Tài liệu Báo cáo khoa học: Control analysis as a tool to understand the formation of the las operon in Lactococcus lactis doc

Control analysis as a tool to understand the formation
of the las operon in Lactococcus lactis
Brian Koebmann, Christian Solem and Peter Ruhdal Jensen
Microbial Physiology and Genetics, BioCentrum-DTU, Technical University of Denmark, Kgs Lyngby, Denmark
Over the last three decades increasing attention has
been paid to how metabolic pathways are controlled.
Metabolic control analysis [1,2] has been applied suc-
cessfully to determine the flux control of many single
enzymes [3–7], but much less attention has been paid
to determine flux control by individual enzymes
cotranscribed in prokaryotic operons.
In Lactococcus lactis, an industrially important
organism used extensively in the fermentation of dairy
products, the three glycolytic enzymes phosphofructo-
kinase (PFK), pyruvate kinase (PK) and lactate dehy-
drogenase (LDH) are clustered in the so-called las
operon [8]. This organization of glycolytic genes is
unique and has given rise to speculation that the three
enzymes might play an important role in the control
and regulation of lactic acid production by this organ-
ism. We have previously shown that small changes
in the activity of PFK result in pronounced changes in
metabolite pools, glycolytic flux and growth rate in
L. lactis, but control by PFK has not been quantified
[9]. LDH was shown to have no control over either
growth or glycolytic flux at wild-type levels, but a
strong negative control over the minor flux to mixed
acids via pyruvate formate lyase (PFL) [10].
In this study, the activities of PFK and PK were
modulated individually by changing expression of the
Keywords

glycolysis; Lactococcus; las; metabolic
control analysis; operon
Correspondence
P. R. Jensen, Microbial Physiology and
Genetics, BioCentrum-DTU, Technical
University of Denmark, Building 301,
DK-2800 Kgs. Lyngby, Denmark
Tel: +45 4525 2510
Fax: +45 4593 2809
E-mail:
(Received 23 December 2004, revised 28
February 2005, accepted 9 March 2005)
doi:10.1111/j.1742-4658.2005.04656.x
In Lactococcus lactis the enzymes phosphofructokinase (PFK), pyruvate
kinase (PK) and lactate dehydrogenase (LDH) are uniquely encoded in the
las operon. We used metabolic control analysis to study the role of this
organization. Earlier studies have shown that, at wild-type levels, LDH has
no control over glycolysis and growth rate, but high negative control over
formate production (C
J
formate
LDH
¼À1:3). We found that PFK and PK exert no
control over glycolysis and growth rate at wild-type enzyme levels but both
enzymes exert strong positive control on the glycolytic flux at reduced
activities. PK exerts high positive control over formate (C
J
formate
PK
¼ 0:9 À 1:1)

and acetate production (C
J
acetate
PK
¼ 0:8 À 1:0), whereas PFK exerts no control
over these fluxes at increased expression. Decreased expression of the entire
las operon resulted in a strong decrease in the growth rate and glycolytic
flux; at 53% expression of the las operon glycolytic flux was reduced to
44% and the flux control coefficient increased towards 3. Increased las
expression resulted in a slight decrease in the glycolytic flux. At wild-type
levels, control was close to zero on both glycolysis and the pyruvate bran-
ches. The sum of control coefficients for the three enzymes individually
was comparable with the control coefficient found for the entire operon;
the strong positive control exerted by PK almost cancels out the negative
control exerted by LDH on formate production. Our analysis suggests that
coregulation of PFK and PK provides a very efficient way to regulate gly-
colysis, and coregulating PK and LDH allows cells to maintain homolactic
fermentation during glycolysis regulation.
Abbreviations
LDH, lactate dehydrogenase; PFK, phosphofructokinase; PFL, pyruvate formate lyase; PK, pyruvate kinase.
2292 FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS
corresponding genes. We measured the control exerted
by each of the las enzymes on the glycolytic flux,
growth rate and product formation. We also studied
strains with modulated expression of the entire las
operon, and show that the data fit well with the indi-
vidual determination of flux control coefficients by
PFK, PK and LDH. The role of the las operon is dis-
cussed on the basis of the distribution of flux control
for PFK, PK and LDH.

