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. 2022 Nov 21;25(2):241–249. doi: 10.1111/1462-2920.16284

Emerging enzyme surface display systems for waste resource recovery

Beth Davenport 1, Steven J Hallam 1,2,3,4,5,6,✉
PMCID: PMC10100002  PMID: 36369958

Abstract

The current century marks an inflection point for human progress, as the developed world increasingly comes to recognize that the ecological and socioeconomic impacts of resource extraction must be balanced with more sustainable modes of growth that are less reliant on non‐renewable sources of energy and materials. This has opened a window of opportunity for cross‐sector development of biotechnologies that harness the metabolic problem‐solving power of microbial communities. In this context, recovery has emerged as an organizing principal to create value from industrial and municipal waste streams, and the search is on for new enzymes and platforms that can be used for waste resource recovery at scale. Enzyme surface display on cells or functionalized materials has emerged as a promising platform for waste valorization. Typically, surface display involves the use of substrate binding or catalytic domains of interest translationally fused with extracellular membrane proteins in a microbial chassis. Novel display systems with improved performance features include S‐layer display with increased protein density, spore display with increased resistance to harsh conditions, and intracellular inclusions including DNA‐free cells or nanoparticles with improved social licence for in situ applications. Combining these display systems with advances in bioprinting, electrospinning and high‐throughput functional screening have potential to transform outmoded extractive paradigms into ‘trans‐metabolic” processes for remediation and waste resource recovery within an emerging circular bioeconomy.

INTRODUCTION

In the present century, closing the carbon cycle through circular bioeconomy development has become a pivotal organizing principle for collective action on a global scale. Bioeconomy, as defined by the Organization for Economic Co‐operation and Development (OECD) represents ‘the aggregate set of economic operations in a society that use the latent value incumbent in biological products and processes to capture new growth and welfare benefits for citizens and nations’ (Arundel & Sawaya, 2009). From a fiscal perspective, the OECD projects that the global bioeconomy will exceed $1 trillion USD by 2030 (2.7% of the GDP of OECD countries) (OECD International Futures Program, 2009). While this potential shift in economic incentives is encouraging, energy and materials produced from renewable resources are not necessarily better for people or the environment if they follow a linear model of production, consumption, and disposal. A truly circular bioeconomy must consider conversion steps to multiple end products while reducing environmental impacts. Beyond climate‐active products such as carbon dioxide, methane, or nitrous oxide, many other waste products can have profound impacts on ecosystem functions and services that translate into negative health effects across multiple trophic levels (Ferner‐Ortner‐Bleckmann et al., 2013; Patel et al., 2010). For example, over 80% of wastewater globally is released into the environment with no or only partial treatment (Anjum et al., 2019; Connor et al., 2017) resulting in release of toxins and carcinogens including heavy metals such as cadmium and lead (Patel et al., 2010), halogenated compounds and phenolic dyes (Gao et al., 2016; Sharma et al., 2018) to name a few. This problem persists across sectors including energy, forestry, agriculture, and mining, necessitating implementation of more efficient ways to recover value from recalcitrant sources such as lignin, silage, or tailings (Naidu et al., 2019; Sanderson, 2011; Selvaraj et al., 2022). In the context of forestry and agriculture, lignocellulosic feedstocks with different chemical properties can be converted to useful bioproducts as replacements for fossil fuel‐derived energy and materials (Isikgor & Becer, 2015; Shahab et al., 2020). In the context of mining, these same feedstocks can be used to support microbial enhanced bioprocesses for waste resource recovery from tailings or other sources (Işıldar et al., 2019; Park et al., 2016; Wellmer et al., 2019).

