CN112391300B - Application of flavone 3 beta-hydroxylase derived from silybum marianum and coenzyme thereof - Google Patents

Abstract

The invention discloses flavone 3 beta-hydroxylase derived from silybum marianum and application of coenzyme thereof, belonging to the field of genetic and metabolic engineering. SmF 3'H and SmCPR with flavone 3' -hydroxylase function are obtained and verified from silybum marianum, and have the capacity of catalyzing naringenin to synthesize eriodictyol. The SmF 3'H and the SmCPR used in the invention, more commonly used GhF 3' H and CrCPR, can improve the yield of eriodictyol generated by converting naringenin by 17.0 percent under the same culture condition. On the basis, promoters with different strengths are used for regulating SmF 3' H and SmCPR, so that the yield of eriodictyol is further improved, the yield of the eriodictyol can reach 805.6mg/L in a 250mL shake flask, and is increased by 302.8% compared with the reported highest yield of 200 mg/L. The silybum marianum-derived flavone 3 beta-hydroxylase and the coenzyme thereof have good performance of converting naringenin into eriodictyol, so that the silybum marianum-derived flavone 3 beta-hydroxylase has wide application prospect.

Description

Silybum marianum-derived flavone 3 beta-hydroxylase and application of coenzyme thereof

Technical Field

The invention relates to flavone 3 beta-hydroxylase derived from silybum marianum and application of coenzyme thereof, belonging to the field of gene and metabolic engineering.

Background

Eriodictyol is a natural flavanone compound widely existing in vegetables, fruits and traditional Chinese medicines, has various pharmacological activities such as oxidation resistance, inflammation resistance, tumor resistance, neuroprotection and the like, is commonly used for treating diseases such as asthma, allergic rhinitis, rheumatism and the like, has very high medicinal value, is also a precursor substance of taxifolin, anthocyanin, silybum marianum, chaulmoogra and the like, and has wide application value. Currently, eriodictyol is mainly extracted from plants directly, but in the extraction process, a large amount of energy is consumed to maintain high temperature, a large amount of chemical reagents are added, and the production efficiency is low, so that the ecological environment is greatly influenced.

At present, biological extraction, particularly microbial transformation, is reported, and although the method is safer and more environment-friendly than plant extraction, the method also has the problems of low yield and low transformation efficiency. The current main microbial transformation method is to catalyze naringenin by flavone 3 beta-hydroxylase F3' H under the combined action of coenzyme CPR to obtain the naringenin. A related study reported that F3 'H catalyzes naringenin to obtain the highest yield of eriodictyol of only 200mg/L (Amor, I.L., et al, Biotransformation of naringenin to eriodicine by Saccharomyces cerevisiae functional expressing flavone 3' hydroxyylase. nat Prod Commun,2010.5(12): p.1893-1898.). The yields of synthetic eriodictyol in E.coli are only 107mg/L at the highest (Zhu, S., et al., effective synthesis of eriodic from L-tyrosine in Escherichia coli. appl. Environ. Microbiol,2014.80(10): p.3072-3080.). This is because F3 'H and CPR need to anchor to the endoplasmic reticulum to transfer electrons efficiently (fig. 1), and therefore most of F3' H and CPR are poorly or even not active in prokaryotic microorganisms.

Therefore, the lack of efficient production of F3' H and CPR still causes the production of eriodictyol to be extremely limited, and the microbiological method cannot be well applied to industrial production.

Disclosure of Invention

For genes involved in a particular metabolic pathway, the expression level of each gene is often not optimal, and promoters of different strengths can be used to initiate each gene in the expression pathway and combined, followed by screening for high producing strains and sequencing of the high producing strains, i.e., the genotype for the optimal promoter combination can be obtained. Therefore, it is very important to find efficient F3 'H/CPR and adjust the expression level of F3' H/CPR by replacing the promoter, so as to obtain the genotype with the optimal expression ratio and the eriodictyol-producing strain thereof.

In order to obtain efficient F3 'H/CPR, the invention provides a pair of high activity F3' H derived from Silybum marianum (Silybum marianum) and coenzyme CPR thereof.

The invention provides a microbial cell expressing flavone 3 beta-hydroxylase and/or flavone 3 beta-hydroxylase coenzyme, wherein the amino acid sequence of the flavone 3 beta-hydroxylase is shown as SEQ ID NO. 2; the amino acid sequence of the flavone 3 beta-hydroxylase coenzyme is shown in SEQ ID NO. 4.

In one embodiment of the invention, the nucleotide sequence encoding said flavone 3 β -hydroxylase is shown in SEQ ID No. 1.

In one embodiment of the invention, the nucleotide sequence encoding the flavone 3 β -hydroxylase coenzyme is shown in SEQ ID No. 3.

In one embodiment of the invention, the promoter P is used _INO1 、P _SED1 、P _TPI1 、P _MET6 、P _FAS2 、P _GAL1 、P _LEU2 、P _ZWF1 、P _ARO7 And P _GLN1 Promoting the expression of flavone 3 beta-hydroxylase.

In one embodiment of the invention, the promoter P _INO1 、P _SED1 、P _TPI1 、P _MET6 、P _FAS2 、P _GAL1 、P _LEU2 、P _ZWF1 、P _ARO7 And P _GLN1 The nucleotide sequence of (A) is shown in SEQ ID NO.5-SEQ ID NO. 14.

In one embodiment of the invention, the promoter P is used _TDH1 、P _PGK1 、P _TDH3 、P _ERG20 、P _ADH6 、P _GAL10 、P _ADE2 、P _PMA1 、P _ADE6 And P _FAD1 Promoting the expression of the coenzyme of the flavone 3 beta-hydroxylase.

In one embodiment of the invention, the promoter P _TDH1 、P _PGK1 、P _TDH3 、P _ERG20 、P _ADH6 、P _GAL10 、P _ADE2 、P _PMA1 、P _ADE6 And P _FAD1 The nucleotide sequence of (A) is shown in SEQ ID NO.15-SEQ ID NO. 24.

In one embodiment of the invention, the promoter P is used _INO1 Promoting expression of flavone 3 beta-hydroxylase by using promoter P _TDH1 Promoting the expression of the coenzyme of the flavone 3 beta-hydroxylase.

In one embodiment of the invention, the promoter P is used _SED1 Promoting expression of flavone 3 beta-hydroxylase by using promoter P _TDH1 Initiating flavone 3 beta-hydroxyExpression of the coenzyme of the chemolase.

In one embodiment of the invention, the microbial cell is a prokaryotic or eukaryotic host.

In one embodiment of the invention, the microbial cell is saccharomyces cerevisiae strain C800.

