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Intracellular Metabolite Pool Changes in Response to Nutrient Depletion Induced Metabolic Switching in Streptomyces coelicolor.

ABSTRACT: A metabolite profiling study of the antibiotic producing bacterium Streptomyces coelicolor A3(2) has been performed. The aim of this study was to monitor intracellular metabolite pool changes occurring as strains of S. coelicolor react to nutrient depletion with metabolic re-modeling, so-called metabolic switching, and transition from growth to secondary metabolite production phase. Two different culture media were applied, providing depletion of the key nutrients phosphate and L-glutamate, respectively, as the triggers for metabolic switching. Targeted GC-MS and LC-MS methods were employed to quantify important primary metabolite groups like amino acids, organic acids, sugar phosphates and other phosphorylated metabolites, and nucleotides in time-course samples withdrawn from fully-controlled batch fermentations. A general decline, starting already in the early growth phase, was observed for nucleotide pools and phosphorylated metabolite pools for both the phosphate and glutamate limited cultures. The change in amino acid and organic acid pools were more scattered, especially in the phosphate limited situation while a general decrease in amino acid and non-amino organic acid pools was observed in the L-glutamate limited situation. A phoP deletion mutant showed basically the same metabolite pool changes as the wild-type strain M145 when cultivated on phosphate limited medium. This implies that the inactivation of the phoP gene has only little effect on the detected metabolite levels in the cell. The energy charge was found to be relatively constant during growth, transition and secondary metabolite production phase. The results of this study and the employed targeted metabolite profiling methodology are directly relevant for the evaluation of precursor metabolite and energy supply for both natural and heterologous production of secondary metabolites in S. coelicolor.

SUBMITTER: Wentzel A

PROVIDER: S-EPMC3901196 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Intracellular Metabolite Pool Changes in Response to Nutrient Depletion Induced Metabolic Switching in Streptomyces coelicolor.

Wentzel Alexander A Sletta Havard H Ellingsen Trond E TE Bruheim Per P

Metabolites 20120217 1

A metabolite profiling study of the antibiotic producing bacterium Streptomyces coelicolor A3(2) has been performed. The aim of this study was to monitor intracellular metabolite pool changes occurring as strains of S. coelicolor react to nutrient depletion with metabolic re-modeling, so-called metabolic switching, and transition from growth to secondary metabolite production phase. Two different culture media were applied, providing depletion of the key nutrients phosphate and L-glutamate, resp ...[more]

PMID: 24957373

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Project description:The well-known secondary metabolite-producing bacterium Streptomyces coelicolor is a natural choice for the development of super-hosts optimized for the heterologous expression of antibiotic biosynthetic gene clusters (BGCs). In this study, we used S. coelicolor M145 and its derivative strain M1146 where all active BGCs have been deleted and generated high-resolution quantitative time series metabolite profiles under two cultivation conditions (phosphate and nitrogen limitation to cease growth and trigger secondary metabolism). Five targeted LC-MS/MS-based methods were used to quantify intracellular primary metabolites covering phosphorylated metabolites, amino acids, organic acids, (deoxy) nucleoside/sugar phosphates, Nicotinamide adenine dinucleotide (NAD), and Coenzyme A (CoA). The nitrogen limitation resulted in a sharp decline in respiration and an immediate drop in the cell mass concentration. Intracellularly, a reduction in the level of the metabolites next to α-ketoglutarate in the tricarboxylic acid cycle and a decrease in the NADH pool were among the most prominent adaptation to this nutrient limitation. Phosphate limitation evoked a different adaptation of the metabolite pools as most of the phosphorylated metabolite pools except 6-phosphogluconic acid (6PG) pool were downregulated. 13C-isotope-labeling experiments revealed the simultaneous activity of both glycolysis and gluconeogenesis during the co-utilization of glucose and glutamate. The S. coelicolor M1146 strain had similar time-series metabolite profile dynamics as the parent M145 strain, except for a visibly increased 6PG pool in the stationary phase. In general, the nutrient limitation had a larger effect on the metabolite pool levels than the absence of secondary metabolite production in M1146. This study provides new insight into the primary carbon metabolism and its link to the secondary metabolism which is needed for further optimization of both super-host genotype and cultivation conditions.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03146-x.

Project description:BackgroundSystems biology approaches to study metabolic switching in Streptomyces coelicolor A3(2) depend on cultivation conditions ensuring high reproducibility and distinct phases of culture growth and secondary metabolite production. In addition, biomass concentrations must be sufficiently high to allow for extensive time-series sampling before occurrence of a given nutrient depletion for transition triggering. The present study describes for the first time the development of a dedicated optimized submerged batch fermentation strategy as the basis for highly time-resolved systems biology studies of metabolic switching in S. coelicolor A3(2).ResultsBy a step-wise approach, cultivation conditions and two fully defined cultivation media were developed and evaluated using strain M145 of S. coelicolor A3(2), providing a high degree of cultivation reproducibility and enabling reliable studies of the effect of phosphate depletion and L-glutamate depletion on the metabolic transition to antibiotic production phase. Interestingly, both of the two carbon sources provided, D-glucose and L-glutamate, were found to be necessary in order to maintain high growth rates and prevent secondary metabolite production before nutrient depletion. Comparative analysis of batch cultivations with (i) both L-glutamate and D-glucose in excess, (ii) L-glutamate depletion and D-glucose in excess, (iii) L-glutamate as the sole source of carbon and (iv) D-glucose as the sole source of carbon, reveal a complex interplay of the two carbon sources in the bacterium's central carbon metabolism.ConclusionsThe present study presents for the first time a dedicated cultivation strategy fulfilling the requirements for systems biology studies of metabolic switching in S. coelicolor A3(2). Key results from labelling and cultivation experiments on either or both of the two carbon sources provided indicate that in the presence of D-glucose, L-glutamate was the preferred carbon source, while D-glucose alone appeared incapable of maintaining culture growth, likely due to a metabolic bottleneck at the oxidation of pyruvate to acetyl-CoA.

Dataset Information

Intracellular Metabolite Pool Changes in Response to Nutrient Depletion Induced Metabolic Switching in Streptomyces coelicolor.

Publications

Intracellular Metabolite Pool Changes in Response to Nutrient Depletion Induced Metabolic Switching in Streptomyces coelicolor.

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