Berberine bridge enzyme mutants and their use in the synthesis of (s)-scolerine

Abstract

This invention discloses a berberine bridging enzyme mutant and its application in the synthesis of (S)-scolerine, belonging to the field of bioengineering technology. This invention utilizes the berberine bridging enzyme BBE mutant to efficiently synthesize the original berberine alkaloid intermediate (S)-scolerine and its derivatives. Furthermore, using *Escherichia coli* BL21(DE3) as the chassis cell, an engineered strain M3a was constructed, and (S)-scolerine was synthesized using M3a, achieving a yield of 3.19 g / L.

Description

Technical Field

[0001] This invention relates to berberine-bridged enzyme mutants and their application in the synthesis of (S)-scolerine, belonging to the field of bioengineering technology. Background Technology

[0002] The misuse and overuse of antibiotics in human and livestock environments 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 widespread attention. Proberberine alkaloids have attracted increasing interest due to their bioactivity and safety against microorganisms and viruses. Proberberine belongs to the phenylisoquinoline alkaloid (BIAs) family, which includes more than 2,500 structurally diverse compounds well-known for their pharmacological properties.

[0003] Within this family, berberine is a prominent proberberine alkaloid, effective against many pathogenic bacteria and viruses. Furthermore, numerous clinical trials of berberine are underway to evaluate its pharmacological properties, including its potential for anti-hyperlipidemia, anti-diabetic, anti-inflammatory, and anti-atherosclerotic effects. Another well-known proberberine alkaloid, palmitin, possesses similar antioxidant and anti-inflammatory properties to berberine and is considered a promising candidate for DNA phototherapy.

[0004] While isolating these compounds from plants is feasible, meeting the growing demand through large-scale plant cultivation is challenging, thus necessitating alternative methods, namely microbial biosynthesis, to reliably and scalably supply these products. In plants, a key step in protoperine biosynthesis is the stereoselective conversion of (S)-reticuline to (S)-scolerine catalyzed by berberine bridging enzyme (BBE). This flavin-dependent oxidase has been identified as the critical rate-limiting enzyme in protoperine biosynthesis, severely limiting the biosynthetic yield of (S)-scolerine and hindering progress in research on the biosynthesis of protoperine alkaloids.

[0005] However, improving the catalytic efficiency of BBE in heterologous microbial hosts is challenging, likely due to differences in the microenvironment between plants and microorganisms. Previous attempts to enhance BBE expression levels through plasmid or multi-chromosome integration have not significantly improved its transformation efficiency in microbial hosts. A key aspect of microbial production is improving enzyme performance to ensure efficient biocatalytic processes. Therefore, the development of a highly efficient berberine-bridged enzyme mutant is crucial for the synthesis of the protoberberine alkaloid intermediate (S)-Scolerine. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a berberine bridging enzyme mutant, using the berberine bridging enzyme with the amino acid sequence shown in PDBID:3D2D_A as the parent, and possessing one and / or more mutations in (a) to (c):

[0007] (a) Mutate the arginine at position 354 of the parental line to phenylalanine or tryptophan;

[0008] (b) Mutate tryptophan at position 165 of the parental line to glycine;

[0009] (c) Mutate the alanine at position 421 of the parental line to phenylalanine or tryptophan.

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

[0011] The present invention also provides an expression vector carrying the berberine bridging enzyme mutant gene, or the gene of berberine bridging enzyme (EcBBE) with the amino acid sequence 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 described above.

[0014] In one embodiment, the recombinant cells also express riboflavin synthase (RibH), riboflavin synthase (RibC), bifunctional riboflavin kinase (RibF), and molecular chaperone Gro7.

[0015] In one embodiment, the recombinant cell is recombinant cell M3a, which expresses the following proteins based on the starting strain: berberine brining 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 brining enzyme (EcBBE) mutant, riboflavin synthase (RibC), bifunctional riboflavin kinase (RibF), and riboflavin synthase (RibH).

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

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

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

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

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

[0022] In one embodiment, the amino acid sequence of the berberine brining 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; and 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 brining enzyme (EcBBE) mutant or the recombinant cell M3a as a catalyst and (S)-Reticuline as a substrate for the reaction.

[0024] In one embodiment, the method includes: mixing the recombinant cells M3a with a substrate 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 them in a culture medium to the logarithmic growth phase, inducing them, and then collecting the bacterial cells.

[0027] In one embodiment, the recombinant cells are prepared as follows: recombinant cells M3a are seeded into LB medium and cultured at 20-40°C for 10-12 hours, then transferred to 2YT medium and cultured at 20-40°C until OD. 600 When the pH reaches approximately 0.6–0.8, cool the temperature to 15–25°C, add IPTG at a final concentration of 0.05–0.5 mM to induce induction for 10–20 h, and then collect the bacterial cells.