Results
PFK has no control over glycolytic flux, growth
rate or product formation
PFK converts fructose 6-phosphate to fructose 1,6-bis-
phosphate and is encoded by the first gene in the las
operon (Fig. 1). To study the control of glycolysis and
formate flux by PFK we used strains with modulated
PFK activities. A library of strains with increased
PFK activities ranging from 1.4 to 11 times the wild-
type level was available from a previous study [11]
(Fig. 2A). Strains with reduced levels of PFK,
HWA217 (39% PFK activity) and HWA232 (60%
PFK activity) were obtained by Andersen et al. [9],
who also showed that such decreases in PFK resulted
in a strong decrease in both growth rate and glycolytic
flux [9]. Together these PFK mutants cover the range
of enzyme activities necessary for studies of flux
control.
Five selected strains with increased PFK activity
were grown exponentially at 30 °C in defined SAL
medium supplemented with glucose and analysed with
respect to growth rate, glycolytic flux and fermentation
products (Fig. 2B,C). At increased PFK activity we
found a slight decrease in both growth rate and glyco-
lytic flux (Fig. 2B,C). The strains remained homolactic
with only a slight decrease in formate production com-
pared with the wild-type strain. The data obtained for
strains with modulated PFK activity above the wild-
type level were fitted to linear curves (Fig. 2B,C) and
the respective flux controls were calculated as des-

cribed in Experimental procedures (Fig. 2D). From
these data it is clear that at the wild-type level PFK
has no control over the glycolytic flux (C
J
glucose
PFK
% 0)or
growth rate (C
J
l
PFK
% 0), and no control over the fluxes
to lactate (C
J
lactate
PFK
% 0), formate (C
J
formate
PFK
% 0) or acetate
(C
J
acetate
PFK
% 0) at the wild-type level and above.
Fig. 1. Glycolysis and the las operon in
Lactococcus lactis.The las operon in L. lactis
consists of the three genes pfk, pyk and
ldh, coding for phosphofructokinase (PFK),

pyruvate kinase (PK) and lactate dehydro-
genase (LDH), respectively.
B. Koebmann et al. Control analysis of the las operon
FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS 2293
PK has no control over glycolysis but full control
over mixed acid production
PK, which converts phosphoenolpyruvate to pyruvate,
is encoded by the second gene in the las operon (Fig. 1).
In order to obtain strains with increased PK activity an
additional copy of the pyk gene was recombined into
the TP901-1 attachment site using the site-specific
recombination vector pLB85 as described in Experimen-
tal procedures. The pyk gene was here preceeded by the
leader of the ald gene, see Experimental Procedures.
This resulted in a library of 37 strains 13 of which were
characterized with respect to PK activities. The charac-
terized strains were found to have PK activities ranging
from 100 to 330% of wild-type level, whereas the activ-
ities of PFK and LDH were reduced compared with the
wild-type level (Fig. 3A).
In order to obtain a strain with lower PK activity
one of the strains with an additional copy of the pyk
gene, CS1897 (120% PK activity), was used. The
native pyk gene in CS1897 was deleted by a double
cross-over event as described in Experimental proce-
dures and shown in Fig. 4. The resulting strain,
CS1929 (37% PK activity), thus contains only a single
pyk gene under the control of a synthetic promoter.
The relative PFK activity in strain CS1929 was found
to increase to over 160% of the wild-type level,

whereas the relative LDH activity was reduced to 80%
of the wild-type level (Fig. 3A).
In order to study the control exerted over the meta-
bolic fluxes by PK, strains with PK activities altered
around the wild-type level were grown in defined SAL
medium supplemented with glucose. A slight decrease
in growth rate and glycolytic flux was observed at
increased PK activities (Figs 3B and 5). For strain
CS1929 we found a strong decrease in growth rate and
glycolytic flux, almost proportional to the change in
PK activity. The data points for growth and glucose
flux were then fitted against the PK activities in order
to determine the control exerted by PK over the
growth rate (Fig. 3B) and glycolytic flux (Fig. 5) from
which we conclude that PK exerted no significant con-
trol over either growth rate or glycolytic flux at the
wild-type level. However, reducing the PK activity to
37% enhances the control exerted by PK over growth
rate to C
J
l
PK
% 1.
Product formation changed significantly as the PK
activity was modulated. At increased PK activity we
found an almost proportional increase in formate and
acetate production and a decrease in lactate produc-
Fig. 2. Modulation of PFK activity and the effects on growth and fluxes. (A) Library of strains with modulated PFK activities. The PFK activit-
ies were measured in extracts from strains in which an additional pfk gene transcribed from synthetic promoters was integrated on the chro-
mosome by site-specific recombination in a phage attachment site. The specific PFK activity in MG1363 was determined to 0.55 UÆmg

)1
protein [11]. Selected strains indicated by white bars were cultivated in SAL medium supplemented with glucose and studied with respect
to (B) growth rate (including flux control by PFK on growth rate), (C) metabolic fluxes and (D) flux control coefficients by PFK on metabolic
fluxes. Curve fitting is described in Experimental procedures.
Control analysis of the las operon B. Koebmann et al.
2294 FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS
tion. These results show that PK activity plays an
important role in the metabolic shift from homolactic
to mixed acid fermentation. The data points for prod-
uct formation were then fitted against the PK activities
in order to determine the control exerted by PK over
the flux to formate. Because only one data point was
available for PK activity below the wild-type level
three or four curves were fitted for each of the studied
metabolic fluxes using the best possible suggestions
obtained using the software curveexpert. The resulting
equations were, as for growth rate and glycolytic flux,
used to calculate the control exerted by PK on the
metabolic fluxes (Figs 3B and 5). Interestingly, we
found a very high positive flux control coefficient by
PK on the flux to formate at the wild-type level
(C
J
formate
PYK
¼ 0:9 À 1:1) (Fig. 5). Similarly, the control
exerted by PK over the flux to acetate was determined
to be C
J
acetate

