KR20110102752A - Microorganisms producing l-amino acids and process for producing l-amino acids using the same - Google Patents

Abstract

The present invention is transformed to use sucrose as the main carbon source, and has the ability to produce L-amino acids, microorganisms using the sucrose non-PTS system and sucrose PTS system at the same time and to produce L-amino acid using the microorganisms It is about a method.

Description

Microorganisms producing L-amino acids and process for producing L-amino acids using the same}

The present invention relates to a microorganism transformed to produce a high yield of L-amino acid using sucrose as the main carbon source and a method for producing L-amino acid using the same. Specifically, a microorganism transformed to use sucrose as a main carbon source and having the ability to produce L-amino acid, and simultaneously using a sucrose non-PTS system and a sucrose PTS system (phosphtransferase system) and L- A method for producing amino acids.

As biofuel development accelerates recently, demand for starch sugar, which is mainly used in the fermentation industry, is increasing rapidly, and grain prices including starch sugar are soaring due to crop failure due to abnormal weather. When molasses containing a large amount of sucrose or sucrose, which is cheaper than starch sugar, is used as a carbon source of the fermentation industry, there is an advantage of securing a higher cost competitiveness. About 50% of Escherichia coli in nature has the ability to metabolize sucrose, but E. coli K12 strains, B strains, and C strains, which are mainly used in the fermentation industry, have no sucrose magnetization ability ( Mol . Microbiol) . . (1988) 2:. 1-8 , Can J. Microbiol (1999) 45:. 418-422). Therefore, identifying the genes related to sucrose magnetization ability and improving them to secure enhanced sucrose magnetization related genes and introducing them into sucrose non-magnetic industrial Escherichia coli to produce the desired metabolite is the most important research in fermentation industry. One of the topics.

Sucrose-enabled systems can be largely divided into sucrose non-PTS system and sucrose PTS system.

First, the sucrose non-PTS system uses a proton symport-type sucrose permease, and the csc gene family including cscB encoding such sucrose permease is well known. Known. The csc gene family includes cscB (proton symport-type sucrose permease), cscK (fructokinase), cscA (sucrose hydrolase), cscR (LacI-related sucrose-specific repressor) ( J. Bacteriol . (2002) 184: 5307-5316).

cscB is a proton simport type sucrose permease, the most characteristic of the sucrose non-PTS system compared to the sucrose PTS system. Unlike the sucrose PTS system, cscB uses PEP as an energy source. External sucrose is introduced into the cells by a concentration gradient of hydrogen ( J. Bacteriol . (2002) 184: 5307-5316). Sucrose introduced into cells is hydrolyzed to glucose and fructose by cscA, a sucrose hydrolase.

Intracellular glucose is converted to glucose-6-phosphate by glucokinase, and fructose is fructose-6-phosphate by fructokinase CscK or mak. (fructose-6-phosphate) is converted into the process together.

ScrK and Mak (mannokinase) of the sucrose PTS system are known as proteins having fructokinase activity such as CscK. CscK has 74% amino acid homology with ScrK derived from pUR 400 ( J. Bacteriol . (2002) 184: 5307-5316) and no homology with Mak. On the other hand, Mak is a gene contained in the chromosome of E. coli K12 strain, and kinase converts hexose, including mannose and fructose, into 6-phospho-ester form. Activity ( J. Mol . Microbiol . Biotechnol. (2001) 3: 355-359, Biochemistry (2005) 44: 10776-10783).

As part of the effort to develop microorganisms that use sucrose efficiently, the inventors of the present invention are searching for genes that are expected to be involved in sucrose metabolism. Mannokinase will play an important role in sucrose metabolism. Turned out to be true. Even when the intact csc gene group was introduced into E. coli, which was inactivated by mannokinase, it was confirmed that the sucrose utilization rate was reduced compared to wild-type E. coli, which was not inactivated by mannokinase (Application No. KR10-2009-0012048).

Next, the sucrose PTS system is known to introduce sucrose into cells with external sucrose using PEP as an energy source. scr gene family is scrK (fructokinase), scrY (sucrose porin), scrA (sucrose -specific EIIBC component), scrB (sucrose-6-phosphate hydrolase), scrR consists of five genes (LacI-related sucrose-specific repressor ) , and, s cr mechanism of action of the genes External sucrose is introduced into the periplasmic space through ScrY, an outer membrane protein (OMP), and the sucrose is sucrose PTS fur containing ScrA (sucrose-specific EIIBC component). Mises enter the cells in the form of sucrose-6-phosphate. Sucrose-6-phosphate is hydrolyzed to glucose-6-phosphate and fructose by sucrose-6-phosphate hydrolase (ScrB), and fructose is ScrK (fructokinase) converts fructose-6-phosphate into glycolysis with glucose-6-phosphate ( J. Biotechnol . (2001) 92: 133-158). Representative examples include the scr gene group present on the chromosome of Gram-negative microorganisms Klebsiella pneumoniae and Erwinia amylovora . I've been doing a lot of research to make sure that Scr Gene groups were transformed into E. coli K12 to give sucrose magnetization ability to produce amino acids (Patent No. KR 10-2009-018127).

In the use of sucrose, it would be more desirable to use a sucrose non-PTS system that does not consume PEP (phosphoenolpyruvate) upon influx of sucrose rather than a sucrose PTS system. PEP is an important intermediate in the central metabolic pathway, which acts as a phosphate donor in the sugar PTS system, converts it into pyruvate and enters the TCA circuit to provide reducing power for ATP production. It also feeds the intermediates produced during metabolism into the carbon backbone for biosynthesis, acting as a direct precursor of several amino acids or oxaloacetate (OAA) ( Metab . Eng . (2002) 4: 124-137, Microb. Cell Fact . (2005) 4:14). In particular, OAA is used as a carbon skeleton of amino acids such as threonine, isoleucine methionine, lysine, asparagine, aspartic acid (US Patent 6960455).

Most of the PEP is known to be consumed most by the sugar PTS system. The minimum amount of PEP consumed by a sugar PTS system in a minimal medium containing glucose as a carbon source amounts to 50% of the total PEP ( Microb . Cell) . Fact . (2005) 4:14). Therefore, if the sugar non-PTS system is used instead of the sugar PTS system, the intracellular PEP pool can be increased and the productivity and yield can be improved by using the increased PEP for the biosynthesis of fermentation products ( Biotechnol . Bioeng) . . (2001) 74:.. 364-375, Appl Microbiol Biotechnol (2001) 57:.. 186-191, Metab Eng (2005) 7:. 70-87).

Sucrose non-PTS systems can improve yield by increasing the PEP pool, but have the disadvantage of not being able to efficiently use low concentrations of sucrose. E. coli introduced with the csc gene family has a logarithmic proliferation (doubling time) of 20 hours in a medium containing less than 0.2% sucrose ( J. Bacteriol . (2002) 184: 5307-5316).

However, the sucrose PTS system has an advantage of efficiently introducing low sucrose to a low concentration. However, sucrose is consumed by inducing sucrose to reduce the PEP pool in the cell. The inventors of the present invention, while continuing to research to solve this problem, invented a variety of strains using the sucrose non-PTS system and the sucrose PTS system at the same time to produce a strain that uses sucrose efficiently at low concentrations. In addition, the present inventors have conducted research to search for genes that supplement oxaloacetate (OAA), which is required for biosynthesis or amino acid production during growth, as part of the development of microorganisms with improved yield and productivity using sucrose efficiently. While performing the pyruvate carboxylase gene was introduced. Pyruvate carboxylase has the activity of catalyzing the production of oxaloacetate from pyruvate. Oxaloacetate production can improve productivity and yield by utilizing the carbon skeleton of amino acids such as threonine, isoleucine methionine, lysine, asparagine and aspartic acid, ie, fermentation products. Patent 6960455).

Under this background, the present inventors have developed strains that can efficiently use sucrose even at low concentrations by using a sucrose non-PTS system and a sucrose PTS system simultaneously, and further, by giving pyruvate carboxylase activity Synthesis of oxaloacetate from pyruvate converted in PEP to form a pyruvate pool that enters the TCA circuit, which is fed to the carbon backbone of the amino acid biosynthetic pathway, confirms that the yield is improved, and completes the present invention. It became.

One object of the present invention is to simultaneously use an Escherichia microorganism, preferably a sucrose non-PTS system and a sucrose PTS system, which produce L-amino acids using sucrose as the main carbon source, and further pyruvate It is to provide an Escherichia microorganism endowed with the activity of carboxylase.

Another object of the present invention is to provide a method for producing L-amino acid using the Escherichia microorganism.

As one aspect for achieving the above object, the present invention is characterized in that the sucrose non-magnetic non-microbial to give the co-availability of the sucrose non-PTS system and the sucrose PTS system, sucrose magnetization and increased L It relates to an Escherichia microorganism having an amino acid producing ability.

In the present invention, the sucrose non-PTS system has the activity of sucrose permease, sucrose hydrolase, sucrose transcriptional regulator, and mannokinase, and the sucrose PTS system is sucrose PTS permease and sucrose. It is said to have cross PTS hydrolase activity. The sucrose permease of the sucrose non-PTS system used in the present invention has an activity of introducing external sucrose into cells, and sucrose hydrolase hydrolyzes sucrose introduced into cells into glucose and fructose. Mannokinase has the activity of converting fructose to fructose-6-phosphate. Sucrose PTS permease of the sucrose PTS system has the activity of introducing external sucrose into the cell in the form of sucrose-6-phosphate, and sucrose PTS hydrolase sucrose-6-phosphate to glucose-6- It has the activity of hydrolysis to phosphate and fructose. The activity of the genes used in the sucrose non-PTS system and the sucrose PTS system is present in microorganisms with sucrose magnetization ability, but not in industrially used industrial E. coli K12 strain, B strain, C strain. Therefore, according to the present invention, by introducing the activity of the genes used in the sucrose non-PTS system and the sucrose PTS system to these industrial E. coli so as to have a sucrose magnetization ability and increased L-amino acid production capacity Can be.

The sucrose non-PTS system used in the present invention includes a gene encoding sucrose permease, a gene encoding sucrose hydrolase, a gene encoding sucrose transcriptional regulator, and a gene encoding mannokinase. .

In the present invention, the gene encoding the sucrose permease, the gene encoding the sucrose hydrolase, and the gene encoding the sucrose transcriptional regulator refers to a gene obtained from a microorganism having sucrose magnetization ability, preferably sucrose Genes obtained from Escherichia microorganisms having Ross magnetizing ability, most preferably genes encoding sucrose permease obtained from Escherichia coli ATCC9637 ( Appl . Environ . Microbiol . (1989) 55: 1943-1948) ( cscB , SEQ ID NO: 4), gene encoding sucrose hydrolase ( cscA , SEQ ID NO: 6), gene encoding sucrose transcriptional regulator ( cscR , SEQ ID NO: 7).

Mannokinase (mannokinase) refers to an enzyme having an activity for converting hexose containing mannose to 6-phospho-ester form using ATP. Genes encoding mannokase are sufficient as long as the gene has a nucleotide sequence that can operate in the Escherichia microorganism. One specific example is derived from Escherichia coli, preferably a polynucleotide having the nucleotide sequence of SEQ ID NO: 13 (GenBank accession number AC000091). In particular, the nucleotide sequence encoding the manokinase can be modified to some extent as long as it maintains its activity. Those skilled in the art will readily understand that the nucleotide sequence of which at least 70% homology is maintained by such an artificial modification is equivalent to that derived from the nucleotide sequence of the present invention as long as it retains the activity of the gene of interest in the present invention. will be.

In the present invention, the sucrose PTS system includes a gene encoding sucrose PTS permease and a gene encoding sucrose PTS hydrolase. The genes encoding sucrose PTS permease and the genes encoding sucrose PTS hydrolase refer to genes obtained from a microorganism having sucrose magnetizing ability, and preferably refers to a microorganism gene of the genus Klebsiella with sucrose magnetizing ability. , More preferably the gene obtained from Klebsiella pneumoniae ATCC700721, most preferably the gene encoding the sucrose PTS permease obtained from Klebsiella pneumoniae ATCC700721 ( scrA , SEQ ID NO: 21), number Genes encoding cross hydrolases ( scrB , SEQ ID NO: 22).

In the present invention, a method of enhancing activity for simultaneously using genes encoding a sucrose non-PTS system and a sucrose PTS system of an Escherichia microorganism having L-amino acid producing ability is various methods well known in the art. It is possible to apply. For example, polynucleotides containing nucleotide sequences encoding non-PTS and sucrose PTS systems can be additionally inserted into chromosomes and introduced into vector systems to increase copy number. There may be a method of replacement, a method of introducing a mutation into a promoter and a method by gene mutation, a method using random mutagenesis or site-directed mutagenesis.

In a specific embodiment of the sucrose non-PTS system according to the invention hydroxylamine in the base sequence comprising a gene encoding a sucrose permease, sucrose hydrolase, sucrose transcriptional regulator, and a mannokase ( how to handle the hydroxylamine) causing random mutations (methods Enzymol . (1991) 194: 302-318) was used to improve the usability in low concentration sucrose medium. The sucrose non-magnetizing Escherichia spp. Microorganism retains the activity of sucrose permease, sucrose hydrolase, a sucrose transcriptional regulator ( cscR ', SEQ ID NO: 17) in which the 130th amino acid is substituted for histidine in tyrosine. The method comprises introducing a sucrose permease, a sucrose hydrolase, a mutated sucrose transcriptional regulator, and a nucleic acid sequence encoding a mannokase into a vector regulator (pAcscBAR'-M, SEQ ID NO: 28). The recombinant vector was prepared by transforming Escherichia microorganisms having sucrose non-magnetic L-amino acid production capacity. A method comprising a nucleotide sequence encoding sucrose permease, sucrose hydrolase, a mutated or unmutated sucrose transcriptional regulator, and a mannokase with enhanced activity is applied by various methods well known in the art. It is possible. Contains a base sequence encoding sucrose permease, sucrose hydrolase, and a mutated or unmodified sucrose transcriptional regulator in an Escherichia genus microorganism already having enhanced activity of mannokinase L-amino acids There may be a method of transforming the vector to. Or an Escherichia microorganism having L-amino acid production capacity transformed with a vector comprising a sucrose permease, sucrose hydrolase, and a nucleotide sequence encoding a mutated or unmuted sucrose transcriptional regulator. There may be a way to enhance mannokinase activity. In a specific embodiment of the present invention, a vector comprising a nucleotide sequence encoding a sucrose permease, a sucrose hydrolase, a mutated or unmuted sucrose transcriptional regulator and a nucleotide sequence encoding a mannokase L- By transforming Escherichia genus microorganisms having amino acid production capacity, the activity of mannokinase was enhanced, and the sucrose permease, sucrose hydrolase, and sucrose transcriptional regulators were retained.

In a specific embodiment of the sucrose PTS system according to the present invention by replacing the promoters of sucrose PTS permease and sucrose PTS hydrolase enhanced activity, encoding sucrose PTS permease, sucrose PTS hydrolase The nucleic acid sequence was introduced into a vector (pC-Ptrc-scrAB, SEQ ID NO: 29), and the recombinant vector was produced by transforming Escherichia microorganisms having sucrose non-magnetic L-amino acid production ability. In addition, additional chromosome insertion, replacement with a strong promoter, introduction of mutations into the promoter and by mutations, random mutagenesis or site-directed mutagenesis ) Can be used.

In a more preferred embodiment, the copy number is increased by introducing a nucleotide sequence encoding these into the vector to enhance the activity of the sucrose non-PTS system and the sucrose PTS system of Escherichia microorganisms having L-amino acid production capacity. Or promoter replacement. Recombinant vectors using nucleotide sequences encoding the sucrose non-PTS system and the sucrose PTS system are transformed into Escherichia microorganisms capable of producing L-amino acids. E. coli microorganisms with enhanced activity can be prepared.

      In another aspect of the present invention, the present invention introduces a nucleotide sequence encoding them to the vector to enhance the activity of the sucrose non-PTS system and the sucrose PTS system, and further inhibits the activity of pyruvate carboxylase The present invention relates to an Escherichia microorganism having sucrose magnetization and increased L-amino acid producing ability. The method of imparting the activity of the pyruvate carboxylase is more enhanced than the existing activities such as inserting into the chromosome and increasing the copy number by introducing into the vector system, replacing it with a strong promoter, gene mutation, and introducing mutation into the promoter. It is included.

In the present invention, pyruvate carboxylase refers to a peptide having an activity that catalyzes the production of oxaloacetate from pyruvate. The activity of the pyruvate carboxylase is Bacillus subtillus , Brevibacterium lactofermentum ( Brevibacterium) lactofermentum) and Corynebacterium Com (corynebacterium glutamicum), the presence of microorganisms, such as, but industrial E. coli K12 strain that is industrially used, B strain, W is absent strains.. (Microbiology 143: 1095-1103 ( 1997), Appl Environ Microbiol (2000) 66:. 1844 -1850). Therefore, the gene encoding the pyruvate carboxylase used in the present invention refers to a microbial gene of Corynebacteria, more preferably to a gene obtained from Corynebacteria, most preferably a lysine producing strain (KFCC 10881, Korea It refers to a gene encoding a pyruvate carboxylase obtained from (Patent No. 25).

In the present invention, the method of enhancing the activity of pyruvate carboxylase is applicable to various methods well known in the art. For example, the method further increases the number of copies by inserting a polynucleotide containing a nucleotide sequence encoding pyruvate carboxylase into a chromosome and introducing it into a vector system, a method of replacing with a strong promoter, There may be a method of introducing a mutation into the promoter and a method by genetic variation.

In a preferred embodiment, the activity of the pyruvate carboxylase was replaced by enhancing the promoter, and as shown in the above specific example, the nucleic acid sequence encoding the pyruvate carboxylase was vectored (pB-Ptrc-pyc, SEQ ID NO: 30). ) And the transformed vector was further transformed into Escherichia microorganisms having enhanced activity of the sucrose non-PTS system and the sucrose PTS system. Or additionally inserted into the chromosome, replaced with a strong promoter, introduced into the promoter and by mutations, random mutagenesis or site-directed mutagenesis Can be used.

In another preferred embodiment, the sucrose non-PTS system and the sucrose PTS system having L-amino acid production capacity are encoded to enhance pyruvate carboxylase activity in Escherichia microorganisms having enhanced activity. By introducing a nucleotide sequence into a vector, a method of increasing the copy number or replacing the promoter can be used. In the same manner as above, a recombinant vector having a base sequence encoding the sucrose non-PTS system and the sucrose PTS system is transformed into an Escherichia microorganism having L-amino acid production capacity, and further pyruvate carbide is produced. Escherichia microorganisms with enhanced cyclase activity can be prepared.

In the present invention, the term "vector" refers to a sucrose permease, sucrose hydrolase, a sucrose transcriptional regulator, and a gene encoding a mannokinase as a host cell, a sucrose non-magnetizing Escherichia spp. Nucleic acid compounds used to provide Escherichia microorganisms, including conventional essential expression elements for expressing a protein of interest. Specifically, it has regulatory sequences such as promoters, operators, initiation codons, termination codons, and polyadebolation sequences, and various modifications thereof are possible. It may also include an enhancer sequence or a secretion sequence. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A can be used as the phage vector or cosmid vector, and pBR, pUC, and pBluescriptII systems are used as plasmid vectors. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. The vector usable in the present invention is not particularly limited and known expression vectors can be used. Preferably, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used. Most preferably, pACYC177, pCL, pCC1BAC vectors can be used.

The term "transformation" in the present invention means to introduce the gene into the host cell to be able to express in the host cell. Transformed genes include all of them, as long as they can be expressed in the host cell, regardless of whether they are inserted into or located outside the chromosome of the host cell. In addition, the gene is a polynucleotide capable of encoding a polypeptide and includes DNA and RNA. The gene may be introduced in any form as long as it can be introduced into and expressed in a host cell. For example, the gene may be introduced into a host cell in the form of an expression cassette, which is a polynucleotide construct containing all the elements necessary for its expression. The expression cassette typically includes a promoter, transcription termination signal, ribosomal binding site and translation termination signal operably linked to the gene. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the gene may be introduced into the host cell in its own form or in the form of a polynucleotide structure, and may be operably linked to a sequence required for expression in the host cell.

The microorganism of the present invention can produce L-amino acid, and can use a sucrose non-PTS system and a sucrose PTS system at the same time, and further endow the activity of pyruvate carboxylase and have a sucrose magnetization ability. Say. Accordingly, the microorganism of the present invention includes both prokaryotic and eukaryotic microorganisms as long as they retain the above characteristics. For example, Escherichia (Escherichia), An air Winiah (Erwinia) genus, Serratia marcescens (Serratia) genus, Providencia (Providencia) genus Corynebacterium (Corynebacterium) in and Brevibacterium (Brevibacterium) Microorganism strains belonging to the genus may be included. Preferably, it is a microorganism belonging to the genus Escherichia, more preferably E. coli.

In the present invention, "L-amino acid" is L-threonine, O-succinyl-homoserine, O-acetyl-homoserine, O-Acetyl-homoserine, L-methionine, L-lysine, L-homoserine, L-isoleucine, L-valine, L-tryptophan and the like. Preferably the L-amino acid is L-threonine, O-succinyl-homoserine, O-acetyl-homoserine and L-tryptophan.

