CN113862290B - Isoflavone 4' -O-methyltransferase from liquorice and application thereof - Google Patents

Isoflavone 4' -O-methyltransferase from liquorice and application thereof Download PDF

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CN113862290B
CN113862290B CN202111098860.XA CN202111098860A CN113862290B CN 113862290 B CN113862290 B CN 113862290B CN 202111098860 A CN202111098860 A CN 202111098860A CN 113862290 B CN113862290 B CN 113862290B
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isoflavone
methyltransferase
naringenin
isosakuranetin
leu
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周景文
陈坚
曾伟主
高松
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Jiangnan University
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    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01046—Isoflavone 4'-O-methyltransferase (2.1.1.46)
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Abstract

The invention discloses isoflavone 4' -O-methyltransferase from liquorice and application thereof, belonging to the field of gene and metabolic engineering. The invention provides a new application of isoflavone 4' -O-methyltransferase shown in SEQ ID No.3, which can be used for catalyzing naringenin to synthesize isosakuranetin. The enzyme is expressed in saccharomyces cerevisiae, a strain capable of synthesizing isosakuranetin by naringenin is constructed, and the purpose of synthesizing isosakuranetin by a microbiological method is achieved. The recombinant saccharomyces cerevisiae constructed by the invention can catalyze 300mg/L naringenin to generate 53.3mg/L isosakuranetin within 72h, and the molar conversion rate reaches 16.9%.

Description

Isoflavone 4' -O-methyltransferase from liquorice and application thereof
Technical Field
The invention relates to isoflavone 4' -O-methyltransferase from liquorice and application thereof, belonging to the field of gene and metabolic engineering.
Background
Isosakuranetin belongs to plant natural products, is generally extracted from Clinopodium chinense, has effects of reducing blood sugar and blood lipid, and is a potential candidate medicine for treating diabetes. However, the low content of isosakuranetin in natural plants causes the high price of isosakuranetin obtained by plant extraction, which restricts the application of isosakuranetin. The microbial method for synthesizing the plant natural product becomes a potential alternative scheme of the plant extraction method due to the advantages of short growth cycle, no seasonal limitation and the like. Isosakuranetin can be obtained by adding a methyl group to 4' position of Naringenin, so the key point of synthesizing isosakuranetin in microorganism lies in obtaining high-efficiency flavone 4' -O-methyl transferase (F4 ' OMT) to catalyze Naringenin (Naringenin) to obtain isosakuranetin.
Disclosure of Invention
The invention expresses isoflavone 4'-O-methyltransferase from licorice (Glycyrrhiza echinata), provides a new application of the isoflavone 4' -O-methyltransferase in catalyzing naringenin to produce isosakuranetin, and the isoflavone 4'-O-methyltransferase can catalyze naringenin to add a methyl group to the 4' position so as to obtain isosakuranetin.
The invention provides isoflavone 4'-O-methyltransferase, which contains an amino acid sequence shown in SEQ ID NO.3, has the catalysis function of the flavone 4' -O-methyltransferase, and can catalyze naringenin to synthesize isosakuranetin.
The invention also provides application of isoflavone 4' -O-methyltransferase in catalyzing naringenin to synthesize isosakuranetin.
The invention provides a gene for coding the isoflavone 4' -O-methyltransferase, which contains a nucleotide sequence shown in SEQ ID NO. 2.
The invention also provides an expression vector carrying the gene.
In one embodiment, the microbial cell is a strain of Saccharomyces cerevisiae C800 (CEN. PK2-1D, MAT α; ura3-52 leu2-3,112 trp1-289 C (ii) a SUC2; gal80:: kanMX) (published in Gao, s.; zhou, h.; zhou, J., et al, promoter-library-based optimization for efficacy (2S) -naringenin production from p-basic acid in Saccharomyces cerevisiae [ J]J aggregate Food Chem 2020,68 (25), 6884-6891. The plasmid is pY26-PGAL7-EGFP (disclosed in Gao, S.; xu, X.Y.; zeng, W.Z., et al., effective biosyntheses of (2S) -eriodic from (2S) -naringenin in Saccharomyces cerevisiae through a combination of promoter addition and direct evolution [ J].ACS Synth Biol 2020,9(12),3288-3297.)。
In one embodiment, the expression vector is constructed by connecting a nucleotide sequence shown in SEQ ID NO.2 to the position of EGFP in pY26-PGAL7-EGFP to replace the EGFP gene, and then transferring the recombinant plasmid into Saccharomyces cerevisiae C800.
