Flux dynamics of C-5 amino acid precursors in fed-batch cultures of Streptomyces clavuligerus during clavulanic acid biosynthesis

Luisa María Gómez-Gaona Howard Ramírez-Malule David Gómez-Ríos   

Open Access   

Published:  Jun 12, 2024

DOI: 10.7324/JAPS.2024.191319

Clavulanic acid (CA) is a well-known β-lactamase inhibitor that is mainly obtained from submerged cultures of Streptomyces clavuligerus. Dynamic genome-scale in silico studies were performed to gain insights into the intracellular metabolic fluxes of C-5 precursors during the cultivation of wild-type S. clavuligerus in batch and fed-batch operation. A preliminary literature screening was conducted for media selection and determination of culture conditions for further cultivation in a 5-L bioreactor. Simulations provided insights into the metabolism of C-5 precursors in CA biosynthesis. In addition, carbon utilization for CA biosynthesis was assessed in terms of the ratio of total carbon used in CA biosynthesis to the total carbon influx in batch and feed media. Based on simulation results, we proposed a modification of the glycerol-sucrose-proline-glutamate medium for fed-batch cultivation to improve the carbon utilization for CA biosynthesis. The proposed fed-batch scenario achieved higher specific CA concentration at lower biomass production, indicating better carbon utilization for its synthesis. The dynamic in silico fluxes suggests that metabolic fluxes in this scenario would be stable, favoring a longer stage of continued antibiotic secretion.

Keyword:     Streptomyces clavulanic acid amino acids dFBA fed-batch


Gómez-Gaona LM, Ramírez-Malule H, Gómez-Ríos D. Flux dynamics of C-5 amino acid precursors in fed-batch cultures of Streptomyces clavuligerus during clavulanic acid biosynthesis. J Appl Pharm Sci. 2024. Online First. http://doi.org/10.7324/JAPS.2024.191319

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. Cuozzo S, de Moreno de LeBlanc A, LeBlanc JG, Hoffmann N, Tortella GR. Streptomyces genus as a source of probiotics and its potential for its use in health. Microbiol Res. 2023 Jan;266:127248.

2. Petkovi? H, Lukeži? T, Šuškovi? J. Biosynthesis of oxytetracycline by streptomyces rimosus: past, present and future directions in the development of tetracycline antibiotics. Food Technol Biotechnol. 2017;55(1):3.

3. Sladic G, Urukalo M, Kirn M, Lešnik U, Magdevska V, Benicki N, et al. Identification of lipstatin-producing ability in Streptomyces virginiae CBS 314.55 using dereplication approach. Food Technol Biotechnol. 2014 Jul 1;52:276–84.

4. Ramirez-Malule H. Bibliometric analysis of global research on clavulanic acid. Antibiotics (Basel) [Internet]. 2018 Nov 26;7(4):102. Available from: https://pubmed.ncbi.nlm.nih.gov/30486255

5. López-Agudelo VA, Gómez-Ríos D, Ramirez-Malule H. Clavulanic acid production by Streptomyces clavuligerus: insights from systems biology, strain engineering, and downstream processing. Antibiotics (Basel) [Internet]. 2021 Jan 18;10(1):84. Available from: https://pubmed.ncbi.nlm.nih.gov/33477401

6. Paradkar A. Clavulanic acid production by Streptomyces clavuligerus: biogenesis, regulation and strain improvement. J Antibiot (Tokyo) [Internet]. 2013;66(7):411–20. Available from: http://dx.doi.org/10.1038/ja.2013.26

7. Elson SW, Oliver RS. Studies on the biosynthesis of clavulanic acid. I. Incorporation of 13C-labelled precursors. J Antibiot (Tokyo) [Internet]. 1978;31(6):586–92. Available from: http://dx.doi.org/10.7164/antibiotics.31.586

8. Elson SW, Oliver RS, Bycroft BW, Faruk EA. Studies on the biosynthesis of clavulanic acid. III. Incorporation of DL-(3,4-13C2) glutamic acid. J Antibiot (Tokyo) [Internet]. 1982;35(1):81–6. Available from: http://dx.doi.org/10.7164/antibiotics.35.81

9. Stirling I, Elson SW. Studies on the biosynthesis of clavulanic acid. II. Chemical degradations of 14C-labelled clavulanic acid. J Antibiot (Tokyo) [Internet]. 1979;32(11):1125–9. Available from: http://dx.doi.org/10.7164/antibiotics.32.1125

10. Elson SW, Baggaley KH, Davison M, Fulston M, Nicholson NH, Risbridger GD, et al. The identification of three new biosynthetic intermediates and one further biosynthetic enzyme in the clavulanic acid pathway. J Chem Soc Chem Commun [Internet]. 1993;(15):1212. Available from: http://dx.doi.org/10.1039/c39930001212

