Streamlined screening of extracellularly expressed PETase libraries for improved polyethylene terephthalate degradation.

Enzyme-mediated polyethylene terephthalate (PET) depolymerization has recently emerged as a sustainable solution for PET recycling. Towards an industrial-scale implementation of this technology, various strategies are being explored to enhance PET depolymerization (PETase) activity and improve enzyme stability, expression, and purification processes. Recently, rational engineering of a known PET hydrolase (LCC-leaf compost cutinase) has resulted in the isolation of a variant harboring four-point mutations (LCC-ICCG), presenting increased PETase activity and thermal stability. Here, we revealed the enzyme's natural extracellular expression and used it to efficiently screen error-prone genetic libraries based on LCC-ICCG for enhanced activity toward consumer-grade PET. Following multiple rounds of mutagenesis and screening, we successfully isolated variants that exhibited up to a 60% increase in PETase activity. Among other mutations, the improved variants showed a histidine to tyrosine substitution at position 218, a residue known to be involved in substrate binding and stabilization. Introducing H218Y mutation on the background of LCC-ICCG (named here LCC-ICCG/H218Y) resulted in a similar level of activity improvement. Analysis of the solved structure of LCC-ICCG/H218Y compared to other known PETases featuring different amino acids at the equivalent position suggests that H218Y substitution promotes enhanced PETase activity. The expression and screening processes developed in this study can be further used to optimize additional enzymatic parameters crucial for efficient enzymatic degradation of consumer-grade PET.

Molecular engineering of PETase for efficient PET biodegradation.

The widespread utilization of polyethylene terephthalate (PET) has caused a variety of environmental and health problems. Compared with traditional thermomechanical or chemical PET cycling, the biodegradation of PET may offer a more feasible solution. Though the PETase from Ideonalla sakaiensis (IsPETase) displays interesting PET degrading performance under mild conditions; the relatively low thermal stability of IsPETase limits its practical application. In this study, enzyme-catalysed PET degradation was investigated with the promising IsPETase mutant HotPETase (HP). On this basis, a carbohydrate-binding module from Bacillus anthracis (BaCBM) was fused to the C-terminus of HP to construct the PETase mutant (HLCB) for increased PET degradation. Furthermore, to effectively improve PET accessibility and PET-degrading activity, the truncated outer membrane hybrid protein (FadL) was used to expose PETase and BaCBM on the surface of E. coli (BL21with) to develop regenerable whole-cell biocatalysts (D-HLCB). Results showed that, among the tested small-molecular weight ester compounds (p-nitrophenyl phosphate (pNPP), p-Nitrophenyl acetate (pNPA), 4-Nitrophenyl butyrate (pNPB)), PETase displayed the highest hydrolysing activity against pNPP. HP displayed the highest catalytic activity (1.94 µM(p-NP)/min) at 50 °C and increased longevity at 40 °C. The fused BaCBM could clearly improve the catalytic performance of PETase by increasing the optimal reaction temperature and improving the thermostability. When HLCB was used for PET degradation, the yield of monomeric products (255.7 µM) was ∼25.5 % greater than that obtained after 50 h of HP-catalysed PET degradation. Moreover, the highest yield of monomeric products from the D-HLCB-mediated system reached 1.03 mM. The whole-cell catalyst D-HLCB displayed good reusability and stability and could maintain more than 54.6 % of its initial activity for nine cycles. Finally, molecular docking simulations were utilized to investigate the binding mechanism and the reaction mechanism of HLCB, which may provide theoretical evidence to further increase the PET-degrading activities of PETases through rational design. The proposed strategy and developed variants show potential for achieving complete biodegradation of PET under mild conditions.

Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

Increasing the Soluble Expression and Whole-Cell Activity of the Plastic-Degrading Enzyme MHETase through Consensus Design.

The mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) from Ideonella sakaiensis carries out the second step in the enzymatic depolymerization of poly(ethylene terephthalate) (PET) plastic into the monomers terephthalic acid (TPA) and ethylene glycol (EG). Despite its potential industrial and environmental applications, poor recombinant expression of MHETase has been an obstacle to its industrial application. To overcome this barrier, we developed an assay allowing for the medium-throughput quantification of MHETase activity in cell lysates and whole-cell suspensions, which allowed us to screen a library of engineered variants. Using consensus design, we generated several improved variants that exhibit over 10-fold greater whole-cell activity than wild-type (WT) MHETase. This is revealed to be largely due to increased soluble expression, which biochemical and structural analysis indicates is due to improved protein folding.