The invention provides application of microbial cells in eriodictyol production, wherein naringenin is used as a substrate.

In one embodiment of the present invention, the microbial cells are added to the reaction system.

The invention provides a method for producing eriodictyol by whole-cell catalysis, which is characterized in that microbial cells are added into a reaction system.

In one embodiment of the present invention, the seed medium obtained by culturing at 25-35 ℃ and 200-250rpm for 16-18h is added to the reaction system in an amount of 1-5mL/100 mL.

The invention provides application of a method for producing eriodictyol by whole-cell catalysis in the production of eriodictyol or products taking eriodictyol as a raw material.

The invention has the beneficial effects that: SmF 3'H and SmCPR with flavone 3' -hydroxylase function are obtained from silybum marianum, and the SmF 3'H and SmCPR used in the invention, which are more commonly used as GhF 3' H and CrCPR, can improve the yield of eriodictyol generated by converting naringenin by 17.0 percent under the same culture condition. On the basis, promoters with different strengths are used for regulating SmF 3' H and SmCPR, so that the yield of eriodictyol is further improved, the yield of the eriodictyol can reach 805.6mg/L in a 250mL shake flask, and is increased by 302.8% compared with the reported highest yield of 200 mg/L. The acquisition of the high-eriodictyol-producing strain is of great significance for promoting the application of the high-eriodictyol in industry.

Drawings

FIG. 1 is a schematic diagram showing catalytic production of eriodictyol from naringenin by SmF 3' H/SmCPR.

FIG. 2 is a schematic diagram of promoter-optimized SmF 3' H/SmCPR.

FIG. 3 is a diagram of functional identification and catalytic capability verification of Silybum marianum source SmF 3' H/SmCPR.

FIG. 4 is a graph showing the yields of the 10 strains with the highest yield, the corresponding promoter genotypes and the promoter expression intensities after the promoter optimization.

Detailed Description

YNB medium: 0.72g/L yeast nitrogen source basic culture medium, 20g/L glucose, 50mg/L leucine, 50mg/L tryptophan and 50mg/L histidine.

YPD medium: 10g/L yeast powder, 20g/L peptone and 20g/L glucose.

2g/L agar powder is added into the solid culture medium.

Saccharomyces cerevisiae C800: the construction is described in Gao, S., et al, product-library-based scheduling for efficacy (2S) -naringenin production from p-homologous acid in Saccharomyces cerevisiae, 2020.68(25): p.6884-6891.

Example 1: amplification and identification of SmF 3' H and SmCPR

Extracting total RNA from silybum marianum pistil according to the kit instruction, and performing reverse transcription to obtain cDNA. Contig37917(SmF 3'H using primers SmF 3' H-F and SmF3 'H-R) and Contig668(SmCPR using primers SmCPR-F and SmCP-R) were each amplified from cDNA using primers to give PCR products, and the resulting PCR products were cloned into pMD19T-simple to give pMD-T-SmCPR and pMD-T-SmF 3' H, respectively.

Because the original plasmid pY26-TEF-GPD has a PmlI restriction site, the original plasmid pY26-TEF-GPF is amplified circularly by using primers mut-F and mut-R, caaggtttataa is obtained by self-ligation, a new restriction site PmlI is introduced into the plasmid pY26-TEF-GPD-mut, and pY26-TEF-GPD-mut is constructed and used for subsequently constructing a plasmid.

The reported genes GhF 3'H (GenBank ID: ABA64468.1) and CrCPR (GenBank ID: KM111538.1) were chemically synthesized, ligated to T vectors, and stored, respectively, as pUC57-GhF 3' H and pUC 57-CrCPR. pMD-T-SmF 3' H and pY26-TEF-GPD-mut are respectively cut by BamHI and PmlI endonucleases, and the cut fragments are respectively recovered and are connected to obtain pY 26-GR. pMD-T-SmCPR and pY26-GR are digested with NotI and PacI endonucleases respectively, and the digested fragments are recovered and ligated to obtain pY 26-THGR. pUC57-GhF 3' H and pY26-TEF-GPD were cut with BamHI and BspDI endonucleases in the same manner, and the cut fragments were recovered and ligated to obtain pY26-GR 2. pUC57-CrCPR and pY26-GR2 were digested with NotI and PacI endonucleases, respectively, and the digested fragments were recovered and ligated to obtain pY26-THGR 2.

Respectively transferring the plasmid vectors pY26-GR, pY26-THGR, pY26-GR2 and pY26-THGR2 obtained by the construction into Escherichia coli JM109, culturing at 37 ℃ until a monoclonal is grown out, selecting the monoclonal for sequencing, and extracting the plasmid from the positive transformant, wherein the sequencing verification is correct, namely the positive transformant. And respectively transforming the extracted plasmids pY26-GR, pY26-THGR, pY26-GR2 and pY26-THGR2 into a saccharomyces cerevisiae strain C800 by a lithium acetate transformation method to sequentially obtain strains C800GR, C800THGR, C800GR2 and C800THGR 2.

The amplification primers are shown in Table 1, and the strains and plasmids involved in the invention are shown in Table 2.

Primer sequences used in Table 1

TABLE 2 strains and plasmids related to the present invention

Example 2: verification of SmF 3' H and SmCPR catalytic function

Conditions for fermentation of Contig37917(SmF 3' H) and Contig668 (smcr) and strains C800GR, C800GR2, C800THGR and C800THGR2 in 250mL shake flasks: a single colony is picked and inoculated in a 250mL shake flask containing 20mL YNB liquid medium, and cultured for 16-18h at 30 ℃ and 220rpm to obtain a seed culture medium. The seed culture medium was transferred at 2mL/100mL into a 250mL shake flask containing 20mL fresh YNB broth containing 250 mg.L ^-1 Naringenin was cultured at 30 ℃ and 220rpm for 72 hours, and then the amount of eriodictyol produced was measured.

And diluting fermentation liquor of strains Contig37917, Contig668, C800GR, C800GR2, C800THGR and C800THGR2 by using methanol for one time, shaking for 30s, uniformly mixing, centrifuging at 13500rpm for 5min, filtering, and detecting the yield of the eriodictyol by using a high performance liquid phase. 250mL shake flask detection: mu.L of the fermentation medium was mixed with 900. mu.L of methanol, vortexed and shaken for 30s, centrifuged at 13500rpm for 5min and then filtered. The supernatant was filtered and prepared for HPLC analysis.

