Energetics of growth and penicillin production in a high-producing strain ofPenicillium chrysogenum

vanGulik, W. M.; Antoniewicz, Maciek R.; deLaat, W. T. A. M.; Vinke, J. L.; Heijnen, Joseph J.

doi:10.1002/1097-0290(20000120)72:2<185::aid-bit7>3.0.co;2-m

Cited by 66 publications

(56 citation statements)

References 26 publications

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“…The theory and practice of metabolic flux balancing has been described well in literature and will not be repeated here (25)(26)(27)(28)(29). For each carbon source the specific rates of growth, substrate consumption, carbon dioxide production, and oxygen consumption during steady-state chemostat cultivation were calculated from the measured concentrations and flow rates from three independent experiments.…”

Section: Methodsmentioning

confidence: 99%

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae

Daran‐Lapujade

Jansen

Daran

et al. 2004

Journal of Biological Chemistry

246

207

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In contrast to batch cultivation, chemostat cultivation allows the identification of carbon source responses without interference by carbon-catabolite repression, accumulation of toxic products, and differences in specific growth rate. This study focuses on the yeast Saccharomyces cerevisiae, grown in aerobic, carbon-limited chemostat cultures. Genome-wide transcript levels and in vivo fluxes were compared for growth on two sugars, glucose and maltose, and for two C2-compounds, ethanol and acetate. In contrast to previous reports on batch cultures, few genes (180 genes) responded to changes of the carbon source by a changed transcript level. Very few transcript levels were changed when glucose as the growth-limiting nutrient was compared with maltose (33 transcripts), or when acetate was compared with ethanol (16 transcripts). Although metabolic flux analysis using a stoichiometric model revealed major changes in the central carbon metabolism, only 117 genes exhibited a significantly different transcript level when sugars and C2-compounds were provided as the growthlimiting nutrient. Despite the extensive knowledge on carbon source regulation in yeast, many of the carbon source-responsive genes encoded proteins with unknown or incompletely characterized biological functions. In silico promoter analysis of carbon source-responsive genes confirmed the involvement of several known transcriptional regulators and suggested the involvement of additional regulators. Transcripts involved in the glyoxylate cycle and gluconeogenesis showed a good correlation with in vivo fluxes. This correlation was, however, not observed for other important pathways, including the pentose-phosphate pathway, tricarboxylic acid cycle, and, in particular, glycolysis. These results indicate that in vivo fluxes in the central carbon metabolism of S. cerevisiae grown in steadystate, carbon-limited chemostat cultures are controlled to a large extent via post-transcriptional mechanisms.The yeast Saccharomyces cerevisiae is widely used as a model organism to study carbon source-dependent metabolic regulation in eukaryotes. Wild-type S. cerevisiae strains have a narrow set of carbon sources that can support fast growth in synthetic media (1). The most widely studied of these are the hexoses glucose, fructose, galactose, and mannose, the disaccharides maltose and sucrose, and the C2-compounds ethanol and acetate. The metabolic networks employed for the metabolism of the hexoses and disaccharides are very similar and differ only in the initial steps of metabolism (Fig. 1). For example, glucose and maltose metabolism differ only with respect to two reactions. The first reaction is the sugar transport through the plasma membrane; maltose uptake is catalyzed by an energy-dependent maltose-proton symport mechanism (Fig. 1, step 30) (2), whereas glucose uptake is catalyzed exclusively by a facilitated diffusion mechanism (step 33) (3). The second reaction is the intracellular breakdown of maltose into glucose, which involves a specific ␣-glucosidase ("m...

