CN108456666A

CN108456666A - A kind of 3- sterones-△1Dehydrogenase and its encoding gene and application

Info

Publication number: CN108456666A
Application number: CN201810200187.8A
Authority: CN
Inventors: 张保国; 张瑞婕; 刘相岑; 韩煜畅; 史吉平; 姜标
Original assignee: Shanghai Advanced Research Institute of CAS
Current assignee: Shanghai Advanced Research Institute of CAS
Priority date: 2018-03-12
Filing date: 2018-03-12
Publication date: 2018-08-28
Anticipated expiration: 2038-03-12
Also published as: CN108456666B

Abstract

The invention belongs to genetic engineering fields, and in particular to 3 sterone △ of one kind¹Dehydrogenase and its encoding gene and application.Present invention research finds a kind of 3 completely new sterone △ for the first time¹Dehydrogenase, it is when 4 AD of substrate is at concentrations up to 0.8% in converting culture medium, conversion ratio may be up to 99%, the concentration of product ADD reaches 7.9 g/L in conversion fluid, significantly larger than other relevant reports, it is effective to reduce substrate 4 AD residuals, improve the content of product ADD, the step of reducing separation and Extraction, has good industrialization prospect.

Description

A kind of 3- sterones-△1Dehydrogenase and its encoding gene and application

Technical field

The invention belongs to genetic engineering fields, and in particular to a kind of 3- sterones-△¹It dehydrogenase and its encoding gene and answers With.

Background technology

Steroidal compounds (steroids) are also known as steroids, are a kind of important days being widely present in bio-tissue Right organic compound.Structurally, steroidal is a kind of compound with cyclopentanoperhy drophenanthrene (Cl7) for parent nucleus, by three six Membered ring and a five-membered ring composition, are referred to as A, B, C, D ring (as shown in Figure 1).Due to substituent group on parent nucleus, position of double bond or The difference of spatial configuration forms miscellaneous steroidal compounds, and very important adjustment effect is played to body.Steroid Drug refers to the steroid hormone class drug for having steroid nucleus structure, including mineralocorticoid, glucocorticoid, sex hormone and albumen are same Change hormone etc..Mineralocorticoid and glucocorticoid have anti-inflammatory, antiallergy and other effects, are widely used in treatment rheumatoid and close The diseases such as section inflammation, bronchial asthma and eczema；Sex hormone is the key agents for treating male organs decline and some gynecological diseases, It is the adjuvant therapy medicaments for the treatment of breast cancer, prostate cancer, and the main component of oral contraceptive in great demand in recent years； Protein anabolic hormone can promote protein to synthesize and inhibit protein alienation, make muscle growth；Korea Spro, dish is promoted to sink in the tissue Product accelerates bone tissue differentiation and growth etc..Since steroidal drug plays very important adjustment effect to body, to being at present Only, the steroidal drug used has been got the Green Light about more than 300 kind, is the second major class drug that yield is only second to antibiotic.

3- sterones-△¹Dehydrogenase (KSDD, EC 1.3.99.4) is the key enzyme in steroidal parent nucleus degradation process, can be situated between Lead the formation of double bond between steroidal parent nucleus C1 and C2.Such structure change can be at double increase drug anti-inflammatory activity, It plays an important role in steroidal drug production process, there is abundant application value, therefore KSDD enzyme genes are deeply ground Study carefully and has great importance to the exploitation of steroid drugs.In recent years, lot of domestic and foreign scholar is at arthrobacterium (Arthrobacter), Mycobacterium (Mycobacterium sp.), Nocard's bacillus (Nocardia sp.), Rhodococcus sp (Rhodococcus sp.) and It is cloned in the microorganisms such as Metarhizium anisopliae (Metarhizium anisopliae) and identifies the (reference of multiple KSDD enzyme genes Document), and the KSDD enzyme genes of some separate sources have been subjected to heterogenous expression.Patent CN106636160A discloses one kind From the KSDD of Mycobacterium neoaurum, overexpression of the gene in e. coli bl21, the gene work of structure are realized It is ADD that journey bacterial strain, which can convert 4-AD, but feed concentrations are only 0.1% (w/v), and substrate conversion efficiency is only 76.5%.Patent CN102168099A discloses one kind and deriving from Metarhizium anisopliae KSDD gene orders, by the gene heterogenous expression to complete red ferment In mother, it is ADD that the gene engineering yeast of structure, which can also convert 4-AD Pichia pastoris, but maximum feed concentrations are also only 0.15% (w/v).Patent CN102703494A discloses a kind of utilization recombined bacillus subtilis resting cell 4-AD generations The method of ADD, applicant is by the KSDD heterogenous expressions to bacillus subtilis of Mycobacterium neoaurum, the genetic engineering bacterium of structure Strain is significantly improved than the enzyme activity of control strain, but when using 0.1%4-AD as substrate, the molar yield of substrate is also only 65.7%.

