Exploring untapped bacterial communities and potential polypropylene-degrading enzymes from mangrove sediment through metagenomics analysis.

The versatility of plastic has resulted in huge amounts being consumed annually. Mismanagement of post-consumption plastic material has led to plastic waste pollution. Biodegradation of plastic by microorganisms has emerged as a potential solution to this problem. Therefore, this study aimed to investigate the microbial communities involved in the biodegradation of polypropylene (PP). Mangrove soil was enriched with virgin PP sheets or chemically pretreated PP comparing between 2 and 4 months enrichment to promote the growth of bacteria involved in PP biodegradation. The diversity of the resulting microbial communities was accessed through 16S metagenomic sequencing. The results indicated that Xanthomonadaceae, unclassified Gaiellales, and Nocardioidaceae were promoted during the enrichment. Additionally, shotgun metagenomics was used to investigate enzymes involved in plastic biodegradation. The results revealed the presence of various putative plastic-degrading enzymes in the mangrove soil, including alcohol dehydrogenase, aldehyde dehydrogenase, and alkane hydroxylase. The degradation of PP plastic was determined using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), and Water Contact Angle measurements. The FTIR spectra showed a reduced peak intensity of enriched and pretreated PP compared to the control. SEM images revealed the presence of bacterial biofilms as well as cracks on the PP surface. Corresponding to the FTIR and SEM analysis, the water contact angle measurement indicated a decrease in the hydrophobicity of PP and pretreated PP surface during the enrichment.

Seasonality, rather than estuarine gradient or particle suspension/sinking dynamics, determines estuarine carbon distributions.

Estuaries are important components of the global carbon cycle; exchanging carbon between aquatic, atmospheric, and terrestrial environments, representing important loci for blue carbon storage and greenhouse gas emissions. However, how estuarine gradients affect sinking/suspended particles, and dissolved organic matter dynamic interactions remains unexplored. We fractionated suspended/sinking particles to assess and characterise carbon fate differences. We investigated bacterial colonisation (SYBR Green I) and exopolymer concentrations (TEP/CSP) with microscopy staining techniques. C/H/N and dry weight analysis identified particle composition differences. Meanwhile, nutrient and carbon analysis, and excitation and emission matrix evaluations with a subsequent parallel factor (PARAFAC) analysis characterised dissolved organic matter. The lack of clear salinity driven patterns in our study are presumably due to strong mixing forces and high particle heterogeneity along the estuary, with only density differences between suspended and sinking particles. Elbe estuary particles' organic portion is made up of marine-like (sinking) and terrestrial-like (suspended) signatures. Salinity did not have a significant role in microbial degradation and carbon composition, although brackish estuary portions were more biologically active. Indicative of increased degradation rates, leading to decreased greenhouse gas emissions, which are especially relevant for estuaries, with their disproportionate greenhouse gas emissions. Bacterial colonisation decreased seawards, indicative of decreased degradation, and shifts in microbial community composition and functions. Our findings span diverse strands of research, concerning steady carbon contributions from both marine and terrestrial sources, carbon aromaticity, humification index, and bioavailability. Their integration highlights the importance of the Elbe estuary as a model system, providing robust information for future policy decisions affecting dissolved and particulate matter dynamics within the Elbe Estuary.

The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

Andean soil-derived lignocellulolytic bacterial consortium as a source of novel taxa and putative plastic-active enzymes.

