Variation in Copolymer Composition and Molecular Weight of Polyhydroxyalkanoate Generated by Saturation Mutagenesis ofAeromonas caviae PHA Synthase (original) (raw)
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A New Wave of Industrialization of PHA Biopolyesters
Bioengineering
The ever-increasing use of plastics, their fossil origin, and especially their persistence in nature have started a wave of new innovations in materials that are renewable, offer the functionalities of plastics, and are biodegradable. One such class of biopolymers, polyhydroxyalkanoates (PHAs), are biosynthesized by numerous microorganisms through the conversion of carbon-rich renewable resources. PHA homo- and heteropolyesters are intracellular products of secondary microbial metabolism. When isolated from microbial biomass, PHA biopolymers mimic the functionalities of many of the top-selling plastics of petrochemical origin, but biodegrade in soil, freshwater, and marine environments, and are both industrial- and home-compostable. Only a handful of PHA biopolymers have been studied in-depth, and five of these reliably match the desired material properties of established fossil plastics. Realizing the positive attributes of PHA biopolymers, several established chemical companies an...
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.
Indian Journal of Microbiology, 2009
Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm−1 for scl-PHA and at 2933 and 2976 cm−1 for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm−1 for HV-copolymer. The copolymers were analyzed by GC and 1H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.
PRODUCTION OF PHA BY RECOMBINANT ORGANISMS
Polyhydroxyalkanoates (PHA) are biodegradable, thermoplastic polyesters produced from renewable carbon sources by a number of bacteria. However, their application is limited by high production costs. One of the strategies aimed to reduce their costs is the development of recombinant strains able to utilize different carbon sources. Many bacteria are naturally capable of accumulating biopolyesters composed of 3-hydroxy fatty acids as intracellular inclusions, which serve as storage granules. Recently, these inclusions have been considered as nano/ microbeads with surface-attached proteins, which can be engineered to display various protein-based functions that are suitable for biotechnological and biomedical applications. Natural PHA producers have become accustomed to accumulating PHA during evolution; they often have a long generation time, relatively low optimal growth temperature, are often hard to lysis and contain pathways for PHA degradation. This led to the development of recombinant PHA producers, capable of high PHA accumulation and/or free of PHA degradative pathways. This review summarizes the production of PHA by recombinant bacteria as well as some higher organisms in brief.
Biosynthesis of polyhydroxyalkanoate homopolymers by Pseudomonas putida
Applied Microbiology and Biotechnology, 2011
Pseudomonas putida KT2442 has been a wellstudied producer of medium-chain-length (mcl) polyhydroxyalkanoate (PHA) copolymers containing C6~C14 monomer units. A mutant was constructed from P. putida KT2442 by deleting its phaG gene encoding R-3-hydroxyacyl-ACP-CoA transacylase and several other β-oxidation related genes including fadB, fadA, fadB2x, and fadAx. This mutant termed P. putida KTHH03 synthesized mcl homopolymers including poly(3-hydroxyhexanoate) (PHHx) and poly(3-hydroxyheptanoate) (PHHp), together with a near homopolymer poly(3-hydroxyoctanoate-co-2 mol% 3hydroxyhexanoate) (PHO*) in presence of hexanoate, heptanoate, and octanoate, respectively. When deleted with its mcl PHA synthase genes phaC1 and phaC2, the recombinant mutant termed P. putida KTHH08 harboring pZWJ4-31 containing PHA synthesis operon phaPCJ from Aeromonas hydrophila 4AK4 accumulated homopolymer poly(3-hydroxyvalerate) (PHV) when valerate was used as carbon source. The phaC deleted recombinant mutant termed P. putida KTHH06 harboring pBHH01 holding PHA synthase PhbC from Ralstonia eutropha produced homopolymers poly(3-hydroxybutyrate) (PHB) and poly(4-hydroxybutyrate) using γ-butyrolactone was added as precursor. All the homopolymers were physically characterized. Their weight average molecular weights ranged from 1.8×10 5 to 1.6×10 6 , their thermal stability changed with side chain lengths. The derivatives of P. putida KT2442 have been developed into a platform for production of various PHA homopolymers.
Journal of Biotechnology, 2007
One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) components into the polymers requires the addition of additional carbon sources, leading to extra costs and complexity. In this study, the production of poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)-co-3-hydroxy-2-methylvalerate (3HMV)), with 7-35 C-mol% of 3HV fractions from acetate as the only carbon source was achieved with the use of glycogen accumulating organisms (GAOs). An enriched GAO culture was obtained in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate fed at the beginning of the anaerobic period. The production of PHAs utilizing the enriched GAO culture was investigated under both aerobic and anaerobic conditions. A polymer content of 14-41% of dry cell weight was obtained. The PHA product accumulated by GAOs under anaerobic conditions contained a relatively constant proportion of non-3HB monomers (30 ± 5 C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction during this aerobic feeding period. The PHAs were characterized by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The data demonstrated that the copolymers possessed similar characteristics to those of commercially available poly(3HB-co-3HV) (PHBV) products. The PHAs produced under solely anaerobic conditions possessed lower melting points and crystallinity, higher molecular weights, and narrower molecular-weight distributions, compared to the aerobically produced polymers. This paper hence demonstrates the significant potential of GAOs to produce high quality polymers from a simple and cheap carbon source, contributing considerably to the growing research body on bacterial PHA production by mixed cultures.