Production of Medium-Chain-Length Polyhydroxyalkanoates by Pseudomonas aeruginosa With Fatty Acids and Alternative Carbon Sources (original) (raw)
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Journal of Bioscience and Bioengineering, 2008
phaJ and fabG genes encoding (R)-specific 2-enoyl-CoA hydratase and 3-ketoacyl-acyl carrier protein reductase, respectively, were cloned from Pseudomonas putida KCTC1639 and amplified in the parent strain to achieve oversupplementation of (R)-3-hydroxyalkanoate monomer units for the biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The comparative effect of the overexpressed fabG and phaJ genes in P. putida KCTC1639 on the biosynthesis of mcl-PHA and the cell growth were elucidated. Overexpression of phaJ enhanced the biosynthesis of mcl-PHA, increasing its content and concentration from 18% to 27% and 0.38 to 0.51 g/l, respectively. Conversely, fabG overexpression tended to depress the biosynthesis of mcl-PHA, possibly due to the reversible conversion of (R)-3-hydroxyalkanoate monomer units into 3-ketoacyl-CoA.
Applied Microbiology and Biotechnology, 2007
Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h −1 but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ=0.15 h −1 ) produced 70 g l −1 biomass containing 75% PHA. At a higher exponential feed rate (μ=0.25 h −1 ), the overall productivity was increased but less biomass (56 g l −1 ) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbonlimited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.
Canadian Journal of Microbiology, 2012
Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P. putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P. putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12 h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48 h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C6-C14 saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20 mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1-68.8 mol%) in P. putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88 mol%).
Journal of Industrial Microbiology & Biotechnology, 2015
commercial interest due to their biocompatibility and biodegradability [14, 18]. PHAs are classified as shortchain-length PHAs (SCL-PHAs) when the pendant group of monomer varies from 0 to 2 carbons, medium-chainlength PHAs (MCL-PHAs) when there are three or more carbons on the pendant group and SCL-MCL-PHAs when polymers consist of both SCL and MCL monomers [19, 22]. MCL-PHAs are of increasing interest because of their low crystallinity, high elasticity [13, 25] and the possibility of having different functional groups, such as alkenes [21], aromatic groups [12], halogen [9, 16, 20], esters [30] and phenoxy groups [27] on their side chains. Since more than 100 different MCL-PHA monomers can be incorporated, MCL-PHAs can exhibit a wide variety of properties with many possible applications including coatings, medical implants, drug delivery, water-based latex paints and others [39, 40]. PHAs are more expensive to produce than conventional plastics with expenditures almost evenly divided between carbon source, fermentation process and separation process [37]. Metabolic engineering may be used to achieve higher PHA cellular content, more effective carbon source usage, and to obtain novel PHAs with valuable properties, thus increasing commercially viability. One such approach is the elimination of PhaZ activity. The phaZ gene encoding PhaZ is located between phaC1 and phaC2 genes of the MCL-PHA metabolism gene cluster which consists of phaC1, phaZ, phaC2, phaD, phaF and phaI genes in Pseudomonas putida KT2440 whose entire genome has been mapped [17, 24]. The PhaZ of P. putida KT2442, a spontaneous rif r mutant of P. putida KT2440 [2], is an intracellular MCL-PHA depolymerase and an MCL-PHA granule surface-associated protein [7, 8]. PhaZ is reported to play a crucial role in the turnover of MCL-PHAs under carbon starvation in P. putida KT2442 [5].
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.
Journal of Industrial Microbiology and Biotechnology, 2015
A medium-chain-length poly-3-hydroxyalkanote (MCL-PHA) depolymerase knockout mutant of Pseudomonas putida KT2440 was produced by double homologous recombination. A carbon-limited shake-flask study confirmed that depolymerase activity was eliminated. Lysis of both mutant and wild-type strains occurred under these conditions. In carbon-limited, fed-batch culture, the yield of unsaturated monomers from unsaturated substrate averaged only 0.62 mol mol−1 for the phaZ minus strain compared to 0.72 mol mol−1 for the wild type. The mutant strain also produced more CO2 and less residual biomass from the same amount of carbon substrate. However, most results indicated that elimination of PHA depolymerase activity had little impact on the overall yield of biomass and PHA.