CN102168098B

CN102168098B - Cholesterol oxidase gene, engineering bacterium and application thereof

Info

Publication number: CN102168098B
Application number: CN201110024406XA
Authority: CN
Inventors: 魏东芝; 王风清; 姚抗; 陶欣艺
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2011-01-21
Filing date: 2011-01-21
Publication date: 2013-03-20
Anticipated expiration: 2031-01-21
Also published as: CN102168098A

Abstract

The invention provides a cholesterol oxidase gene, cholesterol oxidase coded by the cholesterol oxidase gene, an expression vector containing a gene sequence of the cholesterol oxidase gene, a gene engineering recombinant strain containing the expression vector and a method for preparing the cholesterol oxidase. The cholesterol oxidase recombinant strain provided by the invention can be used for performing 3-site hydroxyl dehydrogenation or sterol metabolism on 3-hydroxyl steroids and can also be used for degrading sterols, so that the problem of low utilization ratio of mycobacteria and rhodococcus for degrading sterols in the sterol medicine industry is solved.

Description

Cholesterol oxidase gene from rhodococcus equi and engineering bacteria and application

Technical field

The present invention relates to the genetically engineered field, more specifically, relate to the rCO technical field, refer to especially two kinds of mycobacterium cholesterol oxidase gene from rhodococcus equi and correlation engineering bacterium and application.

Background technology

Steroid (steroids) claim again steroid, and being combined closely by 17 carbon atoms forms structure take perhydrocyclopentanophenanthrene as parent nucleus.Its general structure is suc as formula shown in the I,

Formed by three six-rings and a five-ring, be called A, B, C and D ring, wherein, with * number be chiral carbon atom.On the C10 of general parent nucleus and the C13 position methyl is arranged, C3, C11, hydroxyl may be arranged on the C17 position, and (OH), (C=O) or alkyl, the part position of A ring or B ring contains two keys to ketone group, and the different side chain of length is often arranged on the C17 position.Just because of the difference of substituting group, position of double bond or steric configuration on the steroidal compounds parent nucleus, thereby a series of compounds with unique physiological function have been formed.In the humans and animals body, steroid is main endogenous hormone, and do as one likes organ and adrenal cortex are secreted, with the growth of reproduction, brain and bone, stable state keep and the regulation and control of biological effect etc. closely related.Simultaneously as exogenous hormone, steroid hormone class medicine is an indispensable clinically class medicine, body is played important regulating effect, have extremely important pharmaceutical value, have the effects such as anti-inflammatory, antianaphylaxis, resistance attitude, antishock reaction such as adrenocortical hormone.

Cholesterol is the important composition composition of vertebrate cells film, and it has diversified physiological function.But too much cholesterol but is one of Hazard Factor that cause coronary heart disease, arteriosclerosis and myocardial infarction in the body.The mortality ratio of developed country's coronary heart disease such as present America and Europe has surpassed the summation of all cancer mortalities, accounts for 27.4% of general mortality rate.RCO (CHO) can the catalysis cholesterol be oxidized into the courage Gona-4-en-3-one.Therefore can detect with it the content of Blood Cholesterol.It also can decompose the cholesterol in the food, has been applied in the middle of the treatment of cardiovascular disorder.In addition, the growth and breeding that it can also the establishment lepidopterous insects is a kind of biotic pesticide.Its oxidation products also has the curative effects such as anti-obesity, treatment hepatopathy.

RCO (cholesterol oxidase), it is the C3 position hydroxyl desaturase of steroidal compounds, in microorganism, extensively exist, as shown in Figure 1, it can the catalysis cholesterol, the dehydrogenation reaction of C3 position hydroxyl occurs in the steroidal substances such as plant sterol, is one of of paramount importance reaction in the degradation of steroid compound approach, is bringing into play irreplaceable effect for the degraded of steroidal compounds parent nucleus.Therefore, rCO has important research and using value, as: rCO can be used as a biological detection enzyme, is used for the clinical diagnosis of cholesterol.Its principle is: rCO can with cholesterol the 3-hydroxyl-thaumatropy of 5-alkene is 3-ketone-4-alkene structure, generates simultaneously H ₂O ₂, by detecting H ₂O ₂Amount can calculate the amount of substrate cholesterol.The analytical procedure of rCO, cholesteryl esterase and the unification of catalase three enzymes has been widely used in the clinical diagnosis of total cholesterol level in the blood plasma.Utilize the reaction of rCO catalysis cholesterol generation 3-ketone-4-alkene class steroidal compounds, can be used for detecting the content of Blood Cholesterol, understand and control the generation of the cardiovascular disordeies such as coronary heart disease, arteriosclerosis and myocardial infarction.

