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Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes

Journal of Industrial Microbiology & Biotechnology

Abstract

Cephalosporin C (CPC) is the precursor of a class of antibiotics that were more effective than traditional penicillins. CPC production is performed mainly through fermentation by Acremonium chrysogenum, whose secondary metabolism was sensitive to the environmental changes. In the present work, secondary metabolites were measured by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry, and the disparity of them from two scales of CPC fermentations (pilot and industrial) and also two different post-treatment processes (oxalic acid and formaldehyde added and control) were investigated. When fermentation size was enlarged from pilot scale (50 l) to industrial scale (156,000 l), the remarkable disparities of concentrations and changing trends of the secondary metabolites in A. chrysogenum were observed, which indicated that the productivity of CPC biosynthesis was higher in the large scale of fermentation. Three environmental factors were measured, and the potential reasons that might cause the differences were analyzed. In the post-treatment process after industrial fermentation, the changes of these secondary metabolites in the tank where oxalic acid and formaldehyde were added were much less than the control tank where none was added. This indicated that the quality of the final product was more stable after the oxalic acid and formaldehyde were added in the post-treatment process. These findings provided new insight into industrial CPC production.

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References

  1. Baldwin JE, Singh PD, Yoshida M, Sawada Y, Demain AL (1980) Incorporation of 3H and 14C from (6 alpha-3H) penicillin N and (10–14C,6 alpha-3H) penicillin N into deacetoxycephalosporin C. Biochem J 186:889–895

    PubMed  CAS  Google Scholar 

  2. Basch J, Chiang SJ (1998) Genetic engineering approach to reduce undesirable by-products in cephalosporin C fermentation. J Ind Microbiol Biotechnol 20:344–353

    Article  CAS  Google Scholar 

  3. Behmer CJ, Demain AL (1983) Further studies on carbon catabolite regulation of β-lactam antibiotic synthesis in Cephalosporium acremonium. Curr Microbiol 8:107–114

    Article  CAS  Google Scholar 

  4. Cao YX, Qiao B, Lu H, Chen Y, Yuan YJ (2011) Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations. Appl Microbiol Biotechnol 89:1193–1202

    Article  PubMed  CAS  Google Scholar 

  5. Castro JM, Liras P, Cortés J, Martín JF (1985) Regulation of α-aminoadipyl-cysteinyl-valine, isopenicillin N synthetase, isopenicillin N isomerase and deacetoxycephalosporin C synthetase by nitrogen sources in Streptomyces lactamdurans. Appl Microbiol Biotechnol 22:32–40

    Article  CAS  Google Scholar 

  6. Cohen G, Argaman A, Schreiber R, Mislovati M, Aharonowitz Y (1994) The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis. J Bacteriol 176:973–984

    PubMed  CAS  Google Scholar 

  7. Cole M, Batchelor FR (1963) Aminoadipylpenicillin in penicillin fermentations. Nature 198:383–384

    Article  PubMed  CAS  Google Scholar 

  8. Dotzlaf JE, Yeh WK (1987) Copurification and characterization of deacetoxycephalosporin C synthetase/hydroxylase from Cephalosporium acremonium. J Bacteriol 169:1611–1618

    PubMed  CAS  Google Scholar 

  9. Goo KS, Chua CS, Sim TS (2009) Directed evolution and rational approaches to improving Streptomyces clavuligerus deacetoxycephalosporin C synthase for cephalosporin production. J Ind Microbiol Biotechnol 36:619–633

    Article  PubMed  CAS  Google Scholar 

  10. Heim J, Shen YQ, Wolfe S, Demain AL (1984) Regulation of isopenicillin N synthetase and deacetoxycephalosporin C synthetase by carbon source during the fermentation of Cephalosporium acremonium. Appl Microbiol Biotechnol 19:232–236

    Article  CAS  Google Scholar 

  11. Henriksen CM, Nielsen J, Villadsen J (1997) Influence of the dissolved oxygen concentration on the penicillin biosynthetic pathway in steady state cultures of Penicillium chrysogenum. Biotechnol Prog 13:776–782

    Article  CAS  Google Scholar 

  12. Hilgendorf P, Heiser V, Diekmann H, Thoma M (1987) Constant dissolved oxygen concentrations in cephalosporin C fermentation: applicability of different controllers and effect on fermentation parameters. Appl Microbiol Biotechnol 27:247–251

    Article  CAS  Google Scholar 

  13. Hinnen A, Nuesch J (1976) Enzymatic hydrolysis of cephalosporin C by an extracellular acetylhydrolase of Cephalosporium acremonium. Antimicrob Agents Chemother 9:824–830

