CN114934027A - L-sorbose dehydrogenase mutant capable of improving 2-KLG yield - Google Patents
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Abstract
The invention discloses an L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG, belonging to the technical field of genetic engineering and enzyme engineering. The L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG is obtained by site-directed mutagenesis by taking L-sorbose dehydrogenase derived from gluconobacter oxydans as an initial sequence. Through fermentation detection, the 2-KLG yields of the mutants V368C, V368L, V368S and V368T reach 2.5g/L, 3.1g/L, 2.5g/L and 3.0g/L respectively, which are 1.1, 1.4, 1.1 and 1.3 times of the wild-type L-sorbose dehydrogenase strain in the table.
Description
Technical Field
The invention relates to an L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG, belonging to the technical field of genetic engineering and enzyme engineering.
Background
Vitamin C, also known as ascorbic acid, is a vitamin essential to the human body. Can be widely used in the fields of food, beverage, pharmacy, cosmetics and feed. 2-keto-L-gulonic acid (2-KLG) is a direct precursor for the production of vitamin C. Currently, the industrial production of 2-KLG utilizes a three-bacterium two-step fermentation method. Compared with one-step fermentation, the three-bacterium two-step fermentation method has the disadvantages of high energy consumption, high material consumption, difficulty in accurate regulation and control of mixed bacterium fermentation, difficulty in breeding and the like. The exploration of one-step fermentation for producing 2-KLG becomes a common goal of researchers. Since the production of 2-KLG from sorbitol involves only 3 catalytic enzymes, D-sorbitol dehydrogenase, L-sorbose dehydrogenase and L-sorbosone dehydrogenase, respectively. Therefore, most researchers now conduct one-step bacterial studies through metabolic engineering. The main method is to carry out heterologous expression on key enzyme genes in the synthetic pathway of 2-KLG to produce the 2-KLG, but the effect is not comparable with that of industrial production. Current studies indicate that L-sorbose dehydrogenase may be a key rate-limiting enzyme in the production of 2-KLG. The NCBI alignment shows that L-sorbose dehydrogenase belongs to the glucose-methanol-choline oxidoreductase family. This class of enzymes comprises two domains, the N-terminal domain belonging to the cofactor binding domain and the C-terminal domain belonging to the substrate binding domain. At present, the research on the modification of L-sorbose dehydrogenase is less, and the effective modification of the L-sorbose dehydrogenase can possibly improve the production strength of 2-KLG.
Disclosure of Invention
The inventor obtains a Gluconobacter oxydans WSH-004 capable of directly converting D-sorbitol into 2-keto-L-gulonic acid (2-KLG) through screening in earlier stage, and identifies L-sorbose dehydrogenase and L-sorbosone dehydrogenase in the Gluconobacter oxydans WSH-004. The invention prepares the L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG by means of gene engineering and enzyme engineering.
The invention provides an L-sorbose dehydrogenase mutant capable of improving the yield of 2-KLG, which takes L-sorbose dehydrogenase from Gluconobacter oxydans (Gluconobacter oxydans) WSH-004 as an initial sequence, and mutates valine at position 368 into cysteine, leucine, serine or threonine.
In one embodiment, the amino acid sequence of the starting sequence is shown in SEQ ID NO. 1.
In one embodiment, the mutant is obtained by changing valine (Val) at position 368 to cysteine (Cys), and the mutant is named V368C, and the amino acid sequence thereof is shown in SEQ ID No. 2.
In one embodiment, the mutant is a mutant wherein valine (Val) at position 368 is changed to leucine (Leu), and the mutant is named V368L, and the amino acid sequence is shown in SEQ ID No. 3.
In one embodiment, the mutant is a mutant in which valine (Val) at position 368 is changed to serine (Ser), and the mutant is named V368S, and the amino acid sequence of the mutant is shown in SEQ ID No. 4.
In one embodiment, the mutant is a mutant in which valine (Val) at position 368 is changed to threonine (Thr), and the mutant is named V368T, and the amino acid sequence thereof is shown in SEQ ID No. 5.
The invention also provides a gene encoding the mutant.
In one embodiment, the nucleotide sequence encoding mutant V368C is based on SEQ ID No.6 by replacing codon 368 with GUG for UGC.
In one embodiment, the nucleotide sequence encoding mutant V368L is based on SEQ ID NO.6 by replacing codon 368 with GUG for CUU.
In one embodiment, the nucleotide sequence encoding mutant V368S is based on SEQ ID No.6 with the substitution of codon 368 from GUG to UCG.
In one embodiment, the nucleotide sequence encoding mutant V368T is SEQ ID No.6 with codon 368 replaced by GUG to ACC.
