CN106319007B - A kind of biotransformation method for preparing hair growth promoter - Google Patents

Abstract

The invention belongs to the field of medical technology and synthetic biology, and relates to a biotransformation method for preparing hair growth promoter [γ-hydroxy-N-methyl-L-leucine ⁴ ]-cyclosporin A. Using the constructed highly expressed P450 monooxygenase (CYP‑sb21) and reducing chaperone protein as a hybrid catalytic system, under the action of the electron donor regeneration system, CsA can be converted to CsA‑4‑OH, realizing one-step enzymatic conversion . The preparation of CsA-4-OH by using the method of the invention has the characteristics of high catalytic selectivity, high conversion rate, simple product purification, low cost, environmental protection, etc., and has good industrial application prospects.

Description

A kind of biotransformation method for preparing hair growth promoter

technical field

The invention belongs to the field of medical technology and synthetic biology, and relates to a biotransformation method for preparing hair growth promoter [γ-hydroxy-N-methyl-L-leucine ⁴ ]-cyclosporine A.

Background technique

Alopecia is a common clinical disease, which has a great impact on people's spirit and psychology. Alopecia can be divided into alopecia areata, male pattern alopecia, chemotherapy-induced alopecia, senile alopecia and alopecia caused by diseases, among which alopecia areata and seborrheic alopecia have a high incidence rate and are the most difficult to treat. Since the pathogenesis of alopecia areata is still unclear, there is no clear treatment drug so far. For seborrheic alopecia, the only therapeutic drugs approved by the U.S. Food and Drug Administration (FDA) are minoxidil and 5α-reductase inhibitor finasteride. However, the use of both drugs has limitations: 5% of young female patients with minoxidil develop hirsutism (Rogers and Avram 2008); efficacy (Burton and Marshall 1979); finasteride is not suitable for postmenopausal women (Whiting et al.1999) and so on. In addition, there are some hair growth promoting drugs that have not been approved by the FDA, such as saw palmetto extract (Sawpalmetto extract), L-carnitine (L-Carnitine), cyclosporine A (Cyclosporine A, CsA), etc. . Such drugs have obvious side effects, long treatment time, and are prone to drug resistance (Prager et al. 2002; Foitzik et al. 2007). The production of drugs for the treatment of hair loss is a large industry with annual sales of billions of dollars (Wikipedia), so finding new drugs for treating hair loss with good activity and few side effects has very important research significance and practical application value.

CsA is a cyclic peptide composed of 11 amino acids isolated from the fungus Fusarium solani (Tolypocladium inflatum), which has broad-spectrum biological activities, such as immunosuppressive, antifungal, antiparasitic, anti-HIV and anti-inflammatory effects (Borel et al. 1976). CsA is an important anti-rejection drug for organ transplantation. It was found that the drug can cause abnormal hair growth in patients during clinical application. The study found that adding a hydroxyl group to the γ carbon atom of N-methyl-L-leucine (N-methyl-L-Leu ⁴ ) in CsA can significantly reduce the immunosuppressive activity of CsA, but it does not change its hair growth promotion role (Kim et al. 2004). Therefore, γ-hydroxy-N-methyl-L-leucine ⁴ -cyclosporin A (CsA-4-OH) is a hair growth promoter with potential clinical application value. There are obvious limitations in the chemical synthesis of CsA-4-OH: ① Synthesize γ-hydroxy-N-methyl-L-leucine first, and then there is an efficient and selective cyclization problem through the traditional solid-phase peptide synthesis method; ② If using The chemical semi-synthesis of CsA produced by fermentation method lacks a chemical catalyst capable of selectively hydroxylating the 4-position N-methyl-L-leucine. Therefore, the development of an efficient biotransformation method from CsA to CsA-4-OH has become a key issue whether the latter can enter clinical trials and then be marketed as a hair growth promoter.

By screening the strains that can transform CsA from hundreds of actinomycetes, it was found that the rare actinomycete Sebekiabenihana can selectively oxidize the substrate CsA to CsA-4-OH, and found the catalyst for this through gene knockout and complementation experiments. A reactive cytochrome P450 monooxygenase CYP-sb21. Overexpression of CYP-sb21 in S. benihana and Streptomyces coelicolor did not solve the problem of low conversion rate of CsA, and the highest conversion rate was only 21% (Lee et al. 2013). The lack of optimal reducing partners and the low transmembrane efficiency of substrates and products during the biotransformation process may be the main reasons for the above low conversion rates. In addition, the culture cycle of S. benihana and Streptomyces coelicolor is relatively long, the conversion efficiency of CsA is low, the separation and purification of subsequent products is cumbersome, and the cost is high. Currently, it cannot be applied to the large-scale production of CsA-4-OH.

