CN110229838B - Method for obtaining hydroxylated compound by biotransformation of steroid compound - Google Patents
Method for obtaining hydroxylated compound by biotransformation of steroid compound Download PDFInfo
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Abstract
The invention discloses a method for obtaining a hydroxylated compound by biotransformation of a steroid compound by using a recombinant strain of cytochrome P450 enzyme. The method comprises the steps of firstly preparing coexpression recombinant escherichia coli, then carrying out pre-culture and amplification culture on the recombinant escherichia coli, and collecting thalli for carrying out biotransformation on steroid compounds; the invention adopts cytochrome P450 enzyme CYP105D7 recombinant escherichia coli to biologically convert steroid compounds to generate 2 beta and 16 beta hydroxylated compounds; through site-directed mutagenesis, a more efficient recombinant mutant strain is constructed, and the conversion rate is improved by about 20 times compared with that of a wild type.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for obtaining a hydroxylated compound by biotransformation of a steroid compound (steroid) by using recombinant escherichia coli.
Background
Steroid drugs play an important role in medicine, and besides the wide application of diosgenin natural compounds in steroid drugs, some sterols such as cholesterol, sitosterol, and brassicasterol are also considered as starting materials of steroid hormone drugs with huge potential at present. Steroid hormone drugs play a very important role in regulating the organism, and are known as the 'key of life'. Steroid compounds, also known as steroids, are a general term for cyclopentane polyhydrophenanthrene compounds and are commonly found in animal and plant tissues. Steroids have a basic skeleton consisting of four rings, i.e. a carbon structure fused from three six-membered rings and a five-membered ring, which is the parent nucleus of the steroid, i.e. the cyclopentane polyhydrophenanthrene. The four rings are indicated by letters A, B, C and D, and the 17 carbon atoms are numbered in a particular order. Most steroids also have three side chains on the parent nucleus of the cyclopentanoperhydrophenanthrene, which are attached to the C-10, C-13 and C-17 carbon atoms, respectively.
Many steroids have strong physiological activities, such as corticosterone, hydrocortisone, testosterone, androsterone, progesterone, estradiol, etc., and play very important regulation roles in the body, such as adrenocortical hormone which can treat or relieve intractable or dangerous diseases such as collagen diseases and anaphylactic shock, and is also an indispensable medicament for treating endocrine diseases such as Addison's disease. Various sex hormones are main medicines for treating male organ failure and certain gynecological diseases, are auxiliary medicines for treating breast cancer and prostate cancer, and are also main components of oral contraceptives which are in vigorous demand in recent years. Steroids have also been developed as anesthetics, antiarrhythmics, antibacterial agents, anticholinesterases, anticoagulants, antifungal agents, antitumor agents, antiprotozoal agents, bile secretion agents, diagnostic agents, neuromodulatory agents, gallstone dispersing agents, hemostatic agents, calcium modulators, lipid modulators, neuropathy remedies, laxatives, tranquilizers, and the like. Due to the irreplaceable use of steroid drugs and the continuous expansion of the treatment indications, people pay more and more attention.
Steroid hormone drugs are the second largest class of drugs after antibiotics, and are usually prepared from natural products having a steroid nucleus structure as a raw material because of their very complicated structure and difficulty in chemical synthesis. By means of biotransformation, new compounds with properties not possessed by the precursor can be obtained. Hydroxylases from different microorganisms selectively hydroxylate the methine group of the steroid nucleus, while diastereomers selectively catalyze one hydrogen of the methine group to the alpha-or beta-hydroxyl group. For example, since the discovery that Rhizopus nigricans is capable of converting progesterone into hydroxyprogesterone, Streptomyces argentiformis was found to convert progesterone into 11- α hydroxyprogesterone, followed by the discovery that certain species of bacteria, yeasts, molds and actinomycetes are capable of providing valuable transformations at specific sites of steroids. Wherein, after the 16-position hydroxylation, the anti-inflammatory and sugar metabolism effects can be kept unchanged, and the side effect of the drug salt storage can be eliminated.
Disclosure of Invention
The invention aims to provide a method for obtaining a hydroxylated compound by transforming a recombinant escherichia coli biotransformation steroid compound obtained by using a genetic engineering technology aiming at the defects of the prior art.
