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: 13C-NMR spectroscopy and [1,2-13C2]glucose was 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 13C glutamate fractional enrichments, except for [4- 13C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribute of a [4-13C]ketoglutarate pool not originating from mitochondria was suggested. 13C 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 13C-NMR isotopomer analysis of alanine. Conclusions: 13C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, 13C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalised 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.

13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis / Gorietti, Daniela; Zanni, Elena; Palleschi, Claudio; Delfini, Maurizio; Uccelletti, Daniela; Saliola, Michele; Puccetti, Caterina; Sobolev, A. P.; Mannina, Luisa; Miccheli, Alfredo. - In: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS. - ISSN 0304-4165. - STAMPA. - 1850:11(2015), pp. 2222-2227. [10.1016/j.bbagen.2015.07.008]

13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis

GORIETTI, DANIELA;ZANNI, ELENA;PALLESCHI, Claudio;DELFINI, Maurizio;UCCELLETTI, Daniela;SALIOLA, Michele;PUCCETTI, Caterina;MANNINA, LUISA;MICCHELI, Alfredo
2015

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: 13C-NMR spectroscopy and [1,2-13C2]glucose was 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 13C glutamate fractional enrichments, except for [4- 13C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribute of a [4-13C]ketoglutarate pool not originating from mitochondria was suggested. 13C 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 13C-NMR isotopomer analysis of alanine. Conclusions: 13C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, 13C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalised 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.
13 C NMR; casein kinase; isotopomer analysis; lysine synthesis; metabolic profiling; yeasts; carbon-13 magnetic resonance spectroscopy; glutamic acid; ketoglutaric acids; kluyveromyces; lysine; biochemistry; biophysics; molecular biology; medicine (all)
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13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis / Gorietti, Daniela; Zanni, Elena; Palleschi, Claudio; Delfini, Maurizio; Uccelletti, Daniela; Saliola, Michele; Puccetti, Caterina; Sobolev, A. P.; Mannina, Luisa; Miccheli, Alfredo. - In: BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS. - ISSN 0304-4165. - STAMPA. - 1850:11(2015), pp. 2222-2227. [10.1016/j.bbagen.2015.07.008]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/866622
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