Previous Article | Next Article 
Applied and Environmental Microbiology, September 2000, p. 3835-3841, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Metabolic Engineering of Lactobacillus
helveticus CNRZ32 for Production of Pure
L-(+)-Lactic Acid
Kari
Kylä-Nikkilä,1,2
Mervi
Hujanen,3
Matti
Leisola,3 and
Airi
Palva1,2,*
Agricultural Research Centre of Finland, Food
Research Institute, FIN-31600 Jokioinen,1
Department of Basic Veterinary Sciences, Section of
Microbiology, FIN-00014 University of Helsinki,2
and Laboratory of Bioprocess Engineering, Helsinki
University of Technology, FIN-02015 HUT,3
Finland
Received 17 March 2000/Accepted 6 June 2000
 |
ABSTRACT |
Expression of D-(
)-lactate dehydrogenase
(D-LDH) and L-(+)-LDH genes (ldhD
and ldhL, respectively) and production of
D-()- and L-(+)-lactic acid were studied in
Lactobacillus helveticus CNRZ32. In order to develop a host
for production of pure L-(+)-isomer of lactic acid, two
ldhD-negative L. helveticus CNRZ32 strains were
constructed using gene replacement. One of the strains was constructed
by deleting the promoter region of the ldhD gene, and the
other was constructed by replacing the structural gene of
ldhD with an additional copy of the structural gene
(ldhL) of L-LDH of the same species. The
resulting strains were designated GRL86 and GRL89, respectively. In
strain GRL89, the second copy of the ldhL structural gene
was expressed under the ldhD promoter. The two
D-LDH-negative strains produced only
L-(+)-lactic acid in an amount equal to the total lactate
produced by the wild type. The maximum L-LDH activity was
found to be 53 and 93% higher in GRL86 and GRL89, respectively, than
in the wild-type strain. Furthermore, process variables for
L-(+)-lactic acid production by GRL89 were optimized using
statistical experimental design and response surface methodology. The
temperature and pH optima were 41°C and pH 5.9. At low pH, when the
growth and lactic acid production are uncoupled, strain GRL89 produced
approximately 20% more lactic acid than GRL86.
| |
INTRODUCTION |
Lactobacillus helveticus
is a homofermentative, thermo- and acid-tolerant lactic acid bacterium
with the capacity to produce high levels of lactic acid
(23). It is widely used in the dairy industry and generally
recognized as safe. Further, its behavior in both batch (2,
8) and continuous (1, 19, 21) lactic acid
fermentations has been extensively studied. Lactic acid produced by
L. helveticus is a racemic mixture of L-(+)- and
D-()-isomers. L-(+)-Lactic acid is the
preferred isomer, since D-()-lactic acid is not
metabolized in humans, and for many applications, like manufacturing of
biodegradable plastics and pharmaceutical products, it is more
advantageous than a mixture of both isomers (22).
A few attempts have been made to improve and modify lactic acid
production by metabolic engineering in lactobacilli, producing both
L-(+)- and D-()-lactic acids. In L. helveticus and Lactobacillus plantarum, enhancement of
L-(+)-lactic acid production has been tried by the
inactivation of ldhD and by increasing the copy number of
ldhL, respectively (5, 11). In L. helveticus, inactivation of ldhD led to a twofold
increase in the amount of L-(+)-lactic acid, thus restoring
the amount of total lactic acid to the level in the wild-type strain.
In L. plantarum, overexpression of the ldhL gene
increased the amount of L-(+)-lactate dehydrogenase (L-LDH) but had hardly any effect on lactic acid
production. Furthermore, in L. plantarum, prevention of
D-()-LDH (D-LDH) activity by the inactivation
of ldhD or L-LDH activity by inactivation of
ldhL did not substantially affect the total amount of lactic
acid production (11, 12). Only in Lactococcus
lactis, in which the ldhL gene is located as part of
the las operon, did an increase in the copy number of the
whole operon result in a slight increase in lactic acid production
(10).
According to Savijoki and Palva (25), the L. helveticus ldhL gene is transcribed as a monocistronic unit, and
expression was found to be most abundant in the exponential growth
phase. The ldhD gene of the same species is expressed as a
monocistronic unit as well (18; T. Rantanen and A. Palva, unpublished data). Production levels of D-()- and
L-(+)-lactic acid seem to depend on changes in the
expression of the ldhD and ldhL genes only to a
limited extent. Furthermore, there are no data available revealing whether any coregulation occurs at the transcriptional level between the ldhL and ldhD genes.
In this work our aim was to improve the production of
L-(+)-lactic acid by L. helveticus. For this
purpose, two stable ldhD-negative L. helveticus
derivates were constructed by a gene replacement method, and lactic
acid synthesis in these strains was characterized at the enzyme and end
product levels. In the first construct, transcription of the
ldhD gene was prevented by an internal deletion of the
promoter region. For the second construct, the ldhD gene was
inactivated by replacing the ldhD structural gene with
ldhL, resulting in duplication of the gene dose of
ldhL. The goal of this strategy was to enhance and extend
the synthesis of L-(+)-lactic acid into the stationary
phase with the aid of the ldhD promoter. Finally, the
behavior of genetically engineered strains was studied in a bioreactor,
and the process conditions for lactic acid production by L. helveticus GRL89 were optimized using experimental design and
response surface modeling.
