CN110804618A - Directed evolution construction of ROMT mutant for efficiently converting resveratrol to produce pinqi - Google Patents
Directed evolution construction of ROMT mutant for efficiently converting resveratrol to produce pinqi Download PDFInfo
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Abstract
An oriented evolution construction ROMT mutant for efficiently converting resveratrol to produce pinqi belongs to the technical field of genetic engineering. The invention aims to modify the gene of enzyme by mutagenesis technology such as error-prone PCR and the like, and then according to the specific modification purpose, valuable ROMT mutant which can be directionally evolved and constructed to efficiently convert resveratrol to produce pinostilbene is screened. The invention relates to a method for producing a resveratrol ROMT mutant of pinqi by efficiently transforming resveratrol through directed evolution, which is an ROMT mutant obtained by carrying out directed evolution on a grape-derived VvROMT gene through multiple rounds of recombination and random mutation by using an error-prone PCR method. The improvement of the enzyme activity of the invention can be expressed by the efficiency of the expression mutant type ROMT engineering bacteria for converting resveratrol to produce pinostilbene, and the improvement of the efficiency of the expression mutant type ROMT engineering bacteria for producing pinostilbene represents the improvement of the activity of the mutant type ROMT engineering bacteria for converting resveratrol to produce pinostilbene.
Description
Technical Field
The present invention belongs to the field of gene engineering technology.
Background
Pingqi is a resveratrol derivative, and is found in plants such as grape and blueberry, and has antitoxin and antioxidant effects. Grape and other plants have genes encoding resveratrol O-methyltransferase (ROMT), and the expressed ROMT can methylate resveratrol to produce pinostilbene or pterostilbene. Compared with resveratrol, the pinostilbene and the pterostilbene have stronger stability, wherein the pinostilbene is more effective in preventing and treating cancers than the resveratrol, the market price of the pinostilbene is higher than that of the resveratrol and the pterostilbene, and the efficient biosynthesis method of the pinostilbene has important value.
Most of the currently found ROMTs, whose products are predominantly pterostilbene (Wang Y, Bhuiya M W, Zhou R, et al, Annals of Microbiology, 2015, 65(2):817 826.), and a few of which are predominantly pinostilbene, have very low ROMT activity (Jeong Y J, An C H, Woo S G, et al, Enzyme and microbiological technology, 2014, 54: 8-14.). Escherichia coli engineering bacteria expressing grape source ROMT (VvROMT) can catalyze resveratrol to be converted into pinostilbene and pterostilbene, the product mainly contains pterostilbene, and the pinostilbene is only slightly accumulated. The product of the engineering bacteria of Escherichia coli expressing sorghum source ROMT (SbROMT 3) is mainly composed of loose stilbene, but the yield of the engineering bacteria 48h loose stilbene is only 34 mg/L due to low enzyme activity.
Disclosure of Invention
The invention aims to modify the gene of enzyme by mutagenesis technology such as error-prone PCR and the like, and then according to the specific modification purpose, valuable ROMT mutant which can be directionally evolved and constructed to efficiently convert resveratrol to produce pinostilbene is screened.
The invention relates to a method for producing a resveratrol ROMT mutant of pinqi by efficiently transforming resveratrol through directed evolution, which is an ROMT mutant obtained by carrying out directed evolution on a grape-derived VvROMT gene through multiple rounds of recombination and random mutation by using an error-prone PCR method.
The VvROMT gene nucleotide mutation method comprises the following steps: nucleotide mutations were introduced into the VvROMT gene in vitro using error-prone PCR:
the error-prone PCR reaction conditions were as follows
An upstream primer F: 5'-caGGATCCggatctggcaaatggtgtgatcagtg-3', respectively;
a downstream primer R: 5'-atGCGGCCGCtcacggatacacttcaatcagactgcgcag-3'
PCR amplification conditions: 3min at 94 ℃; 1min at 94 ℃, 1min at 59 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃;
after the error-prone PCR amplification product is purified by a DNA purification kit, the error-prone PCR amplification product and the plasmid pRSF are digested and connected by restriction enzymes BamHI and NotI respectively, and are transformed into competent cells of escherichia coli BL21(DE 3); spread on LB (containing 50. mu.g/mL kanamycin) plate, and cultured at 37 ℃ for 12h to form ROMT mutant library;
using the same method as above, multiple rounds of error-prone PCR were performed using the mutant genes as templates to construct a mutant library.
