A mutant of dihydroflavonol 4-reductase and recombinant escherichia coli expressing the mutant and application thereof

Abstract

This invention discloses a mutant of dihydroflavonol 4-reductase, recombinant Escherichia coli expressing this mutant, and its applications. This invention focuses on dihydroflavonol 4-reductase (FaDFR) from Fragaria x ananassa, selecting 12 sites, including the active site and the vicinity of the active pocket, for saturation mutagenesis. Combined with a well plate screening method, a highly active mutant FaDFR that can tolerate high substrate concentrations was finally obtained. G130C This invention aims to provide a mutant of dihydroflavonol 4-reductase, a recombinant Escherichia coli expressing this mutant, and its applications, providing a research foundation for the industrial production of anthocyanins.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to the modification of dihydroflavonol 4-reductase and its application. Background Technology

[0002] Anthocyanins (C3G) are a class of water-soluble natural pigments widely distributed in the plant kingdom. Belonging to the flavonoid family, they possess a variety of health-promoting effects. In addition to their well-known antioxidant activity, anthocyanins have shown great potential in anti-diabetic, anti-obesity, anti-thrombotic, neuroprotective, and vision-protective applications, and are widely used in food, health products, and cosmetics.

[0003] Currently, anthocyanins are mainly extracted from plants, which is insufficient for large-scale industrial applications. Microbial cell factories offer a novel strategy to address these issues. However, the highest anthocyanin titers reported in current microbial synthesis systems have not yet reached the gram level, and all rely on relatively expensive catechins, which are downstream in the synthesis pathway, making industrialization difficult. Synthesizing anthocyanins from cheaper dihydroflavonols, which are further upstream, holds promise for constructing a lower-cost biocatalytic pathway and providing a reference for industrial production.

[0004] Dihydroflavonol 4-reductase (DFR) is a key enzyme catalyzing the formation of colorless anthocyanin precursors from dihydroflavonols, and its catalytic performance directly affects the types and yields of anthocyanins. DFR is a cofactor NADPH-dependent short-chain dehydrogenase / reductase belonging to the SDR family. Its catalytic efficiency varies significantly for different dihydroflavonol substrates (dihydrokaempferol DHK, dihydroquercetin DHQ, and dihydromyricetin DHM). Furthermore, the catalytic activity of DFR is significantly reduced by both competitive inhibition from the active site and non-competitive inhibition from the regulatory site. Most importantly, our previous studies revealed substrate inhibition of DFR; that is, under high substrate concentrations, DFR is almost completely inactivated.

[0005] This invention utilizes SWISS-MODEL to construct a structural model of FaDFR, performs molecular docking of FaDFR with the substrate DHQ in AutoDockVina, and performs saturation mutagenesis on the active site and sites near the active pocket, screening out mutant FaDFRs with enhanced activity and the ability to relieve substrate inhibition. G130C ; with pRSFDuet-Mi3 sc -FaDFR G130C st -PhANS st -At3GT st -SUOMO-MdGSTF6 stAs an expression plasmid, BL21(DE3)ΔpgiΔcadA::GmSuSy was used as the expression host for fermentation, which increased the C3G yield to 1335.29 mg / L, reaching the gram level. This is the highest C3G titer reported in microorganisms to date. Summary of the Invention

[0006] This invention provides a mutant of dihydroflavonol 4-reductase, a recombinant Escherichia coli expressing the mutant, and its application. The mutant exhibits high activity in the catalytic synthesis of cyanidin-3-O-glucoside (C3G) under high concentrations of dihydroquercetin (DHQ) substrate.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention focuses on dihydroflavonol 4-reductase (FaDFR) from Fragaria x ananassa. Twelve sites, including the active site and the vicinity of the active pocket, were selected for saturation mutagenesis. Combined with plate screening, a mutant FaDFR with high activity and tolerance to high substrate concentrations was finally obtained. G130C Using *Escherichia coli* BL21(DE3)ΔpgiΔcadA::GmSuSy as the expression strain, and pRSFDuet-Mi3 as the expression strain... sc -FaDFR G130C st -PhANS st -At3GT st -SUMO-MdGSTF6 st To express the plasmid, the C3G yield was increased to 1335.29 mg / L, which is the highest titer of C3G reported to date for microbial synthesis.

