Determination of δ‐[<scp>L</scp>‐α‐aminoadipyl]‐<scp>L</scp>‐cysteinyl‐<scp>D</scp>‐valine in cell extracts of Penicillium chrysogenum using ion pair‐RP‐UPLC‐MS/MS

Seifar, Reza M.; Deshmukh, Amit T.; Heijnen, Joseph J.; Gulik, Walter M. van

doi:10.1002/jssc.201100527

Cited by 4 publications

(3 citation statements)

References 29 publications

(33 reference statements)

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“…The pH adjustments were done in order to enable NADH analysis because NADH degrades at neutral pH [ 33 ]. The tubes also contained 500 μL 10 mM maleimide solution to enable quantification of the free reduced form of ACV [ 34 ]. Simultaneously with adding the filter to the heated ethanol tube, 100 μL 13 C pen extract was added as internal standard for the MS analysis.…”

Section: Methodsmentioning

confidence: 99%

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

Janoska

Verheijen

Tang

et al. 2022

Engineering in Life Sciences

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In large-scale bioreactors, there is often insufficient mixing and as a consequence, cells experience uneven substrate and oxygen levels that influence product formation. In this study, the influence of dissolved oxygen (DO) gradients on the primary and secondary metabolism of a high producing industrial strain of Penicillium chrysogenum was investigated. Within a wide range of DO concentrations, obtained under chemostat conditions, we observed different responses from P. chrysogenum: (i) no influence on growth or penicillin production (>0.025 mmol L −1 ); (ii) reduced penicillin production, but no growth limitation (0.013-0.025 mmol L −1 ); and (iii) growth and penicillin production limitations (<0.013 mmol L −1 ). In addition, scale down experiments were performed by oscillating the DO concentration in the bioreactor. We found that during DO oscillation, the penicillin production rate decreased below the value observed when a constant DO equal to the average oscillating DO value was used. To understand and predict the influence of oxygen levels on primary metabolism and penicillin production, we developed a black box model that was linked to a detailed kinetic model of the penicillin pathway. The model simulations represented the experimental data during the step experiments; however, during the oscillation experiments the predictions deviated, indicating the involvement of the central metabolism in penicillin production.

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Section: Methodsmentioning

confidence: 99%

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

Janoska

Verheijen

Tang

et al. 2022

Engineering in Life Sciences

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“…Approximately 1 mL of the broth was transferred from the bioreactor into the syringe for cooling of the sample within a fraction of a second to close to 0 °C and was then rapidly filtered through a Millex HV 0.45 μm filter (Millipore, Billerica, MA, USA). For the absolute determination of extracellular metabolites, 2 μL of 100 m m N ‐ethylmaleimide (NEM) for thiol group protection and 20 μL of U‐ 13 C‐labeled cell extract as an internal standard (IS) were added before the filtrate sample was quickly frozen in liquid nitrogen. Subsequently the sample was stored at −80 °C until analysis.…”

Section: Methodsmentioning

confidence: 99%

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

Wang

et al. 2019

Biotechnology Journal

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During glucose-limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β-lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady-state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose-limited chemostat cultures at the dilution rates (D) of both 0.03 h −1 and 0.05 h −1 are determined to evaluate the quantitative impact on the physiology of a high-yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h −1 , while this is not the case at D = 0.05 h −1 . In addition, a higher cytosolic redox status (NADH/NAD + ), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h −1 , which are virtually absent at D = 0.03 h −1 . In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose-limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial-scale scenario.

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“…Intermediates of the glycolysis, TCA cycles, PP pathway, and amino acids were measured by a GC-MS platform (Cipollina et al, 2009). Adenine nucleotides and penicillin pathway intermediates were analyzed by LC-MS/MS (Douma et al, 2010a;Nasution et al, 2006;Seifar et al, 2012). Extracellular glucose is also measured using the GC-MS platform methodology described by de Jonge et al (2013).…”

Section: Metabolite Analysismentioning

confidence: 99%

A 9‐pool metabolic structured kinetic model describing days to seconds dynamics of growth and product formation by Penicillium chrysogenum

Tang

Deshmukh

Haringa

et al. 2017

Biotech & Bioengineering

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A powerful approach for the optimization of industrial bioprocesses is to perform detailed simulations integrating large-scale computational fluid dynamics (CFD) and cellular reaction dynamics (CRD). However, complex metabolic kinetic models containing a large number of equations pose formidable challenges in CFD-CRD coupling and computation time afterward. This necessitates to formulate a relatively simple but yet representative model structure. Such a kinetic model should be able to reproduce metabolic responses for short-term (mixing time scale of tens of seconds) and long-term (fed-batch cultivation of hours/days) dynamics in industrial bioprocesses. In this paper, we used Penicillium chrysogenum as a model system and developed a metabolically structured kinetic model for growth and production. By lumping the most important intracellular metabolites in 5 pools and 4 intracellular enzyme pools, linked by 10 reactions, we succeeded in maintaining the model structure relatively simple, while providing informative insight into the state of the organism. The performance of this 9-pool model was validated with a periodic glucose feast-famine cycle experiment at the minute time scale. Comparison of this model and a reported black box model for this strain shows the necessity of employing a structured model under feast-famine conditions. This proposed model provides deeper insight into the in vivo kinetics and, most importantly, can be straightforwardly integrated into a computational fluid dynamic framework for simulating complete fermentation performance and cell population dynamics in large scale and small scale fermentors. Biotechnol. Bioeng. 2017;114: 1733-1743. © 2017 Wiley Periodicals, Inc.

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Determination of δ‐[L‐α‐aminoadipyl]‐L‐cysteinyl‐D‐valine in cell extracts of Penicillium chrysogenum using ion pair‐RP‐UPLC‐MS/MS

Cited by 4 publications

References 29 publications

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

Influence of oxygen concentration on the metabolism of Penicillium chrysogenum

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

A 9‐pool metabolic structured kinetic model describing days to seconds dynamics of growth and product formation by Penicillium chrysogenum

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