In one specific embodiment, the inventors of the present invention, Escherichia coli having L-threonine production ability, the vector having the nucleotide sequence of SEQ ID NO: 28 including pAcscBAR'-M gene group, SEQ ID NO: 29 including pC-Ptrc-scrAB gene group E. coli ABA5G strain having the ability to produce L-threonine as a vector having the nucleotide sequence of SEQ ID NO: 30 including pB-Ptrc-pyc gene group Constitution, isoleucine leaky, α-amino-β-hydroxy baric acid resistance, 2-aminoethyl-1-cysteine resistance, 1-azetidine-2-carboxylic acid resistance) Was named Escherichia coli CA03-0392P and deposited on February 5, 2010, with the accession number KCCM11060P at the Korea Microbial Conservation Center, an international depository organization located at 361-221, Hongje 1-dong, Hongdae, Seoul, Korea.

As another aspect of the present invention, the present invention is to produce a L-amino acid by culturing the Escherichia microorganism having the sucrose magnetizing ability and L-amino acid production capacity in a medium containing sucrose as the main carbon source It is about a method.

Specifically, the present invention comprises the steps of inoculating the culture medium containing a part or all of the sucrose as a carbon source by inoculating microorganisms having the above-mentioned sucrose magnetizing ability to produce L-amino acid; And separating the L-amino acid from the culture, and relates to a method for producing L-amino acid using the Escherichia microorganism from a carbon source including sucrose.

The culturing process of the present invention can be made according to suitable media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the strain selected. Examples of the culture method include, but are not limited to, batch, continuous and fed-batch cultivation. The medium used for cultivation should suitably meet the requirements of the particular strain.

In the medium used in the present invention, sucrose may be used as the main carbon source, molasses containing a large amount of sucrose may also be used as the carbon source, and other appropriate amounts of the carbon source may be used in various ways without limitation. In addition, examples of nitrogen sources that can be used are organic nitrogen sources and urea such as peptone, yeast extract, gravy, malt extract, sewage dipping solution, and soybean wheat, such as ammonium sulfate, ammonium chloride, ammonium phosphate, anmonium carbonate, and ammonium nitrate Inorganic nitrogen sources may be included. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as personnel. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins and appropriate precursors may be included. These media or precursors may be added batchwise or continuously to the culture.

During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during culturing, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen, or carbon dioxide gas may be injected without injecting gas to maintain anaerobic and unaerobic conditions. The temperature of the culture is usually 27 ° C to 37 ° C, preferably 30 ° C to 35 ° C. The incubation period can continue until the desired amount of useful material is obtained, preferably 10 to 100 hours.

The method for collecting and recovering the L-amino acid produced in the culturing step of the present invention may be carried out using a suitable method known in the art according to a culture method, for example, a batch, continuous or fed-batch culture method. L-amino acids can be collected.

By using the microorganism of the genus Escherichia which has the ability to make the sucrose according to the present invention as a carbon source efficiently, and has the ability to produce a sucrose non-PTS system and a sucrose PTS system at the same time In the production of L-amino acids, in addition to starch sugar, which is mainly used in the fermentation industry, sucrose can be used as a main carbon source to flexibly cope with rapidly changing world grain prices, and efficiently use sucrose even at low concentrations. L-amino acid can be produced.

1 is a schematic of the recombinant plasmid pAcscBAR'-mak comprising pAcscBAR'-M.
2 is a schematic of recombinant plasmid pC-Ptrc-scrAB comprising Ptrc-scrAB.
Figure 3 is a schematic of the recombinant plasmid pB-Ptrcpyc containing Ptrc-pyc.
Figure 4 shows ABA5G / pAcscBAR'-M, ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, pB-Ptrcpyc as a carbon source under a medium of sucrose 7% When incubated, the hourly growth rate is shown as absorbance.
Figure 5 shows ABA5G / pAcscBAR'-M, ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, pB-Ptrcpyc as a carbon source under a medium of sucrose 7% When incubated, it shows the rate of sugar consumption.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One: Sucrose  Availability genes csc regulon Acquisition and Cloning and  Sequencing

The cscBKAR gene, a sucrose non-PTS system, was amplified by PCR using the chromosome of E. coli W strain (ATCC9637) purchased from the American Type Resource Collection. Using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, all four genes continuously present in the genome were amplified by a single polynucleotide into a single polynucleotide, and the primer of SEQ ID NO: 1 was a restriction enzyme EagI. Has a site, and the primer of SEQ ID NO: 2 is XbaI It has a site.

SEQ ID NO: 5'-CTTACGGCCGGAGTACATTTGAGCGACTGT-3 '

SEQ ID NO: 5'-CGACTCTAGACTCGTTGGCGAGAACAGAGG-3 '

The conditions of the PCR method were denatured at 94 ° C. for 3 minutes, followed by 94 ° C 30 seconds denaturation, 56 ° C. 30 seconds annealing, and 72 ° C. 5 min polymerization 25 times, followed by polymerization at 72 ° C. for 7 minutes. As a result, 5179 bp of polynucleotide could be obtained. After the obtained polynucleotides by a restriction enzyme treatment of the EagI, XbaI plated on Mac konki solid media (MacConkey agar plate) containing 1% sucrose and then was cloned to EagI, XbaI place of pACYC184 vector transformed into E. coli DH5α It was. After colonies having a dark purple color among the colonies were selected, plasmids were obtained using a plasmid miniprep method known in the art.

The obtained plasmid was named pAcscBKAR, and EagI , XbaI When the DNA sequence of the cloned cscBKAR (SEQ ID NO: 3) is determined, the 141st to 1388th sequences are the cscB gene (SEQ ID NO: 4), and the 1460th to 2374th sequences are the cscK gene (SEQ ID NO: 5). , 2590 th to 4023 nucleotides were identified as cscA gene (SEQ ID NO: 6), 4031 th to 5026 nucleotides were identified as the cscR gene (SEQ ID NO: 7).

Example  2. pAcscBAR -M production

The preparation used a method of cloning the ma k gene in pAcscBAR. The preparation of pAcscBAR was amplified with polynucleotides of the cscB site from which the cscK site was removed using the primers of SEQ ID NOs: 8 and 9.

SEQ ID NO: 5'-CGCGATATCTAGCATATGCCGGGTACCGCACTAGTTGAGA

GTAAACGGCGAAGT-3 '

SEQ ID NO: 5'-ATTCGGCCGGAGCCCTGCAGGTGCACGAGTACATTTGA

GCGACTGT-3 '

The PCR method was denatured for 3 minutes at 94 ° C, followed by repeated 25 times of 94 ° C denaturation, 56 ° C 30 seconds annealing, 72 ° C 1 minute 30 seconds polymerization, and polymerization at 72 ° C for 7 minutes. As a result, a polynucleotide of 1521 bp could be obtained. The resulting polynucleotide and pAcscBKAR were cloned after restriction enzyme treatment with EcoRV and EagI , respectively, and transformed into E. coli DH5α. Colonies containing pAcscBAR were selected by PCR using colonies grown in LB medium, and plasmids were obtained using a plasmid miniprep method known in the art. The base sequence (SEQ ID NO: 10) analysis of cscBAR linked to the XbaI and EagI sites of the obtained pAcscBAR plasmid confirmed that there was no mutation.

Subsequently, the primers of SEQ ID NOS: 11 and 12 and the chromosome of E. coli W3110 were used as templates, and then amplified with polynucleotides containing mak , and cloned into the restriction enzyme sites of PstI and EagI of pAcscBAR.

SEQ ID NO: 5'-CACTGCAGTGGGGTAAATGCCATCG-3 '

SEQ ID NO: 12'-AACGGCCGTCTCGGTGCTCATTACT-3 '

The conditions of the PCR method were denatured for 3 minutes at 94 ℃, 94 ℃ 30 seconds denaturation, 56 ℃ 30 seconds annealing, 72 ℃ 1 minute 30 seconds polymerization was repeated 25 times, and then polymerization was carried out at 72 ℃ 7 minutes. As a result, polynucleotide (SEQ ID NO: 13) of 1388 bp could be obtained. The resulting polynucleotide and pAcscBAR were subjected to restriction enzyme treatment with PstI and EagI , respectively, and cloned to transform E. coli DH5α. Colonies containing pAcscBAR-M were selected by PCR using colonies grown in LB medium, and plasmids were obtained using a plasmid miniprep method known in the art. The base sequence (SEQ ID NO: 14) analysis of cscBAR-M linked to the XbaI and EagI sites of the obtained pAcscBAR-M plasmid confirmed that there was no mutation.

Example  3. cscBAR - mak of mutation  Introduction

Chemical random mutagenesis was performed to obtain mutated sucrose availability genes by treatment with hydroxylamine to improve sucrose availability as well as to enhance threonine productivity ( Method Enzymol . 1991) 194: 302-318. First, mutagenisis solution was prepared by adding 1 M hydroxylamine, 2 mM EDTA, 100 mM sodium chloride, and 50 mM sodium pyrophosphate, and then the desired target was added to 1 ml mutagenic solution. 25 μl of DNA was added at a concentration of 0.2 mg / ml. Amplified with a polynucleotide containing cscBAR-M using the primers of SEQ ID NOs: 15 and 16 and the pAcscBAR-M plasmid as a template, a DNA fragment containing 5887 bp of cscBAR-M was used as the target DNA.

SEQ ID NO: 15'-ATATCTAGACTCGTTGGCGAGAACAGAG-3 '

SEQ ID NO: 5'-AAT TCTAGATGCTCATTACTTATTGCCG-3 '

DNA fragments to which the mutagenesis solution was added were purified using a purification column (500 μl) at 75 ° C. for 30 minutes, 1 hour, 2 hours, and 4 hours. The pACYC177 vector, which was treated with hydroxylamine-treated DNA and the restriction enzyme of XbaI , was linked using a T4 DNA ligase and transformed into a threonine producer ABA5G. The transformed strains were plated for exclusion of Macconkey solids containing 0.1% low sucrose to select dark purple colonies after one day incubation at 37 ° C.

The selected colonies were evaluated for threonine productivity and sucrose utilization rate in titer medium. Finally, one strain having the highest threonine productivity and sucrose availability was selected. From the selected strains, plasmids were obtained using a conventional plasmid miniprep method, followed by sequencing. As a result, the 388th base of cscR was substituted for C to T. Thus, the 130th amino acid mutated from histidine to tyrosine, and the sucrose transcriptional regulator with this mutation is encoded by the nucleotide sequence of SEQ ID NO. .

Example  4. Sucrose  Availability gene promoter replaced scrAB Acquisition and Cloning and  Sequence

The scrKYABR gene, a sucrose PTS system, was amplified by PCR using the chromosome of Klebsiella pneumoniae (ATCC700721D-5) purchased from the American Type Center for Biotechnology. ScrAB of Klebsiella pneumoniae amplified all of the two genes continuously present in the genome into a single polynucleotide by PCR using the primers SEQ ID NO: 18 and SEQ ID NO: 19.

SEQ ID NO: 18'-AATA CCATGGATTTTGAACAGATTTCCC-3 '

SEQ ID NO: 19'5'-ATAA TCTAGA GTTTGGTTTTCATCAGCAC-3 '

SEQ ID NO: 18 primer has a restriction enzyme NcoI site, and primer of SEQ ID NO: 19 is XbaI It has a site. Primers used at this time were prepared based on information on scrAB and surrounding sequences of Klebsiella pneumoniae (KEGG organism, kpn) registered in KEGG (Kyoto Encyclopedia of Genes and Genomes).

The conditions of the PCR method were denatured at 94 ° C. for 3 minutes, followed by 94 ° C 30 seconds denaturation, 56 ° C. 30 seconds annealing, and 72 ° C. 2 min polymerization 25 times, followed by polymerization at 72 ° C. for 7 minutes. As a result, in the case of Klebsiella pneumoniae was able to obtain a polynucleotide of 2799 bp. Each polynucleotide Mac konki solid is NcoI, and then the restriction enzyme digestion of the XbaI and cloned in NcoI, XbaI place of pCL-trc vector is transformed into E. coli DH5α 1% including a cross media obtained (MacConkey agar plate ). The colonies having a dark purple color among the colonies were selected and plasmids were obtained using a plasmid miniprep method known in the art.

When the plasmid containing the obtained scrAB of Klebsiella pneumoniae was named pC-Ptrc-scrAB and the DNA sequence of scrAB (SEQ ID NO: 20) was replaced with the trc promoter cloned in place of NcoI and XbaI The 261 th to 1631 nucleotide sequences were identified as the scrA gene (SEQ ID NO: 21), and the 1631 th to 3031 nucleotide sequences were identified as the scrB gene (SEQ ID NO: 22).

Example  5. Promoter replaced pyc Acquisition and Cloning and  Sequence

In order to secure the pyc gene, a strain having a production capacity derived from Corynebacterium glutamicum, preferably a chromosome of KFCC 10881 strain (Korean Patent Publication No. 10-2009-0084099, Korean Patent Registration No. 10-0798118) is used as a template. Genes were amplified on the genome by PCR using the primers 23 and 24. The primer of SEQ ID NO: 23 has a restriction enzyme NcoI site, and the primer of SEQ ID NO: 24 is HindIII It has a site. The primer used at this time secured the nucleotide sequence information of the pyc gene based on the National Institute of Health Gene Bank (NIH GenBank).

SEQ ID NO: 23'-AATACCATGGCGACTCACACATCTTC-3 '

SEQ ID NO: 24'-ATTAAGCTTTAAACCAAGACGGCA-3 '

The conditions of the PCR method were denatured at 94 ° C. for 3 minutes, and then repeated 25 times at 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, annealing at 72 ° C. for 3 minutes, and then polymerized at 72 ° C. for 7 minutes. As a result, in the case of the lysine producing strain, 3423 bp of polynucleotide could be obtained (SEQ ID NO: 25). After then, each of the polynucleotides for NcoI, restriction enzyme digestion of the HindIII obtained by cloning a NcoI, HindIII place of pSE380 vector vector transformed into E. coli DH5α screening the colonies using the plasmid mini-program repbeop known in the normally obtained The plasmid was named pSE380-Ptrc-pyc. Using the prepared pSE380-Ptrc-pyc plasmid as a template, genes were amplified on the genome by PCR using primers of SEQ ID NO: 24 and SEQ ID NO: 26. The primer of SEQ ID NO: 24 has a restriction enzyme HindIII site, and the primer of SEQ ID NO: 26 is HindIII It has a site.

SEQ ID NO: 26'-TATAAGCTTGGCGTCAGGCAGCCATCG-3 '

The conditions of the PCR method were denatured at 94 ° C. for 3 minutes, and then repeated 25 times at 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, annealing at 72 ° C. for 3 minutes, and then polymerized at 72 ° C. for 7 minutes. As a result, in the case of a lysine producing strain, a polynucleotide of 3810 bp could be obtained. Each polynucleotide obtained after the restriction enzyme treatment of HindIII were obtained by using plasmid mini-program repbeop known in the normally then cloned into the HindIII to the seat of the BAC vector is transformed into E. coli DH5α and colonies screened.

The plasmid containing pyc of the obtained lysine producing strain was named pB-Ptrc-pyc, and the DNA sequence (SEQ ID NO: 27) of the trc promoter replaced pyc cloned at the HindIII site was determined.

Example  6. Threonine To the producer pAcscBAR ' -M, pC - Ptrc - scrAB , pB - Ptrcpyc  L- through introduction Threonine  Increased productivity

When pAcscBAR'-M (SEQ ID NO: 28), pC-Ptrc-scrAB (SEQ ID NO: 29) and pB-Ptrc-pyc (SEQ ID NO: 30) were introduced into threonine-producing E. coli, they were grown using sucrose. In addition, threonine producer, ABA5G strain (a strain produced by NTG mutagenesis, methionine requirement, isoleucine leaky) ), α-amino-β-hydroxy baric acid resistance, 2-aminoethyl-1-cysteine resistance, 1-azetidine-2-carboxylic acid resistance) to evaluate flask titer. Strains incubated overnight in LB solid medium in a 33 ° C. incubator were inoculated with one platinum in 25 mL titer medium of Table 1, and then incubated for 51 hours in a 33 ° C., 200 rpm incubator, the results of which are shown in Table 2 It was.

Composition Concentration (per liter) Sucrose        70 g KH ₂ PO ₄ 2 g (NH ₄ ) ₂ SO ₄ 25 g MgSO ₄ ? 7H ₂ O 1 g FeSO ₄ ˜7H ₂ O   5 mg MnSO ₄ ? 4H ₂ O 5 mg Yeast Extract 2 g Calcium carbonate 30 g pH 6.8

Mother strain ABA5G OD Remnant
(g / L) L-Threonine
(g / L) pAcscBAR '-M 10.8 44 12.3 pAcscBAR '-M, pC-Ptrc-scrAB 29 0 22.8 pAcscBAR'-M, pC-Ptrc-scrAB, pB-Ptrcpyc 27 0 24.7

As shown in Table 2, the sucrose non-PTS system strain (ABA5 / pAcscBAR'-M) uses 26 g / L sucrose after 51 hours of incubation and 12.3 g / L L-threonine. However, strains using the sucrose non-PTS system and the sucrose PTS system simultaneously (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB) use 70 g / L sucrose and 22.8 g / L L- Threonine was produced. In addition, 70 g of the strain (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, pB-Ptrcpyc) using the sucrose non-PTS system and the sucrose PTS system at the same time and additionally given pyruvate carboxylase activity / L sucrose was used and 24.7 g / L L-threonine was produced. When grown in a medium of sucrose 7% conditions, the growth rate per hour is shown as absorbance. In the case of sucrose non-PTS strain (ABA5G / pAcscBAR'-M), the OD ₆₀₀ value is about 11 after 51 hours of incubation, and a strain using the sucrose non-PTS system and the sucrose PTS system simultaneously (ABA5G / pAcscBAR'-M). M, pC-Ptrc-scrAB) or strains using the sucrose non-PTS system and the sucrose PTS system at the same time and further conferred the activity of pyruvate carboxylase (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB , pB-Ptrcpyc), the OD ₆₀₀ value was about 28 at 51 hours of incubation, and the hourly growth rate was shown as absorbance (FIG. 4). In the case of the sugar consumption rate, the sucrose non-PTS system strain (ABA5G / pAcscBAR'-M), sucrose remained in the medium of about 40g / L in 51 hours culture, sucrose non-PTS system and water Strains that simultaneously use cross PTS systems (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB), or strains that simultaneously use the sucrose non-PTS system and the sucrose PTS system and further impart pyruvate carboxylase activity In the case of (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, pB-Ptrcpyc), no sucrose remained in the medium using all sucrose at 51 hours of incubation (see FIG. 5).

That is, a strain using both a sucrose non-PTS system and a sucrose PTS system (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB) or a sucrose non-PTS system and a sucrose PTS system simultaneously and additionally The sucrose utilization rate was lower than that of sucrose non-PTS strain (ABA5G / pAcscBAR'-M). 2.6-fold, threonine productivity increased by 10 g / L.

