Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum

ARTICLE IN PRESS

Metabolic Engineering 9 (2007) 112–123 www.elsevier.com/locate/ymben

Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum Roelco J. Kleijn, Feng Liu, Wouter A. van Winden, Walter M. van Gulik, Cor Ras, Joseph J. Heijnen Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands Received 23 May 2006; received in revised form 10 August 2006; accepted 14 August 2006 Available online 18 August 2006

Abstract This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production. r 2006 Elsevier Inc. All rights reserved. Keywords: Penicillium chrysogenum; Flux analysis;

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C tracer; Pentose phosphate pathway; NADPH

1. Introduction In the ongoing quest for strains with higher product titers and yields, metabolic engineering has allowed for a more rational approach to strain improvement (Thykaer and Nielsen, 2003). At the level of cellular metabolism, techniques such as metabolic network and flux analysis provide metabolic engineers with a better understanding of the metabolic network with respect to stoichiometry and flux distribution (Gombert et al., 2001; Blank and Sauer, 2004; Kiefer et al., 2004; van Winden et al., 2003). This knowledge enables a more directed genetic modification of industrial microorganism through recombinant DNA technology, thereby increasing the chance of successfully directing the metabolic fluxes towards the desired end product.

Corresponding author. Fax: +31 (0)15 2782355.

E-mail address: [email protected] (J.J. Heijnen). 1096-7176/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2006.08.004

Metabolic flux analysis (MFA) is frequently applied to study microorganisms that either overproduce primary metabolites (e.g. ethanol, glycerol, lactate) (Zhu and Shimizu, 2005; Sonderegger et al., 2004) or products closely related to the primary metabolism (e.g. amino acids) (Kiefer et al., 2004). MFA can, however, also be used to study the metabolism of microorganisms applied for secondary metabolite production, thereby paying special attention to the interconnectivity between the primary and secondary metabolic pathways. In wild-type microorganisms secondary metabolites are normally produced at levels that are several orders of magnitude lower than the primary metabolites. For this reason, classical strain improvements strategies have primarily focused on increasing the levels of enzymes in the product pathway. However, continuous amplification of the enzyme levels in the secondary metabolism will, at some point, lead to a shift of the metabolic bottleneck from secondary metabolism towards primary metabolism. Particularly in highproducing strains the role of primary metabolism in

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supplying the carbon precursors, cofactors and energy for product formation should not be underestimated. A wellknown example is the filamentous fungus Penicillium chrysogenum, of which the b-lactam antibiotic productivity has been increased with several orders of magnitude as a result of more than 50 years of classical strain improvement. A detailed review on the role of primary metabolism in the production of antibiotics is given by Gunnarsson et al. (2004). In general, three potential limitations in the primary metabolism of P. chrysogenum can be identified when synthesizing large amounts of b-lactam antibiotics (e.g. penicillin-G and penicillin-V). These are the supply of the three precursor amino acids (a-aminoadipate, valine and cysteine); the availability of electrons in the form of NADPH; and the supply of energy in the form of ATP. Note that strictly speaking a-aminoadipate is not a precursor of penicillin as it is split-off and (partially) recycled after the formation of the b-lactam nucleus. Several studies have addressed one or more of these potential bottlenecks by MFA (van Gulik et al., 2000, 2001; Jorgensen et al., 1995; Henriksen et al., 1996; Christensen et al., 2000). However, uncertainties in the metabolism of P. chrysogenum have resulted in different assumptions with respect to the applied stoichiometric models for MFA, making it difficult to unambiguously draw conclusions on potential metabolic bottlenecks. van Gulik et al. (2001), for example, estimated from experimental data a much higher ATP consumption associated with penicillin production than the theoretical value used by Jorgensen et al. (1995), resulting in a much lower theoretical maximum yield of penicillin-G on glucose (0.18 mol/mol glucose versus 0.50 mol/mol glucose, respectively). Similarly, Jorgensen et al. (1995) observed that the supply of the three precursor amino acids a-aminoadipate, valine and cysteine to a fed-batch cultivation increased the penicillin-V production by about 20%, indicating that precursor availability may limit penicillin production at high rates. On the other hand, van Gulik et al. (2000) concluded for a different P. chrysogenum strain that primary carbon metabolism was unlikely to form a potential bottleneck in penicillin-G synthesis, based upon the flexibility of four principal metabolic nodes. The supply of sufficient reducing power in the form of NADPH is especially important for the biosynthesis of the two amino acid precursors of the b-lactam nucleus, cysteine and valine. Using stoichiometric models for the primary and secondary metabolism of P. chrysogenum, several groups have speculated that the flux through the oxidative branch of the pentose phosphate pathway (PPP), being the main source of cytosolic NADPH in P. chrysogenum, is strongly correlated to b-lactam production (Henriksen et al., 1996; Jorgensen et al., 1995). This hypothesis was supported by the results of van Gulik et al. (2000), who showed that a stepwise increase in the total metabolic demand for NADPH resulted in a stepwise decrease of the penicillin-G production.

