Use of the repressor glxR for the synthesis of lysine in corynebacterium glutamicum
Isolated polypeptide sequence having the sequence of SEQ ID NO:1 or muteins thereof having the ability to bind cAMP and repress the expression of the aceB gene of C. glutamicum and which can be obtained from SEQ ID NO:1 by inserting, deleting or substituting up to 20% of the amino acids.
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This application is a continuation of U.S. application Ser. No. 10/514,076, filed Nov. 11, 2004, which is a 35 U.S.C. 371 national stage filing of International Application No. PCT/EP03/004969, filed May 13, 2003, which claims priority to European Application Serial No. 02010924.5, filed May 16, 2002. The entire contents of each of these applications are hereby incorporated by reference herein.
INTRODUCTIONCorynebacterium glutamicum is a gram-positive organism and has been well known as the host organism for the industrial production of amino acids, such as glutamate and lysine [Kinoshita, 1995]. Due to the role of the organism in amino acid production, the catabolic and anabolic pathways leading to the industrially important amino acids have been studied in detail during the past decades [for review see Sahm et al, 1995; Malumbres and Martin, 1996]. Although necessary for the in-depth understanding of the metabolic pathways, the information on the regulatory mechanism of gene expression in the organism is very limited.
The glyoxylate bypass of C. glutamicum is a good candidate for studying the regulatory mechanism of gene expression, because the expression of the isocitrate lyase and malate synthase, which catalyze the bypass (
The present invention provides novel polypeptide molecules which have the ability to repress the gene expression of at least the aceB gene of Corynebacterium glutamicum. These molecules are referred to as glxR molecules (glyoxylate bypass regulators). Another aspect of the invention relates to polynucleotide sequences which code for the above-mentioned glxR molecules. Another aspect of the invention relates to the use of glxR molecules for influencing the synthesis of amino acids in hosts such as Corynebacteria.
DETAILED DESCRIPTION OF THE INVENTIONThe present invention provides novel polypeptide molecules which have the ability to repress the gene expression of at least the aceB gene of Corynebacterium glutamicum. The isolation of the glxR molecule having the polypeptide sequence depicted in SEQ ID NO:1 is described in the experimental section.
The preparation of glxR muteins, having the ability to bind cAMP and repress the transcription of the aceB gene coding for malat synthase of C. glutamicum is described in the experimental section.
Polynucleotide sequences encoding the glxR molecules and glxR muteins can be prepared by back-translation of the respective polypeptide sequences according to the genetic code and chemical synthesis of said polynucleotide sequences. The isolation of the polynucleotide sequence encoding the glxR molecule according to SEQ ID NO:1 is described in the experimental section.
Another aspect of the invention is the use of the glxR molecules and the respective polynucleotide sequences for modulating at least the expression of the aceB gene of C. glutamicum. In one embodiment a polynucleotide sequence encoding a glxR molecule is recombinantly introduced in a host organism of the genus Corynebacterium which has a functional aceB gene. At least the gene expression of this aceB gene is modulated on the transcriptional level compared to the naturally occurring genome organization of Corynebacteria. By expressing the glxR molecules in an amount distinctly higher as the naturally occurring amount, a more effective gene repression of aceB is effected.
In another embodiment of the invention an antisense molecule to the polynucleotide sequence encoding a glxR molecule is introduced to a host organism. This antisense molecule reduces the expression of the glxR gene and effects an amount of glxR molecules in the host which is lower than the naturally occurring amount. This results in a higher transcription rate of the aceB gene.
In another embodiment of the invention the polynucleotide sequence encoding a glxR molecule is deleted partially or completely in the host organism. This results in a higher transcription rate of the aceB gene.
By modulation of the aceB gene expression the glyoxylate bypass is influenced directly. The modulation can be in both directions i.e. up-regulation and down-regulation. By influencing the glyoxylate bypass the metabolic flux of amino acids and intermediates thereof can be influenced effectively in Corynebacterium.
