Enzymatic synthesis of immunosuppressant mycophenolic acid and its derivatives

Abstract

The present invention provides an enzymatic synthesis method for the immunosuppressant mycophenolic acid and its derivatives. This method utilizes a multi-enzyme, multi-cycle cascade reaction strategy to catalyze the conversion of the substrates 4-dienodecanoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide or 4-dienodecanoic acid-3,5-dihydroxy-6-methylphthalide into mycophenolic acid and its derivatives. The multi-enzyme combination comprises an acyl-CoA ligase, an FAD-dependent oxidase, a 2,4-dienoyl-CoA reductase, an enoyl-CoA isomerase, a bifunctional enzyme with hydratase and dehydrogenase functions, and a thiolase; in addition, a CoA hydrolase and an oxygen-methyltransferase. This method, for the first time, achieves the in vitro enzymatic synthesis of (M)FDHMP-3C to MPA, fully elucidating the mycophenolic acid oxidative degradation synthesis pathway and the catalytic sequence of the catalytic enzymes.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis of mycophenolic acid, an immunosuppressant, and particularly relates to an in vitro enzymatic synthesis method of mycophenolic acid and its derivatives. Background Art

[0002] Mycophenolic acid (MPA, chemical structure see Figure 1 ), a class of polyketide compounds commonly produced by Penicillium strains such as Penicillium brevicompactum, is used to reduce immune rejection reactions after organ transplantation and treat autoimmune diseases. It is one of the most widely used immunosuppressants.

[0003] The synthesis strategies of mycophenolic acid include chemical synthesis and biosynthesis. In the field of chemical synthesis, 12 chemical synthesis pathways have been reported in the literature. However, due to the unique six-substituted aromatic ring group in the structure of mycophenolic acid, the chemical synthesis process is lengthy and the yield is low, which seriously limits the industrial application of chemical synthesis methods. At present, the acquisition of mycophenolic acid mainly relies on traditional fungal liquid submerged fermentation. At the genetic and enzymatic levels, the biosynthetic gene cluster and precursor synthesis pathway of mycophenolic acid have been preliminarily elucidated, and its polyketide synthase-based skeleton synthesis and oxidative degradation-based side chain production process have been discovered. However, the side chain oxidative degradation process and catalytic enzyme have not been analyzed and clarified, resulting in blindness and uncertainty in the genetic modification of fungal strains. Summary of the Invention

[0004] The present invention aims to provide an enzymatic synthesis method for the immunosuppressant mycophenolic acid and its derivatives, namely, utilizing a multi-enzyme multi-cycle cascade reaction strategy to catalyze the conversion of a substrate 4-dienodecanoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide (MFDHMP-3C) or 4-dienodecanoic acid-3,5-dihydroxy-6-methylphthalide (FDHMP-3C) into mycophenolic acid and its derivatives.

[0005] The present invention first provides a multi-enzyme combination for catalyzing the synthesis of DMMPA-CoA or MPA-CoA using 4-dienoyldecanoate-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienoyldecanoate-3,5-dihydroxy-6-methylphthalide MFDHMP-3C as a substrate. The multi-enzyme combination comprises PbACL75 (acyl-CoA ligase, SEQ ID NO: 1), PbACOX323 (FAD-dependent oxidase, SEQ ID NO: 2), PbDECR799 (2,4-dienoyl-CoA reductase, SEQ ID NO: 3), PbECI1007 (enoyl-CoA isomerase, SEQ ID NO: 4), PbECH1071 (a bifunctional enzyme with hydratase and dehydrogenase functions, SEQ ID NO: 5), and PbKT298 (thiolase, SEQ ID NO: 6).

