2020; Collins et al. 2002). In addition to constructing ELMs around amyloid proteins, such as those found in biofilm matrices, researchers have explored using non‐amyloid proteins like Spa2, OmcS and RsaA for ELM construction. Non‐amyloid proteins are also present within the extracellular matrices of various other bacteria; for instance, the biofilm matrix of B. subtilis contains the amphipathic surface protein BslA (Morris et al. 2024), while that of Pseudomonas aeruginosa includes lectin proteins (Metelkina et al. 2022). Although these non‐amyloid proteins have been identified, they have not yet been utilised in the development of ELMs. Future engineering efforts focused on utilising non‐amyloid proteins from biofilm matrices promise to significantly expand the diversity and potential applications of ELMs.

FIGURE 3.

Phylogenetic distribution of curli systems and operon structure. The count of strains harbouring the curli system within each genus is denoted alongside the respective taxonomic units. The genera highlighted in red represent those where curli systems had been previously documented prior to August 30, 2012. Reprinted with permission from Ref (Dueholm et al. 2012). The Authors 2012. This article is published with open access at Public Library of Science.

Cellulose is also an important extracellular component in biofilms of many bacterial species (Hung et al. 2013; Simm et al. 2014). BC‐based ELMs have been developed with the ability to regenerate in response to damage. Additional species that can produce cellulose will broaden the uses of BC‐based ELMs. K. rhaeticus is an α‐proteobacteria in the family Acetobacteraceae. In one selected proteobacterial genome, many bacteria have BC synthesis (bcs) genes. Some α‐proteobacteria, such as Gluconacetobacter xylinus E25, Gluconacetobacter hansenii ATCC 23769, Methlobacterium extorquens PA1, and Sinorhizobium melilo GR4, have been shown to produce cellulose under experimental conditions (Figure 4) (Römling and Galperin 2015). However, compared to protein modular engineering, modification of cellulose at the genetic level is difficult due to the complicated regulatory process. Living functional materials based on cellulose are currently produced by utilising BC secreted to encapsulate bacterial cells, yet there have been no reports of genetic modification of the cellulose metabolic pathway at the genomic level. In addition, the structure and morphology of BC closely resemble those of natural collagen, which makes it an enticing option for immobilisation. Immobilising Bacillus subtilis cells in BC using the ‘adsorption‐incubation’ method led to living materials demonstrating high therapeutic efficiency in wound healing models (Savitskaya et al. 2019).

FIGURE 4.

The presence of bcs genes in selected proteobacterial genomes. Note that some bacterial strains in the α‐proteobacteria like Gluconacetobacter xylinus E25, Gluconacetobacter hansenii ATCC 23769, Methylobacterium extorquens PA1, Sinorhizobium melilo GR4 have been shown to produce cellulose in the experimental condition. Reprinted with permission from Ref (Römling and Galperin 2015). Copyright2015 Elsevier Ltd. Published by Elsevier Ltd. All rights reserved.

The strategy of encapsulating cells in polymers to form extracellular matrices effectively solves the problem of low extracellular matrix production of many bacteria under laboratory conditions, thereby expanding the range of bacterial species that can be applied in the field of ELMs. In addition to the species mentioned in the section of ELMs based on naturally synthesised biofilms, the E. coli DH1 pBzntlux, S. oneidensis MR‐1, and B. subtilis PY79 have been reported to be encapsulated in artificial matrix polymers for various purposes (Table 2). The key to the success of this strategy is the selection of polymers and bacteria. Since these polymers are not molecules naturally produced by the corresponding bacteria, the interplay between cells and the extracellular matrix formed by these polymers differs from those when the extracellular matrix is naturally synthesised. It has been reported that when Vibrio cholerae is encapsulated in agarose, the interplay between the cell and the external environment determines the external morphology and internal structure of the cell cluster (Zhang et al. 2021). When cultured in a rigid environment, cells tend to form aggregates that exhibit oblate shapes and exhibit bipolar cellular ordering. In contrast, cells thriving in soft environments exhibit a spherical morphology and a disorganised cellular arrangement. When Staphylococcus aureus is encapsulated in agarose, a distinct oblate shape with a degree of hexagonal ordering has also been observed, differing from naturally occurring morphologies and order found during unconstrained growth. Crucially, multiple research studies have demonstrated a robust correlation between bacterial morphologies and their bioproduction capabilities (Jiang and Chen 2016; Wang et al. 2017; Choi and Lee 1999; Wang et al. 2014). Hence, a profound knowledge of the interplay between bacteria and the matrix would help a better choice of polymers and bacteria. It is commonly recognised that the level of oxygen concentration holds a pivotal position in the development of numerous aerobic or facultative anaerobic microorganisms. When S. aureus and P. aeruginosa are encapsulated in agarose and alginate, respectively, the growth of cells exhibits an oxygen‐dependent phenomenon; aggregates are larger near the oxygen‐supplied interface, and aggregates further from the oxygen‐supplied interface are smaller (Pabst et al. 2016; Sønderholm et al. 2023). Deoxyviolacein (dVio), a natural pigment belonging to the violacein compound class, has garnered significant attention due to its promising potential for applications across diverse fields. When E. coli are encapsulated in agarose for production of dVio, the highest production was observed at the air‐medium interfaces, while the lowest level of production was observed 1.5 mm away from both interfaces (Sankaran et al. 2019). Therefore, it is vital to check whether the pore size in the materials formed by wrapping cells with polymers satisfies the cellular requirement for oxygen.

