CN116411009A - Expression cassette for preparing L-lysine, genetically engineered bacterium and application thereof - Google Patents

Abstract

The invention discloses an expression cassette for preparing L-lysine, genetically engineered bacteria and application thereof. An expression cassette comprising a promoter and xylAB gene, further comprising a transaldolase gene and/or a transketolase gene. After the expression cassette provided by the invention is introduced into a starting strain, the expression transketolase and the transaldolase are in an improved state compared with the starting strain, which is beneficial to strengthening the pentose phosphate pathway, so that the utilization efficiency of xylose and mixed sugar is effectively improved, and the output of L-lysine is further improved. When the straw hydrolysate is used, the lysine yield of the genetically engineered bacterium is obviously improved compared with that of the starting bacterium. The genetically engineered bacterium provided by the invention can effectively utilize agricultural wastes such as straw and the like for fermentation, and has good application prospect.

Description

Expression cassette for preparing L-lysine, genetically engineered bacterium and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to an expression cassette for preparing L-lysine, genetic engineering bacteria and application thereof.

Background

Lysine (lysine), also known as 2, 6-diaminocaproic acid, belongs to the basic amino acid. Lysine has important nutrition physiological functions and is widely applied in the industries of medicine, food and feed. At the same time, it can also be used as a precursor substance for synthesizing nylon polymer materials. The production path of lysine mainly comprises three methods of a protein hydrolysis method, a chemical synthesis method and a fermentation method, wherein the microbial fermentation method has the characteristics of low production cost, high production strength, high specificity, small environmental pollution and the like, and becomes the method with the widest application of industrial production of lysine.

The prokaryotic microorganism for producing lysine mainly includes corynebacteria, brevibacterium, nocardia, pseudomonas, escherichia, bacillus, etc. Corynebacterium glutamicum (Corynebacterium glutamicum) is the most important and safe strain for fermentative production of amino acids, and the ability to increase its excessive synthesis of various amino acids by metabolic engineering has been a focus of research. For example, overexpression of a gene involved in lysine synthesis pathway and a gene involved in desensitization of feedback inhibition, or enhancement of energy supply pathway from glucose metabolism and optimization of lysine transporter on cell membrane, etc. are effective in improving productivity of lysine.

Conventional corynebacterium glutamicum cannot utilize xylose, and therefore, it is a precondition for lignocellulose fermentation to be able to utilize xylose in lignocellulose through metabolic engineering and synthetic biological engineering. Patent CN200510076242.X constructs a xylose-to-lysine synthesis path by introducing xylABFGHR gene locus, but the obtained strain still has the problems of low xylose utilization rate, incapability of normal growth in a lignocellulose system, lysine fermentation and the like. Therefore, the construction of a strain for producing lysine by efficiently utilizing glucose and xylose in lignocellulose requires further research. The prior art lacks genetic engineering bacteria with high yield of lysine.

Disclosure of Invention

In order to solve the technical problems, the invention provides an expression cassette for preparing L-lysine, genetically engineered bacteria and application thereof, and particularly provides an expression cassette capable of improving lysine yield of corynebacterium glutamicum and genetically engineered bacteria capable of efficiently producing lysine by utilizing straw hydrolysate. The genetically engineered bacterium can effectively utilize glucose and xylose in the straw hydrolysate. The invention improves the fermentation efficiency of corynebacterium glutamicum under the conditions of xylose, mixed sugar and hydrolysate by enhancing the functions of ketolase and transaldolase of the corynebacterium glutamicum, obviously improves the yield of L-lysine, and has not been reported at present from the angles of ketolase and transaldolase to study corynebacterium glutamicum to produce L-lysine.

In order to solve the technical problems, the invention provides an expression cassette, which comprises a promoter, xylAB genes, and transaldolase genes and/or transketolase genes.

In the expression cassette, the sequence of the xylAB gene is preferably shown as SEQ ID NO. 1.

In the expression cassette, the sequence of the transaldolase gene is preferably shown as SEQ ID NO. 4.

In the expression cassette, the sequence of the transketolase gene is preferably shown as SEQ ID NO. 3.

In the expression cassette, the promoter is preferably a Peftu promoter, and the sequence of the Peftu promoter is preferably shown as SEQ ID NO. 2.

In another aspect, the invention provides an isolated nucleic acid comprising an expression cassette according to the invention.

In another aspect, the invention provides a recombinant expression vector comprising an expression cassette of the invention, or comprising a nucleic acid of the invention.

In a preferred embodiment, the backbone plasmid of the recombinant expression vector is preferably pK18mob.

The specific information of the pK18mob can be referred to as the following web page:

http://www.biovector.net/product/1089.html。

in another aspect, the invention provides a genetically engineered bacterium, which is obtained by transferring an expression cassette or a nucleic acid or a recombinant expression vector according to the invention into a starting bacterium.

In the genetic engineering, the starting bacterium is preferably Corynebacterium glutamicum (Corynebacterium glutamicum), for example C.glutamicum B253 or C.glutamicum Caths141.

In the genetic engineering, the expression cassette is preferably integrated in the genome of the starting bacterium by homologous recombination or is present in the starting bacterium in non-integrated form.

In the genetically engineered bacterium, it is preferable that lactate dehydrogenase is not expressed, for example, the ldh gene is knocked out.

In a preferred embodiment, the expression cassette is integrated into the ldh gene at the site.

The ldh gene is preferably AJE68497.1 as an accession number in Genbank.

The starting strain can be a wild strain or a modified strain.

In another aspect, the present invention provides a method for producing L-lysine, comprising fermenting the genetically engineered bacterium according to the present invention in a fermentation medium.

In a preferred embodiment, the fermentation medium contains not less than 25g/L glucose and/or not less than 25g/L xylose, e.g., 80-110g/L glucose and 25-40g/L xylose.

The temperature of the fermentation is preferably 28 to 32 ℃, for example 30 ℃.

The aeration rate of the fermentation is preferably 1.0 to 1.7vvm, for example 1.4vvm.

The pH of the fermentation is preferably from 6 to 8, for example 7;

the fermentation is preferably carried out by stirring at a speed of 400 to 800rpm, preferably 600rpm.

In a preferred embodiment, the fermentation medium is a lignocellulosic hydrolysate, such as a straw hydrolysate.

The straw hydrolysate is preferably formed by the enzymolysis and saccharification of crop straw.

In a preferred embodiment, 15-25 g/L ammonium sulfate, 2-8 g/L methionine and 2-8 g/L threonine are added to the straw hydrolysate.

In a preferred embodiment, the straw hydrolysate is subjected to pretreatment prior to the enzymatic saccharification, the pretreatment comprising screening, impurity removal, acid pretreatment and/or detoxification treatment. The pretreatment can improve the saccharification efficiency of crop straws. The detoxication treatment can reduce the content of toxicity inhibitors such as acetic acid, furfural, 5-hydroxybenzaldehyde, furfural, hydroxymethylfurfural, 4-hydroxybenzaldehyde, levulinic acid and the like.

In another aspect, the invention provides an expression cassette, a nucleic acid, a recombinant expression vector or a genetically engineered bacterium, wherein the expression cassette, the nucleic acid, the recombinant expression vector or the genetically engineered bacterium are used for preparing L-lysine.

A method for preparing a genetically engineered bacterium, which is obtained by introducing an expression cassette according to the present invention into a starting bacterium as defined in the present invention.

The preparation method preferably further comprises: blocking the expression of lactate dehydrogenase in the genetically engineered bacterium, e.g., knockout of the ldh gene.

In the preparation method, the expression cassette is preferably integrated into the site of the ldh gene.

Preferably, the ldh gene is AJE68497.1 in Genbank accession number.

Although the strain C.glutamicum Caths141 which is resistant to the straw hydrolysate was selected as the starting strain in the examples of the present invention, it can be seen from examples 5 and 6 that the effect of producing L-lysine by the genetically engineered bacterium of the present invention is also superior to that of the starting strain when the culture medium is a formulated glucose culture medium, i.e., the condition that the inhibitor toxicity to the straw hydrolysate is not necessarily possessed by the starting strain.

The transaldolase gene is also called cgl1575 (the amino acid sequence of the transaldolase coded by the transaldolase gene can be a sequence corresponding to GenBank accession number BAB 98968.1); the transketolase gene is also called cgl1574 (the amino acid sequence of the transketolase coded by the transketolase gene can be a sequence corresponding to GenBank accession number BAB 98967.1).

In a preferred embodiment, the preservation number of the C.glutamicum Caths S141 is CCTCC NO: M20211495.

The C.glutamicum B253 of the present invention was purchased from Shanghai Industrial microorganism.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

after the expression cassette provided by the invention is introduced into a starting strain, the expression transketolase and the transaldolase are in an improved state compared with the starting strain, which is beneficial to strengthening the pentose phosphate pathway, so that the utilization efficiency of xylose and mixed sugar is effectively improved, and the output of L-lysine is further improved. When the straw hydrolysate is used, the lysine yield of the genetically engineered bacterium is obviously improved compared with that of the starting bacterium. The genetically engineered bacterium provided by the invention can effectively utilize agricultural wastes such as straw and the like for fermentation, and has good application prospect.

Biological material preservation information

The corynebacterium glutamicum Caths141 of the present invention has been preserved in China Center for Type Culture Collection (CCTCC) at 11.29 of 2021, and has a preservation address: chinese university of Wuhan, post code 430072, deposit number: CCTCC NO: M20211495, culture name Caths141, class name Corynebacterium glutamicum CathS141.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

1. Strains used in the present invention

Coli E.coli DH 5. Alpha. Was used for construction of expression plasmid and knock-out plasmid, C.glutamicum Caths141 was a strain for lysine production, and C.glutamicum Caths141 was used mainly as a starting strain in this experiment.