[0009] A mutant of dihydroflavonol 4-reductase, said dihydroflavonol 4-reductase is obtained by saturating mutations at positions 128, 129, 130, 133, 163, 167, 190, 191, 192, 193, 204, and 227 of the dihydroflavonol 4-reductase sequence from Fragaria xananassa, or by one or a combination thereof.

[0010] Further, the amino acid sequence of the dihydroflavonol 4-reductase is SEQ ID No. 1; the dihydroflavonol 4-reductase mutant shown is any one of the following:

[0011] (i) The glutamic acid at position 130 is mutated to cysteine, and its amino acid sequence is SEQ ID No 2;

[0012] (ii) The asparagine at position 133 is mutated to aspartic acid, and its amino acid sequence is SEQ ID No. 3;

[0013] (iii) The valine at position 193 is mutated to leucine, and its amino acid sequence is SEQ ID No. 4;

[0014] (iv) The phenylalanine mutant at position 204 of the proline is SEQ ID No. 5;

[0015] (v) The proline at position 204 is mutated to valine, and its amino acid sequence is SEQ ID No. 6;

[0016] (vi) The proline at position 204 is mutated to leucine, and its amino acid sequence is SEQ ID No. 7;

[0017] (vii) Glutamine at position 227 is mutated to asparagine, and its amino acid sequence is SEQ ID No. 8.

[0018] The gene encoding the mutant of the dihydroflavonol 4-reductase.

[0019] This invention discloses a recombinant plasmid containing the aforementioned gene.

[0020] This invention discloses a host cell containing the above-mentioned recombinant plasmid; the host cell contains one of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Lactobacillus, Pichia pastoris, and Saccharomyces cerevisiae.

[0021] This invention discloses a bacterial strain expressing a dihydroflavonol 4-reductase mutant. The dihydroflavonol 4-reductase mutant is used to enhance the activity of dihydroflavonol 4-reductase and its substrate tolerance concentration.

[0022] Application of a dihydroflavonol 4-reductase mutant in the synthesis of anthocyanin C3G: The highly active and tolerant mutant FaDFR was screened out. G130C The plasmid pRSFDuet-Mi3 was obtained by introducing it into a self-assembled artificial protein cage. sc -FaDFR st G130C -PhANS st -At3GT st -SUOMO-MdGSTF6 st The strain was transformed into BL21(DE3)ΔpgiΔcadA::GmSuSy to obtain the high-C3G-producing recombinant Escherichia coli strain S1.

[0023] In the above applications, the recombinant plasmid is obtained by the following method: The mutant FaDFR... G130C Imported into self-assembled artificial protein cages; designed upstream primer C1(5'-accgcgagcgcg) tgc agcgttaacgttgaa-3') and downstream primer C2 (5'-gcacgcgctcgcggtgaaaaccag-3') were used to target the plasmid template pRSFDuet-Mi3 sc -FaDFR st -PhANS st -At3GT st -SUOMO-MdGSTF6 st Perform loop PCR to obtain the plasmid pRSFDuet-Mi3 sc -FaDFR G130C st -PhANS st -At3GT st -SUOMO-MdGSTF6 st ;

[0024] Strain S1 was obtained by the following method: pRSFDuet-Mi3 sc -FaDFR G130C st -PhANS st -At3GT st -SUOMO-MdGSTF6 st Transformation into *E. coli* BL21(DE3)ΔpgiΔcadA::GmSuSy yielded *E. coli* S1, a strain highly efficient at producing anthocyanin C3G, with an anthocyanin C3G production rate 5.6 times that of strain S0. S0 used BL21(DE3)ΔpgiΔcadA::GmSuSy as its host and pRSFDuet-Mi3 as its growth promoter. sc -FaDFR st -PhANS st -At3GT st -SUOMO-MdGSTF6 st For expression plasmids.

