Membrane engineering via trans-unsaturated fatty acids production improves succinic acid production in Mannheimia succiniciproducens

  • Jung Ho Ahn
  • Jong An Lee
  • Junho Bang
  • Sang Yup LeeEmail author
Metabolic Engineering and Synthetic Biology - Original Paper


Engineering of microorganisms to produce desired bio-products with high titer, yield, and productivity is often limited by product toxicity. This is also true for succinic acid (SA), a four carbon dicarboxylic acid of industrial importance. Acid products often cause product toxicity to cells through several different factors, membrane damage being one of the primary factors. In this study, cistrans isomerase from Pseudomonas aeruginosa was expressed in Mannheimia succiniciproducens to produce trans-unsaturated fatty acid (TUFA) and to reinforce the cell membrane of M. succiniciproducens. The engineered strain showed significant decrease in membrane fluidity as production of TUFA enabled tight packing of fatty acids, which made cells to possess more rigid cell membrane. As a result, the membrane-engineered M. succiniciproducens strain showed higher tolerance toward SA and increased production of SA compared with the control strain without membrane engineering. The membrane engineering approach employed in this study will be useful for increasing tolerance to, and consequently enhancing production of acid products.


Mannheimia succiniciproducens Succinic acid Membrane engineering Tolerance Trans-unsaturated fatty acid 



This work was supported by the C1 Gas Refinery Program funded by the Ministry of Science and ICT through the National Research Foundation of Korea (NRF-2016M3D3A1A01913250).

Compliance with ethical standards

Conflict of interest

Authors declare that they have competing financial interests as the M. succiniciproducens strains described in this paper are patented and currently of commercial interest.

Supplementary material

10295_2018_2016_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)