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In the past, the flux through the oxidative branch of the PPP has often been estimated via 13C-labeling-based MFA. Recently, a new method has been developed for estimating the oxidative PPP flux and the PPP split ratio (the fraction of glucose taken up by the cell that enters the oxidative branch of the PPP) (Kleijn et al., 2006). This method is based on the cofeeding of trace amounts of 13C labeled gluconate and produces more accurate estimates for the oxidative PPP flux than the 13C-labeling-based MFA method. In this study the gluconate-tracer method is applied to measure the PPP split ratio, under penicillin-G producing and non-producing conditions in carbon-limited chemostat cultures of P. chrysogenum at two different specific growth rates. By comparing the estimated PPP split ratios with those predicted from stoichiometric models based upon the recently found NADPH-specificity of various enzymes in the primary metabolism of P. chrysogenum (Harris et al., 2006), the role of NADPH in the biosynthesis of b-lactam antibiotics is further investigated. 2. Material and methods 2.1. Strain A high-yielding industrial P. chrysogenum strain (code name DS17690) was kindly donated by DSM AntiInfectives (Delft, The Netherlands). 2.2. Cultivation conditions P. chrysogenum was cultivated in a carbon-limited chemostat system at two different dilution rates (0.020 and 0.052 h1), both in the absence and presence of phenylacetic acid (PAA), the side-chain precursor for penicillin-G biosynthesis. All four cultivations were carried out in a 1 L (working volume 0.6 L) bioreactor (Applikon, Schiedam, The Netherlands) equipped with a six-bladed Rusthon turbine stirrer (D ¼ 45 mm). The stirrer speed was 600 rpm and the aeration rate was 11 L/h (0.30 vvm). Under these conditions the dissolved oxygen tension was always above 50%. Temperature was controlled at 25 1C, the pH at 6.5 and the head space overpressure at 0.1 bar. Effluent was removed discontinuously via an overflow tube by means of a peristaltic pump which was operated at fixed time intervals (1 min every 10 min). Silicone antifoam agent (BDH, Poole, UK) was diluted 1:10 (v/v) with demineralized H2O and fed to the reactor at a fixed rate of 0.2 ml/h. The batch phase was initiated by inoculating the reactor with spores from 2.0 g of rice grains. During the germination phase of the spores (first 30 h), the reactor was run at a stirrer speed of 100 rpm and an aeration rate of 1 L/h. The sole carbon source during the batch phase was 3.3 g/L of glucose H2O. The end of the batch phase was typically reached after 50 h, after which the reactor was switched to continuous mode. The continuous mode consisted of two phases; during the first phase P. chrysogenum was grown

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on an unlabeled medium for two residence times, followed by at least three residence times of feeding on a chemically identical medium, but containing uniformly labeled [u-13C]glucono-d-lactone (Omicron, South Bend, USA) instead of naturally labeled glucono-d-lactone. 2.3. Media Cells were grown on a medium specifically developed for the gluconate-tracer method (Kleijn et al., 2006), containing: 3.30 g/L glucose H2O, 0.15 g/L glucono-d-lactone, 0.68 g/L sodium-acetate 3H2O, 0.35 g/L KH2PO4, 1.54 g/L (NH4)2SO4, 0.22 g/L MgSO4  7H2O and 0.90 ml/L trace element solution. The trace element solution contained 75 g/L Na2-EDTA 2H2O, 2.5 g/L CuSO4  5H2O, 10 g/L ZnSO4  7H2O, 10 g/L MnSO4  H2O, 20 g/L FeSO4  7H2O, and 2.5 g/L CaCl2  2H2O. The small amount of sodiumacetate was added to the medium to compare these experiments with previously performed 13C-labeling experiments in which acetate was added for a better estimation of the fluxes in the lower part of the metabolism. In two of the four chemostat experiments PAA was added to the medium to induce the production of penicillin-G. Depending on the applied specific growth rate (0.020 or 0.052 h1), the medium of these experiments contained 0.533 and 0.461 g/L PAA, respectively. The PAA-level in the medium was chosen such that the steadystate concentration in the chemostat was approximately 3 mM. At this concentration PAA was neither limiting for penicillin-G production nor inhibiting for the growth of P. chrysogenum. For the preparation of the minimal medium the appropriate amount of PAA was dissolved in demineralized water, adjusted to a pH of 5.40 using 1 M KOH and autoclaved at 121 1C for 40 min. The remaining medium components were filter-sterilized using an Acropak20 filter (PALL, East-Hills, NY, USA) and added to the PAA solution. After preparation the minimal medium was stirred for at least 12 h on a magnetic stirrer to ensure that all the added glucono-d-lactone was hydrolyzed to gluconate (Sawyer and Bagger, 1959).

separation of cells and medium by filtration as described by Mashego et al. (2003). Samples for intracellular metabolite determinations were obtained by rapidly withdrawing 1 ml of broth from the bioreactor, followed by direct injection of the sample in 5 ml of a 60% (v/v) methanol/water mixture (40 1C) for instantaneous quenching of the cell metabolism as described by Lange et al. (2001). Intracellular metabolites were extracted from these samples as described previously (Kleijn et al., 2006; Nasution et al., 2006). 2.6. Analytical procedures The mass isotopomer distributions glucose-6-phosphate (g6p), 6-phospho-gluconate (6pg) and gluconic acid (gln) were measured by LC-MS as described by van Winden et al. (2005). The concentrations of glucose, gluconate and acetate in the medium and the bioreactor were determined enzymatically (Enzytec, Scil Diagnostics, Viernheim, Germany). The concentration of PAA, penicillin-G, ortho-hydroxyphenylacetic acid (o-OH-PAA), 6-oxopiperidine2-carboxylic acid (OPC), isopenicillin-N (IPN), 8-hydroxypenillic acid (8-HPA), 6-amino-penicillanic acid (6-APA) and penicilloic acid (PIO) in the filtrate samples was analyzed at 27 1C at 600 MHz on a Bruker Avance 600 nuclear magnetic resonance (NMR) spectrometer equipped with an inverse triple-resonance cryoprobe and a pulse field gradient system. Samples were prepared for analysis by combining 1.0 ml of the filtrate with 0.5 ml of standard solution, containing an exactly known concentration of maleic acid for quantification and EDTA to chelate the paramagnetic metal ions. The combined sample was lyophilized and redissolved in 600 ml D2O. For each sample the 1H-NMR spectrum was recorded using a relaxation delay of 30 s, ensuring full relaxation of all the hydrogen atoms between pulses. The integrals of the characteristic resonances for each component and the internal standard (singlet at 6.1 ppm) were measured, and the contents of the individual components were calculated. 2.7. Gluconate-tracer method