In one embodiment, the expression vector is constructed by ligating the nucleotide sequence shown in SEQ ID NO.2 to a position between BamHI and HindIII in a conventional plasmid pET28a (+), and then transferring the recombinant plasmid into E.coli BL21 (DE 3).
The invention also provides a microbial cell containing the gene or the expression vector.
In one embodiment, the microbial cell has integrated into its genome the gene set forth in SEQ ID No. 2.
In one embodiment, the microbial cell contains a recombinant plasmid carrying the gene.
The invention also provides a method for producing 4' -O-methyltransferase, which comprises the steps of inoculating the escherichia coli into a TB culture medium, culturing at 35-40 ℃ until the OD600 is 0.04-0.06, adding IPTG with the final concentration of 0.01-1 mM, and carrying out induced expression for at least 8h at 28-30 ℃.
In one embodiment, the Saccharomyces cerevisiae is inoculated into YNB medium and cultured to obtain a seed solution, and then the seed solution is transferred into YPD medium and cultured for at least 20h at 28-32 ℃ to obtain 4' -O-methyltransferase.
In one embodiment, the method comprises the steps of inoculating the recombinant saccharomyces cerevisiae into a culture medium for culture, and adding a substrate naringenin every 10 to 14 hours after inoculation for at least 72 hours.
In one embodiment, the method comprises adding the 4' -O-methyltransferase and naringenin substrate at a ratio of 50. Mu.M to 1mg, and reacting at 30 ℃ for at least 1h.
The invention also provides application of the recombinant escherichia coli in preparation of microbial cell catalysts.
The invention also provides application of the recombinant saccharomyces cerevisiae in producing functional food or medicine containing isosakuranetin.
The invention has the beneficial effects that: provides a novel isoflavone 4' -O-methyltransferase which can be used for the heterologous expression and synthesis of isosakuranetin. The recombinant saccharomyces cerevisiae expressing the enzyme is used for producing isosakuranetin, 300mg/L of naringenin can be catalyzed within 72 hours to generate 53.3mg/L of isosakuranetin, and the molar conversion rate is 16.9%. In vitro catalysis experiments using purified enzyme further prove that GeF4'OMT has isoflavone 4' -O-methyltransferase catalytic function.
Drawings
FIG. 1 is a schematic diagram of the catalytic production of isosakuranetin from naringenin by GeF4' OMT.
FIG. 2 is a high performance liquid chromatography detection result of fermentation broth of Saccharomyces cerevisiae GeMT of example 3 with naringenin as a substrate; a: high performance liquid phase result of fermentation liquor; b: naringenin and isosakuranetin yield in the fermentation broth.
FIG. 3 is a graph showing the kinetics of the enzymatic reaction of GeF4' OMT.
Detailed Description
YNB medium: 0.72g/L yeast nitrogen source basal 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.
LB medium: LB culture medium: 5g/L yeast powder, 10g/L peptone and 5g/L sodium chloride. Ampicillin was added as required at a final concentration of 50. Mu.g/mL.
TB culture medium: 24g/L yeast powder, 12g/L peptone and 4mL/L glycerol.
20g/L agar powder is added into the solid culture medium.
Buffer A:20mM K 2 HPO 4 ，20mM KH 2 PO 4 ，50mM NaCl，pH 7.4。
And (3) buffer solution B:20mM K 2 HPO 4 ，20mM KH 2 PO 4 50mM NaCl,500mM imidazole, pH 7.4.
PBS buffer: 160mL of 20mM NaH 2 PO4 and 840mL of 20mM Na 2 HPO 4 Mix (pH 7.5).
