CN107828710B - Mark-free multi-site integrated expression glutaminase strain and construction method thereof - Google Patents
Mark-free multi-site integrated expression glutaminase strain and construction method thereof Download PDFInfo
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Abstract
The invention discloses a unmarked multi-site integrated expression glutaminase strain and a construction method thereof, belonging to the field of genetic engineering. The invention is characterized in that glutaminase genes are integrally expressed at 7 sites of 16S rDNA, nprB, nprE, aprE, spo0A, epr and bpr, and finally a multi-site non-resistance integrally expressed glutaminase strain BSM7 is obtained. After the BSM1 and the BSM7 obtained by the invention are fermented in a shake flask for 24 hours, the enzyme activities are respectively 40.5U/mL and 85.6U/mL, and the mutant enzyme activity is improved by 111.4%. The BSM7 strain is fed-batch fermented, and the highest enzyme activity is reached in about 32h, and is 375.6U/mL. Genetic stability analysis shows that the genetic stability of BSM7 is far greater than the stability of plasmid expression of the modified gene. The invention improves the application potential of glutaminase in food industry.
Description
Technical Field
The invention relates to a unmarked multi-site integrated expression glutaminase strain and a construction method thereof, belonging to the technical field of genetic engineering.
Background
Glutaminase (EC 3.5.1.2) catalyzes the deamidation hydrolysis of glutamine to form glutamic acid and ammonia. In recent years, the application of glutaminase in the fields of food and medicine has been rapidly researched and developed, and reports on the application are more and more. By studying how to reduce glutaminase content or activity in cancer cells, glutamine metabolism in cancer cells is attenuated to inhibit tumor cell growth. The application research of glutaminase in the food industry is also very wide, glutaminase can catalyze glutamine to form glutamic acid, and the glutamic acid has an important function in the aspects of improving the taste and the flavor of food. L-asparaginase is widely available and is found to contain glutaminase in both mammals and microorganisms.
Disclosure of Invention
The invention firstly provides a bacillus subtilis preference glutaminase nucleotide sequence which is a sequence shown in SEQ ID NO. 1.
The invention also provides a genetically engineered bacterium for expressing the glutaminase.
The preparation method of the genetic engineering bacteria comprises the steps of extending 4 parts including homology arms at two ends of 16SrDNA, lox71-zeo-lox66 and Hpa II-Mglu into 1 fragment by an overlapping extension method on the basis of a sequence shown by SEQ ID NO.1 to obtain an integrated fragment, transforming the integrated fragment into host bacteria to obtain B.subtilis genetic engineering bacteria, transforming plasmids containing CRE recombinase into the engineering bacteria, and removing resistance markers to obtain unmarked 16S rDNA site integrated expression glutaminase bacillus subtilis engineering bacteria.
In one embodiment of the present invention, the preparation method specifically comprises (the sequence is shown in table 1):
(1) taking a B.subtilis168 genome as a template, P3/P4 and P9/P10 as primers, obtaining two sections of homologous arms of 16S rDNA by PCR, taking nucleotide sequences shown by pMA5-Mglu and P7Z6 as templates, and P5/P6 and P7/P8 as primers, and obtaining Hpa II-Mglu and lox71-zeo-lox66 by PCR; then, the 4 fragments were subjected to fusion PCR using PrimeSTAR GXLDDNA polymerase to obtain integrated fragments.
(2) Transforming the integrated fragment obtained in the last step into B.subtilis168 to obtain a resistant recombinant bacillus subtilis engineering strain, transforming a plasmid pDG148 containing CRE recombinase into the engineering strain, and then culturing at 51 ℃ for 24h to obtain a non-resistant integrated expression glutaminase strain which is named as BSM 1.
(3) Taking nprB, nprE, aprE, spo0A, epr and bpr genes as integration expression sites, taking P11-P18, P19-P26, P27-P34, P35-P42, P43-P50 and P51-P58 as primers, repeating the steps (1) and (2), and sequentially obtaining 2, 3, 4, 5, 6 and 7 site integration expression glutaminase strain BSM2-BSM 7.
According to the invention, on the basis of a bacillus subtilis preference modified glutaminase nucleic acid sequence, glutaminase is expressed by multi-site integration, and finally, a 7-site integration expression glutaminase strain BSM7 is obtained, and compared with BSM1, the enzyme activity is improved by 120%. Compared with B.subtilis 168/pMA5-Mglu, the BSM7 greatly improves the enzyme activity stability under the condition of no resistance. The invention shows that the integrated expression can effectively improve the enzyme activity of the glutaminase by increasing the integrated expression sites, improve the stability of the glutaminase and improve the industrial application potential of the glutaminase. The invention can be used for improving the mouthfeel and flavor of food and inhibiting the growth of cancer cells.
Detailed Description
EXAMPLE 1 construction of glutaminase-containing recombinant vector
(1) Acquisition of glutaminase Gene: from NCBI website (B) (U.S. Luteus K-3 salt tolerant glutaminase Gene sequences)http://www.ncbi.nlm.nih.gov/nuccore/DQ019448.1) The sequence was queried and optimized for Bacillus subtilis preference by Sangon Biotechnology Inc. (Shanghai, China) and synthesized to obtain E.coli/pUC-Mglu.
(2) Taking P1/P2 as a primer and pUC-Mglu as a template, obtaining a glutaminase gene sequence (SEQ ID NO.1) by PCR, carrying out double digestion on the gene and pMA5 by BamHI and MluI respectively, purifying, and carrying out overnight ligation by T4DNA ligase at 16 ℃. Ligation products were chemically transformed into JM109 competent cells. The transformation liquid is coated on an LB plate containing kanamycin (50mg/L), plasmids are extracted, and the constructed recombinant plasmids are verified by double enzyme digestion and named as pMA 5-Mglu. The sequencing work is completed by Shanghai worker.
Table 1: primer sequence List
Example 2 construction of plasmid-expressed glutaminase Bacillus subtilis engineered bacteria
The recombinant plasmid pMA5-Mglu obtained in example 1 was chemically transformed into B.subtilis168 competent cells by the following specific method:
(1) the solutions required for the conversion experiments were as follows (g/L):
Sp-A：(NH4)2SO44，K2HPO428, sodium citrate 12 Sp-B: MgSO (MgSO)4·7H2O 0.4
100 × CAYE, Casamino acid 20, yeast powder 100 SpI culture medium Sp-A49%, Sp-B49%, 50% glucose 2%, 100 × CAYE 2% Sp II culture medium Sp I culture medium 98%, 50mmol/LCaCl21％，250mmol/L MgCl21 percent. Sterilizing at 115 deg.C by wet heat.