RCO is the key enzyme of the sterol degradation microbiological deterioration 3-hydroxy-steroids such as mycobacterium, rhodococcus, the Degradation and Transformation that can be used for the 3-hydroxy-steroids is produced androstane-4-alkene-3,17-diketone (AD) and androstane-1,4-diene-3, the important steroid drugs precursor substances such as 17-diketone (ADD).RCO catalyzed oxidation steroidal compounds C3 position oh group is most important, application in the steroidal medicine industry is quite extensive, tight related androstane-4-alkene-3 wherein, 17-diketone (AD), androstane-1,4-diene-3, the production of the important steroid drugs precursor substances such as 17-diketone (ADD) and related derivatives.Mycobacterium and rhodococcus are the important microbe of sterol (animals and plants sterol, simple sterol or sterol mixture) preparation AD or ADD of degrading in the industrial production.Often substrate conversion efficiency is on the low side for most of mycobacterium strains and rhodococcus degraded sterol, causes the output of product A D or ADD very low, becomes the maximum bottleneck of industrial applications.

Therefore, be necessary this is studied, to overcome the low problem of mycobacterium and rhodococcus degraded sterol utilization ratio, realize a large amount of generations of important intermediate AD or ADD.

Summary of the invention

The present inventor is for addressing the above problem, rCO has been carried out large quantity research, found under study for action a kind of brand-new rCO, and 2 kinds of high reactivity rCO recombinant bacterial strains have been made up, this recombinant bacterial strain can be used for 3-hydroxy kind steroidal compounds and carries out 3 hydroxyl dehydrogenations or sterol metabolism, and sterol degradation, thereby the aforementioned difficult problem in the solution steroidal medicine industry.Therefore, one of purpose of the present invention is to provide a kind of rCO and gene thereof; The recombinant microorganism that two of purpose provides the expression vector that comprises this rCO and utilizes this carrier to transform is such as engineering strain; Three of purpose provides the application of this cholesterol oxidase gene from rhodococcus equi engineering strain.

RCO of the present invention can derive from mycobacterium, such as mycobacterium ATCC 25795 (NwIB-00 bacterial strain).The technical solution used in the present invention is: by the very high full genomic information of mycobacterium NwIB-01 of homology approximation is analyzed, obtain complete cholesterol oxidase gene from rhodococcus equi, and design pcr amplification primer, from cloning the dna sequence dna of rCO cho-1 the mycobacterium NwIB-00 (shown in SEQ ID NO:1, corresponding aminoacid sequence is shown in SEQ ID NO:2) with the dna sequence dna (shown in SEQ ID NO:3, corresponding aminoacid sequence is shown in SEQ ID NO:4) of cho-2.Certainly, as known to persons skilled in the art, cholesterol oxidase gene from rhodococcus equi of the present invention can also be other nucleotide sequences of the protein that the aminoacid sequence shown in the SEQ ID NO:2 or 4 forms in the coding sequence table; And rCO of the present invention is only for being the protein with composition of the aminoacid sequence shown in the SEQ ID NO:2 or 4 in the sequence table, can also be through replacement, disappearance or the interpolation of one or more amino-acid residues and the protein of being derived by

sequence

2 or 4 with same enzyme activity with the aminoacid sequence in

sequence

2 or 4, such as adding one or several amino acid at C-terminal and/or N-terminal, merge, do not affect the situations such as difference on the modified forms of sequence such as the amino acid with vector encoded.

The expression vector that comprises the nucleotide sequence of cholesterol oxidase gene from rhodococcus equi provided by the invention is with ordinary method the nucleotide sequence of cholesterol oxidase gene from rhodococcus equi of the present invention to be connected in to make up on the various carriers to form, this carrier can be commercially available plasmid, clay, phage or virus vector etc., such as pUC, pBluescript (Stratagene), pET (Novagen, Inc., Madison, Wis), PQE (Qiagen), pREP, pSE420 and pLEX (Invitrogen), but be not limited to these carriers.

