Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris Aina Sola`1,2, Hannu Maaheimo2,3, Katri Ylo¨nen3, Pau Ferrer1and Thomas Szyperski2 1 Department of Chemical Engine
Trang 1Amino acid biosynthesis and metabolic flux profiling of Pichia pastoris Aina Sola`1,2, Hannu Maaheimo2,3, Katri Ylo¨nen3, Pau Ferrer1and Thomas Szyperski2
1 Department of Chemical Engineering, Escola Te`cnica Superior d’Enginyeria (E.T.S.E), Universitat Auto`noma de Barcelona, Bellaterra, Spain; 2 Department of Chemistry, University at Buffalo, The State University of New York at Buffalo, NY, USA;
3 NMR-laboratory and Structural Biology and Biophysics Program, VTT Biotechnology, Helsinki, Finland
Amino acid biosynthesis and central carbon metabolism of
Pichia pastoriswere studied using biosynthetically directed
fractional 13C labeling Cells were grown aerobically in a
chemostat culture fed at two dilution rates (0.05 h)1,
0.16 h)1) with glycerol as the sole carbon source For
investigation of amino acid biosynthesis and comparison
with glycerol cultivations, cells were also grown at 0.16 h)1
on glucose Our results show that, firstly, amino acids are
synthesized as in Saccharomyces cerevisiae Secondly,
bio-synthesis of mitochondrial pyruvate via the malic enzyme
is not registered for any of the three cultivations Thirdly,
transfer of oxaloacetate across the mitochondrial membrane
appears bidirectional, with a smaller fraction of cytosolic
oxaloacetate stemming from the mitochondrial pool at the
higher dilution rate of 0.16 h)1(for glucose or glycerol
cul-tivation) when compared to the glycerol cultivation at 0.05 h)1 Fourthly, the fraction of anaplerotic synthesis of oxaloacetate increases from 33% to 48% when increasing the dilution rate for glycerol supply, while 38% is detected when glucose is fed Finally, the cultivation on glucose also allowed qualitative comparison with the flux ratio profile previously published for Pichia stipitis and S cerevisiae grown on glucose in a chemostat culture at a dilution rate of 0.1 h)1 This provided a first indication that regulation of central carbon metabolism in P pastoris and S cerevisiae might be more similar to each other than to P stipitis Keywords: 13C NMR; central metabolism; flux profiling; metabolic engineering; Pichia pastoris
The methylotrophic yeast Pichia pastoris has emerged as an important host for heterologous protein expression in both biomedical research and industrial biotechnology [1,2] As a eukaryotic organism, P pastoris offers well known advan-tages for protein expression, such as facilitated proteolytic processing as well as efficient protein folding, disulfide bond formation and glycosylation Very recently, the capability of
P pastoristo produce complex human glycoproteins has been demonstrated in a paradigmatic study [3], indicating that this organism might well become the premier choice for future expression of human proteins for medical applica-tions Specific advantages of P pastoris are due to (a) the availability of an unusually tightly regulated promoter from the methanol-regulated alcohol oxidase I gene (AOX1) (b) the system’s efficient protein secretion which, combined with the very low secretion levels of endogenous proteins, is a major advantage for their purification, and (c) P pastoris’ preference to grow in a respiratory mode, which tends to reduce the excretion of fermentation byproducts such as ethanol or acetic acid and allows one to reach exceptionally high cell densities Furthermore, P pastoris expresses pro-teins at high levels when grown on a minimal medium, which also makes this yeast strain attractive for the production of stable isotope labeled proteins [4,5] for NMR-based struc-tural biology and strucstruc-tural genomics [6]
The level of protein expression in P pastoris depends critically on the growth conditions, and the attainment of high cell densities has been shown to improve protein yields substantially [7] Typically, aerobic growth is achieved in two phases First, the cells are grown in a batch culture with glycerol Subsequently, a mixture of glycerol and methanol
Correspondence to P Ferrer, Department of Chemical Engineering,
Escola Te`cnica Superior d’Enginyeria (E.T.S.E), Universitat
Auto`noma de Barcelona, 08193-Bellaterra, Spain.
Fax: +34 935 812013, Tel.: +34 935 812141,
E-mail: pau.ferrer@uab.es and Thomas Szyperski,
Department of Chemistry, University at Buffalo, The State University
of New York at Buffalo, NY 14260, USA.
Fax: + 1 716 6457338, Tel.: + 1 716 6456800 ext 2245,
E-mail: szypersk@chem.buffalo.edu
Abbreviations: [ 13 C, 1 H]-COSY, [ 13 C, 1 H] correlation NMR
spectroscopy; BDF, biosynthetically directed fractional;
METAFoR, metabolic flux ratio; ROL, Rhizopus oryzae lipase;
D, dilution rate; l max , maximum specific growth rate;
cyt, cytosolic; mt, mitochondrial; TCA, tricarboxylic acid;
PEP, phosphoenolpyruvate; PPP, pentose phosphate pathway;
PYR, pyruvate; OAA, oxaloacetate; SHMT, serine
hydroxymethyl-transferase; GCV, glycine cleavage pathway; ICL, isocitrate lyase;
MS, malate synthase.