[0025] A method for producing anthocyanin C3G by fermentation of Escherichia coli includes the following steps:

[0026] (1) Culture induction: Single colonies were picked and cultured in LB medium containing kanamycin at 37°C and 220 rpm for 12 h. The seed culture was then inoculated at 1% into fresh LB medium containing kanamycin and cultured at 37°C and 220 rpm until the recombinant bacterial concentration reached OD200. 600The concentration was 0.6. After cooling to 30℃, isopropyl-β-D-thiogalactoside was added to a final concentration of 1mM, and induction was carried out at 30℃ and 220rpm for 3h.

[0027] (2) Fermentation production of anthocyanin C3G: Collect the bacterial cells and transfer them to a modified M9 medium, and add 1.0 mM Mg(SO4)2, 0.1 mM CaCl2, and 0.1 mM Fe(SO4)2. Add the substrate dihydroquercetin DHQ for fermentation to obtain the final product. The fermentation conditions are 30℃ and 220 rpm for 12-24 h. The fermentation broth is treated with acidified methanol (1% hydrochloric acid).

[0028] In Example 1, using SWISS-MODEL, a structural model of FaDFR was constructed using the crystal structure of VvDFR (PDB:2C29) as a template. FaDFR was docked with DHQ in AutoDock Vina. PyMOL was used to find substrate binding sites and active pockets. Twelve sites, including Ser128, Ala129, Gly130, Asn133, Tyr163, Lys167, Pro190, Thr191, Leu192, Val193, Pro204, and Gln227, were screened for saturation mutagenesis.

[0029] Based on the sites screened in Example 1, mutation primers were designed, and a FaDFR mutant library was constructed by PCR amplification. The mutants were initially screened to identify sites affecting FaDFR activity and substrate inhibition. The experimental results were then verified using shake-flask fermentation, ultimately yielding the beneficial FaDFR mutant. G130C Mutant FaDFR G130C The ability to synthesize C3G at high substrate concentrations is 1.77 times that of WT.

[0030] In Example 2, fixed-point sampling was performed from 0 to 24 hours, and the mutant FaDFR was found. G130C Compared to WT, it exhibits higher catalytic efficiency within 0 to 8 hours. Analysis of the mutant FaDFR... G130C Molecular docking results with DHQ and NADPH revealed that the substrate in this mutant was closer to the active site Ser128 compared to WT. Compared to WT, it is more stable to the substrate and promotes hydrogen transfer, thereby increasing the reaction rate.

[0031] In Example 3, the dominant mutant FaDFR obtained in Example 1 was used. G130C In a self-assembled artificial protein cage constructed in the laboratory, BL21(DE3)ΔpgiΔcadA::GmSuSy was used as the expression host to obtain strain S1, which synthesizes C3G efficiently. The C3G yield was 5.60 times that of the wild-type strain (S0).

[0032] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0033] (1) This invention designs and modifies FaDFR using semi-rational design and saturation mutagenesis techniques, screens out 12 saturation mutation sites, and finally obtains a mutant FaDFR with enhanced activity and substrate inhibition relief. G130C .

[0034] (2) In this invention, the dominant mutant was introduced into the previously obtained self-assembled artificial protein cage strain, and BL21(DE3)ΔpgiΔcadA::GmSuSy was used as the expression host, containing the mutant FaDFR G130C The strain's ability to synthesize C3G was increased by 5.6 times, with a C3G titer of 1335.29 mg / L, which is the highest C3G titer reported in microorganisms to date, and has extremely high industrial application value. Attached Figure Description

[0035] Figure 1 The yield of strain C3G at different substrate concentrations in Example 1 of this invention is shown in the figure.

[0036] Figure 2 The FaDFR in Embodiment 2 of the present invention is shown. G130C A schematic diagram of its WT and DHQ docking.

