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Polyethylene terephthalate (PET) is one of the most important synthetic polymers used today. Unfortunately, the polymers accumulate in nature and to date no highly active enzymes are known that can degrade it at high velocity. Enzymes involved in PET degradation are mainly α- and β-hydrolases, like cutinases and related enzymes (EC 3.1.1). Currently, only a small number of such enzymes are well characterized. In this work, a search algorithm was developed that identified 504 possible PET hydrolase candidate genes from various databases. A further global search that comprised more than 16 Gb of sequence information within 108 marine and 25 terrestrial metagenomes obtained from the Integrated Microbial Genome (IMG) database detected 349 putative PET hydrolases. Heterologous expression of four such candidate enzymes verified the function of these enzymes and confirmed the usefulness of the developed search algorithm. In this way, two novel and thermostable enzymes with high potential for downstream application were partially characterized. Clustering of 504 novel enzyme candidates based on amino acid similarities indicated that PET hydrolases mainly occur in the phyla of Actinobacteria, Proteobacteria, and Bacteroidetes Within the Proteobacteria, the Betaproteobacteria, Deltaproteobacteria, and Gammaproteobacteria were the main hosts. Remarkably enough, in the marine environment, bacteria affiliated with the phylum Bacteroidetes appear to be the main hosts of PET hydrolase genes, rather than Actinobacteria or Proteobacteria, as observed for the terrestrial metagenomes. Our data further imply that PET hydrolases are truly rare enzymes. The highest occurrence of 1.5 hits/Mb was observed in sequences from a sample site containing crude oil.IMPORTANCE Polyethylene terephthalate (PET) accumulates in our environment without significant microbial conversion. Although a few PET hydrolases are already known, it is still unknown how frequently they appear and with which main bacterial phyla they are affiliated. In this study, deep sequence mining of protein databases and metagenomes demonstrated that PET hydrolases indeed occur at very low frequencies in the environment. Furthermore, it was possible to link them to phyla that were previously not known to harbor such enzymes. This work contributes novel knowledge on the phylogenetic relationships, the recent evolution, and the global distribution of PET hydrolases. Finally, we describe the biochemical traits of four novel PET hydrolases.

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the IsPETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as IsPETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation.

Certain members of the Actinobacteria and Proteobacteria are known to degrade polyethylene terephthalate (PET). Here, we describe the first functional PET-active enzymes from the Bacteroidetes phylum. Using a PETase-specific Hidden-Markov-Model- (HMM-) based search algorithm, we identified several PETase candidates from Flavobacteriaceae and Porphyromonadaceae. Among them, two promiscuous and cold-active esterases derived from Aequorivita sp. (PET27) and Kaistella jeonii (PET30) showed depolymerizing activity on polycaprolactone (PCL), amorphous PET foil and on the polyester polyurethane Impranil® DLN. PET27 is a 37.8 kDa enzyme that released an average of 174.4 nmol terephthalic acid (TPA) after 120 h at 30°C from a 7 mg PET foil platelet in a 200 μl reaction volume, 38-times more than PET30 (37.4 kDa) released under the same conditions. The crystal structure of PET30 without its C-terminal Por-domain (PET30ΔPorC) was solved at 2.1 Å and displays high structural similarity to the Is PETase. PET30 shows a Phe-Met-Tyr substrate binding motif, which seems to be a unique feature, as Is PETase, LCC and PET2 all contain Tyr-Met-Trp binding residues, while PET27 possesses a Phe-Met-Trp motif that is identical to Cut190. Microscopic analyses showed that K. jeonii cells are indeed able to bind on and colonize PET surfaces after a few days of incubation. Homologs of PET27 and PET30 were detected in metagenomes, predominantly aquatic habitats, encompassing a wide range of different global climate zones and suggesting a hitherto unknown influence of this bacterial phylum on man-made polymer degradation. [ABSTRACT FROM AUTHOR]

