Dehydrogenase, gene, engineered bacteria and application
By screening and modifying highly active dehydrogenases and constructing engineered bacteria, the problem of low efficiency in the biological dehydrogenation reaction of steroid drugs has been solved, achieving efficient and low-cost production of steroid drugs, especially high conversion rate and high selectivity of methylprednisolone intermediates.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HUNAN NORCHEM PHARMACEUTICAL CO LTD
- Filing Date
- 2023-01-05
- Publication Date
- 2026-06-05
AI Technical Summary
In existing technologies, the biodehydrogenation reaction process of steroid drugs is relatively long, with low conversion rate and low catalytic activity, which cannot meet the needs of industrial production, especially for high-value substrates such as methylprednisolone.
We screened and modified reductases with good stability and broad substrate spectrum, constructed highly active dehydrogenases, and obtained engineered bacteria through genetic modification to catalyze the biological dehydrogenation reaction of steroid hormones. We adopted a whole-cell catalysis method, eliminating the need for enzyme purification steps.
It improves substrate conversion efficiency, reduces costs, significantly enhances industrialization potential, exhibits high dehydrogenation activity, strong stereoselectivity and regioselectivity, and generates no other conformational impurities.
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Figure BDA0004039785830000051
Abstract
Description
Technical Field
[0001] This invention belongs to the field of steroidal drug biocatalytic synthesis technology, specifically involving a dehydrogenase, gene, engineered bacteria and its application. Background Technology
[0002] Steroids, also known as steroids, play an important role in regulating human metabolism, protein synthesis, and reproductive function. They mainly include phytosterols, bile acids, insect metamorphic hormones, cardiac glycosides, steroidal saponins, and steroidal alkaloids. Steroid drugs are the second largest class of drugs used clinically, after antibiotics.
[0003] Currently, the synthesis of steroid drugs using chemical methods often suffers from drawbacks such as cumbersome procedures, high costs, and residual reaction solvents. Compared with chemical synthesis, the biotransformation of steroid drugs has advantages such as simple procedures, high specificity, mild reaction conditions, and no pollution. Therefore, biosynthesis has become an important industrial route for synthesizing steroid drugs.
[0004] Methylprednisolone (11β,17α,21-trihydroxy-6α-methylpregn-1,4-diene-3,20-dione) and its derivatives are a class of highly potent adrenocortical steroids used to combat immune stress and are widely applied in organ transplantation and clinical treatment of immune stress. This drug is a synthetic glucocorticoid with typical glucocorticoid effects, such as anti-inflammatory activity and suppression of the immune response. Compared to hydrocortisone, a representative glucocorticoid, its anti-inflammatory efficacy is four times that of hydrocortisone. The dehydrogenation of the steroid nucleus at the C1,2 position to introduce a double bond can significantly increase its anti-inflammatory effect, and this is an important reaction process in the industrial production of corticosteroids. For example, the anti-inflammatory effect of dehydrocortisone acetate, produced by C1,2 dehydrogenation of cortisone acetate, is 3-4 times higher than that of the original. Chemical methods often use the selenium dioxide method for C1,2 dehydrogenation, but this has significant side effects, often resulting in the presence of selenium, which is harmful to the human body, in the product. Therefore, biological dehydrogenation, with its advantages of being green, environmentally friendly, and highly specific, has gradually become a key step in the synthesis of steroidal anti-inflammatory hormone drugs.