Preferably with pcr amplification to the cholesterol oxidase gene from rhodococcus equi product be connected with expression vector pET-28a+) connect and to obtain recombinant plasmid.

Transformant provided by the invention, it is that the expression vector that will comprise cholesterol oxidase gene from rhodococcus equi nucleotide sequence of the present invention is transformed into host microorganism, such as the competence intestinal bacteria---obtain genetic engineering bacterium in the e. coli bl21 (DE3).

Wherein, this host cell can be protokaryon, eukaryotic microorganisms or insect etc.Preferably intestinal bacteria, obtaining corresponding transformant is above-mentioned engineering strain.

RCO recombinant bacterial strain provided by the invention can be used for 3-hydroxy kind steroidal compounds is carried out the application of 3 hydroxyl dehydrogenations or sterol metabolism, the application of the sterol that also can be used for degrading.

Those of ordinary skill in the art can be by gene order amplification commonly used and the method for analyzing, obtain relatively easily being adjacent from the cholesterol oxidase gene from rhodococcus equi of mycobacterium the fragment of gene, SEQ ID NO:1 and SEQ IDNO:3 are much larger than the nucleotide sequence length of corresponding cholesterol oxidase gene from rhodococcus equi, because the controlling element of corresponding gene and adjacent gene pairs later stage make up highly active genetic engineering bacterium material impact is arranged, therefore whole SEQID NO:1 and SEQ ID NO:3 sequence had both been contained in the present invention, but be not limited to the full length fragment of SEQ ID NO:1 and SEQ ID NO:3, the gene of the 281st～1877 nucleotide coding rCO (cho-2) among the gene of the 307th～2052 nucleotide coding rCO (cho-1) and the SEQ ID NO:3 in the SEQ ID NO:1 sequence wherein, it is core gene sequence of the present invention, in addition, by aminoacid sequence SEQ ID NO:2 and the SEQ ID NO:4 of corresponding rCO coding, it also is core of the present invention.

RCO can be expressed by the additive type expression vector in mycobacterium, and used recombination engineered vector is the mycobacterium expression vector of low copy type or high copy type; RCO can be incorporated into karyomit(e) and express in mycobacterium, integrate by target, unmarked mode; Also can be independent of karyomit(e) with the form of free plasmid expresses outward.

In sum, the invention provides rCO, by making up various expression vectors and transforming relevant bacterial strain, thereby obtained the various engineering bacterial strain, the mycobacterium that comprises intestinal bacteria, streptomycete and hypercholesterolemia oxidase activity adopts these engineering bacterias or its crude enzyme liquid can selectively prepare or the catalytic production steroidal compounds.Above-mentioned these engineering strains, can greatly improve production efficiency and the product quality of steroidal medicine production system, help to reduce the energy consumption in the steroid drugs production process, the utilization ratio that improves the medicine prerequisite, simplification production stage, reduce production costs, and reaction conditions is gentle, environmental friendliness, be suitable for wideling popularize application, have higher economic benefit and social benefit.In addition, these two kinds of rCOs all can as testing tool albumen, can be used for the detection of cholesterol.

Description of drawings

Fig. 1 is the reaction synoptic diagram that rCO catalysis steroidal compounds of the present invention generates 3-ketone-4-alkene structure, and wherein, R1, R3, R4, R6 and R7 are alcohol radical, ketone group, the alcohol radical ether of deriving and ester, halogen group; R2 is alcohol radical, ketone group, hydrocarbon chain, with the hydrocarbon chain of alcohol radical or with the hydrocarbon chain of ketone group; R5 is alcohol radical or halogen group.

Fig. 2 is that two kinds of homology mycobacterium NwIB-00 and NwIB-01 are to two kinds of different metabolic modes of steroidal compounds.

Fig. 3 is the cholesterol oxidase gene from rhodococcus equi cho-2 nucleic acid electrophoresis collection of illustrative plates that amplification obtains among the mycobacterium NwIB-00, and wherein, M is DNA standard Marker; Swimming lane 1 negative control experiment;

Swimming lane

2 and 3 is the pcr amplification result.

Fig. 4 is that the enzyme of recombinant plasmid pET28a-NwIB-00.CHO2 is cut the checking collection of illustrative plates, and wherein, M is DNA standard Marker; Swimming lane 1 is HindIII, EcoRI double digestion the result; Swimming lane 2 is the single double digestion the result of HindIII.