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, and growth conditions.
L.
helveticus was routinely cultivated in MRS medium (Difco) at 37 or
42°C without shaking. Erythromycin (4 µg ml
1) was
added to the medium when the pSA3 plasmid was used. In bioreactor fermentations of L. helveticus, a whey permeate medium
(lactose, 41 g liter1) supplemented with lactose (40 g liter1) and yeast extract (20 g liter1)
was used.
Escherichia coli strains TOP10F' (Invitrogen) DH5
and
DH5F' (16) were grown in Luria broth. Kanamycin (50 µg
ml1), chloramphenicol (100 µg ml1),
tetracycline (10 µg ml1), ampicillin (50 µg
ml1), and erythromycin (300 µg ml1) were
added to the growth medium when needed. IPTG
(isopropylthiogalactopyranoside) was added to the growth medium to a
final concentration of 1 mM when pZeRO-2 vectors with inserts were
screened in E. coli. For the pUC19 and pJDC9 vectors in
E. coli, IPTG and
5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside (X-Gal)
were at the final concentrations of 0.5 mM and 40 µg
ml1, respectively.
Basic DNA techniques and bacterial transformation methods.
Plasmid DNAs from E. coli clones were isolated by using the
Wizard Miniprep (Promega) or FlexiPrep (Pharmacia) kit. Total L. helveticus chromosomal DNA was isolated essentially as described earlier (28) but without guanidine hydrochloride treatment. E. coli and L. helveticus strains were
transformed by electroporation using a Bio-Rad Gene Pulser and the
methods described by Sambrook et al. (24) and Bhowmik and
Steele (4), respectively. All other basic DNA methods were
performed according to established procedures (24). The
oligonucleotides were synthesized with an Applied Biosystems DNA/RNA
synthesizer (model 392) and purified with NAP-10 columns (Pharmacia) or
by ethanol precipitation. DNA was amplified by PCR in reaction
conditions recommended by the manufacturer of Dynazyme DNA polymerase
F-501L (Finnzymes). The labeling of DNA probes was performed with a
digoxigenin (DIG)-DNA labeling kit or DIG-High Prime kit (Boehringer Mannheim).
Colony and Southern hybridizations were performed as follows. A
positive
E. coli clone (ERF573; Table
1) carrying the upstream
region of
ldhD in pZeRO-2 was screened by colony hybridization
(
14). Southern hybridizations with chromosomal DNA (2 µg)
were
performed after gel electrophoresis and transfer to a positively
charged nylon membrane (Boehringer Mannheim), followed by hybrid
detection with a DIG luminescence detection kit (Boehringer Mannheim).
DNA sequencing of the upstream region of the
L. helveticus
CNRZ32
ldhD gene was done by an A.L.F. DNA sequencer
(Pharmacia), and
the sequence analysis was done with the PC/GENE
software (IntelliGenetics).
The BLAST network service was used to
search for homologous protein
sequences.
For RNA isolations and primer extension work, total RNA was isolated
from
L. helveticus cells essentially as described by
Vesanto
et al. (
26) or by using the RNeasy Mini and Midi kits
(Qiagen). The primer extension analysis with a 5'-end-labeled
fluorescein oligonucleotide (O1; Table
2)
was performed with
the A.L.F. DNA sequencer (Pharmacia) according to
Vesanto et al.
(
27).
Construction of an integration vector for L. helveticus
ldhD strain.
An ldhD promoter deletion strain
of L. helveticus was constructed using a gene replacement
method (3). The strategy was to construct an integration
vector containing the ldhD gene and its upstream region with
a 0.6-kb deletion covering the ldhD promoter region.
Briefly, a 0.9-kb fragment from the ldhD upstream region was
amplified using primers O2 and O3 (Table 2), and a 0.8-kb downstream
fragment from the ldhD structural gene was amplified with
primer pair O4 and O5 (Table 2). The 0.9- and 0.8-kb fragments were
ligated at their BamHI sites and amplified by PCR, followed by EcoRI digestion and ligation with pSA3. The resulting
integration vector was designated pKTH2154 after cloning into E. coli.
Construction of an integration vector for L. helveticus
ldhD::ldhL strain.
For the expression
of ldhL under the ldhD promoter, the
SphI-XbaI insert of the integration vector was
formed from four separate DNA fragments (Fig.
1), which were the two flanking regions
of ldhD needed for homologous recombination, the L. helveticus CNRZ32 ldhL fragment, and a transcription
terminator of the Lactobacillus brevis slpA gene
(28). The joint between the ldhD promoter and ldhL fragment was constructed at the transcription start
sites of these two genes. In detail, the steps in constructing the
ldhD::ldhL strain were as follows. The
transcription termination region of slpA (0.1 kb) was
amplified by PCR using primers O6 and O7 (Table 2). The amplified
terminator fragment was ligated with a PCR fragment (0.9 kb) generated
with primers O4 and O8 (Table 2) from the ldhD structural
gene lacking the first 79 nucleotides of the structural gene after
ClaI (authentic site) digestion. The resulting fragment was
cloned into pSA3 as a BamHI-XbaI fragment, resulting in plasmid pKTH2155.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 1.
