Skip to main content
SpringerLink
Log in

Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates

  • Applied microbial and cell physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Ralstonia eutropha H16 is a well-studied bacterium with respect to biosynthesis of polyhydroxyalkanoates (PHAs), which has attracted attentions as biodegradable bio-based plastics. However, this strain shows quite poor growth on glycerol of which bulk supply has been increasing as a major by-product of biodiesel industries. This study examined enhancement of glycerol assimilation ability of R. eutropha H16 by introduction of the genes of aquaglyceroporin (glpF) and glycerol kinase (glpK) from Escherichia coli. Although introduction of glpFK Ec into the strain H16 using a multi-copy vector was not successful, a recombinant strain possessing glpFK Ec within the chromosome showed much faster growth on glycerol than H16. Further analyses clarified that weak expression of glpK Ec alone allowed to establish efficient glycerol assimilation pathway, indicating that the poor growth of H16 on glycerol was caused by insufficient kination activity to glycerol, as well as this strain had a potential ability for uptake of extracellular glycerol. The engineered strains expressing glpFK Ec or glpK Ec produced large amounts of poly[(R)-3-hydroxybutyrate] [P(3HB)] from glycerol with much higher productivity than H16. Unlike other glycerol-utilizable wild strains of R. eutropha, the H16-derived engineered strains accumulated P(3HB) with no significant decrease in molecular weights on glycerol, and the polydispersity index of the glycerol-based P(3HB) synthesized by the strains expressing glpFK Ec was lower than those by the parent strains. The present study demonstrated possibility of R. eutropha H16-based platform for production of useful compounds from inexpensive glycerol.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Austin D, Larson TJ (1991) Nucleotide sequence of the glpD gene encoding aerobic sn-glycerol 3-phosphate dehydrogenase of Escherichia coli K-12. J Bacteriol 173:101–107

    CAS  PubMed Central  PubMed  Google Scholar 

  • Brigham CJ, Speth DR, Rha C, Sinskey AJ (2012) Whole-genome microarray and gene deletion studies reveal regulation of the polyhydroxyalkanoate production cycle by the stringent response in Ralstonia eutropha H16. Appl Environ Microbiol 78:8033–8044

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Budde CF, Riedel SL, Willis LB, Rha C, Sinskey AJ (2011) Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains. Appl Environ Microbiol 77:2847–2854

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Cavalheiro JMBT, de Almeida MCMD, Grandfils C, da Fonseca MMR (2009) Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol. Process Biochem 44:509–515

    Article  CAS  Google Scholar 

  • Cavalheiro JM, Raposo RS, de Almeida MC, Cesario MT, Sevrin C, Grandfils C, da Fonseca MM (2012) Effect of cultivation parameters on the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) by Cupriavidus necator using waste glycerol. Bioresour Technol 111:391–397

    Article  CAS  PubMed  Google Scholar 

  • Dobson R, Gray V, Rumbold K (2012) Microbial utilization of crude glycerol for the production of value-added products. J Ind Microbiol Biotechnol 39:217–226

    Article  CAS  PubMed  Google Scholar 

  • Durnin G, Clomburg J, Yeates Z, Alvarez PJ, Zygourakis K, Campbell P, Gonzalez R (2009) Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli. Biotechnol Bioeng 103:148–161

    Article  CAS  PubMed  Google Scholar 

  • Freedberg WB, Kistler WS, Lin EC (1971) Lethal synthesis of methylglyoxal by Escherichia coli during unregulated glycerol metabolism. J Bacteriol 108:137–144

    CAS  PubMed Central  PubMed  Google Scholar 

  • Fukui T, Chou K, Harada K, Orita I, Nakayama Y, Bamba T, Nakamura S, Fukusaki E (2014) Metabolite profiles of polyhydroxyalkanoate-producing Ralstonia eutropha H16. Metabolomics 10:190–202