2.4. Broth sampling and dry weight determination Duplicate 10 ml samples were withdrawn from the bioreactor for determining the biomass dry weight. Samples were filtered over preweighted glass fiber filters (PALL, East-Hills, NY, USA) and dried at 70 1C for at least 24 h. The collected filtrate was immediately frozen in liquid nitrogen and stored at 80 1C before analysis. 2.5. Rapid sampling, quenching and metabolite extraction Extracellular samples were acquired by rapidly sampling 2 ml of broth into a syringe containing precooled stainless steel beads (18 1C), immediately followed by

The gluconate-tracer method was specifically designed for the accurate determination of the PPP split ratio and has been described in detail by Kleijn et al. (2006). In short, the method is based upon the simultaneous feeding of unlabeled glucose and a trace amount of [u-13C]gluconate to a carbon-limited chemostat cultivation (Fig. 1). Once isotopic steady state is reached, the mass isotopomer distributions of the intracellular metabolites surrounding the 6pg-node and the gluconate-uptake rate are measured. Substitution of these measurements in the mass isotopomer balances of 6pg (Eq. (1)) produces an over-determined system from which the oxidative PPP flux (v2) is calculated using a sequential quadratic programming algorithm. By normalizing v2 with the uptake rate of glucose, the PPP

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n-12C6

glc

determined by Monte Carlo simulation in which the added noise was normally distributed with the measured standard deviations of the mass isotopomer fractions.

u-13C6

gln

115

2.8. MFA

v1 v3

v2 g6p

glycolysis

6pg

PPP

P. chrysogenum

Fig. 1. Schematic diagram of the metabolic network surrounding the g6pnode when growing cells simultaneously on glucose (glc) and gluconate (gln).

split ratio is obtained. 1 1 3 0 20 mþ0 mþ0 C C 7 B 6B Bm þ 1C 7 6B m þ 1 C C C 7 B 6B C B C 7 B v1  6 C C 7 B .. 6B .. C C 7 B. 6B . A A 5 @ 4@ m þ 6 g ln m þ 6 6pg 1 1 3 0 20 mþ0 mþ0 C C 7 B 6B Bm þ 1C 7 6B m þ 1 C C C 7 B 6B C B C 7. B ¼ v2  6 C C 7 B .. 6B .. C C 7 B. 6B . A A 5 @ 4@ m þ 6 6pg m þ 6 g6p

Metabolite balancing in combination with a previously described stoichiometric model for growth and product formation of P. chrysogenum (van Gulik et al., 2000) was used to estimate intracellular metabolic fluxes. Calculations were carried out with the software package MNAv3.0 (SpadIT, Nijmegen, The Netherlands). Four versions of the stoichiometric model were constructed using different assumptions for NADPH consumption and production (see Results section). MFA was carried out using the measured biomass specific rates of glucose, gluconate, acetate, CO2, PAA, penicillin-G, o-OH-PAA, PIO, 8-HPA, 6-APA and OPC, which were calculated from the average steady-state values of the measured flows and concentrations. For each data set the full covariance matrix associated with the calculated conversion rates was used in the flux balancing procedure, which was carried out as described by van Gulik et al. (2000). The number of available rates was sufficient to result in an overdetermined and thus solvable system. 3. Results and discussion 3.1. Macroscopic data

ð1Þ

Using the gluconate-tracer method the PPP split ratio of P. chrysogenum was calculated for four different chemostat cultivations. For an accurate calculation of the PPP split ratio the measured mass isotopomers were corrected for naturally occurring isotopes of hydrogen and oxygen, by inverting the procedure proposed by van Winden et al. (2002). To obtain a measure for the PPP split ratio in a chemostat grown solely on glucose, the estimated PPP split ratios were corrected: (i) for the additional influx of gluconate and (ii) for the fact that per mol of glucose catabolized in the oxidative branch of the PPP, two mol of NADPH are produced, while per mol of catabolized gluconate only one mole of NADPH is produced. The latter correction was based on the assumption that the PPP flux was directly proportional to the cytosolic NADPH demand. Note that the first correction corrects for an underestimation of the PPP, whereas the second correction corrects for an overestimation of the PPP, thereby resulting in only minor changes in the final PPP split ratio. The 95% confidence intervals of the estimated PPP split ratios were

P. chrysogenum was cultivated in a carbon-limited chemostat system at a dilution rate of 0.020 and 0.052 h1, both in the absence and presence of PAA. Steady state was assumed after four residence times. Steady-state biomass concentrations and calculated recoveries of carbon, degree of reduction and PAA for the four cultivations are presented in Table 1A. Measured biomass specific conversion rates in steady state are shown in Table 1B. In all four experiments the carbon recoveries were close to 100%, whereas the degree of reduction recovery was significantly higher than 100%. Furthermore, the calculated respiratory quotients (RQ) where all well below the expected value of 1.07 for all four fermentations, indicating an error in the measured biomass specific O2 consumption rate. Data reconciliation under the constraint of the conservation relations for the elements and gross error detection was carried out according to Vanderheijden et al. (1994) and confirmed this error. Apart from the O2 consumption rate, no other significant discrepancies were observed between the measured and the reconciled conversion rates (see Table 1B). Note that for all four cultivations the gluconate uptake-rate was approximately 5% (w/w) of the glucose uptake-rate. Fig. 2 displays the measured specific consumption rate of PAA and the specific production rate of penicillin-G, b-lactam intermediates (8-HPA, 6-APA, IPN) and side products (OPC and o-OH-PAA) during the course of the

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Table 1A Steady-state biomass concentrations and recovery balances for the four gluconate-tracer experiments Biomass concentration and recovery balances

Unit

Biomass concentration Carbon recovery Degree of reduction PAA recovery

g/L (%) (%) (%)

m ¼ 0.020 h1

m ¼ 0.052 h1

Producing

Non-producing

Producing

Non-producing

1.0470.05 104.072.0 120.074.4 114.7733.5

1.2570.05 94.473.0 128.277.0 –

1.6070.09 97.174.2 108.376.5 100.975.3

1.8870.02 106.972.2 114.671.7 –

Chemostat cultivations were performed under penicillin-G producing and non-producing conditions at two different growth rates.

