Cytochrome p450 enzyme and its application in catalyzing synthesis of anthracycline compounds

Abstract

The application provides a cytochrome P450 enzyme DoxA (CYP129 subfamily), and the amino acid sequence of the cytochrome P450 enzyme DoxA is SEQ ID NO:1. The application also provides a use of the cytochrome P450 enzyme DoxA, which is catalyzing synthesis of 13-dihydrodaunorubicin or 13-dihydrodaunorubicin derivatives from anthracycline compound 13-deoxydaunorubicin; and the method, wherein the cytochrome P450 enzyme DoxA catalyzes 13-deoxydaunorubicin by using NAD(P)H. The application provides a cytochrome P450 enzyme DoxA which can directly use NAD(P)H, can synthesize 13-dihydrodaunorubicin by using NAD(P)H in one step, does not need to use electron transfer protein for assistance, simplifies a reaction system, reduces reaction cost, enhances controllability and stability of the reaction, and the electron transfer process is more direct and efficient, and the catalytic efficiency of the reaction is improved.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering, synthetic biology and biotechnology, and specifically relates to a cytochrome P450 enzyme DoxA (CYP129 subfamily) and its application in the synthesis of anthracycline compounds using NAD(P)H. Background Technology

[0002] P450 enzymes, as versatile biological catalysts, generally require the assistance of electron transport proteins (ferroreductin reductase FdR and ferroreductin Fdx) to transfer electrons from the cofactor NAD(P)H to the heme center, thereby activating molecular oxygen to catalyze the reaction. No P450 protein capable of directly utilizing NAD(P)H has yet been discovered in the prokaryotes.

[0003] The anthracycline compounds 13-deoxydaunorubicin (DOD) and 13-dihydrodaunorubicin (DHD) are important precursors and key intermediates in the synthesis of doxorubicin (DXR) and daunorubicin (DNR), respectively. Currently, research on the biological production process of 13-dihydrodaunorubicin mainly focuses on the biotransformation by Streptomyces, and there are no reports of enzymatic catalysis of the conversion of 13-deoxydaunorubicin to 13-dihydrodaunorubicin. Summary of the Invention

[0004] This invention provides a class of cytochrome P450 enzymes, DoxA (CYP129 subfamily), which directly utilizes NAD(P)H to acquire and transfer electrons, catalyzing the synthesis of 13-dihydrodaunorubicin from 13-deoxydaunorubicin, thus providing a new pathway for the synthesis of doxorubicin.

[0005] This invention discovers that the cytochrome P450 enzyme DoxA can directly utilize NAD(P)H to catalyze the oxidation of 13-deoxydaunorubicin to 13-dihydrodaunorubicin.

[0006] This invention first provides a P450 enzyme DoxA derived from Streptomyces coeruleorubidus, comprising:

[0007] 1) A protease with the amino acid sequence SEQ ID NO:1,

[0008] VAVDPFACPMMTMQRKPEVHDAFREAGPVVEVNAPAGGPAWVITDDALAREVLADPRFVKDPDLAPAAWRGVDDGLDIPVPELRPFTLIAVDGEAHRRLRRIHAPAFNPRRLAERTDRIAAIAGRLLTELADTSGRSGKPAELIGGFAYHFPLLVICELLGVPVTDPAMAREAVSSVLKALGLGGPQSGGGDGTDPAGGVPDTSALESLL LEAVHSARRNDTPTMTRVLYERAQAEFGSVSDDQLVYMITGLIFAGHDTTGSFLGFLLAEVLAGRLAADADEDAVSRFVEEALRYHPPVPYTLWRFAATEVTIGGVRLPRGAPVLVDIEGTNTDGRHHDDPHAFHPDRPSWRRLTFGDGPHYCIGEQLAQLESRTMIGVLRSRFPEARLAVPYDELRWSRKGAQTARLTELPVWLR(SEQ ID NO:1);

[0009] 2) A derived protease having the protease function described in 1) obtained by substituting, deleting, or adding one or more amino groups to a protein with the sequence SEQ ID NO:1.

