From friends to foes: fungi could be emerging marine sponge pathogens under global change scenarios.

Global change, experienced in the form of ocean warming and pollution by man-made goods and xenobiotics, is rapidly affecting reef ecosystems and could have devastating consequences for marine ecology. Due to their critical role in regulating marine food webs and trophic connections, sponges are an essential model for studying and forecasting the impact of global change on reef ecosystems. Microbes are regarded as major contributors to the health and survival of sponges in marine environments. While most culture-independent studies on sponge microbiome composition to date have focused on prokaryotic diversity, the importance of fungi in holobiont behavior has been largely overlooked. Studies focusing on the biology of sponge fungi are uncommon. Thus, our current understanding is quite limited regarding the interactions and "crosstalk" between sponges and their associated fungi. Anthropogenic activities and climate change may reveal sponge-associated fungi as novel emerging pathogens. Global change scenarios could trigger the expression of fungal virulence genes and unearth new opportunistic pathogens, posing a risk to the health of sponges and severely damaging reef ecosystems. Although ambitious, this hypothesis has not yet been proven. Here we also postulate as a pioneering hypothesis that manipulating sponge-associated fungal communities may be a new strategy to cope with the threats posed to sponge health by pathogens and pollutants. Additionally, we anticipate that sponge-derived fungi might be used as novel sponge health promoters and beneficial members of the resident sponge microbiome in order to increase the sponge's resistance to opportunistic fungal infections under a scenario of global change.

Enrichment of native plastic-associated biofilm communities to enhance polyester degrading activity.

Plastic pollution is an increasing worldwide problem urgently requiring a solution. While recycling rates are increasing globally, only 9% of all plastic waste has been recycled, and with the cost and limited downstream uses of recycled plastic, an alternative is needed. Here, we found that expanded polystyrene (EPS) promoted high levels of bacterial biofilm formation and sought out environmental EPS waste to characterize these native communities. We demonstrated that the EPS attached communities had limited plastic degrading activity. We then performed a long-term enrichment experiment where we placed a robust selection pressure on these communities by limiting carbon availability such that the waste plastic was the only carbon source. Seven of the resulting enriched bacterial communities had increased plastic degrading activity compared to the starting bacterial communities. Pseudomonas stutzeri was predominantly identified in six of the seven enriched communities as the strongest polyester degrader. Sequencing of one isolate of P. stutzeri revealed two putative polyesterases and one putative MHETase. This indicates that waste plastic-associated biofilms are a source for bacteria that have plastic-degrading potential, and that this potential can be unlocked through selective pressure and further in vitro enrichment experiments, resulting in biodegradative communities that are better than nature.

Purification and biochemical characterization of SM14est, a PET-hydrolyzing enzyme from the marine sponge-derived Streptomyces sp. SM14.

The successful enzymatic degradation of polyester substrates has fueled worldwide investigation into the treatment of plastic waste using bio-based processes. Within this realm, marine-associated microorganisms have emerged as a promising source of polyester-degrading enzymes. In this work, we describe the hydrolysis of the synthetic polymer PET by SM14est, a polyesterase which was previously identified from Streptomyces sp. SM14, an isolate of the marine sponge Haliclona simulans. The PET hydrolase activity of purified SM14est was assessed using a suspension-based assay and subsequent analysis of reaction products by UV-spectrophotometry and RP-HPLC. SM14est displayed a preference for high salt conditions, with activity significantly increasing at sodium chloride concentrations from 100 mM up to 1,000 mM. The initial rate of PET hydrolysis by SM14est was determined to be 0.004 s-1 at 45°C, which was increased by 5-fold to 0.02 s-1 upon addition of 500 mM sodium chloride. Sequence alignment and structural comparison with known PET hydrolases, including the marine halophile PET6, and the highly efficient, thermophilic PHL7, revealed conserved features of interest. Based on this work, SM14est emerges as a useful enzyme that is more similar to key players in the area of PET hydrolysis, like PHL7 and IsPETase, than it is to its marine counterparts. Salt-tolerant polyesterases such as SM14est are potentially valuable in the biological degradation of plastic particles that readily contaminate marine ecosystems and industrial wastewaters.

Isolation, identification, and biochemical characterization of a novel bifunctional phosphomannomutase/phosphoglucomutase from the metagenome of the brown alga Laminaria digitata.