[0006] Furthermore, the multi-enzyme combination further comprises MpaH' (coenzyme A hydrolase, SEQ ID NO: 7) and MpaG' (oxygen methyltransferase, SEQ ID NO: 8);

[0007] The present invention also provides a use of the multi-enzyme combination, which is to catalyze the synthesis of mycophenolic acid using 4-dienedecanoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienedecanoic acid-3,5-dihydroxy-6-methylphthalide MFDHMP-3C as a substrate;

[0008] The present invention also provides a method for catalyzing the synthesis of mycophenolic acid MPA using 4-dienoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienoic acid-3,5-dihydroxy-6-methylphthalide MFDHMP-3C as a substrate, wherein 4-dienoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienoic acid-3,5-dihydroxy-6-methylphthalide MFDHMP-3C is used as a substrate, and the catalytic synthesis of mycophenolic acid MPA is carried out by PbACL75 (acyl-CoA ligase, SEQ ID NO: 1), PbACOX323 (FAD-dependent oxidase, SEQ ID NO: 2), PbDECR799 (2,4-dienoyl-CoA reductase, SEQ ID NO: 3), PbECI1007 (enoyl-CoA isomerase, SEQ ID NO: 4), and PbECH1071 (a bifunctional enzyme with hydratase and dehydrogenase functions, SEQ ID NO: 5). ID NO: 5) and PbKT298 (thiolase, SEQ ID NO: 6) catalyze the production of MFDHMP-d3-CoA or FDHMP-d3-CoA;

[0009] Furthermore, the method described above uses MFDHMP-d3-CoA or its demethylated product FDHMP-d3-CoA as a substrate, and is catalyzed by PbACOX323 (FAD-dependent oxidase, SEQ ID NO: 2), PbECH1071 (a bifunctional enzyme with hydratase and dehydrogenase functions, SEQ ID NO: 5), and PbKT298 (thiolase, SEQ ID NO: 6) to produce mycophenolic acid-CoA (MPA-CoA) or demethylated mycophenolic acid-CoA (DMMPA-CoA).

[0010] Furthermore, in the method described, the process from DMMPA-CoA to MPA is catalyzed by oxygen methyltransferase MpaG', and the process of MPA-CoA hydrolysis to produce MPA is catalyzed by coenzyme A hydrolase MpaH'.

[0011] The present invention realizes the process of synthesizing MPA from MFDHMP-3C or FDHMP-3C by in vitro enzymatic method for the first time, and clarifies the complete synthesis pathway of mycophenolic acid and the catalytic sequence of the catalytic enzyme. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 :Schematic diagram of the in vitro enzymatic synthesis pathway of MPA;

[0013] Figure 2 : Liquid phase detection results of synthetic MFDHMP-d3-CoA;

[0014] Figure 3 : Liquid phase detection results of synthetic MPA-CoA;

[0015] Figure 4 : Synthetic DMMPA-CoA liquid phase detection result diagram;

[0016] Figure 5 : Liquid phase detection results of synthetic MPA. DETAILED DESCRIPTION

[0017] The present invention uses MFDHMP-3C or FDHMP-3C, a substrate containing a 12-carbon atom side chain, as a substrate to achieve in vitro enzymatic synthesis of MPA and its derivatives. Specifically, MFDHMP-3C or FDHMP-3C is used as the substrate, and PbACL75 (acyl-CoA ligase, SEQ ID NO: 1), PbACOX323 (FAD-dependent oxidase, SEQ ID NO: 2), PbDECR799 (2,4-dienoyl-CoA reductase, SEQ ID NO: 3), PbECI1007 (enoyl-CoA isomerase, SEQ ID NO: 4), PbECH1071 (a bifunctional enzyme with hydratase and dehydrogenase functions, SEQ ID NO: 5), PbKT298 (thiolase, SEQ ID NO: 6), MpaH' (CoA hydrolase, SEQ ID NO: 7), and MpaG' (oxymethyltransferase, SEQ ID NO: 8) are added to achieve the synthesis of MPA.

[0018] The amino acid sequences of the PbACL75, PbACOX323, PbDECR799, PbECI1007, PbECH1071, PbKT298, MpaH', and MpaG' proteases used in the present invention are SEQ ID NOs: 1-8, respectively; however, other homologous proteases, or derivative proteases obtained by replacing, deleting, or adding one or more amino acids may also be used, and the derivative proteases have the same or similar effects as the original proteases.

