# Patent

## Title
Microorganisms of Corynebacterium which can utilize xylose and method for producing L-lysine using same

## Abstract

The present invention relates to microorganisms of corynebacterium which can utilize xylose and to a method for producing L-lysine using same. More particularly, the present invention relates to microorganisms of corynebacterium which are modified, in which genes encoding xylose isomerase and xylulokinase which are xylose synthases are introduced to express the xylose synthase. The present invention also relates to a method for producing L-lysine, comprising a step of culturing the modified microorganisms of corynebacterium using xylose as a carbon source, and recovering L-lysine from the culture.

## Background
STATEMENT REGARDING SEQUENCE LISTING
The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 200187_425C1_SEQUENCE_LISTING.txt. The text file is 29.5 KB, was created on Oct. 26, 2015, and is being submitted electronically via EFS-Web.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a Corynebacterium sp. microorganism modified to utilize xylose and a method for producing L-lysine using the same.
2. Description of the Related Art
Industrial microorganisms utilize sugar such as glucose, fructose, and sucrose as a carbon source. Agricultural products are usually used as feedstock to obtain these carbon sources, but they are expensive and are more valuable as food. Recently, instead of using agricultural products as traditional feedstock, cellulosic biomass including agricultural waste or woody waste, industrial waste, etc. has attracted attention as an ideal sugar raw material for fermentation, because it has the advantages of low cost and abundant supply.
Among them, xylose is the second most abundant lignocellulosic carbohydrate in nature, and is a representative cellulosic biomass. Useful materials have been produced from xylose using industrial microorganisms. For example, a method of producing L-amino acid is known, by culturing a Escherichia sp. strain in a medium containing a mixture of pentoses including glucose and xylose, wherein the stain is modified to increase expression of xylABFGHR gene cluster encoding an enzyme (xylosidase) hydrolyzing xyloside, which is a glycoside derived from xylose, and then recovering L-amino acid from the medium (Japanese Patent. No. 4665567).
On the other hand, a Coryneform bacteria, Corynebacterium glutamicum, is known as a Gram-positive strain used in production of various L-amino acids. As described above, because xylose is the second most abundant lignocellulosic carbohydrate in nature, it is expected that L-amino acids such as L-lysine can be more economically produced from Corynebacterium glutamicum by using xylose. However, Corynebacterium glutamicum does not have important genes which are involved in the metabolic pathway of xylose, which is a pentose, and thus there is a problem that L-amino acid cannot be produced from Corynebacterium glutamicum by using xylose. To solve this problem, there has been a report that Corynebacterium glutamicum is modified to be able to utilize xylose by introducing xylose isomerase (XylA) and xylulokinase (XylB) derived from Escherichia coli (Kawaguchi et al., AEM 72:3418-3428, 2006).
The present inventors have made extensive efforts to produce L-amino acid in a more economical manner, and as a result, they found that when XylA and XylB-encoding genes derived from Erwinia carotovora are introduced into Corynebacterium glutamicum, the variant is able to utilize xylose to produce L-lysine and also shows more improved xylose-utilizing ability than the previously known. Coryneform microorganism introduced with xylA and xylB derived from Escherichia coli, thereby completing the present invention.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a modified Corynebacterium sp. microorganism able to produce L-lysine by utilizing xylose.
Another object of the present invention is to provide a method for producing L-lysine using the modified Corynebacterium sp. microorganism.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a cleavage map of an expression vector pECCG122-pcj7-xylAB(Er) of the present invention;
FIG. 2 shows a graph representing growth characteristics of a parent strain and a transformant introduced with the expression vector according to a carbon source contained in a medium; and
FIG. 3 shows a graph representing growth characteristics of a parent strain and a transformant of which pcj7-xylAB(Er) is inserted on chromosome according to a carbon source contained in a medium.



DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
In one aspect, the present invention provides a modified Corynebacterium sp. microorganism able to produce L-lysine by utilizing xylose, characterized in that activities of xylose isomerase and xylulokinase derived from Erwinia carotovora are introduced thereinto.
As used herein, the term “xylose isomerase (XylA)” means an enzyme catalyzing an isomerization reaction from xylose to xylulose, which is involved in the xylose metabolic pathway, and with respect to the object of the present invention, it may be an enzyme derived from Erwinia carotovora. 
The XylA is xylose isomerase derived from Erwinia carotovora, and may include a sequence capable of providing a xylose-utilizing ability by introducing its activity together with activity of xylulokinase derived from Erwinia carotovora into the Corynebacterium sp. microorganism having no xylose isomerase activity, without limitation. In addition, it is apparent that any sequence having an activity equivalent to that of the above sequence, although it is not derived from Erwinia carotovora, is included in the scope of the present invention.
For example, an amino acid sequence of SEQ ID NO: 1, or an amino acid sequence containing a conserved sequence of the amino acid sequence of SEQ ID NO: 1 and substitution, deletion, insertion, addition or inversion of one amino acid or several amino acids (may vary depending on positions and types of amino acid residues in she three-dimensional structure of the protein, specifically 2 to 20, specifically 2 to 10, more specifically 2 to 5 amino acids) at one or more positions, may be included. As long as it is able to maintain or enhance the XylA activity, an amino acid sequence having 80% or more, specifically 90% or more, more specifically 95% or more, much more specifically 97% or more homology with, the amino acid sequence of SEQ ID NO: 1 may be included, and the substitution, deletion, insertion, addition or inversion of the amino acid also includes a mutated sequence naturally occurring in the microorganism having XylA activity or an artificially modified sequence.
As used herein, the term “homology” refers to identity between two different amino acid sequences or two different nucleotide sequences, and can be determined by a method well known to those skilled in the art, for example, BLAST 2.0, which calculates parameters such as score, identity, and similarity, but is not particularly limited thereto.
As used herein, the term “xylulokinase” means an enzyme catalyzing a production reaction from xylulose to xylulose 5-phosphate, which is involved in the xylose metabolic pathway, and with respect to the object of the present invention, it may be an enzyme derived from Erwinia carotovora. 
The XylB is xylulokinase derived from Erwinia carotovora, and may include a sequence capable of providing a xylose-utilizing ability by introducing its activity together with activity of xylose isomerase derived from Erwinia carotovora into the Corynebacterium sp. microorganism having no xylulokinase activity, without limitation. In addition, it is apparent that any sequence having an activity equivalent to that of the above sequence, although it is not derived, from Erwinia carotovora, is included in the scope of the present invention.
For example, an amino acid sequence of SEQ ID NO: 2, or an amino acid sequence containing a conserved sequence of the amino acid sequence of SEQ ID NO: 2 and substitution, deletion, insertion, addition or inversion of one amino acid or several amino acids (may vary depending on positions and types of amino acid residues in the three-dimensional structure of the protein, specifically 2 to 20, specifically 2 to 10, more specifically 2 to 5 amino acids) at one or more positions, may be included. As long as it is able to maintain or enhance the XylB activity, an amino acid sequence having 80% or more, specifically 90% or more, more specifically 95% or more, much more specifically 97% or more homology with the amino acid sequence of SEQ ID NO: 2 may be included, and the substitution, deletion, insertion, addition or inversion of the amino acid also includes a mutated sequence naturally occurring in the microorganism having XylB activity or an artificially modified sequence.
As used herein, the term “xylose isomerase (XylA)-encoding gene (hereinafter, xylA)” means a polynucleotide encoding the above described XylA.
The gene may include a nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence which can hybridize with a probe derived from the nucleotide sequence of SEQ ID NO: 3 under “stringent conditions”, or a nucleotide sequence, in which one nucleotide or several nucleotides is/are substituted, deleted, inserted, or added at one or more positions of the nucleotide sequence of SEQ ID NO: 3. As long as it is able to maintain or enhance the XylA activity, the gene may include a nucleotide sequence having 80% or more, specifically 90% or more, more specifically 95% or more, much more specifically 97% or more homology with the nucleotide sequence of SEQ ID NO: 3, a nucleotide sequence substituted with anions favored by host cells, a nucleotide sequence of which N-terminus or C-terminus is extended or eliminated, or a nucleotide sequence of which start codon is modified to control expression level, and thus the gene is not particularly limited thereto.
As used herein, the term “xylulokinase (XylB)-encoding gene (hereinafter, xylB)” means a polynucleotide encoding the above described XylB.
The gene may include a nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence which can hybridize with a probe derived from the nucleotide sequence of SEQ ID NO: 4 under “stringent conditions”, or a nucleotide sequence, in which one nucleotide or several nucleotides is/are substituted, deleted, inserted, or added at one or more positions of the nucleotide sequence of SEQ ID NO: 4. As long as it is able to maintain or enhance the XylB activity, the gene may include a nucleotide sequence having 80% or more, specifically 90% or more, more specifically 95% or more, much more specifically 97% or more homology with the nucleotide sequence of SEQ ID NO: 4, a nucleotide sequence substituted with codons favored by host cells, a nucleotide sequence of which N-terminus or C-terminus is extended or eliminated, or a nucleotide sequence of which start codon is modified to control expression level, and thus the gene is not particularly limited thereto.
As used herein, the term “stringent conditions” means conditions which permit a specific hybridization between polynucleotides, for example, hybridization in a hybridization buffer at 65° C. (3.5×SSC (0.15 M NaCl/0.15 M sodium citrate, pH 7.0), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.5% SDS, 2 mM EDTA, 2.5 mM NaH2PO4, pH 7), and a detailed description is disclosed in the related art (Molecular Cloning (A Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989) or Current Protocols in Molecular Biology (F. M. Ausubel et al., Editors, John Wiley & Sons, Inc., New York).
As described above, introduction of XylA and XylB activities into Corynebacterium sp. microorganism may be carried out by various methods well known in the art. For example, there are a method of inserting a polynucleotide including the nucleotide sequences encoding XylA and XylB into a chromosome, a method of introducing a vector system including the polynucleotide into the microorganism, a method of introducing a potent promoter to upstream of the nucleotide sequences encoding XylA and XylB, a method of introducing xylA and xylB with a modified promoter, or a method of introducing a modified nucleotide sequences encoding XylA and XylB, or the like. More specifically, if the nucleotide sequences encoding XylA and XylB are introduced, Corynebacterium ammoniagenes-derived pcj7 promoter (Korean Patent No. 10-0620092) can be used as a promoter for controlling the expression thereof. In one embodiment of the present invention, acquisition of xylose-utilizing ability was confirmed as the activity of the foreign gene absent in the parent strain was observed by introduction of an expression vector or chromosomal insertion.
