Progesterone 5beta-reductase mutant and application thereof in synthesis of 5beta-dihydrosteroid

Abstract

The invention discloses a progesterone 5beta-reductase mutant and application thereof in synthesis of 5beta-dihydrosteroid, relates to the technical field of biology, and in particular relates to application of gene mining and engineering modification of progesterone 5beta-reductase derived from bacteria in asymmetric synthesis of 5beta-dihydrosteroid. According to the progesterone 5 beta-reductase LpP5beta R disclosed by the invention, the progesterone 5 beta-reductase LpP5beta R from bacteria psoromais is obtained by a gene mining method, and the progesterone 5 beta-reductase LpP5beta R can be subjected to soluble expression in escherichia coli. The mutant obtained by mutating the wild type LpP5beta R has obviously improved catalytic ability on progesterone, and can efficiently catalyze delta 4-3-carbonyl steroids such as hydrocortisone, dinorcitol and the like to generate corresponding 5beta-dihydrosteroids. According to the invention, the technical bottlenecks of poor heterologous expression and low catalytic efficiency of plant-derived progesterone 5beta-reductase are solved, and a potential biocatalyst is provided for green manufacturing of 5beta-dihydrosteroid drugs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a progesterone 5β-reductase. Lp P5βR mutant and its application in the asymmetric synthesis of 5β-dihydrosteroid (II). Background Technology

[0002] Steroid compounds are widely found in microorganisms, plants, and animals, and possess anti-inflammatory, antitumor, antibacterial, and immunosuppressive effects. Based on the fusion mode of the A / B rings in the steroid nucleus, steroid compounds can be divided into two types: 5α-steroid nuclei with the AB ring in the trans configuration and 5β-steroid nuclei with the AB ring in the cis configuration. Among them, 5β-dihydrosteroids are important intermediates in the synthesis of cardiac glycosides (such as digoxin) and bile acid drugs (such as ursodeoxycholic acid and chenodeoxycholic acid). Currently, the chemical synthesis methods of 5β-dihydrosteroids mainly utilize hydrogen as a reducing agent, with Pd / C or Pd / CaCO3 catalytic hydrogenation. However, this method has poor stereoselectivity, and the yield of 5β-dihydrosteroids is only about 50%. Steroids 2007, 72, 545-551; WO2016164763Al; WO20160615727Al; WO2015027227Al). To improve the stereoselectivity of catalytic reactions, extensive screening of solvents or catalysts is usually required, or expensive PtO2 or chiral ligands are used. This leads to a significant increase in process production costs, which is detrimental to industrial applications. Compared to traditional chemical synthesis routes, enzymatic synthesis is considered an alternative due to its mild reaction conditions, good stereo / regioselectivity, and environmental friendliness. However, currently reported plant-derived progesterone 5β-reductase (P5βR) and animal-derived steroid 5β-reductase both face bottlenecks such as poor heterologous expression in *E. coli* and low catalytic activity against steroid compounds, making the enzymatic synthesis route of 5β-dihydrosteroids unsuitable for application. J. Agric. Food. Chem. 2015, 63 (46), 10112-10120; Int. J. Biol. Macromol. 2021, 175 , 67-78; Enzyme Microb. Technol. 2020, 134 , 109483; Phytochemistry 2012, 77 , 53-59 Mol. Cell. Endocrinol. 2009, 301(1-2), 191-198.). Considering that no bacterial P5βR has been reported to date, and the advantages of exogenous expression of bacterial P5βR in Escherichia coli, the gene mining and engineering of bacterial P5βR is of great significance. Summary of the Invention

[0003] To address the shortcomings of current enzymatic synthesis of 5β-dihydrosteroids, such as poor heterologous expression and low catalytic activity, the present invention aims to obtain bacterial P5βR with high heterologous expression in Escherichia coli through gene mining and to engineer it for efficient asymmetric synthesis of 5β-dihydrosteroids.

[0004] This invention provides a bacterial progesterone 5β-reductase or a mutant thereof, and its application in the synthesis of 5β-dihydrosteroids.

