N-terminal truncation of PhaCBP-M-CPF4 and its effect on PHA production.

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.

Polyhydroxyalkanoates biopolymers toward decarbonizing economy and sustainable future.

Polymers are synonymous with the modern way of living. However, polymers with a large carbon footprint, especially those derived from nonrenewable petrochemical sources, are increasingly perceived as detrimental to the environment and a sustainable future. Polyhydroxyalkanoate (PHA) is a microbial biopolymer and a plausible alternative for renewable sources. However, PHA in its monomeric forms has very limited applications due to its limited flexibility, tensile strength, and moldability. Herein, the life cycle of PHA molecules, from biosynthesis to commercial utilization for diverse applications is discussed. For clarity, the applications of this bioplastic biocomposite material are further segregated into two domains, namely, the industrial sector and the medical sector. The industry sectors reviewed here include food packaging, textiles, agriculture, automotive, and electronics. High-value addition of PHA for a sustainable future can be foreseen in the medical domain. Properties such as biodegradability and biocompatibility make PHA a suitable candidate for decarbonizing biomaterials during tissue repair, organ reconstruction, drug delivery, bone tissue engineering, and chemotherapeutics.

The optimal combination of Nile red identification, colony polymerase chain reaction, and gas chromatography detection methods in screening for polyhydroxyalkanoicate-producing bacteria.

The study devised a detection process combining Nile red-containing media, polymerase chain reaction (PCR), and gas chromatography (GC) to evaluate the possibility of microbes becoming polyhydroxyalkanoate (PHA) producers. The Nile red and PCR detection steps of designating PHA producers had true positive rates of 39.4% and 40%, respectively, and the use of GC analysis as the final step yielded accurate results for the production types and yields of PHAs. When the number of screening samples was up to 102, connecting all three inspection methods in tandem generated economic benefits. When up to 105 samples were screened, the use of all three detection methods reduced the cost to 3% of the cost and the time consumed 6% of using just Nile red plus GC or PCR plus GC. However, when the sum of samples exceeded 108, the cost of combining the three methods exceeds 1 million US dollars and was excessive; here, the combination of Nile red plus PCR could be considered, even though the true positive rate was only 30.7%.

High natural PHA production from acetate in Cobetia sp. MC34 and Cobetia marina DSM 4741T and in silico analyses of the genus specific PhaC2 polymerase variant.

BACKGROUND: Several members of the bacterial Halomonadacea family are natural producers of polyhydroxyalkanoates (PHA), which are promising materials for use as biodegradable bioplastics. Type-strain species of Cobetia are designated PHA positive, and recent studies have demonstrated relatively high PHA production for a few strains within this genus. Industrially relevant PHA producers may therefore be present among uncharacterized or less explored members. In this study, we characterized PHA production in two marine Cobetia strains. We further analyzed their genomes to elucidate pha genes and metabolic pathways which may facilitate future optimization of PHA production in these strains. RESULTS: Cobetia sp. MC34 and Cobetia marina DSM 4741T were mesophilic, halotolerant, and produced PHA from four pure substrates. Sodium acetate with- and without co-supplementation of sodium valerate resulted in high PHA production titers, with production of up to 2.5 g poly(3-hydroxybutyrate) (PHB)/L and 2.1 g poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)/L in Cobetia sp. MC34, while C. marina DSM 4741T produced 2.4 g PHB/L and 3.7 g PHBV/L. Cobetia marina DSM 4741T also showed production of 2.5 g PHB/L from glycerol. The genome of Cobetia sp. MC34 was sequenced and phylogenetic analyses revealed closest relationship to Cobetia amphilecti. PHA biosynthesis genes were located at separate loci similar to the arrangement in other Halomonadacea. Further genome analyses revealed some differences in acetate- and propanoate metabolism genes between the two strains. Interestingly, only a single PHA polymerase gene (phaC2) was found in Cobetia sp. MC34, in contrast to two copies (phaC1 and phaC2) in C. marina DSM 4741T. In silico analyses based on phaC genes show that the PhaC2 variant is conserved in Cobetia and contains an extended C-terminus with a high isoelectric point and putative DNA-binding domains. CONCLUSIONS: Cobetia sp. MC34 and C. marina DSM 4741T are natural producers of PHB and PHBV from industrially relevant pure substrates including acetate. However, further scale up, optimization of growth conditions, or use of metabolic engineering is required to obtain industrially relevant PHA production titers. The putative role of the Cobetia PhaC2 variant in DNA-binding and the potential implications remains to be addressed by in vitro- or in vivo methods.

