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Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha

  • Applied Genetics and Molecular Biotechnology
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Abstract

Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (Pphb), acetoin (PacoE, PacoD, and PacoX) or pyruvate (PpdhE) metabolism were constructed and tested. The promoters were representative either of the enterobacterial σ70 (Pphb, PacoE, and PpdhE)- or σ54 (PacoD and PacoX)-dependent promoters. To obtain polymers consisting of C4–C12 monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 Ps) tandemly linked to the W. eutropha genes encoding β-ketothiolase (phbA We) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB We) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the PacoD and PacoX promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters—independent of the addition of acetoin—resulted in the production of PHA polymers with high 3HB fractions (90–100 mol%) and with high 3HO contents (70–86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the PacoE promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.

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References

  • Abe H, Doi Y, Fukushima T, Eya H (1994) Biosynthesis from gluconate of a random copolymer consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates by Pseudomonas sp. 61-3. Int J Biol Macromol 16:115–119

    CAS  PubMed  Google Scholar 

  • Anderson AJ, Dawes EA (1990) Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev 54:450–472

    CAS  PubMed  PubMed Central  Google Scholar 

  • Du G, Si Y, Yu J (2001) Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha. Biotechnol Lett 23:1613–1617

    Article  CAS  Google Scholar 

  • Haywood GW, Anderson AJ, Ewing DF, Dawes EA (1990) Accumulation of a polyhydroxyalkanoate containing primarily 3-hydroxydecanoate from simple carbohydrate substrates by Pseudomonas sp. strain NCIMB40135. Appl Environ Microbiol 56:3354–3359

    Article  CAS  Google Scholar 

  • Hein S, Steinbüchel A (1996) Alcaligenes eutrophus possesses a second pyruvate dehydrogenase (E1). Eur J Biochem 237:674–684

    Article  CAS  Google Scholar 

  • Huang W-Y, Tang J (1972) Modification of arginyl residue in pepsin by 2,3-butanedione. J Biol Chem 247:2704–2710

    CAS  PubMed  Google Scholar 

  • Jay JM (1982) Antimicrobial properties of diacetyl. Appl Environ Microbiol 44:525–532

    Article  CAS  Google Scholar 

  • Kabir MM, Shimizu K (2003) Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production. Appl Microbiol Biotechnol 62:244–255

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Kato M, Fukui T, Doi Y (1996b) Biosynthesis of polyester blends by Pseudomonas sp. 61-3 from alkanoic acids. Bull Chem Soc Jpn 69:515–520

    Article  CAS  Google Scholar 

  • Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM (1995) Four new derivatives of the broad-host-range vector cloning vector pBBR1MCS carrying different antibiotic-resistance cassettes. Gene 166:175–176

    Article  CAS  Google Scholar 

  • Krüger N, Steinbüchel A (1992) Identification of acoR, a regulatory gene for the expression of genes essential for acetoin catabolism in Alcaligenes eutrophus H16. J Bacteriol 174:4391–4400

    Article  Google Scholar 

  • Lee IY, Kim MK, Park YH, Lee SY (1996a) Regulatory effects of cellular nicotinamide nucleotides and enzyme activities on poly(3-hydroxybutyrate) synthesis in recombinant Escherichia coli. Biotechnol Bioeng 52:707–712

    Article  CAS  Google Scholar 

  • Lee YH, Kim TW, Park JS, Huh TL (1996b) Effect of the supplementation of metabolites on cell growth and poly-β-hydroxybutyrate biosynthesis of Alcaligenes latus. J Microbiol Biotechnol 6:120–127

    CAS  Google Scholar 

  • Lee J-N, Shin H-D, Lee Y-H (2003) Metabolic engineering of pentose phosphate pathway in Ralstonia eutropha for enhanced biosynthesis of poly-β-hydroxybutyrate. Biotechnol Prog 19:1444–1449

    Article  CAS  Google Scholar 

  • Matsumoto K, Nakae S, Taguchi K, Matsusaki H, Seki M, Doi Y (2001) Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) copolymer from sugars by recombinant Ralstonia eutropha harboring the phaC1 Ps and the phaG Ps genes of Pseudomonas sp. 61-3. Biomacromolecules 2:934–939

    Article  CAS  Google Scholar 

  • Matsusaki H, Manji S, Taguchi K, Kato M, Fukui T, Doi Y (1998) Cloning and molecular analysis of the poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) biosynthesis genes in Pseudomonas sp. strain 61-3. J Bacteriol 180:6459–6467

    Article  CAS  Google Scholar 

  • Matsusaki H, Abe H, Doi Y (2000a) Biosynthesis and properties of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant strains of Pseudomonas sp. 61-3. Biomacromolecules 1:17–22

