Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by during batch culture

Taidi, Behnam; Mansfield, David A.; Anderson, Angelika

doi:10.1016/0378-1097(95)00158-2

Cited by 35 publications

(44 citation statements)

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“…We also observed that PHB molecular weight decreased as culture time increased from 48 to 72 h. Previous work has demonstrated turnover of PHB in R. eutropha, with a concurrent decrease in the molecular weight of the stored polymer (43). The reason for this decrease is not well understood, but it may be related to the finding that at least two PHA depolymerases are expressed in R. eutropha as PHB is being produced (20).…”

Section: Discussionsupporting

confidence: 53%

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

Budde¹,

Mahan²,

Lu³

et al. 2010

J Bacteriol

114

104

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The bacterium Ralstonia eutropha H16 synthesizes polyhydroxybutyrate (PHB) from acetyl coenzyme A (acetyl-CoA) through reactions catalyzed by a ␤-ketothiolase (PhaA), an acetoacetyl-CoA reductase (PhaB), and a polyhydroxyalkanoate synthase (PhaC). An operon of three genes encoding these enzymatic steps was discovered in R. eutropha and has been well studied. Sequencing and analysis of the R. eutropha genome revealed putative isologs for each of the PHB biosynthetic genes, many of which had never been characterized. In addition to the previously identified phaB1 gene, the genome contains the isologs phaB2 and phaB3 as well as 15 other potential acetoacetyl-CoA reductases. We have investigated the roles of the three phaB isologs by deleting them from the genome individually and in combination. It was discovered that the gene products of both phaB1 and phaB3 contribute to PHB biosynthesis in fructose minimal medium but that in plant oil minimal medium and rich medium, phaB3 seems to be unexpressed. This raises interesting questions concerning the regulation of phaB3 expression. Deletion of the gene phaB2 did not result in an observable phenotype under the conditions tested, although this gene does encode an active reductase. Addition of the individual reductase genes to the genome of the ⌬phaB1 ⌬phaB2 ⌬phaB3 strain restored PHB production, and in the course of our complementation experiments, we serendipitously created a PHB-hyperproducing mutant. Measurement of the PhaB and PhaA activities of the mutant strains indicated that the thiolase reaction is the limiting step in PHB biosynthesis in R. eutropha H16 during nitrogen-limited growth on fructose.

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Section: Discussionsupporting

confidence: 53%

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

Budde¹,

Mahan²,

Lu³

et al. 2010

J Bacteriol

114

104

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“…The synthesis and degradation of PHB seem to occur simultaneously, since the double mutant showed an explicit increased PHB deposition in the log phase. Such a turnover of PHB was already pointed out (2,8,28). In view of economy, a quick turnover of PHB seems to be a disadvantage to bacteria.…”

Section: Discussionmentioning

confidence: 86%

Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

Kobayashi

Shiraki²,

Abe³

et al. 2003

J Bacteriol

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An intracellular 3-hydroxybutyrate (3HB)-oligomer hydrolase (PhaZ2 Reu ) of Ralstonia eutropha was purified from Escherichia coli harboring a plasmid containing phaZ2 Reu . The purified enzyme hydrolyzed linear and cyclic 3HB-oligomers. Although it did not degrade crystalline poly(3-hydroxybutyrate) (PHB), the purified enzyme degraded artificial amorphous PHB at a rate similar to that of the previously identified intracellular PHB (iPHB) depolymerase (PhaZ1 Reu ). The enzyme appeared to be an endo-type hydrolase, since it actively hydrolyzed cyclic 3HB-oligomers. However, it degraded various linear 3HB-oligomers and amorphous PHB in the fashion of an exo-type hydrolase, releasing one monomer unit at a time. PhaZ2 was found to bind to PHB inclusion bodies and as a soluble enzyme to cell-free supernatant fractions in R. eutropha; in contrast, PhaZ1 bound exclusively to the inclusion bodies. When R. eutropha H16 was cultivated in a nutrient-rich medium, the transient deposition of PHB was observed: the content of PHB was maximized in the log growth phase (12 h, ca. 14% PHB of dry cell weight) and decreased to a very low level in the stationary phase (ca. 1% of dry cell weight). In each phaZ1-null mutant and phaZ2-null mutant, the PHB content in the cell increased to ca. 5% in the stationary phase. A double mutant lacking both phaZ1 and phaZ2 showed increased PHB content in the log phase (ca. 20%) and also an elevated PHB level (ca. 8%) in the stationary phase. These results indicate that PhaZ2 is a novel iPHB depolymerase, which participates in the mobilization of PHB in R. eutropha along with PhaZ1.Poly(3-hydroxybutyrate) (PHB), a homopolymer of R(Ϫ)-3-hydroxybutyrate (3HB), is a storage material produced by some bacteria under certain conditions (1). In the past few decades, the application of this biopolymer to biodegradable polymers or plastics has been studied extensively (12). In these studies, the extracellular metabolism of PHB has been clarified in many bacteria and some fungi (6, 7). However, only a few studies on the intracellular degradation of PHB have been published (13,14,17,19,20). An intracellular PHB (iPHB) depolymerase system in Rhodospirillum rubrum was first reported in 1964 and consisted of a thermostable activator and a thermolabile esterase (13). This system is still not well understood in spite of a recent reinvestigation (14). The molecular cloning of an iPHB depolymerase from Ralstonia eutropha H16 has been also reported (17). This enzyme (PhaZ1 Reu ) degraded artificial amorphous PHB granules but not crystalline PHB. A mutant lacking PhaZ1 Reu showed a higher PHB content compared to the wild-type in a nutrient-rich medium, but in this mutant the mobilization of PHB was not inhibited completely, suggesting that the cloned depolymerase gene is not the only gene responsible for the biodegradation of PHB in this bacterium (5, 17). In regard to this point, recently we found another esterase (PhaZ2 Reu ) that hydrolyzes 3HB-oligomers and cloned its gene (18). We examined the properties of the ...

