Advertisement

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

Abstract

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.

Keywords

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

Notes

Acknowledgments

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)

References

  1. Alam MT, Merlo ME, The STREAM Consortium, Hodgson DA, Wellington EMH, Takano E, Breitling R (2010) Metabolic modeling and analysis of the metabolic switch in Streptomyces coelicolor. BMC Genomics 11:202CrossRefPubMedPubMedCentralGoogle Scholar
  2. Antoniewicz MR (2015) Methods and advances in metabolic flux analysis: a mini-review. J Ind Microbiol Biotechnol 42:317–325CrossRefPubMedGoogle Scholar
  3. Arulanantham H, Kershaw NJ, Hewitson KS, Hughes CE, Thirkettle JE, Schofield CJ (2006) ORF17 from the clavulanic acid biosynthesis gene cluster catalyzes the ATP-dependent formation of N-glycyl-clavaminic acid. J Biol Chem 281:279–287CrossRefPubMedGoogle Scholar
  4. Bachmann B, Li R, Townsend C (1998) Beta-lactam synthetase: a new biosynthetic enzyme. Proc Natl Acad Sci U S A 95:9082–9086CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bellão C, Antonio T, Araujo MLGC, Badino AC (2013) Production of clavulanic acid and cephamycin c by Streptomyces clavuligerus under different fed-batch conditions. Braz J Chem Eng 30:257–266CrossRefGoogle Scholar
  6. Borodina I, Krabben P, Nielsen J (2005) Genome-scale analysis of Streptomyces coelicolor A3(2) metabolism. Genome Res 15:820–829CrossRefPubMedPubMedCentralGoogle Scholar
  7. Brown A, Butterworth D, Cole M, Hanscomb G, Hood J, Reading C, Rolinson G (1976) Naturally occurring β-lactamase inhibitors with actibacterial activity. J Antibiot (Tokyo) 29:668–669CrossRefGoogle Scholar
  8. Bushell ME, Kirk S, Zhao H-J, Avignone-Rossa CA (2006) Manipulation of the physiology of clavulanic acid biosynthesis with the aid of metabolic flux analysis. Enzym Microb Technol 39:149–157CrossRefGoogle Scholar
  9. Cerri MO, Badino AC (2012) Shear conditions in clavulanic acid production by Streptomyces clavuligerus in stirred tank and airlift bioreactors. Bioprocess Biosyst Eng 35:977–984CrossRefPubMedGoogle Scholar
  10. Chan M, Sim T (1998) Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate. Microbiology 144:3229–3237CrossRefPubMedGoogle Scholar
  11. Colombié V, Bideaux C, Goma G, Uribelarrea JL (2005) Effects of glucose limitation on biomass and spiramycin production by Streptomyces ambofaciens. Bioprocess Biosyst Eng 28:55–61CrossRefPubMedGoogle Scholar
  12. D’Huys PJ, Lule I, Vercammen D, Anné J, Van Impe JF, Bernaerts K (2012) Genome-scale metabolic flux analysis of Streptomyces lividans growing on a complex medium. J Biotechnol 161:1–13CrossRefPubMedGoogle Scholar
  13. Darken MA, Berenson H, Shirk RJ, Sjolander NO (1960) Production of tetracycline by Streptomyces aureofaciens in synthetic media. Appl Microbiol 8:46–51PubMedPubMedCentralGoogle Scholar
  14. Dekleva ML, Strohl WR (1988) Activity of phosphoenolpyruvate carboxylase of an anthracycline-producing streptomycete. Can J Microbiol 34:1241–1246CrossRefPubMedGoogle Scholar
  15. Foulstone M, Reading C (1982) Assay of amoxicillin and clavulanic acid, the components of augmentin, in biological fluids with high-performance liquid chromatography. Antimicrob Agents Chemother 22:753–762CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fulston M, Davison M, Elson SW, Tyler JW, Woroniecki SR (2001) Clavulanic acid biosynthesis; the final steps. J Chem Soc Perkin Trans 1(1):1122–1130CrossRefGoogle Scholar
  17. Haines RJ, Pendleton LC, Eichler DC (2011) Argininosuccinate synthase: at the center of arginine metabolism. Int J Biochem Mol Biol 2:8–23PubMedGoogle Scholar
