Optimization of the Expression of Genes Encoding Poly (3-hydroxyalkanoate) Synthase from Pseudomonas aeruginosa PTCC 1310 in Escherichia coli (original) (raw)
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Journal of Biological Chemistry, 1991
Pseudomonas oleovorans accumulates poly(3-hydroxyalkanoates) (PHAs) after growth on medium chain length hydrocarbons. Large amounts of this polyester are synthesized when cells are grown under nitrogen-limiting conditions. When nitrogen is resupplied in the medium, the accumulated PHA is degraded. In this paper, we describe mutants which are defective in the synthesis or in the degradation of PHA. These mutants were used to select DNA fragments which encode PHA Polymerases and a PHA depolymerase. A 26-kilobase (kb) DNA fragment was isolated from P. oleovorans that complements a Pseudomonas putida mutant unable to accumulate PHA, Subcloning resulted in the assignment of a 6.4-kb EcoRI fragment as the pha locus, containing genetic information for PHA synthesis. Mutants in the PHA degradation pathway were also complemented by this fragment, indicating that genes encoding PHA biosynthetic and degradative enzymes are clustered. Analysis of the DNA sequence of the 6.4-kb fragment revealed the presence of two open reading frames encoding PHA polymerases based on homology to the poly(3-hydroxybutyrate) polymerase from Alcaligenes eutrophus. A third open reading frame complemented the PHA degradation mutation and is likely to encode a PHA depolymerase. The presence of two PHA polymerases is due to a 2098-base pair DNA duplication. The PHA polymerases are 53% identical and show 35-40% identity to the poly(3hydroxybutyrate) polymerase. No clear difference in specificity was found for the PHA polymerases. However, with the pha locus cloned on a multicopy vector, a polymer was accumulated that contains a significantly higher amount of substrate-derived monomers. An increase in the rate of polyester synthesis versus oxidation of the monomers in the &oxidation explains these findings. Poly(3-hydroxybutyrate) (PHB)' is a common reserve material in both Gram-positive and Gram-negative bacteria. It
Letters in Applied Microbiology, 2006
Aims: This study is mainly focused on the heterologous expression and accumulation of polyhydroxyalkanoates (PHA) in Escherichia coli.Methods and Results: PHA synthase gene (phaC1) from indigenous Pseudomonas sp. LDC-5 was amplified by PCR and cloned in E. coli (Qiagen EZ competent cells). The recombinant E. coli was analysed and confirmed for its expression of phaC1 gene by phase contrast microscopy, Western blot analysis and spectral studies (Fourier-transform infrared spectroscopy). It was further evaluated for its accumulation in different carbon and nitrogen sources. The accumulation of PHA (3·4 g l−1) was enhanced in the medium supplemented with glycerol and fish peptone compared to the other carbon and nitrogen sources used in this study.Conclusions: This study would enable the reduction of cost of PHA production.Significance and Impact of the Study: An important part of this study is that E. coli harbouring partial phaC1 gene could accumulate medium chain length PHA significantly. The results demonstrated that the E. coli strain could be a potential candidate for the large-scale production of polymer. The conditions for the higher yield and productivity will be optimized in the next phase using fermentation studies.
Microorganisms, 2021
Pseudomonas strains have a variety of potential uses in bioremediation and biosynthesis of biodegradable plastics. Pseudomonas sp. strain phDV1, a Gram-negative phenol degrading bacterium, has been found to utilize monocyclic aromatic compounds as sole carbon source via the meta-cleavage pathway. The degradation of aromatic compounds comprises an important step in the removal of pollutants. The present study aimed to investigate the ability of the Pseudomonas sp. strain phDV1 to produce polyhydroxyalkanoates (PHAs) and examining the effect of phenol concentration on PHA production. The bacterium was cultivated in minimal medium supplemented with different concentrations of phenol ranging from 200–600 mg/L. The activity of the PHA synthase, the key enzyme which produces PHA, was monitored spectroscopically in cells extracts. Furthermore, the PHA synthase was identified by mass spectrometry in cell extracts analyzed by SDS-PAGE. Transmission electron micrographs revealed abundant elec...
Antonie van Leeuwenhoek, 2008
Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated b-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and 1 H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of b-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.