PK
¼ 0:8 À 1:0 (Fig. 5).
Modulation of the entire las operon
Strains with altered expression of the entire las operon
were previously obtained by replacing the native las
promoter with synthetic promoters in a single cross-
over event [11]. From this library consisting of 50
strains with altered expression of the las operon, the
enzyme activities of PFK, PK and LDH were deter-
mined and eight strains with enzyme activities 0.5–3.5
times the wild-type level were selected for further
analysis (Fig. 6A). Good correlation among relative
enzyme activities of the three enzymes was found.
These strains then allowed us to study the control
exerted by all three las enzymes simultaneously. The
growth rate and metabolic fluxes for the strains were
determined and we also found that growth rate and
glycolytic flux were highest when the activities of the
las enzymes were at wild-type levels (Figs 6B and 7).
The data points were fitted to the equations described
in Experimental procedures and are presented in
Figs 6B and 7 for calculations of flux control coeffi-
cients. The sum of flux control on glycolysis and
growth rate by the las enzymes at wild-type levels is
close to 0 (C
J
glucose
las
% 0 and C
J

l
las
% 0) as can be inferred
from the primary data. However, it is interesting that
a slight reduction in las activity resulted in a very
strong decrease in growth rate and glycolytic flux: at
53% expression the flux was reduced to 44%. At this
level, the flux control was found to be as high as
C
J
glucose
las
% 3 (Fig. 7) and C
J
l
las
% 3 (Fig. 6B).
With respect to the fermentation pattern, little
change was observed around the wild-type level, and
flux control coefficients on the formate flux
(C
J
formate
las
¼À0:26) and acetate flux (C
J
acetate
las
¼À0:26) were
smaller than was observed for strains with individual

modulation of PK and LDH (Fig. 7). Strong negative
flux controls on formate production (C
J
formate
las
%
ðÀ1:4ÞÀðÀ1:7Þ) and acetate production (C
J
acetate
las
%
ðÀ1:7ÞÀðÀ2:0Þ) were observed at reduced activities of
the las enzymes to 50–60% of wild-type level (Fig. 7).
When the activities of the las enzymes were increased
three times we find a flux control coefficient at
C
J
formate
las
%ðÀ0:4Þ for the formate flux and
C
J
acetate
las
%ðÀ0:4Þ for the acetate flux.
A
B
Fig. 3. Modulation of PK activity and the effect on growth rate. (A)
Enzyme activities of PFK, PK and LDH relative to the wild-type level
in strains with modulated PK activities. The enzyme activities were

measured in extracts from strains in which the pyk gene placed
after a range of synthetic promoters with different strengths was
integrated on the chromosome by site-specific recombination in a
phage attachment site. In strain CS1929 the native pyk gene was
deleted from the las operon. The specific PK activity in MG1363
was determined to 0.25 UÆmg
)1
protein. (B) Growth rates of selec-
ted strains (including flux control coefficients). Standard deviations,
indicated by error bars, are based on measurement of three individ-
ual cultures.
B. Koebmann et al. Control analysis of the las operon
FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS 2295
Fig. 4. Construction of a strain with the pyk
gene deleted from the las operon.
Truncated fragments of PFK and LDH were
cloned along each other in pG
+
host8 which
cannot replicate in L. lactis at 37 °C [23].
Because PK is essential for growth, deletion
of pyk was performed in strain CS1897
which contains an additional copy of pyk in
the TP9011-1 phage attachment site. A
double cross-over event of the resulting
plasmid, pCS1919, on the las operon resul-
ted in an operon structure in which the pyk
gene was deleted.
Fig. 5. Flux control coefficients for PK on metabolic fluxes. Flux control coefficients for PK with respect to glycolysis, lactate, acetate and
formate production were determined from the fitted equations as described in Experimental procedures. Standard deviations, indicated by

error bars, are based on measurement of three individual cultures.
Control analysis of the las operon B. Koebmann et al.
2296 FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS
AB
Fig. 6. Modulation of the las operon. (A) Enzyme activities of PFK, PK and LDH relative to the wild-type level. The enzyme activities were
measured in extracts from strains in which the native las promoter was replaced by a library of synthetic promoters with different strengths
[11]. (B) Growth rates of selected strains (including flux control coefficients). Standard deviations, indicated by error bars, are based on meas-
urement of three individual cultures.
Fig. 7. Flux control coefficients for the las enzymes on metabolic fluxes. A selection of strains were analysed with respect to glycolytic flux
and metabolic fluxes. Flux control coefficients with respect to glycolysis, lactate, acetate and formate production were determined from the
fitted equations as described in Experimental procedures. Standard deviations, indicated by error bars, are based on measurement of three
individual cultures.
B. Koebmann et al. Control analysis of the las operon
FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS 2297
Comparison of control by the las enzymes
Based on the data presented here and on earlier
data for LDH [10], it is possible to compare the flux
controls of the individual enzymes with that of a
simultaneous modulation of all the las enzymes, i.e. to
test whether: C
J
las
¼ C
J
PFK
þ C
J
PK
þ C
J