Upcycling wastes into valuable products by mixed microbial communities has recently received considerable attention. Sustainable production of high-value substances from one-carbon (C1) compounds, e.g., methanol supplemented as an external electron donor in bioreactors for wastewater treatment, is a promising application of upcycling. This study undertook a gene-centric approach to screen valuable production potentials from mixed culture biomass, removing organic carbon and nitrogen from landfill leachate. To this end, the microbial community of the activated sludge from a landfill leachate treatment plant and its metabolic potential for the production of seven valuable products were investigated. The DNA extracted from the activated sludge was subjected to shotgun metagenome sequencing to analyze the microbial taxonomy and functions associated with producing the seven products. The functional analysis confirmed that the activated sludge could produce six of the valuable products, ectoine, polyhydroxybutyrate (PHB), zeaxanthin, astaxanthin, acetoin, and 2,3-butanediol. Quantification of the detected functional gene hit numbers for these valuable products as a primary trial identified a potential rate-limiting metabolic pathway, e.g., conversion of L-2,4-diaminobutyrate into N-γ-acetyl-L2,4,-diaminobutyrate during the ectoine biosynthesis. Overall, this study demonstrated that primary screening by the proposed gene-centric approach can be used to evaluate the potential for the production of valuable products using mixed culture or single microbe in engineered systems. The proposed approach can be expanded to sites where water purification is highly required, but resource recovery, or upcycling has not been implemented.

Upcycling wastes into valuable products by mixed microbial communities has recently received considerable attention. Sustainable production of high-value substances from one-carbon (C1) compounds, e.g., methanol supplemented as an external electron donor in bioreactors for wastewater treatment, is a promising application of upcycling. This study undertook a gene-centric approach to screen valuable production potentials from mixed culture biomass, removing organic carbon and nitrogen from landfill leachate. To this end, the microbial community of the activated sludge from a landfill leachate treatment plant and its metabolic potential for the production of seven valuable products were investigated. The DNA extracted from the activated sludge was subjected to shotgun metagenome sequencing to analyze the microbial taxonomy and functions associated with producing the seven products. The functional analysis confirmed that the activated sludge could produce six of the valuable products, ectoine, polyhydroxybutyrate (PHB), zeaxanthin, astaxanthin, acetoin, and 2,3-butanediol. Quantification of the detected functional gene hit numbers for these valuable products as a primary trial identified a potential rate-limiting metabolic pathway, e.g., conversion of L -2,4-diaminobutyrate into N -γ-acetyl-L2,4,-diaminobutyrate during the ectoine biosynthesis. Overall, this study demonstrated that primary screening by the proposed gene-centric approach can be used to evaluate the potential for the production of valuable products using mixed culture or single microbe in engineered systems. The proposed approach can be expanded to sites where water purification is highly required, but resource recovery, or upcycling has not been implemented. [ABSTRACT FROM AUTHOR]

Abstract Determining the effects of antimicrobial therapies on airway microbiology at a population-level is essential. Such analysis allows, for example, surveillance of antibiotic-induced changes in pathogen prevalence, the emergence and spread of antibiotic resistance, and the transmission of multi-resistant organisms. However, current analytical strategies for understanding these processes are limited. Culture- and PCR-based assays for specific microbes require the a priori selection of targets, while antibiotic sensitivity testing typically provides no insight into either the molecular basis of resistance, or the carriage of resistance determinants by the wider commensal microbiota. Shotgun metagenomic sequencing provides an alternative approach that allows the microbial composition of clinical samples to be described in detail, including the prevalence of resistance genes and virulence traits. While highly informative, the application of metagenomics to large patient cohorts can be prohibitively expensive. Using sputum samples from a randomised placebo-controlled trial of erythromycin in adults with bronchiectasis, we describe a novel, cost-effective strategy for screening patient cohorts for changes in resistance gene prevalence. By combining metagenomic screening of pooled DNA extracts with validatory quantitative PCR-based analysis of candidate markers in individual samples, we identify population-level changes in the relative abundance of specific macrolide resistance genes. This approach has the potential to provide an important adjunct to current analytical strategies, particularly within the context of antimicrobial clinical trials.