Discovery and characterization of two novel polyethylene terephthalate hydrolases: One from a bacterium identified in human feces and one from the Streptomyces genus.

Polyethylene terephthalate (PET) is widely used for various industrial applications. However, owing to its extremely slow breakdown rate, PET accumulates as plastic trash, which negatively affects the environment and human health. Here, we report two novel PET hydrolases: PpPETase from Pseudomonas paralcaligenes MRCP1333, identified in human feces, and ScPETase from Streptomyces calvus DSM 41452. These two enzymes can decompose various PET materials, including semicrystalline PET powders (Cry-PET) and low-crystallinity PET films (gf-PET). By structure-guided engineering, two variants, PpPETaseY239R/F244G/Y250G and ScPETaseA212C/T249C/N195H/N243K were obtained that decompose Cry-PET 3.1- and 1.9-fold faster than their wild-type enzymes, respectively. The co-expression of ScPETase and mono-(2-hydroxyethyl) terephthalate hydrolase from Ideonella sakaiensis (IsMHETase) resulted in 1.4-fold more degradation than the single enzyme system. This engineered strain degraded Cry-PET and gf-PET by more than 40% and 6%, respectively, after 30 d. The concentrations of terephthalic acid (TPA) in the Cry-PET and gf-PET degradation products were 37.7% and 25.6%, respectively. The discovery of these two novel PET hydrolases provides opportunities to create more powerful biocatalysts for PET biodegradation.

Novel polyurethane-degrading cutinase BaCut1 from Blastobotrys sp. G-9 with potential role in plastic bio-recycling.

Environmental pollution caused by plastic waste has become global problem that needs to be considered urgently. In the pursuit of a circular plastic economy, biodegradation provides an attractive strategy for managing plastic wastes, whereas effective plastic-degrading microbes and enzymes are required. In this study, we report that Blastobotrys sp. G-9 isolated from discarded plastic in landfills is capable of depolymerizing polyurethanes (PU) and poly (butylene adipate-co-terephthalate) (PBAT). Strain G-9 degrades up to 60% of PU foam after 21 days of incubation at 28 â„ƒ by breaking down carbonyl groups via secretory hydrolase as confirmed by structural characterization of plastics and degradation products identification. Within the supernatant of strain G-9, we identify a novel cutinase BaCut1, belonging to the esterase family, that can reproduce the same effect. BaCut1 demonstrates efficient degradation toward commercial polyester plastics PU foam (0.5 mg enzyme/25 mg plastic) and agricultural film PBAT (0.5 mg enzyme/10 mg plastic) with 50% and 18% weight loss at 37 â„ƒ for 48 h, respectively. BaCut1 hydrolyzes PU into adipic acid as a major end-product with 42.9% recovery via ester bond cleavage, and visible biodegradation is also identified from PBAT, which is a beneficial feature for future recycling economy. Molecular docking, along with products distribution, elucidates a special substrate-binding modes of BaCut1 with plastic substrate analogue. BaCut1-mediated polyester plastic degradation offers an alternative approach for managing PU plastic wastes through possible bio-recycling.

Microbial hitchhikers on microplastics: The exchange of aquatic microbes across distinct aquatic habitats.

Microplastics (MPs) have the potential to modify aquatic microbial communities and distribute microorganisms, including pathogens. This poses a potential risk to aquatic life and human health. Despite this, the fate of 'hitchhiking' microbes on MPs that traverse different aquatic habitats remains largely unknown. To address this, we conducted a 50-day microcosm experiment, manipulating estuarine conditions to study the exchange of bacteria and microeukaryotes between river, sea and plastisphere using a long-read metabarcoding approach. Our findings revealed a significant increase in bacteria on the plastisphere, including Pseudomonas, Sphingomonas, Hyphomonas, Brevundimonas, Aquabacterium and Thalassolituus, all of which are known for their pollutant degradation capabilities, specifically polycyclic aromatic hydrocarbons. We also observed a strong association of plastic-degrading fungi (i.e., Cladosporium and Plectosphaerella) and early-diverging fungi (Cryptomycota, also known as Rozellomycota) with the plastisphere. Sea MPs were primarily colonised by fungi (70%), with a small proportion of river-transported microbes (1%-4%). The mere presence of MPs in seawater increased the relative abundance of planktonic fungi from 2% to 25%, suggesting significant exchanges between planktonic and plastisphere communities. Using microbial source tracking, we discovered that MPs only dispersed 3.5% and 5.5% of river bacterial and microeukaryotic communities into the sea, respectively. Hence, although MPs select and facilitate the dispersal of ecologically significant microorganisms, drastic compositional changes across distinct aquatic habitats are unlikely.

