Polyhydroxyalkanoate Synthase Research Articles

Effect of mcl-PHA synthesis in flax on plant mechanical properties and cell wall composition

The high demand for new biomaterials makes synthesis of polyhydroxyalkanoates (PHA) in plants an interesting and desirable achievement. Production of polymers in plants is an example of application of biotechnology for improving the properties of plants, e.g. industrial properties, but it can also provide knowledge about plant physiology and metabolism. The subject of the present study was an industrially important plant: flax, Linum usitatissimum L., of a fibre cultivar (cv Nike). In the study the gene encoding PHA synthase from Pseudomonas aeruginosa, fused to a peroxisomal targeting signal, was expressed in flax plants with the aim of modifying the mechanical properties of plants. Medium-chain-length (mcl) hydroxy acids in flax plants from tissue cultures were detected by GC-FID and FTIR method. The introduced changes did not affect fatty acid content and composition in generated flax plants. Since mcl-PHA are known as elastomers, the mechanical properties of created plants were examined. Modified plants showed increases in the values of all measured parameters (except strain at break evaluated for one modified line). The largest increase was noted for tensile stiffness, which was 2- to 3-fold higher than in wild-type plants. The values estimated for another parameter, Young’s modulus, was almost at the same level in generated flax plants, and they were about 2.7-fold higher when compared to unmodified plants. The created plants also exhibited up to about 2.4-fold higher tensile strength. The observed changes were accompanied by alterations in the expression of selected genes, related to cell wall metabolism in line with the highest expression of phaC1 gene. Biochemical data were confirmed by spectroscopic methods, which also revealed that crystallinity index values of cellulose in modified flax plants were increased in comparison to wild-type flax plants and correlated with biomechanical properties of plants.

Polyhydroxyalkanoate (PHA) Polymer Accumulation and pha Gene Expression in Phenazine (phz⁻) and Pyrrolnitrin (prn⁻) Defective Mutants of Pseudomonas chlororaphis PA23.

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay’s Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz- (PA23-63) and phz- prn- (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz- prn- (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz- (PA23-63), prn- (PA23-8), and phz- prn- (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz- and prn- mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

Inhibition of Rumen Methanogens by a Novel Archaeal Lytic Enzyme Displayed on Tailored Bionanoparticles.

Methane is a potent greenhouse gas, 25 times more efficient at trapping heat than carbon dioxide. Ruminant methane emissions contribute almost 30% to anthropogenic sources of global atmospheric methane levels and a reduction in methane emissions would significantly contribute to slowing global temperature rises. Here we demonstrate the use of a lytic enyzme, PeiR, from a methanogen virus that infects Methanobrevibacter ruminantium M1 as an effective agent inhibiting a range of rumen methanogen strains in pure culture. We determined the substrate specificity of soluble PeiR and demonstrated that the enzyme is capable of hydrolysing the pseudomurein cell walls of methanogens. Subsequently, peiR was fused to the polyhydroxyalkanoate (PHA) synthase gene phaC and displayed on the surface of PHA bionanoparticles (BNPs) expressed in Eschericia coli via one-step biosynthesis. These tailored BNPs were capable of lysing not only the original methanogen host strain, but a wide range of other rumen methanogen strains in vitro. Methane production was reduced by up to 97% for 5 days post-inoculation in the in vitro assay. We propose that tailored BNPs carrying anti-methanogen enzymes represent a new class of methane inhibitors. Tailored BNPs can be rapidly developed and may be able to modulate the methanogen community in vivo with the aim to lower ruminant methane emissions without impacting animal productivity.

Triple knockout of frdC gltA and pta genes enhanced pHA production in Escherichia coli

Polyhydroxyalkanoate (PHA) is a linear polyester produced through the fermentation of sugar or lipid. Biosynthesis of PHA comprises three enzymes known as acetyl-CoA acetyltransferase (phaA), acetoacetyl-CoA reductase (phaB) and PHA synthase (phaC). Comamonas sp. is one of the strains commonly used for PHA production. In order to develop higher PHA production from bacterial respond strategy, PHA biosynthesis operon of Comamonas sp. EB172 was introduced into Escherichia coli BW25113 through a pGEM-T vector. E. coli was chosen due to the complete genome information available and the absence of depolymerisation gene, phaZ. In this study, the deletion of several single genes, which are frdC, gltA, and pta, was found to be associated with PHA metabolism activity in E. coli BW25113. P1 transduction was performed to construct multiple genes knockout. The engineered strain, E. coli BW25113 frdCgltApta::kan/pGEM’-phaCABCo, yielded the highest PHA production at 64 wt.% with 1.4 fold higher than that of control strain of E. coli BW25113/pGEM’-phaCABCo. This strain is potential for industrial application for higher PHA production from E. coli.