  18. Hamed RB, Gomez-Castellanos JR, Henry L, Ducho C, McDonough MA, Schofield CJ (2013) The enzymes of β-lactam biosynthesis. Nat Prod Rep 30:21–107CrossRefPubMedGoogle Scholar
  19. Hodgson DA (2000) Primary metabolism and its control in Streptomycetes: a most unusual group of bacteria. Adv Microb Physiol 42:47–238CrossRefPubMedGoogle Scholar
  20. Hwang K-S, Kim HU, Charusanti P, Palsson BØ, Lee SY (2014) Systems biology and biotechnology of Streptomyces species for the production of secondary metabolites. Biotechnol Adv 32:255–268CrossRefPubMedGoogle Scholar
  21. Iqbal A, Arunlanantham H, Brown T, Chowdhury R, Clifton IJ, Kershaw NJ, Hewitson KS, McDonough MA, Schofield CJ (2010) Crystallographic and mass spectrometric analyses of a tandem GNAT protein from the clavulanic acid biosynthesis pathway. Proteins 78:1398–1407PubMedGoogle Scholar
  22. Ives PR, Bushell ME (1997) Manipulation of the physiology of clavulanic acid production in Streptomyces clavuligerus. Microbiology 143:3573–3579CrossRefPubMedGoogle Scholar
  23. Iwata-Reuyl D, Townsend CA (1992) Common origin of clavulanic acid and other clavam. J Am Chem Soc 7:2762–2763CrossRefGoogle Scholar
  24. Jamshidi N, Palsson BØ (2007) Investigating the metabolic capabilities of Mycobacterium tuberculosis H37Rv using the in silico strain iNJ661 and proposing alternative drug targets. BMC Syst Biol 1:26CrossRefPubMedPubMedCentralGoogle Scholar
  25. Junne S, Klingner A, Kabisch J, Schweder T, Neubauer P (2011) A two-compartment bioreactor system made of commercial parts for bioprocess scale-down studies: impact of oscillations on Bacillus subtilis fed-batch cultivations. Biotechnol J 6:1009–1017CrossRefPubMedGoogle Scholar
  26. Khaleeli N, Li R, Townsend CA (1999) Origin of the β-lactam carbons in clavulanic acid from an unusual thiamine pyrophosphate-mediated reaction. J Am Chem Soc 121:9223–9224CrossRefGoogle Scholar
  27. Kirk S, Avignone-rossa CA, Bushell ME (2000) Growth limiting substrate affects antibiotic production and associated metabolic fluxes in Streptomyces clavuligerus. Biotechnol Lett 22:1803–1809CrossRefGoogle Scholar
  28. Krol WJ, Basak A, Salowe SP, Townsend CA (1989) Oxidative cyclization chemistry catalyzed by clavaminate synthase. J Am Chem Soc 111:7625–7627CrossRefGoogle Scholar
  29. Kumar P, Dubey KK (2017) Mycelium transformation of Streptomyces toxytricini into pellet: role of culture conditions and kinetics. Bioresour Technol 228:339–347CrossRefPubMedGoogle Scholar
  30. Kurakake M, Hirotsu S, Shibata M, Takenaka Y, Kamioka T, Sakamoto T (2017) Effects of nonionic surfactants on pellet formation and the production of β-fructofuranosidases from Aspergillus oryzae KB. Food Chem 224:139–143CrossRefPubMedGoogle Scholar
  31. 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
  32. Lemoine A, Maya Martnez-Iturralde N, Spann R, Neubauer P, Junne S (2015) Response of Corynebacterium glutamicum exposed to oscillating cultivation conditions in a two- and a novel three-compartment scale-down bioreactor. Biotechnol Bioeng 112:1220–1231CrossRefPubMedGoogle Scholar
  33. Lemoine A, Limberg MH, Kästner S, Oldiges M, Neubauer P, Junne S (2016) Performance loss of Corynebacterium glutamicum cultivations under scale-down conditions using complex media. Eng Life Sci 16:620–632CrossRefGoogle Scholar
  34. Licona-Cassani C, Marcellin E, Quek LE, Jacob S, Nielsen LK (2012) Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production. Antonie van Leeuwenhoek, Int J Gen Mol Microbiol 102:493–502CrossRefGoogle Scholar
  35. Llarrull LI, Testero SA, Fisher JF, Mobashery S (2010) The future of the β-lactams. Curr Opin Microbiol 13:551–557CrossRefPubMedGoogle Scholar
  36. Marsh EN, Chang MD, Townsend CA (1992) Two isozymes of clavaminate synthase central to clavulanic acid formation: cloning and sequencing of both genes from Streptomyces clavuligerus. Biochemistry 31:12648–12657CrossRefPubMedGoogle Scholar