Enhancing PET Degrading Enzymes: A Combinatory Approach.

Plastic waste has become a substantial environmental issue. A potential strategy to mitigate this problem is to use enzymatic hydrolysis of plastics to depolymerize post-consumer waste and allow it to be reused. Over the last few decades, the use of enzymatic PET-degrading enzymes has shown promise as a great solution for creating a circular plastic waste economy. PsPETase from Piscinibacter sakaiensis has been identified as an enzyme with tremendous potential for such applications. But to improve its efficiency, enzyme engineering has been applied aiming at enhancing its thermal stability, enzymatic activity, and ease of production. Here, we combine different strategies such as structure-based rational design, ancestral sequence reconstruction and machine learning to engineer a more highly active Combi-PETase variant with a melting temperature of 70 °C and optimal performance at 60 °C. Furthermore, this study demonstrates that these approaches, commonly used in other works of enzyme engineering, are most effective when utilized in combination, enabling the improvement of enzymes for industrial applications.

Bioinformatic Analysis of Sulfotransferases from an Unexplored Gut Microbe, Sutterella wadsworthensis 3_1_45B: Possible Roles towards Detoxification via Sulfonation by Members of the Human Gut Microbiome.

Sulfonation, primarily facilitated by sulfotransferases, plays a crucial role in the detoxification pathways of endogenous substances and xenobiotics, promoting metabolism and elimination. Traditionally, this bioconversion has been attributed to a family of human cytosolic sulfotransferases (hSULTs) known for their high sequence similarity and dependence on 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as a sulfo donor. However, recent studies have revealed the presence of PAPS-dependent sulfotransferases within gut commensals, indicating that the gut microbiome may harbor a diverse array of sulfotransferase enzymes and contribute to detoxification processes via sulfation. In this study, we investigated the prevalence of sulfotransferases in members of the human gut microbiome. Interestingly, we stumbled upon PAPS-independent sulfotransferases, known as aryl-sulfate sulfotransferases (ASSTs). Our bioinformatics analyses revealed that members of the gut microbial genus Sutterella harbor multiple asst genes, possibly encoding multiple ASST enzymes within its members. Fluctuations in the microbes of the genus Sutterella have been associated with various health conditions. For this reason, we characterized 17 different ASSTs from Sutterella wadsworthensis 3_1_45B. Our findings reveal that SwASSTs share similarities with E. coli ASST but also exhibit significant structural variations and sequence diversity. These differences might drive potential functional diversification and likely reflect an evolutionary divergence from their PAPS-dependent counterparts.

Cross-linked enzyme aggregates of polyethylene terephthalate hydrolyse (PETase) from Ideonella sakaiensis for the improvement of plastic degradation.

Polyethylene terephthalate (PET) is one of the most produced plastics globally and its accumulation in the environment causes harm to the ecosystem. Polyethylene terephthalate hydrolyse (PETase) is an enzyme that can degrade PET into its monomers. However, free PETase lacks operational stabilities and is not reusable. In this study, development of cross-linked enzyme aggregate (CLEA) of PETase using amylopectin (Amy) as cross-linker was introduced to solve the limitations of free PETase. PETase-Amy-CLEA exhibited activity recovery of 81.9 % at its best immobilization condition. Furthermore, PETase-Amy-CLEA exhibited 1.37-, 2.75-, 2.28- and 1.36-fold higher half-lives than free PETase at 50 °C, 45 °C, 40 °C and 35 °C respectively. Moreover, PETase-Amy-CLEA showed broader pH stability from pH 5 to 10 and could be reused up to 5 cycles. PETase-Amy-CLEA retained >70 % of initial activity after 40 days of storage at 4 °C. In addition, lower Km of PETase-Amy-CLEA indicated better substrate affinity than free enzyme. PETase-Amy-CLEA corroded PET better and products yielded was 66.7 % higher than free PETase after 32 h of treatment. Hence, the enhanced operational stabilities, storage stability, reusability and plastic degradation ability are believed to make PETase-Amy-CLEA a promising biocatalyst in plastic degradation.

Analysis of Poly(ethylene terephthalate) degradation kinetics of evolved IsPETase variants using a surface crowding model.