An easy and straightforward way to engineer microbial environmental communities is by setting up liquid enrichment cultures containing a specific substrate as the sole source of carbon. Here, we analyzed twenty single-contig high-quality metagenome-assembled genomes (MAGs) retrieved from a microbial consortium (T6) that was selected by the dilution-to-stimulation approach using Andean soil as inoculum and lignocellulose as a selection pressure. Based on genomic metrics (e.g., average nucleotide and amino acid identities) and phylogenomic analyses, 15 out of 20 MAGs were found to represent novel bacterial species, with one of those (MAG_26) belonging to a novel genus closely related to Caenibius spp. (Sphingomonadaceae). Following the rules and requirements of the SeqCode, we propose the name Andeanibacterium colombiense gen. nov., sp. nov. for this taxon. A subsequent functional annotation of all MAGs revealed that MAG_7 (Pseudobacter hemicellulosilyticus sp. nov.) contains 20, 19 and 16 predicted genes from carbohydrate-active enzymes families GH43, GH2 and GH92, respectively. Its lignocellulolytic gene profile resembles that of MAG_2 (the most abundant member) and MAG_3858, both of which belong to the Sphingobacteriaceae family. Using a database that contains experimentally verified plastic-active enzymes (PAZymes), twenty-seven putative bacterial polyethylene terephthalate (PET)-active enzymes (i.e., alpha/beta-fold hydrolases) were detected in all MAGs. A maximum of five putative PETases were found in MAG_3858, and two PETases were found to be encoded by A. colombiense. In conclusion, we demonstrate that lignocellulose-enriched liquid cultures coupled with genome-resolved metagenomics are suitable approaches to unveil the hidden bacterial diversity and its polymer-degrading potential in Andean soil ecosystems.

New dienelactone hydrolase from microalgae bacterial community-Antibiofilm activity against fish pathogens and potential applications for aquaculture.

Biofilms are resistant to many traditional antibiotics, which has led to search for new antimicrobials from different and unique sources. To harness the potential of aquatic microbial resources, we analyzed the meta-omics datasets of microalgae-bacteria communities and mined them for potential antimicrobial and quorum quenching enzymes. One of the most interesting candidates (Dlh3), a dienelactone hydrolase, is a α/ß-protein with predicted eight α-helices and eight ß-sheets. When it was applied to one of the major fish pathogens, Edwardsiella anguillarum, the biofilm development was reproducibly inhibited by up to 54.5%. The transcriptome dataset in presence of Dlh3 showed an upregulation in functions related to self-defense like active genes for export mechanisms and transport systems. The most interesting point regarding the biotechnological potential for aquaculture applications of Dlh3 are clear evidence of biofilm inhibition and that health and division of a relevant fish cell model (CHSE-214) was not impaired by the enzyme.

Extracellular proteins enhance Cupriavidus pauculus nickel tolerance and cell aggregate formation.

Heavy metal-resistant bacteria secrete extracellular proteins (e-PNs). However, the role of e-PNs in heavy metal resistance remains elusive. Here Fourier Transform Infrared Spectroscopy implied that N-H, C = O and NH2-R played a crucial role in the adsorption and resistance of Ni2+ in the model organism Cuprividus pauculus 1490 (C. pauculus). Proteinase K treatment reduced Ni2+ resistance of C. pauculus underlining the essential role of e-PNs. Further three-dimension excitation-emission matrix fluorescence spectroscopy analysis demonstrated that tryptophan proteins as part of the e-PNs increased significantly with Ni2+ treatment. Proteomic and quantitative real-time polymerase chain reaction data indicated that major changes were induced in the metabolism of C. pauculus in response to Ni2+. Among those lipopolysaccharide biosynthesis, general secretion pathways, Ni2+-affiliated transporters and multidrug efflux play an essential role in Ni2+ resistance. Altogether the results provide a conceptual model for comprehending how e-PNs contribute to bacterial resistance and adsorption of Ni2+.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known, and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific hidden Markov model. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, ß-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

Low-Fouling and Antibacterial Polymer Brushes via Surface-Initiated Polymerization of a Mixed Zwitterionic and Cationic Monomer.