As used herein, the term “vector” refers to a DNA product having a nucleotide sequence of a polynucleotide encoding the target protein, which is operably linked to a suitable regulatory sequence to express the target protein in a suitable host. The regulatory sequence includes a promoter capable of initiating transcription, an arbitrary operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and sequences for regulating the termination of transcription and translation. Once transformed into a suitable host, the vector may replicate or function independently of the host genome, or may integrate into the genome itself.
The vector that is used in the present invention is not specifically limited and may be any vector known in the art, as long as it can replicate in a host. Example of the vector typically used may be natural or recombinant plasmid, cosmid, virus and bacteriophage. For example, as the phage vector or the cosmid vector, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, Charon21A or the like may be used. As the plasmid vector, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type, pET type or the like may be used.
The vector useful in the present invention is not particularly limited, and the known expression vector may be used. Specifically, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector or the like may be used.
Furthermore, the vector used in the present invention may be a vector capable of transforming host cells, to insert the polynucleotide encoding the target protein into the chromosome of the host cell. Specific examples of the vector include, but are not limited to, the shuttle vector pECCG112 that can self-replicate in both directions in E. coli and or Coryne-type bacteria (Korean Patent No. 10-0057684).
As used herein, the term “transformation” means a series of operations for introducing a vector including a polynucleotide encoding a target protein into a host cell so as to express the protein encoded by the polynucleotide in the host cell. The polynucleotide to be introduced into the host cell may have any form, as long as it is introduced into the host cell and expressed therein. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette that is a structure including all elements (a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, a translation termination signal, etc.) required for self-expression. The expression cassette may be in the form of a self-replicable expression vector. In addition, the polynucleotide itself may be introduced into a host cell to be operably linked to a sequence required for expression in the host cell.
The host cell may be any one of prokaryotic microorganisms, as long as it is able to produce L-lysine. Examples of the host cell may include Providencia sp., Corynebacterium sp. and Brevibacterium sp. microorganism, specifically, Corynebacterium sp. microorganism, and more specifically Corynebacterium glutamicum. In one embodiment of the present invention, when KCCM11016P, KCCM10770P, KFCC10750, and CJ3P as Corynebacterium sp. microorganism having no xylose-utilizing ability are introduced with XlyA and XlyB derived from Erwinia carotovora, they are provided with xylose-utilizing ability, and as a result, L-amino acid such as L-lysine can be produced by utilizing xylose as a carbon source (Tables 1 to 6).
Corynebacterium sp. microorganism, having an ability to produce L-lysine may be a variant, resistant to an L-lysine analogue. The L-lysine analogue inhibits growth of Coryneform microorganism, but this inhibition is fully or partially desensitized when L-lysine coexists in a medium. Examples of the L-lysine analogue include, but are not limited to, oxa-L-lysine, L-lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyl L-lysine, α-chlorocaprolactam or the like. Variant having resistance to these L-lysine analogues can be obtained by a conventional artificial mutagenesis treatment to Coryneform microorganism. In addition, when genetic manipulation is conducted to induce L-lysine-producing microorganism, it can be achieved by improving the expression of one or more of genes encoding enzymes involved in the L-lysine biosynthetic system. Examples of these genes may include dihydrodipicolinate synthase gene (dapA), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), diaminopimelate decarboxylase gene (lysA), diaminopimelate dehydrogenase gene (ddh), phosphoenolpyruvate carboxylase gene (ppc), aspartate semialdehyde dehydrogenase gene (asd) and aspartase gene (aspA), but are not limited thereto.
As used herein, the term “L-lysine” is one of the basic α-amino acids, is an essential amino acid that is not synthesized in the body, is one of the L-amino acids, and has a chemical formula of NH2(CH2)4CH(NH2)COOH. L-lysine is synthesized from oxaloacetate through L-lysine biosynthetic pathway, and NADPH-dependent reductase catalyzes an intermediate process for L-lysine biosynthesis. During the biosynthetic process of 1 molecule of L-lysine, 3 molecules of NADPH are directly consumed by the corresponding enzymes, and 1 molecule of NADPH is indirectly used.
As used herein, the term “Corynebacterium sp. microorganism capable of producing L-lysine” means Corynebacterium sp, microorganism modified to produce L-lysine from xylose, which is prepared by introducing genes encoding the enzymes involved in the xylose metabolism and not found in the Corynebacterium sp. microorganism. The Corynebacterium sp. microorganism may be, but is not particularly limited to, Corynebacterium glutamicum, and the enzymes involved in the xylose metabolism may be, but are not particularly limited to, XylA and XylB.
In this regard, the host cell may be Corynebacterium sp. microorganism, in which expressions of one or more of the genes encoding enzymes involved in the L-lysine biosynthetic system are improved, and the genes encoding enzymes involved in the L-lysine biosynthetic system may be, but are not limited to, dihydrodipicolinate synthase gene (dapA), aspartokinase gene (lysC), dihydrodipicolinate reductase gene (dapB), diaminopimelate decarboxylase gene (lysA), diaminopimelate dehydrogenase gene (ddh), phosphoenolpyruvate carboxylase gene (ppc), aspartate semialdehyde dehydrogenase gene (asd), aspartame gene (aspA) or the like.
In addition, the host cell may be a mutant strain resistant to an L-lysine analogue. The mutant strain may be obtained by mutation of Corynebacterium sp. microorganism. The L-lysine analogue inhibits growth of Coryneform microorganism, but this inhibition is fully or partially desensitized when L-lysine coexists in a medium. Examples of the L-lysine analogue may be, but are not particularly limited to, preferably oxa-L-lysine, L-lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyl L-lysine, α-chlorocaprolactam or the like.
Meanwhile, in the present invention, activities of the known enzymes involved in the L-lysine biosynthesis may be additionally controlled in order to further improve the L-lysine production. Specifically, in the present invention, asd, dapB, and ddh genes, each encoding aspartate semialdehyde dehydrogenase, dihydrodipicolinate reductase and diaminopimelate dehydrogenase enzymes, are overexpressed to additionally control activities of the enzymes, thereby improving the L-lysine production.