(2) Inoculating a single colony of B.subtilis168 into 10mL LB culture medium (50mL centrifuge tube), and culturing at 37 ℃ and 180r/min overnight;
(3) putting 200 mu L of culture solution into 10mL of SpI culture medium, culturing at 37 ℃ and 180r/min until logarithmic phase (OD600 value is about 1) lasts for about 5-6 h;
(4) adding 1mL of culture solution into 10mL of Sp II culture medium, culturing at 37 ℃ and 180r/min for 90min, taking out, adding 120 mu L of 10mmol/L EGTA, continuously culturing at 37 ℃ and 180r/min for 10min, subpackaging into 500 mu L of each tube, adding 5 mu L of recombinant plasmid pMA5-Mglu, mixing uniformly, culturing at 37 ℃ and 180r/min for 90min, and coating a bacterial solution on a resistant plate. Culturing at 37 ℃ for 12h, and picking positive transformants for verification. The recombinant strain pMA5-Mglu/B.subtilis 168 is obtained.
Example 316 construction of rDNA site integration expression fragment
(1) Taking B.subtilis168 genome as a template, P3/P4 and P9/P10 as primers, obtaining 16S rDNA two sections of homologous arms (SEQ ID NO.60 and SEQ ID NO.61) by PCR, taking nucleotide sequences shown by pMA5-Mglu and P7Z6 as templates, and taking P3/P4 and P5/P6 as primers, obtaining Hpa II-Mglu (SEQ ID NO.63) and lox71-zeo-lox66(SEQ ID NO.62) by PCR, wherein amplification conditions comprise pre-denaturation at 98 ℃, pre-denaturation at 3min, one cycle, pre-denaturation at 98 ℃, annealing at 10S and 61 ℃, extension at 15S and 72 ℃, extension at 2min and 28 cycles, final extension at 72 ℃, 10min, PCR amplification system of the template 1 mu L, starboard 1 mu L, upstream and downstream primers respectively 1 mu L, dNTP Mix4 mu L, 5 × mu L, amplification system of Buffer 10 mu L, recovery of DNA products by double gel electrophoresis, and recovery of PCR products by using a PCR kit of distilled water for purification and recovery of PCR products.
(2) Then, the 4 fragments are used as templates in equimolar amount, and PCR is performed to obtain integrated fragments initially without changing other systems such as step (1) under the condition of not adding primers. The amplification conditions were: pre-denaturation at 98 ℃, 3min, one cycle; pre-denaturation at 98 ℃, annealing at 61 ℃, extension at 72 ℃ for 5s, 4min and 13 cycles; final extension at 72 ℃ for 20 min.
(3) And (3) then, taking the integrated fragment obtained primarily in the step (2) as a template, taking P3/P10 as a primer, obtaining the integrated fragment by PCR, purifying and recovering the PCR product by adopting a gel recovery kit, and carrying out electrophoresis check on the concentration of the recovered product under the amplification conditions of pre-denaturation at 98 ℃, 3min, one cycle, pre-denaturation at 98 ℃, annealing at 10s and 61 ℃, extension at 15s and 72 ℃, 4min and 28 cycles, and final extension at 72 ℃ for 20min, wherein in the PCR amplification system, the template is 2 mu L, the upstream primer and the downstream primer are 1 mu L respectively, the TPdNmix 4 mu L, the 5 × PrimeSTAR GXL Buffer is 10 mu L, the sterilized double distilled water is 31 mu L, and the PrimeSTAR GXL DNA polymerase is1 mu L, purifying and recovering the PCR product by adopting the gel recovery kit, and carrying out electrophoresis check on the concentration of the recovered product.
EXAMPLE 4 construction of a Strain that expresses glutaminase at multiple sites in an Integrated manner
(1) Methods for making competent cells of subtilis168 are described in example 2
(2) Adding 25 μ L recombinant fragment and B.subtilis168 competent cell, mixing, culturing at 37 deg.C and 180r/min for 90min, and spreading the bacterial liquid on resistant plate. Culturing at 37 ℃ for 12h, and picking positive transformants for verification. Obtaining the resistant integrated expression strain.
(3) Preparing competent cells from the transformant in the step (2), transforming 5 mu L of pDG148 plasmid containing CRE enzyme into the competent cells, mixing uniformly, culturing at 37 ℃ and 180r/min for 90min, and taking bacterial liquid to coat a resistant plate. Culturing at 37 ℃ for 12h, and picking positive transformants for verification. Subsequently, the strain was cultured in an incubator at 51 ℃ for 24 hours to obtain a glutaminase integration expression strain without a resistance gene, which was named BSM 1.
(4) P11-P18, P19-P26, P27-P34, P35-P42, P43-P50 and P51-P58 are used as primers, and nprB, nprE, aprE, spo0A, epr and bpr genes are sequentially constructed as integration expression site non-marker integration expression strains by referring to a 16SrDNA site non-marker integration expression strain construction method, and are respectively named as BSM2-BSM 7.
The sequences of homologous arms at two ends of nprB are shown as SEQ ID NO.64 and SEQ ID NO. 65; hpa II-Mglu is shown as SEQ ID NO.63, and lox71-zeo-lox66 is shown as SEQ ID NO. 62;
the sequences of homologous arms at two ends of nprE are shown as SEQ ID NO.66 and SEQ ID NO. 67; hpa II-Mglu is shown as SEQ ID NO.63, and lox71-zeo-lox66 is shown as SEQ ID NO. 62;
the sequences of homologous arms at two ends of aprE are shown as SEQ ID NO.68 and SEQ ID NO. 69; hpa II-Mglu is shown as SEQ ID NO.76, and lox71-zeo-lox66 is shown as SEQ ID NO. 62;
the sequences of homologous arms at two ends of spo0A are shown as SEQ ID NO.70 and SEQ ID NO. 71; hpa II-Mglu is shown as SEQ ID NO.76, and lox71-zeo-lox66 is shown as SEQ ID NO. 62;
the sequences of homologous arms at two ends of the epr are shown as SEQ ID NO.72 and SEQ ID NO. 73; hpa II-Mglu is shown as SEQ ID NO.76, and lox71-zeo-lox66 is shown as SEQ ID NO. 62;
the sequences of homologous arms at two ends of bpr are shown as SEQ ID NO.74 and SEQ ID NO. 75; hpa II-Mglu is shown as SEQ ID NO.76, and lox71-zeo-lox66 is shown as SEQ ID NO. 62.
Example 5 recombinant bacteria BSM1-BSM7 glutaminase high-efficiency expression and enzyme activity determination.
