Abstract
This review focuses on the factors that determine the molecular weight of polyhydroxyalkanoate (PHA), an aliphatic polyester synthesized by bacteria for carbon and energy storage. PHA is a polymer with good thermoplastic, biodegradable, biocompatible and renewable properties. Therefore, it has attracted considerable attention as an environmentally friendly and biomedical material. Because the mechanical strength of PHA increases with its molecular weight, high-molecular-weight PHA polymers are preferred. The synthesis of high-molecular-weight PHA should consider the following factors: the concentration of PHA synthase, the occurrence of a chain transfer reaction, the catalytic activity of PHA synthase and the simultaneous degradation of PHA during biosynthesis. These factors have a direct impact on the molecular weight of PHA. Furthermore, the factors that affect the molecular weight of PHA during in vitro PHA polymerization and cultivation of PHA native and non-native bacteria are discussed.
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References
Sudesh, K., Abe, H. & Doi, Y. Synthesis, structure, and properties of polyhydroxyalkanoates: biological polyesters. Prog. Polym. Sci. 25, 1503–1555 (2000).
Lu, J., Tappel, R. C. & Nomura, C. T. Mini-review: biosynthesis of poly(hydroxyalkanoates). J. Macromol. Sci. Part C Polym. Rev. 49, 226–248 (2009).
Jendrossek, D. Polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes). J. Bacteriol. 191, 3195–3202 (2009).
Tsuge, T. Metabolic improvements and use of inexpensive carbon sources in microbial production of polyhydroxyalkanoates. J. Biosci. Bioeng. 94, 579–584 (2002).
Kahar, P., Agus, J., Kikkawa, Y., Taguchi, K., Doi, Y. & Tsuge, T. Effective production and kinetic characterization of ultra-high-molecular-weight poly[(R-3-hydroxybutyrate] in recombinant Escherichia coli. Polym. Degrad. Stab. 87, 161–169 (2005).
Iwata, T. Strong fibers and films of microbial polyesters. Macromol. Biosci. 5, 689–701 (2005).
Kusaka, S., Iwata, T. & Doi, Y. Microbial synthesis and physical properties of ultra-high molecular weight poly[(R-3-hydroxybutyrate]. J. Macromol. Sci. Pure Appl. Chem. A 35, 319–335 (1998).
Rehm, B. H. A. Polyester synthases: natural catalysts for plastics. Biochem. J. 376, 15–33 (2003).
Tsuge, T., Hyakutake, M. & Mizuno, M. Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus. Appl. Microbiol. Biotechnol. 99, 6231–6240 (2015).
Taguchi, S. & Tsuge, T. Natural Polyester‐Related Proteins: Structure, Function, Evolution and Engineering, Protein Engineering Handbook Vol. 1 & 2 877–914 (Wiley-VCH, Weinheim, 2008).
Kawaguchi, Y. & Doi, Y. Kinetics and mechanism of synthesis and degradation of poly(3-hydroxybutyrate) in Alcaligenes eutrophus. Macromolecules 25, 2324–2329 (1992).
Yamanaka, K., Kimura, Y., Aoki, T. & Kudo, T. End-group analysis of bacterially produced poly(3-hydroxybutyrate): discovery of succinate as the polymerization starter. Macromolecules 42, 4038–4046 (2009).
Madden, L. A., Anderson, A. J., Shah, D. T. & Asrar, J. Chain termination in polyhydroxyalkanoate synthesis: Involvement of exogenous hydroxy-compounds as chain transfer agents. Int. J. Biol. Macromol. 25, 43–53 (1999).
Gerngross, T. U., Snell, K. D., Peoples, O. P., Sinskey, A. J., Csuhai, E., Masamune, S. & Stubbe, J. Overexpression and purification of the soluble polyhydroxyalkanoate synthase from Alcaligenes eutrophus: Evidence for a required posttranslational modification for catalytic activity. Biochemistry 33, 9311–9320 (1994).
