Applied Microbiology and Biotechnology

, Volume 97, Issue 18, pp 8011–8021 | Cite as

Biosynthetic polyesters consisting of 2-hydroxyalkanoic acids: current challenges and unresolved questions



2-Hydroxyalkanoates (2HAs) have become the new monomeric constituents of bacterial polyhydroxyalkanoates (PHAs). PHAs containing 2HA monomers, lactate (LA), glycolate (GL), and 2-hydroxybutyrate (2HB) can be synthesized by engineered microbes in which the broad substrate specificities of PHA synthase and propionyl-CoA transferase are critical factors for the incorporation of the monomers into the polymer chain. LA-based polymers, such as P[LA-co-3-hydroxybutyrate (3HB)], have the properties of pliability and stretchiness which are distinctly different from those of the rigid poly(lactic acid) (PLA) and P(3HB) homopolymers. This versatile platform is also applicable to the biosynthesis of GL- and 2HB-based polymers. In the case of the synthesis of 2HB-based polymers, the enantiospecificity of PHA synthase enabled the production of isotactic (R)-2HB-based polymers, including P[(R)-2HB], from racemic precursors of 2HB. P(2HB) is a pliable material, in contrast to PLA. Furthermore, to obtain a new 2HA-polymerizing PHA synthase, the class I PHA synthase from Ralstonia eutropha was engineered so as to achieve the first incorporation of LA units. The analysis of the polymer synthesized using this new LA-polymerizing PHA synthase unexpectedly focused a spotlight on the studies on block copolymer biosynthesis.


Poly(lactic acid) Polyhydroxybutyrate Biodegradable plastic Bio-based material Enzyme evolution Xylose 


  1. Auras R, Harte B, Selke S (2004) An overview of polylactides as packaging materials. Macromol Biosci 4:835–864PubMedCrossRefGoogle Scholar
  2. de Kok A, Hengeveld AF, Martin A, Westphal AH (1998) The pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria. Biochim Biophys Acta 1385:353–366PubMedCrossRefGoogle Scholar
  3. Girio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S, Bogel-Lukasik R (2010) Hemicelluloses for fuel ethanol: a review. Bioresour Technol 101:4775–4800PubMedCrossRefGoogle Scholar
  4. Han X, Satoh Y, Satoh T, Matsumoto K, Kakuchi T, Taguchi S, Dairi T, Munekata M, Tajima K (2011) Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha. Appl Microbiol Biotechnol 92:509–517PubMedCrossRefGoogle Scholar
  5. Huang W, Shi X, Ren L, Du C, Wang Y (2010) PHBV microspheres—PLGA matrix composite scaffold for bone tissue engineering. Biomaterials 31:4278–4285PubMedCrossRefGoogle Scholar
  6. Jung YK, Kim TY, Park SJ, Lee SY (2010) Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers. Biotechnol Bioeng 105:161–171PubMedCrossRefGoogle Scholar
  7. Lee SY (1998) Poly(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli. Bioprocess Eng 18:397–399CrossRefGoogle Scholar
  8. Lee SY, Lee Y (2003) Metabolic engineering of Escherichia coli for production of enantiomerically pure (R)-(−)-hydroxycarboxylic acids. Appl Environ Microbiol 69:3421–3426PubMedCrossRefGoogle Scholar
  9. Lindenkamp N, Schurmann M, Steinbüchel A (2013) A propionate CoA-transferase of Ralstonia eutropha H16 with broad substrate specificity catalyzing the CoA thioester formation of various carboxylic acids. Appl Microbiol Biotechnol. doi:10.1007/s00253-012-4624-9 PubMedGoogle Scholar
  10. Lu JN, Tappel RC, Nomura CT (2009) Mini-review: biosynthesis of poly(hydroxyalkanoates). Polym Rev 49:226–248CrossRefGoogle Scholar