As Corynebacterium is used as an organism for the production of amino acids, preferably of lysine, glutamate and methionine, the influence on the glyoxylate bypass via the glxR molecules can be used to shift the production of amino acids in Corynebacterium. In some cases it will be advantageous to shift production of the desired amino acid to higher amounts whereas in some other cases a shift in the opposite direction is desired, e.g. in order to block the synthesis of unwanted intermediates.
The influence of the glyoxylate bypass by the glxR molecules is an effective way to design the metabolic capacity of Corynebacteria.
Experimental SectionIsolation of glxR. To isolate genes whose protein products exert regulatory effect on the expression of the C. glutamicum aceB gene, we utilized pSL145 [Kim et al, 2001] carrying the enteric lac operon fused to the downstream of Corynebacterial aceB as the reporter plasmid. With the plasmid, modulation of aceB expression at the promoter region is reflected as changes in the β-galactosidase activity. E. coli DH5αF′ cells carrying pSL145 (E. coli DH5αF′-145) formed blue colonies on LB plates containing X-gal and were used as the host for screening the Corynebacterial library. The host cell carrying a clone whose protein product has regulatory effect on the promoter region of aceB, thus affecting the expression of lacZ, was expected to form a white colony on the plate. Among a total of 20,000 colonies screened, 4 white colonies were identified. The cloned DNA turned out to contain overlapping inserts. Among the clones, plasmid pSL329 (
Sequence analysis of the glxR gene. The complete nucleotide sequence of the clone was determined using pSL329-5 as the sequencing template. An ORF consisted of 684 bp was found in the central region of the clone. As based on the similarities with other proteins (see below), the GTG was chosen as the start codon (
The putative gene product consisted of 228 amino acids (SEQ ID NO:1) encoding a 24,957 Da protein with the predicted isoelectric point of 7.0. The translated amino-acid sequence of the ORF was compared with the sequences in the protein database. Among the known proteins, a putative transcriptional regulatory protein of Mycobacterium tuberculosis (E70790) and a putative transcriptional regulatory protein of Streptomyces coelicolor (T36556) gave the highest score with the amino acid identity of 78 and 53%, respectively. Among the proteins whose roles are known, the cyclic AMP receptor protein (CRP) of Vibrio choleras (NP232242), Salmonella typhimurium (A26049), and E. coli (J01598) gave the highest score with approximately 27% identity. Close analysis of the amino acid sequences revealed 2 conserved motifs that may be involved in the catalytic activity of the enzyme (
Analysis of the encoded protein. Cloning the glxR coding region including the RBS into the pKK223-3 vector and introducing the resulting vector into E. coli resulted in the expression of Mr of 25,000 protein on SDS-PAGE (
Involvement of cAMP. As the first step to study the role of glxR, plasmid pSL329-5, a glxR clone, was introduced into C. glutamicum AS019E12 and the effect was monitored. As shown in FIG. 6AB, the presence of a glxR clone in multicopy significantly affected the growth of the host cell grown on glucose or acetate as the carbon source. As shown in Table 2, the growth impairment observed in acetate minimal medium was apparently due to the decrease of the glyoxylate bypass enzymes, such as malate synthease and isocitrate lyase. The enzymatic activity of isocitrate dehydrogenase, a TCA cycle enzyme, was unaffected. The reduction in the activity was due to the reduction in the amount of expressed enzymes as judged by SDS-PAGE (data not shown). However, when the growth experiment was carried out in the presence of cAMP, an interesting result was observed. Unlike the growth in glucose minimal medium (
DNA-binding activity of GlxR. Knowing that GlxR is involved in controlling the expression of aceB, we tested binding of purified GlxR on the promoter region of the aceB gene. For the purpose a DNA fragment which carries the promoter region of the aceB gene was utilized (
Muteins of the glxR proteins. Starting from the original glxR polypeptide sequence (SEQ ID NO:1) a lot of functional equivalent glxR muteins can be prepared by substituting, by inserting or by deleting one or more of the amino acids of SEQ ID NO:1. Functional equivalent muteins means that these muteins have still the ability to bind to cAMP and to repress the expression, specifically the transcription of the aceB gene of C. glutamicum in an order of magnitude which is the same as with glxR having the SEQ ID NO:1. The preparation of glxR muteins is preferably done by well known genetic engineering techniques such as site directed mutagenesis of the respective encoding polynucleotide sequences.
