Cephalosporin C acylase: Important role, obstacles, and strategies to optimize expression in E. coli

Muthi’ah Rasyidah Sismindari Sismindari Purwanto Purwanto   

Open Access   

Published:  May 24, 2024

DOI: 10.7324/JAPS.2024.179954

Cephalosporins have gained popularity due to the increasing resistance to penicillin antibiotics. Since the first discovery of cephalosporin C in 1945, the development of cephalosporin-class antibiotics has continued. Cephalosporin C was converted into a 7-aminocephalosporanic acid (7-ACA) compound which became a precursor for producing cephalosporin class antibiotics through side chain modification. The conversion of cephalosporin C to 7-ACA is more beneficial when performed enzymatically using the enzyme cephalosporin C acylase (CA) compared to chemical methods. This article will present the importance of cephalosporin CA enzymes in the development of cephalosporin antibiotics, the obstacles, and strategies to optimize the expression of these enzymes in Escherichia coli hosts.

Keyword:     Cephalosporin 7-ACA cephalosporin C acylase E. coli


Rasyidah M, Sismindari S, Purwanto P. Cephalosporin C acylase: Important role, obstacles, and strategies to optimize expression in E. coli. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.179954

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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1. Barber MS, Giesecke U, Reichert A, Minas W. Industrial enzymatic production of cephalosporin-based beta-lactams. Adv Biochem Eng Biotechnol. 2004;88:179-215. https://doi.org/10.1007/b99261

2. World Health Organization. Critically important antimicrobials for human medicine [Internet]. 6th rev. Geneva, Switzerland: World Health Organization; 2019 [cited 2023 Feb 17]. p. 45. Available from: https://apps.who.int/iris/handle/10665/312266

3. Browne AJ, Chipeta MG, Haines-Woodhouse G, Kumaran EPA, Hamadani BHK, Zaraa S, et al. Global antibiotic consumption and usage in humans, 2000-18: a spatial modelling study. Lancet Planetary Health [Internet]. 2021 Dec [cited 2024 Feb 15];5(12):e893-904. https://doi.org/10.1016/S2542-5196(21)00280-1

4. Aoki T, Yoshizawa H, Yamawaki K, Yokoo K, Sato J, Hisakawa S, et al. Cefiderocol (S-649266), a new siderophore cephalosporin exhibiting potent activities against Pseudomonas aeruginosa and other gram-negative pathogens including multi-drug resistant bacteria: Structure activity relationship. Eur J Med Chem. 2018 Jul 15;155:847-68. https://doi.org/10.1016/j.ejmech.2018.06.014

5. Fong IW. New Cephalosporins: fifth and sixth generations. In: new antimicrobials: for the present and the future [Internet]. Cham, Germany: Springer International Publishing; 2023 [cited 2024 Feb 14]. p. 25-38. (Emerging Infectious Diseases of the 21st Century). https://doi.org/10.1007/978-3-031-26078-0_2

6. Pollegioni L, Rosini E, Molla G. Cephalosporin C acylase: dream and(/or) reality. Appl Microbiol Biotechnol. 2013 Mar;97(6):2341-55. https://doi.org/10.1007/s00253-013-4741-0

7. Aramori I, Fukagawa M, Tsumura M, Iwami M, Yokota Y, Kojo H, et al. Isolation of soil strains producing new cephalosporin acylases. J Ferment Bioeng [Internet]. 1991 [cited 2023 May 24];72(4):227-31. https://doi.org/10.1016/0922-338X(91)90154-9

8. Matsuda A, Toma K, Komatsu K. Nucleotide sequences of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. J Bacteriol [Internet]. 1987 Dec [cited 2023 May 24];169(12):5821-6. https://doi.org/10.1128/jb.169.12.5821-5826.1987

9. Tan Q, Qiu J, Luo X, Zhang Y, Liu Y, Chen Y, et al. Progress in one-pot bioconversion of cephalosporin c to 7-aminocephalosporanic acid. Curr Pharm Biotechnol. 2018;19(1):30-42. https://doi.org/10.2174/1389201019666180509093956

10. Morin RB, Jackson BG, Flynn EH, Roeske RW, Andrews SL. Chemistry of cephalosporin antibiotics. XIV. The reaction of cephalosporin C with nitrosyl chloride. J Am Chem Soc. 1969 Mar 12;91(6):1396-400. https://doi.org/10.1021/ja01034a022

11. Tan Q, Zhang Y, Song Q, Wei D. Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide. World J Microbiol Biotechnol [Internet]. 2010 Jan 1 [cited 2023 May 24];26(1):145-52. https://doi.org/10.1007/s11274-009-0153-9

12. Aretz WDK, Sauber KDS. New D-amino Acid Transaminase and Their Use [Internet]. DE3447023A1, 1986 [cited 2023 May 24]. Available from: https://patents.google.com/patent/DE3447023A1/en

13. Fritz-Wolf K, Koller KP, Lange G, Liesum A, Sauber K, Schreuder H, et al. Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C. Protein Sci. 2002 Jan 1;11(1):92-103. https://doi.org/10.1110/ps.27502

14. Aramori I, Fukagawa M, Tsumura M, Iwami M, Isogai T, Ono H, et al. Cloning and nucleotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains. J Ferment Bioeng [Internet]. 1991 Jan 1 [cited 2023 May 24];72(4):232-43. https://doi.org/10.1016/0922-338X(91)90155-A

15. Matsuda A, Matsuyama K, Yamamoto K, Ichikawa S, Komatsu K. Cloning and characterization of the genes for two distinct cephalosporin acylases from a Pseudomonas strain. J Bacteriol [Internet]. 1987 Dec [cited 2023 May 24];169(12):5815-20. https://doi.org/10.1128/jb.169.12.5815-5820.1987

16. Murzin AG, Brenner SE, Hubbard T, Chothia C. SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol. 1995 Apr 7;247(4):536-40. https://doi.org/10.1016/S0022-2836(05)80134-2

17. Pollegioni L, Lorenzi S, Rosini E, Marcone GL, Molla G, Verga R, et al. Evolution of an acylase active on cephalosporin C. Protein Sci [Internet]. 2005 Dec [cited 2023 May 25];14(12):3064-76. https://doi.org/10.1110/ps.051671705

18. Cho K, Kim KH. Cephalosporin acylase precursor, glutaryl-7-aminocephalosporanic acid acylase precursor. Handbook of Proteolytic Enzymes. Cambridge, MA: Academic Press; 2013 Dec 31. Vol. 3, pp 3659-63. https://doi.org/10.1016/B978-0-12-382219-2.00811-5

19. Kim Y, Kim S, Earnest TN, Hol WGJ. Precursor structure of cephalosporin acylase. Insights into autoproteolytic activation in a new N-terminal hydrolase family. J Biol Chem. 2002 Jan 25;277(4):2823-9. https://doi.org/10.1074/jbc.M108888200

20. Shin YC, Jeon JYJ, Jung KH, Park MR, Kim Y. Cephalosporin C acylase mutant and method for preparing 7-ACA using same [Internet]. US7592168B2, 2009 [cited 2023 Mar 18]. Available from: https://patents.google.com/patent/US7592168B2/pt

21. Wang Y, Yu H, Song W, An M, Zhang J, Luo H, et al. Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel. J Biosci Bioeng. 2012 Jan;113(1):36-41. https://doi.org/10.1016/j.jbiosc.2011.08.027

22. Adrio JL, Demain AL. Recombinant organisms for production of industrial products. Bioeng Bugs [Internet]. 2010 [cited 2023 Jul 1];1(2):116-31. https://doi.org/10.4161/bbug.1.2.10484

23. Demain AL, Vaishnav P. Production of recombinant proteins by microbes and higher organisms. Biotechnol Adv. 2009;27(3):297-306. https://doi.org/10.1016/j.biotechadv.2009.01.008

24. Sahdev S, Khattar SK, Saini KS. Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem. 2008 Jan;307(1-2):249-64. https://doi.org/10.1007/s11010-007-9603-6

25. Lee SY. High cell-density culture of Escherichia coli. Trends Biotechnol. 1996 Mar;14(3):98-105. https://doi.org/10.1016/0167-7799(96)80930-9

26. Pope B, Kent HM. High efficiency 5 min transformation of Escherichia coli. nucleic acids res [Internet]. 1996 Feb 1 [cited 2023 Jul 3];24(3):536-7. https://doi.org/10.1093/nar/24.3.536

27. Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli physiology in luria-bertani broth. J Bacteriol [Internet]. 2007 Dec [cited 2023 Jul 3];189(23):8746-9. https://doi.org/10.1128/JB.01368-07

28. Shiloach J, Fass R. Growing E. coli to high cell density—a historical perspective on method development. Biotechnol Adv. 2005 Jul;23(5):345-57. https://doi.org/10.1016/j.biotechadv.2005.04.004

29. Gottesman S. Proteases and their targets in Escherichia coli. Annu Rev Genet. 1996;30:465-506. https://doi.org/10.1146/annurev.genet.30.1.465