Samples were detected using an Agilent high performance liquid chromatography (Agilent 1100, US), C18 reverse phase chromatography column (4.6 mm. times.250 mm, Thermo), at a column temperature of 25 ℃. The mobile phase is methanol: water (41:59) and 3% per mill phosphoric acid. The flow rate is 1mL/min, the sample injection amount is 10 muL, and the detection wavelength is 290 nm.

Through verification, Contig37917 and Contig668 derived from silybum marianum are identified as having the capability of catalyzing naringenin to synthesize eriodictyol, and the catalytic capability is stronger than that of the conventional GhF 3' H/CrCPR. Under the same 250mL shake flask culture condition, the common GhF3 'H/CrCPR can synthesize 49.5mg/L eriodictyol, while the silybum marianum-derived SmF 3' H/SmCPR can synthesize 57.9mg/L, and the yield is increased by 17.0%. Thus, Contig37917(SmF 3' H) and Contig668(SmCPR) showed good eriodictyol production.

Example 3: construction of promoter-optimized SmF 3' H and SmCPR expression level library

20 gradient promoters were selected from the reported Promoter library (Gao, S., et al., Promoter-library-based pathway optimization for efficacy (2S) -naringenin production from p-Promoter acid in Saccharomyces cerevisiae J agricultural Food Chem,2020.68(25): p.6884-6891.) for optimization of expression levels of SmF 3' H and SmCPRR. The 20 promoters were divided into A, B groups. The starter group A contains P _INO1 、P _SED1 、P _TPI1 、P _MET6 、P _FAS2 、P _GAL1 、P _LEU2 、P _ZWF1 、P _ARO7 And P _GLN1 (the sequence of which corresponds in sequence to SEQ ID NO.5-SEQ ID NO.14) for fusion and initiation of transcription of SmF 3' H. The starter subgroup B contains P _TDH1 、P _PGK1 、P _TDH3 、P _ERG20 、P _ADH6 、P _GAL10 、P _ADE2 、P _PMA1 、P _ADE6 And P _FAD1 (the sequence of which corresponds in turn to SEQ ID NO.15-SEQ ID NO.24) for fusion and initiation of transcription SmCPR.

Vector backbones (expression cassettes containing Promoter amplification sequences and SmcXX-homo-F) were amplified from plasmid pMD19T-XXX (plasmid pMD19T-XXX is the name of the Promoter for ligating the above Promoter to the pMD19T vector, XXX is the name of the Promoter, see Gao, S., et al, Promoter-library-based therapy optimization for efficacy (2S) -naringenin production from p-Promoter acid Saccharomyces cerevisiae J Agric Food Chem,2020.68(25): p.6884-6891.), using primers pY26-THGR-homo-F and pY26-THGR-homo-R, respectively, from plasmid pY26-THGR (expression cassettes containing Promoter amplification sequences and the name of the CPR-3 vector, respectively). The PCR products of the vector backbone, promoter groups A and B were purified separately, and then the three products were mixed to 50. mu.L (promoter group A: promoter group B: vector backbone ═ 2: 2: 1, mol/mol, total about 2-3. mu.g), the mixed system was transformed into s.cerevisiae strain C800 by lithium acetate high efficiency Transformation (specific Transformation methods see: Gietz, R.D. and R.A. woods, Transformation of yeast by little salt acetate/single-stranded carrier DNA/polyethylene glycol method. methods enzyme, 2002.350: p.87-96.), and all the fragments were assembled in s.cerevisiae. The transformation system is coated on an YNB agar plate, and the agar plate is incubated for 3-4 days at 30 ℃ to obtain the random assembled library of the promoter.

Primer sequences used in Table 3

Example 4: screening of promoter-optimized strains

2000 single colonies were picked at random from the randomly assembled library and cultured using 48 deep well plates: the colonies on the plate were automatically inoculated into a 48-deep well plate using an automated colony picking instrument QPix 420. 1.5mL YNB liquid medium was added to each well, and the final concentration of the medium was 250 mg.L ^-1 Naringenin. The deep well plate was transferred to a well plate shaker and incubated at 30 ℃ and 220rpm for 48 hours. The deep well plate was placed on a table for 2 hours to pellet the cells and the supernatant was available for high throughput screening.

High throughput detection method: after the strains in the random assembly library are fermented in a 48-deep-hole plate, 100 mu L of fermentation supernatant is automatically taken and transferred into a 96-shallow-hole enzyme label plate hole by using an automatic workstation equipped with an enzyme label instrument, and then 100 mu L of 4M KOH is added. After about 5min, the mixture turned purple, with the shade of purple being proportional to the concentration of eriodictyol, and the high producing strains could be directly screened visually. Automatically transferring the color-changing elisa plate to an elisa plate reader, detecting the light absorption value of the mixture at 550nm, and selecting the strains with high light absorption value to enter a shake flask for rescreening.

The detection method comprises the following steps: diluting the 48-deep-hole plate fermentation liquor by one time with methanol, shaking for 30s, mixing uniformly, centrifuging at 13500rpm for 5min, filtering, and detecting the yield of eriodictyol by using a high performance liquid phase. 250mL shake flask detection: mu.L of the fermentation medium was mixed with 900. mu.L of methanol, vortexed and shaken for 30s, centrifuged at 13500rpm for 5min and then filtered. The supernatant was filtered and prepared for HPLC analysis.

Samples were detected using an Agilent high performance liquid chromatography (Agilent 1100, US), C18 reverse phase chromatography column (4.6 mm. times.250 mm, Thermo), at a column temperature of 25 ℃. The mobile phase is methanol: water (41:59) and 3% o phosphoric acid. The flow rate is 1mL/min, the sample injection amount is 10 muL, and the detection wavelength is 290 nm.

Through promoter optimization, 10 strains with the highest yield are selected, and the eriodictyol yield accumulated at the level of a 48-deep-well plate is respectively 29.2mg/L, 29.9mg/L, 30.7mg/L, 30.8mg/L, 31.9mg/L, 33.0mg/L, 35.5mg/L, 36.6mg/L, 46.2mg/L and 51.8mg/L from low to high. Subsequent sequencing of the genotypes of the 10 strains, their genotypes and the corresponding promoter expression intensities are listed in figure 4. While the starting strain did not detect the formation of eriodictyol.

The 10 strains with the highest eriodictyol yield are selected for rescreening and sequencing, and 250mL shake flask rescreening is carried out: selecting a single colony, inoculating the single colony in a 250mL shake flask containing 20mL YNB liquid culture medium, culturing at 30 ℃ and 220rpm for 16-18h to obtain a seed culture medium; the seed medium was transferred at 2mL/100mL into a 250mL shake flask containing 20mL fresh YPD liquid medium, and the yield of eriodictyol was measured after culturing at 30 ℃ and 220rpm for 72 hours. At 0h, 12h, 24h and 36h, 375 mg. L are respectively added ^-1 Naringenin is added into YPD culture medium, and eriodictyol content is measured after fermentation.