Table 1B Measured and reconciled biomass-specific conversion rates for the four gluconate-tracer experiments in steady state Biomass specific conversion rates (mmol Cmol1 h1)

Glucose consumption Gluconate consumption Acetate consumption Oxygen consumptionb PAA consumption Carbondioxide production Biomass production Penicillin-G and PIO production IPN production 6-APA production 8-HPA production o-OH-PAA production OPC production

m ¼ 0.020 h1

m ¼ 0.052 h1

Producing (PX0.66)a

Non-producing (PX0.08)a

Producing (PX0.26)a

Non-producing (PX0.73)a

Measured

Reconciled

Measured

Reconciled

Measured

Reconciled

Measured

Reconciled

8.6070.40 0.4270.02 2.6770.09 48.9172.10 0.5370.12 31.3471.57 20.3071.00 0.4870.06 o0.01 o0.01 o0.01 0.0970.01 0.0870.01

8.25 0.42 2.66 30.48 0.57 32.26 20.67 0.48 0.00 0.00 0.00 0.09 0.08

7.1470.29 0.3570.02 2.3070.10 40.9072.94 o0.01 22.6070.93 20.2671.00 o0.01 0.0270.00 o0.01 0.0470.01 o0.01 0.0170.00

6.72 0.35 2.28 23.15 0.00 24.31 21.98 0.00 0.02 0.00 0.04 0.00 0.01

13.9170.81 0.6970.04 4.4070.26 47.4773.65 0.4970.03 41.0071.65 52.2971.00 0.4970.02 0.0170.00 0.0270.01 o0.01 o0.01 0.1470.01

14.23 0.69 4.41 37.37 0.52 40.78 52.21 0.52 0.01 0.02 0.00 0.00 0.14

11.8170.12 0.5870.01 3.7470.04 37.6871.12 o0.01 30.1170.31 52.1871.00 o0.01 0.0470.00 0.0170.00 0.1070.01 o0.01 0.0770.01

11.85 0.58 3.74 26.23 0.00 28.86 51.55 0.00 0.04 0.01 0.10 0.00 0.07

Chemostat cultivations were performed under penicillin-G producing and non-producing conditions at two different growth rates. a P-values were determined via a w2-distribution and denote the probability that the discrepancy between the measured and the reconciled conversion rates is a result of measurement error. P-values p0.05 were considered statistically significant, thus indicating a clear deviation between the measured and reconciled conversion rates. b The erroneously measured O2 consumption rate was omitted when reconciling the conversion rates.

four chemostat cultivations. As expected no penicillin-G formation was observed in the absence of the side-chain precursor PAA. However, the absence of PAA did not completely halt the synthesis of b-lactam intermediates as small amounts of IPN, 6-APA and 8-HPA (the carboxylated form of 6-APA) were produced. Interestingly, the formation of penicillin-G intermediates seems to be growth rate dependent. At a specific growth rate of 0.020 h1 the amount of b-lactam compounds produced in the absence of PAA accounted for 13% of the total b-lactam produced under producing conditions, while this value increased to 28% at a specific growth rate of 0.052 h1. The measured specific penicillin-G production rates at dilution rates of 0.020 and 0.052 h1 corresponded well with the penicillinG production rates reported previously by van Gulik et al. (2000) for this P. chrysogenum strain. From Tables 1 it can be seen that the presence of penicillin-G production coincides with a lower mycelium concentration and increased biomass specific conversion rates of substrates, oxygen and carbon dioxide. These observations are indicative of the burden imposed by

penicillin-G production on the catabolic activity of the cell (formation of ATP and NADPH). Conversely, the absence of penicillin-G production decreases the catabolic demand of the cell in favor of the assimilation of biomass, thereby increasing the steady state mycelium concentration in the chemostat. 3.2. PPP split-ratio determination Biomass samples for intracellular metabolite determination were harvested from the four chemostat cultivations after three residence times of feeding on a medium with [u-13C]gluconate. Table 2 shows the measured mass isotopomer fractions of the gluconate added to the medium and the intracellular metabolites g6p, 6pg and gln. Table 2 shows that for all four chemostat experiments the measured mass isotopomer distributions of intracellular gluconate and the gluconate present in the medium are different, especially with respect to the m+0 and m+6 fractions, indicating that an unidentified reaction causes an inflow of unlabeled carbon into the otherwise uniformly

ARTICLE IN PRESS R.J. Kleijn et al. / Metabolic Engineering 9 (2007) 112–123

No penicillin-G production

1e-4

6e-4

Conversion rate (mmol Cmol-1 hr-1)

µ = 0.020 hr-1 Conversion rate (mmol Cmol-1 hr-1)

Penicillin-G production

4e-4 2e-4

117

-qPAA qPenG q6-APA q8-HPA qIPN qOPC qo-OH-PAA

8e-5 6e-5 4e-5 2e-5 0

0 0

1

(A)

2 3 4 5 Residence Time (-)

6

0

1

2 3 4 5 Residence Time (-)

6

0

1

2 3 4 5 Residence Time (-)

6

(B)

Conversion rate (mmol Cmol-1 hr-1)

µ = 0.052 hr-1 Conversion rate (mmol Cmol-1 hr-1)

1.2e-4 6e-4 4e-4 2e-4

6.0e-5 3.0e-5 0.0

0 0 (C)

9.0e-5

1

2 3 4 5 Residence Time (-)

6 (D)

Fig. 2. Biomass specific conversion rates of penicillin-G, IPN, 8-HPA, 6-APA, OPC, PAA and o-OH-PAA during the course of the four gluconate-tracer experiments. Conversion rates were derived from the measured concentrations of the components in the filtrate. The dashed line represents the point at which steady state was assumed (about four residence times).

