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Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

  • Biotechnological products and process engineering
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Abstract

Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this study, we have used the CYP153A heme domain from Marinobacter aquaeolei fused to the reductase domain of CYP102A1 from Bacillus megaterium (BM3) expressed in Escherichia coli. This self-sufficient protein chimera CYP153A-CPRBM3 G307A mutant is able to selectively hydroxylate medium and long chain length fatty acids at the terminal position. ω-Hydroxylated fatty acids can be used in the field of high-end polymers and in the cosmetic and fragrance industry. Here, we have identified the limitations for implementation of a whole-cell P450-catalyzed reaction by characterizing the chosen biocatalyst as well as the reaction system. Despite a well-studied whole-cell P450 catalyst, low activity and poor stability of the artificial fusion construct are the main identified limitations to reach sufficient biocatalyst yield (mass of product/mass of biocatalyst) and space-time yield (volumetric productivity) essential for an economically feasible process. Substrate and product inhibition are also challenges that need to be addressed, and the application of solid substrate is shown to be a promising option to improve the process.

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References

  • Abe A, Sugiyama K (2005) Growth inhibition and apoptosis induction of human melanoma cells by ω-hydroxy fatty acids. Anti-Cancer Drugs 16:543–549

    Article  PubMed  CAS  Google Scholar 

  • Bernhardt R (2006) Cytochromes P450 as versatile biocatalysts. J Biotechnol 124:128–145

    Article  PubMed  CAS  Google Scholar 

  • Bernhardt R, Urlacher VB (2014) Cytochromes P450 as promising catalysts for biotechnological application: chances and limitations. Appl Microbiol Biotechnol 98:6185–6203

    Article  PubMed  CAS  Google Scholar 

  • Biermann U, Bornscheuer U, Meier MAR, Metzger JO, Schaefer HJ (2011) Oils and fats as renewable raw materials in chemistry. Angew Chem Int Ed 50:3854–3871

    Article  CAS  Google Scholar 

  • Bordeaux M, Galarneau A, Fajula F, Drone J (2011) A regioselective biocatalyst for alkane activation under mild conditions. Angew Chem Int Ed 123:2123–2127

    Article  Google Scholar 

  • ChemSpider (a) CSID:3756, http://www.chemspider.com/Chemical-Structure.3756.html

  • ChemSpider (b) CSID:71366, http://www.chemspider.com/Chemical-Structure.71366.html

  • Cirino PC, Arnold FH (2003) A self‐sufficient peroxide‐driven hydroxylation biocatalyst. Angew Chem Int Ed 115:3421–3423

    Article  Google Scholar 

  • Cornelissen S, Julsing MK, Volmer J, Riechert O, Schmid A, Bühler B (2013) Whole‐cell‐based CYP153A6‐catalyzed (S)‐limonene hydroxylation efficiency depends on host background and profits from monoterpene uptake via AlkL. Biotechnol Bioeng 110:1282–1292

    Article  PubMed  CAS  Google Scholar 

  • Cornish-Bowden A (2012) Fundamentals of enzyme kinetics, 4th edn. Wiley-VCH Verlag & Co, Weinheim, Germany

    Google Scholar 

  • Craft DL, Madduri KM, Eshoo M, Wilson CR (2003) Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to α, ω-dicarboxylic acids. Appl Environ Microbiol 69:5983–5991

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Fujii T, Narikawa T, Sumisa F, Arisawa A, Takeda K, Kato J (2006) Production of α, ω-alkanediols using Escherichia coli expressing a cytochrome P450 from Acinetobacter sp. OC4. Biosci Biotechnol Biochem 70:1379–1385

    Article  PubMed  CAS  Google Scholar 

  • Gudiminchi RK, Randall C, Opperman DJ, Olaofe OA, Harrison STL, Albertyn J, Smit MS (2012) Whole-cell hydroxylation of n-octane by Escherichia coli strains expressing the CYP153A6 operon. Appl Microbiol Biotechnol 96:1507–1516

    Article  PubMed  CAS  Google Scholar 

  • Huf S, Kruegener S, Hirth T, Rupp S, Zibek S (2011) Biotechnological synthesis of long-chain dicarboxylic acids as building blocks for polymers. Eur J Lipid Sci Technol 113:548–561