    Article  PubMed  CAS  Google Scholar 

  14. Huber FM, Baltz RH, Caltrider PG (1968) Formation of desacetylcephalosporin C in cephalosporin C fermentation. Appl Environ Microbiol 16:1011–1014

    CAS  Google Scholar 

  15. Jorgensen H, Nielsen J, Villadsen J, Mollgaard H (1995) Analysis of penicillin V biosynthesis during fed-batch cultivations with a high-yielding strain of Penicillium chrysogenum. Appl Microbiol Biotechnol 43:123–130

    Article  PubMed  CAS  Google Scholar 

  16. Kanzaki T, Fujisawa Y (1976) Biosynthesis of cephalosporins. Adv Appl Microbiol 20:159–201

    Article  PubMed  CAS  Google Scholar 

  17. Lee MS, Lim JS, Kim CO, Oh KK, Hong SI, Kim SW (2001) Effects of nutrients and culture conditions on morphology in the seed culture of Cephalosporium acremonium ATCC 20339. Biotechnol Bioprocess Eng 6:156–160

    Article  CAS  Google Scholar 

  18. Lopez-Nieto MJ, Revilla G, Martin JF (1982) Biosynthesis of the tripeptide δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine in Penicillium chrysogenum and its control by glucose. In: Fourth international symposium on the genetics of industrial microorganisms, Kyoto, pp 11–25

  19. Lu W, Kimball E, Rabinowitz JD (2006) A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J Am Soc Mass Spectrom 17:37–50

    Article  PubMed  CAS  Google Scholar 

  20. Lubbe C, Shen YQ, Demain AL (1986) A simple and rapid chemical method for the determination of cephalosporins. Appl Biochem Biotechnol 12:31–35

    Article  PubMed  CAS  Google Scholar 

  21. Malmberg LH, Hu WS (1992) Identification of rate-limiting steps in cephalosporin C biosynthesis in Cephalosporium acremonium: a theoretical analysis. Appl Microbiol Biotechnol 38:122–128

    Article  PubMed  CAS  Google Scholar 

  22. Mishra P, Srivastava P, Kundu S (2005) A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy. World J Microbiol Biotechnol 21:525–530

    Article  CAS  Google Scholar 

  23. Morin RB, Jackson BG, Flynn EH, Roeske RW (1962) Chemistry of cephalosporin antibiotics. I. 7-aminocephalosporanic acid from cephalosporin C. J Am Chem Soc 84:3400–3401

    Article  CAS  Google Scholar 

  24. O’Sullivan J, Bleaney RC, Huddleston JA, Abraham EP (1979) Incorporation of 3H from delta-(l-alpha-amino (4,5–3H) adipyl)-l-cysteinyl-d-(4,4–3H) valine into isopenicillin N. Biochem J 184:421–426

    PubMed  Google Scholar 

  25. Pang CP, Chakravarti B, Adlington RM, Ting HH, White RL, Jayatilake GS, Baldwin JE, Abraham EP (1984) Purification of isopenicillin N synthetase. Biochem J 222:789–795

    PubMed  CAS  Google Scholar 

  26. Pazouki M, Panda T (2000) Understanding the morphology of fungi. Bioprocess Eng 22:127–143

    Article  CAS  Google Scholar 

  27. Perez-Martinez G, Peberdy JF (1985) Production of cephalosporin C, and its intermediates, by raised-titre strains of Acremonium chrysogenum. Enzyme Microb Technol 7:389–394

    Article  CAS  Google Scholar 

  28. Sawada Y, Konomi T, Solomon NA, Demain AL (1980) Increase in activity of β-lactam synthetases after growth of Cephalosporium acremonium with methionine or norleucine. FEMS Microbiol Lett 9:281–284

    CAS  Google Scholar 

  29. Scheidegger A, Kuenzi MT, Nuesch J (1984) Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of beta-lactams in Cephalosporium acremonium. J Antibiot (Tokyo) 37:522–531

    Article  CAS  Google Scholar 

  30. Seifar RM, Zhao Z, van Dam J, van Winden W, van Gulik W, Heijnen JJ (2008) Quantitative analysis of metabolites in complex biological samples using ion-pair reversed-phase liquid chromatography-isotope dilution tandem mass spectrometry. J Chromatogr A 1187:103–110

    Article  PubMed  CAS  Google Scholar 

  31. Shen YQ, Heim J, Solomon NA, Wolfe S, Demain AL (1984) Repression of β-lactam production in Cephalosporium acremonium by nitrogen sources. J Biomed Biotechnol 37:503–511

