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

Abstract

The invention discloses an expression cassette for preparing L-lysine, genetically engineered bacteria and applications thereof. An expression cassette, which includes a promoter and xylAB gene, and also includes a transaldolase gene and/or a transketolase gene. After the expression cassette provided by the present invention is introduced into the starting strain, the expression of transketolase and transaldolase is in an improved state compared with the starting strain, which helps to strengthen the pentose phosphate pathway, thereby effectively improving the utilization efficiency of xylose and mixed sugars, and further improving Production of L-lysine. When the straw hydrolyzate is used, the lysine yield of the genetically engineered bacteria of the present invention is significantly improved compared with that of the original bacteria. The genetically engineered bacteria provided by the invention can effectively utilize agricultural waste such as straws for fermentation, and has good application prospects.

Description

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

technical field

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

Background technique

Lysine (lysine), also known as 2,6-diaminocaproic acid, is a basic amino acid. Lysine has important nutritional and physiological functions and is widely used in medicine, food and feed industries. At the same time, it can also be used as a precursor substance for the synthesis of nylon polymer materials. The production methods of lysine mainly include proteolysis, chemical synthesis and fermentation. Among them, microbial fermentation has the characteristics of low production cost, high production intensity, high specificity, and low environmental pollution. It has become the industrialization method of lysine. The most widely used method of production.

The prokaryotic microorganisms used to produce lysine mainly include Corynebacterium, Brevibacterium, Nocardia, Pseudomonas, Escherichia, Bacillus, etc. Corynebacterium glutamicum (Corynebacterium glutamicum) is the most important and safe strain for the fermentation of amino acids. Improving its ability to oversynthesize various amino acids through metabolic modification has always been a research hotspot. For example, overexpression of genes related to lysine synthesis pathway and related genes of feedback inhibition desensitization, or the strengthening of energy supply pathways starting from glucose metabolism and optimization of lysine transporters on cell membranes have effectively increased the productivity of lysine .

Conventional Corynebacterium glutamicum cannot utilize xylose, so it is a prerequisite for its lignocellulose fermentation to be able to utilize xylose in lignocellulose through metabolic engineering and synthetic biology. Patent CN200510076242.X constructs a xylose-lysine synthesis pathway by introducing the xylABFGHR locus, but the obtained strain still has problems such as low utilization rate of xylose, inability to grow normally in lignocellulosic system and fermentation of lysine . Therefore, further research is needed to construct strains that efficiently utilize glucose and xylose in lignocellulose to produce lysine. Genetically engineered bacteria with high yields of lysine are lacking in the prior art.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides an expression cassette for preparing L-lysine, genetically engineered bacteria and applications thereof, specifically providing an expression cassette and an Genetically engineered bacteria that can efficiently produce lysine using straw hydrolyzate. The genetically engineered bacteria of the invention can effectively utilize the glucose and xylose in the straw in the straw hydrolyzate. The invention improves the fermentation efficiency of Corynebacterium glutamicum under the conditions of xylose, mixed sugar and hydrolyzate by enhancing the functions of glutamic acid rod-shaped transketolase and transaldolase, and significantly increases the output of L-lysine , and there is no report on the production of L-lysine by Corynebacterium glutamicum from the perspective of transketolase and transaldolase.

In order to solve the above technical problems, the present invention provides an expression cassette on the one hand, which includes a promoter and xylAB gene, and also includes a transaldolase gene and/or a transketolase gene.

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

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

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

In the expression cassette of the present invention, the promoter is preferably a Peftu promoter, and the sequence of the Peftu promoter is preferably shown in SEQ ID NO:2.

Another aspect of the present invention provides an isolated nucleic acid comprising an expression cassette according to the present invention.

Another aspect of the present invention provides a recombinant expression vector, which includes the expression cassette described in the present invention, or includes the nucleic acid described in the present invention.

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

The specific information of the pK18mob can refer to the following webpage:

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

Another aspect of the present invention provides a genetically engineered bacterium, which is transformed into the starting bacterium with the expression cassette as described in the present invention, or the nucleic acid as described in the present invention, or the recombinant expression vector as described in the present invention.

