Improved secondary metabolite production in the genus Streptosporangium by optimization of the fermentation conditions.

The cultivation of strains of the genus Streptosporangium in batch fermentations demonstrated that the optimal conditions for secondary metabolite production are completely different to those of the closely related genus Streptomyces. The dissolved oxygen tension (pO(2)) was identified as an important parameter for optimal production of secondary metabolites in submerged cultures. Extreme variations of this parameter by changes in aeration (gas flow), agitation system and stirrer speed showed a tremendous impact in production yields of all investigated strains. Finally, a 20-fold increase in productivity was observed by conditions of controlled oxygen excess compared to optimal fermentation conditions for Streptomyces strains.

Intracellular glucose 6-phosphate content in Streptomyces coelicolor upon environmental changes in a defined medium.

A new, chemically defined medium providing dispersed growth and high biomass formation and a method for quantitative extraction of intracellular metabolites was used to investigate the cellular response of Streptomyces coelicolor A3(2) during growth and upon changes in nutrient utilization. Fast changes of the glucose 6-phosphate content precisely signaled transitions in the flow of carbon sources. The results indicate that intracellular pool sizes may be used to detect early nutrient limitations in view of the onset of antibiotic production. Additionally the results disclose characteristics of the regulation of maltose and glutamic acid uptake and degradation in S. coelicolor A3(2).

Microbial growth and production kinetics of Streptomyces antibioticus Tü 6040.

Streptomyces antibioticus Tü 6040 is the producer of simocyclinones, which belong to a novel family of angucyclinone antibiotics some of which show antitumor activities. Growth and antibiotic production is dependent on the medium composition, especially on the carbon and nitrogen source, and on the fermentation conditions. The best results with respect to antibiotic productivity were achieved using a chemically defined medium with glycerol and L-lysine as carbon and nitrogen source, respectively, in an airlift fermenter with minimised shear stress at low gas flow rates withour oxygen limitation. These conditions led to a homogeneous formation of pellets of 1-2 mm in diameter and guaranteed reproducible product yields of the main compound, simocyclinone D8, in the range of 300 mg/l.

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae: II. Mathematical model.

A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae : I. Experimental observations.

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.

Fast purification and kinetic studies of the glycerol-3-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae.

The glycerol-3-phosphate dehydrogenase has been purified from Saccharomyces cerevisiae 140-fold to electrophoretic homogeneity by a simple procedure involving affinity and ion exchange chromatography. The purified enzyme was most active at pH 6.8 and 51 degrees C. Its molecular mass was determined to be 45000 +/- 2000 Da by SDS-polyacrylamide gel electrophoresis. At physiological pH values the thermodynamic equilibrium constant was determined to be 3.5 x 10(-3) (M-1). Product inhibition as well as competitive inhibition patterns were found which clearly indicate that the kinetic mechanism of the glycerol-3-phosphate dehydrogenase is random bi-bi with the formation of dead-end complexes. In vivo concentrations of selected metabolites and kinetic expression for G3P-DH were used to explain regulatory properties of this enzyme under conditions of short-term glucose effect in Saccharomyces cerevisiae.

In vivo investigations of glucose transport in Saccharomyces cerevisiae.

In the present study, the glucose transport into the yeast Saccharomyces cerevisiae has been investigated. The approach suggested is based on a rapid sampling technique for studying the dynamic response of the yeast to rapid changes in extracellular glucose concentrations. For this purpose a concentrated glucose solution has been injected into a continuous culture at steady state growth conditions resulting in a shift of the extracellular glucose level. Samples have been taken every 5 s for determination of extracellular glucose and intracellular glucose-6-phosphate concentrations. Attempts to fit the experimental observations with simulations from existing models failed. The mechanism then proposed is based on a facilitated diffusion of glucose superimposed by an inhibition of glucose-6-phosphate. The use of the so-called in vivo approach suggested in this article appears to be proper, because the investigations can be performed at defined physiological states of the microbial cultures. Furthermore, the experimental observations are not being corrupted by the preparation of the samples for the transport studies as it happens during radioactive measurements. (c) 1996 John Wiley & Sons, Inc.

In vivo analysis of glucose-induced fast changes in yeast adenine nucleotide pool applying a rapid sampling technique.

Transition from glucose limitation to glucose excess in Saccharomyces cerevisiae leads to a fast change from complete to partial oxidative metabolism. To determine rapid changes of in vivo concentrations of yeast metabolites, occurring in the range of seconds after a glucose injection, a fast sampling technique was developed. A harvesting device with negligible dead space and highly efficient inactivation and extraction steps was applied to analyze the behavior of the physiological concentrations of adenine nucleotides as the dynamic response to fast changing glucose concentrations. Adenine nucleotides were selected as appropriate references due to their high turnover rates (in the range of seconds). During the first 5 s after a glucose pulse to a continuous chemostat culture, a significant decrease (30%) in ATP concentration and a coincident rise of ADP and AMP values were found. A further advantage of this technique is the high sampling frequency (5 s) and the long-term sterility ensuring recurrent harvesting.