Replacement of the ldhD structural gene
with ldhL. The overlapping oligonucleotides used in
constructing the mRNA joint between the ldhD promoter region
and the ldhL structural gene are shown.
PldhD and tslpA refer to
the ldhD promoter region and slpA transcription
terminator, respectively.
|
|
To create an exact joint at the mRNA start sites of the
ldhD
and
ldhL fragments, the recombinant PCR technique
(
17) was
applied. The data derived from the primer extension
of
ldhD mRNA
and previously published
L. helveticus
ldhL (
25) were used to
construct primers for the joint
region. The DNA fragments to be
annealed were synthesized by PCR with a
Perfect Match PCR-Enhancer
(Stratagene) and subsequently gel purified.
The
ldhD fragment
(1.5 kb) was synthesized using primer pair
O9 and O10 (Table
2)
and the
ldhL fragment (1.0 kb) was
synthesized using oligonucleotides
O11 and O12 (Table
2). The
complementary bases between the two
oligonucleotides used for the
construction of the exact mRNA joint
are presented in Fig.
1. In the
second step of the recombinant
PCR, the two DNA fragments were mixed in
an equimolar ratio, denatured
at 95°C for 30 s, and allowed to
anneal for 1 min (first three
cycles at 54°C and the rest at 65°C)
followed by filling in of
the 3' ends by DNA polymerase (72°C for 3 min). This second step
was repeated nine more times. PCR amplification
of the double-stranded
recombinant molecules was done immediately after
the second step
using primer pair O2 and O12 (Table
2) with 35 cycles.
The amplified
PCR fragment was then digested with
SphI
(authentic site) and
BamHI enzymes. The resulting 1.9-kb
insert was ligated with
SphI-
and
BamHI-treated
pKTH2155, and the integration vector formed
was designated pKTH2157.
The mRNA joint region was DNA sequenced
in order to confirm its
correctness.
Bioreactor cultivations.
Stock cultures of bacteria were
stored in milk-MRS medium with 10% glycerol at 80°C. Precultures
for bioreactor cultivations were grown in 10% skim milk medium at
42°C for 24 h. Four preinoculation steps were required before
bioreactor cultivations. Bioreactor cultivations were performed in a
2-liter bioreactor (Biostat B2; Braun) using 1.5 liters of modified
whey medium and 3% skim milk inoculation at 42°C. During the course
of pH-controlled batch fermentations, the temperature was kept at
42°C and an agitation speed of 200 rpm was maintained. The pH was
kept at 5.9 with automatic addition of 7 M NH4OH.
Separating the supernatant from cells by centrifugation and storing
them at 70°C prepared samples for lactic acid assays. The cell
pellet was washed once with water, frozen in liquid nitrogen, and
stored at 70°C for later use for enzymatic and protein assays. The
cells for RNA isolations were harvested separately, frozen in liquid
nitrogen, and stored at 70°C.
Optimization of pH and temperature was carried out in Biostat Q (B. Braun, Melsungen, Germany) multiple fermentor unit bioreactors
with a
600-ml working volume. Agitation speed was 900 rpm. By
using automatic
addition of 6 M NaOH, the pH was maintained at
desired values. Cultures
were not
aerated.
Statistical experimental design.
A central composite
circumscribed 22 experimental design with two variables,
temperature and pH, four star points, and four replicates at the center
point, resulting in a total of 12 experiments, was used. The real and
coded values of variables are shown in Table
3. Modde for Windows version 4.0 (Umetri
AB, Stockholm, Sweden) software was used for statistical experimental
design, analysis of the results, and drawing of the contour plots.
Lactic acid (Y1) and biomass (Y2) production
could be expressed as a polynomial model of the form:
|
(1)
|
where dependent variable
Y is the lactic acid
concentration or biomass of
L. helveticus GRL89,
a0 to
a5 are regression
coefficients
that are to be calculated with linear regression, and pH
and
T (temperature) are independent variables. The model was
fitted
using multiple linear regression. The calculated model was
validated
by MODDE using so-called
R2 and
Q2 values. The coefficient of determination,
R2, describes the goodness of the fit of the
model. Goodness of
prediction,
Q2, is a result
of cross-validation, where part of the data is left
out of the model
and then predicted by the model.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Real and coded values of independent variables in
optimization of pH and temperature for lactic acid production by
L. helveticus GRL89
|
|
Enzyme, protein, and lactic acid assays.
L. helveticus
cell-free extracts from fermentations were prepared by sonicating
samples in potassium phosphate buffer (10 mM, pH 6.5) followed by
removal of cell debris by centrifugation and use of the supernatants in
LDH assays. The LDH enzyme assays were performed spectrophotometrically
(340 nm) at 30°C using lactate as the substrate. The
L-LDH assays were performed in HEPES buffer (50 mM, pH 8.0)
containing 10 mM NAD+ and 150 mM L-(+)-lactic
acid, and the D-LDH assays were performed in Tris-HCl
buffer (50 mM, pH 9.0) containing 10 mM NAD+ and 100 mM
D-()-lactic acid. Protein content of the same cell-free samples was determined by the method of Bradford (6) with
bovine serum albumin as the standard. For screening of
D-()-lactic acid-negative phenotype, the assay of von
Krusch and Lompe (29) was used. All results are based on
average values measured from two parallel samples.