In conclusion currently with gene engineering method by the KSDD enzyme genes heterogenous expression of separate sources to different hosts In, structure recombinant bacterial strain carries out bioconversion 4-AD preparations ADD and still suffers from the problems such as substrate feed concentrations are small, conversion ratio is low. Therefore KSDD enzyme gene resources are fully excavated, the KSDD enzyme genes engineered strain for building high efficient expression utilizes biotechnology to realizing The industrialized production that conversion AD prepares important steroid bulk pharmaceutical chemicals ADD is of great significance.

Invention content

In order to overcome the problems of in the prior art, the purpose of the present invention is to provide a kind of new 3- sterones- △¹Dehydrogenase and its encoding gene and application, with solve it has been reported that KSDD enzyme gene heterogenous expressions when there are 3- sterones- △¹The problems such as dehydrogenase activity is low, and substrate feed concentrations are small when leading to its application, and conversion ratio is low.

To achieve the goals above and other related purposes, the present invention adopt the following technical scheme that：

The first aspect of the present invention provides a kind of 3- sterones-△ of separation¹Dehydrogenase, the 3- sterones-△¹Dehydrogenase For the protein of following (1) or (2)；(1) amino acid sequence such as SEQ ID NO：Protein shown in 2；(2) with (1) in egg White matter is at least 80% homology and with the protein of protein function in (1).

As cited in the embodiment of the present invention, the 3- sterones-△¹The amino acid sequence of dehydrogenase such as SEQ ID Shown in NO.2.

The second aspect of the present invention provides a kind of polynucleotides of separation, the aforementioned 3- of polynucleotide encoding of the separation Sterone-△¹Dehydrogenase.

The aforementioned 3- sterones-△ of coding of the present invention¹The polynucleotides of dehydrogenase can be DNA form or rna form. DNA forms include cDNA, genomic DNA or artificial synthesized DNA.DNA can be single-stranded or double-strand.

The coding 3- sterones-△ of the present invention¹The polynucleotides of dehydrogenase, can be ripe by those skilled in the art It is prepared by any technology appropriate known.The technology sees the general description of this field, such as《Molecular Cloning:A Laboratory guide》(J. Sas Nurse Brooker etc., Science Press, 1995).Including but not limited to the methods of recombinant DNA technology, chemical synthesis；For example, by using weight Folded extension PCR method.

As cited in the embodiment of the present invention, the aforementioned 3- sterones-△ of coding¹The core of the polynucleotides of dehydrogenase Nucleotide sequence such as SEQ ID NO：Shown in 1.

The third aspect of the present invention provides a kind of recombinant expression carrier, includes the polynucleotides of aforementioned separation.

Method well-known to those having ordinary skill in the art can be used to build the recombinant expression carrier.These methods include recombination DNA technologies, DNA synthetic technologys etc..It can will encode the 3- sterones-△¹The DNA of dehydrogenase is effectively connected to more in carrier On cloning site, to instruct mRNA to synthesize and then express 3- sterones-△¹Dehydrogenase, or it is used for homologous recombination.The present invention's In preferable case, the expression vector uses pET-28a.

The fourth aspect of the present invention, provides a kind of host cell, and the cell contains aforementioned recombinant expression carrier or gene The polynucleotides of the aforementioned separation of external source are integrated in group.

In the preferable case of the present invention, the host cell uses Escherichia coli, bacillus subtilis or Pichia pastoris.Institute The Escherichia coli stated are preferably BL21 (DE3).The bacillus subtilis is preferably BS158.The Pichia pastoris is preferably GS115。

The fifth aspect of the present invention provides a kind of method preparing aforementioned 3- sterones-△ 1- dehydrogenases, including following times One：

(1) structure contains 3- sterones-△¹Then the expression vector of the coded polynucleotide of dehydrogenase carries the expression Body converts into host cell induced expression, is detached from expression product and obtains the 3- sterones-△¹Dehydrogenase.