    Article  CAS  Google Scholar 

  • Kiss FM, Lundemo MT, Zapp J, Woodley JM, Bernhardt R (2015) Process development for the production of 15β-hydroxycyproterone acetate using Bacillus megaterium expressing CYP106A2 as whole-cell biocatalyst. Microb Cell Fact 14

  • Kitazume T, Tanaka A, Takaya N, Nakamura A, Matsuyama S, Suzuki T, Shoun H (2002) Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system. Eur J Biochem 269:2075–2082

    Article  PubMed  CAS  Google Scholar 

  • Labinger JA (2004) Selective alkane oxidation: hot and cold approaches to a hot problem. J Mol Catal A Chem 220:27–35

    Article  CAS  Google Scholar 

  • Law HEM, Baldwin CVF, Chen BH, Woodley JM (2006) Process limitations in a whole-cell catalysed oxidation: sensitivity analysis. Chem Eng Sci 61:6646–6652

    Article  CAS  Google Scholar 

  • Lee W, Kim M, Jin Y, Seo J (2013) Engineering of NADPH regenerators in Escherichia coli for enhanced biotransformation. Appl Microbiol Biotechnol 97:2761–2772

    Article  PubMed  CAS  Google Scholar 

  • Liu C, Liu F, Cai J, Xie W, Long TE, Turner SR, Lyons A, Gross RA (2011) Polymers from fatty acids: poly (ω-hydroxyl tetradecanoic acid) synthesis and physico-mechanical studies. Biomacromolecules 12:3291–3298

    Article  PubMed  CAS  Google Scholar 

  • Lu W, Ness JE, Xie W, Zhang X, Minshull J, Gross RA (2010) Biosynthesis of monomers for plastics from renewable oils. J Am Chem Soc 132:15451–15455

    Article  PubMed  CAS  Google Scholar 

  • Lundemo MT, Woodley JM (2015) Guidelines for development and implementation of biocatalytic P450 processes. Appl Microbiol Biotechnol 99:2465–2483

    Article  PubMed  CAS  Google Scholar 

  • Maier T, Forster H, Asperger O, Hahn U (2001) Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104. Biochem Biophys Res Commun 286:652–658

    Article  PubMed  CAS  Google Scholar 

  • Malca SH, Scheps D, Kühnel L, Venegas-Venegas E, Seifert A, Nestl BM, Hauer B (2012) Bacterial CYP153A monooxygenases for the synthesis of omega-hydroxylated fatty acids. Chem Commun 48:5115–5117

    Article  Google Scholar 

  • Marisch K, Bayer K, Cserjan-Puschmann M, Luchner M, Striedner G (2013) Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Microb Cell Fact 12

  • Narhi LO, Fulco AJ (1987) Identification and characterization of two functional domains in cytochrome P-450BM-3, a catalytically self-sufficient monooxygenase induced by barbiturates in Bacillus megaterium. J Biol Chem 262:6683–6690

    PubMed  CAS  Google Scholar 

  • Nelson DR (2009) The cytochrome P450 homepage. Hum Genomics 4:59–65

    PubMed  CAS  PubMed Central  Google Scholar 

  • Olaofe OA, Fenner CJ, Gudiminchi RK, Smit MS, Harrison STL (2013) The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6. Microb Cell Fact 12:8

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Omura T, Sato R (1964) The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J Biol Chem 239:2370–2378

    PubMed  CAS  Google Scholar 

  • O’Reilly E, Köhler V, Flitsch SL, Turner NJ (2011) Cytochromes P450 as useful biocatalysts: addressing the limitations. Chem Commun 47:2490–2501

    Article  Google Scholar 

  • Paul CE, Gargiulo S, Opperman DJ, Lavandera I, Gotor-Fernandez V, Gotor V, Taglieber A, Arends IWCE, Hollmann F (2013) Mimicking nature: synthetic nicotinamide cofactors for C = C bioreduction using enoate reductases. Org Lett 15:180–183

    Article  PubMed  CAS  Google Scholar 

  • Rammler DH (1967) The effect of DMSO on several enzyme systems. Ann N Y Acad Sci 141:291–299