Total lactic acid and lactose were determined by high-performance
liquid chromatography (HPLC) using a 35-cm HPX-87H
+
cation-exchange column (Bio-Rad Laboratories) at a column temperature
of 40°C with UV and RI detectors. The eluent used was 0.5 mM
H
2SO
4 at a flow rate of 0.6 ml
min
1. The injection volume of the sample was 20 µl.
Furthermore,
L-(+)-
and
D-(
)-lactic acid
concentrations of samples were analyzed
enzymatically using Boehringer
Mannheim's kit (catalog no. 1 112
821). Biomass was determined as dry
weight after centrifuging
10 ml of fermentation medium, washing once
with distilled water,
and drying at 115°C
overnight.
Analysis of the level of ldhD and ldhL
transcripts.
Isolation of total RNA was performed as described
above from the samples withdrawn from bioreactor cultivation of
L. helveticus wild-type strains. The isolated RNA was DNase
treated before dot blotting. A constant amount of total RNA from
different growth phases was used. The DIG-labeled probes chosen were
targeted to hybridize with either the ldhL or
ldhD structural gene of L. helveticus. Hybridizations were done as described by Sambrook et al.
(24) and Hames and Higgins (15). After DIG
detections, intensities of the positive hybridization signals were
quantified from scanned films with Fluor-S equipment and Multianalyst
software (Bio-Rad). The ldh mRNA dot blot analyses were
performed in triplicate.
| |
RESULTS AND DISCUSSION |
Cloning of the upstream region of L. helveticus CNRZ32
ldhD.
For construction of a promoter deletion vector for
ldhD, its upstream region was cloned. The ldhD
upstream region was localized in a HindIII fragment of
L. helveticus CNRZ32 chromosomal DNA by Southern analysis.
The 0.3-kb probe used in the Southern hybridization was designed
according to the L. helveticus CNRZ32 ldhD
nucleotide sequence published previously (GenBank accession number
UO7604). A HindIII- digested DNA fragment pool with
a positive hybridization signal (2.6 kb) was ligated with the vector
and used to transform E. coli cells, followed by screening
of the positive clones by colony hybridization. One positive E. coli clone was chosen for further examination by DNA sequencing.
The sequence analysis confirmed that the plasmid contained the upstream
region of the L. helveticus CNRZ32 ldhD. The
E. coli clone and the positive plasmid were designated ERF573 and pKTH2153, respectively.
Sequence analysis of the 2.6-kb insert in pKTH2153 revealed two open
reading frames (ORF1 and ORF2) in the orientation opposite
that of the
ldhD gene (data not shown). The putative amino acid
sequence
of ORF1 had almost 70% identity with the
Streptococcus pneumoniae exodeoxyribonuclease (P21998), whereas ORF2, located
immediately upstream of the
ldhD promoter region, had
approximately
30% identity with a hypothetical
Synechocystis sp. protein
(BAA18702).
Construction of L. helveticus CNRZ32
ldhD strain.
An ldhD promoter deletion
strain of L. helveticus was constructed using a gene
replacement method (3). After the integration vector
pKTH2154 was constructed (see Materials and Methods) in E. coli, it was transferred to L. helveticus, and
integration by the first homologous recombination step was obtained by
a temperature shift from 37 to 45°C under erythromycin selection. The
second homologous recombination was achieved by growing the cells for 100 generations at 37°C without selection, resulting in 5%
erythromycin-sensitive clones. One clone, designated GRL86, of 15 erythromycin-sensitive clones tested showed a D-()-lactic
acid-negative phenotype.
Determination of the transcription start site of L. helveticus CNRZ32 ldhD.
In order to express the
ldhL structural gene under the ldhD promoter,
applying the exact joint of the ldhL and ldhD
transcription start sites, the transcription initiation site of the
ldhD gene was determined by primer extension. Transcription
of ldhD was found to start from base 34 (a G) upstream of
the first nucleotide of the start codon in the CNRZ32 ldhD
sequence (data not shown).
Construction of L. helveticus CNRZ32
ldhD::ldhL strain.
Construction
of an L. helveticus CNRZ32
ldhD::ldhL strain was achieved using
the gene replacement strategy described above. The integration
vector, designated pKTH2157, was constructed from four separate
fragments (Fig. 1).
The integration of pKTH2157 in the
L. helveticus CNRZ32
chromosome and gene replacement events were achieved as described
above
after confirmation that the first homologous recombination
had taken
place only in the region of the
ldhD gene. After growing
cells for approximately 100 generations at 37°C without selection,
about 5% of clones were erythromycin sensitive. One clone (designated
GRL89) of 25 erythromycin-sensitive clones tested was unable to
produce
D-(
)-lactic
acid.
Southern hybridization.
