title: 13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis.
abstract: BACKGROUND
  The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations.
  
  
  METHODS
  (13)C NMR spectroscopy and [1,2-(13)C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants.
  
  
  RESULTS
  Rag8 showed a decrease of all (13)C glutamate fractional enrichments, except for [4-(13)C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-(13)C]ketoglutarate pool not originating from mitochondria were suggested. (13)C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by (13)C NMR isotopomer analysis of alanine.
  
  
  CONCLUSIONS
  (13)C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, (13)C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool.
  
  
  CONCLUSIONS
  A compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.
authors.affiliation: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: daniela.gorietti@virgilio.it.
authors.forename: D
authors.initials: D
authors.lastname: Gorietti
authors.affiliation: Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: elena.zanni@uniroma1.it.
authors.forename: E
authors.initials: E
authors.lastname: Zanni
authors.affiliation: Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: claudio.palleschi@uniroma1.it.
authors.forename: C
authors.initials: C
authors.lastname: Palleschi
authors.affiliation: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: maurizio.delfini@uniroma1.it.
authors.forename: M
authors.initials: M
authors.lastname: Delfini
authors.affiliation: Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: daniela.uccelletti@uniroma1.it.
authors.forename: D
authors.initials: D
authors.lastname: Uccelletti
authors.affiliation: Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: michele.saliola@uniroma1.it.
authors.forename: M
authors.initials: M
authors.lastname: Saliola
authors.affiliation: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: caterinapuccetti@gmail.com.
authors.forename: C
authors.initials: C
authors.lastname: Puccetti
authors.affiliation: Istituto di Metodologie Chimiche (IMC) CNR, Via Salaria Km 29.300, Monterotondo 00015, Rome, Italy. Electronic address: anatoli.sobolev@cnr.it.
authors.forename: A P
authors.initials: AP
authors.lastname: Sobolev
authors.affiliation: Istituto di Metodologie Chimiche (IMC) CNR, Via Salaria Km 29.300, Monterotondo 00015, Rome, Italy; Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro, 5, I-00185 Rome, Italy. Electronic address: luisa.mannina@uniroma1.it.
authors.forename: L
authors.initials: L
authors.lastname: Mannina
authors.affiliation: Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: alfredo.miccheli@uniroma1.it.
authors.forename: A
authors.initials: A
authors.lastname: Miccheli
mesh_terms: D066241:Carbon-13 Magnetic Resonance Spectroscopy / Q000379:methods*; D018698:Glutamic Acid / Q000096:biosynthesis*; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007716:Kluyveromyces / Q000378:metabolism*; D008239:Lysine / Q000096:biosynthesis*
chemical_list: D007656:Ketoglutaric Acids; D018698:Glutamic Acid; D008239:Lysine
keywords: (13)C NMR; Casein kinase; Isotopomer analysis; Lysine synthesis; Metabolic profiling; Yeasts