Metabolic network analysis on Phaffia rhodozyma yeast using 13C–labeled glucose and gas chromatography-mass spectrometry

ARTICLE IN PRESS

Metabolic Engineering 6 (2004) 340–351

Metabolic network analysis on Phaffia rhodozyma yeast using C–labeled glucose and gas chromatography-mass spectrometry

13

Christopher Cannizzaro,a Bjarke Christensen,b Jens Nielsen,b and Urs von Stockara,* a

Laboratory of Chemical and Biochemical Engineering, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland b Center for Process Biotechnology, Technical University of Denmark, Building 223, DK-2800 Lyngby, Switzerland Received 4 December 2003; received in revised form 14 May 2004; accepted 1 June 2004 Available online 28 July 2004

Abstract Carotenoid production by microorganisms, as opposed to chemical synthesis, could fulfill an ever-increasing demand for ‘all natural’ products. The yeast Phaffia rhodozyma has received considerable attention because it produces the red pigment astaxanthin, commonly used as an animal feed supplement. In order to have a better understanding of its metabolism, labeling experiments with [1-13C]glucose were conducted with the wildtype strain (CBS5905 T) and a hyper-producing carotenoid strain (J4-3) in order to determine their metabolic network structure and estimate intracellular fluxes. Amino acid labeling patterns, as determined by GC–MS, were in accordance with a metabolic network consisting of the Embden– Meyerhof–Parnas pathway, the pentose phosphate pathway, and the TCA cycle. Glucose was mainly consumed along the pentose phosphate pathway (B65% for wildtype strain), which reflected high NADPH requirements for lipid biosynthesis. Although common to other oleaginous yeast, there was no, or very little, malic enzyme activity for carbon-limited growth. In addition, there was no evidence of phosphoketolase activity. The central carbon metabolism of the mutant strain was similar to that of the wildtype strain, though the relative pentose phosphate flux was lower and the TCA cycle flux in accordance with the biomass yield being lower. r 2004 Elsevier Inc. All rights reserved. Keywords: Metabolic network analysis; C-13 labeling; GC–MS; Phaffia rhodozyma; Carotenoids; Astaxanthin

1. Introduction There is an increasing interest in the microbial production of carotenoids (Misawa and Shimada, 1998; Schmidt-Dannert, 2000; Sandmann, 2001). The yeast Phaffia rhodozyma, has been the subject of numerous studies since it naturally produces the carotenoid astaxanthin, albeit at relatively low quantities. Through chemical mutagenesis, strains with enhanced carotogenesis have been produced, but at the cost of decreased biomass yield and growth rate (Meyer et al., 1993). Though it could potentially lead to strains with improved characteristics, there is a general lack of

*Corresponding author. Tel.: 41-21-693-3191; fax: 41-21-693-3680. E-mail address: urs.vonstockar@epfl.ch (U.v. Stockar). 1096-7176/$ - see front matter r 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymben.2004.06.001

fundamental knowledge on P. rhodozyma since it is a non-conventional yeast (Flores et al., 2000). Metabolic flux analysis (MFA) has gained popularity as a technique to better understand microbial processes, with the ultimate aim of improving productivity (Gombert and Nielsen, 2000). MFA provides an estimate of intracellular fluxes based upon a pseudosteady state (PSS) assumption of metabolites. Implicit in these calculations is absolute knowledge of relevant biochemical pathways, i.e. the metabolic network. Clearly, choices must be made with regard to which pathways to include, the strength of which will depend upon how well the organism under study is characterized. The systematic study of network structure in conjunction with MFA is known by the general term metabolic network analysis (MNA). Carbon labeling experiments (CLEs) with gas chromatography–mass

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spectrometry (GC–MS) or nuclear magnetic resonance (NMR) are commonly utilized to elucidate pathway structure (Schmidt et al., 1998; Christensen and Nielsen, 1999; Wiechert, 2001). Furthermore, intracellular fluxes may be calculated by relating the labeling patterns of measured amino acids and/or storage carbohydrates back to central pathway metabolites, of which the concentration is too low for accurate measurement. An advantage of 13C-tracer flux estimates is that they are independent of cofactor requirements (e.g. ATP, NADH, and NADPH), which are often subject to controversy (Schmidt et al., 1998). In this work, 13C-labeling and GC–MS (Christensen and Nielsen, 1999) were employed to identify the metabolic network structure and estimate intracellular fluxes of the carotenoid producing yeast P. rhodozyma. As opposed to the relative complexity of NMR, the method is fast, simple, robust, and highly sensitive. The filamentous fungi Penicillium chrysogenum (Christensen and Nielsen, 2000; Thykaer et al., 2002) and Aspergillus niger (Pedersen et al., 2000), as well as the yeast Saccharomyces cerevisiae (Gombert et al., 2001) and Saccharomyces kluyveri (Moller et al., 2002) and the bacteria Bacillus clausii (Christiansen et al., 2002) and Streptomyces norusei (Jonsbu et al., 2001), have all been studied with this technique. Of the Eukaryotes (P. chrysogenum, A. niger, S. cerevisiae, and S. kluveri), all are members of the Ascomycota phylum. However, P. rhodozyma is a Basidiomycete (Federhen et al., 2001), and hence its metabolism may differ significantly from that of an Ascomycete. Furthermore, P. rhodozyma is oleaginous and can accumulate up to 40 w/w% lipids (Johnson and Schroeder, 1995; Flores-Cotera et al., 2001). Several studies have reported that oleaginous yeast have a high pentose phosphate flux (Ho¨fer, 1968; Ratledge and Botham, 1977) and exhibit malic (Whitworth and Ratledge, 1975), citrate lyase (Botham and Ratledge, 1979; Evans et al., 1983) and phosphoketolase (Whitworth and Ratledge, 1977; Evans and Ratledge, 1984) enzyme activity. An objective of this study was to determine if the metabolism of P. rhodozyma shares these characteristics. The CLEs were conducted in continuous culture on defined medium containing [1-13C]glucose at a single dilution rate for the type strain and at two dilution rates for a hyper-producing carotenoid strain.