2. Reagent and culture medium

Cellulase CTec 2.0 was used to hydrolyze cellulose and hemicellulose in lignocellulose and was purchased from novelin (china) corporation of beijing, china. The cellulase enzyme activity was 203.2FPU/mL according to the method in the NREL LAP-006 guide, the cellobiase activity was 4900.0CBU/mL, and the protein concentration was 87.3mg/mL according to the Bradford method. Restriction enzymes were used to cleave plasmids or gene fragments to generate cohesive ends, available from Thermo Scientific (Wilmington, DE, USA). DNA polymerase is used to amplify the gene fragment, DNA ligase is used to ligate the digested gene fragment and plasmid vector, both of which are available from Takara (Otsu, japan). The seamless cloning kit was used to ligate gene fragments containing homologous fragments to plasmid vectors available from han-heng biotechnology company (nanjin, china). Plasmid extraction kits, PCR product purification recovery kits and gel recovery kits were all purchased from Shanghai swirley biotechnology company (Shanghai, china). Other reagents were purchased from local suppliers.

The culture medium used for culturing the escherichia coli is Luria-Bertani (LB) culture medium, and the specific components are as follows: 10.0g/L sodium chloride, 10.0g/L peptone and 5.0g/L yeast extract.

The specific components of the culture medium used for culturing corynebacterium glutamicum are as follows:

(1) Seed culture medium: 25g/L glucose, 1.5g/L potassium dihydrogen phosphate, 2.5g/L urea and 0.6g/L magnesium sulfate, 25g/L corn steep liquor.

(2) Fermentation medium: 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and optionally glucose as a carbon source.

Example 1: acquisition of the starting bacterium C.glutamicum Caths141

Collecting soil sample of Sinkiang Usu, adding 1g soil sample into 10mL sterile water, vigorously mixing for 1min, standing for precipitation for a period of time, and diluting the sample to 10 ^-3 、10 ^-4 、10 ^-5 Spread on LB agar plates containing 100mg/L nystatin and incubated at 30 ℃. And obtaining a purified single colony through repeated streak separation culture.

The strain capable of producing the L-lysine is obtained through separation and purification, and the characteristics, physiological and biochemical characteristics and the like of the strain are measured by referring to a common bacterial System identification Manual. The strain has the following morphological characteristics: the colony is moist, circular, smooth in surface, neat in edge and pale yellow in color; extracting the DNA of the strain by using a bacterial genome DNA extraction kit, using the DNA as a template, using bacterial universal primers 27F and 1492R, performing PCR amplification by using 16S rDNA, sequencing to obtain a strain 16S rDNA sequence, comparing the strain 16S rDNA sequence in a GenBank database, and identifying the strain as corynebacterium glutamicum (Corynebacterium glutamicum).

The wheat straw is subjected to acid pretreatment, detoxification and enzymatic hydrolysis to obtain wheat straw hydrolysate, and the corynebacterium glutamicum obtained by separation and purification is used as an initial strain, and the stable strain capable of tolerating the normal growth of toxic inhibitors in the straw hydrolysate and lysine production is obtained by using ultraviolet rays, nitrosoguanidine, 5-fluorouracil, ARTP and the like for multiple independent mutagenesis and composite mutagenesis. The strain was named c.glutamicum CathS141 and was now deposited in the chinese collection of typical cultures at the address: chinese university of Wuhan, post code 430072, preservation number CCTCC NO: M20211495, preservation date 2021, 11 months and 29 days.

Example 2: integration of xylAB expression cassette at ldh site

Firstly, constructing an integrative plasmid of xylAB (xylose isomerase and xylulokinase coding gene cluster), wherein the specific construction method is as follows: amplifying the genome of the C.glutamicum serving as a template by using a Peftu-F (shown as SEQ ID NO: 6) primer and a Peftu-R (shown as SEQ ID NO: 7) primer by a PCR method to obtain a Peftu promoter (shown as SEQ ID NO: 2); the E.coli BL21 genome is used as a template, and xylAB-F (shown as SEQ ID NO: 8) and xylAB-R (shown as SEQ ID NO: 9) primers are utilized to amplify by a PCR mode to obtain xylAB fragments (shown as SEQ ID NO: 1); peftu and xylAB are used as templates, and Peftu-F and xylAB-R primers are utilized to obtain a Peftu_xylAB fusion fragment (shown as SEQ ID NO: 10) in an overlap extension PCR mode. Amplifying the genome of the C.glutamicum serving as a template by using a ldh-up-F (shown as SEQ ID NO: 11) primer and a ldh-up-R (shown as SEQ ID NO: 12) primer by a PCR method to obtain a ldh-up fragment (shown as SEQ ID NO: 5); amplifying by PCR method with genome of C.glutamicum Caths141 as template and using ldh-down-F (shown as SEQ ID NO: 13) and ldh-down-R (shown as SEQ ID NO: 14) primer to obtain ldh-down fragment (shown as SEQ ID NO: 15); the fusion fragment of Deltaldh:: xylAB is obtained by overlapping extension PCR by using the ldh-up fragment, peftu_xylAB and ldh-down fragment as templates and using the ldh-up-F and ldh-down-R primers, and is treated by EcoRI and HindIII endonucleases, and then inserted into the pK18mob plasmid by using T4 ligase (see http:// www.biovector.net/product/1089.Html for purchase route), thereby obtaining the plasmid pK 18-Deltaldh:: xylAB. During this time, successfully ligated plasmids can be selected using seed culture plates containing kanamycin resistance. Then, the integrated plasmid pK18-Deltaldh is transferred into C.glutamicum Caths141 by electrotransformation, and then the strain which generates correct homologous recombination is screened by PCR verification, thus obtaining recombinant corynebacterium glutamicum which is named cgl01.

Example 3: construction of xylAB and cgl1574, cgl1575, cgl1574&1575, respectively, combination expression cassettes

Reference example 2, with the genome of C.glutamicum Caths141 as a template, the fragments cgl1574 and cgl1575 were amplified by primers cgl1574-F (shown as SEQ ID NO: 16) and cgl1574-R (shown as SEQ ID NO: 17), and cgl1575-F (shown as SEQ ID NO: 18) and cgl1575-R (shown as SEQ ID NO: 19), respectively; the cgl1574&1575 expression cassette (shown as SEQ ID NO: 20) was obtained by fusion PCR as described in reference example 2; in reference example 2, the fusion PCR method further yielded a combination of xylAB with cgl1574, cgl1575, cgl1574&1575, respectively, as shown in SEQ ID NO:21, AB-1575, as shown in SEQ ID NO:22, and AB-1574&1575, as shown in SEQ ID NO:23, using the P-AB-R primers, as shown in SEQ ID NO: 24.

Example 4: the AB-1574, AB-1575 and AB-1574&1575 expression cassettes were integrated at the ldh site, respectively

Reference example 2 inserts AB-1574, AB-1575 and AB-1574&1575, respectively, into the pK18mob plasmid, resulting in pK 18-. DELTA.ldh: AB-1574, pK 18-. DELTA.ldh:: AB-1575 and pK 18-. DELTA.ldh:: AB-1574&1575, respectively. The integrative plasmids pK 18-. DELTA.ldh:: AB-1574, pK 18-. DELTA.ldh:: AB-1575 and pK 18-. DELTA.ldh::: AB-1574&1575 were transferred to C.glutamicum Caths141, respectively, and the correct genetically engineered strains were designated cgl02, cgl03 and cgl04, respectively.

Example 5: xylose fermentation by different genetic engineering bacteria

The fermentation performance of cgl01, cgl02, cgl03 and cgl04, respectively, and control strains was evaluated by shake flask system fermentation. The fermentation process is as follows: the fermentation medium included 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and 40g/L xylose as a carbon source, 25. Mu.g/mL of kanamycin was added. The fermentation was carried out in 250mL shake flasks at 30℃and 200rpm for 96 hours. As shown in Table 1, the results of fermentation showed that cgl04 had the highest xylose metabolism ability.

TABLE 1 xylose fermentation comparison of different genetically engineered bacteria

Strain	Xylose consumption (%)
		CathS141	0
cgl01	49.65±2.32
		cgl02	56.28±1.76
cgl03	53.27±3.13
		cgl04	68.24±1.11

Example 6: mixed sugar fermentation of different genetically engineered bacteria

Reference example 5 fermentation evaluation was performed in a mixed sugar system, except that the carbon source used here was 25g/L xylose and 25g/L glucose. The fermentation results are shown in Table 2, where the enhancement of transketolase and transaldolase increased lysine conversion, with significant differences between each group and the control group.

TABLE 2 comparison of Mixed sugar fermentation of different genetically engineered bacteria

Strain	Total sugar-lysine conversion (%)
		CathS141	12.04±0.11
cgl01	18.58±0.20
		cgl02	23.66±0.17
cgl03	22.30±0.21
		cgl04	28.24±0.39

Example 7: fermenting different genetically engineered bacteria in lignocellulose hydrolysate

Crushing wheat straw, sieving with a sieve with the diameter of 10 mm, washing the sieved straw with water to remove impurities such as soil, stones and metals, drying in a 105 ℃ oven to constant weight, and storing in a closed plastic bag for later use. Then separating to obtain the wheat straw hydrolysate which contains 95.4g/L glucose and 34.7g/L xylose after acid pretreatment, biological detoxification and enzymolysis saccharification. 20g/L ammonium sulfate and 5g/L methionine and threonine are added into the hydrolysate, the transformed xylose utilization strain cgl04 and the original strain are cultured in the wheat straw hydrolysate for fermentation comparison, the fermentation temperature is 30 ℃, the pH is controlled to 7.0 by ammonia water, the ventilation amount is 1.4vvm, and the rotating speed is 600rpm.