Or

(2) foregoing host cell is cultivated under suitable conditions, is allowed to express the 3- sterones-△¹Dehydrogenase, then Separation and purifying obtain the 3- sterones-△¹Dehydrogenase.

In the preferable case of the present invention, the expression vector uses pET-28a.The host cell uses Escherichia coli. The Escherichia coli are preferably BL21 (DE3).

The sixth aspect of the present invention provides 3- sterones-△ described in first aspect¹Multinuclear described in dehydrogenase, second aspect The host cell described in recombinant expression carrier or fourth aspect described in thuja acid, the third aspect is used to convert 4-AD and generates ADD's Purposes.

The seventh aspect of the present invention provides a kind of transforming agent of 4-AD generations ADD, contains 3- steroids described in aforementioned first aspect Ketone-△¹The host cell described in polynucleotides, the third aspect described in dehydrogenase, second aspect or the place described in fourth aspect Any one of chief cell.

It should be noted that in the present invention, 4-AD is androstenedione, ADD is androsadiendione.

The eighth aspect of the present invention provides a kind of method that conversion 4-AD generates ADD, including step：By the of the present invention 3- sterones-the △ of one side¹Host cell described in dehydrogenase or fourth aspect, acts on 4-AD.

Compared with prior art, the present invention has the advantages that：

1, inventor clones a new 3- sterones-△ from Mycobacterium neoaurum DSM 1381 for the first time¹Dehydrogenase base Because of kstD2.The kstD2 and other 3- sterones-△ reported at present¹Dehydrogenase gene highest similarity is only 85%, is had apparent Otherness, be a kind of new 3- sterones-△¹Dehydrogenase gene provides item further to study the gene family evolution relationship Part also has efficient 3- sterones-△ to be built using technique for gene engineering¹Dehydrogenase recombinant bacterium provides more gene moneys Source.

2, by genetic engineering means, kstD2 genes heterologous overexpression has been subjected in Escherichia coli, have obtained height Effect expression 3- sterones-△¹The recombinant bacterium of dehydrogenase.The recombinant bacterium is established to turn the biology of 3- sterone -4- en steroids class compounds Chemical industry skill may be up to when substrate 4-AD is at concentrations up to 0.8% in converting culture medium using the conversion ratio of the technique substrate 99%, the concentration of product ADD reaches 7.9g/L in conversion fluid, significantly larger than other relevant reports, effective to reduce substrate 4-AD The step of residual improves the content of product ADD, reduces separation and Extraction has good industrialization prospect.

Description of the drawings

Fig. 1：Steroidal compounds general formula, in the present invention, 3- sterones steroidal compounds can be existing 3- sterones -4- alkene steroids Body class compound common are AD, 9 α-OH-AD, 17 Alpha-hydroxy progesterone, epoxy progesterone, mold oxide, cortisone, hydrogen Change cortisone, eplerenone, the pregnant -4- alkene -3- ketone of 20- (methylol).

Fig. 2：Agarose gel electrophoresis detects KstD2 PCR products.

Fig. 3：Recombination bacillus coli BL21-kstD2 AD conversion ratios.

Fig. 4：The surplus of AD in recombination bacillus coli BL21-kstD2AD transformation experiments.

Fig. 5：The yield of ADD in recombination bacillus coli BL21-kstD2 AD transformation experiments.

Specific implementation mode

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The test method of actual conditions is not specified in the following example, Usually according to normal condition, or according to the condition proposed by each manufacturer.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment, Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related field.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987 and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

1 Mycobacterium neoaurum DSM of embodiment, 1381 3- sterones-△¹The acquisition of dehydrogenase coding genes kstD2

Sequencing analysis is carried out to Mycobacterium neoaurum DSM 1381 using Illumina Miseq sequencing technologies, according to gene Annotation information obtains complete 3- sterones-△¹Dehydrogenase coding genes kstD2 sequences, the 3- sterones-△¹Dehydrogenase is compiled The nucleotide sequence such as SEQ ID NO of code gene kstD2：Shown in 1, specially：