Obtaining the ROMT mutant of the invention:
the kanamycin-resistant transformants grown on the LB plates were transferred to LB plates containing 50. mu.g/mL of kanamycin using sterilized toothpicks, while the control bacterium Escherichia coli BL21(DE3)/pRSF-VvROMT containing 50. mu.g/mL of kanamycin was inoculated to LB plates and cultured at 37 ℃ for 12 hours;
and (3) screening by using a 96-well plate: adding 100 μ L of LB (containing 50 μ g/mL kanamycin) liquid medium to 1.8 mL/well 96-well plate, inoculating the strain deposited on LB (containing 50 μ g/mL kanamycin) plate (together with Escherichia coli BL21(DE3)/pRSF-VvROMT as a control), culturing overnight in a shaker at 37 ℃ and 200 rpm, supplementing 400 μ L of LB (containing 50 μ g/mL kanamycin and 0.125 mM IPTG) liquid medium to 96-well plate, culturing for 24 h in a shaker at 18 ℃ and 200 rpm, centrifuging the 96-well plate fermentation broth for 1 day at 4 ℃ and 4000 g for 5 min, removing supernatant medium, suspending the cells with 300 μ L of modified M9 (containing 50 μ g/mL kanamycin, 0.1 mM G, 228 mg/L resveratrol), culturing at 18 ℃, culturing in shaking table at 200 rpm for 48h to obtain fermentation broth for converting Escherichia coli BL21(DE3) with expression ROMT gene into resveratrol, adding 3 μ L of 6M HCl into 300 μ L of fermentation broth for converting Escherichia coli BL21(DE3) with expression ROMT gene into resveratrol, mixing, incubating at 37 deg.C for 30 min, extracting with 300 μ L of ethyl acetate, vacuum rotating and centrifuging to dry 200 μ L of ethyl acetate layer, re-suspending with 5 μ L of anhydrous ethanol, collecting 1 μ L for TLC analysis, performing gray scale analysis on the point of pinostilbene compound in the image by thin layer chromatography with ImageJ software, analyzing pinostilbene yield, under the same conditions, strain with remarkably higher pinostilbene yield than that of reference engineering bacteria BL21(DE 3)/VvSF-ROMT yield is selected target strain, the contrast engineering bacterium BL21(DE3)/pRSF-VvROMT only produces trace pinostilbene;
screening is carried out on the basis of a mutation library constructed by an error-prone PCR method, 8 strains with obviously improved resveratrol pinostilbene-producing efficiency are obtained, an ROMT expression plasmid is extracted, an ROMT nucleotide sequence is determined, a triplet codon is used for presuming an amino acid sequence of ROMT, the change mode of amino acid and the relative activity of pinostilbene-producing are shown in table 1, the relative activity of pinostilbene-producing of engineering bacteria in the ROMT mutation library is determined according to the analysis result of TLC detection images of ImageJ software, wherein the relative activity of pinostilbene-producing of the engineering bacteria in the ROMT mutation library is determined by comparing the relative activity of pinostilbene-producing of the engineering bacteria in DE 21(DE 3)/pRSF-;
TABLE 1 sequencing results of ROMT mutant expressed by high-yield Songqi engineering bacteria (Strain number ROMT 01-08)
The high-yield pinqi engineering bacteria of the invention express the amino acid sequence of the ROMT mutant: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8.
The directed evolution of the invention belongs to irrational design, and refers to that aiming at a certain protease gene, the enzyme gene is modified by mutagenesis technologies such as error-prone PCR and the like, and then valuable natural enzymes are screened according to specific modification purposes. Currently, directed evolution technology has been successfully used to improve catalytic activity of enzymes, improve substrate specificity, improve thermal stability, and enantioselectivity. The improvement of the enzyme activity of the invention can be expressed by the efficiency of the expression mutant type ROMT engineering bacteria for converting resveratrol to produce pinostilbene, and the improvement of the efficiency of the expression mutant type ROMT engineering bacteria for producing pinostilbene represents the improvement of the activity of the mutant type ROMT engineering bacteria for converting resveratrol to produce pinostilbene.