[0005] Therefore, this invention utilizes techniques such as gene mining and rational design to... Lichenihabitans psoromatis Progesterone 5β-reductase (from) Lp P5βR (GenBank: WP_131114925.1) was modified to obtain a target for Δ 4 The aim is to develop mutants with high catalytic activity of -3-carbonyl steroids in order to develop a promising enzymatic synthesis pathway for 5β-dihydrosteroids.

[0006] The first objective of this invention is to provide a progesterone 5β-reductase mutant, which, through the action of bacteria ( Lichenihabitans psoromatis ) progesterone 5β-reductase Lp The mutation was obtained by mutagenesis of P5βR, which was performed on the progesterone 5β-reductase enzyme. Lp The mutant is generated based on the amino acid sequence of P5βR, specifically by mutations at one or more of the following amino acid residue sites: positions 170, 180, 307, and 311; or by mutations occurring at positions 170, 180, 307, and 311 of progesterone 5β-reductase. Lp The P5βR amino acid sequence is obtained by mutating at least one of the above sites in an amino acid sequence with 80% homology.

[0007] All of the above amino acid mutants have the following characteristics: Lp P5βR shows the catalytic function of wild-type progesterone 5β-reductase in progesterone.

[0008] More specifically, the Lp The P5βR mutant is selected from any single amino acid site or any combination of multiple site mutations obtained by mutation of position 170 to V, position 180 to A or V, position 307 to A or L, and position 311 to I.

[0009] The present invention provides a gene encoding the progesterone 5β-reductase mutant, and a recombinant vector containing the gene.

[0010] The present invention further provides a recombinant bacterial strain, which is obtained by transferring the recombinant vector into a host cell. The host cell contains the recombinant vector or a gene containing the progesterone 5β-reductase or a mutant thereof integrated into its genome. Preferably, the starting bacterium used to construct the recombinant strain is *Escherichia coli*, more preferably... E.coli BL21(DE3).

[0011] The present invention therefore provides the progesterone 5β-reductase mutant, the coding gene, the recombinant vector, or the recombinant strain as a biocatalyst, in the asymmetric catalytic substrate Δ 4 The application of -3-carbonyl steroid compound (Ⅰ) in the formation of product 5β-dihydrosteroid compound (Ⅱ) is described in the following process route:

[0012] In the raw material steroid compound (Ⅰ) and the product steroid compound (Ⅱ), R1 is selected from acetyl, carbonyl, hydroxyl, hydrogen atom, ethynyl, β-hydroxyacetyl, β-methylhexyl, and hydroxy-substituted hydrocarbon groups; R2 is selected from acetyl, carbonyl, hydroxyl, and hydrogen atom, or R2 forms an epoxy structure with an adjacent carbon atom; R3 is selected from hydroxyl and hydrogen atom; P5βR represents the progesterone 5β-reductase mutant, the coding gene, the recombinant vector, or the recombinant strain.

[0013] The above reaction system also contains cofactors, cofactor regeneration system, cosolvents and / or buffer solutions.

[0014] In the above reaction system, substrate (Ⅰ) Δ 4 The concentration of the -3-carbonyl steroid compound was 0.1 g / L-100 g / L; the pH of the reaction system was 7.0-9.0; the temperature of the reaction system was 25℃-45℃; the final concentration of the cofactor was 0.1 mM-0.5 mM; and the concentration of the cosolvent was 0.1%-30% (v / v) of the reaction system.

[0015] The cofactor is NADPH.

[0016] The cofactor regeneration system is selected from at least one of glucose dehydrogenase / glucose and formate dehydrogenase / formate compounds; the mass concentration of glucose dehydrogenase or formate dehydrogenase is 0.1 g / L-100 g / L, and the molar equivalent of glucose or formate compounds is 1-10.

[0017] The co-solvent is selected from one or more of isopropanol, methanol, dimethyl sulfoxide, dimethylformamide, and toluene.

[0018] The buffer solution is either phosphate buffer or Tris-HCl buffer.

[0019] The beneficial effects of this invention are: This invention utilizes gene mining technology to obtain genes from bacteria ( Lichenihabitans psoromatis To obtain progesterone 5β-reductase Lp P5βR can be solublely expressed in E. coli. Site-directed mutagenesis can be used to express it in... Lp Mutation was performed on the wild-type P5βR to obtain a progesterone 5β-reductase mutant, which showed Δ responsiveness to progesterone and other hormones. 4 The catalytic activity of -3-carbonyl steroidal compounds is significantly enhanced.

