Applied Microbiology and Biotechnology

, Volume 102, Issue 9, pp 4009–4023 | Cite as

Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis

  • Howard Ramirez-Malule
  • Stefan Junne
  • Mariano Nicolás Cruz-Bournazou
  • Peter Neubauer
  • Rigoberto Ríos-Estepa
Biotechnological products and process engineering


Clavulanic acid (CA) is produced by Streptomyces clavuligerus (S. clavuligerus) as a secondary metabolite. Knowledge about the carbon flux distribution along the various routes that supply CA precursors would certainly provide insights about metabolic performance. In order to evaluate metabolic patterns and the possible accumulation of tricarboxylic acid (TCA) cycle intermediates during CA biosynthesis, batch and subsequent continuous cultures with steadily declining feed rates were performed with glycerol as the main substrate. The data were used to in silico explore the metabolic capabilities and the accumulation of metabolic intermediates in S. clavuligerus. While clavulanic acid accumulated at glycerol excess, it steadily decreased at declining dilution rates; CA synthesis stopped when glycerol became the limiting substrate. A strong association of succinate, oxaloacetate, malate, and acetate accumulation with CA production in S. clavuligerus was observed, and flux balance analysis (FBA) was used to describe the carbon flux distribution in the network. This combined experimental and numerical approach also identified bottlenecks during the synthesis of CA in a batch and subsequent continuous cultivation and demonstrated the importance of this type of methodologies for a more advanced understanding of metabolism; this potentially derives valuable insights for future successful metabolic engineering studies in S. clavuligerus.


Clavulanic acid Streptomyces clavuligerus Continuous cultivation TCA cycle intermediate accumulation Flux balance analysis 



H.R.M. thanks Prof. Sven-Olof Enfors and Victor Lopez-Agudelo for their valuable discussions about in silico simulations. The authors thank Prof. Dr. Rainer Breitling (University of Manchester, England) for providing the genome-scale model of S. clavuligerus, reported by Medema et al. (2010).

Funding information

The authors kindly acknowledge the support of the Federal German Ministry of Education and Research and the Departamento Administrativo de Ciencias, Tecnología e Innovación–COLCENCIAS (grant nos. 01DN16018 and CTO 654-2015, respectively). The German Academic Exchange Service (DAAD) funded the contribution of H.R.M. (grant number A/13/71981).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest

Ethical approval

This article does not contain any studies with human participants or animal performed by any of the authors.

Supplementary material

253_2018_8841_MOESM1_ESM.pdf (2.2 mb)
ESM 1 (PD 2295 kb)


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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Escuela de Ingeniería QuímicaUniversidad del ValleCaliColombia
  2. 2.Chair of Bioprocess Engineering, Department of BiotechnologyTechnische Universität BerlinBerlinGermany
  3. 3.Grupo de Bioprocesos, Departamento de Ingeniería QuímicaUniversidad de Antioquia (UdeA)MedellínColombia

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