CN116121212A - Hydroxylase participating in biosynthesis of benzylisoquinoline alkaloid and application thereof - Google Patents

Abstract

The invention discloses hydroxylase participating in biosynthesis of benzyl isoquinoline alkaloid and application thereof, relating to the field of medical plant genetic engineering, wherein the amino acid sequence is SEQ ID NO.1, or protein with hydroxylase function is obtained by substitution and/or deletion and/or addition of one or more amino acid residues; or a protein having 90% or more identity thereto and having a hydroxylated benzylisoquinoline alkaloid; recombinant expression vectors and recombinant microorganisms thereof; the yeast system composed of the enzyme and the reductase StCPR, the application of the hydroxylase and the recombinant expression vector thereof in-vitro synthesis of heterologously expressed StCYP80B protein, recombinant microorganism and the yeast system in-vivo synthesis of benzyl isoquinoline compounds. The hydroxylase StCYP80B provided by the invention expands the substrate recognition range and is an effective enzymatic tool for hydroxylating monobenzyl isoquinoline alkaloids with different structures.

Description

Hydroxylase participating in biosynthesis of benzylisoquinoline alkaloid and application thereof

Technical Field

The invention relates to the field of medicinal plant genetic engineering, in particular to hydroxylase participating in biosynthesis of benzylisoquinoline alkaloids and application thereof.

Background

Radix Stephaniae Japonicae Stephania tetrandra S.Moore is a Stephania japonica of Menispermaceae, and its root is a well-known traditional Chinese medicinal material radix Stephaniae Tetrandrae, and has effects of inducing diuresis to alleviate edema, dispelling pathogenic wind and relieving pain. The radix stephaniae tetrandrae is rich in Benzyl Isoquinoline Alkaloids (BIAs), mainly comprises monobenzyl isoquinoline type, bisbenzyl isoquinoline type, aporphine type, protoberberine type and the like, and has various pharmacological effects of anti-inflammatory, antibacterial, antiviral, antitumor and the like. However, the existing radix stephaniae tetrandrae has the disadvantages of lack of wild resources, small artificial planting scale, long planting period and low content of active ingredients, and limits the application and development of active BIAs. Biosynthesis is an important means for solving the resource problem, and explaining the key enzyme for BIAs biosynthesis in radix stephaniae tetrandrae can provide a theoretical basis for efficiently producing the effective components by using biotechnology.

Biosynthesis of BIAs generally begins with the conversion of L-tyrosine to dopamine and 4-hydroxyphenylacetaldehyde, catalyzed by norlinderamine synthase (NCS) to produce S-norlinderamine, which is then reacted by 3 methyltransferases (6 OMT, CNMT,4' OMT) and hydroxylases (CYP 80B or NMCH) to form the key intermediate S-palmitin ((S) -reticuline), which is then coupled, isomerised, rearranged, hydroxylated, methylated, demethylated to form various types of BIAs. In the above approach, from S-norlinderane to S-niu-carpine are monobenzyl isoquinoline alkaloids, and structural modification of the compounds plays an important role in downstream various types of BIAs, wherein hydroxylase CYP80B is one of key enzymes for structural modification of monobenzyl isoquinoline.

At present, CYP80B with definite functions in stephania tetrandra has not been reported, CYP80B which is called N-methyl linderamine 3 '-hydroxylase and can only catalyze a single substrate S-N-methyl linderamine to form S-3' -hydroxy-N-methyl linderamine, and the CYP80B which is found in other plants can not catalyze the substrate linderamine (coclaucine) without N-methyl, so that the substrate spectrum is single. Therefore, CYP80B capable of hydroxylating BIA with different structures can be found to widen the substrate spectrum of the enzyme, and has important theoretical and practical values for synthesizing mono-benzyl isoquinoline and downstream multi-type BIAs.

Accordingly, those skilled in the art have focused on developing a new hydroxylase having a diverse substrate spectrum to meet the need for hydroxylation of benzylisoquinoline alkaloids (BIAs) of different structures.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to solve the technical problem of how to develop a hydroxylase with a variety of substrate spectra, which can meet the demands of hydroxylating benzylisoquinoline alkaloids (BIA) with different structures.