The results of rescreening the two strains with the highest yield at the level of a 250mL shake flask show that the two strains can respectively accumulate 720.4mg/L and 805.6mg/L eriodictyol, and the yields are increased by 260.2 percent and 302.8 percent compared with the reported highest yield, the plasmids in the strains are pY26-P04 and pY26-P05 respectively, and the genotypes corresponding to pY26-P05 are pY26-P _INO1 -SmF3′H-P _TDH1 -SmCPR, pY26-P04 corresponds to the genotype pY26-P _SED1 -SmF3′H-P _TDH1 -SmCPR。

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

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<120> flavone 3 beta-hydroxylase of silybum marianum source and application of coenzyme thereof

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ggtcatgctg ttcatgatgc tcaacatcca tgcagatcca atgtggctgt taaaaaggag 960

ctccataccc ctgaatctga tcgctcttgc acgcatctgg aatttgacat ctcacacact 1020

ggactatcat acgaaactgg ggaccatgtt ggtgtctact gtgagaactt aagtgaagtt 1080

gtggaggagg ctgagaggtt aataggttta ccatcggata cttatttctc agttcacacg 1140

gataacgaag atggaacacc acttggtgga gcttccttac tacctccttt ccctccatgc 1200

actttaagaa aagcattggc taattacgca gatgtattga cttctcccaa aaagtcggcc 1260

ttgattgctc tagctgctca tgcttctgat cctactgaag ctgaacgact aaaatttctt 1320

gcatctcctg ctgggaagga tgaatattct caatggatta ttgcaagcca aagaagcctg 1380

cttgaggtca tggaagcttt cccatcggct aagcctccac ttggggtttt ctttgcagct 1440

attgctccac gcttacagcc tcgatactac tctatttctt cctccccgaa gatggcacct 1500

agcaggattc atgttacttg tgcattagtt tatgagaaaa cacctgcagg ccgtctccat 1560

aaaggaatct gttcaacctg gatgaagaat gctgtgccta tgacggaaag tcaggattgc 1620

agctgggcac ctattttcgt tagaacgtct aacttcagac ttcccactga tcctaaagtt 1680

cctgttatca tgattggccc tggaaccgga ttggctccgt tcagaggttt tcttcaagaa 1740

agattagctc tgaaggaagc cggaactgaa ctgggatcat ccattttatt cttcggatgt 1800

agaaatcgca aagtggattt catatatgag aatgaactga aagactttgt tgagaatggt 1860

gctgtttccg agcttattgt tgccttctcc cgtgaaggcc ccaataagga atatgtgcaa 1920

cataaaatga gcgatagggc ttcggatcta tggaacttgc tttcggaggg agcatattta 1980

tacgtttgtg gtgatgccaa aggcatggct aaagatgtac accggaccct tcacacaatt 2040

gtgcaagaac agggatctct agactcgtca aaggcagagc tgtatgtgaa gaatctacaa 2100

atgtcaggaa gatacctccg tgatgtttgg tag 2133

<210> 4

<211> 710

<212> PRT

<213> Silybum marianum

<400> 4

Met Gln Ser Asp Ser Ser Leu Glu Thr Ser Ser Phe Asp Leu Ile Thr

1 5 10 15

Ala Ala Leu Lys Glu Lys Val Ile Asp Thr Ala Asn Ala Ser Asp Ser

20 25 30

Gly Asp Ser Thr Met Pro Pro Ala Leu Ala Met Ile Leu Glu Asn Arg

35 40 45

Glu Leu Phe Met Met Leu Thr Thr Thr Val Ala Leu Leu Leu Gly Phe

50 55 60

Ile Val Val Ser Phe Trp Lys Arg Ser Ser Glu Lys Lys Ser Ala Lys

65 70 75 80

Asp Leu Glu Leu Pro Lys Ile Val Val Pro Lys Arg Gln Gln Glu Gln

85 90 95

Glu Val Asp Asp Gly Lys Lys Lys Val Thr Ile Leu Phe Gly Thr Gln

100 105 110

Thr Gly Thr Ala Glu Gly Phe Ala Lys Ala Leu Leu Glu Glu Ala Lys

115 120 125

Ala Arg Tyr Glu Lys Ala Thr Phe Lys Val Val Asp Leu Asp Asp Tyr

130 135 140

Ala Val Asp Asp Asp Glu Tyr Glu Glu Lys Leu Lys Lys Glu Ser Phe

145 150 155 160

Ala Phe Phe Phe Leu Ala Thr Tyr Gly Asp Gly Glu Pro Thr Asp Asn

165 170 175

Ala Ala Arg Phe Tyr Lys Trp Phe Thr Glu Gly Gly Glu Lys Gly Val

180 185 190

Trp Leu Glu Lys Leu Gln Tyr Gly Val Phe Gly Leu Gly Asn Arg Gln

195 200 205

Tyr Glu His Phe Asn Lys Ile Ala Lys Glu Val Asp Asp Gly Leu Ala

210 215 220

Glu Gln Gly Ala Lys Arg Leu Val Pro Val Gly Leu Gly Asp Asp Asp

225 230 235 240

Gln Ser Ile Glu Asp Asp Phe Thr Ala Trp Lys Glu Leu Val Trp Pro

245 250 255

Glu Leu Asp Glu Leu Leu Arg Asp Glu Asp Asp Lys Gly Val Ala Thr

260 265 270

Pro Tyr Thr Ala Ala Ile Pro Glu Tyr Arg Val Val Phe His Glu Lys

275 280 285

His Asp Thr Ser Ala Glu Asp Gln Ile Gln Thr Asn Gly His Ala Val

290 295 300

His Asp Ala Gln His Pro Cys Arg Ser Asn Val Ala Val Lys Lys Glu

305 310 315 320

Leu His Thr Pro Glu Ser Asp Arg Ser Cys Thr His Leu Glu Phe Asp

325 330 335

Ile Ser His Thr Gly Leu Ser Tyr Glu Thr Gly Asp His Val Gly Val

340 345 350

Tyr Cys Glu Asn Leu Ser Glu Val Val Glu Glu Ala Glu Arg Leu Ile

355 360 365

Gly Leu Pro Ser Asp Thr Tyr Phe Ser Val His Thr Asp Asn Glu Asp

370 375 380

Gly Thr Pro Leu Gly Gly Ala Ser Leu Leu Pro Pro Phe Pro Pro Cys

385 390 395 400

Thr Leu Arg Lys Ala Leu Ala Asn Tyr Ala Asp Val Leu Thr Ser Pro

405 410 415

Lys Lys Ser Ala Leu Ile Ala Leu Ala Ala His Ala Ser Asp Pro Thr

420 425 430

Glu Ala Glu Arg Leu Lys Phe Leu Ala Ser Pro Ala Gly Lys Asp Glu

435 440 445

Tyr Ser Gln Trp Ile Ile Ala Ser Gln Arg Ser Leu Leu Glu Val Met

450 455 460

Glu Ala Phe Pro Ser Ala Lys Pro Pro Leu Gly Val Phe Phe Ala Ala

465 470 475 480

