Light-driven P450 catalytic system and application thereof in pravastatin synthesis

Abstract

The invention belongs to the technical field of biological catalysis, and relates to a light-driven P450 catalytic system and application thereof in pravastatin synthesis. The system comprises a thylakoid membrane, Fdx, P450sca-2 and PC / Cyt C6, and is characterized in that the PC / Cyt C6 is connected with the Fdx; the thylakoid membrane takes H2O as an electron donor under an illumination condition, electrons are generated through a light system II, and the electrons are sequentially transferred to Fdx and are conveyed to P450sca-2 through interaction between proteins to catalyze an enzymatic reaction. According to the invention, light energy and / or water are / is used as an energy source and an electron source, expensive NAD (P) H is completely or partially replaced, and the production cost is greatly reduced. The system lays a certain foundation for constructing a P450 reaction system for photosynthesis and CO2 fixation in the future.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology and relates to a light-driven P450 catalytic system and its application in the synthesis of pravastatin. Background Technology

[0002] Cytochrome P450 enzymes (P450s) are a superfamily of enzymes capable of catalyzing a wide range of oxidation reactions, with enormous application potential in fields such as drug synthesis and natural product modification. However, the vast majority of P450-catalyzed reactions rely on the expensive cofactor NAD(P)H and complex electron transport chaperone proteins (such as ferredoxin Fdx and ferredoxin reductase FdR) to provide the electrons required for the reaction, which greatly limits their industrial application.

[0003] Photosynthetic organisms such as cyanobacteria can convert light energy into chemical energy through their photosynthetic systems on their thylakoid membranes. Photosystem I (PSI) possesses extremely strong reducing power and can theoretically replace NAD(P)H in driving enzymatic reactions requiring strong reducing agents. Previous attempts have attempted to combine P450 with photosynthetic systems, but these have often suffered from low efficiency, complex systems (requiring additional artificial electron donors), or the inability to utilize water as an initial electron donor within the intact thylakoid membrane (TM) in vitro. Therefore, developing a highly efficient, low-cost, light-driven P450 catalytic system that utilizes the complete photosynthetic chain is of great significance.

[0004] Pravastatin is an important cholesterol-lowering drug, and its key industrial production steps are derived from... Streptomyces carbophilus The 6β-hydroxylation of mevastatin was catalyzed by P450sca-2 (CYP105A3). Developing a novel P450sca-2 catalytic process independent of NADPH has significant potential for cost reduction and improved "green catalysis". Summary of the Invention

[0005] This invention aims to address the following key problems encountered in the industrial application of cytochrome P450 enzymes (P450s) in the prior art: 1. Solve the problem of dependence on the expensive cofactor NAD(P)H. The vast majority of P450 catalytic reactions require the continuous consumption of the expensive cofactor NAD(P)H to provide electrons, resulting in extremely high production costs and being the main economic bottleneck limiting its large-scale industrial application; 2. Addressing the issues of complex, inefficient, and poorly universal electron transport systems. P450 enzymes typically require matching with specific natural electron transport chaperone proteins (such as ferroredoxin Fdx and ferroredoxin reductase FdR), and the process of finding and optimizing these chaperone proteins is cumbersome, time-consuming, and uncertain.

[0006] The technical solution adopted by the present invention to solve its technical problem is as follows: a light-driven P450 catalytic system, comprising: a thylakoid membrane, Fdx, P450sca-2, and Cyt C6; the thylakoid membrane contains photosystem I, photosystem II, polyaminoquinone pool, and cytochrome b6f; under light irradiation, the thylakoid membrane uses H2O as an electron donor to generate electrons through photosystem II, which are sequentially transferred to Fdx and transported to P450sca-2 through protein-protein interactions to catalyze the enzymatic reaction.

[0007] Preferably, the Fdx concentration in the system is 20-50 μmol / L.

[0008] Preferably, the concentration of Cyt C6 in the system is 10 μmol / L.

[0009] Preferably, the system further comprises 2,6-dichlorophenolindophenol and sodium ascorbate, wherein the 2,6-dichlorophenolindophenol and sodium ascorbate serve as redox mediators and electron donors, generating electrons through photosystem I.

[0010] Preferably, the system further includes FdR and NADP. + The FdR accepts the transferred electrons to generate NADPH; the generated NADPH serves as an auxiliary electron donor for P450sca-2.