  37. Martín JF (2004) Phosphate control of the biosynthesis of antibiotics and other secondary metabolites is mediated by the PhoR-PhoP system: an unfinished story. J Bacteriol 186:5197–5201CrossRefPubMedPubMedCentralGoogle Scholar
  38. 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
  39. Morris SM (1992) Regulation of enzymes of urea and arginine synthesis. Annu Rev Nutr 12(1):81–101CrossRefPubMedGoogle Scholar
  40. 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
  41. Neto AB, Hirata DB, Filho LCMC, Bellão C, Júnior ACB, Hokka CO (2005) A study on clavulanic acid production by Streptomyces clavuligerus in batch, fed-batch and continuous processes. Braz J Chem Eng 22:557–563CrossRefGoogle Scholar
  42. Neubauer P, Cruz-Bournazou N, Glauche F, Junne S, Knepper A, Raven M (2013) Consistent development of bioprocesses from microliter cultures to the industrial scale. Eng Life Sci 13:224–238CrossRefGoogle Scholar
  43. Olmos E, Mehmood N, Haj Husein L, Goergen JL, Fick M, Delaunay S (2013) Effects of bioreactor hydrodynamics on the physiology of Streptomyces. Bioprocess Biosyst Eng 36:259–272CrossRefPubMedGoogle Scholar
  44. 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
  45. Osadolor OA, Nair RB, Lennartsson PR, Taherzadeh MJ (2017) Empirical and experimental determination of the kinetics of pellet growth in filamentous fungi: a case study using Neurospora intermedia. Biochem Eng J 124:115–121CrossRefGoogle Scholar
  46. Ozcengiz G, Demain AL (2013) Recent advances in the biosynthesis of penicillins, cephalosporins and clavams and its regulation. Biotechnol Adv 31:287–311CrossRefPubMedGoogle Scholar
  47. Paalme T, Anne K, Elken R, Vanatalu K, Tiisma K, Vilu R (1995) The computer-controlled continuous culture of Escherichia coli with smooth change of dilution rate (A-stat). J Microbiol Methods 24:145–153CrossRefGoogle Scholar
  48. Palsson B (2005) Systems biology: properties of reconstructed networks. Cambridge University Press, CambridgeGoogle Scholar
  49. Paradkar A (2013) Clavulanic acid production by Streptomyces clavuligerus: biogenesis, regulation and strain improvement. J Antibiot (Tokyo) 66:411–420CrossRefGoogle Scholar
  50. Pinto LS, Vieira LM, Pons MN, Fonseca MMR, Menezes JC (2004) Morphology and viability analysis of Streptomyces clavuligerus in industrial cultivation systems. Bioprocess Biosyst Eng 26:177–184CrossRefPubMedGoogle Scholar
  51. Qi H, Zhao S, Fu H, Wen J, Jia X (2014) Coupled cell morphology investigation and metabolomics analysis improves rapamycin production in Streptomyces hygroscopicus. Biochem Eng J 91:186–195CrossRefGoogle Scholar
  52. Ramirez-Malule H, Junne S, López C, Zapata J, Sáez A, Neubauer P, Rios-Estepa R (2016a) An improved HPLC-DAD method for clavulanic acid quantification in fermentation broths of Streptomyces clavuligerus. J Pharm Biomed Anal 120:241–247CrossRefPubMedGoogle Scholar
  53. 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
  54. Roubos JA (2002) Bioprocesses modeling and optimization fed-batch clavulanic acid production by Streptomyces clavuligerus. Dissertation, Technische Universiteit DelftGoogle Scholar
  55. Salowe SP, Krol WJ, Iwata-Reuyl D, Townsend CA (1991) Elucidation of the order of oxidations and identification of an intermediate in the multistep clavaminate synthase reaction. Biochemistry 30:2281–2292CrossRefPubMedGoogle Scholar
  56. Sánchez S, Chávez A, Forero A, García-Huante Y, Romero A, Sánchez M, Rocha D, Sánchez B, Valos M, Guzmán-Trampe S, Rodríguez-Sanoja R, Langley E, Ruiz B (2010) Carbon source regulation of antibiotic production. J Antibiot (Tokyo) 63:442–459CrossRefGoogle Scholar
  57. 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