Therefore, there is an urgent need at this stage for a method for synthesizing γ-hydroxy-N-methyl-L-leucine ⁴ -cyclosporin A with high conversion efficiency, easy separation and purification of subsequent products, and low cost.

Contents of the invention

The purpose of the present invention is to provide a biotransformation method for preparing hair growth promoter [γ-hydroxyl-N-methyl-L-leucine ⁴ ]-cyclosporine A.

In order to achieve the above object, the technical solution adopted by the present invention is:

A biotransformation method for preparing a hair growth promoter, using the constructed highly expressed P450 monooxygenase (CYP-sb21) and reduced chaperone protein as a hybrid enzyme catalytic system, hybrid enzyme catalytic system (hybrid enzyme catalytic The system can be pure enzyme, cell lysis crude enzyme solution or immobilized enzyme) under the action of the electron donor regeneration system, catalyze the conversion of CsA to obtain CsA-4-OH, and realize the one-step enzyme conversion.

A whole-cell catalytic biotransformation method for preparing a hair growth promoter, by constructing a recombinant plasmid containing a highly expressed P450 monooxygenase (CYP-sb21), a reduced chaperone protein and an electron donor regeneration system, and then transferring it into a host, The obtained whole cell is used as a catalyst, and the recombinant whole cell catalyst is permeabilized by physical or chemical methods to realize the efficient transformation of the whole cell from CsA to CsA-4-OH.

The P450 monooxygenase (CYP-sb21) is a P450 enzyme CYP-sb21 derived from a rare actinomycete Sebekia benihana strain, which can catalyze CsA to obtain other functional equivalents of CsA-4-OH.

The reduction chaperones are ferredoxin and ferredoxin reductase.

The reduction chaperones are ferredoxin (ferredoxin: SynPcc7942_1499, seFdx) and ferredoxin reductase (ferredoxin reductase: SynPcc7942_0978, seFdR) of cyanobacteria Synechococcus elongates PCC 7942.

The host is Escherichia coli, Bacillus subtilis, yeast or filamentous fungi.

The electron donor regeneration system is a system in which glucose dehydrogenase (GDH) uses glucose as a substrate to catalyze NAD ⁺ or NADP ⁺ as a prosthetic group to generate NADH or NADPH.

The glucose dehydrogenase (GDH) is derived from Bacillus megaterium, Bacillus subtilis or Bacillus pumilus, or glucose dehydrogenase from other sources.

The recombinant plasmid is an inducible plasmid controlled by the T7 promoter, and the recipient plasmid and the target protein gene are connected by restriction enzymes to form a recombinant plasmid, and then transformed into Escherichia coli competent cells to construct highly efficient expression of CYP-sb21 and reduced chaperone protein Escherichia coli recombinant strains of seFdR/seFdR and NADPH regeneration system GDH; the inducible plasmids are pET plasmid series and Duet plasmid series controlled by T7 promoter, such as pET28b or pET22b or pCDFDuet-1 or pACYCDuet1, the large intestine The bacilli are Escherichia coli BL21 (DE3) or other Escherichia coli strains used for heterologous protein expression.

Wherein, the above-mentioned recombinant whole-cell catalyst is preferably: the host is Escherichia coli BL21 (DE3), and the coding gene of CYP-sb21 and NADPH regeneration enzyme GDH in the catalyst is expressed in series from pET28b recombinant plasmid containing T7 promoter or can express CYP-sb21 The other plasmids of sb21 and GDH, the coding genes of reducing chaperones seFdx and seFdR were expressed in tandem from pCDFDuet-1 recombinant plasmid containing T7 promoter or other plasmids capable of expressing seFdx and seFdR.

The whole-cell catalytic protocol is as follows:

1) Induced expression of protein: Escherichia coli genetically engineered bacteria were cultivated overnight in LB medium at 37°C at 250rpm, transferred to fresh LB medium at 10-30% inoculum size, shaken to OD ₆₀₀ at 37°C at 250rpm Add 0.2mM IPTG between 0.6-1.0, culture at 18°C, 250rpm for 24-36h to induce protein expression.

LB medium contains antibiotics required for stable plasmids, and LB medium for CYP-sb21-induced expression also contains 1 mM thiamine and 0.5 mM 5-aminolevulinic acid (5-ALA) as P450 enzyme ferrous Precursors of the heme prosthetic group promote functional expression of CYP-sb21.