Therefore, strains using the sucrose non-PTS system and the sucrose PTS system simultaneously (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB) or the sucrose non-PTS system and the sucrose PTS system simultaneously and additionally In the case of strains (ABA5G / pAcscBAR'-M, pC-Ptrc-scrAB, pB-Ptrcpyc) given pyruvate carboxylase activity, it was confirmed that the sucrose availability and productivity of L-threonine were improved.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the embodiments described above are to be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Korea Microorganism Conservation Center (overseas) KCCM11060 20100205

<110> CJ CheilJedang Corporation <120> ESCHERICHIA COLI USING CARBON SOURCES CONTAINING SUCROSE AND          PROCESS FOR PRODUCING L-AMINO ACIDS USING THEM <130> PA090509 / KR <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of cscBKAR <400> 1 cttacggccg gagtacattt gagcgactgt 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of cscBKAR <400> 2 cgactctaga ctcgttggcg agaacagagg 30 <210> 3 <211> 5179 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (5179) <223> polynucleotide containing cscBKAR regulon <400> 3 gagtacattt gagcgactgt accagaacat gaatgaggcg tttggattag gcgattatta 60 gcagggctaa gcattttact attattattt tccggttgag ggatatagag ctatcgacaa 120 caaccggaaa aagtttacgt ctatattgct gaaggtacag gcgtttccat aactatttgc 180 tcgcgttttt tactcaagaa gaaaatgcca aatagcaaca tcaggcagac aatacccgaa 240 attgcgaaga aaactgtctg gtagcctgcg tggtcaaaga gtatcccagt cggcgttgaa 300 agcagcacaa tcccaagcga actggcaatt tgaaaaccaa tcagaaagat cgtcgacgac 360 aggcgcttat caaagtttgc cacgctgtat ttgaagacgg atatgacaca aagtggaacc 420 tcaatggcat gtaacaactt cactaatgaa ataatccagg ggttaacgaa cagcgcgcag 480 gaaaggatac gcaacgccat aatcacaact ccgataagta atgcattttt tggccctacc 540 cgattcacaa agaaaggaat aatcgccatg cacagcgctt cgagtaccac ctggaatgag 600 ttgagataac catacaggcg cgttcctaca tcgtgtgatt cgaataaacc tgaataaaag 660 acaggaaaaa gttgttgatc aaaaatgtta tagaaagacc acgtccccac aataaatatg 720 acgaaaaccc agaagtttcg atccttgaaa actgcgataa aatcctcttt ttttacccct 780 cccgcatctg ccgctacgca ctggtgatcc ttatctttaa aacgcatgtt gatcatcata 840 aatacagcgc caaatagcga gaccaaccag aagttgatat ggggactgat actaaaaaat 900 atgccggcaa agaacgcgcc aatagcatag ccaaaagatc cccaggcgcg cgctgttcca 960 tattcgaaat gaaaatttcg cgccattttt tcggtgaagc tatcaagcaa accgcatccc 1020 gccagatacc cccagccaaa aaacagcgcc cccagaatta gacctacaga aaaattgctt 1080 tgcagtaacg gttcataaac gtaaatcata aacggtccgg tcaagaccag gatgaaactc 1140 atacaccaga tgagcggttt cttcagaccg agtttatcct gaacgatgcc gtagaacatc 1200 ataaatagaa tgctggtaaa ctggttgacc gaataaagtg tacctaattc cgtccctgtc 1260 aaccctagat gtcctttcag ccaaatagcg tataacgacc accacagcga ccaggaaata 1320 aaaaagagaa atgagtaact ggatgcaaaa cgatagtacg catttctgaa tggaatactc 1380 agtgccataa ttacctgcct gtcgttaaaa aattcacgtc ctatttagag ataagagcga 1440 cttcgccgtt tacttctcac tattccagtt cttgtcgaca tggcagcgct gtcattgccc 1500 ctttcgccgt tactgcaagc gctccgcaac gttgagcgag atcgataatt cgtcgcattt 1560 ctctctcatc tgtagataat cccgtagagg acagacctgt gagtaacccg gcaacgaacg 1620 catctcccgc ccccgtgcta tcgacacaat tcacagacat tccagcaaaa tggtgaactt 1680 gtcctcgata acagaccacc accccttctg cacctttagt caccaacagc atggcgatct 1740 catactcttt tgccagggcg catatatcct gatcgttctg tgtttttcca ctgataagtc 1800 gccattcttc ttccgagagc ttgacgacat ccgccagttg tagcgcctgc cgcaaacaca 1860 agcggagcaa atgctcgtct tgccatagat cttcacgaat attaggatcg aagctgacaa 1920 aacctccggc atgccggatc gccgtcatcg cagtaaatgc gctggtacgc gaaggctcgg 1980 cagacaacgc aattgaacag agatgtaacc attcgccatg tcgccagcag ggcaagtctg 2040 tcgtctctaa aaaaagatcg gcactggggc ggaccataaa cgtaaatgaa cgttcccctt 2100 gatcgttcag atcgacaagc accgtggatg tccggtgaca ttcatcttgc ttcagatacg 2160 tgatatcgac tccctcagtt agcagcgttc tttgcattaa cgcaccaaaa ggatcatccc 2220 ccacccgacc tataaaccca cttgttccgc ctaatctggc gattcccacc gcaacgttag 2280 ctggcgcgcc gccaggacaa ggcagtaggc gcccgtctga ttctggcaag agatctacga 2340 ccgcatcccc taaaacccat actttggctg acattttttt cccttaaatt catctgagtt 2400 acgcatagtg ataaacctct ttttcgcaaa atcgtcatgg atttactaaa acatgcatat 2460 tcgatcacaa aacgtcatag ttaacgttaa catttgtgat attcatcgca tttatgaaag 2520 taagggactt tatttttata aaagttaacg ttaacaattc accaaatttg cttaaccagg 2580 atgattaaaa tgacgcaatc tcgattgcat gcggcgcaaa acgccctagc aaaacttcat 2640 gagcaccggg gtaacacttt ctatccccat tttcacctcg cgcctcctgc cgggtggatg 2700 aacgatccaa acggcctgat ctggtttaac gatcgttatc acgcgtttta tcaacatcat 2760 ccgatgagcg aacactgggg gccaatgcac tggggacatg ccaccagcga cgatatgatc 2820 cactggcagc atgagcctat tgcgctagcg ccaggagacg ataatgacaa agacgggtgt 2880 ttttcaggta gtgctgtcga tgacaatggt gtcctctcac ttatctacac cggacacgtc 2940 tggctcgatg gtgcaggtaa tgacgatgca attcgcgaag tacaatgtct ggctaccagt 3000 cgggatggta ttcatctcga gaaacagggt gtgatcctca ctccaccaga aggaatcatg 3060 cacttccgcg atcctaaagt gtggcgtgaa gccgacacat ggtggatggt agtcggggcg 3120 aaagatccag gcaacacggg gcagatcctg ctttatcgcg gcagttcgtt gcgtgaatgg 3180 accttcgatc gcgtactggc ccacgctgat gcgggtgaaa gctatatgtg ggaatgtccg 3240 gactttttca gccttggcga tcagcattat ctgatgtttt ccccgcaggg aatgaatgcc 3300 gagggataca gttaccgaaa tcgctttcaa agtggcgtaa tacccggaat gtggtcgcca 3360 ggacgacttt ttgcacaatc cgggcatttt actgaacttg ataacgggca tgacttttat 3420 gcaccacaaa gctttttagc gaaggatggt cggcgtattg ttatcggctg gatggatatg 3480 tgggaatcgt caatgccctc aaaacgtgaa ggatgggcag gctgcatgac gctggcgcgc 3540 gagctatcag agagcaatgg caaacttcta caacgcccgg tacacgaagc tgagtcgtta 3600 cgccagcagc atcaatctgt ctctccccgc acaatcagca ataaatatgt tttgcaggaa 3660 aacgcgcaag cagttgagat tcagttgcag tgggcgctga agaacagtga tgccgaacat 3720 tacggattac agctcggcac tggaatgcgg ctgtatattg ataaccaatc tgagcgactt 3780 gttttgtggc ggtattaccc acacgagaat ttagacggct accgtagtat tcccctcccg 3840 cagcgtgaca cgctcgccct aaggatattt atcgatacat catccgtgga agtatttatt 3900 aacgacgggg aagcggtgat gagtagtcga atctatccgc agccagaaga acgggaactg 3960 tcgctttatg cctcccacgg agtggctgtg ctgcaacatg gagcactctg gctactgggt 4020 taacataata tcaggtggaa caacggatca acagcgggca agggatccgc gtcactcttc 4080 ccccttcacg accttcaata atatgcaatg cagcttcccg cccgataatg tcatgtggaa 4140 gctgaattgt ggtcagcggc ggtaaaaaca gatgcccgac gccaaccaga ttatcaaagc 4200 ccattacggc gacatcctgc gggattcgta cccccttcgc cagaagaacc tgataagcca 4260 caaaggctgc gcgatcgtta ccacatatca gaacatcaaa atctggtttg cccggtttga 4320 agtgggcatt gagtaaactt gcgagatcgg tgtagtgatc atcacctgtt gccatgtgaa 4380 attgtttcac ctcagccaga tctcgttcag catcacgcca ggcctgctca aatccctgcc 4440 gacgataccc tgttgccaac gcactttccg gtagccagaa gcataacggt tgacgatagc 4500 ccgccgcgag caaatgctgt gttgattcat attgtgcagt gtaatcatca gggatataac 4560 tgggtaacgc tgggtcatcc gccacacagt tcgccaatac aatattttca ccatacagag 4620 actcaggcag cgtgatatgt cgcagcccca ttgtagtata gataatgcca tccggacggt 4680 gggcaagcag ctgacgtgcc gcgcgggcag cgtcatcttc agaaaaaata ttgattaaaa 4740 aactattcca gccgaactcg ctggcggttt gctcaatggc aagcagaata tcaacagaga 4800 aaggagtggt agccgtgtcc tgcgccagca cggcgagagt cgacggctta cgtccttgag 4860 cgcgcatctt acgggcggaa agatcaggaa cataattcag ggtctggatt gcctgcaata 4920 cgcggtcacg cgttgcagga cgcacagatt ctgcattatg catcacccgg gagactgtca 4980 tcatcgacac tcccgccagg cgtgcgacat cctttaatga agccataccc aagccgtttg 5040 ccgtaaaacg ggcactgtag cagaaacaga cgtcactggc gagatccaac gccctatcac 5100 ctgacacagc aatacaataa aaaataacaa taattcccgg acaattgtcc ccagttccgc 5160 ctctgttctc gccaacgag 5179 <210> 4 <211> 1248 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (1248) <223> cscB <400> 4 atggcactga gtattccatt cagaaatgcg tactatcgtt ttgcatccag ttactcattt 60 ctctttttta tttcctggtc gctgtggtgg tcgttatacg ctatttggct gaaaggacat 120 ctagggttga cagggacgga attaggtaca ctttattcgg tcaaccagtt taccagcatt 180 ctatttatga tgttctacgg catcgttcag gataaactcg gtctgaagaa accgctcatc 240 tggtgtatga gtttcatcct ggtcttgacc ggaccgttta tgatttacgt ttatgaaccg 300 ttactgcaaa gcaatttttc tgtaggtcta attctggggg cgctgttttt tggctggggg 360 tatctggcgg gatgcggttt gcttgatagc ttcaccgaaa aaatggcgcg aaattttcat 420 ttcgaatatg gaacagcgcg cgcctgggga tcttttggct atgctattgg cgcgttcttt 480 gccggcatat tttttagtat cagtccccat atcaacttct ggttggtctc gctatttggc 540 gctgtattta tgatgatcaa catgcgtttt aaagataagg atcaccagtg cgtagcggca 600 gatgcgggag gggtaaaaaa agaggatttt atcgcagttt tcaaggatcg aaacttctgg 660 gttttcgtca tatttattgt ggggacgtgg tctttctata acatttttga tcaacaactt 720 tttcctgtct tttattcagg tttattcgaa tcacacgatg taggaacgcg cctgtatggt 780 tatctcaact cattccaggt ggtactcgaa gcgctgtgca tggcgattat tcctttcttt 840 gtgaatcggg tagggccaaa aaatgcatta cttatcggag ttgtgattat ggcgttgcgt 900 atcctttcct gcgcgctgtt cgttaacccc tggattattt cattagtgaa gttgttacat 960 gccattgagg ttccactttg tgtcatatcc gtcttcaaat acagcgtggc aaactttgat 1020 aagcgcctgt cgtcgacgat ctttctgatt ggttttcaaa ttgccagttc gcttgggatt 1080 gtgctgcttt caacgccgac tgggatactc tttgaccacg caggctacca gacagttttc 1140 ttcgcaattt cgggtattgt ctgcctgatg ttgctatttg gcattttctt cttgagtaaa 1200 aaacgcgagc aaatagttat ggaaacgcct gtaccttcag caatatag 1248 <210> 5 <211> 915 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (915) <223> cscK <400> 5 atgtcagcca aagtatgggt tttaggggat gcggtcgtag atctcttgcc agaatcagac 60 gggcgcctac tgccttgtcc tggcggcgcg ccagctaacg ttgcggtggg aatcgccaga 120 ttaggcggaa caagtgggtt tataggtcgg gtgggggatg atccttttgg tgcgttaatg 180 caaagaacgc tgctaactga gggagtcgat atcacgtatc tgaagcaaga tgaatgtcac 240 cggacatcca cggtgcttgt cgatctgaac gatcaagggg aacgttcatt tacgtttatg 300 gtccgcccca gtgccgatct ttttttagag acgacagact tgccctgctg gcgacatggc 360 gaatggttac atctctgttc aattgcgttg tctgccgagc cttcgcgtac cagcgcattt 420 actgcgatga cggcgatccg gcatgccgga ggttttgtca gcttcgatcc taatattcgt 480 gaagatctat ggcaagacga gcatttgctc cgcttgtgtt tgcggcaggc gctacaactg 540 gcggatgtcg tcaagctctc ggaagaagaa tggcgactta tcagtggaaa aacacagaac 600 gatcaggata tatgcgccct ggcaaaagag tatgagatcg ccatgctgtt ggtgactaaa 660 ggtgcagaag gggtggtggt ctgttatcga ggacaagttc accattttgc tggaatgtct 720 gtgaattgtg tcgatagcac gggggcggga gatgcgttcg ttgccgggtt actcacaggt 780 ctgtcctcta cgggattatc tacagatgag agagaaatgc gacgaattat cgatctcgct 840 caacgttgcg gagcgcttgc agtaacggcg aaaggggcaa tgacagcgct gccatgtcga 900 caagaactgg aatag 915 <210> 6 <211> 1434 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (1434) <223> cscA <400> 6 atgacgcaat ctcgattgca tgcggcgcaa aacgccctag caaaacttca tgagcaccgg 60 ggtaacactt tctatcccca ttttcacctc gcgcctcctg ccgggtggat gaacgatcca 120 aacggcctga tctggtttaa cgatcgttat cacgcgtttt atcaacatca tccgatgagc 180 gaacactggg ggccaatgca ctggggacat gccaccagcg acgatatgat ccactggcag 240 catgagccta ttgcgctagc gccaggagac gataatgaca aagacgggtg tttttcaggt 300 agtgctgtcg atgacaatgg tgtcctctca cttatctaca ccggacacgt ctggctcgat 360 ggtgcaggta atgacgatgc aattcgcgaa gtacaatgtc tggctaccag tcgggatggt 420 attcatctcg agaaacaggg tgtgatcctc actccaccag aaggaatcat gcacttccgc 480 gatcctaaag tgtggcgtga agccgacaca tggtggatgg tagtcggggc gaaagatcca 540 ggcaacacgg ggcagatcct gctttatcgc ggcagttcgt tgcgtgaatg gaccttcgat 600 cgcgtactgg cccacgctga tgcgggtgaa agctatatgt gggaatgtcc ggactttttc 660 agccttggcg atcagcatta tctgatgttt tccccgcagg gaatgaatgc cgagggatac 720 agttaccgaa atcgctttca aagtggcgta atacccggaa tgtggtcgcc aggacgactt 780 tttgcacaat ccgggcattt tactgaactt gataacgggc atgactttta tgcaccacaa 840 agctttttag cgaaggatgg tcggcgtatt gttatcggct ggatggatat gtgggaatcg 900 tcaatgccct caaaacgtga aggatgggca ggctgcatga cgctggcgcg cgagctatca 960 gagagcaatg gcaaacttct acaacgcccg gtacacgaag ctgagtcgtt acgccagcag 1020 catcaatctg tctctccccg cacaatcagc aataaatatg ttttgcagga aaacgcgcaa 1080 gcagttgaga ttcagttgca gtgggcgctg aagaacagtg atgccgaaca ttacggatta 1140 cagctcggca ctggaatgcg gctgtatatt gataaccaat ctgagcgact tgttttgtgg 1200 cggtattacc cacacgagaa tttagacggc taccgtagta ttcccctccc gcagcgtgac 1260 acgctcgccc taaggatatt tatcgataca tcatccgtgg aagtatttat taacgacggg 1320 gaagcggtga tgagtagtcg aatctatccg cagccagaag aacgggaact gtcgctttat 1380 gcctcccacg gagtggctgt gctgcaacat ggagcactct ggctactggg ttaa 1434 <210> 7 <211> 996 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (996) <223> cscR <400> 7 atggcttcat taaaggatgt cgcacgcctg gcgggagtgt cgatgatgac agtctcccgg 60 gtgatgcata atgcagaatc tgtgcgtcct gcaacgcgtg accgcgtatt gcaggcaatc 120 cagaccctga attatgttcc tgatctttcc gcccgtaaga tgcgcgctca aggacgtaag 180 ccgtcgactc tcgccgtgct ggcgcaggac acggctacca ctcctttctc tgttgatatt 240 ctgcttgcca ttgagcaaac cgccagcgag ttcggctgga atagtttttt aatcaatatt 300 ttttctgaag atgacgctgc ccgcgcggca cgtcagctgc ttgcccaccg tccggatggc 360 attatctata ctacaatggg gctgcgacat atcacgctgc ctgagtctct gtatggtgaa 420 aatattgtat tggcgaactg tgtggcggat gacccagcgt tacccagtta tatccctgat 480 gattacactg cacaatatga atcaacacag catttgctcg cggcgggcta tcgtcaaccg 540 ttatgcttct ggctaccgga aagtgcgttg gcaacagggt atcgtcggca gggatttgag 600 caggcctggc gtgatgctga acgagatctg gctgaggtga aacaatttca catggcaaca 660 ggtgatgatc actacaccga tctcgcaagt ttactcaatg cccacttcaa accgggcaaa 720 ccagattttg atgttctgat atgtggtaac gatcgcgcag cctttgtggc ttatcaggtt 780 cttctggcga agggggtacg aatcccgcag gatgtcgccg taatgggctt tgataatctg 840 gttggcgtcg ggcatctgtt tttaccgccg ctgaccacaa ttcagcttcc acatgacatt 900 atcgggcggg aagctgcatt gcatattatt gaaggtcgtg aagggggaag agtgacgcgg 960 atcccttgcc cgctgttgat ccgttgttcc acctga 996 <210> 8 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of cscB to construct pAcscBAR <400> 8 cgcgatatct agcatatgcc gggtaccgca ctagttgaga agtaaacggc gaagt 55 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of cscB to construct pAcscBAR <400> 9 attcggccgg agccctgcag gtgcacgagt acatttgagc gactgt 46 <210> 10 <211> 4520 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (4520) <223> polynucleotide containing cscBAR <400> 10 gagccctgca ggtgcacgag tacatttgag cgactgtacc agaacatgaa tgaggcgttt 60 ggattaggcg attattagca gggctaagca ttttactatt attattttcc ggttgaggga 120 tatagagcta tcgacaacaa ccggaaaaag tttacgtcta tattgctgaa ggtacaggcg 180 tttccataac tatttgctcg cgttttttac tcaagaagaa aatgccaaat agcaacatca 240 ggcagacaat acccgaaatt gcgaagaaaa ctgtctggta gcctgcgtgg tcaaagagta 300 tcccagtcgg cgttgaaagc agcacaatcc caagcgaact ggcaatttga aaaccaatca 360 gaaagatcgt cgacgacagg cgcttatcaa agtttgccac gctgtatttg aagacggata 420 tgacacaaag tggaacctca atggcatgta acaacttcac taatgaaata atccaggggt 480 taacgaacag cgcgcaggaa aggatacgca acgccataat cacaactccg ataagtaatg 540 cattttttgg ccctacccga ttcacaaaga aaggaataat cgccatgcac agcgcttcga 600 gtaccacctg gaatgagttg agataaccat acaggcgcgt tcctacatcg tgtgattcga 660 ataaacctga ataaaagaca ggaaaaagtt gttgatcaaa aatgttatag aaagaccacg 720 tccccacaat aaatatgacg aaaacccaga agtttcgatc cttgaaaact gcgataaaat 780 cctctttttt tacccctccc gcatctgccg ctacgcactg gtgatcctta tctttaaaac 840 gcatgttgat catcataaat acagcgccaa atagcgagac caaccagaag ttgatatggg 900 gactgatact aaaaaatatg ccggcaaaga acgcgccaat agcatagcca aaagatcccc 960 aggcgcgcgc tgttccatat tcgaaatgaa aatttcgcgc cattttttcg gtgaagctat 1020 caagcaaacc gcatcccgcc agataccccc agccaaaaaa cagcgccccc agaattagac 1080 ctacagaaaa attgctttgc agtaacggtt cataaacgta aatcataaac ggtccggtca 1140 agaccaggat gaaactcata caccagatga gcggtttctt cagaccgagt ttatcctgaa 1200 cgatgccgta gaacatcata aatagaatgc tggtaaactg gttgaccgaa taaagtgtac 1260 ctaattccgt ccctgtcaac cctagatgtc ctttcagcca aatagcgtat aacgaccacc 1320 acagcgacca ggaaataaaa aagagaaatg agtaactgga tgcaaaacga tagtacgcat 1380 ttctgaatgg aatactcagt gccataatta cctgcctgtc gttaaaaaat tcacgtccta 1440 tttagagata agagcgactt cgccgtttac ttctcaacta gtgcggtacc cggcatatgc 1500 tagatatcga ctccctcagt tagcagcgtt ctttgcatta acgcaccaaa aggatcatcc 1560 cccacccgac ctataaaccc acttgttccg cctaatctgg cgattcccac cgcaacgtta 1620 gctggcgcgc cgccaggaca aggcagtagg cgcccgtctg attctggcaa gagatctacg 1680 accgcatccc ctaaaaccca tactttggct gacatttttt tcccttaaat tcatctgagt 1740 tacgcatagt gataaacctc tttttcgcaa aatcgtcatg gatttactaa aacatgcata 1800 ttcgatcaca aaacgtcata gttaacgtta acatttgtga tattcatcgc atttatgaaa 1860 gtaagggact ttatttttat aaaagttaac gttaacaatt caccaaattt gcttaaccag 1920 gatgattaaa atgacgcaat ctcgattgca tgcggcgcaa aacgccctag caaaacttca 1980 tgagcaccgg ggtaacactt tctatcccca ttttcacctc gcgcctcctg ccgggtggat 2040 gaacgatcca aacggcctga tctggtttaa cgatcgttat cacgcgtttt atcaacatca 2100 tccgatgagc gaacactggg ggccaatgca ctggggacat gccaccagcg acgatatgat 2160 ccactggcag catgagccta ttgcgctagc gccaggagac gataatgaca aagacgggtg 2220 tttttcaggt agtgctgtcg atgacaatgg tgtcctctca cttatctaca ccggacacgt 2280 ctggctcgat ggtgcaggta atgacgatgc aattcgcgaa gtacaatgtc tggctaccag 2340 tcgggatggt attcatctcg agaaacaggg tgtgatcctc actccaccag aaggaatcat 2400 gcacttccgc gatcctaaag tgtggcgtga agccgacaca tggtggatgg tagtcggggc 2460 gaaagatcca ggcaacacgg ggcagatcct gctttatcgc ggcagttcgt tgcgtgaatg 2520 gaccttcgat cgcgtactgg cccacgctga tgcgggtgaa agctatatgt gggaatgtcc 2580 ggactttttc agccttggcg atcagcatta tctgatgttt tccccgcagg gaatgaatgc 2640 cgagggatac agttaccgaa atcgctttca aagtggcgta atacccggaa tgtggtcgcc 2700 aggacgactt tttgcacaat ccgggcattt tactgaactt gataacgggc atgactttta 2760 tgcaccacaa agctttttag cgaaggatgg tcggcgtatt gttatcggct ggatggatat 2820 gtgggaatcg tcaatgccct caaaacgtga aggatgggca ggctgcatga cgctggcgcg 2880 cgagctatca gagagcaatg gcaaacttct acaacgcccg gtacacgaag ctgagtcgtt 2940 acgccagcag catcaatctg tctctccccg cacaatcagc aataaatatg ttttgcagga 3000 aaacgcgcaa gcagttgaga ttcagttgca gtgggcgctg aagaacagtg atgccgaaca 3060 ttacggatta cagctcggca ctggaatgcg gctgtatatt gataaccaat ctgagcgact 3120 tgttttgtgg cggtattacc cacacgagaa tttagacggc taccgtagta ttcccctccc 3180 gcagcgtgac acgctcgccc taaggatatt tatcgataca tcatccgtgg aagtatttat 3240 taacgacggg gaagcggtga tgagtagtcg aatctatccg cagccagaag aacgggaact 3300 gtcgctttat gcctcccacg gagtggctgt gctgcaacat ggagcactct ggctactggg 3360 ttaacataat atcaggtgga acaacggatc aacagcgggc aagggatccg cgtcactctt 3420 cccccttcac gaccttcaat aatatgcaat gcagcttccc gcccgataat gtcatgtgga 3480 agctgaattg tggtcagcgg cggtaaaaac agatgcccga cgccaaccag attatcaaag 3540 cccattacgg cgacatcctg cgggattcgt acccccttcg ccagaagaac ctgataagcc 3600 acaaaggctg cgcgatcgtt accacatatc agaacatcaa aatctggttt gcccggtttg 3660 aagtgggcat tgagtaaact tgcgagatcg gtgtagtgat catcacctgt tgccatgtga 3720 aattgtttca cctcagccag atctcgttca gcatcacgcc aggcctgctc aaatccctgc 3780 cgacgatacc ctgttgccaa cgcactttcc ggtagccaga agcataacgg ttgacgatag 3840 cccgccgcga gcaaatgctg tgttgattca tattgtgcag tgtaatcatc agggatataa 3900 ctgggtaacg ctgggtcatc cgccacacag ttcgccaata caatattttc