[0037] Figure 3 A comparison chart of C3G yields in strains S0 and S1 in Example 3 of this invention is shown. Detailed Implementation

[0038] The present invention will be further described in detail below through specific embodiments.

[0039] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0041] Example 1: Modification of dihydroflavonol 4-reductase

[0042] In this embodiment, firstly, a structural model of FaDFR was constructed using SWISS-MODEL with the crystal structure of VvDFR (PDB:2C29). The molecular structure of the substrate molecule DHQ was downloaded from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ). Then, FaDFR and DHQ were molecularly docked using AutoDock Vina. The results were analyzed using PyMOL (http: / / www.pymol.org), and 12 sites were screened: Ser128, Ala129, Gly130, Asn133, Tyr163, Lys167, Pro190, Thr191, Leu192, Val193, Pro204, and Gln227. A FaDFR mutant library was constructed using PCR. The primers used are shown in Table 1 (mutation sites are underlined, with Gly130 as an example).

[0043] Table 1. Primer design for constructing the plasmid of dihydroflavonol reductase single-point saturation mutant (Gly130 site).

[0044]

[0045]

[0046]

[0047] The PCR reaction system for amplifying the target fragment is as follows: 0.5 μL DNA template (50-200 ng), 0.4 μL each of forward and reverse primers (10 μM), 10 μL high-fidelity enzyme reaction buffer, and ddH2O to a final volume of 20 μL; the PCR reaction program is: 98℃ pre-denaturation for 3 min, 98℃ for 10 s, 56-62℃ for 5 s, 72℃ for 0.5-3 min, 25 cycles, and a final extension at 72℃ for 10 min; the amplified target fragment is then subjected to agarose gel electrophoresis. The gene fragments were detected and purified using the SanPrep column PCR product purification kit (catalog number B518141-0100). The gene concentration of all DNA samples was calculated using K5600C micro spectrophotometry. The PCR linearized plasmids obtained after DNA fragment recovery were transformed into Escherichia coli DH5α strain. Positive single clones were screened using the kanamycin resistance of the plasmids. Successful ligation was verified by colony PCR and sequenced to obtain 228 FaDFR mutants.

[0048] The specific steps for mutant screening are as follows: Preliminary screening of mutants was performed using 48-well fermentation plates. Single colonies of the constructed mutant strain were inoculated into 1 mL of LB medium containing kanamycin and cultured overnight for 12 h. A 1% seed culture was then transferred to 2 mL of LB medium containing kanamycin and cultured at 37°C and 220 rpm for 3 h until OD (Organic Depth) was reached. 600 The concentration was 0.6. Isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 1 mM and quickly mixed. Induction was performed at 30°C and 220 rpm for 3 h. Cells were collected by centrifugation at 3500 rpm, 4°C, and 15 min. Modified M9 medium was added to 48-well plates for resuspending, with 400 μL per well. After fermentation for 12–24 h, absorbance was measured at 520 nm. The yield of product C3G was calculated using a previously established standard curve. The results are shown below. Figure 1 The dominant strain was then subjected to shake-flask fermentation using the same method as plate fermentation, and the optimal mutant FaDFR was finally screened. G130C FaDFR G130C The yields of C3G produced by fermentation of WT under different substrate concentrations are shown in the figure. Figure 1 .

[0049] Example 2: Analysis of G130C catalytic efficiency and substrate docking results

[0050] In this example, the mutant FaDFR G130C Fermentation with WT strain. Sampling was performed on the fermentation system at fixed points from 0-24 h, and FaDFR was found. G130C Compared to WT, it exhibits higher catalytic efficiency within 0-8 hours. Figure 2 A. Using SWISS-MODEL, FaDFR was constructed based on the crystal structure of FaDFR simulated in Example 1. G130C The structural model, and then using AutoDock Vina to convert FaDFR G130C Molecular docking with DHQ and NADPH was performed, and the results were analyzed using PyMOL. The FaDFR mutant was also analyzed. G130C Molecular docking results with DHQ and NADPH revealed that the substrate in this mutant was closer to the active site Ser128 compared to WT. ( Figure 2 B) is more stable on the substrate and promotes hydrogen transfer than WT, thereby increasing the reaction rate.