The invention also provides a recombinant plasmid carrying the gene.
In one embodiment, the recombinant plasmid is pMD19 as the starting plasmid.
The invention also provides a microbial cell expressing the L-sorbose dehydrogenase mutant or carrying the gene.
In one embodiment, the host cell of the microbial cell is a bacterial or fungal cell.
In one embodiment, the bacterium is a gram-negative or gram-positive bacterium.
In one embodiment, the plasmid is pMD 19-T.
In one embodiment, the microbial cell expresses the L-sorbose dehydrogenase, the L-sorbose dehydrogenase mutant and a key enzyme gene in the synthetic pathway of 2-KLG by taking Escherichia coli as a host and pMD19-T as an expression vector.
In one embodiment, the expression of the L-sorbose dehydrogenase gene and the L-sorbosone dehydrogenase gene is regulated by a cspA promoter.
In one embodiment, the microbial cell is a host of escherichia coli BL21(DE 3).
The invention also provides application of the L-sorbose dehydrogenase mutant in producing 2-keto-L-gulonic acid.
In one embodiment, the application is that the recombinant Escherichia coli expressing the L-sorbose dehydrogenase mutant is cultured in an LB culture medium at 30-37 ℃ and 150-250 rpm until OD is reached 600 Preparing a seed solution, transferring the seed solution into a sorbose culture medium, and fermenting at 30-37 ℃.
The invention also provides recombinant escherichia coli capable of producing 2-KLG, wherein the recombinant escherichia coli is expressed by any one of the L-sorbose dehydrogenase and the L-sorbosone dehydrogenase shown in SEQ ID No. 2-5.
In one embodiment, the gene of the L-sorbosone dehydrogenase is represented by SEQ ID NO. 7.
In one embodiment, the recombinant E.coli is hosted in BL21(DE 3).
The invention also provides application of the microbial cell or the recombinant Escherichia coli in the aspect of producing vitamin C or precursors thereof.
Has the beneficial effects that:
the invention takes L-sorbose dehydrogenase in Gluconobacter oxydans WSH-004 as a target, and modifies an amino acid sequence of the L-sorbose dehydrogenase through site-directed mutagenesis biotechnology to finally obtain 4 strains of L-sorbose dehydrogenase mutants V368C, V368L, V368S and V368T with improved enzyme activity, and the strains expressing the L-sorbose dehydrogenase mutants are fermented to ensure that the yield of 2-KLG reaches 2.5g/L, 3.1g/L, 2.5g/L and 3.0g/L which are respectively 1.1, 1.4, 1.1 and 1.3 times of the strains expressing wild type L-sorbose dehydrogenase.
Drawings
FIG. 1 is a diagram showing the construction of an L-sorbose dehydrogenase expression vector (pMD19-cspA-SNDH-SDH) modified by site-directed mutagenesis.
FIG. 2 is a schematic representation of plasmid pMD19-cspA-SNDH-V368L in one embodiment of the present invention.
FIG. 3 is a graph of high performance liquid chromatography for detecting 2-KLG produced by fermentation of different strains.
FIG. 4 is a graph comparing the production of 2-KLG by different strains.
Detailed Description
1. Culture medium and reagents:
LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. 18g/L agar powder is also needed to be added for preparing the solid culture medium.
Sorbitol culture medium: 10g/L of yeast powder and 50g/L of sorbitol. 18g/L agar powder was added to prepare a sorbitol solid medium.
Sorbose culture medium: sorbose 10g/L, peptone 10g/L, yeast powder 5g/L, sodium chloride 10 g/L.
2. Cloning of the L-sorbose dehydrogenase Gene:
gluconobacter oxydans WSH-004(CCTCC M2019481, Strain disclosed in the paper "High-Throughput Screening of a 2-Keto-L-Gulonic Acid-Producing Gluconobacter oxydans Strain Based on Related microorganisms") was inoculated into sorbitol medium for culture. Collecting thallus, extracting genome with Ezup column type bacterial genome DNA extraction kit (purchased from Biotechnology engineering (Shanghai) Co., Ltd.), amplifying L-sorbose dehydrogenase by PCR, wherein the amplification primer contains homology arm sequence required for connecting plasmid. PCR was performed using 2X Phanta Max Master Mix (purchased from Nanjing Novowed). PCR product recovery Using a DNA gel recovery kit of SanPrep column type (purchased from Biotechnology engineering (Shanghai) Co., Ltd.).
3. Construction of L-sorbose dehydrogenase plasmid and expression of the Gene:
1) the vector was amplified by PCR, linearized, and provided with a sequence of homology arms that could be complementary to the amplified L-sorbose dehydrogenase gene.
