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The International Center for Biotechnology, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Correspondence
Hiroko Kawasaki
ICBKawasakiNakagawa{at}icb.osaka-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Food and Agricultural Product Technology, Gadjah Mada University, Bulaksumur, Yogyakarta 55281, Indonesia. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In order to increase lysine formation in organisms by molecular techniques, information on the regulation of lysine biosynthesis and the availability of the functional genes is required. L-Lysine, together with other members of the aspartate family of amino acids, L-methionine, L-threonine and L-isoleucine, is synthesized via a branched pathway. The consecutive actions of the first two enzymes, aspartokinase and aspartate semialdehyde dehydrogenase, lead to the formation of aspartate semialdehyde, the precursor of all members of the aspartate family. In the lysine branch, aspartate semialdehyde is converted to dihydrodipicolinate by dihydrodipicolinate synthase, which is then reduced to piperidine dicarboxylate by dihydrodipicolinate reductase (Umbarger, 1978
). The first four steps of lysine biosynthesis from aspartate are well conserved in bacteria. From piperidine dicarboxylate, however, there are three variants of the pathway: the succinylase pathway, which uses a succinyl residue as the blocking group, is present in Escherichia coli (Kindler & Gilvarg, 1960); the acetylase pathway, involving acetylated intermediates, is found in certain Bacillus species (Sundharadas & Gilvarg, 1967; Weinberger & Gilvarg, 1970); and the dehydrogenase pathway, in which piperidine dicarboxylate is converted in a single step to the ultimate lysine precursor meso-diaminopimelate, is found in Bacillus sphaericus (Misono et al., 1979; White, 1983).
The regulation of lysine biosynthesis varies among organisms. Only aspartokinase is regulated for the biosynthesis of lysine in Corynebacterium glutamicum. It is inhibited by L-lysine plus L-threonine; either L-lysine or L-threonine alone does not inhibit aspartokinase (Nakayama et al., 1966; Cremer et al., 1988). In E. coli, many enzymes of lysine biosynthesis are regulated by the end-products of the pathway. There are three isozymes of aspartokinase in E. coli: aspartokinase I is repressed by L-threonine plus L-isoleucine and inhibited by L-threonine, aspartokinase II is repressed by L-methionine (Patte et al., 1967), and aspartokinase III is repressed and inhibited by L-lysine (Theze et al., 1974). Aspartokinase I and II are bifunctional enzymes and show both aspartokinase and homoserine dehydrogenase activities. The aspartate semialdehyde dehydrogenase is repressed by L-lysine in E. coli (Cohen & Patte, 1963), as is diaminopimelate decarboxylase, the enzyme that catalyses the last step in the pathway (White, 1976). The activity of dihydrodipicolinate synthase in E. coli is inhibited by L-lysine (Yugari & Gilvarg, 1962).
In L. plantarum, only the regulation of aspartokinase has been reported to date (Adebawo et al., 1997); that study was done with cell extract of L. plantarum. Because aspartokinase in L. plantarum may be present as an isozyme, as indicated in the genome sequence (Kleerebezem et al., 2003), the study of enzyme regulation using cell extracts of L. plantarum may not be appropriate; the aspartokinases should be separated when the regulation of the enzyme is studied. The genome sequence of L. plantarum also reveals that aspartate semialdehyde dehydrogenase and dihydrodipicolinate synthase may be present as isozymes.
We studied here the expression of genes encoding the four key enzymes in lysine biosynthesis, i.e. aspartokinase, aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase, and the effects of the aspartate family of amino acids on those enzymes in L. plantarum. The first step of the pathway and the branching point are potential sites of regulation of carbon flow. Homoserine dehydrogenase was included in this study because this enzyme catalyses the reaction at the branch point and may have a role in the regulation of carbon flow toward L-lysine.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, and E. coli strains were grown in M9 medium.
Gene cloning and expression of the genes.