In nature, microbial communities capture and convert carbon into an incredibly diverse array of compounds that support metabolism, growth and renewal (Mohan et al., 2020). At the same time microbial communities are adept at transporting and extracting metals that can be used in new process designs to extract raw materials for future energy supply, for example, for electrification of the power grid to replace fossil fuels and to stabilize or remediate industry‐impacted sites (Dev et al., 2020). Improved understanding of the diverse metabolic functions contained in microbial communities and their role in driving metabolic fluxes at different levels of biological organization provides a conceptual roadmap for the recovery of biological parts enabling rational design of new platform technologies. Here, we explore the application of enzyme surface display on cells or functionalized materials as a promising biotechnology for waste resource recovery with particular emphasis on S‐layer display, spore display, and intracellular inclusions. We go on to consider integration of these display systems with advances in bioprinting and electrospinning to develop programmable materials for waste resource recovery, and high‐throughput functional metagenomic screening to recover biological devices from uncultivated microbial communities to display.

Enzyme display systems

Enzymes or peptides that bind waste products for remediation or recovery, can now be identified in nature through sequencing or screening and improved in specificity, efficiency and stability through rational design or directed evolution (Alneyadi et al., 2018; Sharma et al., 2018, 2019; Zhu et al., 2019). For an overview of common enzymes/peptides researched for metal remediation and recovery, see Wang and colleagues (Wang, Selvamani, et al., 2021), and for hydrocarbon containing pollutant remediation see Sharma and colleagues (Sharma et al., 2018). Enzyme immobilization or display is essential for practical use at scale increasing enzyme stability, efficiency, and reusability compared to free suspended enzyme (Chen, Mulchandani, & Ge, 2017; Han et al., 2018; Wu et al., 2008; Zhu et al., 2019). Cell surface display (CSD) is an immobilization method that combines substrate binding or catalytic domains of interest translationally fused with extracellular membrane proteins in a microbial chassis that can be expressed constitutively or on demand through use of regulatory circuits (Zhu et al., 2019). This method has been studied extensively for its biomedical potential in whole‐cell vaccines, but its potential in waste resource recovery is only now being recognized (Han et al., 2018; Wu et al., 2008; Zhu et al., 2019). For example, Belcher and colleagues have described a yeast‐based CSD platform for heavy metal hyperaccumulation, and metal re‐extraction through precipitation of metal sulfide nanoparticles (Sun et al., 2019, 2020). Unlike other immobilization methods, CSD combines production, purification, and display within a single microbial cell factory, reducing time, cost and lower enzyme activity or stability from chemical crosslinking reactions (Chen, Mulchandani, & Ge, 2017; Falahati‐Pour et al., 2015; Wu et al., 2008; Zhu et al., 2019). In addition, CSD is more environmentally friendly (enzyme catalysis generates few toxic byproducts, can proceed at moderate temperatures and pressures, and is an easier process to control due to high catalytic specificity), and increases enzyme activity through cell proliferation and regeneration of enzyme cofactors (Chen, Mulchandani, & Ge, 2017; Zhu et al., 2019). Although a number of substrate binding or catalytic domains have been displayed using common CSD systems including fusion to Escherichia coli (Francisco et al., 1993; Sousa et al., 1998) and Saccharomyces cerevisiae (Kondo & Ueda, 2004; Liu et al., 2016; Nam et al., 2002) cell wall components such platforms tend to be economically and practically unsuitable for field application, due to (i) low display densities (ii) size limit of heterologous enzyme insertions, and (iii) presence of recombinant DNA (Duncan et al., 2005; Han et al., 2018; Wu et al., 2008; Zhu et al., 2019). New, biological, self‐assembling surface display platforms are currently in development that show promise for overcoming these application barriers including S‐layer display, spore display, and intracellular inclusions including DNA‐free cell or nanoparticle display (Figure 1).

FIGURE 1.

FIGURE 1

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Biological, self‐assembling surface display platforms currently in development. Each display platform is shown in relation to specific application limits overcome.

