Preloader

Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

  • 1.

    Hughes, G. & Lewis, J. C. Introduction: Biocatalysis in industry. Chem. Rev. 118, 1–3. https://doi.org/10.1021/acs.chemrev.7b00741 (2018).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 2.

    Woodley, J. M. Accelerating the implementation of biocatalysis in industry. Appl. Microbiol. Biotechnol. 103, 4733–4739. https://doi.org/10.1007/s00253-019-09796-x (2019).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 3.

    Aalbers, F. S. & Fraaije, M. W. Enzyme fusions in biocatalysis: Coupling reactions by pairing enzymes. ChemBioChem 20, 20–28. https://doi.org/10.1002/cbic.201800394 (2019).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 4.

    Rabe, K. S., Müller, J., Skoupi, M. & Niemeyer, C. M. Cascades in compartments: En route to machine-assisted biotechnology. Angew. Chem. Int. Ed. Engl. 56, 13574–13589. https://doi.org/10.1002/anie.201703806 (2017).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 5.

    Ellis, G. A. et al. Artificial multienzyme scaffolds: Pursuing in vitro substrate channeling with an overview of current progress. ACS Catal. 9, 10812–10869. https://doi.org/10.1021/acscatal.9b02413 (2019).

    CAS 
    Article 

    Google Scholar 

  • 6.

    Belsare, K. D., Ruff, A. J., Martinez, R. & Schwaneberg, U. Insights on intermolecular FMN-heme domain interaction and the role of linker length in cytochrome P450cin fusion proteins. Biol. Chem. 401, 1249–1255. https://doi.org/10.1515/hsz-2020-0134 (2020).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 7.

    Bakkes, P. J. et al. Design and improvement of artificial redox modules by molecular fusion of flavodoxin and flavodoxin reductase from Escherichia coli. Sci. Rep. 5, 12158. https://doi.org/10.1038/srep12158 (2015).

    ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 8.

    Chen, X., Zaro, J. L. & Shen, W.-C. Fusion protein linkers: Property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369. https://doi.org/10.1016/j.addr.2012.09.039 (2013).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 9.

    Aalbers, F. S. & Fraaije, M. W. Coupled reactions by coupled enzymes: Alcohol to lactone cascade with alcohol dehydrogenase–cyclohexanone monooxygenase fusions. Appl. Microbiol. Biotechnol. 101, 7557–7565. https://doi.org/10.1007/s00253-017-8501-4 (2017).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 10.

    Peters, C., Rudroff, F., Mihovilovic, M. D. & Bornscheuer, U. T. Fusion proteins of an enoate reductase and a Baeyer–Villiger monooxygenase facilitate the synthesis of chiral lactones. Biol. Chem. 398, 31–37. https://doi.org/10.1515/hsz-2016-0150 (2017).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 11.

    Schulz, S., Schumacher, D., Raszkowski, D., Girhard, M. & Urlacher, V. B. Fusion to hydrophobin HFBI improves the catalytic performance of a cytochrome P450 system. Front. Bioeng. Biotechnol. 4, 57–57. https://doi.org/10.3389/fbioe.2016.00057 (2016).

    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 12.

    Bakkes, P. J. et al. Engineering of versatile redox partner fusions that support monooxygenase activity of functionally diverse cytochrome P450s. Sci. Rep. 7, 9570. https://doi.org/10.1038/s41598-017-10075-w (2017).

    ADS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 13.

    Lerchner, A., Daake, M., Jarasch, A. & Skerra, A. Fusion of an alcohol dehydrogenase with an aminotransferase using a pas linker to improve coupled enzymatic alcohol-to-amine conversion. Protein Eng. Des. Sel. 29, 557–562. https://doi.org/10.1093/protein/gzw039 (2016).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 14.

    Sibbesen, O., De Voss, J. J. & Montellano, P. R. Putidaredoxin reductase-putidaredoxin-cytochrome P450cam triple fusion protein. Construction of a self-sufficient Escherichia coli catalytic system. J. Biol. Chem. 271, 22462–22469. https://doi.org/10.1074/jbc.271.37.22462 (1996).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 15.