    Article  CAS  Google Scholar 

  • Garcia IL, Lopez JA, Dorado MP, Kopsahelis N, Alexandri M, Papanikolaou S, Villar MA, Koutinas AA (2013) Evaluation of by-products from the biodiesel industry as fermentation feedstock for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Cupriavidus necator. Bioresour Technol 130:16–22

    Article  CAS  PubMed  Google Scholar 

  • Hiroe A, Hyakutake M, Thomson NM, Sivaniah E, Tsuge T (2013) Endogenous ethanol affects biopolyester molecular weight in recombinant Escherichia coli. ACS Chem Biol 8:2568–2576

    Article  CAS  PubMed  Google Scholar 

  • Insomphun C, Mifune J, Orita I, Numata K, Nakamura S, Fukui T (2013) Modification of β-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil. J Biosci Bioeng 117:184–190

    Article  PubMed  Google Scholar 

  • Kaddor C, Steinbuchel A (2011) Implications of various phosphoenolpyruvate-carbohydrate phosphotransferase system mutations on glycerol utilization and poly(3-hydroxybutyrate) accumulation in Ralstonia eutropha H16. AMB Express 1:16

    Article  PubMed Central  PubMed  Google Scholar 

  • Kato M, Bao HJ, Kang CK, Fukui T, Doi Y (1996) Production of a novel copolyester of 3-hydroxybutyric acid and medium chain length 3-hydroxyalkanaic acids by Pseudomonas sp. 61-3 from sugars. Appl Microbiol Biotechnol 45:363–370

    Article  CAS  Google Scholar 

  • Kawashima Y, Cheng W, Mifune J, Orita I, Nakamura S, Fukui T (2012) Characterization and functional analyses of R-specific enoyl coenzyme A hydratases in polyhydroxyalkanoate-producing Ralstonia eutropha. Appl Environ Microbiol 78:493–502

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Keshavarz T, Roy I (2010) Polyhydroxyalkanoates: bioplastics with a green agenda. Curr Opin Microbiol 13:321–326

    Article  CAS  PubMed  Google Scholar 

  • Loo CY, Lee WH, Tsuge T, Doi Y, Sudesh K (2005) Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from palm oil products in a Wautersia eutropha mutant. Biotechnol Lett 27:1405–1410

    Article  CAS  PubMed  Google Scholar 

  • Lu J, Brigham CJ, Gai CS, Sinskey AJ (2012) Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha. Appl Microbiol Biotechnol 96:283–297

    Article  CAS  PubMed  Google Scholar 

  • Lu J, Brigham CJ, Rha C, Sinskey AJ (2013) Characterization of an extracellular lipase and its chaperone from Ralstonia eutropha H16. Appl Microbiol Biotechnol 97:2443–2454

    Article  CAS  PubMed  Google Scholar 

  • Madden LA, Anderson AJ, Shah DT, Asrar J (1999) Chain termination in polyhydroxyalkanoate synthesis: involvement of exogenous hydroxy-compounds as chain transfer agents. Int J Biol Macromol 25:43–53

    Article  CAS  PubMed  Google Scholar 

  • Meiswinkel TM, Rittmann D, Lindner SN, Wendisch VF (2013) Crude glycerol-based production of amino acids and putrescine by Corynebacterium glutamicum. Bioresour Technol 145:254–258

    Article  CAS  PubMed  Google Scholar 

  • Mifune J, Nakamura S, Fukui T (2008) Targeted engineering of Cupriavidus necator chromosome for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Can J Chem 86:621–627

    Article  Google Scholar 

  • Mifune J, Nakamura S, Fukui T (2010) Engineering of pha operon on Cupriavidus necator chromosome for efficient biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from vegetable oil. Polym Degrad Stab 95:1305–1312

    Article  CAS  Google Scholar 

  • Mothes G, Schnorpfeil C, Ackermann JU (2007) Production of PHB from crude glycerol. Eng Life Sci 7:475–479

