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 Correspond
Trang 1of 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 phosphofructo-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: prj@biocentrum.dtu.dk
(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 (CJformate
LDH ¼ 1:3) 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 (CJformate
PK ¼ 0:9 1:1) and acetate production (CJacetate
PK ¼ 0:8 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.
Trang 2corresponding 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 30C 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 (CJglucose
PFK 0) or growth rate (CJl
PFK 0), and no control over the fluxes
to lactate (CJlactate
PFK 0), formate (CJformate
PFK 0) or acetate (CJacetate
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.
Trang 3PK 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 CJl
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.
Trang 4tion 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 (CJformate
PYK ¼ 0:9 1:1) (Fig 5) Similarly, the control exerted by PK over the flux to acetate was determined
to be CJacetate
PK ¼ 0:8 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 (CJglucose
las 0 and CJl
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
CJglucose las 3 (Fig 7) and CJl
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 (CJformate
las ¼ 0:26) and acetate flux (CJacetate
las ¼ 0:26) were smaller than was observed for strains with individual modulation of PK and LDH (Fig 7) Strong negative flux controls on formate production (CJformate
ð1:4Þ ð1:7Þ) and acetate production (CJacetate
ð1:7Þ ð2:0Þ) 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
CJformate las ð0:4Þ for the formate flux and
CJacetate las ð0:4Þ 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)1protein (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.
Trang 5Fig 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.
Trang 6A B
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.
Trang 7Comparison 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: CJlas¼ CJ
PFKþ CJ
PKþ CJ 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 (CJformate
LDH ¼ 1:3) [10] In this study, we found
that PFK has almost no flux control on formate
pro-duction (CJformate
PFK 0), whereas PK is found to have a
high positive flux control (CJformate
PK 1:0), so addition of these flux control coefficients on formate gives us:
CJformate
PFK þ CJformate
PK þ CJformate
LDH ¼ 0:3 Interestingly, when all enzymes from the las operon were modulated
simul-taneously we found a control of CJformate
las ¼ 0:26 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
Trang 8been 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
(CJformate
PK ¼ 0:9 1:1) over formate production was
almost comparable but of the opposite sign compared
with the negative control found previously for LDH
(CJformate
LDH 1:3) [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
(CJformate
las 0:3), 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–)[KanrMcrA–McrCB–McrF–
Mrr– HsdR (rk mk)][F’proAB lacIqZDM15 Tn10(Tetr)]} (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
Trang 9on 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 37C in Luria–
Bertani broth [24] L lactis strains were routinely cultivated
at 30C without aeration in M17 broth [25] or in
chemic-ally defined SA medium [26] modified by exclusion of
ace-tate and inclusion of 2 lgÆmL)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 30C in 100 mL of SAL medium [26]
supplemented with 0.12% (w⁄ v) of glucose when
determin-ing biomass yield on glucose, Yg, 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 A600were 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 A600¼ 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)1for 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 ElongaseRenzyme 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 A600 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 KH2PO4, 10 mm 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 sonicprepar-ation Following sonication, cell debris and intact cells were removed by
cen-trifugation (10 min, 20 000 g, 4C) As a measure for the degree of cell disruption the A280 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 MgCl2,
10 mm NH4Cl, 0.3 UÆmL)1 triose phosphate isomerase,
1 UÆmL)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 MgCl2, 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,
1 mm fructose 1,6-bisphosphate All measured enzyme activities were related to the A280 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)1of protein, where a unit (U) is defined as the amount of enzyme producing 1 lmol of NADH per
Trang 10minute 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¢) (N ¼ 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 lactischromosome [21] The cells were plated on GM17
plates supplemented with 5 lgÆmL)1 erythromycin and
200 lgÆmL)15-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 (Jglucose), growth rate (Jl) and on the
metabolic fluxes for the entire range of enzyme activity (ax), 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: Jl(aPFK)¼ )0.0077 * aPFK+ 0.886, Jglucose(aPFK)¼)0.234 * aPFK+ 22.9, Jlactate(aPFK)¼)0.414 * aPFK+ 42.7, Jformate(aPFK)¼ )0.0806 * aPFK+ 1.80: Jacetate(aPFK)¼)0.0237 * aPFK+ 1.17, PK: JlðaPKÞ ¼ 0:0298 ð18:7 aPKÞ ð1 e7:9a 3:4
PKÞ þ 0:315, JglucoseðaPKÞ ¼ 53:2 0:5041=a PK a0:641
PK (Modified Hoerl Model), Jglucose(aPK)¼ e3.97)(0.685/aPK ) )0.641*ln(a PK ))
(Vapor Pressure Model), JglucoseðaPKÞ ¼ 0:511 þ 56:2
aPK 35:8 a2
PKþ 6:90 a3
PK(Polynomial Fit), JglucoseðaPKÞ ¼ ð52:4 aPK 0:533Þ=ð1 þ 0:249 aPK þ 0:696 aPKÞ2 (Rat-ional Function), Jlactate(aPK)¼ e4.71)(0.782/aPK ) )0.817*ln(a PK ))
(Vapor Pressure Model), JlactateðaPKÞ ¼ 111 0:4581=a PYK
a0:817
PK (Modified Hoerl Model), JlactateðaPKÞ ¼ 1:21 þ 111
aPK 73:6 a2
PKþ 14:5 a3
PK(Polynomial Fit), JlactateðaPKÞ ¼ ð94:0 aPK 0:984Þ=ð1 þ 0:00179 aPKþ 0:846 a2
PKÞ (Ratio-nal Function), JformateðaPKÞ ¼ 3:58 0:535a PK a1:67
PK (Hoerl model), JformateðaPKÞ ¼ ð1:39 aPK 0:00750Þ=ð1 0:455
aPKþ 0:187 a2
PKÞ (Rational function), JformateðaPKÞ ¼
0:453 þ 2:93 aPK 0:538 a2
PK(Quadratic fit), JacetateðaPKÞ ¼ 0:0329þ 1:178 aPYKþ 0:276 a2
PK 0:165 a3
PK (Polyno-mial fit), JacetateðaPKÞ ¼ 0:0137 þ 1:636 aPK 0:284 a2
PK
(Quadratic fit), JacetateðaPKÞ ¼ 1:91 0:701a PYK a1:177
PK (Hoerl model), JacetateðaPKÞ ¼ ð0:0500 þ 1:034 aPKÞ=ð1 0:350 aPKþ 0:171 a2
PKÞ (Rational function), All las enzymes: JlðalasÞ ¼ 0:0123 ð93:9 alasÞ ð1 e7:1a 3:2
Þ 0:276, JglucoseðalasÞ ¼ 0:693 ð83:3 alasÞ ð1 e6a 2:1
Þ 33:2 (User defined),
JlactateðalasÞ ¼ 0:919 ð129 alasÞ ð1 e6a 2:1
Þ 75:2 (User defined), JacetateðalasÞ ¼ 0:1135 ð30:3 alasÞ ð1 e6a 3:3
lasÞþ 4:66 (User defined), JformateðalasÞ ¼ 0:173 ð23:7 alasÞ ð1 e5:6a 2:3
Þ þ 6:02 (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 CJ
x¼ (dJ(ax)⁄ J(ax))⁄ (d(ax)⁄ (ax) for the entire range
of ax, 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 DanDan-ish Center for Advance Food Studies (LMC)