    CAS  Google Scholar 

  32. Shirafuji H, Fujisawa Y, Kida M, Kanzaki T, Yoneda M (1979) Accumulation of tripeptide derivatives by mutants of Cephalosporium acremonium. Agric Biol Chem 43:155–160

    Article  CAS  Google Scholar 

  33. Skatrud PL, Tietz AJ, Ingolia TD, Cantwell CA, Fisher DL, Chapman JL, Queener SW (1989) Use of recombinant DNA to improve production of cephalosporin C by Cephalosporium acremonium. Nat Biotechnol 7:477–485

    Article  CAS  Google Scholar 

  34. Theilgaard HA, Nielsen J (1999) Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control. Antonie Van Leeuwenhoek 75:145–154

    Article  PubMed  CAS  Google Scholar 

  35. Theilgaard HB, Kristiansen KN, Henriksen CM, Nielsen J (1997) Purification and characterization of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase from Penicillium chrysogenum. Biochem J 327:185–191

    PubMed  CAS  Google Scholar 

  36. Tucker KG, Thomas CR (1992) Mycelial morphology: the effect of spore inoculum level. Biotechnol Lett 14:1071–1074

    Article  Google Scholar 

  37. Yeh WK (1997) Evolving enzyme technology for pharmaceutical applications: case studies. J Ind Microbiol Biotechnol 19:334–343

    Article  PubMed  CAS  Google Scholar 

  38. Yezza A, Tyagi RD, Valèro JR, Surampalli RY, Smith J (2004) Scale-up of biopesticide production processes using wastewater sludge as a raw material. J Ind Microbiol Biotechnol 31:545–552

    Article  PubMed  CAS  Google Scholar 

  39. Yoshida M, Konomi T, Kohsaka M, Baldwin JE, Herchen S, Singh P, Hunt NA, Demain AL (1978) Cell-free ring expansion of penicillin N to deacetoxycephalosporin C by Cephalosporium acremonium CW-19 and its mutants. Proc Natl Acad Sci USA 75:6253–6257

    Article  PubMed  CAS  Google Scholar 

  40. Zanca DM, Martín JF (1983) Carbon catabolite regulation of the conversion of penicillin N into cephalosporin C. J Biomed Biotechnol 36:700–708

    CAS  Google Scholar 

  41. Zhang J, Banko G, Wolfe S, Demain AL (1987) Methionine induction of ACV synthetase in Cephalosporium acremonium. J Ind Microbiol Biotechnol 2:251–255

    CAS  Google Scholar 

  42. Zhang J, Wolfe S, Demain AL (1988) Phosphate repressible and inhibitable β-lactam synthetases in Cephalosporium acremonium strain C-10. Appl Microbiol Biotechnol 29:242–247

    CAS  Google Scholar 

  43. Zhang J, Wolfe S, Demain AL (1989) Carbon source regulation of ACV synthetase in Cephalosporium acremonium C-10. Curr Microbiol 18:361–367

    Article  CAS  Google Scholar 

  44. Zhang J, Demain AL (1992) Regulation of ACV synthetase activity in the beta-lactam biosynthetic pathway by carbon sources and their metabolites. Arch Microbiol 158:364–369

    Article  CAS  Google Scholar 

  45. Zhang J, Demain AL (1992) ACV synthetase. Crit Rev Biotechnol 12:245–260

    Article  PubMed  CAS  Google Scholar 

  46. Zhang JY, Wolfe S, Demain AL (1987) Effect of ammonium as nitrogen source on production of delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine synthetase by Cephalosporium acremonium C-10. J Biomed Biotechnol 40:1746–1750

    CAS  Google Scholar 

  47. Zhou W, Holzhauer-Rieger K, Dors M, Schügerl K (1992) Influence of dissolved oxygen concentration on the biosynthesis of cephalosporin C. Enzyme Microb Technol 14:848–854

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors are grateful for the financial support from the National High-tech R&D Program (863 Program: 2012AA021204, 2012AA02A701), National Basic Research Program of China (973 Program: 2013CB733601), and the National Natural Science Foundation of China (Major International Joint Research Project: 21020102040).

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Correspondence to Hua Lu or Ying-Jin Yuan.

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Cao, YX., Lu, H., Qiao, B. et al. Comparison of the secondary metabolites in two scales of cephalosporin C (CPC) fermentation and two different post-treatment processes. J Ind Microbiol Biotechnol 40, 95–103 (2013). https://doi.org/10.1007/s10295-012-1203-0

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