(1) The recombinant bacteria BSM1-BSM7 constructed in the embodiment 4 are respectively inoculated in l0mL LB culture medium containing kanamycin, the shaking culture is carried out at 37 ℃ for overnight, the recombinant bacteria BSM1-BSM7 are transferred to a Bacillus subtilis fermentation culture medium according to the inoculum size of 4% the next day, the culture is carried out for 24 hours at 24 ℃, the fermentation liquid is taken and centrifuged at 4 ℃ and 10000r/min for l0min, the supernatant is extracellular crude enzyme liquid, and the cell crushing supernatant is intracellular crude enzyme liquid and is used for measuring the enzyme activity.
(2) Bacillus subtilis fermentation medium: angel yeast powder 40g/L, glucose 25g/L, K2HPO43H2O1.875g/L，KH2PO41.125g/L，CaCl22 g/L. Adjusting the pH value to 6.8-7.0.
(3) The enzyme activity is defined as that the enzyme quantity required for catalyzing the conversion of glutamine into 1 mu mol of glutamic acid per minute is one enzyme activity unit under the reaction condition of 37 ℃.
(4) The method for measuring the enzyme activity of the glutaminase comprises the following steps: and (3) directly measuring the content of glutamic acid in the enzyme product by adopting an SBA biosensing analyzer. Glutaminase enzyme activity measurement reaction system (total system 1 mL): 880. mu.L of 50mmol L-1Tris-HCl buffer and 50mmol L-1Preheating L-glutamine mixed solution in a water bath kettle at 37 ℃ for 5min, adding 20 mu L of enzyme solution, reacting in a constant-temperature water bath at 37 ℃ for 5min, rapidly adding 100 mu L of 15% trichloroacetic acid, shaking up and down, and mixing uniformly to terminate the reaction. Placing the enzyme reaction solution into a centrifuge at 10000r min-1Centrifuging for 10min under the condition of (1), removing protein, collecting supernatant, and diluting the product concentration of the reaction solution to 0.3-1.0g L-1Then, 25. mu.L of the reaction solution was subjected to glutamic acid assay.
The result shows that the enzyme activity of the glutaminase expressed by the recombinant strain BSM7 is 85.6U/mL, and is improved by 111.4 percent compared with the enzyme activity of the L-asparaginase of the strain BSM1 (40.5U/mL).
Example 6 genetic stability analysis of recombinant bacteria
BSM7 and B.subtilis 168/pMA5-Mglu recombinant bacteria colonies are respectively picked and inoculated into a non-resistant LB liquid culture medium, and are cultured at 37 ℃ to be used as seed liquid. The next day, transferring the strain to a non-resistant fermentation medium according to the inoculum size of 4%, culturing at 24 ℃ for 12h, sampling and measuring enzyme activity respectively, and simultaneously continuing transferring the strain to a fresh non-resistant fermentation medium according to the inoculum size of 4%. And repeating the steps once every 24 hours, sampling and measuring the enzyme activity simultaneously, and measuring for 120 hours in total. The result shows that the enzyme activity is basically unchanged after the BSM7120h is measured, but the enzyme activity is basically not changed after the B.subtilis 168/pMA5-Mglu is cultured for 96 hours, that is, the genetic stability of the BSM7 recombinant strain is far better than that of the B.subtilis 168/pMA5-Mglu, and a reference is provided for industrial production of food-grade glutaminase. Example 7 fed-batch fermentation of recombinant bacteria BSM7
The BSM7 strain was streaked on LB solid plates and grown at 37 ℃ for 12 h. Single colonies were picked up in 10mL LB liquid medium and cultured at 37 ℃ for 12 h. The cells were transferred to 100mL of LB liquid medium and cultured at 37 ℃ for 12 hours to obtain a seed solution. The seed solution was transferred to a 5L fermentor containing 2L fermentation medium. A feed medium (glucose 1000 g/L; Angel year extract700g/L) was fed to the fermenter while maintaining the glucose concentration at 10-20 g/L. The fed-batch fermentation was carried out at 25 ℃ and pH7.0, at 600 rpm. The pH was adjusted with 50% ammonia and 50% sulfuric acid. The result shows that the BSM7 strain reaches the highest enzyme activity at about 32h, and the enzyme activity is 375.6U/mL.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> a strain for unmarked multi-site integrated expression of glutaminase and a construction method thereof
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<211>30
<212>DNA
<213> Artificial sequence
<400>28
ccgatattgg ttaaacagcg gcgcaatggc 30
<210>29
<211>58
<212>DNA
<213> Artificial sequence
<400>29
ctcaaaaaat caccaccttt aaacccttgc caatgaacct ttccgccttt ttcagaaa 58
<210>30
<211>58
<212>DNA
<213> Artificial sequence
<400>30
ggatgttatt tctgaaaaag gcggaaaggt tcattggcaa gggtttaaag gtggagat 58
<210>31
<211>58
<212>DNA
<213> Artificial sequence
<400>31
cccgggtacc gagctcgaat tcgtaatcat ggtagccggc gaacgtggcg agaaagga 58
<210>32
<211>58
<212>DNA
