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Previous Article | Next Article 
Applied and Environmental Microbiology, November 1998, p. 4328-4332, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Construction of a Proline-Producing Mutant of the
Extremely Thermophilic Eubacterium Thermus
thermophilus HB27
Takehide
Kosuge and
Takayuki
Hoshino*
Institute of Applied Biochemistry, University
of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Received 2 June 1998/Accepted 24 August 1998
 |
ABSTRACT |
Growth of Thermus thermophilus HB27 was inhibited by a
proline analog, 3,4-dehydroproline (DHP). This result suggested that the 
-glutamyl kinase (the product of the proB gene) was
inhibited by feedback inhibition in T. thermophilus.
DHP-resistant mutants were reported previously for Escherichia
coli (A. M. Dandekar and S. L. Uratsu, J. Bacteriol.
170:5943-5945, 1988) and Serratia marcescens (K. Omori, S. Suzuki, Y. Imai, and S. Komatsubara, J. Gen. Microbiol.
138:693-699, 1992), and their mutated sites in the proB
gene were identified. Comparison of the amino acid sequence of T. thermophilus -glutamyl kinase with those of E. coli and S. marcescens mutants revealed that the DHP
resistance mutations occurred in the amino acids conserved among the
three organisms. For eliminating the feedback inhibition, we first
constructed a DHP-resistant mutant, TH401, by site-directed mutagenesis
at the proB gene as reported for the proline-producing
mutant of S. marcescens. The mutant, TH401, excreted about
1 mg of L-proline per liter at 70°C after 12 h of
incubation. It was also suggested that T. thermophilus had
a proline degradation and transport pathway since it was able to grow
in minimal medium containing L-proline as sole nitrogen
source. In order to disrupt the proline degradation or transport genes,
TH401 was mutated by UV irradiation. Seven mutants unable to utilize
L-proline for their growth were isolated. One of the
mutants, TH4017, excreted about 2 mg of L-proline per liter
in minimal medium at 70°C after 12 h of incubation.
| |
INTRODUCTION |
Thermus thermophilus, a
gram-negative aerobic eubacterium, is one of the most widely studied
species of extremely thermophilic microorganisms. We have been working
on the molecular genetics and molecular reproduction of T. thermophilus HB27. We have already cloned and sequenced three
proline biosynthetic genes, proB, proA, and
proC, and reported that the proB and
proA genes exist in tandem (7, 9).
We have also constructed physical maps of the HB27 chromosome and of a
large plasmid, pTT27, and determined the locations of all proline
biosynthetic genes on the chromosomal DNA (20, 21). We have
already succeeded in overproducing carotenoids in T. thermophilus HB27 (6), but at present there is no
report about extracellular production of amino acids in extreme
thermophiles. We have elucidated the consensus sequences for strong
promoters of T. thermophilus (11) and developed a
thermostable antibiotic resistance gene (12). It is also
easy to disrupt or mutate genes on chromosomal DNA in T. thermophilus HB27 (8). Among the extreme thermophiles,
a host-vector system has been established only in T. thermophilus. Generally, the reaction rate of thermostable enzymes
which are produced from T. thermophilus is higher than those
of enzymes from mesophiles. In a fermentation process such as amino
acid production, T. thermophilus may contribute to the improvement of amino acid productivity since fermentation at a high
temperature eliminates the problems of contamination and cooling
procedures. So, we decided to attempt excretion of proline at a high
temperature with T. thermophilus mutants.
L-Proline is synthesized from glutamate by the sequential
reaction of -glutamyl kinase, -glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase in bacteria (1). Genes
proB and proA, which encode -glutamyl kinase
and -glutamyl phosphate reductase, respectively, were found to
comprise an operon in T. thermophilus (9),
Escherichia coli (5), and Serratia
marcescens (13). In E. coli and S. marcescens, -glutamyl kinase is subject to feedback control by
L-proline (3, 13), but -glutamyl phosphate
reductase and pyrroline-5-carboxylate reductase are not inhibited by
proline (3, 15). Meanwhile, E. coli and S. marcescens rapidly degrade proline by proline dehydrogenase (proline oxidase), encoded by the putA gene (3, 14,
22). So far, it has been reported that E. coli mutants
resistant to proline analogs, DL-3,4-dehydroproline and
L-azetidine-2-carboxylic acid, excreted
L-proline into the medium. But the amount of
L-proline excreted was too small for practical use because
of the existence of the proline degradation pathway (2). For
S. marcescens, Sugiura et al. (18, 19) have
constructed a proline-overproducing strain, SP126, as a double mutant
resistant to 3,4-dehydroproline and thiazolidine-4-carboxylate and
derived from a proline dehydrogenase-deficient mutant (18).