[0010] The present invention also provides a gene doxA encoding the cytochrome P450 enzyme DoxA, a specific nucleotide sequence of which is as follows:

[0011]

[0012] The present invention also provides a recombinant expression vector carrying the nucleic acid fragment of the above-mentioned coding gene.

[0013] In another aspect, the present invention provides a recombinant engineered strain carrying the above-described recombinant expression vector.

[0014] This invention also provides an application of the aforementioned P450 enzyme DoxA, which is to catalyze the synthesis of 13-deoxydaunorubicin into 13-dihydrodaunorubicin or 13-dihydrodaunorubicin derivatives.

[0015] In another aspect, the present invention provides a method for catalytic synthesis of 13-dihydrodaunorubicin from 13-deoxydaunorubicin, wherein the method uses the aforementioned P450 enzyme DoxA for catalysis.

[0016] Furthermore, in the method described, the 450 enzyme DoxA utilizes NAD(P)H to catalyze 13-deoxydaunorubicin.

[0017] This invention provides a method for producing anthracycline 13-deoxydaunorubicin or 13-dihydrodaunorubicin derivatives or analogs from DoxA protein or its homologous protein using NAD(P)H catalysis.

[0018] Furthermore, in the method described, the concentration of NADPH added to the reaction system is 2-5 mM.

[0019] This invention provides a cytochrome P450 enzyme, DoxA, capable of directly utilizing NAD(P)H to synthesize 13-dihydrodaunorubicin in a one-step process without the aid of electron transport proteins. This simplifies the reaction system, reduces reaction costs, and enhances the controllability and stability of the reaction. The electron transfer process is more direct and efficient, improving the catalytic efficiency of the reaction. The ability of the cytochrome P450 enzyme DoxA to directly utilize NAD(P)H to synthesize the anthracycline compound 13-dihydrodaunorubicin also provides a new synthetic route for the synthesis of the anticancer drugs doxorubicin and daunorubicin. Attached Figure Description

[0020] Figure 1 SDS-PAGE spectrum of P450 enzyme DoxA protein.

[0021] Figure 2 UPLC chromatogram of P450 enzyme DoxA catalyzing the production of 13-dihydrodaunorubicin from 13-deoxydaunorubicin using NADPH.

[0022] Figure 3UPLC detection chromatogram of 13-dihydrodaunorubicin produced by the P450 enzyme DoxA catalyzing the production of 13-dihydrodaunorubicin from 13-deoxydaunorubicin using NADH. Detailed Implementation

[0023] This invention provides a P450 enzyme DoxA derived from Streptomyces coeruleorubidus, comprising:

[0024] 1) A protease with the amino acid sequence SEQ ID NO:1;

[0025] 2) A derived protease having the protease function described in 1) obtained by substituting, deleting, or adding one or more amino groups to a protein with the sequence SEQ ID NO:1.

[0026] The aforementioned derived protease is one that has at least 90% homology with the protease whose amino acid sequence is SEQ ID NO:1, preferably 99% homology (i.e., a difference of approximately 5 amino acids compared to the amino acid sequence of SEQ ID NO:1).

[0027] The present invention also provides a coding gene for the above-mentioned P450 enzyme DoxA, one specific nucleic acid sequence of which is SEQ ID NO:2; however, the coding gene can be optimized according to different hosts.

[0028] The present invention also provides a recombinant expression vector for recombinantly expressing the above-mentioned P450 enzyme DoxA in the host of engineered bacteria. The recombinant expression vector can be any protein recombinant expression vector, such as the pET28b vector.

[0029] In another aspect, the present invention provides a recombinant engineered strain carrying the above-described recombinant expression vector.

[0030] This invention also provides an application of the aforementioned P450 enzyme DoxA, which is to catalyze the synthesis of 13-deoxydaunorubicin into 13-dihydrodaunorubicin or 13-dihydrodaunorubicin derivatives.

[0031] In another aspect, the present invention provides a method for catalytic synthesis of 13-dihydrodaunorubicin from 13-deoxydaunorubicin, wherein the method uses the aforementioned P450 enzyme DoxA for catalysis.