2) Catalytic reaction of the substrate: collect the bacteria by centrifugation, resuspend the strain with 100mM potassium nitrate buffer (pH 6.5-8.0) to reach 50-200g wet cells/L, add different concentrations of CsA (1-100mM), React at 25-40°C, 150rpm for 12-48h. After the reaction is over, add an equal volume of ethyl acetate or chloroform for extraction to obtain the hair growth promoter CsA-4-OH catalyzed by whole cells.

Compared with the prior art, the present invention has advantages and positive effects: the present invention is based on the method of biocatalytic synthesis of hair growth promoter CsA-4-OH based on cytochrome P450 monooxygenase CYP-sb21, compared with traditional chemical synthesis As well as the reported biotransformation pathway of actinomycetes, the culture cycle is short, the production process is simple, the catalytic selectivity is high, the substrate conversion rate is high, the product purification is simple, the energy consumption is low, and there is no pollution. It is a low-cost, simple-to-operate method The green catalytic system has a good application prospect.

Description of drawings

Fig. 1 is a diagram of the enzyme catalysis process provided by the embodiment of the present invention.

Fig. 2 is a diagram of the whole cell transformation process provided by the embodiment of the present invention.

Fig. 3 is the SDS-PAGE of CYP-sb21, seFdx and seFdR proteins provided by the embodiment of the present invention and the detection of P450 enzyme activity of CYP-sb21 (differential scanning spectrum under carbon monoxide binding and reduction state).

Fig. 4 is the HPLC picture of CsA-4-OH obtained by the enzymatic method and the whole cell conversion method provided by Examples 1-4 of the present invention; wherein, A. (a) Example 2 enzymatic conversion of CsA to obtain CsA-4-OH HPLC figure; (b) Example 1 enzymatic conversion of CsA to obtain the HPLC figure of CsA-4-OH; (c) and (d) are CsA and CsA-4-OH standard respectively. B. (a) Example 3 Whole-cell method transforms CsA to obtain the HPLC figure of CsA-4-OH; (b) Embodiment 4 Whole-cell method transforms CsA to obtain the HPLC figure of CsA-4-OH; (c) Empty plasmid control Group HPLC chart.

Fig. 5 is a diagram of LC-Q-TOF/MS detection results of remaining substrate CsA and product CsA-4-OH in the whole cell transformation experiment provided by the embodiment of the present invention.

Detailed ways

The present invention will be further described below in conjunction with embodiment.

The present invention is based on the rare actinomycete Sebekia benihana cytochrome P450 monooxygenase CYP-sb21 selective hydroxylation immunosuppressant cyclosporine A (Cyclosporine A, CsA) the 4th N-methyl-L-leu amino acid, which is converted into the hair growth promoter γ-hydroxy-N-methyl-L-leu ^{4 -cyclosporine A (γ-hydroxy-N-methyl-L-Leu 4 -Cyclosporine} A, CsA -4-OH) biocatalytic approach (Figure 1).

Example 1

Pure enzyme catalysis method:

(1) Construction of target gene vector and protein purification

cyp-sb21 (SEQ ID NO:1 in the sequence table) was optimized according to the codons of Escherichia coli (SEQ ID NO:2 in the sequence table), artificially synthesized with CYP-sb21-F/CYP-sb21-R (see Table 1 ) is the optimized cyp-sb21 fragment amplified by primers; the PCR amplification system is 100 μL: 10 μM each primer, 50 μM dNTPs, 0.5 μL DNA template, 5×DNA polymerase buffer 20 μL, DNA polymerase 5U; PCR reaction conditions are: Pre-denaturation at 95°C for 5min, followed by denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1min, 30 cycles of reaction, and finally extension at 72°C for 10min).

With Synechococcus elongates PCC 7942 bacterial strain genome as template, respectively with FdR-F/FdR-R, Fdx-F/Fdx-R (see Table 1 for primer sequence) as primers, respectively amplify sefdR and sefdx (sequence table SEQ ID NO: 3 and SEQ ID NO:4), the PCR reaction system and conditions were the same as (1), wherein sefdR was extended at 72°C for 1 min, and sefdR was extended at 72°C for 30s.