TABLE 3 comparison of the fermentation of hydrolysates of different genetically engineered bacteria

Strain	Total sugar-lysine conversion (%)
		CathS141	15.57±0.08
cgl01	20.38±0.15
		cgl04	26.31±0.27

As a result, as shown in table 3, when the wheat straw hydrolysate was used as a medium, the lysine yield of the genetically engineered bacterium cgl04 was significantly higher than that of the strains CathS141 and cgl01. In addition, when Corynebacterium glutamicum B253 was used as the starting strain, the strain obtained by transformation was designated as B253-104 by the same transformation method as that of the strain cgl04, the strain obtained by transformation was designated as B253-101 by the same transformation method as that of the strain cgl01, and the fermentation effect under the same fermentation conditions as described above was confirmed, and the total sugar-lysine conversion rates of B253, B253-101 and B253-104 were 15.27.+ -. 0.09%, 20.40.+ -. 0.15% and 25.77.+ -. 0.23%, respectively. Therefore, the recombinant strain obtained by the invention has stronger inhibitor tolerance and high-efficiency lysine production capacity, and has good application prospect.

The foregoing specifically describes an operation example of the technical solution of the present invention, and is not to be construed as limiting the application of the present invention. All equivalent substitutions of operating conditions are within the scope of the present invention.

SEQUENCE LISTING

<110> Shanghai Kaisei Biotechnology Co., ltd

CIC Energy Center

<120> expression cassette for preparing L-lysine, genetically engineered bacterium and use thereof

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gacgttgcag caatcgaagc tgcagtggct gaggctaaga aggacaccaa gcgacctacc 720

ttcatccgcg ttcgcaccat catcggcttc ccagctccaa ctatgatgaa caccggtgct 780

gtgcacggtg ctgctcttgg cgcagctgag gttgcagcaa ccaagactga gcttggattc 840

gatcctgagg ctcacttcgc gatcgacgat gaggttatcg ctcacacccg ctccctcgca 900

gagcgcgctg cacagaagaa ggctgcatgg caggtcaagt tcgatgagtg ggcagctgcc 960

aaccctgaga acaaggctct gttcgatcgc ctgaactccc gtgagcttcc agcgggctac 1020

gctgacgagc tcccaacatg ggatgcagat gagaagggcg tcgcaactcg taaggcttcc 1080

gaggctgcac ttcaggcact gggcaagacc cttcctgagc tgtggggcgg ttccgctgac 1140

ctcgcaggtt ccaacaacac cgtgatcaag ggctcccctt ccttcggccc tgagtccatc 1200

tccaccgaga cctggtctgc tgagccttac ggccgtaacc tgcacttcgg tatccgtgag 1260

cacgctatgg gatccatcct caacggcatt tccctccacg gtggcacccg cccatacggc 1320

ggaaccttcc tcatcttctc cgactacatg cgtcctgcag ttcgtcttgc agctctcatg 1380

gagaccgacg cttactacgt ctggacccac gactccatcg gtctgggcga agatggccca 1440

acccaccagc ctgttgaaac cttggctgca ctgcgcgcca tcccaggtct gtccgtcctg 1500

cgtcctgcag atgcgaacga gaccgcccag gcttgggctg cagcacttga gtacaaggaa 1560

ggccctaagg gtcttgcact gacccgccag aacgttcctg ttctggaagg caccaaggag 1620

aaggctgctg aaggcgttcg ccgcggtggc tacgtcctgg ttgagggttc caaggaaacc 1680

ccagatgtga tcctcatggg ctccggctcc gaggttcagc ttgcagttaa cgctgcgaag 1740

gctctggaag ctgagggcgt tgcagctcgc gttgtttccg ttccttgcat ggattggttc 1800

caggagcagg acgcagagta catcgagtcc gttctgcctg cagctgtgac cgctcgtgtg 1860

tctgttgaag ctggcatcgc aatgccttgg taccgcttct tgggcaccca gggccgtgct 1920

gtctcccttg agcacttcgg tgcttctgcg gattaccaga ccctgtttga gaagttcggc 1980

atcaccaccg atgcagtcgt ggcagcggcc aaggactcca ttaacggtta a 2031

<210> 4

<211> 1083

<212> DNA

<213> Artificial Sequence

<220>

<223> cgl1575

<400> 4

atgtctcaca ttgatgatct tgcacagctc ggcacttcca cttggctcga cgacctctcc 60

cgcgagcgca ttacttccgg caatctcagc caggttattg aggaaaagtc tgtagtcggt 120

gtcaccacca acccagctat tttcgcagca gcaatgtcca agggcgattc ctacgacgct 180

cagatcgcag agctcaaggc cgctggcgca tctgttgacc aggctgttta cgccatgagc 240

atcgacgacg ttcgcaatgc ttgtgatctg ttcaccggca tcttcgagtc ctccaacggc 300

tacgacggcc gcgtgtccat cgaggttgac ccacgtatct ctgctgaccg cgacgcaacc 360

ctggctcagg ccaaggagct gtgggcaaag gttgatcgtc caaacgtcat gatcaagatc 420

cctgcaaccc caggttcttt gccagcaatc accgacgctt tggctgaggg catcagcgtt 480

aacgtcacct tgatcttctc cgttgctcgc taccgcgagg tcatcgctgc gttcatcgag 540

ggcatcaagc aggctgctgc aaacggccac gacgtctcca agatccactc tgtggcttcc 600

ttcttcgtct cccgcgtcga cgttgagatc gacaagcgcc tcgaggcaat cggatccgat 660

gaggctttgg ctctgcgcgg caaggcaggc gttgccaacg ctcagcgcgc ttacgctgtg 720

tacaaggagc ttttcgacgc cgccgagctg cctgaaggtg ccaacactca gcgcccactg 780

tgggcatcca ccggcgtgaa gaaccctgcg tacgctgcaa ctctttacgt ttccgagctg 840

gctggtccaa acaccgtcaa caccatgcca gaaggcacca tcgacgcggt tctggagcag 900

ggcaacctgc acggtgacac cctgtccaac tccgcggcag aagctgacgc tgtgttctcc 960

cagcttgagg ctctgggcgt tgacttggca gatgtcttcc aggtcctgga gaccgagggt 1020

gtggacaagt tcgttgcttc ttggagcgaa ctgcttgagt ccatggaagc tcgcctgaag 1080

tag 1083

<210> 5

<211> 943

<212> DNA

<213> Artificial Sequence

<220>

<223> ldh-up

<400> 5

ggaacaccat gcgattaagg tgcgctgctt gaattgcaga attatgcaag atgcgccgca 60

acaaaacgcg atcggccaag gtcaaagtgg tcaatgtaat gaccgaaacc gctgcgatga 120

aactaatcca cggcggtaaa aacctctcaa ttaggagctt gacctcatta atgctgtgct 180

gggttaattc gccggtgatc agcagcgcgc cgtaccccaa ggtgccgaca ctaatgcccg 240

cgatcgtctc cttcggtcca aaattcttct gcccaatcag ccggatttgg gtgcgatgcc 300

tgatcaatcc cacaaccgtg gtggtcaacg tgatggcacc agttgcgatg tgggtggcgt 360

tgtaaatttt cctggatacc cgccggttgg ttctggggag gatcgagtgg attcccgtcg 420

ctgacgcatg ccccaccgct tgtaaaacag ccaggttagc agccgtaacc caccacggtt 480

tcggcaacaa tgacggcgag agagcccacc acattgcgat ttccgctccg ataaagccag 540

cgcccatatt tgcagggagg attcgcctgc ggtttggcga cattcggatc cccggaacca 600

gctctgcaat cacctgcgcg ccgagggaag cgaggtgggt ggcaggtttt agtgcgggtt 660

taagcgttgc caggcgagtg gtgagcagag acgctagtct ggggagcgaa accatattga 720

gtcatcttgg cagagcatgc acaattctgc agggcataga ttggttttgc tcgatttaca 780

atgtgatttt ttcaacaaaa ataacacatg gtctgaccac attttcggac ataatcgggc 840

ataattaaag gtgtaacaaa ggaatccggg cacaagctct tgctgatttt ctgagctgct 900

ttgtgggttg tccggttagg gaaatcagga agtgggatcg aaa 943

<210> 6

<211> 47

<212> DNA

<213> Artificial Sequence

<220>

<223> Peftu-F

<400> 6

gaaatcagga agtgggatcg aaacgaaaag caatttgctt ttcgacg 47

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> Peftu-R

<400> 7

tgtatgtcct cctggacttc gtg 23

<210> 8

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> xylAB-F

<400> 8

ccacgaagtc caggaggaca tacaatgcaa gcctattttg accag 45

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> xylAB-R

<400> 9

ttacgccatt aatggcagaa g 21

<210> 10

<211> 3184

<212> DNA

<213> Artificial Sequence

<220>

<223> Peftu_xylAB

<400> 10

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac atacaatgca agcctatttt gaccagctcg 360

atcgcgttcg ttatgaaggc tcaaaatcct caaacccgtt agcattccgt cactacaatc 420

ccgacgaact ggtgttgggt aagcgtatgg aagagcactt gcgttttgcc gcctgctact 480

ggcacacctt ctgctggaac ggggcggata tgtttggtgt gggggcgttt aatcgtccgt 540

ggcagcagcc tggtgaggca ctggcgttgg cgaagcgtaa agcagatgtc gcatttgagt 600

ttttccacaa gttacatgtg ccattttatt gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaaga gagcggcgtg aagctgctgt ggggaaccgc caactgcttt acaaaccctc 780