  1. 1.
    Andersson C, Helmerius J, Hodge D, Berglund KA, Rova U (2009) Inhibition of succinic acid production in metabolically engineered Escherichia coli by neutralizing agent, organic acids, and osmolarity. Biotechnol Prog 25:116–123CrossRefPubMedGoogle Scholar
  2. 2.
    Baysse C, O’Gara F (2007) Role of membrane structure during stress signaling and adaptation in Pseudomonas. In: Ramos JL, Filloux A (eds) Pseudomonas. Springer, Dordrecht, pp 193–224CrossRefGoogle Scholar
  3. 3.
    Cho H, Cronan JE (1995) Defective export of a periplasmic enzyme disrupts regulation of fatty acid synthesis. J Biol Chem 270(9):4216–4219CrossRefPubMedGoogle Scholar
  4. 4.
    Choi S, Song CW, Shin JH, Lee SY (2015) Biorefineries for the production of top building block chemicals and their derivatives. Metab Eng 28:223–239CrossRefPubMedGoogle Scholar
  5. 5.
    Choi S, Song H, Lim SW, Kim TY, Ahn JH, Lee JW, Lee MH, Lee SY (2016) Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification. Biotechnol Bioeng 113:2168–2177CrossRefPubMedGoogle Scholar
  6. 6.
    Engelman DM (2005) Membranes are more mosaic than fluid. Nature 438:578CrossRefPubMedGoogle Scholar
  7. 7.
    Fishov I, Woldringh CL (1999) Visualization of membrane domains in Escherichia coli. Mol Microbiol 32:1166–1172CrossRefPubMedGoogle Scholar
  8. 8.
    Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6:343–345CrossRefPubMedGoogle Scholar
  9. 9.
    Holtwick R, Meinhardt F, Keweloh H (1997) Cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8. Appl Environ Microbiol 63:4292–4297PubMedPubMedCentralGoogle Scholar
  10. 10.
    In’t Veld G, Driessen AJ, den Kamp JAO, Konings WN (1991) Hydrophobic membrane thickness and lipid-protein interactions of the leucine transport system of Lactococcus lactis. BBA Biomemb 1065:203–212CrossRefGoogle Scholar
  11. 11.
    Inui M, Murakami S, Okino S, Kawaguchi H, Vertès AA, Yukawa H (2004) Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J Mol Microbiol Biotechnol 7:182–196CrossRefPubMedGoogle Scholar
  12. 12.
    Jang Y-S, Jung YR, Lee SY, Kim JM, Lee JW, Oh D-B, Kang HA, Kwon O, Jang SH, Song H (2007) Construction and characterization of shuttle vectors for succinic acid-producing rumen bacteria. Appl Envrion Microbiol 73:5411–5420CrossRefGoogle Scholar
  13. 13.
    Jarboe LR, Royce LA, Liu P (2013) Understanding biocatalyst inhibition by carboxylic acids. Front Microbiol.
  14. 14.
    Junker F, Jaun LR (1999) Involvement of the cis/trans Isomerase Cti in Solvent Resistance of Pseudomonas putidaDOT-T1E. J Bacteriol 181(18):5693–5700PubMedPubMedCentralGoogle Scholar
  15. 15.
    Kanehisa M, Goto S, Kawashima S, Nakaya A (2002) The KEGG databases at GenomeNet. Nucl Acids Res 30(1):42–46CrossRefPubMedGoogle Scholar
  16. 16.
    Kim JM, Lee KH, Lee SY (2007) Development of a markerless gene knock-out system for Mannheimia succiniciproducens using a temperature-sensitive plasmid. FEMS Microbiol Lett 278:78–85CrossRefPubMedGoogle Scholar
  17. 17.
    Kim WJ, Ahn JH, Kim HU, Kim TY, Lee SY (2017) Metabolic engineering of Mannheimia succiniciproducens for succinic acid production based on elementary mode analysis with clustering. Biotechnol J. CrossRefPubMedGoogle Scholar
  18. 18.
    Kuhnert P, Scholten E, Haefner S, Mayor D, Frey J (2010) Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen. Int J Syst Evol Microbiol 60:44–50CrossRefPubMedGoogle Scholar
  19. 19.
    Lee JW, Yi J, Kim TY, Choi S, Ahn JH, Song H, Lee M-H, Lee SY (2016) Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens. Metab Eng 38:409–417CrossRefPubMedGoogle Scholar
  20. 20.
    Lee P, Lee S, Hong S, Chang H (2002) Isolation and characterization of a new succinic acid-producing bacterium, Mannheimia succiniciproducens MBEL55E, from bovine rumen. Appl Microbiol Biotechnol 58:663CrossRefPubMedGoogle Scholar
  21. 21.
    Lee SJ, Song H, Lee SY (2006) Genome-based metabolic engineering of Mannheimia succiniciproducens for succinic acid production. Appl Environ Microbiol 72:1939–1948CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Lennen RM, Pfleger BF (2013) Modulating membrane composition alters free fatty acid tolerance in Escherichia coli. PLoS One 8:e54031CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Li Q, Wang D, Wu Y, Yang M, Li W, Xing J, Su Z (2010) Kinetic evaluation of products inhibition to succinic acid producers Escherichia coli NZN111, AFP111, BL21, and Actinobacillus succinogenes 130Z T. J Microbiol 48:290–296CrossRefPubMedGoogle Scholar