<213> Artificial sequence
<400>32
tcttcccttc ctttctcgcc acgttcgccg gctaccatga ttacgaattc gagctcgg 58
<210>33
<211>58
<212>DNA
<213> Artificial sequence
<400>33
acgttccgtt ataagcgccg taagtgcctc cagattctac cgttcgtata atgtatgc 58
<210>34
<211>58
<212>DNA
<213> Artificial sequence
<400>34
ttcgtatagc atacattata cgaacggtag aatctggagg cacttacggc gcttataa 58
<210>35
<211>30
<212>DNA
<213> Artificial sequence
<400>35
tgtcatgaag cacgtttaca tcggtctggc 30
<210>36
<211>30
<212>DNA
<213> Artificial sequence
<400>36
gcgacaaaag gaatggtgtt gaaaattacc 30
<210>37
<211>58
<212>DNA
<213> Artificial sequence
<400>37
ctcaaaaaat caccaccttt aaacccttgc caaaccgcaa gtccgtctag atgcggca 58
<210>38
<211>58
<212>DNA
<213> Artificial sequence
<400>38
tattattatg ccgcatctag acggacttgc ggtttggcaa gggtttaaag gtggagat 58
<210>39
<211>58
<212>DNA
<213> Artificial sequence
<400>39
cccgggtacc gagctcgaat tcgtaatcat ggtagccggc gaacgtggcg agaaagga 58
<210>40
<211>58
<212>DNA
<213> Artificial sequence
<400>40
tcttcccttc ctttctcgcc acgttcgccg gctaccatga ttacgaattc gagctcgg 58
<210>41
<211>58
<212>DNA
<213> Artificial sequence
<400>41
ctacacggct tgcggttgtg ttaaattttt tggattctac cgttcgtata atgtatgc 58
<210>42
<211>58
<212>DNA
<213> Artificial sequence
<400>42
ttcgtatagc atacattata cgaacggtag aatccaaaaa atttaacaca accgcaag 58
<210>43
<211>30
<212>DNA
<213> Artificial sequence
<400>43
ggctgggtgc cttataaaga ttaatgtgca 30
<210>44
<211>30
<212>DNA
<213> Artificial sequence
<400>44
cggcaatttt cccggcgaca ggcattattt 30
<210>45
<211>58
<212>DNA
<213> Artificial sequence
<400>45
ctcaaaaaat caccaccttt aaacccttgc caattcctgt cagtcctgcc ttccaagc 58
<210>46
<211>58
<212>DNA
<213> Artificial sequence
<400>46
tgaaacaggc ttggaaggca ggactgacag gaattggcaa gggtttaaag gtggagat 58
<210>47
<211>58
<212>DNA
<213> Artificial sequence
<400>47
cccgggtacc gagctcgaat tcgtaatcat ggtagccggc gaacgtggcg agaaagga 58
<210>48
<211>58
<212>DNA
<213> Artificial sequence
<400>48
tcttcccttc ctttctcgcc acgttcgccg gctaccatga ttacgaattc gagctcgg 58
<210>49
<211>58
<212>DNA
<213> Artificial sequence
<400>49
gggacgtttt ttctgaactt gcaggcagct tgcattctac cgttcgtataatgtatgc 58
<210>50
<211>58
<212>DNA
<213> Artificial sequence
<400>50
ttcgtatagc atacattata cgaacggtag aatgcaagct gcctgcaagt tcagaaaa 58
<210>51
<211>30
<212>DNA
<213> Artificial sequence
<400>51
gagcatgaga atagggggtt gtccaaggcg 30
<210>52
<211>30
<212>DNA
<213> Artificial sequence
<400>52
gaggaaaaaa acgaaaaaca gactcatcag 30
<210>53
<211>58
<212>DNA
<213> Artificial sequence
<400>53
ctcaaaaaat caccaccttt aaacccttgc caaaccccgg tatcaatgga cgcaacaa 58
<210>54
<211>58
<212>DNA
<213> Artificial sequence
<400>54
tggcacggtt gttgcgtcca ttgataccgg ggtttggcaa gggtttaaag gtggagat 58
<210>55
<211>58
<212>DNA
<213> Artificial sequence
<400>55
cccgggtacc gagctcgaat tcgtaatcat ggtagccggc gaacgtggcg agaaagga 58
<210>56
<211>58
<212>DNA
<213> Artificial sequence
<400>56
tcttcccttc ctttctcgcc acgttcgccg gctaccatga ttacgaattc gagctcgg 58
<210>57
<211>58
<212>DNA
<213> Artificial sequence
<400>57
ttgttgagta tgatgcctgc atggaacgtg ccaattctac cgttcgtata atgtatgc 58
<210>58
<211>58
<212>DNA
<213> Artificial sequence
<400>58
ttcgtatagc atacattata cgaacggtag aattggcacg ttccatgcag gcatcata 58
<210>59
<211>30
<212>DNA
<213> Artificial sequence
<400>59
aattttctgt gttcatatta agttttccat 30
<210>60
<211>654
<212>DNA
<213> Artificial sequence
<400>60
gctggcggcg tgcctaatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg 60
ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120
ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcaaaca taaaaggtgg 180
cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240
caccaaggcg acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420
agaacaagtg ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540
gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600
gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tcca 654
<210>61
<211>842
<212>DNA
<213> Artificial sequence
<400>61
cgtgtagcgg tgaaatgcgt agagatgtgg aggaacacca gtggcgaagg cgactctctg 60
gtctgtaact gacgctgagg agcgaaagcg tggggagcga acaggattag ataccctggt 120
agtccacgcc gtaaacgatg agtgctaagt gttagggggt ttccgcccct tagtgctgca 180
gctaacgcat taagcactcc gcctggggag tacggtcgca agactgaaac tcaaaggaat 240
tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc 300
ttaccaggtc ttgacatcct ctgacaatcc tagagatagg acgtcccctt cgggggcaga 360
gtgacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 420
aacgagcgca acccttgatc ttagttgcca gcattcagtt gggcactcta aggtgactgc 480
cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg 540
gctacacacg tgctacaatg gacagaacaa agggcagcga aaccgcgagg ttaagccaat 600
cccacaaatc tgttctcagt tcggatcgca gtctgcaact cgactgcgtg aagctggaat 660
cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg 720
cccgtcacac cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt ttaggagcca 780
gccgccgaag gtgggacaga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag 840
gt 842
<210>62
<211>547
<212>DNA