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“…If carbon is the only growth-limiting factor, a transient induction of a second i-PHB depolymerase can enable the cells to increase the rate of PHB mobilization and to perform one or two cell divisions. This assumption might explain an apparent contradiction between the results of Doi et al (2) and Taidi et al (13), who found evidence for the simultaneous synthesis and degradation of PHB. It will be necessary to study the expression of both i-PHB depolymerases separately under PHB-accumulating and PHB-mobilizing conditions in the future.…”

mentioning

confidence: 74%

Mobilization of Poly(3-Hydroxybutyrate) inRalstonia eutropha

2000

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Ralstonia eutropha H16 degraded (mobilized) previously accumulated poly(3-hydroxybutyrate) (PHB) in the absence of an exogenous carbon source and used the degradation products for growth and survival. Isolated native PHB granules of mobilized R. eutropha cells released 3-hydroxybutyrate (3HB) at a threefold higher rate than did control granules of nonmobilized bacteria. No 3HB was released by native PHB granules of recombinant Escherichia coli expressing the PHB biosynthetic genes. Native PHB granules isolated from chromosomal knockout mutants of an intracellular PHB (i-PHB) depolymerase gene of R. eutropha H16 and HF210 showed a reduced but not completely eliminated activity of 3HB release and indicated the presence of i-PHB depolymerase isoenzymes.The mechanism of degradation of denatured, exogenous, crystalline poly(3-hydroxybutyrate) (PHB) by extracellular PHB depolymerases has been extensively studied during the last decade (for a recent review, see reference 6). However, intracellular PHB (i-PHB) degradation, i. e., the mobilization of previously accumulated amorphous PHB, is poorly understood. The beneficial effect of accumulated PHB on survival in the absence of a carbon source has been described for several species, including Ralstonia eutropha (4), Legionella pneumophila (5), and Hydrogenophaga pseudoflava (15). Recently, a DNA sequence of a putative i-PHB depolymerase of R. eutropha H16 was determined (GenBank accession no. AB017612). However, the physiological relevance of this gene during mobilization of PHB has not been investigated.PHB-rich cells of R. eutropha H16 (DSM428) were resuspended and incubated in carbon-free mineral-salt medium (12) with (mobilization) or without (control) NH 4 Cl for 6 days. The number of viable cells, in CFU per milliliter, increased about fourfold in the presence of NH 4 Cl within 30 h and remained constantly high for 6 days ( Fig. 1). In contrast, the CFU/ml remained almost unchanged in the absence of a nitrogen source. The PHB content of strain H16 (assayed through gas chromatography [1]) rapidly decreased from 70 to Ϸ30% during the first day of mobilization and decreased further, below 20%, after 6 days (Fig. 1). The PHB content of the control decreased only very little. The CFU/ml of the PHB-free mutant PHB Ϫ 4 (DSM541) decreased rapidly by several orders of magnitude after 3 days regardless of the absence or presence of a nitrogen source. Similar results were obtained with other bacteria such as Acidovorax delafieldii (DSM50403), Alcaligenes faecalis (14), a Comamonas sp. (DSM6781) and two Paracoccus denitrificans strains (DSM1404 and DSM413); however, the amount of PHB accumulation and the increase of the CFU/ml during mobilization of PHB were not as pronounced for these bacteria as for R. eutropha (data not shown). We conclude that R. eutropha H16 and other bacteria are able to mobilize previously accumulated PHB and to use the degradation products for one or two cell divisions even in the absence of an exogenous carbon source. Very little PHB is mobilized if ...

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Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by during batch culture

Cited by 35 publications

References 0 publications

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

Roles of Multiple Acetoacetyl Coenzyme A Reductases in Polyhydroxybutyrate Biosynthesis in Ralstonia eutropha H16

Purification and Properties of an Intracellular 3-Hydroxybutyrate-Oligomer Hydrolase (PhaZ2) in Ralstonia eutropha H16 and Its Identification as a Novel Intracellular Poly(3-Hydroxybutyrate) Depolymerase

Mobilization of Poly(3-Hydroxybutyrate) inRalstonia eutropha

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