PHB Synthase Research Articles

Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5-9), temperature ranges (4-65 °C), and NaCl concentrations (100-350 g L-1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography-mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm-1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli

Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA–04 and pColdTF-phaCABA–04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Revealing of sugar utilization systems in Halomonas sp. YLGW01 and application for poly(3-hydroxybutyrate) production with low-cost medium and easy recovery

Poly(3-hydroxybutyrate) (PHB) is a common polyhydroxyalkanoate (PHA) with potential as an alternative for petroleum-based plastics. Previously, we reported a new strain, Halomonas sp. YLGW01, which hyperproduces PHB with 94% yield using fructose. In this study, we examined the PHB production machinery of Halomonas sp. YLGW01 in more detail by deep-genome sequencing, which revealed a 3,453,067-bp genome with 65.1% guanine-cytosine content and 3054 genes. We found two acetyl-CoA acetyltransferases (Acetoacetyl-CoA thiolase, PhaA), one acetoacetyl-CoA reductase (PhaB), two PHB synthases (PhaC1, PhaC2), PHB depolymerase (PhaZ), and Enoyl-CoA hydratase (PhaJ) in the genome, along with two fructose kinases and fructose transporter systems, including the phosphotransferase system (PTS) and ATP-binding transport genes. We then examined the PHB production by Halomonas sp. YLGW01 using high-fructose corn syrup (HFCS) containing fructose, glucose, and sucrose in sea water medium, resulting in 7.95 ± 0.11 g/L PHB (content, 67.39 ± 0.34%). PHB was recovered from Halomonas sp. YLGW01 using different detergents; the use of Tween 20 and SDS yielded micro-sized granules with high purity. Overall, these results reveal the distribution of PHB synthetic genes and the sugar utilization system in Halomonas sp. YLGW01 and suggest a possible method for PHB recovery.

Bacterially assembled biopolyester nanobeads for removing cadmium from water

Cadmium (Cd)-contaminated waterbodies are a worldwide concern for the environment, impacting human health. To address the need for efficient, sustainable and cost-effective remediation measures, we developed innovative Cd bioremediation agents by engineering Escherichia coli to assemble poly(3-hydroxybutyric acid) (PHB) beads densely coated with Cd-binding peptides. This was accomplished by translational fusion of Cd-binding peptides to the N- or C-terminus of a PHB synthase that catalyzes PHB synthesis and mediates assembly of Cd2 or Cd1 coated PHB beads, respectively. Cd1 beads showed greater Cd adsorption with 441 nmol Cd mg−1 bead mass when compared to Cd2 beads (334 nmol Cd mg−1 bead-mass) and plain beads (238 nmol Cd mg−1 bead-mass). The Cd beads were not ecotoxic and did attenuate Cd-spiked solutions toxicity. Overall, the bioengineered beads provide a means to remediate Cd-contaminated sites, can be cost-effectively produced at large scale, and offer a biodegradable and safe alternative to synthetic ecotoxic treatments.

Considerable increase in Poly(3-hydroxybutyrate) production via phbC gene overexpression in Ralstonia eutropha PTCC 1615.

Introduction: Poly(3-hydroxybutyrate) (PHB) is a well-known biodegradable polymer produced by some microorganisms and can be a suitable alternative for petrochemical plastics. PHB synthase encoded by phb C gene is the main enzyme in PHB biosynthesis pathway in Ralstonia eutropha. The aim of current study was the transformation of R. eutropha PTCC 1615 with its own phb C gene and evaluation of the overexpression effect on PHB accumulation. Methods: DNA fragment including phbC gene and its promoter and terminator regions, was isolated from R. eutropha PTCC 1615, inserted into pET28a(+) vector, and transferred to the competent bacteria using calcium chloride and heat shock method. The effect of the cloned gene expression on PHB production was investigated with absorption of crotonic acid produced through PHB dehydration. Statistical analyses were carried out by SPSS software. Results: PHB content of cells of the engineered strain was 1.4 times more than that of the native bacteria. This significant difference can be an important finding for improvement of biopolymer production. Conclusion: Overexpression of phb C, the critical gene in PHB biosynthesis pathway, in R. eutropha PTCC 1615 had considerable effect on PHB accumulation.

Formation of an Organic-Inorganic Biopolymer: Polyhydroxybutyrate-Polyphosphate.