The use of surface-grafted polymer brushes with combined low-fouling and antibacterial functionality is an attractive strategy to fight biofilm formation. This report describes a new styrene derivative combining a quaternary ammonium group with a sulfobetaine group in one monomer. Surface-initiated polymerization of this monomer on titanium and a polyethylene (PE) base material gave bifunctional polymer brush layers. Grafting was achieved via surface-initiated atom transfer radical polymerization from titanium or heat-induced free-radical polymerization from plasma-activated PE. Both techniques gave charged polymer layers with a thickness of over 750 nm, as confirmed by ToF-SIMS-SPM measurements. The chemical composition of the brush polymers was confirmed by XPS and FT-IR analysis. The surface charge, characterized by the ζ potential, was positive at different pH values, and the number of solvent-accessible excess ammonium groups was found to be ∼1016 N+/cm2. This led to strong antibacterial activity against Gram-positive and Gram-negative bacteria that was superior to a structurally related contact-active polymeric quaternary ammonium brush. In addition to this antibacterial activity, good low-fouling properties of the dual-function polymer brushes against Gram-positive and Gram-negative bacteria were found. This dual functionality is most likely due to the combination of antibacterial quaternary ammonium groups with antifouling sulfobetaines. The combination of both groups in one monomer allows the preparation of bifunctional brush polymers with operationally simple polymerization techniques.

Quantitative Insights and Visualization of Antimicrobial Tolerance in Mixed-Species Biofilms.

Biofilms are a major problem in hard-to-heal wounds. Moreover, they are composed of different species and are often tolerant to antimicrobial agents. At the same time, interspecific synergy and/or competition occurs when some bacterial species clash. For this reason, the tolerance of two dual-species wound biofilm models of Pseudomonas aeruginosa and Staphylococcus aureus or Enterococcus faecium against antimicrobials and antimicrobial dressings were analyzed quantitatively and by confocal laser scanning microscopy (CLSM). The results were compared to findings with planktonic bacteria. Octenidine-dihydrochloride/phenoxyethanol and polyhexamethylene biguanide (PHMB) irrigation solutions showed a significant, albeit delayed reduction in biofilm bacteria, while the PHMB dressing was not able to induce this effect. However, the cadexomer-iodine dressing caused a sustained reduction in and killed almost all bacteria down to 102 cfu/mL within 6 days compared to the control (1010 cfu/mL). By means of CLSM in untreated human biofilm models, it became evident that P. aeruginosa dominates over E. faecium and S. aureus. Additionally, P. aeruginosa appeared as a vast layer at the bottom of the samples, while S. aureus formed grape-like clusters. In the second model, the distribution was even clearer. Only a few E. faecium were visible, in contrast to the vast layer of P. aeruginosa. It seems that the different species avoid each other and seek their respective niches. These mixed-species biofilm models showed that efficacy and tolerance to antimicrobial substances are nearly species-independent. Their frequent application appears to be important. The bacterial wound biofilm remains a challenge in treatment and requires new, combined therapy options.

Stenotrophomonas maltophilia affects the gene expression profiles of the major pathogens Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro multispecies biofilm model.

IMPORTANCE: In the past, studies have focused on bacterial pathogenicity in mono-species infections, in part ignoring the clinical relevance of diseases caused by more than one pathogen (i.e., polymicrobial infections). However, it is now common knowledge that multiple bacteria species are often involved in the course of an infection. For treatment of such infections, it is absolutely important to understand the dynamics of species interactions at possible infection sites and the molecular mechanisms behind these interactions. Here, we studied the impact of Stenotrophomonas maltophilia on its commensals Pseudomonas aeruginosa and Staphylococcus aureus in multispecies biofilms. We analyzed the 3D structural architectures of dual- and triple-species biofilms, niche formation within the biofilms, and the interspecies interactions on a molecular level. RNAseq data identified key genes involved in multispecies biofilm formation and interaction as potential drug targets for the clinical combat of multispecies infection with these major pathogens.

Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia.

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate.

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 Å. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.

Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Pseudomonas aeruginosa responds to altered membrane phospholipid composition by adjusting the production of two-component systems, proteases and iron uptake proteins.