In order to achieve the above purpose, the invention provides a hydroxylase StCYP80B involved in biosynthesis of benzylisoquinoline alkaloids, wherein the hydroxylase StCYP80B has the amino acid sequence as follows:

(a1) The amino acid sequence of the hydroxylase StCYP80B is shown as SEQ ID NO. 1;

or (a 2) an amino acid sequence of a protein having hydroxylase function obtained by substitution and/or deletion and/or addition of one or more amino acid residues to the amino acid sequence of the above (a 1);

or (a 3) an amino acid sequence of a protein having 90% or more identity with the amino acid sequence of the above (a 1) and having a hydroxylated benzylisoquinoline alkaloid.

Further, the hydroxylase StCYP80B nucleotide sequence is:

(b1) The nucleotide sequence is shown as SEQ ID NO. 2;

or (b 2) a DNA sequence obtained by substituting and/or deleting and/or adding one or more nucleotides to the nucleotide sequence of (b 1);

or (B3) a DNA molecule which has 90% or more identity with the nucleotide sequence represented by SEQ ID NO.2 as described above and which encodes a CYP80B protein.

The invention also provides a recombinant expression vector of the hydroxylase StCYP80B gene, which comprises a subcloning vector or a yeast cell expression vector.

Further, the recombinant vector containing the StCYP80B gene comprises a subclone vector and a microbial cell expression vector such as yeast.

The invention also provides a recombinant microorganism of the hydroxylase StCYP80B gene, which comprises a yeast cell.

Further, the recombinant microorganism containing the StCYP80B gene includes microbial cells such as yeast.

The invention also provides a yeast system for coexpression of StCYP80B and StCPR, which comprises hydroxylase StCYP80B and reductase StCPR.

Further, the amino acid sequence of the reductase StCPR is shown as SEQ ID NO.3, and the encoding gene is shown as SEQ ID NO. 4.

The invention also provides application of the hydroxylase StCYP80B in-vitro synthesis of benzyl isoquinoline compounds.

The invention also provides an application of the recombinant expression vector containing the StCYP80B gene in-vitro synthesis of the heterologously expressed StCYP80B protein.

The invention also provides an application of the recombinant microorganism containing the StCYP80B gene in synthesizing benzyl isoquinoline compounds in vivo.

The invention also provides application of the yeast system in synthesizing benzyl isoquinoline compounds in vivo.

Further, the hydroxylase protein is derived from stephania tetrandra Stephania tetrandra, named StCYP80B,

further, a CYP450 Reductase (CPR) is derived from stephania tetrandra Stephania tetrandra, named StCPR.

Further, the conversion rate of the yeast system co-expressing StCYP80B and StCPR to benzyl isoquinoline alkaloids is significantly higher than that of the system expressing only StCYP80B.

Further, the hydroxylase StCYP80B catalyzes the use of the substrate linderamine (coclaucine) without an N-methyl group.

Further, the use of StCYP80B as a hydroxylase.

Further, the StCYP80B gene is applied to the preparation of hydroxylase.

Further, the recombinant expression vector containing the StCYP80B gene is applied to the preparation of hydroxylase.

Further, the use of a recombinant microorganism containing the StCYP80B gene in the preparation of hydroxylase.

Further, the application of StCYP80B protein in the biosynthesis of benzyl isoquinoline alkaloids is disclosed.

Further, the application of StCYP80B protein in preparing benzyl isoquinoline alkaloid.

Further, the hydroxylase StCYP80B can catalyze a single substrate S-N-methyl linderamine to form S-3' -hydroxy-N-methyl linderamine, and can catalyze a substrate linderamine (coclaucine) without N-methyl.

In the preferred embodiment 1 of the present invention, the process of finding a benzylisoquinoline alkaloid 3' -hydroxylase (StCYP 80B) and its encoding gene in radix Stephaniae Tetrandrae is described in detail;

in another preferred embodiment 2 of the present invention, the function of s.cerevisiae in verifying StCYP80B in vivo is described in detail;

in another preferred embodiment 3 of the present invention, the StCYP80B microsomal protein extraction and in vitro catalysis process is described in detail.