[0005] In recent years, domestic research progress on the biodehydrogenation of methylprednisolone has been limited. CN1978457A discloses a method for biodehydrogenation using Arthrobacterium during the synthesis of methylprednisolone, but the biodehydrogenation reaction is lengthy (over 48 hours) and has a low conversion rate (around 85%), making industrial production difficult. Currently reported steroidal C1,2-position dehydrogenases (3-ketosteroid-Δ1-dehydrogenase, KstD) exhibit low catalytic activity for steroidal substrates. Furthermore, the low substrate solubility leads to low substrate concentrations in practical applications, failing to meet the requirements for industrial production. Simultaneously, no steroidal C1,2-position dehydrogenases with high catalytic activity have been found for certain economically valuable substrates such as methylprednisolone. These problems severely restrict the development of this enzyme; therefore, finding novel, highly active, and thermostable steroidal C1,2-position dehydrogenases is particularly important. Summary of the Invention
[0006] The technical problem to be solved by the present invention is to provide a dehydrogenase, gene, engineered bacteria and its application to improve the conversion rate of steroid hormones and reduce the impurity content.
[0007] One embodiment of the present invention includes a dehydrogenase, the amino acid sequence of which is SEQ ID NO.1 or SEQ ID NO.2.
[0008] The sequence of SEQ ID NO.1 is:
[0009] MDWAEEYDVL VAGSGAGGMA GTYTAAREGL SVCLVEAGDK FGGTTAYSGGCGAWFPANPVLLRAGTDDTI EDALEYYRAV VGDRTPADLQ ETYVRGGAGL VAYLEEDDHFSFESYPWPDYFGDAPKARRD GQRHIIPTPL PVPSAPELRE VVRGPLDNDR LGTPQPDDLFIGGRALVARFLTALATYPHA TLVRETALAE LVVEDGVVVG AIVETDGVRR AIRARRGVLLAAGGFEANDELRQKYGVPGV ARDTMGPPTN VGAAHQAAIA VGADTDLMGE AWWSPGLTHPDGRSAFALWFTGGIFVDGAG RRFVNESAPY DRLGRAVIDH LTEGGVTPRY WMVYDHKEGSIPPVRATNVSMVDEEQYVAA GLWHTADTLP ELAALIGVPA DALVATVARF NELVADGYDADFGRGGEAYDRFFSGGEPPL VSIDEGPFHA AAFGISDLGT KGGLRTDTSA RVLTADGTPIGGLYAAGNTMAAPSGTTYPG GGNPIGTSML FSHLAVRHMG TEDAR。
[0010] The sequence of SEQ ID NO.2 is:
[0011] MDWAEEYDVL VAGSGAGGMA GTYTAAREGL SVCLVEAGDK FGGTTAYSGGGGAWFPANPVLLRAGTDDTI EDALEYYRAV VGDRTPADDLQ ETYVRGGAGL VAYLEEDDHFSFESYPWPDYFGDAPKARRD GQRHIIPTPL PVPSAPELRE VVRGPLDNDR LGTPQPDDLFIGGRALVARFLTALATYPHA TLVRETALAE LVVEDGVVVG AIVETDGVRR AIRARRGVLLNAGGFEANDELRQKYGVPGV ARDTMGPPTN VGAAHQAAIA VGADTDLMGE AWWSPGLTHPDGRSAFALWFTGGIFVDGAG RRFVNESAPY DRLGRAVIDH LTEGGVTPRY WMVYDHKEGSIPPVRATNVSMVDEEQYVAA GLWHTADTLP ELAALIGVPA DALVATVARF NELVADGYDADFGRGGEAYDRFFSGGEPPL VSIDEGPFHA AAFGISDLGT KGGLRTDTSA RVLTADGTPIGGLYAAGNTMAAPSGTTYPG GGNPIGTSML FSHLAVRHMG TEDAR.
[0012] The inventors screened and modified numerous stable reductases from plant and microbial sources to obtain the two dehydrogenases mentioned above. The main difference between the two dehydrogenases is that in SEQ ID NO.1, position 51 is C and position 231 is A; while in SEQ ID NO.2, position 51 is G and position 231 is N.
[0013] This invention provides a gene that encodes the aforementioned dehydrogenase. The nucleotide sequence of the gene may be SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6.
