International Microbiology

, Volume 21, Issue 1–2, pp 47–57 | Cite as

Burkholderia xenovorans LB400 possesses a functional polyhydroxyalkanoate anabolic pathway encoded by the pha genes and synthesizes poly(3-hydroxybutyrate) under nitrogen-limiting conditions

  • Viviana Urtuvia
  • Pamela Villegas
  • Sebastián Fuentes
  • Myriam González
  • Michael Seeger
Original Article


Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics that are synthesized by diverse bacteria. In this study, the synthesis of PHAs by the model aromatic-degrading strain Burkholderia xenovorans LB400 was analyzed. Twelve pha genes including three copies of phaC and five copies of the phasin-coding phaP genes are distributed among the three LB400 replicons. The phaC1ABR gene cluster that encodes the enzymes of the PHA anabolic pathway is located at chromosome 1 of strain LB400. During the growth of strain LB400 on glucose under nitrogen limitation, the expression of the phaC1, phaA, phaP1, phaR, and phaZ genes was induced. Under nitrogen limitation, PHA accumulation in LB400 cells was observed by fluorescence microscopy after Nile Red staining. GC-MS analyses revealed that the PHA accumulated under nitrogen limitation was poly(3-hydroxybutyrate) (PHB). LB400 cells grown on glucose as the sole carbon source under nitrogen limitation accumulated 40 ± 0.96% PHB of the cell dry weight, whereas no PHA was observed in cells grown in control medium. The functionality of the phaC1 gene from strain LB400 was further studied using heterologous expression in a Pseudomonas putida KT40C1ZC2 mutant strain derived from P. putida KT2440 that is unable to synthesize PHAs. Interestingly, KT40C1ZC2[pVNC1] cells that express the phaC1 gene from strain LB400 were able to synthesize PHB (33.5% dry weight). This study indicates that B. xenovorans LB400 possesses a functional PHA synthetic pathway that is encoded by the pha genes and is capable of synthesizing PHB.


Burkholderia xenovorans pha gene PHA Bioplastic Poly(3-hydroxybutyrate) 



The authors thank Maria Auxiliadora Prieto for the generous support in heterologous expression assays and Vincent Collins for critical reading of the manuscript.

Funding information

The present work was supported by PhD scholarship CONICYT 21100268 (VU), Programa de Investigación Asociativa (PIA) Anillo ACT172128 GAMBIO (MS), FONDECYT 1151174 and 1110992 (MS), USM 131342 and 131562 (MS), USM Pie>A (PV), USM CN&SB (MS), and CYTED-PRIBOP grants (MS).

Supplementary material

10123_2018_4_MOESM1_ESM.docx (15 kb)
ESM 1 (DOCX 14 kb)