Macroalgae host diverse epiphytic bacterial communities with potential symbiotic roles including important roles influencing morphogenesis and growth of the host, nutrient exchange, and protection of the host from pathogens. Macroalgal cell wall structures, exudates, and intra-cellular environments possess numerous complex and valuable carbohydrates such as cellulose, hemi-cellulose, mannans, alginates, fucoidans, and laminarin. Bacterial colonizers of macroalgae are important carbon cyclers, acquiring nutrition from living macroalgae and also from decaying macroalgae. Seaweed epiphytic communities are a rich source of diverse carbohydrate-active enzymes which may have useful applications in industrial bioprocessing. With this in mind, we constructed a large insert fosmid clone library from the metagenome of Laminaria digitata (Ochrophyta) in which decay was induced. Subsequent sequencing of a fosmid clone insert revealed the presence of a gene encoding a bifunctional phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme 10L6AlgC, closely related to a protein from the halophilic marine bacterium, Cobetia sp. 10L6AlgC was subsequently heterologously expressed in Escherichia coli and biochemically characterized. The enzyme was found to possess both PMM and PGM activity, which had temperature and pH optima of 45°C and 8.0, respectively; for both activities. The PMM activity had a K m of 2.229 mM and V max of 29.35 mM min-1 mg-1, while the PGM activity had a K m of 0.5314 mM and a V max of 644.7 mM min-1 mg-1. Overall characterization of the enzyme including the above parameters as well as the influence of various divalent cations on these activities revealed that 10L6AlgC has a unique biochemical profile when compared to previously characterized PMM/PGM bifunctional enzymes. Thus 10L6AlgC may find utility in enzyme-based production of biochemicals with different potential industrial applications, in which other bacterial PMM/PGMs have previously been used such as in the production of low-calorie sweeteners in the food industry.

Identification of BgP, a Cutinase-Like Polyesterase From a Deep-Sea Sponge-Derived Actinobacterium.

Many marine bacteria produce extracellular enzymes that degrade complex molecules to facilitate their growth in environmental conditions that are often harsh and low in nutrients. Marine bacteria, including those inhabiting sea sponges, have previously been reported to be a promising source of polyesterase enzymes, which have received recent attention due to their potential ability to degrade polyethylene terephthalate (PET) plastic. During the screening of 51 marine bacterial isolates for hydrolytic activities targeting ester and polyester substrates, a Brachybacterium ginsengisoli B129SM11 isolate from the deep-sea sponge Pheronema sp. was identified as a polyesterase producer. Sequence analysis of genomic DNA from strain B129SM11, coupled with a genome "mining" strategy, allowed the identification of potential polyesterases, using a custom database of enzymes that had previously been reported to hydrolyze PET or other synthetic polyesters. This resulted in the identification of a putative PET hydrolase gene, encoding a polyesterase-type enzyme which we named BgP that shared high overall similarity with three well-characterized PET hydrolases-LCC, TfCut2, and Cut190, all of which are key enzymes currently under investigation for the biological recycling of PET. In silico protein analyses and homology protein modeling offered structural and functional insights into BgP, and a detailed comparison with Cut190 revealed highly conserved features with implications for both catalysis and substrate binding. Polyesterase activity was confirmed using an agar-based polycaprolactone (PCL) clearing assay, following heterologous expression of BgP in Escherichia coli. This is the first report of a polyesterase being identified from a deep-sea sponge bacterium such as Brachybacterium ginsengisoli and provides further insights into marine-derived polyesterases, an important family of enzymes for PET plastic hydrolysis. Microorganisms living in association with sponges are likely to have increased exposure to plastics and microplastics given the wide-scale contamination of marine ecosystems with these plastics, and thus they may represent a worthwhile source of enzymes for use in new plastic waste management systems. This study adds to the growing knowledge of microbial polyesterases and endorses further exploration of marine host-associated microorganisms as a potentially valuable source of this family of enzymes for PET plastic hydrolysis.

An assessment of how the properties of pyrochar and process thermodynamics impact pyrochar mediated microbial chain elongation in steering the production of medium-chain fatty acids towards n-caproate.