Lysine biosynthetic genes were amplified from chromosomal DNA of L. plantarum NCIMB 8826 by PCR. The primers were designed based on the genome sequence of L. plantarum and to include the putative promoter sites of the genes. The primers used were ThrA1-1 (5'-TTCAATAAGGACTACTTCTTG-3'), ThrA1-2 (5'-ATCTAGGTCAACAAAAAAGC-3'), ThrA2-1 (5'- AATTAAATTCTCGTGAGTGG-3'), ThrA2-2 (5'-AACACGGCCATAAAAAATG-3'), Hom1-1 (5'-TTAGCAAACGTCGCTGATG-3'), Hom1-2 (5'-TGATATCTACGTTCTCGTG-3'), Hom2-1 (5'-GGGCCAATTAAATTAGTCTG-3'), Hom2-2 (5'-ACACTTGTAGCGTTAGTTGC-3'), Asd1-1 (5'-CATAGGAAGAAAAAAGTGCG-3'), Asd1-2 (5'-AGCCTCCTCAATAATTCTCC-3'), Asd2-1 (5'-ATCTGCGTGTGCAAAACAG-3'), Asd2-2 (5'-GGATCAGCTTTTCATTAC-3'), DapA1-1 (5'-GGTTAGCCGTGATTGTTG-3'), DapA1-2 (5'-AACTGAAGTCCGGAATCAC-3'), DapA2-1 (5'-ATGGCATCACACAACTGTC-3'), DapA2-2 (5'-CTGATCCCTGATGATGAAAC-3'), DapB-1 (5'-AATAGTGTTCGACCGAGCG-3') and DapB-2 (5'-AATATCGACGTCATGTAGGGG-3').
The PCR products were cloned into pCR2.1-TOPO (Invitrogen) to facilitate sequencing of the genes. The genes with the right sequences were then subcloned to pUC18 for heterologous expression of the genes in E. coli mutants. Transformation of pUC18 harbouring the relevant gene into E. coli cells was performed by electroporation using a Bio-Rad Gene Pulser II as described by Dower et al. (1988). Aspartokinase and homoserine dehydrogenase genes were expressed in E. coli Gif 102, whereas aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase genes were expressed in E. coli strains Hfr3000 U482, AT997 and AT999, respectively.
RT-PCR.
Nucleotide sequences inside the genes were used as primers for RT-PCR. RNA of L. plantarum was used as a template. The RNA was isolated by RNeasy (Qiagen). The primers used were thrA1-RTF (5'-AAAGTTCGGCGGCACGTCAG-3'), thrA1-RTR (5'-GAAACTTTAGCACAGTTCGC-3'), thrA2-RTF (5'-GGCACCTGGTAAACGGTTCG-3'), thrA2-RTR (5'-AGTAGTCGTGCGTAAAGTAA-3'), hom1-RTF (5'-TTCAGTTACCGATTGGGACG-3'), hom1-RTR (5'-TGCAACATTTATGGGAGATG-3'), hom2-RTF (5'-AGTTGATTTGGGGGACGTTC-3'), hom2-RTR (5'-TTATAACTCGCTAATAGTTG-3'), asd1-RTF (5'-GTTGATCAGCTCGCTGAATC-3'), asd1-RTR (5'-CTATTCAAATAATACCGCTG-3'), asd2-RTF (5'-ATCAAAATGGTGGAACAGAC-3'), asd2-RTR (5'-TAAGCCCATCTTGTCCATTG-3'), dapA1-RTF (5'-CGAAAAGCGGTTAGCAAGTT-3'), dapA1-RTR (5'-CCACCGGCTGACCCAAATGA-3'), dapA2-RTF (5'-TTTCCACCGCTTAGAGCAAC-3'), dapA2-RTR (5'-CGACTAACCAGTGTTGGGCG-3'), dapB-RTF (5'-GTGGTAAAAGTAATTGTTGC-3'), and dapB-RTR (5'-TCATAGTAAGTGCTCCAAAC-3').
Preparation of cell extracts and enzyme assay.
Cells were harvested by centrifugation, washed twice with deionized water, and resuspended in the buffer used for the enzyme assay. The cells were lysed by sonication (Kaijo Denki Co.) at maximum setting, for 2 min for E. coli and 20 min for L. plantarum, and the cell debris was separated by centrifugation at 10 000 g for 30 min. The supernatant was used as a cell-free extract.
Aspartokinase was measured as described by Black & Wright (1955b). The assay mixture contained 50 mM Tris buffer pH 7·0, 15 mM ATP, 10 mM MgSO4, 200 mM (NH4)2SO4, 500 mM hydroxylamine, 25 mM L-aspartate, and cell extract. After incubation at 37 °C for 1 h, the reaction was stopped by addition of FeCl3. The reaction mixture was centrifuged at 12 000 r.p.m for 5 min and the absorbance of the supernatant was measured at 540 nm. The absorbance of the samples was compared to those of standards obtained with aspartyl hydroxamate. Activity is expressed as the amount of enzyme required to form 1 nmol hydroxamic acid min–1.