<213> Artificial sequence
<400>62
accatgatta cgaattcgag ctcggtaccc ggggatcctc tagagattct accgttcgta 60
tagcatacat tatacgaagt tatcttgata tggcttttta tatgtgttac tctacataca 120
gaaaggagga actaaatatg gccaagttga ccagtgccgt tccggtgctc accgcgcgcg 180
acgtcgccgg agcggtcgag ttctggaccg accggctcgg gttctcccgg gacttcgtgg 240
aggacgactt cgccggtgtg gtccgggacg acgtgaccct gttcatcagc gcggtccagg 300
accaggtggt gccggacaac accctggcct gggtgtgggt gcgcggcctg gacgagctgt 360
acgccgagtg gtcggaggtc gtgtccacga acttccggga cgcctccggg ccggccatga 420
ccgagatcgg cgagcagccg tgggggcggg agttcgccct gcgcgacccg gccggcaact 480
gcgtgcactt cgtggccgag gagcaggact gaataacttc gtatagcata cattatacga 540
gtagaat 547
<210>63
<211>1810
<212>DNA
<213> Artificial sequence
<400>63
taagctagac aaaacggaca aaataaaaat tggcaagggt ttaaaggtgg agattttttg 60
agtgatcttc tcaaaaaata ctacctgtcc cttgctgatt tttaaacgag cacgagagca 120
aaacccccct ttgctgaggt ggcagagggc aggttttttt gtttcttttt tctcgtaaaa 180
aaaagaaagg tcttaaaggt tttatggttt tggtcggcac tgccgacagc ctcgcagagc 240
acacacttta tgaatataaa gtatagtgtg ttatacttta cttggaagtg gttgccggaa 300
agagcgaaaa tgcctcacat ttgtgccacc taaaaaggag cgatttacat atgagttatg 360
cagtttgtag aatgcaaaaa gtgaaatcag ggggaatgcg tcaccctatc ccagactacc 420
tggccagcct ggtaaccgag ctgggtgcag taaaccctgg cgaaaccgct cagtacatcc 480
cggtgctggc agaggcagat ccagaccgtt tcggtatcgc tctggctacc ccgactggtc 540
gtctgcattg cgcaggtgac gctgatgtgg agttcaccat tcagtccgcg tccaaaccgt 600
tcacctacgc ggctgcgctg gtcgaccgtg gtttcgcagc tgtggaccgt caggtaggtc 660
tgaacccgag cggtgaggct ttcaacgagc tgagcctgga ggcagaaagc caccgtccgg 720
acaacgcaat gatcaacgcg ggtgcactgg ctgtacacca gctgctggtc ggtccggaag 780
catctcgtaa ggaacgtctg gaccgtgcag tggaaatcat gtccctgctg gccggtcgtc 840
gtctgtccgt ggattgggaa acgtacgaat ccgaaatggc ggtcagcgac cgcaacctgt 900
ccctggcgca catgctgcgt agctatggcg tgctgcagga ctccgcagaa gaaatcgtgg 960
ccggctacgt ggcacagtgc gcagtcctgg tcactgtcaa agacctggcg gtgatgggcg 1020
catgtctggc aaccggtggt atccacccga tgacgggtga acgtatgctg ccgtctatcg 1080
tggcgcgtcg tgtggtgtct gttatgacct cctctggcat gtatgacgcg gccggccagt 1140
ggctggctga tgtaggcatc ccggctaaat ctggtgttgc gggcggtgtt ctgggtgctc 1200
tgccgggtcg tgttggtatc ggtgttttca gcccgcgcct ggatgaagtt ggcaactctg 1260
cgcgtggcgt tctggcttgt cgtcgcctgt ctgaagactt ccgcctgcat ctgatggacg 1320
gcgactctct gggtggtacc gctgttcgtt ttgttgaacg cgaaggcgac cgcgtttttc 1380
tgcacctgca gggcgttatc cgctttggcg gcgcggaagc ggttctggac gctctgaccg 1440
atctgcgtac gggtgctgag aaaccgggta ctggctggga tgctgctgtt tatccgcgct 1500
ggcaagaagc cgccgccgat cgtgcggctc tgtctgcggc gactggcggc ggtgccgttc 1560
atgaagcggc agccgctgcg gcgcgtgatg agaatgatgg cccaattcgt actgttgttc 1620
tgaatctggc ccgtgttgat cgtattgatg acgtaggtcg ccgcctgatt gccgaaggcg 1680
ttcgccgtct gcaagcggat ggcgtacgcg tagaagtaga agatccggaa cgcattctgc 1740
cgctggaaga agcgggcgcg caccaccacc accaccacca ctaatcctct agagtcgagc 1800
tcaagctagc 1810
<210>64
<211>800
<212>DNA
<213> Artificial sequence
<400>64
ttgcgcaact tgaccaagac atctctatta ctggccggct tatgcacagc ggcccaaatg 60
gtttttgtaa cacatgcctc agctgaagaa agcatcgaat acgaccatac gtatcaaacc 120
ccttcataca tcatcgaaaa gtcaccgcag aagccggtac aaaacacaac ccagaaagaa 180
tcgctatttt cctatcttga caagcatcaa acgcagttta agctcaaagg gaatgcgaac 240
agccattttc gcgtttcgaa aaccataaag gatccaaaga caaaacaaac gttttttaaa 300
ttaacggagg tttacaaagg aattccgatt tacggctttg aacaagcggt cgcgatgaag 360
gaaaacaaac aagtgaaaag tttctttgga aaggtgcatc cgcaaatcaa ggacgtctcc 420
gtcacaccgt ctatttctga gaaaaaagca atacatacag caaggcgtga gctcgaggct 480
tccattggaa aaatcgaata tcttgatggg gaaccaaaag gcgaattata tatctatcca 540
cacgacggtg aatatgatct cgcctacctt gtgagactct cgacatctga acctgagcct 600
ggctattggc attatttcat cgatgccaaa aacggaaagg tcatcgagtc ctttaatgcc 660
attcatgaag cggcaggtac aggaatcggc gtgtcaggtg atgaaaaaag ctttgacgtc 720
acagaacaaa atgggcgctt ttatttggct gacgaaacaa ggggaaaagg gatcaataca 780
tttgacgcga agaacctgaa 800
<210>65
<211>819
<212>DNA
<213> Artificial sequence
<400>65
ccttgtttac gcttttgtct caactgatcg ggtatacggg caaagaaata gtcagcggca 60
cgtccgtatt taatgaacct gcggctgtag acgcacacgc aaatgcgcaa gccgtttacg 120
attattacag caagacattt ggccgtgatt cttttgatca aaacggagca aggattacgt 180
ctaccgtgca tgtcggcaaa caatggaaca atgctgcgtg gaacggtgtc cagatggtat 240
acggggatgg agacggttcg aaatttaagc cgctgtctgg atcgctcgac attgtcgcgc 300
atgaaatcac acacgcagtc acacagtatt ccgccggtct tttatatcaa ggagaacccg 360
gtgcattaaa tgagtccatt tctgacatta tgggcgcgat ggctgaccgt gatgattggg 420
agatcggcga agatgtctat actcctggta ttgcaggaga ttcattgcgg tcattggagg 480
acccatctaa gcagggaaat ccagatcatt actcgaaccg ctacacagga acagaggatt 540
atggcggagt ccatatcaat tcgtccattc acaataaagc agcttatctt cttgcagaag 600
gaggcgtgca ccacggtgta caggttgaag ggattgggcg tgaagcaagt gaacaaattt 660
actatcgggc tttaacatat tatgtaacgg catctacaga tttcagcatg atgaagcaag 720
cggcgattga agctgccaat gatttatacg gtgaaggctc gaagcaatca gcttcagtcg 780
aaaaggcgta tgaggctgtc ggcattctat gagcaaaca 819
<210>66
<211>800
<212>DNA
<213> Artificial sequence
