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Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer.

Commodity polymer materials are now required to be environmentally friendly due to problems associated with resource depletion and low recycling rates, which has promoted the development of circular material systems. Herein, a novel concept is introduced, where a polymer is used as a source of a fertilizer. To demonstrate the viability of this concept, the chemical recycling of poly(isosorbide carbonate) (PIC) is presented as a model for the next generation of plastic-recycling systems. PIC, a bio-based polymer known for its excellent physical properties, undergoes a degradation reaction with aqueous ammonia. Monitoring the degradation behavior by 1H NMR and FT-IR spectroscopy as well as gel-permeation chromatography revealed that PIC completely decomposes within 6 h at 90 °C to afford isosorbide and urea. The utility of isosorbide and urea obtained from the degradation of PIC as fertilizers was demonstrated via plant-growth experiments. The generation of fertilizers via polymer degradation in the present study is expected to lead not only to innovative chemical recycling systems to address the environmental problems associated with polymer materials, but also to provide solutions to the food-production problems associated with the growth of the global population.

Biopolymers are driving the plastic industry to the next generation of environmentally friendly bioproducts, considering green chemistry principles and contemporary economic concepts, such as environmental, social, and governance (ESG) criteria. Hence, microbial biopolymers arise in this context. Resulting from a natural carbon and energy storage process, polyhydroxyalkanoates are the raw material for a range of products based on plastic, with the advantage of being biodegradable in a short period of time. Discovering new biopolymers with different properties, carbon sources and PHA-related enzymes will facilitate market development as well as competition with petrochemical polymers. This work reports the experimental findings of PHA production and genomic data for two bacteria, Ralstonia pickettii and Aquitalea sp., isolated from a blackwater lake located in the ecological reserve of Tupe, Iranduba, AM, Brazil. They were able to produce PHB from carbon sources related to sugar, and R. pickettii also produced PHB from soybean oil and lignin derivatives. Whole-genome sequencing of these isolates enabled the identification of the genetic background to use other oxidizable carbon sources, such as lactic and malonic acids, amino acids, and lignin. Bacteria possibly capable of polyhydroxyalkanoate synthesis from lignin were investigated by genomics and chemistry of biomaterials. Aquitalea sp and Ralstonia pickettii isolated from a blackwater lake rich humic substances, showed great potential to be developed for this purpose. Canonical lignin degradation pathways, such as protocatechuate 4,5-dioxygenase-dependent pathway, CoA-dependent non-β-oxidation pathway of ferulic acid and β-ketoadipate pathway were mapped in their genomes. In addition, traces of P(3HB) were found when using vanillic and gallic acids as sole carbon source, which indicates that the downstream β-ketoadipate pathway is functional.

Among the various types of polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] has a high potential to serve as commercial bioplastic due to its striking resemblance to petroleum-based plastics. In this study, five different genotypes of Cupriavidusnecator transformants harbouring the phaCBP-M-CPF4 gene (including PHB¯4/pBBR1-CBP-M-CPF4) were developed to evaluate the efficiency of 3HHx monomer incorporation. The fraction of 3-hydroxyhexanoate (3HHx) monomer that was incorporated into the PHA synthesized by these C. necator transformants using palm oil as the sole carbon source, was examined. Overall, co-expression of enoyl-CoA hydratase gene (phaJ1) from Pseudomonas aeruginosa, along with PHA synthase (PhaC), increased the 3HHx composition in the PHA copolymer. The differences in the enzyme activities of β-ketothiolase (PhaACn) and NADPH-dependent acetoacetyl-CoA reductase (PhaBCn) of the C. necator mutant hosts used in this study, were observed to alter the 3HHx composition and molecular weight of the PHA copolymer produced. The 3HHx fractions in the P(3HB-co-3HHx) produced by these C. necator transformants ranged between 1 and 18 mol%, while the weight-average molecular weight ranged from 0.7 × 106 to 1.8 × 106 Da. PhaCBP-M-CPF4 displayed a typical initial lag-phase and a relatively low synthase activity in the in vitro enzyme assay, which is thought to be the reason for the higher molecular weights of PHA obtained in this study.

A novel polyhydroxyalkanoate (PHA)-producing bacterium, Jeongeupia sp. USM3 (JCM 19920) was isolated from the limestone soil at Gua Tempurung, Perak, Malaysia. This is the first report on the complete genome sequence for the genus Jeongeupia. This genome consists of a circular chromosome with a size of 3,788,814 bp and contains 3557 genes. Two PHA synthase (phaC) genes encoding for the key enzyme in the polymerization of PHA monomers and other PHA-associated genes were identified from the genome. Phylogenetic analysis of the PhaC protein sequences has revealed that both PhaC1 and PhaC2 of Jeongeupia sp. USM3 are categorized as Class I PHA synthases with 56% similarity to each other. Both of the PHA synthase genes of this isolate were cloned and heterologously expressed in a PHA mutant strain Cupriavidus necator PHB−4. The ability of the transformants to accumulate PHA showed that both PhaC1 and PhaC2 were functional.