To confirm that the desired gene
replacements had taken place, Southern hybridizations were performed
with chromosomal DNAs isolated from L. helveticus strains
CNRZ32, GRL86, and GRL89 using both ldhD and ldhL
probes. The same 0.8- and 1.0-kb fragments used for construction of the
GRL86 and GRL89 strains, respectively, were utilized. The Southern blot
data shown in Fig. 2 confirmed that the
desired modifications had taken place. In GRL86 there is a 0.6-kb
deletion of the ldhD gene (Fig. 2, lanes 1 and 2), and in
GRL89 the 1-kb insertion can be demonstrated (Fig. 2, lanes 3 and 4).
Furthermore, in GRL89 (Fig. 2, lanes 5 and 6), the extra copy of the
ldhL gene is present in the chromosome, and no changes had
occurred in the authentic ldhL locus.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 2.
Analysis of gene replacements in the ldhD
locus of L. helveticus GRL86 and GRL89 chromosomal DNAs by
Southern hybridization. Chromosomal DNAs were from the wild-type
L. helveticus CNRZ32 (lane 1) and mutant GRL86 (lane 2)
digested with SphI and XbaI and from CNRZ32 (lane
3) and GRL89 (lane 4) digested with DraI. DNAs in lanes 1 to
4 were hybridized with an ldhD gene probe. In lanes 5 and 6, the ldhL copies in L. helveticus CNRZ32 and
GRL89, respectively, are shown after digestion of the DNAs with
DraI and XbaI and hybridization in the presence
of an ldhL gene probe.
|
|
Lactic acid production.
To assay the expression kinetics of
the ldhL and ldhD genes and production of
L-(+)- and D-()-lactic acid as a function of growth, mRNA analysis, LDH assays, and lactic acid assays were conducted with samples withdrawn from L. helveticus
fermentation. The ratio of L-(+)- and
D-()-lactic acids produced by the wild-type strain was
4:3 at the end of growth (Fig. 3).
Production of L-(+)-lactic acid was mainly at the
exponential phase of growth, whereas that of D-()-lactic
acid started later and reached its maximum at the stationary phase
(Fig. 3). This is in accordance with earlier observations of
homofermentative lactobacilli, where D-()-lactic acid
production was associated with the stationary phase and low pH of the
medium and L-(+)-lactic acid production was associated with
earlier growth phases (13). Since the enzyme assays for L-LDH and D-LDH were performed under different
conditions, the specific activities obtained could not be
quantitatively compared. However, the profiles of L-LDH and
D-LDH activities are in agreement with those of lactic acid
production. There seemed to be, however, a difference in the stability
of D-LDH and L-LDH activities, with a sharp
decrease in the D-LDH activity at the stationary phase (Fig. 3). The relative amounts of ldhL mRNA decreased prior
to ldhD transcripts. The decreases in the relative amounts
of both mRNAs preceded the rises in amounts of their respective acids (Fig. 3). The abrupt cessation of L-(+)-lactic acid
production when about 85% of the maximum level of lactic acid
production is reached cannot be explained by the lack of
L-LDH at that time point. This would rather suggest that
the intracellular conditions have changed in such a way that either the
affinity of L-LDH for pyruvate is diminished or the
catalytic activity of L-LDH is inhibited, resulting in a
flow of pyruvate through D-LDH.
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 3.
Expression of the ldh genes and production of
lactic acid in L. helveticus CNRZ32 as a function of growth.
Symbols:  , cell density;  , L-LDH activity;  ,
D-LDH activity;  , L-(+)-lactic acid;  ,
D-()-lactic acid;  , relative ldhL mRNA
intensities with an ldhL gene probe;  , relative
ldhD mRNA intensities with an ldhD gene probe.
|
|
To study the effects of the gene replacements on growth and lactic acid
production in the recombinant
L. helveticus strains
GRL86
and GRL89, they were cultivated in a pH-controlled bioreactor
similarly
to the wild-type strain. Minor, insignificant differences
between the
growth profiles of the recombinant strains and that
of the wild type
were found (Fig.
4A). Also, the total
amount
of lactic acid produced, as assayed by HPLC, was very similar
in
all cases (Fig.
4A). Furthermore, the enzymatic lactic acid
assay
confirmed that no
D-(
)- lactic acid was produced by
either
of the mutant strains. This in agreement with earlier results
obtained with the
ldhD insertion inactivation strain by
Bhowmik
and Steele (
5) and further confirms the lack of a
lactic acid
isomerase in
L. helveticus. Similar results have
been obtained
by inactivation of either the
ldhL or
ldhD gene in
L. plantarum (
11,
12).
View larger version (14K):
[in this window]
[in a new window]
|
FIG. 4.
Growth, L-LDH activity, and lactic acid
production in L. helveticus strains. Circles, CNRZ32;
triangles, GRL86; squares, GRL89. (A) Open symbols refer to growth, and
solid symbols refer to total lactic acid. (B) L-LDH
activity.
|
|
The promoter deletion strain GRL86 grew consistently slightly faster
than the wild-type strain, with an accompanying increase
in the
production rate of
L-(+)-lactic acid. This may be due to
a
lower energy burden of the cell as the result of the lack of
ldhD transcription and/or translation. Alternatively, this
faster
growth may be due to inactivation of the unknown
orf2
gene. The
production kinetics of
L-(+)-lactic acid in
mutant strains GRL86
and GRL89 did not differ significantly from each
other or from
that of total lactic acid in the wild-type CNRZ32 in
these cultivation
conditions. However, the production phase of
L-(+)-lactic acid
in the mutant strains was prolonged
compared to
L-(+)-lactic acid
production by the wild type
(Fig.