Enzymes: alanine:glyoxylate amino transferase (EC 2.6.1.44), glycine
cleavage system (EC 2.1.2.10), glycerol kinase (EC 2.7.1.30),
FAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.99.5),
isocitrate lyase (EC 4.1.3.1), lipase [triacylglycerol acylhydrolase]
(EC 3.1.1.3), malic enzyme (EC 1.1.1.39/1.1.40), malate synthase
(EC 2.3.3.9), pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate
carboxykinase (EC 4.1.1.49), serine hydroxymethyltransferase
(EC 2.1.2.1), succinate dehydrogenase (EC 1.1.1.42), threonine
aldo-lase (EC 4.1.2.5), transaldoaldo-lase (EC 2.2.1.2), transketoaldo-lase
(EC 2.2.1.1).
(Received 19 December 2003, revised 23 March 2004,
accepted 20 April 2004)
Trang 2is fed to the culture in a fed-batch mode During this second
growth phase, the production of the recombinant protein is
induced by methanol, which activates the AOX1 promoter
controlling the heterologous gene Notably, adaptation to
growth on methanol leads to the induction of several key
enzymes, e.g alcohol oxidase, catalase, formaldehyde
dehydrogenase and dihydroxyacetone synthase, as well as
peroxisome biosynthesis [8] Although methanol
assimil-ation is rather strongly repressed by multicarbon sources
such as glucose and glycerol, coassimilation of a
multicar-bon source and methanol can be triggered at certain growth
conditions [9] In turn, this allows one to generate a
significant fraction of the heterologous protein from the
cheap C1 source methanol
Although the central metabolic bioreaction network is
quite similar for all yeast strains, important variations exist
with respect to its regulation [10,11] It is, for example, well
documented that during aerobic growth of Saccharomyces
cerevisiae, catabolism of glucose and related sugars causes
a strong impairment in respiratory capacity (the so called
Crabtree-effect) In contrast, most non-Saccharomyces
yeasts grow under aerobic conditions in a respiratory mode,
that is, reduction equivalents are used to reduce oxygen to
water In fact, variations in the regulation of central carbon
metabolism in non-S cerevisiae genera are essentially
unexplored Moreover, even more comprehensive
investi-gations of amino acid metabolism have so far been pursued
only for S cerevisiae (for example [12]) In view of the
outstanding role of P pastoris for biotechnology research,
this organism represents an obvious target for studies of its
metabolism
Stable isotope labeling experiments employed in
con-junction with NMR spectroscopy and/or mass spectrometry
[13] are a powerful tool for metabolic studies In particular,
biosynthetically directed fractional (BDF)13C labeling of
proteinogenic amino acids has been developed into a
cost-effective approach to assess the topology of active
bioreac-tions (i.e the active pathways) and to quantify metabolic
flux ratios [14] BDFlabeling has been applied to study
central carbon metabolism of eubacteria [14–16] as well as
eukaryotic yeast cells [17,18] Moreover, when feeding
glycerol as the sole carbon source, such labeling enabled one
to explore amino acid biosynthesis pathways in the
halo-philic archaeon Haloarcula hispanica [19]
In this publication, we employ BDF 13C-labeling to
elucidate the central carbon metabolism of P pastoris cells
growing under different dilution rates in chemostat cultures
Specifically, this study focuses on characterizing P pastoris
cells growing on glycerol For comprehensive investigation
of amino acid biosynthesis and comparison with flux ratios
obtained for growth on glycerol, P pastoris was also grown
with glucose as the sole carbon source
Materials and methods
Strain and media
A prototrophic P pastoris strain expressing a heterologous
protein – a Rhizopus oryzae lipase (ROL) – under the
transcriptional control of the AOX1 promoter has been
chosen for metabolic flux ratio profiling Pichia pastoris
x-33/pPICZaA-ROL [20] is the wild type x-33 strain
(Invitrogen, Carlsbad, CA, USA) with the pPICZaA-derived expression vector (Invitrogen) containing the ROL gene, pPICZaA-ROL, integrated in its AOX1 locus Chemostat cultures were fed with a defined minimal medium containing per litre of deionized water: yeast nitrogen base (YNB; Difco, Detroit, MI, USA), 0.17 g; (NH4)2SO4, 5 g; glycerol
or glucose, 10 g; Antifoam Mazu DF7960 (Mazer Chem-icals, PPG Industries, Gurnee, IL, USA), 0.1 mL The YNB components were sterilized separately by microfiltration and then added to the bioreactor The medium used for starter cultures was YPD medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose Chemostat cultivations