In the genetic engineering, the starting bacterium is preferably Corynebacterium glutamicum, such as C. glutamicum B253 or C. glutamicum CathS141.

In the genetic engineering, the expression cassette is preferably integrated into the genome of the starting bacterium through homologous recombination, or exists in the starting bacterium in a non-integrated form.

In the genetically engineered bacteria, lactate dehydrogenase is preferably not expressed, for example, the ldh gene is knocked out.

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

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

The starting bacterium described in the present invention can be a wild-type strain or a modified strain.

Another aspect of the present invention provides a method for preparing L-lysine, the method comprising fermenting the genetically engineered bacterium according to the present invention in a fermentation medium.

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

The fermentation temperature is preferably 28-32°C, such as 30°C.

The aeration volume of the fermentation is preferably 1.0-1.7vvm, such as 1.4vvm.

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

The fermentation is preferably carried out by stirring, and the rotation speed of the stirring is 400-800 rpm, preferably 600 rpm.

In a preferred embodiment, the fermentation medium is lignocellulose hydrolyzate such as straw hydrolyzate.

The straw hydrolyzate is preferably formed after enzymatic hydrolysis and saccharification of crop straws.

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

In a preferred embodiment, the straw hydrolyzate is pretreated before the enzymatic saccharification, and the pretreatment includes screening, impurity removal, acid pretreatment and/or detoxification treatment. Pretreatment can improve the saccharification efficiency of crop straw. The detoxification treatment can reduce the content of toxic inhibitors such as acetic acid, furfural, 5-hydroxybenzaldehyde, furfural, hydroxymethylfurfural, 4-hydroxybenzaldehyde, and levulinic acid.

Another aspect of the present invention provides an expression cassette as described in the present invention, nucleic acid as described in the present invention, recombinant expression vector as described in the present invention, or genetically engineered bacteria as described in the present invention in the preparation of L-lysine Application in amino acids.

A method for preparing a genetically engineered bacterium, which obtains the genetically engineered bacterium by introducing the expression cassette as described in the present invention into the starting bacterium as defined in the present invention.

The preparation method preferably further includes: blocking the expression of lactate dehydrogenase in the genetically engineered bacteria, such as knocking out the ldh gene.

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

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

Although the starting strain in the embodiment of the present invention has selected the bacterial strain C.glutamicumCathS141 tolerant to straw hydrolyzate, it can be seen from Examples 5 and 6 that when the medium is a prepared glucose medium, the genetically engineered bacteria of the present invention The effect of producing L-lysine is also better than that of the starting strain, that is, tolerance to the inhibitor toxicity of the straw hydrolyzate is not a necessary condition for the starting strain.

Transaldolase gene of the present invention is also referred to as cgl1575 (the amino acid sequence of the transaldolase of its encoding can be the sequence corresponding to GenBank accession number BAB98968.1); Transketolase gene of the present invention is also referred to as It is cgl1574 (the amino acid sequence of the transketolase encoded by it can be the sequence corresponding to GenBank accession number BAB98967.1).

In a certain preferred embodiment, the deposit number of C. glutamicum CathS141 described in the present invention is CCTCC NO: M 20211495.

C. glutamicum B253 described in the present invention was purchased from Shanghai Institute of Industrial Microbiology.

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

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

The positive progress effect of the present invention is:

After the expression cassette provided by the present invention is introduced into the starting strain, the expression of transketolase and transaldolase is in an improved state compared with the starting strain, which helps to strengthen the pentose phosphate pathway, thereby effectively improving the utilization efficiency of xylose and mixed sugars, and then improving Production of L-lysine. When the straw hydrolyzate is used, the lysine yield of the genetically engineered bacteria of the present invention is significantly improved compared with that of the original bacteria. The genetically engineered bacteria provided by the invention can effectively utilize agricultural waste such as straws for fermentation, and has good application prospects.