2) The linearized vector and L-sorbose dehydrogenase were seamlessly ligated using an Infusion-Cloning kit (purchased from Nanjing Novonoprazan) to construct a complete plasmid.
3) The constructed plasmid is transferred into a target competence, and is coated on a plate containing corresponding antibiotics, and positive clones are selected for sequencing.
4. Fermenting the strain expressing L-sorbose dehydrogenase and mutant plasmids thereof:
1) the correct single colony was inoculated in LB medium and cultured overnight to prepare a seed solution.
2) Inoculating the seed liquid into a sorbose culture medium in an inoculation amount of 5%, fermenting and culturing for 72 hours, and collecting fermentation liquid.
3) The collected fermentation broth was centrifuged at 12000 Xg for 3 minutes, and the supernatant was collected for examination of 2-KLG production.
5. Detecting the yield of 2-KLG in the fermentation liquor by using high performance liquid chromatography: the measurement was performed by using high performance liquid chromatography. Detection conditions of high performance liquid chromatography: a chromatographic column: aminex HPX-87H column (300 mm. times.7.8 mm; Bio-Rad, Hercules, Calif.); column temperature: 40 ℃; mobile phase: 5mM H 2 SO 4 (ii) a Flow rate: 0.5mL/min, detected using a parallax detector.
The enzyme activity determination of the L-sorbose dehydrogenase and the mutant thereof adopts a spectrophotometer capable of controlling temperature and monitoring absorbance in real time, a cuvette with the light path of 1.0cm, and a reaction system is as follows: 25mM of substrate, 2.5mM of Phenazine Methosulfate (PMS), 0.5mM of 2, 6-dichloroindophenol sodium (DCIP), 0.04g/L of enzyme final concentration and 50mM Tris-HCl with pH7.0 as buffer. The unit enzyme activity (U) is defined as the amount of enzyme required to reduce 1. mu. mol of DCIP in 1min at 37 ℃ and pH 7.0.
Example 1: cloning of wild L-sorbose dehydrogenase gene, construction and expression of plasmid
(1) Construction of L-sorbose dehydrogenase expression vector:
inoculating Gluconobacter oxydans WSH-004 in a glycerol tube, culturing at 37 ℃, culturing at 220rpm for 48h, centrifuging at 4000rpm, collecting thalli, extracting a Gluconobacter oxydans WSH-004 genome, and amplifying an L-sorbose dehydrogenase gene (the gene sequence is shown in SEQ ID NO.6) by using a primer pair F1/R1. And recovering the PCR product. Primer pair F1/R1 was as follows:
F1：GGATTTCGTAATGACGAGCGGTTTTGATTACATCGTTGTCG；
R1：ATCTGCAGAATTCTCAGGCGTTCCCCTGAATGAAATCCGC。
the primer pair F2/R2 is used for amplifying the L-sorbosone dehydrogenase (the gene sequence is shown in SEQ ID NO.7) which is used for producing the key gene of 2-KLG, and a PCR product is recovered. Primer pair F2/R2 was as follows:
F2：AAAGGTAATACACTATGAATGTTGTCTCAAAGACTGTATCTTTACCG；
R2：AAACCGCTCGTCATTACGAAATCCAGTGCGAACGTTTG。
the pMD19 plasmid (pMD19-cspA) carrying the cspA promoter (SEQ ID NO.8) (plasmid disclosed in "High through Screening plasmid for a FAD-Dependent L-Sorbose Dehydrogenase") was linearized with the primer pair F3/R3, and the PCR product was recovered. Primer pair F3/R3 was as follows:
F3：GGGGAACGCCTGAGAATTCTGCAGATATCCATCACACTGG；
R3：CAACATTCATAGTGTATTACCTTTAATAATTAAGTGTGCCTTTCGG。
the PCR reaction systems are as follows: 25 μ L of 2 × Phanta Max Master Mix, 1 μ L of forward primer (10 μmol. L) -1 ) 1 μ L reverse primer (10 μmol. L) -1 ) mu.L of template DNA was added to 50. mu.L of distilled water. The PCR amplification program of the L-sorbose dehydrogenase and the L-sorbosone dehydrogenase is set as follows: first, pre-denaturation at 95 ℃ for 3 min; then 30 cycles were entered: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1 min; finally, extension is carried out for 5min at 72 ℃, and heat preservation is carried out at 4 ℃. The PCR amplification program of pMD19-cspA linearized vector is set as: firstly, performing pre-denaturation at 95 ℃ for 3 min; then 25 cycles were entered: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 3 min; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.
