阅读说明：本技术 一种基于丙二酰辅酶a再生的柚皮素体外酶促合成方法 (Naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration ) 是由 张新跃 聂也森 何妍之 张智萍 丁笠 陈磊 廖凯 赵晨宏 于 2021-07-30 设计创作，主要内容包括：本发明设计一种基于丙二酰辅酶A再生的柚皮素体外酶促合成方法,在构建乙酰辅酶A合成酶ACS基因与乙酰辅酶A羧化酶ACC1基因的重组表达质粒的基础上,将重组质粒分别转化大肠杆菌和酵母细胞,表达目的蛋白,并将纯化的ACS与ACC1重组蛋白加入柚皮素体外酶促合成体系中,实现了丙二酰辅酶A的再生,以及以4-香豆酸为底物在体外低成本酶促合成柚皮素。本发明设计了一个新的反应体系,无需添加昂贵的丙二酰辅酶A便能对其进行再生利用,并最终生成柚皮素。(The invention designs a naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration, which is used for constructing acetyl coenzyme A synthetase ACS Gene and acetyl-CoA carboxylase ACC1 On the basis of the recombinant expression plasmid of the gene, the recombinant plasmid is respectively transformed into escherichia coli and yeast cells to express target protein, and the purified ACS and ACC1 recombinant protein is added into a naringenin in-vitro enzymatic synthesis system to realize the regeneration of malonyl coenzyme A and 4-fragranceThe naringenin is synthesized by enzymatic synthesis of the leguminous acid serving as a substrate in vitro at low cost. The invention designs a new reaction system, which can be regenerated and utilized without adding expensive malonyl coenzyme A, and finally generate naringenin.)

1. A naringenin in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration is characterized by comprising the following steps:

step 1 cloning from E.coliACSGene, construction of recombinant plasmid pET-32a-ACSCloning from Pichia pastorisACC1Gene, construction of recombinant plasmid pGAP-Neo-ACC1；

Step 2, recombining the plasmid pET-32a-ACSTransforming competent escherichia coli, and performing IPTG induced expression and purification to obtain recombinant protein ACS; at the same time, the recombinant plasmid pGAP-Neo-ACC1Transforming pichia pastoris, and obtaining a recombinant protein ACC1 through expression and purification;

step 3, adding the 4-coumaroyl-CoA ligase gene of the soybean4CL1And chalcone isomerase geneCHIAnd chalcone synthase gene of sorghumCHS2With flexible linker peptide (GGGGS)₂Connecting, constructing prokaryotic expression plasmid pET-32a-SUMO-4CL1- (GGGGS)₂-CHS2-(GGGGS)₂CHI, prokaryotic expression plasmid pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHITransforming escherichia coli, and performing IPTG induced expression and purification to obtain a recombinant three-function enzyme;

step 4, establishing an in vitro enzymatic synthesis system based on the regeneration of malonyl coenzyme A, wherein the system at least comprises Tris-HCl, potassium phosphate buffer solution, glycerol and MgCl₂ATP, CoA, sodium acetate, NaHCO₃ACS, ACC1 and recombinant trifunctional enzyme, and reacting under certain conditions to obtain a reaction product.

2. The method of malonyl-coa regeneration-based enzymatic synthesis of naringenin in vitro according to claim 1, wherein: in the step 3, the 4-coumaroyl-CoA ligase gene of the soybean is subjected to overlap extension PCR technology4CL1And chalcone isomerase geneCHIJowar chalcone synthase geneCHS2Cloning to prokaryotic expression vector pET-32a-SUMO, constructing recombinant plasmid

pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHIThe recombinant plasmid is transformed into escherichia coli BL21(DE3) or Rosetta (DE3) according to a conventional method, and the recombinant trifunctional enzyme protein is obtained by IPTG induced expression and nickel column affinity purification.

3. The method of claim 2, wherein the recombinant plasmid pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHIIn the process, cloning4CL1When the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5' -GAACAGATTGGTGGT GGATCC ATGGCACCTTCTCCACAA-3'; the reverse primer was 5'-CGACCCACCTCCGCCCGACCCACCTCCGCCACTAGTATTGGCCACCACCAAACC-3'.

4. The method of claim 2, wherein the malonyl-CoA regeneration-based naringenin in vitro enzymatic synthesis is performed by a specific method,

cloningCHS2When the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5'-GGCGGAGGTGGGTCGGCTAGCATGGCCGGCGCGACTGTG-3'; the reverse primer was 5'-ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCGGCGGTGATGGCCGCTCCG-3'.

5. The method of claim 2, wherein the malonyl-coa regeneration-based naringenin in vitro enzymatic synthesis is clonedCHIWhen the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5'-GGTGGCGGGGGCAGTGAATTCATGGCAACGATCAGCGCG-3';

the reverse primer is 5' -GTGGTGGTGGTGGTG CTCGAG TCAGACTATAATGCCGTGGC-3’。

6. The method of claim 1, wherein in step 4, a malonyl-CoA regeneration-based naringenin in-vitro enzymatic synthesis system is designed, and the system comprises Tris-HCl, potassium phosphate buffer, glycerol, MgCl and MgCl₂BSA, DMSO, beta-mercaptoethanol, ATP, CoA, mM sodium acetate, NaHCO₃4-coumaric acid, ACS, ACC1 and recombinant trifunctional enzyme.

7. According to claim6 the method for the in-vitro enzymatic synthesis of the pomelo peel element based on the regeneration of the malonyl coenzyme A, which is characterized in that, in the step 4, a system for the in-vitro enzymatic synthesis of the pomelo peel element based on the regeneration of the malonyl coenzyme A is designed, and the system comprises 100 mM Tris-HCl, 100 mM potassium phosphate buffer solution, 10% glycerol and 5 mM MgCl₂0.1 mg/mL BSA, 1% DMSO, 1 mM beta-mercaptoethanol, 6 mM ATP, 1 mM CoA, 1-20 mM sodium acetate, 5-65 mM NaHCO₃0.3-1.5 mM 4-coumaric acid, 60 mu g/mL ACS, 60 mu g/mL ACC1 and 60 mu g/mL recombinant trifunctional enzyme.

8. The method of claim 7, wherein the prepared synthesis system is placed in a constant temperature shaking table, and reacted for 6 hours at 25-35 ℃ and 600 rpm, NaOH is added to a final concentration of 1M, and the reaction is terminated.

9. The method of claim 1, wherein in step 1, naringenin is cloned by in vitro enzymatic synthesis based on malonyl-CoA regenerationACSWhen the gene is expressed, a pair of PCR primers is designed: the forward primer is 5' -GCTGATATCGGATCC G AATTC atgagccaaattcacaaacac-3'; the reverse primer is 5' -GTGGTGGTGGTGGTG CTCGAG ttacgatggcatcgcgatag-3’。

10. The method of claim 1, wherein in step 1, naringenin is cloned by in vitro enzymatic synthesis based on malonyl-CoA regenerationACC1When the gene is expressed, a pair of PCR primers is designed: the forward primer is 5' -CTATTTCGAAACGAG GAATTC ATGAGCGAAGAAAGCTTATTC-3'; the reverse primer is 5' -TCGGGCCCAAGCTG GCGGCCGC CTTTCAAAGTCTTCAACAATTTTTC-3’。

Technical Field

The invention relates to an in vitro enzymatic synthesis method of naringenin based on malonyl coenzyme A regeneration, which has a classification number of C12N9 and belongs to the technical field of biological medicines.