The amplified fragments CYP-sb21, seFdx and seFdR obtained above were connected to the pET28b plasmid using restriction endonucleases NdeI/EcoRI (CYP-sb21), NdeI/XhoI (seFdx), BamHI/HindIII (seFdR) respectively, and then The obtained plasmids were respectively expressed in Escherichia coli BL21(DE3) cells. The correct target strain was verified by sequencing, and the obtained strain was cultivated in LB liquid medium containing 1mM thiamine (thiamine) and 0.5mM 5-aminolevulinic acid (5-ALA), and 0.2mM IPTG induced protein For expression, the cells were collected and crushed with a cell disruptor, and centrifuged in a high-speed refrigerated centrifuge. The supernatants were passed through the Ni ²⁺ -NTA affinity column, 20-300mM imidazole gradient elution, 250mM imidazole can elute the target proteins CYP-sb21, seFdx and seFdR, and the collected proteins were removed by incubation with thrombin N-terminal histidine tag, then concentrated with Amicon-Ultra-15 ultrafiltration tube, and finally desalted with GE PD-10 desalting column to obtain purified CYP-sb21, seFdx and seFdR, refer to the method of Omura and Sato for active CYP- sb21 was tested for P450 activity (Omura and Sato, 1964). After CYP-sb21 was reduced by the reducing agent sodium dithionite and combined with CO, it had a typical absorption peak at 450nm, indicating that the protein was actively expressed in E. coli (Figure 3).

(2) Construction of in vitro enzyme reaction system

The total in vitro enzyme reaction system is 100 μL, add 10 μM CYP-sb21, 10 μM reduction partners seFdx and seFdR, 0.5 mM NADPH, 0.2 mM substrate CsA, react overnight at 30 ° C, and then add methanol equal to the volume of the reaction solution to the reaction system, After high-speed centrifugation, the supernatant was collected for HPLC, LC-MS or LC-Q-TOF/MS analysis. CsA is insoluble in water, and 10% co-solvent methanol is added to the reaction system to improve the conversion rate. The conversion of CsA to CsA-4-OH was 53% (Fig. 4A-b).

In addition, the pure enzyme purified by the hybrid enzyme catalytic system in step (1) can be replaced by the crude enzyme solution obtained by direct cell lysis of the hybrid enzyme catalytic system or the immobilized enzyme obtained by immobilization treatment of the hybrid enzyme catalytic system.

Table 1 Primer list

Example 2

The difference from Example 1 is:

In the in vitro enzyme reaction system, the electron regeneration system was replaced by 5U GDH and 20mM glucose, NADPH regeneration was achieved by oxidizing glucose with GDH, and the conversion rate of CsA into CsA-4-OH was 83.5% (Fig. 4A-a).

Example 3

Whole-cell catalytic approach:

(1) Construction of genetically engineered bacteria: the codon-optimized cyp-sb21 gene and gdh were respectively digested and connected to the pET28b plasmid by restriction enzymes EcoRI/XhoI and NdeI/BamHI, and the cyanobacterium Synechococcuselongatus PCC 7942 The P450 reducing partners sefdx and sefdR were connected to the pCDFDuet-1 plasmid, and the resulting two recombinant plasmids were transformed into E. coli competent cells E.coli BL21(DE3), using a method containing 50 μg/mL kanamycin and 50 μg/mL streptavidin The positive transformants were screened on the LB resistance plate of the protein, and the recombinant Escherichia coli genetically engineered strains highly expressing CYP-sb21, GDH, seFdx and seFdR were obtained.

Among them, the codon-optimized cyp-sb21 gene (see Sequence Table 2) uses CYP-sb21-Duet-F/CYP-sb21-Duet-R as primers to amplify cyp-sb21, connect pCDFDuet-1, and use EcoRI/ XhoI excised the cyp-sb21 fragment with T7 promoter;

gdh (refer to the sequence listing SEQ ID NO: 5) uses GDH-F/GDH-R as primers to amplify gdh. (Amplification conditions and system refer to the record of step (1) of Example 1)

(2) Construction of whole cell reaction system: inoculate single colonies of recombinant genetically engineered strains screened above into 50 mL shake flasks containing 10 mL of LB (containing 50 μg/mL kanamycin and 50 μg/mL streptomycin) medium, Cultivate overnight at 250 rpm on a shaker at 37°C, inoculate the overnight seed solution into 500 mL LB (containing 50 μg/mL kanamycin and 50 μg/mL streptomycin, 1 mM sulfur Amine, 0.5mM 5-ALA) in a 2L shake flask, cultivated at 37°C, 250rpm shaker, when the cell density reached OD ₆₀₀ =0.6-1.0, added 0.2mM IPTG, induced the expression of the target protein at 18°C , cultivated for 20 hours, centrifuged at 6000g for 20 minutes to collect the bacteria, resuspended the strain with 100mM potassium nitrate buffer (pH 7.0) to reach 200g wet cells/L, added 100μM CsA, 0.3% (w/v) hexadecyltrimethyl Ammonium bromide (N-cetyl-N,N,N,N-trimethylammonium bromide, CTAB), 100mM glucose, 150rpm shaker, 30°C for 16h. After the reaction was completed, an equal volume of ethyl acetate or chloroform was added for extraction, and after drying with nitrogen, an equal volume of methanol was added and analyzed by HPLC. The conversion rate of CsA into CsA-4-OH was 54% (Fig. 4B-a) .

