Applied Microbiology and Biotechnology

, Volume 87, Issue 6, pp 2247–2256 | Cite as

Production of polyhydroxyalkanoates by Escherichia coli mutants with defected mixed acid fermentation pathways

Applied Microbial and Cell Physiology


A series of Escherichia coli BW25113 mutants with reduced mixed acid fermentation were constructed. Genes ackA-pta, poxB, ldhA, adhE, and pflB encoding acetate kinase, phosphate acetyltransferase, pyruvate oxidase, d-lactate dehydrogenase, acetaldehyde dehydrogenase, and pyruvate formate-lyase, respectively, were deleted successively. When grown under microaerobic condition, the mutants reduced approximately 90% acetate excretion after the deletion of genes ackA-pta and poxB. Production of lactate, ethanol, and formate was also significantly reduced after the deletion of genes ldhA, adhE, and pflB, respectively. The accumulation of biomass and poly(3-hydroxybutyrate) (PHB) were significantly enhanced after deleting the mixed acid fermentation. E. coli mutant BWapld with deletions of ackA-pta, poxB, ldhA, and adhE produced twice the cell dry weight (CDW) and 3.5 times of PHB compared with its wild-type under microaerobic conditions. E. coli mutant BWapl with deletions of ackA-pta, poxB, and ldhA also achieved nearly twice CDW and three times of PHB content in comparison to the wild-type during 48 h static cultivation. Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was observed in the mutants under static cultivation. E. coli mutant BWapld could produce approximately 50 wt.% P(3HB-co-3HV) consisting of 5 mol% of 3-hydroxyvalerate (3HV) under aerobic conditions, when the seed culture was inoculated at an appropriate time. When ackA-pta, poxB, ldhA, adhE, and pflB were deleted, E. coli mutant BWapldf accumulated over 70 wt.% P(3HB-co-3HV) consisting of 8 mol% 3HV under aerobic conditions.


PHB P(3HB-co-3HV) Escherichia coli Polyhydroxyalkanoates Mixed acid fermentation Metabolic engineering 