GTGACCGATCAGAACAACATCACCGTCGACCTCGTCGTCGTCGGCTCGGGTACC GGGATGGCGGCAGCATTGGCTGCCCACGAGCTGGGAATGTCGACGCTGATCGTCGAG AAGAGCGCCTATGTCGGTGGTTCGACGGCTCGCTCCGGCGGTGCCTTCTGGCTTCCCG GCAGCTCCATTCTCAAGGACGCCGGTTCGGCGGACACTCCGGCCAAGGCGCGCACCT ACCTTGAAGCACTCGTCGGTGACGACGTCTCACCCGAACGCGCACGCACTTTCATCGA TCAGATCCCCGCGACCATCGACATGTTGCGTCGCACCACCCCGATGAAGTTCATGTGG GCCAAGGGATATTCGGACTACCACCCGGAGAGGCCAGGAGGCAGTGCGGTGGGCCGG ACCTGTGAGTGTCGCCCGTTCGACACTGCGGTCCTCGGTCCAGAGCTGGCGCGGCTAC GACCTGGAGTGATGAAGTCATCGTTCCCGATGCCGGTCACCGGCGCCGATTACCGTTG GCTGAACCTGATGGCCCGCACCCCGCGCAAGTCCTGGCCGCGGATCATGCTGCGGGC CATGCAGGGTGTCGGCGGTTTGGCCCTGCGGCGCCGGTACGCCGCAGGCGGCCAGGC CTTGGCGGCCGGGATGTTCGCCGGCGTGCTGCAGGCGGGGATCCCGGTGTGGACCGA TTCGACGGTGACCGAGCTCATCACCGATGGTGGGCGGGTGACCGGCGCGCGGGTGCT GCGCGAGGGATCGGCCGTGACCGTCACCGCACGCCGTGGCATCGTGCTGGCCACCGG CGGTTTCGACCACGAGATGAATTGGCGGCGGAAGTTCCAGTCCGAGCTCCTCGGTGA ACATCTCAGCCTTGGGGCCGAGAGCAATACCGGCGATGGCATCCGGCTCGCCCAGGA CCTGGGCGCAGGCACCGGACTGATGGACCAGGCATGGTGGTTTCCGGCCTTTGCTCCG CTGCCTGGCGGGGATCCCACCGTGATGCTGGCCGAGCGGTCGCTGCCCGGCTGCCTGC TGGTAGACCAGACCGGTGAGCGCTTCATCAACGAGGCCACCGACTACATGTCCTTCGG ACAGCAGCTGCTGCGTCGCGAACACGCGGGCAATCCGGTCGAGACGATGTGGATGAT CTTCGATCAGCGCTACCGGAACAGCTATCTGCTTGCCGCCGAACTATTTCCACGAATG CCGATCCCACAGAGTTGGTACGACGCCGGGATCGCGCACCGCGGCACGGATGCGGAA GCACTGGGCCGCCAGATCGGTTTCGATCCCGCGACGTTGGTCGCCACGATCGAGCGGT TCAACGGACTCGCCGATGCCGGTGTCGACGCCGACTTCCAGCGCGGCGCGAGCGCCT ACGACCGCTACTACGGCGACCCGACGATCACGCCCAACCCGAACCTGCGACCGCTGG ATCCCGGCCCGCTGTACGCCGTCAAGGTCGTGCTGAGCGACCTGGGCACCTGTGGTGG GGTCCTGTGCGACGTGAACGGCCGGGTTCTGCGCGAAGACGGAGTGCCCATCGACGG TCTGTACGCGATCGGCAATACCGCGGCCAACGCATTCGGCAAGACCTACCCGGGCGC GGGCGCGACCATCGCGCAGGGGCTGGTGTACGGCCATGTTGCCGCGCAGCATGCCGC CGGACACACCTGA。

3- sterones-the △¹3- sterones-△ corresponding to dehydrogenase coding genes kstD2¹The amino acid sequence of dehydrogenase Row such as SEQ ID NO：Shown in 2, specially：