Continuous cultivations were carried out at a working volume of 0.8 L in a 1.5 L bench-top bioreactor (BiofloIII, New Brunswick, NJ, USA) at 30C, and a minimum of 30% dissolved oxygen tension Cultivations using glycerol
as the sole carbon source were performed at two different dilution rates, D (defined as volumetric flow rate/working volume) of 0.05 h)1 and 0.16 h)1 The cultivation using glucose as sole carbon source was performed at
D¼ 0.16 h)1 The maximum specific growth rates, lmax,
of P pastoris on excess glycerol or glucose are virtually identical (0.17 and 0.18 h)1, respectively) Hence, at
D¼ 0.16 h)1cells are growing at around 90% of lmax, for both the glycerol and the glucose cultivation This ensures comparability of metabolic flux ratios Medium feeding was controlled by a Masterflex pump (Cole-Parmer, Vernon Hills, IL, USA) The working volume was kept constant by removal of effluent from the center of the culture volume by use of a peristaltic pump (B Braun Biotech Int., Melsungen, Germany) The pH of the culture was maintained at 5.5 by addition of 1M KOH, and the airflow was maintained at 1 LÆmin)1with filter-sterilized air using a mass flow controller (Brooks Instruments B.V., Veenendaal, the Netherlands) The agitation speed was set
to 500 r.p.m Starter cultures (100 mL) were grown in 1 L baffled shake flasks at 200 r.p.m., 30C for 24 h Cells were harvested by centrifugation (4000 g, 10 mins) and resus-pended in fresh medium prior to the inoculation of the bioreactor
Analytical procedures Cell biomass was monitored by measuring the attenuance at
600 nm For cellular dry weight, a known volume of cultivation broth was filtered using preweighted filters; these were washed with two volumes of distilled water and dried
to constant weight Samples for extracellular metabolite analyses were centrifuged at 12 000 g for 2 min in a microcentrifuge to remove the cells Glycerol, glucose, organic acids and ethanol were analyzed by HPLC (Hewlett Packard 1050, Wilmington, DE, USA) analysis using an ionic exchange column, Aminex HPX-87H (Bio-Rad, Hercules, CA, USA) The mobile phase was 15 mMsulfuric acid The injection volume was 20 lL Data was quantified
by theMILLENIUM2.15.10 software (Waters, Milford, MA, USA) The exhaust gas of the bioreactor was cooled in a condenser at 2–4C (Frigomix R; B Braun Biotech Int., Melsungen, Germany) and dried through a silica gel
Trang 3column Concentrations of oxygen and carbon dioxide in
the exhaust gas of bioreactor cultivations were determined
on line with a mass spectrometer (Omnistar; Balzers
Instruments, Liechtenstein)
Biosynthetically directed fractional (BDF)13C-labeling
P pastoriscells were fed with a minimal medium containing
either 10 gÆL)1glycerol or glucose for five volume changes to
reach a metabolic steady state, as is indicated by a constant
cell density and constant oxygen and carbon dioxide
concentrations in the bioreactor exhaust gas BDF 13
C-labeling was achieved as described [16,18], that is, by feeding
the medium containing about 10% (w/w) of uniformly13
C-labeled and 90% (w/w) unC-labeled substrate for one volume
change Uniformly13C-labeled glycerol and glucose (isotopic
enrichment of > 98%) were purchased from Martek
Biosciences (Columbia, MD, USA) and Spectra Stable
Isotopes (Columbia, MD, USA), respectively Cells were
then harvested by centrifugation at 4000 g for 10 min,
resuspended in 20 mMTris/HCl (pH 7.6) and centrifuged
again Finally, the washed cell pellets were lyophilized
(Benchtop 5L Virtis Sentry, Virtis Co., Gardiner, NY,
USA), of which 200 mg were resuspended in 3 mL of 20 mM
Tris/HCl (pH 7.6) After addition of 6 mL of 6MHCl, the
biomass was hydrolyzed in sealed glass tubes at 110C for
24 h, the solutions were filtered using 0.2 lm filters
(Millex-GP, Millipore, Bedford, MA, USA) and lyophilized
NMR spectroscopy and data analysis
The lyophilized hydrolysates were dissolved in 0.1M DCl
in D2O, and 2D [13C,1H]-COSY spectra were acquired for
both aliphatic and aromatic resonances as described [14] at
40C on a Varian Inova spectrometer (Varian, Inc., Palo
Alto, CA, USA) operating at a1H resonance frequency of
600 MHz The spectra were processed using the program
PROSA[21] or standard Varian spectrometer softwareVNMR
(version 6.1, C) The programFCAL2.3.1 [22] was used for
the integration of 13C-13C scalar fine structures in 2D
[13C,1H]-COSY, for the calculation of relative abundances
(f-values) of intact carbon fragments arising from a single
carbon source molecule [14], and for the calculation of the
resulting flux ratios through several key pathways in central
metabolism [14,17]