The beneficial technical effects of the invention are as follows:

the invention discloses a novel hydroxylase StCYP80B and a coding gene thereof. StCYP80B can catalyze N-methyl linderane (N-methylcoclaurine) to form 3 '-hydroxy-N-methyllinderane (3' -OH-N-methylcoclaurine), and can catalyze substrate linderane (coclaucine) without N-methyl to form 3 '-hydroxy-linderane (3' -OH-coclaucine), compared with reported CYP80B, the substrate recognition range is enlarged, and the method is an effective enzymatic tool for hydroxylating monobenzyl isoquinoline alkaloids with different structures, and has important significance for synthesizing monobenzyl isoquinoline and downstream multi-type BIAs.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a LC-MS and LC-MS/MS analysis chromatogram of StCYP80B catalyzed (R, S) -N-methyl linderane to form hydroxylation products in a yeast system of a preferred embodiment 2 of the invention;

FIG. 2 is a LC-MS and LC-MS/MS analysis chromatogram of StCYP80B catalyzed (R, S) -linderane to form hydroxylation products in a yeast system of a preferred embodiment 2 of the invention.

Detailed Description

The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.

EXAMPLE 1 discovery of benzyl isoquinoline alkaloid 3' -hydroxylase (StCYP 80B) and its encoding Gene in Fangji

The radix stephaniae tetrandrae transcriptome database is obtained by transcriptome sequencing of radix stephaniae tetrandrae and leaf tissues. And performing Blast screening and sequence comparison peer-to-peer analysis on sequences of public data such as NCBI and KEGG, and mining to 6 CYP80B candidate sequences. After Saccharomyces cerevisiae heterologous expression and substrate feeding, one protein is determined to have the effect of catalyzing 3' -hydroxylation of (R, S) -N-methyl linderane and (R, S) -linderane. The protein is named StCYP80B, and the amino acid of the protein is shown as SEQ ID NO.1 of a sequence table and consists of 490 amino acids. The gene for coding the StCYP80B protein is named as StCYP80B gene, and the open reading frame cDNA sequence of the gene is shown as a sequence SEQ ID NO.2 of a sequence table.

By the same method, 1 CYP450 reductase is mined from the tetrandra root transcriptome, the protein of which is named StCPR and consists of 690 amino acids, and the amino acid sequence of the CYP450 reductase is shown in SEQ ID NO.3 of a sequence table. The gene encoding StCPR protein is named StCPR, and the open reading frame cDNA sequence is shown as SEQ ID NO.4 of the sequence table. In the Saccharomyces cerevisiae heterologous expression system, the hydroxylation conversion rate of the system which co-expresses StCYP80B and StCPR on (R, S) -N-methyl linderane and (R, S) -linderane is obviously higher than that of the system which expresses only StCYP80B.

Example 2 in vivo validation of StCYP80B function in Saccharomyces cerevisiae

1. Construction of heterologous expression plasmids

Saccharomyces cerevisiae episomal plasmid pESC-Leu was digested with restriction enzymes NotI and SacI, and the linearized plasmid was recovered in a gel. Designing primers (F1 and R1) containing homology arms at two ends of the plasmid, carrying out PCR amplification from a tetrandra root cDNA sample to obtain a StCYP80B gene with homology arms, and recovering and purifying a PCR product.

The primer sequence F1 is shown as the sequence SEQ ID NO. 5: 5'-CTCACTAAAGGGCGGCCGCATGGAGATAGTCTCTGC-3'; r1 is shown as a sequence SEQ ID NO. 6: 5'-TCACTAAAGGGCGGCCGCATGGATCAAACCATCCTCTC-3'.