Example 4

The difference from Example 3 is that

(1) Construction of genetically engineered bacteria: the codon-optimized cyp-sb21 gene was connected to the pET28b plasmid by enzyme-cut ligation, and the P450 reducing partner sefdx and sefdR of the cyanobacterium Synechococcus elongatus PCC 7942 were connected to the pCDFDuet-1 plasmid, The obtained two recombinant plasmids were transformed into Escherichia coli competent cells Escherichiacoli BL21(DE3), and the positive transformants were screened with LB resistance plates containing 50 μg/mL kanamycin and 50 μg/mL streptomycin, respectively to obtain highly expressed CYP - Recombinant E. coli genetically engineered strains of sb21, seFdx and seFdR.

(2) Construction of the whole cell reaction system: Same as in Example 3, the conversion rate of CsA into CsA-4-OH is 45% (Fig. 4B-b), which is lower than the conversion rate of the above-mentioned co-expressed GDH corresponding strain, indicating the use of Necessity of NADPH regeneration system to increase whole cell conversion rate.

SEQ ID NO: 1:

ATGGACACCGTCAACCTGATGGATCCCGCACTGATGACCGACCCCTTCAGGGGCTTCTCACGAATCAGGGAGGAGGCGCCCATCGCGCGCGCCTGCTTCCCCGGTCAGGACACACCCATCTGGCTGGTCACCCGGTACGACGACGTCAAGACCGTGCTGGGCGAGCACCGCTTCGTCAACAATCCGGCCTCCATTCCCGGCGGCGACATCCCCGACCTGCGCGAGAAGCTCATGAAGGCGCGCGGGATCCCCGACGACTACGTCGTCTACCTCACCGACGGCATCCTCGACCTCGACGGCGATGACCACCTCAGGCTGCGCCGACTCGTCTCGCGGGCCTTCACCGCCAGGCGCGTCATGGAGATGCGTCCGCGGGTCGAGGAGATCAGTGGCAGGCTGCTCGACGCGCTGCCGGGCGACCGCGTCGTGGACCTGGTCGAGGAGTACGCCTACCCGCTGCCGATCACGGTCATCTGCGAGCTCGTCGGCATCCCCGAGAGCGACCGTCCGCTGTGGCGCGAGTGGGGCGCGAAGATGGTGTCTCTGTCACCTGGCGCCATGGCCGAACCTGTGATCAGCATGGTCGACTACATCCACGACCTGATCCCCCGCCGCCGCGCCGCGCCCGCCGACGACCTGCTCACCGGGCTGATCAAAGCGCACGACGACGACGGCGACCGCTTCACCGACACCGAGCTGATCACGATGGTGCTCACGCTCGTCCTGGCTGGACACGAGACCACCGCCCACCTGATCGGCAACGGCACCGCGGCCCTGCTCACCCACCCCGGCCAGCTTGCCATGCTGCGCGCGCGGCCCGAGCTCATGCCGCGCGCCGTCCACGAGCTCATGCGCTGGTGCGGGCCGGTGCAGGGCACCAGGGTCCGCTACGCGGCCGAGGACGTGGAGCTCGGTGGCATGACGGTGAAGCGGGGCGAGGCCGTGATGGCCGTACTGGTCTCGGCCAACTACGACCCTCGCCGCTTCGAGCGCCCCGACC GGCTCGACCTCACCCGCGAGGAGGACGGCAGGCGCGAGGTCCACGTCGGCTTCGGGCACGGCCTGCACTACTGCCTCGGCGCGGCGCTGCCAGGCAGGAGGGCGAGGTCGCCTTCGCCGGGCTGCTCTCACGCTTCCCGAAGCTGCGCCTCGCCGTCGCACCCGAGGAGCTCGAACGCAGCTCATGACCGCCTCGTACGTCGGCTCGCGAGCTCG

(a) Sequence features:

●Length: 1233bp

●Type: base sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule type: DNA

(c) Assumption: No

(d) Antisense: no

(e) Original source: rare actinomycetes Sebekia benihana

(f) Specific name: cyp-sb21

Structural features: The encoded protein contains heme.