2. Materials and methods 2.1. Microorganism and medium The yeast P. rhodozyma (CBS5905 T) was ordered directly from the CBS culture collection (Delft, Netherlands). The J4–3 mutant strain was a kind gift from Prof. James du Preez at the University of Orange Free

341

State, South Africa. The J4-3 strain is an astaxanthin over-producer obtained by successive N-Methyl-N0 Nitro-N-Nitrosoguanidine (NTG) mutation of the CBS5905 type strain (Meyer et al., 1993). The cells were stored frozen at 80 C in 1.8 ml aliquots. The bioreactor inoculum was prepared by adding a single aliquot to a 500 ml Erlenmeyer flask containing 100 ml of medium (0.67 g/l Yeast Nitrogen Base (Becton Dickinson, Sparks, MD); 10 g/L glucose). The flask was grown for 48 h at 22 C and 150 rpm. The bioreactor was than inoculated with 50 ml from this flask. The defined medium for the experiments was adapted from Verduyn et al. (1992). Unless otherwise noted, all chemicals were from Fluka (Buchs, Switzerland). Medium was sterilized by filtration and contained per liter: 5 g (NH4)2SO4, 3 g KH2PO4, 0.5 g MgSO4  7H2O. The medium also contained following vitamins and trace elements: 0.01 g CaCl2  2H20, 2.67 mg H3BO3, 0.8 mg CuSO4  5H2O, 0.27 mg KI, 2.67 mg MnCl2, 1.07 mg Na2MoO4  2H2O,  12 mg ZnSO4  7H2O, 0.8 mg CoCl2, 8 mg FeSO4  7H2O, 2.67 Ca pentothenate, 0.13 mg biotin, 66.67 mg m-inositol, 2.67 mg nicotinic acid, 0.53 mg para-amino benzoic acid (PABA), 2.67 mg pyridoxine hydrochloride, 2.67 mg thiamine hydrochloride. External sources of carbon were eliminated by replacing PPG2000 antifoam (Fluka) with silicone-based Antifoam A (Sigma, St. Louis, MO), by passing inlet air through 2 M NaOH to remove CO2, and by removing EDTA from medium. Carbon isotope: D-[1-13C]glucose, was purchased from Omicron Biochemicals Inc. (South Bends, Indiana). Isotopic abundance was 99%. 2.2. Cultivation conditions Cultivations were performed in a RC1 calorimeter modified for use as bioreactor (Marison and von Stockar, 1985). The pH was controlled at 5.0 with 1.0 M NaOH. Temperature and aeration were maintained constant at 22 C and 0.5 l/min, respectively. The reactor working volume was 530 ml for the experiment with the CBS5905 strain and 500 ml for the experiments with the J4–3 strain. Dissolved oxygen tension was monitored with a pO2 probe (Mettler-Toledo, Greifensee, Switzerland). Samples were taken by collecting 20 ml on ice from the bioreactor overflow tube. The culture broth was then centrifuged and washed twice at 3000 g. The supernatant was discarded, and the tube with the biomass pellet was placed in liquid nitrogen for 10 min. The samples were stored at 40 C until they were lyophilized. CBS5905 T labeling experiment: For the batch phase and the start of the continuous culture, the sole carbon source was 3.0 g/l of glucose. The dilution rate was 0.114/h. After ten residence times (t), the feed was switched to 1.5 g/l [1-13C]glucose and 1.5 g/l naturally

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labeled glucose. After 5.6t; feeding was stopped and the biomass harvested. According to first-order washout kinetics, 99.6% of the biomass was labeled. J4-3 labeling experiments: For the batch phase and the start of the continuous culture, the sole carbon source was 3.0 g/l of glucose. The initial dilution rate was 0.064/ h. After 6t the feed was switched to 1.5 g/l [1-13C]glucose and 1.5 g/l naturally labeled glucose. After 3.78t; the feed was switched back to naturally labeled glucose for 5t; before the dilution rate was increased to 0.082/h. Once a new steady state was established, the feed was switched once more to 50% [1-13C]glucose for a further 3.49t: According to first order washout kinetics, 97.7% of the biomass became labeled for the experiment at 0.064/h, and 96.9% for the experiment at 0.082/h.

2.3. Analysis of the

13

C-labeling patterns of amino acids

The isotopic labeling patterns were determined by gas chromatography–mass spectrometry (GC–MS, model HP-G1723A, Hewlett-Packard, Palo Alto, CA). For all analyses, a JW-1701 column with the following dimensions was used: length, 30 m; inner diameter, 250 mm; film thickness, 0.15 mm. The MS had a quadropole mass selective detector and was operated at 70 eV. Sample preparation (e.g. derivatization) and analysis were done according to methods established by Christensen and Nielsen (1999, 2000): Hydrolysis: Approximately 20 mg of lyophilized biomass was hydrolyzed with 800 ml of 6 M HCL at 105 C for 12–20 h. Residue was then dried with a steady stream of air. For the glucose measurements, B5 mg of biomass was hydrolyzed with 100 ml of 6 M HCl at 105 C for 30 min. Ethylchloroformate (ECF) derivatization: The dried hydrolysate was first dissolved in 600 ml 20 mM HCl and 400 ml pyridin:ethanol, 1:4. Then 50 ml of ECF was added. The derivatives were extracted into 1 ml of dichloromethane once carbon dioxide production had ceased. (N,N)-Dimethylformamide dimethyl acetal (DMFDMA) derivatization: The dried hydrolysate was dissolved in 100 ml of methanol, and then 200 ml of acetonitrile was added. The mixture was then derivatized by adding 600 ml DMFDMA. Acetylation of glucose: Crude biomass hydrolysate was dissolved in 50 ml of distilled water, and then 1 ml of acetic anhydride and 50 ml of acetylchloride were added. The prepared samples were injected into the GC–MS, whereby the derivatized amino acids (or glucose) were first separated by the GC, then ionized, and subsequently fragmented in the MS. Depending upon the derivatization procedure, the fragments contained different subsets of the original carbon skeleton. The mass isotopomer distribution of an ion cluster was converted