Zhang, S., Yasuo, T., Lenz, R. W. & Goodwin, S. Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha. Biomacromolecules 1, 244–251 (2000).
Stubbe, J. & Tian, J. Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase. Nat. Prod. Rep. 20, 445–457 (2003).
Koizumi, F., Abe, H. & Doi, Y. Molecular weight of poly(3-hydroxybutyrate) during biological polymerization in Alcaligenes eutrophus. J. Macromol. Sci. A 32, 759–774 (1995).
Hiroe, A., Hyakutake, M., Thomson, N. M., Sivaniah, E. & Tsuge, T. Endogenous ethanol affects biopolyester molecular weight in recombinant Escherichia coli. ACS Chem. Biol. 8, 2568–2576 (2013).
Shi, F., Ashby, R. & Gross, R. A. Use of poly(ethylene glycol)s to regulate poly(3-hydroxybutyrate) molecular weight during Alcaligenes eutrophus cultivations. Macromolecules 29, 7753–7758 (1996).
Shi, F., Gross, R. A. & Rutherford, D. R. Microbial polyester synthesis: effects of poly(ethylene glycol) on product composition, repeat unit sequence, and end group structure. Macromolecules 29, 10–17 (1996).
Sanguanchaipaiwong, V., Gabelish, C. L., Hook, J., Scholz, C. & Foster, L. J. R. Biosynthesis of natural-synthetic hybrid copolymers polyhydroxyoctanoate-diethylene glycol. Biomacromolecules 5, 643–649 (2004).
Foster, L. J. R. Biosynthesis, properties and potential of natural synthetic hybrids of polyhydroxyalkanoates and polyethylene glycols. Appl. Microbiol. Biotechnol. 75, 1241–1247 (2007).
Kim, O. Biological effects of poly(ethylene glycol) on the microbial poly(β-hydroxyalkanoates) produced by Pseudomonas microorganisms. J. Polym. Res. 7, 91–96 (2000).
Tomizawa, S., Saito, Y., Hyakutake, M., Nakamura, Y., Abe, H. & Tsuge, T. Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent. Appl. Microbiol. Biotechnol. 87, 1427–1435 (2010).
Thomson, N. M., Hiroe, A., Tsuge, T., Summers, D. K. & Sivaniah, E. Efficient molecular weight control of bacterially synthesized polyesters by alcohol supplementation. J. Chem. Technol. Biotechnol. 89, 1110–1114 (2014).
Tsuge, T., Ko, T., Tago, M. & Abe, H. Effect of glycerol and its analogs on polyhydroxyalkanoate biosynthesis by recombinant Ralstonia eutropha: a quantitative structure-activity relationship study of chain transfer agents. Polym. Degrad. Stab. 98, 1586–1590 (2013).
Kusaka, S., Abe, H., Lee, S. Y. & Doi, Y. Molecular mass of poly[(R-3-hydroxybutyric acid] produced in a recombinant Escherichia coli. Appl. Microbiol. Biotechnol. 47, 140–143 (1997).
Choi, J. & Lee, S. Y. High level production of supra molecular weight poly((R-3hydroxybutyrate) by metabolically engineered Escherichia coli. Biotechnol. Bioprocess Eng. 9, 196–200 (2004).
Arikawa, H., Sato, S., Fujiki, T. & Matsumoto, K. A study on the relation between poly(3-hydroxybutyrate) depolymerases or oligomer hydrolases and molecular weight of polyhydroxyalkanoates accumulating in Cupriavidus necator H16. J. Biotechnol. 227, 94–102 (2016).
Sim, S. J., Snell, K. D., Hogan, S. A., Stubbe, J., Rha, C. & Sinskey, A. J. PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat. Biotechnol. 15, 63–67 (1997).
Sim, S. J., Sneel, K. D., Kim, B. W., Rha, K. C. & Sinskey, A. J. Increased poy-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli. Biotechnol. Lett. 23, 2057–2061 (2001).