  1. Acevedo F, Villegas P, Urtuvia V, Hermosilla J, Navia R, Seeger M (2018) Bacterial polyhydroxybutyrate for electrospun fiber production. Int J Biol Macromol 106:692–697CrossRefPubMedGoogle Scholar
  2. Agulló L, Romero-Silva MJ, Domenech M, Seeger M (2017) p-Cymene promotes its catabolism through the p-cymene and the p-cumate pathways, activates a stress response and reduces the biofilm formation in Burkholderia xenovorans LB400. PLoS ONE 12:e0169544CrossRefPubMedPubMedCentralGoogle Scholar
  3. Aldor LS, Keasling JD (2003) Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates. Curr Opin Biotechnol 14:475−483CrossRefGoogle Scholar
  4. Anderson A, Dawes EA (1990) Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472PubMedPubMedCentralGoogle Scholar
  5. Ayub ND, Pettinari MJ, Méndez BS, López NI (2006) Impaired polyhydroxybutyrate biosynthesis from glucose in Pseudomonas sp. 14−3 is due to a defective β−ketothiolase gene. FEMS Microbiol Lett 264:125−131CrossRefGoogle Scholar
  6. Cai L, Yuan MQ, Liu F, Jian J, Chen GQ (2009) Enhanced production of medium-chain-length polyhydroxyalkanoates (PHA) by PHA depolymerase knockout mutant of Pseudomonas putida KT2442. Bioresour Technol 100:2265–2270CrossRefPubMedGoogle Scholar
  7. Chain PSG, Denef VJ, Konstantinidis KT, Vergez LM, Agulló L, Reyes VL, Hauser L, Córdova M, Gómez L, González M, Land M, Lao V, Larimer F, LiPuma JJ, Mahenthiralingam E, Malfatti SA, Marx CJ, Parnell JJ, Ramette A, Richardson P, Seeger M, Smith D, Spilker T, Sul WJ, Tsoi TV, Ulrich LE, Zhulin IB, Tiedje JM (2006) Burkholderia xenovorans LB400 harbors a multi−replicon 9.73−Mbp genome shaped for versatility. Proc Natl Acad Sci 103:15280–15287CrossRefPubMedGoogle Scholar
  8. Chirino B, Strahsburger E, Agulló L, González M, Seeger M (2013) Genomic and functional analyses of the 2−aminophenol catabolic pathway and partial conversion of its substrate into picolinic acid in Burkholderia xenovorans LB400. PLoS ONE 8:e75746CrossRefPubMedPubMedCentralGoogle Scholar
  9. De Eugenio LI, Escapa IF, Morales V, Dinjaski N, Galán B, García JL, Prieto MA (2010) The turnover of medium-chain-length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance. Environ Microbiol 12:207–221CrossRefPubMedGoogle Scholar
  10. Díaz-Barrera A, Andler R, Martínes I, Peña C (2016) Poly-3-hydroxybutyrate production by Azotobacter vinelandii strains in batch cultures at different oxygen transfer rates. J Chem Technol Biotechnol 91:1063–1071CrossRefGoogle Scholar
  11. Eggers J, Steinbüchel A (2013) Poly(3-hydroxybutyrate) degradation in Ralstonia eutropha H16 is mediated stereoselectively to (S)-3-hydroxybutyryl coenzyme A (CoA) via crotonyl-CoA. J Bacteriol 195:3213–3223CrossRefPubMedPubMedCentralGoogle Scholar
  12. Encarnación S, Vargas M, Dunn MF, Dávalos A, Mendoza G, Mora Y, Mora J (2002) AniA regulates reserve polymer accumulation and global protein expression in Rhizobium etli. J Bacteriol 184:2287–2295CrossRefPubMedPubMedCentralGoogle Scholar
  13. Hyakutake M, Saito Y, Tomizawa S, Mizuno K, Tsuge T (2011) Polyhydroxyalkanoate (PHA) synthesis by class IV PHA synthases employing Ralstonia eutropha PHB-4 as host strain. Biosci Biotechnol Biochem 75:1615–1617CrossRefPubMedGoogle Scholar
  14. Livak K, Schmittgen T (2001) Analysis of relative gene expression data using real−time quantitative PCR and 2-ΔΔCt method. Methods 25:402−408CrossRefGoogle Scholar
  15. Lopes M, Gosset G, Rocha R, Gomez J, Ferreira da Silva L (2011) PHB biosynthesis in catabolite repression mutant of Burkholderia sacchari. Curr Microbiol 63:319–326CrossRefPubMedGoogle Scholar
  16. Madison LL, Huisman GW (1999) Metabolic engineering of poly (3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev 63:21–53PubMedPubMedCentralGoogle Scholar
  17. Maehara A, Taguchi S, Nishiyama T, Yamane T, Doi Y (2002) A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA. J Bacteriol 184:3992–4002CrossRefPubMedPubMedCentralGoogle Scholar