gctacggcgc gggtgcggcg acgaacccag atcctgaagt cttcagctgg gcggcaacgc 840

aagttgttac agcgatggaa gcaacccata aattgggcgg tgaaaactat gtcctgtggg 900

gcggtcgtga aggttacgaa acgctgttaa ataccgactt gcgtcaggag cgtgaacaac 960

tgggccgctt tatgcagatg gtggttgagc ataaacataa aatcggtttc cagggcacgt 1020

tgcttatcga accgaaaccg caagaaccga ccaaacatca atatgattac gatgccgcga 1080

cggtctatgg cttcctgaaa cagtttggtc tggaaaaaga gattaaactg aacattgaag 1140

ctaaccacgc gacgctggca ggtcactctt tccatcatga aatagccacc gccattgcgc 1200

ttggcctgtt cggttctgtc gacgccaacc gtggcgatgc gcaactgggc tgggacaccg 1260

accagttccc gaacagtgtg gaagagaatg cgctggtgat gtatgaaatt ctcaaagcag 1320

gcggtttcac caccggtggt ctgaacttcg atgccaaagt acgtcgtcaa agtactgata 1380

aatatgatct gttttacggt catatcggcg cgatggatac gatggcactg gcgctgaaaa 1440

ttgcagcgcg catgattgaa gatggcgagc tggataaacg catcgcgcag cgttattccg 1500

gctggaatag cgaattgggc cagcaaatcc tgaaaggcca aatgtcactg gcagatttag 1560

ccaaatatgc tcaggaacat aatttgtctc cggtgcatca gagtggtcgc caggagcaac 1620

tggaaaatct ggtaaatcat tatctgttcg acaaataacg gctaactgtg cagtccgttg 1680

gcccggttat cggtagcgat accgggcatt tttttaagga acgatcgata tgtatatcgg 1740

gatagatctt ggcacctcgg gcgtaaaagt tattttgctc aacgagcagg gtgaggtggt 1800

tgcttcgcaa acggaaaagc tgaccgtttc gcgcccgcat ccactctggt cggaacaaga 1860

cccggaacag tggtggcagg caactgatcg cgcaatgaaa gctctgggcg atcagcattc 1920

tctgcaggac gttaaagcat tgggtattgc cggccagatg catggagcaa ccttactgga 1980

tgctcaacaa cgggtattgc gccctgccat tttgtggaac gacgggcgct gtgcgcaaga 2040

gtgcactttg ctggaagcga gagttccgca atcacgagtg attaccggca acctgatgat 2100

gcccggattt actgcgccta aattgctatg ggttcagcgg catgagccgg agatattccg 2160

tcaaatcgac aaagtattat taccgaaaga ttacttgcgt ctgcgtatga cgggggagtt 2220

tgccagcgat atgtctgacg cagctggcac catgtggctg gatgtcgcaa agcgtgactg 2280

gagtgacgtc atgctgcagg cttgcgactt atctcgtgac cagatgcccg cattatacga 2340

aggcagcgaa attactggtg ctttgttacc tgaagttgcg aaagcgtggg gtatggcgac 2400

ggtgccagtt gtcgcaggcg gtggcgacaa tgcagctggt gcagttggtg tgggaatggt 2460

tgatgctaat caggcaatgt tatcgctggg gacgtcgggg gtctattttg ctgtcagcga 2520

agggttctta agcaagccag aaagcgccgt acatagcttt tgccatgcgc taccgcaacg 2580

ttggcattta atgtctgtga tgctgagtgc agcgtcgtgt ctggattggg ccgcgaaatt 2640

aaccggcctg agcaatgtcc cagctttaat cgctgcagct caacaggctg atgaaagtgc 2700

cgagccagtt tggtttctgc cttatctttc cggcgagcgt acgccacaca ataatcccca 2760

ggcgaagggg gttttctttg gtttgactca tcaacatggc cccaatgaac tggcgcgagc 2820

agtgctggaa ggcgtgggtt atgcgctggc agatggcatg gatgtcgtgc atgcctgcgg 2880

tattaaaccg caaagtgtta cgttgattgg gggcggggcg cgtagtgagt actggcgtca 2940

gatgctggcg gatatcagcg gtcagcagct cgattaccgt acgggagggg atgtggggcc 3000

agcactgggc gcagcaaggc tggcgcagat cgcggcgaat ccagagaaat cgctcattga 3060

attgttgccg caactaccgt tagaacagtc gcatctacca gatgcgcagc gttatgccgc 3120

ttatcagcca cgacgagaaa cgttccgtcg cctctatcag caacttctgc cattaatggc 3180

gtaa 3184

<210> 11

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<223> ldh-up-F

<400> 11

tcccccgggg gaacaccatg cgattaaggt gc 32

<210> 12

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> ldh-up-R

<400> 12

caaattgctt ttcgtttcga tcccacttcc tgatttccct aac 43

<210> 13

<211> 42

<212> DNA

<213> Artificial Sequence

<220>

<223> ldh-down-F

<400> 13

caaggactcc attaacggtt aaatctttgg cgcctagttg gc 42

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> ldh-down-R

<400> 14

gtaagcttgt ctgggacgtt gatgacgctg 30

<210> 15

<211> 959

<212> DNA

<213> Artificial Sequence

<220>

<223> ldh-down

<400> 15

atctttggcg cctagttggc gacgcaagtg tttcattgga acacttgcgc tgccaacttt 60

ttggtttacg ggcaaaatga aactgttgga tggaatttaa agtgtttgta gcttaaggag 120

ctcaaatgaa tgagtttgac caggacattc tccaggagat caagactgaa ctcgacgagt 180

taattctaga acttgatgag gtgacacaaa ctcacagcga ggccatcggg caggtctccc 240

caacccatta cgttggtgcc cgcaacctca tgcattacgc gcatcttcgc accaaagacc 300

tccgtggcct gcagcaacgc ctctcctctg tgggagctac ccgcttgact accaccgaac 360

cagcagtgca ggcccgcctc aaggccgccc gcaatgttat cggagctttc gcaggtgaag 420

gcccacttta tccaccctca gatgtcgtcg atgccttcga agatgccgat gagattctcg 480

acgagcacgc cgaaattctc cttggcgaac ccctaccgga tactccatcc tgcatcatgg 540

tcaccctgcc caccgaagcc gccaccgaca ttgaacttgt ccgtggcttc gccaaaagcg 600

gcatgaatct agctcgcatc aactgtgcac acgacgatga aaccgtctgg aagcagatga 660

tcgacaacgt ccacaccgtt gcagaagaag ttggccggga aatccgcgtc agcatggacc 720

ttgccggacc aaaagtacgc accggcgaaa tcgccccagg cgcagaagta ggtcgcgcac 780

gagtaacccg cgacgaaacc ggaaaagtac tgacgcccgc aaaactgtgg atcaccgccc 840

acggctccga accagtccca gcccccgaaa gcctgcccgg tcgccccgct ctgccgattg 900

aagtcacccc agaatggttc gacaaactag aaatcggcag cgtcatcaac gtcccagac 959

<210> 16

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> cgl1574-F

<400> 16

ccacgaagtc caggaggaca tacaatggac accaaggctg tag 43

<210> 17

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> cgl1574-R

<400> 17

ttaaccgtta atggagtcct tgg 23

<210> 18

<211> 47

<212> DNA

<213> Artificial Sequence

<220>

<223> cgl1575-F

<400> 18

ccacgaagtc caggaggaca tacaatgtct cacattgatg atcttgc 47

<210> 19

<211> 19

<212> DNA

<213> Artificial Sequence

<220>

<223> cgl1575-R

<400> 19

ctacttcagg cgagcttcc 19

<210> 20

<211> 3784

<212> DNA

<213> Artificial Sequence

<220>

<223> cgl1574&1575

<400> 20

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac atacaatgga caccaaggct gtagacactg 360