The detection method comprises the following steps:
the samples were tested using a Shimadzu high performance liquid chromatography (Prominence LC-20A instrument), C18 reverse phase chromatography column (4.6 mm. Times.250mm, thermo), at a column temperature of 25 ℃. The mobile phase was acetonitrile (a): water (B) and 1 ‰ trifluoroacetic acid. The flow rate is 1mL/min, the sample injection amount is 10 mu L, and the detection wavelength is 350nm. The mobile phase conditions are 0-10min (B: 10-40%), 10-30min (B: 40-80%), 30-35min (B: 80%), 35-37min (B: 80-10%), 37-40min (B: 10%). Naringenin standard sample showed peak at 15.833min, and isosakuranetin standard sample showed peak at 20.676 min.
The method for detecting the enzyme activity of isoflavone 4' -O-methyltransferase comprises the following steps: 400. Mu.L of the protein solution was put into a 1.5mL centrifuge tube, and 400. Mu.L of a PBS solution containing 5mM S-adenosyl-L-methionine and 200. Mu.L of a PBS solution containing 2.5mM naringenin were added in this order. Blowing, stirring uniformly, and placing in a water bath kettle at 30 ℃ for reaction for 1h. And putting 800 mu L of reaction solution into a 5mL centrifuge tube, adding 800 mu L of ethyl acetate, fully and uniformly mixing, centrifuging at 14000rpm for 5min, and taking supernatant for liquid phase detection.
The amount of enzyme required to catalyze the conversion of 1. Mu. Mol of naringenin, a substrate, to the product isosakuranetin, per minute in PBS buffer at pH7.5 at 30 ℃ is defined as 1 enzyme activity unit (1U).
The genotypes of the strains and plasmids used in embodiments of the invention are shown in table 1. The original nucleotide sequence of the isoflavone 4'-O-methyltransferase gene is shown in a sequence table SEQ ID NO.1, the sequence after codon optimization is shown in a sequence table SEQ ID NO.2, and the amino acid sequence of the isoflavone 4' -O-methyltransferase is shown in a sequence table SEQ ID NO. 3.
TABLE 1 genotypes of strains and plasmids
Figure BDA0003270106370000031
Figure BDA0003270106370000041
Example 1: construction of recombinant plasmid
(1) Isoflavone 4'-O-methyltransferase GeF4' OMT gene from Glycyrrhiza glabra (Glycyrrhiza echinata) was synthesized based on the codon preference of Saccharomyces cerevisiae. The original nucleotide sequence is shown in a sequence table SEQ ID NO.1, the sequence after codon optimization is shown in a sequence table SEQ ID NO.2, and the corresponding amino acid sequence is shown in a sequence table SEQ ID NO. 3.
Amplifying a sequence SEQ ID NO.2 by using a primer P1/P2, amplifying a vector pY26-PGAL7-EGFP by using a primer P3/P4, recovering a correct fragment by using a DNA recovery kit, integrating the two fragments on the plasmid pY26-PGAL7-EGFP in a seamless manner by Gibson assembly, and completely replacing the DNA sequence of the EGFP by the DNA sequence of the SEQ ID NO.2 so that the gene GeF4' OMT is amplified by a strong promoter P GAL7 Transcription is initiated to obtain a recombinant plasmid pY26-GeF4' OMT. Similarly, SEQ ID NO.2 and the plasmid pET28a (+) were amplified using P5/P6 and P7/P8, respectively, and the target fragment was recovered using a DNA recovery kit, and then integrated tracelessly at the BamHI/HindIII position of the plasmid pET28a (+) by Gibson assembly to obtain a recombinant plasmid pET28-GeF4' OMT.
TABLE 2 base and amino acid sequences of all genes
Figure BDA0003270106370000042
Figure BDA0003270106370000051
TABLE 3 base and amino acid sequences of all genes
Figure BDA0003270106370000052
The homology arm sequences are underlined for Gibson assembly.
Example 2: construction of recombinant Saccharomyces cerevisiae and recombinant Escherichia coli
Through a high-efficiency transformation method of saccharomyces cerevisiae, the recombinant plasmid pY26-GeF4' OMT constructed in the embodiment 1 is transformed into a saccharomyces cerevisiae strain C800, a transformation liquid is coated on a YNB solid plate, and after 3-5 days of culture at 30 ℃, a single colony is grown, namely a recombinant strain. The recombinant strain was named GeMT.