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

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

[0030] In one embodiment, the amount of recombinant cell M3a added 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, or the recombinant cell, or the method in the preparation of (S)-Scolerine.

[0033] Beneficial effects:

[0034] (1) This invention provides a berberine brining enzyme mutant capable of achieving efficient synthesis of (S)-Scolerine in vitro and / or in vivo. Figure 1 The mutant R354W constructed in this invention showed a 132-fold increase in enzyme activity compared to the wild-type enzyme; the mutant W165G showed a 3-fold increase in enzyme activity compared to the wild-type enzyme; the A421W showed a 7-fold increase in enzyme activity compared to the wild-type enzyme; and the A421F showed a 16-fold increase in enzyme activity compared to the wild-type enzyme.

[0035] (2) This invention provides a recombinant strain M3a that can enhance intracellular FAD production. It enhances the expression of berberine brining enzyme BBE by introducing the endogenous constitutive promoter ssrA from *E. coli* and co-expressing the molecular chaperone Gro7, while simultaneously achieving carbon-oxidative 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, wherein the yield of (S)-Scolerine is 3.19 g / L. Attached Figure Description

[0037] Figure 1 The synthetic route for (S)-Scolerine is shown.

[0038] Figure 2 To modify EcBBE for efficient synthesis of (S)-Scolerine; (a) the relative activity of the EcBBE mutant for the substrate (S)-Reticuline, with the catalytic activity of the parent for the substrate (S)-Reticuline set to 1; (b) liquid phase analysis chromatogram of the conversion of the substrate (S)-Reticuline to the product (S)-Scolerine via in vitro reaction.

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

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

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

[0042] Figure 6 The time curve for (S)-Scolerine synthesis using strain M3a is shown. Detailed Implementation

[0043] Sources of reagents and materials: The antibiotics such as ampicillin sodium, kanamycin sulfate, and streptomycin sulfate used in this invention are all from Shanghai Sangon Biotech; the PCR enzymes and homologous recombinant enzymes involved in the molecular experiments in this invention are all purchased from Takara Bio Engineering (Dalian) Co., Ltd.; the chemical reagents used in this 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 16g / L, yeast extract 10g / L, sodium chloride 5g / L.

[0046] Berberine-bridged enzyme (EcBBE) activity assay: A 200 μL reaction system consisted of 50 mM Tris-HCl buffer (pH 9.0), 5 μM EcBBE enzyme or mutant, 2 mM (S)-Reticuline, and 10 mM sodium ascorbate. The entire system was run in a shaker at 30 °C for 4 h. After the reaction, 4 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20,000 g for 5 min. 200 μL of the sample was then loaded onto the chromatographic column.

[0047] High-performance liquid chromatography (HPLC) detection conditions: ZORBAX Eclipse XDB-C18 column, column temperature 30℃, detection wavelength 280nm. The detection method employed a gradient separation using 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), with a mobile phase flow rate of 1 mL / min. -1The linear gradient is as follows: first, elute isocratically with 5% acetonitrile for 1 min, then elute with a gradient of 5%-50% acetonitrile for 25 min.

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

[0049] 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)-Scolerine was selected based on the previously reported complex structure containing the substrate (S)-Scolerine and the BBE protein. The amino acids within the specified range were modified. The nucleotide sequence shown in SEQ ID NO.1 was ligated into the vector pCDFDuet-1, and the mutation was introduced using the primers shown in Table 1. PCR was performed using the upstream and downstream primers corresponding to the mutation points, followed by ligation 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 the EcBBE mutant

[0051]

[0052]

[0053]

[0054] The recombinant bacteria expressing the mutant were cultured at 37°C for 2 h, followed by induction at 16°C for 12 h. Cells were collected and resuspended in lysis buffer (50 mmol / L Tris-HCl, 300 mmol / L NaCl, 20 mM imidazole, pH 8). The resuspended cells were then homogenized using a high-pressure homogenizer. The homogenized resuspended cells were centrifuged at low temperature and high speed (4°C, 10,000 rpm for 30 min) to obtain the crude enzyme solution. The solution was then desalted using nickel affinity chromatography and a Histrap™ 5 mL desalting column to obtain the purified protein.

[0055] The enzyme protein was used to catalyze the preparation of (S)-Scolerine. The reaction system consisted of 50 mM Tris-HCl buffer (pH 9.0), 5 μM EcBBE mutant, 2 mM (S)-Reticuline, and 10 mM sodium ascorbate. The entire reaction was carried out in a shaker at 30 °C for 12 h. After the reaction was complete, 4 volumes of methanol were added to quench the reaction, followed by shaking, low-temperature high-speed centrifugation, and centrifugation at 20,000 g for 5 min. 200 μL of the sample was then loaded onto the chromatographic column.