Homoserine dehydrogenase activity was assayed as described by Black (1962) at 30 °C in 1 ml 100 mM potassium phosphate buffer pH 7·0 containing 0·25 mM NADPH and 0·35 mM L-aspartate semialdehyde as substrate. The activity was determined by following the decrease in absorbance at 340 nm. Activity is expressed as the amount of enzyme that oxidized 1 nmol NADPH min–1.
Aspartate semialdehyde dehydrogenase was measured as described by Hegeman et al. (1970) in the reverse of the biosynthetic reaction. The assay mixture consisted of 30 mM diethanolamine buffer pH 9·0, 0·8 mM NADP, 40 mM Na2HAsO4, 120 mM NaCl, 0·3 mM L-aspartate semialdehyde, and cell extract. L-Aspartate semialdehyde was synthesized by ozonization of allylglycine as described by Black & Wright (1955a). The rate of reduction of NADP+ to NADPH was measured spectrophotometrically at 30 °C and 340 nm. Activity is expressed as the amount of enzyme required to form 1 nmol NADPH min–1.
Dihydropicolinate synthase was assayed according to the method of Yamakura et al. (1974). The assay mixture consisted of 200 mM Tris/HCl buffer pH 8·0, 4 mM sodium pyruvate, 2 mM L-aspartate semialdehyde, and cell extract. After incubation at 37 °C for 10 min, the 1 ml assay mixture was mixed with 1 ml 1 M HCl and 1 ml o-aminobenzaldehyde (2 mg ml–1 in ethanol). The assay mixture was incubated at 37 °C for an additional 30 min in the dark and then centrifuged. The absorbance of the supernatant was determined at 540 nm. Activity is expressed as the amount of enzyme required to convert 1 nmol aspartate semialdehyde min–1.
Dihydropicolinate reductase was assayed by a coupled method as described by Cremer et al. (1988). The gene encoding DHPS from L. plantarum IAM 12477 was transformed into DHPR-deficient E. coli AT999, and the extract of this strain was used to synthesize the reductase substrate, i.e. 2,3-dihydrodipicolinate. The reaction mixture contained 100 mM Tris/HCl buffer pH 7·5, 5 mM sodium pyruvate, 1·5 mM L-aspartate semialdehyde, 1 mM NADPH, and cell extract of E. coli AT999 containing 0·4 units of DHPS. After incubation at 30 °C for 10 min, the reductase reaction was started by addition of the cell extract. The NADPH-dependent reduction was followed at 340 nm and 30 °C. Activity is expressed as the amount of enzyme required to oxidize 1 nmol NADPH per minute.
Specific activity of the enzymes is expressed as the activity of the enzyme per mg protein. Protein was measured by the method of Bradford (1976) with bovine serum albumin as a standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Cloning and expression of lysine biosynthetic genes
Seven genes involved in lysine biosynthesis, i.e. thrA1, thrA2, asd1, asd2, dapA1, dapA2 and dapB, together with homoserine dehydrogenase genes hom1 and hom2, were amplified by PCR from the DNA of L. plantarum NCIMB 8826. The primers used to amplify the genes were designed to include the predicted promoter regions of the genes. The cloned genes with nucleotide sequence identical to the one reported in the genome project were used for further work.