Poly(ethylene terephthalate) (PET) is a major plastic polymer utilized in the single-use and textile industries. The discovery of PET-degrading enzymes (PETases) has led to an increased interest in the biological recycling of PET in addition to mechanical recycling. IsPETase from Ideonella sakaiensis is a candidate catalyst, but little is understood about its structure-function relationships with regards to PET degradation. To understand the effects of mutations on IsPETase productivity, we develop a directed evolution assay to identify mutations beneficial to PET film degradation at 30 °C. IsPETase also displays enzyme concentration-dependent inhibition effects, and surface crowding has been proposed as a causal phenomenon. Based on total internal reflectance fluorescence microscopy and adsorption experiments, IsPETase is likely experiencing crowded conditions on PET films. Molecular dynamics simulations of IsPETase variants reveal a decrease in active site flexibility in free enzymes and reduced probability of productive active site formation in substrate-bound enzymes under crowding. Hence, we develop a surface crowding model to analyze the biochemical effects of three hit mutations (T116P, S238N, S290P) that enhanced ambient temperature activity and/or thermostability. We find that T116P decreases susceptibility to crowding, resulting in higher PET degradation product accumulation despite no change in intrinsic catalytic rate. In conclusion, we show that a macromolecular crowding-based biochemical model can be used to analyze the effects of mutations on properties of PETases and that crowding behavior is a major property to be targeted for enzyme engineering for improved PET degradation.

Surface-active antibiotic production as a multifunctional adaptation for postfire microorganisms.

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments.

Relationships of crystallinity and reaction rates for enzymatic degradation of poly (ethylene terephthalate), PET.

Biocatalytic degradation of plastic waste is anticipated to play an important role in future recycling systems. However, enzymatic degradation of crystalline poly (ethylene terephthalate) (PET) remains consistently poor. Herein, we employed functional assays to elucidate the molecular underpinnings of this limitation. This included utilizing complementary activity assays to monitor the degradation of PET disks with varying crystallinity (XC), as well as determining enzymatic kinetic parameters for soluble PET fragments. The results indicate that an efficient PET-hydrolase, LCCICCG, operates through an endolytic mode of action, and that its activity is limited by conformational constraints in the PET polymer. Such constraints become more pronounced at high XC values, and this limits the density of productive sites on the PET surface. Endolytic chain-scissions are the dominant reaction type in the initial stage, and this means that little or no soluble organic product are released. However, endolytic cuts gradually and locally promote chain mobility and hence the density of attack sites on the surface. This leads to an upward concave progress curve; a behavior sometimes termed lag-phase kinetics.

Antagonistic actions of Paucibacter aquatile B51 and its lasso peptide paucinodin toward cyanobacterial bloom-forming Microcystis aeruginosa PCC7806.

Superior antagonistic activity against axenic Microcystis aeruginosa PCC7806 was observed with Paucibacter sp. B51 isolated from cyanobacterial bloom samples among 43 tested freshwater bacterial species. Complete genome sequencing, analyzing average nucleotide identity and digital DNA-DNA hybridization, designated the B51 strain as Paucibacter aquatile. Electron and fluorescence microscopic image analyses revealed the presence of the B51 strain in the vicinity of M. aeruginosa cells, which might provoke direct inhibition of the photosynthetic activity of the PCC7806 cells, leading to perturbation of cellular metabolisms and consequent cell death. Our speculation was supported by the findings that growth failure of the PCC7806 cells led to low pH conditions with fewer chlorophylls and down-regulation of photosystem genes (e.g., psbD and psaB) during their 48-h co-culture condition. Interestingly, the concentrated ethyl acetate extracts obtained from B51-grown supernatant exhibited a growth-inhibitory effect on PCC7806. The physical separation of both strains by a filter system led to no inhibitory activity of the B51 cells, suggesting that contact-mediated anti-cyanobacterial compounds might also be responsible for hampering the growth of the PCC7806 cells. Bioinformatic tools identified 12 gene clusters that possibly produce secondary metabolites, including a class II lasso peptide in the B51 genome. Further chemical analysis demonstrated anti-cyanobacterial activity from fractionated samples having a rubrivinodin-like lasso peptide, named paucinodin. Taken together, both contact-mediated inhibition of photosynthesis and the lasso peptide secretion of the B51 strain are responsible for the anti-cyanobacterial activity of P. aquatile B51.

