Appl Microbiol Biotechnol (1997) 47: 600±603
SHORT CONTRIBUTION
H. Dominguez á M. Cocaign-Bousquet á N. D. Lindley
Simultaneous consumption of glucose and fructose from sugar mixtures
during batch growth of Corynebacterium glutamicum
Received: 24 October 1996 / Received revision: 10 January 1997 / Accepted: 10 January 1997
Abstract Growth of Corynebacterium glutamicum on
mixtures of glucose and fructose leads to simultaneous
consumption of both sugars in which the uptake of
each sugar is directly related to the expression of the
corresponding sugar uptake mechanism. The overall
rate of sugar uptake was higher on sugar mixtures than
on either glucose or fructose alone and was similar to
that observed during sucrose metabolism. The results
suggest that sugar uptake limits metabolic rates though,
in the case of fructose, over¯ow metabolism of both
lactate and dihydroxyacetone was observed. Such
products could re¯ect a higher ¯ux through glycolysis
rather than the pentose pathway during catabolism of
fructose.
Introduction
The gram-positive soil bacterium, Corynebacterium glu-
tamicum, has been used industrially for the production
of various amino acids for many years. Gains in both
yields and rates of amino acid production under various
culture conditions have been achieved by empirical
modi®cation of the process conditions together with
genetic improvements of the strains employed. Such
genetic intervention has been principally of two types:
random mutagenesis followed by strain selection to
yield industrial strains, the modi®ed metabolism of
which remains, in general, non-characterised, and more
H. Dominguez á M. Cocaign-Bousquet á N. D. Lindley (&)
Centre de BioingeÂnierie Gilbert Durand,
UMR CNRS/INSA and Laboratoire AssocieÂe INRA,
Institut National des Sciences AppliqueÂes,
Complexe Scienti®que de Rangueil,
F-31077 Toulouse cedex 4, France
Tel.: (33) 5 61 55 94 89
Fax: (33) 5 61 55 94 00
e-mail: lindley@insatlse.insa-tlse.fr
Ó Springer-Verlag 1997
recently by molecular genetics to overcome speci®c
regulatory bottlenecks associated with particular bio-
synthetic pathways (e.g. Ikeda et al. 1993; Reinscheid et
al. 1994). A somewhat unusual distribution of carbon
¯ux within the central metabolic network (Hollander
1994; Vallino and Stephanopoulos 1993) and the ab-
sence of detectable NADH:NADP transhydrogenase
activity (Cocaign-Bousquet et al. 1996) necessitate that
the NADPH required for the anabolic pathways is
generated within the central pathways either via the
pentose pathway (Marx et al. 1996; Rollin et al. 1995) or
by alternative NADPH-generating pathways involving
malic enzyme (Cocaign-Bousquet and Lindley 1995).
This incapacity to interconvert NADH and NADPH
probably explains why amino acids are the typical
over¯ow metabolites when growth is perturbed. Yields
are, therefore, dependent upon the central pathways and
improved performance will be dependent upon our ca-
pacity to control carbon ¯ux through the primary
metabolic network. One way of achieving this is by use
of substrate mixtures.
Though simultaneous consumption of glucose and
organic acids has been described (Cocaign et al. 1993)
little is known of the regulation of sugar mixture con-
sumption or the e€ect this may have on carbon ¯ux
through the central pathways. It has been postulated
that fructose metabolism involved a diminished ¯ux
through the pentose pathway in Brevibacterium ¯avum.
They attributed this behaviour to the fructose transport
system, a typical fructose phosphotransferase system
(PTSFru) with a concomitant uptake and phosphoryla-
tion that lead to fructose 1-phosphate as the ®rst intra-
cellular product (Sugimoto and Shiio 1989). Recent
work in our own laboratory has con®rmed that a similar
PTSFru exists in C. glutamicum (Dominguez and Lindley
1996) and that expression of this transporter occurs
during growth on other sugars. We have undertaken a
preliminary investigation of the capacity of C. glutam-
icum to co-metabolise sugar (glucose and fructose)
mixtures.
 601

between fructose and xylitol uptake rates in strains of C. glutam-

Materials and methods icum lacking PTSMan activity, selected for their resistance to 2-
deoxyglucose (Dominguez and Lindley 1996).
Bacterial strain, media and growth conditions

