D Gorietti1, E Zanni2, C Palleschi3, M Delfini4, D Uccelletti5, M Saliola6, C Puccetti7, A P Sobolev8, L Mannina9, A Miccheli10. 1. Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: daniela.gorietti@virgilio.it. 2. Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: elena.zanni@uniroma1.it. 3. Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: claudio.palleschi@uniroma1.it. 4. Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: maurizio.delfini@uniroma1.it. 5. Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: daniela.uccelletti@uniroma1.it. 6. Dipartimento di Biologia e Biotecnologie C. Darwin, Sapienza Università di Roma, Piazzale A. Moro, 5, 00185 Rome, Italy. Electronic address: michele.saliola@uniroma1.it. 7. Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: caterinapuccetti@gmail.com. 8. Istituto di Metodologie Chimiche (IMC) CNR, Via Salaria Km 29.300, Monterotondo 00015, Rome, Italy. Electronic address: anatoli.sobolev@cnr.it. 9. 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. 10. Dipartimento di Chimica, Sapienza Università di Roma, Piazzale Aldo Moro, 5, 00185 Rome, Italy. Electronic address: alfredo.miccheli@uniroma1.it.
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. GENERAL SIGNIFICANCE: 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.
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 lactisyeast 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. lactisyeast 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. GENERAL SIGNIFICANCE: 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.