Detailed Description
The invention relates to an ROMT mutant for producing pinqi by efficiently converting resveratrol through directed evolution construction, which is an ROMT mutant obtained by directed evolution through multiple rounds of recombination and random mutation by using a grape-derived VvROMT gene (GenBank accession number FM 178870) SEQ ID No.1 and applying an error-prone PCR method and through directed evolution, wherein the amino acid sequence of the ROMT mutant comprises Phe311Trp, Phe311Trp/Ser67Thr/Gly300Ala, Phe311Trp/Val19Leu, Phe311Tyr/Glu330Gln, Phe311Tyr/Ser9Thr, Phe311Tyr, Phe311Arg/Asn46Gln and Phe311Arg/Cys69Asn/Val118 Lle. The yield of the resveratrol-producing pinostilbene transformed by the engineering bacteria expressing the corresponding ROMT mutants is shown, and compared with the control engineering bacteria expressing the wild VvROMT, the efficiency of the mutants for transforming the resveratrol-producing pinostilbene is improved.
The formula of the culture medium related by the invention is as follows:
LB liquid medium (tryptone 1%; Yeast extract 0.5%; NaCl 1%; pH 7.0)
LB solid medium (tryptone 1%; Yeast extract 0.5%; NaCl 1%; agar 2%; pH 7.0)
Modified M9 medium (Na)2HPO4·12H20 1.71%； KH2PO40.3%； NaCl 0.05%； NH4Cl 0.1%；MgSO40.036%； CaCl20.0011%; 0.5% of glycerin; yeast extract 0.125%)
The medium has a medium unit of% (W/V)
Thin Layer Chromatography (TLC) conditions: the thin-layer chromatography silica gel plate of Yangtze river friend silica gel development Co., Ltd is used, the model is HSGF254, and the developing agent is dichloromethane: the acetone contained 0.1% formic acid at a volume ratio of 40: 2.
Liquid chromatography-mass spectrometry(LC-MS) conditions: the qualitative and quantitative analysis of resveratrol and pinostilbene in the extracted compounds was carried out using Agilent 1260-6120 LC-MS system, and the products were separated by reverse chromatography ZORBAX SB-C18 (particle size 1.8 μm; 2.1X 50 mm) in 100% acetonitrile containing 0.1% (v/v) formic acid (solution A) and 0.1% (v/v) formic acid (solution B). The sample was diluted with 50% absolute ethanol and the gradient elution procedure was as follows: eluting 20% solution A for 8min, linearly eluting 20% -50% solution A for 7 min, eluting 20% solution A for 13 min, with flow rate of 0.2mL/min, sample injection volume of 1 μ L, detection mode positive ion mode, detection voltage of 1.56 kV, and atomizing gas (N)2) Flow rate 1.5L/min, dry gas (N)2) The pressure is 100 kPa, the ion collection time is 30 MS, the collision energy is 50 percent, the MS scanning range is 100-600 m/z, and the sample is quantitatively analyzed by an external standard method.
The invention relates to a method for producing a resveratrol ROMT mutant of pinqi by efficiently transforming resveratrol through directed evolution, which is an ROMT mutant obtained by carrying out directed evolution on a grape-derived VvROMT gene through multiple rounds of recombination and random mutation by using an error-prone PCR method.
The VvROMT gene nucleotide mutation method comprises the following steps: nucleotide mutations were introduced into the VvROMT gene in vitro using error-prone PCR:
the error-prone PCR reaction conditions were as follows
An upstream primer F: 5'-caGGATCCggatctggcaaatggtgtgatcagtg-3', respectively;
a downstream primer R: 5'-atGCGGCCGCtcacggatacacttcaatcagactgcgcag-3'
PCR amplification conditions: 3min at 94 ℃; 1min at 94 ℃, 1min at 59 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃;
after the error-prone PCR amplification product is purified by a DNA purification kit, the error-prone PCR amplification product and the plasmid pRSF are digested and connected by restriction enzymes BamHI and NotI respectively, and are transformed into competent cells of escherichia coli BL21(DE 3); spread on LB (containing 50. mu.g/mL kanamycin) plate, and cultured at 37 ℃ for 12h to form ROMT mutant library;
using the same method as above, multiple rounds of error-prone PCR were performed using the mutant genes as templates to construct a mutant library.