  1. Aldor AS, Kim SW, Prather KLJ, Keasling JD (2002) Metabolic engineering of a novel propionate-independent pathway for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Salmonella enterica serovar typhimurium. Appl Environ Microbiol 68:3848–3854CrossRefGoogle Scholar
  2. Atsumi S, Cann AF, Connor MR, Shen CR, Smith KM, Brynildsen MP, Chou KJY, Hanai T, Liao JC (2008) Metabolic engineering of Escherichia coli for 1-butanol production. Metab Eng 10:305–311CrossRefGoogle Scholar
  3. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008CrossRefGoogle Scholar
  4. Carlson R, Wlaschin A, Srienc F (2005) Kinetic studies and biochemical pathway analysis of anaerobic poly-(R)-3-hydroxybutyric acid synthesis in Escherichia coli. Appl Environ Microbiol 71:713–720CrossRefGoogle Scholar
  5. Chang DE, Shin S, Rhee JS, Pan JG (1999) Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme a flux for growth and survival. J Bacteriol 181:6656–6663Google Scholar
  6. Chen GQ (2009) A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem Soc Rev 38:2434–2446CrossRefGoogle Scholar
  7. Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158:9–14CrossRefGoogle Scholar
  8. Cherrington CA, Hinton M, Chopra I (1990) Effect of short-chain organic-acids on macromolecular-synthesis in Escherichia coli. J Appl Bacteriol 68:69–74Google Scholar
  9. Clark DP (1989) The fermentation pathways of Escherichia coli. FEMS Microbiol Lett 63:223–234CrossRefGoogle Scholar
  10. Contiero J, Beatty C, Kumari S, DeSanti CL, Strohl WR, Wolfe A (2000) Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli. J Ind Microbiol Biotechnol 24:421–430CrossRefGoogle Scholar
  11. Cox SJ, Levanon SS, Sanchez A, Lin H, Peercy B, Bennett GN, San KY (2006) Development of a metabolic network design and optimization framework incorporating implementation constraints: a succinate production case study. Metab Eng 8:46–57CrossRefGoogle Scholar
  12. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645CrossRefGoogle Scholar
  13. Dittrich CR, Bennett GN, San KY (2005) Characterization of the acetate-producing pathways in Escherichia coli. Biotechnol Progr 21:1062–1067CrossRefGoogle Scholar
  14. El-Mansi M (2004) Flux to acetate and lactate excretions in industrial fermentations: physiological and biochemical implications. J Ind Microbiol Biotechnol 31:295–300CrossRefGoogle Scholar
  15. Eschenlauer AC, Stoup SK, Srienc F, Somers DA (1996) Production of heteropolymeric polyhydroxyalkanoate in Escherichia coli from a single carbon source. Int J Biol Macromol 19:121–130CrossRefGoogle Scholar
  16. Fidler S, Dennis D (1992) Polyhydroxyalkanoate production in recombinant Escherichia coli. FEMS Microbiol Rev 9:231–235Google Scholar
  17. Froese DS, Dobson CM, White AP, Wu X, Padovani D, Banerjee R, Haller T, Gerlt JA, Surette MG, Gravel RA (2009) Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli. Microbiol Res 164:1–8CrossRefGoogle Scholar
  18. Haller T, Buckel T, Retey J, Gerlt JA (2000) Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 39:4622–4629CrossRefGoogle Scholar
  19. Jensen EB, Carlsen S (1990) Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts. Biotechnol Bioeng 36:1–11CrossRefGoogle Scholar
  20. Kadouri D, Jurkevitch E, Okon Y, Castro-Sowinski S (2005) Ecological and agricultural significance of bacterial polyhydroxyalkanoates. Crit Rev Microbiol 31:55–67CrossRefGoogle Scholar
  21. Kang Z, Geng Y, Yz X, Kang J, Qi Q (2009) Engineering Escherichia coli for an efficient aerobic fermentation platform. J Biotechnol 144:58–63CrossRefGoogle Scholar
  22. Li M, Yao SJ, Shimizu K (2007a) Effect of poxB gene knockout on metabolism in Escherichia coli based on growth characteristics and enzyme activities. World J Microbiol Biotechnol 23:573–580CrossRefGoogle Scholar
  23. Li R, Zhang HX, Qi QS (2007b) The production of polyhydroxyalkanoates in recombinant Escherichia coli. Bioresour Technol 98:2313–2320CrossRefGoogle Scholar
  24. Nakamura K, Goto Y, Yoshie N, Inoue Y, Chujo R (1992) Biosynthesis of poly(3-hydroxyalkanoates) from threonine. J Ind Microbiol Biotechnol 14:117–118Google Scholar
  25. Qiu YZ, Ouyang SP, Shen ZY, Wu Q, Chen GQ (2004) Metabolic engineering for the production of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonas hydrophila. Macromol Biosci 4:255–261CrossRefGoogle Scholar
  26. Rodgers M, Wu GX (2010) Production of polyhydroxybutyrate by activated sludge performing enhanced biological phosphorus removal. Bioresour Technol 101:1049–1053CrossRefGoogle Scholar
  27. Schubert P, Steinbuchel A, Schlegel HG (1988) Cloning of the Alcaligenes eutrophus genes for synthesis of poly-β-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. J Bacteriol 170:5837–5847Google Scholar
  28. Slater S, Gallaher T, Dennis D (1992) Production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) in a recombinant Escherichia coli strain. Appl Environ Microbiol 58:1089–1094Google Scholar
  29. Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinbuchel A (1999) A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch Microbiol 171:73–80CrossRefGoogle Scholar
  30. Steinbüchel A (2001) Perspectives for biotechnological production and utilization of biopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Macromol Biosci 1:1–24CrossRefGoogle Scholar
  31. Steinbüchel A, Pieper U (1992) Production of a copolyester of 3-hydroxybutyric acid and 3-hydroxyvaleric acid from single unrelated carbon-sources by a mutant of Alcaligenes eutrophus. Appl Microbiol Biotechnol 37:1–6Google Scholar
  32. Suriyamongkol P, Weselake R, Narine S, Moloney M, Shah S (2007) Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants—a review. Biotechnol Adv 25:148–175CrossRefGoogle Scholar
  33. Trinh CT, Unrean P, Srienc F (2008) Minimal Escherichia coli cell for the most efficient production of ethanol from hexoses and pentoses. Appl Environ Microbiol 74:3634–3643CrossRefGoogle Scholar
  34. Verlinden RAJ, Hill DJ, Kenward MA, Williams CD, Radecka I (2007) Bacterial synthesis of biodegradable polyhydroxyalkanoates. J Appl Microbiol 102:1437–1449CrossRefGoogle Scholar
  35. Xie YP, Kohls D, Noda I, Schaefer DW, Akpalu YA (2009) Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) nanocomposites with optimal mechanical properties. Polymer 50:4656–4670CrossRefGoogle Scholar
  36. Xu B, Jahic M, Blomsten G, Enfors SO (1999) Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli. Appl Microbiol Biotechnol 51:564–571CrossRefGoogle Scholar
  37. Zhang XJ, Luo RC, Wang Z, Deng Y, Chen GQ (2009) Applications of (R)-3-hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as Novel Biofuel. Biomacromolecules 10:707–711CrossRefGoogle Scholar
  38. 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 64:367–375CrossRefGoogle Scholar
  39. Zhu HF, Shimizu K (2005) Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for d-lactate production under microaerobic condition. Metab Eng 7:104–115CrossRefGoogle Scholar
  40. Zou XH, Chen GQ (2007) Metabolic engineering for microbial production and applications of copolyesters consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates. Macromol Biosci 7:174–182CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  1. 1.Protein Science Laboratory of the Ministry of Education, Department of Biology, School of Life SciencesTsinghua UniversityBeijingChina
  2. 2.Department of ChemistryUniversity of MelbourneMelbourneAustralia
  3. 3.Life Science Division, Graduate School at ShenzhenTsinghua UniversityShenzhenChina

Personalised recommendations