Ile Ala Pro Arg Leu Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro

485 490 495

Lys Met Ala Pro Ser Arg Ile His Val Thr Cys Ala Leu Val Tyr Glu

500 505 510

Lys Thr Pro Ala Gly Arg Leu His Lys Gly Ile Cys Ser Thr Trp Met

515 520 525

Lys Asn Ala Val Pro Met Thr Glu Ser Gln Asp Cys Ser Trp Ala Pro

530 535 540

Ile Phe Val Arg Thr Ser Asn Phe Arg Leu Pro Thr Asp Pro Lys Val

545 550 555 560

Pro Val Ile Met Ile Gly Pro Gly Thr Gly Leu Ala Pro Phe Arg Gly

565 570 575

Phe Leu Gln Glu Arg Leu Ala Leu Lys Glu Ala Gly Thr Glu Leu Gly

580 585 590

Ser Ser Ile Leu Phe Phe Gly Cys Arg Asn Arg Lys Val Asp Phe Ile

595 600 605

Tyr Glu Asn Glu Leu Lys Asp Phe Val Glu Asn Gly Ala Val Ser Glu

610 615 620

Leu Ile Val Ala Phe Ser Arg Glu Gly Pro Asn Lys Glu Tyr Val Gln

625 630 635 640

His Lys Met Ser Asp Arg Ala Ser Asp Leu Trp Asn Leu Leu Ser Glu

645 650 655

Gly Ala Tyr Leu Tyr Val Cys Gly Asp Ala Lys Gly Met Ala Lys Asp

660 665 670

Val His Arg Thr Leu His Thr Ile Val Gln Glu Gln Gly Ser Leu Asp

675 680 685

Ser Ser Lys Ala Glu Leu Tyr Val Lys Asn Leu Gln Met Ser Gly Arg

690 695 700

Tyr Leu Arg Asp Val Trp

705 710

<210> 5

<211> 510

<212> DNA

<213> Artificial sequence

<400> 5

gaagacgatg aggccggtgc cgatgtgccc ttgatggaca acaaacaaca gctctcttcc 60

ggccgtactt agtgatcgga acgagctctt tatcaccgta gttctaaata acacatagag 120

taaattattg cctttttctt cgttcctttt gttcttcacg tcctttttat gaaatacgtg 180

ccggtgttcc ggggttggat gcggaatcga aagtgttgaa tgtgaaatat gcggaggcca 240

agtatgcgct tcggcggcta aatgcggcat gtgaaaagta ttgtctattt tatcttcatc 300

cttctttccc agaatattga acttatttaa ttcacatgga gcagagaaag cgcacctctg 360

cgttggcggc aatgttaatt tgagacgtat ataaattgga gctttcgtca cctttttttg 420

gcttgttctg ttgtcgggtt cctaatgtta gttttatcct tgatttattc tgtttcattc 480

cctttttttt ccagtgaaaa agaagtaaca 510

<210> 6

<211> 550

<212> DNA

<213> Artificial sequence

<400> 6

ctaccttcca tacaccactg attgctccac gtcatgcggc cttctttcga ggacaaaaag 60

gcatatatcg ctaaaattag ccatcagaac cgttattgtt attatatttt cattacgaaa 120

gaggagaggg cccagcgcgc cagagcacac acggtcattg attactttat ttggctaaag 180

atccatccct tctcgatgtc atctctttcc attcttgtgt atttttgatt gaaaatgatt 240

ttttgtccac taatttctaa aaataagaca aaaagccttt aagcagtttt tcatccattt 300

tactacggta aaatgaatta gtacggtatg gctcccagtc gcattatttt tagattggcc 360

gtaggggctg gggtagaact agagtaagga acattgctct gccctctttt gaactgtcat 420

ataaatacct gacctatttt attctccatt atcgtattat ctcacctctc tttttctatt 480

ctcttgtaat tattgattta tagtcgtaac tacaaagaca agcaaaataa aatacgttcg 540

ctctattaag 550

<210> 7

<211> 623

<212> DNA

<213> Artificial sequence

<400> 7

acccaaatgg actgattgtg agggagacct aactacatag tgtttaaaga ttacggatat 60

ttaacttact tagaataatg ccattttttt gagttataat aatcctacgt tagtgtgagc 120

gggatttaaa ctgtgaggac cttaatacat tcagacactt ctgcggtatc accctactta 180

ttcccttcga gattatatct aggaacccat caggttggtg gaagattacc cgttctaaga 240

cttttcagct tcctctattg atgttacacc tggacacccc ttttctggca tccagttttt 300

aatcttcagt ggcatgtgag attctccgaa attaattaaa gcaatcacac aattctctcg 360

gataccacct cggttgaaac tgacaggtgg tttgttacgc atgctaatgc aaaggagcct 420

atataccttt ggctcggctg ctgtaacagg gaatataaag ggcagcataa tttaggagtt 480

tagtgaactt gcaacattta ctattttccc ttcttacgta aatatttttc tttttaattc 540

taaatcaatc tttttcaatt ttttgtttgt attcttttct tgcttaaatc tataactaca 600

aaaaacacat acataaacta aaa 623

<210> 8

<211> 580

<212> DNA

<213> Artificial sequence

<400> 8

catgaaccag ggtcccgcac tccgggtaaa ggaccatcac gccacatcac gtgcacatta 60

ctagtaaaag ccacaggaaa tatttcacgt gacttacaaa cagagtcgta cgtcaggacc 120

ggagtcaggt gaaaaaatgt gggccggtaa agggaaaaaa ccagaaacgg gactactatc 180

gaactcgttt agtcgcgaac gtgcaaaagg ccaatatttt tcgctagagt catcgcagtc 240

atggcagctc tttcgctcta tctcccggtc gcaaaactgt ggtagtcata gctcgttctg 300

ctcaattgag aactgtgaat gtgaatatgg aacaaatgcg atagatgcac taatttaagg 360

gaagctagct agttttccca actgcgaaag aaaaaaagga aagaaaaaaa aattctatat 420

aagtgataga tatttccatc tttactagca ttagtttctc ttttacgtat tcaatatttt 480

tgttaaactc ttcctttatc ataaaaaagc aagcatctaa gagcattgac aacactctaa 540

gaaacaaaat accaatataa tttcaaagta catatcaaaa 580

<210> 9

<211> 543

<212> DNA

<213> Artificial sequence

<400> 9

gttgtcgttg ttgtcccagc cgttgtcaaa acgcgttaat tccaactatt ttctatattt 60

ctattctatc cgaactcccc ttttgtatat caatatatct taatactttc gcctattctt 120

tactatattt cctaaatttt ctctggtctg caggccaaaa acaacaactt actactgaat 180

catggacgtg tatttagttt agccaagcaa tatttaaata tcactcttcc taaaaataca 240

ttgggcatta cccgcaaact aacccatcgc ttagcaaaat ccaaccattt tttttttatc 300

tcccgcgttt tcacatgcta cctcattcgc ctcgtaacgt tacgaccgaa atctcactaa 360

ggcacggttt gttgggcagt ttacagatgt tggataacca gttgtttcta aacggttatg 420