Design of Modular Polyhydroxyalkanoate Scaffolds for Protein Immobilization by Directed Ligation.

In vivo-assembled polyhydroxyalkanoate (PHA) particles have been successfully bioengineered to display foreign protein functions toward high-value applications in medicine and industry. To further expand the design space of PHA particles toward immobilization of various functional proteins, we developed a tunable modular protein immobilization method implementing the SpyCatcher/SpyTag chemistry. We successfully displayed the SpyCatcher protein using translational fusion with the Ralstonia eutropha PHA synthase (PhaC). The SpyCatcher domain displayed on the surface of PHA particles was accessible for cross-linker-free ligation with SpyTag-bearing proteins. We demonstrated tunable protein immobilization of various SpyTagged proteins on SpyCatcher-PHA particles, which ultimately enabled assembly of multiple proteins coating the surface of PHA particles. Overall, the functionality, stability, and reusability of proteins immobilized to SpyCatcher-PHA particles were either retained or enhanced in comparison to the soluble forms. This modular platform can be implemented as a generic tool for protein immobilization in an array of applications.

Polyhydroxybutyrate production by an extremely halotolerant Halomonas elongata strain isolated from the hypersaline meromictic Fără Fund Lake (Transylvanian Basin, Romania).

This study aimed at unprecedented physical and chemical evaluation of the 'green plastics' polyhydroxyalkanoates (PHAs), in an extremely halotolerant Halomonas elongata strain 2FF under high-salt concentration. The investigated bacterial strain was isolated from the surface water of the hypersaline Fără Fund Lake. The 16S rRNA gene sequence phylogeny and phenotypic analysis indicated that the isolate belonged to H. elongata. PHA inclusions were observed by Sudan Black B, Nile Red staining, and transmission electron microscopy during growth at high salinity (10%, w/v, NaCl) on 1% (w/v) d-glucose. The produced polymer was quantitatively and qualitatively assessed using crotonic acid assay, elemental analysis, Fourier transform infrared and Raman spectroscopies. Additionally, X-ray powder diffraction, 1 H-NMR spectroscopy, and differential scanning calorimetry were applied. The investigations showed that the intracellular polymer was polyhydroxybutyrate (PHB) of which the strain produced up to 40 wt% of total cell dry weight after 48h. The analysis of phaC gene from the isolated H. elongata strain indicated that the encoded PHA synthase belongs to Class I PHA synthase family. Overall, our investigations pointed out that the halotolerant H.elongata strain 2FF was capable to produce significant amounts of PHB fromd-glucose, and PHAs from various carbon substrates at high-salt concentrations. The tested strain showed the ability for significant production of natural, biodegradable polymers under nutrient limitation and hypersaline conditions suggesting its potentiality for further metabolic and molecular investigations towards enhanced biopolymer production. Additionally, this study reports on the unprecedented use of Raman and XPRD techniques to investigate PHAs of an extremely halotolerant bacterium, thus expanding the repertoire of physical methods to study green plastics derived from extremophilic microorganisms.

Identification and Analysis of Putative Polyhydroxyalkanoate Synthase (PhaC) in Pseudomonas fluorescens.

Pseudomonas fluorescens KLR101 was found to be capable of producing polyhydroxyalkanoate (PHA) using various sugars and fatty acids with carbon numbers ranging from 2 to 6. The PHA granules consisted mainly of a poly(3-hydroxybutyrate) homopolymer and/or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. Genomic DNA of P. fluorescens was fractionated and cloned into a lambda library, in which a 5.8-kb fragment that hybridized to a heterologous phaC probe from Ralstonia eutropha was identified. In vivo expression in Klebsiella aerogenes KC2671 (pUMS), restriction mapping, Southern hybridization experiments, and sequencing data revealed that PHA biosynthesis by P. fluorescens relied upon a polypeptide encoded by a 1,683-bp non-operonal ORF, which was preceded by a possible -24/-12 promoter and highly similar to DNA sequences of a gene encoding PHA synthase in the genus Pseudomonas. In vivo expression of the putative PHA synthase gene (phaCPf) in a recombinant Escherichia coli strain was investigated by using glucose and decanoate as substrates. E. coli (phaCPf+, pUMS) grown in medium containing glucose accumulated PHA granules consisting mainly of 3-hydroxybutyrate, whereas only a trace amount of 3-hydroxydecanoate was detected from an E. coli fadR mutant (phaCPf+) grown in medium containing decanoate. In vitro enzymatic assessment experiments showed that 3-hydroxybutyryl-CoA was efficiently used as a substrate of purified PhaCPf, suggesting that the putative PHA synthase of P. fluorescens utilizes mainly short-chain-length PHA precursors as a substrate.