VTDQNNITVDLVVVGSGTGMAAALAAHELGMSTLIVEKSAYVGGSTARSGGAFWL PGSSILKDAGSADTPAKARTYLEALVGDDVSPERARTFIDQIPATIDMLRRTTPMKFMWA KGYSDYHPERPGGSAVGRTCECRPFDTAVLGPELARLRPGVMKSSFPMPVTGADYRWLN LMARTPRKSWPRIMLRAMQGVGGLALRRRYAAGGQALAAGMFAGVLQAGIPVWTDST VTELITDGGRVTGARVLREGSAVTVTARRGIVLATGGFDHEMNWRRKFQSELLGEHLSL GAESNTGDGIRLAQDLGAGTGLMDQAWWFPAFAPLPGGDPTVMLAERSLPGCLLVDQT GERFINEATDYMSFGQQLLRREHAGNPVETMWMIFDQRYRNSYLLAAELFPRMPIPQSW YDAGIAHRGTDAEALGRQIGFDPATLVATIERFNGLADAGVDADFQRGASAYDRYYGDP TITPNPNLRPLDPGPLYAVKVVLSDLGTCGGVLCDVNGRVLREDGVPIDGLYAIGNTAAN AFGKTYPGAGATIAQGLVYGHVAAQHAAGHT。

With the other 3- sterones-△ reported at present¹Dehydrogenase gene highest similarity is only 85%, and it is apparent poor to have The opposite sex, therefore kstD2 is a kind of new 3- sterones-△¹Dehydrogenase gene.It is as follows using 5.0 design primers of Primer：

kstD2-F 5’cagcaagcttGTGACCGATCAGAACAACATCACC(SEQ ID NO：3)；

kstD2-R 5’ctcgaagcttTCAGGTGTGTCCGGCGGC(SEQ ID NO：4).

Wherein, EcoRI restriction enzyme sites are introduced on primer kstD2-F, and Hind III digestions are introduced on primer kstD2-R Site.

Using the total DNA of Mycobacterium neoaurum DSM 1381 as template, using primer kstD2-F and kstD2-R as amplimer, High-fidelity PrimeSTAR HS DNA Polymerase with GC Buffer (TaKaRa) are selected to carry out the specific item of PCR amplification Part is as follows：

95 DEG C of pre-degeneration 3min, 95 DEG C of denaturation 30sec, 62 DEG C of annealing 30sec, 72 DEG C of extension 2min, 30 recycle；72℃ Finally extend 10min；PCR product is detected with 1.5% agarose gel electrophoresis, the results are shown in Figure 2.Target DNA fragments size For 1674bp, always with target gene fragment length.Through sequencing company sequencing analysis, 3- sterones-△ is obtained¹Dehydrogenase encodes Gene kstD2 sequences, length are 1674bp (SEQ ID NO：1).

2 3- sterones-△ of embodiment¹The structure of dehydrogenase coding genes kstD2 expression vectors and conversion

The segment that embodiment 1 is obtained is through EcoRI and Hind III double digestions, and also with EcoRI and Hind III The expression vector pET-28a for carrying out double digestion is attached, and the inverted bacillus coli DH 5 alpha competent cell of connection product (is purchased from TAKARA companies), screening verification obtains recombinant expression.Recombinant plasmid is sent to the Suzhou bio tech ltd Jin Weizhi Sequencing analysis is carried out, through the 3- sterones-△ is sequenced¹Dehydrogenase coding genes kstD2 sequences are SEQ ID NO in sequence table： Nucleotide sequence shown in 1.The recombinant expression of structure is conventionally transformed into e. coli bl21 (DE3) bacterial strain Competent cell in (laboratory preservation), obtain production 3- sterones-△¹The genetic engineering bacterium BL21-kstD2 of dehydrogenase.

The enzyme activity determination of 3 genetic engineering bacterium of embodiment