Fig. 5 is the SDS-PAGE electrophorogram of genetic engineering bacterium expression product, wherein, M is protein electrophoresis standard Marker, swimming lane 1 adds whole-cell protein electrophorogram after IPTG induces 6 hours for BL21/NwIB-00.CHO2, and swimming lane 2 adds whole-cell protein electrophorogram after IPTG induces 6 hours for BL21/NwIB-00.CHO1.

Fig. 6 finally obtains rCO (cho-2) deletion mycopremna for using existing screening principle by mycobacterium mycobacterium NwIB transformant being carried out the screening of two steps, and wherein, Marker is DNA standard Marker; Sco is the amplification of single recon of sequence after having simultaneously full length sequence and lacking; Wt is the amplification of cho-2 among the wild-type NwIB-00; MutDCO-1 is double exchange 1 that screens ^#(NwIB-00) amplification; MutDCO-2 is double exchange 2 that screens ^#The amplification of (not carrying out substrate conversion research).

Fig. 7 is the result that PCR confirms NwIB-00.CHO1-405 genetically engineered deletion mycopremna, and wherein, Marker is DNA standard Marker; Swimming lane 1～4 is the pcr amplification situation of corresponding cho-1 gene and cho-2 gene.

Fig. 8 is the as a result figure of two kinds of engineering strain transformation phytosterins in the shaking flask, chart has reflected: 1, during at 0.7g/l, conversion rate is with the comparison of original mycobacterium NwIB-01 in plant sterol concentration for mycobacterium NwIB-01.CHO1-405 and NwIB-01.CHO2-405; 2, mycobacterium NwIB-01.CHO1-405 and NwIB-01.CHO2-405 are when different concentration of substrate 1g/l～4g/l, and transformation efficiency is with the comparison of original mycobacterium NwIB-01.

Fig. 9 is that the GC of tunning analyzed collection of illustrative plates when engineering strain NwIB-01.CHO2-405 transformed the 2.0g/l plant sterol in the shaking flask.

Figure 10 is the situation that NwIB-01.CHO2-405 transforms the 15g/L plant sterol in 10 liters of fermentor tanks.

Embodiment

Content for a better understanding of the present invention is described further below in conjunction with specific embodiment.Should be understood that following examples are only for explanation the present invention but not for limiting scope of the present invention.

The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in " molecular cloning: laboratory manual " (New York:Cold Spring Harbor Laboratory Press, 1989) is carried out.

The gene manipulation techniques that the application adopts mainly contains: gene expression technique and unmarked enzyme functionally inactive technology.Gene expression technique mainly contains: the low copy expression plasmid that non-chromosome is integrated or the phraseology of high copy expression plasmid; Single copy cholesterol oxidase gene from rhodococcus equi of chromosomal integration formula or the phraseology of multiple copied cholesterol oxidase gene from rhodococcus equi.Unmarked enzyme functionally inactive technology comprises that the cholesterol oxidase gene from rhodococcus equi of non-resistant mark lacks mode with frame.

The e. coli bl21 that uses in an embodiment of the present invention, JM109, DH5a, TOP10, and pET22, pET28, pET32 carrier all available from Novagen company, primer is synthetic by precious biological (Takara) company in Dalian.

The substrate of indication steroidal compounds of the present invention is 3-alcohol-5-en steroids compound, only so that a class 3-alcohol-5-en steroids compound---" sterol " is as example, sterol is divided into again cholesterol, ergosterol and plant sterol etc., plant sterol is comparatively commonly used, plant sterol is that the sterol mixture is comprised of multiple sterols such as Sitosterol, Stigmasterol, campesterols, comes from the industry byproducts such as Tall oil of grease deodorized distillate, pulping and papermaking industry more.

Indication steroidal compounds product of the present invention is 3-ketone-4-en steroids, be generally AD, ADD and 9 α-derivatives such as OHAD, the present invention is only take the engineering strain that produces ADD as example, all the other engineering strains of producing AD and 9 α-derivatives such as OHAD are relating to rCO related fields of the present invention without any difference with the engineering strain that produces ADD, therefore, though the present invention is not limited to the engineering strain that produces ADD take the engineering strain that produces ADD as example.Any have common genetic manipulation technical ability can according to content of the present invention, make up product AD, the ADD relevant with rCO and the engineering strain of 9 α-derivatives such as OHAD per capita.