Background

Malonyl-coenzyme a (malonyl-coenzyme a) is a coenzyme a derivative. Malonyl-coa, a key intermediate metabolite, plays an important role in a variety of metabolic pathways, being an important precursor molecule for intracellular synthesis of fatty acids and triglycerides (Kastaniotis a J, et al biochem biophysis Acta Mol Cell Biol Lipids, 2017, 1862(1): 39-48.), and also being a raw material for the synthesis of polyketides (Shimizu Y, et al Chembiochem, 2017, 18(1): 50-65.) and many platform compounds (kildogaard K R, et al micro Cell Fact, 2016, 15: 53.).

In general, intracellular CoA is catalyzed by acetyl-CoA synthetase (ACS) followed by acetyl-CoA carboxylase (ACC) to form malonyl-CoA (Qu Q, et al, Cell Death Dis, 2016, 7(5): e 2226.). malonyl-CoA is decarboxylated while participating in the cellular reaction and consumed to regenerate coenzyme A (Waki T, et al. Nat Commun, 2020, 11(1): 870.). Thus, malonyl-coa is in a dynamic equilibrium within the cell. Because the intracellular content of malonyl-coenzyme A is low, the separation and purification difficulty is high, and the malonyl-coenzyme A is difficult to synthesize by a chemical method, the market price of the malonyl-coenzyme A is usually far higher than that of downstream products, and the extensive application of the cofactor in industrial production is restricted.

In recent years, researchers have attempted to increase the amount of malonyl-CoA in microbial cells by various means, such as increasing the intracellular level of acetyl-CoA (Krivoruchko A, et al. Metab Eng, 2015, 28: 28-42.) or the level of acetyl-CoA carboxylase expression (Wang J C, et al. Biosci Biotechnol Biochem, 2016, 80(6): 1214;) to promote the formation of malonyl-CoA while limiting the flow of malonyl-CoA to pathways unrelated to the target molecule (Yang D, et al. Proc Natl Acad Sci U S A, 2018, 115(40): 9835;. 9844.), to increase the yield of the target molecule. However, there is no similar strategy to reduce the high production cost due to malonyl-coa in vitro synthesis systems.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a naringenin enzymatic synthesis method based on malonyl coenzyme A regeneration, the method firstly establishes a regeneration system of malonyl coenzyme A to obviously reduce the high cost caused by expensive malonyl coenzyme A when the naringenin is synthesized by the in vitro enzymatic synthesis, and the system is also suitable for other biochemical reactions which need to take malonyl coenzyme A as a substrate; meanwhile, a high-activity three-function enzyme is constructed, and the yield of target molecules and the total conversion rate of substrates are obviously improved by optimizing in-vitro reaction conditions.

The invention provides an in vitro enzymatic synthesis method of naringenin based on malonyl coenzyme A regeneration, which specifically comprises the following steps:

step 1, cloning key enzyme gene in malonyl coenzyme A regeneration system

Cloning from E.coliACSGene, construction of recombinant plasmid pET-32a-ACSCloning from Pichia pastorisACC1Gene, construction of recombinant plasmid pGAP-Neo-ACC1；

Step 2, inducible expression and purification of recombinant protein ACS and ACC1

Recombinant plasmid pET-32a-ACSTransforming competent escherichia coli, and performing IPTG induced expression and purification to obtain recombinant protein ACS; at the same time, the recombinant plasmid pGAP-Neo-ACC1Transforming pichia pastoris, and obtaining a recombinant protein ACC1 through expression and purification;

step 3, construction, expression and purification of recombinant three-function enzyme for de novo synthesis of naringenin

The 4-coumaroyl-CoA ligase gene of soybean is subjected to overlap extension PCR technology4CL1And chalcone isomerase geneCHIAnd chalcone synthase gene of sorghumCHS2With flexible linker peptide (GGGGS)₂Connecting, constructing prokaryotic expression plasmid pET-32a-SUMO-4CL1- (GGGGS)₂-CHS2-(GGGGS)₂CHI, prokaryotic expression plasmid pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHITransforming escherichia coli, and performing IPTG induced expression and purification to obtain a recombinant three-function enzyme;

step 4, establishing an in vitro enzymatic synthesis system based on malonyl coenzyme A regeneration

The in vitro enzymatic synthesis system based on the regeneration of malonyl-CoA comprises at least Tris-HCl, potassium phosphate buffer, glycerol, MgCl₂ATP, CoA, sodium acetate, NaHCO₃ACS and ACC1, adding recombinant tri-functional enzyme for synthesizing naringenin into the system, and reacting under certain conditions to obtain a reaction product.

The invention designs a reaction system for regenerating malonyl coenzyme A, and acetyl coenzyme A synthetase is constructedACSGene and acetyl-CoA carboxylaseACC1On the basis of the recombinant expression plasmid of the gene, the recombinant plasmid is respectively transformed into escherichia coli and yeast cells to express target protein, and the purified ACS and ACC1 recombinant protein is added into a naringenin in-vitro enzymatic synthesis system, so that the regeneration of malonyl coenzyme A is realized, and naringenin is synthesized in vitro in an enzymatic manner at low cost by taking 4-coumaric acid as a substrate. The invention designs a new reaction system, which can be regenerated and utilized without adding expensive malonyl coenzyme A, and finally generate naringenin. The system only needs to add low-cost inorganic salt, ATP and coenzyme A which can maintain the cyclic generation of malonyl coenzyme A, the reaction product is single, the separation and purification are easy, the cost is controllable, the external synthesis of the pomelo peel element becomes possible in the production level, and a new idea is provided for other schemes of utilizing malonyl coenzyme A to produce.

In said step 1, cloningACSWhen the gene is expressed, a pair of PCR primers is designed: is justThe primer is 5' -GCTGATATCGGATCC GAATTC atgagccaaattcacaaacac-3', the italic base shows the restriction site EcoRI; the reverse primer is 5' -GTGGTGGTGGTGGTG CTCGAG ttacgatggcatcgcgatag-3', the italic base shows the restriction site XhoI;

cloningACC1When the gene is expressed, a pair of PCR primers is designed: the forward primer is 5' -CTATTTCGAAACGAG GAATTC ATGAGCGAAGAAAGCTTATTC-3', the italic base shows the restriction site EcoRI; the reverse primer is 5' -TCGGGCCCAAGCTG GCG GCCGC CTTTCAAAGTCTTCAACAATTTTTC-3', the italic base shows the cleavage site NotI.

PCR fragments were cloned into the prokaryotic expression plasmid pET-32a (+) and the yeast expression plasmid pGAP-Neo, respectively, using the ligase-independent single-fragment rapid Cloning Kit (Clonexpress II One Step Cloning Kit) of Nanjing Novowed Biotechnology Ltd.

In the step 2, the recombinant plasmid pET-32a-ACSConventionally, competent Escherichia coli BL21(DE3) was cultured overnight at 37 ℃ and 220 rpm; selecting 3-5 colonies, inoculating the colonies to an LB culture medium containing 100 mug/mL ampicillin, and culturing overnight at 37 ℃ and 250 rpm; transferring to 300 mL LB culture medium containing 100 mug/mL ampicillin according to the ratio of 1:100, culturing at 37 ℃ and 250 rpm for about 2 h until bacterial liquid OD_600nm0.4 to 0.6; adding IPTG into the bacterial liquid to a final concentration of 0.1 mM, carrying out induced expression for 4 h at 20 ℃, and centrifugally collecting thalli at 4 ℃; purifying the recombinant protein according to the Beyogold His-tag Purification Resin reagent instruction of Byunying; dialyzing purified recombinant protein with Tris-Cl buffer (pH 7.5) by conventional method, concentrating with PEG 20000, collecting protein solution, adding glycerol to final concentration of 10%, packaging, and storing at-80 deg.C.