[0011] Preferably, the system also includes catalase.

[0012] Furthermore, the present invention also provides the application of the aforementioned light-driven P450 catalytic system in the synthesis of pravastatin.

[0013] Furthermore, the present invention also provides a method for synthesizing pravastatin, which utilizes the aforementioned light-driven P450 catalytic system to catalyze the conversion of mevastatin to pravastatin.

[0014] Compared with the prior art, the beneficial effects of the present invention are reflected in: (1) Green economy: This invention uses light energy and / or water as energy and electron sources, which completely or partially replaces the expensive NAD(P)H, significantly reducing production costs and conforming to the principles of green chemistry; (2) High efficiency and versatility: This invention successfully utilizes the intact cyanobacterial thylakoid membrane as an electron donor, achieving efficient coupling with P450 enzymes. Through optimization of three pathways, the yield of pravastatin was significantly increased from 12.7% to 70.1%; (3) Avoiding bottlenecks: This invention avoids the problem of finding and matching a natural and efficient electron transport partner for a specific P450, and provides a universal P450 optical driving solution; (4) Great application potential: This in vitro system lays a certain foundation for the future construction of P450 reaction systems for photosynthesis and CO2 fixation. Attached Figure Description

[0015] Figure 1 For SDS-PAGE gel analysis; M: marker; 1: P450sca2 (45 kDa); 2: Fdx (12.8 kDa); 3: FdR (47.6 kDa); Figure 2 Electron transfer pathways for the photosynthetic system of cyanobacteria and the light-driven P450sca-2 system designed in this invention; wherein, PSII: photosystem II; PQ: plastoquinone; Cyt b6f: cytochrome b6f; PC: plastocyanin; C6: cytochrome C6; 6-dichlorophenolindopheno-DCPIP: 2; Asc: sodium ascorbate; PSI: photosystem I; Fdx: ferricoxin; FdR: ferricoxin reductase; Figure 3 For product analysis and reaction optimization of pathway i; A. UPLC analysis of pathway i reaction and related control reactions; i: Pravastatin standard; ii: Pathway i reaction under light conditions; iii: Pathway i reaction under dark conditions; iv: TMe inactivated by boiling as a control; v: P450sca-2 reaction supported by Fdx-FdR-NADPH; B. Optimized Fdx concentration results; C. Optimized Cyt C6 concentration results; D. Time gradient experimental results of pathway i reaction at optimal Fdx and Cyt C6 concentrations; Figure 4 For product analysis and reaction optimization of pathway ii; A. UPLC analysis of pathway ii reaction and related control reactions; i: Pravastatin standard; ii: Pathway i reaction under light conditions; iii: Pathway i reaction under dark conditions; iv: TMe inactivated by boiling as a control; v: P450sca-2 reaction supported by Fdx-FdR-NADPH; B. Optimized Fdx concentration results; C. Optimized CytC6 concentration results; D. Time gradient experimental results of pathway ii reaction at optimal Fdx and CytC6 concentrations; Figure 5Product analysis and reaction optimization for pathway iii; A. NADPH formation analysis; B. UPLC analysis of pathway ii reaction and related control reactions; i: Pravastatin standard; ii: Pathway i reaction under light conditions; iii: Pathway i reaction under dark conditions; iv: TMe inactivated by boiling as a control; v: P450sca-2 reaction supported by Fdx-FdR-NADPH; C. Optimized Fdx concentration results; D. Optimized Cyt C6 concentration results; E. Optimized FdR concentration results; F. Time gradient experimental results of pathway iii reaction at optimal concentrations of Fdx, Cyt C6, and FdR. Detailed Implementation

[0016] To more clearly understand the technical content of this invention, its solution will now be described in detail with reference to the accompanying drawings and specific embodiments. The accompanying drawings show a preferred embodiment of this invention, but its scope of protection is not limited thereto, and can be flexibly adjusted according to specific usage requirements in practical applications. This embodiment aims to help those skilled in the art to fully and deeply understand the inventive concept of this solution.

[0017] 1. Extraction and concentration detection of thylakoid membranes: Cyanobacteria strains Synechococcus elongatus PCC 7942 was obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. This strain was inoculated in BG-11 medium and cultured under visible light at 25°C. When OD... 730 When the concentration reaches 0.6-0.8, cells are collected for subsequent extraction of thylakoid membrane (TMe).

