# Patent

## Title
L-lysine-inducible promoter

## Abstract

Disclosed is a novel L-lysine-inducible promoter nucleic acid molecule. Also disclosed are a vector containing the nucleic acid molecule, a host cell transformed with the vector, and a method of inducing expression of a target gene using the L-lysine-inducible promoter nucleic acid molecule.

## Background
TECHNICAL FIELD
The present invention relates to a novel L-lysine-inducible promoter nucleic acid molecule, a vector containing such a nucleic acid molecule, a host cell transformed with the vector, and a method of inducing expression of a target gene using the L-lysine-inducible promoter nucleic acid molecule.
BACKGROUND ART
Coryneform bacteria are industrial microorganisms that produce chemical substances having various applications in the industry of animal feed, drugs, food, and the like, which contain L-lysine, L-threonine and various nucleic acids. To develop high titer strains from such coryneform bacteria by genetic engineering or metabolic engineering, the expression of genes involving several metabolic pathways in coryneform bacteria should be selectively regulated. Thus, promoter sequences useful for this gene regulation are required.
Upon the gene expression in coryneform bacteria, genes are generally expressed from their own promoters (Vasicova, P., et al., J. Bacteriol. 181, 6188-6191, (1999), etc.). However, unlike other industrial microorganisms such as Escherichia coli and Bacillus subtilis, there is no information on the basic structure of promoter sequences for gene expression in coryneform bacteria. For this reason, promoters have been developed by eliminating promoter regions from genes associated with resistance to antibiotics such as chloramphenicol, introducing chromosomal DNA isolated from coryneform bacteria and digested with suitable restriction enzymes into the promoter sites, transforming coryneform bacteria with the resulting DNA molecules, assessing antibiotic resistance of obtained strains (Eikmanns, B. J., et al., Gene, 102, 93-98, (1991); Patek, M., et al., Microbiology, 142, 1297-1309, (1996)). However, previously developed promoter sequences still need to be improved with respect to selective expression and expression efficiency of genes of interest.
Conventional methods involving promoter isolation include (1) the use of a promoter probe vector system in the random cloning of genomic DNA fragments upstream of a reporter gene expressed only when a cloned fragment contains promoter activity; (2) gene-specific probe-based hybridization by which a gene and its promoter are isolated from a genomic library; and (3) differential hybridization of an inducible cDNA probe and a non-inducible cDNA probe to a gene bank.
Recently developed comparative proteome analysis by two-dimensional (2-D) gel electrophoresis is a technology that identifies proteins differentially expressed at different physiological states. Based on differentially expressed proteins, many studies have been performed to identify regulatory genes capable of increasing expression of the proteins.
Based on this background, the present inventors conducted 2-D gel electrophoresis of proteins whose expression was induced in the presence of lysine, and detected and identified proteins displaying differential expression patterns by comparative analysis of proteomes. When polynucleotides corresponding to the putative promoter regions of the identified proteins were amplified by PCR and were introduced upstream of a promoter-deficient lacZ gene, a remarkable increase in beta-galactosidase activity was observed in the presence of lysine, thereby leading to the present invention, which provides novel promoter nucleic acid molecules capable of inducing gene expression in the presence of lysine.
DISCLOSURE OF THE INVENTION
It is therefore an object of the present invention to provide a novel L-lysine-inducible promoter nucleic acid molecule.
It is another object of the present invention to provide a vector comprising the novel promoter nucleic acid molecule.
It is a further object of the present invention to provide a host cell transformed with the vector.
It is yet another object of the present invention to provide a method of inducing expression of a target gene using the novel promoter nucleic acid molecule.



BRIEF DESCRIPTION OF THE DRAWINGS
The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the results of 2-D gel electrophoresis of whole-cell lysates of C. glutamicum cultured in either the absence or presence of lysine, wherein differentially expressed proteins are indicated by arrows; and
FIG. 2 is a schematic presentation of a process of constructing vectors for monitoring promoter activity, wherein beta-galactosidase was used for detecting promoter activity.