Taking the recombinant plasmid pGAP-Neo-ACC1Transforming Pichia pastoris GS115 by a LiCl method, plating a YPD plate containing 500 mug/mL G-418, and carrying out inverted culture in a 30 ℃ constant temperature incubator; after 3 d, picking single colonies, inoculating the single colonies into 2 mL YPD liquid culture medium containing 500 mug/mL G-418, and culturing at 30 ℃ and 250 rpm for 24 h; according to the following steps: transferring 300 mL YPD liquid culture medium containing 500 mug/mL G-418 to 100, and continuously culturing at 30 ℃ and 250 rpm for 12 h; at 4 deg.C,Centrifuging at 5000 g for 10 min, and collecting thallus; adding 16.5 g of 0.4 mm glass beads and 21 mL of non-denatured lysate, and crushing yeast in a vortex oscillator; centrifuging, collecting the cracked supernatant, and performing ammonium sulfate precipitation to obtain ammonium sulfate with a final concentration of 40% saturation; centrifuging at 4 deg.C and 7000 g for 20 min, discarding supernatant, and dissolving precipitate with non-denaturing lysis solution; purifying the recombinant protein according to the Beyogold His-tag Purification Resin reagent instruction of Byunying; preparing a pH 7.5 dialysis buffer (containing 100 mM Tris-HCl and 100 mM potassium phosphate), dialyzing the purified protein for 3 times and 3 h/time; concentrating dialyzed protein solution with PEG 20000, adding glycerol to final concentration of 10%, packaging, and storing at-80 deg.C.

In the step 3, the gene segments are spliced by utilizing the overlap extension PCR technology, and the 4-coumaroyl coenzyme A ligase gene of the soybean4CL1And chalcone isomerase geneCHIJowar chalcone synthase geneCHS2Recombining into a single sequence, cloning to a prokaryotic expression vector pET-32a-SUMO, and constructing a recombinant plasmid

pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHIEach key enzyme is connected with each other through a connecting peptide (GGGGS)₂Are connected. Transforming the recombinant plasmid into Escherichia coli BL21(DE3) or Rosetta (DE3) by conventional method, performing induced expression at 16 deg.C for 12 h with 0.05 mM IPTG, performing affinity purification by nickel column to obtain recombinant trifunctional enzyme protein, dialyzing, concentrating PEG 20000, adding glycerol to final concentration of 10%, packaging, and storing at-80 deg.C.

Construction of recombinant plasmid pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHIIn the process, cloning4CL1When the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5' -GAACAGATTGGTGGT GGATCC ATGGCACCTTCTCCACAA-3', restriction sites in italic baseBamHI; the reverse primer was 5'-CGACCCACCTCCGCCCGACCCACCTCCGCCACTAGTATTGGCCACCACCAAACC-3'.

CloningCHS2When the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5'-GGCGGAGGTGGGTCGGCTAGCATGGCCGGCGCGACTGTG-3'; the reverse primer is 5'-ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCGGCGGTGATGGCCGCTCCG-3';

cloningCHIWhen the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5'-GGTGGCGGGGGCAGTGAATTCATGGCAACGATCAGCGCG-3';

the reverse primer is 5' -GTGGTGGTGGTGGTG CTCGAG TCAGACTATAATGCCGTGGC-3', restriction sites in italic baseXhoI。

Each gene fragment was spliced by overlap extension PCR technique, and the PCR fragment was cloned into the prokaryotic expression plasmid pET-32a-SUMO using the ligase independent single-fragment rapid Cloning Kit (Clonexpress II One Step Cloning Kit) from Nanjing Novowed Biotechnology Ltd.

In the step 4, a naringenin in-vitro enzymatic synthesis system based on malonyl coenzyme A regeneration is designed, and the system comprises Tris-HCl, potassium phosphate buffer solution, glycerol and MgCl₂BSA, DMSO, beta-mercaptoethanol, ATP, CoA, mM sodium acetate, NaHCO₃4-coumaric acid, ACS, ACC1 and recombinant trifunctional enzyme.

In the step 4, a malonyl-CoA regeneration-based naringenin in-vitro enzymatic synthesis system comprising 100 mM Tris-HCl (pH 6.8-8.1), 100 mM potassium phosphate buffer (pH 6.8-8.1), 10% glycerol, 5 mM MgCl was designed₂0.1 mg/mL BSA, 1% DMSO, 1 mM beta-mercaptoethanol, 6 mM ATP, 1 mM CoA, 1-20 mM sodium acetate, 5-65 mM NaHCO₃0.3-1.5 mM 4-coumaric acid, 60 mug/mL ACS, 60 mug/mL ACC1, 60 mug/mL 4CC, 4CC is a recombinant trifunctional enzyme; and (3) placing the prepared synthesis system in a constant-temperature shaking table, reacting for 6 hours at the temperature of 25-35 ℃ and at the speed of 600 rpm, adding a NaOH solution until the final concentration is 1M, and stopping the reaction.

Detection of reaction products: 100. mu.L of the reaction solution after the reaction was terminated was added with an equal volume of ethyl acetate, extracted for 2 hours, and the upper organic phase was extracted. Performing extraction again as above, mixing organic phases, air drying, dissolving with 75 μ L HPLC grade methanol, adding 75 μ L50% HPLC grade acetonitrile, mixing, filtering with 0.22 μm organic microporous membrane, and performing High Performance Liquid Chromatography (HPLC).

The invention establishes an in vitro regeneration system of malonyl coenzyme A, and takes 4-coumaric acid as a raw material to synthesize naringenin de novo under the catalysis of a recombinant three-function enzyme on the basis, wherein the yield is as high as 3.99 mg/L; the shaddock peel in-vitro enzymatic synthesis system has simple components, does not have complex regulation and control function in engineering bacteria cells, is easy to accurately control the reaction process, has few byproducts, is relatively simple in product separation, obviously shortens the production period and obviously reduces the production cost.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of a purified protein of a trifunctional enzyme of the present invention. In the figure: 1 for ACS, 2 for ACC1, and 3 for 4 CC.

FIG. 2 is a graph showing the results of HPLC analysis of the trifunctional enzyme activity in the present invention. In the figure: a represents 4-coumaric acid standard substance, and the peak emergence time is 6.11 min; b represents naringenin standard substance, and the peak time is 11.22 min; c represents the experimental group.

Detailed Description

The technical scheme of the invention is further explained in detail by combining the embodiment as follows: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.

Sources of materials involved in the examples:

g-418 was purchased from Gibco; 0.4 mm glass beads were purchased from EASYBIO, Boolyje. Pichia pastoris GS115 was purchased from Invitrogen; the non-denaturing lysis solution is prepared by the laboratory, each liter contains 6.9 g of NaH2PO 4. H2O and 17.54 g of NaCl, and the pH value is adjusted to 8.0 by using 10M sodium hydroxide after the distilled water is dissolved; pET-32a-SUMO vector was constructed by this laboratory: replacing thioredoxin TrxA in a pET-32a (+) vector of Novagen company with small molecule ubiquitin-like modified protein SUMO to obtain the thioredoxin; the pGAP-Neo vector was constructed by this laboratory: the bleomycin resistance gene of the pGAPZ A vector of Invitrogen company was replaced with a neomycin resistance gene.