The bacterial strain used in this study was Corynebacterium glu-

tamicum ATTC 17965. The growth medium was that previously
described (Dominguez et al. 1993) except as regards the carbon
source (glucose, fructose, sucrose or various glucose + fructose
mixtures), which was added to obtain an initial sugar concentration
of approximately 15 g/l (500 mM carbon equivalent). The fer-
mentations were performed in a 3.5-l fermentor (Chemap) of 2.5-l
working volume. The temperature was maintained at 30 °C, pH
was regulated by automatic addition of KOH (5 M) and both the
aeration rate and stirrer speed were modulated to avoid the dis-
solved oxygen concentration falling below 50% saturation. Shake-
¯ask cultivation for inoculum preparation used the same medium as
that used for fermentor studies except that the KH2PO4/K2HPO4
concentration was increased to 4.86 g/l to improve pH-bu€ering
capacity. The growth medium was inoculated (4% v/v.) with an
overnight culture in exponential phase (A ˆ 3 at 650 nm) grown on
the same sugar(s) as used in the fermentor.
Substrate and product analysis
Cell concentration and fermentor o€-gas, concentrations were
measured as described previously (Dominguez et al. 1993). Sugars
and organic acid concentrations were determined by HPLC, using
a BioRad H+ column maintained at 48 °C with H2SO4 (5 mM) as
eluant. In all cases detection was by refractometry.
Sugar phosphotransferase system
PTS assays were performed on toluenized cells by a method based
on that of Mori and Shiio (1987) as described by Cocaign-Bousquet
et al. (1996) involving the detection of NADH oxidation in the
enzyme-coupled transformation of pyruvate to lactate. Control
assays were undertaken to verify that this reaction rate was de-
pendent upon the presence of both sugar and phosphoenolpyruvate
in the assay mixture. Similar speci®c activities were obtained for
glucose-transporting PTS activity when rates were obtained by
measuring the glucose 6-phosphate concentration in samples taken
from assay mixtures containing glucose-6-phosphate dehydroge-
nase and NADP (0.3 mM), the reduction of which to NADPH was
followed at 340 nm. The proportion of fructose uptake by the
PTSFru was assessed from a previously established relationship
Results
Growth kinetics
Growth of C. glutamicum on either glucose or sucrose
was more rapid than on fructose and was characterised
by an exponential growth curve maintained throughout
the culture until the sugar was depleted. During growth
on either glucose or sucrose, cell material and CO2 were
the only products, though dihydroxyacetone and lactate
were also synthesised during exponential growth on
fructose, as has previously been described (Dominguez
and Lindley 1996). Lactate was reconsumed, together
with the remaining fructose, during a decelerating
growth phase prior to stationary phase. During this
period, in which both lactate and fructose were con-
sumed, CO2 production relative to substrate consump-
tion was higher than during growth on fructose alone.
The production of over¯ow metabolites, together with a
globally increased production of CO2, resulted in a
somewhat diminished biomass yield on fructose com-
pared to growth on either glucose or sucrose.
When C. glutamicum was grown on mixtures of glu-
cose and fructose co-consumption was always observed
irrespective of the relative importance of each sugar
within the mixture. The speci®c rates of consumption of
each sugar and the presence of over¯ow metabolites
were dependent upon the mixture composition. It is in-
teresting to note that a growth rate equivalent to that
obtained on glucose alone was reached on the equimolar
mixture, though a slightly higher value, similar to that
on sucrose, was obtained in the mixture composed of
75% glucose and 25% fructose. It should also be noted
that the overall rate of sugar consumption was signi®-
cantly higher during the co-consumption periods,

Table 1 Kinetics parameters during exponential growth of Cor-
ynebacterium glutamicum in batch cultures on glucose, fructose,
sucrose and glucose + fructose mixtures (percentage compositions
are shown) and the corresponding phosphotransferase system
Parameter Growth substrate
(PTS) activity levels. Sugar consumption rates (q) and PTS activ-
ities are given as mmol substrate (g dry cells))1h)1 and were mea-
sured with an estimated error of < 5% in all cases. Values in
parentheses indicate the estimated PTSMan activity for fructose