    Lu, P. & Feng, M. G. Bifunctional enhancement of a β-glucanase-xylanase fusion enzyme by optimization of peptide linkers. Appl. Microbiol. Biotechnol. 79, 579–587. https://doi.org/10.1007/s00253-008-1468-4 (2008).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 16.

    Spivey, H. O. & Ovádi, J. Substrate channeling. Methods 19, 306–321. https://doi.org/10.1006/meth.1999.0858 (1999).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 17.

    Jorgensen, K. et al. Metabolon formation and metabolic channeling in the biosynthesis of plant natural products. Curr. Opin. Plant Biol. 8, 280–291. https://doi.org/10.1016/j.pbi.2005.03.014 (2005).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 18.

    Sweetlove, L. J. & Fernie, A. R. The role of dynamic enzyme assemblies and substrate channelling in metabolic regulation. Nat. Commun. 9, 2136–2136. https://doi.org/10.1038/s41467-018-04543-8 (2018).

    ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 19.

    Zhang, Y., Tsitkov, S. & Hess, H. Proximity does not contribute to activity enhancement in the glucose oxidase–horseradish peroxidase cascade. Nat. Commun. 7, 13982. https://doi.org/10.1038/ncomms13982 (2016).

    ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 20.

    Kuzmak, A., Carmali, S., von Lieres, E., Russell, A. J. & Kondrat, S. Can enzyme proximity accelerate cascade reactions?. Sci. Rep. 9, 455. https://doi.org/10.1038/s41598-018-37034-3 (2019).

    ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 21.

    Poshyvailo, L., von Lieres, E. & Kondrat, S. Does metabolite channeling accelerate enzyme-catalyzed cascade reactions?. PLoS One 12, e0172673. https://doi.org/10.1371/journal.pone.0172673 (2017).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 22.

    Jeon, E.-Y., Baek, A. H., Bornscheuer, U. T. & Park, J.-B. Enzyme fusion for whole-cell biotransformation of long-chain sec-alcohols into esters. Appl. Microbiol. Biotechnol. 99, 6267–6275. https://doi.org/10.1007/s00253-015-6392-9 (2015).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 23.

    Iturrate, L., Sánchez-Moreno, I., Oroz-Guinea, I., Pérez-Gil, J. & García-Junceda, E. Preparation and characterization of a bifunctional aldolase/kinase enzyme: A more efficient biocatalyst for C–C bond formation. Chem. A Eur. J. 16, 4018–4030. https://doi.org/10.1002/chem.200903096 (2010).

    CAS 
    Article 

    Google Scholar 

  • 24.

    Mourelle-Insua, Á., Aalbers, F. S., Lavandera, I., Gotor-Fernández, V. & Fraaije, M. W. What to sacrifice? Fusions of cofactor regenerating enzymes with Baeyer–Villiger monooxygenases and alcohol dehydrogenases for self-sufficient redox biocatalysis. Tetrahedron 75, 1832–1839. https://doi.org/10.1016/j.tet.2019.02.015 (2019).

    CAS 
    Article 

    Google Scholar 

  • 25.

    Lau, P. C. K. et al. Sustained development in Baeyer–Villiger biooxidation technology. In Green Polymer Chemistry: Biocatalysis And Biomaterials vol. 1043 ACS Symposium Series Ch. 24, 343–372 (American Chemical Society, 2010).

  • 26.

    Beyer, N. et al. P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations. Appl. Microbiol. Biotechnol. 101, 2319–2331. https://doi.org/10.1007/s00253-016-7993-7 (2017).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 27.

    Noble, M. A. et al. Roles of key active-site residues in flavocytochrome P450 BM3. Biochem. J. 339(Pt 2), 371–379. https://doi.org/10.1042/bj3390371 (1999).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 28.