TABLE 2.

Bacterial species used in hybrid living materials.

Strain name	Year	Description	Features	Advantages	Application
E. coli DH1 pBzntlux (Affi et al. 2009)	2009	A bioluminescent E. coli strain that can be used to detect the presence of cadmium	Contains lux CDABE genes	High sensitivity	Biosensing
S. oneidensis MR‐1 (Knoll et al. 2022)	2022	Model organism for exoelectrogenic electron transfer, low biofilm formation ability on electrodes	Diverse electron acceptor spectrun	Versatile use of electron acceptors	Bioelectronics
B. subtilis PY79 (González et al. 2020; Jo and Sim 2022)	2020, 2022	A derivative of B. subtilis ATCC23857	Spore‐forming capability	Tolerance to harsh conditions	3D printing; biosensing
L. lactis NZ9020 (Witte et al. 2019)	2019	A strain lacks the ldhA and ldhB genes for lactate dehydrogenase	Lacks lactate dehydrogenase	Reduced lactic acid accumulation	3D printing
M. radiotolerans (Strobel et al. 2018)	2018	Pink‐pigmented methylobacteria	Radiation tolerance, ability to colonise nodules and plant tissues	Promotion of plant growth, strong environmental adaptability	Agriculture use
Bradyrhizobium sp. SEMIA6144, A. brasilense Az39 (Cesari et al. 2020)	2020	Rhizobacteria that are commonly utilised as liquid commercial inoculants for peanut plants	The ability to fix atmospheric nitrogen	Improve crop yields	Agriculture use
S. elongatus PCC 7942 (Datta et al. 2023)	2023	Model cyanobacteria	Photoautotrophic	Cost‐effective cultivation, high genetic tractability	3D printing, biosensing, bioremediation
B. subtilis 3610 (Lufton et al. 2018)	2018	Natural wild‐type, nonmodified strain	Antifungal capability	High safety profile	Biotherapy
E. coli DH5αPRO (Liu et al. 2018)	2017	Molecular cloning strain	High efficiency in uptaking foreign DNA	High transformation efficiency	3D printing
P. aeruginosa PA14 (Connell et al. 2014)	2014	Laboratory reference strain	Possesses complex QS system	Enhanced therapeutic development	Micro‐3D printing
L. casei ATCC 393 (Li et al. 2009)	2009	Probiotic bacteria	Wide pH tolerance	Enhances immune function	Probiotic products
L. casei 01 (Ta et al. 2021)	2021	Probiotic bacteria	Wide pH tolerance, susceptibility to antibiotics	Safety for consumption	Probiotic products
Lactobacillus casei ssp. Casei NCFB 161, L. plantarum DSM 12028, L. acidophilus NCFB 1748, L. gasseri NCFB 2233, L. bulgaricus NCFB1489, B. adolescentis NCIMB 2204, Lactococcus lactis ssp. lactis NCIMB 6681 (Donthidi et al. 2010)	2010	Probiotic bacteria	Adaptation to gut environment, metabolic diversity	Promotion of gut health	Probiotic products

The 3D structure of a natural biofilm, composed of polysaccharides, proteins, and DNA, affords physical protection to bacteria contained within. The barrier formed by the biofilm presents a formidable obstacle for antibiotics and other antimicrobial agents, hindering their penetration and access to the bacterial interior. This impediment results in a reduced effective concentration of these medications, diminishing their potency. Furthermore, certain bacteria embedded within the biofilm may exist in a state of slow growth or dormancy, rendering many antibiotics, which are primarily designed to target rapidly reproducing bacteria, less effective against them (Gebreyohannes et al. 2019; Stewart and Costerton 2001). In parallel, experiments have demonstrated that when encapsulated within an alginate matrix, E. coli exhibits substantial protection against antibiotics. Specifically, they can withstand concentrations up to 1000 times the minimum inhibitory concentration for ciprofloxacin and chloramphenicol (Pham et al. 2023). Additionally, it is noteworthy that the bacterial strains commonly utilised in the development of living materials are often genetically modified microorganisms (GMMs). Consequently, there is a critical need to thoroughly assess and manage the potential environmental release of such engineered cells. To keep bacteria from escaping the living materials and surviving in the environment, both chemical and physical containment measures have been implemented as barriers. One strategy that has been developed for chemical containment involves the use of S. elongatus. In S. elongatus , the gene Synpcc7942_0766 is regulated by a theophylline‐inducible riboswitch. When the substance theophylline is added, it induces overexpression of gene Synpcc7942_0766. The overexpression of the gene Synpcc7942_0766 can lead to the excision of prophage, ultimately resulting in cellular lysis (Datta et al. 2023). Additional methodologies have been formulated for chemical confinement of GMMs, which could be implemented in living materials (Lee et al. 2018; Foo et al. 2014; Hirota et al. 2017). However, in certain instances, the successful implementation of these strategies necessitates further genetic manipulation of the engineered bacteria. Multiple genetic operation steps are always labour‐intensive and time‐consuming. Interestingly, prophage‐related genetic elements and residual prophage sequences have been widely identified in virtually all sequenced bacterial genomes, and it is not uncommon for bacteria to harbour multiple prophages within their chromosomal structures (Canchaya et al. 2003). Experimental evidence has shown that prophage‐induced cell lysis has occurred in P. aeruginosa and G. sulfurreducens biofilms upon exposure to a quorum‐sensing signal molecule, referred to as 2‐heptyl‐4‐hydroxyquinoline, and mitomycin C, a known lytic inducer, respectively (Giallonardi et al. 2023; Liu, Ye, et al. 2023). Based on these findings, it is speculated that such a mechanism could potentially be harnessed in the future for containing bacteria escape in this field.