[0014] The dehydrogenase sequence corresponding to the gene with nucleotide sequences SEQ ID NO.3 and SEQ ID NO.4 is SEQ ID NO.1, and the dehydrogenase sequence corresponding to the gene with nucleotide sequences SEQ ID NO.5 and SEQ ID NO.6 is SEQ ID NO.2.
[0015] The sequence of SEQ ID NO.3 is:
[0016] ATGGACTGGG CAGAGGAGTA CGACGTACTG GTGGCGGGCT CCGGCGCCGG CGGCATGGCCGGGACCTACA CCGCGGCCCG CGAGGGGCTC AGCGTGTGCC TGGTCGAGGC CGGGGACAAG
[0017] TTCGGCGGGA CGACCGCCTA CTCCGGCGGC TGTGGGGCCT GGTTCCCCGCGAACCCGGTGCTGCTGCGGG CGGGCACCGA CGACACGATC GAGGACGCTC TCGAGTACTACCGAGCGGTCGTCGGCGACC GCACCCCCGC GGACCTGCAG GAGACCTACG TCCGCGGCGGCGCCGGCCTGGTCGCCTACC TCGAGGAGGA CGACCACTTC TCCTTCGAGT CCTACCCGTGGCCGGACTACTTCGGCGACG CCCCCAAGGC CCGTCGCGAC GGCCAGCGGC ACATCATCCCGACGCCGCTGCCGGTGCCCT CCGCACCCGA GCTGCGCGAG GTGGTCCGCG GGCCGCTCGACAACGACCGGCTCGGCACGC CGCAGCCCGA CGACCTGTTC ATCGGCGGAC GGGCGCTCGTCGCCCGCTTCCTGACCGCGC TCGCGACCTA CCCCCACGCC ACGCTCGTGC GCGAGACCGCACTGGCCGAGCTCGTCGTCG AGGACGGCGT CGTGGTCGGC GCGATCGTCG AGACCGACGGCGTCCGCCGCGCGATCCGGG CCCGCCGCGG CGTCCTCCTG GCCGCGGGCG GCTTCGAGGCCAATGACGAGCTCCGCCAGA AGTACGGCGT CCCCGGCGTC GCGCGCGACA CGATGGGCCCGCCGACCAACGTCGGCGCCG CGCACCAGGC CGCGATCGCG GTCGGCGCCG ACACCGACCTGATGGGCGAGGCCTGGTGGT CCCCCGGGCT GACCCACCCC GACGGACGAT CGGCGTTCGCGCTCTGGTTCACCGGCGGCA TCTTCGTCGA CGGCGCCGGC CGGCGCTTCG TCAACGAGTCGGCGCCGTACGACCGGCTCG GCCGCGCCGT CATCGACCAC CTCACCGAGG GCGGCGTCACCCCGCGGTACTGGATGGTCT ACGACCACAA GGAGGGCTCGATCCCCCCGG TGCGCGCCACCAACGTCTCGATGGTCGACG AGGAGCAGTA CGTCGCCGCG GGCCTGTGGC ACACCGCCGACACGCTGCCCGAGCTGGCCG CGCTGATCGG CGTCCCCGCC GACGCGCTGG TCGCCACGGTCGCGCGCTTCAACGAGCTCG TCGCCGACGG GTACGACGCG GACTTCGGCC GCGGCGGCGAGGCCTACGACCGGTTCTTCT CCGGCGGCGA GCCGCCGCTG GTGAGCATCG ACGAGGGGCCGTTCCACGCGGCCGCCTTCG GCATCTCCGA CCTCGGCACC AAGGGCGGGC TGCGCACCGACACGTCCGCGCGCGTGCTGA CCGCGGACGG CACGCCGATC GGGGGCCTCT ACGCAGCCGGCAATACGATGGCGGCGCCGA GCGGCACCAC CTACCCGGGC GGTGGCAACC CGATCGGGACAAGCATGCTCTTCAGCCACC TCGCCGTGCG GCACATGGGC ACCGAGGACG CGCGATGA。
[0018] The sequence of SEQ ID NO.4 is that the sequence at positions 151 - 153 is replaced with TGC based on SEQ ID NO.3.