The expression of the genes in L. plantarum was determined by RT-PCR. The results showed that six genes (thrA1, thrA2, hom2, asd2, dapA1 and dapB) were strongly expressed to mRNA, and the other genes (hom1, asd1 and dapA2) were weakly expressed (Fig. 1). The enzyme activities encoded by the cloned genes were assayed in cell extracts of E. coli harbouring the gene. As a control, cell extract of E. coli harbouring pUC18 was used. For E. coli harbouring aspartokinase or homoserine dehydrogenase genes, the cell extract was assayed for both aspartokinase and homoserine dehydrogenase activities. The aspartokinase activity of the cell extract of E. coli Gif 102 harbouring thrA2 was considerably higher than the control (Table 1). However, the aspartokinase activity of the cell extract of E. coli Gif 102 harbouring thrA1 was not significantly different from the control, although thrA1 was strongly expressed to mRNA in L. plantarum. There were two homoserine dehydrogenase genes indicated in the genome sequence of L. plantarum; hom2 was strongly expressed to mRNA, but the expression of hom1 was weak. However, activity of homoserine dehydrogenase was detected in the cell extract of E. coli Gif 106 harbouring hom1, although it was relatively low. The homoserine dehydrogenase activities in the cell extracts of E. coli Gif 106 harbouring hom1 and hom2 were 7·6 and 1667 nmol min–1 mg–1, respectively, compared to 2·4 nmol min–1 mg–1 for the control. Surprisingly, one of the homoserine dehydrogenase genes, hom1, also exhibited aspartokinase activity, although the activity (12·8 nmol min–1 mg–1) was considerably lower than that of ThrA2 (162·1 nmol min–1 mg–1). Thus, Hom1 was a bifunctional enzyme, with both aspartokinase and homoserine dehydrogenase activities. There are two copies of aspartate semialdehyde dehydrogenase genes in L. plantarum as shown in the genome sequence. The RT-PCR data showed that only one of them, i.e. asd2, was strongly expressed to mRNA. The enzyme activity assay also showed that only Asd2 exhibited strong aspartate semialdehyde dehydrogenase activity. The aspartate semialdehyde dehydrogenase activity of Asd1 was not significantly different from that of the control. The aspartate semialdehyde dehydrogenase activities of cell extract of E. coli Hfr3000 U482 harbouring asd1 and asd2 were 215 and 2149 nmol min–1 mg–1, respectively, compared to 207 nmol min–1 mg–1 for the control. Although there are two genes encoding dihydrodipicolinate synthase in L. plantarum as indicated in the genome sequence, only dapA1 showed strong expression on RT-PCR and also gave detectable activity (125·2 nmol min–1 mg–1) of dihydrodipicolinate synthase in the cell extract of the recombinant E. coli. Dihydrodipicolinate synthase activity in the cell extract of E. coli AT997 harbouring dapA2 was undetectable, as it was in the control. The genome sequence of L. plantarum shows only one copy of the dapB gene. This gene was able to be expressed in E. coli AT999, giving dihydrodipicolinate reductase activity of 1153 nmol min–1 mg–1 in the cell extract.
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Aspartokinase and homoserine dehydrogenase.
Although two enzymes exhibited aspartokinase activity, only the activity of ThrA2 was inhibited by the end-product of the pathway. L-Lysine inhibited the activity of ThrA2 by 87 % at 1 mM (data not shown) and by 96 % at 10 mM (Table 2). The concerted inhibition of aspartokinase by L-lysine plus L-threonine as found in C. glutamicum was not observed in L. plantarum (data not shown). Study of the regulation of aspartokinase using L. plantarum cell extract showed inhibition of the enzyme by L-lysine and repression by L-threonine (Table 3). The repression of aspartokinase by L-threonine in L. plantarum may contribute to the reduced growth rate when the cells were grown in the medium supplemented with L-threonine (Fig. 2).
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, who found that aspartokinase in L. plantarum was not inhibited by amino acids of the aspartate family. Despite that difference, the activation of aspartokinase by L-methionine observed in this study (Tables 2 and 3
) was also reported by Adebawo et al. (1997). The regulation of aspartokinase in L. plantarum was found to differ from that in other organisms such as E. coli (Patte et al., 1967; Theze et al., 1974), Bacillus subtilis (Graves & Switzer, 1990; Rosner & Paulus, 1971) and C. glutamicum (Nakayama et al., 1966).
The activity of homoserine dehydrogenase in L. plantarum was contributed by Hom1 and Hom2, although the activity of Hom1 was considerably lower than that of Hom2. The activity of Hom1 was not inhibited by amino acids of the aspartate family, though the activity of Hom2 was inhibited by L-threonine; at 1 mm L-threonine, the homoserine dehydrogenase activity of Hom2 was reduced by 90 % (data not shown), and at 10 mM L-threonine, the activity of Hom2 was inhibited completely (Table 2). The inhibition of homoserine dehydrogenase by L-threonine was also observed when we used cell extract of L. plantarum (Table 3).
Aspartate semialdehyde dehydrogenase.
Study of the regulation of aspartate semialdehyde dehydrogenase in the cell extracts of both E. coli Hfr3000 U482 harbouring asd2 and L. plantarum did not show any influence by amino acids of the aspartate family. Such a lack of effect was also observed in C. glutamicum (Cremer et al., 1988). However, aspartate semialdehyde dehydrogenase in E. coli is repressed by L-lysine (Cohen & Patte, 1963).
Dihydrodipicolinate synthase.