Modelling of polyhydroxyalkanoate synthase from Aquitalea sp. USM4 suggests a novel mechanism for polymer elongation

Polyhydroxyalkanoate (PHA) synthase, PhaC, is a key enzyme in the biosynthesis of PHA, a type of bioplastics with huge potential to replace petroleum-based plastics. While two structures have been determined, the exact mechanism remains unclear partly due to the absence of a tunnel for product passage. A model of the class I PhaC from Aquitalea sp. USM4, characterised with Km of 394 μM and kcat of 476 s−1 on 3-(R)-hydroxybutyryl-CoA, revealed a three-branched channel at the dimeric interface. Two of them are opened to the solvent and are expected to serve as the putative routes for substrate entrance and product exit, while the third is elongated in the class II PhaC1 model from Pseudomonas aeruginosa, indicating a role in accommodating the hydroxyalkanoate (HA) moiety of a HA-CoA substrate. Docking of the two tetrahedral intermediates, formed during the transfer of the growing PHA chain from the catalytic Cys to a new molecule of substrate and back to Cys, suggests a common elongation mechanism requiring the HA moiety of the ligand to rotate ~180°. Substrate specificity is determined in part by a bulky Phe/Tyr/Trp residue in the third branch in class I, which is conserved as Ala in class II to create room for longer substrates.

Increased synthesis of poly(3-hydroxydodecanoate) by random mutagenesis of polyhydroxyalkanoate synthase.

Poly(3-hydroxydodecanoate) [P(3HDD)], a medium-chain-length polyhydroxyalkanoate (PHA), is expected to be used as a novel type of bioplastic characterized by a soft and transparent nature. In this study, to achieve a high yield of P(3HDD), PHA synthase was modified through random mutagenesis of a region of the PHA synthase 1 gene from Pseudomonas putida KT2440 (phaC1Pp). Screening of the mutant library using a β-oxidation-deficient Escherichia coli LSBJ was performed. As a result, four mutants, designated w10, w14, w309, and w311, were selected from 10,000 mutants. The w311 mutant had two amino acid replacements (E358G and N398S), and showed the highest production of P(3HDD) with increased polymer molecular weights when compared to the native enzyme. Saturation mutagenesis at the N398 position, which was found to be highly conserved among Pseudomonas PhaCs, revealed that amino acids with hydrophobic and smaller residues either retained or increased P(3HDD) production. This study demonstrates the benefit of using the PHA synthase mutants to enhance the production of P(3HDD).

Disentangling the drivers of functional complexity at the metagenomic level in Shark Bay microbial mat microbiomes.

The functional metagenomic potential of Shark Bay microbial mats was examined for the first time at a millimeter scale, employing shotgun sequencing of communities via the Illumina NextSeq 500 platform in conjunction with defined chemical analyses. A detailed functional metagenomic profile has elucidated key pathways and facilitated inference of critical microbial interactions. In addition, 87 medium-to-high-quality metagenome-assembled genomes (MAG) were assembled, including potentially novel bins under the deep-branching archaeal Asgard group (Thorarchaetoa and Lokiarchaeota). A range of pathways involved in carbon, nitrogen, sulfur, and phosphorus cycles were identified in mat metagenomes, with the Wood-Ljungdahl pathway over-represented and inferred as a major carbon fixation mode. The top five sets of genes were affiliated to sulfate assimilation (cysNC cysNCD, sat), methanogenesis (hdrABC), Wood-Ljungdahl pathways (cooS, coxSML), phosphate transport (pstB), and copper efflux (copA). Polyhydroxyalkanoate (PHA) synthase genes were over-represented at the surface, with PHA serving as a potential storage of fixed carbon. Sulfur metabolism genes were highly represented, in particular complete sets of genes responsible for both assimilatory and dissimilatory sulfate reduction. Pathways of environmental adaptation (UV, hypersalinity, oxidative stress, and heavy metal resistance) were also delineated, as well as putative viral defensive mechanisms (core genes of the CRISPR, BREX, and DISARM systems). This study provides new metagenome-based models of how biogeochemical cycles and adaptive responses may be partitioned in the microbial mats of Shark Bay.