SEQ ID NO: 2:

ATGGACACCGTTAATCTGATGGACCCAGCGCTGATGACCGATCCATTCCGTGGTTTCAGCCGTATTCGCGAAGAAGCGCCTATTGCCCGCGCTTGCTTCCCTGGCCAAGATACCCCGATCTGGCTGGTGACGCGCTACGATGATGTAAAAACTGTCCTGGGCGAGCACCGTTTCGTTAACAACCCGGCGTCCATCCCTGGCGGCGACATTCCTGATCTGCGTGAGAAACTGATGAAAGCGCGTGGCATTCCGGATGACTACGTTGTTTACCTGACCGATGGCATCCTGGACCTGGATGGTGACGACCATCTGCGTCTGCGTCGTCTGGTGTCCCGTGCGTTCACCGCTCGCCGTGTAATGGAAATGCGTCCGCGCGTAGAAGAAATCTCTGGTCGCCTGCTGGACGCTCTGCCGGGTGACCGTGTCGTCGACCTGGTTGAAGAGTACGCCTACCCACTGCCGATCACCGTGATCTGTGAACTGGTGGGCATCCCGGAAAGCGACCGTCCGCTGTGGCGCGAATGGGGTGCTAAAATGGTATCCCTGTCTCCGGGTGCAATGGCCGAGCCGGTTATCAGCATGGTTGATTACATCCACGATCTGATTCCACGTCGCCGCGCCGCCCCGGCGGATGATCTGCTGACTGGCCTGATCAAAGCGCACGACGACGACGGCGACCGCTTTACTGACACGGAGCTGATTACGATGGTACTGACCCTGGTTCTGGCAGGTCACGAAACCACTGCTCACCTGATCGGTAACGGTACCGCTGCGCTGCTGACTCATCCGGGTCAGCTGGCAATGCTGCGTGCGCGTCCTGAACTGATGCCGCGTGCGGTGCACGAGCTGATGCGTTGGTGTGGTCCGGTGCAGGGTACTCGTGTTCGTTATGCAGCAGAGGATGTCGAACTGGGTGGCATGACCGTTAAGCGTGGCGAAGCAGTGATGGCAGTACTGGTATCTGCGAACTATGATCCGCGTCGCTTTGAACGTCCGGACC GTCTGGACCTGACCCGTGAAGAAGATGGCCGTCGCGAAGTTCATGTGGGTTTCGGCCACGGTCTGCACTATTGCCTGGGCGCTGCACTGGCTCGCCAGGAAGGTGAAGTGGCTTTTGCTGGTCTGCTGTCTCGTTTCCCGAAACTGCGTCTGGCTGTTGCGCCGGAAGAACTGGAACGTCAGCTGATGCCGGCCTCTTGGCGCCTGGCATGACT

(a) Sequence features:

●Length: 1233bp

●Type: base sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule type: DNA

(c) Assumption: No

(d) Antisense: no

(e) Original source: rare actinomycetes Sebekia benihana

(f) Specific name: cyp-sb21

SEQ ID NO: 3:

ATGTTGAATGCGAGTGTGGCTGGCGGAGCAGCTACCACCACCTATGGCAACCGGCTCTTTATCTATGAAGTGATCGGTCTGCGCCAAGCCGAGGGCGAACCGTCCGACAGCTCAATCCGCCGTAGTGGCAGCACCTTCTTCAAGGTGCCTTACAGCCGGATGAATCAAGAAATGCAACGGATTTTGCGCCTTGGCGGCAAAATCGTTAGCATCCGGCCTGCGGAGGAAGCAGCCGCGAATAATGGTGCGGCTCCTCTACAGGCAGCAGCTGAAGAACCTGCTGCAGCACCAACCCCCGCTCCGGCTGCCAAAAAACATTCAGCCGAAGACGTGCCTGTCAATATCTACCGGCCTAACAAGCCTTTCGTAGGCAAGGTGCTCTCGAACGAGCCCTTGGTTCAAGAAGGCGGGATTGGTGTTGTGCAGCACCTCACCTTCGATATTTCGGAAGGCGATCTGCGCTACATCGAAGGTCAAAGTATCGGGATTATCCCGGATGGCACCGATGACAAAGGCAAGCCGCACAAGCTCCGTCTTTACTCGATCGCATCCACTCGCCACGGCGACCACGTGGATGACAAAACCGTCTCGCTGTGCGTGCGCCAGCTGCAGTACCAGAACGAAGCCGGCGAAACGATTAATGGCGTCTGCTCGACTTTCCTCTGTGGTCTGAAGCCAGGCGATGACGTCAAGATCACCGGTCCTGTGGGCAAAGAAATGCTCCTACCGGCGGACACAGACGCCAACGTGATCATGATGGGTACTGGCACCGGGATTGCTCCGTTCCGAGCCTACCTATGGCGGATGTTTAAAGACAACGAGCGAGCCATCAACAGCGAGTATCAATTCAACGGCAAGGCTTGGTTGATCTTCGGGATTCCGACGACCGCCAACATCCTCTACAAAGAGGAGCTGGAAGCGCTGCAGGCTCAGTATCCAGATAACTTCCGCCTGACCTACGCGATCAGCCGCGAGCAGAAAAATGAAGCGGGCGGCCGGA TGTACATCCAAAGACCGCGTCGCTGAACATGCTGACGAGATCTGGAACCTACTCAAGGACGAAAAAACCCACGTCTATATCTGTGGTTTGCGTGGCATGGAAGATGGGATCGATCAAGCCATGACCGTCGCAGCTGCCAAGGAAGATGTGGTTTGGTCTGACTACCAACGCACCCTCAAGAAAGCGGGTCGTTGGCATGTTGAAACCTACTAG

