Steroid hormone dehydrogenases, genes, mutants, construction methods and applications

Abstract

This invention belongs to the field of steroid hormone drug technology, specifically relating to a steroid hormone dehydrogenase, gene, mutant, construction method, and application. The amino acid sequence of the steroid hormone dehydrogenase is SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14, or a protein with equivalent activity to the above amino acid sequence through substitution, deletion, or addition of one or more amino acid residues. The dehydrogenase exhibits significantly enhanced enzyme activity, resulting in a higher conversion rate during steroid hormone fermentation.

Description

Technical Field

[0001] This invention belongs to the field of steroid hormone drug technology, specifically relating to a steroid hormone dehydrogenase, gene, mutant, construction method, and application. Background Technology

[0002] Steroids, also known as steroids, are cyclopentane-polyhydrophenanthrene compounds containing three six-membered rings and one five-membered ring. They are ubiquitous in nature and are essential components of living organisms. Based on their physiological activities, they are classified into sex hormones, adrenaline, and anabolic steroids. Sex hormones primarily promote the development and maturation of sex organs; adrenaline plays a major role in saline metabolism, protein breakdown, anti-shock, and anti-allergic effects; anabolic steroids accelerate calcium uptake and growth in bone tissue, promote protein synthesis, and the formation of new tissue and granulation tissue. Based on the effects of these steroids, hormone drugs with similar structures can be developed to treat related diseases. This is of great significance for improving human health and curing various diseases caused by hormonal deficiencies or dysfunctions.

[0003] Steroid C1,2-position dehydrogenase is a membrane protein with a flavin adenine dinucleotide binding site (i.e., a FAD binding site), belonging to the flavoprotein family. Its active site is anchored to the inner cell membrane and consists of a transmembrane region composed of two hydrophobic amino acid segments. It primarily acts on steroids with a ketone group at the 3-position. The dehydrogenation product forms a double bond, increasing water solubility and affinity for receptors.

[0004] Steroid compounds have multiple sites capable of dehydrogenation reactions, primarily at the 1 and 2 positions, which are also the most studied in steroid dehydrogenation. Chemical dehydrogenation uses heavy metals as catalysis, but residues remain after treatment, posing harm to the environment and human health. Biocatalysis has been studied using various bacterial genera, including Aspergillus, Bacillus, Mycobacterium, Pseudomonas, Arthrobacter, and Nocardia. The substrates for biocatalysis are sterols and their derivatives or modifications from plant and animal sources. A major challenge is that sterols are hydrophobic compounds that are almost insoluble in water. This leads to prolonged conversion times, increased byproducts, and reduced substrate feed rates in biotransformation. Therefore, research on the biotransformation or catalysis of steroid compounds focuses on improving substrate solubility and cell permeability. Recent breakthroughs in this area include dissolving substrates in organic solvents or using surfactants to aid substrate dispersion in the conversion system. Furthermore, studies suggest that cyclodextrins and their derivatives can increase both cell permeability and substrate solubility. More research is focused on system transformation, including two-phase systems, cloud point systems, microemulsion systems, and immobilized systems.

[0005] The conversion method reported in CN111349677A involves adding the substrate to the fermentation broth. The fermentation broth has a complex composition, and the post-processing may be cumbersome. It requires a significant amount of organic solvent. The substrate feed concentration is low, with a maximum of 3%; the amount of co-solvent added is relatively high, ranging from 10% to 60%. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a steroid hormone dehydrogenase, gene, mutant, construction method and application, which significantly improves the enzyme activity of the dehydrogenase.

[0007] The present invention includes a steroid hormone dehydrogenase having an amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 or SEQ ID NO:14, or a protein having an activity equivalent to the above amino acid sequence by substitution, deletion or addition of one or more amino acid residues.

[0008] The present invention provides a gene encoding the aforementioned steroid hormone dehydrogenase; preferably, the nucleotide sequence of the gene is SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:13.

[0009] The sequence of SEQ ID NO:6 is:

[0010] MQDWTSECDV LVVGSGGGAL TGAYTAAAQG LTTIVLEKTD RFGGTSAYSG

[0011] ASIWLPGTQV QERAGLPDST ENARTYLRAL LGDAESERQD AYVETAPAVV

[0012] ALLEQNPNIE FEFRAFPDYYKAEGRMDTGR SINPLDLDPA DIGDLAGKVR

[0013] PELDQDRTGQDHAPGPMIGG RALIGRLLAA VQSTGKAELR TESGLTSLIV

[0014] EDGRVVGAEV ESGGETQRIK ANRGVLMAAG GIEGNAEMRE QAGTPGKAIW

[0015] SMGPFGANTG DAISAGIAVG GATALLDQAW FCPGVEQPDG SAAFMVGVRG

[0016] GLVVDSAGER YLNESLPWDQ FGRAMDAHDD NGSAVPSFMI FDSREGGGLP

[0017] AICIPNTAPA KHLEAGTWVG ADTLEELAAK TGLPADALRS TVEKFNDAAK

[0018] LGVDEEFHRG EDPYDAFFCP PNGGANAALT AIENGPFYAA RIVLSDLGTK

[0019] GGLVTDVNGR VLRADGSAID GLYAAGNTSA SLSGRFYPGP GVPLGTAMVF

[0020] SYRAAQDMAK。

[0021] The sequence of SEQ ID NO:5 is:

[0022] atgcaggact ggaccagcga gtgcgacgtg ttggtagtcg gctccggcgg cggagcgctg

[0023] accggcgcat ataccgccgc tgctcaggga ttgacgacga tcgtcctcga gaaaaccgat

[0024] cgtttcggcg ggacctccgc ctactcgggc gcctcgatct ggctcccagg tacccaggtg

[0025] caggaacgcg ccggacttcc cgactcgacc gagaatgccc gcacctatct gcgcgcgttg

[0026] ctcggtgacg ccgagtccga gcgccaggac gcctacgtcg agaccgctcc cgctgtcgtc

[0027] gctctactcg agcagaaccc gaacatcgaa ttcgagttcc gtgcgttccc cgactactac

[0028] aaagccgaag gccggatgga cacgggacgc tccatcaacc ctctcgatct cgatcccgcc

[0029] gacatcggtg acctcgccgg caaggtgcgt ccggaactgg accaagaccg caccggtcag

[0030] gatcatgctc ccggcccgat gatcggtggg cgcgcactga tcggccgtct gctggccgca

[0031] gttcagagca ccggtaaggc agaacttcgc accgaatccg gcctcacctc cctgatcgtg

[0032] gaagacggcc gtgttgtcgg cgccgaggtc gaatccggcg gcgaaaccca gcgaatcaag

[0033] gcgaaccgcg gtgtcctgat ggcagcaggc ggcatcgaag gcaacgccga gatgcgtgag

[0034] caggccggca cccccggcaa ggcgatctgg agtatgggtc ccttcggcgc caacaccggc

[0035] gacgcgatct ccgccggaat tgccgtcggc ggcgcaacag ccttgctcga tcaggcgtgg

[0036] ttctgccccg gcgtcgagca gcccgacggc agcgccgcct tcatggtcgg cgttcgcggt

[0037] gggctcgtcg tcgacagcgc cggtgagcgc tacctcaacg agtcgcttcc gtgggaccag

[0038] ttcggacgag ccatggatgc tcacgacgac aacggttctg ccgtgccgtc gttcatgatc

[0039] ttcgactcgc gcgagggtgg cggactgccc gccatctgca tcccgaacac ggcgcccgcc

[0040] aagcacctcg aagccggaac ctgggtcggt gccgacactc tcgaagaact cgctgccaag

[0041] accggactac cggccgacgc attgcgcagc actgtcgaaa agttcaacga tgccgcaaaa

[0042] ctgggcgtcg acgaagagtt ccatcgcggc gaagacccgt acgacgcgtt cttctgccca

[0043] cccaacggtg gtgcgaatgc ggcactgacg gccatcgaga acggtccgtt ctacgcggcc

[0044] cgcatcgtcc tcagtgacct cggcaccaag ggcggattgg tcaccgacgt caacggccga

[0045] gtcctgcgtg ctgacggcag cgccatcgac ggcctttacg ccgcaggcaa cacgagcgcg

[0046] tcactgagcg gccgcttcta ccccggcccc ggagttccac tcggcacggc catggtcttc

[0047] tcgtaccgag cagcccagga catggcgaag taa。

[0048] Nucleotide sequence of SEQ ID NO:7 M1:

[0049] 1 atgcaggact ggaccagcga gtgcgacgtg ttggtagtcg gctccggcgg cggagcgctg

[0050] 61 accggcgcat ataccgccgc tgctcaggga ttgacgacga tcgtctccga gaaaaccgat

[0051] 121 cgtttcggcg ggacctccgc ctactcgggc gcctcgatct ggctcccagg tacccaggtg

[0052] 181 caggaacgcg ccggacttcc cgactcgacc gagaatgccc gcacctatct gcgcgcgttg

[0053] 241 ctcggtgacg ccgagtccga gcgccaggac gcctacgtcg agaccgctcc cgctgtcgtc

[0054] 301 gctctactcg agcagaaccc gaacatcgaa ttcgagttcc gtgcgttccc cgactactac

[0055] 361 aaagccgaag gccggatgga cacgggacgc tccatcaacc ctctcgatct cgatcccgcc

[0056] 421 gacatcggtg acctcgccgg caaggtgcgt ccggaactgg accaagaccg caccggtcag

[0057] 481 gatcatgctc ccggcccgat gatcggtggg cgcgcactga tcggccgtct gctggccgca

[0058] 541 gttcagagca ccggtaaggc agaacttcgc accgaatccg tcctcacctc cctgatcgtg

[0059] 601 gaagacggcc gtgttgtcgg cgccgaggtc gaatccggcg gcgaaaccca gcgaatcaag

[0060] 661 gcgaaccgcg gtgtcctgat ggcagcaggc ggcatcgaag gcaacgccga gatgcgtgag

[0061] 721 cagggcggca cccccggcaa ggcgatctgg agtatgggtc ccttcggcgc caacaccggc

[0062] 781 gacgcgatct ccgccggaat tgccgtcggc ggcgcaacag ccttgctcga tcaggcgtgg

[0063] 841 ttctgccccg gcgtcgagca gcccgacggc agcgccgcct tcatggtcgg cgttcgcggt

[0064] 901 gggctcgtcg tcgacagcgc cggtgagcgc tacctcaacg agtcgcttcc gtacgaccag

[0065] 961 ttcggacgag ccatggatgc tcacgacgac aacggttctg ccgtgccgtc gttcatgatc

[0066] 1021 ttcgactcgc gcgagggtgg cggactgccc gccatctgca tcccgaacacggcgcccgcc

[0067] 1081 aagcacctcg aagccggaac ctgggtcggt gccgacactc tcgaagaactcgctgccaag

[0068] 1141 accggactac cggccgacgc attgcgcagc actgtcgaaa agttcaacgatgccgcaaaa

[0069] 1201 ctgggcgtcg acgaagagtt ccatcgcggc gaagacccgt acgacgcgttcttctgccca

[0070] 1261 cccaacggtg gtgcgaatgc ggcactgacg gccatcgaga acggtccgttctacgcggcc

[0071] 1321 cgcatcgtcc tcagtgacct caagaccaag ggcggattgg tcaccgacgtcaacggccga

[0072] 1381 gtcctgcgtg ctgacggcag cgccatcgac ggcctttacg ccgcaggcaacacgagcgcg

[0073] 1441 tcactgagcg gccgcttcta ccccggcccc ggagttccac tcggcacggccatggtcttc

[0074] 1501 tcgtaccgag cagcccagga catggcgaag taa。

[0075] SEQ ID NO:8M1

[0076] MQDWTSECDV LVVGSGGGAL TGAYTAAAQG LTTIVSEKTD RFGGTSAYSG

[0077] ASIWLPGTQV QERAGLPDST ENARTYLRAL LGDAESERQD AYVETAPAVV

[0078] ALLEQNPNIE FEFRAFPDYKAEGRMDTGR SINPLDLDPA DIGDLAGKVR

[0079] PELDQDRTGQ DHAPGPMIGG RALIGRLLAA VQSTGKAELR TESVLTSLIV

[0080] EDGRVVGAEV ESGGETQRIK ANRGVLMAAG GIEGNAEMRE QGGTPGKAIW

[0081] SMGPFGANTG DAISAGIAVG GATALLDQAW FCPGVEQPDG SAAFMVGVRG

[0082] GLVVDSAGER YLNESLPYDQ FGRAMDAHDD NGSAVPSFMI FDSREGGGLP

[0083] AICIPNTAPA KHLEAGTWVG ADTLEELAAK TGLPADALRS TVEKFNDAAK

[0084] LGVDEEFHRG EDPYDAFFCP PNGGANAALT AIENGPFYAA RIVLSDLKTK

[0085] GGLVTDVNGR VLRADGSAID GLYAAGNTSA SLSGRFYPGP GVPLGTAMVF

[0086] SYRAAQDMAK。

[0087] SEQ ID NO:9 M2 nucleotide sequence

[0088] 1 atgcaggact ggaccagcga gtgcgacgtg ttggtagtcg gctccggcgg cggagcgctg

[0089] 61 accggcgcat ataccgccgc tgctcaggga ttgacgacga tcgtcctcga gaaaaccgat

[0090] 121 cgtttcggcg ggacctccgc ctactcgggc gcctcgatct ggctcccagg tacccaggtg

[0091] 181 caggaacgcg ccggacttcc cgactcgacc gagaatgccc gcacctatct gcgcgcgttg

[0092] 241 ctcggtgacg ccgagtccga gcgccaggac gcctacgtcg agaccgctcc cgctgtcgtc

[0093] 301 gctctactcg agcagaaccc gaacatcgaa ttcgagttcc gtgcgttccc cgactactac

[0094] 361 aaagccgaag gccggatgga cacgggacgc tccatcaacc ctctcgatct cgatcccgcc

[0095] 421 gacatcggtg acctcgccgg caaggtgcgt ccggaactgg accaagaccg caccggtcag

[0096] 481 gatcatgctc ccggcccgat gatcggtggg cgcgcactga tcggccgtct gctggccgca

[0097] 541 gttcagagca ccggtaaggc agaacttcgc accgaaaacg tcctcacctc cctgatcgtg

[0098] 601 gaagacggcc gtgttgtcgg cgccgaggtc gaatccggcg gcgaaaccca gcgaatcaag

[0099] 661 gcgaaccgcg gtgtcctgat ggcagcaggc ggcatcgaag gcaacgccga gatgcgtgag