3 and
4). Thus, there was no
decrease in the rate of
L-(+)-lactate synthesis at a lactic acid
concentration at
which
L-lactate excretion ceased completely in
the
wild-type strain. This suggests that the rate of
L-LDH
catalysis
is not likely to depend on the external lactic acid
concentration.
Instead, the change of flow from pyruvate to
D-lactate may be
due to changes in substrate binding of
these two
enzymes.
Assays of LDH activity showed that the maximum levels of
L-LDH activity in GRL86 and GRL89 were 53 and 93%,
respectively,
higher than that in CNRZ32 (Fig.
4B). This is in contrast
to what
was observed earlier with
L-LDHs from
L. plantarum. In this host,
inactivation of neither the
ldhL nor the
ldhD gene seemed to affect
the
remaining LDH activity markedly (
11,
12). In all three
strains, maximum
L-LDH activity was reached at the end of
the
exponential growth phase. Furthermore, as expected, with mutant
strains GRL86 and GRL89 no
D-LDH activity could be
shown.
Optimization by response surface methodology.
In order to
determine the effect of pH and temperature on lactic acid production, a
response surface methodology was applied for the GRL89 strain. The
results of the experimental design are summarized in Table
4. These results were fitted to equation 1 using multilinear regression. Resulting polynomes are plotted as the
response surface in Fig. 5.
View this table:
[in this window]
[in a new window]
|
TABLE 4.
Lactic acid production by L. helveticus GRL89
at 25 h of fermentation using pH and temperature as
independent variables
|
|
View larger version (53K):
[in this window]
[in a new window]
|
FIG. 5.
Effect of pH and temperature on lactic acid production
(A) and biomass formation (B) in L. helveticus GRL89, shown
as response surfaces.
|
|
A response surface for lactic acid production as a function of pH and
temperature by
L. helveticus GRL89 is presented in Fig.
5A.
As shown, the optimal conditions for lactic acid production
are pH 5.6 to 6.0, and the optimal temperature is 40 to 42°C.
The statistical
values for the model were
R2 = 0.875 and
Q2 = 0.504. Significant terms in the model
are the second-order
term for pH, with a probability (
P)
value of 0.005, and the second-order
term for temperature (
P = 0.0024).
Figure
5B shows the response surface for the model predicting
L. helveticus GRL89 biomass formation as function of temperature
and
pH. In this model also, both temperature and pH were very
significant
(
P = 0.00035 and 0.00018, respectively). The optimum
conditions for biomass were at the center of the experimental
domain,
41°C and pH 5.8. These values are very close to the conditions
used
in the primary batch fermentations and are also in good agreement
with
earlier studies (
20) where unmodified
L. helveticus strains
have been used. The steep curvature of the
response surface is
due to high values of the coefficients of
second-order terms.
Consequently, the optimum area for the growth of
L. helveticus GRL89 is quite small. The statistical values
for this model were
R2 = 0.9435 and
Q2 = 0.6197.
Lactic acid production at low pH.
Coupling of growth and acid
production in L. helveticus has been shown to be controlled
mainly by cultivation pH (2). To test whether there is any
difference in the behavior of the ldhD and ldhL
promoters under such conditions, strains GRL86 and GRL89 were
cultivated at 44°C and pH 5.4. Our preliminary experiments indicated
(results not shown) that in these conditions, biomass growth and lactic
acid production are uncoupled.
Lactic acid production started in strain GRL86 slightly earlier than in
strain GRL89 (Fig.
6A). This was due to
faster growth
of the deletion strain, as shown by the biomass data from
these
fermentations (Fig.
6B). The maximal volumetric productivities
of
the strains were 3.34 and 3.21 g liter
1
h
1, respectively. However, a distinctive increase in
lactic acid
production with GRL89 was obtained at the late stationary
phase.
The final lactic acid yield with strain GRL89 was 91.6%,
whereas
that of strain GRL86 was only 76.2% (Fig.
6A).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 6.
Batch fermentations of genetically engineered L. helveticus strains GRL86 (open symbols) and GRL89 (solid symbols):
concentrations of lactose (circles) and lactic acid (squares) (A) and
change in dry weight (B) as a function of time in two parallel
cultivations at pH 5.4 and 44°C.
|
|
| |
ACKNOWLEDGMENTS |
This work was supported by the National Technology Agency, Finland.
We are grateful to Ilkka Palva for valuable discussion and critical
reading of the manuscript. We also thank Anneli Virta for the
sequencing work and Jaana Jalava for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Veterinary Medicine, Department of Basic Veterinary Sciences, Section
of Microbiology, P.O. Box 57, FIN-00014 University of Helsinki,
Finland. Phone: 358 9 191 49531. Fax: 358 9 191 49799. E-mail:
Airi.Palva{at}helsinki.fi.
| |
REFERENCES |
| 1.
|
Amrane, A., and Y. Prigent.
1996.
A novel concept of bioreactor: specialized function two-stage continuous reactor, and its application to lactose conversion into lactic acid.
J. Biotechnol.
45:195-203.
|
| 2.
|
Amrane, A., and Y. Prigent.