As described previously [13–19], the calculation of the
flux ratios when using fractional13C-labeling of amino acids
is based on assuming both a metabolic (see above) and an
isotopomeric steady state To establish an affordable
protocol for13C-labeling, it has been proposed to feed a
chemostat, which is operating in metabolic steady state, for
the duration of one volume change with the medium
containing the13C-labeled substrate [16,18] before
harvest-ing the biomass Then, the fraction of unlabeled biomass
produced prior to the start of the supply with13C-labeled
medium can be calculated following simple wash-out
kinetics [16,18,23–25] When chemostats are fed in a carbon
limited manner, one usually finds that (a) the steady-state
concentration of labeled carbon source in the bioreactor is
small (or even zero), and (b) only small metabolic
byprod-ucts, which could be potentially re-imported into the cell
after some time, are synthesized Both conditions are met
for the chemostat cultivations of the present study Hence, after the supply was switched from unlabeled to labeled substrate, only smaller intracellular pools of metabolites had to reach isotopic steady state, which is usually attained for yeast within about half an hour or less [25] (Notably, bacterial cells reach isotopic steady state within a few minutes or even faster [26], except when very large intracellular pools are present in high-yield overproducing strains [24,27–29].) At D¼ 0.16 h)1(0.05 h)1) one volume change requires 6.25 (20) hours, and we thus find that only a small fraction of the labeled biomass is generated while intracellular metabolism is far from isotopic equilibrium In fact, van Winden et al [25] determined in their experimental setup that the slowest wash-in rate in yeast cells growing in
a chemostat was 0.63· 10)3s)1 Even at D¼ 0.16 h)1this
is much faster than the turnover rate of biomass (4.4· 10)5h)1) Hence, the errors of flux ratios resulting from biomass that was fractionally labeled under isotopic nonsteady state conditions were neglected In agreement with this proposition, we do not observe inconsistencies for labeling patterns that serve to validate the bioreaction network (Fig 1) Such inconsistencies are expected if isotopomeric steady state was not reached before the majority of fractional13C labeled biomass was generated,
as the pools of different metabolite approach isotopic steady state at different rates [25]
A special technical comment relates to the fact that the formalism developed for calculating flux ratios for cells that were grown on glucose is also readily applicable for experiments where glycerol is fed This is because the catabolic breakdown of uniformly labeled [13C]glycerol does not generate isotopomers which differ from those created when glucose is fed A more detailed discussion can be found in a recent article describing a study in which fractional labeling of biomass was achieved with glycerol as the sole carbon source [19]
Biochemical reaction network model forP pastoris Because the central carbon metabolism of P pastoris has so far not been comprehensively characterized, the biochemical reaction network model taken for data interpretation was the one recently identified for S cerevisiae [17,18], which has also been shown to be suitable for Pichia stipitis [18] Following consideration of published data [10,30], only the pathway for glycerol metabolism was added (Fig 1) This involves glycerol phosphorylation by a cytosolic glycerol kinase to 3-phosphoglycerol which is subsequently oxidized
by a mitochondrial (membrane) FAD-dependent glycerol phosphate ubiquitone oxidoreductase in order to yield dihydroxyacetone phosphate The thus generated dihyd-roxyacetone phosphate serves for both pyruvate synthesis and gluconeogenesis [10] In principle, the glyoxylate cycle also had to be included (and is thus indicated in grey in Fig 1) The two characteristic reactions of this cycle are catalyzed by malate synthase (MS) and isocitrate lyase (ICL), which are subject to catabolite repression in S cere-visiae [31] Notably, repression occurs to a lesser extent when S cerevisiae grows on a medium containing a nonfermentable carbon source such as glycerol as the sole carbon source [32,33] Although ICL and MS are most probably cytosolic in S cerevisiae [10], they are assumed to
Trang 4be located in peroxisomes in methylotrophic yeasts such as
P pastoris[34] However, the13C-labeling pattern arising
from the action of the glyoxylate cycle and the efflux of
oxaloacetate (OAA) from the mitochondria cannot be
