Kinetics of medium-chain-length polyhydroxyalkanoate production by a novel isolate of Pseudomonas putida LS46 (original) (raw)
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Polyhydroxyalkanoate production in Pseudomonas putida from alkanoic acids of varying lengths
PLOS ONE, 2023
Many studies have been conducted to produce microbial polyhydroxyalkanoates (PHA), a biopolymer, from Pseudomonas sp. fed with various alkanoic acids. Because this previous data was collected using methodologies that varied in critical aspects, such as culture media and size range of alkanoic acids, it has been difficult to compare the results for a thorough understanding of the relationship between feedstock and PHA production. Therefore, this study utilized consistent culture media with a wide range of alkanoic acids (C7-C14) to produce medium chain length PHAs. Three strains of Pseudomonas putida (NRRL B-14875, KT2440, and GN112) were used, and growth, cell dry weight, PHA titer, monomer distribution, and molecular weights were all examined. It was determined that although all the strains produced similar PHA titers using C7-C9 alkanoic acids, significant differences were observed with the use of longer chain feedstocks. Specifically, KT2440 and its derivative GN112 produced higher PHA titers compared to B-14875 when fed longer chain alkanoates. We also compared several analytical techniques for determining amounts of PHA and found they produced different results. In addition, the use of an internal standard had a higher risk of calculating inaccurate concentrations compared to an external standard. These observations highlight the importance of considering this aspect of analysis when evaluating different studies.
PHAs accumulation in Pseudomonas putida P5 (wild type and mutants) in lipid containing media
Romanian …, 2010
Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by various bacteria as intracellular carbon and energy storage compounds and accumulated as granules in the cytoplasm of cells. In this work, we describe the isolation and characterization of Pseudomans putida P5 strain, as well as of some of its derivatives (mutants and recombinants) regarding the accumulation of PHAs. All the strains were subjected to fermentation and showed PHA accumulation with glucose, gluconate or octanoate as carbon source. In order to obtain PHA produced by these strains, a special procedure was developed, which allowed 80% PHA recovered. Differences in msc-PHA accumulation were observed in chemically induced mutants. RAPD analysis of these mutants proved that some rearrangements are produced as result of mutagenesis. The best results concerning msc-PHA accumulation were obtained when 0.4% octanoate was used. Different other lipid substrates (vegetal oils) were tested in order to establish the best substrate that allowed the largest accumulation of PHA. The results showed that the selected strains of P.putida (wild type or mutant) could utilize oil waste in the medium as a carbon source better than that of glucose and thus could substantially lower the cost of production of PHA.
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.
Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001
Brazilian Journal of Microbiology
Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L(-1) to 15.45 g L(-1), respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (T m) of 42.0 (± 0.2) °C, glass transition temperature (T g) of -1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g(-1). The molecular weight (M w) range of the polymer was relatively narrow between 55 to 7...
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.
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.
BMC Microbiology, 2010
Background: Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/ [CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary. Results: Two in vitro assays were developed which allow the measurement of PhaC and PhaZ activities in crude cell extracts of Pseudomonas putida U. Using the assays, it was demonstrated that the activity of PhaC decreased 5-fold upon exponential growth on nitrogen limited medium and octanoate. In contrast, the activity of PhaZ increased only 1.5-fold during growth. One reason for the changes in the enzymatic activity of PhaC and PhaZ could relate to a change in interaction with the phasin surface proteins on the PHA granule. SDS-PAGE analysis of isolated PHA granules demonstrated that during growth, the ratio of [phasins]/[PHA] decreased. In addition, it was found that after eliminating phasins (PhaF and PhaI) from the granules PhaC activity decreased further. Conclusion: Using the assays developed in this study, we followed the enzymatic activities of PhaC and PhaZ during growth and correlated them to the amount of phasins on the PHA granules. It was found that in P. putida PhaC and PhaZ are concomitantly active, resulting in parallel synthesis and degradation of PHA. Moreover PhaC activity was found to be decreased, whereas PhaZ activity increased during growth. Availability of phasins on PHA granules affected the activity of PhaC.