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

Production of β-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·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·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 β-lactams.     

Engineering NADH metabolism in Saccharomyces cerevisiae: formate as an electron donor for glycerol production by anaerobic, glucose-limited chemostat cultures

Anaerobic Saccharomyces cerevisiae cultures reoxidize the excess NADH formed in biosynthesis via glycerol production. This study investigates whether cometabolism of formate, a well-known NADH-generating substrate in aerobic cultures, can increase glycerol production in anaerobic S. cerevisiae cultures. In anaerobic, glucose-limited chemostat sultures (D=0.10 h(-1)) with molar formate-to-glucose ratios of 0 to 0.5, only a small fraction of the formate added to the cultures was consumed. To investigate whether incomplete formate consumption was by the unfavourable kinetics of yeast formate dehydrogenase (high k(M) for formate at low intracellular NAD(+) concentrations) strains were constructed in which the FDH1 and/or GPD2 genes, encoding formate dehydrogenase and glycerol-3-phosphate dehydrogenase, respectively, were overexpressed. The engineered strains consumed up to 70% of the formate added to the feed, thereby increasing glycerol yields to 0.3 mol mol(-1) glucose at a formate-to-glucose ratio of 0.34. In all strains tested, the molar ratio between formate consumption and additional glycerol production relative to a reference culture equalled one. While demonstrating that that format can be use to enhance glycerol yields in anaerobic S. cerevisiae cultures, This study also reveals kinetic constraints of yeast formate dehydrogenase as an NADH-generating system in yeast mediated reduction processes.     

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

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.     

Alginate oligosaccharides as enhancers of penicillin production in cultures of penicillium chrysogenum

Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 mug/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. (c) 1997 John Wiley & Sons, Inc.     

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

The results of a large number of carbon-limited chemostat cultures of Penicillium chrysogenum carried out on glucose, ethanol, and acetate as the growth limiting substrate have been used to obtain an estimation of the adenosine triphosphate (ATP) costs for mycelium growth, penicillin production, and maintenance and the overall stoichiometry of oxidative phosphorylation of the fungus. It was found that penicillin production was accompanied by a significant additional energy drain (73 mol of ATP per mole of penicillin-G) from primary metabolism. This finding has been confirmed in independent experiments and has been shown to result in a significantly lower estimate for the maximum theoretical yield of penicillin-G on the carbon source.     

Thermodynamic evaluation of energy metabolism in mixed substrate catabolism: modeling studies of stationary and oscillatory states

Thermodynamic and kinetic calculations were performed in a model of mixed substrate metabolism. The model simulates the catabolic breakdown of a first substrate, glucose (S(1)), in the presence of a second substrate, formate (S(2)), which acts as an additional source of free energy. The principal results obtained with different relative rates of uptake of S(2) allow to predict and interpret the following experimental observations: (1) the existence of increased ATP yields by mixed substrate utilization with a maximum ATP yield and optimum input (or molar) ratio for both substrates; (2) a greater assimilation of S(1) which may be interpreted as a decreasing fraction of energy required for assimilation; (3) a decrease in ATP yields due to increasing energy demand for transport; (4) an increased assimilation of the carbon source (S(1)) as a function of increasing inputs of the additional energy source; (5) thermodynamic efficiency (eta) defined as the ratio between the output power of ATP synthesis and the input catabolic power, increases for S(2)/S(1) ratios ranging between 0.08 and 2 while for ratios higher than two a slight decrease of eta was noticed; and (6) the observed maximum in ATP yield for optimum molar ratio of the two substrates corresponds to high eta predicting that higher biomass yields may be obtained through a variable, high, eta by chanelling fluxes through catabolic pathways with different ATP yields. During oscillatory behavior, maxima in fluxes were almost coincident with maxima in forces. Thus, the pattern of dissipation was not so advantageous as in the single substrate model under starvation conditions.     

Growth and energetics of Leuconostoc oenos during cometabolism of glucose with citrate or fructose.

The metabolic and energetic characterization of the growth of Leuconostoc oenos on glucose-citrate or glucose-fructose mixtures enables the potential role of this bacterium in the wine-making process to be ascertained. Moreover, mixotrophic conditions remain a suitable means for improving biomass productivities of malolactic starter cultures. When the malolactic bacterium L. oenos was grown in batch cultures on complex medium at pH 5.0 with glucose-citrate or glucose-fructose mixtures, enhancement of both the specific growth rate and biomass production yields was observed. While growth was possible on fructose as the sole source of energy, citrate alone did not allow subsequent biomass production. The metabolic interactions between the catabolic pathways of the glucose cosubstrates and the heterofermentation of hexoses led to an increased acetate yield as a result of modified NADH oxidation. However, the calculated global coenzyme regeneration showed that the reducing equivalent balance was never equilibrated. The stimulatory effects of these glucose cosubstrates on growth resulted from increased ATP synthesis by substrate-level phosphorylation via acetate kinase. While the energetic efficiency remained close to 10 g of biomass produced per mol of ATP, the increase in the specific growth rate and biomass production yields was directly related to the rate and yield of ATP generation.     

