Berberine bridging enzyme mutant and application of berberine bridging enzyme mutant in synthesis of (S)-scoleine

Abstract

The invention discloses a berberine bridging enzyme mutant and application of the berberine bridging enzyme mutant in synthesis of (S)-scoleine, and belongs to the technical field of biological engineering. According to the invention, a berberine bridging enzyme BBE mutant is utilized to efficiently synthesize the protoberberine alkaloid intermediate (S)-Scoleroine and the derivative thereof. Escherichia coli BL21 (DE3) is used as a chassis cell to construct an engineering strain M3a, the M3a is used for synthesizing (S)-Scoleroine, and the yield can reach 3.19 g / L.

Description

Technical Field

[0001] The invention relates to a berberine bridge enzyme mutant and application thereof in synthesizing (S)-scolerine, belonging to the technical field of bioengineering. Background Art

[0002] Misuse and overuse of antibiotics in both human and livestock settings has led to the emergence of antimicrobial resistance, posing a serious threat to global public health. As an alternative approach, the exploration of plant-derived natural products with antimicrobial properties has received extensive attention. Protoberberine alkaloids have attracted increasing interest due to their bioactivity and safety against microorganisms and viruses. Protoberberine belongs to the family of phenylisoquinoline alkaloids (BIAs), which includes more than 2,500 structurally diverse compounds that are well known for their pharmacological properties.

[0003] In this family, berberine is a prominent protoberberine alkaloid that has shown efficacy against many pathogenic bacteria and viruses. In addition, many clinical trials on berberine are ongoing to evaluate its pharmacological properties, including its anti-hyperlipidemic, anti-diabetic, anti-inflammatory and atherosclerosis-preventive potential. Another well-known protoberberine alkaloid, palmitoyl ester, has antioxidant and anti-inflammatory properties similar to berberine and is considered a promising DNA phototherapy agent.

[0004] Although it is feasible to isolate these compounds from plants, it is challenging to meet the growing demand through large-scale plant cultivation, so alternative methods, namely microbial biosynthesis, are urgently needed to reliably and scalably supply these products. In plants, the key step in protoberberine biosynthesis is the stereoselective conversion of (S)-reticuline to (S)-scolerine catalyzed by berberine bridge enzyme (BBE). This flavin-dependent oxidase has been identified as the key rate-limiting enzyme in protoberberine biosynthesis, severely limiting the biosynthetic yield of (S)-scolerine and hindering the progress of protoberberine alkaloid biosynthesis research.

[0005] However, improving the catalytic efficiency of BBE in heterologous microbial hosts is challenging, probably due to the differences in the microenvironment between plants and microorganisms. Previous attempts to increase the expression level of BBE via plasmid or multi-chromosomal integration did not significantly improve its conversion efficiency in microbial hosts. The key to microbial production is to improve the performance of the enzyme to ensure an efficient biocatalytic process. Therefore, the development of an efficient berberine bridge enzyme mutant is key to the synthesis of protoberberine alkaloid intermediate (S)-Scolerine. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a berberine bridge enzyme mutant, which uses the berberine bridge enzyme with an amino acid sequence as shown in PDBID: 3D2D_A as a parent and has one and / or more mutations in (a) to (c):

[0007] (a) mutating the arginine at position 354 of the parent to phenylalanine or tryptophan;

[0008] (b) mutating the tryptophan at position 165 of the parent to glycine;

[0009] (c) The alanine at position 421 of the parent gene is mutated to phenylalanine or tryptophan.

[0010] The present invention also provides a gene encoding the berberine bridge enzyme mutant.

[0011] The present invention also provides an expression vector carrying the berberine bridge enzyme mutant gene, or a gene having an amino acid sequence of berberine bridge enzyme (EcBBE) shown in SEQ ID NO.1.

[0012] In one embodiment, the vector used for the overexpression includes but is not limited to pET series plasmids, or pRSFDuet-1 plasmid, or pCDFDuet-1 plasmid, or pETDuet-1 plasmid, or pACYCDuet-1 plasmid, preferably pCDFDuet-1.