The recombinant plasmid pET28-GeF4' OMT constructed in example 1 is transformed into Escherichia coli BL21 (DE 3) by an Escherichia coli chemical transformation method, the transformation liquid is coated on an LB solid culture medium plate containing 50 mug/mL, after overnight culture at 37 ℃, a single colony is grown, namely recombinant Escherichia coli which is named as EGeMT.
Example 3 production of Isosakuranetin by recombinant Saccharomyces cerevisiae GeMT
Fermentation verification is carried out on the recombinant saccharomyces cerevisiae GeMT constructed in the embodiment 2, naringenin, dihydrokaempferol, apigenin, kaempferol and 8-isopentenyl kaempferol are respectively used as substrates, and whether substrate consumption and new substance generation exist or not is detected.
The fermentation conditions were as follows: fermentation conditions in 250mL shake flasks: at least 10 single colonies were picked and inoculated in a 250mL shake flask containing 20mL YNB liquid medium, incubated at 30 ℃ and 220rpm for 16-18h to obtain OD 600 Between 2 and 3 seed culture fluid. Seed culture broth was transferred at 2% (V/V) to 250mL shake flasks (initial OD) containing 20mL fresh YPD broth 600 Around 0.05),at 12 th, 24 th, 36 th and 48 th hour, 75mg/L of substrate was added to the medium, respectively, for a total of 300mg/L. The fermentation was terminated after culturing at 30 ℃ and 220rpm for 72 hours. And putting 500 mu L of fermentation liquor into a 2mL centrifuge tube, adding methanol with the same volume, shaking and uniformly mixing, centrifuging at 13500rpm for 5min, taking supernatant, and passing through a 0.22 mu m nylon membrane for later use for liquid phase and liquid quality detection. Liquid phase results prove that GeF4' OMT has the capability of catalyzing naringenin to synthesize isosakuranetin, and cannot identify other 4 substrates (dihydrokaempferol, apigenin, kaempferol and 8-isopentenyl kaempferol). After 72h fermentation, 300mg/L naringenin can be catalyzed to obtain 53.3mg/L isosakuranetin.
Example 4: identification of enzymatic Properties of GeF4' OMT
Inoculating recombinant Escherichia coli strain EGeMT single colony into 250mL shake flask containing 25mL LB culture medium, adding kanamycin to final concentration of 50 μ g/mL, culturing at 37 deg.C and 220rpm for 10-12 hr, and performing initial OD 600 =0.01 transfer to 250mL shake flask containing 50mL fresh TB Medium, adding kanamycin to the medium to a final concentration of 50. Mu.g/mL, culturing at 37 ℃,220rpm for 3-4h, and allowing OD 600 When the temperature reached 2-3 ℃, IPTG was added to a final concentration of 1mM for induction of expression, and the temperature was lowered to 30 ℃. After 8h of induction expression, the fermentation broth was centrifuged at 4 ℃ and 5000rpm for 10min, and then the supernatant was removed, and the pellet was resuspended 2 times in 50mL of PBS buffer, and then centrifuged at 4 ℃ and 5000rpm for 10min, and then the supernatant was removed. The pellet was resuspended in 50mL of PBS and added to a high pressure homogenizer that had been pre-cooled to 4 ℃. The high-pressure homogenizer is adjusted to 850bar, the machine is started and the lysate is collected after 2 times of crushing. The lysate was centrifuged at 14000rpm for 10min at 4 ℃ to retain the supernatant. The supernatant was passed through a 0.45m filter for further use. The filtrate is the crude enzyme solution.
The His-tagged GeF4' OMT protein was recovered by affinity chromatography. The 1mL nickel column was equilibrated with buffer A, followed by washing 10 volumes of buffer A at a flow rate of 1mL/min with crude enzyme solution at a flow rate of 1mL/min, followed by washing the nickel column with buffer B at a flow rate of 1mL/min, and the elution peaks were collected according to the UV value. The collected elution peak was desalted using a Desalting column (5 mL), and the target protein was recovered by elution with PBS (pH 7.5) at a flow rate of 5 mL/min. The recovered target protein was quantified using NanoDrop, and diluted with PBS (pH 7.5) to a protein concentration of 2.5mg/mL, and the enzyme activity of the protein solution was 15U/mg protein, and the solution was placed on ice at 4 ℃ for use.
