Skip to main content
SpringerLink
Log in

Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing

  • Biocatalysis - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Fatty acid methyl esters (FAMEs) originating from plant oils are most interesting renewable feedstocks for biofuels and bio-based materials. FAMEs can also be produced and/or functionalized by engineered microbes to give access to, e.g., polymer building blocks. Yet, they are often subject to hydrolysis yielding free fatty acids, which typically are degraded by microbes. We identified BioH as the key enzyme responsible for the hydrolysis of medium-chain length FAME derivatives in different E. coli K-12 strains. E. coli ΔbioH strains showed up to 22-fold reduced FAME hydrolysis rates in comparison with respective wild-type strains. Knockout strains showed, beside the expected biotin auxotrophy, unchanged growth behavior and biocatalytic activity. Thus, high specific rates (~80 U g −1CDW ) for terminal FAME oxyfunctionalization catalyzed by a recombinant alkane monooxygenase could be combined with reduced hydrolysis. Biotransformations in process-relevant two-liquid phase systems profited from reduced fatty acid accumulation and/or reduced substrate loss via free fatty acid metabolization. The BioH knockout strategy was beneficial in all tested strains, although its effect was found to differ according to specific strain properties, such as FAME hydrolysis and FFA degradation activities. BioH or functional analogs can be found in virtually all microorganisms, making bioH deletion a broadly applicable strategy for efficient microbial bioprocessing involving FAMEs.

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
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Akatsuka H, Kawai E, Sakurai N, Omori K (2003) The Serratia marcescens bioH gene encodes an esterase. Gene 302:185–192. doi:10.1016/S0378111902011502

    Article  CAS  PubMed  Google Scholar 

  2. Baba T, Ara T, Hasegawa M et al (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008. doi:10.1038/msb4100050

    Article  PubMed  PubMed Central  Google Scholar 

  3. Bae JH, Park BG, Jung E, Lee P-G, Kim B-G (2014) fadD deletion and fadL overexpression in Escherichia coli increase hydroxy long-chain fatty acid productivity. Appl Microbiol Biotechnol 98:8917–8925. doi:10.1007/s00253-014-5974-2

    Article  CAS  PubMed  Google Scholar 

  4. Beller HR, Lee TS, Katz L (2015) Natural products as biofuels and bio-based chemicals: fatty acids and isoprenoids. Nat Prod Rep 32:1508–1526. doi:10.1039/C5NP00068H

    Article  CAS  PubMed  Google Scholar 

  5. Blank LM, Ebert BE, Bühler B, Schmid A (2008) Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint-based modeling and experimental verification. Biotechnol Bioeng 100:1050–1065. doi:10.1002/bit.21837

    Article  CAS  PubMed  Google Scholar 

  6. Brandenbusch C, Glonke S, Collins J, Hoffrogge R, Grunwald K, Bühler B, Schmid A, Sadowski G (2015) Process boundaries of irreversible scCO2-assisted phase separation in biphasic whole-cell biocatalysis. Biotechnol Bioeng 112:2316–2323. doi:10.1002/bit.25655

    Article  CAS  PubMed  Google Scholar 

  7. Bühler B, Bollhalder I, Hauer B, Witholt B, Schmid A (2003) Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale. Biotechnol Bioeng 82:833–842. doi:10.1002/bit.10637

    Article  PubMed  Google Scholar 

  8. Campbell JW, Morgan-Kiss RM, Cronan EJ (2003) A new Escherichia coli metabolic competency: growth on fatty acids by a novel anaerobic β-oxidation pathway. Mol Microbiol 47:793–805. doi:10.1046/j.1365-2958.2003.03341.x

    Article  CAS  PubMed  Google Scholar 

  9. Cao Z, Gao H, Liu M, Jiao P (2006) Engineering the acetyl-CoA transportation system of Candida tropicalis enhances the production of dicarboxylic acid. Biotechnol J 1:68–74. doi:10.1002/biot.200500008

    Article  CAS  PubMed  Google Scholar 

  10. Clomburg JM, Blankschien MD, Vick JE, Chou A, Kim S, Gonzalez R (2015) Integrated engineering of β-oxidation reversal and ω-oxidation pathways for the synthesis of medium chain ω-functionalized carboxylic acids. Metab Eng 28:202–212. doi:10.1016/j.ymben.2015.01.007