(a) Sequence features:

●Length: 1212bp

●Type: base sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule type: DNA

(c) Assumption: No

(d) Antisense: no

(e) Original source: Cyanobacterium Synechococcus elongates PCC 7942

(f) Specific name: sefdR

SEQ ID NO: 4:

ATGGCAACCTACAAGGTTACGCTCGTCAATGCTGCCGAAGGCTTGAACACCACGATCGACGTGGCTGACGATACCTACATCTTGGACGCCGCTGAAGAGCAAGGCATTGACCTGCCTTACTCCTGCCGTGCTGGTGCTTGCTCGACCTGTGCTGGCAAAGTCGTCTCTGGTACCGTCGACCAATCGGATCAATCCTTCTTGGATGACGACCAAATTGCAGCAGGCTTTGTCCTGACCTGCGTCGCCTATCCGACCTCCGATGTGACGATCGAAACCCACAAAGAAGAAGACCTCTACTAA(a)序列特征：

●Length: 300bp

●Type: base sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule type: DNA

(c) Assumption: No

(d) Antisense: No

(e) Original source: Cyanobacterium Synechococcus elongates PCC 7942

(f) Specific name: sefdx

SEQ ID NO: 5:

ATGTATAAAGATTTAGAAGGAAAAGTAGTGGTCATAACAGGTTCATCTACAGGTTTGGGAAAATCAATGGCGATTCGTTTTGCGACAGAAAAAGCTAAAGTAGTTGTGAATTATCGTTCTAAGGAAGACGAAGCTAACAGCGTTTTAGAAGAAATTAAAAGAGTTGGCGGAGAGGCTATTGCCGTTAAAGGTGACGTAACAGTTGAGTCTGATGTAATCAATTTAGTTCAATCTGCAATTAAAGAATTTGGAAAGCTAGACGTTATGATTAACAACGCAGGACTAGAAAATCCGGTTTCATCTCATGAAATGTCTTTAAGCGATTGGAATAAAGTAATTGATACGAACTTAACGGGAGCTTTCTTAGGTAGTCGTGAAGCGATTAAATATTTTGTTGAAAATGATATTAAGGGAACAGTTATTAACATGTCGAGTGTTCACGAGAAAATTCCTTGGCCATTATTTGTTCATTATGCAGCAAGTAAAGGCGGTATGAAGCTTATGACTGAAACACTGGCATTAGAATACGCTCCAAAAGGTATTCGTGTAAATAACATTGGACCGGGAGCGATTAATACACCGATTAACGCTGAGAAATTTGCTGATCCTGAGCAGCGTGCAGATGTAGAAAGCATGATTCCAATGGGATACATCGGAGAGCCGGAAGAAATTGCAGCAGTTGCTGCATGGCTAGCTTCTTCAGAGGCGAGTTATGTAACAGGAATTACGCTCTTTGCTGACGGCGGTATGACACTGTACCCATCATTCCAAGCAGGACGCGGATAA

(a) Sequence features:

●Length: 786bp

●Type: base sequence

●Chain type: single chain

●Topological structure: linear

(b) Molecule type: DNA

(c) Assumption: No

(d) Antisense: No

(e) Original source: Bacillus megaterium

(f) Specific name: gdh

references

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2. Burton JL, Marshall A. Hypertrichosis due to minoxidil. Br JDermatol. 1979, 101(5): 593-5.