A considerable variety of different biopolymers is formed by the entirety of organisms present on earth. Most of these compounds are organic polymers such as polysaccharides, polyamino acids, polynucleotides, polyisoprenes or polyhydroxyalkanoates (PHAs), but some biopolymers can consist of solely inorganic monomers such as phosphate in polyphosphates (polyPs). In this contribution, we describe the formation of an organic-inorganic block copolymer consisting of poly(3-hydroxybutyrate) (PHB) and polyP. This was achieved by the expression of a fusion of the polyP kinase gene (ppk2c) with the PHB synthase gene (phaC) of Ralstonia eutropha in a polyP-free and PHB-free mutant background of R. eutropha. The fusion protein catalyzed both the formation of polyP by its polyP kinase domain and the formation of PHB by its PHB synthase domain. It was also possible to synthesize the polyP-PHB polymer in vitro with purified Ppk2c-PhaC, if the monomers, adenosine triphosphate (ATP) and 3-hydroxybutyryl-CoA (3HB-CoA), were provided. Most likely, the formed block copolymer (polyP-protein-PHB) turns into a blend of polyP and PHB after release from the enzyme.

Poly(3-Hydroxybutyrate) (PHB) Polymerase PhaC1 and PHB Depolymerase PhaZa1 of Ralstonia eutropha Are Phosphorylated In Vivo.

In this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 of Ralstonia eutropha for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability of R. eutropha to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase.IMPORTANCE Poly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in Ralstonia eutropha (alternative designation, Cupriavidus necator) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conducted in vitro and in vivo analyses of PHB synthase activity and PHB contents.

Engineering Bacillus megaterium for production of functional intracellular materials

BackgroundOver the last 10–15 years, a technology has been developed to engineer bacterial poly(3-hydroxybutyrate) (PHB) inclusions as functionalized beads, for applications such as vaccines, diagnostics and enzyme immobilization. This has been achieved by translational fusion of foreign proteins to the PHB synthase (PhaC). The respective fusion protein mediates self-assembly of PHB inclusions displaying the desired protein function. So far, beads have mainly been produced in recombinant Escherichia coli, which is problematic for some applications as the lipopolysaccharides (LPS) co-purified with such inclusions are toxic to humans and animals.ResultsIn this study, we have bioengineered the formation of functional PHB inclusions in the Gram-positive bacterium Bacillus megaterium, an LPS-free and established industrial production host. As B. megaterium is a natural PHB producer, the PHB-negative strain PHA05 was used to avoid any background PHB production. Plasmid-mediated T7 promoter-driven expression of the genes encoding β-ketothiolase (phaA), acetoacetyl-CoA-reductase (phaB) and PHB synthase (phaC) enabled PHB production in B. megaterium PHA05. To produce functionalized PHB inclusions, the N- and C-terminus of PhaC was fused to four and two IgG binding Z-domains from Staphylococcus aureus, respectively. The ZZ-domain PhaC fusion protein was strongly overproduced at the surface of the PHB inclusions and the corresponding isolated ZZ-domain displaying PHB beads were found to purify IgG with a binding capacity of 40–50 mg IgG/g beads. As B. megaterium has the ability to sporulate and respective endospores could co-purify with cellular inclusions, a sporulation negative production strain was generated by disrupting the spoIIE gene in PHA05. This strain did not produce spores when tested under sporulation inducing conditions and it was still able to synthesize ZZ-domain displaying PHB beads.ConclusionsThis study provides proof of concept for the successful genetic engineering of B. megaterium as a host for the production of functionalized PHB beads. Disruption of the spoIIE gene rendered B. megaterium incapable of sporulation but particularly suitable for production of functionalized PHB beads. This sporulation-negative mutant represents an improved industrial production strain for biotechnological processes otherwise impaired by the possibility of endospore formation.