40ng of L-sorbose dehydrogenase and 40ng of pcr product of L-sorbosone dehydrogenase were mixed with 20ng of pMD19-cspA linearized vector, and ligated by using an Infusion-Cloning kit to construct plasmid pMD19-cspA-SNDH-SDH (FIG. 1). Transferring 10 μ L pMD19-cspA-SNDH-SDH ligation product into the competence of Escherichia coli BL21(DE3), ice-cooling for 30min, heat shock for 90s at 42 ℃, ice-cooling for 5min, adding 1ml LB culture medium, culturing at 37 ℃, 220rpm for 45min, centrifuging at 3000rpm for 3min, removing supernatant, suspending thallus in 100 μ L LB culture medium, spreading in LB plate containing 100mg/L ampicillin, and culturing overnight. The next day, positive clones were selected for sequencing to verify that the plasmid was correct. The correctly sequenced strain was designated WT.
(2) Expression of L-sorbose dehydrogenase:
the correctly sequenced clones were transferred to 10ml LB containing 100mg/L ampicillin and cultured overnight to OD 600 (3), a seed solution was prepared. The cultured seed solution was transferred at 5% to 25ml of a sorbose medium (OD after inoculation) containing 100mg/L ampicillin 600 0.153), culturing at 30 ℃ for 72 hours, collecting fermentation liquor and detecting the product yield.
Example 2: preparation of L-sorbose dehydrogenase mutant
(1) Preparation of Single mutations
Primers for introducing V368C, V368L, V368S and V368T mutations are designed and synthesized respectively according to a pMD19-cspA-SNDH-SDH plasmid sequence constructed in example 1, site-directed mutagenesis is carried out on a gene sequence of the L-sorbose dehydrogenase, and sequencing is carried out respectively to confirm whether a coding gene of the L-sorbose dehydrogenase mutant is correct or not; the vector carrying the mutant gene was introduced into E.coli BL21(DE3) and fermented.
PCR amplification of site-directed mutant coding gene: the recombinant plasmid carrying mutant gene is amplified by PCR technology with the expression vector pMD19-cspA-SNDH-SDH plasmid carrying wild L-sorbose dehydrogenase gene as template.
The site-directed mutagenesis primer pair F4/R4 for introducing the V368C mutation is:
F4：GAGGCTGGGtgcACGTCCGTTCCCAAGGGAGCG (underlined with mutated bases)
R4：TGGGAACGGACGTgcaCCCAGCCTCAGCCCCAGC (underlined with mutated bases)
The site-directed mutagenesis primer pair F5/R5 for introducing the V368L mutation is:
F5：GAGGCTGGGcttACGTCCGTTCCCAAGGGAGCG (underlined with mutated bases)
R5：TGGGAACGGACGTaagCCCAGCCTCAGCCCCAGC (underlined with mutated bases)
The site-directed mutagenesis primer pair F6/R6 for introducing the V368S mutation is:
F6：GAGGCTGGGtcgACGTCCGTTCCCAAGGGAGCG (underlined with mutated bases)
R6：TGGGAACGGACGTcgaCCCAGCCTCAGCCCCAGC (base mutation is underlined)
The site-directed mutagenesis primer pair for introducing the V368T mutation is F7/R7:
F7：GAGGCTGGGaccACGTCCGTTCCCAAGGGAGCG (base mutation is underlined)
R7：TGGGAACGGACGTggtCCCAGCCTCAGCCCCAGC (base mutation is underlined)
The PCR system was the same as in example 1. The mutant plasmid PCR amplification program was set up as: first, pre-denaturation at 95 ℃ for 3 min; then 25 cycles were entered: denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 5 min; finally, extension is carried out for 10min at 72 ℃, and heat preservation is carried out at 4 ℃.
(2) Expression and validation of mutants
The L-sorbose dehydrogenase mutant was verified by the method of step (1) in example 1. The strain expressing the mutant containing V368C was named M1, and the strain expressing the mutant containing V368The strain expressing the L mutant (plasmid map, see FIG. 2) was designated M2, the strain expressing the mutant containing V368S was designated M3, and the strain expressing the mutant containing V368T was designated M4. Culturing the recombinant strain in TB medium at 37 deg.C to OD 600 When the temperature is reduced to 20 ℃, IPTG (isopropyl thiogalactoside) with the final concentration of 0.5mM/L is added for further culture for 20 hours, thalli are collected by centrifugation, the enzymes are purified at 37 ℃ and pH7.0 after the cells are crushed, and the enzyme activity test is carried out on the purified wild type enzyme and the mutant in vitro, the specific enzyme activity of WT is 5400 +/-400U/mg, and the specific enzyme activities of V368C, V368L, V368S and V368T are respectively 1.43, 1.83, 1.39 and 1.74 times of WT.
