13C NMR based profiling unveils different α-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: (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.

Monitoring of metabolic profiling and water status of Hayward kiwifruits by nuclear magnetic resonance.

The metabolic profiling of kiwifruit (Actinidia deliciosa, Hayward cultivar) aqueous extracts and the water status of entire kiwifruits were monitored over the season (June-December) using nuclear magnetic resonance (NMR) methodologies. The metabolic profiling of aqueous kiwifruit extracts was investigated by means of high field NMR spectroscopy. A large number of water-soluble metabolites were assigned by means of 1D and 2D NMR experiments. The change in the metabolic profiles monitored over the season allowed the kiwifruit development to be investigated. Specific temporal trends of aminoacids, sugars, organic acids and other metabolites were observed. The water status of kiwifruits was monitored directly on the intact fruit measuring the T(2) spin-spin relaxation time by means of a portable unilateral NMR instrument, fully non-invasive. Again, clear trends of the relaxation time were observed during the monitoring period. The results show that the monitoring of the metabolic profiling and the monitoring of the water status are two complementary means suitable to have a complete view of the investigated fruit.

NMR and chemometrics in tracing European olive oils: the case study of Ligurian samples.

An NMR and chemometric analytical approach to classify extra virgin olive oils according to their geographical origin was developed within the European TRACE project (FP6-2003-FOOD-2-A, contract number: 0060942). Olive oils (896 samples) of three consecutive harvesting years (2005, 2006, and 2007) coming from Mediterranean areas were analyzed by (1)H NMR spectroscopy. Olive oil samples from Liguria, an Italian region, were chosen as a case study and PLS-DA and SIMCA modeling analyses were used to build up statistical models both to discriminate between Ligurian and non-Ligurian olive oils and to define the Ligurian olive oil class to confirm the declared provenience.

NMR metabolic profiling of organic and aqueous sea bass extracts: implications in the discrimination of wild and cultured sea bass.

The nuclear magnetic resonance (NMR) technique was used as analytical tool to determine the complete metabolic profiling of sea bass extracts: water-soluble metabolites belonging to different classes such as sugars, amino acids, dipeptides and organic acids as well as metabolites soluble in organic solvent such as lipids, sterols and fatty acids were identified. The metabolite profiling together with a suitable statistical analysis were used to discriminate between wild and cultured sea bass samples. Preliminary results show that discrimination between wild and cultured sea bass was obtained not only using fatty acid composition but also cholesterol and phosphatidylethanolamine and some water-soluble metabolites such as choline, trimethylamine oxide, glutamine, fumaric and malic acids.