Strain SP126 produced about 20 mg of L-proline per ml in
the fermentation medium (18).
In T. thermophilus, the control system in proline
biosynthesis has not been elucidated. However, we thought that the
feedback control of proline biosynthesis in T. thermophilus
should be similar to that of E. coli and S. marcescens, since the amino acid sequences of proline biosynthetic
enzymes in T. thermophilus show a high similarity to
sequences of those of E. coli and S. marcescens (7, 9). E. coli and S. marcescens
mutants resistant to 3,4-dehydroproline have already been determined to
be proB mutants (4, 14). The comparison of the
amino acid sequences of -glutamyl kinases in E. coli,
S. marcescens, and T. thermophilus showed that
these mutations occurred in the positions conserved among the three microorganisms (Fig. 1). We thought that
it was possible to construct a 3,4-dehydroproline-resistant mutant of
T. thermophilus by introducing the same mutations into the
proB gene found in the mutants of E. coli and
S. marcescens. We determined the strategy for construction of a proline-producing strain of T. thermophilus by
following two steps: first, construction of a
3,4-dehydroproline-resistant mutant by introduction of mutations into
the proB gene, and second, isolation of a mutant which
cannot utilize proline for its growth by mutagenizing the
dehydroproline-resistant mutant.
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FIG. 1.
Comparison of the amino acid sequences of -glutamyl
kinases in E. coli, S. marcescens, and T. thermophilus. The amino acid substitutions found in E. coli (4) and S. marcescens (14)
are shown by arrows. Asterisks show the amino acid residues conserved
in the three microorganisms.
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|
| |
MATERIALS AND METHODS |
Chemicals.
Restriction endonucleases and DNA modification
enzymes were purchased from Toyobo (Tokyo, Japan) or Takara Shuzo
(Kyoto, Japan). L-Proline and
DL-3,4-dehydroproline were purchased from Sigma Chemical
Co. (St. Louis, Mo.). All the other reagents used were of the purest
grade available.
Bacterial strains and growth conditions.
T.
thermophilus HB27 (17) and its proline auxotrophic
mutant, TH104 (7), were used. TM medium (10) was
used for routine cultivation of T. thermophilus. Minimal
medium (MM) (10) was also used. When necessary,
3,4-dehydroproline and L-proline were added to MM at the
concentration of 1 mM. MM-proline plates which contained 10 mM
L-proline instead of
(NH4)2SO4 were used to isolate proline-producing mutants. The growth curve of T. thermophilus was measured as follows. T. thermophilus
HB27 was grown in 10 ml of TM medium at 70°C for 16 h. A 0.1-ml
aliquot of the culture was inoculated into 10 ml of a fresh medium. The
growth was monitored by measuring absorbance at 580 nm. E. coli JM109 (23) was also used in the cloning experiments.
Preparation of the fragment containing the mutated
proB genes.
Four primers, PROBDNF
(5'-GTCCTCCTCACCGCCGAGAACCTC-3'), PROBDNR
(5'-GAGGTTCTCGGCGGTGAGGAGGAC-3'), PROBAVF
(5'-AGGAGCCGCTACCTGAACGTCAAG-3'), and PROBAVR
(5'-CTTGACGTTCAGGTAGCGGCTCCT) (Fig.
2), were prepared to introduce the
mutations into the proB gene. Two primers, PROB_F (5'-GGGAATTCCCGAGGCCATGCCGGAGGC-3') and PROB_R
(5'-GAAGCTTTCATGCCTCCTCCTTCAAGGC-3') (Fig. 2),
were prepared to amplify the fragment containing the entire mutated
proB gene. The primers contained restriction endonuclease sites for EcoRI (italics) and HindIII
(underlined).