Biological Material Deposit Information

The Corynebacterium glutamicum CathS141 of the present invention has been preserved in the China Center for Type Culture Collection (CCTCC) on November 29, 2021, the preservation address: Wuhan, China, Wuhan University, postcode 430072, and the preservation number is: CCTCC NO: M20211495 , the culture name is CathS141, and the taxonomic name is Corynebacterium glutamicum CathS141.

Detailed ways

The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.

One, the bacterial strain used in the present invention

Escherichia coli E.coli DH5α is used for the construction of expression plasmids and knockout plasmids. Corynebacterium glutamicum C. glutamicum CathS141 is a strain used to produce lysine. In this experiment, C. glutamicum CathS141 is mainly used as the starting strain use.

2. Reagents and media

Cellulase CTec 2.0 was used to hydrolyze cellulose and hemicellulose in lignocellulose and was purchased from Novozymes (China) Company, Beijing, China. According to the method in the NREL LAP-006 guideline, the filter paper enzyme activity of cellulase was 203.2 FPU/mL, the cellobiase activity was 4900.0 CBU/mL, and the protein concentration was 87.3 mg/mL according to the Bradford method. Restriction endonucleases were used to cleave plasmid or gene fragments to generate cohesive ends and were purchased from Thermo Scientific (Wilmington, DE, USA). DNA polymerase was used to amplify gene fragments, and DNA ligase was used to ligate enzyme-digested gene fragments and plasmid vectors, both of which were purchased from Takara (Otsu, Japan). The seamless cloning kit was used to connect gene fragments containing homologous fragments and plasmid vectors, and was purchased from Hanbio Technology Co., Ltd. (Nanjing, China). Plasmid Extraction Kit, PCR Product Purification and Recovery Kit and Gel Recovery Kit were all purchased from Shanghai Jierui Biotechnology Company (Shanghai, China). Other reagents were purchased from local suppliers.

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

The specific composition of the medium used for cultivating Corynebacterium glutamicum is as follows:

(1) Seed 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 glucose as a carbon source as appropriate.

Example 1: Obtaining the starting bacterium C. glutamicum CathS141

Collect soil samples from Wusu, Xinjiang, add each 1g soil sample to 10mL sterile water, mix vigorously for 1min, let it settle for a period of time, and then dilute the original samples to 10 ^-3 , 10 ^-4 , 10 ^-5 Spread on LB agar plates containing 100mg/L nystatin and culture at 30°C. Purified single colonies were obtained by streaking and culturing several times.

A bacterial strain capable of producing L-lysine was obtained through separation and purification, and the bacterial characteristics, physiological and biochemical characteristics, etc. of the bacterial strain were determined by referring to the "Common Bacterial System Identification Manual". The bacterial strain has the following morphological characteristics: the colony is moist, round, smooth, with neat edges, and light yellow in color; the DNA of the bacterial strain is extracted with a bacterial genome DNA extraction kit, and the DNA of the bacterial strain is used as a template. Primers 27F and 1492R were used for PCR amplification with 16S rDNA, and the 16S rDNA sequence of the strain was obtained after sequencing. The 16S rDNA sequence of the strain was compared in the GenBank database, and the strain was identified as Corynebacterium glutamicum.

Wheat straw is subjected to acid pretreatment, detoxification and enzymatic hydrolysis to obtain wheat straw hydrolyzate. The Corynebacterium glutamicum obtained from the above separation and purification is used as the starting strain, through the use of ultraviolet light, nitrosoguanidine, 5-fluorouracil, ARTP, etc. A single mutagenesis and compound mutagenesis were carried out to obtain a stable strain that could tolerate the toxic inhibitors in the straw hydrolyzate for normal growth and lysine production. The strain is named C. glutamicum CathS141, and it is now preserved in the China Center for Type Culture Collection, address: Wuhan, China, Wuhan University, zip code 430072, preservation number CCTCC NO:M 20211495, preservation date November 29, 2021.