[0019] Example 1: In vitro enzymatic synthesis of MFDHMP-d3-CoA

[0020] In this example, the synthesis of MFDHMP-d3-CoA from MFDHMP-3C was carried out in the presence of proteases PbACL75, PbACOX323, PbDECR799, PbECI1007, PbECH1071, and PbKT298.

[0021] The process from MFDHMP-3C to 4-dienedecanoate-3-hydroxy-5-hydroxymethyl-6-methylphthalide coenzyme A (MFDHMP-3C-CoA) is catalyzed by the coenzyme A ligase PbACL75, which functions to connect CoA to the carboxyl group of MFDHMP-3C.

[0022] From MFDHMP-3C-CoA to trans-Δ 2,4,8 -trienoyl-MFDHMP-3C-CoA (trans-MFDHMP-d6-CoA), which is catalyzed by the FAD-dependent oxidase PbACOX323;

[0023] From trans-MFDHMP-d6-CoA to trans-Δ 2,8 The production of -dienoyl-MFDHMP-3C-CoA (trans-MFDHMP-d8-CoA) is catalyzed by 2,4-dienoyl-CoA reductase PbDECR799 and enoyl-CoA isomerase PbECI1007;

[0024] The process from trans-MFDHMP-d8-CoA to 3-ketone-MFDHMP-d1-CoA is catalyzed by the bifunctional enzyme PbECH1071 with hydratase and dehydrogenase functions;

[0025] The process from 3-ketone-MFDHMP-d1-CoA to MFDHMP-d3-CoA is catalyzed by thiolase PbKT298.

[0026] The following is a detailed description of the enzymatic process for synthesizing MFDHMP-d3-CoA from MFDHMP-3C.

[0027] 1) Protein induction expression

[0028] E. coli BL21 (DE3) strains that efficiently expressed PbACL75, PbACOX323, PbDECR799, PbECI1007, PbECH1071, and PbKT298 proteins were activated and cultured on solid LB plates (containing 50 μg / mL kanamycin). The single clones grown on the LB solid plates were transferred into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C, 220 rpm to prepare seed liquid. The seed liquid was transferred to a sterile 0.5 L TB medium at a 1% inoculum volume, and kanamycin was added at a final concentration of 50 μg / mL and cultured at 37°C, 220 rpm until the OD 600 Between 0.6 and 0.8, add 0.2mM isopropyl-β- D -IPTG, culture at 18°C, 150 rpm for 18-22 h to induce protein expression.

[0029] 2) Target protein purification

[0030] The cells were collected by centrifugation, resuspended in 30-40 mL Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), and the cells were disrupted by ultrasound; the fragments were centrifuged at high speed (10,000 rpm, 4 ° C), and the supernatant was incubated with Ni-NTA for 1 h; impurities were washed with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) (the elution volume was based on the Coomassie Brilliant Blue G-250 detection solution not turning blue); the target proteins were eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0), and the protein solution was concentrated using an ultrafiltration tube of appropriate size according to the size of the protein. Finally, the imidazole was removed using a PD-10 desalting column, and the buffer solution was desalted using a Desalting column. buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0).

[0031] 3) In vitro enzymatic reaction

[0032] The reaction buffer was Tris-HCl solution (50 mM, pH 8.0), 200 μM MFDHMP-3C was used as the substrate, and the final concentrations of 10 μM PbACL75, 10 μM PbACOX323, 10 μM PbDECR799, 10 μM PbECI1007, 10 μM PbECH1071, 10 μM PbKT298, 2 mM ATP, 2 mM CoA, 10 mM MgCl2, 1 mM DTT, 10 mM NADPH, and 10 mM NAD were added. + The reaction was carried out with 2 mM FAD at 30°C for 4 h. An equal volume of methanol was added to the reaction system to quench the reaction. The enzyme reaction products were detected using high-performance liquid chromatography (HPLC) at a wavelength of λ = 254 nm, a flow rate of 1 mL / min, a column temperature of 30°C, and a mobile phase of A:B = water:acetonitrile (containing 10 mM ammonium acetate). The detection procedure was as follows: 20% B for 0-3 min, 20%-100% B for 3-20 min, 100% B for 20-28 min, 100% B for 28-28.5 min, 100%-20% B for 28.5-35 min, and 20% B for 30 μL injection volume.