ttcgtgtcct cgctgcagac gctgtagaaa actgtggctc cggccaccca ggcaccgcaa 420

tgagcctggc tccccttgca tacaccttgt accagcgggt tatgaacgta gatccacagg 480

acaccaactg ggcaggccgt gaccgcttcg ttctttcttg tggccactcc tctttgaccc 540

agtacatcca gctttacttg ggtggattcg gccttgagat ggatgacctg aaggctctgc 600

gcacctggga ttccttgacc ccaggacacc ctgagtaccg ccacaccaag ggcgttgaga 660

tcaccactgg ccctcttggc cagggtcttg catctgcagt tggtatggcc atggctgctc 720

gtcgtgagcg tggcctattc gacccaaccg ctgctgaggg cgaatcccca ttcgaccacc 780

acatctacgt cattgcttct gatggtgacc tgcaggaagg tgtcacctct gaggcatcct 840

ccatcgctgg cacccagcag ctgggcaacc tcatcgtgtt ctgggatgac aaccgcatct 900

ccatcgaaga caacactgag atcgctttca acgaggacgt tgttgctcgt tacaaggctt 960

acggctggca gaccattgag gttgaggctg gcgaggacgt tgcagcaatc gaagctgcag 1020

tggctgaggc taagaaggac accaagcgac ctaccttcat ccgcgttcgc accatcatcg 1080

gcttcccagc tccaactatg atgaacaccg gtgctgtgca cggtgctgct cttggcgcag 1140

ctgaggttgc agcaaccaag actgagcttg gattcgatcc tgaggctcac ttcgcgatcg 1200

acgatgaggt tatcgctcac acccgctccc tcgcagagcg cgctgcacag aagaaggctg 1260

catggcaggt caagttcgat gagtgggcag ctgccaaccc tgagaacaag gctctgttcg 1320

atcgcctgaa ctcccgtgag cttccagcgg gctacgctga cgagctccca acatgggatg 1380

cagatgagaa gggcgtcgca actcgtaagg cttccgaggc tgcacttcag gcactgggca 1440

agacccttcc tgagctgtgg ggcggttccg ctgacctcgc aggttccaac aacaccgtga 1500

tcaagggctc cccttccttc ggccctgagt ccatctccac cgagacctgg tctgctgagc 1560

cttacggccg taacctgcac ttcggtatcc gtgagcacgc tatgggatcc atcctcaacg 1620

gcatttccct ccacggtggc acccgcccat acggcggaac cttcctcatc ttctccgact 1680

acatgcgtcc tgcagttcgt cttgcagctc tcatggagac cgacgcttac tacgtctgga 1740

cccacgactc catcggtctg ggcgaagatg gcccaaccca ccagcctgtt gaaaccttgg 1800

ctgcactgcg cgccatccca ggtctgtccg tcctgcgtcc tgcagatgcg aacgagaccg 1860

cccaggcttg ggctgcagca cttgagtaca aggaaggccc taagggtctt gcactgaccc 1920

gccagaacgt tcctgttctg gaaggcacca aggagaaggc tgctgaaggc gttcgccgcg 1980

gtggctacgt cctggttgag ggttccaagg aaaccccaga tgtgatcctc atgggctccg 2040

gctccgaggt tcagcttgca gttaacgctg cgaaggctct ggaagctgag ggcgttgcag 2100

ctcgcgttgt ttccgttcct tgcatggatt ggttccagga gcaggacgca gagtacatcg 2160

agtccgttct gcctgcagct gtgaccgctc gtgtgtctgt tgaagctggc atcgcaatgc 2220

cttggtaccg cttcttgggc acccagggcc gtgctgtctc ccttgagcac ttcggtgctt 2280

ctgcggatta ccagaccctg tttgagaagt tcggcatcac caccgatgca gtcgtggcag 2340

cggccaagga ctccattaac ggttaacgaa aagcaatttg cttttcgacg ccccaccccg 2400

cgcgttttag cgtgtcagta ggcgcgtagg gtaagtgggg tagcggcttg ttagatatct 2460

tgaaatcggc tttcaacagc attgatttcg atgtatttag ctggccgtta ccctgcgaat 2520

gtccacaggg tagctggtag tttgaaaatc aacgccgttg cccttaggat tcagtaactg 2580

gcacattttg taatgcgcta gatctgtgtg ctcagtcttc caggctgctt atcacagtga 2640

aagcaaaacc aattcgtggc tgcgaaagtc gtagccacca cgaagtccag gaggacatac 2700

aatgtctcac attgatgatc ttgcacagct cggcacttcc acttggctcg acgacctctc 2760

ccgcgagcgc attacttccg gcaatctcag ccaggttatt gaggaaaagt ctgtagtcgg 2820

tgtcaccacc aacccagcta ttttcgcagc agcaatgtcc aagggcgatt cctacgacgc 2880

tcagatcgca gagctcaagg ccgctggcgc atctgttgac caggctgttt acgccatgag 2940

catcgacgac gttcgcaatg cttgtgatct gttcaccggc atcttcgagt cctccaacgg 3000

ctacgacggc cgcgtgtcca tcgaggttga cccacgtatc tctgctgacc gcgacgcaac 3060

cctggctcag gccaaggagc tgtgggcaaa ggttgatcgt ccaaacgtca tgatcaagat 3120

ccctgcaacc ccaggttctt tgccagcaat caccgacgct ttggctgagg gcatcagcgt 3180

taacgtcacc ttgatcttct ccgttgctcg ctaccgcgag gtcatcgctg cgttcatcga 3240

gggcatcaag caggctgctg caaacggcca cgacgtctcc aagatccact ctgtggcttc 3300

cttcttcgtc tcccgcgtcg acgttgagat cgacaagcgc ctcgaggcaa tcggatccga 3360

tgaggctttg gctctgcgcg gcaaggcagg cgttgccaac gctcagcgcg cttacgctgt 3420

gtacaaggag cttttcgacg ccgccgagct gcctgaaggt gccaacactc agcgcccact 3480

gtgggcatcc accggcgtga agaaccctgc gtacgctgca actctttacg tttccgagct 3540

ggctggtcca aacaccgtca acaccatgcc agaaggcacc atcgacgcgg ttctggagca 3600

gggcaacctg cacggtgaca ccctgtccaa ctccgcggca gaagctgacg ctgtgttctc 3660

ccagcttgag gctctgggcg ttgacttggc agatgtcttc caggtcctgg agaccgaggg 3720

tgtggacaag ttcgttgctt cttggagcga actgcttgag tccatggaag ctcgcctgaa 3780

gtag 3784

<210> 21

<211> 5550

<212> DNA

<213> Artificial Sequence

<220>

<223> AB-1574

<400> 21

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac atacaatgca agcctatttt gaccagctcg 360

atcgcgttcg ttatgaaggc tcaaaatcct caaacccgtt agcattccgt cactacaatc 420

ccgacgaact ggtgttgggt aagcgtatgg aagagcactt gcgttttgcc gcctgctact 480

ggcacacctt ctgctggaac ggggcggata tgtttggtgt gggggcgttt aatcgtccgt 540

ggcagcagcc tggtgaggca ctggcgttgg cgaagcgtaa agcagatgtc gcatttgagt 600

ttttccacaa gttacatgtg ccattttatt gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaaga gagcggcgtg aagctgctgt ggggaaccgc caactgcttt acaaaccctc 780

gctacggcgc gggtgcggcg acgaacccag atcctgaagt cttcagctgg gcggcaacgc 840

aagttgttac agcgatggaa gcaacccata aattgggcgg tgaaaactat gtcctgtggg 900

gcggtcgtga aggttacgaa acgctgttaa ataccgactt gcgtcaggag cgtgaacaac 960

tgggccgctt tatgcagatg gtggttgagc ataaacataa aatcggtttc cagggcacgt 1020

tgcttatcga accgaaaccg caagaaccga ccaaacatca atatgattac gatgccgcga 1080

cggtctatgg cttcctgaaa cagtttggtc tggaaaaaga gattaaactg aacattgaag 1140

ctaaccacgc gacgctggca ggtcactctt tccatcatga aatagccacc gccattgcgc 1200

ttggcctgtt cggttctgtc gacgccaacc gtggcgatgc gcaactgggc tgggacaccg 1260

accagttccc gaacagtgtg gaagagaatg cgctggtgat gtatgaaatt ctcaaagcag 1320

gcggtttcac caccggtggt ctgaacttcg atgccaaagt acgtcgtcaa agtactgata 1380

aatatgatct gttttacggt catatcggcg cgatggatac gatggcactg gcgctgaaaa 1440

ttgcagcgcg catgattgaa gatggcgagc tggataaacg catcgcgcag cgttattccg 1500

gctggaatag cgaattgggc cagcaaatcc tgaaaggcca aatgtcactg gcagatttag 1560

ccaaatatgc tcaggaacat aatttgtctc cggtgcatca gagtggtcgc caggagcaac 1620

tggaaaatct ggtaaatcat tatctgttcg acaaataacg gctaactgtg cagtccgttg 1680

gcccggttat cggtagcgat accgggcatt tttttaagga acgatcgata tgtatatcgg 1740

gatagatctt ggcacctcgg gcgtaaaagt tattttgctc aacgagcagg gtgaggtggt 1800

tgcttcgcaa acggaaaagc tgaccgtttc gcgcccgcat ccactctggt cggaacaaga 1860

cccggaacag tggtggcagg caactgatcg cgcaatgaaa gctctgggcg atcagcattc 1920

tctgcaggac gttaaagcat tgggtattgc cggccagatg catggagcaa ccttactgga 1980

tgctcaacaa cgggtattgc gccctgccat tttgtggaac gacgggcgct gtgcgcaaga 2040

gtgcactttg ctggaagcga gagttccgca atcacgagtg attaccggca acctgatgat 2100

gcccggattt actgcgccta aattgctatg ggttcagcgg catgagccgg agatattccg 2160

tcaaatcgac aaagtattat taccgaaaga ttacttgcgt ctgcgtatga cgggggagtt 2220

tgccagcgat atgtctgacg cagctggcac catgtggctg gatgtcgcaa agcgtgactg 2280

gagtgacgtc atgctgcagg cttgcgactt atctcgtgac cagatgcccg cattatacga 2340

aggcagcgaa attactggtg ctttgttacc tgaagttgcg aaagcgtggg gtatggcgac 2400

ggtgccagtt gtcgcaggcg gtggcgacaa tgcagctggt gcagttggtg tgggaatggt 2460

tgatgctaat caggcaatgt tatcgctggg gacgtcgggg gtctattttg ctgtcagcga 2520

agggttctta agcaagccag aaagcgccgt acatagcttt tgccatgcgc taccgcaacg 2580

ttggcattta atgtctgtga tgctgagtgc agcgtcgtgt ctggattggg ccgcgaaatt 2640

aaccggcctg agcaatgtcc cagctttaat cgctgcagct caacaggctg atgaaagtgc 2700

cgagccagtt tggtttctgc cttatctttc cggcgagcgt acgccacaca ataatcccca 2760

ggcgaagggg gttttctttg gtttgactca tcaacatggc cccaatgaac tggcgcgagc 2820

agtgctggaa ggcgtgggtt atgcgctggc agatggcatg gatgtcgtgc atgcctgcgg 2880

tattaaaccg caaagtgtta cgttgattgg gggcggggcg cgtagtgagt actggcgtca 2940

gatgctggcg gatatcagcg gtcagcagct cgattaccgt acgggagggg atgtggggcc 3000

agcactgggc gcagcaaggc tggcgcagat cgcggcgaat ccagagaaat cgctcattga 3060

attgttgccg caactaccgt tagaacagtc gcatctacca gatgcgcagc gttatgccgc 3120

ttatcagcca cgacgagaaa cgttccgtcg cctctatcag caacttctgc cattaatggc 3180

gtaacgaaaa gcaatttgct tttcgacgcc ccaccccgcg cgttttagcg tgtcagtagg 3240

cgcgtagggt aagtggggta gcggcttgtt agatatcttg aaatcggctt tcaacagcat 3300

tgatttcgat gtatttagct ggccgttacc ctgcgaatgt ccacagggta gctggtagtt 3360

tgaaaatcaa cgccgttgcc cttaggattc agtaactggc acattttgta atgcgctaga 3420

tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa gcaaaaccaa ttcgtggctg 3480