cctcatatat aacttgttaa ctgaaggtta cacaagacca catcaccact gtcgtgcttt 480

tctaataacc gctatattag acgtttaaag ggctacagca acaccaattg aaataccatc 540

att 543

<210> 10

<211> 668

<212> DNA

<213> Artificial sequence

<400> 10

ttatattgaa ttttcaaaaa ttcttacttt ttttttggat ggacgcaaag aagtttaata 60

atcatattac atggcattac caccatatac atatccatat ctaatcttac ttatatgttg 120

tggaaatgta aagagcccca ttatcttagc ctaaaaaaac cttctctttg gaactttcag 180

taatacgctt aactgctcat tgctatattg aagtacggat tagaagccgc cgagcgggcg 240

acagccctcc gacggaagac tctcctccgt gcgtcctcgt cttcaccggt cgcgttcctg 300

aaacgcagat gtgcctcgcg ccgcactgct ccgaacaata aagattctac aatactagct 360

tttatggtta tgaagaggaa aaattggcag taacctggcc ccacaaacct tcaaattaac 420

gaatcaaatt aacaaccata ggatgataat gcgattagtt ttttagcctt atttctgggg 480

taattaatca gcgaagcgat gatttttgat ctattaacag atatataaat ggaaaagctg 540

cataaccact ttaactaata ctttcaacat tttcagtttg tattacttct tattcaaatg 600

tcataaaagt atcaacaaaa aattgttaat atacctctat actttaacgt caaggagaaa 660

aaactata 668

<210> 11

<211> 544

<212> DNA

<213> Artificial sequence

<400> 11

tcctgtactt ccttgttcat gtgtgttcaa aaacgttata tttataggat aattatactc 60

tatttctcaa caagtaattg gttgtttggc cgagcggtct aaggcgcctg attcaagaaa 120

tatcttgacc gcagttaact gtgggaatac tcaggtatcg taagatgcaa gagttcgaat 180

ctcttagcaa ccattatttt tttcctcaac ataacgagaa cacacagggg cgctatcgca 240

cagaatcaaa ttcgatgact ggaaattttt tgttaatttc agaggtcgcc tgacgcatat 300

acctttttca actgaaaaat tgggagaaaa aggaaaggtg agagcgccgg aaccggcttt 360

tcatatagaa tagagaagcg ttcatgacta aatgcttgca tcacaatact tgaagttgac 420

aatattattt aaggacctat tgttttttcc aataggtggt tagcaatcgt cttactttct 480

aacttttctt accttttaca tttcagcaat atatatatat atatttcaag gatataccat 540

tcta 544

<210> 12

<211> 600

<212> DNA

<213> Artificial sequence

<400> 12

gccgtcgaaa aggatctcgt ctctgttggg agcacctggt aagtaaggtg tagttttgca 60

cccgtgtaca taagcgtgaa atcaccacaa actgtgtgta tcaagtacat agtgacattt 120

aaataatagc aagaacaaca ataatagtag cgctactgga agcaccacgt aatagtggaa 180

aagaactgga aaaaccgcta taagatgcat actccggcgg tcttacgcgg agatacaagc 240

ttccaacggt gctaaaagcc cggtttcggc tcggccggag gaggaagaga gacgaaaaaa 300

aaaaaaatga ctaaaaaaaa aatggaatat tattaatgtg ggatttttgg ctcaaggtgt 360

ggtggcccct tttctaaggg tggcgaattc ttcaatgtac ggaaaactcg ccaaggctat 420

cccatatata agcaaactgt gggttcatct atataccgac acataacacc taaagtggct 480

tcctcctgcc cctctctccc ttttctccac tcacccctcc ttctccccct tccccctctc 540

caattggctg tatagacaga aagagtaaat ccaatagaat agaaaaccac ataaggcaag 600

<210> 13

<211> 520

<212> DNA

<213> Artificial sequence

<400> 13

tggattacat ttgattcagt catacacgaa ttatggtctt gatactgaca aattttccag 60

attgaggcgg ttcttatggt ttagaacttg gggactttac aagtcgaaag aggatttaga 120

tagagaagcc aagatcaatg aagaaatgat acgcaaactg aaagcagcta aatgaaatca 180

cctattgcgc cgctcgcgga atacaattac taaattttat atatattctt taaaaatgca 240

tctatacatt cgtttttcca cgtataccaa attcgaaaaa agttgttaaa ccatcgtttt 300

cacgtttttt aatttttttt tggttctctt tttttttttt tttcaatatc aacttttttt 360

caaacttcgt gttgcatttc ctttatcgta aattttcaat ggatctctat aatcttcgaa 420

gttcgaagaa aagaagaaaa aaagtattga aaagttgaaa catcgattcc gttttgctaa 480

caaatagcac tcagcatcct gcataaaatt ggtataagat 520

<210> 14

<211> 553

<212> DNA

<213> Artificial sequence

<400> 14

ggtccgcaca gacgatgcca gacggtgttt tatcgaaaat ttttttcgca tcatagtgcc 60

atttgtggtc attattattc cccaaatatg cgaaaatagt acactatttt tggcaggaga 120

gtaggctgat atgccgcatt gatgtcctgt gtagcgaaac acaaacaaaa aaagaaaaag 180

taggatgaaa aaaagaaaag taatatgaaa aaagagtgaa aaattaattc atttgttagt 240

gtaagcggtc aggtgtaagt agtaggcttg ataatgaatt aaagatgact ccgacgcata 300

ttgtttgcca tgtttttatt ttagtttgta gatttctttt tttgtaatat ataagggagt 360

gattctatat atcgaattct caggcttggt tggttcgtag gttgttctgt ctttgttttc 420

gttaggtaag aacatcacac aaagataact atagaatcac atacatattt gtgagaaatt 480

aacttcattt catttataga agaagttcaa ccgaaacaaa aattaaacat aatataatat 540

aatataatca aaa 553

<210> 15

<211> 530

<212> DNA

<213> Artificial sequence

<400> 15

gaaaccacac cgtggggcct tgttgcgcta ggaataggat atgcgacgaa gacgcttctg 60

cttagtaacc acaccacatt ttcagggggt cgatctgctt gcttccttta ctgtcacgag 120

cggcccataa tcgcgctttt tttttaaaag gcgcgagaca gcaaacagga agctcgggtt 180

tcaaccttcg gagtggtcgc agatctggag actggatctt tacaatacag taaggcaagc 240

caccatctgc ttcttaggtg catgcgacgg tatccacgtg cagaacaaca tagtctgaag 300

aaggggggga ggagcatgtt cattctctgt agcagtaaga gcttggtgat aatgaccaaa 360

actggagtct cgaaatcata taaatagaca atatattttc acacaatgag atttgtagta 420

cagttctatt ctctctcttg cataaataag aaattcatca agaacttggt ttgatatttc 480

accaacacac acaaaaaaca gtacttcact aaatttacac acaaaacaaa 530

<210> 16

<211> 700

<212> DNA

<213> Artificial sequence

<400> 16