400. Mu.L of the protein solution (enzyme activity: 15U) was put into a 1.5mL centrifuge tube, and 400. Mu.L of a PBS solution containing 5mM S-adenosyl-L-methionine dissolved therein and 200. Mu.L of a PBS solution containing 2.5mM naringenin dissolved therein were sequentially added (enzyme substrate ratio: 110.21U/g substrate). Blowing, beating and mixing evenly, and then placing in a 30 ℃ water bath kettle for reaction for 1h. And putting 800 mu L of reaction solution into a 5mL centrifuge tube, adding 800 mu L of ethyl acetate, fully and uniformly mixing, centrifuging at 14000rpm for 5min, and taking supernatant for liquid phase detection. The result shows that in a pure enzyme catalysis system, 15U enzyme protein can catalyze 500 mu M naringenin (136.1 mg/L) to obtain 400.2 mu M isosakuranetin (126.0 mg/L), and the conversion rate is 88.0%. The Vmax, km, kcat and Kcat/Km of GeF4' OMT were calculated to be 15.00. Mu. Mol/(min. Mg), 522.30. Mu. Mol/L, 10.23/s and 0.0196. Mu. Mol/(L. S) in this order.
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> isoflavone 4' -O-methyltransferase from licorice and application thereof
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gaagaaaccg cttatgcatt gactccacca tctaaattgt tggttaaagg taaaccaaca 360
tgtttagctt ctattgttag aggtgctttg cacccatcta gtttagatat gtggagatct 420
tctgaaaaat ggtttaaaga agataaagaa ttgactttat ttgaatcagc tacaggtgaa 480
tctttttggg atttcttgaa caaagattct gaatcaggta cattatctat gtttcaagaa 540
gctatggctg ctgattcaca aatgtttaaa ttagctttaa aagaatgtag acatgttttt 600
gaaggtttag aatctttggt tgatgttggt ggtggtactg gtggtgttac taaattaatt 660
catgaagaat ttccacattt gaaatgtact gtttttgatc aaccacaagt tgttggtaac 720
ttgtcaggta atgaaaactt aaaatttgtc ggtggtgaca tgtttaaatc tatacctcca 780
gctgatgctg ttttgttaaa atgggttttg catgattgga atgatgaatt atctttaaaa 840
attttaaaaa attctaaaga agctatttct ggtaaaggta aagaaggtaa agttataatt 900
atagatatat caattgatga agcttctggt gacagagaat taactgaatt acaattagat 960
tatgacttag ttatgttaac catgtttaat ggtaaagaaa gagaaaagaa agaatgggaa 1020
aaattgatta gtgatgctgg tttttcttct tataaaatta ctccaatttg tggttttaaa 1080
tctttgattg aagtttttcc ataa 1104
<210> 3
<211> 367
<212> PRT
<213> Glycyrrhiza echinata
<400> 3
Met Ala Phe Ser Thr Asn Gly Ser Glu Glu Ile Glu Leu Tyr His Ala
1 5 10 15
Gln Ile His Leu Tyr Lys His Val Tyr Asn Phe Val Ser Ser Met Ala
20 25 30
Leu Lys Ser Ala Met Glu Leu Gly Ile Ala Asp Val Ile His Asn His
35 40 45
Gly Lys Pro Ile Thr Leu Pro Glu Leu Ala Ser Ala Leu Lys Leu His
50 55 60
Pro Ser Lys Val Gly Ile Leu Tyr Arg Phe Leu Arg Leu Leu Thr His
65 70 75 80
Asn Gly Phe Phe Ala Lys Thr Thr Val Pro Ser Gln Asn Gly Lys Asp
85 90 95
Gly Glu Glu Glu Glu Glu Thr Ala Tyr Ala Leu Thr Pro Pro Ser Lys
100 105 110
Leu Leu Val Lys Gly Lys Pro Thr Cys Leu Ala Ser Ile Val Arg Gly
115 120 125
Ala Leu His Pro Ser Ser Leu Asp Met Trp Arg Ser Ser Glu Lys Trp
130 135 140
Phe Lys Glu Asp Lys Glu Leu Thr Leu Phe Glu Ser Ala Thr Gly Glu
145 150 155 160
Ser Phe Trp Asp Phe Leu Asn Lys Asp Ser Glu Ser Gly Thr Leu Ser
165 170 175
Met Phe Gln Glu Ala Met Ala Ala Asp Ser Gln Met Phe Lys Leu Ala