[0018] Collect 1 liter of cultured cyanobacterial cells, wash twice with BG11 medium, and then resuspend in 15 mL of buffer A (25 mmol / L MES-NaOH, pH 6.5, 10 mmol / L CaCl2, 10 mmol / L MgCl2, 25% glycerol). Add isoweighted glass beads (221–300 μm) and vortex at maximum speed for 20 seconds, followed by cooling for 1 minute. Repeat the cycle 5 times to disrupt the cell wall. Incubate the suspension at 4°C and 3,000 mL. g Centrifuge for 2 minutes to collect the supernatant. Then, add 20 mL of buffer A, shake twice, and centrifuge again to collect the supernatant. Combine the two supernatants and centrifuge to remove intact cells and glass beads. Afterward, incubate the supernatant at 4°C and 180,000 mL. g Centrifuge for 45 minutes to collect the precipitate. The precipitate containing the thylakoid membrane is then resuspended in buffer A for concentration and enzymatic assays.

[0019] The chlorophyll (Chl) content in the thylakoid membrane suspension was measured as follows: 13.5 μL of suspension was mixed with 1.5 μL of 10% n-dodecyl β-D maltodextrin, and then... g Centrifuge for 5 minutes, then transfer 10 μL of supernatant to 990 μL of methanol. After vortexing, the mixture is discharged at 16,000 mL / min. g Centrifuge for 5 minutes and collect the supernatant. Detect the absorption spectrum at 350–750 nm, and then calculate the concentration using the following formula: C TMe (μg Chl / mL) = (12.25 × A663–2.79 × A645) × dilution factor.

[0020] 2. Expression and purification of Escherichia coli proteins Recombinant *E. coli* BL21(DE3) strain for P450sca-2, Fdx, and FdR protein expression was cultured in 10 mL of LB medium containing a specific antibiotic at 37°C and 220 rpm for approximately 12 hours to obtain seed culture. Then, 5 mL aliquots of the seed culture were inoculated into 500 mL of LB medium and further cultured at 37°C and 220 rpm for 4–6 hours. When OD... 600 When the protein concentration reached 0.6-0.8, 0.2 mmol / L IPTG (final concentration) was added to induce protein expression; for P450sca-2, 0.25 mmol / L 5-ALA (final concentration) was further added as a precursor for heme biosynthesis. The mixture was then cultured at 16°C and 160 rpm for 16-20 hours, followed by incubation at 4°C and 6,000 rpm. g Centrifuge for 10 minutes under the specified conditions to harvest cells.

[0021] Protein purification of P450sca-2, Fdx, and FdR involved resuspending collected cells in lysis buffer (50 mmol / L NaH2PO4, 300 mmol / L NaCl, 10 mmol / L imidazole, 10% glycerol, pH 8.0) and sonicating on ice. The lysis buffer was centrifuged at 4°C. 10,000 gThe reaction was carried out for 1 hour, then the supernatant was collected, Ni-NTA was added, and the mixture was incubated at 4°C for 1 hour to bind the His6-tagged protein. Next, the mixture was transferred to a gravity flow column and washed with wash buffer (50 mmol / L NaH₂PO₄, 300 mmol / L NaCl, 20 mmol / L imidazole, 10% glycerol, pH 8.0) to remove most nonspecific proteins. Then, the target protein was eluted with 5 mL of elution buffer (50 mmol / L NaH₂PO₄, 300 mmol / L NaCl, 250 mmol / L imidazole, 10% glycerol, pH 8.0). The collected protein solution was concentrated using an ultrafiltration tube of appropriate molecular weight, and then desalted using a PD-10 column and desalting buffer (50 mmol / L NaH₂PO₄, 10% glycerol, pH 7.4). The collected protein solution was further concentrated using ultrafiltration to obtain the desired protein, and the protein was analyzed by SDS-PAGE. Figure 1 As shown.

[0022] The concentration of cytochrome P450 is calculated using the following formula: C P450sca2 (μmol / L) = (ΔA450–ΔA490) / 91000 × 10 6 ×Dilution factor; The formulas used for Fdx and FdR are as follows: C Fdx (μmol / L) = A420 / 9700 × 10 6 ×Dilution factor; C FdR (μmol / L) = A456 / 10400 × 10 6 × Dilution factor.

