Isolation and characterization of polyhydroxyalkanoate-degrading bacteria in seawater at two different depths from Suruga Bay.

IMPORTANCE: Polyhydroxyalkanoate (PHA) is a highly biodegradable microbial polyester, even in marine environments. In this study, we incorporated an enrichment culture-like approach in the process of isolating marine PHA-degrading bacteria. The resulting 91 isolates were suggested to fall into five genera (Alloalcanivorax, Alteromonas, Arenicella, Microbacterium, and Pseudoalteromonas) based on 16S rRNA analysis, including two novel genera (Arenicella and Microbacterium) as marine PHA-degrading bacteria. Microbacterium schleiferi (DSM 20489) and Alteromonas macleodii (NBRC 102226), the type strains closest to the several isolates, have an extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase homolog that does not fit a marine-type domain composition. However, A. macleodii exhibited no PHA degradation ability, unlike M. schleiferi. This result demonstrates that the isolated Alteromonas spp. are different species from A. macleodii. P(3HB) depolymerase homologs in the genus Alteromonas should be scrutinized in the future, particularly about which ones work as the depolymerase.

Exploring Class I polyhydroxyalkanoate synthases with broad substrate specificity for polymerization of structurally diverse monomer units.

Polyhydroxyalkanoate (PHA) synthases (PhaCs) are key enzymes in PHA polymerization. PhaCs with broad substrate specificity are attractive for synthesizing structurally diverse PHAs. In the PHA family, 3-hydroxybutyrate (3HB)-based copolymers are industrially produced using Class I PhaCs and can be used as practical biodegradable thermoplastics. However, Class I PhaCs with broad substrate specificities are scarce, prompting our search for novel PhaCs. In this study, four new PhaCs from the bacteria Ferrimonas marina, Plesiomonas shigelloides, Shewanella pealeana, and Vibrio metschnikovii were selected via a homology search against the GenBank database, using the amino acid sequence of Aeromonas caviae PHA synthase (PhaCAc), a Class I enzyme with a wide range of substrate specificities, as a template. The four PhaCs were characterized in terms of their polymerization ability and substrate specificity, using Escherichia coli as a host for PHA production. All the new PhaCs were able to synthesize P(3HB) in E. coli with a high molecular weight, surpassing PhaCAc. The substrate specificity of PhaCs was evaluated by synthesizing 3HB-based copolymers with 3-hydroxyhexanoate, 3-hydroxy-4-methylvalerate, 3-hydroxy-2-methylbutyrate, and 3-hydroxypivalate monomers. Interestingly, PhaC from P. shigelloides (PhaCPs) exhibited relatively broad substrate specificity. PhaCPs was further engineered through site-directed mutagenesis, and the variant resulted in an enzyme with improved polymerization ability and substrate specificity.

School-based online survey on chronic headache, migraine, and medication-overuse headache prevalence among children and adolescents in Japanese one city - Itoigawa Benizuwaigani study.

BACKGROUND: We investigated the prevalence of headache, migraine, and medication-overuse headache (MOH) among children and adolescents through a school-based online questionnaire. We also investigated the triggers for migraine among them and the effect of the COVID-19 pandemic on headache frequency. METHODS: Children and adolescents aged 6-17 y.o. completed an online questionnaire. Migraine, MOH was defined as The International Classification of Headache Disorders Third edition. Factor and clustering analyses were performed for migraine triggers. The effect of the coronavirus disease 2019 (COVID-19) pandemic on headache frequency was also asked. RESULTS: Of the 2489 respondents, the prevalence of headache, migraine, and MOH were 36.44%, 9.48%, and 0.44%, respectively. Up to 70% of the respondents with headaches complained of the disturbance to daily life, but about 30% consulted doctors. The migraine triggers were grouped into 5 factors by factor analysis. The sensitivities of the migraineurs against the factors were divided into 3 clusters. Cluster 1 had stronger sensitivity for several triggers. Cluster 2 was sensitive to weather, smartphones, and video games. Cluster 3 had less sensitivity for triggers. Cluster 2 less consulted doctors even though the burden of migraine was enormous. During the COVID-19 pandemic, 10.25% of respondents increased headache attacks, while 3.97% decreased. CONCLUSIONS: This is the first detailed study on headache prevalence in Japanese students from elementary school to high school in one region. The burden of headaches is large among children and adolescents, and the unmet needs of its clinical practice should be corrected.

Headache education by leaflet distribution during COVID-19 vaccination and school-based on-demand e-learning: Itoigawa Geopark Headache Awareness Campaign.