Biosynthesis of Polyhydroxyalkanoate from Steamed Soybean Wastewater by a Recombinant Strain of Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulates a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer, poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) [P(3HB-co-3HA)], consisting of 3HA units of 4–12 carbon atoms. Pseudomonas sp. 61-3 possesses two types of PHA synthases, PHB synthase (PhbC) and PHA synthases (PhaC1 and PhaC2), encoded by the phb and pha loci, respectively. The P(94 mol% 3HB-co-6 mol% 3HA) copolymer synthesized by the recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of phaC1 gene is known to have desirable physical properties and to be a flexible material with moderate toughness, similar to low-density polyethylene. In this study, we focused on the production of the P(3HB-co-3HA) copolymer using steamed soybean wastewater, a by-product in brewing miso, which is a traditional Japanese seasoning. The steamed soybean wastewater was spray-dried to produce a powder (SWP) and used as the sole nitrogen source for the synthesis of P(3HB-co-3HA) by the Pseudomonas sp. 61-3 recombinant strain. Hydrolyzed SWP (HSWP) was also used as a carbon and nitrogen source. P(3HB-co-3HA)s with relatively high 3HB fractions could be synthesized by a recombinant strain of Pseudomonas sp. 61-3 (phbC::tet) harboring additional copies of the phaC1 gene in the presence of 2% glucose and 10–20 g/L SWP as the sole nitrogen source, producing a PHA concentration of 1.0–1.4 g/L. When HSWP was added to a nitrogen- and carbon-free medium, the recombinant strain could synthesize PHA without glucose as a carbon source. The recombinant strain accumulated 32 wt% P(3HB-co-3HA) containing 80 mol% 3HB and 20 mol% medium-chain-length 3HA with a PHA concentration of 1.0 g/L when 50 g/L of HSWP was used. The PHA production yield was estimated as 20 mg-PHA/g-HSWP, which equates to approximately 1.0 g-PHA per liter of soybean wastewater.

Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly(3-hydroxybutyrate) (PHB) production

PHB biosynthesis pathway, consisting of three open reading frames (ORFs) that encode for β-ketothiolase (phaA Cma , 1179bp), acetoacetyl-CoA reductase (phaB Cma , 738bp), and PHA synthase (phaC Cma , 1694bp), of Caldimonas manganoxidans was identified. The functions of PhaA, PhaB, and PhaC were demonstrated by successfully reconstructing PHB biosynthesis pathway of C. manganoxidans in Escherichia coli, where PHB production was confirmed by OD600, gas chromatography, Nile blue stain, and transmission electron microscope (TEM). The protein sequence alignment of PHB synthases revealed that phaC Cma shares at least 60% identity with those of class I PHB synthase. The effects of PhaA, PhaB, and PhaC expression levels on PHB production were investigated. While the overexpression of PhaB is found to be important in recombinant E. coli, performances of PHB production can be quantified as follows: PHB concentration of 16.8±0.6g/L, yield of 0.28g/g glucose, content of 74%, productivity of 0.28g/L/h, and Mw of 1.41MDa.

Клонирование и характеристика полигидроксибутиратсинтазы из Methylobacterium extorquens AM1

Multiple genes encoding putative PHB synthases (up to 5 in single strain) were found in Methylobacterium genomes. As a result of phylogenetic analysis proteins PhaC1, PhaC2, PhaC3 were identified as class I PHB synthases, PhaC4 proteins were identified as class III PHB synthases, while PhaC5 apparently belongs to uncharacterized class of PHB synthases. Firstly, the recombinant class I PBH synthase (EC 2.3.1.B2) encoded by phaC1 gene from Methylobacterium extorquens AM1 was purified and characterized. Molecular mass of enzyme monomer was 78 kDa. Michaelis constant (Km) for PhaC1 was 1,3 mM and maximal reaction rate (Vmax) was 0,1 μmol∙min–1∙mg–1. The deletion mutant of Methylobacterium extorquens in the phaC gene was obtained which is promising for further study of peculiarities of methylobacteria’s PHB biosynthesis.

Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens.

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase, and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4 Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK2 (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK2 IMPORTANCE: In this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer and the network responsible for microoxic metabolism through the interaction between the gene regulators phaR and fixK2 These results contribute to the understanding of the physiological conditions required for polyhydroxybutyrate biosynthesis. The interaction between these two main metabolic pathways is also reflected in the symbiotic phenotypes of soybeans inoculated with phaR mutants, which were more competitive for nodulation and enhanced dry matter production by the plants. Therefore, this knowledge may be applied to the development of superior strains to be used as improved inoculants for soybean crops.

PhaM Is the Physiological Activator of Poly(3-Hydroxybutyrate) (PHB) Synthase (PhaC1) in Ralstonia eutropha