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C-labeled gluconate pool. Kleijn et al. (2006) also observed this phenomenon when developing the gluconate-tracer method and proposed three candidate reactions to explain the presence of unlabeled intracellular gluconate; the oxidation of glucose to gluconate by either glucose oxidase or glucose dehydrogenase and the dephosphorylation of intracellular 6pg to gluconate by a phosphatase. Furthermore, Kleijn et al. (2006) found the enzyme activities of the three proposed enzymes to be below the detection limit of the applied assays, which was not unexpected considering the low flux estimates for the three proposed reactions. Examination of the effect of these metabolic scenarios on the actual PPP split ratio showed that an active phosphatase had no effect, while the oxidation of unlabeled glucose to gluconate led to a slightly altered PPP split ratio. Appendix A shows that for all four chemostat cultivations a small glucose-oxidation flux suffices to explain the changed labeling of intracellular gluconate. From these calculations it was concluded that the possible presence of a glucose-oxidizing reaction could be safely neglected in the PPP split-ratio estimations presented below. Table 3 shows the calculated PPP split ratios with their 95% confidence intervals. For both specific growth rates a difference is observed between the PPP split ratio of mycelia cultivated in the absence and presence of PAA. It has been reported previously that the uncoupling effect of PAA (by dissipating the proton-motive force across the plasma membrane) in this strain is negligible under the applied PAA concentration and pH (van Gulik et al., 2000). Furthermore, PAA is not catabolized by the cell, as can be inferred from the mass balance for PAA (Table 1).

Therefore the observed difference in flux distribution can be completely attributed to the formation of penicillin-G. Contrary to the above findings, Christensen et al. (2000) did not observe a correlation between the PPP split ratio and penicillin production when investigating the metabolism of a high- and a low-yielding strain of P. chrysogenum cultivated in a chemostat at a specific growth rate of 0.06–0.07 h1. In this study the PPP split ratio was determined by growing the cells on specifically labeled [1-13C] glucose, followed by a 13C-based MFA in which metabolite balances were combined with labeling patterns of proteinogenic amino acids measured with GC-MS. Using phenoxyacetic acid (POA) as the side-chain precursor, the PPP split ratio under penicillin-V producing conditions in the high- and low-yielding strain was estimated to be 0.70 and 0.66, respectively. Cultivation of the high-yielding strain in a medium without POA resulted in an estimated PPP split ratio of 0.71. Based upon these findings it was speculated that the flux through the oxidative branch of the PPP might be strain specific and not a result of metabolic burden. The rigidity of the PPP split ratio observed by Christensen et al. (2000) can be partly ascribed to the use of a different P. chrysogenum strain that had a 30% lower penicillin production rate compared to this study (0.012 versus 0.017 mmol/g biomass/h) under the applied cultivation conditions. Due to this lower production rate less NADPH was needed for penicillin synthesis, resulting in a diminished split-ratio difference between the producing and non-producing chemostat culture. Furthermore, the relatively high specific growth rate at which Christensen et al. (2000) performed their chemostat

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Table 2 Measured mass isotopomer fractions and standard deviations for the four gluconate-tracer experiments Measured metabolite

Mass isotopomer

m ¼ 0.020

m ¼ 0.052

Penicillin-G production

No penicillin-G production

Penicillin-G production

No penicillin-G production

gln in mediuma

m+0 m+1 m+2 m+3 m+4 m+5 m+6

0.00070.000 0.00070.000 0.00070.000 0.00070.000 0.00270.000 0.05970.001 0.93970.001

0.00070.000 0.00070.000 0.00070.000 0.00070.000 0.00270.000 0.05970.001 0.93970.001

0.00070.000 0.00070.000 0.00070.000 0.00070.000 0.00270.000 0.05970.001 0.93970.001

0.00070.000 0.00070.000 0.00070.000 0.00070.000 0.00270.000 0.05970.001 0.93970.001

glnb

m+0 m+1 m+2 m+3 m+4 m+5 m+6

0.21770.006 0.01970.001 0.00370.001 0.00170.000 0.00270.001 0.04070.003 0.71870.007

0.25770.005 0.02270.003 0.00270.001 0.00370.001 0.00670.001 0.04370.004 0.66970.005

0.10070.003 0.01070.001 0.00270.000 0.00170.000 0.00270.000 0.05270.002 0.83370.005

0.09670.005 0.01070.001 0.00270.001 0.00170.000 0.00270.000 0.05370.003 0.83670.007

g6pb

m+0 m+1 m+2 m+3 m+4 m+5 m+6

0.86670.003 0.09470.002 0.01770.002 0.01570.000 0.00770.000 0.00170.000 0.00170.000

0.87370.001 0.08470.001 0.01470.000 0.01570.000 0.00970.000 0.00270.000 0.00270.000

0.86470.002 0.09570.002 0.01970.001 0.01470.000 0.00870.000 0.00170.000 0.00170.000

0.86070.003 0.09370.003 0.02070.001 0.01670.000 0.00970.000 0.00170.000 0.00170.000

6pgb

m+0 m+1 m+2 m+3 m+4 m+5 m+6

0.78670.003 0.08270.003 0.01570.001 0.01170.000 0.01670.003 0.00870.001 0.08170.003

0.77270.003 0.07370.001 0.01270.000 0.01170.000 0.00970.001 0.01170.000 0.11170.002

0.78370.002 0.08470.001 0.01770.001 0.01070.001 0.01170.001 0.01070.001 0.08670.003

0.76770.002 0.08270.002 0.01770.001 0.01370.000 0.00870.001 0.01170.001 0.10370.001

Presented mass fractions have been corrected for the natural isotopes of the elements hydrogen and oxygen as described in the materials and methods section. a The mass isotopomer fractions of pure gluconate were measured once. The same gluconate was used for all four gluconate-tracer experiments. b Mass isotopomer fractions of the intracellular metabolite.