Deletion of the pps-like gene activates the cryptic phaC genes in Haloferax mediterranei.

Haloferax mediterranei, a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) producing haloarchaeon, possesses four PHA synthase encoding genes, phaC, phaC1, phaC2, and phaC3. In the wild-type strain, except phaC, the other three genes are cryptic and not transcribed under PHA-accumulating conditions. The PhaC protein together with PhaE subunit forms the active PHA synthase and catalyzes PHBV polymerization. Previously, it was observed that the deletion of a gene named pps-like significantly enhanced PHBV accumulation probably resulted from the upregulation of pha cluster genes (phaR-phaP-phaE-phaC). The present study demonstrated the influence of pps-like gene deletion on the cryptic phaC genes. As revealed by qRT-PCR, the expression level of the three cryptic genes was upregulated in the ΔEPSΔpps-like geneΔphaC mutant. Sequential knockout of the cryptic phaC genes and fermentation experiments showed that PhaC1 followed by PhaC3 had the ability to synthesize PHBV in ΔEPSΔpps-like geneΔphaC mutant. Both PhaC1 and PhaC3 could complex with PhaE to form functionally active PHA synthase. However, the expression of phaC2 did not lead to PHBV synthesis. Moreover, PhaC, PhaC1, and PhaC3 exhibited distinct substrate specificity as the 3HV content in PHBV copolymers was different. The EMSA result showed that PPS-like protein might be a negative regulator of phaC1 gene by binding to its promoter region. Taken together, PhaC1 had the most pronounced effect on PHBV synthesis in ΔEPSΔpps-like geneΔphaC mutant and deletion of pps-like gene released the negative effect from phaC1 expression and thereby restored PHBV accumulating ability in ΔphaC mutant. KEY POINTS: • Cryptic phaC genes were activated by pps-like gene deletion. • PPS-like protein probably regulated phaC1 expression by binding to its promoter. • Both PhaC1 and PhaC3 formed active PHA synthase with PhaE.

Occurence and Prioritization of Pharmaceutical Active Compounds in Domestic/Municipal Wastewater Treatment Plants.

In this study, pharmaceutically active compounds (PhACs) were analyzed in the influent and effluent of a domestic wastewater treatment plant in Turkey and a municipal wastewater treatment plant in Germany and the toxicity of these wastewaters were estimated using a toxic unit (TU) approach. A total of 21 and 32 PhACs were detected in the domestic wastewater and the municipal wastewater, respectively. The TUs estimated for PhACs in municipal wastewater were higher than the TUs estimated for PhACs present in domestic wastewater. The levels of the anti-anxiety drug, oxazepam were estimated to be in the high risk category (HQ > 10) in both wastewaters. In bench-scale tests with ozonation, the removals of four PhACs in the municipal wastewater were investigated. At a dose of 2 mg/L ozone, 97%-98% of diclofenac and carbamazepine were removed. The lowest removal rate at 71% was observed for metoprolol.

Understanding the intracellular-to-extracellular localization switch of polyhydroxybutyrate polymerase in pseudomonas backgrounds as a microevolutionary process.

After gene duplication, paralogous genes evolve independently, and consequently, the new proteins encoded by these duplicated genes are exposed to changes in their subcellular location. Although there are increasing evidence that phylogenetically related proteins play different functions in different subcellular compartments, the number of evolutionary steps required for the emergence of a novel protein with a novel subcellular localization remains unclear. Regarding this intriguing topic, here we examine in depth our previous reports describing both intracellular and extracellular polyhydroxybutyrate polymerases (PhaC) in the Pseudomonadales group. The recapitulation of the intracellular-to-extracellular localization switch of PhaC in these strains shows a gradual evolution from a simple cytosolic PhaC form to a complex extracellular PhaC form specifically secreted via the type 1 secretion system. This gradual evolution includes several adaptive and pre-adaptive changes at the genomic, genetic and enzymatic levels, which are intimately related to the lifestyle of organisms during the evolution of protein localization. We conclude that the protein localization switch can be an extremely complex process in nature.