Genomic Insights into Paucibacter aquatile DH15, a Cyanobactericidal Bacterium, and Comparative Genomics of the Genus Paucibacter.

Microcystis blooms threaten ecosystem function and cause substantial economic losses. Microorganism-based methods, mainly using cyanobactericidal bacteria, are considered one of the most ecologically sound methods to control Microcystis blooms. This study focused on gaining genomic insights into Paucibacter aquatile DH15 that exhibited excellent cyanobactericidal effects against Microcystis. Additionally, a pan-genome analysis of the genus Paucibacter was conducted to enhance our understanding of the ecophysiological significance of this genus. Based on phylogenomic analyses, strain DH15 was classified as a member of the species Paucibacter aquatile. The genome analysis supported that strain DH15 can effectively destroy Microcystis, possibly due to the specific genes involved in the flagellar synthesis, cell wall degradation, and the production of cyanobactericidal compounds. The pan-genome analysis revealed the diversity and adaptability of the genus Paucibacter, highlighting its potential to absorb external genetic elements. Paucibacter species were anticipated to play a vital role in the ecosystem by potentially providing essential nutrients, such as vitamins B7, B12, and heme, to auxotrophic microbial groups. Overall, our findings contribute to understanding the molecular mechanisms underlying the action of cyanobactericidal bacteria against Microcystis and shed light on the ecological significance of the genus Paucibacter.

Ideonella oryzae sp. nov., isolated from soil, and Spirosoma liriopis sp. nov., isolated from fruits of Liriope platyphylla.

Gram-negative, aerobic, motile by means of two or more polar or subpolar flagella, rod-shaped strain NS12-5T and Gram-negative, facultatively anaerobic, yellow-coloured, rod-shaped strain RP8T were isolated from rice rhizosphere soil and fermented fruits of Liriope platyphylla in the Republic of Korea, respectively. The result of phylogenetic analyses based on 16S rRNA gene sequences showed that strain NS12-5T was most closely related to Ideonella aquatica 4Y11T with 99.79 % sequence similarity. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain NS12-5T and species of the genus Ideonella were 75.6-91.7 % and 20.3-43.9 %, respectively. Growth occurred at 15-40 °C and pH 5-11, and NaCl was not needed for growth. The major fatty acids of strain NS12-5T were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c) and C16 : 0, and the major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C content of strain NS12-5T was 69.03 mol%. The result of phylogenetic analyses based on 16S rRNA gene sequences revealed that strain RP8T was most closely related to Spirosoma aureum BT328T with 96.01 % sequence similarity. The ANI and dDDH values between strain RP8T and reference strains of the genus Spirosoma were 72.9-76.4 % and 18.6-20.0 %, respectively. Growth occurred at 15-37 °C and pH 5-11, and NaCl was not needed for growth. The major fatty acids of strain RP8T were summed feature 3 (comprising C16 : 1 ω7c and/or C16 : 1 ω6c), C16 : 1 ω5c and iso-C15 : 0. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. The DNA G+C contents of strain RP8T were 54.9 mol%. Based on phenotypic, genomic and phylogenetic results, strains NS12-5T and RP8T represent novel species in the genus Ideonella and Spirosoma, respectively, and the names Ideonella oryzae sp. nov. and Spirosoma liriopis sp. nov. are proposed. The type strain of I. oryzae sp. nov. is NS12-5T (=KACC 22691T=TBRC 16346T) and the type strain of S. liriopis is RP8T (=KACC 22688T=TBRC 16345T).

Molecular mechanism underlying high-affinity terephthalate binding and conformational change of TBP from Ideonella sakaiensis.

Ideonella sakaiensis is the bacterium that can survive by degrading polyethylene terephthalate (PET) plastic, and terephthalic acid (TPA) binding protein (IsTBP) is an essential periplasmic protein for uptake of TPA into the cytosol for complete degradation of PET. Here, we demonstrated that IsTBP has remarkably high specificity for TPA among 33 monophenolic compounds and two 1,6-dicarboxylic acids tested. Structural comparisons with 6-carboxylic acid binding protein (RpAdpC) and TBP from Comamonas sp. E6 (CsTphC) revealed the key structural features that contribute to high TPA specificity and affinity of IsTBP. We also elucidated the molecular mechanism underlying the conformational change upon TPA binding. In addition, we developed the IsTBP variant with enhanced TPA sensitivity, which can be expanded for the use of TBP as a biosensor for PET degradation.