[0051] Example 3: Application of dihydroflavonol 4-reductase mutant

[0052] Optimal mutant strain FaDFR G130C The artificial protein cage scaffold designed and assembled in the laboratory before being introduced into the PCR amplification artificial protein cage Mi3 scThe gene and the pathway enzyme are fused to SpyTag via the N-terminal linker (GGGGS)3, and are named FaDFR, respectively. st PhANS st At3GT st SUMO-MdGSTF6 st The above fragments were ligated into the vector pRSFDuet using the Gibson seamless ligation method to obtain the plasmid pRSFDuet-Mi3. sc -FaDFR st -PhANS st -At3GT st -SUMO-MdGS TF6 st Then, using plasmid pRSFDuet-Mi3 sc -FaDFR st -PhANS st -At3GT st -SUMO-MdGSTF6 st Using this as a template, design the upstream primer C1(5'-accgcgagcgcg) tgc agcgttaacgttgaa-3') and downstream primer C2 (5'- gca The plasmid template was subjected to loop PCR using cgcgctcgcggtgaaaaccag-3') to obtain the recombinant plasmid pRSFDuet-Mi3. sc -FaDFR st -PhANS st -At3GT st -SUMO-MdGSTF6 st and pRSFDuet-Mi3 sc -FaDFR G130C st -PhANS st -At3GT st -SUMO-MdGSTF6 st The recombinant expression strains S0 and S1 were obtained by transforming the *E. coli* expression strain BL21(DE3)ΔpgiΔcadA::GmSuSy. Single colonies of strains S0 and S1 were cultured in 10 mL of LB medium containing kanamycin at 37°C and 220 rpm for 12 hours, then inoculated into fresh LB medium containing kanamycin and cultured at 37°C and 220 rpm until the recombinant bacterial concentration reached the OD value. 600The concentration was set to 0.6, and the temperature was lowered to 30℃. Isopropyl-β-D-thiogalactoside was added to a final concentration of 1mM for induction for 3 hours. After collecting the cells by centrifugation at 4℃, 3500 rpm for 15 minutes, the cells were transferred to M9 medium, with the addition of 1mM Mg(SO4)2, 0.1mM CaCl2, and 0.1mM Fe(SO4)2. DHQ was then added as a substrate, and fermentation was carried out at 30℃ and 220 rpm for 12-24 hours to obtain the final product. 1 mL of the fermentation broth was taken, and an equal volume of acidified methanol (1% hydrochloric acid) was added. The mixture was shaken for 1 minute to fully extract the flavonoids. After centrifugation at 12000 rpm for 5 minutes, the supernatant was collected using a 1 mL syringe and filtered through a 0.22 μm nylon membrane for HPLC analysis. C3G was detected using an LC-16 high-performance liquid chromatograph equipped with an SP D-16 dual-wavelength UV detector. The chromatographic column was a Titank C18, 250 × 4.6 mm. Chromatographic conditions: gradient elution, phase A was ultrapure water (0.1% trifluoroacetic acid), phase B was acetonitrile; 0-1 min, phase B: 10%; 1-11 min, phase B: 10-40%; 11-21 min, phase B: 40-60%; 21-23 min, phase B: 60%-10%; 23-28 min, phase B: 10%. Detection wavelengths were 290 nm, 314 nm, 512 nm, and 520 nm, and the column temperature was 35 °C.

[0053] Ultimately, the C3G production of S1 was 5.6 times that of S0. Figure 3 The concentration was 1335.29 mg / L, reaching the gram level, which is the highest C3G titer reported in microorganisms to date.