  18. Méndez V, Agulló L, González M, Seeger M (2011) The homogentisate and homoprotocatechuate central pathways are involved in 3− and 4−hydroxyphenylacetate degradation by Burkholderia xenovorans LB400. PLoS ONE 6:e17583CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mezzina MP, Pettinari MJ (2016) Phasins, multifaceted polyhydroxyalkanoate granule-associated proteins. Appl Environ Microbiol 82:5060–5067CrossRefPubMedPubMedCentralGoogle Scholar
  20. Mozejko J, Ciesielski S (2013) Saponified waste palm oil as an attractive renewable resource for mcl-polyhydroxyalkanoate synthesis. J Biosci Bioeng 116:485−492CrossRefGoogle Scholar
  21. Nikodinovic-Runic J, Guzik M, Kenny ST, Babu R, Werker A, O’Connor KE (2013) Carbon-rich wastes as feedstocks for biodegradable polymer (polyhydroxyalkanoate) production using bacteria. Adv Appl Microbiol 84:139–200CrossRefPubMedGoogle Scholar
  22. Overwin H, Standfuß-Gabisch C, González M, Méndez V, Seeger M, Reichelt J, Wray V, Hofer B (2015) Permissivity of the biphenyl-specific aerobic bacterial metabolic pathway towards analogues with various steric requirements. Microbiology 161(9):1844–1856Google Scholar
  23. Peoples OP, Sinskey AJ (1989) Poly−β−hydroxybutirate (PHB) biosynthesis in Alcaligenes eutrophus H16. J Biol Chem 264:15298–15303PubMedGoogle Scholar
  24. Pfeiffer D, Jendrossek D (2012) Localization of poly (3-hydroxybutyrate)(PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha H16. J Bacteriol 194:5909–5921CrossRefPubMedPubMedCentralGoogle Scholar
  25. Poltronieri P, Kumar P (2017) Polyhydroxyalkanoates (PHAs) in Industrial Applications. In: Torres Martínez L., Kharissova O., Kharisov B. (Eds) Handbook of Ecomaterials. Springer, Cham, pp 1–30.
  26. Pötter M, Steinbüchel A (2005) Poly (3-hydroxybutyrate) granule-associated proteins: impacts on poly (3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6:552–560CrossRefPubMedGoogle Scholar
  27. Pötter M, Madkour MH, Mayer F, Steinbüchel A (2002) Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology 148:2413− 2426CrossRefGoogle Scholar
  28. Pötter M, Müller H, Reinecke F, Wieczorek R, Fricke F, Bowien B, Friedrich B, Steinbüchel A (2004) The complex structure of polyhydroxybutyrate (PHB) granules: four orthologous and paralogous phasins occur in Ralstonia eutropha. Microbiology 150:2301–2311CrossRefPubMedGoogle Scholar
  29. Pötter M, Müller H, Steinbüchel A (2005) Influence of homologous phasins (PhaP) on PHA accumulation and regulation of their expression by the transcriptional repressor PhaR in Ralstonia eutropha H16. Microbiology 151:825–833CrossRefPubMedGoogle Scholar
  30. Prieto MA, Bühler B, Jung K, Witholt B, Kessler B (1999) PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes. J Bacteriol 181:858–868PubMedPubMedCentralGoogle Scholar
  31. Ramsay JA, Hassan MCA, Ramsay BA (1995) Hemicellulose as a potential substrate for production of poly (β-hydroxyalkanoates). Can J Microbiol 41:262–266CrossRefGoogle Scholar
  32. Reddy CSK, Ghai R, Kalia V (2003) Polyhydroxyalkanoates: an overview. Bioresour Technol 87:137–146CrossRefPubMedGoogle Scholar
  33. Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376:15–33CrossRefPubMedPubMedCentralGoogle Scholar
  34. Rehm BHA (2007) Biogenesis of microbial polyhydroxyalkanoate granules: a platform technology for the production of tailor−made bioparticles. Curr Issues Mol Biol 9:41–62PubMedGoogle Scholar
  35. Rehm CSK, Steinbüchel A (1999) Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. Int J Biol Macromol 25:3–19CrossRefPubMedGoogle Scholar
  36. Riis V, Mai W (1988) Gas chromatographic determination of poly−β−hydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis. J Chromatogr 445:285–289CrossRefGoogle Scholar
  37. Romero−Silva MJ, Méndez V, Agulló L, Seeger M (2013) Genomic and functional analyses of the gentisate and protocatechuate ring−cleavage pathways and related 3−hydroxybenzoate and 4−hydroxybenzoate peripheral pathways in Burkholderia xenovorans LB400. PLoS ONE 8:e56038CrossRefPubMedPubMedCentralGoogle Scholar