[0032] Furthermore, in the method described, the P450 enzyme DoxA utilizes NAD(P)H to catalyze 13-deoxydaunorubicin.

[0033] This invention provides a method for producing 13-deoxydaunorubicin or 13-dihydrodaunorubicin derivatives or analogs from DoxA protein or its homologous proteins using NAD(P)H catalysis.

[0034] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0035] Example 1: Recombinant expression of P450 DoxA protein

[0036] The doxA gene from *Streptomyces coeruleus* was codon-optimized by BGI Genomics (its amino acid sequence is SEQ ID NO:1, and the nucleotide sequence encoding the gene is SEQ ID NO:2), cloned into the pET28b vector, and transformed into *E. coli* strain BL21(DE3). *E. coli* strains capable of efficiently expressing the DoxA protein were streaked onto LB agar plates (containing 50 μg / mL Kan) and incubated at 37°C for 24 h. Single colonies were picked and cultured overnight at 37°C and 220 rpm in 50 mL LB agar (containing 50 μg / mL Kan). A 1% inoculum was then inoculated into 2L Erlenmeyer flasks containing 500 mL TB or LB agar (containing 50 μg / mL Kan) and incubated at 37°C and 200 rpm until OD (out of control). 600 Between 0.8 and 1, add IPTG at a final concentration of 200 μM, 500 μM of 5-aminolevulinic acid (5-ALA), and 500 μM of vitamin B1 (VB1), and then incubate at 18°C ​​and 150 rpm for 18-20 h to induce protein expression.

[0037] The bacterial cells were collected by centrifugation, and resuspended in 30-40 mL of Lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 10% glycerol, pH 8.0) by vortexing. After sonication to disrupt the cells, the cells were centrifuged at 10,000 rpm for 60 min at 4°C. The supernatant was incubated with Ni-NTA at 4°C for 60 min. Then, contaminating proteins were eluted with Wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, 10% glycerol, pH 8.0); the target protein was eluted with Elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, 10% glycerol, pH 8.0); the protein solution was concentrated using a 50 kDa ultrafiltration tube; and soluble DoxA protein was obtained by SDS-PAGE analysis. Figure 1 ).

[0038] Example 2: Direct catalytic oxidation of 13-deoxydaunorubicin to 13-dihydrodaunorubicin by DoxA

[0039] Using 100 μM 13-deoxydaunorubicin as the substrate, 2 μM DoxA was added. The reaction buffer consisted of 50 mM NaH2PO4, 300 mM NaCl, pH 7.5, with a total volume of 100 μL. Boiled DoxA was used as a control. The reaction was carried out at 30 °C for 2 h. The reaction was terminated by adding 2 times the volume of methanol to the reaction solution. The mixture was centrifuged at 14,000 × g for 10 min, and the supernatant was collected for UPLC analysis. Detection procedure: Mobile phase A is water containing 0.1% formic acid, mobile phase B is acetonitrile containing 0.1% formic acid, elution gradient is 0-0.5 min, 20% B; 0.5-5 min, 20-90% B; 5-5.1 min, 90-100% B; 5.1-5.6 min, 100% B; 5.6-5.7 min, 100-20% B; 5.7-6.5 min, 20% B; flow rate is 0.4 mL / min, detection wavelength is 470 nm.

[0040] The results showed that, without the addition of exogenous cofactors, the target product 13-dihydrodaunorubicin was produced (retention time 3.1 min), with a yield of approximately 12%. Figure 2 ii), indicating that DoxA can utilize its own bound NAD(P)H to achieve the synthesis of 13-dihydrodaunorubicin.

