Colletotrichum linum P450 enzyme mutant and application thereof

Abstract

The invention discloses a colletotrichum linum P450 enzyme mutant and application thereof, the mutant is obtained by mutating 121st aspartic acid of an amino acid sequence of P450 enzyme CYP68J derived from a colletotrichum linum ST-1 strain into histidine, and the amino acid sequence is shown as SEQ ID NO: 1. Compared with a wild type P450 enzyme, the P450 enzyme mutant disclosed by the invention has the advantages that dehydroepiandrosterone can be efficiently converted into 7 alpha-hydroxy-dehydroepiandrosterone, and the ratio of 7 alpha-hydroxy-dehydroepiandrosterone to 7 alpha, 15 alpha-dihydroxy-dehydroepiandrosterone in a hydroxylation product can be increased from about 4: 5 to about 14: 1; an important way is provided for the biological conversion of the 7alpha-hydroxyl-dehydroepiandrosterone, and the method has important significance on the industrial production of the 7alpha-hydroxyl-dehydroepiandrosterone.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering technology, specifically relating to a P450 enzyme mutant of *Colletotrichum flaxensis* and its application. Background Technology

[0002] Steroid compounds are a collective term for compounds with a cyclopentane-polyhydrophenanthrene core structure. These steroid compounds are used to treat various diseases, including inflammation, tumors, cardiovascular diseases, and endocrine disorders, depending on their specific functions. Steroid drugs constitute the second largest class of drugs in the global pharmaceutical market, after antibiotics. Currently, there are nearly 500 commercially available steroid drugs, among which steroid hormones are a very important category.

[0003] Dehydroepiandrosterone (DHEA, CAS No. 53-43-0) is an intermediate in the production of steroid hormone drugs. DHEA itself also possesses anti-aging and anabolic effects. 7α-hydroxy-dehydroepiandrosterone (DHEA, CAS No. 53-00-9), synthesized by adding a hydroxyl group at the 7-position to DHEA, is also a very important pharmaceutical intermediate and can be used to synthesize 7-keto-dehydroepiandrosterone, 7β-hydroxy-dehydroepiandrosterone, etc. Studies have shown that 7α-hydroxy-dehydroepiandrosterone is highly effective and has low toxicity, making it suitable for the treatment of autoimmune diseases, rheumatoid arthritis, Alzheimer's disease, and related conditions.

[0004] Colchicum lini is one of the important strains reported in recent years that can efficiently complete the hydroxylation of dehydroepiandrosterone (DHEA). The hydroxylation reaction in this strain is carried out by a P450 enzyme, the protein sequence of which has been publicly reported and successfully expressed in yeast for the transformation of DHEA (see Chinese patent document CN106350528A).

[0005] However, the hydroxylation of dehydroepiandrosterone by this wild-type P450 enzyme occurs not only at the C7 position but also at the C15 position. Therefore, the hydroxylated product obtained is mainly 7α,15α-dihydroxy-dehydroepiandrosterone. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned problems and provide a P450 enzyme mutant of *Colletotrichum flabellulatum* capable of efficiently converting dehydroepiandrosterone to 7α-hydroxy-dehydroepiandrosterone and its application.

[0007] One of the technical solutions to achieve the objective of this invention is: a P450 enzyme mutant of Colchicum flax, wherein the 121st amino acid sequence of the P450 enzyme CYP68J from Colchicum flax ST-1 strain is mutated to histidine, denoted as D121H.

[0008] The amino acid sequence of the P450 enzyme CYP68J of the above-mentioned *Colletotrichum flaxensis* ST-1 strain can be found in reference CN106350528A.

[0009] The amino acid sequence of the above-mentioned *Colletotrichum flavomarginata* P450 enzyme mutant is shown in SEQ ID NO: 1: 1 MASYAPLDPD FSLYTIPSPA EKPIPYLITA FLFIALVYAV 41 GDHGSKLPEV NPLRAFEFTN RRRMNEFVQH SKDIMLKGKA 81 TFAGSFYKMY SDWGEVVVLP PQFIHELRNE PALEFLEPAK 121 HDSHGYVPGF GPFAGNPAIP KVITKYLTKA LTKLTKPLSE 161 EATLVIRHVL TDSTDWHQIV PQKDIIRIVS RLSSRVFMGE 201 ELCRDEEWVR VSGDYTATAF SVANELNQWP RRVRPVVHWF 241 LPSCWRVRRLLAECHKSLKPHLEERNARKMAAAARGETKA 281 VFDDSIEWFD LESKTKHDPA TDQITLSLVA IHTTTDLLQQ 321 TMLDLASHPE LFQPLREELV RVLSTEGLKK TALYNLKLMD 361 SVLKESQRMK PVLLSTWRRL VKKDIKLSNG FILRRGQKVI 401 ATNTHMWDAE SYENPLTFDG YRFLNMRSTD EEKHAHLVST 441 SVNHPAFGHG AHACPGRFFA ANEIKIALAH LLLKYEWKLP 481 EGSSFKPMPY GMAFLPDPTA TLWIRRRKEE LDLESLDC.

[0010] The second technical solution to achieve the objective of this invention is: a gene encoding the above-mentioned *Colletotrichum flammatus* P450 enzyme mutant, the nucleotide sequence of which is shown in SEQ ID NO: 2. 1 ATGGCTTCTT ATGCCTCCATT GGATCCTTGAT TTTTCTTTGT ATACTATTCC ATCTCCTGCT 61 GAAAAACCAA TTCCATATTT GATTACTGCT TTTTTGTTTA TTGCTTTGGT TTATGCTGTT 121 GGTGATCATG GTTCTAAGTT ACCTGAAGTT AATCCATTGA GGGCTTTCGA ATTTACTAAT 181 AGGAGAAGAA TGAATGAATT TGTTCAACAC TCTAAGGATA TTATGTTGAA AGGTAAAGCT 241 ACTTTTGCTG GTTCTTTTTA TAAAATGTAT TCTGATTGGG GTGAGGTTGT CGTTTTGCCA 301 CCACAATTTA TTCATGAATT GAGAAATGAA CCTGCTTTGG AATTTTTGGA ACCTGCTAAA 361 CATGATTCTC ATGGTTATGT TCCTGGTTTT GGTCCATTTG CTGGTAATCC TGCTATTCCT 421 AAGGTTATTA CTAAATACTT GACTAAAGCT TTGACTAAAT TGACTAAACC ATTGTCTGAA 481 GAAGCTACTTGGTTATTAG ACATGTTTTG ACTGATTCAA CTGATTGGCA TCAGATTGTT 541 CCACAAAAGG ATATTATTAG AATTGTTTCT AGATTGTCTT CTAGAGTTTT TATGGGTGAA 601 GAATTGTGTA GAGATGAAGA ATGGGTTAGA GTTTCTGGTG ATTATACTGC TACTGCTTTT 661 TCTGTTGCTA ATGAATTGAA TCAATGGCCA AGAAGAGTTA GACCTGTTGT TCATTGGTTT 721 TTGCCATCTT GTTGGAGAGT TAGAAGATTG TTGGCTGAAT GTCATAAATC TTTGAAACCA 781 CATTTGGAAG AAAGAAATGC TAGAAAAATG GCTGCAGCTG CTAGAGGTGA AACTAAAGCT 841 GTTTTTGATG ATTCTATTGA ATGGTTTGAT TTGGAATCTA AAACTAAACA TGATCCTGCT 901 ACTGATCAAA TTACTTTGTC TTTGGTTGCT ATTCATACTA CAACTGATTT GTTGCAACAA 961 ACTATGTTGG ATTTGGCTTC TCATCCTGAA TTGTTCAAC CATTGAGAGA AGAATTGGTT 1021AGAGTCTTAT CTACTGAAGG TTTGAAAAAA ACTGCTTTGT ATAACTTGAA GTTGATGGAT 1081TCTGTTTTGA AAGAATCTCA AAGAATGAAA CCTGTTTTGT TGTCTACTTG GAGGAGATTG 1141GTTAAAAAAG ACATCAAATT GTCAAATGGA TTCATTTTGA GAAGAGGTCA AAAAGTTATT 1201GCTACTAATA CTCATATGTG GGATGCTGAA TCTTATGAAA ATCCATTGAC TTTTGATGGA 1261TACAGATTTT TGAATATGAG ATCTACTGAT GAAGAAAAAC ATGCTCATTT GGTTTCTACT 1321TCTGTTAATC ATCCTGCTTT TGGTCATGGT GCTCATGCTT GTCCTGGTAG ATTTTTTGCT 1381GCTAATGAAA TTAAAATTGC TTTGGCTCAT TTGTTATTGA AATATGAATG GAAATTGCCT 1441GAAGGATCTT CTTTAAACC AATGCCATAT GGTATGGCTT TTTTGCCTGA TCCAACTGCT 1501ACTTTGTGGA TTAGAAGGAG AAAAGAAGAA TTGGATTTGG AGTCTTTGGA TTGTTAA.

[0011] The third technical solution to achieve the objective of this invention is a recombinant vector containing all the genes encoded above.

[0012] Furthermore, the recombinant vector was constructed using pYES2 as the plasmid vector.

[0013] The fourth technical solution to achieve the objective of this invention is a recombinant bacterium comprising the above-mentioned recombinant vector.

[0014] Furthermore, the recombinant strain was constructed using Saccharomyces cerevisiae INVSC1.

[0015] The fifth technical solution to achieve the objective of this invention is the application of the above-mentioned recombinant bacteria in the efficient conversion of dehydroepiandrosterone (DHEA) to 7α-hydroxy-dehydroepiandrosterone (DHEA).

[0016] The positive effects of this invention are as follows: This invention mutates the aspartic acid at position 121 of the wild-type P450 enzyme to histidine to obtain a P450 enzyme mutant. Compared with the wild-type P450 enzyme, this P450 enzyme mutant can achieve efficient conversion of dehydroepiandrosterone (DHEA) to 7α-hydroxy-dehydroepiandrosterone (DHEA). It can increase the ratio of 7α-hydroxy-dehydroepiandrosterone to 7α,15α-dihydroxy-dehydroepiandrosterone in the hydroxylation product from about 4:5 to about 14:1, providing an important pathway for the biotransformation of 7α-hydroxy-dehydroepiandrosterone and is of great significance for the industrial production of 7α-hydroxy-dehydroepiandrosterone. Attached Figure Description

[0017] Figure 1 The HPLC spectrum of the hydroxylated product is shown in the example.

[0018] Figure 2 The HPLC spectrum of the hydroxylated product in Application Example 1 is shown for comparison.

[0019] Figure 3 To compare the HPLC chromatogram of the blank experiment in Application Example 2.

[0020] Figure 4 The image shows the TLC analysis results for the test case. Detailed Implementation

[0021] (Comparative example) This comparative example demonstrates the construction of a wild-type P450 enzyme-recombinant Saccharomyces cerevisiae.

[0022] S1. The amino acid sequence of the P450 enzyme CYP68J from the ST-1 strain of *Colletotrichum flabellulatum* (as shown in SEQ ID NO: 3) was submitted to Suzhou Genewiz Biotechnology Co., Ltd. The company optimized the codons based on the protein sequence. The optimized codons can be expressed in *Saccharomyces cerevisiae*, and its nucleotide sequence is shown in SEQ ID NO: 4. The optimized sequence was cloned into the yeast expression vector pPIC9K, and the company subcloned the vector into the *E. coli* Top10 strain.

[0023] The amino acid sequence of the P450 enzyme CYP68J of *Colletotrichum flaxensis* strain ST-1 is shown in SEQ ID NO: 3. 1 MASYAPLDPD FSLYTIPSPA EKPIPYLITA FLFIALVYAV 41 GDHGSKLPEV NPLRAFEFTN RRRMNEFVQH SKDIMLKGKA 81 TFAGSFYKMY SDWGEVVVLP PQFIHELRNE PALEFLEPAK 121 DDSHGYVPGF GPFAGNPAIP KVITKYLTKA LTKLTKPLSE 161 EATLVIRHVL TDSTDWHQIV PQKDIIRIVS RLSSRVFMGE 201 ELCRDEEWVR VSGDYTATAF SVANELNQWP RRVRPVVHWF 241 LPSCWRVRRLLAECHKSLKPHLEERNARKMAAAARGETKA 281 VFDDSIEWFD LESKTKHDPA TDQITLSLVA IHTTTDLLQQ 321 TMLDLASHPE LFQPLREELV RVLSTEGLKK TALYNLKLMD 361 SVLKESQRMK PVLLSTWRRL VKKDIKLSNG FILRRGQKVI 401 ATNTHMWDAE SYENPLTFDG YRFLNMRSTD EEKHAHLVST 441 SVNHPAFGHG AHACPGRFFA ANEIKIALAH LLLKYEWKLP 481 EGSSFKPMPY GMAFLPDPTA TLWIRRRKEE LDLESLDC。

[0024] SEQ ID NO: 4:00:00:00:00 00:00 00:00 1 ATGGCTTCTT ATGCTCCATT GGATCCTGAT TTTTCTTTGT ATACTATTCC ATCTCCTGCT 61 GAAAAACCAA TTCCATATTT GATTACTGCT TTTTTGTTTA TTGCTTTGGT TTATGCTGTT 121 GGTGATCATG GTTCTAAGTT ACCTGAAGTT AATCCATTGA GGGCTTTCGA ATTTACTAAT 181 AGGAGAAGAA TGAATGAATT TGTTCAACAC TCTAAGGATA TTATGTTGAA AGGTAAAGCT 241 ACTTTTGCTG GTTCTTTTTA TAAAATGTAT TCTGATTGGG GTGAGGTTGT CGTTTTGCCA 301 CCACAATTTA TTCATGAATT GAGAAATGAA CCTGCTTTGG AATTTTTGGA ACCTGCTAAA 361 GATGATTCTC ATGGTTATGT TCCTGGTTTT GGTCCATTTG CTGGTAATCC TGCTATTCCT 421 AAGGTTATTA CTAAATACTT GACTAAAGCT TTGACTAAAT TGACTAAACC ATTGTCTGAA 481 GAAGCTACTT TGGTTATTAG ACATGTTTTG ACTGATTCAA CTGATTGGCA TCAGATTGTT 541 CCACAAAAGG ATATTATTAG AATTGTTTCT AGATTGTCTT CTAGAGTTTT TATGGGTGAA 601 GAATTGTGTA GAGATGAAGA ATGGGTTAGA GTTTCTGGTG ATTATACTGC TACTGCTTTT 661 TCTGTTGCTA ATGAATTGAA TCAATGGCCA AGAAGAGTTA GACCTGTTGT TCATTGGTTT 721 TTGCCATCTT GTTGGAGAGT TAGAAGATTG TTGGCTGAAT GTCATAAATC TTTGAAACCA 781 CATTTGGAAG AAAGAAATGC TAGAAAAATG GCTGCAGCTG CTAGAGGTGA AACTAAAGCT 841 GTTTTTGATG ATTCTATTGA ATGGTTTGAT TTGGAATCTA AAACTAAACA TGATCCTGCT 901 ACTGATCAAA TTACTTTGTC TTTGGTTGCT ATTCATACTA CAACTGATTT GTTGCAACAA 961 ACTATGTTGG ATTTGGCTTC TCATCCTGAA TTGTTCAAC CATTGAGAGA AGAATTGGTT 1021AGAGTCTTAT CTACTGAAGG TTTGAAAAAA ACTGCTTTGT ATAACTTGAA GTTGATGGAT 1081TCTGTTTTGA AAGAATCTCA AAGAATGAAA CCTGTTTTGT TGTCTACTTG GAGGAGATTG 1141GTTAAAAAAG ACATCAAATT GTCAAATGGA TTCATTTTGA GAAGAGGTCA AAAAGTTATT 1201GCTACTAATA CTCATATGTG GGATGCTGAA TCTTATGAAA ATCCATTGAC TTTTGATGGA 1261TACAGATTTT TGAATATGAG ATCTACTGAT GAAGAAAAAC ATGCTCATTT GGTTTCTACT 1321TCTGTTAATC ATCCTGCTTT TGGTCATGGT GCTCATGCTT GTCCTGGTAG ATTTTTTGCT 1381GCTAATGAAA TTAAAATTGC TTTGGCTCAT TTGTTATTGA AATATGAATG GAAATTGCCT 1441GAAGGATCTT CTTTAAACC AATGCCATAT GGTATGGCTT TTTTGCCTGA TCCAACTGCT 1501ACTTTGTGGA TTAGAAGGAG AAAAGAAGAA TTGGATTTGG AGTCTTTGGA TTGTTAA.

[0025] S2, Construction of the recombinant vector pYES2-P450.

[0026] Plasmids were extracted using a plasmid extraction kit. Using the plasmid as a template, PCR was performed using the primers shown in Table 1 and PCR system 1 shown in Table 2 to obtain the P450 enzyme gene fragment. This gene fragment was then cloned into the EcoRI and NotI restriction endonuclease sites of the pYES2 vector to construct the pYES2-P450 recombinant vector. Positive clones were screened on kanamycin-resistant plates, and transformants were sent to Genewiz Biotechnology Co., Ltd. for sequencing to confirm their correctness. Strains with correct sequencing were stored at -80℃.

[0027] Table 1 Primer name Primer uses Primers (5–3') P450F Obtain the P450 enzyme gene CGGAATTCATGGCTTCTTATGCTCCATTG P450R Obtain the P450 enzyme gene ATAAGAATGCGGCCGCTTAACAATCCAAAGACTCCA MutF Obtain the P450 enzyme mutant expression vector GAACCTGCTAAACATGATTCTCATG MutR Obtain the P450 enzyme mutant expression vector CATGAGAATCATGTTTAGCAGGTTC

[0028] Table 2 Reagent Name Volume μL template 2 PCR Buffer 5 dNTPs (10mM) 1 Upstream / downstream primers (100mM) 0.25 each Pfu DNA polymerase 1.5 <![CDATA[ddH2O]]> 40 Total volume 50

[0029] S3, Construction of recombinant bacteria INVSC1-P450.

[0030] Competent cells of Saccharomyces cerevisiae were prepared using the lithium acetate method. 10 μg of pYES2-P450 recombinant vector and 10 μL of salmon sperm DNA were mixed with the competent cells. 700 μL of sterilized and pre-cooled 1×LiAc / 40%PEG3350 / 1×TE solution was added to the mixture and mixed by pipetting. The mixture was then incubated at 30°C for 0.5 h. Subsequently, 88 μL of DMSO was added to the mixture and the cells were incubated at 42°C for 7 min. Then, pre-cooled 1×TE solution was added and mixed. The mixture was centrifuged again at 10,000 rpm for 10 s, the supernatant was discarded, and approximately 80 μL of supernatant was used to resuspend the cells. The cells were then plated on SC plates containing kanamycin resistance and incubated at 30°C for 2 days until transformants appeared.

[0031] Single colonies from SC medium were picked and transferred to YPD plates containing kanamycin. The plates were inverted and incubated at 30°C for 2 days until single colonies grew. These colonies were then transferred to YPD liquid medium and incubated at 30°C for 24 hours. Chromosomal DNA was extracted from the transformants using a yeast chromosome extraction kit. Primers P450F and P450R were used to verify whether the recombinant vector had integrated into the *Saccharomyces cerevisiae* chromosomal DNA. The correctly verified strain was stored at -80°C and named the recombinant strain INVSC1-P450.

[0032] The above reagents and culture media were prepared as follows: 10×TE solution: Weigh 1.21g of Tris and 0.37g of EDTA and dissolve them in 90mL of water. Adjust the pH to 7.5 with hydrochloric acid, bring the volume to 100mL, and then sterilize.

[0033] 10×LiAc: Weigh 10.2g of lithium acetate, dissolve it in 80mL of water, adjust the pH to 7.5 with glacial acetic acid, and then sterilize after bringing the volume to 100mL.

[0034] 1×LiAc / 40%PEG3350 / 1×TE solution: Weigh 40g of PEG3350 into a beaker, add sterile water to dissolve and bring the volume to 80mL, then add 10mL of 10×LiAc solution and 10mL of 10×TE solution, bring the volume to 100mL and sterilize.

[0035] YPD medium: 2% glucose, 2% peptone, 1% yeast extract. If it is a solid medium, add 2% agar powder. Sterilize in an autoclave at 115°C for 20 min.

[0036] SC medium: YNB 6.7g, glucose 20g, adenine 0.1g, arginine 0.1g, cysteine ​​0.1g, leucine 0.1g, lysine 0.1g, threonine 0.1g, tryptophan 0.1g, isoleucine 0.05g, methionine 0.05g, proline 0.05g, serine 0.05g, tyrosine 0.05g, phenylalanine 0.05g, aspartic acid 0.05g, valine 0.05g, histidine 0.05g, agar 20g, dissolved in 800mL deionized water and brought to a final volume of 1L, sterilized at 115℃ for 20min.

[0037] (Example) This example describes the construction of a recombinant Saccharomyces cerevisiae strain with the P450 enzyme mutant.

[0038] Construction of S1, P450 enzyme mutant of Colletotrichum flaxensis.

[0039] Plasmid pYES2-P450 was extracted using a plasmid extraction kit. Using this plasmid as a template, and with MutF and MutR primers (Table 1) as the mutation primers, a reverse PCR reaction was performed using the PCR system shown in Table 3 to obtain the mutant vector. The vector was removed by Dpn I treatment for 2 hours. 10 μL of the reaction mixture was transformed into *E. coli* DH5α competent cells. Transformants grown on kanamycin plates were sent to Genewiz Biotechnology Co., Ltd. for sequencing to determine if mutations had occurred. Strains with correct sequencing results were stored at -80℃.

[0040] Table 3 Reagent Name Volume μL template 1.5 PCR Buffer 5 dNTPs (10mM) 1 Upstream / downstream primers (100mM) 0.5 each Pfu DNA polymerase 1.5 <![CDATA[ddH2O]]> 40 Total volume 50

[0041] S2. A recombinant Saccharomyces cerevisiae strain with the P450 enzyme mutant was constructed according to the method in Example 1 and named INVSC1-Mut.

[0042] (Application example) This application example demonstrates the use of recombinant bacteria INVSC1-Mut in DHEA hydroxylation.

[0043] The positive clones screened in the examples were streaked on YPD plates and cultured at 30°C for 2 days. Transformants were picked and cultured in 10 mL of YPD liquid medium at 30°C and 180 rpm for 24 hours to obtain seed culture.

[0044] The seed culture was transferred to 50 mL of YPD liquid medium at an inoculum of 5%, and cultured at 30 °C and 180 rpm for 24 h. Then, DHEA (to reach 3 g / L), 1 mL of methanol and 3.75 mL of 20% galactose solution were added to induce the expression of the target protein.

[0045] Add 1 mL of methanol every 24 hours. After 72 hours of conversion, take 1000 μL of bacterial culture and extract with 300 μL of ethyl acetate for 30 min. Then centrifuge at 10000 rpm for 10 min. Take the upper organic phase for HPLC detection and TLC analysis (see test example).

[0046] The HPLC detection conditions were as follows: the column was an Agilent ZORBAx Eclipse plus C18 (4.6×250mm, 5μm); the column temperature was 30℃; the detection wavelength was 210nm; the flow rate was 1.0mL / min; and the injection volume was 20μL.

[0047] Take 20 μL of the upper organic phase, dilute it to 2.0 mL with 60% acetonitrile solution, and use this as the test solution. Inject 20 μL of the test solution for HPLC detection. The results are shown in the figure. Figure 1 .

[0048] Depend on Figure 1 It can be seen that the content of 7α-OH-DHEA (peak time 11.214 min) in the hydroxylation products of the P450 enzyme mutant is significantly higher than that of 7α,15α-diOH-DHEA (peak time 9.793 min), with a ratio of approximately 14:1.

[0049] (Compare with application example 1) This comparative application example demonstrates the use of recombinant bacteria INVSC1-P450 in DHEA hydroxylation.

[0050] For specific methods, please refer to the application examples. The HPLC chromatogram of the hydroxylated product is shown below. Figure 2 .

[0051] Depend on Figure 2 It can be seen that the content of 7α-OH-DHEA (peak time 11.207 min) in the hydroxylation products of wild-type P450 enzyme is lower than that of 7α,15α-diOH-DHEA (peak time 9.789 min), with a ratio of about 4:5.

[0052] (Compare with application example 2) This comparative application example is a blank experiment, that is, without the addition of any recombinant bacteria. The HPLC chromatogram is shown below. Figure 3 .

[0053] (Test example) This test example is a TLC analysis: Take 10 mg each of DHEA standard, 7α-OH-DHEA standard and 7α,15α-diOH-DHEA standard into the same 50 mL volumetric flask, add ethanol to make up to volume, and obtain a mixed standard solution.

[0054] Take 2 μL of the mixed standard solution and spot it on a TLC plate (the silica gel plate used is a GF254 high-efficiency silica gel plate). Then, take 2 μL of the upper organic phase obtained from the application example and the comparative application example and spot them respectively.

[0055] After thoroughly drying the spotted silica gel plate, place it in a chromatography tank for full development (development solvent: n-hexane:methanol:water = 90:60:15). Remove the silica gel plate and dry it with a hair dryer. Then, evenly spray the colorimetric reagent onto the silica gel plate (colorimetric solution: concentrated sulfuric acid:ethanol = 1:1). Dry it in a 100℃ oven until color development occurs. The results are shown in the figure. Figure 4 .

Claims

1. A P450 enzyme mutant of *Colletotrichum flammatus*, characterized in that: The mutant is formed by mutating the 121st position of the amino acid sequence of the P450 enzyme CYP68J from the *Colletotrichum flavomarginata* strain ST-1 to histidine; the amino acid sequence of the *Colletotrichum flavomarginata* P450 enzyme mutant is shown in SEQ ID NO:

1.

2. A gene encoding the P450 enzyme mutant of *Colletotrichum flabellulatum* as described in claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant vector, characterized in that: It includes all the genes encoded by claim 2.

4. A recombinant bacterium, characterized in that: It includes the recombinant vector as described in claim 3.

5. The application of the recombinant bacteria according to claim 4 in the efficient conversion of dehydroepiandrosterone to 7α-hydroxy-dehydroepiandrosterone.

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