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

Abstract

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 β‐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. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 602–618, 2000.

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of β-lactam production

“…For all cultivations, including the ones with the constructed strains, steady states with regard to biomass were achieved (data not shown) and the biomass yields were comparable with yields found in previous studies (van Gulik et al, 2000;Robin et al, 2003c). The specific productivities of the reference strains were estimated to be 17(±2) mmol b-lactam (g dry wt)…”

Impact of Thermodynamic Principles in Systems Biology

“…A general problem is the ATP-balance which contains uncertain ATP stoichiometric parameters (P/O ratio, unknown growth related ATP ð¼ K X Þ and the unknown growth unrelated ATP ðm ATP Þ. Van Gulik and Heijnen [7] and Van Gulik et al [8] have shown how in vivo values for these ATP-parameters can be obtained using extensive experiments. For many organisms this ATP-information is not available.…”

“…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.…”

Integrated Approaches for Assessment of Cellular Performance in Industrially Relevant Filamentous Fungi

“…The standard for detailed physiological characterization was set in the late 1990s and is exemplified in studies that employ different cultivation modes to study various physiological states. [56][57][58] Such studies provided highly reproducible data on growth rates, yield coefficients, and productivity, allowing an assessment of conditions for optimal cellular performance.…”