[0013] The present invention also provides a recombinant cell expressing the mutant or the gene, or carrying the expression vector.

[0014] In one embodiment, the recombinant cell further expresses riboflavin synthase (RibH), riboflavin synthase (RibC), bifunctional riboflavin kinase (RibF) and molecular chaperone Gro7.

[0015] In one embodiment, the recombinant cell is the recombinant cell M3a, which expresses the following proteins based on the starting strain: berberine bridge enzyme (EcBBE) mutant, riboflavin synthase (RibH), riboflavin synthase (RibC), bifunctional riboflavin kinase (RibF) and molecular chaperone Gro7; the starting strain is Escherichia coli BL21 (DE3).

[0016] In one embodiment, pCDFDuet is used as a vector to express berberine bridge enzyme (EcBBE) mutant, riboflavin synthase (RibC), bifunctional riboflavin kinase (RibF) and riboflavin synthase (RibH).

[0017] In one embodiment, the expression of the berberine bridge enzyme mutant is regulated by the promoter ssrA.

[0018] In one embodiment, the nucleotide sequence of the promoter ssrA is shown as SEQ ID NO.5.

[0019] In one embodiment, the berberine bridge enzyme mutant is truncated at 22 amino acids at the N-terminus and fused with an MBP tag.

[0020] In one embodiment, the nucleotide sequence encoding the MBP tag is shown as SEQ ID NO.6.

[0021] In one embodiment, plasmid pGro7 is transformed into E. coli BL21(DE3).

[0022] In one embodiment, the amino acid sequence of the berberine bridge enzyme (EcBBE) is shown in PDB: 3D2D_A; the nucleotide sequence encoding the riboflavin synthase (RibH) is shown in SEQ ID NO.4; the nucleotide sequence encoding the riboflavin synthase (RibC) is shown in SEQ ID NO.2; the nucleotide sequence encoding the bifunctional riboflavin kinase (RibF) is shown in SEQ ID NO.3.

[0023] The present invention also provides a method for synthesizing (S)-Scolerine, which uses the berberine bridge enzyme (EcBBE) mutant or the recombinant cell M3a as a catalyst and (S)-Reticuline as a substrate to carry out the reaction.

[0024] In one embodiment, the method comprises: mixing the recombinant cell M3a with a substrate for reaction, and collecting the reaction supernatant; the substrate is a solution containing (S)-Reticuline.

[0025] In one embodiment, the reaction system contains 1-10 mM ascorbate, preferably sodium ascorbate.

[0026] In one embodiment, the recombinant cells are prepared by culturing in a culture medium to a logarithmic growth phase and collecting the cells after induction.

[0027] In one embodiment, the recombinant cells are prepared as follows: the recombinant cells M3a are inoculated into LB medium, cultured at 20-40°C for 10-12 hours, transferred to 2YT medium, and cultured at 20-40°C until OD 600 When the value reaches about 0.6-0.8, cool down to 15-25℃, add IPTG with a final concentration of 0.05-0.5mM to induce for 10-20h, and collect the bacteria.

[0028] In one embodiment, the reaction temperature is 20-40° C., and the reaction pH is 7.5-10.5.

[0029] In one embodiment, the reaction time is 4 to 20 hours.

[0030] In one embodiment, the added amount of the recombinant cell M3a is 10-80 g / L, preferably 70-80 g / L.

[0031] In one embodiment, the structural formula of the substrate (S)-Reticuline is

[0032] The present invention also provides the use of the mutant, the recombinant cell, or the method in preparing (S)-Scolerine.