  58. Schuetz R, Kuepfer L, Sauer U (2007) Systematic evaluation of objective functions for predicting intracellular fluxes in Escherichia coli. Mol Syst Biol 3:119CrossRefPubMedPubMedCentralGoogle Scholar
  59. Ser H, Law JW, Chaiyakunapruk N, Jacop SA, Palanisamy UD, Chan K-G, Goh B-H, Lee L (2016) Fermentation conditions that affect clavulanic acid production in Streptomyces clavuligerus: a systematic review. Front Microbiol 7:522PubMedPubMedCentralGoogle Scholar
  60. Soh BS, Loke P, Sim T (2001) Cloning, heterologous expression and purification of an isocitrate lyase from Streptomyces clavuligerus NRRL 3585. Biochim Biophys Acta 1522:112–117CrossRefPubMedGoogle Scholar
  61. Solomon EI, Brunold TC, Davis MI, Kemsley JN, Lee S-K, Lehnert N, Neese F, Skulan AJ, Yang Y-S, Zhou J (2000) Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem Rev 100:235–350CrossRefPubMedGoogle Scholar
  62. Stephanopoulos G, Aristidou A, Nielsen J (1998) Metabolic engineering: principles and methodologies. Academic Press, CambridgeGoogle Scholar
  63. Tahlan K, Anders C, Wong A, Mosher RH, Beatty PH, Brumlik MJ, Griffin A, Hughes C, Griffin J, Barton B, Jensen SE (2007) 5S clavam biosynthetic genes are located in both the clavam and paralog gene clusters in Streptomyces clavuligerus. Chem Biol 14:131–142CrossRefPubMedGoogle Scholar
  64. Townsend CA (2002) New reactions in clavulanic acid biosynthesis. Curr Opin Chem Biol 6:583–589CrossRefPubMedGoogle Scholar
  65. Villadsen J, Nielsen J, Lidén G (2011) Bioreaction engineering principles, 3rd edn. Springer, New YorkCrossRefGoogle Scholar
  66. Viollier PH, Minas W, Dale GE, Folcher M, Thompson CJ (2001) Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis. J Bacteriol 183:3184–3192CrossRefPubMedPubMedCentralGoogle Scholar
  67. Vorisek J, Powell A, Vanek Z (1969) Regulation of biosynthesis of secondary metabolites iv. purification and properties of phosphoenolpyruvate carboxylase in Streptomyces aureofaciens. Folia Microbiol (Praha) 14:398–405CrossRefGoogle Scholar
  68. Wang C, Liu J, Liu H, Wang J, Wen J (2017) A genome-scale dynamic flux balance analysis model of Streptomyces tsukubaensis NRRL18488 to predict the targets for increasing FK506 production. Biochem Eng J 123:45–56CrossRefGoogle Scholar
  69. 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
  70. Xia X, Lin S, Xia XX, Cong FS, Zhong JJ (2014) Significance of agitation-induced shear stress on mycelium morphology and lavendamycin production by engineered Streptomyces flocculus. Appl Microbiol Biotechnol 98:4399–4407CrossRefPubMedGoogle Scholar
  71. Yousofshahi M, Ullah E, Stern R, Hassoun S (2013) MC3: a steady-state model and constraint consistency checker for biochemical networks. BMC Syst Biol 7:129CrossRefPubMedPubMedCentralGoogle Scholar
  72. Zelyas NJ, Cai H, Kwong T, Jensen SE (2008) Alanylclavam biosynthetic genes are clustered together with one group of clavulanic acid biosynthetic genes in Streptomyces clavuligerus. J Bacteriol 190:7957–7965CrossRefPubMedPubMedCentralGoogle Scholar
  73. Zhang Z, Ren JS, Harlos K, McKinnon CH, Clifton IJ, Schofield CJ (2002) Crystal structure of a clavaminate synthase-Fe(II)-2-oxoglutarate-substrate-NO complex: evidence for metal centered rearrangements. FEBS Lett 517:7–12CrossRefPubMedGoogle Scholar
  74. Zhou J, Gunsior M, Bachmann BO, Townsend CA, Solomon EI (1998) Substrate binding to the α-ketoglutarate-dependent non-heme iron enzyme clavaminate synthase 2: coupling mechanism of oxidative decarboxylation and hydroxylation. J Am Chem Soc 120:13539–13540CrossRefGoogle Scholar
  75. 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

Copyright information

© 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

Personalised recommendations