OBJECTIVE: We prospectively performed the Itoigawa Headache Awareness Campaign from August 2021 to June 2022, with two main interventions, and evaluated its effectiveness. BACKGROUND: Headache is a common public health problem, but its burden could be reduced by raising awareness about headache and the appropriate use of acute and prophylactic medication. However, few studies on raising headache awareness in the general public have been reported. METHODS: The target group was the general public aged 15-64. We performed two main interventions synergistically supported by other small interventions. Intervention 1 included leaflet distribution and a paper-based questionnaire about headache during COVID-19 vaccination, and intervention 2 included on-demand e-learning and online survey through schools. In these interventions, we emphasize the six important topics for the general public that were described in the Clinical Practice Guideline for Headache Disorders 2021. Each response among the two interventions' cohorts was collected on pre and post occasions. The awareness of the six topics before and after the campaign was evaluated. RESULTS: We obtained 4016 valid responses from 6382 individuals who underwent vaccination in intervention 1 and 2577 from 594 students and 1983 parents in intervention 2; thus, 6593 of 20,458 (32.2%) of the overall working-age population in Itoigawa city experienced these interventions. The percentage of individuals' aware of the six topics significantly increased after the two main interventions ranging from 6.6% (39/594)-40.0% (1606/4016) to 64.1% (381/594)-92.6% (1836/1983) (p < 0.001, all). CONCLUSIONS: We conducted this campaign through two main interventions with an improved percentage of individuals who know about headache. The two methods of community-based interventions could raise headache awareness effectively. Furthermore, we can achieve outstanding results by doing something to raise disease awareness during mass vaccination, when almost all residents gather in a certain place, and school-based e-learning without face-to-face instruction due to the COVID-19 pandemic.

Oxidation of methionine-derived 2-hydroxyalkanoate unit in biosynthesized polyhydroxyalkanoate copolymers.

In this study, 2-hydroxy-4-methylthiobutyrate (2H4MTB)-containing polyhydroxyalkanoate (PHA) copolymers were biosynthesized using methionine as the 2H4MTB precursor. 2H4MTB is a novel monomer unit that contains a sulfur atom in its side chain. The 2H4MTB-containing PHA was biosynthesized by functionalizing the leucine degradation and PHA synthesis pathways in recombinant Escherichia coli. The 2H4MTB fraction in the PHA copolymer was increased up to 30.6 mol% by increasing the methionine concentration in the medium. Using purified polymers containing 10.9 mol% 2H4MTB unit, the sulfide group of the 2H4MTB side chain was oxidized with hydrogen peroxide or peracetic acid, which resulted in the conversion of sulfide to sulfoxide and sulfone groups. The oxidized polymer was relatively hydrophilic, as revealed by water contact angle measurements, and swelled slightly when soaked in water. These results suggest that the 2H4MTB unit can be used as an oxidation site to impart hydrophilicity to the PHA copolymers.

Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli.

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

The influence of medium composition on the microbial secretory production of hydroxyalkanoate oligomers.

With the aid of a chain transfer (CT) reaction, hydroxyalkanoate (HA) oligomers can be secreted by recombinant Escherichia coli carrying the gene encoding a lactate-polymerizing enzyme (PhaC1PsSTQK) in Luria-Bertani (LB) medium supplemented with a carbon source and CT agent. In this study, HA oligomers were produced through microbial secretion using a mineral-based medium instead of LB medium, and the impact of medium composition on HA oligomer secretion was investigated. The focused targets were medium composition and NaCl concentration related to osmotic conditions. It was observed that 4.21 g/L HA oligomer was secreted by recombinant E. coli in LB medium, but the amount secreted in the mineral-based modified R (MR) medium was negligible. However, when the MR medium was supplemented with 5 g/L yeast extract, 3.75 g/L HA oligomer was secreted. This can be accounted for by the enhanced expression and activity of PhaC1PsSTQK upon supplementation with growth-activated nutrients as supplementation with yeast extract also promoted cell growth and intracellular growth-associated polymer accumulation. Furthermore, upon adding 10 g/L NaCl to the yeast extract-supplemented MR medium, HA oligomer secretion increased to 6.86 g/L, implying that NaCl-induced osmotic pressure promotes HA oligomer secretion. These findings may facilitate the secretory production of HA oligomers using an inexpensive medium.

Microbial oversecretion of (R)-3-hydroxybutyrate oligomer with diethylene glycol terminal as a macromonomer for polyurethane synthesis.