Controlling microbial PHB synthesis via CRISPRi.

Microbial polyhydroxyalkanoates (PHA) are a family of biopolyesters with properties similar to petroleum plastics such as polyethylene (PE) or polypropylene (PP). Polyhydroxybutyrate (PHB) is the most common PHA known so far. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a technology recently developed to control gene expression levels in eukaryotic and prokaryotic genomes, was employed to regulate PHB synthase activity influencing PHB synthesis. Recombinant Escherichia coli harboring an operon of three PHB synthesis genes phaCAB cloned from Ralstonia eutropha, was transformed with various single guided RNA (sgRNA with its guide sequence of 20-23 bases) able to bind to various locations of the PHB synthase PhaC, respectively. Depending on the binding location and the number of sgRNA on phaC, CRISPRi was able to control the phaC transcription and thus PhaC activity. It was found that PHB content, molecular weight, and polydispersity were approximately in direct and reverse proportion to the PhaC activity, respectively. The higher the PhaC activity, the more the intracellular PHB accumulation, yet the less the PHB molecular weights and the wider the polydispersity. This study allowed the PHB contents to be controlled in the ranges of 1.47-75.21% cell dry weights, molecular weights from 2 to 6 millions Dalton and polydispersity of 1.2 to 1.43 in 48 h shake flask studies. This result will be very important for future development of ultrahigh molecular weight PHA useful to meet high strength application requirements.

Covalent-display of an active chimeric-recombinant tissue plasminogen activator on polyhydroxybutyrate granules surface.

OBJECTIVE: To develop a deliberately engineered expression and purification system for an active chimeric-recombinant tissue plasminogen activator (crtPA) using co-expression with polyhydroxybutyrate (PHB) operon genes. RESULTS: Fusion of crtPA with PhaC-synthase simplified the purification steps through crtPA sedimentation with PHB particles. Moreover, the covalently immobilized crtPA was biologically active as shown in a chromogenic assay. Upon WELQut-protease activity, the released single-chain crtPA converted to the two-chain form which produced a pattern of bands with approx. MW of 32 and 11 kDa in addition to the full length crtPA. CONCLUSION: Fusion of crtPA with PhaC-synthase not only simplifies purification from the bacterial host lysate, but also co-expression of PHB operon genes creates an oxidative environment, thereby reducing the inclusion body formation possibility. The isolated crtPA-PHB granules exhibited crtPA serine protease activity. Thus, fusion with the PhaC protein could be used as a scaffold for covalent displaying of functional disulfide-rich proteins.

Polyhydroxyalkanoate (PHA) accumulation potential and PHA-accumulating microbial communities in various activated sludge processes of municipal wastewater treatment plants.

AIMS: To clarify the polyhydroxyalkanoate (PHA) accumulation potential and the PHA-accumulating microbial community structure in activated sludge in municipal wastewater treatment plants (WWTPs) and to identify their influential factors. METHODS AND RESULTS: Nine activated sludge samples were collected from municipal WWTPs employing various biological treatment processes. In acetate-fed 24-h batch experiments under aerobic and nitrogen- and phosphorus-limited conditions, polyhydroxybutyrate (PHB) content of activated sludge increased from 0-1·3 wt% to 7·9-24 wt%, with PHB yields of 0·22-0·50 C-mol 3-hydroxybutyrate (C-mol acetate)(-1). Microbial community analyses found that activated sludge samples that accumulated >20 wt% of PHB after 24-h PHA accumulation experiments had >5·0 × 10(8) copies g(-1)-mixed liquor-suspended solid of phaC genes. CONCLUSIONS: Results indicated that (i) activated sludge in municipal WWTPs can accumulate up to approx. 20 wt% of PHA without enrichment processes, (ii) PHA accumulation potential of activated sludge varied depending on the operational conditions (treatment processes) of WWTPs, and (iii) phaC gene number can provide a simple indication of PHA accumulation potential. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to compare the PHA accumulation potential and PHA-accumulating microbial communities in activated sludge of various treatment processes. Our findings may be useful for enhancing the resource recovery potential of wastewater treatment systems.

Exogenous Trehalose Improves Growth, Glycogen and Poly-3-Hydroxybutyrate (PHB) Contents in Photoautotrophically Grown Arthrospira platensis under Nitrogen Deprivation.