11. Khaleeli N, Li R, Townsend CA. Origin of the β-lactam carbons in clavulanic acid from an unusual thiamine pyrophosphate-mediated reaction. J Am Chem Soc [Internet]. 1999;121(39):9223–4. Available from: http://dx.doi.org/10.1021/ja9923134

12. Gómez-Ríos D, Ramírez-Malule H, López-Agudelo VA. Dynamic in silico assessment of potential gene targets for enhancing clavulanic acid production in Streptomyces clavuligerus submerged cultures. J Appl Pharm Sci [Internet]. 2022; Available from: http://dx.doi.org/10.7324/japs.2023.130207

13. Kurt-Kizildo?an A, Vanli-Jaccard G, Mutlu A, Sertdemir I, Özcengiz G. Genetic engineering of an industrial strain of Streptomyces clavuligerus for further enhancement of clavulanic acid production. Turk J Biol [Internet]. 2017;41:342–53. Available from: http://dx.doi.org/10.3906/biy-1608-17

14. Gómez-Ríos D, Ramírez-Malule H, Neubauer P, Junne S, Ríos-Estepa R. Degradation kinetics of clavulanic acid in fermentation broths at low temperatures. Antibiotics (Basel) [Internet]. 2019 Jan 17;8(1):6. Available from: https://pubmed.ncbi.nlm.nih.gov/30658482

15. Marques DAV, Oliveira RPS, Perego P, Porto ALF, Pessoa A, Converti A. Kinetic and thermodynamic investigation on clavulanic acid formation and degradation during glycerol fermentation by Streptomyces DAUFPE 3060. Enzyme Microb Technol [Internet]. 2009;45(2):169–73. Available from: http://dx.doi.org/10.1016/j.enzmictec.2009.03.005

16. Fu J, Xie X, Zhang S, Kang N, Zong G, Zhang P, et al. Rich organic nitrogen impacts clavulanic acid biosynthesis through the arginine metabolic pathway in Streptomyces clavuligerus F613-1. Microbiol Spectr [Internet]. 2022/12/14. 2023 Feb 14;11(1):e0201722–e0201722. Available from: https://pubmed.ncbi.nlm.nih.gov/36515504

17. Feng T, Zhao J, Chu J, Wang YH, Zhuang YP. Statistical optimizing of medium for clavulanic acid production by Streptomyces clavuligerus using response surface methodology. Appl Biochem Biotechnol [Internet]. 2021;193(12):3936–48. Available from: http://dx.doi.org/10.1007/s12010-021-03627-4

18. Ramirez-Malule H, Junne S, Nicolás Cruz-Bournazou M, Neubauer P, Ríos-Estepa R. Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis. Appl Microbiol Biotechnol [Internet]. 2018;102(9):4009–23. Available from: http://dx.doi.org/10.1007/s00253-018-8841-8

19. Ramirez-Malule H, López-Agudelo VA, Gómez-Ríos D, Ochoa S, Ríos-Estepa R, Junne S, et al. TCA cycle and its relationship with clavulanic acid production: a further interpretation by using a reduced genome-scale metabolic model of Streptomyces clavuligerus. Bioengineering (Basel) [Internet]. 2021 Jul 22;8(8):103. Available from: https://pubmed.ncbi.nlm.nih.gov/34436106

20. Bushell ME, Kirk S, Zhao HJ, Avignone-Rossa CA. Manipulation of the physiology of clavulanic acid biosynthesis with the aid of metabolic flux analysis. Enzyme Microb Technol [Internet]. 2006;39(1):149–57. Available from: http://dx.doi.org/10.1016/j.enzmictec.2006.01.017

21. Teodoro JC, Baptista-Neto A, Araujo MLGC, Hokka CO, Badino AC. Influence of glycerol and ornithine feeding on clavulanic acid production by Streptomyces clavuligerus. Braz J Chem Eng [Internet]. 2010;27(4):499–506. Available from: http://dx.doi.org/10.1590/s0104-66322010000400001

22. Ser HL, Law JWF, Chaiyakunapruk N, Jacob SA, Palanisamy UD, Chan KG, et al. Fermentation conditions that affect clavulanic acid production in Streptomyces clavuligerus: a systematic review. Front Microbiol [Internet]. 2016 Apr 22;7:522. Available from: https://pubmed.ncbi.nlm.nih.gov/27148211

23. Gómez-Rios D, Ramírez-Malule H, Ochoa S, Ríos-Estepa R. Rational selection of culture medium for clavulanic acid production by Streptomyces clavuligerus based on a metabolic modeling approach. Agric Nat Resour [Internet]. 2022;56(2):267–76. Available from: http://dx.doi.org/10.34044/j.anres.2022.56.2.05

24. Gómez-Ríos D, López-Agudelo VA, Ramírez-Malule H, Neubauer P, Junne S, Ochoa S, et al. A genome-scale insight into the effect of shear stress during the fed-batch production of clavulanic acid by Streptomyces clavuligerus. Microorganisms [Internet]. 2020 Aug 19;8(9):1255. Available from: https://pubmed.ncbi.nlm.nih.gov/32824882