  11. Martin CH, Dhamankar H, Tseng HC, Sheppard MJ, Reisch CR, Prather KL (2013) A platform pathway for production of 3-hydroxyacids provides a biosynthetic route to 3-hydroxy-γ-butyrolactone. Nat Commun 4:1414PubMedCrossRefGoogle Scholar
  12. Matsumoto K, Taguchi S (2010) Enzymatic and whole-cell synthesis of lactate-containing polyesters: toward the complete biological production of polylactate. Appl Microbiol Biotechnol 85:921–932PubMedCrossRefGoogle Scholar
  13. Matsumoto K, Taguchi S (2013) Enzyme and metabolic engineering for the production of novel biopolymers: crossover of biological and chemical processes. Curr Opin Biotechnol. doi:10.1016/j.copbio.2013.02.021 PubMedGoogle Scholar
  14. Matsumoto K, Aoki E, Takase K, Doi Y, Taguchi S (2006) 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 7:2436–2442PubMedCrossRefGoogle Scholar
  15. Matsumoto K, Shozui F, Satoh Y, Tajima K, Munekata M, Taguchi S (2009) Kinetic analysis of engineered polyhydroxyalkanoate synthases with broad substrate specificity. Polym J 41:237–240CrossRefGoogle Scholar
  16. Matsumoto K, Ishiyama A, Sakai K, Shiba T, Taguchi S (2011) Biosynthesis of glycolate-based polyesters containing medium-chain-length 3-hydroxyalkanoates in recombinant Escherichia coli expressing engineered polyhydroxyalkanoate synthase. J Biotechnol 156:214–217PubMedCrossRefGoogle Scholar
  17. Matsumoto K, Okei T, Honma I, Ooi T, Aoki H, Taguchi S (2012) Efficient (R)-3-hydroxybutyrate production using acetyl CoA-regenerating pathway catalyzed by coenzyme A transferase. Appl Microbiol Biotechnol 97:205–210PubMedCrossRefGoogle Scholar
  18. Matsumoto K, Terai S, Ishiyama A, Sun J, Kabe T, Song Y, Nduko JM, Iwata T, Taguchi S (2013) One-pot microbial production, mechanical properties and enzymatic degradation of isotactic P[(R)-2-hydroxybutyrate] and its copolymer with (R)-lactate. Biomacromolecules 14:1913–1918Google Scholar
  19. Mutsuga M, Kawamura Y, Tanamoto K (2007) Studies on polylactide properties. Jpn J Food Chem 14:87–92Google Scholar
  20. Nduko JM, Matsumoto K, Ooi T, Taguchi S (2013) Effectiveness of xylose utilization for high yield production of lactate-enriched P(lactate-co-3-hydroxybutyrate) using a lactate-overproducing strain of Escherichia coli and an evolved lactate-polymerizing enzyme. Metab Eng 15:159–166PubMedCrossRefGoogle Scholar
  21. Normi YM, Hiraishi T, Taguchi S, Sudesh K, Najimudin N, Doi Y (2005) Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase. Biotechnol Lett 27:705–712PubMedCrossRefGoogle Scholar
  22. Ochi A, Matsumoto K, Ooba T, Sakai K, Tsuge T, Taguchi S (2013) Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature. Appl Microbiol Biotechnol 97:3441–3447PubMedCrossRefGoogle Scholar
  23. Park SJ, Lee SY, Kim TW, Jung YK, Yang TH (2012a) Biosynthesis of lactate-containing polyesters by metabolically engineered bacteria. Biotechnol J 7:199–212PubMedCrossRefGoogle Scholar
  24. Park SJ, Lee TW, Lim SC, Kim TW, Lee H, Kim MK, Lee SH, Song BK, Lee SY (2012b) Biosynthesis of polyhydroxyalkanoates containing 2-hydroxybutyrate from unrelated carbon source by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol 93:273–283PubMedCrossRefGoogle Scholar
  25. Park SJ, Kang KH, Lee H, Park AR, Yang JE, Oh YH, Song BK, Jegal J, Lee SH, Lee SY (2013) Propionyl-CoA dependent biosynthesis of 2-hydroxybutyrate containing polyhydroxyalkanoates in metabolically engineered Escherichia coli. J Biotechnol 165:93–98PubMedCrossRefGoogle Scholar