Microbial chain elongation fermentation is an alternative technology for medium-chain fatty acid (MCFA) production. This paper proposed the addition of pyrochar and graphene in chain elongation to improve MCFA production using ethanol and acetate as substrates. Results showed that the yield of, and selectivity towards, C6 n-caproate were significantly enhanced with pyrochar addition. At the optimal mass ratio of pyrochar to substrate of 2 g/g, the maximum n-caproate yield of 13.67 g chemical oxygen demand/L and the corresponding selectivity of 56.8% were obtained; this represents an increase of 115% and 128% respectively as compared with no pyrochar addition. Such improvements were postulated as due to the high electrical conductivity and surface redox groups of pyrochar. The optimal ethanol to acetate molar ratio of 2 mol/mol achieved the highest MCFA yield under pyrochar mediated chain elongation conditions. Thermodynamic calculations modelled an energy benefit of 93.50 kJ/mol reaction for pyrochar mediated n-caproate production.

First identification and characterization of detoxifying plastic-degrading DBP hydrolases in the marine diatom Cylindrotheca closterium.

Diatoms are photosynthetic organisms with potential biotechnological applications in the bioremediation sector, having shown the capacity to reduce environmental concentrations of different pollutants. The diatom Cylindrotheca closterium is known to degrade di-n-butyl phthalate (DBP), one of the most abundant phthalate esters in aquatic environments and a known endocrine-disrupting chemical. In this study, we present for the first time the in silico identification of two putative DBP hydrolases (provisionally called DBPH1 and DBPH2) in the transcriptome of C. closterium. We modeled the structure of both DBPH1-2 and their proposed interactions with the substrate to gain insights into their mechanism of action. Finally, we analyzed the expression levels of the two putative hydrolases upon exposure of C. closterium to different concentrations of DBP (5 and 10 mg/l) for 24 and 48 h. The data showed a DBP concentration-dependent increase in expression levels of both dbph1 and 2 genes, further highlighting their potential involvement in phthalates degradation. This is the first identification of phthalate-degrading enzymes in microalgae, providing new insights into the possible use of diatoms in bioremediation strategies targeting phthalates.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11.

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

Genomic characterization of three marine fungi, including Emericellopsis atlantica sp. nov. with signatures of a generalist lifestyle and marine biomass degradation.

Marine fungi remain poorly covered in global genome sequencing campaigns; the 1000 fungal genomes (1KFG) project attempts to shed light on the diversity, ecology and potential industrial use of overlooked and poorly resolved fungal taxa. This study characterizes the genomes of three marine fungi: Emericellopsis sp. TS7, wood-associated Amylocarpus encephaloides and algae-associated Calycina marina. These species were genome sequenced to study their genomic features, biosynthetic potential and phylogenetic placement using multilocus data. Amylocarpus encephaloides and C. marina were placed in the Helotiaceae and Pezizellaceae (Helotiales), respectively, based on a 15-gene phylogenetic analysis. These two genomes had fewer biosynthetic gene clusters (BGCs) and carbohydrate active enzymes (CAZymes) than Emericellopsis sp. TS7 isolate. Emericellopsis sp. TS7 (Hypocreales, Ascomycota) was isolated from the sponge Stelletta normani. A six-gene phylogenetic analysis placed the isolate in the marine Emericellopsis clade and morphological examination confirmed that the isolate represents a new species, which is described here as E. atlantica. Analysis of its CAZyme repertoire and a culturing experiment on three marine and one terrestrial substrates indicated that E. atlantica is a psychrotrophic generalist fungus that is able to degrade several types of marine biomass. FungiSMASH analysis revealed the presence of 35 BGCs including, eight non-ribosomal peptide synthases (NRPSs), six NRPS-like, six polyketide synthases, nine terpenes and six hybrid, mixed or other clusters. Of these BGCs, only five were homologous with characterized BGCs. The presence of unknown BGCs sets and large CAZyme repertoire set stage for further investigations of E. atlantica. The Pezizellaceae genome and the genome of the monotypic Amylocarpus genus represent the first published genomes of filamentous fungi that are restricted in their occurrence to the marine habitat and form thus a valuable resource for the community that can be used in studying ecological adaptions of fungi using comparative genomics.

Microbial Polyethylene Terephthalate Hydrolases: Current and Future Perspectives.

