Identification and recombinant expression of a cutinase from Papiliotrema laurentii that hydrolyzes natural and synthetic polyesters.

Given the multitude of extracellular enzymes at their disposal, many of which are designed to degrade nature's polymers (lignin, cutin, cellulose, etc.), fungi are adept at targeting synthetic polyesters with similar chemical composition. Microbial-influenced deterioration of xenobiotic polymeric surfaces is an area of interest for material scientists as these are important for the conservation of the underlying structural materials. Here, we describe the isolation and characterization of the Papiliotrema laurentii 5307AH (P. laurentii) cutinase, Plcut1. P. laurentii is basidiomycete yeast with the ability to disperse Impranil-DLN (Impranil), a colloidal polyester polyurethane, in agar plates. To test whether the fungal factor involved in this clearing was a secreted enzyme, we screened the ability of P. laurentii culture supernatants to disperse Impranil. Using size exclusion chromatography (SEC), we isolated fractions that contained Impranil-clearing activity. These fractions harbored a single ~22 kD band, which was excised and subjected to peptide sequencing. Homology searches using the peptide sequences identified, revealed that the protein Papla1 543643 (Plcut1) displays similarities to serine esterase and cutinase family of proteins. Biochemical assays using recombinant Plcut1 confirmed that this enzyme has the capability to hydrolyze Impranil, soluble esterase substrates, and apple cutin. Finally, we confirmed the presence of the Plcut1 in culture supernatants using a custom antibody that specifically recognizes this protein. The work shown here supports a major role for the Plcut1 in the fungal degradation of natural polyesters and xenobiotic polymer surfaces.IMPORTANCEFungi play a vital role in the execution of a broad range of biological processes that drive ecosystem function through production of a diverse arsenal of enzymes. However, the universal reactivity of these enzymes is a current problem for the built environment and the undesired degradation of polymeric materials in protective coatings. Here, we report the identification and characterization of a hydrolase from Papiliotrema laurentii 5307AH, an aircraft-derived fungal isolate found colonizing a biodeteriorated polymer-coated surface. We show that P. laurentii secretes a cutinase capable of hydrolyzing soluble esters as well as ester-based compounds forming solid surface coatings. These findings indicate that this fungus plays a significant role in biodeterioration through the production of a cutinase adept at degrading ester-based polymers, some of which form the backbone of protective surface coatings. The work shown here provides insights into the mechanisms employed by fungi to degrade xenobiotic polymers.

Draft genome sequence of potentially dikaryotic black fungus Aureobasidium melanogenum isolated from aircraft.

The Ascomycota yeast Aureobasidium melanogenum strain W12 was isolated from an aircraft polymer-coated surface. The genome size is 53,160,883 bp with a G + C content of 50.13%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases potentially used for survival on polymer coatings on aircraft.

Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

Meeting report of the sixth annual tri-service microbiome consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among DoD organizations and to facilitate resource, material and information sharing amongst consortium members, which includes collaborators in academia and industry. The 6th Annual TSMC Symposium was a hybrid meeting held in Fairlee, Vermont on 27-28 September 2022 with presentations and discussions centered on microbiome-related topics within seven broad thematic areas: (1) Human Microbiomes: Stress Response; (2) Microbiome Analysis & Surveillance; (3) Human Microbiomes Enablers & Engineering; (4) Human Microbiomes: Countermeasures; (5) Human Microbiomes Discovery - Earth & Space; (6) Environmental Micro & Myco-biome; and (7) Environmental Microbiome Analysis & Engineering. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the activities and outcomes from the 6th annual TSMC symposium.

Screening Enzymatic Degradation of Polyester Polyurethane with Fluorescent Single-walled Carbon Nanotube and Polymer Nanoparticle Conjugates.

Enzymatic biodegradation is a promising method to reclaim plastic materials. However, to date, a high-throughput method for screening potential enzyme candidates for biodegradation is still lacking. Here, we propose a single-walled carbon nanotube (SWCNT) fluorescence sensor for screening the enzymatic degradation of polyester polyurethane nanoparticles. Through wrapping the SWCNT with cationic chitosan, an electrostatic bond is formed between the SWCNT and Impranil, a widely applied model substrate of polyester polyurethane. As Impranil is being degraded by the enzymes, a characteristic quenching at a short reaction time followed by a brightening at a longer reaction time in the fluorescence signal is observed. The time-dependent fluorescence response is compared with turbidity measurement, and we conclude that the brightening in fluorescence results from the binding of the degradation product with the SWCNT. The proposed SWCNT sensor design has the potential to screen enzyme candidates for selective degradation of other plastic particles.