[0019] The sequence of SEQ ID NO.5 is:
[0020] ATGGACTGGG CAGAGGAGTA CGACGTACTG GTGGCGGGCT CCGGCGCCGGCGGCATGGCCGGGACCTACA CCGCGGCCCG CGAGGGGCTC AGCGTGTGCC TGGTCGAGGCCGGGGACAAGTTCGGCGGGA CGACCGCCTA CTCCGGCGGC GGTGGGGCCT GGTTCCCCGCGAACCCGGTGCTGCTGCGGG CGGGCACCGA CGACACGATC GAGGACGCTC TCGAGTACTACCGAGCGGTCGTCGGCGACC GCACCCCCGC GGACCTGCAG GAGACCTACG TCCGCGGCGGCGCCGGCCTGGTCGCCTACC TCGAGGAGGA CGACCACTTC TCCTTCGAGT CCTACCCGTG GCCGGACTAC
[0021] TTCGGCGACG CCCCCAAGGC CCGTCGCGAC GGCCAGCGGC ACATCATCCCGACGCCGCTGCCGGTGCCCT CCGCACCCGA GCTGCGCGAG GTGGTCCGCG GGCCGCTCGACAACGACCGGCTCGGCACGC CGCAGCCCGA CGACCTGTTC ATCGGCGGAC GGGCGCTCGTCGCCCGCTTCCTGACCGCGC TCGCGACCTA CCCCCACGCC ACGCTCGTGC GCGAGACCGCACTGGCCGAGCTCGTCGTCG AGGACGGCGT CGTGGTCGGC GCGATCGTCG AGACCGACGGCGTCCGCCGCGCGATCCGGG CCCGCCGCGG CGTCCTCCTG AACGCGGGCG GCTTCGAGGCCAATGACGAGCTCCGCCAGA AGTACGGCGT CCCCGGCGTC GCGCGCGACA CGATGGGCCCGCCGACCAACGTCGGCGCCG CGCACCAGGC CGCGATCGCG GTCGGCGCCG ACACCGACCTGATGGGCGAGGCCTGGTGGT CCCCCGGGCT GACCCACCCC GACGGACGAT CGGCGTTCGCGCTCTGGTTCACCGGCGGCA TCTTCGTCGA CGGCGCCGGC CGGCGCTTCG TCAACGAGTCGGCGCCGTACGACCGGCTCG GCCGCGCCGT CATCGACCAC CTCACCGAGG GCGGCGTCACCCCGCGGTACTGGATGGTCT ACGACCACAA GGAGGGCTCG ATCCCCCCGG TGCGCGCCACCAACGTCTCGATGGTCGACG AGGAGCAGTA CGTCGCCGCG GGCCTGTGGC ACACCGCCGACACGCTGCCCGAGCTGGCCG CGCTGATCGG CGTCCCCGCC GACGCGCTGG TCGCCACGGTCGCGCGCTTCAACGAGCTCG TCGCCGACGG GTACGACGCG GACTTCGGCC GCGGCGGCGAGGCCTACGACCGGTTCTTCT CCGGCGGCGA GCCGCCGCTGGTGAGCATCG ACGAGGGGCCGTTCCACGCGGCCGCCTTCG GCATCTCCGA CCTCGGCACC AAGGGCGGGC TGCGCACCGACACGTCCGCGCGTGCTGA CCGCGGACGG CACGCCGATC GGGGGCCTCT ACGCAGCCGGCAATACGATGGCGGCGCCGA GCGGCACCAC CTACCCGGGC GGTGGCAACC CGATCGGGACAAGCATGCTCTTCAGCCACC TCGCCGTGCG GCACATGGGC ACCGAGGACG CGCGATGA.