According to one embodiment of the present invention, the present inventors selected Erwinia carotovora (SCRI1043)-derived ECA0097(xylA) (SEQ ID NO: 1) and ECA0096(xylB) (SEQ ID NO: 2) as suitable genes encoding XylA and XylB to introduce into Corynebacterium sp. microorganism (Example 1), and they cloned the selected genes encoding XylA and XylB so as to construct an expression vector pECCG122-pcj7-xylA-xylB (hereinafter, pECCG122-pcj7-xylAB(Er)) expressing xylA and xylB (hereinafter, xylAB(Er)) at the same time. The expression vector was introduced into Corynebacterium glutamicum KCCM11016P (this microorganism, was disclosed as KFCC10881, and re-deposited to an International Depositary Authority under the Budapest Treaty with Accession No. KCCM11016P, Korean Patent Nos. 10-0159812 and 10-0397322) to prepare a transformant overexpressing xylAB(Er). It was confirmed that the prepared transformant grows by utilizing xylose as a carbon source (FIG. 2), and produces L-lysine by utilizing each of glucose and xylose, or by utilizing glucose and xylose at the same time (Table 1). In addition, in order to express xylAB(Er) on the chromosome, a recombinant vector for chromosomal insertion, pDZTn-pcj7-xylAB(Er) was constructed, and transformed into KCCM11016P, and through second crossover, a transformant KCCM11016P-pcj7-xylAB(Er) having xylAB(Er) operably linked to pcj7 promoter inside the transposon on the chromosome was constructed. It was also confirmed that the transformant grows by utilizing xylose as a carbon source (FIG. 3), and produces L-lysine by utilizing each of glucose and xylose, or by utilizing glucose and xylose at the same time (Table 2). Furthermore, in order to compare the effects of improving xylose-utilizing ability between introduction of the previously reported E. coli-derived xylAB (hereinafter, xylAB(Ec)) and introduction of Erwinia carotovora-derived xylAB(Er) of the present invention, a strain (KCCM11016P-pcj7-xylAB(Ec)) was prepared by introducing xylAB(Ec) into KCCM11016P, and its xylose-utilizing ability and L-lysine production characteristics were compared with, those of the prepared KCCM11016P-pcj7-xylAB(Er). As a result, it was found that xylose consumption rate of KCCM11016P-pcj7-xylAB(Er) was remarkably increased, compared to that of KCCM11016P-pcj7-xylAB(Ec), indicating improvement in fermentative production of L-lysine (Table 3). In addition, in order to confirm whether various Corynebacterium sp. microorganisms show the same results, pDZTn-pcj7-xylAB(Er) was introduced to an L-lysine-producing strain KCCM10770P to prepare a transformant KCCM10770P-pcj7-xylAB(Er), and it was confirmed that the transformant is able to produce L-lysine by utilizing each of glucose and xylose, or by utilizing glucose and xylose at the same time (Table 4). pDZTn-pcj7-xylAB(Er) was also introduced into another L-lysine-producing strain KFCC10750 (this microorganism was disclosed as KFCC10750, and re-deposited to an International Depositary Authority under the Budapest Treaty with Accession No. KCCM11347P, Korean Patent No. 10-0073610) to prepare a transformant KFCC10750-pcj7-xylAB(Er). It was also confirmed that this transformant is able to produce L-lysine by utilizing each of glucose and xylose, or by utilizing glucose and xylose at the same time (Table 5). Further, pDZTn-pcj7-xylAB(Er) was also introduced into the other L-lysine-producing strain CJ3P to prepare a transformant CJ3P-pcj7-xylAB(Er). It was also confirmed that this transformant is able to produce L-lysine by utilizing each of glucose and xylose, or by utilizing glucose and xylose at the same time (Table 6).
Accordingly, the present inventors designated the transformant as “CA01-2195”, which grows by utilizing xylose in a medium and also produces L-lysine by utilizing xylose and glucose in the medium, and deposited it under the Budapest Treaty at the Korean Culture Center of Microorganisms (KCCM, located on Hongjae 1-Dong, Seodaemun-Gu, Seoul, Korea) on Dec. 29, 2011 with Accession No. KCCM11242P. That is, this deposit is recognized by an International Depositary Authority under the Budapest Treaty.
In another aspect, the present invention provides a method for producing L-lysine, including the steps of (i) culturing the modified Corynebacterium sp. microorganism able to produce L-lysine by utilizing xylose in a culture medium containing xylose as a carbon source so as to obtain a culture broth; and (ii) recovering L-lysine from the culture broth.
As used herein, the term “culturing” means that a microorganism is cultured under artificially controlled environmental conditions. In the present invention, the method for culturing Corynebacterium sp. microorganism may be conducted using a method widely known in the art. Specifically, examples of the culturing method include batch process, fed batch or repeated fed batch process in a continuous manner, but are not limited thereto.
The medium used for she culture has to meet the requirements of a specific microorganism in a proper manner while controlling temperature, pH, etc. under aerobic conditions in a typical medium containing a proper carbon source, nitrogen source, amino acids, vitamins, etc. The culture media for Corynebacterium strain are disclosed (e.g., Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington D.C., USA, 1981). Possible carbon sources may include sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose, in addition to a mixture of glucose and xylose as a main carbon source, oils and fats such as soy bean oil, sunflower oil, castor oil, and coconut fat, fatty acids such, as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These substances may be used individually or as mixtures. Possible nitrogen sources may include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate and ammonium nitrate; amino acids such as glutamic acid, methionine, and glutamine; and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysates, fish or decomposition products thereof, and defatted soybean cake or decomposition products thereof. These nitrogen sources may be used individually or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or the corresponding sodium-containing salts as phosphorus sources. Possible phosphorus sources may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate or the corresponding sodium-containing salts. Further, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. In addition to the above substances, essential growth substances, such as amino acids and vitamins, may be included.
Appropriate precursors may be also added to the culture media. The above-mentioned, substances may be suitably added to the culture medium in batch, fed-batch or continuous mode during cultivation, but are not particularly limited thereto. pH of the culture may be adjusted by suitably adding basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acidic compounds such as phosphoric acid and sulfuric acid.
An anti-foaming agent such as fatty acid polyglycol esters may be used to suppress the development of foam. In order to maintain aerobic condition, oxygen or oxygen-containing gas (e.g., air) may be introduced into the culture broth. The temperature of the culture broth is normally 27° C. to 37° C., specifically 30° C. to 35° C. The cultivation may be continued until the production of L-lysine reaches a desired level. This objective may be normally achieved within 10 to 100 hours. L-lysine may be released into the culture medium or included within the cells.
Furthermore, the step of recovering L-lysine from the culture broth may be performed by a known method known in the art. Specifically, the known method for recovering L-lysine is, but not particularly limited to, specifically centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential solubility (e.g., ammonium, sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion) or the like.
Hereinafter, the constitutions and effects of 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 invention is not intended to be limited by these Examples.
Example 1
Selection of Foreign Gene
Erwinia carotovora (SCRI1043)-derived ECA0097(xylA) (amino acid: SEQ ID NO: 1, nucleotide: SEQ ID NO: 3) and ECA0096(xylB) (amino acid: SEQ ID NO: 2, nucleotide: SEQ ID NO: 4) were selected as foreign genes to prepare a modified Corynebacterium sp. microorganism provided with a xylose-utilizing ability.
Example 2
Construction of Erwinia carotovora-Derived xylAB Expressing Vector
Erwinia carotovora-derived XylA and XylB-encoding genes selected in Example 1 are located close to each other. Information (Accession NO. BX950851) about xylA and xylB(Er) and surrounding nucleotide sequence was obtained from US NIH GenBank, and based on the obtained nucleotide sequence, primers for amplification of Erwinia carotovora-derived. xylAB(Er) were synthesized.