Constructing a recombinant plasmid pESC-Leu-StCYP80B by adopting a homologous recombination method, transforming escherichia coli DH10B by adopting a heat shock method, picking up an ampicillin-resistant transformant, amplifying by adopting an LB culture medium, extracting the plasmid, and sequentially carrying out PCR, enzyme digestion and sequencing verification to obtain the recombinant plasmid pESC-Leu-StCYP80B with correct sequencing.

Saccharomyces cerevisiae episomal plasmid pESC-Leu-StCYP80B was digested simultaneously with restriction enzymes XhoI and NheI, and the linearized plasmid was recovered in a gel. Designing primers (F2 and R2) containing homology arms at two ends of the plasmid, carrying out PCR amplification from the tetrandra root cDNA sample to obtain a StCPR gene with the homology arms, and recovering and purifying a PCR product.

The primer sequence F2 is shown as the sequence SEQ ID NO. 7: 5'-TTTCCGAAGAAGACCTCGAGATGGCTTCCAAGTACGCGAA-3'; r2 is shown as a sequence SEQ ID NO. 8: 5'-TAGAGCGGATCTTAGCTAGCTCACCAAACGTCCCTGAGAT-3'.

The recombinant plasmid pESC-Leu-StCYP80B-StCPR with correct sequencing is obtained by adopting the same method steps as those for constructing the plasmid pESC-Leu-StCYP80B.

2. Acquisition of recombinant strains of Yeast

And (3) transforming the recombinant plasmid pESC-Leu-StCYP80B or pESC-Leu-StCYP80B-StCPR obtained in the step (A) into a Saccharomyces cerevisiae YPH499 strain by adopting a Frozen-EZ yeast Transformation II kit yeast transformation kit. The bacterial liquid is coated on a defect culture medium SD-Leu flat plate, and single colony can be picked after standing culture for 2d at 30 ℃. At the same time, the empty plasmid was transformed into YPH499 strain to obtain a control strain.

3. Yeast whole cell catalysis

And (3) picking the monoclonal colony grown on the defect plate in the second step to 15ml of SD-Leu liquid culture medium, and shake culturing at 30 ℃ and 220rpm for overnight. Controlling OD600 to 0.8-1.2, centrifuging (3000 rpm,5 min), removing SD-Leu, changing induction medium SG-Leu, performing induction culture for 20h, and centrifuging to obtain bacterial cell precipitate.

The cells were washed once with 50mM PBS and resuspended to a final volume of 500. Mu.l. The substrate (R, S) -N-methyl linderane and (R, S) -linderane were fed to a final concentration of 0.25mM. Whole cells were catalyzed at 30℃and 220rpm for 24h.

4. Treatment and detection of reaction liquid

Adding an equal volume of methanol into the reaction solution obtained in the step three to terminate the reaction, ultrasonically extracting for 15min, centrifuging (12000 rpm,10 min) to remove precipitate, filtering the supernatant with a 0.22 μm organic filter membrane, and performing LC-UV-MS and LC-MS/MS analysis.

LC-UV-MS detection conditions were as follows: the liquid phase system is Shimadzu LCMS-2020; the column was a Shim-pack XR-ODS III (2.0 mm. Times.75 mm,1.6 μm); the column temperature was room temperature (about 25 ℃); the flow rate is 0.2ml/min; mobile phase a (0.1% aqueous formic acid) -B (methanol) was eluted with a gradient, time program: 0min,5% (v/v) B%;10min,45% (v/v) B%;11min,95% (v/v) B%;12min,95% (v/v) B%; PDA detection, wherein the monitoring wavelength is 282nm; the mass spectrum detection adopts an electrospray ionization ion source (ESI) to collect positive ions, and the scanning range is 100-800m/z; the interface voltage is 4.5kv.