[0023] As for Cyt C6, its encoding gene is from S. elongatus The gene fragment cloned from the genomic DNA of PCC 7942 was cloned into the pET-28b expression vector and, after sequence verification by DNA sequencing, transferred into *E. coli* BL21(DE3). Since the Cyt C6 protein is naturally secreted into the periplasmic space guided by its N-terminal sequence, the Cyt C6 protein present in the periplasmic space was extracted. Briefly, the cell pellet was suspended in hypertonic buffer (50 mmol / L Tris-HCl, 5 mmol / L MgSO4, 18% sucrose, 0.1 mmol / L EDTA, pH 8.0) and thoroughly mixed with a magnetic stirrer at 4°C for 10 minutes. The mixture was then incubated at 4°C and 16,000 mL. g Centrifuge for 15 minutes and collect the periplasmic protein supernatant containing Cyt C6. The concentration detection formula is as follows: C Cyt C6 (μmol / L) = A553 / 26200 × 10 6 × Dilution factor.

[0024] 3. Construction of the reaction system All reactions were performed in 500 μL quartz tubes using Rishang Lighting YZ08-75 fluorescent tubes equipped with two 8-watt light sources. The reaction buffer contained 1 mmol / L EDTA, 50 mmol / L NaH₂PO₄, 0.5 mmol / L dithiothreitol (DTT), and 10% glycerol (pH 7.4). All reactions were performed at 25°C in 100 μL reaction volumes, with boiling used as a negative control to inactivate the enzyme. Each reaction was divided into two parts, reacting for 12 hours under light and dark conditions, respectively. Subsequently, the mixture was centrifuged at high speed (12,000 rpm) after mixing with 200 μL of methanol. g The supernatant was subjected to a 10-minute, 16°C process to remove denatured proteins. The supernatant was analyzed using high-performance liquid chromatography (HPLC) or UPLC. Substrate and product were quantified using standards. The product yield was calculated using the following formula: Yield (%) = (mol of product / mol of expected product) × 100%.

[0025] 4. Design of the P450 External Optical Drive System Sunlight is an abundant and renewable energy source that drives enzymatic reactions in nature. For example, widely distributed cyanobacteria can capture sunlight and perform photosynthesis through their photosynthetic systems. In cyanobacterial cells, light-driven reactions occur within the thylakoid membrane (TMe), converting light energy into chemical energy in the forms of NADPH and ATP. Figure 2 As shown, in this process, H2O, acting as the primary electron donor, is oxidized by photosystem II (PSII) to produce oxygen, protons, and electrons. The generated electrons are then transferred to photosystem I (PSI) via the polyaminoquinone (PQ) pool, cytochrome b6f (Cyt b6f), and cytocyanin (PC, a cascade of cytochrome C6 (Cyt C6) in some cyanobacteria). Subsequently, the single-electron carrier Fdx (which can also act as a redox partner for type I P450s) transfers electrons from PSI to FdR, carrying NADP... + It is reduced to NADPH, which further promotes the production of ATP.

[0026] Interestingly, both the photosynthetic pathway and the P450 catalytic system utilize Fdx and FdR to transfer electrons, albeit in opposite directions. Therefore, in principle, shared Fdx can be utilized to transfer electrons generated during photosynthesis from the electron transport chain to a specific P450, achieving a "light-driven P450 reaction," thus utilizing light as an energy source and H2O as an electron donor. Previously, several attempts in this invention have dabbled in driving the P450 reaction by expressing exogenous P450 in vivo onto cyanobacteria TMe, or by binding extracted PSI to P450s in vitro, along with 2,6-dichlorophenolindophenol (DCPIP) and sodium ascorbate (Asc), as an artificial electron source. However, due to the complexity of the in vivo reaction environment, it remains questionable whether the introduced P450 enzyme specifically utilizes electrons provided by the photosystem or electrons transferred from intracellular redox-associated proteins (or both). Furthermore, there is currently a lack of evidence to suggest whether the entire TMe can support the P450 reaction in vitro using water as an electron source. This invention attempts to utilize... S. elongatus TME extracted from cyanobacteria by PCC 7942 drives the P450sca-2-catalyzed reaction of mevastatin to pravastatin.

