Coexpression of Genetically Engineered 3-Ketoacyl-ACP Synthase III (fabH) and Polyhydroxyalkanoate Synthase (phaC) Genes Leads to Short-Chain-Length-Medium-Chain-Length Polyhydroxyalkanoate Copolymer Production from Glucose in Escherichia coli JM109 (original) (raw)
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Applied and Environmental Microbiology, 2005
Polyhydroxyalkanoates (PHAs) are biologically produced polyesters that have potential application as biodegradable plastics. Especially important are the short-chain-length-medium-chain-length (SCL-MCL) PHA copolymers, which have properties ranging from thermoplastic to elastomeric, depending on the ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of applications for SCL-MCL PHA copolymers, it is important to develop and characterize metabolic pathways for SCL-MCL PHA production. In previous studies, coexpression of PHA synthase genes and the 3-ketoacyl-acyl carrier protein reductase gene ( fabG ) in recombinant Escherichia coli has been shown to enhance PHA production from related carbon sources such as fatty acids. In this study, a new fabG gene from Pseudomonas sp. 61-3 was cloned and its gene product characterized. Results indicate that the Pseudomonas sp. 61-3 and E. coli FabG proteins have different substrate specificities in vi...
Biomacromolecules, 2004
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that have a wide variety of physical properties dependent on the lengths of the pendant groups of the monomer units in the polymer. PHAs composed of mostly short-chain-length (SCL) monomers are often stiff and brittle, whereas PHAs composed of mostly medium-chain-length (MCL) monomers are elastomeric in nature. SCL-MCL PHA copolymers can have properties between the two states, dependent on the ratio of SCL and MCL monomers in the copolymer. It is desirable to elucidate new and low cost ways to produce PHA composed of mostly SCL monomer units with a small mol % of MCL monomers from renewable resources, since this type of SCL-MCL PHA copolymer has superior qualities compared to SCL homopolymer. To address this issue, we have created strains of recombinant E. coli capable of producing -ketothiolase (PhbA) and acetoacetyl-CoA synthase (PhbB) from Ralstonia eutropha, genetically engineered 3-ketoacyl-ACP synthase III (FabH) from Escherichia coli, and genetically engineered PHA synthases (PhaC) from Pseudomonas sp. 61-3 to enhance the production of SCL-MCL PHA copolymers from glucose. The cumulative effect of having two monomer-supplying pathways and genetically engineered PHA synthases resulted in higher accumulated amounts of SCL-MCL PHA copolymer from glucose. Polymers were isolated from two recombinant E. coli strains, the first harboring the phbAB, fabH(F87T), and phaC1(SCQM) genes and the second harboring the phbAB, fabH(F87W), and phaC1(SCQM) genes. The thermal and physical properties of the isolated polymers were characterized. It was found that even a very low mol % of MCL monomer in a SCL-MCL PHA copolymer had dramatic effects on the thermal properties of the copolymers.
Journal of Bioprocess Engineering and Biorefinery, 2012
Polyhydroxyalkanoates (PHAs) are aliphatic polyesters produced by a wide variety of bacteria as carbon and energy storage sources. Copolymers of the short chain length (SCL) 3-hydroxybutyrate (3HB) repeating unit and medium chain length hydroxyalkanoate (MCL-HA) repeating units have better thermal and mechanical properties than poly-3HB homopolymers. In this study, a MCL monomer supplier gene phaJ4 from Pseudomonas putida KT2440, an engineered PHA synthase gene (phaC1) from Pseudomonas sp. 61-3, and the SCL monomer supplier genes, phaA and phaB from Ralstonia eutropha, were coexpressed under the lac promoter in recombinant E. coli LS5218 to produce SCL-MCL copolymers. The recombinant strains were co-fed lauric acid (a fatty acid) and glucose as a carbon source and the ratio of SCL to MCL monomers varied dependant on differences in the timing of the addition of each carbon source during the fermentation. The PHA copolymers produced exhibited a range from 0.4 mol% to 35 mol% MCL repeating units giving rise to wide range of physical properties. The molecular weights and thermal properties of different polymers were studied by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The results suggested the polymers produced by co-feeding with lauric acid and glucose were mixtures of 3HB homopolymers and 3HB-co-MCL copolymers. This conclusion was confirmed by acetone fractionation.
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.
ACS Sustainable Chemistry & Engineering, 2014
Polyhydroxyalkanoates (PHAs) are biorenewable and biodegradable polyesters that have garnered attention as alternatives to more common petroleum-based polymers. One of the current limitations for the widespread use of PHAs is the inability to produce PHA polymers with desired material properties. Previous studies have shown that PHA copolymers consisting primarily of one short-chain-length (SCL) repeating unit and a small concentration of medium-chain-length (MCL) repeating units have physical properties resembling the petroleum-based plastic polyethylene. In addition, these SCL-co-MCL PHA copolymers have been investigated for biomedical applications such as tissue engineering. However, bacterial production of these SCL-co-MCL PHA copolymers is often at a much lower yield compared to SCL PHA biosynthesis produced from simple sugars such as glucose. Here, we report the highest yield to date of SCL-co-MCL PHA copolymers produced from glucose. Two separate biosynthetic pathways for SCL and MCL PHAs were introduced into Escherichia coli LS5218, and copolymer production experiments were carried out in batch fermentations. The PHA copolymers produced consisted of repeating units with 4, 6, 8, 10, and 12 carbons at mol % concentrations similar to that of other SCL-co-MCL PHA copolymers reported to have desirable physical properties. The PHA repeating unit compositions, structures, and linkages between individual repeating unit types were analyzed by GC and NMR. The thermal properties of purified PHA copolymers were also examined. The engineered strain developed in this study (E. coli LS5218-STQKABGK) provides a platform to further increase PHA copolymer yields from unrelated carbon sources in a non-native PHA producing bacterial strain.
Applied Sciences, 2017
A recombinant of Pseudomonas putida LS461 (deletion of the phaC1phaZphaC2 genes) was constructed by introducing cosmid JC123 carrying a novel phaC1 16 gene from a metagenomic clone. The resulting strain, P. putida LS46123, was able to synthesize polyhydroxyalkanoate (PHA) polymers with novel monomer compositions when cultured on glucose or free fatty acids, and accumulated PHAs from 9.24% to 27.09% of cell dry weight. The PHAs synthesized by P. putida LS46123 contained up to 50 mol % short chain length subunits (3-hydroxybutyrate and 3-hydroxyvalerate), with the remaining monomers consisting of various medium chain length subunits. The PhaC1 16 protein expressed by P. putida LS46123 had an amino acid sequence similarity of 45% with the PhaC1 protein of the parent strain, P. putida LS46. Predicted 3D structures of the PhaC1 16 proteins from P. putida LS46123 and P. putida LS46 revealed several differences in the numbers and locations of protein secondary structures. The physical and thermal properties of the novel polymers synthesized by P. putida LS46123 cultured with glucose or free fatty acids differed significantly from those produced by P. putida LS46 grown on the same substrates. PHA polymers with different subunit compositions, and hence different physical and thermal properties, can be tailor-made using novel PHA synthase for specific applications.