[0054] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A mutant of dihydroflavonol 4-reductase, characterized in that: The amino acid sequence of the dihydroflavonol 4-reductase is SEQ ID No. 1, and the amino acid sequence of the dihydroflavonol 4-reductase mutant is shown in SEQ ID No.

2.

2. A gene encoding a mutant of the dihydroflavonol 4-reductase as described in claim 1.

3. A recombinant plasmid, characterized in that: The recombinant plasmid contains the gene described in claim 2.

4. A host cell, characterized in that: The host cell comprises the recombinant plasmid as described in claim 3; the host cell is Escherichia coli.

5. A strain expressing the dihydroflavonol 4-reductase mutant of claim 1.

6. The application of the dihydroflavonol 4-reductase mutant according to claim 1 in enhancing the activity of dihydroflavonol 4-reductase and substrate tolerance concentration.

7. The application of the mutant of dihydroflavonol 4-reductase according to claim 1 in the synthesis of anthocyanin C3G, characterized in that: The selected mutants are highly active and can tolerate high substrate concentrations. Fa DFR G130C The plasmid pRSFDuet-Mi3 was obtained by introducing it into a self-assembled artificial protein cage. sc - Fa DFR st G130C - Ph ANS st - At 3GT st -SUOMO -Md GSTF6 st Transformed into BL21(DE3)Δ pgi Δ cadA :: Gm SuSy yielded a high-yield C3G-producing recombinant Escherichia coli strain S1.

8. The application according to claim 7, characterized in that: The recombinant plasmid was obtained by the following method, using the mutant FaDFR G130C Imported into self-assembled artificial protein cages; designed upstream primer C1(5'-accgcgagcgcg) tgc agcgttaacgttgaa-3') and downstream primer C2 (5'-gcacgcgctcgcggtgaaaaccag-3') were used to target the plasmid template pRSFDuet-Mi3 sc - Fa DFR st - Ph ANS st - At 3GT st -SUOMO -Md GSTF6 st Perform loop PCR to obtain the plasmid pRSFDuet-Mi3 sc - Fa DFR G130C st - Ph ANS st - At 3GT st -SUOMO -Md GSTF6 st ; Strain S1 was obtained by the following method: pRSFDuet-Mi3 sc - Fa DFR G130C st - Ph ANS st - At 3GT st -SUOMO - Md GSTF6 st Transformed into Escherichia coli BL21(DE3)Δ pgi Δ cadA :: Gm SuSy obtained Escherichia coli S1, which produced anthocyanin C3G at a high efficiency, with an anthocyanin C3G production rate 5.6 times that of strain S0; strain S0 produced anthocyanin C3G using BL21(DE3)Δ pgi Δ cadA :: Gm SuSy as the host, pRSFDuet-Mi3 sc - Fa DFR st - Ph ANS st - At 3GT st -SUOMO -Md GSTF6 st For expression plasmids.

9. A method for producing anthocyanin C3G by fermentation of Escherichia coli, comprising fermentation using the high-C3G-producing recombinant Escherichia coli strain S1 as described in claim 7 or 8, characterized in that: The process includes the following steps: (1) Culture induction: single colonies are picked and cultured in LB medium containing kanamycin at 37 ℃ and 220 rpm for 12 h. Seed culture is inoculated at 1% in fresh LB medium containing kanamycin and cultured at 37 ℃ and 220 rpm until the recombinant bacterial concentration reaches OD600 of 0.

6. After cooling to 30 ℃, isopropyl-β-D-thiogalactoside is added to a final concentration of 1 mM and induced at 30 ℃ and 220 rpm for 3 h; (2) Fermentation to produce anthocyanin C3G: bacterial cells are collected and transferred to modified M9 medium, and 1.0 mM Mg(SO4)2, 0.1 mM CaCl2, and 0.1 mM Fe(SO4)2 are added. Dihydroquercetin DHQ is added for fermentation to obtain the final product. The fermentation conditions are 30 ℃ and 220 rpm for 12-24 hours. h; The fermentation broth was treated with acidified methanol (1% hydrochloric acid).

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