[0100] 721 caggccggca cccccggcaa ggcgatctgg agtatgggtc ccttcggcgc caacaccggc

[0101] 781 gacgcgatct ccgccggaat tgccgtcggc ggcgcaacag ccttgctcga tcaggcgtgg

[0102] 841 ttctgccccg gcgtcgagca gcccgacggc agcgccgcct tcatggtcgg cgttcgcggt

[0103] 901 gggctcgtcg tcgacagcgc cggtgagcgc tacctcaacg agtcgcttcc gtacgaccag

[0104] 961 ttcggacgag ccatggatgc tcacgacgac aacggttctg ccgtgccgtc gttcatgatc

[0105] 1021 ttcgactcgc gcgagggtgg cggactgccc gccatctgca tcccgaacacggcgcccgcc

[0106] 1081 aagcacctcg aagccggaac ctgggtcggt gccgacactc tcgaagaactcgctgccaag

[0107] 1141 accggactac cggccgacgc attgcgcagc actgtcgaaa agttcaacgatgccgcaaaa

[0108] 1201 ctgggcgtcg acgaagagtt ccatcgcggc gaagacccgt acgacgcgttcttctgccca

[0109] 1261 cccaacggtg gtgcgaatgc ggcactgacg gccatcgaga acggtccgttctacgcggcc

[0110] 1321 cgcatcgtcc tcagtgacct cggcaccaag ggcggattgg tcaccgacgtcaacggccga

[0111] 1381 gtcctgcgtg ctgacggcag cgccatcgac ggcctttacg ccgcaggcaacacgagcgcg

[0112] 1441 tcactgagcg gccgcttcta ccccggcccc ggagttccac tcggcacggccatggtcttc

[0113] 1501 tcgtaccgag cagcccagga catggcgaag taa。

[0114] SEQ ID NO:10 M2 Amino Acid Sequence

[0115] MQDWTSECDV LVVGSGGGAL TGAYTAAAQG LTTIVLEKTD RFGGTSAYSG

[0116] ASIWLPGTQV QERAGLPDST ENARTYLRAL LGDAESERQD AYVETAPAVV

[0117] ALLEQNPNIE FEFRAFPDYYKAEGRMDTGR SINPLDLDPA DIGDLAGKVR

[0118] PELDQDRTGQ DHAPGPMIGG RALIGRLLAA VQSTGKAELR TENVLTSLIV<C

[0119] EDGRVVGAEV ESGGETQRIK ANRGVLMAAG GIEGNAEMRE QAGTPGKAIW

[0120] SMGPFGANTG DAISAGIAVG GATALLDQAW FCPGVEQPDG SAAFMVGVRG

[0121] GLVVDSAGER YLNESLPYDQ FGRAMDAHDD NGSAVPSFMI FDSREGGGLP

[0122] AICIPNTAPA KHLEAGTWVG ADTLEELAAK TGLPADALRS TVEKFNDAAK

[0123] LGVDEEFHRG EDPYDAFFCP PNGGANAALT AIENGPFYAA RIVLSDLGTK

[0124] GGLVTDVNGR VLRADGSAID GLYAAGNTSA SLSGRFYPGP GVPLGTAMVF <00002C52>SYRAAQDMAK。

[0126] SEQ ID NO:11M4 nucleotide sequence

[0127] 1 atgcaggact ggaccagcga gtgcgacgtg ttggtagtcg gctccggcgg cggagcgctg

[0128] 61 accggcgcat ataccgccgc tgctcaggga ttgacgacga tcgtcctcga gaaaaccgat

[0129] 121 cgtttcggcg ggacctccgc ctactcgggc gcctcgatct ggctcccagg tacccaggtg

[0130] 181 caggaacgcg ccggacttcc cgactcgacc gagaatgccc gcacctatct gcgcgcgttg

[0131] 241 ctcggtgacg ccgagtccga gcgccaggac gcctacgtcg agaccgctcc cgctgtcgtc

[0132] 301 gctctactcg agcagaaccc gaacatcgaa ttcgagttcc gtgcgttccc cgactactac

[0133] 361 aaagccgaag gccggatgga cacgggacgc tccatcaacc ctctcgatct cgatcccgcc

[0134] 421 gacatcggtg acctcgccgg caaggtgcgt ccggaactgg accaagaccg caccggtcag

[0135] 481 gatcatgctc ccggcccgat gatcggtggg cgcgcactga tcggccgtct gctggccgca

[0136] 541 gttcagagca ccggtaaggc acaacttcgc accgaatccg tcctcacctc cctgatcgtg

[0137] 601 gaagacggcc gtgttgtcgg cgccgaggtc gaatccggcg gcgaaaccca gcgaatcaag

[0138] 661 gcgaaccgcg gtgtcctgat ggcagcaggc ggcatcgaag gcaacgccga gatgcgtgag

[0139] 721 caggccggca cccccggcaa ggcgatctgg agtatgggtc ccttcggcgc caacaccggc

[0140] 781 gacgcgatct ccgccggaat tgccgtcggc ggcgcaacag ccttgctcga tcaggcgtgg

[0141] 841 ttctgccccg gcgtcgagca gcccgacggc agcgccgcct tcatggtcgg cgttcgcggt

[0142] 901 gggctcgtcg tcgacagcgc cggtgagcgc tacctcaacg agtcgcttcc gtacgaccag

[0143] 961 ttcggacgag ccatggatgc tcacgacgac aacggttctg ccgtgccgtc gttcatgatc

[0144] 1021 ttcgactcgc gcgagggtgg cggactgccc gccatctgca tcccgaacacggcgcccgcc

[0145] 1081 aagcacctcg aagccggaac ctgggtcggt gccgacactc tcgaagaactcgctgccaag

[0146] 1141 accggactac cggccgacgc attgcgcagc actgtcgaaa agttcaacgatgccgcaaaa

[0147] 1201 ctgggcgtcg acgaagagtt ccatcgcggc gaagacccgt acgacgcgttcttctgccca

[0148] 1261 cccaacggtg gtgcgaatgc ggcactgacg gccatcgaga acggtccgttctacgcggcc

[0149] 1321 cgcatcgtcc tcagtgacct cggcaccaag ggcggattgg tcaccgacgacaacggccga