LC-MS/MS detection conditions were as follows: the liquid phase system is Shimadzu LCMS-8060; the column was Venusil XBP PH (2.1X100 mm,5 μm); column temperature 40 ℃; the flow rate is 0.5ml/min; mobile phase a (0.1% aqueous formic acid) -B (acetonitrile), time program: 0min,5% (v/v) B%;5min,25% (v/v) B%;7min,95% (v/v) B%;9min,95% (v/v) B%; the mass spectrum detection adopts ESI to collect positive ions, and the interface voltage is 4.0kV; the scanning mode is multi-reaction monitoring (MRM), the following ion pairs are selected for detection: substrate N-methyl linderane m/z 300.25- & gt 107.15 and product 3 '-hydroxy-N-methyl linderane m/z 316.15- & gt 123.05, substrate linderane m/z 286.25- & gt 107.15 and product 3' -hydroxy-linderane m/z 302.15- & gt 123.05.

The result of the substrate (R, S) -N-methyl linderane is shown in figure 1, figure 1 is an LC-MS and LC-MS/MS analysis chromatogram of the (R, S) -N-methyl linderane catalyzed by StCYP80B to form hydroxylation product in a yeast system, wherein the first structural formula in the upper part of figure 1 is 3' -hydroxy-N-methyl linderane; the second structural formula is N-methyl linderane; the part 1A is LC-MS extraction ion chromatogram of blank control with culture solution and enzymatic reaction solution, the part 1B is LC-MS/MS chromatogram of control and enzymatic reaction solution, chromatographic peak "1" represents compound 3' -hydroxy-N-methyl linderane, and chromatographic peak "2" represents compound N-methyl linderane; the culture solution blank is a culture solution with a substrate but without StCYP80B, and the enzymatic reaction solution is a sample of a recombinant Saccharomyces cerevisiae whole-cell catalytic substrate for co-expressing StCYP80B and StCPR. . The mass spectrum extraction ion chromatogram detected by LC-MS of the 1A part shows that the saccharomyces cerevisiae with heterologously expressed StCYP80B can catalyze N-methyl linderane to generate a hydroxylation Product 1 with m/z increased by 16 Da; the LC-MS/MS chromatogram of the 1B part shows that the ion pair m/z and the retention time of the Product 1 generated in the enzymatic reaction liquid are consistent with those of the 3' -hydroxy-N-methyl linderane reference substance, which indicates that the 3' -hydroxy-N-methyl linderane is formed by hydroxylation of the 3' -position.

The result of the substrate being (R, S) -linderane is shown in figure 2, figure 2 is an LC-MS and LC-MS/MS analysis chromatogram of the (R, S) -linderane catalyzed by StCYP80B in a yeast system to form hydroxylation products, wherein the first structural formula in the upper part of figure 2 is 3' -hydroxy-linderane; the second structural formula is linderane; the part 2A is the LC-MS extraction ion chromatogram of the culture solution blank control and the enzymatic reaction solution, the part 2B is the LC-MS/MS chromatogram of the control and the enzymatic reaction solution, the chromatographic peak ' 3' represents the compound 3' -hydroxy-linderaine, and the chromatographic peak ' 4' represents the compound linderaine. The culture solution blank is a culture solution with a substrate but without StCYP80B, and the enzymatic reaction solution is a sample of a recombinant Saccharomyces cerevisiae whole-cell catalytic substrate for co-expressing StCYP80B and StCPR. . The mass spectrum extraction ion chromatogram detected by LC-MS of the 2A part shows that the saccharomyces cerevisiae with heterologously expressed StCYP80B can catalyze the linderane to generate a hydroxylation Product 2 with m/z increased by 16 Da; the LC-MS/MS chromatogram of the 2B part shows that the ion pair m/z and the retention time of the Product 2 generated in the enzymatic reaction liquid are consistent with those of the 3' -hydroxy-linderane reference substance, which indicates that the 3' -hydroxy-linderane is formed by hydroxylation of the 3' -position.

The transformation plasmid pESC-Leu-StCYP80B-StCPR is compared with two yeast systems of pESC-Leu-StCYP80B, the hydroxylation conversion rate of the former to the substrates N-methyl linderaine and linderaine is 3-100 times that of the latter, which shows that the catalysis effect of the StCPR can be enhanced by the co-expression of the StCPR in the yeast.

The results show that StCYP80B has the function of catalyzing the hydroxylation of the 3' -positions of the N-methyl linderane and the linderane; stCPR can effectively improve the catalytic activity of StCYP80B in a heterologous expression system.