BEST MODE FOR CARRYING OUT THE INVENTION
In one aspect, the present invention relates to a novel L-lysine-inducible promoter nucleic acid molecule.
In detail, the L-lysine-inducible promoter nucleic acid molecule of the present invention has the nucleotide sequence of SEQ ID No. 1, 2, 3, 4 or 5.
The nucleotide sequence of the promoter nucleic acid molecule according to the present invention may be modified to a certain degree by any one of several recently developed methods, for example, directed evolution or site-directed mutagenesis. Those skilled in the art readily appreciate that a nucleotide sequence having 70% or higher homology by such artificial modification is derived from and is an equivalent to the nucleotide sequence of the present invention, as long as it retains promoter activity for expressing a target gene.
Thus, the L-lysine-inducible promoter nucleic acid molecule of the present invention includes nucleotide sequences that have 70% or higher homology to the aforementioned nucleotide sequence and are available as L-lysine-inducible promoters.
The term “homology”, as used herein, is intended to indicate sequence similarity to the native nucleic acid sequence. The “homology” includes a DNA sequence preferably 75% or higher, more preferably 85% or higher, even more preferably 90% or higher, and most preferably 95% or higher, identical to the nucleotide sequence of the L-lysine-inducible promoter nucleic acid molecule of the present invention. The homology evaluation may be done with the naked eye or using commercially available software. Using a commercially available computer program, the homology between two or more sequences may be expressed as a percentage (%), and the homology (%) between adjacent sequences may be evaluated.
In addition, the L-lysine-inducible promoter nucleic acid molecule of the present invention includes an L-lysine-inducible promoter nucleic acid molecule selected from the group consisting of nucleotide sequences complementary to the aforementioned nucleotide sequence.
The term “complementary”, as used herein, refers to the hybridization or base-pairing between nucleotides or nucleic acids, such as, for example, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid to be sequenced or amplified.
The L-lysine-inducible promoter nucleic acid molecule of the present invention also includes a functional equivalent that exerts activity identical to the L-lysine-inducible promoter nucleic acid molecule. The functional equivalent, which includes a functional fragment, may include variants in which one or more nucleotide bases are altered by substitutions, deletions, insertions or combinations thereof.
The L-lysine-inducible promoter nucleic acid molecule of the present invention is derived from coryneform bacteria, and is useful as a promoter for gene expression in prokaryotic cells, particularly E. coli and coryneform bacteria.
The term “promoter”, as used herein, refers to a DNA region to which RNA polymerase binds to initiate gene transcription, and positions at the 5′ direction of an mRNA transcription initiation site. With respect to the objects of the present invention, the promoter of the present invention indicates an L-lysine-inducible promoter that is sufficient to direct promoter-dependent gene expression in the presence of an external signal, L-lysine.
The L-lysine-inducible promoter nucleic acid molecule of the present invention may be isolated or prepared using a standard molecular biology technique. For example, it may be isolated by PCR using proper primer sequences. Also, it may be prepared by a standard synthesis technique using an automated DNA synthesizer.
The term “coryneform bacteria”, as used herein, includes microorganisms belonging to the genus Corynebacterium or Brevibacterium. Such coryneform bacteria include Corynebacterium glutamicum ATCC 13032; other wild-type strains known as suitable strains of the genus Corynebacterium, particularly the species Corynebacterium glutamicum, Corynebacterium thermoaminogenes FERM BP-1539, Brevibacterium flavum ATCC 14067, and Brevibacterium lactofermentum ATCC 13869; and L-amino acid-producing mutants or strains prepared therefrom, for example Corynebacterium glutamicum KFCC 10881 and Corynebacterium glutamicum KFCC 11001.
In another aspect, the present invention relates to a vector comprising the novel L-lysine-inducible promoter nucleic acid molecule.