Polyhydroxyalkanoate Producing Potential of Saccharomyces cerevisiae

Polyhydroxyalkanoates (PHAs) are inclusion bodies accumulated by some microorganisms as reserve material under unbalanced growth condition such as limited oxygen, nitrogen, phosphorous or sulphur and excess carbon source. Saccharomyces cerevisiae strains were screened for Polyhydroxyalkanoates (PHA) producing potential using plate assay technique with Sudan Black B stain. Saccharomyces cerevisiae efficiency for PHA production was analysed utilizing sawdust as carbon source and the production rate was compared with glucose (control). Thus, PHA synthesising enzymes, PHA carbon chain distributions and concentrations were determined using gas chromatography flame ionization detector (GCFID) while microbial growth (biomass yield) during production was determined using Uv-Visible spectrophotometric technique at 600 nm. PHA was extracted from Saccharomyces cerevisiae cells using solvent extraction technique and the extract was further dissolved using acetone derivatisation process. PHA-synthesising enzymes such as Glyceraldehyde-3-phosphate dehydrogenase, Pyruvate dehydrogenase, 3-Ketothiolase, Acetoacetyl-CoA reductase, 3-Ketoacyl reductase and Polyhydroxyalkanoate synthase were extracted from cell-free supernatant obtained from glucose and sawdust media. Saccharomyces cerevisiae cells grown in sawdust medium had the highest PHA yield of 75.12 % while 64.66 % PHA was obtained from cells grown in glucose medium. Consequently, utilization of Saccharomyces cerevisiae in limiting condition using agricultural residue could reduce the cost of industrial production of PHA required for plastic production due to its ability to accumulate the polymer intracellularly.

Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico.

Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs) in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs), which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to a Halomonas sp. In addition, Paracoccus sp., Planomicrobium sp. and Staphylococcus sp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those of Pseudomonas putida, suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence of phaC and phaC1 sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

In Vitro Analysis of d-Lactyl-CoA-Polymerizing Polyhydroxyalkanoate Synthase in Polylactate and Poly(lactate- co-3-hydroxybutyrate) Syntheses.

Engineered d-lactyl-coenzyme A (LA-CoA)-polymerizing polyhydroxyalkanoate synthase (PhaC1PsSTQK) efficiently produces poly(lactate- co-3-hydroxybutyrate) [P(LA- co-3HB]) copolymer in recombinant Escherichia coli, while synthesizing tiny amounts of poly(lactate) (PLA)-like polymers in recombinant Corynebacterium glutamicum. To elucidate the mechanisms underlying the interesting phenomena, in vitro analysis of PhaC1PsSTQK was performed using homo- and copolymerization conditions of LA-CoA and 3-hydroxybutyryl-CoA. PhaC1PsSTQK polymerized LA-CoA as a sole substrate. However, the extension of PLA chains completely stalled at a molecular weight of ∼3000, presumably due to the low mobility of the generated polymer. The copolymerization of these substrates only proceeded with a low concentration of LA-CoA. In fact, the intracellular LA-CoA concentration in P(LA- co-3HB)-producing E. coli was below the detection limit, while that in C. glutamicum was as high as acetyl-CoA levels. Therefore, it was concluded that the mobility of polymerized products and LA-CoA concentration are dominant factors characterizing PLA and P(LA- co-3HB) biosynthetic systems.

Site-directed saturation mutagenesis of polyhydroxylalkanoate synthase for efficient microbial production of poly[(R)-2-hydroxybutyrate

In our previous study, artificial polyhydroxyalkanoate (PHA) poly[(R)-2-hydroxybutyrate] [P(2HB)] was successfully biosynthesized from racemic 2HB in recombinant Escherichia coli using an engineered PHA synthase, PhaC1Ps(S325T/Q481K). Although P(2HB) has promising material properties, the low level of polymer production was a drawback. In this study, we performed directed evolution of PhaC1Ps towards enhanced P(2HB) accumulation in E.coli by site-directed dual saturation mutagenesis at the positions 477 and 481, which was known for their potential in enhancing natural PHA accumulation. By using a screening on agar plates with Nile red, eight colonies were isolated which produced a greater amount of P(2HB) compared to a colony expressing the parent enzyme PhaC1Ps(S325T/Q481K). Among them, the cells expressing PhaC1Ps(S325T/S477R/Q481G) [ST/SR/QG] accumulated polymer at the highest level (up to 2.9-fold). As seen in PhaC1Ps(ST/SR/QG), glycine and basic amino acid residues (K or R) were frequently found at the two positions of the select mutated enzymes. The enzymatic activity of PhaC1Ps(ST/SR/QG) toward 2HB-CoA was approximately 3-fold higher than that of the parent enzyme. Additionally, expression levels of the select mutated enzymes were lower than the parent. These results indicated that PhaC1Ps mutagenesis at the positions 477 and 481 increased specific activity toward 2HB-CoA and it could result in the enhanced production of P(2HB).

