Manfred Rizzi

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.

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.

A kinetic study of immobilized lipase catalysing the synthesis of isoamyl acetate by transesterification in n-hexane

Isoamyl acetate was synthesized by lipase-catalyzed transesterification of ethyl acetate in n-hexane. The selectivity and rates of ester formation decreased when water content of the immobilized enzyme exceeded 3% (w/w). Experimental observations clearly indicate that the substrates as well as the product (ethanol) act as dead-end inhibitors. A ping-pong bi-bi mechanism with competitive inhibition by substrates and products is proposed that predicts the experimental observation satisfactorily.

Screening of yeasts for production of xylitol from d-xylose

Abstract Candida mogii ATCC 18364 was selected for a xylitol producer ( Y P S =0.62 g/g ) from 11 strains of d -xylose utilizing yeasts. Systematic kinetic studies are presented for growth and xylitol formation in synthetic medium using d -xylose as the carbon and energy source. Xylitol is produced from d -xylose under aerobic as well as oxygen-limiting conditions, but not in the absence of oxygen. It can be seen from the exprimental results that the concentrations of d -xylose and dissolved oxygen have a strong influence on the yield and rate for product formation. A maximum product yield was obtained when initial d -xylose concentration and specific oxygen uptake rate were 53 g/l and 0.5 mmol O2/g/h, respectively. Kinetic studies of d -xylose uptake, d -xylose reductase and xylitol dehydrogenase were performed to explain the complex regulatory properties of C. mogii during aerobic and oxygen-limited xylitol formation.

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.

Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture

The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied. This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2. In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2. During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant. None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain. A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500). In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2). In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested. A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate. The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B12. Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium. When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium. In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.

Rapid and highly automated determination of adenine and pyridine nucleotides in extracts of Saccharomyces cerevisiae using a micro robotic sample preparation-HPLC system

An ion-pair reversed-phase chromatography method was adapted for the simultaneous separation and quantification of adenine and pyridine nucleotide concentrations in cell extracts of Saccharomyces cerevisiae. Microbial extracts including metabolites, macromolecular constituents and inorganic compounds were loaded onto a ODS pre-column in the presence of triethylamine phosphate (TEA-Pi) resulting in a selective binding of the nucleotides and removing of interfering compounds. After washing the enrichment cartridge with 30 mM TEA-Pi buffer, adenine and pyridine nucleotides were eluted with a gradient of Mg(II), the competing hetaeron. This combined cleaning and concentration step leads to remarkable improvement of the detection limit for all nucleotides of interest and column lifetimes. The clean up and separation procedures were performed automatically with a micro robotic-system and a control software package written in PASCAL. The paper reports about the application of the proposed method to separation of adenine and pyridine nucleotides in cells extracts of S. cerevisiae grown anaerobically in a continuous culture (D = 0.1 h-1). Rapidity of analysis, high sensitivity as well as reproducibility of the system and the accurate evaluation of the adenine and pyridine nucleotide concentrations make this method particularly useful for routine analysis.

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.

Determination of in-vivo cytoplasmic orthophosphate concentration in yeast

To investigate in-vivo concentrations of cytoplasmic phosphate, especially during dynamic conditions, a method has been developed that enables reproducible determination of cytoplasmic phosphate from 5 μM up to 30 μM. The method involves fast sampling, spontaneous inactivation of cell metabolism and differential extraction procedure to gain porosity of the outer cell membrane exclusively. To determine very low cytoplasmic phosphate concentrations, an enzymatic assay was linked to a sensitive spectrophotometric cycling method to increase the detection limit.

Dynamics of orthophosphate in yeast cytoplasm

SummaryOrthophosphate concentration of bakers yeast Saccharomyces cerevisiae was investigated during dynamic conditions. As an example for those dynamics in cell metabolism the transition from glucose limitation to glucose excess (Crabtree-effect) was choosen. As a result of the metabolic switch from complete to partial oxidative metabolism, the cytoplasmic phosphate concentration increased suddenly from 8.4 mM to a maximum of 17.5 mM and transiently decreased to a minimum of 7.0 mM.