[0020] The optimal mutant obtained by the method of this invention Lp P5βR-M180V / H307A / D311I / T170V significantly enhances the catalytic activity of progesterone and can also efficiently catalyze the decomposition of hydrocortisone, dichlorvos, and other Δ-type hormones. 4 -3-carbonyl steroids generate the corresponding 5β-dihydrosteroids.

[0021] This invention solves the technical bottleneck of poor heterologous expression and low catalytic efficiency of plant-derived progesterone 5β-reductase, and provides a promising biocatalyst for the green manufacturing of 5β-dihydrosteroid drugs. Attached Figure Description

[0022] Figure 1 Progesterone 5β-reductase Lp Polyacrylamide gel electrophoresis image of P5βR-WT expression and purification in Escherichia coli. M represents Marker; L1 represents... Lp The supernatant after ultrasonic disruption and centrifugation of P5βR-WT; L2 represents... Lp The precipitate after ultrasonic disruption and centrifugation of P5βR-WT.

[0023] Figure 2 Progesterone 5β-reductase Lp Polyacrylamide gel electrophoresis image of purified P5βR-WT enzyme. M represents Marker; L1 represents... Lp Pure enzyme of P5βR-WT.

[0024] Figure 3 Progesterone 5β-reductase Lp Time-conversion curve of progesterone catalyzed by P5βR-M180V / H307A / D311I / T170V.

[0025] Figure 4Progesterone 5β-reductase Lp GC detection results of progesterone catalyzed by P5βR-M180V / H307A / D311I / T170V.

[0026] Figure 5 The 1H NMR spectrum of 5β-pregnane-3,20-dione.

[0027] Figure 6 The carbon NMR spectrum of 5β-pregnane-3,20-dione.

[0028] Figure 7 Mass spectra of 5β-pregnane-3,20-dione. Detailed Implementation

[0029] The following description, in conjunction with the accompanying drawings and specific embodiments, further explains and illustrates a progesterone 5β-reductase mutant of the present invention and its application in the synthesis of 5β-dihydrosteroids.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0031] Example 1 Progesterone 5β-reductase Lp P5βR gene mining: Obtained from the NCBI database Lichenihabitans psoromatis Progesterone 5β-reductase source Lp P5βR (GenBank ID: WP_131114925.1), whole genome synthesis Lp P5βR was ligated into the pET-28a(+) expression vector that had been double-digested with NdeⅠ and XhoⅠ to obtain pET-28a- Lp Recombinant expression vector of P5βR.

[0032] Progesterone 5β-reductase LpP5βR amino acid sequence (SEQ ID NO: 1): MTKKNALVVGVTGISGSNVADLLVAEGWTVHGLARRPPQDRPGIHPVTADLLDPASVAEAVTGLDIGHVFFCTWARQETEDENCRVNGAMLQNLLDAVTKTSNIEHVALVTGLKHYLGPFESYGKVKPDTPFREDQARLPYQNFYYDQEDILFAAATRSNFAWSVHRPHTLIGWALGN AMNMGVTLAVYAAICKETGRDFLFPGTLQQHEAVTDVTDARILARQLLWASTTPSAYNQAFNIVNGDVFRWRRLWGTIAAYFGVSVAPYPGHPTPLAEQMAGEDATWDRIVEKHGLRPYRLKTLASWWHTDADLGRELETFTDMTKSREFGFMDLQVTERSFTELFDRLRAEKIIPPRED.

[0033] The recombinant expression vector was transformed into a suitable microbial host. The host microorganism can be any conventional microorganism in the art, as long as it can stably replicate on its own and support the progesterone 5β-reductase gene. Lp P5βR can be effectively expressed. In this embodiment, the aforementioned recombinant expression plasmid is introduced via electroporation. E.coli BL21(DE3) competent cells were incubated upside down on LB agar plates containing kanamycin resistance for 12-16 hours. Positive transformants were selected for DNA sequencing verification. Correctly verified transformants were identified as progesterone 5β-reductase. Lp The P5βR genetically engineered strain was then analyzed using SDS-PAGE. Lp The expression of the P5βR gene-engineered strain was found to be soluble in Escherichia coli. Figure 1 ).