[0033] HPLC detection results showed that MFDHMP-3C was catalyzed by coenzyme A ligase (PbACL75) to connect with CoA to generate product MFDHMP-3C-CoA; MFDHMP-3C-CoA was catalyzed by FAD-dependent oxidase (PbACOX323) to generate product MFDHMP-3C-CoA. αand C β A carbon-carbon double bond is formed to generate trans-MFDHMP-d6-CoA; further, trans-MFDHMP-d6-CoA is catalyzed by 2,4-dienoyl-CoA reductase PbDECR799 to reduce the conjugated double bonds at C2 and C4 to a double bond at C3, and the enoyl-CoA isomerase PbECI1007 further isomerizes the double bond at C3 to C2 to generate trans-MFDHMP-d8-CoA. Trans-MFDHMP-d8-CoA is catalyzed by a bifunctional enzyme (PbECH1071) with hydratase and dehydrogenase functions. The hydratase can catalyze the reversible hydration of the C2 double bond to generate a hydroxyl group; the dehydrogenase catalyzes the dehydrogenation of the hydroxyl group to generate a ketone group, generating 3-ketone-MFDHMP-d1-CoA. Finally, 3-ketone-MFDHMP-d1-CoA is converted to MFDHMP-d3-CoA, which is catalyzed by thiolase (PbKT298) to generate MFDHMP-d3-CoA. The conversion efficiency of MFDHMP-3C to MFDHMP-d3-CoA is approximately 31.3% ( Figure 2 ).

[0034] Example 2: In vitro enzymatic synthesis of mycophenolic acid coenzyme A (MPA-CoA)

[0035] In this example, MPA-CoA was synthesized using MFDHMP-d3-CoA as a substrate in the presence of PbACOX323, PbECH1071, and PbKT298 proteases.

[0036] Among them, the process from MFDHMP-d3-CoA to MFDHMP-d2-CoA is catalyzed by the FAD-dependent oxidase PbACOX323;

[0037] The synthesis of 3-ketone-MFDHMP-d2-CoA from MFDHMP-d2-CoA is catalyzed by the bifunctional enzyme PbECH1071, which has both hydratase and dehydrogenase functions.

[0038] MPA-CoA is synthesized from 3-ketone-MFDHMP-d2-CoA, a process catalyzed by the thiolase PbKT298.

[0039] The process of synthesizing MPA-CoA from MFDHMP-d3-CoA is described in detail below.

[0040] 1) Protein induction expression

[0041] E. coli BL21 (DE3) strains expressing PbACOX323, PbECH1071, and PbKT298 were activated and cultured on solid LB plates (containing 50 μg / mL kanamycin). The single clones grown on the LB solid plates were transferred into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C, 220 rpm to prepare seed liquid. The seed liquid was transferred to sterile 0.5 L TB medium at a 1% inoculum size, and kanamycin was added at a final concentration of 50 μg / mL and cultured at 37°C, 220 rpm until the OD 600 The pH value was between 0.6 and 0.8, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was carried out at 18°C and 150 rpm for 18 to 22 h to induce protein expression.

[0042] 2) Target protein purification

[0043] The cells were collected by centrifugation, resuspended in 30-40 mL Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), and the cells were disrupted by ultrasound; the fragments were centrifuged at high speed (10,000 rpm, 4 ° C), and the supernatant was incubated with Ni-NTA for 1 h; impurities were washed with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) (the elution volume was based on the Coomassie Brilliant Blue G-250 detection solution not turning blue); the target proteins were eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0), and the protein solution was concentrated using an ultrafiltration tube of appropriate size according to the size of the protein. Finally, the imidazole was removed using a PD-10 desalting column, and the buffer solution was desalted using a Desalting column. buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0).