Historically, microbes from the environment have been a reliable source for novel bio-active compounds. Cloning and expression of metagenomic DNA in heterologous strains of bacteria has broadened the range of potential compounds accessible. However, such metagenomic libraries have been under-exploited for applications in mammalian cells because of a lack of integrated methods. We present an innovative platform to systematically mine natural resources for pro-apoptotic compounds that relies on the combination of bacterial delivery and droplet microfluidics. Using the violacein operon from C. violaceum as a model, we demonstrate that E. coli modified to be invasive can serve as an efficient delivery vehicle of natural compounds. This approach permits the seamless screening of metagenomic libraries with mammalian cell assays and alleviates the need for laborious extraction of natural compounds. In addition, we leverage the unique properties of droplet microfluidics to amplify bacterial clones and perform clonal screening at high-throughput in place of one-compound-per-well assays in multi-well format. We also use droplet microfluidics to establish a cell aggregate strategy that overcomes the issue of background apoptosis. Altogether, this work forms the foundation of a versatile platform to efficiently mine the metagenome for compounds with therapeutic potential. [ABSTRACT FROM AUTHOR]

Metagenomes constitute a major source for the identification of novel enzymes for industrial applications. However, current functional screening methods are hindered by the limited transcription efficiency of foreign metagenomic genes. To overcome this constraint, we introduced the ‘Enforced Transcription’ technique, which involves the random insertion of the bi-directional T7 promoter into a metagenomic fosmid library. Then the effect of enforced transcription was quantitatively assessed by screening for metagenomic lipolytic genes encoding enzymes whose catalytic activity forms halos on tributyrin agar plates. The metagenomic library containing the enforced transcription system yielded a significantly increased number of screening hits with lipolytic activity compared to the library without random T7 promoter insertions. Additional sequence analysis revealed that the hits from the enforced transcription library had greater genetic diversity than those from the original metagenome library. Enhancing heterologous expression using the T7 promoter should enable the identification of greater numbers of diverse novel biocatalysts from the metagenome than possible using conventional metagenome screening approaches. [ABSTRACT FROM AUTHOR]

Historically, microbes from the environment have been a reliable source for novel bio-active compounds. Cloning and expression of metagenomic DNA in heterologous strains of bacteria has broadened the range of potential compounds accessible. However, such metagenomic libraries have been under-exploited for applications in mammalian cells because of a lack of integrated methods. We present an innovative platform to systematically mine natural resources for pro-apoptotic compounds that relies on the combination of bacterial delivery and droplet microfluidics. Using the violacein operon from C. violaceum as a model, we demonstrate that E. coli modified to be invasive can serve as an efficient delivery vehicle of natural compounds. This approach permits the seamless screening of metagenomic libraries with mammalian cell assays and alleviates the need for laborious extraction of natural compounds. In addition, we leverage the unique properties of droplet microfluidics to amplify bacterial clones and perform clonal screening at high-throughput in place of one-compound-per-well assays in multi-well format. We also use droplet microfluidics to establish a cell aggregate strategy that overcomes the issue of background apoptosis. Altogether, this work forms the foundation of a versatile platform to efficiently mine the metagenome for compounds with therapeutic potential.

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Determining the effects of antimicrobial therapies on airway microbiology at a population-level is essential. Such analysis allows, for example, surveillance of antibiotic-induced changes in pathogen prevalence, the emergence and spread of antibiotic resistance, and the transmission of multi-resistant organisms. However, current analytical strategies for understanding these processes are limited. Culture- and PCR-based assays for specific microbes require the a priori selection of targets, while antibiotic sensitivity testing typically provides no insight into either the molecular basis of resistance, or the carriage of resistance determinants by the wider commensal microbiota. Shotgun metagenomic sequencing provides an alternative approach that allows the microbial composition of clinical samples to be described in detail, including the prevalence of resistance genes and virulence traits. While highly informative, the application of metagenomics to large patient cohorts can be prohibitively expensive. Using sputum samples from a randomised placebo-controlled trial of erythromycin in adults with bronchiectasis, we describe a novel, cost-effective strategy for screening patient cohorts for changes in resistance gene prevalence. By combining metagenomic screening of pooled DNA extracts with validatory quantitative PCR-based analysis of candidate markers in individual samples, we identify population-level changes in the relative abundance of specific macrolide resistance genes. This approach has the potential to provide an important adjunct to current analytical strategies, particularly within the context of antimicrobial clinical trials. [ABSTRACT FROM AUTHOR]