Accurate prediction by AlphaFold2 for ligand binding in a reductive dehalogenase and implications for PFAS (per- and polyfluoroalkyl substance) biodegradation.

Despite the success of AlphaFold2 (AF2), it is unclear how AF2 models accommodate for ligand binding. Here, we start with a protein sequence from Acidimicrobiaceae TMED77 (T7RdhA) with potential for catalyzing the degradation of per- and polyfluoroalkyl substances (PFASs). AF2 models and experiments identified T7RdhA as a corrinoid iron-sulfur protein (CoFeSP) which uses a norpseudo-cobalamin (BVQ) cofactor and two Fe4S4 iron-sulfur clusters for catalysis. Docking and molecular dynamics simulations suggest that T7RdhA uses perfluorooctanoic acetate (PFOA) as a substrate, supporting the reported defluorination activity of its homolog, A6RdhA. We showed that AF2 provides processual (dynamic) predictions for the binding pockets of ligands (cofactors and/or substrates). Because the pLDDT scores provided by AF2 reflect the protein native states in complex with ligands as the evolutionary constraints, the Evoformer network of AF2 predicts protein structures and residue flexibility in complex with the ligands, i.e., in their native states. Therefore, an apo-protein predicted by AF2 is actually a holo-protein awaiting ligands.

Draft Genome Sequence of the Nonmotile Tremellomycetes Yeast Naganishia albida, Isolated from Aircraft.

The Basidiomycota yeast Naganishia albida strain 5307AI was isolated from an aircraft polymer-coated surface. The genome size is 20,642,279 bp, with a G+C content of 53.99%. The genome contains fatty acid transporters, cutinases, hydroxylases, and lipases that are likely used for survival on polymer coatings on aircraft.

AlphaFold2 models indicate that protein sequence determines both structure and dynamics.

AlphaFold 2 (AF2) has placed Molecular Biology in a new era where we can visualize, analyze and interpret the structures and functions of all proteins solely from their primary sequences. We performed AF2 structure predictions for various protein systems, including globular proteins, a multi-domain protein, an intrinsically disordered protein (IDP), a randomized protein, two larger proteins (> 1000 AA), a heterodimer and a homodimer protein complex. Our results show that along with the three dimensional (3D) structures, AF2 also decodes protein sequences into residue flexibilities via both the predicted local distance difference test (pLDDT) scores of the models, and the predicted aligned error (PAE) maps. We show that PAE maps from AF2 are correlated with the distance variation (DV) matrices from molecular dynamics (MD) simulations, which reveals that the PAE maps can predict the dynamical nature of protein residues. Here, we introduce the AF2-scores, which are simply derived from pLDDT scores and are in the range of [0, 1]. We found that for most protein models, including large proteins and protein complexes, the AF2-scores are highly correlated with the root mean square fluctuations (RMSF) calculated from MD simulations. However, for an IDP and a randomized protein, the AF2-scores do not correlate with the RMSF from MD, especially for the IDP. Our results indicate that the protein structures predicted by AF2 also convey information of the residue flexibility, i.e., protein dynamics.

Comparison of two diphenyl polyenes as acid-sensitive additives during the biodegradation of a thermoset polyester polyurethane coating.