[0033] Beneficial effects:

[0034] (1) The present invention provides a berberine bridge enzyme mutant capable of efficiently synthesizing (S)-Scolerine in vitro and / or in vivo Figure 1 ). The mutant R354W constructed by the present invention has an enzyme activity increased by 132 times compared with the wild-type enzyme; the mutant W165G has an enzyme activity increased by 3 times compared with the wild-type enzyme; the A421W has an enzyme activity increased by 7 times compared with the wild-type enzyme; the A421F has an enzyme activity increased by 16 times compared with the wild-type enzyme;

[0035] (2) The present invention provides a recombinant strain M3a, which can increase the production of intracellular FAD, increase the expression of berberine bridge enzyme BBE by introducing the endogenous constitutive promoter ssrA of Escherichia coli, and co-expression of the molecular chaperone Gro7, while achieving the oxidative carbon-carbon coupling of the substrate (see Figure 1 );

[0036] (3) The present invention also provides a method for synthesizing (S)-Scolerine using (S)-Reticuline as a raw material, and the yield of (S)-Scolerine is 3.19 g / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The synthetic route of (S)-Scolerine.

[0038] Figure 2 To transform EcBBE to efficiently synthesize (S)-Scolerine; (a) The relative activity of EcBBE mutants towards the substrate (S)-Reticuline, the catalytic activity of the parent towards the substrate (S)-Reticuline was set to 1; (b) Liquid phase analysis diagram of the conversion of substrate (S)-Reticuline into product (S)-Scolerine through in vitro reaction.

[0039] Figure 3 This is the standard curve of the product (S)-Scolerine.

[0040] Figure 4 HPLC (a) and MS (b) identification of (S)-Scolerine synthesized by the recombinant strain EcBBE-RibC-RibH-RibF;Gro7 (strain M3a).

[0041] Figure 5 This is the H spectrum of (S)-Scolerine in deuterated chloroform.

[0042] Figure 6 This is the time curve of (S)-Scolerine synthesis using strain M3a. DETAILED DESCRIPTION

[0043] Purchase sources of reagents and materials: The antibiotics such as ampicillin sodium, kanamycin sulfate, and streptomycin sulfate used in the present invention are all from Shanghai Bioengineering; the PCR enzyme and homologous recombination enzyme involved in the molecular experiments in the present invention are all purchased from Bao Bioengineering (Dalian) Co., Ltd.; the chemical reagents used in the present invention are all purchased from Sinopharm Chemical Reagent Co., Ltd. and Shanghai Titan Technology Co., Ltd.

[0044] LB medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L.

[0045] 2YT medium: tryptone 16 g / L, yeast extract 10 g / L, sodium chloride 5 g / L.

[0046] Berberine bridge enzyme (EcBBE) enzyme activity detection: 200μL reaction system includes 50mM Tris-HCl buffer (pH9.0), 5μM EcBBE enzyme or mutant, 2mM (S)-Reticuline and 10mM sodium ascorbate. The whole system is carried out in a reaction shaker, the reaction temperature is 30℃, and the reaction time is 4h. After the reaction is completed, 4 times the volume of methanol is added to quench the reaction, shake, centrifuge at low temperature and high speed, centrifuge at 20000g for 5min, and take 200μL sample to load the chromatographic column.

[0047] High performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature of 30°C, detection wavelength of 280nm. The detection method uses a two-phase solvent system of acetonitrile (containing 0.1% (v / v) trifluoroacetic acid) and double distilled water (containing 0.1% (v / v) trifluoroacetic acid) for gradient separation, and the mobile phase flow rate is 1mL min -1The linear gradient was as follows: isocratic elution with 5% acetonitrile for 1 min, followed by gradient elution with 5%-50% acetonitrile for 25 min.

[0048] Example 1: Modification of the key rate-limiting enzyme (berberine bridge enzyme BBE) in the protoberberine alkaloid pathway

[0049] In order to increase the yield of the product (S)-Scolerine, the rate-limiting enzyme BBE (PDB ID: 3D2D_A) in the reaction was modified. First, the substrate (S)-Reticuline was selected based on the reported complex structure containing the substrate (S)-Reticuline and BBE protein. The nucleotide sequence shown in SEQ ID NO.1 was connected to the vector pCDFDuet-1, and the primers shown in Table 1 were used to introduce mutations. PCR was performed using the upstream and downstream primers corresponding to the mutation points, respectively, and then connected to construct a vector containing the target mutation point. The corresponding vector was then transformed into Escherichia coli BL21 to construct a recombinant strain.