[0044] 3) In vitro enzymatic reaction

[0045] The reaction buffer was Tris-HCl solution (50 mM, pH 8.0), MFDHMP-d3-CoA was used as substrate, and the final concentrations of 10 μM PbACOX323, 10 μM PbECH1071, 10 μM PbKT298, 2 mM CoA, and 10 mM NAD were added. + The mixture was reacted with 2 mM FAD at 30° C. for 4 h, and then an equal volume of methanol was added to the reaction system to quench the reaction. The enzyme reaction product was detected by high performance liquid chromatography (HPLC) using the same detection method as described in Example 1.

[0046] HPLC detection results showed that the substrate MFDHMP-d3-CoA was catalyzed by FAD-dependent oxidase PbACOX323 and α and C β A carbon-carbon double bond is formed to generate MFDHMP-d2-CoA. MFDHMP-d2-CoA is further catalyzed by the bifunctional enzyme PbECH1071, which has both hydratase and dehydrogenase functions. The hydratase can catalyze the reversible hydration of the C2 double bond to generate a hydroxyl group. The dehydrogenase catalyzes the dehydrogenation of the hydroxyl group to generate a ketone group, generating 3-ketone-MFDHMP-d2-CoA. Finally, 3-ketone-MFDHMP-d2-CoA is catalyzed by the thiolase PbKT298 to reduce three carbon atoms to generate MPA-CoA, with a conversion efficiency of approximately 3.6% ( Figure 3 ).

[0047] Example 3: In vitro enzymatic synthesis of demethylmycophenolic acid coenzyme A (DMMPA-CoA)

[0048] In this example, the demethylation product of MFDHMP-d3-CoA, FDHMP-d3-CoA, was used as a substrate to synthesize DMMPA-CoA in the presence of PbACOX323, PbECH1071, and PbKT298 proteases.

[0049] Among them, the process from FDHMP-d3-CoA to FDHMP-d2-CoA is catalyzed by the FAD-dependent oxidase PbACOX323;

[0050] The synthesis of 3-ketone-FDHMP-d2-CoA from FDHMP-d2-CoA is catalyzed by the bifunctional enzyme PbECH1071, which has both hydratase and dehydrogenase functions.

[0051] DMMPA-CoA is synthesized from 3-ketone-FDHMP-d2-CoA, a process catalyzed by the thiolase PbKT298.

[0052] The process of synthesizing DMMPA-CoA from FDHMP-d3-CoA is described in detail below.

[0053] 1) Protein induction expression

[0054] E. coli BL21 (DE3) strains expressing PbACOX323, PbECH1071, and PbKT298 were inoculated onto solid LB medium (containing 50 μg / mL kanamycin) plates for activation culture. The single clones grown on the LB solid plates were transferred into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C, 220 rpm to prepare seed liquid. The seed liquid was transferred to sterile 0.5 L TB medium at a 1% inoculum size, and kanamycin was added at a final concentration of 50 μg / mL and cultured at 37°C, 220 rpm until OD 600 The pH value was between 0.6 and 0.8, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was carried out at 18°C and 150 rpm for 18 to 22 h to induce protein expression.

[0055] 2) Target protein purification

[0056] The cells were collected by centrifugation, resuspended in 30-40 mL Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), and the cells were disrupted by ultrasound; the fragments were centrifuged at high speed (10,000 rpm, 4 ° C), and the supernatant was incubated with Ni-NTA for 1 h; impurities were eluted with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) (the elution volume was based on the Coomassie Brilliant Blue G-250 detection solution not turning blue); the target proteins were eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0), and the protein solution was concentrated using an ultrafiltration tube of appropriate size according to the size of the protein. Finally, the imidazole was removed using a PD-10 desalting column, and the buffer solution was desalted using a Desalting column. buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0).