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Section: Methodsmentioning

confidence: 99%

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae

Daran‐Lapujade

Jansen

Daran

et al. 2004

Journal of Biological Chemistry

246

207

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“…Previous studies on penicillin G production in a high-producing industrial strain of P. chrysogenum have shown that constraints in central metabolism may reside in the supply and regeneration of the cofactor NADPH rather than in the supply of the carbon precursors, ␣-aminoadipic acid, cysteine, and valine (40). Moreover, a careful model-based analysis of chemostat data revealed that penicillin G production in this strain appeared to be associated with an unexpectedly high additional energy dissipation (corresponding to 73 mol of ATP per mol penicillin G) (39).…”

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confidence: 89%

“…For a quantitative evaluation of the effect of coconsuming formate as an auxiliary energy source, metabolic modeling was performed using a previously developed stoichiometric metabolic network model for this strain (40). Calculations were based on previously published P/O ratios for cytosolic and mitochondrial NADH (39). For these calculations, it was assumed that the biomass-specific penicillin G production rate was not affected by formate coconsumption.…”

Section: Downloaded Frommentioning

confidence: 99%

“…The model was extended with a cytosolic NAD ϩ -specific FDH reaction and two alternative formate uptake systems, either passive diffusion of the undissociated acid or active transport requiring 1 mol of ATP per mol of formate imported. The ATP stoichiometry parameters which were used in the model were the same as those estimated previously for this P. chrysogenum strain (39). The determined stoichiometric model required eight measured net conversion rates to calculate all reaction rates and remaining net conversion rates, namely, biomass growth rate, penicillin G production, formate consumption, formation of by-products associated with penicillin production (6-aminopenicillanic acid, 8-hydroxypenillic acid, and 6-oxopiperidine-2-carboxylic acid), and formation of by-products associated with biomass growth (peptides and polysaccharides).…”

mentioning

confidence: 99%

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Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum : Impact on Penicillin G Production and Biomass Yield

Harris

Krogt

Gulik

et al. 2007

Appl Environ Microbiol

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Production of ␤-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol ⅐ mol ؊1 , an increasing rate of formate oxidation via a cytosolic NAD ؉ -dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol ⅐ mol ؊1 , the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as ␤-lactams.The filamentous fungus Penicillium chrysogenum is applied on a large scale (Ͼ60,000 tons year Ϫ1 ) (8, 36) for the industrial production of ␤-lactam antibiotics, such as penicillin G and penicillin V, and for the production of the cephalosporin precursor adipoyl-7-ADCA. ␤-Lactam antibiotics are formed in a multistep process in which the first two steps are common for penicillins and cephalosporins. The three amino acids cysteine, valine, and ␣-aminoadipic acid, derived from central metabolism, are condensed to form the tripeptide ACV (␣-aminoadipyl-cysteinyl-valine). The next step is a ring closure that leads to the characteristic penam structure of isopenicillin N, the branch point intermediate at which penicillin biosynthesis diverges from cephalosporin biosynthesis. Penicillin G is formed from isopenicillin N by exchanging its ␣-aminoadipic acid side chain for phenylacetic acid, using phenylacetyl-coenzyme A as a side chain donor.Overproduction of secondary metabolites can have a large impact on central metabolism if it requires significant amounts of carbon precursors, reducing equivalents (NADH and NADPH), and free energy equivalents (ATP). Previous studies on penicillin G production in a high-producing industrial strain of P. chrysogenum have shown that constraints in central metabolism may reside in the supply and regeneration of the cofactor NADPH rather than in the supply of the carbon precursors, ␣-aminoadipic aci...

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“…A very nice example is the model for penicillin production [8,16] and the model developed for biological P-removal using mixed cultures in a cyclic process [17]. These black box models show that highly complex biological systems, comprising thousands of reactions, can be effectively modeled with a reduced model containing only about 6-12 parameters.…”

Section: Black Box Models For Design Of Biotechnological Processes: Fmentioning

confidence: 99%

Impact of Thermodynamic Principles in Systems Biology

Heijnen

2010

Biosystems Engineering II

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Energetics of growth and penicillin production in a high-producing strain ofPenicillium chrysogenum

Cited by 66 publications

References 26 publications

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae

Role of Transcriptional Regulation in Controlling Fluxes in Central Carbon Metabolism of Saccharomyces cerevisiae

Formate as an Auxiliary Substrate for Glucose-Limited Cultivation of Penicillium chrysogenum : Impact on Penicillin G Production and Biomass Yield

Impact of Thermodynamic Principles in Systems Biology

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