    Solé, J., Caminal, G., Schürmann, M., Álvaro, G. & Guillén, M. Co-immobilization of P450 BM3 and glucose dehydrogenase on different supports for application as a self-sufficient oxidative biocatalyst. J. Chem. Technol. Biotechnol. 94, 244–255. https://doi.org/10.1002/jctb.5770 (2019).

    CAS 
    Article 

    Google Scholar 

  • 29.

    Tan, C. Y., Hirakawa, H. & Nagamune, T. Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel. Sci. Rep. 5, 8648. https://doi.org/10.1038/srep08648 (2015).

    ADS 
    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 30.

    Maurer, S. C., Schulze, H., Schmid, R. D. & Urlacher, V. Immobilisation of P450 BM-3 and an NADP+ cofactor recycling system: Towards a technical application of heme-containing monooxygenases in fine chemical synthesis. Adv. Synth. Catal. 345, 802–810. https://doi.org/10.1002/adsc.200303021 (2003).

    CAS 
    Article 

    Google Scholar 

  • 31.

    Tishkov, V. I. et al. Pilot scale production and isolation of recombinant NAD+– and NADP+-specific formate dehydrogenases. Biotechnol. Bioeng. 64, 187–193 (1999).

    CAS 
    Article 

    Google Scholar 

  • 32.

    Schwarz-Linek, U. et al. Synthesis of natural product precursors by Baeyer–Villiger oxidation with cyclohexanone monooxygenase from Acinetobacter. Synthesis 0947–0951, 2001. https://doi.org/10.1055/s-2001-13394 (2001).

    Article 

    Google Scholar 

  • 33.

    Rissom, S., Schwarz-Linek, U., Vogel, M., Tishkov, V. I. & Kragl, U. Synthesis of chiral ε-lactones in a two-enzyme system of cyclohexanone mono-oxygenase and formate dehydrogenase with integrated bubble-free aeration. Tetrahedron Asymmetry 8, 2523–2526. https://doi.org/10.1016/S0957-4166(97)00311-X (1997).

    CAS 
    Article 

    Google Scholar 

  • 34.

    Tishkov, V. I. & Popov, V. O. Protein engineering of formate dehydrogenase. Biomol. Eng. 23, 89–110. https://doi.org/10.1016/j.bioeng.2006.02.003 (2006).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 35.

    Alekseeva, A. A. et al. The role of Ala198 in the stability and coenzyme specificity of bacterial formate dehydrogenases. Acta Nat. 7, 60–69. https://doi.org/10.32607/20758251-2015-7-1-60-69 (2015).

    CAS 
    Article 

    Google Scholar 

  • 36.

    Zhang, Y., Wang, Y., Wang, S. & Fang, B. Engineering bi-functional enzyme complex of formate dehydrogenase and leucine dehydrogenase by peptide linker mediated fusion for accelerating cofactor regeneration. Eng. Life Sci. 17, 989–996. https://doi.org/10.1002/elsc.201600232 (2017).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 37.

    Hölsch, K. & Weuster-Botz, D. Enantioselective reduction of prochiral ketones by engineered bifunctional fusion proteins. Biotechnol. Appl. Biochem. 56, 131–140. https://doi.org/10.1042/BA20100143 (2010).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 38.

    Girvan, H. M. et al. Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3. J. Biol. Chem. 282, 564–572. https://doi.org/10.1074/jbc.M607949200 (2007).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 39.

    Hecht, A. et al. Measurements of translation initiation from all 64 codons in E. coli. Nucleic Acids Res. 45, 3615–3626. https://doi.org/10.1093/nar/gkx070 (2017).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 40.

    Sührer, I., Haslbeck, M. & Castiglione, K. Asymmetric synthesis of a fluoxetine precursor with an artificial fusion protein of a ketoreductase and a formate dehydrogenase. Process Biochem. 49, 1527–1532. https://doi.org/10.1016/j.procbio.2014.06.001 (2014).

    CAS 
    Article 

    Google Scholar 

  • 41.