    Article  PubMed  CAS  Google Scholar 

  • 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 97:8317–8327

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Sadeghi SJ, Fantuzzi A, Gilardi G (2011) Breakthrough in P 450 bioelectrochemistry and future perspectives. BBA-Protein Proteomics 1814:237–248

    Article  CAS  Google Scholar 

  • Salazar O, Cirino PC, Arnold FH (2003) Thermostabilization of a cytochrome P450 peroxygenase. Chembiochem 4:891–893

    Article  PubMed  CAS  Google Scholar 

  • Scheps D, Malca SH, Hoffmann H, Nestl BM, Hauer B (2011) Regioselective ω-hydroxylation of medium-chain n-alkanes and primary alcohols by CYP153 enzymes from Mycobacterium marinum and Polaromonas sp. strain JS666. Org Biomol Chem 9:6727–6733

    Article  PubMed  CAS  Google Scholar 

  • Scheps D, Honda Malca S, Richter SM, Marisch K, Nestl BM, Hauer B (2013) Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct. Microb Biotechnol 6:694–707

    PubMed  CAS  PubMed Central  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Siriphongphaew A, Pisnupong P, Wongkongkatep J, Inprakhon P, Vangnai AS, Honda K, Ohtake H, Kato J, Ogawa J, Shimizu S (2012) Development of a whole-cell biocatalyst co-expressing P450 monooxygenase and glucose dehydrogenase for synthesis of epoxyhexane. Appl Microbiol Biotechnol 95:357–367

    Article  PubMed  CAS  Google Scholar 

  • Sligar SG, Cinti DL, Gibson GG, Schenkman JB (1979) Spin state control of the hepatic cytochrome P450 redox potential. Biochem Biophys Res Commun 90:925–932

    Article  PubMed  CAS  Google Scholar 

  • Stumpp T, Wilms B, Altenbuchner J (2000) Ein neues L-rhamnose-induzierbares expressionssystem für Escherichia coli. Biospektrum 6:33–36

    CAS  Google Scholar 

  • Tran N, Nguyen D, Dwaraknath S, Mahadevan S, Chavez G, Nguyen A, Dao T, Mullen S, Nguyen T, Cheruzel LE (2013) An efficient light-driven P450 BM3 biocatalyst. J Am Chem Soc 135:14484–14487

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Vallon T, Glemser M, Honda Malca S, Scheps D, Schmid J, Siemann-Herzberg M, Hauer B, Takors R (2013) Production of 1-octanol from n-octane by Pseudomonas putida KT2440. Chem Ing Tech 85:841–848

    Article  CAS  Google Scholar 

  • van Beilen JB, Smits THM, Whyte LG, Schorcht S, Roethlisberger M, Plaggemeier T, Engesser K, Witholt B (2002) Alkane hydroxylase homologues in gram-positive strains. Environ Microbiol 4:676–682

    Article  PubMed  Google Scholar 

  • van Beilen JB, Duetz WA, Schmid A, Witholt B (2003) Practical issues in the application of oxygenases. Trends Biotechnol 21:170–177

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We gratefully acknowledge the funding from the People Programme (Marie Curie Actions) of the European Union’s 7th Framework Programme (FP7/2007-2013) under REA Grant Agreement 289217.

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    Authors and Affiliations

    1. Department of Chemical and Biochemical Engineering, Technical University of Denmark, Søltofts Plads, Building 229, DK-2800, Kgs. Lyngby, Denmark

      M. T. Lundemo & J. M. Woodley

    2. Institute of Technical Biochemistry, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

      S. Notonier & B. Hauer

    3. Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria

      G. Striedner

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    1. M. T. Lundemo
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    2. S. Notonier
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    5. J. M. Woodley
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    Correspondence to J. M. Woodley.

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    We dedicate this work to Prof. Dr. Bernard Witholt.

    M. T. Lundemo and S. Notonier contributed equally to this work.

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    Lundemo, M.T., Notonier, S., Striedner, G. et al. Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli . Appl Microbiol Biotechnol 100, 1197–1208 (2016). https://doi.org/10.1007/s00253-015-6999-x

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    • DOI: https://doi.org/10.1007/s00253-015-6999-x

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