The term “vector”, as used herein, refers to a DNA construct that contains a DNA sequence which is operably linked to a suitable control sequence. Such control sequences may include a promoter to direct transcription, a certain operator sequence to control such transcription, a sequence encoding a suitable ribosome-binding site on the mRNA, and a sequence to control termination of transcription and translation. The vector may be a plasmid, a phage particle, or simply a potential genome insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some cases, integrate into the genome itself.
The term “operably linked”, as used herein, means that a gene to be expressed is linked to its control sequences in a manner as to be expressed.
After performing 2-D gel electrophoresis, the present inventors identified proteins displayed increased expression by comparative analysis of proteomes, and employed a reporter gene to search promoter sequences from such proteins. A reporter gene is a gene that allows the activity of a promoter of interest to be readily detected through its expression. Available reporter genes include beta-galactosidase, beta-glucuronidase, luciferase, chloramphenicol transacetylase, and fluorescent proteins (e.g., green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, cyan fluorescent protein, etc.).
In a detailed practice of the present invention, to determine whether the novel promoter nucleic acid molecule of the present invention is L-lysine-inducible by measuring the activity of beta-galactosidase, expressed from a lacZ gene, a recombinant vector, pLCP, was constructed. In another detailed practice of the present invention, the pLCP vector containing the L-lysine-inducible promoter nucleic acid molecule of the present invention displayed two or more times higher beta-galactosidase activity in the presence of 250 mM of L-lysine than in the absence of L-lysine.
The present vector containing the L-lysine-inducible promoter nucleic acid molecule is operably linked to any one of genes encoding various proteins in order to recombinantly produce target genes. The target genes to be readily expressed using the present vector include, but are not limited to, asd, dapA, dapc, dapF, fbp, lysC and pyc. Of the target genes, asd, dapA, lysC and pyc are particularly preferred.
In a further aspect, the present invention relates to host cells transformed with the vector.
The aforementioned vector containing the L-lysine-inducible promoter nucleic acid molecule is operably linked to a gene encoding a target protein to induce expression of the target protein. The vector constructed in such a way is transformed into host cells, for example, prokaryotic cells, preferably E. coli and coryneform bacteria, and more preferably coryneform bacteria.
The term “transformation”, as used herein, means the introduction of DNA into a host in such a way that it can replicate either as an extrachromosomal element or by chromosomal integration.
The cultivation of the thus transformed host cells (transformants) may be performed according to ordinary methods in the art. The known cultivation methods are described by Chmiel (Bioprozesstechnik 1. Einfuhrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)); and Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
In yet another aspect, the present invention relates to a method of inducing expression of a target gene, which is based on using the L-lysine-inducible promoter nucleic acid molecule.
The novel L-lysine-inducible promoter nucleic acid molecule of the present invention is able to induce high-level expression of a target gene operably linked thereto, and is thus useful in the mass production of a target protein.
A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
Example 1
Screening and Identification of Proteins Whose Expression is Induced by L-Lysine in Corynebacterium glutamicum 
(1) Preparation of Protein Samples from Corynebacterium glutamicum ATCC 13032 Cultured at Various Concentrations of L-Lysine
Corynebacterium glutamicum ATCC 13032 was cultured in the absence or presence of L-lysine (0, 250 and 500 nM), and cells were harvested by centrifugation. The cells were washed three times with 10 mM Tris-HCl buffer (pH 7.5).