1999.
Differentiation of pH and free lactic acid effects on the various growth and production phases on Lactobacillus helveticus.
J. Chem. Technol. Biotechnol.
74:33-40[CrossRef].
|
| 3.
|
Bhowmik, T.,
L. Fernández, and J. L. Steele.
1993.
Gene replacement in Lactobacillus helveticus.
J. Bacteriol.
175:6341-6344[Abstract/Free Full Text].
|
| 4.
|
Bhowmik, T., and J. L. Steele.
1993.
Development of an electroporation procedure for gene disruption in Lactobacillus helveticus CNRZ 32.
J. Gen. Microbiol.
139:1433-1439.
|
| 5.
|
Bhowmik, T., and J. L. Steele.
1994.
Cloning, characterization and insertional inactivation of the Lactobacillus helveticus D-()-lactate dehydrogenase.
Appl. Microbiol. Biotechnol.
41:432-439[Medline].
|
| 6.
|
Bradford, M. M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[CrossRef][Medline].
|
| 7.
|
Chen, J. D., and D. A. Morrison.
1988.
Construction and properties of a new insertion vector, pJDC9, that is protected by transcriptional terminators and useful for cloning of DNA from Streptococcus pneumoniae.
Gene
64:155-164[CrossRef][Medline].
|
| 8.
|
Chiarini, L.,
L. Mara, and S. Tabacchioni.
1992.
Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus.
Appl. Microbiol. Biotechnol.
36:461-464.
|
| 9.
|
Dao, M. L., and J. J. Ferretti.
1985.
Streptococcus-Escherichia coli shuttle vector pSA3 and its use in the cloning of streptococcal genes.
Appl. Environ. Microbiol.
49:115-119[Abstract/Free Full Text].
|
| 10.
|
Davidson, B. E.,
R. M. Llanos,
M. R. Cancilla,
N. C. Redman, and A. J. Hillier.
1995.
Current research on the genetics of lactic acid production in lactic acid bacteria.
Int. Dairy J.
5:763-784[CrossRef].
|
| 11.
|
Ferain, T.,
D. Garmyn,
N. Bernard,
P. Hols, and J. Delcour.
1994.
Lactobacillus plantarum ldhL gene: overexpression and deletion.
J. Bacteriol.
176:596-601[Abstract/Free Full Text].
|
| 12.
|
Ferain, T.,
J. N. Hobbs, Jr.,
J. Richardson,
N. Bernard,
D. Garmyn,
P. Hols,
N. E. Allen, and J. Delcour.
1996.
Knockout of the two ldh genes has a major impact on peptidoglycan precursor synthesis in Lactobacillus plantarum.
J. Bacteriol.
178:5431-5437[Abstract/Free Full Text].
|
| 13.
|
Garvie, E. I.
1980.
Bacterial lactate dehydrogenases.
Microbiol. Rev.
44:106-139[Free Full Text].
|
| 14.
|
Grünstein, M., and D. S. Hogness.
1975.
Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene.
Proc. Natl. Acad. Sci. USA
72:3961-3965[Abstract/Free Full Text].
|
| 15.
|
Hames, B., and S. Higgins.
1985.
Nucleic acid hybridization: a practical approach.
IRL Press, Oxford, England.
|
| 16.
|
Hanahan, D.
1983.
Studies on transformation of Escherichia coli with plasmids.
J. Mol. Biol.
166:557-580[Medline].
|
| 17.
|
Higuchi, R.
1990.
Recombinant PCR, p. 177-183.
In
M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White (ed.), PCR protocols. Academic Press, San Diego, Calif.
|
| 18.
|
Kochhar, S.,
H. Hottinger,
N. Chuard,
P. G. Taylor,
T. Atkinson,
M. D. Scawen, and D. J. Nicholls.
1992.
Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.
Eur. J. Biochem.
208:799-805[Medline].
|
| 19.
|
Kulozik, U.
1998.
Physiological aspects of continuous lactic acid fermentations at high dilution rates.
Appl. Microbiol. Biotechnol.
49:506-510[CrossRef].
|
| 20.
|
Kulozik, U., and J. Wilde.
1999.
Rapid lactic acid production at high cell concentrations in whey ultrafiltrate by Lactobacillus helveticus.
Enzyme Microbiol. Technol.
24:297-302[CrossRef].
|
| 21.
|
Øyaas, J.,
I. Storrø, and D. W. Levin.
1996.
Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate.
Appl. Microbiol. Biotechnol.
46:240-249[CrossRef].
|
| 22.
|
Potera, C.
1992.
Natural lactic acid produced from whey by Ecochem.
Genet. Eng. News
12:1-22.
|
| 23.
|
Roy, D.,
J. Goulet, and A. Le Duy.
1986.
Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production.
Appl. Microbiol. Biotechnol.
24:206-213.
|
| 24.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 25.
|
Savijoki, K., and A. Palva.
1997.
Molecular genetic characterization of the L-lactate dehydrogenase gene (ldhL) of Lactobacillus helveticus and biochemical characterization of the enzyme.
Appl. Environ. Microbiol.
63:2850-2856[Abstract].
|
| 26.
|
Vesanto, E.,
P. Varmanen,
J. L. Steele, and A. Palva.
1994.