into a summed fractional labeling (SFL) with: Pn i  mi ; SFL ¼ i¼0 n P mj j¼0

ð1Þ

ðm þ 1Þ-m þ i; where mi is the intensity of the isotopomer with mass (m þ i) and m0 is the mass of the unlabeled fragment. The summed fractional labeling denotes the number of labeled C-atoms in a given fragment. If the fragment contains n C-atoms, the SFL can range from zero to n. The SFLs were corrected for naturally labeled species using a matrix-based method (Wittmann and Heinzle, 1999). As an example, consider the SFL calculations for glycine. From DMFDMA derivatization, two fragments are seen in the mass spectrum, one with an unlabeled molecular weight m0 of 144 and the other an unlabeled molecular weight of 85. The Gly144 fragment contains two carbon atoms from glycine and the SFL is calculated as Glyð1  2Þ ¼ Glyð1Þ þ Glyð2Þ ¼

0  m12 þ 1  m12 þ 2  m12 0 1 2 ; m12 þ m12 þ m12 0 1 2

ð2Þ

where Gly(1–2) is the fractional labeling of the C-1 and C-2 atoms. In the case of the Gly85 fragment, the first carbon atom has been lost, and thus the fractional labeling of the C-2 atom is given explicitly: Glyð2Þ ¼

0  m20 þ 1  m21 : m20 þ m21

ð3Þ

The fractional labeling of C-1 can then be calculated by the subtraction of Gly(1–2) from Gly(2). In this manner, the fractional labeling of both atoms of glycine can be determined. For larger amino acids, fractional labeling of individual carbon positions could not always be found, and therefore the SFLs were used in the flux calculation algorithm. 2.4. Metabolic flux estimation The mathematical framework for flux estimation with the summed fractional labels was outlined in Christensen and Nielsen (1999, 2000). Briefly, from mass balances over the metabolites of the central metabolism, the fractional labeling of amino acids was predicted based upon an initial estimate of fluxes. The calculated labelings were then compared with the measured values and new flux estimates generated using an evolutionary algorithm (Schmidt, 1998). This iterative procedure continued until the error between the calculated and measured values was below a certain criterion. The algorithm was run multiple times with multiple initial guesses to confirm that it would always converge on the same solution.

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Table 1 Yield coefficients and washout correction factor for continuous culture cultivations Strain —

D 1/h

D/mmaxa —

Ysxb g/g

MWc g/C-mol

Ysx C-mol/C-mol

td —

wcfe —

CBS5905T J4-3 J4-3

0.114 0.064 0.082

0.60 0.58 0.75

0.52 0.41 0.41

25.92 25.92 25.92

0.60 0.47 0.47

5.61 3.78 3.49

0.004 0.023 0.031

a

mmax=0.19/h for CBS5905 T and 0.11/h for J4-3. Values from continuous culture in 2.0 l reactor with 20 g/l glucose. c Elemental composition (C1H1.75O0.588N0.131) determined at D¼ 0:12=h for CBS5905 T in 2.0 l reactor. d Residence time t of labeling for sample used to calculate fluxes. e Washout correction factor is wcf=(1-et)1. b

A metabolic flux model (Table 5) previously developed for S. cerevisiae (Gombert et al., 2001) was used in the flux estimation algorithm. Inputs to the model were the biomass composition (taken to be same as S. cerevisiae: 42% protein, 7% RNA, 7% lipid, 40% carbohydrate), the 13C-labeling patterns of amino acids and glucose, and the steady state biomass yield. Since the glucose feed concentration was only 3.0 g/l, the biomass concentration in reactor was very low. As a result, most measurements, both and on- and off-line, were either too noisy or not sensitive enough for quantitative analysis. It was therefore decided to use biomass yield data from continuous culture experiments with a glucose feed of 20 g/l (Table 1). It was previously demonstrated that in continuous culture, metabolite production (e.g. ethanol, glycerol) by P. rhodoyzma was very low or nonexistent (Cannizzaro, 2002), and hence was not included in the model. Labeling patterns were adjusted with a washout correction factor (wcf) to account for unlabeled biomass (see Table 1).

3. Results and discussion 3.1. Isotopic steady state and label incorporation An advantage of conducting labeling experiments in continuous culture is that growth conditions are well defined. In these experiments, it was first checked that a steady state was attained, and then once the feed was switched to [1-13C]glucose, that an isotopic steady state was achieved. The base addition for two of the labeling experiments is shown in Fig. 1. The rate of base addition was constant before the start of labeling, and was not perturbed by the subsequent switch to the labeled feed. With defined medium, and provided organic acid formation is low, there is a 1:1 relation between base and ammonia uptake. Thus, the ammonia consumed over one dilution rate was 7.56 mmol for CBS5905 T strain experiment and 5.76 mmol for J4–3 strain at 0.064/h. If the nitrogen content of the biomass is assumed constant at 0.13 mol/C-mol, then the biomass yield of mutant strain was only B76% of the wildtype

