Abstract
Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.
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References
Abe H, Doi Y (2002) Side-chain effect of second monomer units on crystalline morphology, thermal properties, and enzymatic degradability for random copolyesters of (R)-3-hydroxybutyric acid with (R)-3-hydroxyalkanoic acids. Biomacromolecules 3:133–138
Amara AA, Steinbüchel A, Rehm BH (2002) In vivo evolution of the Aeromonas punctata polyhydroxyalkanoate (PHA) synthase: isolation and characterization of modified PHA synthases with enhanced activity. Appl Microbiol Biotechnol 59:477–482
Arai Y, Shikanai T, Doi Y, Yoshida S, Yamaguchi I, Nakashita H (2004) Production of polyhydroxybutyrate by polycistronic expression of bacterial genes in tobacco plastid. Plant Cell Physiol 45:1176–1184
Bohmert K, Balbo I, Kopka J, Mittendorf VV, Nawrath C, Poirier Y, Tischendorf G, Trethewey RN, Willmitzer L (2000) Transgenic Arabidopsis plants can accumulate polyhydroxybutyrate to up to 4% of their fresh weight. Planta 211:841–845
Doi Y (1990) Microbial Polyesters. Wiley, New York
Doi Y, Kitamura S, Abe H (1995) Microbial synthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28:4822–4828
Houmiel KL, Slater S, Broyles D, Casagrande L, Colburn S, Gonzalez K, Mitsky TA, Reiser SE, Shah D, Taylor NB et al (1999) Poly(beta-hydroxybutyrate) production in oilseed leukoplasts of Brassica napus. Planta 209:547–550
Jia Y, Yuan W, Wodzinska J, Park C, Sinskey A, Stubbe J (2001) Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: class I and III synthases share a similar catalytic mechanism. Biochemistry 40:1011–1019
Kichise T, Taguchi S, Doi Y (2002) Enhanced accumulation and changed monomer composition in polyhydroxyalkanoate (PHA) copolyester by in vitro evolution of Aeromonas caviae PHA synthase. Appl Environ Microbiol 68:2411–2419
Klinke S, Ren Q, Witholt B, Kessler B (1999) Production of medium-chain-length poly(3-hydroxyalkanaotes) from gluconate by recombinant Escherichia coli. Appl Environ Microbiol 65:540–548
Lee SY (1996) Bacterial polyhydroxyalkanoates. Biotechnol Bioeng 49:1–14
Madison LL, Huisman GW (1999) Metabolic engineering of poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53
Matsumoto K, Nagao R, Murata T, Arai Y, Kichise T, Nakashita H, Taguchi S, Shimada H, Doi Y (2005a) Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae. Biomacromolecules 6:2126–2130
Matsumoto K, Takase K, Aoki E, Doi Y, Taguchi S (2005b) Synergistic effects of Glu130Asp substitution in the type II polyhydroxyalkanoate (PHA) synthase: enhancement of PHA production and alteration of polymer molecular weight. Biomacromolecules 6:99–104
Matsumoto K, Aoki E, Takase K, Doi Y, Taguchi S (2006a) In vivo and in vitro characterization of Ser477X mutations in polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. 61-3: effects of beneficial mutations on enzymatic activity, substrate specificity, and molecular weight of PHA. Biomacromolecules 7(8):2436–2442
Matsumoto K, Arai Y, Nagao R, Murata T, Kakase K, Nakashita H, Taguchi S, Shimada H, Doi Y (2006b) Synthesis of short-chain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymers in peroxisome of the transgenic Arabidopsis thaliana harboring the PHA synthase gene from Pseudomonas sp. 61-3. J Polym Environ (in press)
Matsusaki H, Abe H, Doi Y (2000) Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant strains of Pseudomonas sp. 61-3. Biomacromolecules 1:17–22