[0022] The sequence of SEQ ID NO.6 is based on SEQ ID NO.5, with the sequences 691-693 replaced by AAT.
[0023] This invention provides an engineered bacterium containing the aforementioned gene. The gene can be integrated into the chromosome of a host microorganism, or the gene can be placed in a recombinant plasmid, which is then placed within the host microorganism. The preferred host microorganism for the engineered bacterium is *Escherichia coli*.
[0024] This invention provides the application of the engineered bacteria described above in the conversion of steroid hormones into dehydrogenation products.
[0025] The steroid hormone is 11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione.
[0026] A bacterial culture containing engineered bacteria is mixed with a substrate steroid hormone, an electron acceptor, and an organic solvent. The mixture is then subjected to a catalytic reaction and further processed to obtain the dehydrogenation product, 6-methyldehydrogenase, which is the dehydrogenation product of the substrate and is typically C6-3. 1,2 Dehydrogenation products.
[0027] The electron acceptor is methyl phenazine sulfate or 2,6-dichlorophenolindophenol, and the organic solvent is isopropanol.
[0028] The method for preparing the bacterial suspension containing the engineered bacteria is as follows: The engineered bacteria are cultured for a period of time under the induction of IPTG (isopropyl-β-D-thiogalactoside), the bacterial cells are collected, and a buffer solution is added to obtain the bacterial suspension containing the engineered bacteria. The IPTG concentration is preferably 0.5 mM, the culture temperature is room temperature, the rotation speed is 180 rpm, and the culture time is 8-12 h. Before adding the buffer solution, the bacterial cells can be washed with a 0.9% sodium chloride solution; the buffer solution is preferably Tris-HCl with a pH of 7.0.
[0029] When the engineered bacteria of this application are used for dehydrogenation, the reaction formula for the conversion of the substrate 6-methyl dehydrogenase to the product is as follows:
[0030]
[0031] The beneficial effects of this invention are that the dehydrogenase used in this invention exhibits high dehydrogenation activity, strong stereoselectivity and regioselectivity, and no other conformational impurities are generated. The engineered bacteria constructed can efficiently secrete the above-mentioned dehydrogenase, effectively improving the substrate conversion efficiency, especially for the substrate 6-methylglycate (11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione), which is inexpensive and has significant industrialization potential. The dehydrogenase mutant of this invention has well-defined physicochemical properties, and the whole-cell catalysis method eliminates the cumbersome steps of enzyme purification, reducing costs and simplifying operation.
[0032] We conducted research on the biodehydrogenation of 6-methoxygenated compound (11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione), an intermediate of methylprednisolone, and constructed a mutant engineered strain that efficiently reacts to the dehydrogenation of 6-methoxygenated compound. Detailed Implementation
[0033] Comparative Example 1
[0034] The following steps were taken to construct the engineered strain E. coli BL21(DE3) / pET28a-PsKstD, which contains an unmutated steroidal C1,2-position dehydrogenase:
[0035] Using nucleotide SEQ ID NO:7 (NCBI: NZ_BJMC01000016.1) as a template, the amino acid sequence encoded by nucleotide SEQ ID NO:7 is nucleotide SEQ ID NO:8. Codon optimization was performed on the expression vector pET-28a(+), and the entire gene sequence was synthesized. Enzyme restriction sites BamHI and XhoI were designed at both ends and subcloned into the corresponding sites on the vector pET28a(+), thus obtaining the recombinant plasmid pET28a-PsKstD.
[0036] The constructed recombinant plasmid pET28a-PsKstD was transformed into competent E. coli BL21(DE3) cells prepared by the calcium chloride method. The cells were plated on LB medium containing 50 mg / L kanamycin (containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L NaCl) and cultured overnight. Positive clones were selected and sent to a sequencing company for verification to obtain recombinant E. coli BL21(DE3) / pET28a-PsKstD expressing wild-type steroid C1,2-position dehydrogenase.
