Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus (original) (raw)
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Applied and Environmental Microbiology, 2004
Polyhydroxyalkanoates (PHAs) can be divided into three main types based on the sizes of the monomers incorporated into the polymer. Short-chain-length (SCL) PHAs consist of monomer units of C 3 to C 5 , mediumchain-length (MCL) PHAs consist of monomer units of C 6 to C 14 , and SCL-MCL PHAs consist of monomers ranging in size from C 4 to C 14 . Although previous studies using recombinant Escherichia coli have shown that either SCL or MCL PHA polymers could be produced from glucose, this study presents the first evidence that an SCL-MCL PHA copolymer can be made from glucose in recombinant E. coli. The 3-ketoacyl-acyl carrier protein synthase III gene (fabH) from E. coli was modified by saturation point mutagenesis at the codon encoding amino acid 87 of the FabH protein sequence, and the resulting plasmids were cotransformed with either the pAPAC plasmid, which harbors the Aeromonas caviae PHA synthase gene (phaC), or the pPPAC plasmid, which harbors the Pseudomonas sp. strain 61-3 PHA synthase gene (phaC1), and the abilities of these strains to accumulate PHA from glucose were assessed. It was found that overexpression of several of the mutant fabH genes enabled recombinant E. coli to induce the production of monomers of C 4 to C 10 and subsequently to produce unusual PHA copolymers containing SCL and MCL units. The results indicate that the composition of PHA copolymers may be controlled by the monomer-supplying enzyme and further reinforce the idea that fatty acid biosynthesis may be used to supply monomers for PHA production.
Antonie van Leeuwenhoek, 2008
Four (R)-specific enoyl CoA hydratases (PhaJ) interconnect the b-oxidation pathway with PHA biosynthesis in Pseudomonas aeruginosa. The use of antisense technique and over-expression to delineate the role of two of these enzymes, PhaJ1 and PhaJ4 forms the basis of this study. It has been observed that P. aeruginosa recombinant with phaJ1 antisense construct, fed with different fatty acids, produces PHA with less hydroxy octanoate (7-11% reduction) and a proportionate increase in other monomer fractions, compared to that of the control. Recombinants bearing phaJ4 antisense construct are found to contain less hydroxy decanoate (10-11% reduction) and more or less equal amount of hydroxy octanoate, compared to that of the control. P. aeruginosa produced PHA with more hydroxy octanoate and decanoate (6-17% increase), respectively, when PhaJ1 and PhaJ4 have been overexpressed individually or along with PhaC1. PhaJ1 and PhaJ4 are found to be involved mainly in the production of hydroxy octanoyl CoA and hydroxy decanoyl CoA, respectively, in P. aeruginosa. The strongest accumulation of hydroxy octanoate and hydroxy decanoate has been observed along with hydroxy butyrate, in PHA, produced by E. coli, when PhaC1 has been co-expressed with PhaJ1 and PhaJ4, respectively. We have demonstrated, for the first time, the polymerization of hydroxy butyryl CoA monomers in recombinant E. coli by PhaC1 of P. aeruginosa.
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.
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.
Applied microbiology and biotechnology, 2016
Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.
FEMS Microbiology Letters, 1999
Polyhydroxyalkanoate biosynthesis genes of Aeromonas caviae were expressed in Escherichia coli LS5218 (fadR atoC(Con)), and the polyhydroxyalkanoate-producing ability of the recombinants was investigated. A LS5218 strain harboring only phaC e (polyhydroxyalkanoate synthase gene) did not accumulate any polyhydroxyalkanoate from dodecanoate in spite of the existence of translated polyhydroxyalkanoate synthase protein, whereas co-expression phaC e and phaJ e ((R)-specific enoyl-CoA hydratase gene) resulted in the accumulation of P(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer up to 7^11 wt% of dry cell weight from octanoate and dodecanoate. These results indicated that both phaC e and phaJ e are essential for E. coli LS5218 to establish the polyhydroxyalkanoate biosynthesis pathway from alkanoic acids. The copolyester content in the strain expressing both the genes under the lac promoter control reached to 38 wt% from dodecanoate. Enzyme assays suggest that efficient monomer formation via L-oxidation by a high level expression of phaJ e was important to achieve a high polyhydroxyalkanoate content in the recombinant E. coli.
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...