Polyhydroxyalkanoate (PHA) Synthesis by Class IV PHA Synthases Employing<i>Ralstonia eutropha</i>PHB<sup>−</sup>4 as Host Strain

Hyakutake, Manami; Saito, Yuta; Tomizawa, Satoshi; Mizuno, Kouhei; Tsuge, Takeharu

doi:10.1271/bbb.110229

Cited by 34 publications

(25 citation statements)

References 15 publications

(16 reference statements)

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“…Thus, scission activity, as well as polymerization activity, was shown to require both the PhaC YB-4 and PhaR YB-4 subunits. Unlike E. coli, the PHA-negative mutant R. eutropha PHB Ϫ 4 produces high-molecular-weight P(3HB) when expressing PhaR-C YB-4 (12). Because the ethanol produced by this R. eutropha strain remains at undetectable levels (data not shown), the alcoholytic activity of PhaRC YB-4 could not be induced.…”

Section: Discussionmentioning

confidence: 95%

“…A subunit recombination study of PhaR and PhaC from B. cereus YB-4 and B. megaterium revealed that the PhaC YB-4 subunit is responsible for the scission activity. However, unlike E. coli, the PHA-negative mutant R. eutropha PHB Ϫ 4 produced high-molecular-weight P(3HB) even when PhaRC YB-4 was expressed (12). From these observations, we hypothesized that there might be regulatory mechanisms governing the P(3HB) scission activity of PhaC .…”

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confidence: 99%

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Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Hyakutake

Tomizawa

Mizuno

et al. 2014

Appl Environ Microbiol

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c Polyhydroxyalkanoate (PHA)-producing Bacillus strains express class IV PHA synthase, which is composed of the subunits PhaR and PhaC. Recombinant Escherichia coli expressing PHA synthase from Bacillus cereus strain YB-4 (PhaRC YB-4 ) showed an unusual reduction of the molecular weight of PHA produced during the stationary phase of growth. Nuclear magnetic resonance analysis of the low-molecular-weight PHA revealed that its carboxy end structure was capped by ethanol, suggesting that the molecular weight reduction was the result of alcoholytic cleavage of PHA chains by PhaRC YB-4 induced by endogenous ethanol. This scission reaction was also induced by exogenous ethanol in both in vivo and in vitro assays. In addition, PhaRC YB-4 was observed to have alcoholysis activity for PHA chains synthesized by other synthases. The PHA synthase from Bacillus megaterium (PhaRC Bm ) from another subgroup of class IV synthases was also assayed and was shown to have weak alcoholysis activity for PHA chains. These results suggest that class IV synthases may commonly share alcoholysis activity as an inherent feature.

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Section: Discussionmentioning

confidence: 95%

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confidence: 99%

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Hyakutake

Tomizawa

Mizuno

et al. 2014

Appl Environ Microbiol

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“…Previous researches consistently indicate that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but it can also polymerize some unusual monomers as minor components [52][53][54]. There is an increasing interest in class IV PHA synthase, due to the possible alcoholysis activity as an inherent feature among these enzymes [55]. This alcoholysis reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus, which can be manipulated to produce more promising PHA materials with more beneficial properties [56,57].…”

Section: Discussionmentioning

confidence: 99%

Genome Structure ofBacillus cereustsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

Zhou

Johnson

et al. 2017

International Journal of Polymer Science

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In previous work, we reported on the isolation and genome sequence analysis of Bacillus cereus strain tsu1 NCBI accession number JPYN00000000. The 36 scaffolds in the assembled tsu1 genome were all aligned with B. cereus B4264 genome with variations. Genes encoding for xylanase and cellulase and the cluster of genes in the poly-3-hydroxybutyrate (PHB) biosynthesis pathway were identified in tsu1 genome. The PHB accumulation in B. cereus tsu1 was initially identified using Sudan Black staining and then confirmed using high-performance liquid chromatography. Physical properties of these PHB extracts, when analyzed with Raman spectra and Fourier transform infrared spectroscopy, were found to be comparable to the standard compound. The five PHB genes in tsu1 (phaA, phaB, phaR, phaC, and phaP) were cloned and expressed with TOPO cloning, and the recombinant proteins were validated using peptide mapping of in-gel trypsin digestion followed by mass spectrometry analysis. The recombinant E. coli BL21 (DE3) (over)expressing phaC was found to accumulate PHB particles. The cellulolytic activity of tsu1 was detected using carboxymethylcellulose (CMC) plate Congo red assay and the shift towards low-molecular size forms of CMC revealed by gel permeation chromatography in CMC liquid culture and the identification of a cellulase in the secreted proteome.

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“…Bacillus strains belong to class IV synthase that comprises PhaC and its subunit PhaR [24]. The most important genes that involves in PHB production in Bacillus strains were phaA and phaR, phaB, phaC [3,23].…”

Section: Discussionmentioning

confidence: 99%

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Rabey¹

2017

JIG

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Poly 3-hydroxybutyrate (PHB) is the most known degradable biopolymers, produced by some genera of bacteria under unfavorable growth conditions. Isolation, cloning, and expression of PHB genes from PHB producing Bacillus cereus was achieved. A highest Poly 3-hydroxybutyrate producer bacterium was selected and identified as Bacillus cereus MM7, based on morphological and physiological characters in addition to 16S rRNA gene sequences. The four genes of Bacillus cereus MM7 that involved in Poly 3-hydroxybutyrate production were successfully amplified using specific primers which were designed depending on the sequence alignment of the known PHA biosynthetic genes from Bacillus cereus, cloned in E. coli DH5α and completely sequenced. Moreover, the expression of the four genes responsible for PHA biosynthetic in E. coli XL1-Blue and E coli MJ 109 was assayed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the current study Bacillus cereus was able to synthesize PHB. PHB genes were isolated, cloned and expressed in E. coli DH5α. The PHB encoding proteins were analyzed in SDS-PAGE and the PHB corresponding protein bands were analyzed using mass spectrometry to nominate their amino acid sequence. Keywords: Pre cultureThe pre culture was prepared in 250 ml Erlenmeyer flasks containing 50 ml of nutrient broth [16]. Each flask was inoculated with a loop of the tested cultures and incubated in a 130 rpm speed rotary shaker at 30˚C for 24h.

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Polyhydroxyalkanoate (PHA) Synthesis by Class IV PHA Synthases EmployingRalstonia eutrophaPHB⁻4 as Host Strain

Cited by 34 publications

References 15 publications

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Genome Structure ofBacillus cereustsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

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Polyhydroxyalkanoate (PHA) Synthesis by Class IV PHA Synthases EmployingRalstonia eutrophaPHB−4 as Host Strain

Cited by 34 publications

References 15 publications

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Alcoholytic Cleavage of Polyhydroxyalkanoate Chains by Class IV Synthases Induced by Endogenous and Exogenous Ethanol

Genome Structure ofBacillus cereustsu1 and Genes Involved in Cellulose Degradation and Poly-3-Hydroxybutyrate Synthesis

Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

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Polyhydroxyalkanoate (PHA) Synthesis by Class IV PHA Synthases EmployingRalstonia eutrophaPHB⁻4 as Host Strain