Glycogen and poly-3-hydroxybutyrate (PHB) are excellent biopolymer products from cyanobacteria. In this study, we demonstrate that nitrogen metabolism is positively influenced by the exogenous application of trehalose (Tre) in Arthrospira platensis under nitrogen-deprived (-N) conditions. Cells were cultivated photoautotrophically for 5 days under -N conditions, with or without the addition of exogenous Tre. The results revealed that biomass and chlorophyll-a content of A. platensis experienced enhancement with the addition of 0.003 M and 0.03 M Tre in the -N medium after one day, indicating relief from growth inhibition caused by nitrogen deprivation. The highest glycogen content (54.09 ± 1.6% (w/w) DW) was observed in cells grown for 2 days under the -N + 0.003 M Tre condition (p < 0.05), while the highest PHB content (15.2 ± 0.2% (w/w) DW) was observed in cells grown for 3 days under the -N + 0.03 M Tre condition (p < 0.05). The RT-PCR analysis showed a significant increase in glgA and phaC transcript levels, representing approximately 1.2- and 1.3-fold increases, respectively, in A. platensis grown under -N + 0.003 M Tre and -N + 0.03 M Tre conditions. This was accompanied by the induction of enzyme activities, including glycogen synthase and PHA synthase with maximal values of 89.15 and 0.68 µmol min-1 mg-1 protein, respectively. The chemical structure identification of glycogen and PHB from A. platensis was confirmed by FTIR and NMR analysis. This research represents the first study examining the performance of trehalose in promoting glycogen and PHB production in cyanobacteria under nitrogen-deprived conditions.

Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

Production of polyhydroxyalkanoates using sewage and cheese whey.

Recently, polyhydroxyalkanoates (PHAs) have been produced using raw sewage in our laboratory; however, the production concentrations are low. Therefore, this study aimed to enhance PHA production by applying different strategies. PHA production was higher in sewage-containing medium than in mineral salt medium and was enhanced 22-fold after glucose supplementation. A relatively high degree of glucose consumption (83.6 ± 1.59 %) was also achieved. Bacteria incubated with cheese whey diluted with sewage showed higher PHA production than bacteria incubated with cheese whey diluted with distilled water did. The expression of the PHA synthase gene (phaC) was evaluated via real-time polymerase chain reaction using low- and high-carbon-containing sewage. Relatively higher phaC expression levels were observed in high-carbon-containing sewage but at lower nitrogen concentrations. The characteristics of the produced PHA were comparable to those of standard PHA. Therefore, this study revealed that the bacterium Bacillus sp. CYR1 can produce PHA from low- or high-carbon-containing wastewater.

Ultrafiltration and Nanofiltration for the Removal of Pharmaceutically Active Compounds from Water: The Effect of Operating Pressure on Electrostatic Solute-Membrane Interactions.

The present work investigates nanofiltration (NF) and ultrafiltration (UF) for the removal of three widely used pharmaceutically active compounds (PhACs), namely atenolol, sulfamethoxazole, and rosuvastatin. Four membranes, two polyamide NF membranes (NF90 and NF270) and two polyethersulfone UF membranes (XT and ST), were evaluated in terms of productivity (permeate flux) and selectivity (rejection of PhACs) at pressures from 2 to 8 bar. Although the UF membranes have a much higher molecular weight cut-off (1000 and 10,000 Da), when compared to the molecular weight of the PhACs (253-482 Da), moderate rejections were observed. For UF, rejections were dependent on the molecular weight and charge of the PhACs, membrane molecular weight cut-off (MWCO), and operating pressure, demonstrating that electrostatic interactions play an important role in the removal of PhACs, especially at low operating pressures. On the other hand, both NF membranes displayed high rejections for all PhACs studied (75-98%). Hence, considering the optimal operating conditions, the NF270 membrane (MWCO = 400 Da) presented the best performance, achieving permeate fluxes of about 100 kg h-1 m-2 and rejections above 80% at a pressure of 8 bar, that is, a productivity of about twice that of the NF90 membrane (MWCO = 200 Da). Therefore, NF270 was the most suitable membrane for this application, although the tight UF membranes under low operating pressures displayed satisfactory results.