  38. Roy I, Visakh PM (2014) Polyhydroxyalkanoate (PHA) based blends, composites and nanocomposites. The Royal Society of Chemistry, Cambridge.
  39. Sabbagh F, Muhamad II (2017) Production of poly-hydroxyalkanoate as secondary metabolite with main focus on sustainable energy. Renew Sust Energ Rev 72:95–104CrossRefGoogle Scholar
  40. Savenkova L, Gercberga Z, Nikolaeva V, Dzene A, Bibers I, Kalnin M (2000) Mechanical properties and biodegradation characteristics of PHB-based films. Process Biochem 35:573–579CrossRefGoogle Scholar
  41. Sawana A, Adeolu M, Gupta RS (2014) Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species. Front Genet 5:429CrossRefPubMedPubMedCentralGoogle Scholar
  42. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108CrossRefPubMedGoogle Scholar
  43. Schwartz E, Henne A, Cramm R, Eitinger T, Friedrich B, Gottschalk G (2003) Complete nucleotide sequence of pHG1: a Ralstonia eutropha H16 megaplasmid encoding key enzymes of H 2-based lithoautotrophy and anaerobiosis. J Mol Biol 332:369–383CrossRefPubMedGoogle Scholar
  44. Schwartz E, Voigt B, Zühlke D, Pohlmann A, Lenz O, Albrecht D, Schwarze A, Kohlmann Y, Krause C, Heker M, Friedrich B (2009) A proteomic view of the facultatively chemolithoautotrophic lifestyle of Rasltonia eutropha H16. Proteomics 9:5132−5142CrossRefGoogle Scholar
  45. Seeger M, Cámara B, Hofer B (2001) Dehalogenation, denitration, dehydroxylation, and angular attack on substituted biphenyls and related compounds by a biphenyl dioxygenase. J Bacteriol 183:3548−3555CrossRefPubMedCentralGoogle Scholar
  46. Silva-Rocha R, Martínez-García E, Calles B, Chavarría M, Arce-Rodríguez A, de Las Heras A, Paéz-Espina D, Durante-Rodriguez G, Kim J, Nikel P I, Platero R, de Lorenzo V (2012) The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res, 41(D1):D666−D675Google Scholar
  47. Steinbüchel A (2001) Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Macromol Biosci 1(1):1–24CrossRefGoogle Scholar
  48. Steinbüchel A, Lütke-Eversloh T (2003) Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem Eng J 16:81−96CrossRefGoogle Scholar
  49. Urtuvia V, Villegas P, González M, Seeger M (2014) Bacterial production of the biodegradable plastics polyhydroxyalkanoates. Int J Biol Macromol 70:208−213CrossRefGoogle Scholar
  50. Ushimaru K, Motoda Y, Numata K, Tsuge T (2014) Phasin proteins activate Aeromonas caviae polyhydroxyalkanoate (PHA) synthase but not Ralstonia eutropha PHA synthase. Appl Environ Microbiol 80:2867–2873CrossRefPubMedPubMedCentralGoogle Scholar
  51. Valappil SP, Boccaccini AR, Bucke C, Roy I (2007) Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera Bacillus and Streptomyces. Antonie Van Leeuwenhoek 91:1–17CrossRefPubMedGoogle Scholar
  52. Wahl A, Schuth N, Pfeiffer D, Nussberger S, Jendrossek D (2012) PHB granules are attached to the nucleoid via PhaM in Ralstonia eutropha. BMC Microbiol 12:262–273CrossRefPubMedPubMedCentralGoogle Scholar
  53. Ward PG, O’Connor KE (2005) Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3. Int J Biol Macromol 35:127−133CrossRefGoogle Scholar
  54. Yagi K, Miyawaki I, Kayashita A, Kondo M, Kitano Y, Murakami Y, Mizoguchi T (1996) Biosynthesis of poly (3-hydroxyalkanoic acid) copolymer from CO (inf2) in Pseudomonas acidophila through introduction of the DNA fragment responsible for chemolithoautotrophic growth of Alcaligenes hydrogenophilus. Appl Environ Microbiol 62:1004–1007PubMedPubMedCentralGoogle Scholar
  55. Zou H, Shi M, Zhang T, Li L, Li L, Xian M (2017) Natural and engineered polyhydroxyalkanoate (PHA) synthase: key enzyme in biopolyester production. Appl Microbiol Biotechnol 101:7417–7426CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química & Centro de BiotecnologíaUniversidad Técnica Federico Santa MaríaValparaísoChile

Personalised recommendations