Table 3 Estimated PPP split ratios for two penicillin-G producing and two non-producing chemostat cultivations of P. chrysogenum operated at a growth rate of 0.020 and 0.052 h1 Chemostat cultivation m (h1)

Penicillin-G production

0.020 0.020 0.052 0.052

+  + 

95% lower boundary

PPP split ratio

95% upper boundary

0.472 0.355 0.447 0.376

0.517 0.381 0.494 0.411

0.559 0.407 0.538 0.443

The 95% confidence interval for the PPP split ratios was determined using Monte Carlo simulations. To obtain the PPP split ratio in a chemostat grown solely on glucose, the estimated PPP split ratios were corrected for the additional influx of gluconate and for the slightly lower NADPH production rate in the oxidative branch of the PPP as a result of gluconate uptake.

cultures reduced the chance of observing a measurable difference in split ratio between a producing and a nonproducing chemostat-culture. In general, the PPP split ratio of a cell is positively correlated with its specific growth rate because at low specific growth rates most substrate entering a cell is converted into energy (ATP) for

maintenance requirements, causing its metabolism to be dominated by catabolism. At increased growth rates the corresponding increase in anabolic activity causes an increase in the cell’s biosynthetic NADPH-demand, which increases the PPP split ratio. This effect is visualized in Fig. 3, where the relationship between the specific growth

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the PPP split ratio for the 13C-based MFA method is 70.02. Based upon these confidence intervals and Fig. 3, it was checked whether the two methods are sensitive enough to distinguish between penicillin producing and nonproducing conditions. At the specific growth rates used by Christensen et al. (2000) the difference in PPP split ratio between penicillin producing and non-producing conditions may be hard to distinguish (0.04 at m ¼ 0:07 h1 ), but at the lower specific growth rates used in this study the difference can be reliably observed by both methods (0.12 at m ¼ 0:02 h1 ).

0.6 0.5 PPP split-ratio (-)

119

0.4 0.3 0.2 0.1 qp= 0.017 mmol/g biomass/hr qp= 0 mmol/g biomass/hr

0.0 0.00

0.02

0.04 0.06 Growth rate (1/hr)

0.08

0.10

Fig. 3. Theoretical relation between the PPP split ratio and specific growth rate in a penicillin-G producing and non-producing chemostat cultivation (Eq. (B.4)). The derivation of the relation is described in Appendix B.

rate and the PPP split ratio is plotted for both a penicillinG producing and non-producing chemostat culture. Split ratios were calculated using the relation presented in Appendix B, based upon the stoichiometric model and the energetic parameters proposed by van Gulik et al. (2000, 2001). Cysteine synthesis was modeled according to the transsulfuration pathway and the maximal biomass specific penicillin-G production rate (qp) was set at a constant value of 0.017 mmol/g biomass/h independent of the specific growth rate. Note that Eq. (B.5) is based upon assumptions with respect to the yield parameters of P. chrysogenum, making it difficult to draw conclusions based upon the absolute values presented in Fig. 3. Nonetheless, the two curves clearly demonstrate that at increasing specific growth rates the difference in PPP split ratio between a b-lactam producing and a non-producing chemostat culture decreases. The relation plotted in Fig. 3 is in good accordance with the results of the present study. Table 3 shows that a smaller difference in PPP split ratio is observed between the producing and non-producing culture for the experiments performed at a specific growth rate of 0.052 h1 compared to those at 0.020 h1. Since the maximal specific penicillin production rates were practically identical for the two tested specific growth rates (Table 1), the observed difference can be fully attributed to the expected higher biosynthetic NADPH-demand at increased specific growth rates. Previous studies have shown that both the gluconatetracer method and the 13C-labeling-based MFA produce accurate estimates for the PPP split ratio. Kleijn et al. (2006) and Table 3 show that the 95% confidence interval of the PPP split ratio for the gluconate-tracer method is 70.04, while Christensen et al. (2002) show that in Saccharomyces cerevisiae the 95% confidence interval of

3.3. Sources and sinks of NADPH The increased PPP split ratio under penicillin-producing conditions indicates a higher demand for cytosolic NADPH, which is primarily caused by the synthesis of the two amino acid precursors of the b-lactam nucleus: valine and cysteine. It is known that the biosynthesis of 1 mol of valine requires 1 mol of cytosolic NADPH and 1 mol of mitochondrial NADPH. The biggest burden on the cytosolic NADPH pool is, however, imposed by the biosynthesis of cysteine. This is a direct consequence of the fact that cysteine is a sulfur-containing amino acid, requiring the active uptake and reduction of sulfate (SO3 4 ) from the medium. The reduction of sulfate to sulfide (H2S) requires four cytosolic NADPH equivalents. Cysteine is formed by coupling sulfide to the carbon backbone of serine. In P. chrysogenum two different cysteine biosynthesis pathways exist (Ostergaard et al., 1998): (i) the direct sulfhydrylation pathway where sulfide is coupled in a single step to serine and (ii) the transsulfuration pathway where sulfide is first coupled to acetyl-homoserine forming homocysteine (an intermediate in methionine biosynthesis), then coupled to serine to form cystathionine and subsequently, split into a-ketobutyrate and cysteine. Because the cytosolic NADPH requirement for the synthesis of serine is 1 mol/mol and for homocysteine 3 mol/mol, the total NADPH requirement per mol of synthesized cysteine is 5 mol for the direct sulfhydrylation pathway and 8 mol for the transsulfuration pathway. Recently, Harris et al. (2006) identified the enzymes involved in the cytosolic NADPH metabolism of an aerobic glucose-limited P. chrysogenum chemostat culture. Enzyme assays confirmed the strict NADP+-specificity of the g6p and 6pg dehydrogenases that catalyze the oxidative branch of the PPP. In addition, a cytosolic NADP+dependent isocitrate dehydrogenase was detected. No NAD+-dependent activities were detected for these three enzymes in cell-free extracts. Other cytosolic redox enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and acetaldehyde dehydrogenase, were not NADPHspecific. The observations of Harris et al. (2006) confirm to a large extent the assumptions on the cytosolic NADPH metabolism of P. chrysogenum made in the stoichiometric model of van Gulik et al. (2000).