  26. Pederson EN, McChalicher CW, Srienc F (2006) Bacterial synthesis of PHA block copolymers. Biomacromolecules 7:1904–1911PubMedCrossRefGoogle Scholar
  27. Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33PubMedCrossRefGoogle Scholar
  28. Sato S, Fujiki T, Matsumoto K (2013) Construction of a stable plasmid vector for industrial production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by a recombinant Cupriavidus necator H16 strain. J Biosci Bioeng. doi:10.1016/j.jbiosc.2013.05.026 Google Scholar
  29. Schacher FH, Rupar PA, Manners I (2012) Functional block copolymers: nanostructured materials with emerging applications. Angew Chem Int Ed 51:7898–7921CrossRefGoogle Scholar
  30. Selmer T, Willanzheimer A, Hetzel M (2002) Propionate CoA-transferase from Clostridium propionicum—cloning of the gene and identification of glutamate 324 at the active site. Eur J Biochem 269:372–380PubMedCrossRefGoogle Scholar
  31. Shiraki M, Endo T, Saito T (2006) Fermentative production of (R)-(−)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli. J Biosci Bioeng 102:529–534PubMedCrossRefGoogle Scholar
  32. Shozui F, Matsumoto K, Nakai T, Yamada M, Taguchi S (2010) Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate. Appl Microbiol Biotechnol 85:949–954PubMedCrossRefGoogle Scholar
  33. Shozui F, Matsumoto K, Motohashi R, Sun JA, Satoh T, Kakuchi T, Taguchi S (2011) Biosynthesis of a lactate (LA)-based polyester with a 96 mol% LA fraction and its application to stereocomplex formation. Polym Degrad Stab 96:499–504CrossRefGoogle Scholar
  34. Song YY, Matsumoto K, Yamada M, Gohda A, Brigham CJ, Sinskey AJ, Taguchi S (2012) Engineered Corynebacterium glutamicum as an endotoxin-free platform strain for lactate-based polyester production. Appl Microbiol Biotechnol 93:1917–1925PubMedCrossRefGoogle Scholar
  35. Taguchi S (2010) Current advances in microbial cell factories for lactate-based polyesters driven by lactate-polymerizing enzymes: towards the further creation of new LA-based polyesters. Polym Degrad Stab 95:1421–1428CrossRefGoogle Scholar
  36. Taguchi S, Doi Y (2004) Evolution of polyhydroxyalkanoate (PHA) production system by “enzyme evolution”: successful case studies of directed evolution. Macromol Biosci 4:145–156CrossRefGoogle Scholar
  37. 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 131:801–806PubMedCrossRefGoogle Scholar
  38. Taguchi S, Yamada M, Matsumoto K, Tajima K, Satoh Y, Munekata M, Ohno K, Kohda K, Shimamura T, Kambe H, Obata S (2008) A microbial factory for lactate-based polyesters using a lactate-polymerizing enzyme. Proc Natl Acad Sci U S A 105:17323–17327PubMedCrossRefGoogle Scholar
  39. Tajima K, Satoh Y, Satoh T, Itoh R, Han XR, Taguchi S, Kakuchi T, Munekata M (2009) Chemo-enzymatic synthesis of poly(lactate-co-(3-hydroxybutyrate)) by a lactate-polymerizing enzyme. Macromolecules 42:1985–1989CrossRefGoogle Scholar
  40. 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 5:480–485PubMedCrossRefGoogle Scholar
  41. Tripathi L, Wu LP, Chen J, Chen GQ (2012) Synthesis of diblock copolymer poly-3-hydroxybutyrate-block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a beta-oxidation weakened Pseudomonas putida KT2442. Microb Cell Factories 11:44CrossRefGoogle Scholar
  42. Tseng HC, Martin CH, Nielsen DR, Prather KL (2009) Metabolic engineering of Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate. Appl Environ Microbiol 75:3137–3145PubMedCrossRefGoogle Scholar