Polyhydroxyalkanoates (PHAs) are a group of microbial polyesters that are widely studied due to their versatile physicochemical and mechanical properties. The biosynthesis of PHAs is governed by the enzyme PHA synthase. We have previously reported the discovery of a novel wide substrate specificity PHA synthase isolated from a mangrove environment. In this study, we report the biosynthesis, via the novel PHA synthase, of various PHA co- and terpolymers containing 4-hydroxybutyrate (4HB) and 5-hydroxyvalerate (5HV) monomers, which are lipase degradable. The biosynthesized polymers have varying 4HB and 5HV contents between 5 and 70 mol % depending on the concentration of precursor compounds used. All the polymers exhibit low melting temperature (Tm) and crystallinity and elastomeric behaviour due to high 4HB and 5HV monomer contents. Supplementation of crude palm kernel oil (CPKO) as carbon source with appropriate precursors enabled the biosynthesis of interesting terpolymers and copolymers containing 3-hydroxyhexanoate (3HHx) along with 5HV and/or 4HB monomers. Lipase degradation assays indicated that co- and ter-polymers containing 4HB and 5HV monomers were successfully hydrolysed by various lipases, but addition of 3HHx monomers in the polymer chain reduced its degradability. These properties of the polymers suggest potential uses as biomaterials.

Bacteria of the genus Bacillus have been investigated due to the ability that many species have of accumulating polyhydroxyalkanoates (PHA) via a wide variety of raw materials as their carbon source. Herein, we report the draft whole-genome sequence of the putative PHA-accumulating strain Bacillus paramycoides LB_RP2, isolated from an Amazonian river.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 &deg, C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks., 光合成細菌を用いてクモ糸を作ることに成功 --天然資源を利用した物質生産のモデル微生物--. 京都大学プレスリリース. 2020-07-13.

The polyhydroxyalkanoate (PHA) producing capability of four bacterial strains isolated from Antarctica was reported in a previous study. This study analyzed the PHA synthase genes and the PHA-associated gene clusters from the two antarctic Pseudomonas isolates (UMAB-08 and UMAB-40) and the two antarctic Janthinobacterium isolates (UMAB-56 and UMAB-60) through whole-genome sequence analysis. The Pseudomonas isolates were found to carry PHA synthase genes which fall into two different PHA gene clusters, namely Class I and Class II, which are involved in the biosynthesis of short-chain-length-PHA (SCL-PHA) and medium-chain-length-PHA (MCL-PHA), respectively. On the other hand, the Janthinobacterium isolates carry a Class I and an uncharacterized putative PHA synthase genes. No other gene involved in PHA synthesis was detected in close proximity to the uncharacterized putative PHA synthase gene in the Janthinobacterium isolates, therefore it falls into a separate clade from the ordinary Class I, II, III and IV clades of PHA synthase (PhaC) phylogenetic tree. Multiple sequence alignment showed that the uncharacterized putative PHA synthase gene contains all the highly conserved amino acid residues and the proposed catalytic triad of PHA synthase. PHA biosynthesis and in vitro PhaC enzymatic assay results showed that this uncharacterized putative PHA synthase from Janthinobacterium sp. UMAB-60 is funtional. This report adds new knowledge to the PHA synthase database as we describe scarce information of PHA synthase genes and PHA-associated gene clusters from the antarctic bacterial isolates (extreme and geographically isolated environment) and comparing with those from non-antarctic PHA-producing bacteria.

A filamentous benthic cyanobacteria, strain USMAC16, was isolated from the High Arctic Svalbard archipelago, Norway, and a combination of morphological, ultrastructural and molecular characterisation (16S rRNA gene sequence) used to identify to species level. Cell dimensions, thylakoid arrangement and apical cell shape are consistent with the Pseudanabaena genus description. The molecular characterisation of P. catenata gave 100% similarity with Pseudanabaena catenata SAG 1464-1, originally reported from Germany. Strain USMAC16 was cultured under a range of temperature and photoperiod conditions, in solid and liquid media, and harvested at exponential phase to examine its phenotypic plasticity. Under different culture conditions, we observed considerable variations in cell dimensions. The longest cell (5.91±0.13 μm) was observed at 15°C under 12:12 light:dark, and the widest cell (3.24±0.06 μm) at 4°C under 12:12 light: dark in liquid media. The study provides baseline data documenting the morphological variation of P. catenata in response to changing temperature regimes.