[0034] Example 2 Progesterone 5β-reductase Lp Obtaining the P5βR mutant gene: The mutant was obtained using overlap extension PCR. The mutant containing the above-mentioned mutant was obtained. Lp Using the pET-28a(+) plasmid of the P5βR gene as a template, the mutant gene was obtained by overlapping extension PCR. The primers for each mutant are shown in Table 1.

[0035] The following describes the preparation of the corresponding mutant using the M180V mutant as an example: Using primers Lp P5βR-Nde I-F Lp P5βR-M180V-R and Lp P5βR- Xho I-R Lp P5βR-M180V-F was subjected to one round of PCR according to the PCR system described in Table 2 to amplify the mutant gene fragment.

[0036] Table 1. Primers involved in the mutations

[0037] The PCR amplification system is shown in Table 2: Table 2 PCR amplification system

[0038] Amplification program: 94℃: 10 min, (94℃: 30 s, 48℃: 30 s, 72℃: 90 s) 35 cycles, 72℃: 10 min.

[0039] Two fragments were obtained from the first round of PCR. After verifying the correct band size by agarose gel electrophoresis, the two fragments were recovered using a standard DNA product gel extraction kit and used as templates for the second round of PCR. The second round of PCR utilized primers at both ends. Lp P5βR- Nde I-F Lp P5βR- Xho I-R was performed to amplify the mutant gene according to the system described in Table 2. After two rounds of PCR, the product was recovered using a DNA purification kit to obtain the DNA product containing the mutation site.

[0040] Example 3 Construction of recombinant engineered plasmids: The purified products were then reacted with the carrier pET-28a(+) using... Nde I and Xho I. Double enzyme digestion was performed (Table 3). After incubation at 37°C for 4-6 hours, the digested products were recovered using a DNA product gel extraction kit to obtain mutant DNA fragments and vector fragments with the same sticky ends. The digested target gene was ligated to the vector plasmid using T4 DNA ligase to construct recombinant engineered plasmids of different mutants. The recombinant engineered plasmids were then transformed into competent cells of E. coli BL21(DE3) and plated on LB agar plates containing kanamycin resistance, and incubated overnight at 37°C.

[0041] Table 3 Enzyme digestion system

[0042] Example 4 Progesterone 5β-reductaseLp Expression of the P5βR mutant: Different positive transformants verified on LB agar plates were inoculated into 4 mL of LB liquid medium containing kanamycin resistance and cultured at 37°C and 200 rpm for 6-8 h to obtain seed culture of different mutants. 1 mL of the seed culture of different mutants was then inoculated into 100 mL of LB liquid medium containing kanamycin resistance and cultured at 37°C and 200 rpm until the culture medium reached OD. 600 When the concentration reaches 0.8-1.0, add IPTG solution to a final concentration of 0.1 mM, and lower the temperature to 15-20℃ to induce expression for 15-20 h. Centrifuge at 3000 r / min for 10 min to collect the bacterial cells. After washing with physiological saline, centrifuge again to collect the bacterial cells and obtain the whole-cell biocatalyst.

[0043] Example 5 Progesterone 5β-reductase Lp Isolation and purification of the P5βR mutant: Cells overexpressing the target protein were resuspended in 100 mM Na₂HPO₄-NaH₂PO₄ buffer (pH 7.0) and lysed by sonication in an ice bath. The supernatant was collected by centrifugation at 4°C (12000 rpm, 15 min). The supernatant was loaded onto a Ni-NTA affinity column and equilibrated with binding buffer (20 mM sodium phosphate, 0.5 M NaCl, and 20 mM imidazole, pH 7.4). The column was sequentially washed with washing buffer (20 mM sodium phosphate, 0.5 M NaCl, 50 mM imidazole, pH 7.4) and elution buffer (20 mM sodium phosphate, 0.5 M NaCl, 250 mM imidazole, pH 7.4), and the elution buffer was collected. Salts in the elution buffer were then removed using an Amicon ultracentrifuge filter, and progesterone 5β-reductase was detected by SDS-PAGE. Lp P5βR purification status (e.g.) Figure 2 (As shown). The concentration of purified protein was determined using the BCA protein concentration assay kit.

