title: Metabolomics investigation of recombinant mTNFα production in Streptomyces lividans.
abstract: BACKGROUND
  Whilst undergoing differentiation, Streptomyces produce a large quantity of hydrolytic enzymes and secondary metabolites, and it is this very ability that has focussed increasing interest on the use of these bacteria as hosts for the production of various heterologous proteins. However, within this genus, the exploration and understanding of the metabolic burden associated with such bio-products has only just begun. In this study our overall aim was to apply metabolomics approaches as tools to get a glimpse of the metabolic alterations within S. lividans TK24 when this industrially relevant microbe is producing recombinant murine tumour necrosis factor alpha (mTNFα), in comparison to wild type and empty (non-recombinant protein containing) plasmid-carrying strains as controls.
  
  
  RESULTS
  Whilst growth profiles of all strains demonstrated comparable trends, principal component-discriminant function analysis of Fourier transform infrared (FT-IR) spectral data, showed clear separation of wild type from empty plasmid and mTNFα-producing strains, throughout the time course of incubation. Analysis of intra- and extra-cellular metabolic profiles using gas chromatography-mass spectrometry (GC-MS) displayed similar trends to the FT-IR data. Although the strain carrying the empty plasmid demonstrated metabolic changes due to the maintenance of the plasmid, the metabolic behaviour of the recombinant mTNFα-producing strain appeared to be the most significantly affected. GC-MS results also demonstrated a significant overflow of several organic acids (pyruvate, 2-ketoglutarate and propanoate) and sugars (xylitol, mannose and fructose) in the mTNFα-producing strain.
  
  
  CONCLUSIONS
  The results obtained in this study have clearly demonstrated the metabolic impacts of producing mTNFα in S. lividans TK24, while displaying profound metabolic effects of harbouring the empty PIJ486 plasmid. In addition, the level of mTNFα produced in this study, further highlights the key role of media composition towards the efficiency of a bioprocess and metabolic behaviour of the host cells, which directly influences the yield of the recombinant product.
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. Howbeer.muhamadali@manchester.ac.uk.
authors.forename: Howbeer
authors.initials: H
authors.lastname: Muhamadali
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. Yun.Xu-2@manchester.ac.uk.
authors.forename: Yun
authors.initials: Y
authors.lastname: Xu
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. D.Ellis@manchester.ac.uk.
authors.forename: David I
authors.initials: DI
authors.lastname: Ellis
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. drupad.trivedi@manchester.ac.uk.
authors.forename: Drupad K
authors.initials: DK
authors.lastname: Trivedi
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. nicholas.rattray@manchester.ac.uk.
authors.forename: Nicholas J W
authors.initials: NJ
authors.lastname: Rattray
authors.affiliation: Bio- and Chemical Systems Technology, Reactor Engineering and Safety, Department of Chemical Engineering, KU Leuven (University of Leuven), Leuven Chem&Tech, Celestijnenlaan 200F (bus 2424), 3001, Leuven, Belgium. Kristel.Bernaerts@cit.kuleuven.be.
authors.forename: Kristel
authors.initials: K
authors.lastname: Bernaerts
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester, UK. roy.goodacre@manchester.ac.uk.
authors.forename: Royston
authors.initials: R
authors.lastname: Goodacre
mesh_terms: D000818:Animals; D016002:Discriminant Analysis; D008401:Gas Chromatography-Mass Spectrometry; D055442:Metabolome; D055432:Metabolomics; D051379:Mice; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D025341:Principal Component Analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D048170:Streptomyces lividans / Q000254:growth & development / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha