Application of fungal cytochrome P450 enzyme

Abstract

The invention discloses application of fungal cytochrome P450 enzyme, and belongs to the technical field of biological enzyme catalysis. The fungal cytochrome P450 enzyme is derived from Gibberella fujikuroi, and the amino acid of the fungal cytochrome P450 enzyme is as shown in SEQ ID NO. 1 or has at least 80% similarity with the sequence as shown in SEQ ID NO. 1; the fungal cytochrome P450 enzyme at least can catalyze 7beta hydroxylation of five steroids L1, L2, L3, L4 and L5, and has excellent selectivity and high yield, and corresponding hydroxylation products can directly or indirectly generate ursodesoxycholic acid. The invention provides a good prospect for synthesizing ursodesoxycholic acid more environmentally and efficiently by using a biological enzyme method.

Description

Technical Field

[0001] This invention belongs to the field of bio-enzyme catalysis technology, specifically relating to the application of a fungal cytochrome P450 enzyme. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid with diverse biological activities. It increases bile secretion and is an important clinical drug for treating hepatobiliary diseases. In 1997, the US FDA approved UDCA as the only first-line drug for treating primary biliary cholangitis (PBC), and it remained the only drug used to treat PBC until the advent of obeticholic acid. Recent studies have found that UDCA has significant efficacy in cancer treatment. It is closely related to various cancer-inducing factors, mediating and influencing the regulation of oncogenic signaling pathways in oncogenes, such as controlling the cell cycle. Furthermore, UDCA can specifically regulate the apoptosis threshold, inhibit cancer cell growth, and induce autophagy and apoptosis. Moreover, UDCA can improve damaged mitochondrial function, exerting a neuroprotective effect and playing an important role in the treatment of neurological diseases.

[0003] Currently, the industrial synthetic route for producing UDCA using chenodeoxycholic acid (CDCA) as a raw material is relatively mature. The prominent problems of the chemical route using CDCA as a raw material are high raw material costs and significant pollution. In contrast, the bio-enzymatic catalytic route using CDAC as a raw material mainly includes… 7α- HSDH and 7 β- The two key enzymes, HSDH and 7α-HSDH, are reacted together in a reactor using a "one-pot, two-step" method, with the substrate and cofactors added together. Currently, this method achieves a space-time yield of 942 g / L / d. However, the high cost of NADPH hinders its practical application. Recent studies have shown that NADPH can be replaced with the more economical NADH, and protein engineering has significantly increased the yield of Rt7. β The specific activity and half-life of HSDH increased its space-time yield to 192 g / L / d, and its production cost was reduced by 22% compared to when NADPH was used as a cofactor, but the cost of CDCA is still relatively high.

[0004] Lithocholic acid (LCA) is an attractive bile acid steroid that uses LCA as a substrate to catalyze the degradation of its C7 molecule via enzymatic processes. βHydroxylation can generate UDCA. Existing research indicates that steroid hydroxylases in filamentous fungi belong to the cytochrome P450 (CYP) enzyme family. Fungal cytochrome P450 enzymes are primarily anchored on the endoplasmic reticulum and inner mitochondrial membrane; therefore, fungal cytochrome P450 enzymes are typically heterologously expressed in yeast and Aspergillus. In 2013, Kollerov et al. conducted a systematic screening of filamentous fungi and found that some filamentous fungi can hydroxylate the inert C atoms of bile acid steroid nuclei. Gibberella zeae The VKM F-2600 strain exhibited high catalytic activity against LCA, achieving a conversion rate of 90% at a substrate concentration of 1 g / L. Subsequently, Kollerov subjected this strain to UV mutagenesis to obtain a mutant strain. G.zeae M23, with a substrate concentration of 4 g / L, achieved a conversion rate of 88%; however, this study did not clarify the fungal cytochrome P450 enzyme involved. Furthermore, Uwe T. Bornscheuer discovered that CYP107D1 (Olep) derived from *Streptomyces* can achieve C6 LCA. β Selective hydroxylation produces mouse deoxycholic acid (MDCA). In 2021, they obtained a triple mutant CYP107D1-M3 (CYP107D1-F84Q / S204A / V291G) through protein engineering, achieving C7 hydroxylation of LCA. β Selective hydroxylation yielded UDCA, but only 67 μmol of UDCA was produced with excellent selectivity. In summary, the production of UDCA using LCA as a substrate via enzymatic methods has two shortcomings: firstly, the key enzyme characterization is unclear, and secondly, the characterized enzymes have low activity and poor selectivity. Summary of the Invention

[0005] In view of this, the present invention aims to explore novel fungal cytochrome P450 enzymes to achieve the goal of green and efficient synthesis of UDCA using steroidal compounds, including LCA, as substrates through a bio-enzymatic method, thereby accelerating the domestic production capacity of this drug.

[0006] The specific technical solution of the present invention is as follows: A fungal cytochrome P450 enzyme catalyzes steroid compound 7 β Applications in hydroxylation, wherein the amino acid sequence of the fungal cytochrome P450 enzyme is as shown in SEQ ID NO.1 or has at least 80% sequence similarity to the sequence shown in SEQ ID NO.1.

[0007] Preferably, in the above applications, the amino acid sequence of the fungal cytochrome P450 enzyme specifically falls into the following two categories: As shown in SEQ ID NO.1; Alternatively, a protein tag sequence may be added to the N-terminus or C-terminus shown in SEQ ID NO.1; it is understood that the protein tag sequence does not alter the protein's catalytic activity against steroid compound 7. β Hydroxylation is used specifically to achieve protein expression, purification, detection, and tracing.

[0008] The fungal cytochrome P450 enzyme provided by this invention is mined from Fumigranobacter fusiforme (… Gibberella fujikuroi Therefore, it is recorded as fungal cytochrome P450 enzyme P450gf.

[0009] Preferably, in the above applications, the steroidal compounds are selected from L1-L5, and the structural formulas of each compound are shown below: .

[0010] The experimental data of this invention show that the fungal cytochrome P450 enzyme P450gf can catalyze the direct synthesis of UDCA from L1 (i.e., LCA), exhibiting excellent conversion rate and selectivity; when L2, L3, L4, and L5 are used as substrates, respectively, the fungal cytochrome P450 enzyme P450gf catalyzes the production of 7 β The hydroxylated products can serve as precursors for the synthesis of UDCA, enriching the bioenzymatic synthesis routes for UDCA.

[0011] Preferably, in the above applications, recombinant strains heterologously expressing fungal cytochrome P450 enzyme P450gf are used for the extraction of steroidal compounds. β Hydroxylation reaction. The specific procedure can be as follows: culturing the recombinant strain to induce expression, collecting the bacterial cells, and constructing a reaction system containing the bacterial cells and steroidal compounds for the reaction.

[0012] More preferably, in the above applications, the method for preparing the recombinant strain includes the following steps: S1. Construct a recombinant plasmid containing the gene encoding the fungal cytochrome P450 enzyme P450gf; S2. Transform the recombinant plasmid into the host cell to obtain the recombinant strain.

[0013] In the application of this invention, the nucleotide sequence of the gene encoding the fungal cytochrome P450 enzyme P450gf can be optimized and adjusted according to actual needs, and it is preferably as shown in SEQ ID NO.2 or has more than 80% sequence similarity with the sequence shown in SEQ ID NO.2.

[0014] In this invention, the expression vector for the recombinant plasmid includes, but is not limited to, pPICZA. The encoding gene can be synthesized into the vector using conventional techniques to obtain the recombinant plasmid. For example, in some embodiments of this invention, the encoding gene is directly synthesized into the pPICZA vector by a gene synthesis company to obtain the recombinant plasmid pPICZA-P450gf. Furthermore, a unique PmeI restriction site can be retained on the AOX promoter in the recombinant plasmid pPICZA-P450gf for subsequent construction of recombinant strains.

[0015] In the application of this invention, the host cell includes, but is not limited to, Pichia pastoris; however, experimental data indicates that Pichia pastoris is preferred. In some embodiments of this invention, the recombinant plasmid pPICZA-P450gf was transformed into Pichia pastoris X33 competent cells by electroporation, and the recombinant strain X33-pPICZA-P450gf was obtained through screening. It should be noted that the recombinant plasmid pPICZA-P450gf does not exist freely in the recombinant strain.

[0016] The beneficial effects of this invention are as follows: This invention, through in-depth mining of fungal cytochrome P450 enzymes in the Uniprot database, followed by phylogenetic tree analysis and cluster analysis for screening, and confirmation of the protein structure of candidate enzymes through AlphaFold2 homology modeling, successfully identified enzymes derived from *Fujikura scabica*. Gibberella fujikuroi The fungal cytochrome P450 enzyme P450gf exhibits excellent selectivity and activity for the 7β-hydroxylation of steroidal compounds L1, L2, L3, L4, and L5, and can be used for the direct or indirect synthesis of UDCA, enriching the bioenzymatic synthesis routes of UDCA. Attached Figure Description

[0017] Figure 1 The steroid compounds L1-L5 and their corresponding 7 in this invention β Schematic diagram of the structure of the hydroxylated product; Figure 2 These are two route diagrams for the synthesis of ursodeoxycholic acid in this invention; Figure 3 The HPLC-ELSD chromatogram of the product obtained by recombinant strain X33-pPICZA-P450gf catalyzing L1 in Example 2 is shown. Figure 4 The image shows the HPLC-ELSD chromatogram of the product obtained by L2 catalyzed by the recombinant strain X33-pPICZA-P450gf in Example 3. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The term "comprising" and any variations thereof in the specification and claims of this invention are intended to cover a non-exclusive inclusion.

[0019] In this invention, sequence similarity refers to the comparison between two amino acid sequences or nucleotide sequences. It is confirmed by using the best alignment sequence within a comparison window. Compared to the reference sequence, the comparison sequence may have additions or deletions to achieve optimal alignment between the two sequences. The similarity percentage is calculated by counting the number of identical amino acid or nucleotide positions in the two sequences within the comparison window, dividing this number by the total number of positions in the comparison window to obtain the sequence similarity percentage. The sequence length of the comparison window is at least 20.

[0020] Currently, it can catalyze steroid compound 7 β Cytochrome P450 enzymes hydroxylated to ursodeoxycholic acid are rarely reported, and a common problem with reported cytochrome P450 enzymes is poor selectivity and / or low activity. Patent application 202410719504.2 describes the synthesis of cytochrome P450 enzymes from Fusarium solani. FL The modification yielded mutants with significantly improved selectivity and yield. However, it should be noted that the optimal mutant only exhibited superior conversion rates for LCA; the conversion rates for other steroidal compounds still require further improvement. In the long term, exploring this type of mutant with 7... β The discovery of novel wild-type cytochrome P450 enzymes with hydroxylation function, and their further structural modification, are of significant practical importance for achieving green and efficient synthesis of UDCA.

[0021] The inventors have long been engaged in the discovery of novel fungal cytochrome P450 enzymes. Based on a review of all reported fungal cytochrome P450 enzymes catalyzing steroid compounds, they conducted in-depth mining of fungal cytochrome P450 enzymes in the Uniprot database by combining sequence similarity and species information. Further screening was performed using phylogenetic tree analysis and cluster analysis, and the protein structures of candidate enzymes were confirmed using AlphaFold2 homology modeling. Ultimately, enzymes originating from *Fujikura scabica* were discovered. Gibberella fujikuroi The fungal cytochrome P450 enzyme P450gf is described. Experimental data from this invention show that the fungal cytochrome P450 enzyme P450gf can catalyze the formation of corresponding 7-hydroxyl groups from steroidal compounds L1, L2, L3, L4, and L5. β Hydroxylated products ( Figure 1 It is selective and highly active.

[0022] It is understandable that the fungal cytochrome P450 enzyme P450gf can effectively catalyze the formation of steroid compounds L1, L2, L3, L4, and L5 into their corresponding 7-hydroxyl groups. β The detection results of the hydroxylation products show that the present invention can be used for the efficient synthesis of UDCA, and specifically includes the following two routes ( Figure 2 ): Using L1 as a substrate, fungal cytochrome P450 enzyme P450gf catalyzes the direct synthesis of UDCA; or, using L2, L3, L4 and L5 as substrates, fungal cytochrome P450 enzyme P450gf catalyzes the generation of precursor materials, which are then subjected to chemical and / or biological reactions to obtain UDCA.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be understood that the embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] Unless otherwise specified, the techniques or conditions used in the following examples were performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions; and all reagents used in the following examples are commercially available.

[0025] Example 1 Based on the discovery of Fumigranobacter fumonis (based on this invention) Gibberella fujikuroi The fungal cytochrome P450 enzyme P450gf (amino acid sequence as shown in SEQ ID NO.1, Uniprot accession number SOE5D6) is described in this example. A recombinant bacterial cell heterologously expressing this enzyme is provided, and its preparation process includes the following steps: The P450gf sequence of Pichia pastoris with optimized codons (as shown in SEQ ID NO.2) was artificially synthesized (Jinkairui Biotechnology Co., Ltd.), and then ligated into the pPICZA plasmid to obtain the recombinant plasmid pPICZA-P450gf.

[0026] The recombinant plasmid pPICZA-P450gf was linearized using the restriction endonuclease PmeI, digested overnight at 37°C, and the linearized product was recovered after detection by 1% agarose gel electrophoresis. After recovery, 1-5 μg of the linearized recombinant plasmid fragment was added to competent Pichia pastoris cells and incubated on ice for 5 min. The competent cell mixture was then transferred to an electroporation cuvette and electroporated (1.5 kV). Immediately after electroporation, 200 μL of a premixed solution of YPD and 200 μL of 1M sorbitol was added to the cuvette, mixed well, and incubated at 30°C and 220 rpm for 2 h. After incubation, an appropriate amount of culture medium was plated onto YPD plates containing bleomycin resistance (100 μg / mL) (YPD+Z). The plates were incubated upside down in a 30°C incubator for 2-3 days, and positive transformants of the recombinant strain were obtained through resistance screening.

[0027] The positive transformants of the recombinant strain were confirmed by colony PCR verification of Pichia pastoris, and the specific procedures are as follows: After the colonies grew on the YPD+Z plates, a single colony was picked and inoculated into 2 mL of YPD+Z culture medium and cultured overnight at 30°C and 220 rpm. The genome of the transformant was then extracted and used as a template for PCR verification. Genome extraction method: Add 100 μL of bacterial culture to a 1.5 mL EP tube, centrifuge at 12000 rpm for 2 min, and discard the supernatant; add 100 μL of 0.2 M LiAc solution containing 1% SDS, heat in a metal bath at 75℃ for 10 min; add 300 μL of 100% ethanol and vortex thoroughly, centrifuge at 12000 rpm for 3 min, and discard the supernatant; add 150 μL of 70% ethanol, centrifuge at 12000 rpm for 3 min, and discard the supernatant; open the cap of the EP tube and place it at 37℃ until the remaining ethanol is dried; add 50 μL of sterile water and vortex for 1-2 min, centrifuge at 12000 rpm for 3 min, and collect the supernatant. The Pichia pastoris genome is present in the supernatant, which can be used as a template for colony PCR.

[0028] PCR amplification system (10 μL): 1 μL of *Pichia pastoris* single colony genome, 0.5 μL each of primers p450gf-F and p450gf-R, 5 μL of 2× Rapid Taq Master Mix DNA polymerase, and finally, deionized water to bring the total to 10 μL. PCR amplification program: 95℃ pre-denaturation, 3 min; 95℃ denaturation, 15 sec; 56℃ annealing, 15 sec; 72℃ extension, 30 sec; 30 cycles; 72℃ extension, 5 min; 12℃ incubation, 10 min. The nucleotide sequences of the primers are shown in Table 1. The PCR products were detected by 1% agarose gel electrophoresis. The *Pichia pastoris* single colony represented by the brighter lane, where the product band size matches the target band size, is the target recombinant strain X33-pPICZA-P450gf.

[0029] Table 1 Primer sequences for Pichia pastoris single colony genome PCR

[0030] Example 2 Using the recombinant strain X33-pPICZA-P450gf obtained in Example 1, this example uses L1 (LCA) as the substrate for 7... β The hydroxylation reaction specifically includes the following steps: The recombinant strain X33-pPICZA-P450gf was inoculated into a 100 mL Erlenmeyer flask containing 20 mL of BMGY medium and cultured at 30℃ and 220 rpm for 2 days on a shaker. The bacterial culture was then collected in a sterile centrifuge tube under a laminar flow hood, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected. 10 mL of BMGY medium was added, and the culture was induced at 25℃ and 220 rpm. Every 24 h, 1% methanol (100 μL) was added to 10 mL of the bacterial culture for continuous induction.

[0031] After induction for 48 h, the bacterial culture was collected in sterile centrifuge tubes in a clean bench, centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the bacterial cells were collected. The bacterial cells were resuspended in 5 mL of 100 mM phosphate buffer (pH 7.0), and 50 μL of 100 mM lithocholic acid solution (steroid compound L1) dissolved in DMSO was added to the resuspended bacterial culture. The reaction system was incubated at 30 °C and 220 rpm for 48 h.

[0032] After the reaction was complete, the reaction solution was thoroughly mixed. 900 μL of the reaction solution was taken, and 100 μL of HCl of a certain concentration was added. The mixture was extracted twice with an equal volume of ethyl acetate. The extracts were combined and air-dried in a fume hood to remove the ethyl acetate. Then, 900 μL of methanol was added and thoroughly mixed. The sample was filtered through a 0.22 μm organic filter membrane.

[0033] Example 3 The only difference from Example 2 is that this example uses L2 as the substrate for 7... β Hydroxylation reaction.