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FIG. 2.
Outline of the preparation of DNA fragments which
contain three types of mutated proB genes. The detailed
explanations are given in Materials and Methods. The numbers shown
below or above each primer correspond to the nucleotide numbers of the
proBA region.
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|
In the first PCR, the four DNA fragments which would be the DNA
template for construction of the mutated proB gene were
prepared (Fig. 2). For the preparation of fragments A, B, C, and D,
primers PROB_F and PROBDNR, PROB_F and PROBAVR, PROB_R and PROBDNF, and PROB_R and PROBAVF were used, respectively. The PCR mixture contained PCR buffer with 2 mM MgCl2 (Takara Shuzo), 0.2 mM
deoxynucleoside triphosphates, 0.1 µM (each) primer, 0.1 µg of
template (pUC-pro3,5+ [9]), and 2.5 U of
rTaq DNA polymerase (Takara Shuzo). A DNA thermal cycler
(Perkin-Elmer Cetus) was used with the following conditions: melting
temperature, 96°C (30 s); annealing temperature, 62°C (1 s); and
polymerization at 72°C (150 s). Twenty-five cycles were run with a
subsequent polymerization period of 10 min at 72°C. The amplified
fragments A (610 bp), B (637 bp), C (814 bp), and D (787 bp) were
recovered from 1.0% agarose gels and used for the second PCR.
In the second PCR, three types of DNA fragments containing the mutated
proB genes were prepared. For the amino acid change of
Asp-115 to Asn in the proB gene, fragments A and C were used for the DNA template (Fig. 2, fragment 1). For the amino acid change of
Ala-125 to Val (Fig. 2, fragment 2), fragments B and D were used. For
the two amino acid changes of Asp-115 and Ala-125 to Asn and Val,
respectively, fragments B and C were used (Fig. 2, fragment 3). The
second PCR mixture contained PCR buffer with 2 mM MgCl2
(Takara Shuzo), 0.2 mM deoxynucleoside triphosphates, 0.1 µM (each)
primer PROB_F and PROB_R, 10 ng of each amplified fragment, and 2.5 U
of rTaq DNA polymerase (Takara Shuzo). PCR was performed
under the following conditions: 96°C for 30 s, gradual decrease
of the temperature to 62°C for 45 s, and immediate increase of
the temperature from 62 to 72°C, and maintenance for 150 s. Twenty-five cycles were run with a subsequent polymerization period of
10 min at 72°C. The three types of amplified DNA fragments were
recovered from 1.0% agarose gels and filled and phosphorylated with T4
DNA polymerase and T4 polynucleotide kinase. Each fragment was ligated
to SmaI-digested and dephosphorylated pUC19, and the ligation mixtures were used for E. coli JM109
transformation. Three types of plasmids which contained D115N, V125A,
and D115N and V125A replacements were prepared, and their nucleotide
sequences were determined.
Construction of a 3,4-dehydroproline-resistant mutant.
T.
thermophilus HB27 was transformed with 3 µg each of three types
of plasmid (containing fragment 1, 2, or 3 [Fig. 2]) as described
previously (10). The transformed cells were washed with
0.85% NaCl (saline) and resuspended in saline. The diluted suspensions
were spread on MM plates containing DL-3,4-dehydroproline at the concentration of 1 mM. When the plasmid containing fragment 2 which included the amino acid change in the proB gene of
Ala-125 to Val was used for the transformation, many
3,4-dehydroproline-resistant mutants were obtained. Eight transformants
among them were randomly selected, and total DNAs were prepared by the
method of Saito and Miura (16). Total DNA from the eight
transformants was digested with SphI, and DNA fragments
including the proBA genes of 2.8 kb in size were recovered
by agarose gel electrophoresis. Each DNA fragment was ligated with
SphI-digested and dephosphorylated pUC19 and introduced into
E. coli JM109. The clones containing the entire mutant
proB genes were screened by colony hybridization using the
inserted fragment of pUC-pro3,5+ as a probe. The entire
proB genes of eight transformants were sequenced and checked
for the amino acid change of Ala-125 to Val. The eight transformants
showed the same growth curves in MM containing 1 mM 3,4-dehydroproline.