Example 3: l-sorbose dehydrogenase and its mutant fermentation for producing 2-KLG
The strains constructed in example 1 and example 2 (WT, M1, M2, M3 and M4) were inoculated into LB supplemented with 100mg/L ampicillin, respectively, and cultured overnight at 37 ℃ and 220rpm to OD 600 (vi) 3, prepare seed liquid. The next day, the seed liquid was inoculated at 5% into a sorbose medium and fermented at 30 ℃ for 72 hours, the fermentation broth was collected and centrifuged at 12000 Xg for 3 minutes, the precipitate was discarded, and the supernatant was collected for the detection of 2-KLG yield.
And (3) determining the yield of the 2-KLG in the fermentation liquor by using a parallax detector of high performance liquid chromatography. 5mM H were used 2 SO 4 As the mobile phase, the flow rate was 0.5mL/min, and the detection was performed on an Aminex HPX-87H column (300 mm. times.7.8 mm; Bio-Rad, Hercules, Calif.) at a column temperature of 40 ℃ and the detection results are shown in FIG. 3. The results showed that the yields of 2-KLG after 72 hours fermentation of M1, M2, M3 and M4 reached 2.5g/L, 3.1g/L, 2.5g/L and 3.0g/L (OD) 600 3.733, 3.796, 3.859 and 3.805), the yield of 2-KLG was WT (2-KLG yield was 2.2g/L, OD 600 Up to 1.1, 1.4, 1.1 and 1.3 times of 3.799) (FIG. 4).
Comparative example 1:
the primers were designed according to the same strategy as in example 2 to construct mutants V368D, V368E, V368F, V368G, V368K, V368M, V368P, V368Q, V368R, V368W, and V368Y, respectively. Mutants were prepared and expressed according to the method of example 2, and fermentation and detection were performed according to the method of example 3. The results are shown in Table 1.
TABLE 1 yield of 2-KLG of different single mutants
| Mutants | 2-KLG(g/L) |
| V368D | 2.06297 |
| V368E | 0.85952 |
| V368F | 2.067019 |
| V368G | 0.677537 |
| V368K | 2.006918 |
| V368M | 2.175933 |
| V368P | 1.803614 |
| V368Q | 0.700799 |
| V368R | 1.535407 |
| V368W | 2.18846 |
| V368Y | 1.811543 |
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> L-sorbose dehydrogenase mutant capable of increasing 2-KLG production
<130> BAA220025A
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 531
<212> PRT
<213> Gluconobacter oxydans
<400> 1
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Val
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 2
<211> 531
<212> PRT
<213> Artificial sequence
<400> 2
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Cys
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 3
<211> 531
<212> PRT
<213> Artificial sequence
<400> 3
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Leu
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 4
<211> 531
<212> PRT
<213> Artificial sequence
<400> 4
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Ser
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 5
<211> 531
<212> PRT
<213> Artificial sequence
<400> 5
Met Thr Ser Gly Phe Asp Tyr Ile Val Val Gly Gly Gly Ser Ala Gly
1 5 10 15
Cys Val Leu Ala Ala Arg Leu Ser Glu Asn Pro Ser Val Arg Val Cys
20 25 30
Leu Ile Glu Ala Gly Arg Arg Asp Thr His Pro Leu Ile His Met Pro
35 40 45
Val Gly Phe Ala Lys Met Thr Thr Gly Pro His Thr Trp Asp Leu Leu
50 55 60
Thr Glu Pro Gln Lys His Ala Asn Asn Arg Gln Ile Pro Tyr Val Gln