    Article  CAS  PubMed  Google Scholar 

  11. Demirbas A (2009) Progress and recent trends in biodiesel fuels. Energy Convers Manag 50:14–34. doi:10.1016/j.enconman.2008.09.001

    Article  CAS  Google Scholar 

  12. Fujita Y, Matsuoka H, Hirooka K (2007) Regulation of fatty acid metabolism in bacteria. Mol Microbiol 66:829–839. doi:10.1111/j.1365-2958.2007.05947.x

    Article  CAS  PubMed  Google Scholar 

  13. Garg S, Rizhsky L, Jin H, Yu X, Jing F, Yandeau-Nelson MD, Nikolau BJ (2016) Microbial production of bi-functional molecules by diversification of the fatty acid pathway. Metab Eng 35:9–20. doi:10.1016/j.ymben.2016.01.003

    Article  CAS  PubMed  Google Scholar 

  14. Grant C, Deszcz D, Wei Y-C et al (2014) Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes. Sci Rep. doi:10.1038/srep05844

    Google Scholar 

  15. Gross R, Lang K, Buehler K, Schmid A (2010) Characterization of a biofilm membrane reactor and its prospects for fine chemical synthesis. Biotechnol Bioeng 105:705–717. doi:10.1002/bit.22584

    CAS  PubMed  Google Scholar 

  16. Heeres AS, Picone CSF, van der Wielen LAM, Cunha RL, Cuellar MC (2014) Microbial advanced biofuels production: overcoming emulsification challenges for large-scale operation. Trends Biotechnol 32:221–229. doi:10.1016/j.tibtech.2014.02.002

    Article  CAS  PubMed  Google Scholar 

  17. Heipieper HJ, Weber FJ, Sikkema J, Keweloh H, de Bont JAM (1994) Mechanisms of resistance of whole cells to toxic organic solvents. Trends Biotechnol 12:409–415. doi:10.1016/0167-7799(94)90029-9

    Article  CAS  Google Scholar 

  18. Honda Malca S (2013) Substrate characterization and protein engineering of bacterial cytochrome P450 monooxygenases for the bio-based synthesis of omega-hydroxylated aliphatic compounds. PhD thesis, University of Stuttgart. doi:10.18419/opus-1388

  19. Jang H-Y, Jeon E-Y, Baek A-H, Lee S-M, Park J-B (2014) Production of ω-hydroxyundec-9-enoic acid and n-heptanoic acid from ricinoleic acid by recombinant Escherichia coli-based biocatalyst. Process Biochem 49:617–622. doi:10.1016/j.procbio.2014.01.025

    Article  CAS  Google Scholar 

  20. Julsing MK, Schrewe M, Cornelissen S, Hermann I, Schmid A, Bühler B (2012) Outer membrane protein AlkL boosts biocatalytic oxyfunctionalization of hydrophobic substrates in Escherichia coli. Appl Environ Microbiol 78:5724–5733. doi:10.1128/AEM.00949-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kalscheuer R, Stölting T, Steinbüchel A (2006) Microdiesel: Escherichia coli engineered for fuel production. Microbiology 152:2529–2536. doi:10.1099/mic.0.29028-0

    Article  CAS  PubMed  Google Scholar 

  22. Klein K, Steinberg R, Fiethen B, Overath P (1971) Fatty acid degradation in Escherichia coli. An inducible system for the uptake of fatty acids and further characterization of old mutants. Eur J Biochem 19:442–450. doi:10.1111/j.1432-1033.1971.tb01334.x

    Article  CAS  PubMed  Google Scholar 

  23. Kusunose M, Coon MJ, Kusunose E (1964) Enzymatic ω-oxidation of fatty acids: I. Products of octanoate, decanoate, and laurate oxidation. J Biol Chem 239:1374–1380

    CAS  PubMed  Google Scholar 

  24. Laane C, Boeren S, Vos K, Veeger C (1987) Rules for optimization of biocatalysis in organic solvents. Biotechnol Bioeng 30:81–87. doi:10.1002/bit.260300112

    Article  CAS  PubMed  Google Scholar 

  25. Ladkau N, Assmann M, Schrewe M, Julsing MK, Schmid A, Bühler B (2016) Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli. Metab Eng 36:1–9. doi:10.1016/j.ymben.2016.02.011

    Article  CAS  PubMed  Google Scholar 

  26. Leive L (1974) The barrier function of the Gram-negative envelope. Ann N Y Acad Sci 235:109–129. doi:10.1111/j.1749-6632.1974.tb43261.x