SEQ ID NO: 5: 5′-ACACATATGCAAGCCTATTTTGAACAGATC-3′


 


SEQ ID NO: 6: 5′-AGAACTAGTGCCTTTTGGTGGTGTTTAAGT-3′






In order to obtain the xylAB(Er) fragment, PGR was conducted using chromosomal DNA of Erwinia carotovora strain SCRI1043 as a template and a pair of primers (SEQ ID NOs: 5 and 6). PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and PGR was conducted with denaturation at 94° C. for 5 minutes, followed by repeating the cycle 30 times including denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C. for 3 minutes, and then polymerization at 72° C. for 7 minutes. As a result, a gene fragment of 3122 bp containing xylAB(Er) (SEQ ID NO: 17) of 2844 bp was obtained (SEQ ID NO: 18). In order to obtain Corynebacterium ammoniagenes-derived pcj7 promoter (KR0620092), PGR was conducted using genomic DNA of Corynebacterium ammoniagenes CJHB100 (KR0620092) as a template and a pair of primers (SEQ ID NOs: 15 and 16). PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and PGR was conducted with denaturation at 94° C. for 5 minutes, followed by repeating the cycle 30 times including denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C. for 1 minute, and then polymerization at 72° C. for 7 minutes. As a result, a polynucleotide of 318 bp was obtained (SEQ ID NO: 14).







SEQ ID NO: 15: 5′-AATCTAGAAACATCCCAGCGCTA-3′


 


SEQ ID NO: 16: 5′-AAACTAGTCATATGTGTTTCCTTTCGTTG-3′






pcj7 was cloned into E. coli-Corynebacterium shuttle vector pECCG122 using restriction enzymes, XbaI and SpeI, and then xylAB(Er) fragment was cloned using NdeI and SpeI, thereby obtaining a pECCG122-pcj7-xylAB(Er) vector (FIG. 1). FIG. 1 is a cleavage map of the expression vector pECCG122-pcj7-xylAB(Er) of the present invention.
Example 3
Development of L-Lysine-Producing Strain Introduced with Erwinia carotovora-Derived xylAB and Examination of Xylose-Utilizing Ability
Each, expression vector pECCG122-pcj7-xylAB(Er) obtained in Example 2 was introduced into Corynebacterium glutamicum KCCM11016P (Korean Patent Nos. 10-0159812 and 10-0397322) to prepare a xylAB(Er)-expressing transformant, Corynebacterium glutamicum CA01-2195.
In order to compare the xylose-utilizing ability between KCCM11016P and CA01-2195, the strains were cultured in a seed medium containing glucose or xylose as a carbon source and their growth characteristics were compared. They were also cultured in a production medium containing glucose or xylose as a carbon source and their L-lysine production characteristics were compared.
First, in order to compare the growth characteristics, the strains were inoculated in 25 ml of seed medium [carbon source (glucose or xylose) 10 g/l, peptone 10 g/l, yeast extract 10 g/l, urea 5 g/l, KH2PO4 4 g/l, K2HPO4 8 g/l, MgSO47H2O 0.5 g/l, biotin 100 μg/l thiamine-HCl 1 mg/l, pH 7.0], respectively. Absorbance (OD600) of the culture broth was measured while culturing the strains at 30° C. for 32 hours, and compared to each other (FIG. 2). FIG. 2 is a graph showing growth, characteristics of KCCM11016P and CA01-2195 according to carbon source contained in the medium, in which (♦) indicates KCCM11016P cultured in the medium containing glucose, (▪) indicates KCCM11016P cultured in the medium containing xylose, (▴) indicates CA01-2195 cultured in the medium containing glucose, and (×) indicates CA01-2195 cultured in the medium containing xylose.
As shown in FIG. 2, there was no difference in growth characteristics between KCCM11016P and CA01-2195 in the seed medium containing glucose as a carbon source. However, in the seed medium containing xylose as a carbon source, CA01-2195 grew to a certain level whereas KCCM11016P hardly grew. Therefore, it can be seen that CA01-2195 is able to grow by utilizing xylose contained in the medium as a sole carbon source.
Next, to compare L-lysine production characteristics between KCCM11016P and CA01-2195, 1 ml of seed, culture broth was inoculated in 24 ml of production medium [carbon source, (NH4)2SO4 40 g/l, soybean protein 2.5 g/l, corn steep solids 5 g/l, urea 3 g/l, KH2PO4 1 g/l, MgSO47H2O 0.5 g/l, biotin 100 μg/l, thiamine-HCl 1 mg/l, CaCO3 30 g/l, pH 7.0], and cultured at 35° C. and 200 rpm for 7.2 hours. At this time, glucose 100 g/l, glucose 50 g/l+xylose 50 g/l, and glucose 70 g/l+xylose 30 g/l were determined to be used as the carbon source. Next, concentration of L-lysine, concentration of residual xylose, and concentration of residual glucose in each culture broth were measured and compared (Table 1).