accatacaga 3960 gactcaggca gcgtgatatg tcgcagcccc attgtagtat agataatgcc atccggacgg 4020 tgggcaagca gctgacgtgc cgcgcgggca gcgtcatctt cagaaaaaat attgattaaa 4080 aaactattcc agccgaactc gctggcggtt tgctcaatgg caagcagaat atcaacagag 4140 aaaggagtgg tagccgtgtc ctgcgccagc acggcgagag tcgacggctt acgtccttga 4200 gcgcgcatct tacgggcgga aagatcagga acataattca gggtctggat tgcctgcaat 4260 acgcggtcac gcgttgcagg acgcacagat tctgcattat gcatcacccg ggagactgtc 4320 atcatcgaca ctcccgccag gcgtgcgaca tcctttaatg aagccatacc caagccgttt 4380 gccgtaaaac gggcactgta gcagaaacag acgtcactgg cgagatccaa cgccctatca 4440 cctgacacag caatacaata aaaaataaca ataattcccg gacaattgtc cccagttccg 4500 cctctgttct cgccaacgag 4520 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of mak released from reference sequence,          AP009048 on GeneBank <400> 11 cactgcagtg gggtaaatgc catcg 25 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of mak released from reference sequence,          AP009048 on GeneBank <400> 12 aacggccgtc tcggtgctca ttact 25 <210> 13 <211> 1388 <212> DNA <213> Escherichia coli K-12 W3110 <220> <221> gene (222) (1) .. (1388) <223> polynucleotide containing mak <400> 13 cactgcagtg gggtaaatgc catcgaggct agctgttttt ccatctcttc tgcacgcagc 60 gaaatctcgc ggctaagacg gtaaaccatt aaatttttga accacagcat gataatttcc 120 acggccttgt cgttaaattt agcgggcatg ataacgaatt gtcggcggcc ttgcattgcc 180 aatccggttg tccgtctcta cgctattgat attgaaaaaa ataaggagag taccgtgcgt 240 ataggtatcg atttaggcgg caccaaaact gaagtgattg cactgggcga tgcaggggag 300 cagttgtacc gccatcgtct gcccacgccg cgtgatgatt accggcagac tattgaaacg 360 atcgccacgt tggttgatat ggcggagcag gcgacggggc agcgcggaac ggtaggtatg 420 ggcattcctg gctcaatttc gccttacacc ggtgtggtga agaatgccaa ttcaacctgg 480 ctcaacggtc agccattcga taaagactta agcgcgaggt tgcagcggga agtgcggctg 540 gcaaatgacg ctaactgtct ggcggtttca gaagcagtag atggcgcggc agcgggagcg 600 cagacggtat ttgccgtgat tatcggcacg ggatgcggcg cgggcgtggc attcaatggg 660 cgggcgcata tcggcggcaa tggcacggca ggtgagtggg gacacaatcc gctaccgtgg 720 atggacgaag acgaactgcg ttatcgcgag gaagtccctt gttattgcgg taaacaaggt 780 tgtattgaaa cctttatttc gggcacggga ttcgcgatgg attatcgtcg tttgagcgga 840 catgcgctga aaggcagtga aattatccgc ctggttgaag aaagcgatcc ggtagcggaa 900 ctggcattgc gtcgctacga gctgcggctg gcaaaatcgc tggcacatgt cgtgaatatt 960 ctcgatccgg atgtgattgt cctggggggc gggatgagca atgtagaccg tttatatcaa 1020 acggttgggc agttgattaa acaatttgtc ttcggcggcg aatgtgaaac gccggtgcgt 1080 aaggcgaagc acggtgattc cagcggcgta cgcggcgctg cgtggttatg gccacaagag 1140 taaaaaacgt aggcaattgg cgcatcatgc ctgatgcgac gcttgccgcg tcttatcagg 1200 cctacaaaag gtgccagaac cgtaggccgg ataaggcgtt cacgccgcat ccggcaataa 1260 gtgctccgat gcctgatgcg acgcttgccg cgtcttatca ggcctgcaaa atgtgccaga 1320 accgcgtagg gcggataagg cgttcacgcc gcatccggca ataagtaatg agcaccgaga 1380 cggccgtt 1388 <210> 14 <211> 5887 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide containing cscBAR-mak (Escherichia coli (ATCC9637)          and Escherichia coli K-12 W3110) <400> 14 tctcggtgct cattacttat tgccggatgc ggcgtgaacg ccttatccgc cctacgcggt 60 tctggcacat tttgcaggcc tgataagacg cggcaagcgt cgcatcaggc atcggagcac 120 ttattgccgg atgcggcgtg aacgccttat ccggcctacg gttctggcac cttttgtagg 180 cctgataaga cgcggcaagc gtcgcatcag gcatgatgcg ccaattgcct acgtttttta 240 ctcttgtggc cataaccacg cagcgccgcg tacgccgctg gaatcaccgt gcttcgcctt 300 acgcaccggc gtttcacatt cgccgccgaa gacaaattgt ttaatcaact gcccaaccgt 360 ttgatataaa cggtctacat tgctcatccc gccccccagg acaatcacat ccggatcgag 420 aatattcacg acatgtgcca gcgattttgc cagccgcagc tcgtagcgac gcaatgccag 480 ttccgctacc ggatcgcttt cttcaaccag gcggataatt tcactgcctt tcagcgcatg 540 tccgctcaaa cgacgataat ccatcgcgaa tcccgtgccc gaaataaagg tttcaataca 600 accttgttta ccgcaataac aagggacttc ctcgcgataa cgcagttcgt cttcgtccat 660 ccacggtagc ggattgtgtc cccactcacc tgccgtgcca ttgccgccga tatgcgcccg 720 cccattgaat gccacgcccg cgccgcatcc cgtgccgata atcacggcaa ataccgtctg 780 cgctcccgct gccgcgccat ctactgcttc tgaaaccgcc agacagttag cgtcatttgc 840 cagccgcact tcccgctgca acctcgcgct taagtcttta tcgaatggct gaccgttgag 900 ccaggttgaa ttggcattct tcaccacacc ggtgtaaggc gaaattgagc caggaatgcc 960 catacctacc gttccgcgct gccccgtcgc ctgctccgcc atatcaacca acgtggcgat 1020 cgtttcaata gtctgccggt aatcatcacg cggcgtgggc agacgatggc ggtacaactg 1080 ctcccctgca tcgcccagtg caatcacttc agttttggtg ccgcctaaat cgatacctat 1140 acgcacggta ctctccttat ttttttcaat atcaatagcg tagagacgga caaccggatt 1200 ggcaatgcaa ggccgccgac aattcgttat catgcccgct aaatttaacg acaaggccgt 1260 ggaaattatc atgctgtggt tcaaaaattt aatggtttac cgtcttagcc gcgagatttc 1320 gctgcgtgca gaagagatgg aaaaacagct agcctcgatg gcatttaccc cactgcaggt 1380 gcacgagtac atttgagcga ctgtaccaga acatgaatga ggcgtttgga ttaggcgatt 1440 attagcaggg ctaagcattt tactattatt attttccggt tgagggatat agagctatcg 1500 acaacaaccg gaaaaagttt acgtctatat tgctgaaggt acaggcgttt ccataactat 1560 ttgctcgcgt tttttactca agaagaaaat gccaaatagc aacatcaggc agacaatacc 1620 cgaaattgcg aagaaaactg tctggtagcc tgcgtggtca aagagtatcc cagtcggcgt 1680 tgaaagcagc acaatcccaa gcgaactggc aatttgaaaa ccaatcagaa agatcgtcga 1740 cgacaggcgc ttatcaaagt ttgccacgct gtatttgaag acggatatga cacaaagtgg 1800 aacctcaatg gcatgtaaca acttcactaa tgaaataatc caggggttaa cgaacagcgc 1860 gcaggaaagg atacgcaacg ccataatcac aactccgata agtaatgcat tttttggccc 1920 tacccgattc acaaagaaag gaataatcgc catgcacagc gcttcgagta ccacctggaa 1980 tgagttgaga taaccataca ggcgcgttcc tacatcgtgt gattcgaata aacctgaata 2040 aaagacagga aaaagttgtt gatcaaaaat gttatagaaa gaccacgtcc ccacaataaa 2100 tatgacgaaa acccagaagt ttcgatcctt gaaaactgcg ataaaatcct ctttttttac 2160 ccctcccgca tctgccgcta cgcactggtg atccttatct ttaaaacgca tgttgatcat 2220 cataaataca gcgccaaata gcgagaccaa ccagaagttg atatggggac tgatactaaa 2280 aaatatgccg gcaaagaacg cgccaatagc atagccaaaa gatccccagg cgcgcgctgt 2340 tccatattcg aaatgaaaat ttcgcgccat tttttcggtg aagctatcaa gcaaaccgca 2400 tcccgccaga tacccccagc caaaaaacag cgcccccaga attagaccta cagaaaaatt 2460 gctttgcagt aacggttcat aaacgtaaat cataaacggt ccggtcaaga ccaggatgaa 2520 actcatacac cagatgagcg gtttcttcag accgagttta tcctgaacga tgccgtagaa 2580 catcataaat agaatgctgg taaactggtt gaccgaataa agtgtaccta attccgtccc 2640 tgtcaaccct agatgtcctt tcagccaaat agcgtataac gaccaccaca gcgaccagga 2700 aataaaaaag agaaatgagt aactggatgc aaaacgatag tacgcatttc tgaatggaat 2760 actcagtgcc ataattacct gcctgtcgtt aaaaaattca cgtcctattt agagataaga 2820 gcgacttcgc cgtttacttc tcaactagtg cggtacccgg catatgctag atatcgactc 2880 cctcagttag cagcgttctt tgcattaacg caccaaaagg atcatccccc acccgaccta 2940 taaacccact tgttccgcct aatctggcga ttcccaccgc aacgttagct ggcgcgccgc 3000 caggacaagg cagtaggcgc ccgtctgatt ctggcaagag atctacgacc gcatccccta 3060 aaacccatac tttggctgac atttttttcc cttaaattca tctgagttac gcatagtgat 3120 aaacctcttt ttcgcaaaat cgtcatggat ttactaaaac atgcatattc gatcacaaaa 3180 cgtcatagtt aacgttaaca tttgtgatat tcatcgcatt tatgaaagta agggacttta 3240 tttttataaa agttaacgtt aacaattcac caaatttgct taaccaggat gattaaaatg 3300 acgcaatctc gattgcatgc ggcgcaaaac gccctagcaa aacttcatga gcaccggggt 3360 aacactttct atccccattt tcacctcgcg cctcctgccg ggtggatgaa cgatccaaac 3420 ggcctgatct ggtttaacga tcgttatcac gcgttttatc aacatcatcc gatgagcgaa 3480 cactgggggc caatgcactg gggacatgcc accagcgacg atatgatcca ctggcagcat 3540 gagcctattg cgctagcgcc aggagacgat aatgacaaag acgggtgttt ttcaggtagt 3600 gctgtcgatg acaatggtgt cctctcactt atctacaccg gacacgtctg gctcgatggt 3660 gcaggtaatg acgatgcaat tcgcgaagta caatgtctgg ctaccagtcg ggatggtatt 3720 catctcgaga aacagggtgt gatcctcact ccaccagaag gaatcatgca cttccgcgat 3780 cctaaagtgt ggcgtgaagc cgacacatgg tggatggtag tcggggcgaa agatccaggc 3840 aacacggggc agatcctgct ttatcgcggc agttcgttgc gtgaatggac cttcgatcgc 3900 gtactggccc acgctgatgc gggtgaaagc tatatgtggg aatgtccgga ctttttcagc 3960 cttggcgatc agcattatct gatgttttcc ccgcagggaa tgaatgccga gggatacagt 4020 taccgaaatc gctttcaaag tggcgtaata cccggaatgt ggtcgccagg acgacttttt 4080 gcacaatccg ggcattttac tgaacttgat aacgggcatg acttttatgc accacaaagc 4140 tttttagcga aggatggtcg gcgtattgtt atcggctgga tggatatgtg ggaatcgtca 4200 atgccctcaa aacgtgaagg atgggcaggc tgcatgacgc tggcgcgcga gctatcagag 4260 agcaatggca aacttctaca acgcccggta cacgaagctg agtcgttacg ccagcagcat 4320 caatctgtct ctccccgcac aatcagcaat aaatatgttt tgcaggaaaa cgcgcaagca 4380 gttgagattc agttgcagtg ggcgctgaag aacagtgatg ccgaacatta cggattacag 4440 ctcggcactg gaatgcggct gtatattgat aaccaatctg agcgacttgt tttgtggcgg 4500 tattacccac acgagaattt agacggctac cgtagtattc ccctcccgca gcgtgacacg 4560 ctcgccctaa ggatatttat cgatacatca tccgtggaag tatttattaa cgacggggaa 4620 gcggtgatga gtagtcgaat ctatccgcag ccagaagaac gggaactgtc gctttatgcc 4680 tcccacggag tggctgtgct gcaacatgga gcactctggc tactgggtta acataatatc 4740 aggtggaaca acggatcaac agcgggcaag ggatccgcgt cactcttccc ccttcacgac 4800 cttcaataat atgcaatgca gcttcccgcc cgataatgtc atgtggaagc tgaattgtgg 4860 tcagcggcgg taaaaacaga tgcccgacgc caaccagatt atcaaagccc attacggcga 4920 catcctgcgg gattcgtacc cccttcgcca gaagaacctg ataagccaca aaggctgcgc 4980 gatcgttacc acatatcaga acatcaaaat ctggtttgcc cggtttgaag tgggcattga 5040 gtaaacttgc gagatcggtg tagtgatcat cacctgttgc catgtgaaat tgtttcacct 5100 cagccagatc tcgttcagca tcacgccagg cctgctcaaa tccctgccga cgataccctg 5160 ttgccaacgc actttccggt agccagaagc ataacggttg acgatagccc gccgcgagca 5220 aatgctgtgt tgattcatat tgtgcagtgt aatcatcagg gatataactg ggtaacgctg 5280 ggtcatccgc cacacagttc gccaatacaa tattttcacc atacagagac tcaggcagcg 5340 tgatatgtcg cagccccatt gtagtataga taatgccatc cggacggtgg gcaagcagct 5400 gacgtgccgc gcgggcagcg tcatcttcag aaaaaatatt gattaaaaaa ctattccagc 5460 cgaactcgct ggcggtttgc tcaatggcaa gcagaatatc aacagagaaa ggagtggtag 5520 ccgtgtcctg cgccagcacg gcgagagtcg acggcttacg tccttgagcg cgcatcttac 5580 gggcggaaag atcaggaaca taattcaggg tctggattgc ctgcaatacg cggtcacgcg 5640 ttgcaggacg cacagattct gcattatgca tcacccggga gactgtcatc atcgacactc 5700 ccgccaggcg tgcgacatcc tttaatgaag ccatacccaa gccgtttgcc gtaaaacggg 5760 cactgtagca gaaacagacg tcactggcga gatccaacgc cctatcacct gacacagcaa 5820 tacaataaaa aataacaata attcccggac aattgtcccc agttccgcct ctgttctcgc 5880 caacgag 5887 <210> 15 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of cscBAR-M <400> 15 atatctagac tcgttggcga gaacagag 28 <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of cscBAR-M <400> 16 aattctagat gctcattact tattgccg 28 <210> 17 <211> 996 <212> DNA Escherichia coli (ATCC9637) <220> <221> gene (222) (1) .. (996) <223> cscR mutated tyrosine on 388th amino acid <400> 17 atggcttcat taaaggatgt cgcacgcctg gcgggagtgt cgatgatgac agtctcccgg 60 gtgatgcata atgcagaatc tgtgcgtcct gcaacgcgtg accgcgtatt gcaggcaatc 120 cagaccctga attatgttcc tgatctttcc gcccgtaaga tgcgcgctca aggacgtaag 180 ccgtcgactc tcgccgtgct ggcgcaggac acggctacca ctcctttctc tgttgatatt 240 ctgcttgcca ttgagcaaac cgccagcgag ttcggctgga atagtttttt aatcaatatt 300 ttttctgaag atgacgctgc ccgcgcggca cgtcagctgc ttgcccaccg tccggatggc 360 attatctata ctacaatggg gctgcgatat atcacgctgc ctgagtctct gtatggtgaa 420 aatattgtat tggcgaactg tgtggcggat gacccagcgt tacccagtta tatccctgat 480 gattacactg cacaatatga atcaacacag catttgctcg cggcgggcta tcgtcaaccg 540 ttatgcttct ggctaccgga aagtgcgttg gcaacagggt atcgtcggca gggatttgag 600 caggcctggc gtgatgctga acgagatctg gctgaggtga aacaatttca catggcaaca 660 ggtgatgatc actacaccga tctcgcaagt ttactcaatg cccacttcaa accgggcaaa 720 ccagattttg atgttctgat atgtggtaac gatcgcgcag cctttgtggc ttatcaggtt 780 cttctggcga agggggtacg aatcccgcag gatgtcgccg taatgggctt tgataatctg 840 gttggcgtcg ggcatctgtt tttaccgccg ctgaccacaa ttcagcttcc acatgacatt 900 atcgggcggg aagctgcatt gcatattatt gaaggtcgtg aagggggaag agtgacgcgg 960 atcccttgcc cgctgttgat ccgttgttcc acctga 996 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of scrAB from Klebsiella pneumoniae <400> 18 aataccatgg attttgaaca gatttccc 28 <210> 19 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of scrAB from Klebsiella pneumoniae <400> 19 ataatctaga gtttggtttt catcagcac 29 <210> 20 <211> 3074 <212> DNA (213) Klebsiella pneumoniae (ATCC700721) <220> <221> gene (222) (1) .. (3074) <223> polynucleotide containing trc promoter and scrAB operon <400> 20 ctagatatca tcgactgcac ggtgcaccaa tgcttctggc gtcaggcagc catcggaagc 60 tgtggtatgg ctgtgcaggt cgtaaatcac tgcataattc gtgtcgctca aggcgcactc 120 ccgttctgga taatgttttt tgcgccgaca tcataacggt tctggcaaat attctgaaat 180 gagctgttga caattaatca tccggctcgt ataatgtgtg gaattgtgag cggataacaa 240 tttcacacag gaaacagacc atggattttg aacagatttc ccgctcactg cttcccctgc 300 tgggcggcaa ggaaaatatc gccagcgccg cgcactgcgc cacccgcctg cggctggtgc 360 tggtcgacga cgcgctcgcc gatcagcagg cgattggcaa aatcgacggg gtgaaaggct 420 gctttcgcaa tgccggacag atgcagatca tcttcggcac cggggtggtc aataaagtct 480 atgccgcctt tatccaggcc gcaggcatca gcgaatcgag caaatccgaa gccgccgacc 540 tggcggcgaa aaagctgaac ccgttccagc gcatcgcccg cctgctgtcc aacatcttcg 600 tgccgattat tccggccatc gtcgcctccg gcctgctgat gggcctgctg gggatggtga 660 aaacctacgg ttgggtcgac ccgagcaacg ctctctatat catgctggat atgtgcagtt 720 cggcggcgtt tatcattctg ccgatcctga tcggctttac cgccgcacgc gaatttggcg 780 gtaaccccta tctgggcgcg accctcggcg ggatcctcac ccatccggcg ctgaccaacg 840 cctggggcgt cgccgccggc ttccacacca tgaatttctt cggcatcgaa gtggcgatga 900 tcggctacca gggcaccgtc ttcccggtgc tgctggcggt gtggtttatg agcatggtcg 960 agaagcggct gcgccgcgtg atccctgacg cgctggacct gatcctcact ccgttcctga 1020 cggtgattat ctccggcttt atcgccctgc tgctgatcgg cccggccggt cgcgcgctcg 1080 gcgacggcat ttcgtttatc ctcagcacgc ttatcagcca tgccggctgg ctggcgggcc 1140 tgctgttcgg cggcctctat tcggtgatcg tcattaccgg tatccatcac agcttccatg 1200 ccatcgaggc cggactgctg ggcaacccat cgattggcgt caacttcctg ctgccgatct 1260 gggcgatggc caacgtcgcc cagggcggcg cctgctttgc ggtgtggttt aaaaccaaag 1320 atgccaaaat aaaagccatc accctgccgt cggcgttttc ggcgatgctg gggatcaccg 1380 aggcggcaat cttcgggatt aacctgcgct ttgtgaaacc gtttatcgcc gcgctggtgg 1440 gcggtgccgc cggcggcgcc tgggtggtgt cgatgcacgt ctacatgacc gcggtgggcc 1500 tgacggcgat cccggggatg gctatcgtgc aggccagctc gctgctgaac tacattatcg 1560 gaatggcgat cgccttcgcc gtggccttcg cgctctctct gacgctgaaa tacaaaacgg 1620 acgctgaata atgtcattac cgtcacgcct gcctgcgatc ctgcaggccg ttatgcaggg 1680 ccagccgcag gcgctggccg acagccatta tccgcaatgg catctggcgc cggtcaacgg 1740 actgctgaac gatcctaacg gcttttgcca ggtcgccggg cgttaccacc tgttttatca 1800 gtggaacccg ctcgcctgcg accataccta taagtgctgg ggacactgga gctctgccga 1860 tctgctgcac tggcggcacg aacctatcgc cctgatgccg gatgaagagt atgaccgcaa 1920 cggctgctac tctggcagcg cggtcgagtt cgagggcgcc ctgactctgt gctacaccgg 1980 caacgtgaaa ttccccgacg gcgggcgcac cgcctggcaa tgtctggcga ccgagaatgc 2040 cgatggcacc ttccgcaagc tggggccggt gctgccgctg ccagaaggct ataccggcca 2100 tgtgcgcgac cctaaagtgt ggcggcagga cgggcgctgg tacatggttc ttggggcgca 2160 ggatgtgcaa cagcgcggca aagtgctgct gtttaccgcc agcgacctgc gggagtggcg 2220 cctggtgggc gagattgccg ggcacgacgt aaacggcctg gcgaacgccg gctacatgtg 2280 ggagtgcccg gatctctttc cgctggcgga cacccacctg ctgatctgct gcccgcaggg 2340 gctggcccgc gaagcgcagc gctttctcaa tacctatccg gcggtgtgga tggcaggccg 2400 cttcgacgcc gaacgcggga tcttcgacca cggcccgctg cacgagctgg acagcggatt 2460 tgagttctac gcgccgcaga ccatgcaggc cgatgatggc cgccgcctgc tggttggctg 2520 gatgggcgtc cccgacgggg acgagatgca tcagcccacc cgcgcgcagg gctggatcca 2580 tcagatgacc tgcgtgcgtg agctggagtg gcaggctggc actctgtatc agcgtccgct 2640 gcgcgagctg gtcgccctgc gcggggaagc ccagggctgg tgcggacaga ccctgcccct 2700 cgccccgatg gagctggcct ttgacatcgc ccccaacagc acgctggggc tggactttgc 2760 cggcgccctg cagctcaccg tcaatcgcga cggcctgcgt ctgtcgcgtc gcggcctgca 2820 gacggcggag ctgcatcacc gctactggcg cggcgaggcg cgacgcctgc ggatctttat 2880 cgaccgctcc agcgtggaga ttttcatcaa cgatggcgag ggggtgatga gcagccgctt 2940 cttcccgggc tatccggggc agctcatctt cagcggtgcg acgccggtag cattctgccg 3000 ctggctgctg cggccatgca tggtagaata agcgttttgc tttcaggctc ccgggtgctg 3060 atgaaaacca aact 3074 <210> 21 <211> 1371 <212> DNA (213) Klebsiella pneumoniae (ATCC700721) <220> <221> gene (222) (1) .. (1371) <223> scrA <400> 21 atggattttg aacagatttc ccgctcactg cttcccctgc tgggcggcaa ggaaaatatc 60 gccagcgccg cgcactgcgc cacccgcctg cggctggtgc tggtcgacga cgcgctcgcc 120 gatcagcagg cgattggcaa aatcgacggg gtgaaaggct gctttcgcaa tgccggacag 180 atgcagatca tcttcggcac cggggtggtc aataaagtct atgccgcctt tatccaggcc 240 gcaggcatca gcgaatcgag caaatccgaa gccgccgacc tggcggcgaa aaagctgaac 300 ccgttccagc gcatcgcccg cctgctgtcc aacatcttcg tgccgattat tccggccatc 360 gtcgcctccg gcctgctgat gggcctgctg gggatggtga aaacctacgg ttgggtcgac 420 ccgagcaacg ctctctatat catgctggat atgtgcagtt cggcggcgtt tatcattctg 480 ccgatcctga tcggctttac cgccgcacgc gaatttggcg gtaaccccta tctgggcgcg 540 accctcggcg ggatcctcac ccatccggcg ctgaccaacg cctggggcgt cgccgccggc 600 ttccacacca tgaatttctt cggcatcgaa gtggcgatga tcggctacca gggcaccgtc 660 ttcccggtgc tgctggcggt gtggtttatg agcatggtcg agaagcggct gcgccgcgtg 720 atccctgacg cgctggacct gatcctcact ccgttcctga cggtgattat ctccggcttt 780 atcgccctgc tgctgatcgg cccggccggt cgcgcgctcg gcgacggcat ttcgtttatc 840 ctcagcacgc ttatcagcca tgccggctgg ctggcgggcc tgctgttcgg cggcctctat 900 tcggtgatcg tcattaccgg tatccatcac agcttccatg ccatcgaggc cggactgctg 960 ggcaacccat cgattggcgt caacttcctg ctgccgatct gggcgatggc caacgtcgcc 1020 cagggcggcg cctgctttgc ggtgtggttt aaaaccaaag atgccaaaat aaaagccatc 1080 accctgccgt cggcgttttc ggcgatgctg gggatcaccg aggcggcaat cttcgggatt 1140 aacctgcgct ttgtgaaacc gtttatcgcc gcgctggtgg gcggtgccgc cggcggcgcc 1200 tgggtggtgt cgatgcacgt ctacatgacc gcggtgggcc tgacggcgat cccggggatg 1260 gctatcgtgc aggccagctc gctgctgaac tacattatcg gaatggcgat cgccttcgcc 1320 gtggccttcg cgctctctct gacgctgaaa tacaaaacgg acgctgaata a 1371 <210> 22 <211> 1401 <212> DNA (213) Klebsiella pneumoniae (ATCC700721) <220> <221> gene (222) (1) .. (1401) <223> scrB <400> 22 atgtcattac cgtcacgcct gcctgcgatc ctgcaggccg ttatgcaggg ccagccgcag 60 gcgctggccg acagccatta tccgcaatgg catctggcgc cggtcaacgg actgctgaac 120 gatcctaacg gcttttgcca ggtcgccggg cgttaccacc tgttttatca gtggaacccg 180 ctcgcctgcg accataccta taagtgctgg ggacactgga gctctgccga tctgctgcac 240 tggcggcacg aacctatcgc cctgatgccg gatgaagagt atgaccgcaa cggctgctac 300 tctggcagcg cggtcgagtt cgagggcgcc ctgactctgt gctacaccgg caacgtgaaa 360 ttccccgacg gcgggcgcac cgcctggcaa tgtctggcga ccgagaatgc cgatggcacc 420 ttccgcaagc tggggccggt gctgccgctg ccagaaggct ataccggcca tgtgcgcgac 480 cctaaagtgt ggcggcagga cgggcgctgg tacatggttc ttggggcgca ggatgtgcaa 540 cagcgcggca aagtgctgct gtttaccgcc agcgacctgc gggagtggcg cctggtgggc 600 gagattgccg ggcacgacgt aaacggcctg gcgaacgccg gctacatgtg