[0037] The sequence of SEQ ID NO:7 is:
[0038] ATGGACTGGG CAGAGGAGTA CGACGTACTG GTGGCGGGCT CCGGCGCCGGCGGCATGGCCGGGACCTACA CCGCGGCCCG CGAGGGGCTC AGCGTGTGCC TGGTCGAGGCCGGGGACAAGTTCGGCGGGA CGACCGCCTA CTCCGGCGGC GGTGGGGCCT GGTTCCCCGCGAACCCGGTGCTGCTGCGGG CGGGCACCGA CGACACGATC GAGGACGCTC TCGAGTACTA CCGAGCGGTC
[0039] GTCGGCGACC GCACCCCCGC GGACCTGCAG GAGACCTACG TCCGCGGCGGCGCCGGCCTGGTCGCCTACC TCGAGGAGGA CGACCACTTC TCCTTCGAGT CCTACCCGTGGCCGGACTACTTCGGCGACG CCCCCAAGGC CCGTCGCGAC GGCCAGCGGC ACATCATCCCGACGCCGCTGCCGGTGCCCT CCGCACCCGA GCTGCGCGAG GTGGTCCGCG GGCCGCTCGACAACGACCGGCTCGGCACGC CGCAGCCCGA CGACCTGTTC ATCGGCGGAC GGGCGCTCGTCGCCCGCTTCCTGACCGCGC TCGCGACCTA CCCCCACGCC ACGCTCGTGC GCGAGACCGCACTGGCCGAGCTCGTCGTCG AGGACGGCGT CGTGGTCGGC GCGATCGTCG AGACCGACGGCGTCCGCCGCGCGATCCGGG CCCGCCGCGG CGTCCTCCTG GCCGCGGGCG GCTTCGAGGCCAATGACGAGCTCCGCCAGA AGTACGGCGT CCCCGGCGTC GCGCGCGACA CGATGGGCCCGCCGACCAACGTCGGCGCCG CGCACCAGGC CGCGATCGCG GTCGGCGCCG ACACCGACCTGATGGGCGAGGCCTGGTGGT CCCCCGGGCT GACCCACCCC GACGGACGAT CGGCGTTCGCGCTCTGGTTCACCGGCGGCA TCTTCGTCGA CGGCGCCGGC CGGCGCTTCG TCAACGAGTCGGCGCCGTACGACCGGCTCG GCCGCGCCGT CATCGACCAC CTCACCGAGG GCGGCGTCACCCCGCGGTACTGGATGGTCT ACGACCACAA GGAGGGCTCG ATCCCCCCGG TGCGCGCCACCAACGTCTCGATGGTCGACG AGGAGCAGTA CGTCGCCGCG GGCCTGTGGC ACACCGCCGACACGCTGCCCGAGCTGGCCG CGCTGATCGG CGTCCCCGCCGACGCGCTGG TCGCCACGGTCGCGCGCTTCAACGAGCTCG TCGCCGACGG GTACGACGCG GACTTCGGCC GCGGCGGCGAGGCCTACGACCGGTTCTTCT CCGGCGGCGA GCCGCCGCTG GTGAGCATCG ACGAGGGGCCGTTCCACGCGGCCGCCTTCG GCATCTCCGA CCTCGGCACC AAGGGCGGGC TGCGCACCGACACGTCCGCGCGCGTGCTGA CCGCGGACGG CACGCCGATC GGGGGCCTCT ACGCAGCCGGCAATACGATGGCGGCGCCGA GCGGCACCAC CTACCCGGGC GGTGGCAACC CGATCGGGACAAGCATGCTCTTCAGCCACC TCGCCGTGCG GCACATGGGC ACCGAGGACG CGCGATGA。
[0040] The sequence of SEQ ID NO:8 is:
[0041] MDWAEEYDVL VAGSGAGGMA GTYTAAREGL SVCLVEAGDK FGGTTAYSGGGGAWFPANPVLLRAGTDDTI EDALEYYRAV VGDRTPADDLQ ETYVRGGAGL VAYLEEDDHFSFESYPWPDYFGDAPKARRD GQRHIIPTPL PVPSAPELRE VVRGPLDNDR LGTPQPDDLFIGGRALVARFLTALATYPHA TLVRETALAE LVVEDGVVVG AIVETDGVRR AIRARRGVLLAAGGFEANDELRQKYGVPGV ARDTMGPPTN VGAAHQAAIA VGADTDLMGE AWWSPGLTHPDGRSAFALWFTGGIFVDGAG RRFVNESAPY DRLGRAVIDH LTEGGVTPRY WMVYDHKEGSIPPVRATNVSMVDEEQYVAA GLWHTADTLP ELAALIGVPA DALVATVARF NELVADGYDADFGRGGEAYDRFFSGGEPPL VSIDEGPFHA AAFGISDLGT KGGLRTDTSA RVLTADGTPIGGLYAAGNTMAAPSGTTYPG GGNPIGTSML FSHLAVRHMG TEDAR.