The purpose of the invention is realized by the following technical scheme: a method for obtaining a hydroxylated compound by transforming a recombinant escherichia coli biotransformation steroid compound obtained by using a genetic engineering technology comprises the following steps:
(1) preparation of co-expressed recombinant E.coli: carrying out double mutation on the sav7469 gene of the streptomyces avermitilis MA4680 coding CYP105D7 at the position R70A/R190A by using a site-directed mutagenesis kit to obtain a double mutation gene sequence sav7469R70A/R190AThe double mutant gene sequence obtained by enzyme digestion of Nde I and Spe I and the vector pT7NS-camAB are connected to obtain a recombinant vector pET11:: sav7469R70A/R190AThe recombinant vector is introduced into Escherichia coli BL21 (DE)3) Finally obtaining the coexpression recombinant escherichia coli; the sav7469 of the encoding CYP105D7 of the streptomyces avermitilis MA4680R70A/R190AThe gene sequence of the gene is shown as SEQ ID NO.2, the gene sequence of the vector pT7NS-camAB is shown as SEQ ID NO.4, and the recombinant vector pET11 is sav7469R70A/R190AThe gene sequence of camA-camB is shown as SEQ ID NO. 5;
(2) pre-culture and scale-up culture of recombinant E.coli: adding the recombinant escherichia coli into an LB culture medium, and culturing for 18 hours under the conditions that the pH is 7.4-7.6, the temperature is 37 ℃ and the rotating speed is 200rpm to obtain a seed solution of the recombinant escherichia coli; inoculating the seed liquid of the recombinant escherichia coli into an M9 culture medium for amplification culture and fermentation; the volume ratio of the seed solution to the M9 culture medium is 1:100, after inoculation, the recombinant Escherichia coli is cultured at 37 ℃ until the OD of the bacterial solution600The value reaches about 0.6-0.8, then IPTG and 5-aminolevulinic acid salt are added to induce the thalli to express a large amount of protein, the final concentration of the IPTG is 0.2mM, and the final concentration of the 5-aminolevulinic acid salt is 0.5 mM; then reducing the temperature to 22 ℃, reducing the rotating speed to 180rpm, and continuing culturing for 20 h;
(3) and (3) collecting thalli: centrifuging at 4 deg.C and 4000rpm for 10min, and collecting the precipitated thallus; washing the thalli twice by adopting a sodium phosphate buffer solution at the temperature of 4 ℃, collecting the washed thalli, and suspending the thalli in the sodium phosphate buffer solution to obtain a reaction solution for performing biotransformation on naringenin;
(4) biotransformation of steroid compounds: dissolving a compound in methanol, and adding the compound into the reaction liquid obtained in the step 3 for biotransformation to obtain transformation liquid; the final concentration of the compound is 0.1mM, and the biotransformation conditions are 180rpm and 30 ℃ for 20 h; after the reaction is finished, adding ethyl acetate with the same amount as the conversion solution to stop the reaction, fully mixing the ethyl acetate and the conversion solution to ensure that the product completely enters an ethyl acetate organic phase, shaking the mixture for 2min, centrifuging the mixed solution at 13000rpm for 10min, and collecting supernatant; and drying the supernatant to obtain the hydroxylated compound.
The invention has the beneficial effects that: the invention establishes a method for biosynthesizing 2 beta-site and 16 beta-site hydroxylation products by using recombinant escherichia coli of cytochrome P450(CYP105D7) by using a steroid compound as a substrate. The hydroxylation product was isolated using cytochrome P450 enzymes to convert the steroid compounds and ethyl acetate as extractant. This method demonstrates that the bacterial P450 enzymes can also regioselectively monooxygenase testosterone, progesterone and androstenedione and direct the production of hydroxylated compounds. The invention has the advantages that: compared with the traditional chemical synthesis method, the method can be carried out under the conditions of low pollution, low energy consumption and high selectivity; recombinant bacteria of cytochrome P450 enzyme CYP105D7 can biologically convert steroid compounds such as testosterone, progesterone, androstenedione and the like and generate 2 beta-site and 16 beta-site hydroxylation products; thirdly, through site-directed mutagenesis, a more efficient recombinant mutant strain is constructed, and the conversion rate is improved by about 20 times compared with that of a wild type; and fourthly, by utilizing the biotransformation of the recombinant bacteria, side reaction products can be greatly reduced, and the desired product is easy to separate, thereby achieving high efficiency.
Drawings
FIG. 1 is a flow chart of a method of the present invention;
FIG. 2 is an HPLC profile of an experimental sample, wherein A is testosterone, B is progesterone, and C is androstenedione;
figure 3 is a mass spectrum corresponding to the substrate testosterone and its product after 20 hours of reaction: a is testosterone, b is 2 beta-hydroxytestosterone;
fig. 4 shows the mass spectra corresponding to the substrate progesterone and its product after 20 hours of reaction: a is progesterone, b is 2 beta-hydroxyprogesterone, c is 16 beta-hydroxyprogesterone, d is 2 beta, 16 beta-dihydroxyprogesterone;
FIG. 5 shows the mass spectra of the substrate androstenedione and its reaction product for 20 hours, where a is androstenedione, b is 2 β -hydroxyandrostenedione, and c is 16 β -hydroxyandrostenedione.
Detailed Description
The cytochrome P450 enzyme can regioselectively introduce oxygen atoms into an inactive hydrocarbon complex compound under mild conditions, carries out structural modification or modification, and has the advantage that a chemical catalyst is difficult to compare with. Especially, the microorganism-derived P450 enzyme is concerned about due to good stability and high catalytic activity, and is widely applied to the production of important industrial products such as medical industry intermediates, antibiotics, vitamins and the like at present.