Polyhydroxyalkanoate (PHA) Biopolymer Synthesis by Marine Bacteria of the Malaysian Coral Triangle Region and Mining for PHA Synthase Genes.

Polyhydroxyalkanoate (PHA), a biodegradable and plastic-like biopolymer, has been receiving research and industrial attention due to severe plastic pollution, resource depletion, and global waste issues. This has spurred the isolation and characterisation of novel PHA-producing strains through cultivation and non-cultivation approaches, with a particular interest in genes encoding PHA synthesis pathways. Since sea sponges and sediment are marine benthic habitats known to be rich in microbial diversity, sponge tissues (Xestospongia muta and Aaptos aaptos) and sediment samples were collected in this study from Redang and Bidong islands located in the Malaysian Coral Triangle region. PHA synthase (phaC) genes were identified from sediment-associated bacterial strains using a cultivation approach and from sponge-associated bacterial metagenomes using a non-cultivation approach. In addition, phylogenetic diversity profiling was performed for the sponge-associated bacterial community using 16S ribosomal ribonucleic acid (16S rRNA) amplicon sequencing to screen for the potential presence of PHA-producer taxa. A total of three phaC genes from the bacterial metagenome of Aaptos and three phaC genes from sediment isolates (Sphingobacterium mizutaii UMTKB-6, Alcaligenes faecalis UMTKB-7, Acinetobacter calcoaceticus UMTKB-8) were identified. Produced PHA polymers were shown to be composed of 5C to nC monomers, with previously unreported PHA-producing ability of the S. mizutaii strain, as well as a 3-hydroxyvalerate-synthesising ability without precursor addition by the A. calcoaceticus strain.

Enhanced Antitumor Efficacy of PhAc-ALGP-Dox, an Enzyme-Activated Doxorubicin Prodrug, in a Panel of THOP1-Expressing Patient-Derived Xenografts of Soft Tissue Sarcoma.

Despite poor response rates and dose-limiting cardiotoxicity, doxorubicin (doxo) remains the standard-of-care for patients with advanced soft tissue sarcoma. We evaluated the efficacy of two tetrapeptidic doxo prodrugs (PhAc-ALGP-Dox or CBR-049 and CBR-050) that are locally activated by enzymes expressed in the tumor environment, in ten sarcoma patient-derived xenografts. Xenograft models were selected based on expression of the main activating enzyme, i.e., thimet oligopeptidase (THOP1). Mice were either randomized to vehicle, doxo, CBR-049 and CBR-050 or control, doxo, aldoxorubicin (aldoxo) and CBR-049. Treatment efficacy was assessed by tumor volume measurement and histological assessment of ex-mouse tumors. CBR-049 showed significant tumor growth delay compared to control in all xenografts investigated and was superior compared to doxo in all but one. At the same time, CBR-049 showed comparable efficacy to aldoxo but the latter was found to have a complex safety profile in mice. CBR-050 demonstrated tumor growth delay compared to control in one xenograft but was not superior to doxo. For both experimental prodrugs, strong immunostaining for THOP1 was found to predict better antitumor efficacy. The prodrugs were well tolerated without any adverse events, even though molar doses were 17-fold higher than those administered and tolerated for doxo.

Biosynthesis of P(3HB-co-3HHx) Copolymers by a Newly Engineered Strain of Cupriavidus necator PHB-4/pBBR_CnPro-phaCRp for Skin Tissue Engineering Application.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers synthesized by certain bacteria and archaea with functions comparable to conventional plastics. Previously, our research group reported a newly PHA-producing bacterial strain, Rhodococcus pyridinivorans BSRT1-1, from the soil in Thailand. However, this strain's PHA synthase (phaCRp) gene has not yet been characterized. Thus, this study aims to synthesize PHA using a newly engineered bacterial strain, Cupriavidus necator PHB-4/pBBR_CnPro-phaCRp, which harbors the phaCRp from strain BSRT1-1, and characterize the properties of PHA for skin tissue engineering application. To the best of our knowledge, this is the first study on the characterization of the PhaC from R. pyridinivorans species. The results demonstrated that the expression of the phaCRp in C. necator PHB-4 had developed in PHA production up to 3.1 ± 0.3 g/L when using 10 g/L of crude palm kernel oil (CPKO) as a sole carbon source. Interestingly, the engineered strain produced a 3-hydroxybutyrate (3HB) with 2 mol% of 3-hydroxyhexanoate (3HHx) monomer without adding precursor substrates. In addition, the 70 L stirrer bioreactor improved P(3HB-co-2 mol% 3HHx) yield 1.4-fold over the flask scale without altering monomer composition. Furthermore, the characterization of copolymer properties showed that this copolymer is promising for skin tissue engineering applications.