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3.4. Stoichiometric modeling The first MFA for P. chrysogenum was performed by Jorgensen et al. (1995) based upon a stoichiometric model containing the transsulfuration pathway for cysteine synthesis and a NAD+-dependent isocitrate dehydrogenase. Jorgensen et al. (1995) showed that the PPP split ratio increases when the metabolism shifts from rapid growth to slow growth and mainly penicillin production. Furthermore, they showed that the maximum theoretical yield of penicillin on glucose increased by 20% if cysteine is synthesized by direct sulfhydrylation rather than transsulfuration. Additional insight in the NADPH metabolism of P. chrysogenum was obtained by performing a conventional MFA for different assumptions of the NADPH stoichiometry, using the stoichiometric model of van Gulik et al. (2000) and comparing the estimated PPP split ratios to the experimentally obtained results of the gluconatetracer method (Fig. 3). Four stoichiometric models were constructed, each containing (i) a cytosolic NADP+- or a NAD+-dependent isocitrate dehydrogenase, and (ii) a cysteine synthesis route based upon the transsulfuration or the direct sulfhydrylation pathway. As input for the MFA the measured extracellular rates obtained from the four chemostat conditions were used, with the exception of the erroneous oxygen consumption rate (Table 1). All presented MFA-derived PPP split ratios were statistically acceptable within the 95% confidence interval. The incorporation of a solely NAD+-dependent isocitrate dehydrogenase lead to MFA-derived PPP split ratios which were much higher than the results obtained with the gluconate-tracer method, irrespective of the used pathway for cysteine synthesis (Fig. 4B–D). This is in

agreement with the findings of Harris et al. (2006) that the cytosolic isocitrate dehydrogenase is NADP+-specific. Flux analysis shows that the relative contribution of the NADP+-specific isocitrate dehydrogenase to the total cytosolic NADPH synthesis rate was about 13%. The majority (87%) of cytosolic NADPH thus has to be synthesized by the oxidative branch of the PPP. The results for the two alternative pathways for cysteine synthesis are less straightforward to interpret, but tend to support the transsulfuration pathway. At a specific growth rate of 0.020 h1 similar PPP split ratios were estimated for the gluconate-tracer method and the transsulfurationbased model, while at a specific growth rate of 0.052 h1 a somewhat better correspondence was observed for the direct sulfhydrylation-based model. For both specific growth rates the difference in PPP split ratio between a producing and non-producing chemostat culture was best explained by the transsulfuration pathway. At a specific growth rate of 0.020 and 0.052 h1 the difference in PPP split ratio for the gluconate-tracer method was measured to be 0.136 and 0.083, respectively. Slightly smaller differences were calculated for the transsulfuration pathway (0.116 and 0.040, respectively), while much smaller differences were observed for the direct sulfhydrylation pathway (0.045 and 0.010, respectively). Both cysteine biosynthesis pathways have been identified in P. chrysogenum by Ostergaard et al. (1998). In fact, Evers et al. (2004) provided proof that each pathway has its own distinctive role and compartmentation: the mitochondrial direct sulfhydrylation pathway produces growthrelated cysteine, and the cytosolic transsulfuration pathway produces cysteine for penicillin synthesis. This implies that in the absence of penicillin synthesis only the direct No penicillin-G production

Penicillin-G production

70

(A) PPP split-ratio (%)

µ = 0.020 hr-1 PPP split-ratio (%)

70 60 50 40

50 40 30

50 40

70 (C) PPP split-ratio (%)

µ = 0.052 hr-1 PPP split-ratio (%)

60

60

Gluconate-tracer TS & Icdh (NADP+) DSH & Icdh (NADP+) TS & Icdh (NAD+) DSH & Icdh (NAD+)

30

30 70

(B)

(D)

60 50 40 30

Fig. 4. Comparison of the PPP split ratio estimated via the gluconate-tracer method with the MFA-derived PPP split ratios for four different stoichiometric models of P. chrysogenum. The four stoichiometric models each contained (i) a cytosolic NAD+-dependent or NADP+-dependent isocitrate dehydrogenase (Icdh), and (ii) a cysteine biosynthesis pathway based upon either transsulfuration (TS) or direct sulfhydrylation (DSH). PPP split ratios were derived via MFA using the measured conversion rates in Table 1B.

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sulfhydrylation pathway is active. Verification of this hypothesis using the results in this study was impossible, as only marginal differences were observed between the two cysteine-biosynthesis pathways in the MFA-derived PPP split ratios of Fig. 4B and D. These small differences were caused by the fact that very little cysteine is needed for biomass synthesis (0.15  103 mol cysteine/Cmol biomass). An implication of the low flux through the direct sulfhydrylation pathway is that the cysteine biosynthesis is predominated by the transsulfuration pathway under penicillin-producing conditions. This is in accordance with the findings of this study as explained in the previous paragraph. Apart from identifying the cytosolic NADPH-producing enzymes in P. chrysogenum, Harris et al. (2006) also revealed the presence of a mitochondrial NADPH dehydrogenase that oxidizes cytosolic NADPH via the mitochondrial respiration chain. Active expression of this enzyme causes an increased cytosolic NADPH-consumption, which requires an increased PPP split ratio. Analysis of the MFA-derived PPP split ratios depicted in Fig. 4 shows that there is little room for this increase, indicating that under the applied cultivation conditions the mitochondrial NADPH dehydrogenase only fulfills a minor catabolic function. A quantitative determination of the mitochondrial NADPH dehydrogenase flux was not possible, since its inclusion in the stoichiometric models of Fig. 4 led to a parallel route. 4. Conclusions Quantification of the PPP split ratio in penicillin-G producing and non-producing chemostat cultures of P. chrysogenum confirmed that the flux through the oxidative branch of the PPP is strongly correlated to b-lactam antibiotic production. Furthermore, it was shown through MFA that the oxidative branch of the PPP produces the majority of the cytosolic NADPH needed for penicillin synthesis; the only other supplier of cytosolic NADPH being the recently identified NADP+-dependent isocitrate

