Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Modification of N-Terminal Amino Acids of Fungal Benzoate Hydroxylase (CYP53A15) for the Production of p-Hydroxybenzoate and Optimization of Bioproduction Conditions in Escherichia coli

  • Tamaki, Shun (Division of Signal Responses, Biosignal Research Center, Kobe University) ;
  • Yagi, Mitsuhiko (Division of Signal Responses, Biosignal Research Center, Kobe University) ;
  • Nishihata, Yuki (Division of Signal Responses, Biosignal Research Center, Kobe University) ;
  • Yamaji, Hideki (Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University) ;
  • Shigeri, Yasushi (Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST)) ;
  • Uno, Tomohide (Laboratory of Biochemistry, Graduate School of Agricultural Science, Kobe University) ;
  • Imaishi, Hiromasa (Division of Signal Responses, Biosignal Research Center, Kobe University)
  • Received : 2017.11.16
  • Accepted : 2018.01.30
  • Published : 2018.03.28
Download PDF
⟨ Previous Next ⟩

Abstract

The aromatic compound p-hydroxybenzoate (PHBA) is an important material with multiple applications, including as a building block of liquid crystal polymers in chemical industries. The cytochrome P450 (CYP) enzymes are beneficial monooxygenases for the synthesis of chemicals, and CYP53A15 from fungus Cochliobolus lunatus is capable of executing the hydroxylation from benzoate to PHBA. Here, we constructed a system for the bioconversion of benzoate to PHBA in Escherichia coli cells coexpressing CYP53A15 and human NADPH-P450 oxidoreductase (CPR) genes as a redox partner. For suitable coexpression of CYP53A15 and CPR, we originally constructed five plasmids in which we replaced the N-terminal transmembrane region of CYP53A15 with a portion of the N-terminus of various mammalian P450s. PHBA productivity was the greatest when CYP53A15 expression was induced at $20^{\circ}C$ in $2{\times}YT$ medium in host E. coli strain ${\Delta}gcvR$ transformed with an N-terminal transmembrane region of rabbit CYP2C3. By optimizing each reaction condition (reaction temperature, substrate concentration, reaction time, and E. coli cell concentration), we achieved 90% whole-cell conversion of benzoate. Our data demonstrate that the described novel E. coli bioconversion system is a more efficient tool for PHBA production from benzoate than the previously described yeast system.