[0042] Comparative Example 2
[0043] Based on SEQ ID NO:7 of Comparative Example 1, site-directed saturation mutations were performed at positions 12, 14, 17, 38, 45, 196, 255, 263, 449, 488, and 495 on SEQ ID NO:7, resulting in amino acid sequences containing 19 amino acids other than the original amino acids.
[0044] The genes corresponding to all the mutated amino acids in Comparative Example 2 were used to obtain whole-cell catalytic solutions in the same manner as in Comparative Example 1. The 6-methylformate was then catalytically converted in the manner described in Example 4, and the conversion rate was 0.
[0045] Examples 1-2
[0046] Based on SEQ ID NO:7 of Comparative Example 1, mutations were made at positions 51 and 231 of SEQ ID NO:7 to obtain SEQ ID NO:3-6, respectively. The sequences of the mutated dehydrogenases are shown in SEQ ID NO:1-2, respectively.
[0047] The site-directed mutagenesis primers are shown in Table 1. Reverse PCR was used to perform site-directed mutagenesis on the recombinant plasmid pET28a-PsKstD in Comparative Example 1, yielding the dehydrogenase gene as shown in Examples 1-2. The reverse PCR product was treated with DpnI enzyme (purchased from Takara) and purified, then transformed into competent E. coli BL21(DE3) cells. Positive clones were screened and sequenced correctly to obtain the mutant strains E. coli BL21(DE3) / pET28a-PsKstD-G51C and E. coli BL21(DE3) / pET28a-PsKstD-A231N.
[0048] Table 1 Primer sequences
[0049] Primer name Primer sequence Serial Number G51C-F TACTCCGGCGGCTGCGGGGCCTGGTTC SEQ ID NO:9 G51C-R GAACCAGGCCCCGCAGCCGCCGGAGTA SEQ ID NO:10 A231N-F GGCGTCCTCCTGAACGCGGGCGGCTTC SEQ ID NO:11 A231N-R GAAGCCGCCCGCGTTCAGGAGGACGCC SEQ ID NO:12
[0050] Example 3
[0051] Preparation of whole-cell catalytic solution
[0052] (1) The engineered Escherichia coli strains E. coli BL21(DE3) / pET28a-PsKstD-G51C and E. coli BL21(DE3) / pET28a-PsKstD-A231N obtained in Example 2 were induced and cultured at 25°C with IPTG at a final concentration of 0.5 mM. After fermentation for 8 h, the bacterial cells were collected and the wet weight of the bacterial cells was weighed.
[0053] The wet-weight bacterial cells were washed three times with 0.9% NaCl solution, and then resuspended in a borate-borax buffer solution containing 0.2M boric acid and 0.05M borax at pH 7.0 to obtain whole-cell catalytic solutions of engineered E. coli strains E. coli BL21(DE3) / pET28a-PsKstD-G51C and E. coli BL21(DE3) / pET28a-PsKstD-A231N.