180 185 190
Leu Lys Glu Cys Arg His Val Phe Glu Gly Leu Glu Ser Leu Val Asp
195 200 205
Val Gly Gly Gly Thr Gly Gly Val Thr Lys Leu Ile His Glu Glu Phe
210 215 220
Pro His Leu Lys Cys Thr Val Phe Asp Gln Pro Gln Val Val Gly Asn
225 230 235 240
Leu Ser Gly Asn Glu Asn Leu Lys Phe Val Gly Gly Asp Met Phe Lys
245 250 255
Ser Ile Pro Pro Ala Asp Ala Val Leu Leu Lys Trp Val Leu His Asp
260 265 270
Trp Asn Asp Glu Leu Ser Leu Lys Ile Leu Lys Asn Ser Lys Glu Ala
275 280 285
Ile Ser Gly Lys Gly Lys Glu Gly Lys Val Ile Ile Ile Asp Ile Ser
290 295 300
Ile Asp Glu Ala Ser Gly Asp Arg Glu Leu Thr Glu Leu Gln Leu Asp
305 310 315 320
Tyr Asp Leu Val Met Leu Thr Met Phe Asn Gly Lys Glu Arg Glu Lys
325 330 335
Lys Glu Trp Glu Lys Leu Ile Ser Asp Ala Gly Phe Ser Ser Tyr Lys
340 345 350
Ile Thr Pro Ile Cys Gly Phe Lys Ser Leu Ile Glu Val Phe Pro
355 360 365

Claims (7)

1. Isoflavone 4OUse of a methyltransferase in the catalysis of the synthesis of isosakuranetin from naringenin, characterized in that said isoflavone 4 ″)OThe amino acid sequence of the methyltransferase is shown in SEQ ID NO. 3.
2. A method for producing isosakuranetin, characterized in that isoflavone 4' -is preparedO-methyltransferase or expressing said isoflavone 4-O-the microbial cells of methyltransferase convert naringenin to isosakuranetin in a medium containing naringenin; said isoflavone 4-OThe amino acid sequence of the methyltransferase is shown as SEQ ID NO. 3.
3. Method according to claim 2, characterized in that said isoflavone is 4 ″' -enrichedO-the methyltransferase is produced by fermentation of a microorganism; the microbial fermentation specifically comprises the following steps: the expression of isoflavone 4-OCulturing Escherichia coli of-methyltransferase in a culture medium for a certain period of time, and collecting isoflavone 4-O-a methyltransferase.
4. The method according to claim 3, wherein the Escherichia coli is inoculated into a TB medium and cultured at 35 to 40 ℃ to OD 600 And (3) 0.04 to 0.06, adding IPTG (isopropyl thiogalactoside) with the final concentration of 0.01 to 1mM, and inducing and expressing for at least 8 hours at the temperature of 28 to 30 ℃.
5. The method according to claim 2, characterized in that the expression of isoflavone 4-O-culturing Saccharomyces cerevisiae of methyl transferase in a naringenin-containing culture medium at 28 to 32 ℃ for at least 20 h.
6. The method according to claim 2, characterized in that the expression of isoflavone 4-O-inoculating Saccharomyces cerevisiae of methyltransferase into a culture medium, culturing at 28-32 ℃, and after inoculation, culturing every 10-14h, adding naringenin into the culture medium, and culturing for at least 72h.
7. Isoflavone 4' -with the amino acid sequence shown as SEQ ID NO.3O-methyltransferase or expressing said isoflavone 4-O-use of recombinant microbial cells of methyltransferase for the production of medical nutrition or pharmaceuticals containing isosakuranetin.
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