Example 2: Integration of the xylAB expression cassette at the ldh site

First construct the integrated plasmid of xylAB (xylose isomerase and xylulokinase encoding gene cluster), the specific construction method is as follows: using the genome of C. glutamicum as a template, using Peftu-F (as shown in SEQ ID NO: 6) and Peftu-R (as shown in SEQ ID NO: 7) primers are amplified by PCR to obtain the Peftu promoter (as shown in SEQ ID NO: 2); with the E.coli BL21 genome as a template, using xylAB-F ( As shown in SEQ ID NO: 8) and xylAB-R (as shown in SEQ ID NO: 9) primers are amplified by PCR to obtain xylAB fragments (as shown in SEQ ID NO: 1); with Peftu and xylAB as templates , using Peftu-F and xylAB-R primers, the Peftu_xylAB fusion fragment (as shown in SEQ ID NO: 10) was obtained by overlap extension PCR. Using the genome of C. glutamicum as a template, ldh-up-F (as shown in SEQ ID NO: 11) and ldh-up-R (as shown in SEQ ID NO: 12) primers were used to amplify ldh by PCR -up fragment (as shown in SEQ ID NO:5); With the genome of C.glutamicum CathS141 as template, utilize ldh-down-F (as shown in SEQ ID NO:13) and ldh-down-R (as shown in SEQ ID NO Shown in: 14) primer is amplified by the method for PCR and obtains ldh-down fragment (as shown in SEQ ID NO:15); With ldh-up fragment, Peftu_xylAB and ldh-down fragment are templates, utilize ldh-up-F and The ldh-down-R primer was used to obtain the Δldh::xylAB fusion fragment by overlapping extension PCR, and it was treated with EcoRI and HindIII endonucleases, and then inserted into the pK18mob plasmid using T4 ligase (see http: //www.biovector.net/product/1089.html), the pK18-Δldh::xylAB plasmid was obtained. During this period, the successfully connected plasmids can be screened using a seed culture plate containing kanamycin resistance. Then, the integrated plasmid pK18-Δldh::xylAB was transferred into C. glutamicum CathS141 by electroporation, and then the strain with correct homologous recombination was screened out by PCR verification, and the recombinant Corynebacterium glutamicum was obtained, which was named cgl01.

Example 3: Construction of xylAB combined expression cassettes with cgl1574, cgl1575, cgl1574&1575 respectively

Referring to Example 2, using the genome of C. glutamicum CathS141 as a template, primers cgl1574-F (shown in SEQ ID NO: 16) and cgl1574-R (shown in SEQ ID NO: 17), and cgl1575-F ( As shown in SEQ ID NO: 18) and cgl1575-R (as shown in SEQ ID NO: 19) were amplified respectively to obtain cgl1574 and cgl1575 fragments; the cgl1574 & 1575 expression cassette (as shown in SEQ ID NO: 20 Shown); In the reference example 2, the fusion PCR method further obtains xylAB respectively with cgl1574, cgl1575, cgl1574 & 1575 combined expression cassette AB-1574 (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), the primer used during this period is P-AB-R (as shown in SEQ ID NO: 24).

Example 4: Integration of AB-1574, AB-1575 and AB-1574&1575 expression cassettes at the ldh site

Reference Example 2 Insert AB-1574, AB-1575 and AB-1574&1575 into the pK18mob plasmid respectively to obtain pK18-Δldh::AB-1574, pK18-Δldh::AB-1575 and pK18-Δldh::AB-1574&1575 . Referring to the electroporation method in Example 2, the integrated plasmids pK18-Δldh::AB-1574, pK18-Δldh::AB-1575 and pK18-Δldh::AB-1574&1575 were respectively transferred into C. glutamicum CathS141, the correct genetic engineering strain They were named cgl02, cgl03 and cgl04, respectively.

Embodiment 5: the xylose fermentation of different genetically engineered bacteria

The fermentation performance of cgl01, cgl02, cgl03 and cgl04 and control strains were evaluated by shake flask system fermentation. The fermentation process is: the fermentation medium includes 1g/L potassium dihydrogen phosphate, 3g/L urea, 0.6g/L magnesium sulfate, 20g/L corn steep liquor, and 40g/L xylose is added as a carbon source, and 25μg/mL of Kanamycin. The fermentation was carried out in a 250mL shake flask, the culture conditions were 30°C, 200rpm, and the fermentation time was 96 hours. The fermentation results are shown in Table 1, it can be seen that cgl04 has the strongest ability to metabolize xylose.