Example 3 StCYP80B microsomal protein extraction and in vitro catalysis

1. Extraction of microsomal proteins

Bacterial cell pellets were obtained as in steps one to three of example two. 1ml of TESB buffer is added into each gram of thalli, the thalli is crushed by adopting a vibrating cell crusher, the vibrating speed is 6m/s, the time is 10 s/time, the thalli is placed on ice for 3min, and the process is repeated for 6 times. Centrifuging (10,000 g,4 ℃ C., 20 min), collecting supernatant, and ultracentrifugating (100,000 g,4 ℃ C., 1 h) to obtain microsomal precipitate, and dissolving in a proper amount of TEG buffer. Microsomal protein concentration was determined using the modified Bradford protein assay kit.

2. In vitro enzymatic reactions

Taking microsome sediment to prepare an enzymatic reaction system. Each reaction system was 0.5ml, containing microsomal protein 1mg,0.05mM FAD,0.05mM FMN,1mM NADPH, appropriate amounts of substrate (R, S) -N-methyl linderane and (R, S) -linderane, TEG buffer (pH: 7.5) to 0.5ml. The reaction is carried out for 2 hours at 30 ℃ and 220rpm, and then enzymatic reaction liquid is obtained.

3. Treatment and detection of reaction liquid

Adding equal volume of methanol into the enzymatic reaction solution to terminate the reaction, mixing by vortex, centrifuging (12000 rpm,4 ℃ C., 5 min), and taking supernatant for LC-MS/MS analysis. The detection conditions were the same as in the LC-MS/MS method of step four of example two.

The result shows that the StCYP80B protein can catalyze N-methyl linderane and 3 '-carbon atom of the linderane to carry out hydroxylation reaction to generate a 3' -OH product.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (10)

1. A hydroxylase StCYP80B involved in biosynthesis of benzylisoquinoline alkaloids, characterized in that the hydroxylase StCYP80B has the amino acid sequence:

(a1) The amino acid sequence of the hydroxylase StCYP80B is shown as SEQ ID NO. 1;

or (a 2) the amino acid sequence of the protein with hydroxylase function obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of (a 1);

or (a 3) an amino acid sequence of a protein having 90% or more identity to the amino acid sequence of (a 1) and having a hydroxylated benzylisoquinoline alkaloid.

2. The hydroxylase StCYP80B of claim 1, wherein said hydroxylase StCYP80B nucleotide sequence is:

(b1) The nucleotide sequence is shown as SEQ ID NO. 2;

or (b 2) a DNA sequence obtained by substituting and/or deleting and/or adding one or more nucleotides to the nucleotide sequence of (b 1);

or (B3) a DNA sequence which has 90% or more identity with the nucleotide sequence shown in SEQ ID NO.2 and which encodes a CYP80B protein.

3. A recombinant expression vector comprising the hydroxylase StCYP80B gene of claim 1 or 2, wherein said recombinant vector comprises a subclone vector or a yeast cell expression vector.

4. A recombinant microorganism comprising the hydroxylase StCYP80B gene of claim 1 or 2, wherein said recombinant microorganism comprises a yeast cell.

5. A yeast system that co-expresses StCYP80B and StCPR, wherein the yeast system comprises hydroxylase StCYP80B and reductase StCPR.

6. The yeast system of claim 5, wherein the reductase StCPR has an amino acid sequence shown in SEQ ID NO.3 and a coding gene shown in SEQ ID NO. 4.

7. Use of hydroxylase StCYP80B according to claim 1 or 2 for the in vitro synthesis of benzylisoquinoline compounds.

8. Use of the recombinant expression vector according to claim 3 for in vitro synthesis of benzylisoquinoline compounds by heterologous expression of StCYP80B protein.

9. The use of the recombinant microorganism according to claim 4 for in vivo synthesis of benzylisoquinoline compounds.

10. The use of the yeast system of claim 5 for in vivo synthesis of benzylisoquinoline compounds.

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