Obtaining the ROMT mutant of the invention:
the kanamycin-resistant transformants grown on the LB plates were transferred to LB plates containing 50. mu.g/mL of kanamycin using sterilized toothpicks, while the control bacterium Escherichia coli BL21(DE3)/pRSF-VvROMT containing 50. mu.g/mL of kanamycin was inoculated to LB plates and cultured at 37 ℃ for 12 hours;
and (3) screening by using a 96-well plate: adding 100 μ L of LB (containing 50 μ g/mL kanamycin) liquid medium to 1.8 mL/well 96-well plate, inoculating the strain deposited on LB (containing 50 μ g/mL kanamycin) plate (together with Escherichia coli BL21(DE3)/pRSF-VvROMT as a control), culturing overnight in a shaker at 37 ℃ and 200 rpm, supplementing 400 μ L of LB (containing 50 μ g/mL kanamycin and 0.125 mM IPTG) liquid medium to 96-well plate, culturing for 24 h in a shaker at 18 ℃ and 200 rpm, centrifuging the 96-well plate fermentation broth for 1 day at 4 ℃ and 4000 g for 5 min, removing supernatant medium, suspending the cells with 300 μ L of modified M9 (containing 50 μ g/mL kanamycin, 0.1 mM G, 228 mg/L resveratrol), culturing at 18 ℃, culturing in shaking table at 200 rpm for 48h to obtain fermentation broth for converting Escherichia coli BL21(DE3) with expression ROMT gene into resveratrol, adding 3 μ L of 6M HCl into 300 μ L of fermentation broth for converting Escherichia coli BL21(DE3) with expression ROMT gene into resveratrol, mixing, incubating at 37 deg.C for 30 min, extracting with 300 μ L of ethyl acetate, vacuum rotating and centrifuging to dry 200 μ L of ethyl acetate layer, re-suspending with 5 μ L of anhydrous ethanol, collecting 1 μ L for TLC analysis, performing gray scale analysis on the point of pinostilbene compound in the image by thin layer chromatography with ImageJ software, analyzing pinostilbene yield, under the same conditions, strain with remarkably higher pinostilbene yield than that of reference engineering bacteria BL21(DE 3)/VvSF-ROMT yield is selected target strain, the contrast engineering bacterium BL21(DE3)/pRSF-VvROMT only produces trace pinostilbene;
screening is carried out on the basis of a mutation library constructed by an error-prone PCR method, 8 strains with obviously improved resveratrol pinostilbene-producing efficiency are obtained, an ROMT expression plasmid is extracted, an ROMT nucleotide sequence is determined, a triplet codon is used for presuming an amino acid sequence of ROMT, the change mode of amino acid and the relative activity of pinostilbene-producing are shown in table 1, the relative activity of pinostilbene-producing of engineering bacteria in the ROMT mutation library is determined according to the analysis result of TLC detection images of ImageJ software, wherein the relative activity of pinostilbene-producing of the engineering bacteria in the ROMT mutation library is determined by comparing the relative activity of pinostilbene-producing of the engineering bacteria in DE 21(DE 3)/pRSF-;
TABLE 1 sequencing results of ROMT mutant expressed by high-yield Songqi engineering bacteria (Strain number ROMT 01-08)
The invention relates to a method for measuring the yield of pinostilbene produced by converting resveratrol by engineering bacteria expressing pinostilbene ROMT mutant efficiently
And (3) determining the yield of the resveratrol-produced pinostilbene converted by the engineering bacteria expressing the high-efficiency pinostilbene ROMT mutant in a 50 mL culture system.
And (3) shaking flask fermentation: control bacteria, namely escherichia coli BL21(DE3)/pRSF-VvROMT and 8 engineering bacteria expressing a high-efficiency pinostilbene ROMT mutant are respectively inoculated into 5 mL of LB (containing 50 mu g/mL of kanamycin) liquid culture medium in a 50 mL centrifuge tube, cultured for 12 hours in a shaker at 37 ℃ and 200 rpm to serve as seed liquid, 0.5 mL of the seed liquid is inoculated into 50 mL of LB (containing 50 mu g/mL of kanamycin) culture medium in a 250 mL triangular flask, and fermented and cultured in the shaker at 37 ℃ and 200 rpm. And detecting the absorbance of the fermentation liquor by using a spectrophotometer at the wavelength of 600 nm, and adding IPTG (isopropyl-beta-thiogalactoside) into the fermentation liquor to the final concentration of 100 mu M when the absorbance of the fermentation liquor at the wavelength of 600 nm reaches 0.6. Culturing the fermentation liquor in a shaking table at 18 ℃ and 200 rpm for 8 hours to obtain a control strain BL21(DE3)/pRSF-VvROMT and 8 fermentation liquor expressing the high-efficiency pinostilbene ROMT mutant engineering bacteria, centrifuging at 4 ℃ and 4000 g for 15 min, removing a supernatant culture medium, suspending the bacteria by using 50 mL of improved M9 (containing 50 mu g/mL of kanamycin, 0.1 mM of IPTG and 228 mg/L of resveratrol) culture medium, and culturing in the shaking table at 18 ℃ and 200 rpm for 12 hours to obtain the fermentation liquor expressing the control strain BL21(DE3)/pRSF-VvROMT and the high-efficiency pinostilbene ROMT engineering bacteria. Respectively adding 500 μ L of fermentation liquid into 500 μ L of anhydrous ethanol, vortex oscillating for 30 s, centrifuging at 12000 rpm for 10min in a centrifuge, filtering the supernatant with 0.45 μm organic filter membrane, and determining the yield of pinqi by LC-MS. The multiple improvement of the yield of pinqi in 12h by expressing the high-efficiency pinqi-producing ROMT mutant engineering bacteria (the strain number is ROMT 01-08) compared with the yield of a control strain BL21(DE3)/pRSF-VvROMT (the strain number is VvROMT) is shown in Table 2.