According to our research, the cytochrome P450 enzyme CYP105D7 from streptomyces avermitilis can selectively catalyze the hydroxylation reaction of 2 beta position and 16 beta position of the steroid compound. According to research, 16-position hydroxylation of the steroid compound can not only keep the anti-inflammatory and sugar metabolism effects unchanged, but also eliminate the side effects of the medicine. The recombinant escherichia coli is constructed for biotransformation, so that side reaction products can be greatly reduced, a desired product is easy to separate, the production efficiency is improved, and the method is an effective method for efficiently producing the steroid hydroxylation product.
The present invention will be further described with reference to specific embodiments.
The method for biotransforming the steroid compound to obtain the hydroxylated compound comprises the following steps:
1. obtaining a coexpression recombinant E.coli
sav7469 R70A/R190AThe gene is characterized in that the gene is a sav7469 gene (the gene sequence of which is shown in SEQ ID NO. 1) of the streptomyces avermitilis MA4680 coding CYP105D7 (http:// avermitilis. ls. kitasato-u.ac. jp), a site-directed mutagenesis kit is utilized, and three pairs of primers of SEQ ID NO. 3: 5'-AGCGGCTTTCCTCCGACGCGACGCTGCCCAGGTTCCC-3' and reverse primer SEQ ID NO. 4: 5'-GGGAACCTGGGCAGCGTCGCGTCGGAGGAAAGCCGCT-3', respectively; SEQ ID No. 5: 5'-CCGAGGTCCAGGACGCCGCGGCCCAACTGGACGACTA-3' and reverse primer SEQ ID NO. 6: 5'-TAGTCGTCCAGTTGGGCCGCGGCGTCCTGGACCTCGG-3', respectively; SEQ ID NO. 7: 5'-GGAATTCCATATGACAGAGCCCGGTACGT-3' and reverse primer SEQ ID NO. 8: 5'-CCGCTCGAGTTACCAGGTCACGGGGAG-3' obtained by amplification. CarrierThe camA gene and camB gene in pT7NS-camAB (International patent No. US 2006/0234337A1, the gene sequence is shown in SEQ ID NO. 9) encode Pseudomonas putida redox protein reductase and Pseudomonas redox protein, respectively. The Nde I and Spe I are used for enzyme digestion of the PCR amplification product and a vector pT7NS-camAB, and the enzyme digestion and the ligation are carried out to obtain a recombinant vector pET11:: sav7469R70A/R190ANamely camA-camB (the gene sequence is shown as SEQ ID NO. 10). The recombinant vector was introduced into E.coli BL21 (DE)3) Finally, the coexpression recombinant Escherichia coli is obtained.
2. Pre-and expanded culture of recombinant E.coli
Adding the recombinant Escherichia coli into LB culture medium, and culturing at pH 7.4-7.6, 37 deg.C and rotation speed of 200rpm for 18 hr to obtain seed solution of recombinant Escherichia coli. Inoculating the seed liquid of the recombinant escherichia coli into an M9 culture medium for amplification culture and fermentation; the volume ratio of the seed solution to the M9 culture medium is 1:100, after inoculation, the recombinant Escherichia coli is cultured at 37 ℃ until the OD of the bacterial solution600The value reaches about 0.6-0.8, then IPTG and 5-aminolevulinic acid salt are added to induce the thalli to express a large amount of protein, the final concentration of the IPTG is 0.2mM, and the final concentration of the 5-aminolevulinic acid salt is 0.5 mM; then the temperature is reduced to 22 ℃, the rotating speed is reduced to 180rpm, and the culture is continued for 20 h.
The M9 medium can be prepared by the following method: casein amino acid with the mass concentration of 1%, glucose with the mass concentration of 0.4% and 0.1mMCaCl are added into the M9 salt solution2、1mM MgCl2、0.1mM FeCl3。
The LB medium can be prepared by the following method: 10g tryptone, 5g yeast extract, 10g NaCl were added to 950ml deionized water, the pH was adjusted to 7.0 with 5mol/LNaOH, the volume was adjusted to 1L with deionized water, and steam sterilized at 15psi for 20 min.
3. Collection of cells
After the recombinant Escherichia coli is subjected to amplification culture for 20h, the recombinant Escherichia coli is centrifuged at 4000rpm at 4 ℃ for 10min, and the precipitated bacteria are collected. And the thalli is washed twice by adopting a sodium phosphate buffer solution (comprising 1mM of EDTA, 2mM of dithiothreitol and 10% of glycerol by volume, the solvent is water, and the pH value is 7.2) at 4 ℃, the washed thalli is collected and suspended in the sodium phosphate buffer solution, and a reaction solution for biotransformation of naringenin is obtained.