Phylogenetic analyses of Candidatus Branchiomonas cysticola refine the taxonomic classification of Betaproteobacteria associated with epitheliocystis in fish.

Candidatus Branchiomonas cysticola is recognized as the most prevalent bacterial agent causing epitheliocystis in Atlantic salmon (Salmo salar). Based on its partial 16S rRNA sequence, the bacterium has previously been found to be a member of Burkholderiales in the class Betaproteobacteria. Multilocus Sequence Analysis (MLSA) of the bacterium and 60 type strains of Betaproteobacteria using newly identified housekeeping genes (dnaK, rpoC, and fusA) and ribosomal subunit sequences (16S and 23S), instead supported the bacterium's affiliation to Nitrosomodales. Taxonomic rank normalization by Relative Evolutionary Divergence (RED) showed the phylogenetic distinction between Cand. B. cysticola and its closest related type strain to be at the family level. A novel bacterial family named Branchiomonaceae has thus been proposed to include a monophyletic clade of Betaproteobacteria exclusively associated with epitheliocystis in fish.

Rational Design of Disulfide Bridges in BbPETaseCD for Enhancing the Enzymatic Performance in PET Degradation.

Polyethylene terephthalate (PET) is one of the most prevalent transparent thermoplastics. It is commonly utilized due to its low cost and high durability. With the massive accumulation of waste PET, however, serious environmental pollution has become a global problem. Compared to traditional chemical degradation, biodegradation of PET catalyzed by PET hydrolase (PETase) is more environmentally friendly and energy-efficient. BbPETaseCD from the Burkholderiales bacterium is a PETase that shows favorable properties for application in the biodegradation of PET. To enhance the enzymatic performance of this enzyme, this work focuses on the rational design of disulfide bridges in BbPETaseCD. We utilized two computational algorithms to predict the probable disulfide-bridge mutations in BbPETaseCD, and five variants were acquired from the computations. Among these, the N364C/D418C variant with one additional disulfide bond showed higher expression than the wild-type enzyme (WT) and the best enzymatic performance. The melting temperature (Tm) of the N364C/D418C variant presented an increase of 14.8 °C over that of WT (56.5 °C), indicating that the additional disulfide bond significantly raised the thermodynamic stability of the enzyme. Kinetic experiments at different temperatures also demonstrated the thermal stability increase of the variant. The variant also showed significantly increased activity over WT when using bis(hydroxyethyl) terephthalate (BHET) as the substrate. More remarkably, the N364C/D418C variant exhibited approximately an 11-fold increase over the WT enzyme in the long-term (14 days) degradation of PET films. The results prove that the rationally designed disulfide bond significantly improved the enzymatic performance of the enzyme for PET degradation.

Transcriptome and Deletion Mutant Analyses Revealed that an RpoH Family Sigma Factor Is Essential for Photosystem Production in Roseateles depolymerans under Carbon Starvation.

Roseateles depolymerans is an obligately aerobic bacterium that produces a photosynthetic apparatus only under the scarcity of carbon substrates. We herein examined changes in the transcriptomes of R. depolymerans cells to clarify the expression of photosynthesis genes and their upstream regulatory factors under carbon starvation. Transcriptomes 0, 1, and 6| |h after the depletion of a carbon substrate indicated that transcripts showing the greatest variations (a 500-fold increase [6 h/0 h]) were light-harvesting proteins (PufA and PufB). Moreover, loci with more than 50-fold increases (6 h/0| |h) were fully related to the photosynthetic gene cluster. Among 13 sigma factor genes, the transcripts of a sigma 70 family sigma factor related to RpoH (SP70) increased along photosynthesis genes under starvation; therefore, a knockout experiment of SP70 was performed. ΔSP70 mutants were found to lack photosynthetic pigments (carotenoids and bacteriochlo-rophyll a) regardless of carbon starvation. We also examined the effects of heat stress on ΔSP70 mutants, and found that SP70 was also related to heat stress tolerance, similar to other RpoH sigma factors (while heat stress did not trigger photosystem production). The deficient accumulation of photosynthetic pigments and the heat stress tolerance of ΔSP70 mutants were both complemented by the introduction of an intact SP70 gene. Furthermore, the transcription of photosynthetic gene operons (puf, puh, and bch) was markedly reduced in the ΔSP70 mutant. The RpoH homologue SP70 was concluded to be a sigma factor that is essential for the transcription of photosynthetic gene operons in R. depolymerans.