Poly(3-hydroxybutyrate) (PHB) synthase (PhaC1) is the key enzyme of PHB synthesis in Ralstonia eutropha and other PHB-accumulating bacteria and catalyzes the polymerization of 3-hydroxybutyryl-CoA to PHB. Activity assays of R. eutropha PHB synthase are characterized by the presence of lag phases and by low specific activity. It is assumed that the lag phase is caused by the time necessary to convert the inactive PhaC1 monomer into the active dimeric form by an unknown priming process. The lag phase can be reduced by addition of nonionic detergents such as hecameg [6-O-(N-heptyl-carbamoyl)-methyl-α-D-glucopyranoside], which apparently accelerates the formation of PhaC1 dimers. We identified the PHB granule-associated protein (PGAP) PhaM as the natural primer (activator) of PHB synthase activity. PhaM was recently discovered as a novel type of PGAP with multiple functions in PHB metabolism. Addition of PhaM to PHB synthase assays resulted in immediate polymerization of 3HB coenzyme A with high specific activity and without a significant lag phase. The effect of PhaM on (i) PhaC1 activity, (ii) oligomerization of PhaC1, (iii) complex formation with PhaC1, and (iv) PHB granule formation in vitro and in vivo was shown by cross-linking experiments of purified proteins (PhaM, PhaC1) with glutardialdehyde, by size exclusion chromatography, and by fluorescence microscopic detection of de novo-synthesized PHB granules.

Effect of AI crude extract on PHB accumulation and hydrogen photoproduction in Rhodobacter sphaeroides

Poly-β-hydroxy butyric acid (PHB) accumulation and gaseous H2 release are regarded as alternatives for expending reducing power. Some researchers suggested that quorum-sensing system affects PHB accumulation in Rhodobacter sphaeroides, but whether the system plays regulation role between hydrogen producing and PHB synthesis is still unknown. By adding autoinducer of R. sphaeroides into its culture solutions, measuring its total hydrogen production, PHB content and PHB synthase activity, the function of quorum-sensing on PHB accumulation and hydrogen production was preliminarily investigated. Compared with the control, the total gas productions in experimental groups increased accompanying slight decrease of PHB contents, which was partially caused by the reduction of PHB synthase activities. Biolog tests indicated the carbon source utilization profiles, especially those involving fatty acids and butanoate metabolism, had partly changed after exogenous signal molecules added. These results suggest that quorum-sensing is involved in signal regulation between PHB accumulation and hydrogen production in R. sphaeroides.

Development of a Transferable Bimolecular Fluorescence Complementation System for the Investigation of Interactions between Poly(3-Hydroxybutyrate) Granule-Associated Proteins in Gram-Negative Bacteria

Poly(3-hydroxybutyrate) (PHB) granules are organelle-like multienzyme-polymer complexes (carbonosomes) and are widespread storage compounds in prokaryotes. The interaction of three PHB granule-bound proteins (PHB synthase PhaC1, phasin PhaP5, and PHB/DNA binding protein PhaM) was studied in vivo by bimolecular fluorescence complementation (BiFC) microscopy in Ralstonia eutropha. To this end, a mobilizable 2-plasmid system for arabinose-controlled expression of protein fusions with the N-terminal (YN) and C-terminal (YC) parts of the enhanced yellow fluorescent protein (eYfp) in Gram-negative bacteria was developed. Both plasmids were stably expressed in Escherichia coli and in transconjugants of R. eutropha. Homo-oligomerization of PhaC1, PhaP5, and PhaM and interactions between PhaC1 and PhaM and between PhaM and PhaP5 were detected in R. eutropha and colocalized with PHB granules under PHB-permissive conditions. PhaM-PhaC1 complexes were detected near the midcell/nucleoid region in the absence of PHB. Expression of BiFC complexes in R. eutropha with PhaM (PhaM homo-oligomers or PhaM-PhaC1 or PhaM-PhaP5 complexes) resulted in substantial cell elongation compared to wild-type cells and in BiFC signals that were generally located near the midcell/nucleoid region. Western blot analysis of wild-type cell extracts and proteome analysis of PHB granule-bound proteins revealed that PhaM and PhaP5 are expressed in R. eutropha and that PhaM is constitutively expressed independently of the presence or absence of PHB. Size exclusion chromatography analysis in combination with cross-linking experiments of purified PhaP5-His6 and PhaM-His6 showed that PhaP5 forms dimers and that PhaM is present in oligomeric (dodecamer) form. Implications of this finding for subcellular PHB localization and initiation of PHB granule formation in R. eutropha will be discussed.