strain, which is in agreement with measured yields from cultures on 20 g/l glucose. In a chemostat with defined medium, amino acids are labeled according to first-order kinetics if there is no exchange of the free amino acid pool. In fact, the labeling of the val144, ala116, asp188, and ile158 fragments from the P. rhodozyma J4–3 experiment at 0.064/h did follow 1st first-order kinetics, as shown in Fig. 2. However, the label was incorporated into, and subsequently washed out of, alanine faster than predicted. This may indicate that there was some exchange of alanine with its precursor metabolite, i.e. pyruvate in the cytosol. It then follows that alanine synthesis was via reversible alanine transaminase, rather than irreversible alanine dehydrogenase. By contrast, the SFL of valine144 followed the predicted first-order kinetics more closely. Valine is also formed from pyruvate, but in the mitochondria not the cytoplasm. The SFL of aspartate188, serine132, and threonine175 fragments also followed 1st order kinetics very well, while the SFL of some fragments (isoleucine158, leucine158, lysine156, and proline142) were labeled slightly slower than expected (results not shown). The delayed labeling of isoleucine and proline may be due to the fact that they are formed from other amino acids (aspartate/threonine and glutamate, respectively). Leucine and lysine have in common that they both depend on acetyl-CoA for their formation. In general, these results indicate that an isotopic steady state was achieved. Similar results were found for the other two experiments. 3.2. Metabolic network analysis 3.2.1. Summed fractional labels In Table 2, the summed fractional labels for the three P. rhodozyma experiments at steady state are directly compared with those from S. cerevisiae (Gombert et al., 2001) and P. chrysogenum (Christensen and Nielsen, 2000) continuous culture experiments. The labeling patterns were in accordance with a metabolic network consisting of the EMP pathway, the PP pathway, and the TCA cycle, and indicated that all amino acids were

ARTICLE IN PRESS C. Cannizzaro et al. / Metabolic Engineering 6 (2004) 340–351

344 140

100

τ = 5.6

τ = 9.8

τ = 3.8

τ = 6.0

90

τ = 6.2

120

τ = 2.8

80 60 rNaOH = 0.869 mmol/h r2 = 1.000

40

NaOH addition [mmol]

NaOH addition [mmol]

80 100

60 50 40

rNaOH = 0.369 mmol/h

30

r2 = 0.996

20

batch

20

70

batch 13

C start

10

13

C start

0

0 0

50

100 time [h]

150

0

200

50

100

150

200

250

time [h]

Fig. 1. Check on metabolic and isotopic steady state for continuous cultures. Feed was switched to labeled glucose at 140 h for CBS5905 T strain at D¼ 0:115=h (left) and at 144 h for J4–3 strain at D¼ 0:064=h (right). Metabolic fluxes were calculated based upon labeling patterns after 5.6 (CBS5905) and 3.8 (J4–3) residence time.

18%

18% 0.5*val144 1st order kinetic

14% 12% 10% 8% 6% 4% 2%

14% 12% 10% 8% 6% 4% 2%

0%

0% -25

0

25 50 time [h]

75

100

-25

0

25 50 time [h]

75

100

75

100

50%

30% Asp188 1st order kinetic

Ile158 1st order kinetic

45% SFL of ile158 fragment [%]

25% SFL of asp188 fragment [%]

ala116 1st order kinetic

16% SFL of ala116 fragment [%]

SFL of 0.5*val144 fragment [%]

16%

20%

15%

10%

40% 35% 30% 25% 20% 15% 10%

5%

5% 0%

0% -25

0

25

50

75

100

time [h]

-25

0

25 time [h]

50

Fig. 2. 13C-glucose incorporation into val144, ala116, asp188, and ile158 fragments for experiment with P. rhodozyma J4–3 at D¼ 0:064=h: At time zero, the medium was switched from naturally labeled glucose to 50% [1-13C]glucose, and at 59 h, the feed was switched back to naturally labeled glucose. Solid line represents 13C- incorporation into fragment according to first-order kinetics, SFL=SFLfinal(1-et).

synthesized according to standard biochemistry. Furthermore, there was no evidence of the Entner– Doudoroff (ED) pathway. This is in agreement with the fact that P. rhodozyma does not grow on inositol (CBS culture collection, 1999), which is catabolized along the ED pathway. The ED pathway was also

absent in Candida 107 (Ratledge and Botham, 1977) and Rhodotorula gratinis (Ho¨fer et al., 1971), both oleaginous yeast like P. rhodozyma. Phosphoketolase enzyme activity, if present, was very low. However, phosphoketolase has been found in several oleaginous yeast including Rodotorula graminis (Whitworth and

Table 2 Measured summed fractional labeling (SFL) Derivatized component

1,2,3,4,5,6 1,2 1,2 2 1,2 2,3 2,3 2,3 1,2,3 2,3,4,5,6 2,3,4,5 1,2 2,3,4,5 1,2,3,4,5 2,3,4 2 1,2,3,4 1,2 2,3,4,5,6 1,2 1,2,3,4,5 2,3,4,5 2,3,4,5,6 1,2 2,3,4,5,6,7,8,9

Precursor

G6P G3P G3P G3P G3P G3P PYR PYR PYR PYR+AcCoA PYR PYR PYR PYR OAA OAA OAA OAA OAA+PYR AKG AKG AKG AKG+AcCoA PEP PEP+E4P

C-atom correspondance

1,2,3,4,5,6 1,2 1,2 2 1,2 2,3 2,3 2,3 1,2,3 2,2,3,3+2 2,2,3,3 1,2 2,2,3,3 1,2,2,3,3 2,3,4 2 1,2,3,4 1,2 2,3,4+2,3 1,2 1,2,3,4,5 2,3,4,5 2,3,4,5+2 1,2 1,2,3,4+2,2,3,3

SFL (%) Sca

Pcb

P. rhodozymac

RS

Novo

5905 T

J4-3

J4-3

0.11/h

0.08 h

0.11 h

0.06 h

0.08 h

91.0 5.9 6.2 3.7 3.1 33.8 36.0 36.0 38.5 106.1 73.3 6.9 73.1 74.9 57.1 12.0 64.4 17.8 93.7 39.9 100.1 85.5 117.2 3.0 90.6