Dynamic Changes of Intracellular Monomer Levels Regulate Block Sequence of Polyhydroxyalkanoates in Engineered Escherichia coli.

Biological polymer synthetic systems, which utilize no template molecules, normally synthesize random copolymers. We report an exception, a synthesis of block polyhydroxyalkanoates (PHAs) in an engineered Escherichia coli. Using an engineered PHA synthase, block copolymers poly[(R)-2-hydroxybutyrate(2HB)-b-(R)-3-hydroxybutyrate(3HB)] were produced in E. coli. The covalent linkage between P(2HB) and P(3HB) segments was verified with solvent fractionation and microphase separation. Notably, the block sequence was generated under the simultaneous consumption of two monomer precursors, indicating the existence of a rapid monomer switching mechanism during polymerization. Based on in vivo metabolic intermediate analysis and the relevant in vitro enzymatic activities, we propose a model in which the rapid intracellular 3HB-CoA fluctuation during polymer synthesis is a major factor in generating block sequences. The dynamic change of intracellular monomer levels is a novel regulatory principle of monomer sequences of biopolymers.

One-step fermentative production of aromatic polyesters from glucose by metabolically engineered Escherichia coli strains

Aromatic polyesters are widely used plastics currently produced from petroleum. Here we engineer Escherichia coli strains for the production of aromatic polyesters from glucose by one-step fermentation. When the Clostridium difficile isocaprenoyl-CoA:2-hydroxyisocaproate CoA-transferase (HadA) and evolved polyhydroxyalkanoate (PHA) synthase genes are overexpressed in a d-phenyllactate-producing strain, poly(52.3 mol% 3-hydroxybutyrate (3HB)-co-47.7 mol% d-phenyllactate) can be produced from glucose and sodium 3HB. Also, various poly(3HB-co-d-phenyllactate) polymers having 11.0, 15.8, 20.0, 70.8, and 84.5 mol% of d-phenyllactate are produced from glucose as a sole carbon source by additional expression of Ralstonia eutropha β-ketothiolase (phaA) and reductase (phaB) genes. Fed-batch culture of this engineered strain produces 13.9 g l−1 of poly(61.9 mol% 3HB-co-38.1 mol% d-phenyllactate). Furthermore, different aromatic polyesters containing d-mandelate and d-3-hydroxy-3-phenylpropionate are produced from glucose when feeding the corresponding monomers. The engineered bacterial system will be useful for one-step fermentative production of aromatic polyesters from renewable resources.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHA_MCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HA_MCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HA_MCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

A novel and wide substrate specific polyhydroxyalkanoate (PHA) synthase from unculturable bacteria found in mangrove soil

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.

Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions

BackgroundRecombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction.ResultsBy translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).ConclusionsHere a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.

Polyhydroxyalkanoate accumulation ability and associated microbial community in activated sludge-derived acetate-fed microbial cultures enriched under different temperature and pH conditions

The influence of temperature and pH during enrichment on the polyhydroxyalkanoate (PHA) accumulation ability and composition of PHA-accumulating microorganisms (PHAAMOs) in enrichment cultures was investigated. Enrichment of PHAAMOs from activated sludge was conducted in acetate-fed sequencing batch reactors using a feast-famine regime under different temperature (20°C, 28°C, and 36°C) and pH (controlled at 7.2 or not) conditions. PHA accumulation ability, which was evaluated in nitrogen- and phosphorus-deficient 24-h single-batch cultures, was greatly enhanced by enrichment, irrespective of the temperature and pH. Enrichment at 20°C or 28°C and without pH control seemed most appropriate for strong PHA accumulation. Analyses of the PHAAMO composition by the clone library method targeting phaC genes, which encode the class I and II PHA synthases, revealed that Burkholderiales were the dominant PHAAMOs in the seed sludge, while Rhodocyclales, specifically Azoarcus spp. and Thauera spp., were dominant after enrichment without pH control, showing a strong ability to accumulate PHA. The results indicated that Azoarcus spp. and Thauera spp. are key PHAAMOs in an enrichment culture based on the feast-famine method, with high PHA accumulation ability.