The remaining reagents and materials involved in this example are commercially available and are not listed here. In the present example, "%" is usually used as a mass percentage unless otherwise specified.

Example 1

1. Construction of recombinant plasmid

Escherichia coli acetyl-CoA synthetase gene provided according to GenBank databaseACSAnd yeast acetyl-CoA carboxylase geneACC1Nucleotide sequence information of (a), designing two pairs of primer sequences forACSCloning the coding region of the gene to an Escherichia coli expression vector pET-32a (+), and carrying outACC1The coding region of the gene is cloned to a pichia pastoris expression vector pGAP-Neo:

PCR amplificationACSThe forward primer of the gene is 5' -GCTGATATCGGATCCGAATTCatgagccaaattcacaaacac-3' (shown in SEQ ID No. 3), wherein the italic base represents the restriction site EcoRI; the reverse primer is 5' -GTGGTGGTGGTGGTGCTCGAGttacgatggcatcgcgatag-3' (shown in SEQ ID No. 4), the italic base indicates the cleavage site XhoI.

PCR amplificationACC1The forward primer of the gene is

5’-CTATTTCGAAACGAGGAATTCATGAGCGAAGAAAGCTTATTC-3' (shown in SEQ ID No. 5), wherein the italic base represents the restriction site EcoRI; the reverse primer is 5' -TCGGGCCCAAGCTGGCGGCCGCCTTTCAAAGTCTTCAACAATTTTTC-3' (as shown in SEQ ID No. 6), the italic base indicates the cleavage site NotI.

The following PCR amplification system was established according to the SuperPfx DNA Polymerase protocol of Kangji century:

reagent	100 μ L reaction System
		5×Super Pfx HF Buffer	20 μL
dNTP Mix，10 mM each	2 μL
		Forward Primer，10 μM	5 μL
Reverse Primer，10 μM	5 μL
		Template DNA	1 μL
Super Pfx DNA Polymerase	1 μL
		ddH2O	66 μL

Amplification ofACSThe gene template DNA was 1. mu.L of Escherichia coli DH 5. alpha. cell suspension cultured for 4 hours, and amplifiedACC1The template DNA of the gene is cDNA of Pichia pastoris GS115, and the PCR reaction conditions are as follows:

step (ii) of	Temperature of	Time
			Pre-denaturation	98 ℃	3 min
Denaturation of the material	98 ℃	5 s
			Annealing	45 ℃	15 s
Extension	72 ℃	2 min 30 s
			Final extension	72 ℃	10 min

The PCR reaction product was subjected to DNA gel electrophoresis and the band of interest was recovered as a gel. The vectors pET-32a (+) and pGAP-Neo are respectively subjected toEcoRI/XhoI andEcoRI/Noti, double enzyme digestion, DNA gel electrophoresis and gel recovery of target bands. The target fragment and the vector were ligated using a ligase-independent single-fragment rapid Cloning Kit (Clonexpress II One Step Cloning Kit) of Nanjing Novowed Biotechnology Ltd, and competent E.coli DH 5. alpha. was transformed conventionally, spread evenly on ampicillin-resistant LB plates, and cultured overnight at 37 ℃. Selecting a single colony on the next day, inoculating the single colony into 3 mL LB liquid culture medium with ampicillin resistance, culturing the single colony at 37 ℃ and 250 rpm for 15 h, extracting the plasmid, and then performing sequencing identification to obtain a recombinant plasmid pET-32a-ACSAnd pGAP-Neo-ACC1。

Splicing key enzyme gene by overlapping extension PCR technology4CL1、CHS2AndCHIcloning PCR fragment to prokaryotic expression plasmid pET-32a-SUMO by using ligase-independent single-fragment rapid Cloning Kit (Clonexpress II One Step Cloning Kit) of Nanjing Novowed Biotechnology Ltd to construct recombinant plasmid pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHI. Structure of the organizationConstruction of recombinant plasmid pET-32a-SUMO-4CL1-(GGGGS)₂-CHS2-(GGGGS)₂-CHIIn the process, the 4-coumaroyl-CoA ligase gene of soybean is used as a basis4CL1And chalcone isomerase geneCHIAnd chalcone synthase gene of sorghumCHS2Design of primers and cloning4CL1When the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5' -GAACAGATTGGTGGT GGATCC ATGGCACCTTCTCCACAA-3', restriction sites in italic baseBamHI; the reverse primer was 5'-CGACCCACCTCCGCCCGACCCACCTCCGCCACTAGTATTGGCCACCACCAAACC-3'.

CloningCHS2When the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5'-GGCGGAGGTGGGTCGGCTAGCATGGCCGGCGCGACTGTG-3'; the reverse primer is 5'-ACTGCCCCCGCCACCCGATCCCCCGCCACCGGATCCGGCGGTGATGGCCGCTCCG-3';

cloningCHIWhen the gene is expressed, a pair of PCR primers is designed:

the forward primer is 5'-GGTGGCGGGGGCAGTGAATTCATGGCAACGATCAGCGCG-3';

the reverse primer is 5' -GTGGTGGTGGTGGTG CTCGAG TCAGACTATAATGCCGTGGC-3', restriction sites in italic baseXhoI。

2. Induced expression and purification of recombinant proteins

2.1 inducible expression and purification of ACS

Taking the recombinant plasmid pET-32a-ACSConventionally, competent Escherichia coli BL21(DE3) was cultured overnight at 37 ℃ and 220 rpm; selecting 3-5 colonies, inoculating the colonies to an LB culture medium containing 100 mug/mL ampicillin, and culturing overnight at 37 ℃ and 250 rpm; transferring to 300 mL LB culture medium containing 100 mug/mL ampicillin according to the ratio of 1:100, culturing at 37 ℃ and 250 rpm for about 2 h until bacterial liquid OD_600nm0.4 to 0.6; adding IPTG into the bacterial liquid to a final concentration of 0.1 mM, carrying out induced expression for 4 h at 20 ℃, and centrifugally collecting thalli at 4 ℃; purifying the recombinant protein according to the Beyogold His-tag Purification Resin reagent instruction of Byunying; dialyzing purified recombinant protein with Tris-Cl buffer (pH 7.5) by conventional method, concentrating with PEG 20000, collecting protein solution, adding glycerol to final concentration of 10%, packaging,storing at-80 deg.C for use. The amino acid sequence of the recombinant protein ACS is shown in SEQ ID No. 1.