[0050] Table 1 Primer sequences of EcBBE mutants

[0051]

[0052]

[0053]

[0054] The recombinant bacteria expressing the mutant were cultured at 37°C for 2h, then induced at 16°C for 12h, and the bacterial cells were collected. The collected bacteria were resuspended in lysis buffer (50mmol / L Tris-HCl, 300mmol / L NaCl, 20mM imidazole, pH=8), and then the resuspension was broken by a high-pressure homogenizer. The resuspension after the break was subjected to low-temperature high-speed centrifugation, centrifuged at 4°C and 10000rpm for 30min to obtain a crude enzyme solution. The purified protein was finally obtained by nickel column affinity chromatography and desalting column (HistrapTM 5mLDesalting).

[0055] The enzyme protein was used to catalyze the preparation of (S)-Scolerine. The reaction system was prepared as follows: 50mM Tris-HCl buffer (pH9.0), 5μM EcBBE mutant, 2mM (S)-Reticuline and 10mM sodium ascorbate. The whole system was carried out in a reaction shaker at a reaction temperature of 30°C for 12h. After the reaction, 4 times the volume of methanol was added to quench the reaction, shaken, centrifuged at low temperature and high speed, centrifuged at 20000g for 5min, and 200μL of the sample was loaded onto the chromatographic column.

[0056] According to the standard curve of product (S)-Scolerine ( Figure 3 ) to analyze the yield of each mutant. Using the starting sequence as a control, the relative yield of the product (S)-Scolerine after the mutant reaction was used to indicate the relative activity of different mutants. Figure 2 As shown, multiple single mutants EcBBE with higher activity were obtained R354W , EcBBE W165G , EcBBE L282W , EcBBE R354F , EcBBE F386W , EcBBE A421F , EcBBE A421W , EcBBE I419F , EcBBE W165G / R354W , EcBBE R354W / A421F Compared with wild-type EcBBE, mutant EcBBE R354W The activity was increased by 132 times.

[0057] Table 2 Relative enzyme activity of berberine bridge enzyme EcBBE mutant

[0058]

[0059]

[0060] Example 2: Engineered strain EcBBE R354W - Construction, protein expression and product identification of RibC-RibH-RibF; Gro7 (strain M3a)

[0061] The encoding EcBBE constructed in Example 1 R354W On the basis of the above, 22 amino acids at the N-terminus of EcBBE were truncated and an MBP tag was fused to the N-terminus (the sequence encoding MBP is shown in SEQ ID NO.6), and then its gene was synthesized and connected to the first multiple cloning site (MCS) of the pCDFDuet vector, and the promoter T7 of the first multiple cloning site was replaced with the endogenous promoter ssrA of Escherichia coli (the sequence of ssrA is shown in SEQ ID NO.5), and the plasmid pCDFDuet-TssrA-MBP-EcBBE was obtained. R354W (NΔ22AA), MBP-EcBBE R354W (NΔ22AA) was named EcBBE#.

[0062] The RibC gene fragment (Genbank: CAD6006425.1) was amplified from the genome of Escherichia coli and connected to the pET-21b(+) vector to obtain the plasmid pET-21b-RibC.

[0063] The RibF gene fragment (Genbank: ADD75835.1) was amplified from the genome of Escherichia coli and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b-RibF.

[0064] The RibH gene fragment (Genbank: CAD6020726.1) was amplified from the genome of Escherichia coli and ligated to the pET-21b(+) vector to obtain the plasmid pET-21b-RibH.