84.7 9.3 9.0 4.8 6.3 26.1 29.0 28.3 31.2 73.3 55.8 5.6 54.9 57.0 45.9 15.5 55.3 23.9 71.6 30.2 72.7 60.9 79.1 ND 75.8

86.8 4.6 4.8 1.6 0.4 25.2 28.4 28.8 27.8 76.8 53.2 2.4 52.2 58.0 46.6 14.0 51.2 21.8 72.8 29.8 76.2 62.0 86.2 2.6 ND

92.6 6.6 6.2 3.8 1.6 27.8 30.8 30.2 34.0 81.4 57.4 4.0 56.4 60.8 53.4 15.8 64.6 24.2 79.8 37.6 87.0 71.6 97.6 2.0 ND

88.6 7.2 7.2 3.4 1.4 26.6 29.4 29.4 31.6 80.2 55.6 4.0 56.6 64.0 50.8 15.2 58.6 25.0 77.8 35.0 84.2 69.0 94.0 3.2 74.0

ND: no data. a Saccharomyces cerevisiae RS (CEN.PK113-7D), Gombert et al. (2001). b Penicillium chrysogenum, Novo Nordisk strain, Christensen and Nielsen (2000). c SFLs corrected for 50% [1-13C]glucose according to: SFL=2 measured SFL–nC 1.1, where nC is the number of carbon atoms in fragment. d Ser174 fragment for Pc. e Er228 fragment for Pc.

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331 175 144 85 175d 132e 116 99 158 158 144 143 127 186 188 115 216 175 158 143 230 142 156 143 192

C atoms in measured cluster

C. Cannizzaro et al. / Metabolic Engineering 6 (2004) 340–351

Glucose Glycine Glycine Glycine Serine Serine Alanine Alanine Alanine Leucine Valine Valine Valine Valine Aspartate Aspartate Aspartate Threonine Isoleucine Glutatmate Glutatmate Proline Lysine Phenylalanine Phenylalanine

Ion cluster measured in mass spec. (m/z)

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C. Cannizzaro et al. / Metabolic Engineering 6 (2004) 340–351

Ratledge, 1977) and Candida 107 (Ratledge and Botham, 1977). Phosphoketolase catalyzes the conversion of xylulose-5P to glyceraldehyde-3P and acetyl-P, which is then subsequently converted to acetyl-CoA via phosphate acetyltransferase. If active, phosphoketolase would supply a significant quantity acetyl-CoA to the cytoplasm. The P. rhodozyma samples were on average labeled less than those from S. cerevisiae. An overall reduced labeling pattern is indicative of a high pentose phosphate pathway (PPP) flux since the C-1 label is lost to carbon dioxide upon conversion of xylulose-5Pribulose-5P. The SFLs of the P. rhodozyma type strain were less than for the mutant strain. This result is partially explained by the different dilution rates at which the experiments were conducted. The PPP flux generally increases as a function of dilution rate in order to compensate for a relative increase in protein content. Thus the PPP flux for mutant strain at 0.064 and 0.082/h may have been less than for the wildtype strain at 0.112/ h. As already mentioned, the overall labeling of amino acids will be reduced as PPP flux increases. In fact, the SFLs of mutant strain at 0.082/h were less than those at 0.064/h. Optimally, the labeling experiments for both strains should have been conducted at the same dilution rate. However, the experiment with the wildtype strain

at 0.112/h was done first, and the J4–3 strain cannot grow at dilution rates above 0.09/h. The SFL for P. rhodozyma was more consistent with P. chrysogenum than S. cerevisae. Interestingly, physiological (e.g. life cycle, ubiquinone system) and genetic data indicate that P. rhodozyma is more closely related to higher order fungi (Sugiyama et al., 1985; Boekhout et al., 1993; Fontana et al., 1997; Nakase et al., 2000). A phylogenetic analysis on glyceraldehyde-3P (gpd) sequences from different yeast and filamentous fungi found that the P. rhodozyma gene clustered with the corresponding genes of filamentous Basidiomycetes and Ascomyetes (Verdoes et al., 1997). 3.2.2. Network identification Summed fractional labeling is a very powerful technique to resolve questions concerning the network structure. As seen in the example on the SFL calculation for glycine fragments (Eqs. (2) and (3)), the fractional labeling of individual isotopomers can often be resolved by combining the information from several fragments. The labeling of these isotopomers can then be related back to their precursor metabolites. The relationship between precursor labeling and network structure is shown for several examples in Table 3.

Table 3 Network identification with measured SFLs Fragments

Glycine formation

Malic enzyme

Compartmentation of pyruvate

Compartmentation of AcCoA Compartmentation of OAA TCA cycle labeling

a

gly175 ser175 gly175-ser175 phe143 val143 val143-phe143 ala116 ala99 0.5 val144 0.5 val127 lys156-pro142 leu158-val127 asp188 ile158-0.5 val144 glu230-pro142 glu143-(glu230-pro142) asp216-asp118 asp115 asp216-asp118+asp115 asp118-asp115

Precursor

G3P G3P G3P-G3P PEP PYR PYR-PEP PYR PYR PYR PYR AcCoA AcCoA OAA OAA AKG AKG OAA OAA OAA OAA

C-atom

1,2 1,2 1,2 1,2 1,2 1,2 2,3 2,3 2,3 2,3 2 2 2,3,4 2,3,4 1 2 1 2 1,2 3,4

Compartmentd

Cytoplasm Cytoplasm Cytoplasm Cytoplasm Mitochondria — Cytoplasm Cytoplasm Mitochondria Mitochondria Cytoplasm Mitochondria Cytoplasm Mitochondria Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm

SFL (%) Sca

Pcb

P. rhodozymac

RS

Pc

5905T

J4-3

J4-3

0.11/h

0.08/h

0.11/h

0.06/h

0.08/h

5.9 3.1 2.8 3.0 6.9 3.9 36.0 36.0 36.7 36.6 31.7 33.0 57.1 57.1 14.6 25.3 7.3 12.0 19.3 45.1

9.3 6.3 3.0 ND 5.6 ND 29.0 28.3 27.9 27.5 18.2 18.4 45.9 43.7 11.8 18.4 9.4 15.5 24.9 30.4

4.6 0.4 4.2 2.6 2.4 0.2 28.4 28.8 26.6 26.1 24.2 24.6 46.6 46.2 14.2 15.6 4.6 14.0 18.6 32.6