Table 2 shows that the yield of pinostilbene produced by converting resveratrol by engineering bacteria for efficiently producing pinostilbene ROMT and the fold increase of the yield
The high-yield pinqi engineering bacteria of the invention express the amino acid sequence of the ROMT mutant: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8.
Sequence listing
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Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Ala Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Trp Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Glu Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
<210>4
<211>357
<212>PRT
<213>ROMT 03
<400>4
Met Asp Leu Ala Asn Gly Val Ile Ser Ala Glu Leu Leu His Ala Gln
1 5 10 15
Ala His Leu Trp Asn His Ile Phe Asn Phe Ile Lys Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Gln Leu Gly Ile Pro Asp Ile Ile His Asn His Gly
35 40 45
Lys Pro Met Thr Leu Pro Glu Leu Val Ala Lys Leu Pro Val His Pro
50 5560
Lys Arg Ser Gln Cys Val Tyr Arg Leu Met Arg Ile Leu Val His Ser
65 70 75 80
Gly Phe Leu Ala Ala Gln Arg Val Gln Gln Gly Lys Glu Glu Glu Gly
85 90 95
Tyr Val Leu Thr Asp Ala Ser Arg Leu Leu Leu Met Asp Asp Ser Leu
100 105 110
Ser Ile Arg Pro Leu Val Leu Ala Met Leu Asp Pro Ile Leu Thr Lys
115 120 125
Pro Trp His Tyr Leu Ser Ala Trp Phe Gln Asn Asp Asp Pro Thr Pro
130 135 140
Phe His Thr Ala His Glu Arg Ser Phe Trp Asp Tyr Ala Gly His Glu
145 150 155 160
Pro Gln Leu Asn Asn Ser Phe Asn Glu Ala Met Ala Ser Asp Ala Arg
165 170 175
Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275 280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Gly Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Trp Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Glu Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
<210>5
<211>357
<212>PRT
<213>ROMT 04
<400>5
Met Asp Leu Ala Asn Gly Val Ile Ser Ala Glu Leu Leu His Ala Gln
1 5 10 15
Ala His Val Trp Asn His Ile Phe Asn Phe Ile Lys Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Gln Leu Gly Ile Pro Asp Ile Ile His Asn His Gly
35 40 45
Lys Pro Met Thr Leu Pro Glu Leu Val Ala Lys Leu Pro Val His Pro
50 55 60
Lys Arg Ser Gln Cys Val Tyr Arg Leu Met Arg Ile Leu Val His Ser
65 70 75 80
Gly Phe Leu Ala Ala Gln Arg Val Gln Gln Gly Lys Glu Glu Glu Gly
85 90 95
Tyr Val Leu Thr Asp Ala Ser Arg Leu Leu Leu Met Asp Asp Ser Leu
100 105 110
Ser Ile Arg Pro Leu Val Leu Ala Met Leu Asp Pro Ile Leu Thr Lys
115 120 125
Pro Trp His Tyr Leu Ser Ala Trp Phe Gln Asn Asp Asp Pro Thr Pro
130 135 140
Phe His Thr Ala His Glu Arg Ser Phe Trp Asp Tyr Ala Gly His Glu
145 150 155 160
Pro Gln Leu Asn Asn Ser Phe Asn Glu Ala Met Ala Ser Asp Ala Arg
165 170 175
Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275 280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Gly Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Tyr Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Gln Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
<210>6
<211>357
<212>PRT
<213>ROMT 05
<400>6
Met Asp Leu Ala Asn Gly Val Ile Thr Ala Glu Leu Leu His Ala Gln
1 5 10 15
Ala His Val Trp Asn His Ile Phe Asn Phe Ile Lys Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Gln Leu Gly Ile Pro Asp Ile Ile His Asn His Gly
35 40 45
Lys Pro Met Thr Leu Pro Glu Leu Val Ala Lys Leu Pro Val His Pro