Table 1 Comparison of xylose fermentation of different genetically engineered bacteria

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

Embodiment 6: the mixed sugar fermentation of different genetically engineered bacteria

Reference Example 5 carried out fermentation evaluation in a mixed sugar system, the difference being that the carbon source used here was 25g/L xylose and 25g/L glucose. The fermentation results are shown in Table 2. The enhancement of transketolase and transaldolase can increase the conversion rate of lysine, and there are 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 rate (%) 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

Embodiment 7: Fermentation of different genetically engineered bacteria in lignocellulose hydrolyzate

After the wheat straw is crushed, it is then sieved through a 10 mm diameter sieve, and the sieved straw is washed with water to remove impurities such as soil, stones and metals, dried in an oven at 105°C until it reaches a constant weight, and stored Reserve in an airtight plastic bag. After acid pretreatment, biological detoxification and enzymatic saccharification, the wheat straw hydrolyzate was separated, which contained 95.4g/L glucose and 34.7g/L xylose. 20g/L ammonium sulfate and 5g/L methionine and threonine were added to the hydrolyzate, and the modified xylose-utilizing strain cgl04 and the original strain were cultured in the wheat straw hydrolyzate for fermentation comparison, and the fermentation temperature was 30 ℃, the pH is controlled at 7.0 with ammonia water, the ventilation volume is 1.4vvm, and the rotation speed is 600rpm.

Table 3 Comparison of hydrolyzate fermentation of different genetically engineered bacteria

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

The results are shown in Table 3. When the wheat straw hydrolyzate was used as the medium, the lysine production of the genetically engineered strain cgl04 was significantly higher than that of the strains CathS141 and cgl01. In addition, when Corynebacterium glutamicum B253 was used as the starting bacterium, the strain was named B253-104 according to the same transformation method as the strain cgl04, and the strain was named B253-101 according to the same transformation method as the strain cgl01. Under the same fermentation conditions as above, 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. Accordingly, it can be seen that the recombinant strain obtained in the present invention has strong inhibitor tolerance and high-efficiency lysine production capacity, and has a good application prospect.

The operation examples of the technical solutions of the present invention have been specifically described above, which are not considered as limitations on the application of the present invention. All equivalent replacements of operating conditions are within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shanghai Kaisai Biotechnology Co., Ltd.

CIBT America Inc.

<120> Expression cassette for preparing L-lysine, genetically engineered bacteria and application thereof

<130> P21018723C

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<170> PatentIn version 3.5

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gacgttgcag caatcgaagc tgcagtggct gaggctaaga aggaccacaa 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

accacccagc 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 aattgcgat 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 tcgattaca 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 ccattttat gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaagaaga 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

gtagatctt 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 catttatacga 2340

aggcagcgaa attackggtg 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

ccttacggccg 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

gtccacagggg 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 attacktccg gcaatctcag ccaggttat 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 aacaccatgcc 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 ccattttat gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaagaaga 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

gtagatctt 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 catttatacga 2340

aggcagcgaa attackggtg 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 aattttgta atgcgctaga 3420

tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa gcaaaaccaa ttcgtggctg 3480

cgaaagtcgt agccaccacg aagtccagga ggacatacaa tggaccacaa ggctgtagac 3540

actgttcgtg tcctcgctgc agacgctgta gaaaactgtg gctccggcca cccaggcacc 3600

gcaatgagcc tggctcccct tgcatacacc ttgtaccagc gggttatgaa cgtagatcca 3660

caggaccacca 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 ccattttat gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaagaaga 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

gtagatctt 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 catttatacga 2340

aggcagcgaa attackggtg 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 ccattttat gcttccacga tgtggatgtt tcccctgagg 660