6.6 1.6 5.0 2.0 4.0 2.0 30.8 30.2 28.7 28.2 26.0 25.0 53.4 51.1 15.4 22.2 11.2 15.8 27.0 37.6

7.2 1.4 5.8 3.2 4.0 0.8 29.4 29.4 27.8 28.3 25.0 23.6 50.8 50.0 15.2 19.8 7.8 15.2 23.0 35.6

Saccharomyce cerevisiae RS (CEN.PK113-7D), Gombert et al. (2001). Penicillium chrysogenum, Novo Nordisk strain, Christensen and Nielsen (2000). c SFLs corrected for 50% [1-13C]glucose. d Assumes ala, asp, gly, lys, phe, pro, ser synthesized in cytoplasm and ile, leu, val synthesized in mitochondria. b

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Glycine formation: As was previously reported for P. chrysogenum (Christensen and Nielsen, 2000) and S. cerevisiae (Gombert et al., 2001), glycine was labeled to a greater extent than if it was synthesized only from the standard route, i.e. from serine via glycine hydroxymethyl-transferase. This can be clearly seen by the subtraction of the gly175 fragment from the ser175 fragment. Hence another pathway for glycine synthesis was active, likely that from threonine via threonine aldolase. Malic enzyme: Malic enzyme plays a central role in the ability of oleaginous yeast (Whitworth and Ratledge, 1975; Botham and Ratledge, 1979) and filamentous fungi to accumulate lipids (Wynn et al., 1999). Malic enzyme catalyzes the conversion of malate to pyruvate, is NADP+ dependent, and is located in the cytoplasm (Holdsworth et al., 1988; Wynn et al., 1999). For nonoleaginous yeast (i.e. S. cerevisiae), malic enzyme is located in the mitochondria (Boles et al., 1998). From the SFL data, the presence of malic enzyme activity can be discerned by comparing the labeling of the C-1 and C-2 carbon atoms of pyruvate with those of phosphoenolpyruvate. Since pyruvate is derived directly from phosphoenolpyruvate, they should be labeled equivalently if there are no other routes towards pyruvate formation. In contrast to S. cerevisiae, pyruvate and phosphoenolpyruvate were almost identically labeled for the P. rhodozyma type strain, which indicates little or no malic enzyme activity. Its activity was also low for the mutant strain. Even though P. rhodozyma is oleaginous, malic enzyme apparently plays only a small role under carbon-limited growth. Labeling experiments should be conducted under nitrogen-limited growth to observe if its activity increases in order to provide NADPH for increased lipid synthesis. Holdsworth et al. (1988) found that in oleaginous yeast, malic enzyme activity was high under conditions favorable to lipid accumulation, i.e. nitrogen limited growth. Furthermore, its activity remained high even upon carbonstarvation whereby endogenous lipid reserves were utilized. Compartmentation of pyruvate: Pyruvate is the precursor for alanine synthesis in the cytoplasm and valine synthesis in the mitochondria. Since the SFL of the ala116 and ala99 fragments was greater than the onehalf the SFL of the val144 and val127 fragments, cytoplasmic pyruvate was labeled to a greater extent than mitochondrial pyruvate. Labeling patterns were similar for P. chrysogenum, but not for S. cerevisiae. It is interesting to note that there is a high degree of consistency in the data as the SFL of ala116 and val144 fragments from the ECF derivatization were very similar to the SFL of ala99 and val127 fragments from the DMFDMA derivatization. Compartmentation of acetyl-CoA: The fractional labeling of C-2 atom from acetyl-CoA formed in the

347

cytoplasm (lys156-pro142) was not significantly different from acetyl-CoA formed in the mitochondria (leu158-val127). Thus it was not possible to ascertain if the route to cytosolic acetyl-CoA was via citrate as in other oleaginous yeast or via pyruvate as is the case for S. cerevisiae. Compartmentation of oxaloacetate: The fractional labeling of oxaloacetate in the C2, C3 and C4 positions was slightly greater when calculated from the asp188 fragment than from subtraction of the ile158 fragment from 0.5 times the val144 fragment. The deviation was very small for the wildtype strain, but somewhat larger for the mutant strain. Similar findings have been seen in P. chrysogenum, but not S. cerevisiae. TCA cycle labeling: From the labeling patterns of amino acids synthesized in the mitochondria it is clearly seen that the TCA cycle is operating as a cycle and not as two branches as found for batch growth of S. cerevisiae at high glucose concentrations (Gombert et al., 2001). 3.2.3. Model performance As described in the materials and methods, the intracellular fluxes were estimated by minimizing the error between measured the SFLs and the SFLs calculated from the stoichiometric model shown in Table 5. Acetyl-CoA, pyruvate, and oxaloacetate were compartmentalized, even though cytoplasm SFLs were not very different from mitochondrial SFLs. Most of the SFLs fit the model within the method error (o1% standard deviation, (Christensen and Nielsen, 1999), even though precursor requirements were taken from S. cerevisiae (Table 4). However, the fractional labeling of alanine and glu230 fragments was underestimated by the model, while aspartate and isoleucine fragment labeling was overestimated. These deviations may be due to incorrect pathway assumptions or to a P. rhodozyma biomass composition significantly different from S. cerevisiae. 3.2.4. Flux estimates The relative and specific fluxes for all three experiments are shown in Fig. 3. The major fluxes were more similar to those calculated for P. chrysogenum (Christensen and Nielsen, 2000) and A. niger (Pedersen et al., 2000) than S. cerevisiae (Gombert et al., 2001). As previously concluded from the reduced labeling pattern, the PPP flux was quite high. For the CBS5905 T strain it represented 66% of the glucose flux, versus 44% for S. cerevisiae respiratory metabolism while the PPP flux for P. chrysogenum and A. niger was estimated to be 60– 70%. A high PPP flux for P. rhodozyma is in agreement with other studies on oleaginous yeast. The PPP flux was estimated to be 63% for the red-pigmented R. glutinis using 14C-labeling (Ho¨fer, 1968). In a later 14C-labeling study by Ratledge and Botham (1977), a value of 63%