The glxR muteins differ from the SEQ ID NO:1 sequence in up to 20%, preferred up to 15%, most preferred up to 10% and very most preferred up to 5% of the amino acid sequence.
It is important, that the glxR muteins possess an intact cAMP binding domain and an intact helix-turn-helix DNA binding motif as defined for example in
Bacterial strains, plasmids, and growth conditions. All strains and plasmids used in this study are listed in Table 1. B. coli DH5αF′ was used for the construction and propagation of plasmids. E. coli JM105 was used to express GlxR from pKK-glxR. Operon fusion of PaceB-lacZ constructed on plasmid pRS415 was transferred to the chromosome of E. coli strains using λRS415 lambda phage as described by Simons et al. [Simons et al, 1987]. Otherwise stated, E. coli and C. glutamicum cells were cultured at 37° C. in LB [Sambrook et al, 1989] and at 30° C. in MB [Follettie et al, 1993], respectively. Minimal media for E. coli and C. glutamicum were M9 [Sambrook et al, 1989] and MCGC [von der Osten, 1989], respectively. Carbon sources were added to the minimal medium in following amounts: glucose, 1%; acetate, 2%. Antibiotics were added in following amounts: ampicillin, 50 μg/ml; tetracycline, 20 μg/ml; kanamycin, 20 μg/ml. X-gal and cAMP were added to medium at 40 μg/ml and 8 mM, respectively. For the expression of proteins, IPTG was added to the final concentration of 1 mM or 0.3 mM.
DMA technology. Standard molecular cloning, transformation, and electrophoresis procedures were used [Sambrook et al, 1989]. Plasmids were introduced into C. glutamicum cells by electroporation [Follettie et al, 1993]. Mini plasmid preparation for C. glutamicum cells was performed as described [Yoshihama et al, 1985]. Chromosomal DNA from C. glutamicum AS019 was prepared as described [Tomioka et al, 1981]. Restriction enzymes and DNA modifying enzymes were purchased from Takara Shuzo Co. (Shiga, Japan) and New England Biolabs (Beverly, USA) and used as recommended by the manufacturer.
Cloning and sequencing. The genomic library of C. glutamicum AS019 was constructed as previously described [Lee and Sinskey, 1994]. It was made of 4 to 13 kb MboI fragments cloned into the E. coli-Corynebacterium shuttle vector pMT1. E. coli DH5αF′ cells were transformed with pSL145 [Kim et al, 2001], and the resulting E. coli DH5αF′-145 strain was used as the host for screening the library. Plasmid pSL 145 carries lacZYA fused to the downstream of the aceB promoter region.
For nucleotide sequence analysis of glxR, plasmids pSL329-5 was used as the template. The complete nucleotide sequence of glxR was determined commercially at the Korea Research Center for Basic Sciences (Taejon, Korea) using universal and synthetic oligonucleotide primers. A sequence similarity search of nucleotide and amino acid sequences was performed at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/) using BLAST [Altschul et al, 1990; Altschul et al, 1997]. Pairwise sequence alignments were performed at the website of ExPASy Proteomics Tools (http://www.expasy.ch/tools/) using the ClustalW alignment method [Thompson et al, 1994].