  24. 24.
    Lin SKC, Du C, Koutinas A, Wang R, Webb C (2008) Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes. Biochem Eng J 41:128–135CrossRefGoogle Scholar
  25. 25.
    Loffhagen N, Claus H, Wolfgang B (2004) Pseudomonas putida NCTC 10936 balances membrane fluidity in response to physical and chemical stress by changing the saturation degree and the trans/cis ratio of fatty acids. Biosci Biotechnol Biochem 68(2):317–323CrossRefPubMedGoogle Scholar
  26. 26.
    Magnuson K, Jackowski S, Rock CO, Cronan JE (1993) Regulation of fatty acid biosynthesis in Escherichia coli. Microbiol Rev 57(3):522–542PubMedPubMedCentralGoogle Scholar
  27. 27.
    McKinlay JB, Vieille C, Zeikus JG (2007) Prospects for a bio-based succinate industry. Appl Microbiol Biotechnol 76:727–740CrossRefPubMedGoogle Scholar
  28. 28.
    Millard CS, Chao Y-P, Liao JC, Donnelly MI (1996) Enhanced production of succinic acid by overexpression of phosphoenolpyruvate carboxylase in Escherichia coli. Appl Environ Microbiol 62:1808–1810PubMedPubMedCentralGoogle Scholar
  29. 29.
    Mykytczuk N, Trevors J, Leduc L, Ferroni G (2007) Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog Biophys Mol Biol 95:60–82CrossRefPubMedGoogle Scholar
  30. 30.
    Royce LA, Boggess E, Fu Y, Liu P, Shanks JV, Dickerson J, Jarboe LR (2014) Transcriptomic analysis of carboxylic acid challenge in Escherichia coli: beyond membrane damage. PLoS One 9:e89580CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Royce LA, Liu P, Stebbins MJ, Hanson BC, Jarboe LR (2013) The damaging effects of short chain fatty acids on Escherichia coli membranes. Appl Microbiol Biotechnol 97:8317–8327CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Royce LA, Yoon JM, Chen Y, Rickenbach E, Shanks JV, Jarboe LR (2015) Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity. Metab Eng 29:180–188CrossRefPubMedGoogle Scholar
  33. 33.
    Samuelov N, Lamed R, Lowe S, Zeikus J (1991) Influence of CO2–HCO3 levels and pH on growth, succinate production, and enzyme activities of Anaerobiospirillum succiniciproducens. Appl Environ Microbiol 57:3013–3019PubMedPubMedCentralGoogle Scholar
  34. 34.
    Sohlenkamp C, Otto G (2016) Bacterial membrane lipids: diversity in structures and pathways. FEMS Microbiol Rev 40(1):133–159CrossRefPubMedGoogle Scholar
  35. 35.
    Song H, Lee SY (2006) Production of succinic acid by bacterial fermentation. Enzyme Microbiol Technol 39:352–361CrossRefGoogle Scholar
  36. 36.
    Tan Z, Yoon JM, Nielsen DR, Shanks JV, Jarboe LR (2016) Membrane engineering via trans-unsaturated fatty acids production improves Escherichia coli robustness and production of biorenewables. Metab Eng 35:105–113CrossRefPubMedGoogle Scholar
  37. 37.
    Thakker C, Martínez I, San KY, Bennett GN (2012) Succinate production in Escherichia coli. Biotechnol J 7:213–224CrossRefPubMedGoogle Scholar
  38. 38.
    Ukey R, Holmes WE, Bajpai R, Chistoserdov AY (2017) Evaluation of thioesterases from Acinetobacter baylyi for production of free fatty acids. Can J Microbiol 63(4):321–329CrossRefPubMedGoogle Scholar
  39. 39.
    Van Der Werf MJ, Guettler MV, Jain MK, Zeikus JG (1997) Environmental and physiological factors affecting the succinate product ratio during carbohydrate fermentation by Actinobacillus sp. 130Z. Arch Microbiol 167:332–342CrossRefPubMedGoogle Scholar
  40. 40.
    Von Wallbrunn A, Richnow HH, Neumann G, Meinhardt F, Heipieper HJ (2003) Mechanism of cis-trans isomerization of unsaturated fatty acids in Pseudomonas putida. J Bacteriol 185:1730–1733CrossRefGoogle Scholar
  41. 41.
    Xia T, Altman E, Eiteman MA (2015) Succinate production from xylose-glucose mixtures using a consortium of engineered Escherichia coli. Eng Life Sci 15:65–72CrossRefGoogle Scholar
  42. 42.
    Zeikus J, Jain M, Elankovan P (1999) Biotechnology of succinic acid production and markets for derived industrial products. Appl Microbiol Biotechnol 51:545–552CrossRefGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2018

Authors and Affiliations

  1. 1.Metabolic and Biomolecular Engineering National Research Laboratory, Department of Chemical and Biomolecular Engineering (BK21 Plus Program), BioProcess Engineering Research Center, and Center for Systems and Synthetic Biotechnology, Institute for the BioCenturyKAISTDaejeonRepublic of Korea

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