Agus, J., Kahar, P., Abe, H., Doi, Y. & Tsuge, T. Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R-3-hydroxybutyrate] synthesis in recombinant Escherichia coli. Polym. Degrad. Stab. 91, 1645–1650 (2006).
Agus, J., Kahar, P., Abe, H., Doi, Y. & Tsuge, T. Molecular weight characterization of poly[(R-3-hydroxybutyrate] synthesized by genetically engineered strains of Escherichia coli. Polym. Degrad. Stab. 91, 1138–1146 (2006).
Hiroe, A., Ushimaru, K. & Tsuge, T. Characterization of polyhydroxyalkanoate (PHA) synthase derived from Delftia acidovorans DS-17 and the influence of PHA production in Escherichia coli. J. Biosci. Bioeng. 115, 633–638 (2013).
Thomson, N. M., Saika, A., Ushimaru, K., Sangiambut, S., Tsuge, T., Summers, D. K. & Sivaniah, E. Efficient production of active polyhydroxyalkanoate synthase in Escherichia coli by coexpression of molecular chaperones. Appl. Environ. Microbiol. 79, 1948–1955 (2013).
Bocanegra, J. K., da Cruz Pradella, J. G., Da Silva, L. F., Taciro, M. K. & Gomez, J. G. C. Influence of pH on the molecular weight of poly-3-hydroxybutyric acid (P3HB) produced by recombinant Escherichia coli. Appl. Biochem. Biotechnol. 170, 1336–1347 (2013).
Hiroe, A., Shiraishi, M., Mizuno, K. & Tsuge, T. Behavior of different polyhydroxyalkanoate synthases in response to the ethanol level in Escherichia coli cultures. Polym. J. 47, 767–770 (2015).
Ushimaru, K., Watanabe, Y., Hiroe, A. & Tsuge, T. A single-nucleotide substitution in phasin gene leads to enhanced accumulation of polyhydroxyalkanoate (PHA) in Escherichia coli harboring Aeromonas caviae PHA biosynthetic operon. J. Gen. Appl. Microbiol. 61, 63–66 (2015).
Tsuge, T., Takase, K., Taguchi, S. & Doi, Y. An extra large insertion in the polyhydroxyalkanoate synthase from Delftia acidovorans DS-17: its deletion effects and relation to cellular proteolysis. FEMS Microbiol. Lett. 231, 77–83 (2004).
Agus, J., Kahar, P., Hyakutake, M., Tomizawa, S., Abe, H., Tsuge, T., Satoh, Y. & Tajima, K. Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli. Polym. Degrad. Stab. 95, 2250–2254 (2010).
Tomizawa, S., Hyakutake, M., Saito, Y., Agus, J., Mizuno, K., Abe, H. & Tsuge, T. Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from Bacillus cereus YB-4 in recombinant Escherichia coli. Biomacromolecules 12, 2660–2666 (2011).
Hyakutake, M., Tomizawa, S., Mizuno, K., Abe, H. & Tsuge, T. Alcoholytic cleavage of polyhydroxyalkanoate chains by class IV synthases induced by endogenous and exogenous ethanol. Appl. Environ. Microbiol. 80, 1421–1429 (2014).
Hyakutake, M., Tomizawa, S., Mizuno, K., Hisano, T., Abe, H. & Tsuge, T. A common active site of polyhydroxyalkanoate synthase from Bacillus cereus YB-4 is involved in polymerization and alcoholysis reactions. Appl. Microbiol. Biotechnol. 99, 4701–4711 (2015).
Hyakutake, M., Tomizawa, S., Sugahara, I., Murata, E., Mizuno, K., Abe, H. & Tsuge, T. Carboxy-terminal modification of polyhydroxyalkanoate (PHA) via alcoholysis reaction catalyzed by class IV PHA synthase. Polym. Degrad. Stab. 117, 90–96 (2015).