4. Biotransformation of steroid compounds
Dissolving the steroid compound in methanol, and adding the dissolved steroid compound into the reaction liquid obtained in the step 3 for biotransformation to obtain transformation liquid; the final concentration of the steroid compound was 0.1 mM. The biotransformation condition is that the culture is carried out for 20 hours under the conditions of 180rpm and 30 ℃; and after the reaction is finished, adding ethyl acetate with the same volume as the conversion solution to stop the reaction, fully mixing the ethyl acetate and the conversion solution to ensure that the product completely enters an ethyl acetate organic phase, shaking the mixture for 2min, centrifuging the mixed solution at 13000rpm for 10min, and collecting the supernatant. And drying the supernatant to obtain the hydroxylated compound.
In this embodiment, three steroid compounds including testosterone, progesterone and androstenedione are biologically converted, and the reaction scheme of testosterone, progesterone and androstenedione and the finally obtained hydroxylated compound are shown in fig. 1.
The above dried product was dissolved in methanol and subjected to HPLC detection analysis and LC/MS analysis. The HPLC analysis in the invention specifically adopts a Japanese LC-20AT HPLC system and a COSMOSIL packed column, the mobile phase adopts 35-100% methanol water gradient elution for 20min, the detection wavelength is 245nm, and the flow rate is 1 ml/min. The specific implementation conditions of LC/MS are Agilent 6230 flight time mass spectrum, Agilent reverse ODS column as chromatographic column, 35% -100% methanol water gradient elution for 40min as mobile phase, 0.6mL/min of flow rate and 245nm of detection wavelength.
FIG. 2 is an HPLC chromatogram of an experimental sample, wherein A is a standard testosterone sample and a reaction time of 20 hours, retention times are 18.128min and 15.180min, B is a standard progesterone sample and a reaction time of 20 hours, retention times are 20.218min, 17.350min, 16.727min and 12.624min, C is a standard androstenedione sample and a reaction time of 20 hours, and retention times are 17.151min, 14.107min and 13.534min, respectively.
The position of the end of the first path in fig. 3, fig. 4,FIG. 5 is a mass spectrum of the substrates testosterone, progesterone and androstenedione and their products reacted for 20 hours, respectively. The MS analysis uses positive ion mode. FIG. 3 is a mass spectrum of testosterone and its products after 20 hours reaction, [ M + Na ] with retention times of 18.128min and 15.180min, respectively]+Molecular weights 311.1980 and 327.1931, respectively; FIG. 4 is a mass spectrum of progesterone and its products after 20 hours reaction, [ M + Na ] with retention times of 20.218min, 17.350min, 16.727min and 12.624min, respectively]+Molecular weights 337.2136,353.2089 and 353.3088, 369.2030, respectively; FIG. 5 shows the mass spectra of androstenedione and its reaction product for 20 hours, [ M + Na ] with retention times of 17.151min, 14.107min and 13.534min, respectively]+Molecular weights are 309.1824, 325.1779 and 325.1781, respectively. 3-5, the products after 20 hours of reaction all increased the molecular weight of one hydroxyl group relative to the substrate, so the conversion products were presumed to be hydroxylated products, and the nuclear magnetic identification of the compounds after 20 hours of reaction confirmed that both 2 β and 16 β specific hydroxylated compounds, of which testosterone produced 2 β -hydroxytestosterone; progesterone produces 2 β -hydroxyprogesterone, 16 β -hydroxyprogesterone and 2 β, 16 β -dihydroxyprogesterone; androstenedione produces 2 β -hydroxyandrostenedione and 16 β -hydroxyandrostenedione.
Compared with the traditional chemical synthesis method, the method can be carried out under the conditions of low pollution, low energy consumption and high selectivity; the recombinant bacteria of cytochrome P450 enzyme CYP105D7 can biologically convert testosterone, progesterone, androstenedione and other steroid compounds and generate 2 beta site and 16 beta site hydroxylation products or 2 beta site and 16 beta site simultaneous hydroxylation compounds; thirdly, through site-directed mutagenesis, a more efficient recombinant mutant strain is constructed, and the conversion rate is improved by about 20 times compared with that of a wild type; and fourthly, by utilizing the biotransformation of the recombinant bacteria, side reaction products can be greatly reduced, and the desired product is easy to separate, thereby achieving high efficiency.