AIMS: Biochemical hydrolysis and chemical catalysis are involved in the successful biodegradation of polymers. In order to evaluate the potential separation between biochemical and chemical catalysis during the biodegradation process, we report the use of two diphenylpolyenes (DPPs), all trans-1,4-diphenylbutadiene (DPB) and all trans-1,6-diphenylhexatriene (DPH), as potential acid-sensitive indicators in polymers. METHODS AND RESULTS: 1,4-Diphenylbutadiene and DPH (0.1% w/w) were melt-cast successfully with poly(ethylene succinate) hexamethylene (PES-HM) polyurethane (thermoset polyester polyurethane) coatings above 80℃. When these two DPP/PES-HM coatings were exposed to a concentrated supernatant with significant esterase activity resulting from the growth of a recently isolated and identified strain of Tremellomycetes yeast (Naganishia albida 5307AI), the DPB coatings exhibited a measurable and reproducible localized decrease in the blue fluorescence emission in regions below where hydrolytic biodegradation was initiated in contrast with DPH blended coatings. The fluorescence changes observed in the biodegraded DPB coating were similar to exposing them to concentrated acids and not bases. CONCLUSIONS: Our experiments resulted in (1) a method to blend DPP additives into thermoset coatings, (2) the first report of the biodegradation of polyester polyurethane coating by N. albida, and (3) demonstration that hydrolytic supernatants from this strain generate acidic region within degrading polyester coatings using DPB as the indicator. SIGNIFICANCE AND IMPACT OF THE STUDY: Our experiments confirm that N. albida is an active polyester degrader and that DPB is a promising acid sensitive polymer coating additive.

Draft Genome Sequence of the Bacterium Delftia acidovorans Strain D4B, Isolated from Soil.

Delftia acidovorans strain D4B is an aerobic bacterium within the Betaproteobacteria lineage that was isolated from soil. The genome size is 6.26 Mbp, with a G+C content of 67%. The genome encodes enzymes potentially involved in the degradation of fluorinated compounds.

Meeting report of the fourth annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations. The annual TSMC symposium is designed to enable information sharing between DoD scientists and leaders in the field of microbiome science, thereby keeping DoD consortium members informed of the latest advances within the microbiome community and facilitating the development of new collaborative research opportunities. The 2020 annual symposium was held virtually on 24-25 September 2020. Presentations and discussions centered on microbiome-related topics within four broad thematic areas: (1) Enabling Technologies; (2) Microbiome for Health and Performance; (3) Environmental Microbiome; and (4) Microbiome Analysis and Discovery. This report summarizes the presentations and outcomes of the 4th annual TSMC symposium.

Meeting report of the third annual Tri-Service Microbiome Consortium symposium.

The Tri-Service Microbiome Consortium (TSMC) was founded to enhance collaboration, coordination, and communication of microbiome research among U.S. Department of Defense (DoD) organizations and to facilitate resource, material and information sharing among consortium members. The 2019 annual symposium was held 22-24 October 2019 at Wright-Patterson Air Force Base in Dayton, OH. Presentations and discussions centered on microbiome-related topics within five broad thematic areas: 1) human microbiomes; 2) transitioning products into Warfighter solutions; 3) environmental microbiomes; 4) engineering microbiomes; and 5) microbiome simulation and characterization. Collectively, the symposium provided an update on the scope of current DoD microbiome research efforts, highlighted innovative research being done in academia and industry that can be leveraged by the DoD, and fostered collaborative opportunities. This report summarizes the presentations and outcomes of the 3rd annual TSMC symposium.

Organism Engineering for the Bioproduction of the Triaminotrinitrobenzene (TATB) Precursor Phloroglucinol (PG).

Organism engineering requires the selection of an appropriate chassis, editing its genome, combining traits from different source species, and controlling genes with synthetic circuits. When a strain is needed for a new target objective, for example, to produce a chemical-of-need, the best strains, genes, techniques, software, and expertise may be distributed across laboratories. Here, we report a project where we were assigned phloroglucinol (PG) as a target, and then combined unique capabilities across the United States Army, Navy, and Air Force service laboratories with the shared goal of designing an organism to produce this molecule. In addition to the laboratory strain Escherichia coli, organisms were screened from soil and seawater. Putative PG-producing enzymes were mined from a strain bank of bacteria isolated from aircraft and fuel depots. The best enzyme was introduced into the ocean strain Marinobacter atlanticus CP1 with its genome edited to redirect carbon flux from natural fatty acid ester (FAE) production. PG production was also attempted in Bacillus subtilis and Clostridium acetobutylicum. A genetic circuit was constructed in E. coli that responds to PG accumulation, which was then ported to an in vitro paper-based system that could serve as a platform for future low-cost strain screening or for in-field sensing. Collectively, these efforts show how distributed biotechnology laboratories with domain-specific expertise can be marshalled to quickly provide a solution for a targeted organism engineering project, and highlights data and material sharing protocols needed to accelerate future efforts.

Structural Perturbations of Rhodopseudomonas palustris Form II RuBisCO Mutant Enzymes That Affect CO2 Fixation.