TABLE 1










Glucose 50 g/l +
Glucose 70 g/l +



Glucose 100 g/l
Xylose 50 g/l
Xylose 30 g/l
















Strain
L-lysine
R.X
R.G
L-lysine
R.X
R.G
L-lysine
R.X
R.G



















KCCM11016P
42
—
0
21
50
0
29
30
0


CA01-2195
42
—
0
40
0
0
41
0
0





R.X: residual xylose (concentration of residual xylose after reaction termination) (unit: g/l)


R.G: residual glucose (concentration of residual glucose after reaction termination)






As shown in Table 1, when the medium containing no xylose (glucose 100 g/l) was used, concentration of L-lysine produced in the parent strain was equivalent to that of CA01-2195. However, when the medium containing xylose (glucose 50 g/l+xylose 50 g/l, and glucose 70 g/l+xylose 30 g/l) was used, CA01-2195 produced L-lysine by consuming both glucose and xylose whereas the parent strain produced L-lysine by consuming no xylose but glucose only.
This result indicates that Corynebacterium sp. microorganism having no xylose-utilizing ability is able to consume xylose by introducing with Erwinia carotovora-derived xylAB.
Example 4
Construction of Recombinant Vector for Chromosomal Insertion of Erwinia carotovora-Derived xylAB (pDZTn-pcj7-xylAB(Er)) and Recombinant Vector for Chromosomal Insertion of E. coli-Derived xylAB (pDZTn-pcj7-xylAB(Ec))
To express the xylAB(Er) on chromosome, a recombinant vector pDZTn-pcj7-xylAB(Er) for chromosomal insertion was constructed. To obtain the pcj7-xylAB(Er) fragment, PCR was conducted using pECCG122-pcj7-xylAB(Er) obtained in Example as a template and a pair of primers (SEQ ID NOs: 7 and 8). PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted with denaturation at 94° C. for 5 minutes, followed by repeating the cycle 30 times including denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C. for 3 minutes, and then polymerization at 72° C. for 7 minutes. As a result, a gene fragment of 3440 bp was obtained (SEQ ID NO: 9). Consequently, pcj7-xylAB(Er) of 3440 bp was cloned into pDZTn vector (Korean Patent No. 10-1126041) treated with restriction enzyme SpeI using a BD In-Fusion kit, thereby obtaining a (pDZTn-pcj7-xylAB(Er)) recombinant vector.









SEQ ID NO: 7:



5′-GAGTTCCTCGAGACTAGTAGAAACATCCCAGCGCTA-3′



 



SEQ ID NO: 8:



5′-GATGTCGGGCCCACTAGGCCTTTTTGGTGGTGTTTA-3′






Next, to express E. coli-derived xylAB on chromosome, recombinant pDZTn-pcj7-xylAB(Ec) for chromosomal insertion was constructed. To obtain pcj7 promoter, PCR was conducted using the pcj7 fragment obtained in Example 2 as a template and a pair of primers (SEQ ID NOs: 7 and 10). PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted with denaturation at 94° C. for 5 minutes, followed by repeating the cycle 30 times including denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C. for 1 minute, and then polymerization at 72° C. for 7 minutes. As a result, a gene fragment of 318 bp was obtained. To obtain xylAB(Ec) fragment, PCR was conducted using chromosomal DNA of E. coli K12 as a template and a pair of primers (SEQ ID NOs: 11 and 12). PfuUltraTM high-fidelity DNA polymerase (Stratagene) was used as the polymerase, and PCR was conducted with denaturation at 94° C. for 5 minutes, followed by repeating the cycle 30 times including denaturation at 94° C. for 30 seconds, annealing at 56° C. for 30 seconds and polymerization at 72° C. for 3 minutes, and then polymerization at 72° C. for 7 minutes. As a result, a xylAB(Ec) fragment of 3145 bp was obtained (SEQ ID NO: 13). Consequently, the PCR products of pcj7 region of 318 bp and xylAB(Ec) of 3145 bp were cloned into pDZTn vector treated with restriction enzyme SpeI using a BD In-Fusion kit, thereby a final pDZTn-pcj7-xylAB(Ec) recombinant vector.









SEQ ID NO: 10:



5′-TCAAAATAGGCTTGCATGAGTGTTTCCTTTCGTTG-3′



 



SEQ ID NO: 11:



5′-CAACGAAAGGAAACACATGCAAGCCTATTTTGAC-3′



 



SEQ ID NO: 12:



5′-GATGTCGGGCCCACTAGTGCTGTCATTAACACGCCA-3′






Example 5
Development of L-Lysine-Producing Strain Inserted with Erwinia-Derived xylAB and Examination of Xylose-Utilizing Ability
The prepared pDZTn-pcj7-xylAB(Er) vector was transformed into KCCM11016P, and through second crossover, a KCCM11016P-pcj7-xylAB(Er) strain having xylAB(Er) with substitution of one copy of pcj7 promoter inside the transposon on the chromosome was obtained.
To compare the xylose-utilizing ability between KCCM11016P-pcj7-xylAB(Er) and KCCM11016P, they were cultured in a seed medium containing glucose or xylose as a carbon source and their growth characteristics were compared, and they were cultured in a production medium containing glucose or xylose as a carbon source and their L-lysine production characteristics were compared, in the same manner as in Example 3.
FIG. 3 is a graph showing growth characteristics of the strains according to carbon source contained in the medium, in which (♦) indicates KCCM11016P cultured in the medium containing glucose, (▪) indicates KCCM11016P cultured in the medium containing xylose, (▴) indicates KCCM11016P-pcj7-xylAB(Er) cultured in the medium containing glucose, and (×) indicates KCCM11016P-pcj7-xylAB(Er) cultured in the medium containing xylose.
There was no difference in growth characteristics between KCCM11016P-pcj7-xylAB(Er) and KCCM11016P in the seed medium containing glucose as a carbon source. However, in the seed medium containing xylose as a carbon source, KCCM11016P-pcj7-xylAB(Er) grew to a certain level whereas KCCM11016P hardly grew. Therefore, it can be seen that when xylAB(Er) is inserted into the chromosome, the strain is able to grow by utilizing xylose contained in the medium.
Next, L-lysine production characteristics of KCCM11016P-pcj7-xylAB(Er) and KCCM11016P were examined and compared with each other (Table 2).