ggagtgcccg 660 gatctctttc cgctggcgga cacccacctg ctgatctgct gcccgcaggg gctggcccgc 720 gaagcgcagc gctttctcaa tacctatccg gcggtgtgga tggcaggccg cttcgacgcc 780 gaacgcggga tcttcgacca cggcccgctg cacgagctgg acagcggatt tgagttctac 840 gcgccgcaga ccatgcaggc cgatgatggc cgccgcctgc tggttggctg gatgggcgtc 900 cccgacgggg acgagatgca tcagcccacc cgcgcgcagg gctggatcca tcagatgacc 960 tgcgtgcgtg agctggagtg gcaggctggc actctgtatc agcgtccgct gcgcgagctg 1020 gtcgccctgc gcggggaagc ccagggctgg tgcggacaga ccctgcccct cgccccgatg 1080 gagctggcct ttgacatcgc ccccaacagc acgctggggc tggactttgc cggcgccctg 1140 cagctcaccg tcaatcgcga cggcctgcgt ctgtcgcgtc gcggcctgca gacggcggag 1200 ctgcatcacc gctactggcg cggcgaggcg cgacgcctgc ggatctttat cgaccgctcc 1260 agcgtggaga ttttcatcaa cgatggcgag ggggtgatga gcagccgctt cttcccgggc 1320 tatccggggc agctcatctt cagcggtgcg acgccggtag cattctgccg ctggctgctg 1380 cggccatgca tggtagaata a 1401 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of pyc <400> 23 aataccatgg cgactcacac atcttc 26 <210> 24 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of pyc <400> 24 attaagcttt aaaccaagac ggca 24 <210> 25 <211> 3423 <212> DNA <213> corynebacterium glutamicum (KFCC 10881) <220> <221> gene (222) (1) .. (3423) <223> pyc <400> 25 gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60 ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120 atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180 attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240 gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 300 cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360 cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 420 gttttggcgg aatccacccc gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc 480 cagacttacc ccatctttgt gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt 540 gttgcttcac ctgatgagct tcgcaaatta gcaacagaag catctcgtga agctgaagcg 600 gctttcggcg atggcgcggt atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa 660 gtgcagatcc ttggcgatca cactggagaa gttgtacacc tttatgaacg tgactgctca 720 ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780 ctgcgtgatc gcatttgtgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840 gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat cgaaatgaac 900 ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960 gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020 atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080 ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140 cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200 aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260 gctgagttca ccgtgtctgg tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgg 1320 gaagaggact tcacttccaa gcgcatcgcc accggattca ttgccgatca ctcgcacctc 1380 cttcaggctc cacctgctga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440 accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagctcctat cgataagctg 1500 cctaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560 ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620 ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680 gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740 gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800 ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860 ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccagctcc 1920 ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980 atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040 gatctctctg atccaaatga aaagctctac accctggatt actacctaaa gatggcagag 2100 gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160 ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220 gtgcacaccc acgacactgc gggtggccag ctggcaacct actttgctgc agctcaagct 2280 ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340 ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400 gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460 tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagttg 2520 tccaacctgc gtgcacaggc caccgcactg ggccttgcgg atcgtttcga actcatcgaa 2580 gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640 aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700 gctgccgatc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760 cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820 tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880 gatgattcca aggaacgtcg caatagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940 gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000 ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060 ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120 gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180 accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240 ggtgttgtca ccgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300 atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360 cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420 taa 3423 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification containing trc promoter and pyc <400> 26 tataagcttg gcgtcaggca gccatcg 27 <210> 27 <211> 3810 <212> DNA <213> corynebacterium glutamicum (KFCC 10881) <220> <221> gene (222) (1) .. (3810) <223> polynucleotide containing trc promoter and pyc <400> 27 ggcgtcaggc agccatcgga agctgtggta tggctgtgca ggtcgtaaat cactgcataa 60 ttcgtgtcgc tcaaggcgca ctcccgttct ggataatgtt ttttgcgccg acatcataac 120 ggttctggca aatattctga aatgagctgt tgacaattaa tcatccggct cgtataatgt 180 gtggaattgt gagcggataa caatttcaca caggaaacag accatggcga ctcacacatc 240 ttcaacgctt ccagcattca aaaagatctt ggtagcaaac cgcggcgaaa tcgcggtccg 300 tgctttccgt gcagcactcg aaaccggtgc agccacggta gctatttacc cccgtgaaga 360 tcggggatca ttccaccgct cttttgcttc tgaagctgtc cgcattggta ccgaaggctc 420 accagtcaag gcgtacctgg acatcgatga aattatcggt gcagctaaaa aagttaaagc 480 agatgccatt tacccgggat acggcttcct gtctgaaaat gcccagcttg cccgcgagtg 540 tgcggaaaac ggcattactt ttattggccc aaccccagag gttcttgatc tcaccggtga 600 taagtctcgc gcggtaaccg ccgcgaagaa ggctggtctg ccagttttgg cggaatccac 660 cccgagcaaa aacatcgatg agatcgttaa aagcgctgaa ggccagactt accccatctt 720 tgtgaaggca gttgccggtg gtggcggacg cggtatgcgt tttgttgctt cacctgatga 780 gcttcgcaaa ttagcaacag aagcatctcg tgaagctgaa gcggctttcg gcgatggcgc 840 ggtatatgtc gaacgtgctg tgattaaccc tcagcatatt gaagtgcaga tccttggcga 900 tcacactgga gaagttgtac acctttatga acgtgactgc tcactgcagc gtcgtcacca 960 aaaagttgtc gaaattgcgc cagcacagca tttggatcca gaactgcgtg atcgcatttg 1020 tgcggatgca gtaaagttct gccgctccat tggttaccag ggcgcgggaa ccgtggaatt 1080 cttggtcgat gaaaagggca accacgtctt catcgaaatg aacccacgta tccaggttga 1140 gcacaccgtg actgaagaag tcaccgaggt ggacctggtg aaggcgcaga tgcgcttggc 1200 tgctggtgca accttgaagg aattgggtct gacccaagat aagatcaaga cccacggtgc 1260 agcactgcag tgccgcatca ccacggaaga tccaaacaac ggcttccgcc cagataccgg 1320 aactatcacc gcgtaccgct caccaggcgg agctggcgtt cgtcttgacg gtgcagctca 1380 gctcggtggc gaaatcaccg cacactttga ctccatgctg gtgaaaatga cctgccgtgg 1440 ttccgacttt gaaactgctg ttgctcgtgc acagcgcgcg ttggctgagt tcaccgtgtc 1500 tggtgttgca accaacattg gtttcttgcg tgcgttgctg cgggaagagg acttcacttc 1560 caagcgcatc gccaccggat tcattgccga tcactcgcac ctccttcagg ctccacctgc 1620 tgatgatgag cagggacgca tcctggatta cttggcagat gtcaccgtga acaagcctca 1680 tggtgtgcgt ccaaaggatg ttgcagctcc tatcgataag ctgcctaaca tcaaggatct 1740 gccactgcca cgcggttccc gtgaccgcct gaagcagctt ggcccagccg cgtttgctcg 1800 tgatctccgt gagcaggacg cactggcagt tactgatacc accttccgcg atgcacacca 1860 gtctttgctt gcgacccgag tccgctcatt cgcactgaag cctgcggcag aggccgtcgc 1920 aaagctgact cctgagcttt tgtccgtgga ggcctggggc ggcgcgacct acgatgtggc 1980 gatgcgtttc ctctttgagg atccgtggga caggctcgac gagctgcgcg aggcgatgcc 2040 gaatgtaaac attcagatgc tgcttcgcgg ccgcaacacc gtgggataca ccccgtaccc 2100 agactccgtc tgccgcgcgt ttgttaagga agctgccagc tccggcgtgg acatcttccg 2160 catcttcgac gcgcttaacg acgtctccca gatgcgtcca gcaatcgacg cagtcctgga 2220 gaccaacacc gcggtagccg aggtggctat ggcttattct ggtgatctct ctgatccaaa 2280 tgaaaagctc tacaccctgg attactacct aaagatggca gaggagatcg tcaagtctgg 2340 cgctcacatc ttggccatta aggatatggc tggtctgctt cgcccagctg cggtaaccaa 2400 gctggtcacc gcactgcgcc gtgaattcga tctgccagtg cacgtgcaca cccacgacac 2460 tgcgggtggc cagctggcaa cctactttgc tgcagctcaa gctggtgcag atgctgttga 2520 cggtgcttcc gcaccactgt ctggcaccac ctcccagcca tccctgtctg ccattgttgc 2580 tgcattcgcg cacacccgtc gcgataccgg tttgagcctc gaggctgttt ctgacctcga 2640 gccgtactgg gaagcagtgc gcggactgta cctgccattt gagtctggaa ccccaggccc 2700 aaccggtcgc gtctaccgcc acgaaatccc aggcggacag ttgtccaacc tgcgtgcaca 2760 ggccaccgca ctgggccttg cggatcgttt cgaactcatc gaagacaact acgcagccgt 2820 taatgagatg ctgggacgcc caaccaaggt caccccatcc tccaaggttg ttggcgacct 2880 cgcactccac ctcgttggtg cgggtgtgga tccagcagac tttgctgccg atccacaaaa 2940 gtacgacatc ccagactctg tcatcgcgtt cctgcgcggc gagcttggta accctccagg 3000 tggctggcca gagccactgc gcacccgcgc actggaaggc cgctccgaag gcaaggcacc 3060 tctgacggaa gttcctgagg aagagcaggc gcacctcgac gctgatgatt ccaaggaacg 3120 tcgcaatagc ctcaaccgcc tgctgttccc gaagccaacc gaagagttcc tcgagcaccg 3180 tcgccgcttc ggcaacacct ctgcgctgga tgatcgtgaa ttcttctacg gcctggtcga 3240 aggccgcgag actttgatcc gcctgccaga tgtgcgcacc ccactgcttg ttcgcctgga 3300 tgcgatctct gagccagacg ataagggtat gcgcaatgtt gtggccaacg tcaacggcca 3360 gatccgccca atgcgtgtgc gtgaccgctc cgttgagtct gtcaccgcaa ccgcagaaaa 3420 ggcagattcc tccaacaagg gccatgttgc tgcaccattc gctggtgttg tcaccgtgac 3480 tgttgctgaa ggtgatgagg tcaaggctgg agatgcagtc gcaatcatcg aggctatgaa 3540 gatggaagca acaatcactg cttctgttga cggcaaaatc gatcgcgttg tggttcctgc 3600 tgcaacgaag gtggaaggtg gcgacttgat cgtcgtcgtt tcctaaacct ttctgtaaaa 3660 agccccgcgt cttcctcatg gaggaggcgg ggctttttgg gccaagatgg gagatgggtg 3720 agttggattt ggtctgattc gacactttta agggcagaga tttgaagatg gagaccaagg 3780 ctcaaaggga atccatgccg tcttggttta 3810 <210> 28 <211> 15191 <212> DNA <213> Artificial Sequence <220> PAcscBAR (mutated) -M (Escherichia coli (ATCC9637) and Escherichia          coli K-12 W3110) <400> 28 gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt 60 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc 120 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgcg 180 gccggatatc cggagattac gcgatgagaa cgacggttgt tattctccgc aatcctaatc 240 tctaagttac cgcatttatt gagaatttct cctttttccc tgtcgttttt ctccataatt 300 tcttcataaa aaagccgggc tgcataaaag caaacccggc ctgattgaga taatgaatag 360 attttactga tgattcatca tcaatttacg caacgcagca aaatcggcgg gcagattatg 420 tgaaagcaag ggtaaatcag cacgttctgc cagctctttt ggcagatcca acgtttcacc 480 gagaatcgct tccacgctct ctttaaattt cgccggatgc gcggtgccga ggaacaagcc 540 atattcgcct ggattcaact ggtcacgcag cgcacgatac gctacggcag cgtgcggctc 600 cgaggtgtag cccagttctt ttaactcacg cattgtctgt tgcgtggttt catcatccac 660 ggctgcataa cccagctctt tcagttgcca gattttgcgg cggaacaact cttccacacg 720 cggccagttg ttcggctgac tcacgtccat cgcgttggat aacgtcgcct gagtcgcttt 780 gggtgaccac tgaccgtcgt gcaggaaacg tggcacggta tcgttcacgt tggtcgcagc 840 aataaaacgt ttcaccggca gaccgagtga cttcgccagc agacccgccg tcaaatcgcc 900 gaagtttccg cttggcaccg agacaaccag ctggttgcgc gcttcctgcg gcagctgcgc 960 aactgcttca aagtagtagc aaatctgcgc cagcaaacgg ctgatgttaa tcgagttagc 1020 tgagtttaac cccagcgcca ctttcagctc ttcatcatca aacgcctgct tcgccagcgc 1080 ctgacaggca tcgaaatcgc cgtcgatggc aacagtttcg atattgccgc ccaatgtaca 1140 gaacagtttt tcttgcagtg gactgatttt gcctcgtgga tagaggataa ccactttcac 1200 attcggtaaa ccgtagaaag catgagccac tgccgctccg gtatcaccgg aggtcgcggt 1260 cagaatggtc actggcttat cgcccgcaat atgggtcagc atttgtgcca taaagcgacc 1320 gccgaaatct ttaaatgcca gcgttggccc gtggaacaat tccagacaac cgacatcgct 1380 ttcaacattg gcgaccggag ccgggaaggc aaacgccgcg cgcacgcgct cttccaggat 1440 ttcctgcggg atttcatcac caataaacgc cgagaggatc ttcgcactgc gggtgacaaa 1500 atccagcttc agcatctcat caatttcagt caggctgaat tccggcaggt catgcggaaa 1560 aaacagcccc tgatttttgc ccaacccctg ggttacggct tgcgcaaagc tgacctgctc 1620 gttgtgatct ttcagattgt agagtttcat ttagttttcc agtactcgtg cgcccgccgt 1680 atccagccgg caaatatgaa caaaaccttc ctgattttgc aggtagttct tacccaacca 1740 gtcggcaacg cgctgggcgg tatccggctt gtcacataga gcgaacaagg tcgggccgga 1800 gccggagata ccgctcgcta ccgcgccgat ttccgcgacc gcccgccgcg cctgccggaa 1860 gccaggcagt aaccgttcac ggtagggttc agcgataaca tctttcatca gcttcgcggc 1920 aagctcaggc tgacgggaat agcaggcgtg aatgaagcca gccagatgtc gcccgtgcgc 1980 aatgcaatcc tggcggcgat actgcgccgg taaaatagcc ctggcttctg ccgtcgagac 2040 tttaatcccc ggatacgcca gcacccacag ccactcatca aaccctggca cttgctggct 2100 gatgatgtcg ttttcttcga tcatcaactg cataccacca agaaaacacg gtgccacgtt 2160 gtcgtaatga atgctgccgg agatacgtcc ttccagctcg cccatcaaag ccagcaaacg 2220 agtgtcatta agcggcttgc cacagtgttc attcatcgcc atcagcgccg cgaccaccga 2280 acaggcgctg gagcctaagc ccgaaccgat tggcatattc ttttccagag tcatcgccac 2340 tggaatttgc ttgccaagct cctggcaaaa acgctcccag cactgataaa cgatattttc 2400 ccgtggttct gacggcagct tatcggcaaa gcgtccgagg ttgttgagac tgaatgtctc 2460 tgccgcctca accgtgacta catctccgag caatgcacca tcaacaggtg tcaccgccgc 2520 cccgagcaca tcaaacccga cgctcatatt ggcactggaa gccggggcat aaactttaac 2580 catgtcagac tcctaacttc catgagaggg tacgtagcag atcggcaaag acaccggcag 2640 ctgtaacgtc attgcccgca ccatatccgc gcagcaccaa cggcagcggc tgataatagt 2700 ggctataaaa ggccagggcg ttttcgccat ttttcacttt gaacagcgga tcattaccat 2760 ccacttcggc aatcttcacg cggcaggcac catcttcatc aatattgcca acatagcgca 2820 aaacctttcc ttcatcacgg gccttcgcca cgcgcgcggc aaagagatcg tcgagctgtg 2880 acagattcgc cataaaagcg gcaacatcac cctcagcgtc aaactctgcg ggcagcacag 2940 gttcaatttc aatatccgcc agctccagtt cacgtcccgt ttcacgagcg agaatcaata 3000 gcttacgcgc tacatccata ccagaaagat catctcgcgg atccggttcg gtataaccca 3060 tttcccgcgc cagagtagtc gcctcggaga aactcatgcc ttcgtctaac ttgccgaaga 3120 tataagaaag cgaacctgaa agaatgccgg agaacttcat caattcatca ccagcattga 3180 gcagattttg caggttctca ataaccggta atccagcccc aacgttggtg tcatagagga 3240 atttacgccg cgatttttcc gccgcatgac gcaacagatg gtagtaatcc atcgacgagg 3300 tgttggcctt tttgttcggc gtgacaacgt ggaaaccttc gcgcaagaag tcggcatatt 3360 gatccgccac tgcctggctg gaggtgcagt caacaatgac cgggttcagc agatgatatt 3420 ctttcacgag gcgaattaag cgcccgagat taaacggctc tttggcttgc gccagttctt 3480 cctgccggtt ttccagattt aggccatgca cattggtgag cagtgccttc gagttggcaa 3540 caccgcagac acgtaagtcg atatgcttat tcttcagcca gctttgttga cgcttcagtt 3600 gctccagcag cgcaccgcca acgccaccga cgccaatcac aaacacttcg ataacctgat 3660 cggtattgaa cagcatctga tgagtaacgc gcacgccagt ggtcgcatca tcgttattta 3720 ccacgacaga gattgagcgt tcagaagatc tctgagcaat ggcgacaatg ttgatattgg 3780 cgcgggccag cgcggcaaag aatttcgccg agatcccacg caaggtgcgc ataccatcac 3840 ctaccaccga gataatggcc agccgttccg tcaccgccag cggctccagt aagccttctt 3900 tcagttccag gtagaactct tcctgcattg cccgttcagc ccgcacacag tcgctttgcg 3960 gaacgcagaa actgatactg tattcggaag atggttgcgt aatcagcacc acggaaatac 4020 gggcgcgcga catcgcggca aagacgcgcg ccgccatgcc gaccattcct ttcatccccg 4080 ggccggaaac gctgaacatt gccatgttat tcagattgga aatgcccttg accggtaatt 4140 cgtcttcatc acgactggca ccaatgagcg tacctggtgc ttgaggattt ccggtatttt 4200 taatcaggca agggatctgg aactgggcga tgggggtaat ggtgcggggg tgaagaactt 4260 tagcgccgaa gtaggaaagc tccatcgctt cctggtagga catcgacttc aacaacctcg 4320 catcgggcac ctgacgcggg tcgcaggtat agaccccgtc aacgtccgtc caaatctcgc 4380 aacaatcggc gcgtaaacag gcagccagca ccgccgcgga gtagtcggaa ccgttgcgtc 4440 caagtaccac cagttcgcct ttttcattac cggcggtgaa acctgccatc aacaccatgt 4500 gatcagccgg aatgcggctt gccgcaatac ggcgggtgga ctcagcaata tcgacggtag 4560 attcgaggta atgccccact gccagcagtt tttcgaccgg atcgataacg gtaacgttgt 4620 gaccacgcgc ttctaacacg ccggccataa tggcgatcga cattttctcg ccacggcaaa 4680 tcagcgcagc gttgatgcta tccgggcact gccccaacaa actaatgcca tgcaggacat 4740 gttttatttg ggcaaattcc tgatcgacga aagttttcaa ttgcgccagc gggaaccccg 4800 gctgggcggc ggcgagtccc gtcaaaagtt cggcaaaaat acgttcggca tcgctgatat 4860 tgggtaaagc atcccggccg ctaatggttt tttcaatcat cgccaccagg tggttggtga 4920 ttttggcggg ggcagagagg acggtggcca cctgcccctg cctggcattg ctttccagaa 4980 tatcggcaac acgcagaaaa cgttctgcat ttgccactga tgtaccgccg aacttcaaca 5040 ctcgcatggt tgttacctcg ttaccttttt tctgtgtttc ctgtacgcgt cagcccgcac 5100 cgttacctgt ggtaatggtg atggtggtgg taatggtggt gctaatgcgt ttcatggatg 5160 ttgtgtactc tgtaattttt atctgtctgt gcgctatgcc tatattggtt gaagtattta 5220 gtgacctaag tcaataaaat tttaatttac tcacggcagg taaccagttc agaagctgct 5280 atcagacact ctttttttaa tccacacaga gacatattgc ccgttgcagt cagaatgaaa 5340 agctgaaaaa tacttactaa ggcgtttttt atttggtgat atttttttca atatcatgca 5400 gcaaacggtg caacattgcc gtgtctcgtt gctctaaaag ccccaggcgt tgttgtaacc 5460 agtcgaccag aatctgaatt cgtcgacaag cttctcgagc ctaggctagc tctagactcg 5520 ttggcgagaa cagaggcgga actggggaca attgtccggg aattattgtt attttttatt 5580 gtattgctgt gtcaggtgat agggcgttgg atctcgccag tgacgtctgt ttctgctaca 5640 gtgcccgttt tacggcaaac ggcttgggta tggcttcatt aaaggatgtc gcacgcctgg 5700 cgggagtgtc gatgatgaca gtctcccggg tgatgcataa tgcagaatct gtgcgtcctg 5760 caacgcgtga ccgcgtattg caggcaatcc agaccctgaa ttatgttcct gatctttccg 5820 cccgtaagat gcgcgctcaa ggacgtaagc cgtcgactct cgccgtgctg gcgcaggaca 5880 cggctaccac tcctttctct gttgatattc tgcttgccat tgagcaaacc gccagcgagt 5940 tcggctggaa tagtttttta atcaatattt tttctgaaga tgacgctgcc cgcgcggcac 6000 gtcagctgct tgcccaccgt ccggatggca ttatctatac tacaatgggg ctgcgatata 6060 tcacgctgcc tgagtctctg tatggtgaaa atattgtatt ggcgaactgt gtggcggatg 6120 acccagcgtt acccagttat atccctgatg attacactgc acaatatgaa tcaacacagc 6180 atttgctcgc ggcgggctat cgtcaaccgt tatgcttctg gctaccggaa agtgcgttgg 6240 caacagggta tcgtcggcag ggatttgagc aggcctggcg tgatgctgaa cgagatctgg 6300 ctgaggtgaa acaatttcac atggcaacag gtgatgatca ctacaccgat ctcgcaagtt 6360 tactcaatgc ccacttcaaa ccgggcaaac cagattttga tgttctgata tgtggtaacg 6420 atcgcgcagc ctttgtggct tatcaggttc ttctggcgaa gggggtacga atcccgcagg 6480 atgtcgccgt aatgggcttt gataatctgg ttggcgtcgg gcatctgttt ttaccgccgc 6540 tgaccacaat tcagcttcca catgacatta tcgggcggga agctgcattg catattattg 6600 aaggtcgtga agggggaaga gtgacgcgga tcccttgccc gctgttgatc cgttgttcca 6660 cctgatatta tgttaaccca gtagccagag tgctccatgt tgcagcacag ccactccgtg 6720 ggaggcataa agcgacagtt cccgttcttc tggctgcgga tagattcgac tactcatcac 6780 cgcttccccg tcgttaataa atacttccac ggatgatgta tcgataaata tccttagggc 6840 gagcgtgtca cgctgcggga ggggaatact acggtagccg tctaaattct cgtgtgggta 6900 ataccgccac aaaacaagtc gctcagattg gttatcaata tacagccgca ttccagtgcc 6960 gagctgtaat ccgtaatgtt cggcatcact gttcttcagc gcccactgca actgaatctc 7020 aactgcttgc gcgttttcct gcaaaacata tttattgctg attgtgcggg gagagacaga 