Gerngross, T. U. & Martin, D. P. Enzyme-catalyzed synthesis of poly[(R-(−)-3-hydroxybutyrate]: formation of macroscopic granules in vitro. Proc. Natl Acad. Sci. USA 92, 6279–6283 (1995).
Jossek, R. & Steinbüchel, A. In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system. FEMS Microbiol. Lett. 168, 319–324 (1998).
Han, X., Satoh, Y., Tajima, K., Matsushima, T. & Munekata, M. Chemo-enzymatic synthesis of polyhydroxyalkanoate by an improved two-phase reaction system (TPRS). J. Biosci. Bioeng. 108, 517–523 (2009).
Tomizawa, S., Sato, S., Lan, J. C. W., Nakamura, Y., Abe, H. & Tsuge, T. In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate. Appl. Microbiol. Biotechnol. 97, 4821–4829 (2013).
Tomizawa, S., Yoshioka, M., Ushimaru, K. & Tsuge, T. Preparative synthesis of poly[(R-3-hydroxybutyrate]; monomer for enzymatic cell-free polymerization. Polym. J. 44, 982–985 (2012).
Saldivar-Guerra, E. & Vivaldo-Lima, E. Handbook of Polymer Synthesis, Characterization, and Processing. Chapter 7, (John Wiley and Sons, Hoboken, 2013).
Jossek, R., Reichelt, R. & Steinbüchel, A. In vitro biosynthesis of poly (3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum. Appl. Microbiol. Biotechnol. 49, 258–266 (1998).
Chen, C., Cao, R., Shrestha, R., Ward, C., B. Katz, B. B., Fischer, C. J., Tomich, J. M. & Li, P. Trapping of intermediates with substrate analog HBOCoA in the polymerizations catalyzed by Class III polyhydroxybutyrate (PHB) synthase from Allochromatium vinosum. ACS Chem. Biol. 10, 1330–1339 (2015).
Zhang, W., Shrestha, R., Buckley, R. M., Jewell, J., Bossmann, S. H., Stubbe, J. & Li, P. Mechanistic insight with HBCH2CoA as a probe to polyhydroxybutyrate (PHB) synthases. ACS Chem. Biol. 9, 1773–1779 (2014).
Tian, J., Sinskey, A. J. & Stubbe, J. Class III polyhydroxybutyrate synthase: involvement in chain termination and reinitiation. Biochemistry 44, 8369–8377 (2005).
Ilham, M., Nakanomori, S., Kihara, T., Hokamura, A., Matsusaki, H., Tsuge, T. & Mizuno, K. Characterization of polyhydroxyalkanoate synthases from Halomonas sp. O-1 and Halomonas elongata DSM2581: Site-directed mutagenesis and recombinant expression. Polym. Degrad. Stab. 109, 416–423 (2014).
Ushimaru, K., Sangiambut, S., Thomson, N., Sivaniah, E. & Tsuge, T. New insights into activation and substrate recognition of polyhydroxyalkanoate synthase from Ralstonia eutropha. Appl. Microbiol. Biotechnol. 97, 1175–1182 (2013).
Cho, M., Brigham, C. J., Sinskey, A. J. & Stubbe, J. Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo. Biochemistry 51, 2276–2288 (2012).
Ushimaru, K., Motoda, Y., Numata, K. & Tsuge, T. Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase. Appl. Environ. Microbiol. 80, 2867–2873 (2014).
Pfeiffer, D. & Jendrossek, D. PhaM is the physiological activator of PHB synthase (PhaC1) in Ralstonia eutropha. Appl. Environ. Microbiol. 80, 555–563 (2014).
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Tsuge, T. Fundamental factors determining the molecular weight of polyhydroxyalkanoate during biosynthesis. Polym J 48, 1051–1057 (2016). https://doi.org/10.1038/pj.2016.78
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DOI: https://doi.org/10.1038/pj.2016.78
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