TABLE 2










Glucose 50 g/l +
Glucose 70 g/l +



Glucose 100 g/l
Xylose 50 g/l
Xylose 30 g/l
















Strain
L-lysine
R.X
R.G
L-lysine
R.X
R.G
L-lysine
R.X
R.G



















KCCM11016P
43.0
—
0
22.6
50
0
29.2
30
0



42.5
—
0
21.9
50
0
29.6
30
0


KCCM11016P-
42.8
—
0
42.1
0
0
42.6
0
0


pcj7-xylAB(Er)
43.1
—
0
42.0
0
0
42.2
0
0





R.X: residual xylose (concentration of residual xylose after reaction termination) (unit: g/l)


R.G: residual glucose (concentration of residual glucose after reaction termination)






As shown in Table 2, when the medium containing no xylose (glucose 100 g/l) was used, concentration of L-lysine produced in KCCM11016P was equivalent to that of KCCM11016P-pcj7-xylAB(Er). However, when the medium containing xylose (glucose 50 g/l+xylose 50 g/l, and glucose 70 g/l+xylose 30 g/l) was used, the KCCM11016P strain produced L-lysine by consuming only glucose whereas KCCM11016P-pcj7-xylAB(Er) produced L-lysine by consuming both glucose and xylose.
Example 6
Preparation of L-Lysine-Producing Strain Inserted with E. coli-Derived xylAB and Comparison of its Xylose-Utilizing Ability with that of KCCM11016P-pcj7-xylAB(Ec) Strain
To compare the effects of improving xylose-utilizing ability between introduction of the previously reported E. coli-derived xylAB and introduction of Erwinia carotovora-derived XylAB of the present invention, a strain was prepared by introducing xylAB(Ec) into KCCM11016P, and its xylose-utilizing ability and L-lysine production characteristics were compared with those of the prepared KCCM11016P-pcj7-xylAB(Er).
To prepare a xylAB(Ec)-introduced strain, the pDZTn-pcj7-xylAB(Ec) recombinant vector prepared in Example 4 was transformed into KCCM11016P, and through second crossover, a KCCM11016P-pcj7-xylAB(Ec) strain having xylAB(Ec) operably linked to pcj7 promoter inside the transposon on the chromosome was obtained.
In the same manner as in example 3, to compare the xylose-utilizing ability between KCCM11016P-pcj7-xylAB(Er) and KCCM11016P-pcj7-xylAB(Ec), they were cultured in the production medium containing glucose 50 g/l+xylose 50 g/l as a carbon source, and their L-lysine production characteristics were compared, and to examine the xylose-utilizing ability, concentration of residual xylose in the culture broth was measured at 15 hours (Table 3).










TABLE 3









R.X (g/L)
Lysine











45 h
72 h
72 h
















KCCM11016P-pcj7-xylAB(Er)
8.0
0
42.8




7.2
0
42.2



KCCM11016P-pcj7-xylAB(Ec)
11.2
0
42.7




12.1
0
42.4







R.X: residual xylose (concentration of residual xylose after reaction termination) (unit: g/l)






As shown in Table 3, the two strains showed equivalent L-lysine productivity. The xylose consumption rate of KCCM11016P-pcj7-xylAB(Er) was faster than that of KCCM11016P-pcj7-xylAB(Ec), indicating improvement in fermentation productivity. That is, this result indicates that introduction of Erwinia carotovora-derived xylAB (Er) of the present invention shows more excellent effect of improving L-lysine fermentation productivity than introduction of the previous E. coli-derived xylAB (Ec).
Example 7
Development of KCCM10770P-Derived Strain Inserted with Erwinia carotovora-derived xylAB and Examination of Xylose-Utilizing Ability
To examine whether xylAB(Er) introduction exhibits the same effect in other L-lysine-producing strains than KCCM11016P, pDZTn-pcj7-xylAB(Er) was introduced into an L-lysine-producing strain KCCM10770P (Korean Patent No. 10-0924065) which was deposited to an International Depositary Authority under the Budapest Treaty. After introduction by an electric pulse method, a strain having xylAB(Er) with, substitution of one copy of pcj7 promoter inside the transposon on the chromosome was obtained through second crossover, and the strain was named as Corynebacterium glutamicum KCCM10770P-pcj7-xylAB(Er).
Xylose-utilizing ability and L-lysine productivity of Corynebacterium glutamicum. KCCM10770P and Corynebacterium glutamicum. KCCM10770P-pcj7-xylAB(Er) of the present invention were measured in the same manner as in Example 3 (Table 4).












TABLE 4







Strain
R.G
R.X
L-lysine





















KCCM10770P
0
50
23.8




0
50
24.4



KCCM10770P-pcj7-xylAB(Er)
0
0
47.6




0
0
47.5







R.X: residual xylose (concentration of residual xylose after reaction termination) (unit: g/l)



R.G: residual glucose (concentration of residual glucose after reaction termination)






As shown in Table 4, when the L-lysine-producing strain KCCM10770P was introduced with xylAB(Er), it completely consumed xylose, unlike the parent strain that cannot utilize xylose, and its L-lysine productivity was also increased.
This result supports that when Erwinia carotovora-derived xylAB is introduced into various Corynebacterium sp. microorganisms as well as Corynebacterium sp. microorganism specified by a certain Accession Number, they completely consume xylose as a carbon source, thereby efficiently producing amino acids such as L-lysine.
Example 8
Development of KFCC10750-Derived Strain Inserted with Erwinia carotovora-Derived xylAB and Examination of Xylose-Utilizing Ability
To examine whether xylAB(Er) introduction exhibits the same effect in other L-lysine-producing strains than KCCM11016P, pDZTn-pcj1-xylAB(Er) was introduced into an L-lysine-producing strain KFCC10750 (Korean Patent No. 10-0073610). After introduction by an electric pulse method, a strain having xylAB(Er) with substitution of one copy of pcj7 promoter inside the transposon on the chromosome was obtained through second crossover, and the strain was named as Corynebacterium glutamicum KFCC10750-pcj7-xylAB(Er).
Xylose-utilizing ability and L-lysine productivity of Corynebacterium glutamicum KFCC10750 and Corynebacterium glutamicum KFCC10750-pcj7-xylAB(Er) of the present invention were measured in the same manner as in Example 3 (Table 5).