Human Drug Pollution in the Aquatic System: The Biochemical Responses of Danio rerio Adults.

To date, drug pollution in aquatic systems is an urgent issue, and Danio rerio is a model organism to study the toxicological effects of environmental pollutants. The scientific literature has analyzed the effect of human drug pollution on the biochemical responses in the tissues of D. rerio adults. However, the information is still scarce and conflicting, making it difficult to understand its real impact. The scientific studies are not consistent with each other and, until now, no one has grouped their results to create a baseline of knowledge of the possible impacts. In this review, the analysis of literature data highlights that the effects of drugs on adult zebrafishes depend on various factors, such as the tissue analyzed, the drug concentration and the sex of the individuals. Furthermore, the most influenced biochemical responses concern enzymes (e.g., antioxidants and hydrolase enzymes) and total protein and hormonal levels. Pinpointing the situation to date would improve the understanding of the chronic effects of human drug pollution, helping both to reduce it in the aquatic systems and then to draw up regulations to control this type of pollution.

Identification of regions affecting enzyme activity, substrate binding, dimer stabilization and polyhydroxyalkanoate (PHA) granule morphology in the PHA synthase of Aquitalea sp. USM4.

Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized by microorganisms as intracellular energy reservoirs under stressful environmental conditions. PHA synthase (PhaC) is the key enzyme responsible for PHA biosynthesis, but the importance of its N- and C-terminal ends still remains elusive. Six plasmid constructs expressing truncation variants of Aquitalea sp. USM4 PhaC (PhaC1As) were generated and heterologously expressed in Cupriavidus necator PHB-4. Removal of the first six residues at the N-terminus enabled the modulation of PHA composition without altering the PHA content in cells. Meanwhile, deletion of 13 amino acids from the C-terminus greatly affected the catalytic activity of PhaC1As, retaining only 1.1-7.4% of the total activity. Truncation(s) at the N- and/or C-terminus of PhaC1As gradually diminished the incorporation of comonomer units, and revealed that the N-terminal region is essential for PhaC1As dimerization whereas the C-terminal region is required for stabilization. Notably, transmission electron microscopy analysis showed that PhaC modification affected the morphology of intracellular PHA granules, which until now is only known to be regulated by phasins. This study provided substantial evidence and highlighted the significance of both the N- and C-termini of PhaC1As in regulating intracellular granule morphology, activity, substrate specificity, dimerization and stability of the synthase.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in metabolically recombinant Escherichia coli.

In this study, a phaCR gene encoding PHA synthase was identified in Rhodoligotrophos defluvii which was adjacent to ß-ketothiolase encoded by phaAR gene and acetoacetyl-CoA reductase encoded by phaBR gene. Amino acid comparison of PhaCR showed the highest homology of 65.98% with PhaC of R. appendicifer, while its homology with typical class I PHA synthase in Cupriavidus necator was only 42.54%. PHA synthesis genes were then transformed into E. coli harboring phaCABR and phaCRABC which were cultured with 15 g/L glucose respectively, and 20.46 wt% and 16.95 wt% of CDW for poly(3-hydroxybutyrate) (PHB) were accumulated respectively. To further explore the effect of substrate specificity for PHA production, the ptsG gene was then deleted and 15 g/L glucose and 1.5 g/L propionate were co-employed as carbon sources, which enabled the synthesis of poly(3HB-co-3HV) copolymer. As a result, poly(3HB-co-3HV) was accumulated up to 24.74 wt% of CDW, and the highest content of 3-hydroxyvalerate (3HV) was 10.86 mol%. The Td5 was 260 °C, which implied that it possessed good thermal stability, and the Mw of GPC in recombinant strains were between 22 and 26 × 104 g/mol, and the highest PDI was 3.771. The structure of poly (3HB-co-3HV) copolymer was determined through 1H NMR analysis.