ARTICLE IN PRESS C. Cannizzaro et al. / Metabolic Engineering 6 (2004) 340–351

348

Table 4 Measured and calculated summed fractional labeling for P. rhodozyma CBS5905 and J4-3 Fragmentb

Glc331 Glc314 Glc149 Gly175 Gly144 Gly85 Ser175 Ser132 Ala116 Ala99 Ala158 Leu158 Val144 Val143 Val127 Val186 Asp188 Asp115 Asp216 Ile158 Glu143 Glu230 Pro142 Lys156 Phe143

SFL (%)a CBS 5905T

J4-3

J4-3

D=0.114/h (60% mmax)

D=0.064/h (58% mmax)

D=0.082/h (75% mmax)

Measured

Calculated

Measured

Calculated

Measured

Calculated

47.0 42.7 42.5 3.5 3.6 1.4 1.4 13.8 15.4 15.6 15.7 41.4 29.0 2.4 28.5 32.0 25.1 7.6 28.0 39.4 16.1 41.1 33.4 46.1 2.5

46.4 42.1 42.6 3.5 3.5 1.9 2.2 13.7 13.7 13.7 14.8 41.1 28.6 3.7 28.6

49.9

49.8

47.9

47.6

















4.5 4.3 2.5 2.0 15.1 16.6 16.3 18.8 43.7 31.1 3.2 30.6 33.4 28.5 8.5 34.7 42.9 20.0 46.5 38.2 51.8 2.2

4.5 4.5 2.3 2.9 15.0 15.0 15.0 16.7 44.6 30.8 3.6 30.8

4.8 4.8 2.3 1.9 14.5 15.9 15.9 17.6 43.1 30.2 3.2 30.7 35.0 27.2 8.2 31.7 41.9 18.7 45.1 36.9 50.0 2.8

4.7 4.7 2.5 2.8 14.6 14.6 14.6 16.2 43.6 30.3 3.8 30.3



26.2 6.7 30.5 40.5 16.7 40.1 33.6 46.1 2.2



29.3 8.6 34.5 44.7 19.8 45.5 38.0 51.8 2.9



27.8 7.3 32.5 43.0 19.0 44.1 36.9 50.2 2.8



Indicates that fragment was not included in flux minimization routine. Bold indicates SFLs that were overestimated; ital indicates SFLs that were underestimated. a Calculation of SFL as described in Gombert et al. (2001). b Nomenclature and explanation of the fragments are given in Christensen et al. (2000) for Glc314 and Glc149, and in Gombert et al. (2000) for the rest of the fragments.

was found for Candida 107, and as a control only 25% for S. cerevisiae. As expected, the relative PPP flux estimate for the mutant strain was higher at D¼ 0:082=h than D¼ 0:064=h; 56% versus 50%, respectively. Relative fluxes are lower than for the wildtype strain, but if fluxes are scaled to compensate for lower biomass yield, then the PPP flux at 0.064/h is approximately equal to the wildtype strain at 66% and the PPP flux at D¼ 0:082=h is higher at 74%. The PPP was the main source of cytoplasmic NADPH, since ‘malic enzyme’ activity was very low. Both carotenoid and lipid production pathways are especially NADPH intensive which may account for the high PPP value. The lipid content of the wildtype strain at B12% is higher than the 4–8% commonly reported for S. cerevisiae (Gombert et al., 2001). The lipid content of the mutant strain is not known, though it is most likely higher than the wildtype strain. R. glutinis, which is taxonomically related to P. rhodozyma (Voigt and Wo¨stermeyer, 2001), can accumulate over 50 w/w% of lipid material (Granger et al.,

1993). The increased energetic demand of lipid synthesis is the putative cause of the lower biomass yield and growth rate of the mutant strain since the PPP flux, once scaled for decreased biomass yield, was similar to that of wildtype strain but at much lower dilution rates. The carbon dioxide evolved for the wildtype strain was calculated to be 239.9 mmol/100 mmol of glucose, which corresponds to a yield Ysc of 0.40 C-mol/C-mol. Similarly, values of 315.7, and 317.0 were found for the mutant strain, corresponding to a C-molar yield of 0.53 for both cases. The model predicted that the increased CO2 flux for the mutant strain was due to an elevated TCA cycle flux, and that the degree of reversibility between oxaloacetate and fumarate was much less than for the mutant strain.

4. Conclusions 13

C-labeling in continuous culture of P. rhodozyma, and subsequent GC–MS analysis of the samples, led

ARTICLE IN PRESS C. Cannizzaro et al. / Metabolic Engineering 6 (2004) 340–351 fluxes are in mmol / 100 mmol glucose taken up 66 50 56

PPP

1.5 1.4

Ser

3.5 2.8 2.9

4.8 3.9 3.7

53 64 62

PPP

120 137 135

0.5 0.4 0.6

Thr 20 16 18

OAA

PEP 116 133 131

AcCoA

Ser

biomass

22 17 17 60 41 48

PYR 23 67 59

PYR 41.6 69.6 39.3

1.3 1.5

6.8 3.6 4.5

55.7 81.1 52.4

48 (0.7) 81 (0.3) 80 (0.4)

3.9 3.7 5.4

FUM

59 89 88

48 81 80

AKG mitochondrion

0 0 0

CIT 10 7.8 7.7

0.6 0.3 0.7

25 13 19

OAA

59 89 88

65 53 65

0.9 0.6 0.7

G3P 147 114 143

biomass

6.0 3.3 4.1 20 11 13

PEP 141 110 139

AcCoA

26 14 17 73 34 50 29 55 63

PYR 26.6 54.0 30.4

ME

OAA

35.7 62.9 40.6

59 (0.7) 67 (0.3) 85 (0.4)

71 74 93 71 74 93

59 67 85

AKG

biomass

AcCoA 46 20 33

AcCoA

4.8 3.1 5.8

FUM

2.8 2.2 2.2

(A)