    Article  CAS  Google Scholar 

  • Matsusaki H, Abe H, Taguchi K, Fukui T, Doi Y (2000b) Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3. Appl Microbiol Biotechnol 53:401–409

    Article  CAS  Google Scholar 

  • Park J-S, Lee Y-H (1996) Metabolic characteristics of isocitrate dehydrogenase leaky mutant of Alcaligenes eutrophus and its utilization for poly-β-hydroxybutyrate production. J Ferment Bioeng 81:197–205

    Article  CAS  Google Scholar 

  • Peoples OP, Sinskey AJ (1989) Poly-β-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding b-ketothiolase and acetoacetyl-CoA reductase. J Biol Chem 264:15293–15297

    CAS  PubMed  Google Scholar 

  • Pouton CW, Akhtar S (1996) Biosynthetic polyhydroxyalkanoates and their potential in drug delivery. Adv Drug Deliv Rev 18:133–162

    Article  CAS  Google Scholar 

  • Priefert H, Steinbüchel A (1992) Identification and molecular characterization of the acetyl coenzyme A synthetase gene (acoE) of Alcaligenes eutrophus. J Bacteriol 174:6590–6599

    Article  CAS  Google Scholar 

  • Priefert H, Hein S, Krüger N, Zeh K, Schmidt B, Steinbüchel A (1991) Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism. J Bacteriol 173:4056–4071

    Article  CAS  Google Scholar 

  • Priefert H, Krüger N, Jendrossek D, Schmidt B, Steinbüchel A (1992) Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus. J Bacteriol 174:899–907

    Article  CAS  Google Scholar 

  • Saad B, Neuenschwander P, Uhlschmid GK, Suter UW (1999) New versatile, elastomeric, degradable polymeric materials for medicine. Int J Biol Macromol 25:293–301

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Schlegel HG, Kaltwasser H, Gottschalk G (1961) Ein Submersverfahren zur Kultur Wasserstoff oxydierender Bakterien: Wachstumsphysiologische Untersuchungen. Arch Mikrobiol 38:209–222

    Article  CAS  Google Scholar 

  • Schlegel HG, Lafferty R, Krauss I (1970) Isolation of mutants not accumulating poly-β-hydroxybutyric acid. Arch Mikrobiol 71:283–294

    Article  CAS  Google Scholar 

  • Schubert P, Kruger N, Steinbüchel A (1991) Molecular analysis of the Alcaligenes eutrophus poly(3-hydroxyburyrate) biosynthesis operon: identification of the N terminus of poly(3-hydroxybutyrate) synthase and identification of the promoter. J Bacteriol 173:168–175

    Article  CAS  Google Scholar 

  • Steinbüchel A (1991) Polyhydroxyalkanoic acids. In: Byrom D (ed) Biomaterials. Macmillan, Basingstoke, pp 123–213

    Chapter  Google Scholar 

  • Steinbüchel A, Füchtenbusch B, Gorenflo V, Hein S, Jossek R, Langenbach S, Rehm BHA (1998) Biosynthesis of polyesters in bacteria and recombinant organisms. Polym Degrad Stab 59:177–182

    Article  Google Scholar 

  • Taguchi S, Nakamura H, Kichise T, Tsuge T, Yamato I, Doi Y (2003) Production of polyhydroxyalkanoate (PHA) from renewable carbon sources in recombinant Ralstonia eutropha using mutants of original PHA synthase. Biochem Eng J 16:107–113

    Article  CAS  Google Scholar 

  • Williams SF, Martin DP, Horowitz DM, Peoples OP (1999) PHA applications: addressing the price performance issue. I. Tissue engineering. Int J Biol Macromol 25:111–121

    Article  CAS  Google Scholar 

  • Yu J, Wang J (2001) Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels. Biotechnol Bioeng 73:458–464

    Article  CAS  Google Scholar 

  • Zinn M, Witholt B, Egli T (2001) Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate. Adv Drug Deliv Rev 53:5–21

    Article  CAS  Google Scholar 

Download references

Acknowledgement

We thank Procter & Gamble (Cincinnati, USA) for supporting this work and providing us with the pBSEB50 and pBBR1MCS-2/Pphb constructs.

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Authors and Affiliations

  1. Department of Food Science, Stocking Hall, Cornell University, Ithaca, NY, 14853, USA

    Soazig C. Delamarre & Carl A. Batt

  2. Field of Microbiology, Cornell University, Ithaca, NY, 14853, USA

    Carl A. Batt

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  1. Soazig C. Delamarre
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  2. Carl A. Batt
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Correspondence to Carl A. Batt.

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Delamarre, S.C., Batt, C.A. Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha . Appl Microbiol Biotechnol 71, 668–679 (2006). https://doi.org/10.1007/s00253-005-0217-1

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  • DOI: https://doi.org/10.1007/s00253-005-0217-1

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