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. To achieve these, bacteria use adaptation mechanisms involving covalent modification and remodelling of the acyl chain length of PLs. However, little is known about bacterial pathways regulated by PLs. Here, we investigated proteomic changes in the biofilm of P. aeruginosa phospholipase mutant (∆plaF) with altered membrane PL composition. The results revealed profound alterations in the abundance of many biofilm-related two-component systems (TCSs), including accumulation of PprAB, a key regulator of the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of several proteases, in ∆plaF, indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional response. Moreover, proteomics and biochemical assays revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in ∆plaF, while proteins from alternative iron-uptake systems were accumulated. These suggest that PlaF may function as a switch between different iron-acquisition pathways. The observation that PL-acyl chain modifying and PL synthesis enzymes were overproduced in ∆plaF reveals the interconnection of degradation, synthesis and modification of PLs for proper membrane homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that alteration of PL composition in ∆plaF plays a role for the global adaptive response in P. aeruginosa mediated by TCSs and proteases. Our study revealed the global regulation of virulence and biofilm by PlaF and suggests that targeting this enzyme may have therapeutic potential.

Zwitterionic surface modification of polyethylene via atmospheric plasma-induced polymerization of (vinylbenzyl-)sulfobetaine and evaluation of antifouling properties.

Zwitterionic polymer brushes were grafted from bulk polyethylene (PE) by air plasma activation of the PE surface followed by radical polymerization of the zwitterionic styrene derivative (vinylbenzyl)sulfobetaine (VBSB). Successful formation of dense poly-(VBSB)-brush layers was confirmed by goniometry, IR spectroscopy, XPS and ToF-SIMS analysis. The resulting zwitterionic layers are about 50-100 nm thick and cause extremely low contact angles of 10° (water) on the material. Correspondingly we determined a high density of > 1.0 × 1016 solvent accessible zwitterions/cm2 (corresponding to 2,0 *10-8 mol/cm2) by a UV-based ion-exchange assay with crystal violet. The elemental composition as determined by XPS and characteristic absorption bands in the IR spectra confirmed the presence of zwitterionic sulfobetaine polymer brushes. The antifouling properties of the resulting materials were evaluated in a bacterial adhesion test against gram-positive bacteria (S. aureus). We observed significantly reduced cellular adhesion of the zwitterionic material compared to pristine PE. These microbiological tests were complemented by tests in natural seawater. During a test period of 21 days, confocal microscopy revealed excellent antifouling properties and confirmed the operating antifouling mechanism. The procedure reported herein allows the efficient surface modification of bulk PE with zwitterionic sulfobetaine polymer brushes via a scalable approach. The resulting modified PE retains important properties of the bulk material and has excellent and durable antifouling properties.

Microbial enzymes will offer limited solutions to the global plastic pollution crisis.

Global economies depend on the use of fossil-fuel-based polymers with 360-400 million metric tons of synthetic polymers being produced per year. Unfortunately, an estimated 60% of the global production is disposed into the environment. Within this framework, microbiologists have tried to identify plastic-active enzymes over the past decade. Until now, this research has largely failed to deliver functional biocatalysts acting on the commodity polymers such as polyethylene (PE), polypropylene (PP), polyvinylchloride (PVC), ether-based polyurethane (PUR), polyamide (PA), polystyrene (PS) and synthetic rubber (SR). However, few enzymes are known to act on low-density and low-crystalline (amorphous) polyethylene terephthalate (PET) and ester-based PUR. These above-mentioned polymers represent >95% of all synthetic plastics produced. Therefore, the main challenge microbiologists are currently facing is in finding polymer-active enzymes targeting the majority of fossil-fuel-based plastics. However, identifying plastic-active enzymes either to implement them in biotechnological processes or to understand their potential role in nature is an emerging research field. The application of these enzymes is still in its infancy. Here, we summarize the current knowledge on microbial plastic-active enzymes, their global distribution and potential impact on plastic degradation in industrial processes and nature. We further outline major challenges in finding novel plastic-active enzymes, optimizing known ones by synthetic approaches and problems arising through falsely annotated and unfiltered use of database entries. Finally, we highlight potential biotechnological applications and possible re- and upcycling concepts using microorganisms.

Screening Metagenomes for Algae Cell Wall Carbohydrates Degrading Hydrolases in Enrichment Cultures.

Sustainable use of natural products is one of the key challenges for the future. An increasing focus is on marine organic matter, mostly algae. New biotechnological tools for processing high amounts of micro- and macroalgae are necessary for efficient industrial degradation of marine matter. Secreted glycosyl hydrolases can be enriched and tested on the specific algae cell wall polymers of all algae groups (Rhodophyta; Phaeophyceae; Chlorophyta/Charophyta). Metagenomic analyses established new possibilities to screen algae-associated microbiomes for novel degrading enzymes in combination with sequence-based function prediction.