121

dehydrogenase in P. chrysogenum. The observed increase in PPP split ratio under penicillin producing conditions is best explained by a stoichiometric model in which cysteine is synthesized via the transsulfuration pathway, requiring 9 mol of cytosolic NADPH for every mole of penicillin produced from glucose. The P. chrysogenum strains currently used by industry have penicillin titers that are several factors higher than those measured in this study, meaning that the metabolic burden on the supply of cytosolic NADPH will be even higher. Limiting the cytosolic NADPH demand for penicillin synthesis, by for example introducing a cytosolic direct sulfhydrylation pathway for cysteine synthesis as earlier proposed by Jorgensen et al. (1995) and Ostergaard et al. (1998), can thus form an interesting target for future metabolic engineering of P. chrysogenum. Acknowledgments This work was financially supported by the Dutch EET program (Project No. EETK20002) and DSM. Appendix A Two candidate reactions were hypothesized for explaining the difference in 13C-labeling between the gluconate added to the medium and the intracellular gluconate (Table 2); the oxidation of glucose to gluconate by either glucose oxidase or glucose dehydrogenase and the dephosphorylation of intracellular 6pg to gluconate by a phosphatase. By setting up a labeling balance around the intracellular gluconate pool, estimates for the 6pg dephosphorylation flux and the glucose-oxidation flux were determined. Corresponding PPP split ratios were determined by means of a second labeling balance around the 6pg-pool (similar to Eq. (1)). The applied labeling balances are described in detail by Kleijn et al. (2006). In all four chemostat experiments the estimated glucose oxidation and 6pg dephosphorylation flux was only a fraction of the uptake rate of glucose (Table 4). P-values

Table 4 Flux estimates and corresponding PPP split ratios for the two metabolic pathways hypothesized to explain the unlabeled mass fraction of intracellular gluconate: the oxidation of glucose and the dephosphorylation of 6pg Chemostat cultivation

Glucose oxidation

6pg dephosphorylation

m (h1)

Penicillin-G production

Flux-estimate (mmol/Cmol/h)

P-valuea

PPP split ratio

P-valuea

Flux estimate (mmol/Cmol/h)

P-valuea

PPP split ratio

P-valuea

0.020 0.020 0.052 0.052

+  + 

0.13 0.14 0.09 0.07

o0.01 0.19 o0.01 0.13

0.511 0.371 0.490 0.408

0.32 0.18 0.13 0.41

0.15 0.17 0.10 0.08

o0.01 0.49 0.79 0.92

0.517 0.381 0.494 0.411

0.46 0.11 0.13 0.38

To obtain the PPP split ratio in a chemostat grown solely on glucose, the estimated PPP split ratios were corrected for the additional influx of gluconate and for the slightly lower NADPH production rate in the oxidative branch of the PPP as a result of gluconate uptake. a P-values for the flux fits were determined via a w2-distribution and denote the probability that the discrepancy between the measured and simulated mass isotopomer distributions in the labeling balance is a result of measurement error. A P-value p0.05 was considered as statistically significant and thus indicates a clear deviation between the measured and simulated mass isotopomer distributions.

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for the flux-estimates indicate that the unlabeled mass fraction of intracellular gluconate is somewhat better explained by the dephosphorylation of 6pg to gluconate. Note that the dephosphorylation of 6pg has no effect on the label-inflow into the 6pg-pool, resulting in unchanged PPP split ratios. Due to the small size of the glucose oxidation flux only marginally different PPP split ratios were observed for this metabolic scenario. PPP split ratios were statistically acceptable for both candidate reactions in all four chemostat experiments (P-value40.05). Appendix B The biomass specific glucose uptake-rate ðqs Þ is derived from the well-known Herbert–Pirt equation for substrate consumption: qp m qs ¼ max þ max þ ms , (B.1) Y sx Y sp where Y max sx is the maximum theoretical yield of biomass on glucose, Y max is the maximum theoretical yield of sp penicillin-G on glucose and ms is the non-growth related maintenance coefficient. These parameters have been determined and reported for P. chrysogenum by van Gulik et al. (2001). A similar relation can be derived for the biomass specific cytosolic NADPH consumption rate ðqnadph Þ, neglecting the NADPH required for maintenance: qp m (B.2) qnadph ¼ max þ max . Y nx Y np Y max is the maximum theoretical yield of biomass on nx cytosolic NADPH (3.93 Cmol biomass/mol NADPH) which can be derived from the stoichiometric model proposed by van Gulik et al. (2000), Y max np is the maximum theoretical yield of penicillin-G on cytosolic NADPH (0.111 mol penicillin-G/mol NADPH, see main text) based upon cysteine synthesis via the transsulfuration pathway. Taking into account that the oxidative branch of the PPP produces two molecules of NADPH per cycle, the PPP split ratio (PPPSR) can be defined as the ratio between qnadph and qs : qnadpha , (B.3) PPPSR ¼ 2qs where a is a correction factor for the amount of NADPH synthesized via the oxidative branch of the PPP with respect to the cytosolic NADP+-dependent isocitrate dehydrogenase (70.87, see main text). By combining Eqs. (B.1)–(B.3) and substituting the appropriate values for the yield parameters the PPP split ratio as a function of the specific growth rate and the biomass specific penicillin-G production rate is obtained:

PPPSR ¼

0:21m þ 7:50qp . 0:50m þ 11:5  qp þ 0:0030

(B.4)

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