Polyhydroxyalkanoates (PHAs) are a group of natural biopolyesters that resemble petroleum-derived plastics in terms of physical properties but are less harmful biologically to the environment and humans. Most of the current PHA producers are heterotrophs, which require expensive feeding materials and thus contribute to the high price of PHAs. Marine photosynthetic bacteria are promising alternative microbial cell factories for cost-effective, carbon neutral and sustainable production of PHAs. In this study,Rhodovulum sulfidophilum, a marine photosynthetic purple nonsulfur bacterium with a high metabolic versatility, was evaluated for cell growth and PHA production under the influence of various media components found in previous studies. We evaluated iron, using ferric citrate, as another essential factor for cell growth and efficient PHA production and confirmed that PHA production inR. sulfidophilumwas growth-associated under microaerobic and photoheterotrophic conditions. In fact, a subtle amount of iron (1 to 2 μM) was sufficient to promote rapid cell growth and biomass accumulation, as well as a high PHA yield during the logarithmic phase. However, an excess amount of iron did not enhance the growth rate or PHA productivity. Thus, we successfully confirmed that an optimum concentration of iron, an essential nutrient, promotes cell growth inR. sulfidophilumand also enhances PHA utilization.

Polyhydroxyalkanoates (PHAs) are a group of natural biopolyesters that resemble petroleum-derived plastics in terms of physical properties but are less harmful biologically to the environment and humans. Most of the current PHA producers are heterotrophs, which require expensive feeding materials and thus contribute to the high price of PHAs. Marine photosynthetic bacteria are promising alternative microbial cell factories for cost-effective, carbon neutral and sustainable production of PHAs. In this study, Rhodovulum sulfidophilum, a marine photosynthetic purple nonsulfur bacterium with a high metabolic versatility, was evaluated for cell growth and PHA production under the influence of various media components found in previous studies. We evaluated iron, using ferric citrate, as another essential factor for cell growth and efficient PHA production and confirmed that PHA production in R. sulfidophilum was growth-associated under microaerobic and photoheterotrophic conditions. In fact, a subtle amount of iron (1 to 2 μM) was sufficient to promote rapid cell growth and biomass accumulation, as well as a high PHA volumetric productivity during the logarithmic phase. However, an excess amount of iron did not enhance the growth rate or PHA productivity. Thus, we successfully confirmed that an optimum concentration of iron, an essential nutrient, promotes cell growth in R. sulfidophilum and also enhances PHA utilization.

A filamentous benthic cyanobacteria strain isolated from a tropical man-made pond in Malaysia was characterised using combined phenotypic and genetic approaches. Morphological and ultrastructural observations were performed together with growth measurements. Cell dimensions, thylakoid arrangement and apical cell shape with aerotopes were consistent with the description of Pseudanabaena amphigranulata (Goor) Anagnostidis. Molecular characterisation of the16S rRNA gene gave 94% pairwise sequence identity with Pseudanabaena sp. PCC 6802,which corresponds to the genus identification threshold value while also suggesting that the strain is distinctly different to the species of Pseudanabaena currently represented in available databases. The strain showed identical 16S-23S ITS configuration with other strains of Pseudanabaena apart from having a larger spacer region. Cultures of the strain were exposed to various temperature and photoperiod treatments and harvested at exponential phase in order to examine phenotypic plasticity. Significant relationships between environmental conditions and morphological characteristics (cell dimensions and shape) were identified for the first time within the genus Pseudanabaena. The maximum cell length (5.7 ± 0.07 μm) was observed at 25 °C under 12:12 light to dark, while the greatest cell width (3.2 ± 0.11 μm) was also observed at 25 °C but under 16:8 light to dark. The strain showed high plasticity in cell dimensions and shape under different temperature and photoperiod treatments, with25 °C under 12:12 light to dark providing the optimal conditions for its growth.

This study reports the discovery of a polyhydroxyalkanoate (PHA) synthase (PhaC) possessing very wide substrate specificity from a mangrove soil metagenome. For the first time, putative PhaCs were identified from a metagenome using next-generation sequencing (NGS) and bioinformatic approaches. High-throughput shotgun metagenomic sequencing was conducted using the Illumina HiSeq 2000 platform. Sequence annotation and bioinformatic analyses were performed using the MG-RAST metagenomic pipeline. Reads annotated as PhaC against the NCBI RefSeq database were retrieved using the MG-RAST RESTful API (Application Programming Interface). PhaC gene sequence assembly was accomplished using the SPAdes assembler. A total of two de novo assembled contigs were subjected to sequence verification. A putative PhaC sequence, “BP-M-CPF4”, was selected for functional assessment by in vivo PHA biosynthesis in a PHA-negative mutant. An artificial stop codon was added at the 3′-end of the incomplete coding gene sequence. This novel PhaC showed very broad substrate specificity with the ability to incorporate six types of PHA monomers, 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 4-hydroxybutyrate (4HB), 3-hydroxy-4-methylvalerate (3H4MV), 5-hydroxyvalerate (5HV) and 3-hydroxyhexanoate (3HHx) in the presence of suitable precursors. This PHA synthase is suitable for the biosynthesis of PHAs that can be used in various biomedical applications due to its ability to incorporate the lipase-degradable monomer sequences of 4HB and 5HV. This study demonstrates that a functional metagenomic approach using next-generation sequencing can be used to mine novel PHA synthases with interesting substrate specificities from unculturable microorganisms.