[0044] Example 6 Progesterone 5β-reductase Lp Activity screening of purified enzymes from the P5βR mutant: 1 mL reaction system: 0.25 mg / mL of different mutant purified enzymes as described in Example 5, 0.1 mg / mL of glucose dehydrogenase (BsGDH purified enzyme), and 0.5 mM NADP. +The reaction mixture consisted of 0.64 mM progesterone, 5 mM glucose, and 0.1 M PB buffer (pH 7.5). The reaction conditions were 35°C, 220 rpm shaker, and 1 h. The results are shown in Table 4.

[0045] After the reaction was complete, 500 μL of ethyl acetate was added to 1 mL of the reaction solution. After thorough extraction, the mixture was centrifuged at 12000 rpm for 1 min. The upper ethyl acetate layer was transferred to an ampoule, and the organic phase was removed by rotary evaporation. A suitable amount of ethyl acetate was added again to dissolve the mixture to the appropriate concentration. The solution was filtered through a microporous membrane and then analyzed by GC. GC detection conditions for steroidal compounds: initial temperature 70℃, held for 0 min; then increased to 230℃ at a rate of 10℃ / min, held for 0 min; then increased to 250℃ at a rate of 2℃ / min, held for 0 min; then increased to 280℃ at a rate of 10℃ / min, held for 6 min; total run time was 39.5 min. An Rtx®-5 column (0.32 mm × 30 m, 0.25 μm; Restek) was used with helium as the carrier gas at a flow rate of 1 mL / min and an injection temperature of 220℃.

[0046] Table 4. Asymmetric catalysis of progesterone by wild-type and mutant progesterone 5β-reductase

[0047] Example 7 Progesterone 5β-reductase Lp Substrate spectral analysis of P5βR: In order to explore Lp P5βR Δ other than progesterone 4 The catalytic ability of 3-carbonyl steroid compounds, utilizing Lp P5βR-WT and its mutants Lp Substrate spectra were studied using P5βR-M180V / H307A / D311I / T170V. 1 mL reaction system: 10 g / L Lp P5βR-WT or Lp Wet cell lysis buffer of P5βR-M180V / H307A / D311I / T170V, wet cell lysis buffer of 5 g / L BsGDH, 2 g / L steroidal compound, 4 g / L glucose, 0.1 mM NADP + The reaction was carried out using 0.1M PB buffer (pH 7.5). The reaction conditions were 35°C, 220 rpm shaker, and 2 h. Post-treatment of the reaction solution and GC detection conditions are described in Example 6. The reaction results are shown in Table 5. Mutant Lp Compared to the wild type, P5βR-M180V / H307A / D311I / T170V has a lower Δ 4The catalytic activity of -3-carbonyl steroid compounds is generally enhanced.

[0048] Table 5. Progesterone 5β-reductase substrate profile analysis

[0049] Example 8 Asymmetric catalysis of progesterone amplification reaction by progesterone 5β-reductase mutant: To further explore the application potential of progesterone 5β-reductase mutants catalyzing progesterone, superior mutants were screened. Lp P5βR-M180V / H307A / D311I / T170V was used as a biocatalyst for the catalytic reaction of progesterone to obtain the product 5β-pregnane-3,20-dione. The 100 mL reaction system is as follows: 40 g / L of… Lp P5βR-M180V / H307A / D311I / T170V wet cell lysis buffer, 5 g / L BsGDH wet cell lysis buffer, 28 g / L progesterone, 50 g / L glucose, 0.1 mM NADP + pH 7.5 PB buffer (0.1M), 10% DMSO. Reaction conditions: 35℃, 220 rpm shaker, 2 h. During the reaction, every 15 min, 50 μL of the reaction solution was added to 1 mL of ethyl acetate, mixed thoroughly, and centrifuged at 1200 rpm for 5 min. An appropriate amount of the organic layer was transferred to an ampoule, and the organic phase was removed using a rotary evaporator. Ethyl acetate was then added to dissolve the organic layer to an appropriate concentration. The mixture was filtered through a microporous membrane and analyzed by GC. Progesterone 5β-reductase Lp The time-conversion curve of progesterone catalyzed by P5βR-M180V / H307A / D311I / T170V is as follows: Figure 3 As shown, the GC detection results are as follows: Figure 4 As shown (standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd.). The 1H, 1C, and mass spectra of the product 5β-pregnane-3,20-dione are shown below. Figures 5 - 7 As shown, the spectral data is as follows: 1 H NMR (400 MHz, in CDCl3). δ 2.69 (dd, J = 15.3,13.2 Hz, 1H), 2.55 (t, J = 8.9 Hz, 1H), 2.34 (td, J = 14.6, 5.4 Hz, 1H), 2.21-1.09 (m, 20H), 2.12 (s, 3H), 1.03 (s, 3H), 0.64 (s, 3H). 13C NMR (101 MHz,in CDCl3). δ 213.05, 209.42, 63.78, 56.65, 44.30, 44.18, 42.31, 40.77, 39.13,37.18, 36.97, 35.54, 34.94, 31.56, 26.53, 25.78, 24.41, 22.94, 22.64, 21.21,13.46. (ESI + ) m / z : calcd for C 21 H 32 O2[M+H] + : 317.2, found 317.2。