Brazilian Archives of Biology and Technology, 2013
In this study, a variety of samples were screened for the presence of PHA synthase gene. Results showed that 16 out of 102 isolated were positive for PHA respective genes. The highest prevalence was observed in Pseudomonas aeruginosa. The capability of PHA production was also shown by growing these strains on the defined medium and subsequent analysis using intracellular granules staining and Fourier transform infrared spectroscopy (FT-IR). The microscopic analysis showed that the positive strains accumulated PHA in the cell. The FT-IR analysis showed the presence of PHA peaks in the dried cells as well as in extraction product. P aeruginosa strain P7 showed higher concentration of PHA compared to the others as demonstrated by the highest respective peaks in FT-IR.
Applied Microbiology and Biotechnology, 2006
In this study we examined polyhydroxyalkanoate (PHA) synthases phaC1 and phaC2 gene expression in two strains of Pseudomonas corrugata (Pc) grown in a minimum mineral medium with related (oleic acid and octanoate) or unrelated (glucose) carbon sources. Analysis of transcription was performed by Northern blot and conventional reverse transcriptase (RT) polymerase chain reaction (PCR). In addition, we developed a RT-real-time
PCR cloning of type II polyhydroxyalkanoate biosynthesis genes from two Pseudomonas strains
Fems Microbiology Letters, 2001
Two polyhydroxyalkanoate synthase genes, phaC1 from Pseudomonas pseudoalcaligenes HBQ06 and phaC2 from Pseudomonas nitroreducens 0802, were cloned using a PCR cloning strategy based on the type II pha loci property of Pseudomonas strains. The complete open reading frames (ORFs) of phaC1 (P. nitroreducens HBQ06) and phaC2 (P. nitroreducens 0802) were identified from the PCR products. Using the sequence information, the complete PHA synthase genes were PCR cloned directly from the genomic DNA and expressed in Escherichia coli as confirmed by Fourier transform-infrared spectroscopy and gas chromatography. The differences between PhaC1 and PhaC2 were analyzed and the two proteins were suggested to contain different functions and evolution history.
Applied Microbiology and Biotechnology, 2005
We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.
Applied Microbiology and Biotechnology, 2005
A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha Ps-1 and pha Ps-2 ) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha Ps-2 locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1 Ps-2 -phaC1 Ps-2 -phaZ Ps-2 -phaC2 Ps-2 -phaD Ps-2 . The pha Ps-1 locus that contains phaC1 Ps-1 -phaZ Ps-1 appears to have arisen by a duplication event that placed it downstream of a gene (ORF1 Ps-1 ), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 Ps-1 and phaC1 Ps-2 were investigated by heterologous expression in Wautersia eutropha PHB − 4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1 Ps-1 to confer PHA synthesis, however, appeared reduced compared to that of PhaC1 Ps-2 , since cells harboring PhaC1 Ps-1 accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1 Ps-2 . Primary sequence analysis revealed that PhaC1 Ps-1 had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1 Ps-2 ). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 Ps-1 produces a protein product in the native strain, it is likely that PhaC1 Ps-1 may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.
Screening of PHA (poly hydroxyalkanoate) producing bacteria from diverse sources
International Journal of …, 2011
Synthetic plastics are non-degradable and cause waste disposal problems leading to environmental pollution. Bioplastics (polyhydroxyalkanoates) are considered good substitutes for petroleum derived synthetic plastics because of their similar physical and chemical properties. Main advantage of bioplastics is that they are of biological origin and can get degraded completely to CO2 and water under natural environment by the enzymatic activities of microorganisms. Poly-β-hydroxyalkanoates (PHA) are polyesters of various hydroxyalkanoates, synthesized by numerous bacteria as an intracellular carbon and energy storage compound under limited nutrient conditions and with excess carbon. Poly-β-hydroxy butyrate (PHB) is the best known polyhydroxyalkanoate. Considering the industrial interest of PHA, this work has been undertaken for the screening of PHA producing bacteria from diverse sources. In the present study, an attempt was made to isolate efficient PHB producing bacteria from diverse environmental samples. Different industrial wastes and soil samples were screened for bacteria possessing the ability to accumulate poly hydroxyalkanoate (PHA) granules. About 23 bacterial isolates were found to be promising PHA accumulating bacteria. Screening for PHA producers was performed by using E 2 medium. Accumulation of PHB granules in the organisms was analyzed by Sudan black method.