Sequence listing
<110> Zhejiang university of science and engineering
<120> a method for obtaining a hydroxylated compound by biotransformation of a steroid compound
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cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 420
agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 540
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 600
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 660
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 720
accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 840
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 900
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 960
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1020
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1080
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1140
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1200
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1260
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1320
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1380
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1440
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1500
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1560
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1620
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1680
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1740
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1800
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1860
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1920
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1980
cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2040
tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2100
gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2160
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2220
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2280
cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2340
tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2400
ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2460
tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2520
cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2580
tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2640
ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2700
acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2760
agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2820
gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2880
tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2940
tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3000
tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3060
cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3120
acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3180
ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3240
taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3300
cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3360
aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3420
ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3480
gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3540
cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3600
tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3660
cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 3720
tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3780
gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3840
gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3900
ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3960
ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4020
cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4080
gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4140
cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4200
ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4260
gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4320
ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4380
gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4440
caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4500
gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4560
ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4620
cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4680
gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4740
gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4800
caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4860
gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4920
ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4980
ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5040
agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5100
aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5160
cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5220
ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5280
aaataatttt gtttaacttt aagaaggaga tatacatatg cgtcactagt cgggagtgcg 5340
ttatatgaac gcaaacgaca acgtggtcat cgtcggtacc ggactggctg gcgttgaggt 5400
cgccttcggc ctgcgcgcca gcggctggga aggcaatatc cggttggtgg gggatgcgac 5460
ggtaattccc catcacctac caccgctatc caaagcttac ttggccggca aagccacagc 5520
ggaaagcctg tacctgagaa ccccagatgc ctatgcagcg cagaacatcc aactactcgg 5580
aggcacacag gtaacggcta tcaaccgcga ccgacagcaa gtaatcctat cggatggccg 5640