    Galkin, A., Kulakova, L., Tishkov, V., Esaki, N. & Soda, K. Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10. Appl. Microbiol. Biotechnol. 44, 479–483. https://doi.org/10.1007/bf00169947 (1995).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 42.

    Boddupalli, S. S., Pramanik, B. C., Slaughter, C. A., Estabrook, R. W. & Peterson, J. A. Fatty acid monooxygenation by P450BM-3: Product identification and proposed mechanisms for the sequential hydroxylation reactions. Arch. Biochem. Biophys. 292, 20–28. https://doi.org/10.1016/0003-9861(92)90045-X (1992).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 43.

    Urlacher, V. B., Makhsumkhanov, A. & Schmid, R. D. Biotransformation of β-ionone by engineered cytochrome P450 BM-3. Appl. Microbiol. Biotechnol. 70, 53–59. https://doi.org/10.1007/s00253-005-0028-4 (2006).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 44.

    Zehentgruber, D., Urlacher, V. B. & Lütz, S. Studies on the enantioselective oxidation of β-ionone with a whole E. coli system expressing cytochrome P450 monooxygenase BM3. J. Mol. Catal. B Enzym. 84, 62–64. https://doi.org/10.1016/j.molcatb.2012.05.014 (2012).

    CAS 
    Article 

    Google Scholar 

  • 45.

    Bernhardt, R. Cytochrome P450: Structure, function, and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol. 127, 137–221. https://doi.org/10.1007/BFb0048267 (1996).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 46.

    Neeli, R. et al. The dimeric form of flavocytochrome P450 BM3 is catalytically functional as a fatty acid hydroxylase. FEBS Lett. 579, 5582–5588. https://doi.org/10.1016/j.febslet.2005.09.023 (2005).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 47.

    Munro, A. W., Gordon Lindsay, J., Coggins, J. R., Kelly, S. M. & Price, N. C. Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3. Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1296, 127–137. https://doi.org/10.1016/0167-4838(96)00061-1 (1996).

    Article 

    Google Scholar 

  • 48.

    Forneris, F., Orru, R., Bonivento, D., Chiarelli, L. R. & Mattevi, A. ThermoFAD, a Thermofluor®-adapted flavin ad hoc detection system for protein folding and ligand binding. FEBS J. 276, 2833–2840. https://doi.org/10.1111/j.1742-4658.2009.07006.x (2009).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 49.

    Sadykhov, E. G. et al. A comparative study of the thermal stability of formate dehydrogenases from microorganisms and plants. Appl. Biochem. Microbiol. 42, 236–240. https://doi.org/10.1134/S0003683806030021 (2006).

    CAS 
    Article 

    Google Scholar 

  • 50.

    Jamakhandi, A. P., Jeffus, B. C., Dass, V. R. & Miller, G. P. Thermal inactivation of the reductase domain of cytochrome P450 BM3. Arch. Biochem. Biophys. 439, 165–174. https://doi.org/10.1016/j.abb.2005.04.022 (2005).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 51.

    Hummel, W. New alcohol dehydrogenases for the synthesis of chiral compounds. Adv. Biochem. Eng. Biotechnol. 58, 145–184. https://doi.org/10.1007/bfb0103304 (1997).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 52.

    Yang, H., Liu, L. & Xu, F. The promises and challenges of fusion constructs in protein biochemistry and enzymology. Appl. Microbiol. Biotechnol. 100, 8273–8281. https://doi.org/10.1007/s00253-016-7795-y (2016).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 53.

    Elleuche, S. Bringing functions together with fusion enzymes—From nature’s inventions to biotechnological applications. Appl. Microbiol. Biotechnol. 99, 1545–1556. https://doi.org/10.1007/s00253-014-6315-1 (2015).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 54.

    Jiang, W. & Fang, B.-S. Construction and evaluation of a novel bifunctional phenylalanine–formate dehydrogenase fusion protein for bienzyme system with cofactor regeneration. J. Ind. Microbiol. Biotechnol. 43, 577–584. https://doi.org/10.1007/s10295-016-1738-6 (2016).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 55.