[0056] Based on the standard curve of product (S)-Scolerine ( Figure 3 The yields of each mutant were analyzed. Using the starting sequence as a control, the relative activity of different mutants was expressed based on the relative yields of the product (S)-Scolerine after the mutant reaction. Figure 2 As shown, several single mutants with high activity, EcBBE, were obtained. R354W EcBBE W165G EcBBE L282W EcBBE R354F EcBBE F386W EcBBE A421F EcBBE A421W EcBBE I419F EcBBE W165G / R354W EcBBE R354W / A421F Compared to wild-type EcBBE, mutant EcBBE R354W The activity was increased by 132 times.

[0057] Table 2. Relative enzyme activities of berberine briningase 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 encoded EcBBE constructed in Example 1 R354W Based on this, the 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). The gene was then synthesized and ligated into the first multiple cloning site (MCS) of the pCDFDuet vector. The promoter T7 at the first multiple cloning site was replaced with the endogenous E. coli promoter ssrA (the ssrA sequence is shown in SEQ ID NO. 5), resulting in the plasmid pCDFDuet-TssrA-MBP-EcBBE. R354W (NΔ22AA), MBP-EcBBE R354W (NΔ22AA) is named EcBBE#.

[0062] The RibC gene fragment (Genbank: CAD6006425.1) was amplified from the genome of Escherichia coli and ligated into 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 into 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 into 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 The MCS1 site of (NΔ22AA) was used to integrate RibF and RibH into pCDFDuet-TssrA-MBP-EcBBE. R354W The plasmid pCDFDuet-TssrA-rbs-EcBBE#-rbs-RibC-T7-rbs-RibH-rbs-RibF (where the rbs sequence is AAGGAG) was obtained by targeting the MCS2 site of (NΔ22AA). The recombinant plasmid and plasmid pGro7 were co-transformed into *E. 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] Single clones of strain EcBBE#-RibC-RibH-RibF;Gro7 (strain M3a) were selected and transferred to 5 ml of LB medium, incubated at 250 rpm and 37°C for 10–12 h. Subsequently, they were transferred to 500 ml of 2YT medium and incubated at 120 rpm and 37°C. When the bacterial OD... 600When the pH reaches approximately 0.6-0.8, cool the temperature to 18℃ and incubate for 30 minutes. Then add IPTG to a final concentration of 0.2 mM and induce culture at 18℃ for 16 hours. Collect the bacterial cells after centrifugation. Wash the bacterial cells three times with a buffer containing 50 mM Tris-HCl (pH 9.0) and 100 mM NaCl, and obtain the bacterial cells by centrifugation.

[0070] Subsequently, a 5 ml reaction system was prepared to verify the synthesis of the product (S)-Scolerine. The reaction system consisted of 50 mM Tris-HCl (pH 9.0), 100 mM NaCl buffer containing 13 mM (S)-Reticuline, and 15 mM sodium ascorbate. The bacterial OD value in the reaction system was [not specified]. 600 The reaction was carried out in a shaker at 30°C. After 8 hours, 200 μL of 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 through a 0.22 μm filter membrane.

[0071] The synthesis of the product (S)-Scolerine was detected using an HPLC system. For example... Figure 4 As shown, the compound obtained by whole-cell catalysis had the same elution time as the standard, and the formation 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 above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Berberine brining enzyme mutant, using berberine brining enzyme with the amino acid sequence shown in SEQ ID NO.7 as the parent, and the mutation mode is any one of the following (a) to (e): (a) Mutate the arginine at position 354 of the parental line to phenylalanine or tryptophan; (b) Mutate tryptophan at position 165 of the parental stem to glycine; (c) Mutate the alanine at position 421 of the parental stem to phenylalanine or tryptophan; (d) Mutate tryptophan at position 165 of the parental stem to glycine and arginine at position 354 to tryptophan; (d) Mutate arginine at position 354 of the parent to tryptophan and alanine at position 421 to phenylalanine.

2. The gene encoding the berberine brining enzyme mutant of claim 1.

3. A recombinant cell expressing the mutant of claim 1 or the gene of 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, With Escherichia coli Escherichia coli BL21 (DE3) is the host.

6. Synthesis of S-golden corydaline ( S The method of )-Scoulerine is characterized by, Using the berberine brining enzyme mutant as described in claim 1 or the recombinant cells as described in any of claims 3-5 as catalysts, and S-boscin ( S The reaction proceeds using )-Reticuline as the 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℃, and the reaction pH is 7.5~10.

5.

9. The mutant of claim 1, or the recombinant cell of any one of claims 3-5, or the method of any one of claims 6-8 in the preparation of S-golden corydaline ( S Applications in Scoulerine.

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