ggcactggat tacgaccggc tggtattggc taccggaggg cgtccaagac ccctaccggt 5700
ggccagtggc gcagttggaa aggcgaacaa ctttcgatac ctgcgcacac tcgaggacgc 5760
cgagtgcatt cgccggcagc tgattgcgga taaccgtctg gtggtgattg gtggcggcta 5820
cattggcctt gaagtggctg ccaccgccat caaggcgaac atgcacgtca ccctgcttga 5880
tacggcagcc cgggttctgg agcgggttac cgccccgccg gtatcggcct tttacgagca 5940
cctacaccgc gaagccggcg ttgacatacg aaccggcacg caggtgtgcg ggttcgagat 6000
gtcgaccgac caacagaagg ttaccgccgt cctctgcgag gacggcacaa ggctgccagc 6060
ggatctggta atcgccggga ttggcctgat accaaactgc gagttggcca gtgcggccgg 6120
cctgcaggtt gataacggca tcgtgatcaa cgaacacatg cagacctctg atcccttgat 6180
catggccgtc ggcgactgtg cccgatttca cagtcagctc tatgaccgct gggtgcgtat 6240
cgaatcagtg cccaatgcct tggagcaggc acgaaagatc gccgccatcc tctgtggcaa 6300
ggtgccacgc gatgaggcgg cgccctggtt ctggtccgat cagtatgaga tcggattgaa 6360
gatggtcgga ctgtccgaag ggtacgaccg gatcattgtc cgcggctctt tggcgcaacc 6420
cgacttcagc gttttctacc tgcagggaga ccgggtattg gcggtcgata cagtgaaccg 6480
tccagtggag ttcaaccagt caaaacaaat aatcacggat cgtttgccgg ttgaaccaaa 6540
cctactcggt gacgaaagcg tgccgttaaa ggaaatcatc gccgccgcca aagctgaact 6600
gagtagtgcc tgaaatctat acccacaata aatcaccgtt ttgccccata gcgtgtgagg 6660
ataaacagat gtctaaagta gtgtatgtgt cacatgatgg aacgcgtcgc gaactggatg 6720
tggcggatgg cgtcagcctg atgcaggctg cagtctccaa tggtatctac gatattgtcg 6780
gtgattgtgg cggcagcgcc agctgtgcca cctgccatgt ctatgtgaac gaagcgttca 6840
cggacaaggt gcccgccgcc aacgagcggg aaatcggcat gctggagtgc gtcacggccg 6900
aactgaagcc gaacagcagg ctctgctgcc agatcatcat gacgcccgag ctggatggca 6960
tcgtggtcga tgttcccgat aggcaatggg gatccggctg ctaacaaagc ccgaaaggaa 7020
gctgagttgg ctgctgccac cgctgagcaa taactagcat aaccccttgg ggcctctaaa 7080
cgggtcttga ggggtttttt gctgaaagga ggaactatat ccggatatcc cgcaagaggc 7140
ccggcagtac cggcataacc aagcctatgc ctacagcatc cagggtgacg gtgccgagga 7200
tgacgatgag cgcattgtta gatttcatac acggtgcctg actgcgttag caatttaact 7260
gtgataaact accgcattaa agcttatcga tgataagctg tcaaacatga gaa 7313
<210> 10
<211> 8812
<212> DNA
<213> Artificial design (Unknown)
<400> 10
ttcttgaaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat 60
aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg 120
tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat 180
gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat 240
tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt 300
aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag 360
cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa 420
agttctgcta tgtggcgcgg tattatcccg tgttgacgcc gggcaagagc aactcggtcg 480
ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct 540
tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac 600
tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca 660
caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat 720
accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg gcaacaacgt tgcgcaaact 780
attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc 840
ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga 900
taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg 960
taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg 1020
aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca 1080
agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta 1140
ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca 1200
ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg 1260
cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga 1320
tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa 1380
tactgtcctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc 1440
tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg 1500
tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac 1560
ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct 1620
acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc 1680
ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg 1740
gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg 1800
ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct 1860
ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga 1920
taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg 1980
cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca 2040
tctgtgcggt atttcacacc gcatatatgg tgcactctca gtacaatctg ctctgatgcc 2100
gcatagttaa gccagtatac actccgctat cgctacgtga ctgggtcatg gctgcgcccc 2160
gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt 2220
acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac 2280
cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg gtcgtgaagc gattcacaga 2340
tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc cagaagcgtt aatgtctggc 2400
ttctgataaa gcgggccatg ttaagggcgg ttttttcctg tttggtcact gatgcctccg 2460
tgtaaggggg atttctgttc atgggggtaa tgataccgat gaaacgagag aggatgctca 2520
cgatacgggt tactgatgat gaacatgccc ggttactgga acgttgtgag ggtaaacaac 2580
tggcggtatg gatgcggcgg gaccagagaa aaatcactca gggtcaatgc cagcgcttcg 2640
ttaatacaga tgtaggtgtt ccacagggta gccagcagca tcctgcgatg cagatccgga 2700
acataatggt gcagggcgct gacttccgcg tttccagact ttacgaaaca cggaaaccga 2760
agaccattca tgttgttgct caggtcgcag acgttttgca gcagcagtcg cttcacgttc 2820
gctcgcgtat cggtgattca ttctgctaac cagtaaggca accccgccag cctagccggg 2880
tcctcaacga caggagcacg atcatgcgca cccgtggcca ggacccaacg ctgcccgaga 2940
tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga tatgttctgc caagggttgg 3000