2.2 expression and purification of ACC1

Taking the recombinant plasmid pGAP-Neo-ACC1, transforming Pichia pastoris GS115 (purchased from Invitrogen company) by a LiCl method, coating a YPD plate containing 500 mug/mL geneticin G-418 (purchased from Gibco company), and carrying out inverted culture in a 30 ℃ constant temperature incubator; after 3 d, picking single colonies, inoculating the single colonies into 2 mL YPD liquid culture medium containing 500 mug/mL G-418, and culturing at 30 ℃ and 250 rpm for 24 h; according to the mass ratio of 1: transferring 100 mL YPD liquid culture medium containing 500 mug/mL G-418 to 300 mL YPD liquid culture medium, and continuously culturing at 30 ℃ and 250 rpm for 12 h; centrifuging at 4 deg.C and 5000 g for 10 min, and collecting thallus; 16.5 g of 0.4 mm glass beads (from EASYBIO, Paoyijie) and 21 mL of a non-denaturing lysis buffer (prepared in the laboratory and weighed 6.9 g of NaH)₂PO₄•H₂Dissolving O and 17.54 g NaCl in 800 mL of distilled water, adjusting the pH value to 8.0 by using 10M sodium hydroxide, then fixing the volume to 1L by using the distilled water, and crushing yeast cells in a vortex oscillation instrument; centrifuging, collecting the cracked supernatant, and performing ammonium sulfate precipitation to obtain ammonium sulfate with a final concentration of 40% saturation; centrifuging at 4 deg.C and 7000 g for 20 min, discarding supernatant, and dissolving precipitate with non-denaturing lysis solution; purifying the recombinant protein according to the Beyogold His-tag Purification Resin reagent instruction of Byunying; preparing a pH 7.5 dialysis buffer (containing 100 mM Tris-HCl and 100 mM potassium phosphate), dialyzing the purified protein for 3 times and 3 h/time; concentrating dialyzed protein solution with PEG 20000, adding glycerol to final concentration of 10%, packaging, and storing at-80 deg.C. The amino acid sequence of the recombinant protein ACC1 is shown in SEQ ID No. 2.

2.3 oil duct expression and purification of recombinant trifunctional enzymes

Taking prokaryotic expression plasmid pET-32a-SUMO-4CL1- (GGGGS)₂-CHS2-(GGGGS)₂CHI, transforming Escherichia coli BL21(DE3) or Rosetta (DE3) by conventional method, inducing expression for 12 h at 16 deg.C with 0.05 mM IPTG, affinity purifying recombinant trifunctional enzyme protein 4CC with nickel column, dialyzing, concentrating PEG 20000, adding glycerol to 10% of final concentration, and packaging at-80 deg.C.

The results of SDS-PAGE of the three purified proteins are shown in FIG. 1, in which 1 represents ACS, 2 represents ACC1, and 3 represents 4 CC.

3. Establishment of naringenin in-vitro enzyme-promoted synthesis system based on regeneration of malonyl coenzyme A

In vitro Synthesis System for malonyl-CoA comprising 100 mM Tris-HCl (pH 7.5), 100 mM potassium phosphate buffer (pH 7.5), 10% glycerol, 5 mM MgCl₂0.1 mg/mL BSA, 1% DMSO, 1 mM beta-mercaptoethanol, 6 mM ATP, 1 mM CoA, 10 mM sodium acetate, 50 mM NaHCO₃0.6 mM 4-coumaric acid, 60 μ g/mL ACS, 60 μ g/mL ACC1, 60 μ g/mL 4 CC. Placing the prepared synthetic system in a constant temperature shaking table, reacting for 6 h at 30 ℃ and 600 rpm, adding NaOH solution until the final concentration is 1M, and terminating the reaction.

4. Detection condition of naringenin product

100 μ L of the reaction solution after the reaction was terminated was taken, an equal volume of ethyl acetate was added thereto, extraction was performed for 2 hours, and the upper organic phase was extracted. Extracting again as above, mixing organic phases, air drying, dissolving with 75 μ L HPLC grade methanol, adding 75 μ L50% HPLC grade acetonitrile, mixing, filtering with 0.22 μm organic microporous membrane, and performing High Performance Liquid Chromatography (HPLC), with HPLC detection result as shown in figure 2, wherein A represents 4-coumaric acid standard, and the peak time is 6.11 min; b represents naringenin standard substance, and the peak time is 11.22 min; c represents the experimental group (i.e., the product after the reaction of the in vitro malonyl-CoA synthesis system described above). The HPLC detection conditions were as follows:

the instrument equipment comprises: agilent 1260 liquid chromatography-mass spectrometry (Agilent 1260) Inc., USA.

A chromatographic column: agilent C18column (150X 4.6 mm, 5 μm, Thermo Fisher scientific. Inc., San Jose, Calif., USA). Column temperature: 35 ℃ is carried out.

Mobile phase: a (acetonitrile), B (Milli Q water), gradient elution: 0-4 min, A: 20% -22%, B: 80% -78%; 4-7 min, A: 22% -42%, B: 78% -58%; 7-10 min, A: 42% -38%, B: 58% -62%; 10-15 min, A: 38% -95%, B: 62 to 5 percent; 15-24 min, A: 95%, B: 5 percent; 24-25 min, A: 95% -20%, B: 5 to 80 percent.

Sample introduction amount: 20 mu L of the solution; flow rate: 1 mL/min;

a detector: an ultraviolet detector (VWD);

detection wavelength: λ 280 nm; 330 nm.

The yield of naringenin is 3.99 mg/L through measurement.

Naringenin has various pharmacological and physiological activities and has wide application prospect in the field of biological medicine. Compared with a microbial cell factory fermentation method, the invention provides the preparation method which is simple and convenient to operate, relatively simple in product separation, obviously shortened in production period and obviously reduced in production cost. Taking the production of naringenin by saccharomyces cerevisiae as an example, fermentation culture generally exceeds 48 hours, but the preparation method of the invention consumes only 3 hours each time, is less limited by raw materials and manpower, and is easy to enlarge. If the labor cost is calculated, the relative production cost is further reduced, and therefore the potential economic benefit is considerable.

The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can understand that the modifications or substitutions within the technical scope of the present invention are included in the scope of the present invention, and therefore, the scope of the present invention should be subject to the protection scope of the claims.

Sequence listing

<110> Yangzhou university

<120> shaddock peel in-vitro enzymatic synthesis method based on malonyl coenzyme A regeneration

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Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

1 5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Glu Phe Met Ser Gln Ile His Lys His Thr Ile