50 55 60
Lys Arg Ser Gln Cys Val Tyr Arg Leu Met Arg Ile Leu Val His Ser
65 70 75 80
Gly Phe Leu Ala Ala Gln Arg Val Gln Gln Gly Lys Glu Glu Glu Gly
85 90 95
Tyr Val Leu Thr Asp Ala Ser Arg Leu Leu Leu Met Asp Asp Ser Leu
100 105 110
Ser Ile Arg Pro Leu Val Leu Ala Met Leu Asp Pro Ile Leu Thr Lys
115 120 125
Pro Trp His Tyr Leu Ser Ala Trp Phe Gln Asn Asp Asp Pro Thr Pro
130 135 140
Phe His Thr Ala His Glu Arg Ser Phe Trp Asp Tyr Ala Gly His Glu
145 150 155 160
Pro Gln Leu Asn Asn Ser Phe Asn Glu Ala Met Ala Ser Asp Ala Arg
165 170 175
Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275 280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Gly Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Tyr Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Glu Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
<210>7
<211>357
<212>PRT
<213>ROMT 06
<400>7
Met Asp Leu Ala Asn Gly Val Ile Ser Ala Glu Leu Leu His Ala Gln
1 5 10 15
Ala His Val Trp Asn His Ile Phe Asn Phe Ile Lys Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Gln Leu Gly Ile Pro Asp Ile Ile His Asn His Gly
35 40 45
Lys Pro Met Thr Leu Pro Glu Leu Val Ala Lys Leu Pro Val His Pro
50 55 60
Lys Arg Ser Gln Cys Val Tyr Arg Leu Met Arg Ile Leu Val His Ser
65 70 75 80
Gly Phe Leu Ala Ala Gln Arg Val Gln Gln Gly Lys Glu Glu Glu Gly
85 90 95
Tyr Val Leu Thr Asp Ala Ser Arg Leu Leu Leu Met Asp Asp Ser Leu
100 105 110
Ser Ile Arg Pro Leu Val Leu Ala Met Leu Asp Pro Ile Leu Thr Lys
115 120 125
Pro Trp His Tyr Leu Ser Ala Trp Phe Gln Asn Asp Asp Pro Thr Pro
130 135 140
Phe His Thr Ala His Glu Arg Ser Phe Trp Asp Tyr Ala Gly His Glu
145 150 155 160
Pro Gln Leu Asn Asn Ser Phe Asn Glu Ala Met Ala Ser Asp Ala Arg
165 170 175
Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275 280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Gly Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Tyr Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Glu Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
<210>8
<211>357
<212>PRT
<213>ROMT 07
<400>8
Met Asp Leu Ala Asn Gly Val Ile Ser Ala Glu Leu Leu His Ala Gln
1 5 10 15
Ala His Val Trp Asn His Ile Phe Asn Phe Ile Lys Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Gln Leu Gly Ile Pro Asp Ile Ile His Gln His Gly
35 40 45
Lys Pro Met Thr Leu Pro Glu Leu Val Ala Lys Leu Pro Val His Pro
50 55 60
Lys Arg Ser Gln Cys Val Tyr Arg Leu Met Arg Ile Leu Val His Ser
65 70 75 80
Gly Phe Leu Ala Ala Gln Arg Val Gln Gln Gly Lys Glu Glu Glu Gly
85 90 95
Tyr Val Leu Thr Asp Ala Ser Arg Leu Leu Leu Met Asp Asp Ser Leu
100 105 110
Ser Ile Arg Pro Leu Val Leu Ala Met Leu Asp Pro Ile Leu Thr Lys
115 120 125
Pro Trp His Tyr Leu Ser Ala Trp Phe Gln Asn Asp Asp Pro Thr Pro
130 135 140
Phe His Thr Ala His Glu Arg Ser Phe Trp Asp Tyr Ala Gly His Glu
145 150 155 160
Pro Gln Leu Asn Asn Ser Phe Asn Glu Ala Met Ala Ser Asp Ala Arg
165 170 175
Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275 280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Gly Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Arg Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Glu Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
<210>9
<211>357
<212>PRT
<213>ROMT 08
<400>9
Met Asp Leu Ala Asn Gly Val Ile Ser Ala Glu Leu Leu His Ala Gln