Claims

1. A progesterone 5β-reductase mutant, characterized in that, The mutant is obtained by mutating at least one site in the amino acid sequence of progesterone 5β-reductase shown in SEQ ID NO.1 or an amino acid sequence that has at least 80% homology with the amino acid sequence of progesterone 5β-reductase shown in SEQ ID NO.1; wherein the mutation site is one or more of positions 170, 180, 307, and 311.

2. The progesterone 5β-reductase mutant according to claim 1, characterized in that, The mutant is selected from any single amino acid site or any combination of multiple site mutations obtained by mutating position 170 to V, position 180 to A or V, position 307 to A or L, and position 311 to I.

3. The encoding gene of the progesterone 5β-reductase mutant according to claim 1 or 2.

4. A recombinant vector comprising the encoding gene of claim 3.

5. A recombinant strain comprising the recombinant vector of claim 4.

6. The recombinant strain according to claim 5, characterized in that, The starting bacteria used to construct this recombinant strain was Escherichia coli.

7. A progesterone 5β-reductase mutant as described in claim 1 or 2, or the encoding gene as described in claim 3, or the recombinant vector as described in claim 4, or the recombinant strain as described in claim 5 or 6, as a biocatalyst in asymmetric catalysis Δ 4 The application of -3-carbonyl steroid compound (Ⅰ) in the preparation of 5β-dihydrosteroid compound (Ⅱ) is characterized by, The process route is as follows: ; Where, Δ 4 In the -3-carbonyl steroid compound (Ⅰ) and the 5β-dihydrosteroid compound (Ⅱ), R1 is selected from acetyl, carbonyl, hydroxyl, hydrogen atom, ethynyl, β-hydroxyacetyl, β-methylhexyl, and hydroxy-substituted hydrocarbon groups; R2 is selected from acetyl, carbonyl, hydroxyl, and hydrogen atom, or R2 forms an epoxy structure with an adjacent carbon atom; R3 is selected from hydroxyl and hydrogen atom; P5βR represents a progesterone 5β-reductase mutant, encoding gene, recombinant vector, or recombinant strain.

8. The application according to claim 7, characterized in that, The reaction system also contains cofactors, cofactor regeneration system, cosolvents and / or buffer solutions.

9. The application according to claim 7, characterized in that, In the reaction system, the substrate Δ 4 The concentration of the -3-carbonyl steroid compound (Ⅰ) was 0.1 g / L-100 g / L; the pH of the reaction system was 7.0-9.0; the temperature of the reaction system was 25℃-45℃; the final concentration of the cofactor was 0.1 mM-0.5 mM; and the concentration of the cosolvent was 0.1%-30% of the reaction system, v / v.

10. The application according to claim 8 or 9, characterized in that, The cofactor is NADPH; the cofactor regeneration system is selected from at least one of glucose dehydrogenase / glucose and formate dehydrogenase / formate compounds; the mass concentration of glucose dehydrogenase or formate dehydrogenase is 0.1 g / L-100 g / L, and the molar equivalent of glucose or formate compounds is 1-10; the cosolvent is selected from one or more of isopropanol, methanol, dimethyl sulfoxide, dimethylformamide, and toluene; the buffer is phosphate buffer or Tris-HCl buffer.

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