    Article  CAS  Google Scholar 

  • Mukoyama M (2007) Method of imparting glycerol-assimilation ability to bacterium. International Patent Publication WO2007/013695

  • 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 84:139–200

    Article  CAS  PubMed  Google Scholar 

  • Orita I, Iwazawa R, Nakamura S, Fukui T (2011) Identification of mutation points in Cupriavidus necator NCIMB 11599 and genetic reconstitution of glucose-utilization ability in wild strain H16 for polyhydroxyalkanoate production. J Biosci Bioeng 113:63–69

    Article  PubMed  Google Scholar 

  • Pan P, Inoue Y (2009) Polymorphism and isomorphism in biodegradable polyesters. Prog Polym Sci 34:605–640

    Article  CAS  Google Scholar 

  • Peplinski K, Ehrenreich A, Doring C, Bomeke M, Reinecke F, Hutmacher C, Steinbuchel A (2010) Genome-wide transcriptome analyses of the 'Knallgas' bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism. Microbiology 156:2136–2152

    Article  CAS  PubMed  Google Scholar 

  • Pohlmann A, Fricke WF, Reinecke F, Kusian B, Liesegang H, Cramm R, Eitinger T, Ewering C, Potter M, Schwartz E, Strittmatter A, Voss I, Gottschalk G, Steinbuchel A, Friedrich B, Bowien B (2006) Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16. Nat Biotechnol 24:1257–1262

    Article  PubMed  Google Scholar 

  • Raberg M, Peplinski K, Heiss S, Ehrenreich A, Voigt B, Doring C, Bomeke M, Hecker M, Steinbuchel A (2011) Proteomic and transcriptomic elucidation of the mutant Ralstonia eutropha G+1 with regard to glucose utilization. Appl Environ Microbiol 77:2058–2070

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Raberg M, Kaddor C, Kusian B, Stahlhut G, Budinova R, Kolev N, Bowien B, Steinbuchel A (2012) Impact of each individual component of the mutated PTSNag on glucose uptake and phosphorylation in Ralstonia eutropha G+1. Appl Microbiol Biotechnol 95:735–744

  • Riedel SL, Bader J, Brigham CJ, Budde CF, Yusof ZA, Rha C, Sinskey AJ (2011) Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations. Biotechnol Bioeng 109:74–83

    Article  PubMed  Google Scholar 

  • Rittmann D, Lindner SN, Wendisch VF (2008) Engineering of a glycerol utilization pathway for amino acid production by Corynebacterium glutamicum. Appl Environ Microbiol 74:6216–6222

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    Article  CAS  PubMed  Google Scholar 

  • Shimizu R, Chou K, Orita I, Suzuki Y, Nakamura S, Fukui T (2013) Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly(3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses. BMC Microbiol 13:169

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sichwart S, Hetzler S, Broker D, Steinbuchel A (2011) Extension of the substrate utilization range of Ralstonia eutropha strain H16 by metabolic engineering to include mannose and glucose. Appl Environ Microbiol 77:1325–1334

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering. Transposon mutagenesis in Gram negative bacteria. Bio/Technology 1:784–791

    Article  CAS  Google Scholar 

  • Spoljaric IV, Lopar M, Koller M, Muhr A, Salerno A, Reiterer A, Horvat P (2013a) In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol. J Biotechnol 168:625–635

    Article  CAS  PubMed  Google Scholar 

  • Spoljaric IV, Lopar M, Koller M, Muhr A, Salerno A, Reiterer A, Malli K, Angerer H, Strohmeier K, Schober S, Mittelbach M, Horvat P (2013b) Mathematical modeling of poly[(R)-3-hydroxyalkanoate] synthesis by Cupriavidus necator DSM 545 on substrates stemming from biodiesel production. Bioresour Technol 133:482–494