Poly((R)-3-hydroxybutyrate) (P(3HB)) is a polyester that is synthesized and accumulated in many prokaryotic cells. Recently, a new culture method for the secretion of the intracellularly synthesized (R)-3-hydroxybutyrate oligomer (3HBO) from recombinant Escherichia coli cells was developed. In this study, we attempted to produce microbial 3HBO capped with a diethylene glycol terminal (3HBO-DEG) as a macromonomer for polymeric materials. First, we prepared recombinant E. coli strains harboring genes encoding various polyhydroxyalkanoate (PHA) synthases (PhaC, PhaEC or PhaRC) that can incorporate chain transfer (CT) agents such as DEG into the polymer's terminal and generate CT end-capped oligomers. To this end, each strain was cultivated under DEG supplemental conditions, and the synthesis of 3HBO-DEG was confirmed. As a result, the highest secretory production of 3HBO-DEG was observed for the PHA synthase derived from Bacillus cereus YB-4 (PhaRCYB4). To evaluate the usability of the secreted 3HBO-DEG as a macromonomer, 3HBO-DEG was purified from the culture medium and polymerized with 4,4'-diphenylmethane diisocyanate as a spacer compound. Characterization of the polymeric products revealed that 3HBO-based polyurethane was successfully obtained and was a flexible and transparent noncrystalline polymer, unlike P(3HB). These results suggested that microbial 3HBO-DEG is a promising platform building block for synthesizing polyurethane and various other polymers.

Autotrophic biosynthesis of polyhydroxyalkanoate by Ralstonia eutropha from non-combustible gas mixture with low hydrogen content.

OBJECTIVES: To autotrophically produce polyhydroxyalkanoate (PHA) by Ralstonia eutropha without the risk of gas explosion, the feasibility of using a non-combustible gas mixture with low hydrogen content was investigated. RESULTS: A non-combustible gas mixture (H2: O2: CO2: N2 = 3.6: 7.6: 12.3: 76.5) was used for a 144-hour flask cultivation of two R. eutropha strains. Initially, using strain H16, the production conditions for poly(3-hydroxybutyrate) [P(3HB)] were explored by examining nutrient deficiency. Of these, a nitrogen source-deficient culture medium yielded the highest polymer content of 70 wt% in cells. Next, to produce PHA copolymer, the recombinant strain 1F2 was cultured under the nitrogen source-deficient autotrophic condition. As a result, the accumulation of 3HB-based copolymer containing of 1.2 mol% 3-hydroxyvalerate unit and 1.2 mol% 3-hydroxy-4-methylvalerate unit was observed with 57 wt% of the cell content. CONCLUSIONS: The use of a non-combustible gas with low hydrogen content is beneficial for PHA production in eliminating the risk of explosion due to hydrogen leakage.

Synergy of valine and threonine supplementation on poly(2-hydroxybutyrate-block-3-hydroxybutyrate) synthesis in engineered Escherichia coli expressing chimeric polyhydroxyalkanoate synthase.

The engineered chimeric polyhydroxyalkanoate (PHA) synthase PhaCAR is composed of N-terminal portion of Aeromonas caviae PHA synthase and C-terminal portion of Ralstonia eutropha (Cupriavidus necator) PHA synthase. PhaCAR has a unique and useful capacity to synthesize the block PHA copolymer poly(2-hydroxybutyrate-block-3-hydroxybutyrate) [P(2HB-b-3HB)] in engineered Escherichia coli from exogenous 2HB and 3HB. In the present study, we initially attempted to incorporate the amino acid-derived 2-hydroxyalkanoate (2HA) units using PhaCAR and the 2HA-CoA-supplying enzymes lactate dehydrogenase (LdhA) and CoA transferase (HadA). Cells harboring the genes for PhaCAR, LdhA, and HadA, as well as for the 3HB-CoA-supplying enzymes ß-ketothiolase and acetoacetyl-CoA reductase, were cultivated with supplementation of four hydrophobic amino acids, i.e., leucine, valine (Val), isoleucine (Ile), and phenylalanine, in the medium. No hydrophobic amino acid-derived monomers were incorporated into the polymer, which was most likely because of the strict substrate specificity of PhaCAR; however, P(2HB-co-3HB) was unexpectedly produced with Val supplementation. The copolymer was likely P(2HB-b-3HB) based on proton nuclear magnetic resonance analysis. Based on the endogenous pathways in E. coli, 2HB units are likely derived from threonine (Thr) through deamination and dihydroxylation. In fact, dual supplementation with Thr and Val showed synergy on the 2HB fraction of the polymer. Val supplementation promoted the 2HB synthesis likely by inhibiting the metabolism of 2-ketobutyrate into Ile and/or activating Thr dehydratase. In conclusion, the LdhA/HadA/PhaCAR pathway served as the system for the synthesis of P(2HB-b-3HB) from biomass-derived carbon sources.

Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases.

Polyhydroxyalkanoate (PHA) synthases catalyze the polymerization reaction of the acyl moiety of hydroxyacyl-coenzyme A into polyester. The catalytic subunit PhaC of PHA synthase has the PhaC box sequence at the active site that is typically described as G-X-C-X-G-G (X is an arbitrary amino acid), and cysteine is an active center. In this study, an amino acid replacement was introduced into the PhaC box of the PHA synthase derived from Ralstonia eutropha (PhaCRe ) to investigate the importance of highly conserved residues in polymerizing activity. Point mutagenesis revealed that PhaCRe mutants with the expanded PhaC box sequence ([GAST]-X-C-X-[GASV]-[GA]) are functional PHA synthases. These findings highlight the low mutational robustness of the last glycine residue in the PhaC box as well as that of the active center cysteine.

In vivo and in vitro characterization of hydrophilic protein tag-fused Ralstonia eutropha polyhydroxyalkanoate synthase.

Polyhydroxyalkanoates (PHAs) are synthesized by bacteria as an intracellular storage polyester, where PHA synthase (PhaC) catalyzes the polymerization of its substrate hydroxyacyl-coenzyme A (HA-CoA) to form PHA. When PhaC is overexpressed in Escherichia coli, most PhaC protein is produced as insoluble inclusion bodies due to its low aqueous solubility. This study aimed to improve the solubility of Ralstonia eutropha PHA synthase (PhaCRe) by fusing a hydrophilic tag, glutathione S-transferase (GST), to the protein's N-terminus. In in vivo assays, the GST tag had no obvious effect on solubility and enzymatic activity of PhaCRe. However, an in vitro assay revealed that the surface of GST-fused PhaCRe (GST-PhaCRe) had increased hydrophilicity, and tended to form correct PhaCRe dimers when added to the (R)-3-hydroxybutyryl-CoA substrate. Although GST-PhaCRe displayed a long lag phase at the start of a polymerization reaction, granule-associated GST-PhaCRe showed higher catalytic activity than PhaCRe in kinetic analysis. The results are discussed in light of the dimerization mechanisms of PhaCRe.

Low Carbon Concentration Feeding Improves Medium-Chain-Length Polyhydroxyalkanoate Production in Escherichia coli Strains With Defective ß-Oxidation.

Medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) of near homopolymeric composition are unnatural polymers, having almost identical repeating units throughout the polymer chain. These homopolymeric PHAs can be produced by ß-oxidation defective bacterial hosts. Escherichia coli is an attractive workhorse for the production of such genetically engineered PHAs; however, achieving efficient production of the near homopolymers by ß-oxidation defective strains is a major challenge because of a lack of process development studies. In order to address this issue, we investigated the optimization of carbon feeding for efficient production of MCL-PHAs by an E. coli strain with defective ß-oxidation, LSBJ. Engineered bacteria were cultured in shake-flasks with intermittent feeding of a fatty acid substrate [either decanoate (C10) or dodecanoate (C12)] at various concentrations together with a co-carbon source (glucose, glycerol, or xylose) in order to support cell growth. It was found that feeding low concentrations of both fatty acids and co-carbon sources led to an enhanced production of MCL-PHAs. Additionally, the supplementation of yeast extract improved cell growth, resulting in achieving higher titers of MCL-PHA. As a result, poly(3-hydroxydecanoate) [P(3HD)] and poly(3-hydroxydodecanoate) [P(3HDD)] were produced up to 5.44 g/L and 3.50 g/L, respectively, as near homopolymers by employing the developed feeding strategy. To the best of our knowledge, we record the highest titer of P(3HD) ever reported so far.

Gas chromatography-mass spectrometry-based monomer composition analysis of medium-chain-length polyhydroxyalkanoates biosynthesized by Pseudomonas spp.