The washed cells were resuspended in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS (3-[(3-chloamidopropyl)dimethyl-ammonio]-1-propanesulfonate), 0.4% dithiothreitol), were disrupted by sonication on ice using a sonicator, and were centrifuged at 12,000×g for 15 min at 4° C. The thus obtained cell lysate was mixed with endonuclease (Sigma) at a final concentration of 700 units/ml and 1× protease inhibitor cocktail (Roche). Protein concentrations were determined by the Bradford method.
(2) Protein Separation by 2-D Gel Electrophoresis
500 μg of each of the obtained protein samples was mixed with solubilization buffer (7 M urea, 2 M thiourea, 4% CHAPS (3-[(3-chloamidopropyl)dimethyl-ammonio]-1-propanesulfonate), 0.4% dithiothreitol, 1% bromophenol blue) to give a final volume of 500 μl, and was centrifuged at 20,000×g for 15 min at 4° C. The supernatant was mixed with 0.5% immobiline pH gradient (IPG) buffer (pH 4.5-5.5 carrier ampolyte, Amersham Pharmacia Biotech). The resulting solution was loaded onto an IPG dry strip rehydration tray, and an 18-cm IPG dry strip with a linear gradient of pH 4.5-5.5 was rehydrated with the protein sample on the rehydration tray for 12 hrs.
The rehydrated strip was subjected to isoelectric focusing on a Multiphor II electrophoresis chamber (Amersham Pharmacia Biotech) for 90 kVh.
After the first-dimension isoelectric focusing was completed, the focused IPG strip was equilibrated with equilibration buffer (0.2 mM tributylphosphine, 6 M urea, 2% SDS, 375 mM Tris-HCl, pH 8.8, 20% glycerol and 2.5% acrylamide) for 15 min with agitation.
The equilibrated IPG strip was slightly moistened with 1× SDS-PAGE running buffer (192 mM glycine, 0.1% SDS, 24.8 mM Trisma Base), and was then subjected to second-dimension electrophoresis, as follows.
The equilibrated IPG strip was placed onto the top of an 8-16% gradient polyacrylamide gel, and was run at 15 mA per gel for 16 hrs to separate proteins.
(3) Detection of Protein Spots Displaying Increased Expression Levels
The two-dimension gel was washed with 250 ml of triple-distilled water, and was incubated in a fixing solution (40% methanol, 5% phosphoric acid) for 1 hr. The gel was stained with Coomassie brilliant G-250 (Sigma) for 5 hrs and then destained in a destaining solution (1% acetic acid) for 12 hrs to remove excessive staining.
The two-dimension gels, prepared using protein samples obtained from cells cultured in the presence of L-lysine of 0, 250 and 500 mM, were compared with each other using PDQuest software (Bio-Rad). The results are given in FIG. 1. Five protein spots displaying increased expression were detected.
Example 2
Identification of Proteins Whose Expression is Induced by L-Lysine
(1) Peptide Extraction from Protein Spots
The interested protein spots shown in FIG. 1 were excised from the gels, and were transferred into microtubes. The gel pieces were destained three times with 120 μl of 50% acetonitrile in 25 mM ammonium bicarbonate (pH 7.8,) for 15 min with gentle agitation, and were completely dried. The dried gel pieces were incubated in a trypsin digestion solution (25 mM ammonium bicarbonate, pH 7.8, 0.015 μg/μl trypsin) at 37° C. for 16 hrs to digest proteins in gels. After the in-gel trypsin digestion was completed, the digested gel pieces were covered with 8 μl of a peptide extraction solution (a mixture of 50% acetonitrile and 0.5% trifluoroacetic acid) and was sonicated for 10 min to extract peptides from gels. Then, the gel pieces were centrifuged, and supernatants containing peptides released from gels were recovered and concentrated to a volume of about 5 μl using a freeze dryer.
(2) Protein Identification by Mass Spectrometry
1 μl of each of the concentrated sample was mixed with 1 μl of a matrix solution (10 mg/ml α-cyano-4-hydroxy cinnamic acid, 0.5% trifluoroacetic acid, 50% acetonitrile), was loaded onto a sampling plate for Matrix Assisted Laser Desorption Ionization-Time Of Flight (MALDI-TOF) mass spectrometry, and was dried to be crystallized. Masses of the crystallized peptides were analyzed using a MALDI-TOF mass spectrometer. For protein identification, the measured peptide masses were searched against protein databases of National Center for Biotechnology Information (NCBI) and other sources using MS-Fit software or Profound software. The results are given in Table 1, below. As shown in Table 1, the five protein spots were identified to be encoded by Cgl1910 for spot a, Cgl2134 for spot b, Cgl2754 for spot c, Cgl2818 for spot d, and Cgl3029 for spot e.