[0027] like Figure 2 As shown, the TMe-related photosynthetic electron transport chain includes PSI, PSII, PQ, and Cyt. b6f PC / CytC6 and Fdx. The membrane-bound PSI, PSII, and Cyt b6f, as well as the PQ cell, should be tightly immobilized within the TMe, while PC / Cyt C6 and Fdx participate in the electron transport chain through dynamic interactions. These components may be lost during TMe preparation. Therefore, this invention provides exogenous Cyt C6, Fdx, and FdR during the construction of the reaction system to reconstruct the entire photosynthetic pathway in vitro.

[0028] 5. Photoelectric drive P450sca-2 reaction using H2O as an electron donor To achieve the photocatalytic reaction from pravastatin to P450sca-2, this invention initially uses only TMe to provide electrons to P450sca-2. In this pathway, PSII in TMe first photolyzes H2O to generate electrons, which are then sequentially transferred to Fdx and transported to P450sca-2 via protein-protein interactions. Figure 2(Path i). In the experiment, 100 μmol / L methylphenidate was incubated with 1 μmol / L P450sca-2, 10 μmol / L Fdx, 10 μmol / L Cyt C6, and 20 μg chlorophyll equivalent TMe. Additionally, 50 mg / mL catalase was added to the reaction mixture to avoid the influence of reactive oxygen species (ROS) on P450. The reaction was incubated at 25°C for 12 hours under illumination. The negative control was simultaneously inactivated by boiling or incubated in the dark. For the positive control, this invention uses... S. elongatus PCC 7942-derived Fdx1499 and FdR0978 act as redox partners for P450sac-2, driving the P450sca-2 reaction using NADPH as an electron source.

[0029] like Figure 3 As shown, this invention continuously optimizes the optimal parameters for each component in the system. Results show that the product yield stabilizes after 8 hours and reaches its maximum value (12.7 ± 0.2%) after 12 hours. These results collectively confirm the feasibility of using TMe as an electron donor to support the P450 reaction, and that the catalytic efficiency of the light-driven system can be improved by adjusting the cofactor concentration.

[0030] 6. Improve product yield by adding artificial electron donors. A mixture of DCPIP and Asc can serve as a redox medium and electron donor for PSI. Therefore, to further improve the catalytic efficiency of the photodriven P450sca-2 reaction, this invention incorporates a DCPIP / Asc solution into the reaction of P450sca-2 with mevastatin. In this alternative system, in addition to the electrons generated by PSII, PSI (under light conditions) can also transport exogenous electrons from DCPIP / ASC to PC / Cyt C6 (see...). Figure 2 (Pathway ii). As expected, the DCPIP / Asc reaction under light conditions resulted in higher pravastatin yields than in path i.

[0031] like Figure 4As shown, this invention attempts to optimize the concentrations of Fdx and Cyt C6 in the system. Concentration gradient experiments indicate that the optimal concentration of Fdx is 30 μmol / L, consistent with the concentration in pathway i. However, experiments involving the Cyt C6 concentration gradient show that the product yield decreases significantly when the Cyt C6 concentration increases from 0 to 10 μmol / L. These findings suggest that the addition of Cyt C6 consumes DCPIP / ASC in a protein concentration-dependent manner, and also imply that the naturally bound PC / Cyt C6 in TMe should be sufficient to promote electron transfer from DCPIP / Asc to PSI. Finally, time-cycle experiments with optimized protein concentrations show that the pravastatin yield reaches 19.2 ± 0.4% after 4 hours and then remains stable. These findings highlight that the catalytic efficiency of the light-driven P450 reaction can be improved by supplementing with a low-cost artificial electron donor.

[0032] 7. Improve catalytic efficiency through in-situ NADPH production Although the catalytic efficiency of the TMe-supported P450sca-2 reaction was improved by optimizing the reaction system and adding an artificial electron donor, the substrate conversion was still significantly lower than that of the redox chaperone system that could completely convert mevastatin to pravastatin. This invention attributes the low catalytic efficiency to the imperfect coupling efficiency between PSI-anchored Fdx and P450sca-2 through protein-protein interactions. To address this issue, this invention proposes constructing a complete NADPH photosynthetic pathway as an electron donor to provide additional electrons and enhance the catalytic activity of P450sca-2 (see...). Figure 2 (Pathway iii). In this pathway, some electrons transferred by Fdx are directed to P450sca-2, while the remainder are transferred to FdR to generate NADPH. The in-situ generated NADPH can serve as an auxiliary electron donor for P450sca-2, thanks to the electron transport bridge composed of FdR and Fdx.