<400>66
gtgggtttag gtaagaaatt gtctgttgct gtcgctgctt cgtttatgag tttatcaatc 60
agcctgccag gtgttcaggc tgctgaaggt catcagctta aagagaatca aacaaatttc 120
ctctccaaaa acgcgattgc gcaatcagaa ctctctgcac caaatgacaa ggctgtcaag 180
cagtttttga aaaagaacag caacattttt aaaggtgacc cttccaaaag gctgaagctt 240
gttgaaagca cgactgatgc ccttggatac aagcactttc gatatgcgcc tgtcgttaac 300
ggagtgccaa ttaaagattc gcaagtgatc gttcacgtcg ataaatccga taatgtctat 360
gcggtcaatg gtgaattaca caatcaatct gctgcaaaaa cagataacag ccaaaaagtc 420
tcttctgaaa aagcgctggc actcgctttc aaagctatcg gcaaatcacc agacgctgtt 480
tctaacggag cggccaaaaa cagcaataaa gccgaattaa aagcgataga aacaaaagac 540
ggcagctatc gtcttgctta cgacgtgacg attcgctatg tcgagcctga acctgcaaac 600
tgggaagtct tagttgacgc cgaaacaggc agcattttaa aacagcaaaa taaagtagaa 660
catgccgccg ccactggaag cggaacaacg ctaaagggcg caactgttcc tttgaacatc 720
tcttatgaag gcggaaaata tgttctaaga gatctttcaa aaccaacagg cacccaaatc 780
atcacatatg atttgcaaaa 800
<210>67
<211>766
<212>DNA
<213> Artificial sequence
<400>67
cagacaaagc cgccttccgg gcacgcttgt ctcaagcaca acgaaaacat ttacatcttc60
atcacagcgg gcagccgttg acgcacacta taacctcggt aaagtgtacg attattttta 120
ttcaaacttt aaacgaaaca gctatgataa caaaggcagt aaaatcgttt cttccgttca 180
ctacggcact caatacaata acgctgcatg gacaggagac cagatgattt acggtgatgg 240
cgacggttca ttcttctctc cgctttccgg ctcattagat gtgacagcgc atgaaatgac 300
acatggcgtc acccaagaaa cagccaactt gatttatgaa aatcagccag gtgcattaaa 360
cgagtctttc tctgacgtat tcgggtattt taacgataca gaagactggg acatcggtga 420
agacattacg gtcagccagc ctgctcttcg cagcctgtcc aaccctacaa aatacaacca 480
gcctgacaat tacgccaatt accgaaacct tccaaacaca gatgaaggcg attatggcgg 540
tgtacacaca aacagcggaa ttccaaacaa agccgcttac aacaccatca caaaacttgg 600
tgtatctaaa tcacagcaaa tctattaccg tgcgttaaca acgtacctca cgccttcttc 660
cacgttcaaa gatgccaagg cagctctcat tcagtctgcc cgtgacctct acggctcaac 720
tgatgccgct aaagttgaag cagcctggaa tgctgttgga ttgtaa 766
<210>68
<211>700
<212>DNA
<213> Artificial sequence
<400>68
ccgatattgg ttaaacagcg gcgcaatggc ggccgcatct gatgtctttg cttggcgaat 60
gttcatctta tttcttcctc cctctcaata attttttcat tctatccctt ttctgtaaag 120
tttatttttc agaatacttt tatcatcatg ctttgaaaaa atatcacgat aatatccatt 180
gttctcacgg aagcacacgc aggtcatttg aacgaatttt ttcgacagga atttgccggg 240
actcaggagc atttaaccta aaaaagcatg acatttcagc ataatgaaca tttactcatg 300
tctattttcg ttcttttctg tatgaaaata gttatttcga gtctctacgg aaatagcgag 360
agatgatata cctaaataga gataaaatca tctcaaaaaa atgggtctac taaaatatta 420
ttccatctat tacaataaat tcacagaata gtcttttaag taagtctact ctgaattttt 480
ttaaaaggag agggtaaaga gtgagaagca aaaaattgtg gatcagcttg ttgtttgcgt 540
taacgttaat ctttacgatg gcgttcagca acatgtctgc gcaggctgcc ggaaaaagca 600
gtacagaaaa gaaatacatt gtcggattta aacagacaat gagtgccatg agttccgcca 660
agaaaaagga tgttatttct gaaaaaggcg gaaaggttca 700
<210>69
<211>683
<212>DNA
<213> Artificial sequence
<400>69
ctggaggcac ttacggcgct tataacggaa cgtccatggc gactcctcac gttgccggag 60
cagcagcgtt aattctttct aagcacccga cttggacaaa cgcgcaagtc cgtgatcgtt 120
tagaaagcac tgcaacatat cttggaaact ctttctacta tggaaaaggg ttaatcaacg 180
tacaagcagc tgcacaataa tagtaaaaag aagcaggttc ctccatacct gcttcttttt 240
atttgtcagc atcctgatgt tccggcgcat tctcttcttt ctccgcatgt tgaatccgtt 300
ccatgatcga cggatggctg cctctgaaaa tcttcacaag caccggagga tcaacctggc 360
tcagccccgt cacggccaaa tcctgaaacg ttttaacagc ggcttctctg ttctctgtca 420
actcgatccc atactggtca gccttattct cctgataacg cgagacagca ttagaaaaag 480
gcgtaaccgc aaagctcaaa acagaaaaca aaagcaataa cagcggaagt gccgcaagat 540
catgccgccc ttctaaatga aacatgctgc gggttaggcg aaccgtccgc ttgtaaagct 600
tatcaatgac ataaaatccg gcgagcgaca cgagcaaata gccagccaga ccgatgtaaa 660
cgtgcttcat gacataatgg ccc 683
<210>70
<211>700
<212>DNA
<213> Artificial sequence
<400>70
gcgacaaaag gaatggtgtt gaaaattacc gatccaagac tgttgaaaga aacaggaggc 60
atcgtacagg ggatgagcgg aagcccgatc attcaaaatg gaaaagtgat cggtgctgtc 120
acccatgtat ttgtaaatga cccgacaagc ggctacggtg ttcatattga atggatgctg 180
tcagaagcag gaatcgatat ttatggaaaa gaaaaagcaa gctgactgcc ggagtttccg 240
gcagtttttt tattttgatc cctcttcact tctcagaata catacggtaa aatatacaaa 300
agaagatttt tcgacaaatt cacgtttcct tgtttgtcaa atttcatttt tagtcgaaaa 360
acagagaaaa acatagaata acaaagatat gccactaata ttggtgatta tgattttttt 420
agagggtata tagcggtttt gtcgaatgta aacatgtagc aagggtgaat cctgttaact 480
acatttgggg aggaagaaac gtggagaaaa ttaaagtttg tgttgctgat gataatcgag 540
agctggtaag cctgttaagt gaatatatag aaggacagga agacatggaa gtgatcggcg 600
ttgcttataa cggacaggaa tgcctgtcgc tgtttaaaga aaaagatccc gatgtgctcg 660
tattagatat tattatgccg catctagacg gacttgcggt 700
<210>71
<211>688
<212>DNA
<213> Artificial sequence
<400>71
ccaaaaaatt taacacaacc gcaagccgtg tagaaagagc gatccgccat gcaattgaag 60
tggcatggag cagaggaaac attgattcca tttcctcgtt gtttggttat actgtcagca 120
tgacaaaagc taaacctacc aacagtgaat tcattgcaat ggttgcggat aagctgaggt 180
tagagcataa ggcttcttaa acatgagctt attaagtggt cattaaatca aacgtctttt 240
atttattagt ttgcgctgat aaataggagg cgttttgttt tggggacatt tgtagtatgg 300