[0057] 3) In vitro enzymatic reaction

[0058] The reaction buffer was Tris-HCl solution (50 mM, pH 8.0), and the demethylated product of MFDHMP-d3, FDHMP-d3 (1 mM), was used as the substrate. The final concentrations of 10 μM PbACL75, 10 μM PbACOX323, 10 μM PbECH1071, 10 μM PbKT298, 2 mM CoA, and 10 mM NAD were added. +The mixture was reacted with 2 mM FAD at 30° C. for 4 h, and then an equal volume of methanol was added to the reaction system to quench the reaction. The enzyme reaction product was detected by high performance liquid chromatography (HPLC) using the same detection method as in Example 1.

[0059] HPLC detection results showed that the substrate FDHMP-d3 produced FDHMP-d3-CoA under the catalysis of coenzyme A ligase PbACL75; FDHMP-d3-CoA was catalyzed by FAD-dependent oxidase (PbACOX323) and α and C β A carbon-carbon double bond is formed to generate FDHMP-d2-CoA. Further, FDHMP-d2-CoA is catalyzed by the bifunctional enzyme PbECH1071, which has the functions of hydratase and dehydrogenase. The hydratase can catalyze the reversible hydration of the C2 double bond to generate a hydroxyl group; the dehydrogenase catalyzes the dehydrogenation of the hydroxyl group to generate a ketone group, generating 3-ketone-FDHMP-d2-CoA. Finally, 3-ketone-FDHMP-d2-CoA is catalyzed by the thiolase PbKT298 to reduce three carbon atoms to generate DMMPA-CoA, with a conversion efficiency of approximately 5.3% ( Figure 4 ).

[0060] Example 4: In vitro enzymatic synthesis of MPA

[0061] In this example, MPA was synthesized using DMMPA-CoA as a substrate with the participation of coenzyme A hydrolase MpaH' and oxygen methyltransferase MpaG'.

[0062] Among them, DMMPA-CoA is synthesized into MPA-CoA, which is catalyzed by the oxygen methylase MpaG';

[0063] Furthermore, MPA-CoA is synthesized into MPA, which is catalyzed by coenzyme A hydrolase MPaH'.

[0064] The synthesis of MPA from DMMPA-CoA is described in detail below.

[0065] 1) Protein induction expression

[0066] E. coli BL21 (DE3) strains that efficiently express coenzyme A hydrolase MpaH' and oxygen methyltransferase MpaG' were activated and cultured on solid LB plates (containing 50 μg / mL kanamycin). Single clones grown on the LB solid plates were transferred into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C, 220 rpm to prepare seed liquid. The seed liquid was transferred to 0.5 L TB medium at a 1% inoculum volume, and kanamycin was added at a final concentration of 50 μg / mL. The culture was continued at 37°C, 220 rpm until the OD 600The pH value was between 0.6 and 0.8, 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture was carried out at 18°C and 150 rpm for 18 to 22 h to induce protein expression.

[0067] 2) Target protein purification

[0068] The cells were collected by centrifugation, resuspended in 30-40 mL Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0), and the cells were disrupted by ultrasound; the fragments were centrifuged at high speed (10,000 rpm, 4 ° C), and the supernatant was incubated with Ni-NTA for 1 h; impurities were eluted with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0) (the elution volume was based on the Coomassie Brilliant Blue G-250 detection solution not turning blue); the target protein was eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0), and the protein solution was concentrated using an ultrafiltration tube of appropriate size according to the size of the protein. Finally, the imidazole was removed using a PD-10 desalting column, and the buffer solution was desalted using a Desalting column. buffer (50 mM NaH2PO4, 300 mM NaCl, 10% glycerol, pH 8.0).