distinguished [18] Hence, the exchange of OAA between the cytosol and mitochondria was likewise considered to
be bidirectional, as discussed for yeast cells growing in glucose-limited chemostat cultivations [18] (Note, that
Fig 1 Network of active biochemical pathways constructed for P pastoris cells grown with either glycerol or glucose as the sole carbon source The network is based on the networks recently identified for S cerevisiae [17] and P stipitis [18] and on the literature on P pastoris metabolism [10,30] (see text) The central carbon metabolism of P pastoris is dissected into cytosolic and mitochondrial subnetworks In addition, the glyoxylate cycle reactions are supposed to reside in peroxisomes in methylotrophic yeast like P pastoris Because the reactions of the glyoxylate cannot be identified with the currently employed13C-labeling strategy (see text), its reactions are depicted in grey The amino acids and the carbon fragments originating from a single intermediate of the central carbon metabolism are represented in rectangular boxes Thin lines between the amino acid carbon atoms denote carbon bonds that are formed between fragments originating from different precursor molecules, while thick lines indicate the intact carbon connectivities in the fragments arising from a single precursor molecule The carbon skeletons of the intermediates of the glycolysis, TCA cycle and pentose phosphate pathway are represented by circles, squares and triangles, respectively The numbering of the carbon atoms refers to the corresponding atoms in the precursor molecule Abbreviations: AcCoA, Acetyl-Coenzyme A; DHAP, dihydroxyacetone phosphate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate; Fum, fumarate; G6P, glucose 6-phosphate; Glc, glucose; Glyox, glyoxylate; G3P, glyceralde-hyde 3-phosphate; 3PG, 3-phosphoglycerate; Mae, malic enzyme; Mal, malate; OAA, oxaloacetate; 2Og, 2-oxoglutarate; PYR, pyruvate; PEP, phosphoenolpyruvate; S7P, seduheptulose-7-phosphate; Ser, serine; Succ, succinate For AcCoA, Fum, OAA, PYR and Succ cytosolic (cyt) and mitochondrial (mt) pools are indicated separately.
Trang 5exchange of C4 intermediates in cytosol and mitochondria
may occur via either shuttle transport mechanisms of the
tricarboxylic acid (TCA) cycle intermediates, e.g
succinate-fumarate shuttle [35,36], or mitochondrial redox shuttles,
e.g malate-oxaloacetate shuttle, malate-aspartate shuttle
and malate-pyruvate shuttle [37])
Results and discussion
P pastoriscultivations in aerobic chemostats using glycerol
as the sole carbon source were performed at dilution rates of
D¼ 0.16 h)1, which is slightly below the maximum specific
growth rate of the organism previously observed in a batch
culture on glycerol (0.17 h)1, A Sola`, unpublished results),
and D¼ 0.05 h)1, where the glycerol supply is
growth-limiting (Table 1) Consistently, some residual glycerol
(3.0 gÆL)1) was detected in the chemostat operating at
D¼ 0.16 h)1, while no glycerol was found at D¼ 0.05 h)1
Biomass yield per gram of glycerol was 0.63 g (cell dry
weight) in both cases An aerobic chemostat cultivation
using glucose as sole carbon source was performed at
D¼ 0.16 h)1(Table 1) In this case, the biomass yield was
0.57 g (cell dry weight) per gram of glucose, slightly lower
than for cells grown on glycerol Moreover, cells growing on
glucose exhibited higher CO2 production rates compared
with cells growing on glycerol at the same growth rate The
residual glucose concentration in the corresponding
culti-vation was 0.5 gÆL)1, indicating that the cells were likewise
growing close to the maximum specific growth rate (which
was 0.18 h)1for P pastoris grown in a batch culture with
excess glucose) Notably, ethanol, acetate, succinate and
pyruvate were not detected by HPLC in any of the
cultivations, and carbon balances closed within 5% Hence,
P pastoris used both glycerol and glucose entirely to
generate biomass and CO2 This supports the notion that
P pastoris cells grow exclusively in a respiratory manner
and are thus efficient biomass and protein producers
The metabolic flux ratio analyses were performed with
hydrolyzed biomass samples that were harvested from these
chemostat cultures in physiological steady-state 2D
[13C,1H]-COSY data were analyzed as described [17],
yielding the desired relative abundances (f-values) of intact
carbon fragments arising from a single source molecule of
glycerol or glucose (Table 2)
Biosynthesis of proteinogenic amino acids and C1
metabolism inP pastoris
As expected, the f-values obtained for the glucose and
glycerol cultivations (Table 2) show that the proteinogenic