[0065] After obtaining the above four plasmids, the gene fragment RibC was integrated into pCDFDuet-TssrA-MBP-EcBBE using homologous recombination technology. R354W (NΔ22AA) at the MCS1 site and RibF and RibH were integrated into pCDFDuet-TssrA-MBP-EcBBE R354W The MCS2 site of (NΔ22AA) was inserted into the ribosome to obtain the plasmid pCDFDuet-TssrA-rbs-EcBBE#-rbs-RibC-T7-rbs-RibH-rbs-RibF (wherein the rbs sequence is AAGGAG). The recombinant plasmid and the plasmid pGro7 were co-transformed into Escherichia coli to obtain the recombinant strain EcBBE#-RibC-RibH-RibF;Gro7 (strain M3a). The primers used for plasmid construction are shown in Table 2.

[0066] Table 3 Primer sequences for constructing recombinant strains

[0067]

[0068]

[0069] The single clone strain EcBBE#-RibC-RibH-RibF;Gro7 (strain M3a) was transferred to 5 ml LB medium and cultured at 250 rpm and 37°C for 10-12 h. Subsequently, it was transferred to 500 ml 2YT medium and cultured at 120 rpm and 37°C. When the bacterial OD 600When the value reaches about 0.6-0.8, cool down to 18℃, culture for 30min, add IPTG with a final concentration of 0.2mM, induce culture at 18℃ for 16h, collect the cells after centrifugation, wash the cells three times with buffer containing 50mM Tris-HCl (pH 9.0) and 100mM NaCl, and obtain the cells by centrifugation.

[0070] Subsequently, a 5 ml reaction system was prepared to verify the synthesis of the product (S)-Scolerine. The reaction system was: 50 mM Tris-HCl (pH 9.0), 100 mM NaCl buffer containing 13 mM (S)-Reticuline, 15 mM sodium ascorbate. The bacterial OD in the reaction system was 600 The reaction was carried out in an oscillator at 30°C. After 8 h, 200 μL of the sample was taken and 800 μL of methanol was added to quench the reaction. The sample was centrifuged at 20,000 g for 5 min and filtered with a 0.22 μm filter membrane.

[0071] The synthesis of the product (S)-Scolerine was detected by HPLC system. Figure 4 As shown, the compound obtained by whole-cell catalysis had the same peak time as the standard, and the generation of the product was further confirmed by high-resolution mass spectrometry. The yield of (S)-Scolerine was 3.19 g / L.

[0072] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A berberine bridge enzyme mutant, with the berberine bridge enzyme with the amino acid sequence as shown in PDB ID: 3D2D_A as a parent, having one and / or more mutations in (a) to (c): (a) mutating the arginine at position 354 of the parent to phenylalanine or tryptophan; (b) mutating the tryptophan at position 165 of the parent to glycine; (c) The alanine at position 421 of the parent gene is mutated to phenylalanine or tryptophan.

2. A gene encoding the berberine bridge enzyme mutant according to claim 1.

3. A recombinant cell expressing the mutant according to claim 1 or the gene according to claim 2.

4. The recombinant cell according to claim 3, characterized in that The recombinant cells also expressed riboflavin synthase RibH, riboflavin synthase RibC, bifunctional riboflavin kinase RibF and molecular chaperone Gro7.

5. The recombinant cell according to claim 3 or 4, characterized in that Escherichia coli BL21 (DE3) was used as the host.

6. A method for synthesizing (S)-Scolerine, characterized in that: The reaction is carried out using the berberine bridge enzyme mutant according to claim 1 or the recombinant cell according to any one of claims 3 to 5 as a catalyst and (S)-Reticuline as a substrate.

7. The method according to claim 6, characterized in that The reaction system contains 1-10 mM ascorbate.

8. The method according to claim 6 or 7, characterized in that: The reaction temperature is 20-40°C, and the reaction pH is 7.5-10.

5.

9. The method according to any one of claims 6 to 8, characterized in that: The structural formula of the substrate (S)-Reticuline is 10. Use of the mutant according to claim 1, or the recombinant cell according to any one of claims 3 to 5, or the method according to any one of claims 6 to 9 in preparing (S)-Scolerine.

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