Glucose Fructose Sucrose Glu/Fru Glu/Fru Glu/Fru

100 100 100 25/75 50/50 75/25

Speci®c kinetic rates

Growth (h)1) 0.59 0.48 0.61 0.53 0.58 0.59
qGlu (mmol g)1h)1) 5.25 ± ± 2.12 2.62 3.55
qFru (mmol g)1h)1) ± 4.42 ± 3.10 2.45 2.26
qSuc (mmol g)1h)1) ± ± 2.90 ± ± ±
Sugar uptake capacity
PTSMan 4.46 2.24 2.08 2.11 2.79 3.61
(1.29) (0.66) (0.61) (0.63) (0.68) (0.75)
Fru
PTS 1.73 3.85 2.99 3.05 2.59 2.22
PTSSuc 1.94 2.06 2.97 2.24 2.28 2.17
602
exceeding that obtained on glucose alone by as much as
20%, though similar to that seen for sucrose.
Sugar uptake capacities
Speci®c rates of glucose consumption were similar to the
PTS transport activity measured with permeabilised cells
under all mixture compositions (Table 1) once values
had been corrected to take into account the overesti-
mation of the fructose transport capacity. A signi®cant
proportion of the measured fructose-transporting
Man
PTS
activity could be attributed to the PTS , which has
some capacity to transport fructose (Dominguez and
Lindley 1996). Only during growth on fructose alone
was the PTSMan expectedFruto participate signi®cantly in
fructose uptake. The PTS activity was a linear function
of the proportion of fructose in the mixture, though
expressed to signi®cant amounts (50% of maximal spe-
ci®c activity) even in the absence of fructose, as indeed
was the case for all PTS uptake systems examined in this
study.
Discussion
The uptake rate of each sugar from various glucose +
fructose mixtures was always dependent upon sugar
transport capacities. However, growth rates varied be-
cause of the production of over¯ow metabolites and a
somewhat higher production of CO2. This implies that
the manner in which sugar mixtures are catabolised
varies signi®cantly from that of either sugar alone. In
Fru
this light it should be remembered that the PTS yields
fructoseMan
1-phosphate (Hanson and Anderson 1968) while
the PTS phosphorylates the transported sugar to yield
a hexose 6-phosphate (Mori and Shiio 1987). This
double entry into central metabolism was probably the
cause of the high catabolic activity, which appears to be
unable to generate improved synthesis of cell material
via the anabolic pathways. Growth yields of C. glu-
tamicum have been reported to be dependent upon the
micro-organism's capacity to generate adequate
NADPH for the anabolic pathways, a demand met by
the high ¯ux through the pentose pathway during
growth on glucose (Rollin et al. 1995; Cocaign-Bousquet
et al. 1996). Thus, it would appear that co-consumption
of glucose and fructose modi®ed carbon ¯ux through the
central metabolic pathways in such a manner as to di-
minish NADPH generation relative to sugar consump-
tion. Such an e€ect would be obtained by a decreased
¯ux through the pentose pathway (as suggested for
growth on fructose alone; Shiio et al. 1990) with a cor-
responding increase in tricarboxylic acid cycle activity.
Clearly such a restructured central metabolism yields a
corresponding increase in NADH, possibly explaining
the production of reduced over¯ow metabolites. Man
When
Fru
the total
Suc
PTS activity represented by the PTS ,
PTS and PTS are examined under the various sugar
mixtures employed in this study it is seen that an ap-
proximately constant level of total PTS activity was
maintained. In response to the sugar present, each in-
dividual PTS varied with a potential twofold modulation
of transport capacity. This response is somewhat dif-
ferent from that described for many enteric bacteria
(Postma et al. 1993) and it may prove interesting to see if
the catabolite repression mechanism of some gram-
positive bacteria, involving phosphorylation of the HPr
transport protein by fructose-bisphosphate-activated
HPr kinase (Saier et al. 1996) operates in C. glutamicum.
In the light of the constant overall PTS activity de-
scribed here, it is tempting to speculate that EI (the only
transport protein common to all three PTS mechanisms)
may have a controlling in¯uence on uptake capacity,
though a detailed study of the puri®ed transporters and
the corresponding genes will be necessary to elucidate
this aspect.
It has been suggested (Linton 1990) that the principal
factor limiting the rates of amino acid production in
C. glutamicum is the potential of this bacterium to
transport sugars. The use of sugar mixtures may help
overcome this limitation, though rates of sugar uptake
from glucose/fructose mixtures, while higher than those
obtained on each sugar alone, were not higher than
those achieved on sucrose. However, the modi®ed ¯ux
distribution associated with the use of sugar mixtures
may enable some gains in amino acid yields to be
achieved and merits further attention.
References
Cocaign-Bousquet M, Lindley ND (1995) Pyruvate over¯ow and
carbon ¯ux within the central metabolic pathways of Coryne-
bacterium glutamicum during growth on lactate. Enzyme
Microb Technol 17: 260±267
Cocaign M, Monnet C, Lindley ND (1993) Batch kinetics of Co-
rynebacterium glutamicum during growth on various carbon
substrates: use of substrate mixtures to localise metabolic bot-
tlenecks. Appl Microbiol Biotechnol 40: 526±530
Cocaign-Bousquet M, Guyonvarch A, Lindley ND (1996) Growth
rate dependent modulation of carbon ¯ux through central me-
tabolism and the kinetic consequences for glucose-limited che-
mostat cultures of Corynebacterium glutamicum. Appl Environ
Microbiol 62: 429±436
Dominguez H, Lindley ND (1996) Complete sucrose metabolism
requires fructose phosphotransferase activity in Corynebacteri-
um glutamicum to ensure phosphorylation of liberated fructose.
Appl Environ Microbiol 62: 3878±3880
Dominguez H, Nezondet C, Lindley ND, Cocaign M (1993)
Modi®ed carbon ¯ux during oxygen limited growth of Co-
rynebacterium glutamicum and the consequences for amino acid
overproduction. Biotechnol Lett 15: 449±454
Hanson TE, Anderson RL (1968) Phosphoenolpyruvate-dependent
formation of fructose-1-phosphate by a four-component phos-
photransferase system. Proc Natl Acad Sci USA 61: 269±276
Hollander JA (1994) Potential metabolic limitations in lysine pro-
duction by Corynebacterium glutamicum as revealed by meta-
bolic network analysis. Appl Microbiol Biotechnol 42: 508±515
Ikeda M, Ozaki A, Katsumata R (1993) Phenylalanine production
by metabolically engineered Corynebacterium glutamicum with
the pheA gene of Escherichia coli. Appl Microbiol Biotechnol
39: 318±323