TABLE 1







Proteins displaying increased expression


levels in the presence of lysine









Spot
Protein name
Cgl No.





a
Transcriptional regulator
1910


b
Dehydrogenases with different specificities
2134


c
NADPH-dependent glutamate synthase beta chain and
2754



related oxidoreductases


d
NADPH-dependent glutamate synthase beta chain and
2818



related oxidoreductases


e
Anthranilate synthase component I
3029









Example 3
Construction of Recombinant Vectors Containing Promoter Sequences
(1) Amplification of DNA Fragments Corresponding to Putative Promoter Regions
The nucleotide sequences of genomic genes of Corynebacterium glutamicum have been already fully determined and are well known. Genetic information (Cgl1910, Cgl2134, Cgl2754, Cgl2818 and Cgl3029) of proteins listed in Table 1 was obtained from the NIH GenBank database. In order to amplify putative promoter regions (SEQ ID No. 1: promoter region of Cgl1910, SEQ ID No. 2: promoter region of Cgl2134, SEQ ID No. 3: promoter region of Cgl2754, SEQ ID No. 4: promoter region of Cgl2818, and SEQ ID No. 5: promoter region of Cgl3029) located upstream of each protein ORF, as shown in Table-2, below, primers were designed to contain a BamHI site based on the reported nucleotide sequences, and were synthesized. To amplify the five promoter regions, PCR was carried out using chromosomal DNA of Corynebacterium glutamicum ATCC 13032 as a template with the synthesized primers [Sambrook et al., Molecular Cloning, a Laboratory Manual (1989), Cold Spring Harbor Laboratories]. PCR conditions included 25 cycles of denaturation at 94° C. for 1 min, annealing at 58° C. for 1 min and polymerization at 68° C. for 30 sec.








TABLE 2





Promoter
Primer sequences



















Cg11910
Primer 1
(SEQ ID No. 6):
TTGGATCC AACCCCGTAGCAACCAGC




Primer 2
(SEQ ID No. 7):
GAGGATCC GCGCCCAAGTGTACAAATT


 


Cg12134
Primer 3
(SEQ ID No. 8):
ATGGATCC TTGCCTCTAGTGGGACTG



Primer 4
(SEQ ID No. 9):
TAGGATCC TGAAATCATGCCTTTCTCG


 


Cg12754
Primer 5
(SEQ ID No. 10):
TAGGATCC ATTTCAGCCTGAACCTTC



Primer 6
(SEQ ID No. 11):
CAGGATCC ATAAAGTTCGATTCCTTAAA


 


Cg12818
Primer 7
(SEQ ID No. 12):
ACGGATCC TGCTTAATTTCCTCGGCA



Primer 8
(SEQ ID No. 13):
ATGGATCC GTGTTTGAAGTTGCCTTT


 