    Article  CAS  PubMed  Google Scholar 

  27. Lennen RM, Kruziki MA, Kumar K et al (2011) Membrane stresses induced by overproduction of free fatty acids in Escherichia coli. Appl Environ Microbiol 77:8114–8128. doi:10.1128/AEM.05421-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Lin S, Cronan JE (2011) Closing in on complete pathways of biotin biosynthesis. Mol BioSyst 7:1811–1821. doi:10.1039/c1mb05022b

    Article  CAS  PubMed  Google Scholar 

  29. Lin S, Hanson RE, Cronan JE (2010) Biotin synthesis begins by hijacking the fatty acid synthetic pathway. Nat Chem Biol 6:682–688. doi:10.1038/nchembio.420

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lundemo MT, Woodley JM (2015) Guidelines for development and implementation of biocatalytic P450 processes. Appl Microbiol Biotechnol 99:2465–2483. doi:10.1007/s00253-015-6403-x

    Article  CAS  PubMed  Google Scholar 

  31. McKenna EJ, Coon MJ (1970) Enzymatic ω-oxidation: IV. Purification and properties of the ω-hydroxylase of Pseudomonas oleovorans. J Biol Chem 245:3882–3889

    CAS  PubMed  Google Scholar 

  32. Nawabi P, Bauer S, Kyrpides N, Lykidis A (2011) Engineering Escherichia coli for biodiesel production utilizing a bacterial fatty acid methyltransferase. Appl Environ Microbiol 77:8052–8061. doi:10.1128/AEM.05046-11

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Nieboer M, Vis AJ, Witholt B (1996) Overproduction of a foreign membrane protein in Escherichia coli stimulates and depends on phospholipid synthesis. Eur J Biochem 241:691–696. doi:10.1111/j.1432-1033.1996.00691.x

    Article  CAS  PubMed  Google Scholar 

  34. Nikaido H (2003) Molecular basis of bacterial outer membrane permeability revisited. Microbiol Mol Biol Rev 67:593–656. doi:10.1128/mmbr.67.4.593-656.2003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Nunn WD (1986) A molecular view of fatty acid catabolism in Escherichia coli. Microbiol Rev 50:179–192

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Panke S, Meyer A, Huber CM, Witholt B, Wubbolts MG (1999) An alkane-responsive expression system for the production of fine chemicals. Appl Environ Microbiol 65:2324–2332

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Park JB, Bühler B, Habicher T, Hauer B, Panke S, Witholt B, Schmid A (2006) The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation. Biotechnol Bioeng 95:501–512. doi:10.1002/bit.21037

    Article  CAS  PubMed  Google Scholar 

  38. Peralta-Yahya PP, Zhang F, del Cardayre SB, Keasling JD (2012) Microbial engineering for the production of advanced biofuels. Nature 488:320–328. doi:10.1038/nature11478

    Article  CAS  PubMed  Google Scholar 

  39. Picataggio S, Rohrer T, Deanda K, Lanning D, Reynolds R, Mielenz J, Eirich LD (1992) Metabolic engineering of Candida tropicalis for the production of long–chain dicarboxylic acids. Bio/Technology 10:894–898. doi:10.1038/nbt0892-894

    Article  CAS  PubMed  Google Scholar 

  40. Riesenberg D (1991) High cell density cultivation of Escherichia coli. Curr Opin Biotechnol 2:380–384. doi:10.1016/S0958-1669(05)80142-9

    Article  CAS  PubMed  Google Scholar 

  41. Sambrook J, Russell DW (2001) Molecular cloning—a laboratory manual, 3rd edn. Cold Spring harbor Laboratory Press, New York

    Google Scholar 

  42. Sanishvili R, Yakunin AF, Laskowski RA et al (2003) Integrating structure, bioinformatics, and enzymology to discover function: BioH, a new carboxylesterase from Escherichia coli. J Biol Chem 278:26039–26045. doi:10.1074/jbc.M303867200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Sayers EW, Barrett T, Benson DA et al (2009) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res 37:D5–D15. doi:10.1093/nar/gkn741

    Article  CAS  PubMed  Google Scholar 

  44. Schrewe M, Julsing MK, Bühler B, Schmid A (2013) Whole-cell biocatalysis for selective and productive C–O functional group introduction and modification. Chem Soc Rev 42:6346–6377. doi:10.1039/c3cs60011d

