W.M. van Gulik

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G.

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.

Measuring enzyme activities under standardized in vivo-like conditions for systems biology

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113‐7D grown in aerobic glucose‐limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo‐like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme‐specific, optimal conditions.

Growth of a Catharanthus roseus cell suspension culture in a modified chemostat under glucose-limiting conditions

SummaryA system for the continuous cultivation of plant cells has been developed, based on a commercially available 3–1 turbine-stirred fermentor. A special device was constructed to provide for homogeneous effluent from the culture at low dilution rates. Two steady states with Catharanthus roseus cells growing under glucose limitation are described with respect to biomass yield on the carbon and energy source glucose, specific oxygen consumption, specific carbon dioxide production and (by)product formation. From a carbon balance for each steady state it is shown that the flow of carbon to the culture (as glucose) practically equalled the flow of carbon from the culture (as biomass, carbon dioxide and (by)product). Biomass yields on glucose were 0.31 g/g and 0.35 g/g at dilution rates of 0.0060 l/h and 0.0081 l/h respectively. The striking difference between the obtained yield coefficients and biomass yield commonly found for batch-cultured plant cells is discussed.

The application of continuous culture for plant cell suspensions

Continuous culture of plant cell suspensions has been developed during the last 35 years. Starting from rather imperfect set-ups, nowadays much better equipment is used for studies on growth and production kinetics or cell physiology. In this review the development of equipment and theory, as well as the applications are discussed.

Energetic and metabolic transient response of Saccharomyces cerevisiae to benzoic acid

Saccharomyces cerevisiae is known to be able to adapt to the presence of the commonly used food preservative benzoic acid with a large energy expenditure. Some mechanisms for the adaptation process have been suggested, but its quantitative energetic and metabolic aspects have rarely been discussed. This study discusses use of the stimulus response approach to quantitatively study the energetic and metabolic aspects of the transient adaptation of S. cerevisiae to a shift in benzoic acid concentration, from 0 to 0.8 mm. The information obtained also serves as the basis for further utilization of benzoic acid as a tool for targeted perturbation of the energy system, which is important in studying the kinetics and regulation of central carbon metabolism in S. cerevisiae. Using this experimental set‐up, we found significant fast‐transient (< 3000 s) increases in O2 consumption and CO2 production rates, of ∼ 50%, which reflect a high energy requirement for the adaptation process. We also found that with a longer exposure time to benzoic acid, S. cerevisiae decreases the cell membrane permeability for this weak acid by a factor of 10 and decreases the cell size to ∼ 80% of the initial value. The intracellular metabolite profile in the new steady‐state indicates increases in the glycolytic and tricarboxylic acid cycle fluxes, which are in agreement with the observed increases in specific glucose and O2 uptake rates.

Continuous culture of suspended plant cells

SummaryContinuous culture is an attractive research tool in physiologic and growth and production kinetics research. However, fulfillment of the basic assumptions of continuous culture in the experimental set-up may cause problems. The homogeneity of plant cell cultures and effluent, particularly, may cause problems. This paper presents an experimental set-up which solves these problems and describes the use of this equipment in a study of the growth kinetics of plant cells. Industrial application of the continuous culture of plant cells in the production of secondary metabolites seems to be profitable when compared with batch or fed-batch cultures. However, various problems such as uncoupled product formation and strain instability make fed-batch culture a better choice.

Protease and phenol production of Cynara cardunculus L. cell suspension in a chemostat

Abstract Proteases from Cynara cardunculus L. are used in cheese-making. The growth of C. cardunculus cell suspension culture and the production of proteases and phenols were studied in sucrose-limited chemostat cultures at two dilution rates. Biomass yields and biomass and protease productivities in chemostat cultures were compared with those in batch cultures. C. cardunculus cells showed a high ability to adapt to chemostat conditions. Phenolic production was not observed in the steady-state cultures of C. cardunculus cell cultures in contrast to batch cultures. Protease production was linked to the exponential growth phase and was higher and more stable in sucrose-limited chemostats than in batch culture.

Use of sequential-batch fermentations to characterize the impact of mild hypothermic temperatures on the anaerobic stoichiometry and kinetics of Saccharomyces cerevisiae

This work presents a characterization of the stoichiometry and kinetics of anaerobic batch growth of Saccharomyces cerevisiae at cultivation temperatures between 12 and 30°C. To minimize the influence of the inoculum condition and ensure full adaptation to the cultivation temperature, the experiments were carried out in sequencing batch reactors. It was observed that the growth rate obtained in the first batch performed after each temperature shift was 10–30% different compared with the subsequent batches at the same temperature, which were much more reproducible. This indicates that the sequencing batch approach provides accurate and reproducible growth rate data. Data reconciliation was applied to the measured time patterns of substrate, biomass, carbon dioxide and byproducts with the constraint that the elemental conservation relations were satisfied, allowing to obtain consistent best estimates of all uptake and secretion rates. Subsequently, it was attempted to obtain an appropriate model description of the temperature dependency of these rates. It was found that the Ratkowsky model provided a better description of the temperature dependency of growth, uptake and secretion rates than the Arrhenius law. Most interesting was to find that most of the biomass‐specific rates have the same temperature dependency, leading to a near temperature independent batch stoichiometry. Biotechnol. Bioeng. 2012; 109:1735–1744.

Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions, using alcohol dehydrogenase as sensor reaction.

With the current quantitative metabolomics techniques, only whole‐cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH‐catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose‐limited chemostats under steady‐state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose‐limited steady‐state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright

Production of secondary metabolites in cell cultures of some terpenoid-indole alkaloids producing plants

Within the group of terpenoid-indole alkaloids and related compounds quite a few have pharmaceutical interest. As most of them have quite high prices on the market, the production by means of biotechnological processes seems attractive. Thus in the past years a number of studies have been dealing with plant cell, tissue and organ cultures of terpenoid-indole alkaloids producing plants from genera like Rauwolfia, Catharanthus, Vinca, Tabernaemontana, Voacanga, Amsonia and Cinchona. Three of these are at present studied in our Biotechnology Delft Leiden (BDL) project group, viz. Catharanthus, Tabernaemontana and Cinchona with the aim to improve yields of alkaloids in the cell cultures. Recently we have reviewed the work on Cinchona cell cultures (Verpoorte et al., 1985; Wijnsma and Verpoorte, 1986 e). Here we shall confine ourselves to a brief review of some of the results recently in our laboratories.