30. Grodberg J, Dunn JJ. ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol. 1988 Mar;170(3):1245-53. https://doi.org/10.1128/jb.170.3.1245-1253.1988

31. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol [Internet]. 2014 [cited 2023 May 29];5. https://doi.org/10.3389/fmicb.2014.00172

32. Francis DM, Page R. Strategies to optimize protein expression in E. coli. Curr Protoc Protein Sci [Internet]. 2010 Aug [cited 2023 Jul 4];61(1):5-24. https://doi.org/10.1002/0471140864.ps0524s61

33. Gustafsson C, Govindarajan S, Minshull J. Codon bias and heterologous protein expression. Trends Biotechnol. 2004 Jul;22(7):346-53. https://doi.org/10.1016/j.tibtech.2004.04.006

34. Bhatwa A, Wang W, Hassan YI, Abraham N, Li XZ, Zhou T. Challenges associated with the formation of recombinant protein inclusion bodies in Escherichia coli and strategies to address them for industrial applications. Front Bioeng Biotechnol [Internet]. 2021 [cited 2023 Mar 18];9. https://doi.org/10.3389/fbioe.2021.630551

35. Carrió MM, Villaverde A. Construction and deconstruction of bacterial inclusion bodies. J Biotechnol. 2002 Jun 13;96(1):3-12. https://doi.org/10.1016/S0168-1656(02)00032-9

36. Hartley DL, Kane JF. Properties of inclusion bodies from recombinant Escherichia coli. Biochem Soc Trans. 1988 Apr;16(2):101-2. https://doi.org/10.1042/bst0160101

37. Stewart EJ, Aslund F, Beckwith J. Disulfide bond formation in the Escherichia coli cytoplasm: an in vivo role reversal for the thioredoxins. EMBO J [Internet]. 1998 Oct 1 [cited 2023 Jul 4];17(19):5543-50. https://doi.org/10.1093/emboj/17.19.5543

38. Hartl FU, Hayer-Hartl M. Molecular chaperones in the cytosol: from nascent chain to folded protein. Science. 2002 Mar 8;295(5561):1852-8. https://doi.org/10.1126/science.1068408

39. Chen Y, Song J, Sui SF, Wang DN. DnaK and DnaJ facilitated the folding process and reduced inclusion body formation of magnesium transporter CorA overexpressed in Escherichia coli. Protein Expr Purif. 2003 Dec;32(2):221-31. https://doi.org/10.1016/S1046-5928(03)00233-X

40. Singh D, Tripathi P, Sharma R, Grover S, Batra JK. Role of a substrate binding pocket in the amino terminal domain of Mycobacterium tuberculosis caseinolytic protease B (ClpB) in its function. J Biomole Struct Dyn [Internet]. 2023 Jul 7 [cited 2024 Feb 14];1-11. https://doi.org/10.1080/07391102.2023.2232032

41. de Marco A, Vigh L, Diamant S, Goloubinoff P. Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones. Cell Stress Chaperones. 2005;10(4):329-39. https://doi.org/10.1379/CSC-139R.1

42. Diamant S, Rosenthal D, Azem A, Eliahu N, Ben-Zvi AP, Goloubinoff P. Dicarboxylic amino acids and glycine-betaine regulate chaperone-mediated protein-disaggregation under stress. Mol Microbiol. 2003 Jul;49(2):401-10. https://doi.org/10.1046/j.1365-2958.2003.03553.x

43. Deutch CE. Growth of Escherichia coli K-12 on L-proline in the absence of known proline transporters. J Adv Microbiol Res [Internet]. 2023 Jan 1 [cited 2024 Feb 15];4(1):01-10. https://doi.org/10.22271/micro.2022.v3.i2a.57

44. Hernandez-Leon SG, Valenzuela-Soto EM. Glycine betaine is a phytohormone-like plant growth and development regulator under stress conditions. J Plant Growth Regul [Internet]. 2023 Aug [cited 2024 Feb 15];42(8):5029-40. https://doi.org/10.1007/s00344-022-10855-3

45. Lucht JM, Bremer E. Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system proU. FEMS Microbiol Rev. 1994 May;14(1):3-20. https://doi.org/10.1016/0168-6445(94)90008-6

46. Diamant S, Eliahu N, Rosenthal D, Goloubinoff P. Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses. J Biol Chem. 2001 Oct 26;276(43):39586-91. https://doi.org/10.1074/jbc.M103081200