LDH
. With respect
to glycolysis, growth rate and lactate flux, the flux con-
trol coefficients of the three individual enzymes PFK,
PK and LDH added up to a value close to 0, which is
in accordance with the low control over the glycolytic
flux found for all enzymes in the las operon.
With respect to control over the formate flux, LDH
has previously been found to exert a high negative
control (C
J
formate
LDH
¼À1:3) [10]. In this study, we found
that PFK has almost no flux control on formate pro-
duction (C
J
formate
PFK
% 0), whereas PK is found to have a
high positive flux control (C
J
formate
PK
% 1:0), so addition of
these flux control coefficients on formate gives us:
C
J
formate
PFK

þ C
J
formate
PK
þ C
J
formate
LDH
¼À0:3. Interestingly, when
all enzymes from the las operon were modulated simul-
taneously we found a control of C
J
formate
las
¼À0:26 on the
formate flux, which again fits very well with the sum
of the control by the individual enzymes.
A similar comparison of flux control was not poss-
ible for the acetate flux because this was not measured
in the earlier study on LDH [10]. However, we expect
the sum of the individual flux control coefficients to
add up to that found for the combined change of the
las enzymes, because mixed acid metabolism under
anaerobic conditions is expected to result in equal
amounts of formate and acetyl-CoA and the resulting
acetyl-CoA is then metabolized into equal amount of
ethanol and acetate to maintain the redox balance.
Discussion
In this study we quantified the control exerted by the
las enzymes on the metabolic fluxes under conditions

where any autoregulation of the modulated enzyme in
question that might occur in a wild-type cell was elim-
inated by the introduction of synthetic promoters. The
method measures so-called ‘inherent control coeffi-
cients’ and has previously been applied successfully to
the study of DNA supercoiling in Escherichia coli
[12,13] and more recently to a study of the control
exerted by CTP synthase on the nucleotide pools in
Lactococcus [14].
The three enzymes PFK, PK and LDH encoded by
the las operon in L. lactis MG1363 were modulated
both individually and simultaneous by changing the
expression of the respective genes. We found that nei-
ther the individual enzymes nor the sum of the las
enzymes had significant control on the glycolytic flux at
wild-type levels. The sum of the flux control coefficients
determined for the individual enzymes on glycolysis
and on the formate flux fits very well with the coeffi-
cients obtained from modulating the entire operon,
which demonstrates the solidity of the approach used
here.
Both PFK and PK were found to exert very strong
positive control on glycolysis at reduced activities
around half the normal enzyme level. When expression
of the las operon was reduced to 53% the glycolytic
flux was reduced to 44%, which amounts to more than
a proportional decrease in the flux. Moreover, by look-
ing at the las expression range from 53 to 61% of the
wild-type level we observe a relative change in the gly-
colytic flux of 34%. In terms of flux control based on

the fitted equations, this amounts to a flux control
coefficient approaching 3! This is significantly higher
than the flux control coefficients for the individual las
enzymes at comparable levels. From the data for PFK
given in Andersen et al. [9], the flux control coefficient
on the glycolytic flux of PFK activity at 50% of wild-
type level can be estimated to 0.45 by fitting the data
to a linear curve. According to Andersen et al. [10],
the flux control coefficient on the glycolytic flux for
LDH at 50% of wild-type activity was found to be
around 0.1–0.2. In this study we found the flux control
coefficient on the glycolytic flux by PK at 50% of
wild-type activity to be around 1.0. Thus, the sum of
the individual enzymes amounts to only 1.6–1.7.
The dramatic reduction in growth rate and glycolytic
flux at reduced las enzyme activity may be explained
by perturbations in metabolite pools. In the previous
study by Andersen et al. it was suggested that the
strong effect on growth and glycolytic flux observed
when reducing PFK activity could be due to an accu-
mulation of hexose phosphates [9]. The stronger effect
on the growth rate and glycolytic flux observed in this
study when all the las enzymes were reduced to 50%
of wild-type levels may then be the result of decreased
PK activity which would result in an increased
phosphoenolpyruvate pool, which in turn would
enhance the activity of the PTS system and thereby
result in further increases in hexose phosphate pools.
The decreased LDH activity may contribute further to
this effect by causing an accumulation of pyruvate and