1 5 10 15
Ala His Val Trp Asn His Ile Phe Asn Phe Ile Lys Ser Met Ser Leu
20 25 30
Lys Cys Ala Ile Gln Leu Gly Ile Pro Asp Ile Ile His Asn His Gly
35 40 45
Lys Pro Met Thr Leu Pro Glu Leu Val Ala Lys Leu Pro Val His Pro
50 55 60
Lys Arg Ser Gln Asn Val Tyr Arg Leu Met Arg Ile Leu Val HisSer
65 70 75 80
Gly Phe Leu Ala Ala Gln Arg Val Gln Gln Gly Lys Glu Glu Glu Gly
85 90 95
Tyr Val Leu Thr Asp Ala Ser Arg Leu Leu Leu Met Asp Asp Ser Leu
100 105 110
Ser Ile Arg Pro Leu Ile Leu Ala Met Leu Asp Pro Ile Leu Thr Lys
115 120 125
Pro Trp His Tyr Leu Ser Ala Trp Phe Gln Asn Asp Asp Pro Thr Pro
130 135 140
Phe His Thr Ala His Glu Arg Ser Phe Trp Asp Tyr Ala Gly His Glu
145 150 155 160
Pro Gln Leu Asn Asn Ser Phe Asn Glu Ala Met Ala Ser Asp Ala Arg
165 170 175
Leu Leu Thr Ser Val Leu Leu Lys Glu Gly Gln Gly Val Phe Ala Gly
180 185 190
Leu Asn Ser Leu Val Asp Val Gly Gly Gly Thr Gly Lys Val Ala Lys
195 200 205
Ala Ile Ala Asn Ala Phe Pro His Leu Asn Cys Thr Val Leu Asp Leu
210 215 220
Pro His Val Val Ala Gly Leu Gln Gly Ser Lys Asn Leu Asn Tyr Phe
225 230 235 240
Ala Gly Asp Met Phe Glu Ala Ile Pro Pro Ala Asp Ala Ile Leu Leu
245 250 255
Lys Trp Ile Leu His Asp Trp Ser Asp Glu Glu Cys Val Lys Ile Leu
260 265 270
Lys Arg Cys Arg Glu Ala Ile Pro Ser Lys Glu Asn Gly Gly Lys Val
275 280 285
Ile Ile Ile Asp Met Ile Met Met Lys Asn Gln Gly Asp Tyr Lys Ser
290 295 300
Thr Glu Thr Gln Leu Phe Arg Asp Met Thr Met Met Ile Phe Ala Pro
305 310 315 320
Gly Arg Glu Arg Asp Glu Asn Glu Trp Glu Lys Leu Phe Leu Asp Ala
325 330 335
Gly Phe Ser His Tyr Lys Ile Thr Pro Ile Leu Gly Leu Arg Ser Leu
340 345 350
Ile Glu Val Tyr Pro
355
Claims (4)
1. An ROMT mutant for efficiently converting resveratrol to produce pinqi by directed evolution is characterized in that: the ROMT mutant of pinqi is produced by efficiently transforming resveratrol constructed by directed evolution, and the ROMT mutant is obtained by carrying out directed evolution on a VvROMT gene from a grape source by applying an error-prone PCR method through multiple rounds of recombination and random mutation.
2. The directed evolution construction of ROMT mutants capable of efficiently transforming resveratrol to produce pinostilbene according to claim 1, which is characterized in that: VvROMT gene nucleotide mutation method: nucleotide mutations were introduced into the VvROMT gene in vitro using error-prone PCR:
the error-prone PCR reaction conditions were as follows
An upstream primer F: 5'-caGGATCCggatctggcaaatggtgtgatcagtg-3', respectively;
a downstream primer R: 5'-atGCGGCCGCtcacggatacacttcaatcagactgcgcag-3'
PCR amplification conditions: 3min at 94 ℃; 1min at 94 ℃, 1min at 59 ℃, 2min at 72 ℃ and 30 cycles; 10min at 72 ℃;
after the error-prone PCR amplification product is purified by a DNA purification kit, the error-prone PCR amplification product and the plasmid pRSF are digested and connected by restriction enzymes BamHI and NotI respectively, and are transformed into competent cells of escherichia coli BL21(DE 3); spread on LB (containing 50. mu.g/mL kanamycin) plate, and cultured at 37 ℃ for 12h to form ROMT mutant library;
using the same method as above, multiple rounds of error-prone PCR were performed using the mutant genes as templates to construct a mutant library.