tttgcgcatt cacagttctc cgcaagaatt gattggctcc aattcttgga gtggtgaatc 3060
cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc atgcaccgcg 3120
acgcaacgcg gggaggcaga caaggtatag ggcggcgcct acaatccatg ccaacccgtt 3180
ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc agcggtccag tgatcgaagt 3240
taggctggta agagccgcga gcgatccttg aagctgtccc tgatggtcgt catctacctg 3300
cctggacagc atggcctgca acgcgggcat cccgatgccg ccggaagcga gaagaatcat 3360
aatggggaag gccatccagc ctcgcgtcgc gaacgccagc aagacgtagc ccagcgcgtc 3420
ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa cgtttggtgg cgggaccagt 3480
gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc gcaagcgaca ggccgatcat 3540
cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc cagagcgctg ccggcacctg 3600
tcctacgagt tgcatgataa agaagacagt cataagtgcg gcgacgatag tcatgccccg 3660
cgcccaccgg aaggagctga ctgggttgaa ggctctcaag ggcatcggtc gagatcccgg 3720
tgcctaatga gtgagctaac ttacattaat tgcgttgcgc tcactgcccg ctttccagtc 3780
gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 3840
gcgtattggg cgccagggtg gtttttcttt tcaccagtga gacgggcaac agctgattgc 3900
ccttcaccgc ctggccctga gagagttgca gcaagcggtc cacgctggtt tgccccagca 3960
ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata acatgagctg tcttcggtat 4020
cgtcgtatcc cactaccgag atatccgcac caacgcgcag cccggactcg gtaatggcgc 4080
gcattgcgcc cagcgccatc tgatcgttgg caaccagcat cgcagtggga acgatgccct 4140
cattcagcat ttgcatggtt tgttgaaaac cggacatggc actccagtcg ccttcccgtt 4200
ccgctatcgg ctgaatttga ttgcgagtga gatatttatg ccagccagcc agacgcagac 4260
gcgccgagac agaacttaat gggcccgcta acagcgcgat ttgctggtga cccaatgcga 4320
ccagatgctc cacgcccagt cgcgtaccgt cttcatggga gaaaataata ctgttgatgg 4380
gtgtctggtc agagacatca agaaataacg ccggaacatt agtgcaggca gcttccacag 4440
caatggcatc ctggtcatcc agcggatagt taatgatcag cccactgacg cgttgcgcga 4500
gaagattgtg caccgccgct ttacaggctt cgacgccgct tcgttctacc atcgacacca 4560
ccacgctggc acccagttga tcggcgcgag atttaatcgc cgcgacaatt tgcgacggcg 4620
cgtgcagggc cagactggag gtggcaacgc caatcagcaa cgactgtttg cccgccagtt 4680
gttgtgccac gcggttggga atgtaattca gctccgccat cgccgcttcc actttttccc 4740
gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg ggaaacggtc tgataagaga 4800
caccggcata ctctgcgaca tcgtataacg ttactggttt cacattcacc accctgaatt 4860
gactctcttc cgggcgctat catgccatac cgcgaaaggt tttgcgccat tcgatggtgt 4920
ccgggatctc gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg 4980
ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac 5040
agtcccccgg ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg 5100
aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca 5160
cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc 5220
ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag 5280
aaataatttt gtttaacttt aagaaggaga tatacatatg acagagcccg gtacgtccgt 5340
gtcagcgccc gtcgccttcc cccaggaccg cacgtgtccc tacgacccgc ccacagccta 5400
cgacccgctg cgcgaggggc gtccgctgtc gcgggtctcc ctctacgacg gacgcagcgt 5460
gtgggtggtc accgggcacg ccgccgcccg tgcactcctc tccgaccagc ggctttcctc 5520
cgacgcgacg ctgcccaggt tccccgcgac caccgagcgg ttcgaggccg tacgcacccg 5580
ccgggtggcg ctgctcggtg tggacgaccc cgagcaccgc acccagcgcc gcatgctggt 5640
cccgagcttt accctcaagc gggccgccgc cctgcgcccg cgcatccagg agaccgtcga 5700
cgggctgctc gacgccatgg aggcacaggg cccgccggcc gagctggtga gcgcgttcgc 5760
gctgccgctg ccctcgatgg tgatctgcgc cctgctcggc gtcccgtacg ccgaccacga 5820
cttcttcgag tcccagtccc gcaggctgct gcggggcccc gggatcgccg aggtccagga 5880
cgccgcggcc caactggacg actacctgta cgcgttgatc gaccggaagc ggaaggaacc 5940
cggggacggg ctcctggacg atctcatcca ggagcagctg aaccggggca cggtggatcg 6000
cgccgagctg gtctccctgg cgacgctcct gctgatcgcc ggacacgaga cgacggcgaa 6060
catgatctcg ctcggtacgt tcaccctgct ccggcatccc gaacagctgg ccgagctgcg 6120
ggccgagccg ggcctcatgc ccgccgccgt ggaggagctg ctgcgtttcc tgtccatcgc 6180
cgacgggctg ctgcgggtgg ccaccgagga catcgaagtg gccggtacga ccatcagggc 6240
cgatgagggc gtcgtcttcg cgacctccgt catcaaccgc gacgcggccg gcttcgccga 6300
gcccgacgcc ctggactggc atcgctcggc ccgccaccat gtcgccttcg gtttcggcat 6360
ccaccagtgc ctcgggcaga acctggccag ggccgagatg gagatcgccc tgggcacgct 6420
ctcgagcggc tgcccggcct gcggctggcg gcgccggccg acgagatccc cttcaaaccg 6480
ggcgacacga tccaggggat gctggaactc cccgtgacct ggtaagaggc tgcggtccat 6540
gcacaacgac agcaacaaca gcaacggcaa caactcccac agcaacggca acgtcaacag 6600
caacggcagc aacagcgacg acaacgtgat cgtgatcgac cgggacctct gcatcggtgc 6660
cgggcagtgc gccctgaccg cgcccggtgt cttcacccag gacgacgacg gattcagcga 6720
actgctgccc ggccgggagg acggggcagg cgacccgatg gtgcgcgagg ccgcacggtc 6780
ctgcccggtg ggggccatca cggtcccgcg gtcggcaagc tgaactagtc gggagtgcgt 6840
tatatgaacg caaacgacaa cgtggtcatc gtcggtaccg gactggctgg cgttgaggtc 6900
gccttcggcc tgcgcgccag cggctgggaa ggcaatatcc ggttggtggg ggatgcgacg 6960
gtaattcccc atcacctacc accgctatcc aaagcttact tggccggcaa agccacagcg 7020
gaaagcctgt acctgagaac cccagatgcc tatgcagcgc agaacatcca actactcgga 7080
ggcacacagg taacggctat caaccgcgac cgacagcaag taatcctatc ggatggccgg 7140
gcactggatt acgaccggct ggtattggct accggagggc gtccaagacc cctaccggtg 7200
gccagtggcg cagttggaaa ggcgaacaac tttcgatacc tgcgcacact cgaggacgcc 7260
gagtgcattc gccggcagct gattgcggat aaccgtctgg tggtgattgg tggcggctac 7320
attggccttg aagtggctgc caccgccatc aaggcgaaca tgcacgtcac cctgcttgat 7380
acggcagccc gggttctgga gcgggttacc gccccgccgg tatcggcctt ttacgagcac 7440
ctacaccgcg aagccggcgt tgacatacga accggcacgc aggtgtgcgg gttcgagatg 7500