25. Gómez-Ríos D, Ramírez-Malule H, Neubauer P, Junne S, Ríos-Estepa R, Ochoa S. Tuning of fed-batch cultivation of Streptomyces clavuligerus for enhanced clavulanic acid production based on genome-scale dynamic modeling. Biochem Eng J [Internet]. 2022;185:108534. Available from: http://dx.doi.org/10.1016/j.bej.2022.108534

26. Mahadevan R, Edwards JS, Doyle J. Dynamic flux balance analysis of diauxic growth in Escherichia coli. Biophys J [Internet]. 2002 Sep;83(3):1331–40. Available from: https://pubmed.ncbi.nlm.nih.gov/12202358

27. da Veiga Moreira J, Jolicoeur M, Schwartz L, Peres S. Fine-tuning mitochondrial activity in Yarrowia lipolytica for citrate overproduction. Sci Rep [Internet]. 2021 Jan 13;11(1):878. Available from: https://pubmed.ncbi.nlm.nih.gov/33441687

28. Gómez-Ríos D, Junne S, Neubauer P, Ochoa S, Ríos-Estepa R, Ramírez-Malule H. Characterization of the metabolic response of Streptomyces clavuligerus to shear stress in stirred tanks and single-use 2D rocking motion bioreactors for clavulanic acid production. Antibiotics (Basel) [Internet]. 2019 Sep 27;8(4):168. Available from: https://pubmed.ncbi.nlm.nih.gov/31569725

29. Rodrigues KCS, Costa CLL, Badino AC, Pedrolli DB, Pereira JFB, Cerri MO. Application of acid and cold stresses to enhance the production of clavulanic acid by Streptomyces clavuligerus. Appl Biochem Biotechnol [Internet]. 2019;188(3):706–19. Available from: http://dx.doi.org/10.1007/s12010-019-02953-y

30. Ribeiro RMMGP, Esperança MN, Sousa APA, Neto ÁB, Cerri MO. Individual effect of shear rate and oxygen transfer on clavulanic acid production by Streptomyces clavuligerus. Bioprocess Biosyst Eng [Internet]. 2021;44(8):1721–32. Available from: http://dx.doi.org/10.1007/s00449-021-02555-1

31. Saudagar PS, Singhal RS. Optimization of nutritional requirements and feeding strategies for clavulanic acid production by Streptomyces clavuligerus. Bioresour Technol [Internet]. 2007;98(10):2010–7. Available from: http://dx.doi.org/10.1016/j.biortech.2006.08.003

32. Saudagar PS, Singhal RS. A statistical approach using L25 orthogonal array method to study fermentative production of clavulanic acid by Streptomyces clavuligerus MTCC 1142. Appl Biochem Biotechnol [Internet]. 2007;136(3):345–59. Available from: http://dx.doi.org/10.1007/s12010-007-9030-x

33. Bellão C, Antonio T, Araujo MLGC, Badino AC. Production of clavulanic acid and cephamycin C by Streptomyces clavuligerus under different fed-batch conditions. Braz J Chem Eng [Internet]. 2013;30(2):257–66. Available from: http://dx.doi.org/10.1590/s0104-66322013000200004

34. Kunath S, Kühn M, Völker M, Schmidt T, Rühl P, Heidel G. MILP performance improvement strategies for short-term batch production scheduling: a chemical industry use case. SN Appl Sci [Internet]. 2022;4(4):87. Available from: http://dx.doi.org/10.1007/s42452-022-04969-2

35. Rodrigues KC da S, Souza AT de, Badino AC, Pedrolli DB, Cerri MO. Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus. Braz J Microbiol [Internet]. 2018/03/15. 2018;49(4):832–9. Available from: https://pubmed.ncbi.nlm.nih.gov/29588197

36. Saudagar PS, Survase SA, Singhal RS. Clavulanic acid: a review. Biotechnol Adv [Internet]. 2008;26(4):335–51. Available from: http://dx.doi.org/10.1016/j.biotechadv.2008.03.002

37. Virolle MJ. A challenging view: antibiotics play a role in the regulation of the energetic metabolism of the producing bacteria. Antibiotics (Basel) [Internet]. 2020 Feb 13;9(2):83. Available from: https://pubmed.ncbi.nlm.nih.gov/32069930

38. Millan-Oropeza A, Henry C, Lejeune C, David M, Virolle MJ. Expression of genes of the Pho regulon is altered in Streptomyces coelicolor. Sci Rep [Internet]. 2020 May 22;10(1):8492. Available from: https://pubmed.ncbi.nlm.nih.gov/32444655

39. Esnault C, Dulermo T, Smirnov A, Askora A, David M, Deniset-Besseau A, et al. Strong antibiotic production is correlated with highly active oxidative metabolism in Streptomyces coelicolor M145. Sci Rep [Internet]. 2017 Mar 15;7(1):200. Available from: https://pubmed.ncbi.nlm.nih.gov/28298624

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