3. The directed evolution construction of ROMT mutants capable of efficiently transforming resveratrol to produce pinostilbene according to claim 1, which is characterized in that: acquisition of ROMT mutants:
the kanamycin-resistant transformants grown on the LB plates were transferred to LB plates containing 50. mu.g/mL of kanamycin using sterilized toothpicks, while the control bacterium Escherichia coli BL21(DE3)/pRSF-VvROMT containing 50. mu.g/mL of kanamycin was inoculated to LB plates and cultured at 37 ℃ for 12 hours;
and (3) screening by using a 96-well plate: adding 100 μ L of LB (containing 50 μ g/mL kanamycin) liquid medium to 1.8 mL/well 96-well plate, inoculating the strain deposited on LB (containing 50 μ g/mL kanamycin) plate (together with Escherichia coli BL21(DE3)/pRSF-VvROMT as a control), culturing overnight in a shaker at 37 ℃ and 200 rpm, supplementing 400 μ L of LB (containing 50 μ g/mL kanamycin and 0.125 mM IPTG) liquid medium to 96-well plate, culturing for 24 h in a shaker at 18 ℃ and 200 rpm, centrifuging the 96-well plate fermentation broth for 1 day at 4 ℃ and 4000 g for 5 min, removing supernatant medium, suspending the cells with 300 μ L of modified M9 (containing 50 μ g/mL kanamycin, 0.1 mM G, 228 mg/L resveratrol), culturing at 18 ℃, culturing in shaking table at 200 rpm for 48h to obtain fermentation broth for converting Escherichia coli BL21(DE3) with expression ROMT gene into resveratrol, adding 3 μ L of 6M HCl into 300 μ L of fermentation broth for converting Escherichia coli BL21(DE3) with expression ROMT gene into resveratrol, mixing, incubating at 37 deg.C for 30 min, extracting with 300 μ L of ethyl acetate, vacuum rotating and centrifuging to dry 200 μ L of ethyl acetate layer, re-suspending with 5 μ L of anhydrous ethanol, collecting 1 μ L for TLC analysis, performing gray scale analysis on the point of pinostilbene compound in the image by thin layer chromatography with ImageJ software, analyzing pinostilbene yield, under the same conditions, strain with remarkably higher pinostilbene yield than that of reference engineering bacteria BL21(DE 3)/VvSF-ROMT yield is selected target strain, the contrast engineering bacterium BL21(DE3)/pRSF-VvROMT only produces trace pinostilbene;
screening is carried out on the basis of a mutation library constructed by an error-prone PCR method, 8 strains with obviously improved resveratrol pinostilbene-producing efficiency are obtained, an ROMT expression plasmid is extracted, an ROMT nucleotide sequence is determined, a triplet codon is used for presuming an amino acid sequence of ROMT, the change mode of amino acid and the relative activity of pinostilbene-producing are shown in table 1, the relative activity of pinostilbene-producing of engineering bacteria in the ROMT mutation library is determined according to the analysis result of TLC detection images of ImageJ software, wherein the relative activity of pinostilbene-producing of the engineering bacteria in the ROMT mutation library is determined by comparing the relative activity of pinostilbene-producing of the engineering bacteria in DE 21(DE 3)/pRSF-;
TABLE 1 sequencing results of ROMT mutant expressed by high-yield Songqi engineering bacteria (Strain number ROMT 01-08)
4. The directed evolution construction of ROMT mutants capable of efficiently transforming resveratrol to produce pinostilbene according to claim 1, which is characterized in that: the high-yield pinqi engineering bacteria express the amino acid sequence of the ROMT mutant: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No. 8.
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| WO2021223545A1 (en) * | 2020-05-07 | 2021-11-11 | 中国科学院遗传与发育生物学研究所 | Directed evolution method based on primary and secondary replicon of gemini virus |
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| WO2021223545A1 (en) * | 2020-05-07 | 2021-11-11 | 中国科学院遗传与发育生物学研究所 | Directed evolution method based on primary and secondary replicon of gemini virus |
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