tcgaccgacc aacagaaggt taccgccgtc ctctgcgagg acggcacaag gctgccagcg 7560
gatctggtaa tcgccgggat tggcctgata ccaaactgcg agttggccag tgcggccggc 7620
ctgcaggttg ataacggcat cgtgatcaac gaacacatgc agacctctga tcccttgatc 7680
atggccgtcg gcgactgtgc ccgatttcac agtcagctct atgaccgctg ggtgcgtatc 7740
gaatcagtgc ccaatgcctt ggagcaggca cgaaagatcg ccgccatcct ctgtggcaag 7800
gtgccacgcg atgaggcggc gccctggttc tggtccgatc agtatgagat cggattgaag 7860
atggtcggac tgtccgaagg gtacgaccgg atcattgtcc gcggctcttt ggcgcaaccc 7920
gacttcagcg ttttctacct gcagggagac cgggtattgg cggtcgatac agtgaaccgt 7980
ccagtggagt tcaaccagtc aaaacaaata atcacggatc gtttgccggt tgaaccaaac 8040
ctactcggtg acgaaagcgt gccgttaaag gaaatcatcg ccgccgccaa agctgaactg 8100
agtagtgcct gaaatctata cccacaataa atcaccgttt tgccccatag cgtgtgagga 8160
taaacagatg tctaaagtag tgtatgtgtc acatgatgga acgcgtcgcg aactggatgt 8220
ggcggatggc gtcagcctga tgcaggctgc agtctccaat ggtatctacg atattgtcgg 8280
tgattgtggc ggcagcgcca gctgtgccac ctgccatgtc tatgtgaacg aagcgttcac 8340
ggacaaggtg cccgccgcca acgagcggga aatcggcatg ctggagtgcg tcacggccga 8400
actgaagccg aacagcaggc tctgctgcca gatcatcatg acgcccgagc tggatggcat 8460
cgtggtcgat gttcccgata ggcaatgggg atccggctgc taacaaagcc cgaaaggaag 8520
ctgagttggc tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac 8580
gggtcttgag gggttttttg ctgaaaggag gaactatatc cggatatccc gcaagaggcc 8640
cggcagtacc ggcataacca agcctatgcc tacagcatcc agggtgacgg tgccgaggat 8700
gacgatgagc gcattgttag atttcataca cggtgcctga ctgcgttagc aatttaactg 8760
tgataaacta ccgcattaaa gcttatcgat gataagctgt caaacatgag aa 8812
Claims (2)
1. A process for biotransformation of steroid compounds to obtain hydroxylated compounds, characterized in that it comprises the following steps:
(1) preparation of co-expressed recombinant E.coli: carrying out double mutation on the sav7469 gene of the streptomyces avermitilis MA4680 coding CYP105D7 at the position R70A/R190A by using a site-directed mutagenesis kit to obtain a double mutation gene sequence sav7469R70A/R190AThe Nde I and Spe I are used for enzyme digestion to obtain a double mutation gene sequence and a vector pT7NS-camAB, and the double mutation gene sequence and the vector pT7NS-camAB are connected to obtain a recombinant vector pET11:: sav7469R70A/R190AThe recombinant vector is introduced into Escherichia coli BL21 (DE)3) Finally obtaining the coexpression recombinant escherichia coli; the sav7469 of the encoding CYP105D7 of the streptomyces avermitilis MA4680R70A/R190AThe double mutation gene sequence is shown as SEQ ID NO.2, the gene sequence of the vector pT7NS-camAB is shown as SEQ ID NO.9, the recombinant vector pET11 is sav7469R70A/R190AThe gene sequence of camA-camB is shown as SEQ ID NO. 10;
(2) pre-culture and scale-up culture of recombinant E.coli: adding the recombinant escherichia coli into an LB culture medium, and culturing for 18 hours under the conditions that the pH is 7.4-7.6, the temperature is 37 ℃ and the rotating speed is 200rpm to obtain a seed solution of the recombinant escherichia coli; inoculating the seed liquid of the recombinant escherichia coli into an M9 culture medium for amplification culture and fermentation; the volume ratio of the seed solution to the M9 culture medium is 1:100, after inoculation, the recombinant Escherichia coli is cultured at 37 ℃ until the OD of the bacterial solution600The value reaches 0.6-0.8, then IPTG and 5-aminolevulinic acid salt are added to induce the thallus to promote protein expression, the final concentration of the IPTG is 0.2mM, and the final concentration of the 5-aminolevulinic acid salt is 0.5 mM; then reducing the temperature to 22 ℃, reducing the rotating speed to 180rpm, and continuing culturing for 20 h;
(3) and (3) collecting thalli: centrifuging at 4 deg.C and 4000rpm for 10min, and collecting the precipitated thallus; washing the thalli by adopting a sodium phosphate buffer solution at 4 ℃, collecting the washed thalli, and suspending the thalli in the sodium phosphate buffer solution to obtain a reaction solution for carrying out biotransformation on the steroid compound;
(4) biotransformation of steroid compounds: dissolving a steroid compound in methanol, and adding the methanol to the reaction solution obtained in the step (3) for biotransformation to obtain a transformation solution; the final concentration of the steroid compound is 0.1mM, and the biotransformation condition is 180rpm, and the temperature is 30 ℃ for 20 h; after the reaction is finished, adding ethyl acetate with the same volume as the conversion solution to stop the reaction, fully mixing the ethyl acetate and the conversion solution to ensure that the product completely enters an ethyl acetate organic phase, shaking the mixture for 2min, centrifuging the mixed solution at 13000rpm for 10min, and collecting supernatant; drying the supernatant to obtain 2 beta position and 16 beta position hydroxylated compounds or 2 beta position and 16 beta position hydroxylated compounds simultaneously;
wherein the steroid compound is testosterone, progesterone or androstenedione.
2. The method of claim 1, wherein in step (3), the sodium phosphate buffer comprises 1mM EDTA, 2mM dithiothreitol and 10% by volume glycerol, the solvent is water, and the pH is 7.2.
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