then back-pressure on PK.
We therefore conclude that by placing the pfk and
pyk genes together in an operon, L. lactis is provided
with a very efficient tool for regulating glycolysis: by
regulating expression of the las operon two- to three-
fold the glycolytic flux will be dramatically affected.
Indeed, regulation of expression of the las operon has
Control analysis of the las operon B. Koebmann et al.
2298 FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS
been shown to take place at the transcriptional level
[15]. Deletion of the entire pyk gene from the las
operon resulted in a slight disturbance in the relative
levels of PFK and LDH, which were altered to 167
and 76% of the wild-type level, respectively (Fig. 3A).
The mechanism behind these effects is unclear but may
reflect a combination of a hierarchical up-regulation
[16] of the las operon at low PK activity and a polar
effect of the pyk deletion on expression of ldh.An
important question is whether this affects the conclu-
sions drawn from our control analysis. But we already
know that PFK has no control over growth rate, gly-
colytic flux or formate flux at wild-type levels and
above and therefore changes in PFK should not affect
the control by PK. Furthermore, if LDH expression is
decreased due to a polar effect, this would result in an
underestimation of the flux control by PK on mixed
acid production. We therefore believe that it is safe to
use strain CS1929 for the current metabolic control
analysis.
Overexpression of pyk resulted in a proportional

increase in the flux to mixed acid products. In a recent
study by Ramos et al. [17] it was found that the
fermentation pattern in a PK-overproducing strain
showed a typical homolactic metabolism under anaer-
obic conditions. At first, this seems to contradict our
results. However, in practice, the flux to formate at the
wild-type level amounts to only 3.5% of the pyruvate
metabolism, and a doubling in formate flux would
amount to only 7%, which would still be considered to
be homolactic fermentation.
The magnitude of the control exerted by PK
(C
J
formate
PK
¼ 0:9 À 1:1) over formate production was
almost comparable but of the opposite sign compared
with the negative control found previously for LDH
(C
J
formate
LDH
%À1:3) [10]. Because the control by PFK on
the flux to formate was found to be 0, the sum of
control on the formate flux was only slightly negative
(C
J
formate
las
%À0:3), which explains why changing expres-

sion of the las operon around the normal level led to
little change in the production pattern. By coregulating
PK and LDH cells can maintain homolactic fermenta-
tion.
The fact that the effects of PK and LDH almost
cancel each other out may also add to the explanation
of why the genes are organized in an operon in L. lac-
tis. When L. lactis needs to up- or down-regulate the
glycolytic flux it can do so without interfering with the
pattern of product formation. Indeed, L. lactis appears
to strongly favour the homolactic route despite the fact
that significantly less ATP is gained compared with
mixed acid production. Because L. lactis is resistant to
high concentrations of lactic acid it may benefit from
homolactic fermentation by efficiently inhibiting the
growth of its competitors.
In this analysis we have considered only metabolic
fluxes, flux control coefficients and, to some extent,
external metabolite concentrations. However, organiza-
tion of the las operon may also play an important
role in keeping internal metabolite pools constant, by
coregulating enzymes early and late in glycolysis when
changes in the flux are required [18].
A simple explanation for why prokaryotic genes are
organized in operons could be to efficiently regulate
pathways by regulating only a few genes, for example,
in order to save energy and protein synthesizing capa-
city. This would be preferable to placing all the genes
involved in the pathway in the same operon; the cell
can then respond quickly to changes in the environ-

ment by changing the expression of only a few genes
and using the protein-synthesizing capacity to express
these genes when needed. Here we have studied a set
of enzymes that are needed by these cells under all
growth conditions, because glycolysis is the energy-
producing pathway. Indeed, in contrast to many other
systems, only a few fold regulations of the genes have
been shown to take place [15].
Metabolic control analysis has helped us to charac-
terize the role of the individual genes in an operon
and, to some extent, explain why L. lactis may benefit
from the way in which the las operon is organized. We
believe that such analysis would not have been possible
using traditional functional analysis with gene knock-
outs and overexpression of enzymes from a plasmid.
Experimental procedures
Bacterial strains and plasmids
For cloning purposes was used Escherichia coli strain
ABLE-C {E. coli C lac(LacZ
–
)[Kan
r
McrA
–
McrCB
–
McrF
–
Mrr
–

HsdR (r
k
–
m
k
–
)][F’proAB lacI
q
ZDM15 Tn10(Tet
r
)]}
(Stratagene) or KW1 {metB, strA, purB(aad-uid-man), hsr,
hsm
+
,gusA
–
} [19]. L. lactis ssp. cremoris MG1363, a pro-
phage-cured and plasmid-free derivative of NCDO712 [20],
was used as a model organism for modulating gene expres-
sion. L. lactis LB436 is a derivative of MG1363 containing
a plasmid, pLB65, that harbours a gene coding for the tem-
perate lactococcal bacteriophage TP901-1 integrase [21].
The strain was used as the host for site-specific integration
in the chromosomal attB site of phage TP901. The E. coli
vector plasmid pRC1 [22] was used for integration of syn-
thetic promoters upstream to the las operon. The plasmid
pLB85 harbouring attP of TP901-1 and a promotorless
gusA gene encoding b-glucuronidase [21] was used as plas-
mid vector for site-specific integration of extra gene copies
B. Koebmann et al. Control analysis of the las operon

FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS 2299
on the TP901-1 attB locus on the chromosome of MG1363.
The replication-thermosensitive plasmid pG
+
host8, which
contains a gene for tetracycline resistance [23], was used to
delete the pyk gene from the las operon on the chromo-
some.
Growth media and growth conditions
E. coli strains were grown aerobically at 37 °C in Luria–
Bertani broth [24]. L. lactis strains were routinely cultivated
at 30 °C without aeration in M17 broth [25] or in chemic-
ally defined SA medium [26] modified by exclusion of ace-
tate and inclusion of 2 lgÆ mL
)1
lipoic acid (SAL medium).
The media were supplemented with 1 or 10 gÆL
)1
glucose
and appropriate selective antibiotics.
L. lactis growth experiments were performed as batch
cultures (flasks) at 30 °C in 100 mL of SAL medium [26]
supplemented with 0.12% (w ⁄ v) of glucose when determin-
ing biomass yield on glucose, Y
g
, or else 1% (w ⁄ v) of glu-
cose. Antibiotics were only used in precultures and not in
the growth experiments. Enzyme activities and product for-
mation were determined by using the same cultures thereby
assuring that genetic constructions were intact. A slow stir

with magnets was used to keep the culture homogenous.
Regular measurements of A
600
were made, and HPLC sam-
ples taken for measuring the product formation and glyco-
lytic flux. Cell density was correlated to the cell mass of
L. lactis to be 0.36 gdwÆL
)1
SA medium for A
600
¼ 1 [10].
All fluxes were calculated from changes in concentration of
metabolites measured by HPLC from Shimadza Corp.
(Kyoto, Japan) as previously described [9].
Antibiotics
Antibiotics were used at the following concentrations: Ery-
thromycin: 5 lgÆmL
)1
for L. lactis and 200 lgÆmL
)1
for
E. coli. Tetracycline: 5 lgÆmL
)1
for L. lactis and 8 lgÆmL
)1
for E. coli.
Enzymes
All enzymes used in the enzymatic assays for PFK, PK and
LDH were purchased from Roche A ⁄ S (Hvidovre, Denmark).
DNA techniques

All manipulations were performed as described by
Sambrook et al. [24]. Taq DNA polymerase (New England
Biolabs, Frankfurt am Main, Germany) was applied
for analytical purposes and PCR products intended for
cloning were generated using Elongase
R
enzyme mix (Invi-
trogen, Ta
˚
strup, Denmark). Chromosomal DNA from
L. lactis was isolated using a method described previously
[27] with the modification that cells were treated with
20 lg lysozyme per mL for 2 h before lysis. Digestion with
restriction enzymes (Fermentas, St Leon, Germany; Amer-
sham, Hillerød, Denmark), treatment with T4 DNA ligase
(Fermentas) and shrimp alkaline phosphatase (Fermentas)
were carried out as prescribed by the manufacturers. DNA
fragments were purified from agarose gels using GFX
PCR DNA and Gel Band Purification Kit (Amersham).
E. coli was transformed by electroporation. Cells were
plated on Luria–Bertani plates supplemented with appro-
priate antibiotics. Plasmid DNA was isolated from E. coli
by using Qiaprep Spin Miniprep Kit (Qiagen, Hilden, Ger-
many). Cells of L. lactis were made electrocompetent by
growth in GM17 medium containing 1% glycine, and
DNA was introduced by electroporation as previously des-
cribed by Holo and Nes [28]. After electroporation cells
were plated on GM17 supplemented with appropriate anti-
biotics.
Enzyme measurements

The activities of PFK, PK and LDH were measured in cell
extracts obtained by sonication. Cells were grown in SAL
medium and harvested at A
600
% 0.5. The cells were washed
twice with ice-cold 0.2% (w ⁄ v) KCl and then resuspended
in ice-cold sonication buffer. Sonication buffer for LDH
and PK activity measurements: 50 mm triethanolamine,
10 mm KH
2
PO
4
,10mm EDTA, 50% (v ⁄ v) glycerol,
pH 4.7; sonication buffer for PFK activity measurements,
50 mm Tris ⁄ HCl, 0.1 mm EDTA, 50% (v ⁄ v) glycerol, 1 mm
dithiothrietol, pH 7.5. The cell suspension was sonicated
three times for 45 s with an interval of 30 s. The prepar-
ation was kept on ice during the sonication. Following
sonication, cell debris and intact cells were removed by cen-
trifugation (10 min, 20 000 g,4°C). As a measure for the
degree of cell disruption the A
280
was used. The enzyme
activities were determined from the consumption of NADH
using a Zeiss M500 spectrophotometer. PFK was assayed
according to Fordyce et al. [29] with the following modifi-
cations. Final concentrations in assay: 1 mm ATP, 1 mm
fructose 6-phosphate, 0.2 mm NADH, 10 mm MgCl
2
,