We named one of the eight transformants TH401 and used it for further experiments.
Construction of a proline-producing strain.
Strain TH401 was
grown in TM medium for 3 h at 70°C. The culture was diluted 1/10
with saline and irradiated for 3 min under a 15-W UV lamp. Five
milliliters of the culture was added to 5 ml of fresh TM medium and
incubated at 70°C for 60 min in the dark. This culture was diluted
and spread on TM plates. A total of 8,217 colonies on TM plates were
replica plated onto MM and MM-proline plates. Seven strains which were
able to grow on MM plates but not on MM-proline plates were isolated.
We named them TH4011, TH4012, TH4013, TH4014, TH4015, TH4016 and TH4017.
Bioassay of proline production.
An overnight TM culture of
each strain was diluted 1/10 with saline (0.85% NaCl), and 5 µl was
spotted onto the MM plates onto which TH104 had been spread. The plates
were incubated at 70°C for 2.5 days. After incubation, the growth of
TH104 around the spots was checked.
Analysis of amino acid production.
Each strain was incubated
in 10 ml of MM at 70°C for 12 h. All cells were removed by
centrifugation (18,000 × g, 10 min), and the amino acids in
the culture supernatant were analyzed by ion-exchange chromatography
with a Hitachi L-8500 amino acid analysis system (Hitachi, Tokyo, Japan).
Nucleotide sequence accession number.
The nucleotide
sequence of the proBA genes in T. thermophilus is
in the EMBL, GenBank, and DDBJ databases under accession no. D29973.
| |
RESULTS AND DISCUSSION |
Analysis of the regulation system of proline biosynthesis in
T. thermophilus.
The growth of T. thermophilus in
3,4-dehydroproline-containing MM was first assayed. As shown in Fig.
3A, the growth of T. thermophilus was clearly inhibited by dehydroproline at the
concentration of 1 mM. This result indicated that proline biosynthesis
was controlled by feedback inhibition and that -glutamyl kinase was
inhibited by proline in T. thermophilus.
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FIG. 3.
Growth curves of T. thermophilus HB27. HB27
was grown in MM with or without 3,4-dehydroproline (A) and in
(NH4)2SO4-minus MM (MM') with or
without L-proline (B). O.D.580, optical density at 580 nm.
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|
Analysis of the existence of a proline degradation system in
T. thermophilus.
The proline degradation system in T. thermophilus was examined since it disturbs overproduction of
proline. In E. coli, two proline degradative genes, the
putA and putP genes, have been reported
previously. The putA gene encodes proline dehydrogenase, which catalyzes the conversion of proline to pyrroline-5-carboxylate. The putP gene encodes proline permease, which is necessary
for uptake of extracellular proline into the cell. If T. thermophilus has PutA and PutP activity, it is able to grow in MM
which contains proline as a sole nitrogen source. We checked whether
T. thermophilus was able to utilize proline for its growth.
As shown in Fig. 3B, T. thermophilus was able to grow in MM
containing proline as sole nitrogen source. This result indicated that
T. thermophilus had a proline degradation system.
Site-directed mutagenesis of the chromosomal proB gene
in T. thermophilus.
The mutation sites of the
proline-overproducing mutants have been determined previously in
E. coli and S. marcescens (3, 4, 14).
These proline-overproducing mutants showed 3,4-dehydroproline resistance, and their -glutamyl kinases were not inhibited by proline. We compared the amino acid sequences of -glutamyl kinases among T. thermophilus and two proline-overproducing mutants.
As shown in Fig. 1, the mutations occurred at the sites which were conserved in the three -glutamyl kinases.
Three kinds of plasmids including the mutated proB gene were
prepared by recombinant PCR (Fig. 2). Fragment 1 had the amino acid
substitution of Asn for Asp115 in the proB gene, fragment 2 had the amino acid substitution of Val for Ala125, and fragment 3 had
both amino acid substitutions (Fig. 2). We transformed T. thermophilus HB27 by using these plasmids. Many
dehydroproline-resistant transformants were obtained when the plasmid
including fragment 2 was used for the donor DNA whereas no colony was
obtained when the plasmid including fragment 1 or fragment 3 was used.