3. Foitzik K, Hoting E, Forster T, Pertile P, Paus R. L-carnitine-L-tartrate promotes human hair growth in vitro. Exp Dermatol. 2007,16(11):936-45.

4. Kim SN, Ahn HJ, Lee CW, Lee MH, Kim JH, Kim JI, Kim SJ, Cho HS, Lee HS, KimHJ. The use of nonimmunosuppressive[γ-hydroxy-N-methyl-L-leucine4]cyclosporin derivatives for treating hair loss. 2004, Patent DE-1392224, WO02/092033A1.

5. Lee MJ, Kim HB, Yoon YJ, Han K, Kim ES. Identification of acyclosporine-specific P450 hydroxylase gene through targeted cytochrome P450 complement (CYPome) disruption in Sebekia benihana. Appl Environ Microbiol. 2013, 79(7): 2253-62.

6. Omura T, Sato R. The carbon monoxide-binding pigment of livermicrosomes. II. Solubilization, purification, and properties. J Biol Chem. 1964, 239: 2379-85.

7. Prager N, Bickett K, French N, Marcovici G.A randomized, double-blind, placebo-controlled trial to determine the effectiveness of botanically derived inhibitors of 5-alpha-reductase in the treatment of androgenetic opecia. J Altern Complement Med. 2002, 8 (2):143-52.

8. Rogers NE, Avram MR. Medical treatments for male and female pattern hair loss. J Am Acad Dermatol. 2008,59(4):547-66.

9. Whiting DA, Waldstreicher J, Sanchez M, Kaufman KD. Measuring reversal of hair miniaturization in androgenetic alopecia by focal counts horizontal sections of serial scalp biopsies: results of finasteride 1 mgtreatment of men and postmenopausal women. J Investig 9 Prompt 4(3):282-4.

Claims (4)

1. a kind of biotransformation method preparing hair growth promoter CsA-4-OH is characterized in that: utilize P450 monooxygenase and reduction chaperone protein as hybrid enzyme catalytic system in vitro, hybrid enzyme catalytic system is in electron supply Under the action of the body regeneration system, it catalyzes the conversion of CsA to obtain CsA-4-OH, realizing one-step enzymatic conversion; The P450 monooxygenase is the P450 enzyme CYP-sb21 or other functional equivalents derived from the rare actinomycete Sebekia benihana strain; The reduced chaperone protein is a ferredoxin (ferredoxin se Fdx) of cyanobacteria Synechococcus elongates PCC 7942: SynPcc7942_1499 and a ferredoxin reductase (ferredoxin reductase se FdR): SynPcc7942_0978; The electron donor regeneration system is a system in which glucose dehydrogenase (GDH) uses glucose as a substrate to catalyze NAD ⁺ or NADP ⁺ as a prosthetic group to generate NADH or NADPH. 2. A whole-cell catalytic biotransformation method for preparing hair growth promoter CsA-4-OH, characterized in that: by constructing a highly expressed P450 monooxygenase CYP-sb21, reduced chaperone protein and electron donor regeneration system The recombinant plasmid is then transferred into the host, and the resulting whole cell is used as a catalyst, and the recombinant whole cell catalyst is permeabilized by physical or chemical methods, so as to realize the efficient conversion of whole cells from CsA to CsA-4-OH; The P450 monooxygenase is the P450 enzyme CYP-sb21 or other functional equivalents derived from the rare actinomycete Sebekia benihana strain; The reduced chaperone protein is a ferredoxin (ferredoxin se Fdx) of cyanobacteria Synechococcus elongates PCC 7942: SynPcc7942_1499 and a ferredoxin reductase (ferredoxin reductase se FdR): SynPcc7942_0978; The electron donor regeneration system is a system in which glucose dehydrogenase (GDH) uses glucose as a substrate to catalyze NAD ⁺ or NADP ⁺ as a prosthetic group to generate NADH or NADPH; The recombinant plasmid is an inducible plasmid controlled by a T7 promoter. 3. The method according to claim 1 or 2, characterized in that: the glucose dehydrogenase (GDH) is derived from Bacillus megaterium , Bacillus subtilis or Bacillus pumilus ), or glucose dehydrogenase from other sources. 4. The whole-cell catalytic biotransformation method for preparing hair growth promoter CsA-4-OH according to claim 2, characterized in that: the host is Escherichia coli, Bacillus subtilis, yeast or filamentous fungi.