cgaaagtcgt agccaccacg aagtccagga ggacatacaa tggacaccaa ggctgtagac 3540

actgttcgtg tcctcgctgc agacgctgta gaaaactgtg gctccggcca cccaggcacc 3600

gcaatgagcc tggctcccct tgcatacacc ttgtaccagc gggttatgaa cgtagatcca 3660

caggacacca actgggcagg ccgtgaccgc ttcgttcttt cttgtggcca ctcctctttg 3720

acccagtaca tccagcttta cttgggtgga ttcggccttg agatggatga cctgaaggct 3780

ctgcgcacct gggattcctt gaccccagga caccctgagt accgccacac caagggcgtt 3840

gagatcacca ctggccctct tggccagggt cttgcatctg cagttggtat ggccatggct 3900

gctcgtcgtg agcgtggcct attcgaccca accgctgctg agggcgaatc cccattcgac 3960

caccacatct acgtcattgc ttctgatggt gacctgcagg aaggtgtcac ctctgaggca 4020

tcctccatcg ctggcaccca gcagctgggc aacctcatcg tgttctggga tgacaaccgc 4080

atctccatcg aagacaacac tgagatcgct ttcaacgagg acgttgttgc tcgttacaag 4140

gcttacggct ggcagaccat tgaggttgag gctggcgagg acgttgcagc aatcgaagct 4200

gcagtggctg aggctaagaa ggacaccaag cgacctacct tcatccgcgt tcgcaccatc 4260

atcggcttcc cagctccaac tatgatgaac accggtgctg tgcacggtgc tgctcttggc 4320

gcagctgagg ttgcagcaac caagactgag cttggattcg atcctgaggc tcacttcgcg 4380

atcgacgatg aggttatcgc tcacacccgc tccctcgcag agcgcgctgc acagaagaag 4440

gctgcatggc aggtcaagtt cgatgagtgg gcagctgcca accctgagaa caaggctctg 4500

ttcgatcgcc tgaactcccg tgagcttcca gcgggctacg ctgacgagct cccaacatgg 4560

gatgcagatg agaagggcgt cgcaactcgt aaggcttccg aggctgcact tcaggcactg 4620

ggcaagaccc ttcctgagct gtggggcggt tccgctgacc tcgcaggttc caacaacacc 4680

gtgatcaagg gctccccttc cttcggccct gagtccatct ccaccgagac ctggtctgct 4740

gagccttacg gccgtaacct gcacttcggt atccgtgagc acgctatggg atccatcctc 4800

aacggcattt ccctccacgg tggcacccgc ccatacggcg gaaccttcct catcttctcc 4860

gactacatgc gtcctgcagt tcgtcttgca gctctcatgg agaccgacgc ttactacgtc 4920

tggacccacg actccatcgg tctgggcgaa gatggcccaa cccaccagcc tgttgaaacc 4980

ttggctgcac tgcgcgccat cccaggtctg tccgtcctgc gtcctgcaga tgcgaacgag 5040

accgcccagg cttgggctgc agcacttgag tacaaggaag gccctaaggg tcttgcactg 5100

acccgccaga acgttcctgt tctggaaggc accaaggaga aggctgctga aggcgttcgc 5160

cgcggtggct acgtcctggt tgagggttcc aaggaaaccc cagatgtgat cctcatgggc 5220

tccggctccg aggttcagct tgcagttaac gctgcgaagg ctctggaagc tgagggcgtt 5280

gcagctcgcg ttgtttccgt tccttgcatg gattggttcc aggagcagga cgcagagtac 5340

atcgagtccg ttctgcctgc agctgtgacc gctcgtgtgt ctgttgaagc tggcatcgca 5400

atgccttggt accgcttctt gggcacccag ggccgtgctg tctcccttga gcacttcggt 5460

gcttctgcgg attaccagac cctgtttgag aagttcggca tcaccaccga tgcagtcgtg 5520

gcagcggcca aggactccat taacggttaa 5550

<210> 22

<211> 4601

<212> DNA

<213> Artificial Sequence

<220>

<223> AB-1575

<400> 22

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac atacaatgca agcctatttt gaccagctcg 360

atcgcgttcg ttatgaaggc tcaaaatcct caaacccgtt agcattccgt cactacaatc 420

ccgacgaact ggtgttgggt aagcgtatgg aagagcactt gcgttttgcc gcctgctact 480

ggcacacctt ctgctggaac ggggcggata tgtttggtgt gggggcgttt aatcgtccgt 540

ggcagcagcc tggtgaggca ctggcgttgg cgaagcgtaa agcagatgtc gcatttgagt 600

ttttccacaa gttacatgtg ccattttatt gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaaga gagcggcgtg aagctgctgt ggggaaccgc caactgcttt acaaaccctc 780