S‐LAYER DISPLAY PLATFORMS

S‐layers are geometric, monomolecular, highly stable protein lattices encasing the outside of many bacterial and archaeal cells (Nomellini et al., 2004; Sleytr et al., 2014). Native S‐layer proteins are secreted to self‐assemble extracellularly through C‐terminal interaction, while N‐terminal regions anchor to cell wall components (Nomellini et al., 2004; Sleytr et al., 2014). Some S‐layers have been shown to tolerate insertion of heterologous functional domains without compromising self‐assembly capacity, including S‐layers of Lysinibacillus sphaericus, Geobacillus stearothermophilus, and Caulobacter crescentus (Ilk et al., 2011). Modified S‐layers are under active development as one‐step immobilization scaffolds for many life and material science applications (Sleytr et al., 2014). S‐layer display offers several advantages, including superior display density (25% of cell protein synthesis in C. crescentus is for its S‐layer protein, displayed at >20,000 copies/μm2), periodicity and orientation (which ensures standardized functionality) (Figure 1) (Charrier et al., 2018; Duncan et al., 2005; Nomellini et al., 2004; Smit, 2008).

Caulobacter spp. are suitable for remediation applications, as they are non‐toxic and thrive under oligotrophic or low nutrient conditions (Nomellini et al., 2004; Xu et al., 2010). They also show promise for heavy metal and rare earth element (REE) recovery (Park et al., 2016; Xu et al., 2010). To date, S‐layer metal‐binding peptide display has facilitated whole cell specific adsorption of dissolved cadmium Cd(II) (Patel et al., 2010) and lead Pb(II) in the context of wastewater treatment (Cappucio et al., unpublished data). S‐layer display of elastin‐like polypeptides has also been shown to non‐specifically absorb heavy metals albeit to a lesser extent (Molinari et al., 2021). Park and colleagues demonstrated that S‐layer lanthanide binding tag display can adsorb REEs with high efficiency (>90%), affinity and selectivity when treated with rare earth mine soil samples (Park et al., 2016). Moreover, citrate or other acid treatment enables platform recycling and metal recovery between extraction rounds (Pallares et al., 2022; Park et al., 2016; Patel et al., 2010). S‐layer display also has potential application in lignocellulosic biomass conversion. Although sugar platforms have been developed to effectively hydrolyze cellulose and hemicellulose components, lignin remains difficult to utilize (Isikgor & Becer, 2015; Sanderson, 2011). C. crescentus S‐layer has been used to display different cellulases encompassing the three functional classes needed for synergistic cellulose degradation (Duncan et al., 2005; UBC iGEM Team 2016, unpublished data; Davenport et al., unpublished data) and could form the basis of a combinatorial platform in which different hydrolytic enzymes are displayed with small laccases or peroxidases to convert and funnel lignocellulosic biomass into defined product profiles. One potential disadvantage relates to insert size restriction approaching 40 kDa and steric hindrance limiting multi‐subunit complex formation (Charrier et al., 2018; Nomellini et al., 2004).

Recent development of S‐layer bioconjugation platforms for post‐assembly attachment of functional domains including S‐layer‐Spytag‐peptide fusion proteins which irreversibly bind SpyCatcher‐modified proteins could overcome these limitations, especially for laccase or peroxidase display systems (Charrier et al., 2018; Molinari et al., 2021; Orozco‐Hidalgo et al., 2021). Interestingly, S‐layers can reassemble into crystalline 2D regular arrays after removal from their host using a disrupting treatment, enabling their potential application as functional nanomaterials that require no crosslinking step for immobilization (Figure 1) (Pallares et al., 2022; Sleytr et al., 2014). Re‐assembly of L. sphaericus S‐layer fusion proteins has been used to display functional laccases (Ferner‐Ortner‐Bleckmann et al., 2011), and other multimeric enzymes with linker inclusions (Ferner‐Ortner‐Bleckmann et al., 2013). Although S‐layer bioconjugation and 2D regular arrays overcome insertion size limits of the C. crescentus S‐layer platform, they require additional immobilization or recrystallization steps adding complexity and cost to bioprocess development.