7080 ttgatgctgc tggcgtaacg actcagcttc gtgtaccggg cgttgtagaa gtttgccatt 7140 gctctctgat agctcgcgcg ccagcgtcat gcagcctgcc catccttcac gttttgaggg 7200 cattgacgat tcccacatat ccatccagcc gataacaata cgccgaccat ccttcgctaa 7260 aaagctttgt ggtgcataaa agtcatgccc gttatcaagt tcagtaaaat gcccggattg 7320 tgcaaaaagt cgtcctggcg accacattcc gggtattacg ccactttgaa agcgatttcg 7380 gtaactgtat ccctcggcat tcattccctg cggggaaaac atcagataat gctgatcgcc 7440 aaggctgaaa aagtccggac attcccacat atagctttca cccgcatcag cgtgggccag 7500 tacgcgatcg aaggtccatt cacgcaacga actgccgcga taaagcagga tctgccccgt 7560 gttgcctgga tctttcgccc cgactaccat ccaccatgtg tcggcttcac gccacacttt 7620 aggatcgcgg aagtgcatga ttccttctgg tggagtgagg atcacaccct gtttctcgag 7680 atgaatacca tcccgactgg tagccagaca ttgtacttcg cgaattgcat cgtcattacc 7740 tgcaccatcg agccagacgt gtccggtgta gataagtgag aggacaccat tgtcatcgac 7800 agcactacct gaaaaacacc cgtctttgtc attatcgtct cctggcgcta gcgcaatagg 7860 ctcatgctgc cagtggatca tatcgtcgct ggtggcatgt ccccagtgca ttggccccca 7920 gtgttcgctc atcggatgat gttgataaaa cgcgtgataa cgatcgttaa accagatcag 7980 gccgtttgga tcgttcatcc acccggcagg aggcgcgagg tgaaaatggg gatagaaagt 8040 gttaccccgg tgctcatgaa gttttgctag ggcgttttgc gccgcatgca atcgagattg 8100 cgtcatttta atcatcctgg ttaagcaaat ttggtgaatt gttaacgtta acttttataa 8160 aaataaagtc ccttactttc ataaatgcga tgaatatcac aaatgttaac gttaactatg 8220 acgttttgtg atcgaatatg catgttttag taaatccatg acgattttgc gaaaaagagg 8280 tttatcacta tgcgtaactc agatgaattt aagggaaaaa aatgtcagcc aaagtatggg 8340 ttttagggga tgcggtcgta gatctcttgc cagaatcaga cgggcgccta ctgccttgtc 8400 ctggcggcgc gccagctaac gttgcggtgg gaatcgccag attaggcgga acaagtgggt 8460 ttataggtcg ggtgggggat gatccttttg gtgcgttaat gcaaagaacg ctgctaactg 8520 agggagtcga tatctagcat atgccgggta ccgcactagt tgagaagtaa acggcgaagt 8580 cgctcttatc tctaaatagg acgtgaattt tttaacgaca ggcaggtaat tatggcactg 8640 agtattccat tcagaaatgc gtactatcgt tttgcatcca gttactcatt tctctttttt 8700 atttcctggt cgctgtggtg gtcgttatac gctatttggc tgaaaggaca tctagggttg 8760 acagggacgg aattaggtac actttattcg gtcaaccagt ttaccagcat tctatttatg 8820 atgttctacg gcatcgttca ggataaactc ggtctgaaga aaccgctcat ctggtgtatg 8880 agtttcatcc tggtcttgac cggaccgttt atgatttacg tttatgaacc gttactgcaa 8940 agcaattttt ctgtaggtct aattctgggg gcgctgtttt ttggctgggg gtatctggcg 9000 ggatgcggtt tgcttgatag cttcaccgaa aaaatggcgc gaaattttca tttcgaatat 9060 ggaacagcgc gcgcctgggg atcttttggc tatgctattg gcgcgttctt tgccggcata 9120 ttttttagta tcagtcccca tatcaacttc tggttggtct cgctatttgg cgctgtattt 9180 atgatgatca acatgcgttt taaagataag gatcaccagt gcgtagcggc agatgcggga 9240 ggggtaaaaa aagaggattt tatcgcagtt ttcaaggatc gaaacttctg ggttttcgtc 9300 atatttattg tggggacgtg gtctttctat aacatttttg atcaacaact ttttcctgtc 9360 ttttattcag gtttattcga atcacacgat gtaggaacgc gcctgtatgg ttatctcaac 9420 tcattccagg tggtactcga agcgctgtgc atggcgatta ttcctttctt tgtgaatcgg 9480 gtagggccaa aaaatgcatt acttatcgga gttgtgatta tggcgttgcg tatcctttcc 9540 tgcgcgctgt tcgttaaccc ctggattatt tcattagtga agttgttaca tgccattgag 9600 gttccacttt gtgtcatatc cgtcttcaaa tacagcgtgg caaactttga taagcgcctg 9660 tcgtcgacga tctttctgat tggttttcaa attgccagtt cgcttgggat tgtgctgctt 9720 tcaacgccga ctgggatact ctttgaccac gcaggctacc agacagtttt cttcgcaatt 9780 tcgggtattg tctgcctgat gttgctattt ggcattttct tcttgagtaa aaaacgcgag 9840 caaatagtta tggaaacgcc tgtaccttca gcaatataga cgtaaacttt ttccggttgt 9900 tgtcgatagc tctatatccc tcaaccggaa aataataata gtaaaatgct tagccctgct 9960 aataatcgcc taatccaaac gcctcattca tgttctggta cagtcgctca aatgtactcg 10020 tgcacctgca gtggggtaaa tgccatcgag gctagctgtt tttccatctc ttctgcacgc 10080 agcgaaatct cgcggctaag acggtaaacc attaaatttt tgaaccacag catgataatt 10140 tccacggcct tgtcgttaaa tttagcgggc atgataacga attgtcggcg gccttgcatt 10200 gccaatccgg ttgtccgtct ctacgctatt gatattgaaa aaaataagga gagtaccgtg 10260 cgtataggta tcgatttagg cggcaccaaa actgaagtga ttgcactggg cgatgcaggg 10320 gagcagttgt accgccatcg tctgcccacg ccgcgtgatg attaccggca gactattgaa 10380 acgatcgcca cgttggttga tatggcggag caggcgacgg ggcagcgcgg aacggtaggt 10440 atgggcattc ctggctcaat ttcgccttac accggtgtgg tgaagaatgc caattcaacc 10500 tggctcaacg gtcagccatt cgataaagac ttaagcgcga ggttgcagcg ggaagtgcgg 10560 ctggcaaatg acgctaactg tctggcggtt tcagaagcag tagatggcgc ggcagcggga 10620 gcgcagacgg tatttgccgt gattatcggc acgggatgcg gcgcgggcgt ggcattcaat 10680 gggcgggcgc atatcggcgg caatggcacg gcaggtgagt ggggacacaa tccgctaccg 10740 tggatggacg aagacgaact gcgttatcgc gaggaagtcc cttgttattg cggtaaacaa 10800 ggttgtattg aaacctttat ttcgggcacg ggattcgcga tggattatcg tcgtttgagc 10860 ggacatgcgc tgaaaggcag tgaaattatc cgcctggttg aagaaagcga tccggtagcg 10920 gaactggcat tgcgtcgcta cgagctgcgg ctggcaaaat cgctggcaca tgtcgtgaat 10980 attctcgatc cggatgtgat tgtcctgggg ggcgggatga gcaatgtaga ccgtttatat 11040 caaacggttg ggcagttgat taaacaattt gtcttcggcg gcgaatgtga aacgccggtg 11100 cgtaaggcga agcacggtga ttccagcggc gtacgcggcg ctgcgtggtt atggccacaa 11160 gagtaaaaaa cgtaggcaat tggcgcatca tgcctgatgc gacgcttgcc gcgtcttatc 11220 aggcctacaa aaggtgccag aaccgtaggc cggataaggc gttcacgccg catccggcaa 11280 taagtgctcc gatgcctgat gcgacgcttg ccgcgtctta tcaggcctgc aaaatgtgcc 11340 agaaccgcgt agggcggata aggcgttcac gccgcatccg gcaataagta atgagcaccg 11400 agacggccga cgcgctgggc tatctagaga ggaccgaagg agctaaccgc ttttttgcac 11460 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 11520 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta 11580 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 11640 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 11700 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 11760 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 11820 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 11880 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 11940 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 12000 tgagcgtcag accccttaat aagatgatct tcttgagatc gttttggtct gcgcgtaatc 12060 tcttgctctg aaaacgaaaa aaccgccttg cagggcggtt tttcgaaggt tctctgagct 12120 accaactctt tgaaccgagg taactggctt ggaggagcgc agtcaccaaa acttgtcctt 12180 tcagtttagc cttaaccggc gcatgacttc aagactaact cctctaaatc aattaccagt 12240 ggctgctgcc agtggtgctt ttgcatgtct ttccgggttg gactcaagac gatagttacc 12300 ggataaggcg cagcggtcgg actgaacggg gggttcgtgc atacagtcca gcttggagcg 12360 aactgcctac ccggaactga gtgtcaggcg tggaatgaga caaacgcggc cataacagcg 12420 gaatgacacc ggtaaaccga aaggcaggaa caggagagcg cacgagggag ccgccagggg 12480 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca ccactgattt gagcgtcaga 12540 tttcgtgatg cttgtcaggg gggcggagcc tatggaaaaa cggctttgcc gcggccctct 12600 cacttccctg ttaagtatct tcctggcatc ttccaggaaa tctccgcccc gttcgtaagc 12660 catttccgct cgccgcagtc gaacgaccga gcgtagcgag tcagtgagcg aggaagcgga 12720 atatatcctg tatcacatat tctgctgacg caccggtgca gccttttttc tcctgccaca 12780 tgaagcactt cactgacacc ctcatcagtg ccaacatagt aagccagtat acactccgct 12840 agcgctgagg tctgcctcgt gaagaaggtg ttgctgactc ataccaggcc tgaatcgccc 12900 catcatccag ccagaaagtg agggagccac ggttgatgag agctttgttg taggtggacc 12960 agttggtgat tttgaacttt tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg 13020 tgatctgatc cttcaactca gcaaaagttc gatttattca acaaagccac gttgtgtctc 13080 aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt 13140 ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt 13200 gctcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc 13260 gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc 13320 cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg 13380 tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta 13440 ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat 13500 tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc 13560 ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg 13620 ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc 13680 gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac 13740 cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga 13800 aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg 13860 ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa 13920 aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt 13980 ttttctaatc agaattggtt aattggttgt aacactggca gagcattacg ctgacttgac 14040 gggacggcgg ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agatcacgca 14100 tcttcccgac aacgcagacc gttccgtggc aaagcaaaag ttcaaaatca ccaactggtc 14160 cacctacaac aaagctctca tcaaccgtgg ctccctcact ttctggctgg atgatggggc 14220 gattcaggcc tggtatgagt cagcaacacc ttcttcacga ggcagacctc agcgctcaaa 14280 gatgcagggg taaaagctaa ccgcatcttt accgacaagg catccggcag ttcaacagat 14340 cgggaagggc tggatttgct gaggatgaag gtggaggaag gtgatgtcat tctggtgaag 14400 aagctcgacc gtcttggccg cgacaccgcc gacatgatcc aactgataaa agagtttgat 14460 gctcagggtg tagcggttcg gtttattgac gacgggatca gtaccgacgg tgatatgggg 14520 caaatggtgg tcaccatcct gtcggctgtg gcacaggctg aacgccggag gatcctagag 14580 cgcacgaatg agggccgaca ggaagcaaag ctgaaaggaa tcaaatttgg ccgcaggcgt 14640 accgtggaca ggaacgtcgt gctgacgctt catcagaagg gcactggtgc aacggaaatt 14700 gctcatcagc tcagtattgc ccgctccacg gtttataaaa ttcttgaaga cgaaagggcc 14760 tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct tagacgtcag 14820 gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 14880 caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 14940 ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 15000 gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 15060 tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 15120 ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 15180 tattatcccg t 15191 <210> 29 <211> 10728 <212> DNA <213> Artificial Sequence <220> <223> pC-Ptrc-scrAB (Klebsiella pneumoniae (ATCC700721)) <400> 29 agctcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag 60 attgtactga gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa 120 taccgcatca ggcgccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg 180 cgggcctctt cgctattacg ccagctggcg aaagggggat gtgctgcaag gcgattaagt 240 tgggtaacgc cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgaattcgag 300 ctcgggcccc cacacgtgtg gtctagatat catcgactgc acggtgcacc aatgcttctg 360 gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat 420 tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg 480 gttctggcaa atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg 540 tggaattgtg agcggataac aatttcacac aggaaacaga ccatggattt tgaacagatt 600 tcccgctcac tgcttcccct gctgggcggc aaggaaaata tcgccagcgc cgcgcactgc 660 gccacccgcc tgcggctggt gctggtcgac gacgcgctcg ccgatcagca ggcgattggc 720 aaaatcgacg gggtgaaagg ctgctttcgc aatgccggac agatgcagat catcttcggc 780 accggggtgg tcaataaagt ctatgccgcc tttatccagg ccgcaggcat cagcgaatcg 840 agcaaatccg aagccgccga cctggcggcg aaaaagctga acccgttcca gcgcatcgcc 900 cgcctgctgt ccaacatctt cgtgccgatt attccggcca tcgtcgcctc cggcctgctg 960 atgggcctgc tggggatggt gaaaacctac ggttgggtcg acccgagcaa cgctctctat 1020 atcatgctgg atatgtgcag ttcggcggcg tttatcattc tgccgatcct gatcggcttt 1080 accgccgcac gcgaatttgg cggtaacccc tatctgggcg cgaccctcgg cgggatcctc 1140 acccatccgg cgctgaccaa cgcctggggc gtcgccgccg gcttccacac catgaatttc 1200 ttcggcatcg aagtggcgat gatcggctac cagggcaccg tcttcccggt gctgctggcg 1260 gtgtggttta tgagcatggt cgagaagcgg ctgcgccgcg tgatccctga cgcgctggac 1320 ctgatcctca ctccgttcct gacggtgatt atctccggct ttatcgccct gctgctgatc 1380 ggcccggccg gtcgcgcgct cggcgacggc atttcgttta tcctcagcac gcttatcagc 1440 catgccggct ggctggcggg cctgctgttc ggcggcctct attcggtgat cgtcattacc 1500 ggtatccatc acagcttcca tgccatcgag gccggactgc tgggcaaccc atcgattggc 1560 gtcaacttcc tgctgccgat ctgggcgatg gccaacgtcg cccagggcgg cgcctgcttt 1620 gcggtgtggt ttaaaaccaa agatgccaaa ataaaagcca tcaccctgcc gtcggcgttt 1680 tcggcgatgc tggggatcac cgaggcggca atcttcggga ttaacctgcg ctttgtgaaa 1740 ccgtttatcg ccgcgctggt gggcggtgcc gccggcggcg cctgggtggt gtcgatgcac 1800 gtctacatga ccgcggtggg cctgacggcg atcccgggga tggctatcgt gcaggccagc 1860 tcgctgctga actacattat cggaatggcg atcgccttcg ccgtggcctt cgcgctctct 1920 ctgacgctga aatacaaaac ggacgctgaa taatgtcatt accgtcacgc ctgcctgcga 1980 tcctgcaggc cgttatgcag ggccagccgc aggcgctggc cgacagccat tatccgcaat 2040 ggcatctggc gccggtcaac ggactgctga acgatcctaa cggcttttgc caggtcgccg 2100 ggcgttacca cctgttttat cagtggaacc cgctcgcctg cgaccatacc tataagtgct 2160 ggggacactg gagctctgcc gatctgctgc actggcggca cgaacctatc gccctgatgc 2220 cggatgaaga gtatgaccgc aacggctgct actctggcag cgcggtcgag ttcgagggcg 2280 ccctgactct gtgctacacc ggcaacgtga aattccccga cggcgggcgc accgcctggc 2340 aatgtctggc gaccgagaat gccgatggca ccttccgcaa gctggggccg gtgctgccgc 2400 tgccagaagg ctataccggc catgtgcgcg accctaaagt gtggcggcag gacgggcgct 2460 ggtacatggt tcttggggcg caggatgtgc aacagcgcgg caaagtgctg ctgtttaccg 2520 ccagcgacct gcgggagtgg cgcctggtgg gcgagattgc cgggcacgac gtaaacggcc 2580 tggcgaacgc cggctacatg tgggagtgcc cggatctctt tccgctggcg gacacccacc 2640 tgctgatctg ctgcccgcag gggctggccc gcgaagcgca gcgctttctc aatacctatc 2700 cggcggtgtg gatggcaggc cgcttcgacg ccgaacgcgg gatcttcgac cacggcccgc 2760 tgcacgagct ggacagcgga tttgagttct acgcgccgca gaccatgcag gccgatgatg 2820 gccgccgcct gctggttggc tggatgggcg tccccgacgg ggacgagatg catcagccca 2880 cccgcgcgca gggctggatc catcagatga cctgcgtgcg tgagctggag tggcaggctg 2940 gcactctgta tcagcgtccg ctgcgcgagc tggtcgccct gcgcggggaa gcccagggct 3000 ggtgcggaca gaccctgccc ctcgccccga tggagctggc ctttgacatc gcccccaaca 3060 gcacgctggg gctggacttt gccggcgccc tgcagctcac cgtcaatcgc gacggcctgc 3120 gtctgtcgcg tcgcggcctg cagacggcgg agctgcatca ccgctactgg cgcggcgagg 3180 cgcgacgcct gcggatcttt atcgaccgct ccagcgtgga gattttcatc aacgatggcg 3240 agggggtgat gagcagccgc ttcttcccgg gctatccggg gcagctcatc ttcagcggtg 3300 cgacgccggt agcattctgc cgctggctgc tgcggccatg catggtagaa taagcgtttt 3360 gctttcaggc tcccgggtgc tgatgaaaac caaactctag agtacatgca tctatggtac 3420 ctcaatcggt aacgcgtctg cattgtctca tgagcggata catatttgaa tgtatttaga 3480 aaaataaaca aaagagtttg tagaaacgca aaaaggccat ccgtcaggat ggccttctgc 3540 ttaatttgat gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc 3600 ttcgcaacgt tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac 3660 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc 3720 ctggcagttc cctactctcg catggggaga ccccacacta ccatcggcgc tacggcgttt 3780 cacttctgag ttcggcatgg ggtcaggtgg gaccaccgcg ctactgccgc caggcaaatt 3840 ctgttttatc agaccgcttc tgcgttctga tttaatctgt atcaggctga aaatcttctc 3900 tcatccgcca aaacagccaa gcttaaggtg cacggcccac gtggccacta gtacttctcg 3960 agctcttata aaccaggtcg aacccccagc gtatggcaaa tcgcgtaact catttcagca 4020 cggttaagcg tatagaagtg gaaatctttc actccttcac ggcttaaaat cttcaccata 4080 tccatggcaa tattcgcgcc aaccagtttg cgggtttcgg catcatcatc cagaccgtcg 4140 aacatttgcg ccatccacgc cggaatacgc acgttggtca tatcggcaaa tttcttcgcc 4200 tgtttaaagt tagataccgg caaaattccc ggaataattt ccacatcaat gcccgccgat 4260 acacagcggt cacgaaaacg caggtagctt tcgacatcga agaagaactg agtaatcgcg 4320 cggttggctc cggcatccac tttgcgtttc agattaagca aatccgcctg agcgcttttt 4380 gcttccgggt gaacttccgg atacgccgcc acggagatat cgaaatctgc cacttctttt 4440 aacagcgtca ccaggtcaga agcatacatt tctggcttac cacttcccgg cggcagatcg 4500 ccacgcagcg ccacgatatg acgaataccg ttattccagt agtcgcgtgc aatggtgcgc 4560 agctcgtcgg gcgtcgcatc aatgcaagta agatgcggtg ccgcttccag accagtgcga 4620 tctttaatgc ctttaataat gctgtgcgta cggtcgcgct cgccggagtt cgcgccatag 4680 gtcaccgata caaacttcgg tttcaggctg ctaaggcgat cgatggagtt ccacagggtc 4740 tgctccattt cactggtacg cggcgggaaa aactcgaacg aaacgttaat ctgcccctgg 4800 acttctgcca ggctctgatt cagggcatcc cgctggctgg cgtgaaaaaa gctcatagct 4860 agcatgcgca aatttaaagc gctgatatcg atcgcgcgca gatctgtcat gatgatcatt 4920 gcaattggat ccatatatag ggcccgggtt ataattacct gagctcttat gcgtaaaccg 4980 ggtaacgtgc gcagatgtcg agaactttac ctttgatgcg ctcgataacg gcttcatcat 5040 tgatgctgtc cagcacgtca cacatccagc cagccagttc tttcgcttcg gcttctttaa 5100 agccgcgacg ggtaatcgcc ggagtaccta cacgaatacc ggaggtcaca aacgggctct 5160 tcggatcgtt cggtacgctg tttttgttga cggtgatgtt agcacggccc agagcggcgt 5220 ctgcttcttt accggtcagg tttttatcaa ccagatcaac caggaacagg tggttatcag 5280 tgccgccgga aaccactttg tagccgcgct cgaggaacac ttctaccatc gctttagcgt 5340 ttttagcgac ctgctgctgg taagttttga actcaggctc catcgcttct ttcagagcaa 5400 ccgctttacc ggcgattacg agcatcaacg gaccgccctg accaccaggg aaaacggcag 5460 agttcagttt tttgtacagc tcttcgctac cacctttcgc caggatcagg ccgccgcgcg 5520 gacccgccag ggttttgtga gtggtggtag taacaacgtg agcatgagga accgggttcg 5580 ggtagacgcc agcagcaacc aggcccgcaa cgtgcgccat atcaacgaac aggtaagcac 5640 cgatgctgtc agcgatttca cgcattttcg cccagtccac cacgccggaa tatgcagaga 5700 aaccaccgat aatcattttc ggcttgtgtt ctttggcttg tttttccaga tcggcgtagt 5760 cgatatgacc ggtagcatcg ataccgtaag gaacgatgtt gtacagttta ccggagaagt 5820 taaccggaga accgtgagtc aggtgaccgc catgcgccag gttcataccc agaacggtat 5880 cacctggttc cagcagcgcg gtgtagaccg caaagttagc ctgggagccg gagtgcggct 5940 ggacgttagc gtagtcagcg ccgaacagtt ctttcgcacg atcgatcgcc agttgttcaa 6000 cgatatcaac atactcgcaa ccgccgtagt agcgtttgcc cggataacct tcagcatatt 6060 tgttggtcag ctgagaaccc tgcgcctgca ttacgcgcgg gctggtgtag ttttcggagg 6120 cgatcagttc gatgtgctct tcctgacgta ctttttcctg ctccatagcc tgccacagtt 6180 cggcatcata atcggcaatg ttcatttcac gctataacat cgctagcatg cgcaaattta 6240 aagcgctgat atcgatcgcg cgcagatctg tcatgatgat cattgcaatt ggatccatat 6300 atagggcccg ggttataatt acctcaggtc gacgtcccat gtgcaggtgc tgaattcgaa 6360 ggcattccac gacgtggtca ccagccatgg tctgtttcct gtgtgaaatt gttatccgct 6420 cacaattcca cacattatac gagccggatg attaattgtc aacagctcat ttcagaatat 6480 ttgccagaac cgttatgatg tcggcgcaaa tgcaggcatg caagcttggc gtaatcatgg 6540 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc 6600 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg 6660 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc 6720 ggccaacgcg aattcccgac agtaagacgg gtaagcctgt tgatgatacc gctgccttac 6780 tgggtgcatt agccagtctg aatgacctgt cacgggataa tccgaagtgg tcagactgga 6840 aaatcagagg gcaggaactg ctgaacagca aaaagtcaga tagcaccaca tagcagaccc 6900 gccataaaac gccctgagaa gcccgtgacg ggcttttctt gtattatggg tagtttcctt 6960 gcatgaatcc ataaaaggcg cctgtagtgc catttacccc cattcactgc cagagccgtg 7020 agcgcagcga actgaatgtc acgaaaaaga cagcgactca ggtgcctgat ggtcggagac 7080 aaaaggaata ttcagcgatt tgcccgagct tgcgagggtg ctacttaagc ctttagggtt 7140 ttaaggtctg ttttgtagag gagcaaacag cgtttgcgac atccttttgt aatactgcgg 7200 aactgactaa agtagtgagt tatacacagg gctgggatct attcttttta tcttttttta 7260 ttctttcttt attctataaa ttataaccac ttgaatataa acaaaaaaaa cacacaaagg 7320 tctagcggaa