After IPTG inductions for 24 hours, 6000rpm, 4 DEG C of centrifugations collect recombination bacillus coli in 10 minutes from 50Ml cultures 164 thalline of BL21 (DE) and bacillus subtilis, is then cleaned with 50mM Tris-HCl buffering areas (pH value 7.0), finally twice It is resuspended with 4mL buffer solutions.Object is resuspended and carries out ultrasonic disruption processing, then 4 DEG C of 12000rpm is centrifuged 30 minutes.Supernatant is taken to survey Determine KstD enzyme activity.Enzyme activity determination use spectrophotometry, using NanoDrop 2000 measure 600nm (600 nm=18.7 of ε × 103cm^-1M^-1) absorbance change when being in 30 DEG C measures corresponding enzyme activity.(1 milliliter) of reaction mixture includes 50mM Tris-HCl pH7.0,1.5mM phenazine methosulfates (PMS), 0.12mM 2,6- dichloropheno-lindophenols (DCPIP), the 300 thick enzymes of μ L Liquid, 500 μM of AD and 5g/L hydroxypropyl-β-cyclodextrins.It is each to measure in triplicate.The enzyme activity to specific substrates of each sample Property (be free of any steroidal) is calculated by the value of the blank control subtracted.The sequencing of total protein content uses Bradford methods. The enzymatic activity of 1U is defined as restoring 1 μm of required enzyme amount of ol DCPIP in 1min.Measuring the thick enzyme activity of recombinant bacterial strain is 3.46U/mg。

Conversions of the 4 engineering strain BL21-kstD2 of embodiment to AD

Recombination bacillus coli BL21-kstD2 is inoculated in 5% inoculum concentration in TB culture mediums, in 37 DEG C of 200rpm shaking tables Middle culture, when OD600 reaches 0.8 or so, isopropylthio galactolipin former times (IPTG), which is added, makes its final concentration reach 1.0 mM, 4-AD and 0.5% (W/V) is added according to 1.0% (W/V) simultaneously, hydroxypropyl cyclodextrin is added, continues to convert at 37 DEG C.Through HPLC It tests and analyzes, the conversion ratio of substrate A D reaches 99% (see Fig. 3), recombination bacillus coli BL21-kstD2AD conversions after conversion 21h The surplus of AD in experiment is as shown in figure 4, product ADD contents reach 7.9g/L (see Fig. 5).

The above, only presently preferred embodiments of the present invention, not to the present invention in any form with substantial limitation, It should be pointed out that for those skilled in the art, under the premise of not departing from the method for the present invention, can also make Several improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile all substantial technologicals pair according to the present invention The variation, modification and evolution of any equivalent variations made by above-described embodiment, still fall within the range of technical scheme of the present invention It is interior.

Sequence table

<110>Shanghai Advanced Research Institute, Chinese Academy of Sciences

<120>A kind of 3- sterones-△ 1- dehydrogenases and its encoding gene and application

<130> 181606

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<170> SIPOSequenceListing 1.0

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<213>Artificial sequence (Artificial Sequence)

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gtgaccgatc agaacaacat caccgtcgac ctcgtcgtcg tcggctcggg taccgggatg 60