10 mm NH
4
Cl, 0.3 UÆmL
)1
triose phosphate isomerase,
1UÆmL
)1
glycerol 3-phosphate dehydrogenase and 0.3 U
aldolase. PK was assayed as described by Crow and
Pritchard [30]. Final concentrations in assay was: 1 mm
GDP, 1 mm PEP, 1 mm fructose 1,6-bisphosphate, 10 mm
MgCl
2,
0.2 mm NADH and 6.3 UÆmL
)1
LDH. LDH was
measured according to Crow and Pritchard [31]. Final con-
centrations in assay was: 10 mm pyruvate, 0.2 mm NADH,
1mm fructose 1,6-bisphosphate. All measured enzyme
activities were related to the A
280
of the extract, for the
purpose of determining relative activities. The specific activ-
ities of PFK and PK and LDH in MG1363 were deter-
mined as UÆmg
)1
of protein, where a unit (U) is defined as
the amount of enzyme producing 1 lmol of NADH per
Control analysis of the las operon B. Koebmann et al.
2300 FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS

minute. The relative values of simultaneous modulation of
the three las enzymes are calculated as the average of the
three individual relative activities.
Construction of strains with modulated
expression of pyk
Strains with increased PK activity were obtained by intro-
ducing an additional copy of the gene on the chromosome
transcribed from synthetic promoters. At first we tried to
use the natural leader of the pyk gene, but this resulted in
merely 25% increased PK activity. We then inserted the
leader mRNA from the L. lactis ald gene as follows.
A PCR fragment was generated using primer CP-pyk
(5¢-ACGACTAGTGGATCCATNNNNNAGTTTATTCTT
GACANNNNNNNNNNNNNNTGRTATAATNNNNAA
GTAATAAAATATTCGGAGGAATTTTGAAATGAATA
AACGTGTAAAAATCG-3¢)(N¼ A, T, G, C) and pyk-
back (5¢-CTCTACATGCATTTCAACAATAGGGCCTG
TC-3¢) for amplification of pyk. The resulting PCR prod-
ucts, containing synthetic promoters followed by an ald
leader and a full-length pyk gene, were digested with SpeI
and NsiI and cloned in the vector pLB85 digested with
XbaI and PstI. Following ligation the plasmids were intro-
duced directly to L. lactis LB436, carrying plasmid pLB65
in which pLB85 and other plasmids containing the attB
site from TP901-1 will integrate with high frequency at the
corresponding attachment site for phage TP901-1 on the
L. lactis chromosome [21]. The cells were plated on GM17
plates supplemented with 5 lgÆmL
)1
erythromycin and

200 lgÆmL
)1
5-bromo-4-chloro-3-indolyl-beta-d-glucuronide
(X-gluc) (Biosynth AG, Switzerland).
Construction of a strain with reduced PK activity was
performed by deleting the native pyk gene in strain CS1897
which already contains an additional copy of the pyk gene
at the TP901-1 phage attachment site. PCR products
upstream to pyk using primer pyk1 (5¢-TGGTACTCGAG
CAATTTCTGAAGGTATCGAAG-3¢) and pyk2 (5¢-GG
AAGGATCCTTGTGTTTTTCTCCTATAATG-3¢) and
downstream to pyk using primer pyk3 (5¢-GGAAGGA
TCCTTTGTCAATTAATGATCTTAAAAC-3¢) and pyk4
(5¢-CTAGTCTAGATGAGCTCCAGAAGCTTCC-3¢) were
amplified. The PCR products were digested with XhoI ⁄
BamHI and BamHI ⁄ XbaI, respectively, and cloned in iden-
tical restriction sites in plasmid pG
+
host8, using E. coli
KW1 as cloning host. The resulting plasmid, pCS1919, was
used to delete pyk from the las operon by a double cross-
over event as previously described [11].
Curve fitting and calculation of control
coefficients
To estimate the control of PFK, PK and all las enzymes on
the glycolytic flux (J
glucose
), growth rate (J
l
) and on the

metabolic fluxes for the entire range of enzyme activity (a
x
),
the experimental data points were fitted to equations. For
strains with modulated PFK activity the data points were
fitted to linear equations by the least square method using
excel (Microsoft). The experimental data points for strains
with modulated PK activity and las activity were fitted
by the least square method using curveexpert 1.3¢
(Hyams Development, Hixson, TN, USA) using the Leven-
berg–Marquardt regression to solve nonlinear regressions.
This resulted in the following functions: PFK: J
l
(a
PFK
) ¼
)0.0077
*
a
PFK
+ 0.886, J
glucose
(a
PFK
) ¼ )0.234
*
a
PFK
+
22.9, J

lactate
(a
PFK
) ¼ )0.414
*
a
PFK
+42.7, J
formate
(a
PFK
) ¼
)0.0806
*
a
PFK
+ 1.80: J
acetate
(a
PFK
) ¼ ) 0.0237
*
a
PFK
+
1.17, PK: J
l
ða
PK
Þ¼0:0298 Ãð18:7 À a