[0034] Example 4 The only difference from Example 2 is that this example uses L3 as the substrate for 7... β Hydroxylation reaction.

[0035] Example 5 The only difference from Example 2 is that this example uses L4 as the substrate for 7... β Hydroxylation reaction.

[0036] Example 6 The only difference from Example 2 is that this example uses L5 as the substrate for 7... β Hydroxylation reaction.

[0037] HPLC-ELSD analysis was performed on the 7 catalyzed steroid compounds L1, L2, L3, L4, and L5 by the recombinant strain X33-pPICZA-P450gf. β Quantitative analysis of the hydroxylated products was performed. Figure 3 A is the HPLC-ELSD chromatogram of the product obtained by recombinant strain X33-pPICZA-P450gf catalyzing L1 in Example 2. Figure 3 B is the HPLC-ELSD chromatogram of the product after the reaction of the control strain (which differs from X33-pPICZA-P450gf only in that the pPICZA plasmid is used instead of the recombinant plasmid pPICZA-P450gf) with L1. Figure 3 C represents the HPLC-ELSD chromatograms of L1 and UDCA standards. Figure 4 A is the HPLC-ELSD chromatogram of the product obtained by recombinant strain X33-pPICZA-P450gf catalyzing L1 in Example 3. Figure 4 B is the HPLC-ELSD chromatogram of the product after the reaction of the control strain with L1.

[0038] The test results show that the fungal cytochrome p450 enzyme P450gf has a 7% activity against steroid compounds L1, L2, L3, L4, and L5. β Hydroxylation activity was assessed, with lithocholic acid (L1) exhibiting the best catalytic selectivity and activity. However, it also showed significant catalytic potential for other key steroids L2, L3, L4, and L5, demonstrating good catalytic selectivity and activity. Specific conversion rates and selectivities are shown in Table 2 (selectivity = 7). β (Hydrated product / total product × 100%).

[0039] Table 2. Conversion rates and selectivity of the five steroidal compounds in Examples 2-6

[0040] In summary, the fungal cytochrome P450 enzyme provided by this invention can catalyze the oxidation of five steroidal compounds, L1, L2, L3, L4, and L5. β Hydroxylation exhibits excellent selectivity and high yield, and the corresponding hydroxylated products can directly or indirectly generate ursodeoxycholic acid. Furthermore, the wild-type fungal cytochrome P450 enzyme of this invention already demonstrates superior conversion rate and high selectivity for LCA. Based on this, through protein engineering modification and Pichia pastoris chassis strain modification, it is expected to accelerate the industrialization of ursodeoxycholic acid synthesis via enzymatic methods.

[0041] It should be noted that the above embodiments are only some embodiments of the present invention and not all embodiments, and are only used to illustrate the technical solutions of the present invention and not to limit it; based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

Claims

1. Use of a fungal cytochrome P450 enzyme in catalyzing the hydroxylation of a steroidal compound 7 β characterized in that, The amino acid sequence of the fungal cytochrome P450 enzyme is shown in SEQ ID NO. 1 or has at least 80% similarity with the sequence shown in SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The amino acid sequence of the fungal cytochrome P450 enzyme is specifically as shown in SEQ ID NO. 1, or a protein tag sequence is added to the N-terminus or C-terminus of SEQ ID NO.

1.

3. Use according to claim 1, characterized in that, The steroid compound is selected from the following: 。 4. Use according to claim 3, characterized in that, At least one of the steroid compounds L1, L2, L3, L4 and L5 is used as a substrate to directly or indirectly catalyze the synthesis of ursodeoxycholic acid by the fungal cytochrome P450 enzyme.

5. The use according to claim 1, characterized in that, 7 β hydroxylation reactions.

6. Use according to claim 5, characterized in that, Specifically, the recombinant strain is cultured and induced for expression, the bacterial cells are collected, and a reaction system containing the bacterial cells and the steroid compound is constructed for reaction.

7. Use according to claim 5, characterized in that, The preparation method of the recombinant strain comprises the following steps: constructing a recombinant plasmid containing a gene encoding the fungal cytochrome P450 enzyme, transforming the recombinant plasmid into a host cell to obtain a recombinant strain.

8. Use according to claim 7, characterized in that, The host cell is Pichia pastoris.

9. Use according to claim 8, characterized in that, The expression vector used in the recombinant plasmid is pPICZA.

10. Use according to claim 8, characterized in that, The nucleotide sequence of the gene encoding the fungal cytochrome P450 enzyme is shown in SEQ ID NO. 2.

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