Characterization and expression of the Lactobacillus helveticus pepC gene encoding a general aminopeptidase.
Eur. J. Biochem.
24:991-997.
|
| 27.
|
Vesanto, E.,
S. Savijoki,
T. Rantanen,
J. L. Steele, and A. Palva.
1995.
An X-prolyl dipeptidyl aminopeptidase (pepX) gene from Lactobacillus helveticus.
Microbiology
141:3067-3075[Abstract/Free Full Text].
|
| 28.
|
Vidgren, G.,
I. Palva,
R. Pakkanen,
K. Lounatmaa, and A. Palva.
1992.
S-layer protein gene of Lactobacillus brevis: cloning by polymerase chain reaction and determination of the nucleotide sequence.
J. Bacteriol.
174:7419-7427[Abstract/Free Full Text].
|
| 29.
|
von Krusch, U., and A. Lompe.
1982.
Schnelltest zum qualitativen Nachweis von L-(+)- und D-()-Milchsäure für die Bestimmung von Milchsäurebakterien.
Milchwissenschaft
37:65-68.
|
| 30.
|
Yanisch-Perron, C.,
J. Vieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene
33:103-119[CrossRef][Medline].
|
Applied and Environmental Microbiology, September 2000, p. 3835-3841, Vol. 66, No. 9
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Helanto, M., Kiviharju, K., Leisola, M., Nyyssola, A.
(2007). Metabolic Engineering of Lactobacillus plantarum for Production of L-Ribulose. Appl. Environ. Microbiol.
73: 7083-7091
[Abstract]
[Full Text]
-
Saitoh, S., Ishida, N., Onishi, T., Tokuhiro, K., Nagamori, E., Kitamoto, K., Takahashi, H.
(2005). Genetically Engineered Wine Yeast Produces a High Concentration of L-Lactic Acid of Extremely High Optical Purity. Appl. Environ. Microbiol.
71: 2789-2792
[Abstract]
[Full Text]
-
Lindholm, A., Smeds, A., Palva, A.
(2004). Receptor Binding Domain of Escherichia coli F18 Fimbrial Adhesin FedF Can Be both Efficiently Secreted and Surface Displayed in a Functional Form in Lactococcus lactis. Appl. Environ. Microbiol.
70: 2061-2071
[Abstract]
[Full Text]
-
Juarez Tomas, M. S., Ocana, V. S., Wiese, B., Nader-Macias, M. E.
(2003). Growth and lactic acid production by vaginal Lactobacillus acidophilus CRL 1259, and inhibition of uropathogenic Escherichia coli. J Med Microbiol
52: 1117-1124
[Abstract]
[Full Text]
-
Avall-Jaaskelainen, S., Lindholm, A., Palva, A.
(2003). Surface Display of the Receptor-Binding Region of the Lactobacillusbrevis S-Layer Protein in Lactococcuslactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells. Appl. Environ. Microbiol.
69: 2230-2236
[Abstract]
[Full Text]
-
Zhou, S., Shanmugam, K. T., Ingram, L. O.
(2003). Functional Replacement of the Escherichia coliD-(-)-Lactate Dehydrogenase Gene (ldhA) with the L-(+)-Lactate Dehydrogenase Gene (ldhL) from Pediococcus acidilactici. Appl. Environ. Microbiol.
69: 2237-2244
[Abstract]
[Full Text]
-
Smeds, A., Pertovaara, M., Timonen, T., Pohjanvirta, T., Pelkonen, S., Palva, A.
(2003). Mapping the Binding Domain of the F18 Fimbrial Adhesin. Infect. Immun.
71: 2163-2172
[Abstract]
[Full Text]
-
Zhou, S., Causey, T. B., Hasona, A., Shanmugam, K. T., Ingram, L. O.
(2003). Production of Optically Pure D-Lactic Acid in Mineral Salts Medium by Metabolically Engineered Escherichia coli W3110. Appl. Environ. Microbiol.
69: 399-407
[Abstract]
[Full Text]
-
Jakava-Viljanen, M., Avall-Jaaskelainen, S., Messner, P., Sleytr, U. B., Palva, A.
(2002). Isolation of Three New Surface Layer Protein Genes (slp) from Lactobacillus brevis ATCC 14869 and Characterization of the Change in Their Expression under Aerated and Anaerobic Conditions. J. Bacteriol.
184: 6786-6795
[Abstract]
[Full Text]
-
Avall-Jaaskelainen, S., Kyla-Nikkila, K., Kahala, M., Miikkulainen-Lahti, T., Palva, A.
(2002). Surface Display of Foreign Epitopes on the Lactobacillus brevis S-Layer. Appl. Environ. Microbiol.
68: 5943-5951
[Abstract]
[Full Text]
-
Underwood, S. A., Zhou, S., Causey, T. B., Yomano, L. P., Shanmugam, K. T., Ingram, L. O.
(2002). Genetic Changes To Optimize Carbon Partitioning between Ethanol and Biosynthesis in Ethanologenic Escherichia coli. Appl. Environ. Microbiol.
68: 6263-6272
[Abstract]
[Full Text]
Top
Abstract
Introduction