gcggcagcat tggctgccca cgagctggga atgtcgacgc tgatcgtcga gaagagcgcc 120

tatgtcggtg gttcgacggc tcgctccggc ggtgccttct ggcttcccgg cagctccatt 180

ctcaaggacg ccggttcggc ggacactccg gccaaggcgc gcacctacct tgaagcactc 240

gtcggtgacg acgtctcacc cgaacgcgca cgcactttca tcgatcagat ccccgcgacc 300

atcgacatgt tgcgtcgcac caccccgatg aagttcatgt gggccaaggg atattcggac 360

taccacccgg agaggccagg aggcagtgcg gtgggccgga cctgtgagtg tcgcccgttc 420

gacactgcgg tcctcggtcc agagctggcg cggctacgac ctggagtgat gaagtcatcg 480

ttcccgatgc cggtcaccgg cgccgattac cgttggctga acctgatggc ccgcaccccg 540

cgcaagtcct ggccgcggat catgctgcgg gccatgcagg gtgtcggcgg tttggccctg 600

cggcgccggt acgccgcagg cggccaggcc ttggcggccg ggatgttcgc cggcgtgctg 660

caggcgggga tcccggtgtg gaccgattcg acggtgaccg agctcatcac cgatggtggg 720

cgggtgaccg gcgcgcgggt gctgcgcgag ggatcggccg tgaccgtcac cgcacgccgt 780

ggcatcgtgc tggccaccgg cggtttcgac cacgagatga attggcggcg gaagttccag 840

tccgagctcc tcggtgaaca tctcagcctt ggggccgaga gcaataccgg cgatggcatc 900

cggctcgccc aggacctggg cgcaggcacc ggactgatgg accaggcatg gtggtttccg 960

gcctttgctc cgctgcctgg cggggatccc accgtgatgc tggccgagcg gtcgctgccc 1020

ggctgcctgc tggtagacca gaccggtgag cgcttcatca acgaggccac cgactacatg 1080

tccttcggac agcagctgct gcgtcgcgaa cacgcgggca atccggtcga gacgatgtgg 1140

atgatcttcg atcagcgcta ccggaacagc tatctgcttg ccgccgaact atttccacga 1200

atgccgatcc cacagagttg gtacgacgcc gggatcgcgc accgcggcac ggatgcggaa 1260

gcactgggcc gccagatcgg tttcgatccc gcgacgttgg tcgccacgat cgagcggttc 1320

aacggactcg ccgatgccgg tgtcgacgcc gacttccagc gcggcgcgag cgcctacgac 1380

cgctactacg gcgacccgac gatcacgccc aacccgaacc tgcgaccgct ggatcccggc 1440

ccgctgtacg ccgtcaaggt cgtgctgagc gacctgggca cctgtggtgg ggtcctgtgc 1500

gacgtgaacg gccgggttct gcgcgaagac ggagtgccca tcgacggtct gtacgcgatc 1560

ggcaataccg cggccaacgc attcggcaag acctacccgg gcgcgggcgc gaccatcgcg 1620

caggggctgg tgtacggcca tgttgccgcg cagcatgccg ccggacacac ctga 1674

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<211> 557

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Val Thr Asp Gln Asn Asn Ile Thr Val Asp Leu Val Val Val Gly Ser

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Gly Thr Gly Met Ala Ala Ala Leu Ala Ala His Glu Leu Gly Met Ser

20 25 30

Thr Leu Ile Val Glu Lys Ser Ala Tyr Val Gly Gly Ser Thr Ala Arg

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Ser Gly Gly Ala Phe Trp Leu Pro Gly Ser Ser Ile Leu Lys Asp Ala

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Gly Ser Ala Asp Thr Pro Ala Lys Ala Arg Thr Tyr Leu Glu Ala Leu

65 70 75 80

Val Gly Asp Asp Val Ser Pro Glu Arg Ala Arg Thr Phe Ile Asp Gln

85 90 95

Ile Pro Ala Thr Ile Asp Met Leu Arg Arg Thr Thr Pro Met Lys Phe

100 105 110

Met Trp Ala Lys Gly Tyr Ser Asp Tyr His Pro Glu Arg Pro Gly Gly

115 120 125

Ser Ala Val Gly Arg Thr Cys Glu Cys Arg Pro Phe Asp Thr Ala Val

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Leu Gly Pro Glu Leu Ala Arg Leu Arg Pro Gly Val Met Lys Ser Ser

145 150 155 160

Phe Pro Met Pro Val Thr Gly Ala Asp Tyr Arg Trp Leu Asn Leu Met

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Ala Arg Thr Pro Arg Lys Ser Trp Pro Arg Ile Met Leu Arg Ala Met

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Gln Gly Val Gly Gly Leu Ala Leu Arg Arg Arg Tyr Ala Ala Gly Gly

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Gln Ala Leu Ala Ala Gly Met Phe Ala Gly Val Leu Gln Ala Gly Ile

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Pro Val Trp Thr Asp Ser Thr Val Thr Glu Leu Ile Thr Asp Gly Gly