SPORE SURFACE DISPLAY PLATFORMS

Under adverse environmental conditions, some bacterial species undergo sporulation to produce highly resistant dormant spores through asymmetric cell division that are then released from the mother cell on lysis (Hwang et al., 2011; Lin et al., 2020; Wang, Jiang, et al., 2021). Bacillus subtilis is a well‐characterized model organism, and its spores contain an array of coat proteins that can tolerate recombinant insertions (Lin et al., 2020; Wang, Jiang, et al., 2021). Bacillus subtilis spore surface display (BSSD) is a recently developed CSD alternative in which mother cells are engineered to produce spore coat‐fusion proteins to display heterologous domains on spore surfaces during sporulation (Hwang et al., 2011; Isticato et al., 2001; Lin et al., 2020; Wang, Jiang, et al., 2021) (Figure 2). Spore ability to survive harsh conditions (due to their multiple, thick coat layers), easy purification, and generally‐recognized‐as‐safe (GRAS) designation, makes BSSD particularly useful for industrial applications (Chen, Ullah, & Jia, 2017; Cho et al., 2011). Wang and colleagues recently used BSSD to increase haloalkane dehalogenase stability and activity in harsh chemical environments, with implications for remediation of toxic halogenate‐contaminated soils (Wang et al., 2019).

FIGURE 2.

FIGURE 2

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The process of plasmid programmed spore modification for display of heterologous proteins on B. subtilis spore coats. On sporulation, both the forespore and mother cell contain recombinant plasmid copies, but it is only transcribed and translated to fusion proteins in the mother cell for spore coat assembly. After mother cell lysis, recombinant spores are released and can be isolated for whole‐spore biocatalyst application. BSSD can result in recombinant DNA‐free spores if spore plasmids are engineered for nuclease expression (Quijano & Sahin, 2021).

One important advantage of BSSD is its ability to display more complex protein configurations than S‐layer display (Cho et al., 2011; Hwang et al., 2011; Park et al., 2019). As the spore coat forms within the mother cell, the coat‐fusion proteins do not require trans‐membrane secretion before folding, which enables larger, multimeric and co‐factor containing enzymes to assemble correctly (Hwang et al., 2011). For example, functional laccases for degradation of dye effluent have been successfully displayed with potential application in lignocellulosic biomass conversion (Cho et al., 2011; Park et al., 2019). Additionally, recent work shows that heterologous enzymes can be co‐displayed on the same spore using different carrier proteins to create immobilized enzyme cascades that regenerate cofactors (required by oxidoreductase laccases) (Chen, Ullah, & Jia, 2017; Gao et al., 2016; Park et al., 2019). However, utilizing BSSD for cellulase and co‐immobilized cofactor‐regenerating enzyme display to catalyse lignocellulosic biomass conversion would likely come up against spore germination issues induced by sugar monomers in solution. Alternatively, BSSD is under development for drop‐in biodiesel production from waste oils through functional, multimeric lipase display (Karava et al., 2021). Although BSSD shows promise in the display of complex protein configurations under harsh conditions, display density (estimated as up to 4000 copies/μm2, depending on spore surface protein used) (Negri et al., 2013; Rodenburg et al., 2014) and consistency remains a limiting factor when compared to S‐layer display (Lin et al., 2020). Lower display density and predictability decreases enzyme efficiency and may prevent certain applications.

A potential barrier to in situ applications of S‐layer and spore display is the presence of recombinant DNA in the producing cells (Wang, Selvamani, et al., 2021). Although this can in principle be managed through the use of kill switches or other containment strategies in closed bioreactors, open systems present clear containment challenges. Non‐conventional versions of S‐layer and spore display as well as intracellular inclusion display technologies are currently being developed that do not contain recombinant material increasing social licence. For example, Quijano and Sahin (Quijano & Sahin, 2021), demonstrated self‐digesting plasmids post spore formation, to create DNA‐free, phenotypically engineered spores (Figure 2). Such GRAS bioengineered spores could be used for in situ remediation and open wastewater treatment. Bacterial ghost systems may also facilitate the creation of non‐recombinant CSD display systems (Riedmann et al., 2003). These functional membrane inclusions derived from gram‐negative bacteria are devoid of all cytoplasmic content and DNA through E‐mediated lysis and nuclease expression. Riedmann and colleagues demonstrated the combination of ghost display with Bacillus stearothermophilus S‐layer display for vaccine application, but nonbiomedical application remains to be realized (Riedmann et al., 2003).