[0041] Example 3: DoxA utilizes 2mM NADPH to catalyze the oxidation of 13-deoxydaunorubicin to 13-dihydrodaunorubicin

[0042] In a system containing 100 μM 13-deoxydaunorubicin and 2 μM DoxA, 2 mM NADPH was added, and the reaction was carried out at 30 °C for 2 h. The reaction was terminated by adding 2 volumes of methanol to the reaction solution. Aqueous solution (0.1% formic acid) was used as mobile phase A, and acetonitrile (0.1% formic acid) was used as mobile phase B. The elution gradient was: 0–0.5 min, 20% B; 0.5–5 min, 20–90% B; 5–5.1 min, 90–100% B; 5.1–5.6 min, 100% B; 5.6–5.7 min, 100–20% B; 5.7–6.5 min, 20% B. The flow rate was 0.4 mL / min, and the detection wavelength was 470 nm.

[0043] The results showed that under conditions containing 2 mM NADPH, the yield of the product 13-dihydrodaunorubicin increased to approximately 64%. Figure 2 iii); further evidence shows that the P450 enzyme DoxA directly utilizes NADPH to catalyze the oxidation of 13-deoxydaunorubicin to 13-dihydrodaunorubicin.

[0044] Example 4: DoxA utilizes 5mM NADPH to catalyze the oxidation of 13-deoxydaunorubicin to 13-dihydrodaunorubicin

[0045] In a system containing 100 μM 13-deoxydaunorubicin and 2 μM DoxA, 5 mM NADPH was added, and the reaction was carried out at 30 °C for 2 h. The reaction was terminated by adding 2 volumes of methanol to the reaction solution. Aqueous solution (0.1% formic acid) was used as mobile phase A, and acetonitrile (0.1% formic acid) was used as mobile phase B. The elution gradient was: 0–0.5 min, 20% B; 0.5–5 min, 20–90% B; 5–5.1 min, 90–100% B; 5.1–5.6 min, 100% B; 5.6–5.7 min, 100–20% B; 5.7–6.5 min, 20% B; the flow rate was 0.4 mL / min, and the detection wavelength was 470 nm. The results confirmed that under the reaction conditions containing 5 mM NADPH, the yield of the product 13-dihydrodaunorubicin increased to approximately 71%. Figure 2 iv).

[0046] Example 5: DoxA utilizes NADH to catalyze the production of 13-dihydrodaunorubicin from 13-deoxydaunorubicin.

[0047] Add 5 mM NADH to a reaction system containing 100 μM 13-deoxydaunorubicin and 2 μM DoxA, react at 30 °C for 2 h, and terminate the reaction by adding twice the volume of methanol and mixing thoroughly. Use water (0.1% formic acid) as mobile phase A and acetonitrile (0.1% formic acid) as mobile phase B. The elution gradient is: 0–0.5 min, 20% B; 0.5–5 min, 20–90% B; 5–5.1 min, 90–100% B; 5.1–5.6 min, 100% B; 5.6–5.7 min, 100–20% B; 5.7–6.5 min, 20% B. The flow rate is 0.4 mL / min, and the detection wavelength is 470 nm. The results show that the yield of 13-dihydrodaunorubicin is approximately 75%. Figure 3 This demonstrates that the P450 enzyme DoxA can directly utilize NADH to catalyze the oxidation of 1,3-deoxydaunorubicin to produce 1,3-dihydrodaunorubicin.

[0048] In summary, this invention provides a class of cytochrome P450 enzymes, DoxA, which can directly utilize NAD(P)H to catalyze the synthesis of 13-dihydrodaunorubicin, an important intermediate in the synthesis of doxorubicin from 13-deoxydaunorubicin. This enzyme catalytic system is simple and efficient, providing a new approach for the industrial production of daunorubicin and doxorubicin; it enriches the natural repertoire of P450 enzymes and provides important materials for the study of P450 enzymatic mechanisms.

Claims

1. A method for catalytic synthesis of 13-dihydrodaunorubicin from 13-deoxydaunorubicin, characterized in that, The method involves catalysis using the P450 enzyme DoxA with the amino acid sequence SEQ ID NO:1; The reaction buffer solution used in the method is 50 mM NaH2PO4, 300 mM NaCl, pH 7.5, and 2-5 mM NADH or NADPH.

2. The method as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the P450 enzyme DoxA is SEQ ID NO:2.

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