225 230 235 240

Arg Val Thr Gly Ala Arg Val Leu Arg Glu Gly Ser Ala Val Thr Val

245 250 255

Thr Ala Arg Arg Gly Ile Val Leu Ala Thr Gly Gly Phe Asp His Glu

260 265 270

Met Asn Trp Arg Arg Lys Phe Gln Ser Glu Leu Leu Gly Glu His Leu

275 280 285

Ser Leu Gly Ala Glu Ser Asn Thr Gly Asp Gly Ile Arg Leu Ala Gln

290 295 300

Asp Leu Gly Ala Gly Thr Gly Leu Met Asp Gln Ala Trp Trp Phe Pro

305 310 315 320

Ala Phe Ala Pro Leu Pro Gly Gly Asp Pro Thr Val Met Leu Ala Glu

325 330 335

Arg Ser Leu Pro Gly Cys Leu Leu Val Asp Gln Thr Gly Glu Arg Phe

340 345 350

Ile Asn Glu Ala Thr Asp Tyr Met Ser Phe Gly Gln Gln Leu Leu Arg

355 360 365

Arg Glu His Ala Gly Asn Pro Val Glu Thr Met Trp Met Ile Phe Asp

370 375 380

Gln Arg Tyr Arg Asn Ser Tyr Leu Leu Ala Ala Glu Leu Phe Pro Arg

385 390 395 400

Met Pro Ile Pro Gln Ser Trp Tyr Asp Ala Gly Ile Ala His Arg Gly

405 410 415

Thr Asp Ala Glu Ala Leu Gly Arg Gln Ile Gly Phe Asp Pro Ala Thr

420 425 430

Leu Val Ala Thr Ile Glu Arg Phe Asn Gly Leu Ala Asp Ala Gly Val

435 440 445

Asp Ala Asp Phe Gln Arg Gly Ala Ser Ala Tyr Asp Arg Tyr Tyr Gly

450 455 460

Asp Pro Thr Ile Thr Pro Asn Pro Asn Leu Arg Pro Leu Asp Pro Gly

465 470 475 480

Pro Leu Tyr Ala Val Lys Val Val Leu Ser Asp Leu Gly Thr Cys Gly

485 490 495

Gly Val Leu Cys Asp Val Asn Gly Arg Val Leu Arg Glu Asp Gly Val

500 505 510

Pro Ile Asp Gly Leu Tyr Ala Ile Gly Asn Thr Ala Ala Asn Ala Phe

515 520 525

Gly Lys Thr Tyr Pro Gly Ala Gly Ala Thr Ile Ala Gln Gly Leu Val

530 535 540

Tyr Gly His Val Ala Ala Gln His Ala Ala Gly His Thr

545 550 555

<210> 3

<211> 34

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 3

cagcaagctt gtgaccgatc agaacaacat cacc 34

<210> 4

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 4

ctcgaagctt tcaggtgtgt ccggcggc 28

Claims

1. a kind of 3- sterones-△ of separation¹Dehydrogenase, the 3- sterones-△¹Dehydrogenase is the protein of following (1) or (2)； (1) amino acid sequence such as SEQ ID NO：Protein shown in 2；(2) with (1) in protein have at least 80% homology and Protein with protein function in (1).

2. a kind of polynucleotides of separation, the polynucleotide encoding 3- sterones-△ as described in claim 1 of the separation¹It is de- Hydrogen enzyme.

3. the polynucleotides detached as claimed in claim 2, which is characterized in that the nucleotide sequence of the polynucleotides is such as SEQ ID NO：Shown in 1.

4. a kind of recombinant expression carrier, including the polynucleotides detached as claimed in claim 2 or claim 3.

5. recombinant expression carrier as claimed in claim 4, which is characterized in that the recombinant expression carrier is by such as claim 2 Or the polynucleotides of the separation described in 3 are cloned into expression vector and obtain.

6. recombinant expression carrier as claimed in claim 5, which is characterized in that the expression vector is pET-28a.

7. a kind of host cell, the cell contains any one of them recombinant expression carrier or gene such as claim 4~6 The polynucleotides of external source detached such as any one of them of claim 2~3 are integrated in group.

8. a kind of preparing 3- sterones-△ as described in claim 1¹The method of dehydrogenase, includes the following steps：

Host cell as claimed in claim 7 is cultivated under suitable conditions, is allowed to express the 3- sterones-△¹Dehydrogenase, It then detaches and purifies and obtain the 3- sterones-△¹Dehydrogenase.

9. 3- sterones-△ as described in claim 1¹Dehydrogenase, as described in any one of claim 2~3 polynucleotides, such as weigh Profit requires 4~6 any one recombinant expression carrier or host cell as claimed in claim 7 to be generated for converting 4-AD The purposes of ADD.

10. a kind of 4-AD generates the transforming agent of ADD, contain 3- sterones-△ as described in claim 1¹Dehydrogenase, such as claim 2~3 any one polynucleotides, recombinant expression carrier or such as claim 7 as described in any one of claim 4~6 Any one of described host cell.

11. a kind of method that conversion 4-AD generates ADD, including step：By 3- sterones-△ as described in claim 1¹Dehydrogenase Or host cell as claimed in claim 7, act on 4-AD.