EMERGING TRANS‐METABOLIC SYSTEMS

Recent advances in displaying heterologous functional domains on self‐assembling, intracellular inclusion surfaces including polyhydroxyalkanoate (PHA) particles, virus‐like particles and magnetosomes, provide a promising alternative to whole cell display systems resulting in non‐canonical or ‘trans‐metabolic’ systems (Nikel & de Lorenzo, 2018; Wong et al., 2020) (Figure 1). For example, functionalized magnetosomes have been shown to tolerate large insertions with display densities of up to 100 copies/magnetosome (Lang & Schüler, 2008; Mickoleit & Schüler, 2018), which corresponds to up to ~12,000 copies/μm2. Magnetosomes are intracellular inclusions in magnetotactic bacteria, consisting of iron oxide crystals surrounded by a phospholipid bilayer (Kolinko et al., 2014). Functionalized magnetosomes can be engineered by inserting proteins or peptides into magnetosome membrane proteins (Figure 3) (Mickoleit & Schüler, 2018). Interestingly, magnetosome gene clusters can now be transferred to non‐magnetic bacteria enabling efficient magnetosome cultivation (Kolinko et al., 2014; Mickoleit et al., 2021), and rapid, continuous systems for magnetosome purification have been developed to increase scalability using reversible magnetic fields (Guo et al., 2011; Zwiener et al., 2021).

FIGURE 3.

FIGURE 3

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(A) In vivo heterologous protein immobilization using magnetosome display. Recombinant vectors containing a heterologous protein fused to a magnetosome membrane protein are expressed in magnetotactic bacteria (usually Magnetospirillum) under magnetite biomineralization conditions. Functionalised magnetosomes can be isolated through cell disruption treatment and magnetic separation techniques. (B) Heterologous protein gene copy number can be increased within the gene fusion to create immobilized arrays of multiple functional domains, which dramatically increases magnetosome display density per particle and therefore biocatalytic efficiency on application (Mickoleit & Schüler, 2018).

Whole cell, spore or intracellular inclusion immobilization will likely be necessary for application of enzyme display systems at scale. This format maintains a high concentration of enzyme, reduces recombinant contamination of any product outputs, and enables easy removal for replacement or recycling (Polakovič et al., 2017; Zhu et al., 2019). Recent advances in electrospinning and bioprinting enable displayed proteins to be immobilized on porous substrates without decreasing activity (Canbolat et al., 2013; Chen et al., 2018). Based on this demonstration, functionalized magnetosomes could be integrated into porous hydrogel matrices using electrospinning resulting in flow‐through formats for continuous water treatment. As with any multi‐step process, whole cell, spore or intracellular inclusion immobilization is more expensive to implement with potential implications for bioprocess development. However, Molinari and colleagues recently engineered a cell‐immobilizing extracellular matrix that self‐associates on secretion to overcome this problem (Molinari et al., 2021). S‐layer engineered C. crescentus can self‐immobilize naturally (Nomellini et al., 2004; Patel et al., 2010) with adhesive, stalk‐like appendages to form stable, monolayer biofilms. Alternatively, spore co‐display of functional enzymes and anchor domains could facilitate inherent spore binding ability to its support, for example, displaying cellulose binding domains for attachment to cellulose (Francisco et al., 1993; Wang et al., 2002), amenable to fibre brush or spiral sheet bioreactors (Xu et al., 2010).