amino acids are primarily synthesized in P pastoris
accord-ing to the pathways documented for S cerevisiae [12,17,38,39] In particular, the data confirm that (a) Lys synthesis occurs primarily via the a-aminoadipate pathway, (b) Ser is (primarily) synthesized from 3-phosphoglycerate, and (c) the pool of Ser molecules is affected by reversible cleavage by serine hydroxymethyltransferase (SHMT; about 40% were cleaved in all three cultivations) For Gly synthesis, yeasts can cleave either Ser (via SHMT) or Thr (via threonine aldolase) Due to near degeneracy of f-values, however, it is not possible to accurately determine the relative contribution of the two pathways, or to distinguish between cytosolic and mitochondrial SHMT activity Nonetheless, the data prove that the SHMT pathway is active This is consistent with recent 13C-labeling experi-ments with S cerevisiae cells growing on glucose batch cultures, where the threonine aldolase pathway accounts for only about one-third of the Gly biosynthesis, leaving the SHMT pathway as the major route to Gly in these cells [40]
In contrast to the SHMT pathway, Thr cleavage reaction via threonine aldolase is, if present, irreversible This can be readily inferred from the fact that nearly identical f-values were obtained from Thr and Asp Gly may also be synthesized from a C1 unit and CO2via the mitochondrial glycine cleavage (GCV) pathway In contrast to the previous study with S cerevisiae [17] and the present glucose cultivation of P pastoris, we find in the glycerol cultivations
no evidence for the efflux into cytosol of Gly which has been reversibly cleaved by GCV Hence, it may either be that the mitochondrial GCV pathway is operating irreversibly, or that Gly is not exported into the cytosol when cells are grown on glycerol In principle, yeasts can also synthesize Gly from TCA cycle intermediates via ICL and the alanine, glyoxylate aminotransferase [41] However, our data suggest that the activity of the glyoxylate cycle is low (see below), so that this route for Gly synthesis is, if active at all, likely to be
of minor importance
Central carbon metabolism ofP pastoris growing
on glycerol in chemostats The use of the C3 source glycerol for BDF13C-labeling of proteinogenic amino acids enabled the determination of the flux ratios for reactions associated with the TCA cycle, while those related to glycolysis and the pentose phosphate pathway (PPP) cannot be assessed (Table 3) This is because labeled glycerol being metabolized through gluconeogenesis and oxidative PPP does not produce labeling patterns that are sufficiently distinct from those generated when glycerol
is channeled through the nonoxidative PPP In fact, the only information that can be derived with respect to the operation of the PPP is obtained from the f-values of His
Table 1 Growth parameters at steady state chemostat cultivations of P pastoris Y s/x represents the biomass yield, q glyc , q glc and q O2 are specific utilization rates; q CO2 specific production rates, where glyc and glc indicate glycerol and glucose, respectively ND, not determined.
Carbon source and
dilution rate
(D, [h)1])
Residual substrate concentration (gÆL)1)
Y s/x
(g (dry wt)Æg)1)
q glyc , q gluc
(mmolÆg)1Æh)1)
q CO2
(mmolÆg)1Æh)1)
q O2
(mmolÆg)1Æh)1)
Trang 6Cb (Table 2) These reveal the reversible activity of the
transketolase and transaldolase reactions when P pastoris
is grown on glycerol On the other hand, the wealth of
information obtained for pathways associated with the
TCA intermediates can be summarized as follows (Fig 2): (a) Gluconeogenesis from cyt-OAA via phosphoenolpyru-vate (PEP) carboxykinase is not registered (b) Synthesis of mitochondrial pyruvate (mt-PYR) from malate via malic
Table 2 Relative abundances of intact C2 and C3 fragments in proteinogenic amino acids The first column indicates the carbon position for which the 13 C fine structure was observed The f-values were calculated as described [14], and are given for the glycerol chemostat cultivation at
D ¼ 0.05 h)1in columns 2–5, for the glycerol chemostat cultivation at D ¼ 0.16 h)1in columns 6–9 and for the glucose chemostat cultivation at
D ¼ 0.16 h)1in columns 10–13 Note, first, that for terminal carbons f(2*)and f(3)are not defined, second, that in cases where f(2*)is not given for a mid-chain carbon, the carbon-carbon scalar coupling constants are similar and the two doublets cannot be distinguished, and third, that for Tyr the two carbons d 1 and d 2 , and e 1 and e 2 , respectively, give rise to only one 13 C fine structure each [14] ND, not determined.