TABLE 5







Strain
R.G
R.X
L-lysine





















KFCC10750
0
50
19.7




0
50
18.8



KFCC10750-pcj7-xylAB(Er)
0
0
38.3




0
0
38.6







R.X: residual xylose (concentration of residual xylose after reaction termination) (unit: g/l)



R.G: residual glucose (concentration of residual glucose after reaction termination)






As shown in Table 5, when the L-lysine-producing strain KFCC10750 was introduced with xylAB(Er), it completely consumed xylose, unlike the parent strain that cannot utilize xylose, and its L-lysine productivity was also increased.
This result supports that when Erwinia carotovora-derived xylAB is introduced into various Corynebacterium sp. microorganisms as well as Corynebacterium sp. microorganism specified by a certain Accession Number, they completely consume xylose as a carbon source, thereby efficiently producing amino acids such as L-lysine.
Example 9
Development of CJ3P-Derived Strain Inserted with Erwinia carotovora-Derived xylAB and Examination of Xylose-Utilizing Ability
To examine whether xylAB(Er) introduction exhibits the same effect in other L-lysine-producing strains than KCCM11016P, pDZTn-pcj7-xylAB(Er) was introduced into an L-lysine-producing strain CJ3P. CJ3P strain is a Corynebacterium glutamicum strain which has an ability to produce L-lysine by introduction of P458S, V59A, and T311I mutations into 3 types of genes, pyc, horn, and lysC of the wild-type strain, based on the technique reported by Binder et al. (Genome Biology 2102, 13:R40), After introduction by an electric pulse method, a strain having xylAB(Er) with substitution of one copy of pcj7 promoter inside the transposon on the chromosome was obtained through second crossover, and the strain was named as Corynebacterium glutamicum CJ3P-pcj7-xylAB(Er).
Xylose-utilizing ability and L-lysine productivity of Corynebacterium glutamicum CJ3P and Corynebacterium glutamicum CJ3P-pcj7-xylAB(Er) of the present invention were measured in the same manner as in Example 3 (Table 6).












TABLE 6







Strain
R.G
R.X
L-lysine





















CJ3P
0
50
4.0




0
50
4.5



CJ3P-pcj7-xylAB(Er)
0
0
8.5




0
0
9.0







R.X: residual xylose (concentration of residual xylose after reaction termination) (unit: g/l)



R.G: residual glucose (concentration of residual glucose after reaction termination)






As shown in Table 6, when the L-lysine-producing strain CJ3P was introduced with xylAB(Et), it completely consumed xylose, unlike the parent strain that cannot utilize xylose, and its L-lysine productivity was also increased.
This result supports that when Erwinia carotovora-derived xylAB is introduced into various Corynebacterium sp. microorganisms as well as Corynebacterium sp. microorganism specified by a certain Accession Number, they completely consume xylose as a carbon source, thereby efficiently producing amino acids such as L-lysine.
Therefore, Corynebacterium sp. microorganism expressing xylAB(Er) is able to grow by utilizing xylose in a medium, and also able to produce L-lysine by utilizing xylose and glucose in a medium.
EFFECT OF THE INVENTION
When Corynebacterium sp. microorganism of the present invention able to produce L-lysine by utilizing xylose is used, L-lysine can be produced by using xylose as the second most abundant lignocellulosic carbohydrate in nature. Therefore, the microorganism can be widely used for the efficient and economical production of L-lysine.

## Claims

1. A Corynebacterium sp. microorganism, wherein said microorganism is transformed with a polynucleotide encoding an Erwinia carotovora xylose isomerase (XylA) and a polynucleotide encoding an Erwinia carotovora xylulokinase (XylB), and wherein the microorganism expresses the E. carotovora XylA and E. carotovora XylB and utilizes xylose when present in a culture medium to produce L-lysine.

2. The microorganism according to claim 1, wherein the E. carotovora XylA comprises the amino acid sequence of SEQ ID NO: 1, and the E. carotovora XylB comprises the amino acid sequence of SEQ ID NO: 2.

3. The microorganism according to claim 1, wherein the E. carotovora XylA is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3, and the E. carotovora XylB is encoded by a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 4.

4. The microorganism according to claim 1, wherein:
the polynucleotides encoding the E. carotovora XylA and E. carotovora XylB are integrated into the chromosome of the microorganism,
the polynucleotides encoding the E. carotovora XylA and E. carotovora XylB are present within a vector,
the polynucleotides encoding the E. carotovora XylA and E. carotovora XylB are operably linked to a promoter that is heterologous to the polynucleotides, or
a combination thereof.

5. A method for producing L-lysine, comprising the steps of:
(i) culturing the Corynebacterium sp. microorganism of claim 1 in a culture medium containing xylose as a carbon source so as to obtain a culture broth comprising L-lysine; and
(ii) recovering the L-lysine from the culture broth.

6. The method according to claim 5, wherein the E. carotovora XylA comprises the amino acid sequence of SEQ ID NO: 1, and the E. carotovora XylB comprises the amino acid sequence of SEQ ID NO: 2.

7. The microorganism according to claim 4, wherein the promoter comprises the nucleotide sequence of SEQ ID NO: 14.