24 12 17

5.8 3.2 3.9

PYR

38 24 31

AcCoA ME

OAA

14 (0.9) 27 (0.7) 28 (0.8)

3.7 (1.0) 2.0 (1.0) 3.4 (1.0) biomass Gly

4.3 2.3 3.0

Thr

AcCoA

27 14 18

F6P

biomass 2.1

4.9 4.0 3.8 16 13 13

G6P

51 26 37

3.3 2.0 2.6 0.7 0.8 0.6

G3P

3.0 (1.0) 2.4 (1.0) 2.2 (1.0) biomass Gly

122 83 106

81 41 59

12 (0.9) 33 (0.7) 26 (0.8)

20 14 16

Glucose

22 17 17

F6P

biomass 1.7

5.6 4.4 4.3

G6P

42 31 35

2.7 2.4 2.5

fluxes are in mmol /100g DCW / h

Glucose 100 100 100

349

mitochondrion

0 0 0

CIT 12 6.5 8.2

3.4 1.8 2.3 biomass

(B)

Fig. 3. Relative and specific fluxes in the central metabolism of Phaffia rhodozyma. Relative fluxes (A) are in mmole per 100 mmol glucose taken up. Specific fluxes (B) are in mmole per 100 g DCW per hour. Fluxes in upper position correspond to chemostat experiment with CBS5905 type strain at D¼ 0:114=h: Fluxes in italics correspond to chemostat experiment with J4–3 mutant strain at D¼ 0:064=h and D¼ 0:082=h (bold). For reversible reactions, degree of reversibility is shown in parentheses. Dashed line indicates acetyl-CoA synthesis from citrate; reaction was not considered in model. Abbreviations: AcCoA, acetyl-CoA; AKG, a-ketoglutarate; CIT, citrate; E4P, erythrose-4-phosphate; F6P, fructose-6-phosphate; FUM, fumarate; Gly, glycine; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; ME, malic enzyme; OAA, oxaloacetate; P5P, pentose-5phosphate; PEP, phosphenolpyruvate; PYR, pyruvate; PPP, pentose phosphate pathway; Ser, serine; Thr, threonine.

Table 5 Metabolic network model used for flux calculations Glucose uptake Glucose

Glucose 6-P

EMP-pathway Glucose 6-P Fructose 6-P Glyceraldehyde 3-P Phosphoenolpyruvate

Fructose 6-P (reversible) 2 Glyceraldehyde 3-P Phosphoenolpyruvate Pyruvate (cytosolic)

PP-pathway Glucose 6-P 2 Pentose 5-P Sedoheptulose 7-P + Glyceraldehyde 3-P Pentose 5-P + Erythrose 4-P

Pentose 5-P + CO2 Sedoheptulose 7-P + Glyceraldehyde 3-P (reversible) Fructose 6-P + Erythrose 4-P (reversible) Fructose 6-P + Glyceraldehyde 3-P (reversible)

Formation of Acetyl-CoA in the cytosol Pyruvate (cytosolic)

Acetyl-CoA (cytosolic) + CO2

Anaplerotic reaction (cytosolic) Pyruvate (cytosolic) + CO2

Oxaloacetate (cytosolic)

TCA-cycle Pyruvate (mitochondrial) Oxaloacetate (mitochondrial) + Acetyl-CoA (mitochondrial)

Acetyl-CoA (mitochondrial) + CO2 Isocitrate

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Table 5 (continued) Isocitrate 2-Oxoglutarate Fumarate

2-Oxoglutarate + CO2 Fumarate + CO2 Oxaloacetate (mitochondrial, reversible, scrambling included)

Transports Oxaloacetate (mitochondrial) Acetyl-CoA (cytosolic) Pyruvate (cytosolic)

Oxaloacetate (cytosolic, reversible) Acetyl-CoA (mitochondrial) Pyruvate (mitochondrial)

Serine/Glycine metabolism (all enzymes assumed to be cytoplasmic) Glyceraldehyde 3-P Serine Oxaloacetate (cytosolic)

Serine Glycine + C1-Tetrahydrofolate (reversible) Glycine + Acetyl-CoA (cytosolic)

Malic enzyme (oxaloacetate decarboxylation, mitochondrial) Oxaloacetate (mitochondrial)

Pyruvate (mitochondrial) + CO2

Drain of intermediates to macromolecules In the model, the following intracellular metabolites are used for biosynthesis of macromolecules: Glucose 6-P, pentose 5-P, erythrose 4-P, glyceraldehyde 3-P, phosphoenolpyruvate, pyruvate (mitochondrial), pyruvate (cytosolic), oxaloacetate (cytosolic), 2-oxoglutarate, acetyl-CoA (cytosolic), acetyl-CoA (mitochondrial), serine, glycine, and C1tetrahydrofolate. Excreted Products The model includes fluxes representing the production of CO2 as the sole excreted product. Note: The reactions followed by reversible are reversible reactions, and both the forward and the reverse direction of the reaction were included in the calculations.

to several interesting results. Direct observation of the labeling patterns indicated that the central carbon metabolism of P. rhodozyma was more similar to filamentous fungi than to S. cerevisiae. Estimation of the intracellular fluxes based upon summed fractional labeling found that the pentose phosphate flux was approximately 65% for the wildtype strain. Such a high PPP flux is likely due to a high requirement for cytoplasmic NADPH, since the labeling patterns indicated little or no malic enzyme activity. The network structure and flux estimates for the mutant strain were not notably different from the wildtype strain, indicating that chemical mutagenesis (to increase carotenogenesis) did not alter the central carbon metabolism. Further studies should focus upon nitrogen assimilation since there is evidence (Johnson and Schroeder, 1995; An, 2001) that it may be impaired in mutated strains.

Acknowledgments Financial support was provided by the Swiss National Science Foundation.

Appendix A metabolic flux model used in flux estimation is shown in Table 5.

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