Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor

Nasution, U.; Gulik, Walter M. van; Proell, Angela M.; Winden, Wouter A. van; Heijnen, Joseph J.

doi:10.1016/j.ymben.2006.04.002

Cited by 43 publications

(31 citation statements)

References 15 publications

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“…In all chemostat cultivations, the carbon balances closed for more than 90% of the carbon. From previous experiments under comparable conditions, it was calculated that the missing carbon is most likely due to the excretion of polymeric by-products (proteins and polysaccharides) and by-products of penicillin biosynthesis (24,40). Consistent with the stoichiometry of formate oxidation by FDH, the specific CO 2 production rate increased with increasing formate consumption (Fig.…”

Section: Resultssupporting

confidence: 70%

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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Section: Resultssupporting

confidence: 70%

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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“…Dissolved-oxygen tension was monitored but not controlled, and it never dropped below 50% of air saturation during the course of the experiment. O 2 and CO 2 concentrations in the off gas were analyzed using a combined paramagnetic/ infrared NGA 2000 MLT 1 gas analyzer (Fisher-Rosemount GMbH & Co, Hasselroth, Germany) (15,54). After reaching steady state, the nonlabeled phenylalanine medium was switched to a chemically identical medium containing phenylalanine uniformly labeled on carbon as the sole nitrogen source.…”

Section: Methodsmentioning

confidence: 99%

Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

et al. 2012

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The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-13 C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH 4 ) 2 SO 4 as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6⌬ aro10⌬ thi3⌬). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate.

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“…Many examples have been found which show this behaviour in Penicillium chrysogenum [3,4] and in Saccharomyces cerevisiae [26] such as for phospho glucose isomerase, enolase, phosphoglycerate mutase, phosphoglucomutase, fumarase.…”

Section: Pseudo-equilibrium Reactionsmentioning

confidence: 90%

Impact of Thermodynamic Principles in Systems Biology

Heijnen

2010

Biosystems Engineering II

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Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor

Cited by 43 publications

References 15 publications

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

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

Resolving Phenylalanine Metabolism Sheds Light on Natural Synthesis of Penicillin G in Penicillium chrysogenum

Impact of Thermodynamic Principles in Systems Biology

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