Strategies for physical containment of bacteria in living materials involve integrating different types of hydrogels. For instance, leveraging the hydrophilic and biologically inert properties of polyacrylamide (PAAM), it has been combined with alginate to create polyacrylamide‐alginate (PAA) mixed hydrogels, which effectively encapsulated 2‐phenylphenol living sensors (Luisi et al. 2022). The encapsulated E. coli NEB5α cells within the living sensors can remain viable for up to 2 weeks without any leakage. Alternatively, a deployable physical containment strategy (DEPCOS) system has been developed (Tang et al. 2021). This system comprises dual components: a core made of alginate‐based hydrogel and an outer protective shell. The outer shell ingeniously integrates a stretchable polymer matrix (derived from polyacrylamide) with an energy‐dissipating network (also sourced from alginate). This innovative design ensures both robust structural integrity and efficient energy absorption. The results have shown that the DEPCOS system provides an effective means of containing E. coli.

5. Outlook

Bacteria, owing to their simple genomes, rapid reproduction, and the advanced state of genetic engineering technologies available for them, stand at the forefront of ELM research. Bacterial‐based ELMs have garnered diverse applications in areas such as biocatalysis, environmental remediation, electrical conductivity, biosensing, biotherapy, and agriculture. However, much of the existing work has concentrated on single‐cell types, which can complicate practical implementation due to the need for multifunctionality. Looking forward, integrating bacteria with other cell types such as archaea and various eukaryotic organisms like fungi, algae, and animal cells can pave the way for ELMs that exhibit enhanced multifunctionality. For instance, co‐culturing K. rhaeticus with S. cerevisiae or combining L. lactis with human mesenchymal stem cells showcases innovative approaches that combine distinct biological attributes (Gilbert et al. 2021; Hay et al. 2018).

A significant challenge remains the inadequate natural production of extracellular matrix by bacterial cells. To tackle this challenge, future efforts should focus on the following areas. Firstly, the application of artificial intelligence, particularly machine learning techniques, to systematically explore literature for bacteria harbouring the csg operon that can produce increased csgA homologous proteins. Machine learning has proven invaluable in advancing multiple facets of healthcare, contributing to the discovery of new drugs, improving disease diagnostic accuracy, and facilitating disease prognosis (Carracedo‐Reboredo et al. 2021; Yeoh et al. 2021; Silva et al. 2022). Essential genes are crucial for the development and continuance of all organisms, and machine learning techniques have also been employed in identifying essential genes (Aromolaran et al. 2021). Secondly, the identification of small biological molecules that can act as cross‐link polymers for constructing artificial matrix components to envelop bacteria and create hybrid living materials is essential. Currently, the majority of hybrid living materials derived from artificial synthesis involve encapsulating bacteria with a single cross‐link polymer. However, natural biofilms typically consist of a blend of various components such as proteins, cellulose, and DNA in conjunction with bacterial cells. To achieve a more accurate simulation of the structure of natural biofilms, the selection of diverse cross‐link polymers incorporating different extracellular matrix molecules like proteins and DNA will enhance the future applications of ELMs.

Ethical considerations are paramount. While certain bacteria like B. subtilis and L. casei are typically considered GRAS organisms, they can become pathogenic under specific conditions (de Boer and Diderichsen 1991; de Seynes et al. 2018). Genetic manipulation, while innovative, may introduce unforeseen ecological impacts. Therefore, stringent measures and regulatory oversight are essential to ensure safety and compliance. Public engagement through education and outreach is also critical for building trust and promoting acceptance. In conclusion, advancing bacterial‐based ELMs requires a balanced approach that considers human health, environmental protection, and public sentiment. Responsible progress should be pursued with these factors in mind, fostering innovation while safeguarding societal well‐being.

Author Contributions

Hu Wang: conceptualization, investigation, writing – original draft, writing – review and editing. Chunzhong Li: investigation, supervision. Yanmin Wang: investigation, supervision. Huanming Zhang: project administration, supervision, funding acquisition.

Conflicts of Interest

It is hereby stated by the authors that no conflicts of interest exist.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.