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and its central role in capturing atmospheric CO2 via the Calvin-Benson-Bassham (CBB) cycle have been well-studied. Previously, a form II RuBisCO from Rhodopseudomonas palustris, a facultative anaerobic bacterium, was shown to assemble into a hexameric holoenzyme. Unlike previous studies with form II RuBisCO, the R. palustris enzyme could be crystallized in the presence of the transition state analogue 2-carboxyarabinitol 1,5-bisphosphate (CABP), greatly facilitating the structure-function studies reported here. Structural analysis of mutant enzymes with substitutions in form II-specific residues (Ile165 and Met331) and other conserved and semiconserved residues near the enzyme's active site identified subtle structural interactions that may account for functional differences between divergent RuBisCO enzymes. In addition, using a distantly related aerobic bacterial host, further selection of a suppressor mutant enzyme that overcomes negative enzymatic functions was accomplished. Structure-function analyses with negative and suppressor mutant RuBisCOs highlighted the importance of interactions involving different parts of the enzyme's quaternary structure that influenced partial reactions that constitute RuBisCO's carboxylation mechanism. In particular, structural perturbations in an intersubunit interface appear to affect CO2 addition but not the previous step in the enzymatic mechanism, i.e., the enolization of substrate ribulose 1,5-bisphosphate (RuBP). This was further substantiated by the ability of a subset of carboxylation negative mutants to support a previously described sulfur-salvage function, one that appears to rely solely on the enzyme's ability to catalyze the enolization of a substrate analogous to RuBP.

Draft Genome Sequence of Filamentous Fungus Phialemoniopsis curvata, Isolated from Diesel Fuel.

Phialemoniopsis curvata D216 is a filamentous fungus isolated from contaminated diesel fuel. The genome size is 40.3 Mbp with a G+C content of 54.81%. Its genome encodes enzymes and pathways likely involved in the degradation of and survival in fuel, including lipases, fatty acid transporters, and beta oxidation.

An enhanced Lactobacillus reuteri biofilm formulation that increases protection against experimental necrotizing enterocolitis.

One significant drawback of current probiotic therapy for the prevention of necrotizing enterocolitis (NEC) is the need for at least daily administration because of poor probiotic persistence after enteral administration, increasing the risk of the probiotic bacteria causing bacteremia or sepsis if the intestines are already compromised. We previously showed that the effectiveness of Lactobacillus reuteri ( Lr) in preventing NEC is enhanced when Lr is grown as a biofilm on the surface of dextranomer microspheres (DM). Here we sought to test the efficacy of Lr administration by manipulating the Lr biofilm state with the addition of biofilm-promoting substances (sucrose and maltose) to DM or by mutating the Lr gtfW gene (encoding an enzyme central to biofilm production). Using an animal model of NEC, we determined that Lr adhered to sucrose- or maltose-loaded DM significantly reduced histologic injury, improved host survival, decreased intestinal permeability, reduced intestinal inflammation, and altered the gut microbiome compared with Lr adhered to unloaded DM. These effects were abolished when DM or GtfW were absent from the Lr inoculum. This demonstrates that a single dose of Lr in its biofilm state decreases NEC incidence. Importantly, preloading DM with sucrose or maltose further enhances Lr protection against NEC in a GtfW-dependent fashion, demonstrating the tunability of the approach and the potential to use other cargos to enhance future probiotic formulations. NEW & NOTEWORTHY Previous clinical trials of probiotics to prevent necrotizing enterocolitis have had variable results. In these studies, probiotics were delivered in their planktonic, free-living form. We have developed a novel probiotic delivery system in which Lactobacillus reuteri (Lr) is delivered in its biofilm state. In a model of experimental necrotizing enterocolitis, this formulation significantly reduces intestinal inflammation and permeability, improves survival, and preserves the natural gut microflora compared with the administration of Lr in its free-living form.

Finished Genome Sequence of a Polyurethane-Degrading Pseudomonas Isolate.

Pseudomonas sp. strain WP001 is a laboratory isolate capable of polyurethane polymer degradation and harbors a predicted lipase precursor gene. The genome of strain WP001 is 6.15 Mb in size and is composed of seven scaffolds with a G+C content of 60.54%. Strain WP001 is closely related to Pseudomonas fluorescens based on ribosomal DNA comparisons.