Plastic has rapidly transformed our world, with many aspects of human life now relying on a variety of plastic materials. Biological plastic degradation, which employs microorganisms and their degradative enzymes, has emerged as one way to address the unforeseen consequences of the waste streams that have resulted from mass plastic production. The focus of this review is microbial hydrolase enzymes which have been found to act on polyethylene terephthalate (PET) plastic. The best characterized examples are discussed together with the use of genomic and protein engineering technologies to obtain PET hydrolase enzymes for different applications. In addition, the obstacles which are currently limiting the development of efficient PET bioprocessing are presented. By continuing to study the possible mechanisms and the structural elements of key enzymes involved in microbial PET hydrolysis, and by assessing the ability of PET hydrolase enzymes to work under practical conditions, this research will help inform large-scale waste management operations. Finally, the contribution of microbial PET hydrolases in creating a potential circular PET economy will be explored. This review combines the current knowledge on enzymatic PET processing with proposed strategies for optimization and use, to help clarify the next steps in addressing pollution by PET and other plastics.

Not That Close to Mommy: Horizontal Transmission Seeds the Microbiome Associated with the Marine Sponge Plakina cyanorosea.

Marine sponges are excellent examples of invertebrate-microbe symbioses. In this holobiont, the partnership has elegantly evolved by either transmitting key microbial associates through the host germline and/or capturing microorganisms from the surrounding seawater. We report here on the prokaryotic microbiota during different developmental stages of Plakina cyanorosea and their surrounding environmental samples by a 16S rRNA metabarcoding approach. In comparison with their source adults, larvae housed slightly richer and more diverse microbial communities, which are structurally more related to the environmental microbiota. In addition to the thaumarchaeal Nitrosopumilus, parental sponges were broadly dominated by Alpha- and Gamma-proteobacteria, while the offspring were particularly enriched in the Vibrionales, Alteromonodales, Enterobacterales orders and the Clostridia and Bacteroidia classes. An enterobacterial operational taxonomic unit (OTU) was the dominant member of the strict core microbiota. The most abundant and unique OTUs were not significantly enriched amongst the microbiomes from host specimens included in the sponge microbiome project. In a wider context, Oscarella and Plakina are the sponge genera with higher divergence in their associated microbiota compared to their Homoscleromorpha counterparts. Our results indicate that P. cyanorosea is a low microbial abundance sponge (LMA), which appears to heavily depend on the horizontal transmission of its microbial partners that likely help the sponge host in the adaptation to its habitat.

Diversity of bacteria populations associated with different thallus regions of the brown alga Laminaria digitata.

Stipitate kelp species such as Laminaria digitata dominate most cold-water subtidal rocky shores and form underwater forests which are among the most productive coastal systems worldwide. Laminaria also sustains rich bacterial communities which offer a variety of biotechnological applications. However, to date, in-depth studies on the diversity and uniqueness of bacterial communities associated with this macroalgal species, their ecological role and their interactions with the alga are under-represented. To address this, the epibacterial populations associated with different thallus regions (holdfast, stipe, meristem, blade) of this brown seaweed were investigated using high-throughput Illumina sequencing of the 16S rRNA genes. The results show that epibacterial communities of the brown seaweed are significantly different and specific to the thallus region, with the shared bacterial population comprising of only 1.1% of the total amplicon sequence variants. The diverse holdfast and blade tissues formed distinct clusters while the meristem and stipe regions are more closely related. The data obtained further supports the hypothesis that macroalgal bacterial communities are shaped by morphological niches and display specificity.

Harnessing the sponge microbiome for industrial biocatalysts.

Within the marine sphere, host-associated microbiomes are receiving growing attention as prolific sources of novel biocatalysts. Given the known biocatalytic potential of poriferan microbial inhabitants, this review focuses on enzymes from the sponge microbiome, with special attention on their relevant properties and the wide range of their potential biotechnological applications within various industries. Cultivable bacterial and filamentous fungal isolates account for the majority of the enzymatic sources. Hydrolases, mainly glycoside hydrolases and carboxylesterases, are the predominant reported group of enzymes, with varying degrees of tolerance to alkaline pH and growing salt concentrations being common. Prospective areas for the application of these microbial enzymes include biorefinery, detergent, food and effluent treatment industries. Finally, alternative strategies to identify novel biocatalysts from the sponge microbiome are addressed, with an emphasis on modern -omics-based approaches that are currently available in the enzyme research arena. By providing this current overview of the field, we hope to not only increase the appetite of researchers to instigate forthcoming studies but also to stress how basic and applied research can pave the way for new biocatalysts from these symbiotic microbial communities in a productive fashion. KEY POINTS: • The sponge microbiome is a burgeoning source of industrial biocatalysts. • Sponge microbial enzymes have useful habitat-related traits for several industries. • Strategies are provided for the future discovery of microbial enzymes from sponges.