gtgagtaagg aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc 60

gcgaatcctt tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt 120

ttccctcctt cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga 180

aattaccgtc gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc 240

tcgacttcct gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag 300

cgacggctca caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt 360

agtaccacat gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg 420

ttactctctc tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca 480

cacactcttt tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac 540

atttacatat atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt 600

tctaattcgt agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc 660

aaggaagtaa ttatctactt tttacaacaa atataaaaca 700

<210> 17

<211> 698

<212> DNA

<213> Artificial sequence

<400> 17

ataaaaaaca cgctttttca gttcgagttt atcattatca atactgccat ttcaaagaat 60

acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa ttagcctttt 120

aattctgctg taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca 180

tcgtaggtgt ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct 240

ttttaagctg gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc 300

aaccatcagt tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag 360

gcaaaaaacg ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac 420

aaggcaattg acccacgcat gtatctatct cattttctta caccttctat taccttctgc 480

tctctctgat ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc 540

cctacttgac taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat 600

ttcttaaact tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca 660

agaacttagt ttcgaataaa cacacataaa caaacaaa 698

<210> 18

<211> 530

<212> DNA

<213> Artificial sequence

<400> 18

gaaaccacac cgtggggcct tgttgcgcta ggaataggat atgcgacgaa gacgcttctg 60

cttagtaacc acaccacatt ttcagggggt cgatctgctt gcttccttta ctgtcacgag 120

cggcccataa tcgcgctttt tttttaaaag gcgcgagaca gcaaacagga agctcgggtt 180

tcaaccttcg gagtggtcgc agatctggag actggatctt tacaatacag taaggcaagc 240

caccatctgc ttcttaggtg catgcgacgg tatccacgtg cagaacaaca tagtctgaag 300

aaggggggga ggagcatgtt cattctctgt agcagtaaga gcttggtgat aatgaccaaa 360

actggagtct cgaaatcata taaatagaca atatattttc acacaatgag atttgtagta 420

cagttctatt ctctctcttg cataaataag aaattcatca agaacttggt ttgatatttc 480

accaacacac acaaaaaaca gtacttcact aaatttacac acaaaacaaa 530

<210> 19

<211> 807

<212> DNA

<213> Artificial sequence

<400> 19

acgcacgtag gcgcatactt ctatcatacg tagaaaagcg tttcccagag gttatgatgt 60

tggaaaaata gcttcattga acattttagc attagtggga agaattcctc atgttactta 120

tgagggagtt aattttcctt agggatttta ggaatttcta tacggaggta gcgatcgacc 180

ttagaacttt tatttagttt gtacatatac ctcacctgag ttttgctttt ttctctggga 240

gcctaaacca tttaaaatga tatataatag ataataaatc caggataaaa tgtggctaat 300

tgatcttttt tcattttcaa cttggtaatg acgtactgga tactttcgac gctctttttt 360

agtccccgat ccccgtcttc caggaccttg acgtggaatt ccgatcacag ccactctcgt 420

cacggctccg ttaaaatgaa tggttttccg ttacatttac tggtcttttt atctttttac 480

agtaaatggg tgatatactg tgacacaatt tgtgtctcta ctgtgtgaac ttccattgct 540

gactaaagat tccccgctcc gcttatatgt ccggtccgtc cttgaccgaa gatcacattg 600

ccaatttttc acatctggaa gcgatacgac aatataggag aaaaagaaaa gtgaaaggca 660

aaaaagcacc aacagttctc gaggtgaagt gccgtcaatc ttctgtataa attcggccaa 720

ttcaatctaa tttaatagat ttgcgacaga ctttcacatc cacattcgag gaagaaattc 780

aacacaacaa caagaaaagc caaaatc 807

<210> 20

<211> 668

<212> DNA

<213> Artificial sequence

<400> 20

tatagttttt tctccttgac gttaaagtat agaggtatat taacaatttt ttgttgatac 60

ttttatgaca tttgaataag aagtaataca aactgaaaat gttgaaagta ttagttaaag 120

tggttatgca gcttttccat ttatatatct gttaatagat caaaaatcat cgcttcgctg 180

attaattacc ccagaaataa ggctaaaaaa ctaatcgcat tatcatccta tggttgttaa 240

tttgattcgt taatttgaag gtttgtgggg ccaggttact gccaattttt cctcttcata 300

accataaaag ctagtattgt agaatcttta ttgttcggag cagtgcggcg cgaggcacat 360

ctgcgtttca ggaacgcgac cggtgaagac gaggacgcac ggaggagagt cttccgtcgg 420

agggctgtcg cccgctcggc ggcttctaat ccgtacttca atatagcaat gagcagttaa 480

gcgtattact gaaagttcca aagagaaggt ttttttaggc taagataatg gggctcttta 540

catttccaca acatataagt aagattagat atggatatgt atatggtggt aatgccatgt 600

aatatgatta ttaaacttct ttgcgtccat ccaaaaaaaa agtaagaatt tttgaaaatt 660

caatataa 668

<210> 21

<211> 689

<212> DNA

<213> Artificial sequence

<400> 21

ctagtaacgc cgtatcgtga ttaacgtatt acataagtta caggattcat gcttatgggt 60

tagctatttc gcccaatgtg tccatctgac attactattt tgcattttaa tttaattaga 120

acttgactag cgcactacca gtatatcatc tcatttccgt aaataccaaa tgtattatat 180

attgaaagct tttgaccagg ttattataaa agaaacttca tgctcgaaaa agatcatttc 240