Construction of plasmids. Plasmids pSL329-1, pSL329-2, pSL329-3 and pSL329-5 were made by ligating the 5.1 kb EcoRI-XbaI fragment, 5.2 kb XhoI fragment, 2.7 kb SmaI-BcoRI fragment, and 1.8 kb KpnI fragment of pSL329 into the pMT1 vector digested with SmaI-XbaI, XhoI, and KpnI, respectively. Plasmid pSL329-4 was made by deleting the 0.2 kb Sail fragment of pSL329-3. Plasmid pSL08-glxR was constructed by inserting the 1.8 kb KpnI-BamHI fragment of pSL329-5 into pSL08 which was digested with XbaI and blunt-ended with Klenow enzymes. The direction of transcription of the cloned glxR gene was opposite to that of the aceB gene. Plasmid pKK-glxR was constructed by amplifying the 828 bp fragment of pSL329-5 with C1 and C2 primers and inserting the resulting fragment into the EcoRI site of pKK223-3. The 828 bp fragment carries the glxR gene and its ribosome-binding site. Plasmid pMAL-glxR was constructed as follows. The glxR coding region was amplified with primers of D1 and D2 using plasmid pSL329-5 as a template. The 0.89 kb PCR product was treated with EcoRI and Sail, and ligated into the EcoRI-SalI digested and dephosphorylated pMAL-c2.
Purification of GlxR. Plasmid pMAL-glxR was used to express GlxR. The vector carries glxR fused to malE and expresses MBP-GlxR fusion protein. The cleavage of the fusion protein with Factor Xa releases a free GlxR protein with a string of amino acids (Ile-Ser-Val-Phe) attached to the N-terminus of the protein. The fusion protein was expressed and purified as suggested by the manufacturer of the vector pMAL-c2 (New England Biolabs, Beverly, USA). GlxR was separated from MBP by Q-Sepharose Fast Flow column chromatography (Amersham Pharmacia Biotech, 2.8 cm by 11 cm) using a linear NaCl gradient of 25-500 mM. Fractions containing the GlxR were pooled and the protein was concentrated by dialysis against PEG 8,000.
Gel mobility shift assay. The probe DNA was prepared as follows. The 200 bp DNA fragment which includes an upstream region of the aceB gene (from 120 to 320 bp upstream from the ATG start codon) was amplified using E1 and E2 primers and subsequently labeled with [γ-32P]dATP using T4 polynucleotide kinase. A binding reaction mixture of 10 μl contained the labeled DNA fragments, various amount of purified GlxR, 10 mM phosphate buffer, 137 mM NaCl, 2.7 mM KCl, 3 μg of bovine serum albumin, and 1 μg of poly[dI-dC]·poly[dI-dC]. The binding of GlxR to the probe DNA was performed at room temperature for 15 min and the mixture was analyzed in 6% native polyacrylamide gels as described [Sambrook et al, 1989]. When necessary, 0.2 mM cAMP was included in the binding buffer, gels, and the running buffer.
Biochemical analysis and preparation of antibody. Corynebacterial cell extracts were prepared as described [Follettie et al, 1993]. The enzymatic activities of β-galactosidase, malate synthase, isocitrate lyase, isocitrate dehydrogenase, and acetate kinase were determined as previously described. [Miller, 1972; Gubler et al, 1993; Dixon and Romberg, 1959; Garnak and Reeves, 1979; van Dyk and LaRossa, 1987] Protein was measured by the method of Bradford, with bovine serum albumin as the standard [Bradford, 1976]. SDS-PAGE and Western blot analysis were performed as previously described [Laemmli, 1970]. The anti-GlxR antiserum was prepared commercially at Koma Biotech (Seoul, Korea) using a mouse as the host.
GenBank accession number. The nucleotide sequence of glxR was deposited in GenBank under the accession number of AF293334.