The Effects of the Marine-Derived Polysaccharides Laminarin and Chitosan on Aspects of Colonic Health in Pigs Challenged with Dextran Sodium Sulphate.

This study examined the effects of dietary supplementation with laminarin or chitosan on colonic health in pigs challenged with dextran sodium sulphate (DSS). Weaned pigs were assigned to: (1) a basal diet (n = 22); (2) a basal diet + laminarin (n = 10); and (3) a basal diet + chitosan (n = 10). On d35, the basal group was split, creating four groups: (1) the basal diet (control); (2) the basal diet + DSS; (3) the basal diet + laminarin + DSS; and (4) the basal diet + chitosan + DSS. From d39-42, the pigs were orally challenged with DSS. On d44, colonic tissue/digesta samples were collected. The basal DSS group had reduced growth, higher pathology score and an increased expression of MMP1, IL13 and IL23 compared with the controls (p < 0.05); these parameters were similar between the DSS-challenged groups (p > 0.05). In the basal DSS group, the relative abundance of beneficial taxa including Prevotella and Roseburia were reduced while Escherichia/Shigella were increased, compared with the controls (p < 0.05). The relative abundance of Escherichia/Shigella was reduced and the molar proportions of acetate were increased in the laminarin DSS group compared with the basal DSS group (p < 0.01), suggesting that laminarin has potential to prevent pathogen proliferation and enhance the volatile fatty acid profile in the colon in a porcine model of colitis.

Draft Genome Sequences of Three Pseudomonas fluorescens Strains Isolated from Marine Sponges Harvested off the West Coast of Ireland.

Three Pseudomonas sp. strains isolated from marine sponges have shown potential quorum sensing inhibition (QSI) activity. We sequenced the draft genomes of the three strains with the goal of determining which genes or gene cluster(s) could be potentially involved in the QSI activity. Average nucleotide identity (ANI) and phylogenetic analysis classified the three strains as belonging to the Pseudomonas fluorescens species.

Peeling the Layers Away: The Genomic Characterization of Bacillus pumilus 64-1, an Isolate With Antimicrobial Activity From the Marine Sponge Plakina cyanorosea (Porifera, Homoscleromorpha).

Bacillus pumilus 64-1, a bacterial strain isolated from the marine sponge Plakina cyanorosea, which exhibits antimicrobial activity against both pathogenic and drug-resistant Gram-positive and Gram-negative bacteria. This study aimed to conduct an in-depth genomic analysis of this bioactive sponge-derived strain. The nearly complete genome of strain 64-1 consists of 3.6 Mbp (41.5% GC), which includes 3,705 coding sequences (CDS). An open pangenome was observed when limiting to the type strains of the B. pumilus group and aquatic-derived B. pumilus representatives. The genome appears to encode for at least 12 potential biosynthetic gene clusters (BGCs), including both types I and III polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), and one NRPS-T1PKS hybrid, among others. In particular, bacilysin and other bacteriocin-coding genes were found and may be associated with the detected antimicrobial activity. Strain 64-1 also appears to possess a broad repertoire of genes encoding for plant cell wall-degrading carbohydrate-active enzymes (CAZymes). A myriad of genes which may be involved in various process required by the strain in its marine habitat, such as those encoding for osmoprotectory transport systems and the biosynthesis of compatible solutes were also present. Several heavy metal tolerance genes are also present, together with various mobile elements including a region encoding for a type III-B Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) region, four prophage segments and transposase elements. This is the first report on the genomic characterization of a cultivable bacterial member of the Plakina cyanorosea holobiont.

Metagenomics of Atacama Lithobiontic Extremophile Life Unveils Highlights on Fungal Communities, Biogeochemical Cycles and Carbohydrate-Active Enzymes.