Keywords

References

  1. Padias AB, Hall HK. 2011. Mechanism studies of LCP synthesis. Polymers 3: 833-845. https://doi.org/10.3390/polym3020833
  2. Nelson DR. 1999. Cytochrome P450 and the individuality of species. Arch. Biochem. Biophys. 369: 1-10. https://doi.org/10.1006/abbi.1999.1352
  3. Omura T, Takesue S. 1970. New method for simultaneous purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes. J. Biochem. 67: 249-257.
  4. Bernhardt R. 2006. Cytochromes P450 as versatile biocatalysts. J. Biotechnol. 124: 128-145. https://doi.org/10.1016/j.jbiotec.2006.01.026
  5. O'Reilly E, Köhler V, Flitsch SL, Turner NJ. 2011. Cytochromes P450 as useful biocatalysts: addressing the limitations. Chem. Commun. (Camb.) 47: 2490-2501. https://doi.org/10.1039/c0cc03165h
  6. Lah L, Podobnik B, Novak M, Korosec B, Berne S, Vogelsang M, et al. 2011. The versatility of the fungal cytochrome P450 monooxygenase system is instrumental in xenobiotic detoxification. Mol. Microbiol. 81: 1374-1389. https://doi.org/10.1111/j.1365-2958.2011.07772.x
  7. Podobnik B, Stojan J, Lah L, Krasevec N, Seliskar M, Rizner TL, et al. 2008. CYP53A15 of Cochliobolus lunatus, a target for natural antifungal compounds. J. Med. Chem. 51: 3480-3486. https://doi.org/10.1021/jm800030e
  8. Harwood CS, Parales RE. 1996. The $\beta$-ketoadipate pathway and the biology of self-identity. Annu. Rev. Microbiol. 50: 553-590. https://doi.org/10.1146/annurev.micro.50.1.553
  9. Jawallapersand P, Mashele SS, Kovacic L, Stojan J, Komel R, Pakala SB, et al. 2014. Cytochrome P450 monooxygenase CYP53 family in fungi: comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target. PLoS One 9: e107209.
  10. Noda S, Kitazono E, Tanaka T, Ogino C, Kondo A. 2012. Benzoic acid fermentation from starch and cellulose via a plant-like $\beta$-oxidation pathway in Streptomyces maritimus. Microb. Cell Fact. 11: 49. https://doi.org/10.1186/1475-2859-11-49
  11. Jeon H, Durairaj P, Lee D, Ahsan MM, Yun H. 2016. Improved NADPH regeneration for fungal cytochrome P450 monooxygenase by co-expressing bacterial glucose dehydrogenase in resting-cell biotransformation of recombinant yeast. J. Microbiol. Biotechnol. 26: 2076-2086. https://doi.org/10.4014/jmb.1605.05090
  12. Goto T, Moriuchi H, Fu X, Ikegawa T, Matsubara T, Chang G, et al. 2010. The effects of single nucleotide polymorphisms in CYP2A13 on metabolism of 5-methoxypsoralen. Drug Metab. Dispos. 38: 2110-2116. https://doi.org/10.1124/dmd.110.034553
  13. 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.
  14. Yun CH, Yim SK, Kim DH, Ahn T. 2006. Functional expression of human cytochrome P450 enzymes in Escherichia coli. Curr. Drug Metab. 7: 411-429. https://doi.org/10.2174/138920006776873472
  15. Yamazaki S, Sato K, Suhara K, Sakaguchi M, Mihara K, Omura T. 1993. Importance of the proline-rich region following signal-anchor sequence in the formation of correct conformation of microsomal cytochrome P-450s. J. Biochem. 114: 652-657.
  16. Uno T, Nakao A, Masuda S, Taniguchi Y, Kanamaru K, Yamagata H, et al. 2006. Modification of small molecules by using cytochrome P450 expressed in Escherichia coli. J. Ind. Microbiol. Biotechnol. 33: 1043-1050. https://doi.org/10.1007/s10295-006-0146-8
  17. Miki Y, Asano Y. 2014. Biosynthetic pathway for the cyanide-free production of phenylacetonitrile in Escherichia coli by utilizing plant cytochrome P450 79A2 and bacterial aldoxime dehydratase. Appl. Environ. Microbiol. 80: 6828-6836. https://doi.org/10.1128/AEM.01623-14
  18. Zhang JD, Li AT, Xu JH. 2010. Improved expression of recombinant cytochrome P450 monooxygenase in Escherichia coli for asymmetric oxidation of sulfides. Bioprocess Biosyst. Eng. 33: 1043-1049. https://doi.org/10.1007/s00449-010-0429-3
  19. Zhou Y, Minami T, Honda K, Omasa T, Ohtake H. 2010. Systematic screening of Escherichia coli single-gene knockout mutants for improving recombinant whole-cell biocatalysts. Appl. Microbiol. Biotechnol. 87: 647-655. https://doi.org/10.1007/s00253-010-2505-7
  20. De Wulf P, McGuire AM, Liu X, Lin EC. 2002. Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli. J. Biol. Chem. 277: 26652-26661. https://doi.org/10.1074/jbc.M203487200
  21. Plate CA, Seely SA, Laffler TG. 1986. Evidence for a protonmotive force related regulatory system in Escherichia coli and its effects on lactose transport. Biochemistry 25: 6127-6132. https://doi.org/10.1021/bi00368a044
  22. Ghrist AC, Stauffer GV. 1995. Characterization of the Escherichia coli gcvR gene encoding a negative regulator of gcv expression. J. Bacteriol. 177: 4980-4984.
  23. Ginsburg A, Stadtman ER. 1973. Regulation of glutamine synthetase in Escherichia coli, pp. 9-43. In Prusiner S, Stadtman ER (eds.). The Enzymes of Glutamine Metabolism. Academic Press, New York.
  24. Zhou Y, Minami T, Honda K, Omasa T, Ohtake H. 2010. Enhancement of recombinant enzyme activity in cpxA-deficient mutant of Escherichia coli. J. Biosci. Bioeng. 110: 403-407. https://doi.org/10.1016/j.jbiosc.2010.04.013
  25. Cornelissen S, Julsing MK, Schmid A, Buhler B. 2012. Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis. J. Ind. Microbiol. Biotechnol. 39: 275-287. https://doi.org/10.1007/s10295-011-1026-4
  26. Kim DH, Kim BG, Jung NR, Ahn JH. 2009. Production of genistein from naringenin using Escherichia coli containing isoflavone synthase-cytochrome P450 reductase fusion protein. J. Microbiol. Biotechnol. 19: 1612-1616. https://doi.org/10.4014/jmb.0905.05043
  27. Li QS, Schwaneberg U, Fische P, Schmi RD. 2000. Directed evolution of the fatty-acid hydroxylase P450 BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6: 1531-1536. https://doi.org/10.1002/(SICI)1521-3765(20000502)6:9<1531::AID-CHEM1531>3.3.CO;2-4
  28. Lu Y, Mei L. 2007. Co-expression of P450 BM3 and glucose dehydrogenase by recombinant Escherichia coli and its application in an NADPH-dependent indigo production system. J. Ind. Microbiol. Biotechnol. 34: 247-253. https://doi.org/10.1007/s10295-006-0193-1
  29. Degregorio D, D'Avino S, Castrignano S, Di Nardo G, Sadeghi SJ, Catucci G, et al. 2017. Human cytochrome P450 3A4 as a biocatalyst: effects of the engineered linker in modulation of coupling efficiency in 3A4-BMR chimeras. Front. Pharmacol. 8: 121.

Cited by

  1. Genomic and Transcriptomic Study for Screening Genes Involved in the Limonene Biotransformation of Penicillium digitatum DSM 62840 vol.11, pp.None, 2018, https://doi.org/10.3389/fmicb.2020.00744