gagaagaata ttaagtatcc gcatatagta aaggataggt ttgaaaagta aggaaagcaa 360
atggcggtat tacctgcact cttggtccat tcatacatct tataggatca cgaagcatga 420
tcgaccctag atgtgtccgt tataaaatga aacctttctc ctgcaaaatc gtaagtaagg 480
cgtattcatg taaaaaggag acgtacgaat gaaaaagaaa aaagcaagaa agcgtgaggg 540
gtttttcctg gattttcttt tcgaagttgg tggggagctc tttcttttgc tgtttagatg 600
tgtacacaaa ctatttatat aaagggaaaa aagactgccg aatcgtttcg gcagtctttg 660
cacattaatc tttataaggc acccagcc 688
<210>72
<211>688
<212>DNA
<213> Artificial sequence
<400>72
cggcaatttt cccggcgaca ggcattattt tttcctccat cacccgagtg aatgtgctca 60
tcttaaaaac ccccttttct cattgctttg tgaacaacct ccgcaatgtt ttctttatct 120
tattttgaaa acgcttacaa attcatttgg aaaatttcct cttcatgcgg aaaaaatctg 180
cattttgcta aacaaccctg cccatgaaaa attttttcct tcttactatt aatctctctt 240
tttttctccg atatatatat caaacatcat agaaaaagga gatgaatcat gaaaaacatg 300
tcttgcaaac ttgttgtatc agtcactctg tttttcagtt ttctcaccat aggccctctc 360
gctcatgcgc aaaacagcag cgagaaagag gttattgtgg tttataaaaa caaggccgga 420
aaggaaacca tcctggacag tgatgctgat gttgaacagc agtataagca tcttcccgcg 480
gtagcggtca cagcagacca ggagacagta aaagaattaa agcaggatcc tgatattttg 540
tatgtagaaa acaacgtatc atttaccgca gcagacagca cggatttcaa agtgctgtca 600
gacggcactg acacctctga caactttgag caatggaacc ttgagcccat tcaggtgaaa 660
caggcttgga aggcaggact gacaggaa 688
<210>73
<211>692
<212>DNA
<213> Artificial sequence
<400>73
gcaagctgcc tgcaagttca gaaaaaacgt ccctgcagaa acgccttaac aaagtgaaga 60
gcaccaattt gaagacggca cagcaatccg tatctgcggc tgaaaagaaa tcaactgatg 120
caaatgcggc aaaagcacaa tcagccgtca atcagcttca agcaggcaag gacaaaacgg 180
cattgcaaaa acggttagac aaagtgaaga aaaaggtggc ggcggctgaa gcaaaaaaag 240
tggaaactgc aaaggcaaaa gtgaagaaag cggaaaaaga caaaacaaag aaatcaaaga 300
catccgctca gtctgcagtg aatcaattaa aagcatccaa tgaaaaaaca aagctgcaaa 360
aacggctgaa cgccgtcaaa ccgaaaaagt aaccaaaaac ctttaagatt tgcattccaa 420
gtcttaaagg tttttttcat tctaagaaca ccacacacaa cctttttccc atccattgta 480
caggcttttc atactattgc tatacagcca tgaacagcat aaaatgaacg ttattacagt 540
tatcaccaca tatggcggga ttgtgactgg gcaggcaggc aagacccaat gatgcaaagg 600
gagtattaat gcctaaaaaa cagggcattt taactcttct tttcgtgttg ggctcataac 660
ggcgccttgg acaaccccct attctcatgc tc 692
<210>74
<211>687
<212>DNA
<213> Artificial sequence
<400>74
gaggaaaaaa acgaaaaaca gactcatcag ctctgtttta agtacagttg tcatcagttc 60
actgctgttt ccgggagcag ccggggcaag cagtaaagtc acctcacctt ctgttaaaaa 120
ggagcttcaa tctgcggaat ccattcaaaa caagatttcg agttcattaa agaaaagctt 180
taaaaagaaa gaaaaaacga cttttctgat taaatttaaa gatctggcta acccagaaaa 240
agcggcaaaa gcggctgtta aaaaagcgaa atcgaagaag ctgtctgccg ctaagacgga 300
atatcaaaag cgttctgctg ttgtgtcatc tttaaaagtc acagccgatg aatcccagca 360
agatgtccta aaatacttga acacccagaa agataaagga aatgcagacc aaattcattc 420
ttattatgtg gtgaacggga ttgctgttca tgcctcaaaa gaggttatgg aaaaagtggt 480
gcagtttccc gaagtggaaa aggtgcttcc taatgagaaa cggcagcttt ttaagtcatc540
ctccccattt aatatgaaaa aagcacagaa agctattaaa gcaactgacg gtgtggaatg 600
gaatgtagac caaatcgatg ccccaaaagc ttgggcactt ggatatgatg gaactggcac 660
ggttgttgcg tccattgata ccggggt 687
<210>75
<211>695
<212>DNA
<213> Artificial sequence
<400>75
tggcacgttc catgcaggca tcatactcaa caagggtgaa aatgagctga cggcaactgc 60
atcaactgac aacggaacaa cagatgcctc cagcccaatc acggtcacgc ttgatcaaga 120
aaagcctgaa ttaacactgg acaatccaaa ggatggcggg aaaacaaata aagaaacgct 180
gactgtcaaa ggggctgtat ccgatgacaa tctgaaagac gtcaaggtga atggcaaaaa 240
agcaacagta gctgatggtt catactcagc ccgtattctt ttggaaaatg gaagaaatga 300
aatcaaggta attgctacag acttggcagg caacaaaacg acgaaaaaga cagtcattga 360
tgtgaacttt gacaagcctg tcatttccgg cttaattccg ggagaggata aaaacttaaa 420
agccggtgaa tctgtgaaaa tcgctttctc aagcgctgag gatttagatg caacgtttac 480
cattcgtatg ccgctgacca atgcaagagc gagtgtgcaa aatgccaccg aactcccgtt 540
aagagaaatc tctccgggga gatatgaagg ctattggact gccacttctt ctattaaagc 600
aaaaggagca aaagtagaag tgatcgtccg agatgattat ggaaatgaaa caagaaaaac 660
tgcgaatgga aaacttaata tgaacacaga aaatt 695
<210>76
<211>2210
<212>DNA
<213> Artificial sequence
<400>76
ttggcaaggg tttaaaggtg gagatttttt gagtgatctt ctcaaaaaat actacctgtc 60
ccttgctgat ttttaaacga gcacgagagc aaaacccccc tttgctgagg tggcagaggg 120
caggtttttt tgtttctttt ttctcgtaaa aaaaagaaag gtcttaaagg ttttatggtt 180
ttggtcggca ctgccgacag cctcgcagag cacacacttt atgaatataa agtatagtgt 240
gttatacttt acttggaagt ggttgccgga aagagcgaaa atgcctcaca tttgtgccac 300
ctaaaaagga gcgatttaca tatgatgcgt caccctatcc cagactacct ggccagcctg 360
gtaaccgagc tgggtgcagt aaaccctggc gaaaccgctc agtacatccc ggtgctggca 420
gaggcagatc cagaccgttt cggtatcgct ctggctaccc cgactggtcg tctgcattgc 480
gcaggtgacg ctgatgtgga gttcaccatt cagtccgcgt ccaaaccgtt cacctacgcg 540
gctgcgctgg tcgaccgtgg tttcgcagct gtggaccgtc aggtaggtct gaacccgagc 600
ggtgaggctt tcaacgagct gagcctggag gcagaaagcc accgtccgga caacgcaatg 660
atcaacgcgg gtgcactggc tgtacaccag ctgctggtcg gtccggaagc atctcgtaag 720
gaacgtctgg accgtgcagt ggaaatcatg tccctgctgg ccggtcgtcg tctgtccgtg 780
gattgggaaa cgtacgaatc cgaaatggcg gtcagcgacc gcaacctgtc cctggcgcac 840