Continuous and biomass recycle fermentations of Clostridium acetobutylicum

The availability and demand of biosynthetic energy (ATP) is an important factor in the regulation of solvent production in steady state continuous cultures of Clostridium acetobutylicum. The effect of biomass recycle at a variety of dilution rates and recycle ratios under both glucose and non-glucose limited conditions on product yields and selectivities has been investigated. Under conditions of non-glucose limitation, when the ATP supply is not growth-limiting, a lower growth rate imposed by biomass recycle leads to a reduced demand for ATP and substantially higher acetone and butanol yields. When the culture is glucose limited, however, biomass recycle results in lower solvent yields and higher acid yields.List of Symbols A ₆₀₀ absorbance at 600 nm - ATP adenosine triphosphate - C _imol/dm³ concentration of componenti in the fermentor - C _i ⁰ mol/dm³ concentration of componenti in the feed - D h^–1 dilution rate - F dm³/h feed flow rate - FdH₂ ferredoxin, reduced form - NAD nicotinamide adenine dinucleotide, oxidized form - NADH nicotinamide adenine dinucleotide, reduced form - NfF mmol/g/h NADH produced from oxidation of FdH₂ per unit biomass per unit time - P dm³/h filtrate flow during biomass recycle operation - PCRP C-mole carbon per C-mole glucose utilized percent of (substrate) carbon recovered in products - R recycle ratio,P/F - SPR mmol/g/h specific production rate - X _imol product/100 mol glucose utilized product yield - Y _ATP g biomass/mol ATP biomass yield on ATP - Y _GLU g biomass/mol glucose biomass yield on glucose - Y _ig biomass/mol biomass yield on nutrienti - h^–1 specific growth rate     

Growth yield increase linked to caffeate reduction in Acetobacterium woodii

Growth yields were determined with Acetobacterium woodii strain NZva 16 on hydrogen and CO₂, formate, methanol, vanillate, ferulate and fructose in mineral medium in the absence and presence of 0.05% yeast extract. Yeast extract was not essential for growth but enhanced growth yields by 25–100% depending on the substrate fermented. Comparison of yields on formate or methanol allowed calculation of an energy yield in the range of 1.5–2 mol ATP per mol acetate formed during homoacetate fermentation of A. woodii. In the presence of 6 mM caffeate, growth yields were determined with the substrates formate or methanol. Caffeate was reduced to hydrocaffeate and increased growth yields were obtained. An ATP yield of about 1 mol per mol of caffeate reduced was calculated. Cytochromes were not detectable in cell free extracts or membrane preparations.     

Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum.

The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum. Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis. The culture response was assessed chemically and morphologically, using computerised image analysis. High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis. However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss. Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process. Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P. chrysogenum. By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P. chrysogenum.     

Modelling of the protonophoric uncoupling by phenoxyacetic acid of the plasma membrane potential of Penicillium chrysogenum

Physiological effects of phenoxyacetic acid, the penicillin V side-chain precursor, on steady-state continuous cultures of Penicillium chrysogenum have been studied both theoretically and experimentally. Theoretical calculations show that at an extracellular pH of 6.50, phenoxyacetic acid has negligible influence on the growth energetics due to protonophoric uncoupling of membrane potentials by passive diffusive uptake. On the other hand, when the extracellular pH is lowered to 5.00, a severe maintenance-related uncoupling effect of phenoxyacetic acid is calculated. These findings were confirmed experimentally by steady-state continuous cultivations with a high-yielding penicillin strain of P. chrysogenum performed on a chemically defined and glucose-limited medium at pH 6.50 and pH 5.00, both with and without phenoxyacetic acid present. The yield and maintenance coefficients were determined from steady-state measurements of the specific uptake rates of glucose and oxygen and the specific production rate of carbon dioxide as functions of the specific growth rate. Combining these data with a simple stoichiometric model for the primary metabolism of P. chrysogenum allows quantitative information to be extracted on the growth energetics in terms of ATP spent in maintenance- and growth-related processes, i.e. mATP and YxATP. The increased maintenance-related ATP consumption when adding phenoxyacetic acid at pH 5.00 agrees with the theoretical calculations on the uncoupling effect of phenoxyacetic acid. When YxATP is compared with earlier reported values for the theoretical ATP requirement for biosynthesis of P. chrysogenum, i.e. YxATP, growth, it is found that YxATP,growth is only 40-50% of YxATP, which stresses that a large amount of ATP is wasted in turnover of macromolecules, leaks, and futile cycles.     