65 70 75 80
Gly Arg Ile Leu Gly Gly Gly Ser Ser Ile Asn Ala Glu Val Phe Thr
85 90 95
Arg Gly His Pro Ser Asp Phe Asp Arg Trp Ala Ala Glu Gly Ala Asp
100 105 110
Gly Trp Ser Phe Arg Asp Val Gln Lys Tyr Phe Ile Arg Ser Glu Gly
115 120 125
Asn Ala Val Phe Ser Gly Thr Trp His Gly Thr Asn Gly Pro Leu Gly
130 135 140
Val Ser Asn Leu Ala Glu Pro Asn Pro Thr Ser Arg Ala Phe Val Gln
145 150 155 160
Ser Cys Gln Glu Met Gly Leu Pro Tyr Asn Pro Asp Phe Asn Gly Ala
165 170 175
Ser Gln Glu Gly Ala Gly Ile Tyr Gln Met Thr Ile Arg Asn Asn Arg
180 185 190
Arg Cys Ser Thr Ala Val Gly Tyr Leu Arg Pro Ala Leu Gly Arg Lys
195 200 205
Asn Leu Thr Val Val Thr Arg Ala Leu Val Leu Lys Ile Val Phe Asn
210 215 220
Gly Thr Arg Ala Thr Gly Val Gln Tyr Ile Ala Asn Gly Thr Leu Asn
225 230 235 240
Thr Ala Glu Ala Ser Gln Glu Ile Val Val Thr Ala Gly Ala Ile Gly
245 250 255
Thr Pro Lys Leu Met Met Leu Ser Gly Val Gly Pro Ala Ala His Leu
260 265 270
Arg Glu Asn Gly Ile Pro Val Val Gln Asp Leu Pro Gly Val Gly Glu
275 280 285
Asn Leu Gln Asp His Phe Gly Val Asp Ile Val Ala Glu Leu Lys Thr
290 295 300
Asp Glu Ser Phe Asp Lys Tyr Arg Lys Leu His Trp Met Leu Trp Ala
305 310 315 320
Gly Leu Glu Tyr Thr Met Phe Arg Ser Gly Pro Val Ala Ser Asn Val
325 330 335
Val Glu Gly Gly Ala Phe Trp Tyr Ser Asp Pro Ser Ser Gly Val Pro
340 345 350
Asp Leu Gln Phe His Phe Leu Ala Gly Ala Gly Ala Glu Ala Gly Thr
355 360 365
Thr Ser Val Pro Lys Gly Ala Ser Gly Ile Thr Leu Asn Ser Tyr Val
370 375 380
Leu Arg Pro Lys Ser Arg Gly Thr Val Arg Leu Arg Ser Ala Asp Pro
385 390 395 400
Arg Val Asn Pro Met Val Asp Pro Asn Phe Leu Gly Asp Pro Ala Asp
405 410 415
Leu Glu Thr Ser Ala Glu Gly Val Arg Leu Ser Tyr Glu Met Phe Ser
420 425 430
Gln Pro Ser Leu Gln Lys His Ile Arg Lys Thr Cys Phe Phe Ser Gly
435 440 445
Lys Gln Pro Thr Met Gln Met Tyr Arg Asp Tyr Ala Arg Glu His Gly
450 455 460
Arg Thr Ser Tyr His Pro Thr Cys Thr Cys Lys Met Gly Arg Asp Asp
465 470 475 480
Met Ser Val Val Asp Pro Arg Leu Lys Val His Gly Leu Glu Gly Ile
485 490 495
Arg Ile Cys Asp Ser Ser Val Met Pro Ser Leu Leu Gly Ser Asn Thr
500 505 510
Asn Ala Ala Thr Ile Met Ile Ser Glu Arg Ala Ala Asp Phe Ile Gln
515 520 525
Gly Asn Ala
530
<210> 6
<211> 1596
<212> DNA
<213> Artificial sequence
<400> 6
atgacgagcg gttttgatta catcgttgtc ggtggcggtt cggctggctg tgttctcgca 60
gcccgccttt ccgaaaatcc ttccgtccgt gtctgcctca tcgaggcggg ccggcgggac 120
acgcatcccc tgatccacat gccggtcggt ttcgcgaaga tgaccacggg gccgcatacc 180
tgggatcttc tgacggagcc gcagaaacat gcgaacaacc gccagatccc ctatgtgcag 240
ggccggattc tgggcggcgg atcgtccatc aacgcggaag tcttcacgcg gggacaccct 300
tccgacttcg accgctgggc ggcggaaggt gcggatggct ggagcttccg ggatgtccag 360
aagtacttca tccgttccga aggcaatgcc gtgttttcgg gcacctggca tggcacgaac 420
gggccgctcg gggtgtccaa cctcgcggag ccgaacccga ccagccgtgc cttcgtgcag 480
agctgtcagg aaatggggct gccctacaac cctgacttca acggcgcatc gcaggaaggc 540
gcaggcatct atcagatgac gatccgcaac aaccggcgct gctcgacggc tgtggggtat 600
ctgcgtccgg ctctggggcg gaagaacctg acggttgtga cgcgggcgct ggtcctgaag 660
atcgtcttca acggaacgcg ggcgacgggc gtgcagtata tcgccaacgg caccctgaat 720
accgccgaag cgagccagga aatcgttgtg acggccggag cgatcggaac gccgaagctg 780
atgatgctgt cgggcgtcgg gcctgccgcg catcttcgcg aaaatggtat cccggtcgtg 840
caggatctgc cgggcgtggg cgagaacctt caggatcatt tcggtgtgga tatcgtagcc 900