Halites, which are typically found in various Atacama locations, are evaporitic rocks that are considered as micro-scaled salterns. Both structural and functional metagenomic analyses of halite nodules were performed. Structural analyses indicated that the halite microbiota is mainly composed of NaCl-adapted microorganisms. In addition, halites appear to harbor a limited diversity of fungal families together with a biodiverse collection of protozoa. Functional analysis indicated that the halite microbiome possesses the capacity to make an extensive contribution to carbon, nitrogen, and sulfur cycles, but possess a limited capacity to fix nitrogen. The halite metagenome also contains a vast repertory of carbohydrate active enzymes (CAZY) with glycosyl transferases being the most abundant class present, followed by glycosyl hydrolases (GH). Amylases were also present in high abundance, with GH also being identified. Thus, the halite microbiota is a potential useful source of novel enzymes that could have biotechnological applicability. This is the first metagenomic report of fungi and protozoa as endolithobionts of halite nodules, as well as the first attempt to describe the repertoire of CAZY in this community. In addition, we present a comprehensive functional metagenomic analysis of the metabolic capacities of the halite microbiota, providing evidence for the first time on the sulfur cycle in Atacama halites.

A Review on Viral Metagenomics in Extreme Environments.

Viruses are the most abundant biological entities in the biosphere, and have the ability to infect Bacteria, Archaea, and Eukaryotes. The virome is estimated to be at least ten times more abundant than the microbiome with 107 viruses per milliliter and 109 viral particles per gram in marine waters and sediments or soils, respectively. Viruses represent a largely unexplored genetic diversity, having an important role in the genomic plasticity of their hosts. Moreover, they also play a significant role in the dynamics of microbial populations. In recent years, metagenomic approaches have gained increasing popularity in the study of environmental viromes, offering the possibility of extending our knowledge related to both virus diversity and their functional characterization. Extreme environments represent an interesting source of both microbiota and their virome due to their particular physicochemical conditions, such as very high or very low temperatures and >1 atm hydrostatic pressures, among others. Despite the fact that some progress has been made in our understanding of the ecology of the microbiota in these habitats, few metagenomic studies have described the viromes present in extreme ecosystems. Thus, limited advances have been made in our understanding of the virus community structure in extremophilic ecosystems, as well as in their biotechnological potential. In this review, we critically analyze recent progress in metagenomic based approaches to explore the viromes in extreme environments and we discuss the potential for new discoveries, as well as methodological challenges and perspectives.

Corrigendum: Comparative Genomics of Marine Sponge-Derived Streptomyces spp. Isolates SM17 and SM18 With Their Closest Terrestrial Relatives Provides Novel Insights Into Environmental Niche Adaptations and Secondary Metabolite Biosynthesis Potential.

[This corrects the article DOI: 10.3389/fmicb.2019.01713.].

In silico Screening and Heterologous Expression of a Polyethylene Terephthalate Hydrolase (PETase)-Like Enzyme (SM14est) With Polycaprolactone (PCL)-Degrading Activity, From the Marine Sponge-Derived Strain Streptomyces sp. SM14.

Plastics, such as the polyethylene terephthalate (PET), are widely used for various industrial applications, due to their physicochemical properties which are particularly useful in the packaging industry. However, due to improper plastic waste management and difficulties in recycling, post-consumer plastic waste has become a pressing issue for both the environment and for human health. Hence, novel technologies and methods of processing plastic waste are required to address these issues. Enzymatic-assisted hydrolysis of synthetic polymers has been proposed as a potentially more efficient and environment-friendly alternative to the currently employed methods. Recently, a number of PET hydrolases have been described, and in particular a PETase derived from Ideonella sakaiensis 201-F6 (IsPETase), which appears to be the most efficient and substrate-specific bacterial PET hydrolase enzyme discovered to date. In order to further investigate this class of PETase-like enzymes, we employed an in silico-based screening approach on the biotechnologically relevant genus Streptomyces, including terrestrial and marine isolates; in a search for potential PETase homologs. From a total of 52 genomes analyzed, we were able to identify three potential PETase-like enzymes, all of which were derived from marine-sponge associated Streptomyces isolates. A candidate PETase-like gene (SM14est) was identified in Streptomyces sp. SM14. Further in silico characterization of the SM14est protein sequence and its predicted three-dimensional structure were performed and compared to the well-characterized IsPETase. Both the serine hydrolase motif Gly-x1-Ser-x2-Gly and the catalytic triad Ser, Asp, His are conserved in both sequences. Molecular docking experiments indicated that the SM14est enzyme possessed the capacity to bind plastics as substrates. Finally, polyesterase activity was confirmed using a polycaprolactone (PCL) plate clearing assay which is a model substrate for the degradation of plastics; following heterologous expression of SM14est in Escherichia coli, with secretion being facilitated by the native Streptomyces signal peptide. These findings provide further insights into this important class of PETase-like enzymes.