[0033] like Figure 5 As shown, to verify this hypothesis, the present invention first uses FdR and NADP... + The photosynthetic reaction was incorporated, and NADPH production in TMe was measured. The results showed that the photosynthetic system did indeed produce sufficient NADPH. However, the control reaction, incubated in the dark, also produced a small amount of NADPH. Previous reports have indicated that the oxidized pentose phosphate pathway (OPPP) in cyanobacteria can degrade glycogen and generate NADPH under dark conditions. Therefore, the extracted TMe fraction should contain OPPP enzymes and cofactors, which can generate NADPH under dark conditions. Subsequently, this invention further incorporates 1 μmol / L LFdR and 3 mmol / L NADP into the pathway i reaction. +(No artificial electron source). As expected, the yield of pravastatin increased compared to pathways i and ii. Furthermore, trace amounts of pravastatin were detected in the control reaction cultured under dark conditions, which could be reasonably attributed to the NADPH-supported P450sac-2 reaction product generated by OPPP.

[0034] This invention aimed to optimize the protein concentrations of Fdx, Cyt C6, and FdR, respectively. Results showed that, under illumination, the optimal concentrations of Fdx, Cyt C6, and FdR were 30, 10, and 0.5 μmol / L, respectively. Subsequently, this invention utilized the optimized protein concentrations to accelerate the reaction in a time-course experiment. Results showed that pravastatin production plateaued after 4 hours, with a maximum yield of 70.1 ± 0.6%, indicating that the production and cycling of NADPH significantly improved catalytic efficiency. This improvement is attributed to two factors: a) NADPH is a natural product of the TMe photosynthetic system and may accelerate the overall reactivity of the photosynthetic system; b) NADPH also acts as a natural electron donor for P450sca-2, thereby providing a more favorable electron supply chain for P450sca-2 via FdR-Fdx.

[0035] In summary, this invention achieves a photo-driven P450 reaction by in vitro integration of the cyanobacterium TMe with P450sca-2, converting mevastatin into pravastatin. This invention strategically designs three pathways to improve the efficiency of the photo-driven P450 reaction, significantly increasing the product yield from 12.7% to 70.1%. The photo-driven P450 reaction bypasses the need for expensive cofactor NADPH and complex redox partners, resulting in benefits such as cost reduction and CO2 emission mitigation. This invention anticipates that TMe, as a general electron donor in the P450 reaction, will open new avenues for the invention and application of P450.

Claims

1. A light-driven P450 catalytic system, characterized in that, include: Thylakoid membrane, Fdx, P450sca-2, PC / CytC6; Under light conditions, the thylakoid membrane uses H2O as an electron donor to generate electrons through photosystem II. These electrons are then transferred to Fdx and transported to P450sca-2 through protein-protein interactions to catalyze the enzymatic reaction.

2. The light-driven P450 catalytic system according to claim 1, characterized in that, In the system, the Fdx concentration is 20-50 μmol / L.

3. The light-driven P450 catalytic system according to claim 1, characterized in that, In the system, the concentration of Cyt C6 is 10 μmol / L.

4. The light-driven P450 catalytic system according to claim 1, characterized in that, The system also contains 2,6-dichlorophenolindophenol and sodium ascorbate, which act as redox mediators and electron donors to generate electrons through photosystem I.

5. The light-driven P450 catalytic system according to claim 1, characterized in that, The system also includes FdR and NADP. + The FdR accepts the transferred electrons to generate NADPH; the generated NADPH serves as an auxiliary electron donor for P450sca-2.

6. The light-driven P450 catalytic system according to claim 1, characterized in that, The system also contains catalase.

7. The use of the light-driven P450 catalytic system according to any one of claims 1-6 in the synthesis of pravastatin.

8. A method for synthesizing pravastatin, characterized in that: The photo-driven P450 catalytic system according to any one of claims 1-6 is used to catalyze the conversion of mevastatin to pravastatin.

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