[0069] 3) In vivo enzymatic reaction

[0070] The reaction was carried out in a 50 mM Tris-HCl solution (pH 8.0) with 1 mM demethylmycophenolic acid coenzyme A (DMMPA-CoA) as the substrate. MpaH', 10 μM MpaG', and 10 mM SAM were added to a final concentration of 10 μM. The reaction was allowed to proceed at 30°C for 2 h. An equal volume of methanol was added to quench the reaction, and the enzyme reaction product was detected by high-performance liquid chromatography (HPLC) using the same method as in Example 1.

[0071] HPLC analysis revealed that the substrate DMMPA-CoA could be recognized by the methyltransferase MpaG' and catalyze the methylation of the C5 hydroxyl group to produce MPA-CoA. Furthermore, MPA-CoA was hydrolyzed by the coenzyme A hydrolase MpaH' to produce the final product MPA. The conversion efficiency of DMMPA-CoA to MPA was approximately 98.2% ( Figure 5 ).

[0072] In summary, the present invention uses FDHMP-3C or MFDHMP-3C as a substrate, and generates DMMPA-CoA or MPA-CoA through the catalysis of PbACL75, PbACOX323, PbDECR799, PbECI1007, PbECH1071, and PbKT298 enzymes; and then finally generates MPA through the catalysis of methyltransferase MpaG' and coenzyme A hydrolase MpaH'.

Claims

1. A multi-enzyme combination, characterized in that The multi-enzyme combination comprises an acyl-CoA ligase, a FAD-dependent oxidase, a 2,4-dienoyl-CoA reductase, an enoyl-CoA isomerase, a bifunctional enzyme with hydratase and dehydrogenase functions, a thiolase, a CoA hydrolase, and an oxygen methyltransferase; The amino acid sequence of the acyl-CoA ligase is SEQ ID NO: 1, the amino acid sequence of the FAD-dependent oxidase is SEQ ID NO: 2; the amino acid sequence of the 2,4-dienoyl-CoA reductase is SEQ ID NO: 3; the amino acid sequence of the enoyl-CoA isomerase is SEQ ID NO: 4; the amino acid sequence of the bifunctional enzyme having hydratase and dehydrogenase functions is SEQ ID NO: 5; and the amino acid sequence of the thiolase is SEQ ID NO:

6. The amino acid sequence of the coenzyme A hydrolase is SEQ ID NO: 7, and the amino acid sequence of the oxygen methyltransferase is SEQ ID NO:

8.

2. Use of the multi-enzyme combination according to claim 1 in catalyzing the production of mycophenolic acid MPA using 4-dienoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienoic acid-3,5-dihydroxy-6-methylphthalide MFDHMP-3C as a substrate.

3. A method for synthesizing mycophenolic acid MPA, characterized in that: The method is to use the multi-enzyme combination according to claim 1 to catalyze the production of mycophenolic acid MPA using 4-dienoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienoic acid-3,5-dihydroxy-6-methylphthalide MFDHMP-3C as a substrate.

4. The method according to claim 3, wherein In the method, acyl-CoA ligase, FAD-dependent oxidase, 2,4-dienoyl-CoA reductase, enoyl-CoA isomerase, a bifunctional enzyme with hydratase and dehydrogenase functions, and thiolase catalyze the synthesis of 4-dienodecanoic acid-3-hydroxy-5-hydroxymethyl-6-methylphthalide FDHMP-3C or 4-dienodecanoic acid-3,5-dihydroxy-6-methylphthalide MFDHMP-3C to generate MFDHMP-d3-CoA or FDHMP-d3-CoA.

5. The method according to claim 3, wherein In the method, MFDHMP-d3-CoA or FDHMP-d3-CoA is used as a substrate, and mycophenolic acid-CoA or demethylated mycophenolic acid-CoA is generated through catalysis by FAD-dependent oxidase, a bifunctional enzyme with hydratase and dehydrogenase functions, and a thiolase.

6. The method according to claim 3, wherein In the method, mycophenolic acid coenzyme A or demethylated mycophenolic acid coenzyme A is used as a substrate, and mycophenolic acid MPA is produced by catalysis of coenzyme A hydrolase and oxygen methyltransferase.

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