165 170 175

Pro Ala Asn Ile Ala Asp Arg Cys Leu Ile Asn Pro Gln Gln Tyr Glu

180 185 190

Ala Met Tyr Gln Gln Ser Ile Asn Val Pro Asp Thr Phe Trp Gly Glu

195 200 205

Gln Gly Lys Ile Leu Asp Trp Ile Lys Pro Tyr Gln Lys Val Lys Asn

210 215 220

Thr Ser Phe Ala Pro Gly Asn Val Ser Ile Lys Trp Tyr Glu Asp Gly

225 230 235 240

Thr Leu Asn Leu Ala Ala Asn Cys Leu Asp Arg His Leu Gln Glu Asn

245 250 255

Gly Asp Arg Thr Ala Ile Ile Trp Glu Gly Asp Asp Ala Ser Gln Ser

260 265 270

Lys His Ile Ser Tyr Lys Glu Leu His Arg Asp Val Cys Arg Phe Ala

275 280 285

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

290 295 300

Tyr Met Pro Met Val Pro Glu Ala Ala Val Ala Met Leu Ala Cys Ala

305 310 315 320

Arg Ile Gly Ala Val His Ser Val Ile Phe Gly Gly Phe Ser Pro Glu

325 330 335

Ala Val Ala Gly Arg Ile Ile Asp Ser Asn Ser Arg Leu Val Ile Thr

340 345 350

Ser Asp Glu Gly Val Arg Ala Gly Arg Ser Ile Pro Leu Lys Lys Asn

355 360 365

Val Asp Asp Ala Leu Lys Asn Pro Asn Val Thr Ser Val Glu His Val

370 375 380

Val Val Leu Lys Arg Thr Gly Gly Lys Ile Asp Trp Gln Glu Gly Arg

385 390 395 400

Asp Leu Trp Trp His Asp Leu Val Glu Gln Ala Ser Asp Gln His Gln

405 410 415

Ala Glu Glu Met Asn Ala Glu Asp Pro Leu Phe Ile Leu Tyr Thr Ser

420 425 430

Gly Ser Thr Gly Lys Pro Lys Gly Val Leu His Thr Thr Gly Gly Tyr

435 440 445

Leu Val Tyr Ala Ala Leu Thr Phe Lys Tyr Val Phe Asp Tyr His Pro

450 455 460

Gly Asp Ile Tyr Trp Cys Thr Ala Asp Val Gly Trp Val Thr Gly His

465 470 475 480

Ser Tyr Leu Leu Tyr Gly Pro Leu Ala Cys Gly Ala Thr Thr Leu Met

485 490 495

Phe Glu Gly Val Pro Asn Trp Pro Thr Pro Ala Arg Met Ala Gln Val

500 505 510

Val Asp Lys His Gln Val Asn Ile Leu Tyr Thr Ala Pro Thr Ala Ile

515 520 525

Arg Ala Leu Met Ala Glu Gly Asp Lys Ala Ile Glu Gly Thr Asp Arg

530 535 540

Ser Ser Leu Arg Ile Leu Gly Ser Val Gly Glu Pro Ile Asn Pro Glu

545 550 555 560

Ala Trp Glu Trp Tyr Trp Lys Lys Ile Gly Asn Glu Lys Cys Pro Val

565 570 575

Val Asp Thr Trp Trp Gln Thr Glu Thr Gly Gly Phe Met Ile Thr Pro

580 585 590

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

595 600 605

Phe Gly Val Gln Pro Ala Leu Val Asp Asn Glu Gly Asn Pro Leu Glu

610 615 620

Gly Ala Thr Glu Gly Ser Leu Val Ile Thr Asp Ser Trp Pro Gly Gln

625 630 635 640

Ala Arg Thr Leu Phe Gly Asp His Glu Arg Phe Glu Gln Thr Tyr Phe

645 650 655

Ser Thr Phe Lys Asn Met Tyr Phe Ser Gly Asp Gly Ala Arg Arg Asp

660 665 670

Glu Asp Gly Tyr Tyr Trp Ile Thr Gly Arg Val Asp Asp Val Leu Asn

675 680 685

Val Ser Gly His Arg Leu Gly Thr Ala Glu Ile Glu Ser Ala Leu Val

690 695 700

Ala His Pro Lys Ile Ala Glu Ala Ala Val Val Gly Ile Pro His Asn

705 710 715 720

Ile Lys Gly Gln Ala Ile Tyr Ala Tyr Val Thr Leu Asn His Gly Glu

725 730 735

Glu Pro Ser Pro Glu Leu Tyr Ala Glu Val Arg Asn Trp Val Arg Lys

740 745 750

Glu Ile Gly Pro Leu Ala Thr Pro Asp Val Leu His Trp Thr Asp Ser

755 760 765

Leu Pro Lys Thr Arg Ser Gly Lys Ile Met Arg Arg Ile Leu Arg Lys

770 775 780

Ile Ala Ala Gly Asp Thr Ser Asn Leu Gly Asp Thr Ser Thr Leu Ala

785 790 795 800

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

805 810 815

Met Pro Ser

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Met Ser Glu Glu Ser Leu Phe Glu Ser Ser Pro Gln Lys Met Glu Tyr