[0150] 1381 gtcctgcgtg ctgacggcag cgccatcgac ggcctttacg ccgcaggcaacacgagcgcg

[0151] 1441 tcactgagcg gccgcttcta ccccggcccc ggagttccac tcggcacggccatggtcttc

[0152] 1501 tcgtaccgag cagcccagga catggcgaag taa。

[0153] SEQ ID NO:12M4

[0154] MQDWTSECDV LVVGSGGGAL TGAYTAAAQG LTTIVLEKTD RFGGTSAYSG

[0155] ASIWLPGTQV QERAGLPDST ENARTYLRAL LGDAESERQD AYVETAPAVV

[0156] ALLEQNPNIE FEFRAFPDYKAEGRMDTGR SINPLDLDPA DIGDLAGKVR

[0157] PELDQDRTGQ DHAPGPMIGG RALIGRLLAA VQSTGKAQLR TESVLTSLIV

[0158] EDGRVVGAEV ESGGETQRIK ANRGVLMAAG GIEGNAEMRE QAGTPGKAIW

[0159] SMGPFGANTG DAISAGIAVG GATALLDQAW FCPGVEQPDG SAAFMVGVRG

[0160] GLVVDSAGER YLNESLPYDQ FGRAMDAHDD NGSAVPSFMI FDSREGGGLP

[0161] AICIPNTAPA KHLEAGTWVG ADTLEELAAK TGLPADALRS TVEKFNDAAK

[0162] LGVDEEFHRG EDPYDAFFCP PNGGANAALT AIENGPFYAA RIVLSDLGTK

[0163] GGLVTDANGR VLRADGSAID GLYAAGNTSA SLSGRFYPGP GVPLGTAMVF

[0164] SYRAAQDMAK。

[0165] SEQ ID NO:13 M5 nucleotide sequence

[0166] 1 atgcaggact ggaccagcga gtgcgacgtg ttggtagtcg gctccggcgg cggagcgctg

[0167] 61 accggcgcat ataccgccgc tgctcaggga ttgacgacga tcgtcctcga gaaaaccgat

[0168] 121 cgtttcggcg ggacctccgc ctactcgggc gcctcgatct ggctcccagg tacccaggtg

[0169] 181 caggaacgcg ccggacttcc cgactcgacc gagaatgccc gcacctatct gcgcgcgttg

[0170] 241 ctcggtgacg ccgagtccga gcgccaggac gcctacgtcg agaccgctcc cgctgtcgtc

[0171] 301 gctctactcg agcagaaccc gaacatcgaa ttcgagttcc gtgcgttccc cgactactac

[0172] 361 aaagccgaag gccggatgga cacgggacgc tccatcaacc ctctcgatct cgatcccgcc

[0173] 421 gacatcggtg acctcgccgg caaggtgcgt ccggaactgg accaagaccg caccggtcag

[0174] 481 gatcatgctc ccggcccgat gatcggtggg cgcgcactga tcggccgtct gctggccgca

[0175] 541 gttcagagca ccggtaaggc agaacttcgc accgaatccg tcctcacctc cctgatcgtg

[0176] 601 gaagacggcc gtgttgtcgg cgccgaggtc gaatccggcg gcgaaaccca gcgaatcaag

[0177] 661 gcgaaccgcg gtgtcctgat ggcagcaggc ggcatcgaag gcaacgccga gatgcgtgag

[0178] 721 caggccggca cccccggcaa ggcgatctgg agtatgggtc ccttcggcgc caacaccggc

[0179] 781 gacgcgatct ccgccggaat tgccgtcggc ggcgcaacag ccttgctcga tcaggcgtgg

[0180] 841 ttctgccccg gcgtcgagca gcccgacggc agcgccgcct tcatggtcgg cgttcgcggt

[0181] 901 gggctcgtcg tcgacagcgc cggtgagcgc tacctcaacg agtcgcttcc gtacgaccag

[0182] 961 ttcggacgag ccatggatgc tcacgacgac aacggttctg ccgtgccgtc gttcatgatc

[0183] 1021 ttcgactcgc gcgagggtgg cggactgccc gccatctgca tcccgaacacggcgcccgcc

[0184] 1081 aagcacctcg aagccggaac ctgggtcggt gccgacactc tcgaagaactcgctgccaag

[0185] 1141 accggactac cggccgacgc attgcgcagc actgtcgaaa agttcaacgatgccgcaaaa

[0186] 1201 ctgggcgtcg acgaagagtt ccatcgcggc gaagacccgt acgacgcgttcttctgccca

[0187] 1261 cccaacggtg gtgcgaatgc ggcactgacg gccatcgaga acggtccgttctacgcggcc

[0188] 1321 cgcatcgtcc tcagtgacct cggcaccaag ggcggattgg tcaccgacgtccaaggccga

[0189] 1381 gtcctgcgtg ctgacggcag cgccatcgac ggcctttacg ccgcaggcaacacgagcgcg

[0190] 1441 tcactgagcg gccgcttcta ccccggcccc ggagttccac tcggcacggccatggtcttc

[0191] 1501 tcgtaccgag cagcccagga catggcgaag taa。

[0192] SEQ ID NO:14 M5 amino acid sequence

[0193] MQDWTSECDV LVVGSGGGAL TGAYTAAAQG LTTIVLEKTD RFGGTSAYSG

[0194] ASIWLPGTQV QERAGLPDST ENARTYLRAL LGDAESERQD AYVETAPAVV

[0195] ALLEQNPNIE FEFRAFPDYYKAEGRMDTGR SINPLDLDPA DIGDLAGKVR

[0196] PELDQDRTGQDHAPGPMIGG RALIGRLLAA VQSTGKAELR TESVLTSLIV

[0197] EDGRVVGAEV ESGGETQRIK ANRGVLMAAG GIEGNAEMRE QAGTPGKAIW

[0198] SMGPFGANTG DAISAGIAVG GATALLDQAW FCPGVEQPDG SAAFMVGVRG

[0199] GLVVDSAGER YLNESLPYDQFGRAMDAHDDNGSAVPSFMIFDSREGGGLP

[0200] AICIPNTAPA KHLEAGTWVG ADTLEELAAK TGLPADALRS TVEKFNDAAK

[0201] LGVDEEFHRG EDPYDAFFCP PNGGANAALT AIENGPFYAA RIVLSDLGTK

[0202] GGLVTDVQGR VLRADGSAID GLYAAGNTSA SLSGRFYPGP GVPLGTAMVF