Scale-down of penicillin production in Penicillium chrysogenum

In large-scale production reactors the combination of high broth viscosity and large broth volume leads to insufficient liquid-phase mixing, resulting in gradients in, for example, the concentrations of substrate and oxygen. This often leads to differences in productivity of the full-scale process compared with laboratory scale. In this scale-down study of penicillin production, the influence of substrate gradients on process performance and cell physiology was investigated by imposing an intermittent feeding regime on a laboratory-scale culture of a high yielding strain of Penicillium chrysogenum. It was found that penicillin production was reduced by a factor of two in the intermittently fed cultures relative to constant feed cultivations fed with the same amount of glucose per hour, while the biomass yield was the same. Measurement of the levels of the intermediates of the penicillin biosynthesis pathway, along with the enzyme levels, suggested that the reduction of the flux through the penicillin pathway is mainly the result of a lower influx into the pathway, possibly due to inhibitory levels of adenosine monophosphate and pyrophosphate and lower activating levels of adenosine triphosphate during the zero-substrate phase of each cycle of intermittent feeding.     

Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations

Based on a review of the Penicillium chrysogenum biochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo-steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if alpha-aminoadipate is converted to 6-oxo-piperidine-2-carboxylic acid (OPC). If only 40% of the alpha-aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, gamma-aminobutyrate, and 21 amino acids were measured during fed-batch cultivations. The rates of formation of penicillin V, delta-(L-alpha)-aminoadipyl-L-cysteinyl-D-valine (ACV), OPC, and the pool of isopenicillin N, 6-APA, and 8-HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide-adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric model Y(xATP) is calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculations m(ATP) is found to 3 mmol ATP/g DW/h. (c) 1995 John Wiley & Sons, Inc.     

Glucose and penicillin concentrations in agar medium below fungal colonies

The growth of colonies of Rhizoctonia cerealis and Penicillium chrysogenum on solid media in plate cultures was studied. When grown on defined media containing 10-50 mM-glucose, R. cerealis did not cause a significant reduction in the glucose concentration of the medium in advance of colonization, but did cause the formation of a steep glucose concentration gradient in the substrate below the colony; the medium directly below the centre of a 7 cm diameter colony of R. cerealis was exhausted of glucose even when the fungus was grown on medium containing 50 mM-glucose. Penicillin produced by colonies of P. chrysogenum accumulated in the medium in advance of fungal colonization. For a period up to about 18 d after inoculation, the concentration of penicillin in the medium throughout the plate increased with colony development and thereafter, except at the margins of the plate, decreased.     

Fermentation characteristics of Clostridium pasteurianum LMG 3285 grown on glucose and mannitol

Clostridium pasteurianum fermented glucose to acetate, butyrate, CO₂ and H₂. In batch cultures the fermentation pattern was only slightly affected by culture pH over the range 8·0 to 5·5. The acetate/butyrate ratio was always higher than or equal to one. Between 2·14 and 2·33 mol H₂ was produced per mol glucose fermented. At unregulated pH, more butanol and less butyrate was formed. In a carbon-limited chemostat, the steady-state acetate/butyrate ratio was always lower than one. H₂ production was approximately 1·70 mol per mol glucose consumed. Substantial amounts of extracellular protein were formed. With decreasing pH, acetate and formate production decreased, while H₂ production was highest at pH 6.0. With increasing dilution rate ( D ), the product spectrum hardly changed, but more biomass was formed. Y _glucose^max and Y _ATP^max were 55·97 and 31·48 g dry weight per mol glucose or ATP respectively. With increasing glucose input the formation of fatty acids and H₂ slightly decreased.
Continuous cultures fermented mannitol to acetate, butyrate, butanol, CO₂ and H₂. With acetate as co-substrate, butanol production and molar growth yields, Y _mannitol and Y _ATP, markedly decreased, while the butyrate and H₂ production increased. The latter reached a value of 2·21 mol H₂ per mol mannitol consumed.     