1 5 10 15

Glu Ile Thr Asn Tyr Ser Glu Arg His Thr Glu Leu Pro Gly His Phe

20 25 30

Ile Gly Leu Asn Thr Val Asp Lys Leu Glu Glu Ser Pro Leu Arg Asp

35 40 45

Phe Val Lys Ser His Gly Gly His Thr Val Ile Ser Lys Ile Leu Ile

50 55 60

Ala Asn Asn Gly Ile Ala Ala Val Lys Glu Ile Arg Ser Val Arg Lys

65 70 75 80

Trp Ala Tyr Glu Thr Phe Gly Asp Asp Arg Thr Val Gln Phe Val Ala

85 90 95

Met Ala Thr Pro Glu Asp Leu Glu Ala Asn Ala Glu Tyr Ile Arg Met

100 105 110

Ala Asp Gln Tyr Ile Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr

115 120 125

Ala Asn Val Asp Leu Ile Val Asp Ile Ala Glu Arg Ala Asp Val Asp

130 135 140

Ala Val Trp Ala Gly Trp Gly His Ala Ser Glu Asn Pro Leu Leu Pro

145 150 155 160

Glu Lys Leu Ser Gln Ser Lys Arg Lys Val Ile Phe Ile Gly Pro Pro

165 170 175

Gly Asn Ala Met Arg Ser Leu Gly Asp Lys Ile Ser Ser Thr Ile Val

180 185 190

Ala Gln Ser Ala Lys Val Pro Cys Ile Pro Trp Ser Gly Thr Gly Val

195 200 205

Asp Thr Val His Val Asp Glu Lys Thr Gly Leu Val Ser Val Asp Asp

210 215 220

Asp Ile Tyr Gln Lys Gly Cys Cys Thr Ser Pro Glu Asp Gly Leu Gln

225 230 235 240

Lys Ala Lys Arg Ile Gly Phe Pro Val Met Ile Lys Ala Ser Glu Gly

245 250 255

Gly Gly Gly Lys Gly Ile Arg Gln Val Glu Arg Glu Glu Asp Phe Ile

260 265 270

Ala Leu Tyr His Gln Ala Ala Asn Glu Ile Pro Gly Ser Pro Ile Phe

275 280 285

Ile Met Lys Leu Ala Gly Arg Ala Arg His Leu Glu Val Gln Leu Leu

290 295 300

Ala Asp Gln Tyr Gly Thr Asn Ile Ser Leu Phe Gly Arg Asp Cys Ser

305 310 315 320

Val Gln Arg Arg His Gln Lys Ile Ile Glu Glu Ala Pro Val Thr Ile

325 330 335

Ala Lys Ala Glu Thr Phe His Glu Met Glu Lys Ala Ala Val Arg Leu

340 345 350

Gly Lys Leu Val Gly Tyr Val Ser Ala Gly Thr Val Glu Tyr Leu Tyr

355 360 365

Ser His Asp Asp Gly Lys Phe Tyr Phe Leu Glu Leu Asn Pro Arg Leu

370 375 380

Gln Val Glu His Pro Thr Thr Glu Met Val Ser Gly Val Asn Leu Pro

385 390 395 400

Ala Ala Gln Leu Gln Ile Ala Met Gly Ile Pro Met His Arg Ile Ser

405 410 415

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

420 425 430

Asp Phe Glu Phe Lys Thr Gln Asp Ala Thr Lys Lys Gln Arg Arg Pro

435 440 445

Ile Pro Lys Gly His Cys Thr Ala Cys Arg Ile Thr Ser Glu Asp Pro

450 455 460

Asn Asp Gly Phe Lys Pro Ser Gly Gly Thr Leu His Glu Leu Asn Phe

465 470 475 480

Arg Ser Ser Ser Asn Val Trp Gly Tyr Phe Ser Val Gly Asn Asn Gly

485 490 495

Asn Ile His Ser Phe Ser Asp Ser Gln Phe Gly His Ile Phe Ala Phe

500 505 510

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

515 520 525

Glu Leu Ser Ile Arg Gly Asp Phe Arg Thr Thr Val Glu Tyr Leu Ile

530 535 540

Lys Leu Leu Glu Thr Glu Asp Phe Glu Asp Asn Thr Ile Thr Thr Gly

545 550 555 560

Trp Leu Asp Asp Leu Ile Thr His Lys Met Thr Ala Glu Lys Pro Asp

565 570 575

Pro Thr Leu Ala Val Ile Cys Gly Ala Ala Thr Lys Ala Phe Leu Ala

580 585 590

Ser Glu Glu Ala Arg His Lys Tyr Ile Glu Ser Leu Gln Lys Gly Gln

595 600 605

Val Leu Ser Lys Asp Leu Leu Gln Thr Met Phe Pro Val Asp Phe Ile

610 615 620

His Glu Gly Lys Arg Tyr Lys Phe Thr Val Ala Lys Ser Gly Asn Asp

625 630 635 640

Arg Tyr Thr Leu Phe Ile Asn Gly Ser Lys Cys Asp Ile Ile Leu Arg

645 650 655

Gln Leu Ser Asp Gly Gly Leu Leu Ile Ala Ile Gly Gly Lys Ser His

660 665 670

Thr Ile Tyr Trp Lys Glu Glu Val Ala Ala Thr Arg Leu Ser Val Asp

675 680 685

Ser Met Thr Thr Leu Leu Glu Val Glu Asn Asp Pro Thr Gln Leu Arg

690 695 700

Thr Pro Ser Pro Gly Lys Leu Val Lys Phe Leu Val Glu Asn Gly Glu

705 710 715 720

His Ile Ile Lys Gly Gln Pro Tyr Ala Glu Ile Glu Val Met Lys Met

725 730 735

Gln Met Pro Leu Val Ser Gln Glu Asn Gly Ile Val Gln Leu Leu Lys

740 745 750

Gln Pro Gly Ser Thr Ile Val Ala Gly Asp Ile Met Ala Ile Met Thr

755 760 765

Leu Asp Asp Pro Ser Lys Val Lys His Ala Leu Pro Phe Glu Gly Met

770 775 780

Leu Pro Asp Phe Gly Ser Pro Val Ile Glu Gly Thr Lys Pro Ala Tyr

785 790 795 800

Lys Phe Lys Ser Leu Val Ser Thr Leu Glu Asn Ile Leu Lys Gly Tyr

805 810 815

Asp Asn Gln Val Ile Met Asn Ala Ser Leu Gln Gln Leu Ile Glu Val

820 825 830

Leu Arg Asn Pro Lys Leu Pro Tyr Ser Glu Trp Lys Leu His Ile Ser

835 840 845

Ala Leu His Ser Arg Leu Pro Ala Lys Leu Asp Glu Gln Met Glu Glu

850 855 860

Leu Val Ala Arg Ser Leu Arg Arg Gly Ala Val Phe Pro Ala Arg Gln

865 870 875 880

Leu Ser Lys Leu Ile Asp Met Ala Val Lys Asn Pro Glu Tyr Asn Pro

885 890 895

Asp Lys Leu Leu Gly Ala Val Val Glu Pro Leu Ala Asp Ile Ala His

900 905 910

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

915 920 925

Phe Leu Glu Glu Tyr Tyr Glu Val Glu Lys Leu Phe Asn Gly Pro Asn

930 935 940

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

945 950 955 960

Asp Leu Asp Lys Val Ala Leu Thr Val Leu Ser His Ser Lys Val Ser

965 970 975

Ala Lys Asn Asn Leu Ile Leu Ala Ile Leu Lys His Tyr Gln Pro Leu

980 985 990

Cys Lys Leu Ser Ser Lys Val Ser Ala Ile Phe Ser Thr Pro Leu Gln

995 1000 1005

His Ile Val Glu Leu Glu Ser Lys Ala Thr Ala Lys Val Ala Leu Gln

1010 1015 1020

Ala Arg Glu Ile Leu Ile Gln Gly Ala Leu Pro Ser Val Lys Glu Arg

1025 1030 1035 1040

Thr Glu Gln Ile Glu His Ile Leu Lys Ser Ser Val Val Lys Val Ala

1045 1050 1055

Tyr Gly Ser Ser Asn Pro Lys Arg Ser Glu Pro Asp Leu Asn Ile Leu

1060 1065 1070

Lys Asp Leu Ile Asp Ser Asn Tyr Val Val Phe Asp Val Leu Leu Gln

1075 1080 1085

Phe Leu Thr His Gln Asp Pro Val Val Thr Ala Ala Ala Ala Gln Val

1090 1095 1100

Tyr Ile Arg Arg Ala Tyr Arg Ala Tyr Thr Ile Gly Asp Ile Arg Val

1105 1110 1115 1120

His Glu Gly Val Thr Val Pro Ile Val Glu Trp Lys Phe Gln Leu Pro

1125 1130 1135

Ser Ala Ala Phe Ser Thr Phe Pro Thr Val Lys Ser Lys Met Gly Met

1140 1145 1150

Asn Arg Ala Val Ser Val Ser Asp Leu Ser Tyr Val Ala Asn Ser Gln

1155 1160 1165

Ser Ser Pro Leu Arg Glu Gly Ile Leu Met Ala Val Asp His Leu Asp

1170 1175 1180

Asp Val Asp Glu Ile Leu Ser Gln Ser Leu Glu Val Ile Pro Arg His

1185 1190 1195 1200

Gln Ser Ser Ser Asn Gly Pro Ala Pro Asp Arg Ser Gly Ser Ser Ala

1205 1210 1215