DISCUSSIONIn this study, we showed involvement of GlxR in the regulation of the aceB gene which is involved in the utilization of acetate as a carbon source. The evidences were, 1) modulation of the β-galactosidase activity in the reporter-carrying E. coli, 2) presence of a putative DNA-binding domain in the encoded protein sequence, 3) decreased expression of glyoxylate bypass enzymes in the presence of introduced glxR clone, and 4) DNA binding of the purified GlxR on the aceB promoter region. In addition, like other DNA-binding proteins, the finding that the protein present as dimers with relatively basic pI also support regulatory role for the protein. Although our data suggest that the glxR gene might be primarily involved in the regulation of glyoxylate bypass genes, such as aceA and aceB, we can not rule out the possibility that the gene may also be involved in the expression of other genes. The growth retardation by glxR in glucose minimal medium may suggest interaction of the GlxR protein with other promoters, although this could be due to the sequence-independent affinity for DNA as observed with CRP. Despite numerous attempts, failure to construct a glxR mutant strain using the cloned DNA may suggest essentiality of the gene (data not shown), suggesting the involvement of the glxR gene for the expression of other genes. In this respect, like CRP of E. coli, the GlxR protein could function as an activator for other genes, making the gene essential.
It is interesting to know that the encoded protein sequence of the glxR gene shows homology with CRP of enteric bacteria. CRP plays a role in carbon catabolite repression, which is governed by cAMP in enteric bacteria, such as E. coli. The major similarities between GlxR and CRP include, 1) amino acid sequence similarity, 2) the presence of cAMP-binding-motif, 3) the involvement of cAMP for modulation of the DNA-binding activity, and 4) differential sensitivity of the protein to protease in the presence of cAMP. Despite these similarities, however, the role of GlxR appears to be distinct form that of CRP. For instance, the glxR gene could not complement the crp mutant of E. coli (data not shown).
Cyclic AMP is an important signaling molecule controlling the expression of many genes. Involvement of cAMP has been reported in many bacteria, such as enteric bacteria. Bacillus species, and Streptomyces species [Botsford and Harman, 1992], In E. coli, the molecule plays important roles for carbon catabolite genes, such as lactose and arabinose operons [Epstein et al, 1975], Under conditions of carbon starvation, the intracellular concentration of cAMP is elevated. Although this is a general phenomenon, there are also exceptions. For example, Brevibacterium species apparently keep the concentration of cAMP low when grown on glucose minimal media [Peter et al, 1991]. This is similar to what we found with C. glutamicum. As evidenced in the growth experiment (
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Claims
1. Isolated polypeptide sequence having the sequence of SEQ ID NO:1 or muteins thereof having the ability to bind cAMP and repress the expression of the aceB gene of C. glutamicum and which can be obtained from SEQ ID NO:1 by inserting, deleting or substituting up to 20% of the amino acids.
2. Isolated polypeptide sequence according to claim 1 which possesses a cAMP binding domain and helix-turn-helix DNA binding motif.
3. Isolated polynucleotide sequence coding for a polypeptide sequence according to claims 1 to 2.
4. Use of a polynucleotide sequence according to claim 3 for modulating at least the expression of the aceB of Corynebacteria.
5. Use according to claim 4 in order to influence the biosynthesis of amino acids.
6. Use according to claim 5 in order to increase the biosynthesis of amino acids.
7. Use according to claim 6 wherein the amino acid is lysine.
8. Process of producing lysine in a host of genus Corynebacterium by influencing the gene expression of at least the aceB gene by expressing a polynucleotide sequence according to claim 3 in said host in a way which is different from the naturally occurring genome organization of Corynebacterium.
Type: Application
Filed: Feb 8, 2007
Publication Date: Nov 13, 2008
Applicant: BASF AG (Ludwigshafen)
Inventors: Corinna Klopprogge (Mannheim), Oskar Zelder (Speyer), Burkhard Kroger (Limburgerhof), Hartwig Schroder (Nussloch), Stefan Hafner (Ludwigshafen), Heung-Shick Lee (Seoul)
Application Number: 11/704,160
International Classification: C12P 13/08 (20060101); C07K 14/34 (20060101); C07H 21/02 (20060101);