    Article  PubMed  Google Scholar 

  • Taguchi S, Iwata T, Abe H, Doi Y (2012) Poly(hydroxyalkanoate)s. In: Matyjaszewski K, Möller M (eds) Polymer science: a comprehensive reference. Elsevier, Amsterdam, pp 157–182

    Google Scholar 

  • Taidi B, Anderson AJ, Dawes EA, Byrom D (1994) Effect of carbon source and concentration on the molecular mass of poly(3-hydroxybutyrate) produced by Methylobacterium extorquens and Alcaligenes eutrophus. Appl Microbiol Biotechnol 40:786–790

  • Tanadchangsaeng N, Yu J (2012) Microbial synthesis of polyhydroxybutyrate from glycerol: gluconeogenesis, molecular weight and material properties of biopolyester. Biotechnol Bioeng 109:2808–2818

    Article  CAS  PubMed  Google Scholar 

  • Tomizawa S, Saito Y, Hyakutake M, Nakamura Y, Abe H, Tsuge T (2010) Chain transfer reaction catalyzed by various polyhydroxyalkanoate synthases with poly(ethylene glycol) as an exogenous chain transfer agent. Appl Microbiol Biotechnol 87:1427–1435

    Article  CAS  PubMed  Google Scholar 

  • Tomizawa S, Sato S, Lan JC, Nakamura Y, Abe H, Tsuge T (2013) In vitro evidence of chain transfer to tetraethylene glycols in enzymatic polymerization of polyhydroxyalkanoate. Appl Microbiol Biotechnol 97:4821–4829

    Article  CAS  PubMed  Google Scholar 

  • Tsuge T, Watanabe S, Shimada D, Abe H, Doi Y, Taguchi S (2007) Combination of N149S and D171G mutations in Aeromonas caviae polyhydroxyalkanoate synthase and impact on polyhydroxyalkanoate biosynthesis. FEMS Microbiol Lett 277:217–222

    Article  CAS  PubMed  Google Scholar 

  • Verlinden RA, Hill DJ, Kenward MA, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102:1437–1449

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Schulten K, Tajkhorshid E (2005) What makes an aquaporin a glycerol channel? A comparative study of AqpZ and GlpF. Structure 13:1107–1118

    Article  CAS  PubMed  Google Scholar 

  • Weissenborn DL, Wittekindt N, Larson TJ (1992) Structure and regulation of the glpFK operon encoding glycerol diffusion facilitator and glycerol kinase of Escherichia coli K-12. J Biol Chem 267:6122–6131

    CAS  PubMed  Google Scholar 

  • Yang F, Hanna MA, Sun R (2012) Value-added uses for crude glycerol—a byproduct of biodiesel production. Biotechnol Biofuels 5:13

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zhu MM, Skraly FA, Cameron DC (2001) Accumulation of methylglyoxal in anaerobically grown Escherichia coli and its detoxification by expression of the Pseudomonas putida glyoxalase I gene. Metab Eng 3:218–225

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank Assoc. Prof. Takeharu Tsuge and Dr. Ayaka Hiroe (Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Japan) for GPC analysis. This work was supported by JSPS KAKENHI grant number 25292058.

Author information

Authors and Affiliations

  1. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan

    Toshiaki Fukui, Izumi Orita & Satoshi Nakamura

  2. Strategic Technology Research Center, NIPPON SHOKUBAI, 1-25-12 Kannondai, Tsukuba, 305-0856, Japan

    Masaharu Mukoyama

Authors
  1. Toshiaki Fukui
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masaharu Mukoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Izumi Orita
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Satoshi Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Toshiaki Fukui.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Table S1

(PDF 110 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fukui, T., Mukoyama, M., Orita, I. et al. Enhancement of glycerol utilization ability of Ralstonia eutropha H16 for production of polyhydroxyalkanoates. Appl Microbiol Biotechnol 98, 7559–7568 (2014). https://doi.org/10.1007/s00253-014-5831-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-014-5831-3

Keywords

Search

Navigation