tttacagagg gtctagcaga atttacaagt tttccagcaa aggtctagca 7380 gaatttacag atacccacaa ctcaaaggaa aaggactagt aattatcatt gactagccca 7440 tctcaattgg tatagtgatt aaaatcacct agaccaattg agatgtatgt ctgaattagt 7500 tgttttcaaa gcaaatgaac tagcgattag tcgctatgac ttaacggagc atgaaaccaa 7560 gctaatttta tgctgtgtgg cactactcaa ccccacgatt gaaaacccta caaggaaaga 7620 acggacggta tcgttcactt ataaccaata cgctcagatg atgaacatca gtagggaaaa 7680 tgcttatggt gtattagcta aagcaaccag agagctgatg acgagaactg tggaaatcag 7740 gaatcctttg gttaaaggct ttgagatttt ccagtggaca aactatgcca agttctcaag 7800 cgaaaaatta gaattagttt ttagtgaaga gatattgcct tatcttttcc agttaaaaaa 7860 attcataaaa tataatctgg aacatgttaa gtcttttgaa aacaaatact ctatgaggat 7920 ttatgagtgg ttattaaaag aactaacaca aaagaaaact cacaaggcaa atatagagat 7980 tagccttgat gaatttaagt tcatgttaat gcttgaaaat aactaccatg agtttaaaag 8040 gcttaaccaa tgggttttga aaccaataag taaagattta aacacttaca gcaatatgaa 8100 attggtggtt gataagcgag gccgcccgac tgatacgttg attttccaag ttgaactaga 8160 tagacaaatg gatctcgtaa ccgaacttga gaacaaccag ataaaaatga atggtgacaa 8220 aataccaaca accattacat cagattccta cctacataac ggactaagaa aaacactaca 8280 cgatgcttta actgcaaaaa ttcagctcac cagttttgag gcaaaatttt tgagtgacat 8340 gcaaagtaag tatgatctca atggttcgtt ctcatggctc acgcaaaaac aacgaaccac 8400 actagagaac atactggcta aatacggaag gatctgaggt tcttatggct cttgtatcta 8460 tcagtgaagc atcaagacta acaaacaaaa gtagaacaac tgttcaccgt tacatatcaa 8520 agggaaaact gtccatatgc acagatgaaa acggtgtaaa aaagatagat acatcagagc 8580 ttttacgagt ttttggtgca ttcaaagctg ttcaccatga acagatcgac aatgtaacag 8640 atgaacagca tgtaacacct aatagaacag gtgaaaccag taaaacaaag caactagaac 8700 atgaaattga acacctgaga caacttgtta cagctcaaca gtcacacata gacagcctga 8760 aacaggcgat gctgcttatc gaatcaaagc tgccgacaac acgggagcca gtgacgcctc 8820 ccgtggggaa aaaatcatgg caattctgga agaaatagcg ctttcagccg gcaaaccggc 8880 tgaagccgga tctgcgattc tgataacaaa ctagcaacac cagaacagcc cgtttgcggg 8940 cagcaaaacc cgtgggaatt aattcccctg ctcgcgcagg ctgggtgcca agctctcggg 9000 taacatcaag gcccgatcct tggagccctt gccctcccgc acgatgatcg tgccgtgatc 9060 gaaatccaga tccttgaccc gcagttgcaa accctcactg atccgcatgc ccgttccata 9120 cagaagctgg gcgaacaaac gatgctcgcc ttccagaaaa ccgaggatgc gaaccacttc 9180 atccggggtc agcaccaccg gcaagcgccg cgacggccga ggtcttccga tctcctgaag 9240 ccagggcaga tccgtgcaca gcaccttgcc gtagaagaac agcaaggccg ccaatgcctg 9300 acgatgcgtg gagaccgaaa ccttgcgctc gttcgccagc caggacagaa atgcctcgac 9360 ttcgctgctg cccaaggttg ccgggtgacg cacaccgtgg aaacggatga aggcacgaac 9420 ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg 9480 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat 9540 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc 9600 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg 9660 cagtcgccct aaaacaaagt taaacatcat gagggaagcg gtgatcgccg aagtatcgac 9720 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt 9780 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagtgata ttgatttgct 9840 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt 9900 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt 9960 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga 10020 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct 10080 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga 10140 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac 10200 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc 10260 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg 10320 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta 10380 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca 10440 ctacgtgaaa ggcgagatca ccaaggtagt cggcaaataa tgtctaacaa ttcgttcaag 10500 ccgacgccgc ttcgcggcgc ggcttaactc aagcgttaga tgcactaagc acataattgc 10560 tcacagccaa actatcaggt caagtctgct tttattattt ttaagcgtgc ataataagcc 10620 ctacacaaat tgggagatat atcatgaaag gctggctttt tcttgttatc gcaatagttg 10680 gcgaagtaat cgcaacatcc gcattaaaat ctagcgaggg ctttacta 10728 <210> 30 <211> 11944 <212> DNA <213> Artificial Sequence <220> P223-Ptrc-pyc (corynebacterium glutamicum (KFCC 10881)) <400> 30 gcggccgcaa ggggttcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg 60 cggcatcaga gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccacacagat 120 gcgtaaggag aaaataccgc atcaggcgcc attcgccatt cagctgcgca actgttggga 180 agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 240 aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 300 cagtgaattg taatacgact cactataggg cgaattcgag ctcggtaccc ggggatcctc 360 tagagtcgac ctgcaggcat gcaagcttgg cgtcaggcag ccatcggaag ctgtggtatg 420 gctgtgcagg tcgtaaatca ctgcataatt cgtgtcgctc aaggcgcact cccgttctgg 480 ataatgtttt ttgcgccgac atcataacgg ttctggcaaa tattctgaaa tgagctgttg 540 acaattaatc atccggctcg tataatgtgt ggaattgtga gcggataaca atttcacaca 600 ggaaacagac catggcgact cacacatctt caacgcttcc agcattcaaa aagatcttgg 660 tagcaaaccg cggcgaaatc gcggtccgtg ctttccgtgc agcactcgaa accggtgcag 720 ccacggtagc tatttacccc cgtgaagatc ggggatcatt ccaccgctct tttgcttctg 780 aagctgtccg cattggtacc gaaggctcac cagtcaaggc gtacctggac atcgatgaaa 840 ttatcggtgc agctaaaaaa gttaaagcag atgccattta cccgggatac ggcttcctgt 900 ctgaaaatgc ccagcttgcc cgcgagtgtg cggaaaacgg cattactttt attggcccaa 960 ccccagaggt tcttgatctc accggtgata agtctcgcgc ggtaaccgcc gcgaagaagg 1020 ctggtctgcc agttttggcg gaatccaccc cgagcaaaaa catcgatgag atcgttaaaa 1080 gcgctgaagg ccagacttac cccatctttg tgaaggcagt tgccggtggt ggcggacgcg 1140 gtatgcgttt tgttgcttca cctgatgagc ttcgcaaatt agcaacagaa gcatctcgtg 1200 aagctgaagc ggctttcggc gatggcgcgg tatatgtcga acgtgctgtg attaaccctc 1260 agcatattga agtgcagatc cttggcgatc acactggaga agttgtacac ctttatgaac 1320 gtgactgctc actgcagcgt cgtcaccaaa aagttgtcga aattgcgcca gcacagcatt 1380 tggatccaga actgcgtgat cgcatttgtg cggatgcagt aaagttctgc cgctccattg 1440 gttaccaggg cgcgggaacc gtggaattct tggtcgatga aaagggcaac cacgtcttca 1500 tcgaaatgaa cccacgtatc caggttgagc acaccgtgac tgaagaagtc accgaggtgg 1560 acctggtgaa ggcgcagatg cgcttggctg ctggtgcaac cttgaaggaa ttgggtctga 1620 cccaagataa gatcaagacc cacggtgcag cactgcagtg ccgcatcacc acggaagatc 1680 caaacaacgg cttccgccca gataccggaa ctatcaccgc gtaccgctca ccaggcggag 1740 ctggcgttcg tcttgacggt gcagctcagc tcggtggcga aatcaccgca cactttgact 1800 ccatgctggt gaaaatgacc tgccgtggtt ccgactttga aactgctgtt gctcgtgcac 1860 agcgcgcgtt ggctgagttc accgtgtctg gtgttgcaac caacattggt ttcttgcgtg 1920 cgttgctgcg ggaagaggac ttcacttcca agcgcatcgc caccggattc attgccgatc 1980 actcgcacct ccttcaggct ccacctgctg atgatgagca gggacgcatc ctggattact 2040 tggcagatgt caccgtgaac aagcctcatg gtgtgcgtcc aaaggatgtt gcagctccta 2100 tcgataagct gcctaacatc aaggatctgc cactgccacg cggttcccgt gaccgcctga 2160 agcagcttgg cccagccgcg tttgctcgtg atctccgtga gcaggacgca ctggcagtta 2220 ctgataccac cttccgcgat gcacaccagt ctttgcttgc gacccgagtc cgctcattcg 2280 cactgaagcc tgcggcagag gccgtcgcaa agctgactcc tgagcttttg tccgtggagg 2340 cctggggcgg cgcgacctac gatgtggcga tgcgtttcct ctttgaggat ccgtgggaca 2400 ggctcgacga gctgcgcgag gcgatgccga atgtaaacat tcagatgctg cttcgcggcc 2460 gcaacaccgt gggatacacc ccgtacccag actccgtctg ccgcgcgttt gttaaggaag 2520 ctgccagctc cggcgtggac atcttccgca tcttcgacgc gcttaacgac gtctcccaga 2580 tgcgtccagc aatcgacgca gtcctggaga ccaacaccgc ggtagccgag gtggctatgg 2640 cttattctgg tgatctctct gatccaaatg aaaagctcta caccctggat tactacctaa 2700 agatggcaga ggagatcgtc aagtctggcg ctcacatctt ggccattaag gatatggctg 2760 gtctgcttcg cccagctgcg gtaaccaagc tggtcaccgc actgcgccgt gaattcgatc 2820 tgccagtgca cgtgcacacc cacgacactg cgggtggcca gctggcaacc tactttgctg 2880 cagctcaagc tggtgcagat gctgttgacg gtgcttccgc accactgtct ggcaccacct 2940 cccagccatc cctgtctgcc attgttgctg cattcgcgca cacccgtcgc gataccggtt 3000 tgagcctcga ggctgtttct gacctcgagc cgtactggga agcagtgcgc ggactgtacc 3060 tgccatttga gtctggaacc ccaggcccaa ccggtcgcgt ctaccgccac gaaatcccag 3120 gcggacagtt gtccaacctg cgtgcacagg ccaccgcact gggccttgcg gatcgtttcg 3180 aactcatcga agacaactac gcagccgtta atgagatgct gggacgccca accaaggtca 3240 ccccatcctc caaggttgtt ggcgacctcg cactccacct cgttggtgcg ggtgtggatc 3300 cagcagactt tgctgccgat ccacaaaagt acgacatccc agactctgtc atcgcgttcc 3360 tgcgcggcga gcttggtaac cctccaggtg gctggccaga gccactgcgc acccgcgcac 3420 tggaaggccg ctccgaaggc aaggcacctc tgacggaagt tcctgaggaa gagcaggcgc 3480 acctcgacgc tgatgattcc aaggaacgtc gcaatagcct caaccgcctg ctgttcccga 3540 agccaaccga agagttcctc gagcaccgtc gccgcttcgg caacacctct gcgctggatg 3600 atcgtgaatt cttctacggc ctggtcgaag gccgcgagac tttgatccgc ctgccagatg 3660 tgcgcacccc actgcttgtt cgcctggatg cgatctctga gccagacgat aagggtatgc 3720 gcaatgttgt ggccaacgtc aacggccaga tccgcccaat gcgtgtgcgt gaccgctccg 3780 ttgagtctgt caccgcaacc gcagaaaagg cagattcctc caacaagggc catgttgctg 3840 caccattcgc tggtgttgtc accgtgactg ttgctgaagg tgatgaggtc aaggctggag 3900 atgcagtcgc aatcatcgag gctatgaaga tggaagcaac aatcactgct tctgttgacg 3960 gcaaaatcga tcgcgttgtg gttcctgctg caacgaaggt ggaaggtggc gacttgatcg 4020 tcgtcgtttc ctaaaccttt ctgtaaaaag ccccgcgtct tcctcatgga ggaggcgggg 4080 ctttttgggc caagatggga gatgggtgag ttggatttgg tctgattcga cacttttaag 4140 ggcagagatt tgaagatgga gaccaaggct caaagggaat ccatgccgtc ttggtttaaa 4200 gcttgagtat tctatagtct cacctaaata gcttggcgta atcatggtca tagctgtttc 4260 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 4320 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 4380 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgaac 4440 cccttgcggc cgcccgggcc gtcgaccaat tctcatgttt gacagcttat catcgaattt 4500 ctgccattca tccgcttatt atcacttatt caggcgtagc aaccaggcgt ttaagggcac 4560 caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta ctgttgtaat 4620 tcattaagca ttctgccgac atggaagcca tcacaaacgg catgatgaac ctgaatcgcc 4680 agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatggtgaa aacgggggcg 4740 aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac ccagggattg 4800 gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag gttttcaccg 4860 taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc gtggtattca 4920 ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca 4980 ctatcccata tcaccagctc accgtctttc attgccatac gaaattccgg atgagcattc 5040 atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt tttctttacg 5100 gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca ttgagcaact 5160 gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac ggtggtatat 5220 ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga taactcaaaa 5280 aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct tacgtgccga 5340 tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac agggacacca 5400 ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcgcg ataagctcat 5460 ggagcggcgt aaccgtcgca caggaaggac agagaaagcg cggatctggg aagtgacgga 5520 cagaacggtc aggacctgga ttggggaggc ggttgccgcc gctgctgctg acggtgtgac 5580 gttctctgtt ccggtcacac cacatacgtt ccgccattcc tatgcgatgc acatgctgta 5640 tgccggtata ccgctgaaag ttctgcaaag cctgatggga cataagtcca tcagttcaac 5700 ggaagtctac acgaaggttt ttgcgctgga tgtggctgcc cggcaccggg tgcagtttgc 5760 gatgccggag tctgatgcgg ttgcgatgct gaaacaatta tcctgagaat aaatgccttg 5820 gcctttatat ggaaatgtgg aactgagtgg atatgctgtt tttgtctgtt aaacagagaa 5880 gctggctgtt atccactgag aagcgaacga aacagtcggg aaaatctccc attatcgtag 5940 agatccgcat tattaatctc aggagcctgt gtagcgttta taggaagtag tgttctgtca 6000 tgatgcctgc aagcggtaac gaaaacgatt tgaatatgcc ttcaggaaca atagaaatct 6060 tcgtgcggtg ttacgttgaa gtggagcgga ttatgtcagc aatggacaga acaacctaat 6120 gaacacagaa ccatgatgtg gtctgtcctt ttacagccag tagtgctcgc cgcagtcgag 6180 cgacagggcg aagccctcga gctggttgcc ctcgccgctg ggctggcggc cgtctatggc 6240 cctgcaaacg cgccagaaac gccgtcgaag ccgtgtgcga gacaccgcgg ccggccgccg 6300 gcgttgtgga tacctcgcgg aaaacttggc cctcactgac agatgagggg cggacgttga 6360 cacttgaggg gccgactcac ccggcgcggc gttgacagat gaggggcagg ctcgatttcg 6420 gccggcgacg tggagctggc cagcctcgca aatcggcgaa aacgcctgat tttacgcgag 6480 tttcccacag atgatgtgga caagcctggg gataagtgcc ctgcggtatt gacacttgag 6540 gggcgcgact actgacagat gaggggcgcg atccttgaca cttgaggggc agagtgctga 6600 cagatgaggg gcgcacctat tgacatttga ggggctgtcc acaggcagaa aatccagcat 6660 ttgcaagggt ttccgcccgt ttttcggcca ccgctaacct gtcttttaac ctgcttttaa 6720 accaatattt ataaaccttg tttttaacca gggctgcgcc ctgtgcgcgt gaccgcgcac 6780 gccgaagggg ggtgcccccc cttctcgaac cctcccggtc gagtgagcga ggaagcacca 6840 gggaacagca cttatatatt ctgcttacac acgatgcctg aaaaaacttc ccttggggtt 6900 atccacttat ccacggggat atttttataa ttattttttt tatagttttt agatcttctt 6960 ttttagagcg ccttgtaggc ctttatccat gctggttcta gagaaggtgt tgtgacaaat 7020 tgccctttca gtgtgacaaa tcaccctcaa atgacagtcc tgtctgtgac aaattgccct 7080 taaccctgtg acaaattgcc ctcagaagaa gctgtttttt cacaaagtta tccctgctta 7140 ttgactcttt tttatttagt gtgacaatct aaaaacttgt cacacttcac atggatctgt 7200 catggcggaa acagcggtta tcaatcacaa gaaacgtaaa aatagcccgc gaatcgtcca 7260 gtcaaacgac ctcactgagg cggcatatag tctctcccgg gatcaaaaac gtatgctgta 7320 tctgttcgtt gaccagatca gaaaatctga tggcacccta caggaacatg acggtatctg 7380 cgagatccat gttgctaaat atgctgaaat attcggattg acctctgcgg aagccagtaa 7440 ggatatacgg caggcattga agagtttcgc ggggaaggaa gtggtttttt atcgccctga 7500 agaggatgcc ggcgatgaaa aaggctatga atcttttcct tggtttatca aacgtgcgca 7560 cagtccatcc agagggcttt acagtgtaca tatcaaccca tatctcattc ccttctttat 7620 cgggttacag aaccggttta cgcagtttcg gcttagtgaa acaaaagaaa tcaccaatcc 7680 gtatgccatg cgtttatacg aatccctgtg tcagtatcgt aagccggatg gctcaggcat 7740 cgtctctctg aaaatcgact ggatcataga gcgttaccag ctgcctcaaa gttaccagcg 7800 tatgcctgac ttccgccgcc gcttcctgca ggtctgtgtt aatgagatca acagcagaac 7860 tccaatgcgc ctctcataca ttgagaaaaa gaaaggccgc cagacgactc atatcgtatt 7920 ttccttccgc gatatcactt ccatgacgac aggatagtct gagggttatc tgtcacagat 7980 ttgagggtgg ttcgtcacat ttgttctgac ctactgaggg taatttgtca cagttttgct 8040 gtttccttca gcctgcatgg attttctcat actttttgaa ctgtaatttt taaggaagcc 8100 aaatttgagg gcagtttgtc acagttgatt tccttctctt tcccttcgtc atgtgacctg 8160 atatcggggg ttagttcgtc atcattgatg agggttgatt atcacagttt attactctga 8220 attggctatc cgcgtgtgta cctctacctg gagtttttcc cacggtggat atttcttctt 8280 gcgctgagcg taagagctat ctgacagaac agttcttctt tgcttcctcg ccagttcgct 8340 cgctatgctc ggttacacgg ctgcggcgag cgctagtgat aataagtgac tgaggtatgt 8400 gctcttctta tctccttttg tagtgttgct cttattttaa acaactttgc ggttttttga 8460 tgactttgcg attttgttgt tgctttgcag taaattgcaa gatttaataa aaaaacgcaa 8520 agcaatgatt aaaggatgtt cagaatgaaa ctcatggaaa cacttaacca gtgcataaac 8580 gctggtcatg aaatgacgaa ggctatcgcc attgcacagt ttaatgatga cagcccggaa 8640 gcgaggaaaa taacccggcg ctggagaata ggtgaagcag cggatttagt tggggtttct 8700 tctcaggcta tcagagatgc cgagaaagca gggcgactac cgcacccgga tatggaaatt 8760 cgaggacggg ttgagcaacg tgttggttat acaattgaac aaattaatca tatgcgtgat 8820 gtgtttggta cgcgattgcg acgtgctgaa gacgtatttc caccggtgat cggggttgct 8880 gcccataaag gtggcgttta caaaacctca gtttctgttc atcttgctca ggatctggct 8940 ctgaaggggc tacgtgtttt gctcgtggaa ggtaacgacc cccagggaac agcctcaatg 9000 tatcacggat gggtaccaga tcttcatatt catgcagaag acactctcct gcctttctat 9060 cttggggaaa aggacgatgt cacttatgca ataaagccca cttgctggcc ggggcttgac 9120 attattcctt cctgtctggc tctgcaccgt attgaaactg agttaatggg caaatttgat 9180 gaaggtaaac tgcccaccga tccacacctg atgctccgac tggccattga aactgttgct 9240 catgactatg atgtcatagt tattgacagc gcgcctaacc tgggtatcgg cacgattaat 9300 gtcgtatgtg ctgctgatgt gctgattgtt cccacgcctg ctgagttgtt tgactacacc 9360 tccgcactgc agtttttcga tatgcttcgt gatctgctca agaacgttga tcttaaaggg 9420 ttcgagcctg atgtacgtat tttgcttacc aaatacagca atagcaatgg ctctcagtcc 9480 ccgtggatgg aggagcaaat tcgggatgcc tggggaagca tggttctaaa aaatgttgta 9540 cgtgaaacgg atgaagttgg taaaggtcag atccggatga gaactgtttt tgaacaggcc 9600 attgatcaac gctcttcaac tggtgcctgg agaaatgctc tttctatttg ggaacctgtc 9660 tgcaatgaaa ttttcgatcg tctgattaaa ccacgctggg agattagata atgaagcgtg 9720 cgcctgttat tccaaaacat acgctcaata ctcaaccggt tgaagatact tcgttatcga 9780 caccagctgc cccgatggtg gattcgttaa ttgcgcgcgt aggagtaatg gctcgcggta 9840 atgccattac tttgcctgta tgtggtcggg atgtgaagtt tactcttgaa gtgctccggg 9900 gtgatagtgt tgagaagacc tctcgggtat ggtcaggtaa tgaacgtgac caggagctgc 9960 ttactgagga cgcactggat gatctcatcc cttcttttct actgactggt caacagacac 10020 cggcgttcgg tcgaagagta tctggtgtca tagaaattgc cgatgggagt cgccgtcgta 10080 aagctgctgc acttaccgaa agtgattatc gtgttctggt tggcgagctg gatgatgagc 10140 agatggctgc attatccaga ttgggtaacg attatcgccc aacaagtgct tatgaacgtg 10200 gtcagcgtta tgcaagccga ttgcagaatg aatttgctgg aaatatttct gcgctggctg 10260 atgcggaaaa tatttcacgt aagattatta cccgctgtat caacaccgcc aaattgccta 10320 aatcagttgt tgctcttttt tctcaccccg gtgaactatc tgcccggtca ggtgatgcac 10380 ttcaaaaagc ctttacagat aaagaggaat tacttaagca gcaggcatct aaccttcatg 10440 agcagaaaaa agctggggtg atatttgaag ctgaagaagt tatcactctt ttaacttctg 10500 tgcttaaaac gtcatctgca tcaagaacta gtttaagctc acgacatcag tttgctcctg 10560 gagcgacagt attgtataag ggcgataaaa tggtgcttaa cctggacagg tctcgtgttc 10620 caactgagtg tatagagaaa attgaggcca ttcttaagga acttgaaaag ccagcaccct 10680 gatgcgacca cgttttagtc tacgtttatc tgtctttact taatgtcctt tgttacaggc 10740 cagaaagcat aactggcctg aatattctct ctgggcccac tgttccactt gtatcgtcgg 10800 tctgataatc agactgggac cacggtccca ctcgtatcgt cggtctgatt attagtctgg 10860 gaccacggtc ccactcgtat cgtcggtctg attattagtc tgggaccacg gtcccactcg 10920 tatcgtcggt ctgataatca gactgggacc acggtcccac tcgtatcgtc ggtctgatta 10980 ttagtctggg accatggtcc cactcgtatc gtcggtctga ttattagtct gggaccacgg 11040 tcccactcgt atcgtcggtc tgattattag tctggaacca cggtcccact cgtatcgtcg 11100 gtctgattat tagtctggga ccacggtccc actcgtatcg tcggtctgat tattagtctg 11160 ggaccacgat cccactcgtg ttgtcggtct gattatcggt ctgggaccac ggtcccactt 11220 gtattgtcga tcagactatc agcgtgagac tacgattcca tcaatgcctg tcaagggcaa 11280 gtattgacat gtcgtcgtaa cctgtagaac ggagtaacct cggtgtgcgg ttgtatgcct 11340 gctgtggatt gctgctgtgt cctgcttatc cacaacattt tgcgcacggt tatgtggaca 11400 aaatacctgg ttacccaggc cgtgccggca cgttaaccgg gctgcatccg atgcaagtgt 11460 gtcgctgtcg acgagctcgc gagctcggac atgaggttgc cccgtattca gtgtcgctga 11520 tttgtattgt ctgaagttgt ttttacgtta agttgatgca gatcaattaa tacgatacct 11580 gcgtcataat tgattatttg acgtggtttg atggcctcca cgcacgttgt gatatgtaga 11640 tgataatcat tatcacttta cgggtccttt ccggtgatcc gacaggttac ggggcggcga 11700 cctcgcgggt tttcgctatt tatgaaaatt ttccggttta aggcgtttcc gttcttcttc 11760 gtcataactt aatgttttta tttaaaatac cctctgaaaa gaaaggaaac gacaggtgct 11820 gaaagcgagc tttttggcct ctgtcgtttc ctttctctgt ttttgtccgt ggaatgaaca 11880 atggaagtcc gagctcatcg ctaataactt cgtatagcat acattatacg aagttatatt 11940 cgat 11944