47. Iskandaryan MK. Role of glycine-betaine in the growth and hydrogenases activity of ralstonia eutropha H16. Proceedings of the YSU B: chemical and biological sciences [Internet]. 2023 Jul 7 [cited 2024 Feb 15];57(2 (261)):154-63. https://doi.org/10.46991/PYSU:B/2023.57.2.154

48. Hassan MU, Nawaz M, Shah AN, Raza A, Barbanti L, Skalicky M, et al. Trehalose: a key player in plant growth regulation and tolerance to abiotic stresses. J Plant Growth Regul [Internet]. 2023 Aug [cited 2024 Feb 15];42(8):4935-57. https://doi.org/10.1007/s00344-022-10851-7

49. Isdiyono BW, Hardianto D, Ivan FX. Production of recombinant cephalosporin acylase as biocatalyst for 7-aminocephalosporanic acid production. J Bioteknol Biosains Indonesia (JBBI) [Internet]. 2017 Jul 7 [cited 2023 Jun 2];4(1):28-35. https://doi.org/10.29122/jbbi.v4i1.2059

50. Martius E, Wibisana A, Ardiyani Y. The optimization of soluble cephalosporin C acylase expression in E. coli. IJES 2018;7:29-34.

51. Jobanputra AH, Vasait RD. Cephalosporin C acylase from Pseudomonas species: production and enhancement of its activity by optimization of process parameters. Biocatal Agric Biotechnol [Internet]. 2015 Oct 1 [cited 2023 Jun 2];4(4):465-70. https://doi.org/10.1016/j.bcab.2015.06.009

52. Vasait RD, Jobanputra AH. Bacterial conversion of cephalosporin C: optimization in Achromobacter xylosooxidans. Biocatal Agric Biotechnol [Internet]. 2023 Aug [cited 2024 Feb 13];51:102772. https://doi.org/10.1016/j.bcab.2023.102772

53. Sun H, Liu T, Luo H, Nie Z, Chang Y, Yu H, et al. Optimization of cephalosporin C acylase expression in Escherichia coli by high-throughput screening a constitutive promoter mutant library. Appl Biochem Biotechnol. 2021 Apr;193(4):1056-71. https://doi.org/10.1007/s12010-020-03482-9

54. Novy R, Morris B. Use of glucose to control basal expression in the pET System. Novagen Inc [Internet]. n.d.; Available from: http://wolfson.huji.ac.il/expression/procedures/bacterial/Glucose%20supression.pdf

55. Kopp J, Slouka C, Ulonska S, Kager J, Fricke J, Spadiut O, et al. Impact of glycerol as carbon source onto specific sugar and inducer uptake rates and inclusion body productivity in E. coli BL21(DE3). Bioengineering (Basel) [Internet]. 2017 Dec 21 [cited 2023 Aug 8];5(1):1. https://doi.org/10.3390/bioengineering5010001

56. Narayanan N, Hsieh MY, Xu Y, Chou CP. Arabinose-induction of lac-derived promoter systems for penicillin acylase production in Escherichia coli. Biotechnol Prog. 2006;22(3):617-25. https://doi.org/10.1021/bp050367d

57. Kaplan NA, Islam KN, Kanis FC, Verderber JR, Wang X, Jones JA, et al. Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant. Buan NR, editor. Appl Environ Microbiol [Internet]. 2024 Jan 30 [cited 2024 Feb 15];e02169-23.

58. Duggleby HJ, Tolley SP, Hill CP, Dodson EJ, Dodson G, Moody PC. Penicillin acylase has a single-amino-acid catalytic centre. Nature. 1995 Jan 19;373(6511):264-8. https://doi.org/10.1038/373264a0

59. Kim Y, Hol WG. Structure of cephalosporin acylase in complex with glutaryl-7-aminocephalosporanic acid and glutarate: insight into the basis of its substrate specificity. Chem Biol. 2001 Dec;8(12):1253-64. https://doi.org/10.1016/S1074-5521(01)00092-8

60. Kim JK, Yang IS, Shin HJ, Cho KJ, Ryu EK, Kim SH, et al. Insight into autoproteolytic activation from the structure of cephalosporin acylase: a protein with two proteolytic chemistries. Proc Natl Acad Sci USA. 2006 Feb 7;103(6):1732-7. https://doi.org/10.1073/pnas.0507862103

61. Kim JK, Yang IS, Rhee S, Dauter Z, Lee YS, Park SS, et al. Crystal structures of glutaryl 7-aminocephalosporanic acid acylase: insight into autoproteolytic activation. Biochemistry. 2003 Apr 15;42(14):4084-93. https://doi.org/10.1021/bi027181x

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