Localization of Poly(3-Hydroxybutyrate) (PHB) Granule-Associated Proteins during PHB Granule Formation and Identification of Two New Phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16

Poly(3-hydroxybutyrate) (PHB) granules are covered by a surface layer consisting of mainly phasins and other PHB granule-associated proteins (PGAPs). Phasins are small amphiphilic proteins that determine the number and size of accumulated PHB granules. Five phasin proteins (PhaP1 to PhaP5) are known for Ralstonia eutropha. In this study, we identified three additional potential phasin genes (H16_B1988, H16_B2296, and H16_B2326) by inspection of the R. eutropha genome for sequences with "phasin 2 motifs." To determine whether the corresponding proteins represent true PGAPs, fusions with eYFP (enhanced yellow fluorescent protein) were constructed. Similar fusions of eYFP with PhaP1 to PhaP5 as well as fusions with PHB synthase (PhaC1), an inactive PhaC1 variant (PhaC1-C319A), and PhaC2 were also made. All fusions were investigated in wild-type and PHB-negative backgrounds. Colocalization with PHB granules was found for all PhaC variants and for PhaP1 to PhaP5. Additionally, eYFP fusions with H16_B1988 and H16_B2326 colocalized with PHB. Fusions of H16_B2296 with eYFP, however, did not colocalize with PHB granules but did colocalize with the nucleoid region. Notably, all fusions (except H16_B2296) were soluble in a ΔphaC1 strain. These data confirm that H16_B1988 and H16_B2326 but not H16_B2296 encode true PGAPs, for which we propose the designation PhaP6 (H16_B1988) and PhaP7 (H16_B2326). When localization of phasins was investigated at different stages of PHB accumulation, fusions of PhaP6 and PhaP7 were soluble in the first 3 h under PHB-permissive conditions, although PHB granules appeared after 10 min. At later time points, the fusions colocalized with PHB. Remarkably, PHB granules of strains expressing eYFP fusions with PhaP5, PhaP6, or PhaP7 localized predominantly near the cell poles or in the area of future septum formation. This phenomenon was not observed for the other PGAPs (PhaP1 to PhaP4, PhaC1, PhaC1-C319A, and PhaC2) and indicated that some phasins can have additional functions. A chromosomal deletion of phaP6 or phaP7 had no visible effect on formation of PHB granules.

Hyperproduction of poly(4-hydroxybutyrate) from glucose by recombinant Escherichia coli

BackgroundPoly(4-hydroxybutyrate) [poly(4HB)] is a strong thermoplastic biomaterial with remarkable mechanical properties, biocompatibility and biodegradability. However, it is generally synthesized when 4-hydroxybutyrate (4HB) structurally related substrates such as γ-butyrolactone, 4-hydroxybutyrate or 1,4-butanediol (1,4-BD) are provided as precursor which are much more expensive than glucose. At present, high production cost is a big obstacle for large scale production of poly(4HB).ResultsRecombinant Escherichia coli strain was constructed to achieve hyperproduction of poly(4-hydroxybutyrate) [poly(4HB)] using glucose as a sole carbon source. An engineering pathway was established in E. coli containing genes encoding succinate degradation of Clostridium kluyveri and PHB synthase of Ralstonia eutropha. Native succinate semialdehyde dehydrogenase genes sad and gabD in E. coli were both inactivated to enhance the carbon flux to poly(4HB) biosynthesis. Four PHA binding proteins (PhaP or phasins) including PhaP1, PhaP2, PhaP3 and PhaP4 from R. eutropha were heterologously expressed in the recombinant E. coli, respectively, leading to different levels of improvement in poly(4HB) production. Among them PhaP1 exhibited the highest capability for enhanced polymer synthesis. The recombinant E. coli produced 5.5 g L-1 cell dry weight containing 35.4% poly(4HB) using glucose as a sole carbon source in a 48 h shake flask growth. In a 6-L fermentor study, 11.5 g L-1 cell dry weight containing 68.2% poly(4HB) was obtained after 52 h of cultivation. This was the highest poly(4HB) yield using glucose as a sole carbon source reported so far. Poly(4HB) was structurally confirmed by gas chromatographic (GC) as well as 1H and 13C NMR studies.ConclusionsSignificant level of poly(4HB) biosynthesis from glucose can be achieved in sad and gabD genes deficient strain of E. coli JM109 harboring an engineering pathway encoding succinate degradation genes and PHB synthase gene, together with expression of four PHA binding proteins PhaP or phasins, respectively. Over 68% poly(4HB) was produced in a fed-batch fermentation process, demonstrating the feasibility for enhanced poly(4HB) production using the recombinant strain for future cost effective commercial development.

Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16

A two-hybrid approach was applied to screen for proteins with the ability to interact with PHB synthase (PhaC1) of Ralstonia eutropha. The H16_A0141 gene (phaM) was identified in the majority of positive clones. PhaM (26.6 kDa) strongly interacted with PhaC1 and with phasin PhaP5 but not with PhaP1 or other PHB granule-associated proteins. A ΔphaM mutant accumulated only one or two large PHB granules instead of three to six medium-sized PHB granules of the wild type, and distribution of granules to daughter cells was disordered. All three phenotypes (number, size and distribution of PHB granules) were reversed by reintroduction of phaM. Purified PhaM revealed DNA-binding properties in gel mobility shift experiments. Expression of a fusion of the yellow fluorescent protein (eYfp) with PhaM resulted in formation of many small fluorescent granules that were bound to the nucleoid region. Remarkably, an eYfp-PhaP5 fusion localized at the cell poles in a PHB-negative background and overexpression of eYfp-PhaP5 in the wild type conferred binding of PHB granules to the cell poles. In conclusion, subcellular localization of PHB granules in R. eutropha depends on a concerted expression of at least three PHB granule-associated proteins, namely PhaM, PhaP5 and PHB synthase PhaC1.

Interaction between poly(3-hydroxybutyrate) granule-associated proteins as revealed by two-hybrid analysis and identification of a new phasin in Ralstonia eutropha H16

A large number of polypeptides are attached to poly(3-hydroxybutyrate) (PHB) granules of Ralstonia eutropha, such as PHB synthase (PhaC1), several PHB depolymerases (PhaZs) and phasins (PhaPs), the regulator protein PhaR(Reu), and possibly others. In this study we used the bacterial adenylate cyclase-based two-hybrid assay to investigate interactions between known PHB granule-associated proteins (PGAPs) and to screen for new PGAPs. The utility of the system was tested by the in vivo verification of previously postulated interactions of the PHB synthase subunits of R. eutropha (PhaC1 homo-oligomerization) and of Bacillus megaterium (PhaC(Bmeg)-PhaR(Bmeg) hetero-oligomerization). Nine proteins (PhaA, PhaB1, PhaC1, PhaP1-PhaP4, PhaZ1 and PhaR), with established functions in PHB metabolism of R. eutropha, were tested for interaction in all combinations. While no significant interaction was detected between the PHB synthase PhaC1 and any of the other eight tested Pha proteins, strong interactions were found between all phasin proteins, in particular between PhaP2 and PhaP4. When PhaP2 was used as bait in a two-hybrid screening experiment with a genomic library of R. eutropha, the B1934 gene product was identified in 24 out of 53 isolated clones. B1934 encodes a hypothetical protein (15.7 kDa) with similarity to phasins of PHB-accumulating bacteria. A fusion protein of eYfp and the B1934 gene product colocalized with PHB granules, confirming that B1934 represents a new phasin (PhaP5). PhaP5 was not essential for PHB granule formation, but overexpression of PhaP5 increased the number of cells with PHB granules at the cell poles.

Enhancing photo-fermentative hydrogen production by Rhodobacter sphaeroides KD131 and its PHB synthase deleted-mutant from acetate and butyrate

Purple non-sulfur photosynthetic bacterium Rhodobacter sphaeroides KD131 wild type (wt) and its PHB synthase deleted-mutant P1 were evaluated for hydrogen (H 2) production from acetate and butyrate, the most abundant liquid end products of dark fermentation. In the presence of glutamate (8 mM), 60 mM of acetate and 30 mM of butyrate were degraded down to 41.5% and 24.0%, respectively, and achieved a H 2 yield (HY) of 0.65 mol H 2/mol acetate- and 2.50 mol H 2/mol butyrate-consumed, while 30 mM succinate exhibited an HY of 3.29 mol H 2/mol substrate-consumed. The order of HY observed was inversely related to poly-(3-hydroxybutyrate) (PHB) content and pH increase in the broth. When mutant P1 was used, in spite of depressed cell growth and lower substrate degradation compared to those observed in strain KD131 wt, higher H 2 production was observed, achieving around two-fold increase of HY in both acetate and butyrate. A pH control to 7.0 during fermentation was effective in increasing substrate degradation and decreasing PHB content, thereby significantly increasing H 2 production. When pH was controlled to 7.0, strain KD131 wt evolved more H 2 by 2.36 and 1.70 folds in the acetate- and succinate-medium, respectively, compared to those observed in without pH control. The highest H 2 production was observed when the mutant P1 was photo-fermented with a pH control to 7.0 in the medium containing acetate-(NH 4) 2SO 4. It seemed that pH control had an effect not only on the depressed production of PHB but also on soluble microbial products and secondary metabolites, which would compete with H 2 production in expending reducing power.