Carbon atom
Relative abundance of intact carbon fragments Glycerol (0.05 h)1) Glycerol (0.16 h)1) Glucose (0.16 h)1)
f(1) f(2) f(2*) f(3) f(1) f(2) f(2*) f(3) f(1) f(2) f(2*) f(3) Ala-a 0.02 0.06 0 0.92 0.01 0.07 0 0.92 0 0.14 0.01 0.85
Asp-a 0.26 0.09 0.42 0.23 0.14 0.04 0.18 0.64 0.12 0.12 0.16 0.60 Asp-b 0.26 0.23 0.42 0.09 0.14 0.65 0.17 0.04 0.13 0.71 0.14 0.02 Glu-a 0.24 0.21 0.45 0.10 0.15 0.32 0.39 0.14 0.22 0.27 0.41 0.10
Ile-c 1 0.46 0.51 – 0.03 0.76 0.21 – 0.03 0.74 0.19 – 0.07
Lys-a 0.03 0.07 0.87 0.03 0.03 0.1 0.81 0.06 0.06 0 0.92 0.02
Met-a 0.23 0.13 0.39 0.25 0.12 0.08 0.22 0.58 0.14 0 0.21 0.65
Pro-a 0.29 0.15 0.42 0.14 0.27 0.29 0.27 0.17 0.29 0.28 0.37 0.06
Pro-c 0.06 0.90 – 0.04 0.13 0.85 – 0.02 0.12 0.86 – 0.02
Ser-a 0.01 0 0.42 0.57 0.03 0 0.40 0.57 0.13 0.04 0.36 0.47
Thr-a 0.27 0.09 0.40 0.24 0.13 0.05 0.17 0.65 0.16 0.09 0.13 0.62
Tyr-a 0.06 0 0.01 0.93 0.06 0 0.01 0.93 0.03 0.11 0 0.86
Trang 7enzyme is likewise not observed (c) The fraction of
mt-OAA reversibly interconverted to fumarate is about
the same at high and low dilution rates (d) The fraction of
cytosolic-OAA that stems from the mitochondrial pool of
C4 intermediates, e.g via malate-Asp and/or malate-OAA
shuttles [37] (or has possibly been synthesized via the
glyoxylate cycle) is about twice as high at the lower dilution
rate This indicates that at close to maximal growth rates, a
largely unidirectional flux of OAA from the cytosol to the
mitochondria occurs (e) Significant variations were also identified for the anaplerotic supply of the TCA cycle – a higher fraction of mt-OAA arising from PEP at a higher growth rate reflects the increased demand for biosynthetic building blocks
Comparative profiling ofP pastoris grown on glycerol and glucose
At D¼ 0.16 h)1, P pastoris cells grow at about 90% of the maximum specific growth rate with both glycerol and glucose provided as the sole carbon source Hence, the comparison of flux ratios at this dilution rate allows one to assess the impact of the different chemical nature of the two molecules (Fig 1) on metabolic regulation when the same
biomass production objective is reached Overall, flux ratios turn out to be rather similar (Table 3), as could be expected due to the very similar oxidation state of glycerol and glucose The only notable difference is detected for the anaplerotic supply of the TCA cycle: for cells grown on glycerol, the fraction of mt-OAA arising from PEP is about 10% higher than on glucose The ratio [biomass/(bio-mass + CO2)] formation changes accordingly, that is, cells growing on glucose produce CO2 at higher rates with slightly less biomass being formed when compared to cells grown on glycerol Among the invariant flux ratios, we find that mt-PYR synthesis from malate is negligible in all chemostat cultures Consistently, malic enzyme activities were found only at low basal levels in P pastoris cultures grown on either glycerol or glucose This finding is in contrast to the important role of the malic enzyme for mt-PYR metabolism in respiro-fermentative glucose batch cultures of S cerevisiae [17] Due to degeneracy of the labeling patterns, the glyoxylate cycle activity cannot be
Table 3 Origins of metabolic intermediates during aerobic growth of P pastoris in glycerol chemostat cultures at D = 0.05 h-1and D = 0.16 h-1, and
in glucose chemostat culture at D = 0.16 h-1 For comparison, corresponding data previously reported for P stipitis and S cerevisiae glucose-limited chemostat aerobic cultures at D ¼ 0.10 h)1[18] are given in the two right-most columns.
Metabolites
Fraction of total pool [%] (mean ± SD)
P pastoris P stipitis S cerevisiae Glycerol
0.05 h)1
Glycerol 0.16 h)1
Glucose 0.16 h)1
Glucosea 0.10 h)1
Glucosea 0.10 h)1 Cytosol
PEP derived from PPP through at least one
transketolase reaction (upper bound)
P5P from glucose (lower bound) – – 34 ± 2 28 ± 2 41 ± 2 R5P from G3P and S7P (TK reaction) ND ND 66 ± 2 72 ± 2 59 ± 2 R5P from E4P (TK and TA reactions) ND ND 23 ± 2 43 ± 2 33 ± 2
PEP from cyt-OAA (PEP carboxykinase reaction) 0–3 0–6 0–6 0–3 0–10 cyt-OAA from cyt-PYR b 32 ± 2 68 ± 4 63 ± 4 24 ± 3 62 ± 4 cyt-OAA reversibly converted to fumarate at least once
(cytosolic or intercompartmental exchange)
56 ± 13 12 ± 6 6 ± 5 47 ± 16 0–8
Mitochondria
mt-OAA from PEP (anaplerotic supply of TCA cycle) 33 ± 2 48 ± 2 38 ± 2 32 ± 2 31 ± 2 mt-OAA reversibly converted to fumarate at least once 65 ± 14 61 ± 14 52 ± 14 58 ± 14 56 ± 14
a
Data taken from [18].bValues assuming absence of cytosolic-OAA from fumarate conversion.