Linton JD (1990) The relationship between metabolite production
and the growth eciency of the producing organism. FEMS
Microbiol Rev 75: 1±18
Marx A, de Graaf A, Wiechert W, Eggeling L, Sahm H (1996)
Determination of the ¯uxes in the central metabolism of Co-
rynebacterium glutamicum by nuclear magnetic resonance
spectroscopy combined with metabolite balancing. Biotechnol
Bioeng 49: 111±129
Mori M, Shiio I (1987) Phosphoenolpyruvate: sugar phospho-
transferase systems and sugar metabolism in Brevibacterium
¯avum. Agric Biol Chem 51: 2671±2678
Postma PW, Lengeler JW, Jacobson GR (1993) Phospho-
enolpyruvate:carbohydrate phosphotransferase systems of bac-
teria. Microbiol Rev 57:543±594
Reinscheid DJ, Kronemeyer W, Eggeling L, Eikmanns BJ, Sahm H
(1994) Stable expression of hom-1-thrB in Corynebacterium
glutamicum and its e€ect on the carbon ¯ux to threonine
603
and related amino acids. Appl Environ Microbiol 60: 126±
132
Rollin C, Morgant V, Guyonvarch A, Guerquin-Kern JL (1995)
13
C-NMR studies of Corynebacterium melassecola metabolic
pathways. Eur J Biochem 227: 488±493
Saier MH Jr, Chauvaux S, Cook GM, Deutscher J, Reizer J, Ye JJ
(1996) Catabolite repression and inducer control in gram-pos-
itive bacteria. Microbiology 142: 217±230
Shiio I, Sugimoto SI, Kawamura K (1990) E€ects of carbon source
sugars on yield of amino acid production and sucrose metab-
olism in Brevibacterium ¯avum. Agric Biol Chem 54: 1513±1519
Sugimoto SI, Shiio I (1989) Fructose metabolism and regulation of
1-phosphofructokinase and 6-phosphofructokinase in Brevi-
bacterium ¯avum. Agric Biol Chem 53: 1261±1268
Vallino J, Stephanopoulos G (1993) Metabolic ¯ux distributions in
Corynebacterium glutamicum during growth and lysine over-
production. Biotechnol Bioeng 41: 633±646