Mittendorf VV, Robertson EJ, Leech RM, Kruger N, Steinbüchel A, Poirier Y (1998) Synthesis of medium-chain-length polyhydroxyalkanoates in Arabidopsis thaliana using intermediates of peroxisomal fatty acid beta-oxidation. Proc Natl Acad Sci U S A 95:13397–13402
Mittendorf VV, Bongcam V, Allenbach L, Coullerez G, Martini N, Poirier Y (1999) Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation. Plant J 20:45–55
Moire L, Rezzonico E, Goepfert S, Poirier Y (2004) Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants. Plant Physiol 134:432–442
Nakashita H, Arai Y, Shikanai T, Doi Y, Yamaguchi I (2001) Introduction of bacterial metabolism into higher plants by polycistronic transgene expression. Biosci Biotechnol Biochem 65:1688–1691
Nawrath C, Poirier Y, Somerville C (1994) Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation. Proc Natl Acad Sci U S A 91:12760–12764
Niamsiri N, Delamarre S C, Kim YR, Batt, CA (2004) Engineering of chimeric class II polyhydroxyalkanoate synthases. Appl Environ Microbiol 70:6789–6799
Nomura CT, Doi Y (2006) Metabolic engineering of recombinant Escherichia coli for short-chain-length-medium-chain-length polyhydroxyalkanoate biosynthesis. In: Khemani K, Scholz C (eds) Degradable polymers and materials-principles and practice. American Chemical Society, Washington, DC
Nomura CT, Taguchi K, Taguchi S, Doi Y (2004a) 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. Appl Environ Microbiol 70:999–1007
Nomura CT, Tanaka T, Gan Z, Kuwabara K, Abe H, Takase K, Taguchi K, Doi Y (2004b) Effective enhancement of short-chain-length (SCL)-medium-chain-length (MCL) polyhydroxyalkanoate copolymer production by co-expression of genetically engineered 3-ketoacyl-acyl-carrier protein synthase III (fabH) and polyhydroxyalkanoate synthesis genes. Biomacromolecules 5:1457–1464
Nomura CT, Taguchi K, Gan Z, Kuwabara K, Tanaka T, Doi Y (2005) Expression of 3-ketoacyl-ACP reductase (fabG) enhances polyhydroxyalkanoate copolymer production from glucose in recombinant Escherichia coli JM109. Appl Environ Microbiol 71:4297–4306
Normi YM, Hiraishi T, Taguchi S, Abe H, Sudesh K, Najimudin, N, Doi Y (2005a) Characterization and properties of G4X mutants of Ralstonia eutropha PHA synthase for poly(3-hydroxybutyrate) biosynthesis in Escherichia coli. Macromol Biosci 5:197–206
Normi YM, Hiraishi T, Taguchi S, Sudesh K, Najimudin N, Doi Y (2005b) Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase. Biotechnol Lett 27:705–712
Poirier Y (1999) Production of new polymeric compounds in plants. Curr Opin Biotechnol 10:181–185
Poirier Y (2001) Production of polyesters in transgenic plants. Adv Biochem Eng Biotechnol 71:209–240
Poirier Y, Somerville C, Schechtman LA, Satkowski MM, Noda I (1995) Synthesis of high-molecular-weight poly([R]-(−)-3-hydroxybutyrate) in transgenic Arabidopsis thaliana plant cells. Int J Biol Macromol 17:7–12
Rehm BH (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33
Rehm BH, Mitsky TA, Steinbüchel (2001) Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli. Appl Environ Microbiol 67:3102–3109
Rehm BH, Antonio RV, Spiekermann P, Amara AA, Steinbüchel A (2002) Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model. Biochim Biophys Acta 1594:178–190
Sheu DS, Lee CY (2004) Altering the substrate specificity of polyhydroxyalkanoate synthase 1 derived from Pseudomonas putida GPo1 by localized semirandom mutagenesis. J Bacteriol 186:4177–4184
Slater S, Mitsky TA, Houmiel KL, Hao M, Reiser SE, Taylor NB, Tran M, Valentin HE, Rodriguez DJ, Stone DA et al (1999) Metabolic engineering of Arabidopsis and Brassica for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer production. Nat Biotechnol 17:1011–1016