gagctcaaga cggatgagag cttcgacaag taccggaaac tgcactggat gctgtgggca 960
ggtcttgaat ataccatgtt cagatccggt cccgttgcat ccaacgtggt tgagggcggc 1020
gcgttctggt actcggaccc gtcatcgggt gttcctgatc tccagttcca ttttcttgcg 1080
ggggctgggg ctgaggctgg ggtgacgtcc gttcccaagg gagcgtccgg gattacgctg 1140
aacagctatg tgctgcgtcc gaagtctcgt ggaactgtcc ggctgcgttc ggcagatcca 1200
agggtcaatc cgatggtcga tcccaatttc cttggagacc cggccgacct tgagacgtct 1260
gcggaaggtg tgcgcctgag ctacgagatg ttctcccagc cgtctttgca gaagcacatc 1320
cggaaaacct gtttctttag cggtaaacag ccgacgatgc agatgtatcg ggactatgcg 1380
cgggaacatg gccggacgtc ctatcatccg acatgcacct gcaagatggg tcgtgatgac 1440
atgtccgtcg tcgatccgcg tctgaaggtt catggccttg agggcatcag gatctgtgac 1500
agttcggtta tgccgtcgct gctcggttcc aacaccaatg ctgcgacgat catgatcagt 1560
gagcgggcag cggatttcat tcaggggaac gcctga 1596
<210> 7
<211> 1496
<212> DNA
<213> Artificial sequence
<400> 7
atgaatgttg tctcaaagac tgtatcttta ccgttaaagc cgcgtgagtt cggattcttt 60
attgatggag aatggcgcgc aggtaaggat ttcttcgatc gttcctcgcc ggctcatgat 120
gttcccgtca cccgtattcc acgctgcacc cgtgaagacc ttgatgaggc agtcgctgct 180
gcacgtcgtg ctttcgagaa cggaagctgg gcgggtctgg cagccgcgga tcgtgcggcg 240
gttcttctga aagccgcggg ccttctgcgc gagcgccgtg atgacatcgc ttactgggaa 300
gttctcgaaa acgggaagcc catcagccag gcgaaaggtg agatcgatca ctgtatcgcc 360
tgtttcgaga tggcggccgg cgctgcgcgg atgctgcatg gtgatacgtt caacaatctg 420
ggcgaggggc tgtttggcat ggtcctgcgg gagcccatcg gtgtcgtcgg tctgattacg 480
ccgtggaact tcccgttcat gatcctgtgt gagcgggcgc ctttcattct cgcatccggc 540
tgcacgctgg tcgtcaagcc tgccgaagtc acgagtgcca cgacccttct tctggcagaa 600
atccttgccg atgccgggct gccgaagggt gtcttcaatg tcgtgacagg cacggggcgc 660
acggtcggtc aggccatgac cgagcatcag gatatcgaca tgctgtcctt cacgggctcc 720
acgggcgtcg gcaagtcctg tatccacgcg gcggctgaca gcaacctgaa gaaacttggc 780
ctcgaactgg gcggcaagaa cccgattgtc gtgttcgctg acagcaacct tgaggatgcg 840
gccgacgcgg tagccttcgg gatcagcttt aataccgggc agtgctgtgt gtcgtcgagc 900
cgcctgatcg tagagcggtc cgtggcggag aagttcgagc gcctcgtcgt ggcaaaaatg 960
gagaagatcc gcgttggtga tccgtttgat cccgaaacgc agattggcgc catcacgacg 1020
gaagcgcaga acaagaccat tctggactat atcgcgaaag gcaaggccga gggcgccaag 1080
ctgctctgtg gtggcgggat cgtcgatttc ggcaagggac agtatatcca gcccacgctt 1140
ttcacggatg tgaagccctc gatgggcatc gcgcgtgacg agatttttgg gccggttctg 1200
gcgtccttcc acttcgatac cgtcgatgag gcgatcgcga ttgccaatga cacggtttac 1260
ggcttggccg catcggtctg gagcaaggat atcgacaagg cgcttgccgt gacccgtcgt 1320
gttcgtgccg gccgcttctg ggtgaacacc atcatgagcg gtggtcccga gacgccgctg 1380
ggtggtttca agcagtcggg ctggggccgt gaggccggtc tgtacggcgt tgaggaatat 1440
acgcagatca aatctgtcca tatcgaaact ggcaaacgtt cgcactggat ttcgta 1496
<210> 8
<211> 265
<212> DNA
<213> Artificial sequence
<400> 8
attgctgttt acggtcctga tgacaggacc gttttccaac ctattaatca taaatatgaa 60
aaataattgt tgcatcaccc gccaatgcgt ggcttaatgc acatcaacgg tttgacgtac 120
agaccattaa agcagtgtag taaggcaagt cccttcaaga gttatcgttg atacccctcg 180
tagtgcacat tcctttaacg cttcaaaatc tgtaaagcac gccatatcgc cgaaaggcac 240
acttaattat taaaggtaat acact 265
Claims (10)
- An L-sorbose dehydrogenase mutant characterized in that valine at position 368 is mutated to cysteine, leucine, serine or threonine using L-sorbose dehydrogenase derived from Gluconobacter oxydans WSH-004 as a starting sequence.