The PET-Degrading Potential of Global Metagenomes: From In Silico Mining to Active Enzymes.

Against the background of the steadily increasing amount of plastic waste in the sea and on land, it is more important than ever to find ways out of this situation. In recent years, microorganisms have been discovered that are capable of degrading artificial polymers such as polyethylene terephthalate (PET). Even if the turnover rates of the enzymes responsible for this reaction may be too low to solve the global plastic pollution problem, it is still of great societal interest to find microorganisms that are able to degrade the polymer. The corresponding enzymes, PET esterases (PETases) can be used in biotechnological processes and could contribute to a resource-saving circular economy. In this chapter, we present a sequence-based in silico screening method to find new PETases in metagenomic datasets. This method can easily be adapted to find other enzyme classes. We also list a number of assays that can be used to test the enzymes for activity on PET as well as other substrates.

Metagenomic Screening of a Novel PET Esterase via In Vitro Expression System.

Metagenomic screening is a widely applied biotechnological approach for screening of novel industrial enzymes. The traditional method of metagenomic screening is based on the functional analyses of heterologously expressed environmental genes in a suitable host, which is the bottleneck of this method. To avoid limitation from the clone-dependent system, an in vitro expression technology has been developed in combination with next-generation sequencing and bioinformatics. First, the sequence profile of a target enzyme, e.g., poly(ethylene terephthalate) esterase in this protocol, is constructed according to the sequences of well-characterized enzymes. Then, the sequence screening is performed with this computationally generated profile among all available metagenomic databases. Afterwards, the candidate genes are synthesized and expressed in vitro with RNA polymerase and translation machinery from special cell extract. Finally, such in vitro produced enzymes are directly applied for the functional analyses. Comparing to the traditional screening methods, this in vitro screening technology can not only save time and materials, but also be easily developed for high-throughput screening with an automatic pipetting robot.

An Ultra-Sensitive Comamonas thiooxidans Biosensor for the Rapid Detection of Enzymatic Polyethylene Terephthalate (PET) Degradation.

Polyethylene terephthalate (PET) is a prevalent synthetic polymer that is known to contaminate marine and terrestrial environments. Currently, only a limited number of PET-active microorganisms and enzymes (PETases) are known. This is in part linked to the lack of highly sensitive function-based screening assays for PET-active enzymes. Here, we report on the construction of a fluorescent biosensor based on Comamonas thiooxidans strain S23. C. thiooxidans S23 transports and metabolizes TPA, one of the main breakdown products of PET, using a specific tripartite tricarboxylate transporter (TTT) and various mono- and dioxygenases encoded in its genome in a conserved operon ranging from tphC-tphA1. TphR, an IclR-type transcriptional regulator is found upstream of the tphC-tphA1 cluster where TPA induces transcription of tphC-tphA1 up to 88-fold in exponentially growing cells. In the present study, we show that the C. thiooxidans S23 wild-type strain, carrying the sfGFP gene fused to the tphC promoter, senses TPA at concentrations as low as 10 µM. Moreover, a deletion mutant lacking the catabolic genes involved in TPA degradation thphA2-A1 (ΔtphA2A3BA1) is up to 10,000-fold more sensitive and detects TPA concentrations in the nanomolar range. This is, to our knowledge, the most sensitive reporter strain for TPA and we demonstrate that it can be used for the detection of enzymatic PET breakdown products. IMPORTANCE Plastics and microplastics accumulate in all ecological niches. The construction of more sensitive biosensors allows to monitor and screen potential PET degradation in natural environments and industrial samples. These strains will also be a valuable tool for functional screenings of novel PETase candidates and variants or monitoring of PET recycling processes using biocatalysts. Thereby they help us to enrich the known biodiversity and efficiency of PET degrading organisms and enzymes and understand their contribution to environmental plastic degradation.