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

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 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 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 contribution of a [4-¹³C]ketoglutarate pool not originating from mitochondria were suggested. ¹³C lysine enrichments confirmed the presence of two compartmentalized ?-ketoglutarate (?-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 ?-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, ¹³C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized ?-KG pool. General significance A compartmentalization of the ?-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.