PK
ÞÃð1 À e
À7:9Ãa
3:4
PK
Þþ
0:315, J
glucose
ða
PK
Þ¼53:2 Ã 0:504
1=a
PK
à a
À0:641
PK
(Modified
Hoerl Model), J
glucose
(a
PK
) ¼ e
3.97)(0.685/a
PK
))0.641
*
ln(a
PK
))
(Vapor Pressure Model), J

glucose
ða
PK
Þ¼À0:511 þ 56:2 Ã
a
PK
À 35:8 Ã a
2
PK
þ 6:90 Ã a
3
PK
(Polynomial Fit), J
glucose
ða
PK
Þ¼
ð52:4 Ã a
PK
À 0:533Þ=ð1 þ 0:249 Ã a
PK
þ 0:696 Ã a
PK
Þ
2
(Rat-
ional Function), J
lactate
(a
PK

) ¼ e
4.71)(0.782/a
PK
))0.817
*
ln(a
PK
))
(Vapor Pressure Model), J
lactate
ða
PK
Þ¼111 Ã 0:458
1=a
PYK
Ã
a
À0:817
PK
(Modified Hoerl Model), J
lactate
ða
PK
Þ¼À1:21 þ 111Ã
a
PK
À 73:6 Ã a
2
PK
þ 14:5 Ã a

3
PK
(Polynomial Fit), J
lactate
ða
PK
Þ¼
ð94:0 Ã a
PK
À 0:984Þ=ð1 þ 0:00179 Ã a
PK
þ 0:846 Ã a
2
PK
Þ (Ratio-
nal Function), J
formate
ða
PK
Þ¼3:58 Ã 0:535
a
PK
à a
1:67
PK
(Hoerl
model), J
formate
ða
PK

Þ¼ð1:39 à a
PK
À 0:00750Þ=ð1 À 0:455Ã
a
PK
þ 0:187 Ã a
2
PK
Þ (Rational function), J
formate
ða
PK
Þ¼
À0:453 þ 2:93 Ã a
PK
À 0:538 Ã a
2
PK
(Quadratic fit), J
acetate
ða
PK
Þ¼
0:0329 þ 1:178 Ã a
PYK
þ 0:276 Ã a
2
PK
À 0:165 Ã a
3

PK
(Polyno-
mial fit), J
acetate
ða
PK
Þ¼À0:0137 þ 1:636 à a
PK
À 0:284 Ã a
2
PK
(Quadratic fit), J
acetate
ða
PK
Þ¼1:91 Ã 0:701
a
PYK
à a
1:177
PK
(Hoerl
model), J
acetate
ða
PK
Þ¼ð0:0500 þ 1:034 à a
PK
Þ=ð1 À 0:350 Ã a
PK

þ
0:171 Ã a
2
PK
Þ (Rational function), All las enzymes: J
l
ða
las
Þ¼
0:0123 Ãð93:9 À a
las
ÞÃð1 À e
À7:1Ãa
3:2
las
ÞÀ0:276, J
glucose
ða
las
Þ¼
0:693 Ãð83:3 À a
las
ÞÃð1 À e
À6Ãa
2:1
las
ÞÀ33:2 (User defined),
J
lactate
ða

las
Þ¼0:919 Ãð129 À a
las
ÞÃð1 À e
À6Ãa
2:1
las
ÞÀ75:2 (Us er
defined), J
acetate
ða
las
Þ¼0:1135 ÃðÀ30:3 À a
las
ÞÃð1 À e
À6Ãa
3:3
las
Þþ
4:66 (User defined), J
formate
ða
las
Þ¼0:173 ÃðÀ23:7 À a
las
ÞÃ
ð1 À e
À5:6Ãa
2:3
las

Þþ6:02 (User defined).
User-defined equations were also selected as the functions
giving the least sum of squares.
The control coefficients were then calculated from the
equation C
J
x
¼ (dJ(a
x
) ⁄ J(a
x
)) ⁄ (d(a
x
) ⁄ (a
x
) for the entire range
of a
x
, where J refers to either a flux or a growth rate. The
slopes were determined by differentiation of the equations
using the quickmath hosted by Verio Web hosting services
on the Internet.
Acknowledgements
This work was supported by the Danish Dairy
Research Foundation (Danish Dairy Board), the Dan-
ish Research Agency and the Danish Center for
Advance Food Studies (LMC).
FEBS Journal 272 (2005) 2292–2303 ª 2005 FEBS 2301
B. Koebmann et al. Control analysis of the las operon
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