Genome‐derived minimal metabolic models for Escherichia coli MG1655 with estimated in vivo respiratory ATP stoichiometry

Metabolic network models describing growth of Escherichia coli on glucose, glycerol and acetate were derived from a genome scale model of E. coli. One of the uncertainties in the metabolic networks is the exact stoichiometry of energy generating and consuming processes. Accurate estimation of biomass and product yields requires correct information on the ATP stoichiometry. The unknown ATP stoichiometry parameters of the constructed E. coli network were estimated from experimental data of eight different aerobic chemostat experiments carried out with E. coli MG1655, grown at different dilution rates (0.025, 0.05, 0.1, and 0.3 h^?1) and on different carbon substrates (glucose, glycerol, and acetate). Proper estimation of the ATP stoichiometry requires proper information on the biomass composition of the organism as well as accurate assessment of net conversion rates under well‐defined conditions. For this purpose a growth rate dependent biomass composition was derived, based on measurements and literature data. After incorporation of the growth rate dependent biomass composition in a metabolic network model, an effective P/O ratio of 1.49 ± 0.26 mol of ATP/mol of O, K_X (growth dependent maintenance) of 0.46 ± 0.27 mol of ATP/C‐mol of biomass and m_ATP (growth independent maintenance) of 0.075 ± 0.015 mol of ATP/C‐mol of biomass/h were estimated using a newly developed Comprehensive Data Reconciliation (CDR) method, assuming that the three energetic parameters were independent of the growth rate and the used substrate. The resulting metabolic network model only requires the specific rate of growth, µ, as an input in order to accurately predict all other fluxes and yields. Biotechnol. Bioeng. 2010;107: 369–381. © 2010 Wiley Periodicals, Inc.     

A theoretical evaluation of growth yields of yeasts

Growth yields of Saccharomyces cerevisiae and Candida utilis in carbon-limited chemostat cultures were evaluated. The yields on ethanol and acetate were much lower in S. cerevisiae, in line with earlier reports that site I phosphorylation is absent in this yeast. However, during aerobic growth on glucose both organisms had the same cell yield. This can be attributed to two factors: --S. cerevisiae had a lower protein content than C. utilis; --uptake of glucose by C. utilis requires energy whereas in S. cerevisiae it occurs via facilitated diffusion. Theoretical calculations showed that, as a result of these two factors, the ATP requirement for biomass formation in C. utilis is 35% higher than in S. cerevisiae (theoretical YATP values of 20.8 and 28.1, respectively). The experimental YATP for anaerobic growth of S. cerevisiae on glucose was 16 g biomass.mol ATP-1. In vivo P/O-ratios can be calculated for aerobic growth on ethanol and acetate, provided that the gap between the theoretical and experimental ATP requirements as observed for growth on glucose is taken into account. This was done in two ways: --via the assumption that the gap is independent of the growth substrate (i.e. a fixed amount of ATP bridges the difference between the theoretical and experimental values). --alternatively, on the assumption that the difference is a fraction of the total ATP expenditure, that is dependent on the substrate. Calculations of P/O-ratios for growth of both yeasts on glucose, ethanol, and acetate made clear that only by assuming a fixed difference between theoretical and experimental ATP requirements, the P/O-ratios are more or less independent of the growth substrate. These P/O-ratios are approximately 30% lower than the calculated mechanistic values.     

Bioenergetics and end-product regulation of Clostridium thermosaccharolyticum in response to nutrient limitation

Fermentation of xylose by Clostridium thermosaccharolyticum was studied in batch and continuous culture in which the limiting nutrient was either xylose, phosphate, or ammonia. Transient results obtained in continuous cultures with batch grown inoculum and progressively higher feed substrate concentrations exhibited ethanol selectivities (moles ethanol/moles other products) in excess of 11. The hypothesis that this high ethanol selectivity was a general response to mineral nutrient limitation was tested but could not be supported. Growth and substrate consumption were related by the equation q(s)(1 - Y(x) (c))G(ATP) = (mu/Y(ATP) (max)) + m, with q(s) the specific rate of xylose consumption (moles xylose/hour . g cells), Y(x) (c) the carbon based cell yield (g cell carbon/g substrate carbon), G(ATP) the ATP gain (moles ATP produces/mol substrate catabolized), mu the specific growth rate (1/h), Y(ATP) (max) the ATP-based cell yield (g cells/mol ATP), and m the maintenance coefficient (moles ATP/hour . g cells). Y(ATP) (max) was found to be 11.6 g cells/mol ATP, and m 9.3 mol ATP/hour . g cells for growth on defined medium. Different responses to nutrient limitation were observed depending on the mode of cultivation. Batch and immobilized cell continuous cultures decreased G(ATP) by initiating production of the secondary metabolites, propanediol, and in some cases, D-lactate; in addition, batch cultures increased the fractional allocation of ATP to maintenance and/or wastage. Nitrogen-limited continuous free-cell cultures maintained a constant cell yield, whereas phosphate-limited continuous free-cell cultures did not. In the case of phosphate limitation, the decreased ATP demand associated with the lowered cell yield was accompanied by an increased rate of ATP consumption for maintenance and/or wastage. Neither nitrogen or phosphorus-limited continuous free-cell cultures exhibited an altered G(ATP) in response to mineral nutrient limitation, and neither produced secondary metabolites. (c) 1993 John Wiley & Sons, Inc.