    Goodman, D. B., Church, G. M. & Kosuri, S. Causes and effects of n-terminal codon bias in bacterial genes. Science 342, 475–479. https://doi.org/10.1126/science.1241934 (2013).

    ADS 
    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 56.

    Wheeldon, I. et al. Substrate channelling as an approach to cascade reactions. Nat. Chem. 8, 299–309. https://doi.org/10.1038/nchem.2459 (2016).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 57.

    Thomik, T., Wittig, I., Choe, J.-Y., Boles, E. & Oreb, M. An artificial transport metabolon facilitates improved substrate utilization in yeast. Nat. Chem. Biol. 13, 1158–1163. https://doi.org/10.1038/nchembio.2457 (2017).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 58.

    Lamzin, V. S., Dauter, Z., Popov, V. O., Harutyunyan, E. H. & Wilson, K. S. High resolution structures of holo and apo formate dehydrogenase. J. Mol. Biol. 236, 759–785. https://doi.org/10.1006/jmbi.1994.1188 (1994).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 59.

    Corrado, M. L., Knaus, T. & Mutti, F. G. A chimeric styrene monooxygenase with increased efficiency in asymmetric biocatalytic epoxidation. ChemBioChem 19, 679–686. https://doi.org/10.1002/cbic.201700653 (2018).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 60.

    Hollmann, F., Lin, P. C., Witholt, B. & Schmid, A. Stereospecific biocatalytic epoxidation: The first example of direct regeneration of a fad-dependent monooxygenase for catalysis. J. Am. Chem. Soc. 125, 8209–8217. https://doi.org/10.1021/ja034119u (2003).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 61.

    Kuper, J. et al. The role of active-site Phe87 in modulating the organic co-solvent tolerance of cytochrome P450 BM3 monooxygenase. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 68, 1013–1017. https://doi.org/10.1107/S1744309112031570 (2012).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 62.

    Seng Wong, T., Arnold, F. H. & Schwaneberg, U. Laboratory evolution of cytochrome P450 BM-3 monooxygenase for organic cosolvents. Biotechnol. Bioeng. 85, 351–358. https://doi.org/10.1002/bit.10896 (2004).

    CAS 
    Article 

    Google Scholar 

  • 63.

    Zhang, H. et al. The full-length cytochrome P450 enzyme CYP102A1 dimerizes at its reductase domains and has flexible heme domains for efficient catalysis. J. Biol. Chem. 293, 7727–7736. https://doi.org/10.1074/jbc.RA117.000600 (2018).

    CAS 
    Article 
    PubMed 
    PubMed Central 

    Google Scholar 

  • 64.

    Omura, T. & Sato, R. The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J. Biol. Chem. 239, 2379–2385. https://doi.org/10.1016/S0021-9258(20)82245-5 (1964).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 65.

    Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. https://doi.org/10.1006/abio.1976.9999 (1976).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 66.

    Schwaneberg, U., Schmidt-Dannert, C., Schmitt, J. & Schmid, R. D. A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A. Anal. Biochem. 269, 359–366. https://doi.org/10.1006/abio.1999.4047 (1999).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 67.

    Sekuzu, I., Orii, Y. & Okunuki, K. Studies on cytochrome C1*: I. Isolation, purification and properties of cytochrome C1 from heart muscle. J. Biochem. 48, 214–225. https://doi.org/10.1093/oxfordjournals.jbchem.a127162 (1960).

    CAS 
    Article 

    Google Scholar 

  • 68.

    Van Gelder, B. F. & Slater, E. C. The extinction coefficient of cytochrome c. Biochim. Biophys. Acta 58, 593–595. https://doi.org/10.1016/0006-3002(62)90073-2 (1962).

    CAS 
    Article 
    PubMed 

    Google Scholar 

  • 69.

    Hummel, W. & Riebel, B. Alcohol dehydrogenase and its use for the enzymatic production of chiral hydroxy compounds. United States. US 6,225,099 B1 (1999).

  • Source link