Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

Hustede, E.; Steinbüchel, Alexander; Schlegel, Hans-Günter

doi:10.1016/0378-1097(92)90476-5

Cited by 18 publications

(12 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous studies identified and characterized two PHA polymerases (PhaCs) (5,12) in R. rubrum. Using these two sequences as the query, homology searches of the R. rubrum genome of strain ATCC 11170 revealed three genes encoding PHA polymerases, Rru_A0275, Rru_A2413, and Rru_A1816, which we designated phaC1, phaC2, and phaC3, respectively.…”

Section: Resultsmentioning

confidence: 99%

“…This metabolically versatile bacterium can grow under aerobic or anaerobic conditions or in the presence or absence of light, and in the latter condition it can use a variety of different carbon substrates (39,43). Previous studies and genome sequencing have revealed that R. rubrum can express three PHA polymerases (5,12). Because of its metabolic flexibility, R. rubrum offers the potential for converting many different carbon sources to PHA, and therefore there are considerable industrial interests in exploring this flexibility (6,35).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

Jin

Nikolau

2012

J Bacteriol

View full text Add to dashboard Cite

This study investigated the apparent genetic redundancy in the biosynthesis of polyhydroxyalkanoates (PHAs) in the Rhodospirillum rubrum genome revealed by the occurrence of three homologous PHA polymerase genes (phaC1, phaC2, and phaC3). In vitro biochemical assays established that each gene product encodes PHA polymerase. A series of single, double, and triple phaC deletion mutants were characterized with respect to PHA production and growth capabilities on acetate or hexanoate as the sole carbon source. These analyses establish that phaC2 contributes the major capacity to produce PHA, even though the PhaC2 protein is not the most efficient PHA polymerase biocatalyst. In contrast, phaC3 is an insignificant contributor to PHA productivity, and phaC1, the PHA polymerase situated in the PHA biosynthetic operon, plays a minor role in this capability, even though both of these genes encode PHA polymerases that are more efficient enzymes. These observations are consistent with the finding that PhaC1 and PhaC3 occur at undetectable levels, at least 10-fold lower than that of PhaC2. The monomers in the PHA polymer produced by these strains establish that PhaC2 is responsible for the incorporation of the C 5 and C 6 monomers. The in vitro characterizations indicate that heteromeric PHA polymerases composed of mixtures of different PhaC paralogs are more efficient catalysts, suggesting that these proteins form complexes. Finally, the physiological role of PHA accumulation in enhancing the fitness of R. rubrum was indicated by the relationship between PHA content and growth capabilities of the genetically manipulated strains that express different levels of the PHA polymer.

show abstract

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

Jin

Nikolau

2012

J Bacteriol

View full text Add to dashboard Cite

show abstract

“…Transgenic fibers were treated with Nile blue A fluorescent stain and examined at excitation wavelengths of 546 nm (21). PHB granules were also detected by transmission electron microscopy analysis (University of Wisconsin, Madison) (22). PHB was extracted and converted to crotonic acid and detected by HPLC (23).…”

Section: Methodsmentioning

confidence: 99%

“…7148) were examined by transmission electron microscopy, which has been used to visualize electron-lucent PHB granules in plants and in bacteria (9,22). This technique was also used to measure the size and location of PHB in fibers (Fig.…”

Section: Fig 1 (A)mentioning

confidence: 99%

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

John¹,

Keller²

1996

Proc. Natl. Acad. Sci. U.S.A.

204

104

View full text Add to dashboard Cite

Alcaligenes eutrophus genes encoding the enzymes, ␤-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and polyhydroxyalkanoate synthase (phaC) catalyze the production of aliphatic polyester poly-D-(؊)-3-hydroxybutyrate (PHB) from acetyl-CoA. PHB is a thermoplastic polymer that may modify fiber properties when synthesized in cotton. Endogenous ␤-ketothiolase activity is present in cotton fibers. Hence cotton was transformed with engineered phaB and phaC genes by particle bombardment, and transgenic plants were selected based on marker gene, ␤-glucuronidase (GUS), expression. Fibers of 10 transgenic plants expressed phaB gene, while eight plants expressed both phaB and phaC genes. Electron microscopy examination of fibers expressing both genes indicated the presence of electron-lucent granules in the cytoplasm. High pressure liquid chromatography, gas chromatography, and mass spectrometry evidence suggested that the new polymer produced in transgenic fibers is PHB. Sixty-six percent of the PHB in fibers is in the molecular mass range of 0.6 ؋ 10 6 to 1.8 ؋ 10 6 Da. The presence of PHB granules in transgenic fibers resulted in measurable changes of thermal properties. The fibers exhibited better insulating characteristics. The rate of heat uptake and cooling was slower in transgenic fibers, resulting in higher heat capacity. These data show that metabolic pathway engineering in cotton may enhance fiber properties by incorporating new traits from other genetic sources. This is an important step toward producing new generation fibers for the textile industry.

show abstract

“…Recently, the PHB-biosynthetic genes of nonsulfur purple bacteria were also cloned (Hustede et al, 1992). These bacterial polyesters have attracted much academic and industrial interest since they are thermoplastics, completely biodegradable and can be produced from renewable resources.…”

mentioning

confidence: 99%

Cloning and molecular analysis of the poly(3‐hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1

Timm

Steinbüchel

1992

European Journal of Biochemistry

167

View full text Add to dashboard Cite

From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAOl was cloned and characterised at the molecular level. Two genes coding for polyhydroxyalkanoate synthases, phaCl,, and p h~C 2~, , a polyhydroxyalkanoate depolymerase gene, phaDp,, and four adjacent open reading frames (ORFl,ORF2,ORF3 and ORF4) were identified from the nucleotide sequence. Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for C T~~ and g 7 0 promoters, were identified experimentally upstream of phaCl,,, which was shown by Northern blot analysis to constitute an operon together with phaDp,. A third putative promoter resembling the E. coli consensus sequence for ~'O-dependent promoters was proposed upstream of p h~C 2~, , which is in a bicistronic operon with ORF3. Investigations of rpollrnegative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P. aeruginosa. Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate. The P. aeruginosa PAOl polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate-negative mutant Alcaligenes eutrophus PHB -4 and in the poly(3-hydroxybutyrate)-accumulating strain P. oleovorans DSM1045. It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium-chain-length 3-hydroxyalkanoic acids from unrelated substrates in addition to poly(3-hydroxybutyrate). The sequence of the putative translational product of ORFl was similar to those of the leukotoxin repressor of Pmteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P. aeruginosa and to eukaryotic histone H1 proteins. The proteins of ORF2 and ORF3 appear to be previously unidentified.

show abstract

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

Cited by 18 publications

References 26 publications

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

Role of Genetic Redundancy in Polyhydroxyalkanoate (PHA) Polymerases in PHA Biosynthesis in Rhodospirillum rubrum

Metabolic pathway engineering in cotton: Biosynthesis of polyhydroxybutyrate in fiber cells

Cloning and molecular analysis of the poly(3‐hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1

Contact Info

Product

Resources

About