gctacggcgc gggtgcggcg acgaacccag atcctgaagt cttcagctgg gcggcaacgc 840

aagttgttac agcgatggaa gcaacccata aattgggcgg tgaaaactat gtcctgtggg 900

gcggtcgtga aggttacgaa acgctgttaa ataccgactt gcgtcaggag cgtgaacaac 960

tgggccgctt tatgcagatg gtggttgagc ataaacataa aatcggtttc cagggcacgt 1020

tgcttatcga accgaaaccg caagaaccga ccaaacatca atatgattac gatgccgcga 1080

cggtctatgg cttcctgaaa cagtttggtc tggaaaaaga gattaaactg aacattgaag 1140

ctaaccacgc gacgctggca ggtcactctt tccatcatga aatagccacc gccattgcgc 1200

ttggcctgtt cggttctgtc gacgccaacc gtggcgatgc gcaactgggc tgggacaccg 1260

accagttccc gaacagtgtg gaagagaatg cgctggtgat gtatgaaatt ctcaaagcag 1320

gcggtttcac caccggtggt ctgaacttcg atgccaaagt acgtcgtcaa agtactgata 1380

aatatgatct gttttacggt catatcggcg cgatggatac gatggcactg gcgctgaaaa 1440

ttgcagcgcg catgattgaa gatggcgagc tggataaacg catcgcgcag cgttattccg 1500

gctggaatag cgaattgggc cagcaaatcc tgaaaggcca aatgtcactg gcagatttag 1560

ccaaatatgc tcaggaacat aatttgtctc cggtgcatca gagtggtcgc caggagcaac 1620

tggaaaatct ggtaaatcat tatctgttcg acaaataacg gctaactgtg cagtccgttg 1680

gcccggttat cggtagcgat accgggcatt tttttaagga acgatcgata tgtatatcgg 1740

gatagatctt ggcacctcgg gcgtaaaagt tattttgctc aacgagcagg gtgaggtggt 1800

tgcttcgcaa acggaaaagc tgaccgtttc gcgcccgcat ccactctggt cggaacaaga 1860

cccggaacag tggtggcagg caactgatcg cgcaatgaaa gctctgggcg atcagcattc 1920

tctgcaggac gttaaagcat tgggtattgc cggccagatg catggagcaa ccttactgga 1980

tgctcaacaa cgggtattgc gccctgccat tttgtggaac gacgggcgct gtgcgcaaga 2040

gtgcactttg ctggaagcga gagttccgca atcacgagtg attaccggca acctgatgat 2100

gcccggattt actgcgccta aattgctatg ggttcagcgg catgagccgg agatattccg 2160

tcaaatcgac aaagtattat taccgaaaga ttacttgcgt ctgcgtatga cgggggagtt 2220

tgccagcgat atgtctgacg cagctggcac catgtggctg gatgtcgcaa agcgtgactg 2280

gagtgacgtc atgctgcagg cttgcgactt atctcgtgac cagatgcccg cattatacga 2340

aggcagcgaa attactggtg ctttgttacc tgaagttgcg aaagcgtggg gtatggcgac 2400

ggtgccagtt gtcgcaggcg gtggcgacaa tgcagctggt gcagttggtg tgggaatggt 2460

tgatgctaat caggcaatgt tatcgctggg gacgtcgggg gtctattttg ctgtcagcga 2520

agggttctta agcaagccag aaagcgccgt acatagcttt tgccatgcgc taccgcaacg 2580

ttggcattta atgtctgtga tgctgagtgc agcgtcgtgt ctggattggg ccgcgaaatt 2640

aaccggcctg agcaatgtcc cagctttaat cgctgcagct caacaggctg atgaaagtgc 2700

cgagccagtt tggtttctgc cttatctttc cggcgagcgt acgccacaca ataatcccca 2760

ggcgaagggg gttttctttg gtttgactca tcaacatggc cccaatgaac tggcgcgagc 2820

agtgctggaa ggcgtgggtt atgcgctggc agatggcatg gatgtcgtgc atgcctgcgg 2880

tattaaaccg caaagtgtta cgttgattgg gggcggggcg cgtagtgagt actggcgtca 2940

gatgctggcg gatatcagcg gtcagcagct cgattaccgt acgggagggg atgtggggcc 3000

agcactgggc gcagcaaggc tggcgcagat cgcggcgaat ccagagaaat cgctcattga 3060

attgttgccg caactaccgt tagaacagtc gcatctacca gatgcgcagc gttatgccgc 3120

ttatcagcca cgacgagaaa cgttccgtcg cctctatcag caacttctgc cattaatggc 3180

gtaagaaaag caatttgctt ttcgacgccc caccccgcgc gttttagcgt gtcagtaggc 3240

gcgtagggta agtggggtag cggcttgtta gatatcttga aatcggcttt caacagcatt 3300

gatttcgatg tatttagctg gccgttaccc tgcgaatgtc cacagggtag ctggtagttt 3360

gaaaatcaac gccgttgccc ttaggattca gtaactggca cattttgtaa tgcgctagat 3420

ctgtgtgctc agtcttccag gctgcttatc acagtgaaag caaaaccaat tcgtggctgc 3480

gaaagtcgta gccaccacga agtccaggag gacatacaat gtctcacatt gatgatcttg 3540

cacagctcgg cacttccact tggctcgacg acctctcccg cgagcgcatt acttccggca 3600

atctcagcca ggttattgag gaaaagtctg tagtcggtgt caccaccaac ccagctattt 3660

tcgcagcagc aatgtccaag ggcgattcct acgacgctca gatcgcagag ctcaaggccg 3720

ctggcgcatc tgttgaccag gctgtttacg ccatgagcat cgacgacgtt cgcaatgctt 3780

gtgatctgtt caccggcatc ttcgagtcct ccaacggcta cgacggccgc gtgtccatcg 3840

aggttgaccc acgtatctct gctgaccgcg acgcaaccct ggctcaggcc aaggagctgt 3900

gggcaaaggt tgatcgtcca aacgtcatga tcaagatccc tgcaacccca ggttctttgc 3960

cagcaatcac cgacgctttg gctgagggca tcagcgttaa cgtcaccttg atcttctccg 4020

ttgctcgcta ccgcgaggtc atcgctgcgt tcatcgaggg catcaagcag gctgctgcaa 4080

acggccacga cgtctccaag atccactctg tggcttcctt cttcgtctcc cgcgtcgacg 4140

ttgagatcga caagcgcctc gaggcaatcg gatccgatga ggctttggct ctgcgcggca 4200

aggcaggcgt tgccaacgct cagcgcgctt acgctgtgta caaggagctt ttcgacgccg 4260

ccgagctgcc tgaaggtgcc aacactcagc gcccactgtg ggcatccacc ggcgtgaaga 4320

accctgcgta cgctgcaact ctttacgttt ccgagctggc tggtccaaac accgtcaaca 4380

ccatgccaga aggcaccatc gacgcggttc tggagcaggg caacctgcac ggtgacaccc 4440

tgtccaactc cgcggcagaa gctgacgctg tgttctccca gcttgaggct ctgggcgttg 4500

acttggcaga tgtcttccag gtcctggaga ccgagggtgt ggacaagttc gttgcttctt 4560

ggagcgaact gcttgagtcc atggaagctc gcctgaagta g 4601

<210> 23

<211> 6968

<212> DNA

<213> Artificial Sequence

<220>

<223> AB-1574&1575

<400> 23

cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60

tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120

ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180

aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240

tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300

agtcgtagcc accacgaagt ccaggaggac atacaatgca agcctatttt gaccagctcg 360

atcgcgttcg ttatgaaggc tcaaaatcct caaacccgtt agcattccgt cactacaatc 420

ccgacgaact ggtgttgggt aagcgtatgg aagagcactt gcgttttgcc gcctgctact 480

ggcacacctt ctgctggaac ggggcggata tgtttggtgt gggggcgttt aatcgtccgt 540

ggcagcagcc tggtgaggca ctggcgttgg cgaagcgtaa agcagatgtc gcatttgagt 600

ttttccacaa gttacatgtg ccattttatt gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaaga gagcggcgtg aagctgctgt ggggaaccgc caactgcttt acaaaccctc 780

gctacggcgc gggtgcggcg acgaacccag atcctgaagt cttcagctgg gcggcaacgc 840

aagttgttac agcgatggaa gcaacccata aattgggcgg tgaaaactat gtcctgtggg 900

gcggtcgtga aggttacgaa acgctgttaa ataccgactt gcgtcaggag cgtgaacaac 960

tgggccgctt tatgcagatg gtggttgagc ataaacataa aatcggtttc cagggcacgt 1020

tgcttatcga accgaaaccg caagaaccga ccaaacatca atatgattac gatgccgcga 1080

cggtctatgg cttcctgaaa cagtttggtc tggaaaaaga gattaaactg aacattgaag 1140

ctaaccacgc gacgctggca ggtcactctt tccatcatga aatagccacc gccattgcgc 1200

ttggcctgtt cggttctgtc gacgccaacc gtggcgatgc gcaactgggc tgggacaccg 1260

accagttccc gaacagtgtg gaagagaatg cgctggtgat gtatgaaatt ctcaaagcag 1320

gcggtttcac caccggtggt ctgaacttcg atgccaaagt acgtcgtcaa agtactgata 1380

aatatgatct gttttacggt catatcggcg cgatggatac gatggcactg gcgctgaaaa 1440

ttgcagcgcg catgattgaa gatggcgagc tggataaacg catcgcgcag cgttattccg 1500

gctggaatag cgaattgggc cagcaaatcc tgaaaggcca aatgtcactg gcagatttag 1560

ccaaatatgc tcaggaacat aatttgtctc cggtgcatca gagtggtcgc caggagcaac 1620

tggaaaatct ggtaaatcat tatctgttcg acaaataacg gctaactgtg cagtccgttg 1680

gcccggttat cggtagcgat accgggcatt tttttaagga acgatcgata tgtatatcgg 1740

gatagatctt ggcacctcgg gcgtaaaagt tattttgctc aacgagcagg gtgaggtggt 1800

tgcttcgcaa acggaaaagc tgaccgtttc gcgcccgcat ccactctggt cggaacaaga 1860

cccggaacag tggtggcagg caactgatcg cgcaatgaaa gctctgggcg atcagcattc 1920

tctgcaggac gttaaagcat tgggtattgc cggccagatg catggagcaa ccttactgga 1980

tgctcaacaa cgggtattgc gccctgccat tttgtggaac gacgggcgct gtgcgcaaga 2040

gtgcactttg ctggaagcga gagttccgca atcacgagtg attaccggca acctgatgat 2100

gcccggattt actgcgccta aattgctatg ggttcagcgg catgagccgg agatattccg 2160

tcaaatcgac aaagtattat taccgaaaga ttacttgcgt ctgcgtatga cgggggagtt 2220

tgccagcgat atgtctgacg cagctggcac catgtggctg gatgtcgcaa agcgtgactg 2280

gagtgacgtc atgctgcagg cttgcgactt atctcgtgac cagatgcccg cattatacga 2340

aggcagcgaa attactggtg ctttgttacc tgaagttgcg aaagcgtggg gtatggcgac 2400

ggtgccagtt gtcgcaggcg gtggcgacaa tgcagctggt gcagttggtg tgggaatggt 2460

tgatgctaat caggcaatgt tatcgctggg gacgtcgggg gtctattttg ctgtcagcga 2520

agggttctta agcaagccag aaagcgccgt acatagcttt tgccatgcgc taccgcaacg 2580

ttggcattta atgtctgtga tgctgagtgc agcgtcgtgt ctggattggg ccgcgaaatt 2640

aaccggcctg agcaatgtcc cagctttaat cgctgcagct caacaggctg atgaaagtgc 2700

cgagccagtt tggtttctgc cttatctttc cggcgagcgt acgccacaca ataatcccca 2760

ggcgaagggg gttttctttg gtttgactca tcaacatggc cccaatgaac tggcgcgagc 2820

agtgctggaa ggcgtgggtt atgcgctggc agatggcatg gatgtcgtgc atgcctgcgg 2880

tattaaaccg caaagtgtta cgttgattgg gggcggggcg cgtagtgagt actggcgtca 2940

gatgctggcg gatatcagcg gtcagcagct cgattaccgt acgggagggg atgtggggcc 3000

agcactgggc gcagcaaggc tggcgcagat cgcggcgaat ccagagaaat cgctcattga 3060

attgttgccg caactaccgt tagaacagtc gcatctacca gatgcgcagc gttatgccgc 3120

ttatcagcca cgacgagaaa cgttccgtcg cctctatcag caacttctgc cattaatggc 3180

gtaacgaaaa gcaatttgct tttcgacgcc ccaccccgcg cgttttagcg tgtcagtagg 3240

cgcgtagggt aagtggggta gcggcttgtt agatatcttg aaatcggctt tcaacagcat 3300

tgatttcgat gtatttagct ggccgttacc ctgcgaatgt ccacagggta gctggtagtt 3360

tgaaaatcaa cgccgttgcc cttaggattc agtaactggc acattttgta atgcgctaga 3420

tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa gcaaaaccaa ttcgtggctg 3480