Ser Leu Ser Asn Val Ala Asn Val Cys Val Ala Ser Thr Glu Gly Phe

1220 1225 1230

Glu Ser Glu Glu Glu Ile Leu Val Arg Leu Arg Glu Ile Leu Asp Leu

1235 1240 1245

Asn Lys Gln Glu Leu Ile Asn Ala Ser Ile Arg Arg Ile Thr Phe Met

1250 1255 1260

Phe Gly Phe Lys Asp Gly Ser Tyr Pro Lys Tyr Tyr Thr Phe Asn Gly

1265 1270 1275 1280

Pro Asn Tyr Asn Glu Asn Glu Thr Ile Arg His Ile Glu Pro Ala Leu

1285 1290 1295

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

1300 1305 1310

Ile Phe Thr Asp Asn Arg Asn Ile His Val Tyr Glu Ala Val Ser Lys

1315 1320 1325

Thr Ser Pro Leu Asp Lys Arg Phe Phe Thr Arg Gly Ile Ile Arg Thr

1330 1335 1340

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

1345 1350 1355 1360

Ala Asn Arg Leu Met Ser Asp Ile Leu Asp Asn Leu Glu Val Thr Asp

1365 1370 1375

Thr Ser Asn Ser Asp Leu Asn His Ile Phe Ile Asn Phe Ile Ala Val

1380 1385 1390

Phe Asp Ile Ser Pro Glu Asp Val Glu Ala Ala Phe Gly Gly Phe Leu

1395 1400 1405

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

1410 1415 1420

Ile Arg Ile Ile Ile Lys Asp Pro Gln Thr Gly Ala Pro Val Pro Leu

1425 1430 1435 1440

Arg Ala Leu Ile Asn Asn Val Ser Gly Tyr Val Ile Lys Thr Glu Met

1445 1450 1455

Tyr Thr Glu Val Lys Asn Ala Lys Gly Glu Trp Val Phe Lys Ser Leu

1460 1465 1470

Gly Lys Pro Gly Ser Met His Leu Arg Pro Ile Ala Thr Pro Tyr Pro

1475 1480 1485

Val Lys Glu Trp Leu Gln Pro Lys Arg Tyr Lys Ala His Leu Met Gly

1490 1495 1500

Thr Thr Tyr Val Tyr Asp Phe Pro Glu Leu Phe Arg Gln Ala Ser Ser

1505 1510 1515 1520

Ser Gln Trp Lys Asn Phe Ser Ala Asp Val Lys Leu Thr Asp Asp Phe

1525 1530 1535

Phe Ile Ser Asn Glu Leu Ile Glu Asp Glu Asn Gly Glu Leu Thr Glu

1540 1545 1550

Val Glu Arg Glu Pro Gly Ala Asn Ala Ile Gly Met Val Ala Phe Lys

1555 1560 1565

Ile Thr Val Lys Thr Pro Glu Tyr Pro Arg Gly Arg Gln Phe Val Val

1570 1575 1580

Val Ala Asn Asp Ile Thr Phe Lys Ile Gly Ser Phe Gly Pro Gln Glu

1585 1590 1595 1600

Asp Glu Phe Phe Asn Lys Val Thr Glu Tyr Ala Arg Lys Arg Gly Ile

1605 1610 1615

Pro Arg Ile Tyr Leu Ala Ala Asn Ser Gly Ala Arg Ile Gly Met Ala

1620 1625 1630

Glu Glu Ile Val Pro Leu Phe Gln Val Ala Trp Asn Asp Ala Ala Asn

1635 1640 1645

Pro Asp Lys Gly Phe Gln Tyr Leu Tyr Leu Thr Ser Glu Gly Met Glu

1650 1655 1660

Thr Leu Lys Lys Phe Asp Lys Glu Asn Ser Val Leu Thr Glu Arg Thr

1665 1670 1675 1680

Val Ile Asn Gly Glu Glu Arg Phe Val Ile Lys Thr Ile Ile Gly Ser

1685 1690 1695

Glu Asp Gly Leu Gly Val Glu Cys Leu Arg Gly Ser Gly Leu Ile Ala

1700 1705 1710

Gly Ala Thr Ser Arg Ala Tyr His Asp Ile Phe Thr Ile Thr Leu Val

1715 1720 1725

Thr Cys Arg Ser Val Gly Ile Gly Ala Tyr Leu Val Arg Leu Gly Gln

1730 1735 1740

Arg Ala Ile Gln Val Glu Gly Gln Pro Ile Ile Leu Thr Gly Ala Pro

1745 1750 1755 1760

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

1765 1770 1775

Leu Gly Gly Thr Gln Ile Met Tyr Asn Asn Gly Val Ser His Leu Thr

1780 1785 1790

Ala Val Asp Asp Leu Ala Gly Val Glu Lys Ile Val Glu Trp Met Ser

1795 1800 1805

Tyr Val Pro Ala Lys Arg Asn Met Pro Val Pro Ile Leu Glu Thr Lys

1810 1815 1820

Asp Thr Trp Asp Arg Pro Val Asp Phe Thr Pro Thr Asn Asp Glu Thr

1825 1830 1835 1840

Tyr Asp Val Arg Trp Met Ile Glu Gly Arg Glu Thr Glu Ser Gly Phe

1845 1850 1855

Glu Tyr Gly Leu Phe Asp Lys Gly Ser Phe Phe Glu Thr Leu Ser Gly

1860 1865 1870

Trp Ala Lys Gly Val Val Val Gly Arg Ala Arg Leu Gly Gly Ile Pro

1875 1880 1885

Leu Gly Val Ile Gly Val Glu Thr Arg Thr Val Glu Asn Leu Ile Pro

1890 1895 1900

Ala Asp Pro Ala Asn Pro Asn Ser Ala Glu Thr Leu Ile Gln Glu Pro

1905 1910 1915 1920

Gly Gln Val Trp His Pro Asn Ser Ala Phe Lys Thr Ala Gln Ala Ile

1925 1930 1935

Asn Asp Phe Asn Asn Gly Glu Gln Leu Pro Met Met Ile Leu Ala Asn

1940 1945 1950

Trp Arg Gly Phe Ser Gly Gly Gln Arg Asp Met Phe Asn Glu Val Leu

1955 1960 1965

Lys Tyr Gly Ser Phe Ile Val Asp Ala Leu Val Asp Tyr Lys Gln Pro

1970 1975 1980

Ile Ile Ile Tyr Ile Pro Pro Thr Gly Glu Leu Arg Gly Gly Ser Trp

1985 1990 1995 2000

Val Val Val Asp Pro Thr Ile Asn Ala Asp Gln Met Glu Met Tyr Ala

2005 2010 2015

Asp Val Asn Ala Arg Ala Gly Val Leu Glu Pro Gln Gly Met Val Gly

2020 2025 2030

Ile Lys Phe Arg Arg Glu Lys Leu Leu Asp Thr Met Asn Arg Leu Asp

2035 2040 2045

Asp Lys Tyr Arg Glu Leu Arg Ser Gln Leu Ser Asn Lys Ser Leu Ala

2050 2055 2060

Pro Glu Val His Gln Gln Ile Ser Lys Gln Leu Ala Asp Arg Glu Arg

2065 2070 2075 2080

Glu Leu Leu Pro Ile Tyr Gly Gln Ile Ser Leu Gln Phe Ala Asp Leu

2085 2090 2095

His Asp Arg Ser Ser Arg Met Val Ala Lys Gly Val Ile Ser Lys Glu

2100 2105 2110

Leu Glu Trp Thr Glu Ala Arg Arg Phe Phe Phe Trp Arg Leu Arg Arg

2115 2120 2125

Arg Leu Asn Glu Glu Tyr Leu Ile Lys Arg Leu Ser His Gln Val Gly

2130 2135 2140

Glu Ala Ser Arg Leu Glu Lys Ile Ala Arg Ile Arg Ser Trp Tyr Pro

2145 2150 2155 2160

Ala Ser Val Asp His Glu Asp Asp Arg Gln Val Ala Thr Trp Ile Glu

2165 2170 2175

Glu Asn Tyr Lys Thr Leu Asp Asp Lys Leu Lys Gly Leu Lys Leu Glu

2180 2185 2190

Ser Phe Ala Gln Asp Leu Ala Lys Lys Ile Arg Ser Asp His Asp Asn

2195 2200 2205

Ala Ile Asp Gly Leu Ser Glu Val Ile Lys Met Leu Ser Thr Asp Asp

2210 2215 2220

Lys Glu Lys Leu Leu Lys Thr Leu Lys Ala Ala Ala Ser Phe Leu Glu

2225 2230 2235 2240

Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His

2245 2250 2255

His His His His

2260

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<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctgatatcg gatccgaatt catgagccaa attcacaaac ac 42

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<213> Artificial Sequence (Artificial Sequence)

<400> 4

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<213> Artificial Sequence (Artificial Sequence)

<400> 5

ctatttcgaa acgaggaatt catgagcgaa gaaagcttat tc 42

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<213> Artificial Sequence (Artificial Sequence)

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tcgggcccaa gctggcggcc gcctttcaaa gtcttcaaca atttttc 47