- 2. The L-sorbose dehydrogenase mutant according to claim 1, wherein the amino acid sequence of said starting sequence is represented by SEQ ID No. 1.
- 3. A gene encoding the mutant of claim 1 or 2.
- 4. A recombinant plasmid carrying the gene of claim 3.
- 5. A microbial cell expressing the L-sorbose dehydrogenase mutant of claim 1 or 2 or carrying the gene of claim 3.
- 6. The microbial cell of claim 5, wherein the host cell of the microbial cell is a bacterial or fungal cell.
- 7. A recombinant Escherichia coli is characterized in that pMD19-T is used as an expression vector to express the L-sorbose dehydrogenase mutant and key enzymes in a 2-KLG synthesis path.
- 8. The recombinant Escherichia coli of claim 7, wherein the expression of the L-sorbose dehydrogenase gene and the L-sorbosone dehydrogenase gene is controlled by a cspA promoter.
- 9. Use of the L-sorbose dehydrogenase mutant according to any one of claims 1 to 2 or the recombinant escherichia coli according to any one of claims 7 to 8 for producing 2-keto-L-gulonic acid, vitamin C, or a product containing 2-keto-L-gulonic acid.
- 10. A method for producing 2-keto-L-gulonic acid, characterized in that the recombinant Escherichia coli of claim 7 or 8 is fermented at 30 to 37 ℃ in a sorbose-containing medium.
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Citations (5)
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|---|---|---|---|---|
| CN1120350A (en) * | 1993-03-08 | 1996-04-10 | 藤泽药品工业株式会社 | Novel L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
| WO2010100236A1 (en) * | 2009-03-05 | 2010-09-10 | Dsm Ip Assets B.V. | Improved production of 2-keto-l-gulonic acid |
| CN111979259A (en) * | 2020-08-07 | 2020-11-24 | 江南大学 | Gluconobacter oxydans shuttle vector for high-efficiency gene expression |
| CN113913400A (en) * | 2021-11-26 | 2022-01-11 | 江南大学 | L-sorbosone dehydrogenase mutant with improved catalytic activity |
| CN114480236A (en) * | 2022-02-23 | 2022-05-13 | 江南大学 | Construction method of 2-KLG (bacillus gluconicum) production one-step strain chassis cell bank |
-
2022
- 2022-06-29 CN CN202210756716.9A patent/CN114934027B/en active Active
Patent Citations (5)
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|---|---|---|---|---|
| CN1120350A (en) * | 1993-03-08 | 1996-04-10 | 藤泽药品工业株式会社 | Novel L-sorbose dehydrogenase and novel L-sorbosone dehydrogenase obtained from gluconobacter oxydans T-100 |
| WO2010100236A1 (en) * | 2009-03-05 | 2010-09-10 | Dsm Ip Assets B.V. | Improved production of 2-keto-l-gulonic acid |
| CN111979259A (en) * | 2020-08-07 | 2020-11-24 | 江南大学 | Gluconobacter oxydans shuttle vector for high-efficiency gene expression |
| CN113913400A (en) * | 2021-11-26 | 2022-01-11 | 江南大学 | L-sorbosone dehydrogenase mutant with improved catalytic activity |
| CN114480236A (en) * | 2022-02-23 | 2022-05-13 | 江南大学 | Construction method of 2-KLG (bacillus gluconicum) production one-step strain chassis cell bank |
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| "MULTISPECIES: GMC family oxidoreductase N-terminal domain-containing protein [Gluconobacter] NCBI Reference Sequence: WP_062447068.1" * |
| LI,L.等: "alanine-phosphoribitol ligase [Gluconobacter oxydans] GenBank: KXV13300.1" * |
| XIAOYU SHAN等: "High Throughput Screening Platform for a FAD-Dependent L-Sorbose Dehydrogenase" * |
| YUE CHEN等: "High-Throughput Screening of a 2-Keto-L-Gulonic Acid-Producing Gluconobacter oxydans Strain Based on Related Dehydrogenases" * |
| 陈玥等: "维生素C 生物合成相关脱氢酶研究进展" * |
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| Publication number | Publication date |
|---|---|
| CN114934027B (en) | 2023-08-25 |
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