Streptomyces clavuligerus shows a strong association between TCA cycle intermediate accumulation and clavulanic acid biosynthesis
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.
KeywordsClavulanic 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).
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Conflict of interest
The authors declare that they have no conflict of interest
This article does not contain any studies with human participants or animal performed by any of the authors.
- Lee S, Song H, Kim T, Sohn S (2009) Validation of metabolic models. In: Smolke C (ed) The metabolic pathway engineering handbook: fundamentals, 1st edn. CRC Press, Taylor & Francis Group, Boca Raton, pp 20.1–20.12Google Scholar
- Medema MH, Trefzer A, Kovalchuk A, van den Berg M, Müller U, Heijne W, Wu L, Alam MT, Ronning CM, Nierman WC, Bovenberg RAL, Breitling R, Takano E (2010) The sequence of a 1.8-mb bacterial linear plasmid reveals a rich evolutionary reservoir of secondary metabolic pathways. Genome Biol Evol 2:212–224CrossRefPubMedPubMedCentralGoogle Scholar
- Mosher RH, Paradkar AS, Anders C, Barton B, Jensen SE (1999) Genes specific for the biosynthesis of clavam metabolites antipodal to clavulanic acid are clustered with the gene for clavaminate synthase 1 in Streptomyces clavuligerus. Antimicrob Agents Chemother 43:1215–1224PubMedPubMedCentralGoogle Scholar
- Omura S, Ikeda H, Ishikawa J, Hanamoto A, Takahashi C, Shinose M, Takahashi Y, Horikawa H, Nakazawa H, Osonoe T, Kikuchi H, Shiba T, Sakaki Y, Hattori M (2001) Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci 98:12215–12220CrossRefPubMedPubMedCentralGoogle Scholar
- Palsson B (2005) Systems biology: properties of reconstructed networks. Cambridge University Press, CambridgeGoogle Scholar
- Ramirez-Malule H, Restrepo A, Cardona W, Junne S, Neubauer P, Rios-Estepa R (2016b) Inversion of the stereochemical configuration (3S,5S)-clavaminic acid into (3R,5R)-clavulanic acid: a computationally-assisted approach based on experimental evidence. J Theor Biol 395:40–50CrossRefPubMedGoogle Scholar
- Roubos JA (2002) Bioprocesses modeling and optimization fed-batch clavulanic acid production by Streptomyces clavuligerus. Dissertation, Technische Universiteit DelftGoogle Scholar
- Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ (2011) Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nat Protoc 6:1290–1307CrossRefPubMedPubMedCentralGoogle Scholar
- Stephanopoulos G, Aristidou A, Nielsen J (1998) Metabolic engineering: principles and methodologies. Academic Press, CambridgeGoogle Scholar
- Wu TK, Busby RW, Houston TA, Mcilwaine DB, Egan LA, Townsend CA, Wu T, Busby RW, Houston TA, Ilwaine DBMC, Egan LA, Townsend CA (1995) Identification, cloning, sequencing, and overexpression of the gene encoding proclavaminate amidino hydrolase and characterization of protein function in clavulanic acid biosynthesis. J Bacteriol 177:3714–3720CrossRefPubMedPubMedCentralGoogle Scholar
- Zhou J, Kelly WL, Bachmann BO, Gunsior M, Townsend CA, Solomon EI (2001) Spectroscopic studies of substrate interactions with clavaminate synthase 2, a multifunctional α-kg-dependent non-heme iron enzyme: correlation with mechanisms and reactivities spectroscopic studies of substrate interactions with clavaminate synthase 2. J Am Chem Soc 123:7388–7398CrossRefPubMedGoogle Scholar