gaaaagttgc ctagtttcat gaaattttaa agcagtttat ataaatttta ccttttgatg 300

cggaattgac tttttcttga ataatacata acttttctta aaagaatcaa agacagataa 360

aatttaagag atattaaata ttagtgagaa gccgagaatt ttgtaacacc aacataacac 420

tgacatcttt aacaactttt aattatgata catttcttac gtcatgattg attattacag 480

ctatgctgac aaatgactct tgttgcatgg ctacgaaccg ggtaatacta agtgattgac 540

tcttgctgac cttttattaa gaactaaatg gacaatatta tggagcattt catgtataaa 600

ttggtgcgta aaatcgttgg atctctcttc taagtacatc ctactataac aatcaagaaa 660

aacaagaaaa tcggacaaaa caatcaagt 689

<210> 22

<211> 545

<212> DNA

<213> Artificial sequence

<400> 22

ccctcgttca cagaaagtct gaagaagcta tagtagaact atgagctttt tttgtttctg 60

ttttcctttt tttttttttt acctctgtgg aaattgttac tctcacactc tttagttcgt 120

ttgtttgttt tgtttattcc aattatgacc ggtgacgaaa cgtggtcgat ggtgggtacc 180

gcttatgctc ccctccatta gtttcgatta tataaaaagg ccaaatattg tattattttc 240

aaatgtccta tcattatcgt ctaacatcta atttctctta aattttttct ctttctttcc 300

tataacacca atagtgaaaa tctttttttc ttctatatct acaaaaactt tttttttcta 360

tcaacctcgt tgataaattt tttctttaac aatcgttaat aattaattaa ttggaaaata 420

accatttttt ctctctttta tacacacatt caaaagaaag aaaaaaaata taccccagct 480

agttaaagaa aatcattgaa aagaataaga agataagaaa gatttaatta tcaaacaata 540

tcaat 545

<210> 23

<211> 508

<212> DNA

<213> Artificial sequence

<400> 23

ctgaacgtat cgagactcgg ttgtgtcgtt atgctagcaa tgtcctcaca ggctccattc 60

cttctttcgc tctattggat atcatcacag ctattctccc tggtgcaaaa tatcatatta 120

aattggattt atccttacca acgatggtga agctgacgca tagataggat atgtaattct 180

acatcagctt gtaaataaac aaaaatgact ttcaatatcc ttcaaccgtt cctgactctt 240

tcctgctgac ccgtttttcc aaatttctcg tcgaacttga aattgaaaaa aaaaaaaaaa 300

aattgaatga ggactcatta aacagatgat gccgtaataa atgcaatata tcttgctatt 360

taactctttc tttctttgaa aaccttgaca tacgtattta aataattggc tgtccctgcc 420

tcgaagtata tttctcttct acttttatct tagcgatatc cctaagagtt taatcctccc 480

aggtccataa caaaagaagt caagttca 508

<210> 24

<211> 520

<212> DNA

<213> Artificial sequence

<400> 24

cttgaagcag gagaacagct cagtgcagat gaggaagttt cgtccagcgc aaataaaata 60

gtgaatgtag gtgttttatg gaataaagac acgaataatg acttactaat tgttgaaaat 120

tatctcaaaa gcctcaaaaa aaatttaact agagacaggt agaaaattat tcaaaccttg 180

taaatagtgt tatatatatg tgttagactt aaaagcgcta ataaacgttc ctgctcgcat 240

ttaacttctg ttccaatttt cctatttttt agttagcttg ttcggcagat ttcaaattca 300

ttgagtccga ggaaacaaaa ggatgggaag agctcaaaat ttggagatgg gtttcatata 360

caacacgttg gttagcaaga acctaaggat ccgtctatag aaagaacctc gaaaaatctt 420

cgaaagattt tgttgactga gagtggaaaa aactgatatt actttctcgg tatagagggc 480

aacatttgca aaaagtaata aacaaatagg ggagcacaat 520

Claims (10)

1. A microbial cell expressing flavone 3 β -hydroxylase and/or a flavone 3 β -hydroxylase coenzyme, wherein the flavone 3 β -hydroxylase has the amino acid sequence shown in SEQ ID No. 2; the amino acid sequence of the flavone 3 beta-hydroxylase coenzyme is shown in SEQ ID NO. 4.

2. The microbial cell of claim 1, wherein promoter P is used _INO1 、P _SED1 、P _FAS2 、P _GAL1 Or P _LEU2 The expression of flavone 3 beta-hydroxylase is promoted, and the promoters are respectively shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO. 11.

3. The microbial cell of claim 1, wherein the microbial cell is selected from the group consisting ofUsing the promoter P _TDH1 、P _PGK1 、P _ADH6 、P _GAL10 、P _ADE2 Or P _ADE6 Promoting the expression of the coenzyme of the flavone 3 beta-hydroxylase, wherein the promoters are respectively shown as SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21 and SEQ ID NO. 23.

4. The microbial cell of claim 1, wherein the microbial cell is a prokaryotic or eukaryotic cell.

5. Use of the microbial cell of any one of claims 1-4 for the production of eriodictyol, wherein said microbial cell expresses both said flavone 3 β -hydroxylase and said flavone 3 β -hydroxylase co-enzymes.

6. Use according to claim 5, wherein the microbial cells are added to the reaction system.

7. The use of claim 6, characterized by naringenin as a substrate.

8. A method for whole-cell catalytic production of eriodictyol, characterized in that the microbial cells of any one of claims 1 to 4 are added to a reaction system, and the microbial cells express both the flavone 3 β -hydroxylase and the flavone 3 β -hydroxylase coenzyme.

9. The method as claimed in claim 8, wherein the seed solution is obtained by culturing at 25-35 ℃ and 200-250rpm for 16-18h, and the seed solution is added to the reaction system in an amount of 1-5mL/100 mL.

10. Use of the method of claim 8 or 9 for the production of eriodictyol or products starting from eriodictyol.

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