No dehydroproline-resistant colonies appeared on the MM containing
dehydroproline when no DNA was added. This result showed that only the
A125V substitution led to dehydroproline resistance in HB27 and that
the D115N substitution might impair the heat stability of -glutamyl
kinase of T. thermophilus. The eight transformants were
randomly selected, and their growth was assayed in MM containing
3,4-dehydroproline or proline as sole nitrogen source. Every mutant
showed the same growth curve in MM containing 3,4-dehydroproline or
proline as sole nitrogen source. From this result, we confirmed that
the eight mutants had the same mutations only in the proB
gene. One of the eight 3,4-dehydroproline-resistant mutants was named
TH401. The growth of TH401 was not inhibited in MM containing 1 mM
3,4-dehydroproline. The growth curve of TH401 in MM containing 1 mM
3,4-dehydroproline was the same as that of wild type in MM (data not
shown). This fact suggested that TH401 was not subjected to feedback
inhibition in proline biosynthesis.
Construction of a proline-producing mutant.
Strain TH401 was
further mutated by UV irradiation. Seven strains, TH4011, TH4012,
TH4013, TH4014, TH4015, TH4016, and TH4017, which were unable to grow
on MM-proline plates containing proline as a sole nitrogen source were
isolated. The growth curves of these seven mutants in MM were the same
as those of TH401 and HB27 wild type. The proline production levels of
seven mutants and TH401 were tested by bioassay (see Materials and
Methods). The growth of the proline auxotrophic mutant was observed
only around the spot of TH4017. This result showed that TH4017 excreted proline into the medium. The detailed amino acid production levels of
T. thermophilus HB27, TH401, and TH4017 were measured. The amino acid production was measured twice, and a little difference was
observed in two experiments. As shown in Table
1, TH4017 produced about 2 mg of proline
per liter at 70°C after 12 h of incubation in MM but the wild
type did not. TH401 also produced about 1 mg of proline per liter. The
proline production levels of other mutants (TH4011, TH4012, TH4013,
TH4014, TH4015, and TH4016) were the same as that of TH401 (data not
shown). TH4017 showed the highest production of proline among the
mutants. This result indicated that TH4017 was the first T. thermophilus strain excreting proline into the medium.
As shown in Table 1, TH4017 excreted various amino acids into the
medium. The amounts of some of them were larger than that of proline.
Since the production of various amino acids was also observed for
TH401, it is highly likely that this fact is due to the mutation in the
proB gene. In E. coli, proline biosynthesis is
linked to arginine biosynthesis through the interconversion of
pyrroline-5-carboxylate to ornithine (1). No other linkages of proline biosynthesis have been reported. TH401 and TH4017 did not
excrete arginine into the medium, but they produced relatively larger
amounts of alanine, threonine, and valine. It will be interesting if
proline biosynthesis in T. thermophilus has some
relationship to these amino acid biosynthetic pathways.
Until now, the level of L-proline produced from TH4017 has
been very low. We thought this was mainly due to the low expression of
the proline biosynthetic genes and the remaining activity of the
L-proline degradation system in T. thermophilus.
We believed that TH4011, TH4012, TH4013, TH4014, TH4015,
TH4016, and TH4017 were defective for proline uptake but not for
proline dehydrogenase because all mutants could not grow in MM
containing 10 mM proline as sole nitrogen source but could grow in MM
containing 100 mM proline. As already reported, there were no strong
promoter sequences upstream of the proBA and proC
genes in T. thermophilus (7, 9). Increasing
L-proline production by replacing the proline biosynthetic
promoters should be effective. We also plan to attempt to increase
L-proline production by disrupting the putA gene.
| |
ACKNOWLEDGMENT |
We thank Hiroshi Matsui (Ajinomoto Co., Inc., Kawasaki, Japan)
for analysis of the amino acid production levels of various mutants of
T. thermophilus.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Applied Biochemistry, University of Tsukuba, Ten-nodai, Tsukuba,
Ibaraki 305-8572, Japan. Phone: 81-298-53-6782. Fax: 81-298-53-6782. E-mail: takachan{at}sakura.cc.tsukuba.ac.jp.
| |
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Applied and Environmental Microbiology, November 1998, p. 4328-4332, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