[0203] SYRAAQDMAK.

[0204] The present invention provides a mutant containing the steroid hormone dehydrogenase described above, or containing the gene described above.

[0205] This invention provides a method for constructing a mutant, comprising the steps of constructing a plasmid containing the above-mentioned gene, introducing the plasmid into competent cells, screening, and obtaining a mutant.

[0206] This invention provides an application of the mutant in the enzymatic conversion of substrates to generate dehydrogenation products.

[0207] Preferably, the substrate is a steroid hormone, and more preferably a carrier hormone containing a 3-keto-4-ene structure; preferably, the dehydrogenation site of the steroid hormone is at the C1,2 position.

[0208] Preferably, the substrate is one or more of the following substances:

[0209] Androst-4-ene-3,17-dione, Androst-4,9(11)-diene-3,17-dione, 17β-hydroxyandrost-4-ene-3-one, Pregnant-4-ene-3,20-dione, 17α-hydroxypregnant-4-ene-3,20-dione, 17α,21-dihydroxypregnant-4-ene-3,11,20-trione, 11β,17α,21-trihydroxypregnant-4-ene-3,20-dione, 11β,17α,21-trihydroxypregnant-4-ene-3,20-dione, 11β,17α,21-trihydroxypregnant-4-ene-3,20-dione-21-acetate.

[0210] When enzymatically converting the substrate, isopropanol is used as the solvent, and the volume of the solvent does not exceed 20% of the total fermentation solution volume.

[0211] The beneficial effects of this invention are that it constructs a recombinant dehydrogenase with an enzyme activity 2.83 times higher than that of the original recombinant strain, and the enzyme has a broad substrate spectrum, high substrate concentration, and requires less cosolvent, thus reducing costs and enabling industrial production.

[0212] The mutant strains of this invention exhibit good dehydrogenation activity against substrates (especially steroid hormones containing a 3-keto-4-ene structure). The enzyme activity of the mutant strain is 2.83 times higher than that of the original engineered strain. The content of the solubilizer in the transformation system also affects the conversion rate to varying degrees: isopropanol content below 20% promotes the conversion rate, while content of 20% or higher inhibits the enzyme. Increasing the substrate feed rate decreases the conversion rate and prolongs the transformation time within the same time frame. With a higher substrate feed rate (20%), 99% of the substrate can be converted within 50 hours. Detailed Implementation

[0213] Comparative Example 1

[0214] The original method for preparing the C1,2-position dehydrogenase of this invention is as follows:

[0215] A search on NCBI revealed 3-ketosteroid dehydrogenase dehydrogenase from Rhodococcus erythropolis PR4 (NCBI Reference Sequence: NC_012490.1).

[0216] The specific steps are as follows:

[0217] 1. The dehydrogenase nucleotide sequence is SEQ ID NO:1, and the protein sequence is SEQ ID NO:2. The SEQ ID NO:1 sequence was sent to Shanghai Qingke Biotechnology for whole-gene synthesis and constructed into the pET-28a plasmid expression vector to obtain the recombinant plasmid.

[0218] 2. The recombinant plasmid was introduced into 100 μL of LEscherichia Coli BL21(DE3) competent cells using a heat shock method. The cells were then plated onto LB solid medium containing kanamycin resistance and incubated overnight at 37°C (approximately 16 hours). Larger single clones were selected and colony PCR (polymerase chain reaction) amplification was performed using universal primers (T7, T7ter) to screen for positive clones, thereby obtaining the engineered bacteria (PET-KstD).