Solaiman DK (2003) Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases. J Ind Microbiol Biotechnol 30:322–326
Sudesh K, Abe H, Doi Y (2000) Synthesis, structure and properties of polyhydroxyalkanoates: biological polyesters. Prog Polym Sci 25:1503–1555
Taguchi S, Doi Y (2004) Evolution of polyhydroxyalkanoate (PHA) production system by “enzyme evolution”: successful case studies of directed evolution. Macromol Biosci 4:146–156
Taguchi S, Ozaki A, Momose H (1998) Engineering of a cold-adapted protease by sequential random mutagenesis and a screening system. Appl Environ Microbiol 126:689–693
Taguchi S, Komada S, Momose H (2000) The complete amino acid substitutions at position 131 that are positively involved in cold adaptation of subtilisin BPN′. Appl Environ Microbiol 66:1410–1415
Taguchi S, Maehara A, Takase K, Nakahara M, Nakumura H, Doi Y (2001) Analysis of mutational effects of a polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system. FEMS Microbiol Lett 198:65–71
Taguchi S, Nakamura H, Hiraishi T, Yamato I, Doi Y (2002) In vitro evolution of a polyhydroxybutyrate synthase by intragenic suppression-type mutagenesis. J Biochem (Tokyo) 131:801–806
Takase K, Taguchi S, Doi Y (2003) Enhanced synthesis of poly(3-hydroxybutyrate) in recombinant Escherichia coli by means of error-prone PCR mutagenesis, saturation mutagenesis and in vitro recombination of the type II polyhydroxyalkanoate synthase gene. J Biochem 133:139–145
Takase K, Matsumoto K, Taguchi S, Doi Y (2004) Alteration of substrate chain-length specificity of type II synthase for polyhydroxyalkanoate biosynthesis by in vitro evolution: in vivo and in vitro enzyme assays. Biomacromolecules 5:480–485
Tsuge T, Saito Y, Kikkawa Y, Hiraishi T, Doi Y (2004a) Biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant Ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes. Macromol Biosci 4:238–242
Tsuge T, Saito Y, Narike M, Muneta K, Normi YM, Kikkawa Y, Hiraishi T, Doi Y (2004b) Mutation effects of a conserved alanine (Ala510) in type I polyhydroxyalkanoate synthase from Ralstonia eutropha on polyester biosynthesis. Macromol Biosci 4:963–970
Tsuge T, Yano K, Imazu S, Numata K, Kikkawa Y, Abe H, Taguchi S, Doi Y (2005) Biosynthesis of polyhydroxyalkanoate (PHA) copolymer from fructose using wild-type and laboratory-evolved PHA synthases. Macromol Biosci 4:963–970
Acknowledgements
The authors thank Dr. Ken’ichiro Matsumoto (Tokyo University of Science) for valuable discussions and input regarding this review. Also, we are deeply indebted to a great contribution of Dr. Kazuma Takase to the works introduced in the review. Our works described here were partly supported by funding from the following sources: Grant-in-Aid for Scientific Research of Japan (no. 70216828) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to S. Taguchi), Solution Oriented Research for Science, Technology (SORST) of the Japan Science and Technology Corporation (JST), Hokkaido Foundation for the Promotion of Scientific and Industrial Technology, and Industrial Technology Research Grant Program in 2003 from the New Energy and Industrial Technology Development Organization (NEDO).
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Nomura, C.T., Taguchi, S. PHA synthase engineering toward superbiocatalysts for custom-made biopolymers. Appl Microbiol Biotechnol 73, 969–979 (2007). https://doi.org/10.1007/s00253-006-0566-4
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DOI: https://doi.org/10.1007/s00253-006-0566-4