There is little information on the bacterial diversity of the Fildes Peninsula, King George Island. Hence, this study was conducted to determine the bacterial population of sediments and soils from the lakes, river, glacier and an abandoned oil tank area in the Fildes Peninsula, using a metagenomic approach. DNA was extracted from the sediment and soil samples, and analyzed using the 16S rDNA polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE). A total of 299 DNA fragments resolved using the DGGE were sequenced. The results of the analysis provided an overview of the predominant groups of bacteria and the diversity of the bacterial communities. The most abundant phyla of bacteria in Fildes Peninsula were Bacteroidetes, Proteobacteria, Acidobacteria, Gemmatimonadetes, Nitrospira, Firmicutes, Actinobacteria, Chloroflexi, Cyanobacteria, Spirochaetes, Deinococcus-Thermus, WS3 and BRC1. All of the sediment samples from the lakes had different representatives of dominant bacterial species. Interestingly, 15% of the operational taxonomic units (OTUs) did not group into any of the existing phyla in the Ribosomal Database Project (RDP). One of the OTUs had a similarity of

PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated.

Background Special features of the Japanese ocean include its ranges of latitude and depth. This study is the first to examine the diversity of Class I and II PHA synthases (PhaC) in DNA samples from pelagic seawater taken from the Japan Trench and Nankai Trough from a range of depths from 24 m to 5373 m. PhaC is the key enzyme in microorganisms that determines the types of monomer units that are polymerized into polyhydroxyalkanoate (PHA) and thus affects the physicochemical properties of this thermoplastic polymer. Complete putative PhaC sequences were determined via genome walking, and the activities of newly discovered PhaCs were evaluated in a heterologous host. Results A total of 76 putative phaC PCR fragments were amplified from the whole genome amplified seawater DNA. Of these 55 clones contained conserved PhaC domains and were classified into 20 genetic groups depending on their sequence similarity. Eleven genetic groups have undisclosed PhaC activity based on their distinct phylogenetic lineages from known PHA producers. Three complete DNA coding sequences were determined by IAN-PCR, and one PhaC was able to produce poly(3-hydroxybutyrate) in recombinant Cupriavidus necator PHBˉ4 (PHB-negative mutant). Conclusions A new functional PhaC that has close identity to Marinobacter sp. was discovered in this study. Phylogenetic classification for all the phaC genes isolated from uncultured bacteria has revealed that seawater and other environmental resources harbor a great diversity of PhaCs with activities that have not yet been investigated. Functional evaluation of these in silico-based PhaCs via genome walking has provided new insights into the polymerizing ability of these enzymes. Electronic supplementary material The online version of this article (doi:10.1186/s12866-014-0318-z) contains supplementary material, which is available to authorized users.

The photosynthetic cyanobacterium, Synechocystis sp. strain 6803, is a potential platform for the production of various chemicals and biofuels. In this study, direct photosynthetic production of a biopolymer, polyhydroxyalkanoate (PHA), in genetically engineered Synechocystis sp. achieved as high as 14 wt%. This is the highest production reported in Synechocystis sp. under photoautotrophic cultivation conditions without the addition of a carbon source. The addition of acetate increased PHA accumulation to 41 wt%, and this value is comparable to the highest production obtained with cyanobacteria. Transcriptome analysis by RNA-seq coupled with real-time PCR was performed to understand the global changes in transcript levels of cells subjected to conditions suitable for photoautotrophic PHA biosynthesis. There was lower expression of most PHA synthesis-related genes in recombinant Synechocystis sp. with higher PHA accumulation suggesting that the concentration of these enzymes is not the limiting factor to achieving high PHA accumulation. In order to cope with the higher PHA production, cells may utilize enhanced photosynthesis to drive the product formation. Results from this study suggest that the total flux of carbon is the possible driving force for the biosynthesis of PHA and the polymerizing enzyme, PHA synthase, is not the only critical factor affecting PHA-synthesis. Knowledge of the regulation or control points of the biopolymer production pathways will facilitate the further use of cyanobacteria for biotechnological applications.