Earth's microbiome provides a deep reservoir of genomic information that can be harnessed for biotechnology innovation. Indeed, the majority of enzymes used in industrial processes today are of microbial origin (Uchiyama & Miyazaki, 2009), and functional metagenomic screening paradigms have been developed to mine uncultivated microbial diversity for biological parts expressed in heterologous host chassis that can be used in biocatalyst discovery (Ferrer et al., 2009; Steele et al., 2009; Taupp et al., 2011). For example, substrate conversion screens have been used to recover genes encoding cellulases from a variety of environmental sources (Armstrong et al., 2015, 2019; Mewis et al., 2013), and biosensors have been developed for co‐culture based recovery of lignin transforming enzymes (Ho et al., 2018; Strachan et al., 2014). With respect to metals, several studies have used functional metagenomics to recover metal resistance genes related to nickel (Mirete et al., 2007), copper (Xing et al., 2020) and cadmium (Zheng et al., 2019). Alternatively, researchers can combine in silico detection and targeted gene expression to recover metal binding proteins or domains from metagenomes. For example, Li and colleagues used custom database searches to recovery and test metallothionein genes capable of conferring copper, cadmium and zinc resistance through enhanced sorption in E. coli (Li et al., 2020). Ziller and colleagues used a similar approach to find metallothionein's from soil eukaryotic metatranscriptomes conferring cadmium and zinc resistance in yeast (Ziller et al., 2017).

The first CSD systems reported were in filamentous phages (Chiswell & McCaffery, 1992; Scott & Smith, 1990), which were used as a means of screening antibody fragments contained within phage libraries against antigens to great success. Since then, surface display systems have been adopted for use in targeted screening, for example, affinity protein binding or active variant detection (Heyde et al., 2021; Jahns & Rehm, 2012; Kronqvist et al., 2008) but not in functional metagenomic screening of uncultivated microbial communities. A metagenomic library constructed for and transformed into a C. crescentus host using a modified vector system would enable S‐layer display and consequent external screening of its contents. For example, the use of S‐layer display as a screening paradigm (Nomellini et al., 2004) for metal‐binding proteins or domains. Once identified, these biological parts could be used to engineer relevant industrial chassis for use in remediation and waste resource recovery applications.

CONCLUSION

Protein display on cell, spore, intracellular particle, or functionalized material surfaces represents a promising biotechnology innovation for remediation and waste resource recovery within an emerging bioeconomy. This includes the use of ghost cells or nanoparticles with surface attached proteins for in situ application as well as the development of hybrid materials compatible with solid‐state filtration and bioreactor systems. Integration of functional metagenomic screening into compatible display platforms will expand the search space for substrate binding or catalytic domains of interest and enable more rapid prototyping of known or novel recovery applications across sectors. Meanwhile, combinatorial display approaches manifesting multiple proteins on S layers (Nomellini et al., 2004) or spores (Gao et al., 2016), as well as engineered microbial consortia interfacing with CDS cells (Shahab et al., 2020) present new opportunities to harness the metabolic problem solving power of microorganisms based on the same design principals shaping microbial community structure and function in the world around us.

AUTHOR CONTRIBUTIONS

Beth India Davenport: Conceptualization (equal); investigation (equal); visualization (lead); writing – original draft (lead); writing – review and editing (equal). Steven J. Hallam: Conceptualization (supporting); funding acquisition (lead); project administration (lead); supervision (lead); writing – original draft (supporting); writing – review and editing (equal).

CONFLICT OF INTEREST

Steven J. Hallam is a co‐founder of Koonkie Inc., a bioinformatics consulting company that designs and provides scalable algorithmic and data analytics solutions in the cloud.

ACKNOWLEDGEMENTS

The authors thank Kateryna Ievdokymenko and the UBC iGEM 2016 iGEM team, as well as Tom Pfeifer in the Biofactorial automation core facility at UBC for technical advice and support. The authors also thank current members of the Hallam lab including Avery Noonan and Stefanie Sternagle for helpful discussion along the way, and John Smit and John Nomellini for their technical insight. This work was performed under the auspices of the Natural Sciences and Engineering Research Council (NSERC) of Canada, Genome British Columbia, and the Canada Foundation for Innovation (CFI).

Davenport, B. & Hallam, S.J. (2023) Emerging enzyme surface display systems for waste resource recovery. Environmental Microbiology, 25(2), 241–249. Available from: 10.1111/1462-2920.16284

Funding information Genome British Columbia, and the Canada Foundation for Innovation (CFI); Natural Sciences and Engineering Research Council (NSERC) of Canada

DATA AVAILABILITY STATEMENT

No new data was generated in developing the current review.

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