gcgcgtcgtt aaaagagtac atcaataatt ttgcgcaaat ggttgatgtc ctggcaggca 720

agcaagaagaaga 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

gtagatctt 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 catttatacga 2340

aggcagcgaa attackggtg 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 aattttgta atgcgctaga 3420

tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa gcaaaaccaa ttcgtggctg 3480

cgaaagtcgt agccaccacg aagtccagga ggacatacaa tggaccacaa ggctgtagac 3540

actgttcgtg tcctcgctgc agacgctgta gaaaactgtg gctccggcca cccaggcacc 3600

gcaatgagcc tggctcccct tgcatacacc ttgtaccagc gggttatgaa cgtagatcca 3660

caggaccacca 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 ttgaccacg 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, characterized in that, the expression cassette comprises a promoter and a xylAB gene, and also comprises a transaldolase gene and/or a transketolase gene. 2. expression cassette as claimed in claim 1, is characterized in that, the sequence of described xylAB gene is as shown in SEQ ID NO:1; And/or, the sequence of the transaldolase gene is shown in SEQ ID NO:4; And/or, the sequence of the transketolase gene is shown in SEQ ID NO:3. 3. The expression cassette according to 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, characterized in that the nucleic acid comprises the expression cassette according to any one of claims 1-3. 5. A recombinant expression vector, characterized in that, the recombinant expression vector comprises the expression cassette as claimed in any one of claims 1 to 3, or comprises the nucleic acid as claimed in 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 transferred to the expression cassette as claimed in any one of claims 1 to 3, or the nucleic acid as claimed in claim 4, in the starting bacterium, or The recombinant expression vector as claimed in claim 5; Preferably, the starting bacterium is Corynebacterium glutamicum, such as C. glutamicum B253 or C. glutamicum CathS141 with a deposit number of CCTCC NO: M 20211495. 7. The genetically engineered bacterium according to claim 6, wherein the expression cassette is integrated into the genome of the starting bacterium by homologous recombination, or exists in the starting bacterium in a non-integrated form. 8. The genetically engineered bacterium as claimed in 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 site of the ldh gene, more preferably, the accession number of the ldh gene in Genbank is AJE68497.1. 9. A method for preparing L-lysine, characterized in that the method comprises fermenting the genetically engineered bacterium according to any one of claims 6 to 8 in a fermentation medium; Preferably, the fermentation medium contains no less than 25g/L glucose and/or 25g/L xylose; More preferably, the fermentation meets one or more of the following conditions: The fermentation temperature is 28-32°C, preferably 30°C; The ventilation rate of the fermentation is 1.0～1.7vvm, preferably 1.4vvm; The pH of the fermentation is 6-8, preferably 7; The fermentation is carried out by stirring, and the rotation speed of the stirring is 400-800 rpm, preferably 600 rpm. 10. The method according to claim 9, wherein the fermentation medium is lignocellulose hydrolyzate, preferably straw hydrolyzate; preferably, the straw hydrolyzate is formed after enzymatic hydrolysis and saccharification of crop straw; More preferably, 15-25g/L ammonium sulfate, 2-8g/L methionine and 2-8g/L threonine are added to the straw hydrolyzate, and/or, before the enzymatic hydrolysis and saccharification The straw hydrolyzate is pretreated, and the pretreatment includes screening, impurity removal, acid pretreatment and/or detoxification treatment. 11. The expression cassette as claimed in any one of claims 1 to 3, the nucleic acid as claimed in claim 4, the recombinant expression vector as claimed in claim 5 or the gene as described in any one of claims 6 to 8 Application of engineering bacteria in the preparation of L-lysine. 12. A method for preparing a genetically engineered bacterium, characterized in that, the expression cassette according to any one of claims 1 to 3 is introduced into the starting bacterium as defined in any one of claims 6 to 8 to obtain the Genetically engineered bacteria; Preferably, the preparation method further includes: blocking the expression of lactate dehydrogenase in the genetically engineered bacteria, preferably knocking out the ldh gene; More preferably, the expression cassette is integrated into the site of the ldh gene; and/or, the accession number of the ldh gene in Genbank is AJE68497.1.