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Engineering Yarrowia lipolytica for poly-3-hydroxybutyrate production

  • Zheng-Jun Li
  • Kangjian Qiao
  • Nian Liu
  • Gregory Stephanopoulos
Metabolic Engineering and Synthetic Biology - Original Paper

Abstract

Strains of Yarrowia lipolytica were engineered to express the poly-3-hydroxybutyrate (PHB) biosynthetic pathway. The genes for β-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase, and PHB synthase were cloned and inserted into the chromosome of Y. lipolytica. In shake flasks, the engineered strain accumulated PHB to 1.50 and 3.84% of cell dry weight in complex medium supplemented with glucose and acetate as carbon source, respectively. In fed-batch fermentation using acetate as sole carbon source, 7.35 g/l PHB (10.2% of cell dry weight) was produced. Selection of Y. lipolytica as host for PHB synthesis was motivated by the fact that this organism is a good lipids producer, which suggests robust acetyl-CoA supply also the precursor of the PHB pathway. Acetic acid could be supplied by gas fermentation, anaerobic digestion, and other low-cost supply route.

Keywords

Poly-3-hydroxybutyrate PHB Metabolic engineering phaCAB Acetate Yarrowia lipolytica 

Notes

Acknowledgements

We thank Ms. Xue-Mei Che of the School of Life Sciences of Tsinghua University for the assistance of PHB molecular weight assays. This research was financially supported by Grants from the Department of Energy (DE-SC0008744). ZJL was funded by the National Natural Science Foundation of China (21476014 and 31100025).

Supplementary material

10295_2016_1864_MOESM1_ESM.docx (35 kb)
Supplementary material 1 (DOCX 35 kb)

References

  1. 1.
    Carlson R, Srienc F (2006) Effects of recombinant precursor pathway variations on poly [(R)-3-hydroxybutyrate] synthesis in Saccharomyces cerevisiae. J Biotechnol 124:561–573. doi: 10.1016/j.jbiotec.2006.01.035 CrossRefPubMedGoogle Scholar
  2. 2.
    Chu A, Mavinic DS, Ramey WD, Kelly HG (1996) A biochemical model describing volatile fatty acid metabolism in thermophilic aerobic digestion of wastewater sludge. Water Res 30:1759–1770. doi: 10.1016/0043-1354(96)00051-6 CrossRefGoogle Scholar
  3. 3.
    Hu P, Rismani-Yazdi H, Stephanopoulos G (2013) Anaerobic CO2 fixation by the acetogenic bacterium Moorella thermoacetica. AIChE J 59:3176–3183. doi: 10.1002/aic.14127 CrossRefGoogle Scholar
  4. 4.
    Jiang JG, Zhang YJ, Li KM, Wang Q, Gong CX, Li ML (2013) Volatile fatty acids production from food waste: effects of pH, temperature, and organic loading rate. Bioresour Technol 143:525–530. doi: 10.1016/j.biortech.2013.06.025 CrossRefPubMedGoogle Scholar
  5. 5.
    Jiang XR, Wang H, Shen R, Chen GQ (2015) Engineering the bacterial shapes for enhanced inclusion bodies accumulation. Metab Eng 29:227–237. doi: 10.1016/j.ymben.2015.03.017 CrossRefPubMedGoogle Scholar
  6. 6.
    Kocharin K, Chen Y, Siewers V, Nielsen J (2012) Engineering of acetyl-CoA metabolism for the improved production of polyhydroxybutyrate in Saccharomyces cerevisiae. AMB Express 2:52. doi: 10.1186/2191-0855-2-52 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kocharin K, Siewers V, Nielsen J (2013) Improved polyhydroxybutyrate production by Saccharomyces cerevisiae through the use of the phosphoketolase pathway. Biotechnol Bioeng 110:2216–2224. doi: 10.1002/bit.24888 CrossRefPubMedGoogle Scholar
  8. 8.
    Leaf TA, Peterson MS, Stoup SK, Somers D, Srienc F (1996) Saccharomyces cerevisiae expressing bacterial polyhydroxybutyrate synthase produces poly-3-hydroxybutyrate. Microbiol 142:1169–1180. doi: 10.1099/13500872-142-5-1169 CrossRefGoogle Scholar
  9. 9.
    Lian JZ, Si T, Nair NU, Zhao HM (2014) Design and construction of acetyl-CoA overproducing Saccharomyces cerevisiae strains. Metab Eng 24:139–149. doi: 10.1016/j.ymben.2014.05.010 CrossRefPubMedGoogle Scholar
  10. 10.
    Matsusaki H, Abe H, Taguchi K, Fukui T, Doi Y (2000) 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. doi: 10.1007/s002530051633 CrossRefPubMedGoogle Scholar
  11. 11.
    Philip S, Keshavarz T, Roy I (2007) Polyhydroxyalkanoates: biodegradable polymers with a range of applications. J Chem Technol Biotechnol 82:233–247. doi: 10.1002/jctb.1667 CrossRefGoogle Scholar
  12. 12.
    Qiao K, Imam Abidi SH, Liu H, Zhang H, Chakraborty S, Watson N, Kumaran Ajikumar P, Stephanopoulos G (2015) Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng 29:56–65. doi: 10.1016/j.ymben.2015.02.005 CrossRefPubMedGoogle Scholar
  13. 13.
    Sandstrom AG, de Las Munoz, Heras A, Portugal-Nunes D, Gorwa-Grauslund MF (2015) Engineering of Saccharomyces cerevisiae for the production of poly-3-hydroxybutyrate from xylose. AMB Express 5:14. doi: 10.1186/s13568-015-0100-0 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Sim SJ, Snell KD, Hogan SA, Stubbe J, Rha C, Sinskey AJ (1997) PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat Biotech 15:63–67. doi: 10.1038/nbt0197-63 CrossRefGoogle Scholar
  15. 15.
    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 25:148–175. doi: 10.1016/j.biotechadv.2006.11.007 CrossRefPubMedGoogle Scholar
  16. 16.
    Taguchi S, Yamadaa 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 USA 105:17323–17327. doi: 10.1073/pnas.0805653105 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Wu H, Chen J, Chen GQ (2016) Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli. Appl Microbiol Biotechnol. doi: 10.1007/s00253-016-7715-1 Google Scholar
  18. 18.
    Wu H, Fan Z, Jiang X, Chen J, Chen GQ (2016) Enhanced production of polyhydroxybutyrate by multiple dividing E. coli. Microb Cell Fact 15:128. doi: 10.1186/s12934-016-0531-6 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Xue ZX, Sharpe PL, Hong SP, Yadav NS, Xie DM, Short DR, Damude HG, Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, Macool DJ, Hollerbach DH, Zhang HX, Arcilla DM, Bledsoe SA, Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q (2013) Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 31:734–740. doi: 10.1038/nbt.2622 CrossRefPubMedGoogle Scholar
  20. 20.
    Yin J, Wang H, Fu XZ, Gao X, Wu Q, Chen GQ (2015) Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp. Appl Microbiol Biotechnol 99:5523–5534. doi: 10.1007/s00253-015-6510-8 CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  • Zheng-Jun Li
    • 1
    • 2
  • Kangjian Qiao
    • 1
  • Nian Liu
    • 1
  • Gregory Stephanopoulos
    • 1
  1. 1.Department of Chemical EngineeringMassachusetts Institute of TechnologyCambridgeUSA
  2. 2.Beijing Key Laboratory of Bioprocess, College of Life Science and TechnologyBeijing University of Chemical TechnologyBeijingPeople’s Republic of China

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