  43. Tsuji H, Yamamoto S, Okumura A, Sugiura Y (2010) Heterostereocomplexation between biodegradable and optically active polyesters as a versatile preparation method for biodegradable materials. Biomacromolecules 11:252–258PubMedCrossRefGoogle Scholar
  44. Tsuji H, Hosokawa M, Sakamoto Y (2012) Ternary stereocomplex formation of one L-configured and two D-configured optically active polyesters, poly(L-2-hydroxybutanoic acid), poly(D-2-hydroxybutanoic acid), and poly(D-lactic acid). Acs Macro Lett 1:687–691CrossRefGoogle Scholar
  45. Tung KK, Wood WA (1975) Purification, new assay, and properties of coenzyme A transferase from Peptostreptococcus elsdenii. J Bacteriol 124:1462–1474PubMedGoogle Scholar
  46. Uchino K, Saito T, Jendrossek D (2008) Poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 is involved in mobilization of accumulated PHB in Ralstonia eutropha H16. Appl Environ Microbiol 74:1058–1063PubMedCrossRefGoogle Scholar
  47. Valentin HE, Steinbüchel A (1994) Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases. Appl Microbiol Biotechnol 40:699–709CrossRefGoogle Scholar
  48. Vink ET, Rabago KR, Glassner DA, Springs B, O'Connor RP, Kolstad J, Gruber PR (2004) The sustainability of NatureWorks polylactide polymers and Ingeo polylactide fibers: an update of the future. Macromol Biosci 4:551–564PubMedCrossRefGoogle Scholar
  49. Yamada M, Matsumoto K, Nakai T, Taguchi S (2009) Microbial production of lactate-enriched poly[(R)-lactate-co-(R)-3-hydroxybutyrate] with novel thermal properties. Biomacromolecules 10:677–681PubMedCrossRefGoogle Scholar
  50. Yamada M, Matsumoto K, Shimizu K, Uramoto S, Nakai T, Shozui F, Taguchi S (2010) Adjustable mutations in lactate (LA)-polymerizing enzyme for the microbial production of LA-based polyesters with tailor-made monomer composition. Biomacromolecules 11:815–819PubMedCrossRefGoogle Scholar
  51. Yamada M, Matsumoto K, Uramoto S, Motohashi R, Abe H, Taguchi S (2011) Lactate fraction dependent mechanical properties of semitransparent poly(lactate-co-3-hydroxybutyrate)s produced by control of lactyl-CoA monomer fluxes in recombinant Escherichia coli. J Biotechnol 154:255–260PubMedCrossRefGoogle Scholar
  52. Yamane T, Chen XF, Ueda S (1996) Growth-associated production of poly(3-hydroxyvalerate) from n-pentanol by a methylotrophic bacterium, Paracoccus denitrificans. Appl Environ Microbiol 62:380–384PubMedGoogle Scholar
  53. Yin M, Baker GL (1999) Preparation and characterization of substituted polylactides. Macromolecules 32:7711–7718CrossRefGoogle Scholar
  54. Yuan W, Jia Y, Tian JM, Snell KD, Muh U, Sinskey AJ, Lambalot RH, Walsh CT, Stubbe J (2001) Class I and III polyhydroxyalkanoate synthases from Ralstonia eutropha and Allochromatium vinosum: characterization and substrate specificity studies. Arch Biochem Biophys 394:87–98PubMedCrossRefGoogle Scholar
  55. Zhang S, Yasuo T, Lenz RW, Goodwin S (2000) Kientic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha. Biomacromolecules 1:244–251PubMedCrossRefGoogle Scholar
  56. Zhang S, Kamachi M, Takagi Y, Lenz RW, Goodwin S (2001) Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases. Appl Microbiol Biotechnol 56:131–136PubMedCrossRefGoogle Scholar
  57. Zhu J, Shimizu K (2004) The effect of pfl gene knockout on the metabolism for optically pure D-lactate production by Escherichia coli. Appl Microbiol Biotechnol 64:367–375PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Division of Biotechnology and Macromolecular Chemistry, Graduate School of EngineeringHokkaido UniversitySapporoJapan

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