[0054] Example 4
[0055] The 6-methylglycate compound (11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione) was catalyzed by whole-cell catalytic solutions of the engineered E. coli strains E. coli BL21(DE3) / pET28a-PsKstD-G51C and E. coli BL21(DE3) / pET28a-PsKstD-A231N obtained in Example 3. The operation steps are as follows:
[0056] To 1 g / L of whole-cell catalytic buffer of *E. coli* BL21(DE3) / pET28a-PsKstD-G51C and *E. coli* BL21(DE3) / pET28a-PsKstD-A231N, 0.1 g / L of 6-methylglycine, 0.02% (w / v) PMS (methyl phenazine sulfate), and 1% (w / v) of isopropanol were added, respectively. The mixtures were reacted at 30 °C and 220 rpm and at 35 °C and 220 rpm for 12 h, respectively, and then the reaction was stopped. The reaction mixture was extracted several times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The substrate conversions were 99% and 98%, respectively, which were 1.98 times and 1.96 times the conversions of the wild-type enzyme (Comparative Example 1), and the ee and de values of the products were both >98%.
[0057] Example 5
[0058] The 6-methylglycate compound (11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione) was catalyzed by whole-cell catalytic solutions of the engineered E. coli strains E. coli BL21(DE3) / pET28a-PsKstD-G51C and E. coli BL21(DE3) / pET28a-PsKstD-A231N obtained in Example 3. The operation steps are as follows:
[0059] To 1 g / L of whole-cell catalytic buffer of *E. coli* BL21(DE3) / pET28a-PsKstD-G51C and *E. coli* BL21(DE3) / pET28a-PsKstD-A231N, 0.05 g / L of 6-methylformamide, 0.05% (w / v) DCPIP (2,6-dichlorophenolindophenol), and 1% (w / v) of isopropanol were added, respectively. The mixtures were reacted at 30 °C and 220 rpm and at 35 °C and 220 rpm for 12 h, respectively, and then the reactions were stopped. The reaction mixtures were extracted several times with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The substrate conversions were 98% and 95%, respectively, which were 2.45 times and 2.375 times the conversions of the wild-type enzymes, and the ee and de values of the products were both >98%.
[0060] The results showed that, compared with wild-type steroid C 1,2 Compared to dehydrogenases, the steroidal C of the present invention 1,2 The mutant protein of the dehydrogenase can significantly improve the yield, ee, and de value of the dehydrogenase of 6-methylglyoxal (11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione).
[0061] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.
[0062] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.
Claims
1. A dehydrogenase, characterized in that, The amino acid sequence of the dehydrogenase is SEQ ID NO.1 or SEQ ID NO.
2.
2. A gene encoding the dehydrogenase as described in claim 1.
3. An engineered bacterium, characterized in that, The engineered bacteria contain the gene as described in claim 2.
4. The engineered bacteria as described in claim 3, characterized in that, The host microorganism of the engineered bacteria is Escherichia coli.
5. The application of the engineered bacteria as described in claim 3 or 4 in the conversion of steroid hormones into dehydrogenation products; wherein the steroid hormone is 11β,17α-dihydroxy-6α-methylpregn-1,4-diene-3,20-dione; and the specific steps of the conversion are as follows: The bacterial solution containing engineered bacteria is mixed with substrate steroid hormones, electron acceptors and organic solvents, catalyzed, and processed to obtain dehydrogenation products. The electron acceptor is methyl phenazine sulfate or 2,6-dichlorophenolindophenol; The organic solvent is isopropanol.
6. The application as described in claim 5, characterized in that, The method for preparing the bacterial solution containing engineered bacteria is as follows: after culturing the engineered bacteria under the induction of IPTG for a period of time, the bacterial cells are collected, and a buffer solution is added to obtain the bacterial solution containing engineered bacteria.