[0219] 3. Inoculate the engineered bacterial strain into LB medium at a rate of 0.1% and incubate at 37°C for 12-16 hours to activate the strain. Then, inoculate the activated bacterial solution into fresh LB medium (containing 50 μg / ml kanamycin) at a rate of 2% and incubate at 37°C for 4 hours. Add IPTG (isopropyl-β-D-thiogalactoside) inducer to a final concentration of 0.01 mM and induce at 30°C for 16 hours. Collect the bacterial cells by centrifugation at 4°C. The induced recombinant engineered bacteria are obtained.

[0220] 4. Enzyme activity detection of recombinant engineered bacteria (PET-KstD) (unmutated mutant) was performed using enzyme activity detection methods.

[0221] LB medium: 1% by weight (tryptone) peptone, 0.5% by weight yeast (infusion) powder, 1% by weight sodium chloride. For solid medium, add 1.5% agar.

[0222] Enzyme activity assay:

[0223] 4-AD stock solution: Weigh 0.09 g of androstenedione substrate, dissolve it in deionized water by sonication, and dilute to 100 ml in a volumetric flask. Store at 4 °C.

[0224] DCPIP stock solution: Weigh 0.035g of 2,6-dichlorophenolindophenol, dissolve it in distilled water, and bring the volume to 100ml. Store at 4℃.

[0225] 50mM Tris-HCl (pH 8.0): Weigh 5g of Tris, dissolve it in distilled water and bring the volume to 100ml. Store at 4℃.

[0226] Reaction: The experiment consisted of three parallel groups and one control group. 50 mM Tris-HCl solution was added to the experimental and control groups at volumes of 0.2 ml and 0.22 ml, respectively. Then, 10 μl of DCPIP stock solution and 70 μl of 4-AD stock solution were added, mixed thoroughly, and incubated at 30°C for 10 min. For the experimental group, 20 μl of appropriately diluted enzyme solution was added; after thorough mixing, the absorbance was measured at 600 nm using a microplate reader within 1 min.

[0227] Enzyme activity is defined as the amount of enzyme required to reduce 1 μM DCPIP per minute under optimal conditions, which is one unit (U).

[0228] Example 1

[0229] The method for preparing the mutant of the C1,2 position dehydrogenase of the present invention is as follows:

[0230] Based on the nucleotide sequence SEQ ID NO:1, primer 1 sequence is SEQ-3 and primer 2 sequence is SEQ-4. The primers were then sent to Changsha Qingke for synthesis.

[0231] Error-prone PCR amplification was performed using the plasmid of the PET-KstD recombinant bacteria as a template. The reagents used were the error-prone PCR kit from Beijing Bio-Lab, and the PCR system was prepared according to its instructions. The PCR program was 94℃ for 3 min; 94℃ for 30 s; 55℃ for 30 s; 72℃ for 1 min 30 s, repeated 30 times, followed by incubation at 16℃. The first-round PCR products were run on a 1% agarose gel to determine the PCR product length to be approximately 1500 bp, and then purified by gel extraction. A portion of the recovered PCR products and the empty PET-28a vector plasmid were double-digested with restriction enzymes (purchased from Takara) BamHI and HindIII, and the correct bands were identified after gel extraction and plasmid digestion. The target band and plasmid were ligated overnight at 16℃ using T4 ligase (purchased from Takara). The ligation product was introduced into BL21 competent cells using the heat shock method, plated on plates containing Kanamycin resistance, and incubated at 37℃ until single colonies appeared. Single colonies were inoculated into deep-well plates and cultured to induce enzyme production according to shake-flask culture methods. After centrifugation, the supernatant was discarded, and the bacterial cells were resuspended in buffer containing lysozyme and incubated. After centrifugation, the supernatant was diluted to a certain concentration and used for enzyme activity detection. Clones with high enzyme activity were selected for sequencing. Simultaneously, the PCR products recovered from the first round of gel electrophoresis were used as templates for a second ER-PCR. This process continued until strains with high enzyme activity were selected (Table 1), which were then sent for sequencing to determine the mutation sites.

[0232] The nucleotide sequence of the M1 mutant is SEQ ID NO:7, and the encoded amino acid sequence is SEQ ID NO:8; the nucleotide sequence of the M2 mutant is SEQ ID NO:9, and the encoded amino acid sequence is SEQ ID NO:10; the nucleotide sequence of the M3 mutant is SEQ ID NO:5, and the encoded amino acid sequence is SEQ ID NO:6; the nucleotide sequence of the M4 mutant is SEQ ID NO:11, and the encoded amino acid sequence is SEQ ID NO:12; the nucleotide sequence of the M5 mutant is SEQ ID NO:13, and the encoded amino acid sequence is SEQ ID NO:14.

[0233] Table 1 Enzyme activity data of different mutants

[0234]

[0235] Inducible expression of mutant M3:

[0236] Prepare 100 mL of LB liquid medium as seed culture medium and autoclave at 121 °C for 20 min. After cooling, use an inoculation loop to pick a single colony and inoculate it into the medium. Incubate overnight at 37 °C and 200 rpm to prepare the seed culture. Prepare 4 L of fermentation medium using LB liquid medium. Inoculate 100 mL of the overnight seed culture medium into the fermentation medium and incubate at 37 °C and 200 rpm until the O.D600 reaches between 0.6 and 0.8. Add IPTG to a final concentration of 0.01 mM to induce expression for 12 hours. Collect the cells by centrifugation at 4 °C and 8000 rpm.