cgaaagtcgt agccaccacg aagtccagga ggacatacaa tggacaccaa ggctgtagac 3540

actgttcgtg tcctcgctgc agacgctgta gaaaactgtg gctccggcca cccaggcacc 3600

gcaatgagcc tggctcccct tgcatacacc ttgtaccagc gggttatgaa cgtagatcca 3660

caggacacca actgggcagg ccgtgaccgc ttcgttcttt cttgtggcca ctcctctttg 3720

acccagtaca tccagcttta cttgggtgga ttcggccttg agatggatga cctgaaggct 3780

ctgcgcacct gggattcctt gaccccagga caccctgagt accgccacac caagggcgtt 3840

gagatcacca ctggccctct tggccagggt cttgcatctg cagttggtat ggccatggct 3900

gctcgtcgtg agcgtggcct attcgaccca accgctgctg agggcgaatc cccattcgac 3960

caccacatct acgtcattgc ttctgatggt gacctgcagg aaggtgtcac ctctgaggca 4020

tcctccatcg ctggcaccca gcagctgggc aacctcatcg tgttctggga tgacaaccgc 4080

atctccatcg aagacaacac tgagatcgct ttcaacgagg acgttgttgc tcgttacaag 4140

gcttacggct ggcagaccat tgaggttgag gctggcgagg acgttgcagc aatcgaagct 4200

gcagtggctg aggctaagaa ggacaccaag cgacctacct tcatccgcgt tcgcaccatc 4260

atcggcttcc cagctccaac tatgatgaac accggtgctg tgcacggtgc tgctcttggc 4320

gcagctgagg ttgcagcaac caagactgag cttggattcg atcctgaggc tcacttcgcg 4380

atcgacgatg aggttatcgc tcacacccgc tccctcgcag agcgcgctgc acagaagaag 4440

gctgcatggc aggtcaagtt cgatgagtgg gcagctgcca accctgagaa caaggctctg 4500

ttcgatcgcc tgaactcccg tgagcttcca gcgggctacg ctgacgagct cccaacatgg 4560

gatgcagatg agaagggcgt cgcaactcgt aaggcttccg aggctgcact tcaggcactg 4620

ggcaagaccc ttcctgagct gtggggcggt tccgctgacc tcgcaggttc caacaacacc 4680

gtgatcaagg gctccccttc cttcggccct gagtccatct ccaccgagac ctggtctgct 4740

gagccttacg gccgtaacct gcacttcggt atccgtgagc acgctatggg atccatcctc 4800

aacggcattt ccctccacgg tggcacccgc ccatacggcg gaaccttcct catcttctcc 4860

gactacatgc gtcctgcagt tcgtcttgca gctctcatgg agaccgacgc ttactacgtc 4920

tggacccacg actccatcgg tctgggcgaa gatggcccaa cccaccagcc tgttgaaacc 4980

ttggctgcac tgcgcgccat cccaggtctg tccgtcctgc gtcctgcaga tgcgaacgag 5040

accgcccagg cttgggctgc agcacttgag tacaaggaag gccctaaggg tcttgcactg 5100

acccgccaga acgttcctgt tctggaaggc accaaggaga aggctgctga aggcgttcgc 5160

cgcggtggct acgtcctggt tgagggttcc aaggaaaccc cagatgtgat cctcatgggc 5220

tccggctccg aggttcagct tgcagttaac gctgcgaagg ctctggaagc tgagggcgtt 5280

gcagctcgcg ttgtttccgt tccttgcatg gattggttcc aggagcagga cgcagagtac 5340

atcgagtccg ttctgcctgc agctgtgacc gctcgtgtgt ctgttgaagc tggcatcgca 5400

atgccttggt accgcttctt gggcacccag ggccgtgctg tctcccttga gcacttcggt 5460

gcttctgcgg attaccagac cctgtttgag aagttcggca tcaccaccga tgcagtcgtg 5520

gcagcggcca aggactccat taacggttaa cgaaaagcaa tttgcttttc gacgccccac 5580

cccgcgcgtt ttagcgtgtc agtaggcgcg tagggtaagt ggggtagcgg cttgttagat 5640

atcttgaaat cggctttcaa cagcattgat ttcgatgtat ttagctggcc gttaccctgc 5700

gaatgtccac agggtagctg gtagtttgaa aatcaacgcc gttgccctta ggattcagta 5760

actggcacat tttgtaatgc gctagatctg tgtgctcagt cttccaggct gcttatcaca 5820

gtgaaagcaa aaccaattcg tggctgcgaa agtcgtagcc accacgaagt ccaggaggac 5880

atacaatgtc tcacattgat gatcttgcac agctcggcac ttccacttgg ctcgacgacc 5940

tctcccgcga gcgcattact tccggcaatc tcagccaggt tattgaggaa aagtctgtag 6000

tcggtgtcac caccaaccca gctattttcg cagcagcaat gtccaagggc gattcctacg 6060

acgctcagat cgcagagctc aaggccgctg gcgcatctgt tgaccaggct gtttacgcca 6120

tgagcatcga cgacgttcgc aatgcttgtg atctgttcac cggcatcttc gagtcctcca 6180

acggctacga cggccgcgtg tccatcgagg ttgacccacg tatctctgct gaccgcgacg 6240

caaccctggc tcaggccaag gagctgtggg caaaggttga tcgtccaaac gtcatgatca 6300

agatccctgc aaccccaggt tctttgccag caatcaccga cgctttggct gagggcatca 6360

gcgttaacgt caccttgatc ttctccgttg ctcgctaccg cgaggtcatc gctgcgttca 6420

tcgagggcat caagcaggct gctgcaaacg gccacgacgt ctccaagatc cactctgtgg 6480

cttccttctt cgtctcccgc gtcgacgttg agatcgacaa gcgcctcgag gcaatcggat 6540

ccgatgaggc tttggctctg cgcggcaagg caggcgttgc caacgctcag cgcgcttacg 6600

ctgtgtacaa ggagcttttc gacgccgccg agctgcctga aggtgccaac actcagcgcc 6660

cactgtgggc atccaccggc gtgaagaacc ctgcgtacgc tgcaactctt tacgtttccg 6720

agctggctgg tccaaacacc gtcaacacca tgccagaagg caccatcgac gcggttctgg 6780

agcagggcaa cctgcacggt gacaccctgt ccaactccgc ggcagaagct gacgctgtgt 6840

tctcccagct tgaggctctg ggcgttgact tggcagatgt cttccaggtc ctggagaccg 6900

agggtgtgga caagttcgtt gcttcttgga gcgaactgct tgagtccatg gaagctcgcc 6960

tgaagtag 6968

<210> 24

<211> 48

<212> DNA

<213> Artificial Sequence

<220>

<223> P-AB-R

<400> 24

cgtcgaaaag caaattgctt ttcgttacgc cattaatggc agaagttg 48

Claims (12)

1. An expression cassette comprising a promoter and xylAB gene, and further comprising a transaldolase gene and/or a transketolase gene.

2. The expression cassette of claim 1, wherein the xylAB gene has a sequence as shown in SEQ ID NO. 1;

and/or the sequence of the transaldolase gene is shown as SEQ ID NO. 4;

and/or the sequence of the transketolase gene is shown as SEQ ID NO. 3.

3. The expression cassette of claim 1, wherein the promoter is a Peftu promoter, and the sequence of the Peftu promoter is preferably as shown in SEQ ID No. 2.

4. An isolated nucleic acid comprising the expression cassette of any one of claims 1-3.

5. A recombinant expression vector comprising the expression cassette of any one of claims 1-3, or comprising the nucleic acid of claim 4;

preferably, the backbone plasmid of the recombinant expression vector is pK18mob.

6. A genetically engineered bacterium, characterized in that the genetically engineered bacterium is transformed in a starting bacterium with the expression cassette according to any one of claims 1 to 3, or the nucleic acid according to claim 4, or the recombinant expression vector according to claim 5;

preferably, the starting bacterium is Corynebacterium glutamicum (Corynebacterium glutamicum), such as C.glutamicum B253 or C.glutamicum Caths141 with a preservation number of CCTCC NO: M20211495.

7. The genetically engineered bacterium of claim 6, wherein said expression cassette is integrated into the genome of said starting bacterium by homologous recombination or is present in non-integrated form in said starting bacterium.

8. Genetically engineered bacterium according to claim 6 or 7, wherein the genetically engineered bacterium does not express lactate dehydrogenase, preferably the ldh gene is knocked out;

preferably, the expression cassette is integrated into the ldh gene at the site, more preferably, the ldh gene is AJE68497.1 at Genbank accession number.

9. A method for producing L-lysine, comprising fermenting the genetically engineered bacterium of any one of claims 6 to 8 in a fermentation medium;

preferably, the fermentation medium contains not less than 25g/L glucose and/or 25g/L xylose;

more preferably, the fermentation satisfies one or more of the following conditions:

the fermentation temperature is 28-32 ℃, preferably 30 ℃;

the aeration rate of the fermentation is 1.0 to 1.7vvm, preferably 1.4vvm;

the pH of the fermentation is 6-8, preferably 7;

the fermentation is carried out by stirring at a rotational speed of 400 to 800rpm, preferably 600rpm.

10. The method of claim 9, wherein the fermentation medium is a lignocellulosic hydrolysate, preferably a straw hydrolysate; preferably, the straw hydrolysate is formed by performing enzymolysis saccharification on crop straws; more preferably, 15-25 g/L ammonium sulfate, 2-8 g/L methionine and 2-8 g/L threonine are added into the straw hydrolysate, and/or the straw hydrolysate is subjected to pretreatment before enzymolysis saccharification, wherein the pretreatment comprises screening, impurity removal, acid pretreatment and/or detoxification treatment.

11. Use of the expression cassette according to any one of claims 1 to 3, the nucleic acid according to claim 4, the recombinant expression vector according to claim 5 or the genetically engineered bacterium according to any one of claims 6 to 8 for the preparation of L-lysine.

12. A method for producing a genetically engineered bacterium, characterized in that the genetically engineered bacterium is obtained by introducing the expression cassette according to any one of claims 1 to 3 into a starting bacterium as defined in any one of claims 6 to 8;

preferably, the preparation method further comprises: blocking the expression of lactate dehydrogenase in the genetically engineered bacterium, preferably knocking out the ldh gene;

more preferably, the expression cassette is integrated into the site of the ldh gene; and/or the ldh gene has an accession number in Genbank of AJE68497.1.