Cg13029
Primer 9
(SEQ ID No. 14):
AAGGATCC ATGGCTGCGCATACTGTTG



Primer 10
(SEQ ID No. 15):
ATGGATCC GGGGCACCTACCGAGGAA









(2) Construction of Vectors for Monitoring Promoter Activity

FIG. 2 schematically shows a process of constructing recombinant vectors for monitoring the activity of each putative promoter.
The DNA fragments amplified in the (1) of Example 3 were individually cloned into a BamHI site of pRS415 containing a lacZ gene (Simons, R. W., et al., Gene, 53, 85-96 (1987)). Each recombinant vector was digested with HindIII and XbaI to be cleaved both in front of the inserted putative promoter and in the rear of the lacZ gene, and was electrophoresed on an agarose gel. Each DNA fragment was excised from the gel and purified using a Gel Extraction Kit (Qiagen).
The purified DNA fragments were inserted into a pXMJ19 vector (Jakoby, M. J., et al., Biotechniques, 13, 437-441 (1999)), thus generating vectors for monitoring promoter activity, pLCP-1910, pLCP-2134, pLCP-2754, pLCP-2818 and pLCP-3029. These vectors allow detection of the activity of the putative promoters through measurement of the activity of the lacZ gene product, beta-galactosidase.
The recombinant vectors thus obtained were introduced into competent cells of Corynebacterium glutamicum ATCC 13032 according to a method described by van der Rest et al. (Appl. Microbiol. Biotechnol. 52, 541-545, (1999)). The transformed cells were smeared onto LB agar plates (0.5% yeast extract, 1% NaCl, 1% trypton, 1.5% agar) supplemented with 17 mg/ml of chloramphenicol, and were cultured at 30° C. to select grown colonies.
Example 4
Measurement of Activity of Promoter Sequences in C. glutamicum 
(1) Measurement of Activity of Promoter Sequences in Either the Absence or Presence of 250 mM Lysine
The transformed clones were cultured to analyze the activity of promoter sequences, as follows.
Each transformed clone of Corynebacterium glutamicum was inoculated at a ratio of 1:20 in a 250-ml baffle flask containing 25 ml of minimal medium (5 g glucose, 5 g ammonium sulfate, 2 g urea, 0.5 g NaCl, 1 g KH2PO4, 0.5 g MgSO4.7H2O, 200 μg biotin, 100 μg thiamin hydrochloride, 1 ml Trace element solution, in 1 liter distilled water, pH 7.2) either supplemented with 250 mM L-lysine or not, and was incubated at 30° C. with continuous shaking at 200 rpm. When the culture reached the mid-exponential growth phase (an absorbance of 5), the cultured cells were collected by centrifugation, were suspended in 100 mM potassium phosphate buffer (pH 7.0), were lysed by sonication, and then were high speed-centrifuged. 2 μl of the supernatant was used to measure the activity of beta-galactosidase according to Rosenthal's method (Rosenthal, N., Methods Enzymol. 152, 704-720 (1987)). As a result, when cultured in the presence of lysine, the transformed cells displayed beta-galactosidase activity 2.1-fold higher for Cgl1910, 2.1-fold higher for Cgl2134, 2.0-fold higher for Cgl2754, 3.4-fold higher for Cgl2818, and 2.7-fold higher for Cgl3029, than the culturing in the absence of lysine (Table 3). These results indicate that each promoter induces the expression of beta-galactosidase in the presence of lysine.







TABLE 3







Comparison of promoter activity between


the absence and presence of lysine













Beta-galactosidase activity



Promoters
Lysine (mM)
(mmol/min/mg)















Cgl1910
0
0.28




250
0.58



Cgl2134
0
3.30




250
6.99



Cgl2754
0
3.09




250
6.09



Cgl2818
0
2.75




250
9.23



Cgl3029
0
0.50




250
1.43










(2) Measurement of Activity of Promoter Sequences in Either the Absence or Presence of 250 mM NaCl

The activity of each promoter was measured according to the same method as in the (1) of Example 4, except that cells were cultured in either the absence or presence of NaCl instead of lysine. The results are given in Table 4, below. As shown in Table 4, similar activity of beta-galactosidase was observed in the two culture conditions, namely the absence and presence of NaCl. These results excluded the possibility of lacZ gene expression to be induced by factors other than lysine, and thus demonstrate that each promoter induces gene expression in specific response to lysine.







TABLE 4







Comparison of promoter activity between


the absence and presence of NaCl













Beta-galactosidase activity



Promoters
NaCl (mM)
(mmol/min/mg)















Cgl1910
0
0.30




250
0.31



Cgl2134
0
2.59




250
2.77



Cgl2754
0
2.49




250
2.05



Cgl2818
0
1.69




250
2.30



Cgl3029
0
0.87




250
0.99










INDUSTRIAL APPLICABILITY
As described hereinbefore, the present invention provides promoter nucleic acid molecules that induce gene expression in specific response to lysine, and is thus useful for developing high-titer bacterial strains expressing high levels of a gene encoding a protein of interest.

## Claims

1. A method of inducing expression of a target gene operably linked to a L-lysine-inducible promoter, comprising: i) transforming a host cell with a vector comprising the target gene operably linked to the L-lysine-inducible promoter, wherein the L-lysine-inducible promoter consists of the nucleotide sequence of SEQ ID NO: 4; and ii) culturing the transformed host cell in a medium supplemented with L-Lysine under conditions, wherein said target gene is expressed.

2. The method according to claim 1, wherein the host cell is a coryneform bacterium.