[0237] Example 2

[0238] Weigh the substrate into a conversion flask, add 3 mL of isopropanol, and stir to disperse evenly. Then add 24 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.0.5M), and maintain the temperature at 30℃ while continuing to stir until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add methyl phenothiazine sulfate at a molar mass ratio of 1:1 to the substrate. At the start of the reaction, the pH of the system was controlled between 7.8 and 8.2 using 10% sodium hydroxide solution. The substrate residue was monitored by TLC during the reaction, and detected by high-performance liquid chromatography (HPLC) in the later stages of the reaction.

[0239] Table 2. Effects of different substrates and other conditions on conversion rate.

[0240]

[0241] Example 3

[0242] Weigh 1.5 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 3 mL of isopropanol, stir to disperse evenly, then add 24 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05 M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add 0.13 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.1:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 85% at the 9th hour and 99% at the 12th hour.

[0243] Example 4

[0244] Weigh 1.5 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 3 mL of isopropanol, stir to disperse evenly, then add 24 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05 M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add 0.013 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.01:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 70% at the 8th hour and 99% at the 16th hour.

[0245] Example 5

[0246] Weigh 1.5 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 1.5 mL of isopropanol, stir to disperse evenly, then add 25.5 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05 M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add 0.013 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.01:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 90% at 10 hours and 99% at 14 hours.

[0247] Example 6

[0248] Weigh 3 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 3 mL of isopropanol, stir to disperse evenly, then add 24 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cells to a final M3 concentration of 100 g / L, and finally add 0.013 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.01:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 50% at hour 9 and 98% at hour 22.

[0249] Example 7

[0250] Weigh 4.5 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 3 mL of isopropanol, stir to disperse evenly, then add 24 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05 M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add 0.013 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.01:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 45% at the 10th hour; 75% at the 20th hour; and 97% at the 32nd hour.

[0251] Example 8

[0252] Weigh 6 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 3 mL of isopropanol, stir to disperse evenly, then add 24 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05 M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add 0.013 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.01:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 55% at 20 hours and 96% at 46 hours.

[0253] Comparative Example 2

[0254] Weigh 1.5 g of 11β,17α,21-trihydroxypregn-4-ene-3,20-dione into a conversion flask, add 6 mL of isopropanol, stir to disperse evenly, then add 21 mL of dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 8.0, 0.05M), maintain the temperature at 30℃ and continue stirring until homogeneous. Add recombinant mutant bacterial cell M3 to a final concentration of 100 g / L, and finally add 0.013 g of phenothiazine sulfate methyl ester, with a molar mass ratio of 0.01:1 to the substrate. At the start of the reaction, the pH of the system was controlled at 7.8-8.2 using 10% sodium hydroxide solution. During the reaction, the substrate residue was monitored by TLC. Take 0.2 mL of the reaction sample, add 0.3 mL of dichloromethane as the extraction solvent, shake thoroughly, centrifuge for 20 s, and collect the supernatant. The developing solvent was chloroform:methanol:water = 6 mL:1 mL:0.03 mL, and UV spectrophotometry was used for development. The conversion rate was 45% at the 10th hour and 70% at the 20th hour.

[0255] 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.

[0256] 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 steroid hormone dehydrogenase, characterized in that, The amino acid sequence of steroid hormone dehydrogenase is SEQ ID NO:

6.

2. A gene characterized by, The code is for the steroid hormone dehydrogenase as described in claim 1.

3. The gene as described in claim 2, characterized in that, The nucleotide sequence of the gene is SEQ ID NO:

5.

4. A mutant bacterial cell, characterized in that, It contains the steroid hormone dehydrogenase as described in claim 1, or the gene as described in claim 2 or 3.

5. A method for constructing mutant bacteria, characterized in that, A plasmid containing the gene described in claim 2 or 3 was constructed, and the plasmid was introduced into competent cells. After screening, mutant bacterial cells were obtained.

6. An application of the mutant bacterial cell as described in claim 4 in the enzymatic transformation of a substrate to generate a dehydrogenation product, wherein the substrate is one or more of the following substances: androst-4-ene-3,17-dione, androst-4,9(11)-diene-3,17-dione, 17β-hydroxyandrost-4-ene-3-one, pregn-4-ene-3,20-dione, 17α-hydroxypregn-4-ene-3,20-dione, 17α,21-dihydroxypregn-4-ene-3,11,20-trione, 11β,17α,21-trihydroxypregn-4-ene-3,20-dione, 11β,17α,21-trihydroxypregn-4-ene-3,20-dione, 11β,17α,21-trihydroxypregn-4-ene-3,20-dione-21-acetate; The dehydrogenation site is C1,2.

7. The application as described in claim 6, characterized in that, When enzymatically converting the substrate, isopropanol is used as the solvent, and the volume of the solvent does not exceed 20% of the total fermentation solution volume.

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