There was no inhibition of dihydrodipicolinate synthase by any amino acids of the aspartate family as shown in the study using cell extracts of E. coli AT997 harbouring dapA1. In addition, the transcription of dapA1 to mRNA in L. plantarum was not repressed when the cells were grown in medium supplemented with 10 mM L-lysine (data of RT-PCR not shown). The absence of inhibition and repression of dihydrodipicolinate synthase by the end-product of the pathway was also found in Brevibacterium lactofermentum (Tosaka & Takinami, 1978), C. glutamicum (Cremer et al., 1988) and Bacillus stearothermophilus (Selli et al., 1994), but different results were found in E. coli (Yugari & Gilvarg, 1962) or Bacillus sphaericus (Barlett & White, 1986), where dihydrodipicolinate sythase was reported to be inhibited by lysine.
Dihydrodipicolinate reductase.
Dihydrodipicolinate reductase in L. plantarum was not regulated by amino acids of the aspartate family when it was studied in the cell extracts of both E. coli AT999 harbouring dapB and L. plantarum. The dihydrodipicolinate reductases in Br. lactofermentum and C. glutamicum were also not regulated by the end-product of the pathway. However, dihydrodipicolinate reductase in Staphylococcus aureus is repressed by lysine (Barnes et al., 1969).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), whereas the asd1 and dapA2 genes, which did not show enzyme activity in vitro, were weakly expressed as shown by RT-PCR. The asd1 and dapA2 genes might have lost their functions during their evolution. ThrA1 did not show any aspartokinase activity in vitro, though the RT-PCR data showed strong expression of thrA1. When thrA1 was cloned to the expression vector pET25b and expressed in E. coli, it did not give an enzyme activity (data not shown). It is assumed that the degree of evolution of thrA1 is different from that of asd1 or dapA2.
Our study of the inhibition of lysine biosynthetic enzymes of L. plantarum was carried out in cell extracts of E. coli mutants harbouring the relevant gene and of L. plantarum. However, we studied repression of enzyme synthesis in cell extract of L. plantarum only. Repression of foreign enzymes in E. coli may not represent the repression of enzyme synthesis in the species from which the enzymes originate. However, a study of repression of an enzyme in its own cell extract is suitable only if the enzyme is not an isozyme. In lysine biosynthesis of L. plantarum, aspartokinase was regulated by the end-product of the pathway, whereas aspartate semialdehyde dehydrogenase, dihydrodipicolinate synthase and dihydrodipicolinate reductase were not subject to such regulation (Fig. 3). The study using cell extract of E. coli Gif 102 harbouring thrA2 revealed that this aspartokinase was inhibited by L-lysine (Table 2), and the study using the cell extract of L. plantarum showed that aspartokinase was inhibited by L-lysine and repressed by L-threonine (Table 3). The aspartokinase repressed by L-threonine is likely to be ThrA2, based on the finding that the activity of Hom1 is considerably lower than that of ThrA2.
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). However, the flow of L-aspartate semialdehyde at this branching point is controlled by regulating the homoserine dehydrogenase, given that regulation of dihydrodipicolinate synthase has not been observed in L. plantarum. The inhibition of Hom2 by L-threonine leads to less conversion of aspartate semialdehyde toward L-threonine, though the formation of aspartate semialdehyde also becomes reduced due to the repression of ThrA2 by L-threonine, thus allowing a balanced flow of intermediate at the branch point.
In C. glutamicum, the flow of aspartate semialdehyde to L-lysine is enhanced by increasing the dihydrodipicolinate synthase activity (Eggeling et al., 1998) or by decreasing the homoserine dehydrogenase activity (Vrljic et al., 1995), leading to improved L-lysine production. However, this method of redirecting aspartate semialdehyde flow toward L-lysine may not be applicable to L. plantarum because the supply of aspartate may be reduced, due to inhibition of ThrA2 by L-lysine when L-lysine is in excess, unless a mutant strain with L-lysine-insensitive ThrA2 is used.
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ACKNOWLEDGEMENTS |
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This paper represents a portion of the thesis submitted by M. N. Cahyanto to Osaka University as partial fulfilment of the requirements for the PhD degree.
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REFERENCES |
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Barlett, A. T. M. & White, P. J. (1986). Regulation of the enzymes of lysine biosynthesis in Bacillus sphaericus NCTC 9602 during vegetative growth. J Gen Microbiol 132, 3169–3177.
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Received 6 April 2005;
revised 25 August 2005;
accepted 3 October 2005.
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3); ND, not detectable.
), minimal medium plus 20 mM L-lysine (
), minimal medium plus 50 mM L-lysine (
), minimal medium plus 20 mM L-threonine (
) and minimal medium plus 50 mm L-threonine (
).