Claims (14)

An Escherichia microorganism having sucrose magnetization and increased L-amino acid production, characterized by conferring sucrose non-magnetizable microorganisms with simultaneous use of a sucrose non-PTS system and a sucrose PTS system.
The Escherichia microorganism according to claim 1, further having the activity of pyruvate carboxylase.
The method of claim 1, wherein the sucrose non-PTS system comprises the activity of sucrose permease, sucrose hydrolase, sucrose transcriptional regulator, and mannokinase,
Wherein said sucrose PTS system comprises sucrose PTS permease and sucrose PTS hydrolase activity.
The method according to claim 3, wherein the sucrose permease is SEQ ID NO: 4, the sucrose hydrolase is SEQ ID NO: 6, the sucrose transcriptional regulator is SEQ ID NO: 7, manokinase is encoded by the base sequence of SEQ ID NO: 13,
Wherein said sucrose PTS permease is SEQ ID NO: 21, and sucrose PTS hydrolase is encoded by the nucleotide sequence of SEQ ID NO: 22.
The Escherichia microorganism according to claim 4, wherein the sucrose transcriptional regulator is encoded by a nucleotide sequence of SEQ ID NO.
The method according to claim 1, wherein the use of the sucrose non-PTS system and the sucrose PTS system simultaneously increases the number of copies by chromosomal insertion and vector introduction of genes encoding the sucrose non-PTS system and the sucrose PTS system. Escherichia microorganisms, characterized in that by at least one method selected from the group consisting of promoter replacement, promoter mutations and genetic mutations.
The Escherichia microorganism according to claim 2, wherein the pyruvate carboxylase is encoded by the nucleotide sequence of SEQ ID NO: 25.
According to claim 2, Escherichia microorganism transformed with a vector comprising a base sequence encoding the pyruvate carboxylase.
The Escherichia microorganism according to claim 6, wherein the activity is enhanced by a method by promoter replacement.
The Escherichia microorganism according to claim 1, wherein the microorganism is Escherichia coli.
The Escherichia microorganism according to claim 2, wherein the microorganism is Escherichia coli CA03-0392P (Accession No. KCCM11060P).
The L-amino acid of claim 1, wherein the L-amino acid is L-threonine, O-succinyl-homoserine, O-acetylhomoserine, L-methionine, L-lysine, L-homoserine, L-isoleucine, L- Escherichia microorganism selected from the group consisting of valine, and L-tryptophan.
Inoculating and culturing the microorganism of any one of claims 1 to 12 in a culture medium containing some or all of sucrose as a carbon source; And
L-amino acid production method comprising the step of separating the L-amino acid from the culture.
The method of claim 13, wherein the L-amino acid is L-threonine, O-succinyl-homoserine, O-acetylhomoserine, L-methionine, L-lysine, L-homoserine, L-isoleucine, L- L-amino acid production method, characterized in that selected from the group consisting of valine, and L-tryptophan.

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