Fig 2 Summary of flux information involving the pools of TCA
inter-mediates when P pastoris cells are grown in a chemostat The top,
middle and bottom values in the boxes correspond, respectively, to the
glycerol cultivation at D ¼ 0.05 h)1, the glycerol cultivation at
D ¼ 0.16 h)1and the glucose cultivation at D ¼ 0.16 h)1 Note that
values associated with arrows pointing at the same metabolite pool add
up to 100% For abbreviations see the legend of Fig 1.
Trang 8reliably identified from the NMR data However, to
confirm the previously established view that the glyoxylate
cycle is low when yeast cells grow aerobically on glucose
[17,18], we have measured ICL activities in the P pastoris
cultures Indeed, about the same low ICL activities
(0.018 UÆmg)1 of protein and 0.019 UÆmg)1 of protein,
respectively) were detected in the glucose (D¼ 0.16 h)1)
and glycerol (D¼ 0.05 h)1) limited cultures For cells
grown on glycerol at D¼ 0.16 h)1, the ICL activities were
further reduced (5.07· 10)3UÆmg)1of protein) Glucose is
known to repress the glyoxylate pathway in S cerevisiae
[42], and our enzyme assays show that a similar degree of
repression is induced by glycerol in P pastoris
Comparison ofP pastoris with P stipitis and
S cerevisiae
The central carbon metabolism of S cerevisiae and P
stip-itis cells grown on glucose in chemostat cultures at
D¼ 0.1 h)1has been previously characterized [18] Here
we compare (Table 3) these earlier studies with the new data
for P pastoris growing in glucose chemostat cultures at
D¼ 0.16 h)1 The S cerevisiae and P stipitis glucose
limited continuous cultivations were carried out at about
one-third of the lmax, whereas for the present P pastoris
cultivation about 90% of the lmaxwas reached Moreover,
the medium composition was somewhat different in the
earlier studies [18] Nonetheless, in all cultivations we find
that cells exhibit a respiratory metabolism operating to
support comparably fast cell growth with very little or no
byproduct formation Moreover, the same approach for flux
ratio profiling was used [17], which facilitates comparison
Remarkably, we find that, overall, flux ratios of P
pas-torisat D¼ 0.16 h)1are more similar to those of S
cere-visiaethan P stipitis (both at D¼ 0.1 h)1) For example,
the fraction of cyt-OAA stemming from cyt-PYR is the
same for P pastoris and S cerevisiae, but about 2.5 times
larger in P stipitis This might indicate that in P pastoris
and S cerevisiae the cytosolic and mitochondrial PYR
pools are less well equilibrated in the aerobic growth regime
Similarly, the fraction of cyt-OAA reversibly converted to
fumarate is very low for P pastoris and S cerevisiae, while
about half of the cyt-OAA molecules in P stipitis have
undergone such an interconversion at least once Consistent
with the faster growth, a somewhat increased anaplerotic
supply is detected for P pastoris Although any generalized
conclusion is hampered by the fact that the dilution rate and
the growth media were not the same for all cultivations, our
findings might possibly provide a first indication that
regulation of central carbon metabolism in P pastoris
and S cerevisiae are more similar to each other than to
P stipitis
Conclusions
This is the first comprehensive study of amino acid
biosynthesis and central carbon metabolism of the yeast
P pastoris In the framework of this study, we have
established the BDF13C-labeling approach of proteinogenic
amino acids as an analytical tool to study intermediary
metabolism of yeast cells grown on glycerol This approach
allows one to accurately map the metabolic state of the TCA
cycle and associated pathways, thus being an important methodological expansion for investigating the metabo-lism of eukaryotic cells grown with carbon sources other than glucose Specifically, we have shown that (a) the common amino acids are synthesized in P pastoris as previously described for S cerevisiae, and that (b) growth
on glucose and glycerol results in rather similar flux ratio profiles Our investigation can be expected to become a valuable baseline study for future experiments that are geared towards profiling P pastoris cells growing on mixtures of glycerol and methanol Such studies will probably also support the optimization of the larger-scale production of glycosylated human proteins for biomedical applications
Acknowledgements
This work was supported by the University at Buffalo, The State University of New York, the Spanish Ministry of Science and Technology (CICYT project PPQ2001-1908), and the Academy of Finland (projects 52311 and 202409) The authors thank J M Cregg and C Gancedo for useful comments, and O Cos for technical assistance with cultivation off-gas analyses.
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Supplementary material
The following material is available from http://blackwell publishing.com/products/journals/suppmat/EJB/EJB4176/ EJB4176sm.htm
Fig S1 13C Scalar coupling fine structures of BDF
13C-labeled amino acids from the P pastoris cells grown
in glycerol chemostat cultivations at the dilution rates 0.05 h)1and 0.16 h)1
Fig S2 13C Scalar coupling fine structures of BDF
13C-labeled amino acids from the P pastoris cells grown
in glucose chemostat cultivation at dilution rate of 0.16 h)1