Medium-chain-length (mcl)-polyhydroxyalkanoates (PHAs), elastomeric polyesters synthesized by Genus Pseudomonas bacteria, generally have many different monomer components. In this study, PHAs biosynthesized by four type strains of Pseudomonas (P. putida, P. citronellolis, P. oleovorans, and P. pseudoalcaligenes) and a typical PHA producer (P. putida KT2440) were characterized in terms of the monomer structure and composition by gas chromatography-mass spectrometry (GC-MS) analysis. With a thiomethyl pretreatment of PHA methanolysis derivatives, two unsaturated monomers, 3-hydroxy-5-dodecenoate (3H5DD) and 3-hydroxy-5-tetradecenoate (3H5TD), were identified in mcl-PHAs produced by P. putida and P. citronellolis. The quantitative analysis of PHA monomers was performed by employing GC-MS with methanolysis derivatives, and the results coincided with those obtained by performing nuclear magnetic resonance spectroscopy. Only poly(3-hydroxybutyrate) was detected from the P. oleovorans and P. pseudoalcaligenes type strains. These analytical results would be useful as a reference standard for phenotyping of new PHA-producing bacteria.

Biosynthesis and Characteristics of Aromatic Polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHAs) are polyesters synthesized by bacteria as a carbon and energy storage material. PHAs are characterized by thermoplasticity, biodegradability, and biocompatibility, and thus have attracted considerable attention for use in medical, agricultural, and marine applications. The properties of PHAs depend on the monomer composition and many types of PHA monomers have been reported. This review focuses on biosynthesized PHAs bearing aromatic groups as side chains. Aromatic PHAs show characteristics different from those of aliphatic PHAs. This review summarizes the types of aromatic PHAs and their characteristics, including their thermal and mechanical properties and degradation behavior. Furthermore, the effect of the introduction of an aromatic monomer on the glass transition temperature (Tg) of PHAs is discussed. The introduction of aromatic monomers into PHA chains is a promising method for improving the properties of PHAs, as the characteristics of aromatic PHAs differ from those of aliphatic PHAs.

Biosynthesis of polyhydroxyalkanoates containing 2-hydroxy-4-methylvalerate and 2-hydroxy-3-phenylpropionate units from a related or unrelated carbon source.

The discovery of the lactate-polymerizing enzyme (LPE) enabled the biosynthesis of a polyhydroxyalkanoate (PHA) containing 2-hydroxyalkanoate (2HA). Amino acids are potential precursors of 2HA with various side chain structures if appropriate enzymes are used to convert amino acids to 2HA-coenzyme A (CoA) as the substrate for LPE. In this study, the suitability and utility of (R)-2-hydroxy-4-methylvalerate (2H4MV) dehydrogenase (LdhA) and 2H4MV-CoA transferase (HadA) from Clostridium difficile as 2HA-CoA-supplying enzymes were investigated. By expressing LPE, LdhA, and HadA in Escherichia coli DH5α, we successfully produced poly(3-hydroxybutyrate-co-2HA) [P(3HB-co-2HA)] from a related or unrelated carbon source. The 2HA units incorporated into PHA from unrelated carbon sources were primarily 2H4MV and 2-hydroxy-3-phenylpropionate (2H3PhP), which were assumed to be derived from endogenous leucine and phenylalanine, respectively. Furthermore, P(3HB-co-22 mol% 2HA) synthesis was demonstrated by means of saccharified sugars, which are an abundant and renewable feedstock for polymer production from hemicellulosic biomass (Japanese cedar) as the carbon source. Our study shows that several types of 2HA units such as 2H4MV and 2H3PhP are endogenous monomers for PHA biosynthesis in E. coli expressing LdhA and HadA.

Real-Time Observation of Enzymatic Polyhydroxyalkanoate Polymerization Using High-Speed Scanning Atomic Force Microscopy.

The initial stage of in vitro polyhydroxyalkanoate (PHA) polymerization by PHA synthase from Ralstonia eutropha (PhaCRe) on a mica substrate in water was observed using high-speed scanning atomic force microscopy (HS-AFM). Before PHA polymerization, the adsorption-desorption cycle of the PhaCRe molecule on mica was observed in real time. For approximately 30 s after the addition of the PHA monomer, no significant change was observed on the mica substrate, but PhaCRe could be transformed into an active enzyme in water upon contact with the monomer during this period. Subsequently, linearly elongating rod-shaped objects were observed on the mica substrate, plausibly as a result of the polymerization reaction. The height of these elongating objects was considerably larger than the expected height for a single PHA chain. This observation suggests that PHA chains generated during the reported experiments might form some kind of a semiregular structure.

Genome-based analysis and gene dosage studies provide new insight into 3-hydroxy-4-methylvalerate biosynthesis in Ralstonia eutropha.

Recombinant Ralstonia eutropha strain PHB(-)4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB(-)4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB(-)4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.