    Article  CAS  PubMed  Google Scholar 

  45. Schrewe M, Julsing MK, Lange K, Czarnotta E, Schmid A, Bühler B (2014) Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization. Biotechnol Bioeng 111:1820–1830. doi:10.1002/bit.25248

    Article  CAS  PubMed  Google Scholar 

  46. Schrewe M, Ladkau N, Bühler B, Schmid A (2013) Direct terminal alkylamino-functionalization via multistep biocatalysis in one recombinant whole-cell catalyst. Adv Synth Catal 355:1693–1697. doi:10.1002/adsc.201200958

    Article  CAS  Google Scholar 

  47. Schrewe M, Magnusson AO, Willrodt C, Bühler B, Schmid A (2011) Kinetic analysis of terminal and unactivated C–H bond oxyfunctionalization in fatty acid methyl esters by monooxygenase-based whole-cell biocatalysis. Adv Synth Catal 353:3485–3495. doi:10.1002/adsc.201100440

    Article  CAS  Google Scholar 

  48. Shapiro MM, Chakravartty V, Cronan JE (2012) Remarkable diversity in the enzymes catalyzing the last step in synthesis of the pimelate moiety of biotin. PLoS One 7:e49440. doi:10.1371/journal.pone.0049440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Sherkhanov S, Korman TP, Bowie JU (2014) Improving the tolerance of Escherichia coli to medium-chain fatty acid production. Metab Eng 25:1–7. doi:10.1016/j.ymben.2014.06.003

    Article  CAS  PubMed  Google Scholar 

  50. Sherkhanov S, Korman TP, Clarke SG, Bowie JU (2016) Production of FAME biodiesel in E. coli by direct methylation with an insect enzyme. Sci Rep 6:24239. doi:10.1038/srep24239

  51. Shi Y, Pan Y, Li B, He W, She Q, Chen L (2013) Molecular cloning of a novel bioH gene from an environmental metagenome encoding a carboxylesterase with exceptional tolerance to organic solvents. BMC Biotechnol 13:13. doi:10.1186/1472-6750-13-13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Steen EJ, Kang YS, Bokinsky G, Hu ZH, Schirmer A, McClure A, del Cardayre SB, Keasling JD (2010) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463:559–563. doi:10.1038/nature08721

    Article  CAS  PubMed  Google Scholar 

  53. Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM (2011) Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Process Res Dev 15:266–274. doi:10.1021/op1002165

    Article  CAS  Google Scholar 

  54. Xie X, Wong WW, Tang Y (2007) Improving simvastatin bioconversion in Escherichia coli by deletion of bioH. Metab Eng 9:379–386. doi:10.1016/j.ymben.2007.05.006

    Article  CAS  PubMed  Google Scholar 

  55. Yan Y, Li X, Wang G, Gui X, Li G, Su F, Wang X, Liu T (2014) Biotechnological preparation of biodiesel and its high-valued derivatives: a review. Appl Energy 113:1614–1631. doi:10.1016/j.apenergy.2013.09.029

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The results contribute to the programme topic Solar Fuels funded by the Helmholtz Research Programme, co-financed by the German Federal Ministry of Education and Research (BMBF, Grant Number 0316044A). The authors are grateful for financial support of the Centre for Biocatalysis (MiKat) at the Helmholtz Centre for Environmental Research by European Regional Development Funds (EFRE—Europe funds Saxony) and the Helmholtz Association. We thank Britta Dettweiler (former Laboratory of Chemical Biotechnology, TU Dortmund University) for experimental support. MK, AS, and BB are thankful for using infrastructure of TU Dortmund University at the former Laboratory of Chemical Biotechnology.

Author information

Authors and Affiliations

  1. Department Solar Materials, Helmholtz Centre for Environmental Research–UFZ, Permoserstr. 15, 04318, Leipzig, Germany

    Marvin Kadisch, Andreas Schmid & Bruno Bühler

Authors
  1. Marvin Kadisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Schmid
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bruno Bühler
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bruno Bühler.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kadisch, M., Schmid, A. & Bühler, B. Hydrolase BioH knockout in E. coli enables efficient fatty acid methyl ester bioprocessing. J Ind Microbiol Biotechnol 44, 339–351 (2017). https://doi.org/10.1007/s10295-016-1890-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-016-1890-z

Keywords

Search

Navigation