atgctgcgta gctatggcgt gctgcaggac tccgcagaag aaatcgtggc cggctacgtg 900
gcacagtgcg cagtcctggt cactgtcaaa gacctggcgg tgatgggcgc atgtctggca 960
accggtggta tccacccgat gacgggtgaa cgtatgctgc cgtctatcgt ggcgcgtcgt 1020
gtggtgtctg ttatgacctc ctctggcatg tatgacgcgg ccggccagtg gctggctgat 1080
gtaggcatcc cggctaaatc tggtgttgcg ggcggtgttc tgggtgctct gccgggtcgt 1140
gttggtatcg gtgttttcag cccgcgcctg gatgaagttg gcaactctgc gcgtggcgtt 1200
ctggcttgtc gtcgcctgtc tgaagacttc cgcctgcatc tgatggacgg cgactctctg 1260
ggtggtaccg ctgttcgttt tgttgaacgc gaaggcgacc gcgtttttct gcacctgcag 1320
ggcgttatcc gctttggcgg cgcggaagcg gttctggacg ctctgaccga tctgcgtacg 1380
ggtgctgaga aaccgggtac tggctgggat gctgctgttt atccgcgctg gcaagaagcc 1440
gccgccgatc gtgcggctct gtctgcggcg actggcggcg gtgccgttca tgaagcggca 1500
gccgctgcgg cgcgtgatga gaatgatggc ccaattcgta ctgttgttct gaatctggcc 1560
cgtgttgatc gtattgatga cgtaggtcgc cgcctgattg ccgaaggcgt tcgccgtctg 1620
caagcggatg gcgtacgcgt agaagtagaa gatccggaac gcattctgcc gctggaagaa 1680
gcgggcgcgc actaaggatc ctctagagtc gagctcaagc tagcttggta cgtaccagat 1740
ctgagatcac gcgttctaga ggtcgaaatt cacctcgaaa gcaagctgat aaaccgatac 1800
aattaaaggc tccttttgga gccttttttt ttggagattt tcaacgtgaa aaaattatta 1860
ttcgcaattc caagctaatt cacctcgaaa gcaagctgat aaaccgatac aattaaaggc 1920
tccttttgga gccttttttt ttggagattt tcaacgtgaa aaaattatta ttcgcaattc 1980
caagctctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc 2040
ggagacggtc acagcttgtc tgtaagcgga tgcagatcac gcgccctgta gcggcgcatt 2100
aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 2160
gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct 2210
Claims (5)
1. A gene engineering bacterium for the unmarked multi-site integrated expression of glutaminase is characterized in that bacillus subtilis is used as a host to simultaneously integrate seven sites of 16S rDNA, nprB, nprE, aprE, spo0A, epr and bpr, and integrate and express a gene sequence shown as SEQ ID No. 1.
2. A method for preparing the genetically engineered bacterium of claim 1,
(1) construction of 16SrDNA site-integrated expression fragment: extending two ends of 16S rDNA homology arms, lox71-zeo-lox66 and HpaII-Mglu4 into 1 segment by an overlapping extension method to obtain an integrated segment of 16SrDNA site integrated expression, transforming the integrated segment into host bacteria, transforming a plasmid containing CRE recombinase into the host bacteria, and removing resistance markers to obtain unmarked 16SrDNA site integrated expression glutaminase Bacillus subtilis engineering bacteria BSM 1;
(2) construction of other site integration expression fragments: referring to the step (1), selecting nprB, nprE, aprE, spo0A, epr and bpr as integration expression sites, and respectively constructing integration expression fragments; and (3) transforming the integrated fragment into BSM1, transforming a plasmid containing CRE recombinase into BSM1, and removing the resistance marker to obtain the B.subtiliss 168 genetically engineered bacterium BSM 7.
3. The method for producing glutaminase by fermentation using the genetically engineered bacterium according to claim 1, wherein the strain is activated to prepare a seed solution, the seed solution is transferred to a fermentation tank containing 40% of the fermentation medium, and fed-batch fermentation is performed at 25 ℃, pH7.0 and a rotation speed of 600rpm while maintaining the glucose concentration at 10 to 20 g/L.
4. The method as claimed in claim 3, wherein the composition of the feed medium is glucose 1000g/L and yeast extract700 g/L.
5. The use of the genetically engineered bacteria of claim 1 to improve the mouthfeel and flavor of food.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101084302A (en) * | 2004-12-20 | 2007-12-05 | 花王株式会社 | Recombinant microorganism |
| CN106399218A (en) * | 2016-12-16 | 2017-02-15 | 山东省食品发酵工业研究设计院 | Bacillus subtilis engineering bacteria and application thereof |
| US10370654B2 (en) * | 2013-08-02 | 2019-08-06 | Enevolv, Inc. | Processes and host cells for genome, pathway, and biomolecular engineering |
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|---|---|---|---|---|
| CN101084302A (en) * | 2004-12-20 | 2007-12-05 | 花王株式会社 | Recombinant microorganism |
| US10370654B2 (en) * | 2013-08-02 | 2019-08-06 | Enevolv, Inc. | Processes and host cells for genome, pathway, and biomolecular engineering |
| CN106399218A (en) * | 2016-12-16 | 2017-02-15 | 山东省食品发酵工业研究设计院 | Bacillus subtilis engineering bacteria and application thereof |
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| Accession No.:DQ019448.1, Micrococcus luteus strain K-3 salt-tolerant glutaminase gene, complete cds;Wakayama,M.;《GenBank》;20050521;第1页 * |
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| 高活性谷氨酰胺酶基因glsA2在枯草芽孢杆菌BJ3-2染色体上的定点整合;卢彪;《食品科学》;20141231;第35卷(第01期);第141-144页 * |
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