The preserving number of the mycobacterium NwIB-00 bacterial strain that uses among the present invention is: ATCC 25795 (available from ATCC); The preserving number of NwIB-01 bacterial strain is: CCTCC M 209094.As shown in Figure 2, this bacterial strain degradable transforms sterol and generates ADD, and product A DD only has a little being degraded, in the following embodiments, only take the NwIB-01 bacterial strain as example, the bacterial strain that all the other produce AD and 9 α-OHAD and their derivatives should be considered as being equal to the NwIB-01 bacterial strain.

Embodiment 1, mycobacterium NwIB-00 cholesterol oxidase gene from rhodococcus equi the colibacillary structure of acquisition, Molecular Identification and heterogenous expression

Present embodiment is only take PCR method as example, by the full genomic information of mycobacterium NwIB-01 is analyzed, obtain complete cholesterol oxidase gene from rhodococcus equi, and design pcr amplification primer, from cloning the dna sequence dna of rCO cho-1 the mycobacterium NwIB-00 (shown in SEQ ID NO:1, corresponding aminoacid sequence is shown in SEQ ID NO:2) with the dna sequence dna of cho-2 (shown in SEQ ID NO:3, corresponding aminoacid sequence is shown in SEQ ID NO:4), detailed process is as follows:

1.1, the acquisition of total length cholesterol oxidase gene from rhodococcus equi

Participating in the cholesterol metabolic involved enzyme among the present known NwIB-00 is respectively: 3-sterone-Δ 1-dehydrogenase gene (KSDD) and 3-sterone-9 Alpha-hydroxylation enzyme gene (KSH), compare in twos discovery by software Clustal W1.8 and NCBI Blastn with corresponding gene among the NwIB-01, similarity is all more than 95%, and also there is high similarity in two kinds of cholesterol associated oxidase genes of bacterium of rational conjecture.

NwIB-01 checks order to mycobacterium, and according to its order-checking information, in genome annotation information, retrieve, find entire reading frame sequence corresponding to mycobacterium NwIB-01 cholesterol oxidase gene from rhodococcus equi, and simultaneously in the whole CDS information of mycobacterium NwIB-01, follow the tracks of obtaining two kinds and may be cholesterol oxidase gene from rhodococcus equi upstream and downstream flanking sequence.Utilize thus software Oligo 6.0 and Primer 5.0 design following primer: NwIB-00.CHO1-F1 (shown in SEQ IDNO:5), NwIB-00.CHO1-R1 (shown in SEQ ID NO:6), NwIB-00.CHO2-F1 (shown in SEQ ID NO:7) and NwIB-00.CHO2-R1 (shown in SEQ ID NO:8).

Carry out pcr amplification take the genomic dna of mycobacterium NwIB-00 as template, wherein,

The PCR reaction system is as follows:

Get an amount of template DNA 0.2 μ L, 2 * GC BufferI10 μ L, 2.5mM dNTP 3.2 μ L, each 0.4 μ L of primer (20 μ mol/L), Taq archaeal dna polymerase 1U, moisturizing is to cumulative volume 20 μ L.

The PCR response procedures is as follows: 94 ℃ 30 seconds, 65 ℃ 30 seconds, 72 ℃ 2 minutes, 30 circulations.

The PCR product is detected, and the result shows that SYBR GREENI dyeing band is unique, and few dimer produces, and after glue reclaimed, two kinds of enzyme genes all connected the order-checking of pMD19-T carrier.

Obtain respectively the gene fragment of 1746bp (called after cho-1) and 1617bp (called after cho-2) size after the order-checking, complete coding 581 and 538 amino acid, compare with corresponding cholesterol oxidase gene from rhodococcus equi among the known NwIB-01 by software Clustal W1.8, similarity all reaches 99%, and the amino acid consistence has also reached 99%; By NCBI Blastn comparison, rCO (cho-1) gene order with clearly the rCO of report the highest 85% consistence is arranged, and the cho-2 gene only has 51% consistence with rCO like recently; By NCBI Blastx comparison, rCO (cho-1) amino acid similarity can conclude that it is rCO, and cho-2 only has the highest similarity 55% with Streptomyces Virginia, is a new gene fragment up to about 90%.

Aminoacid sequence according to cho-2 among the NwIB-00 that has obtained, cholesterol oxidase gene from rhodococcus equi (cho-1) in the contrast mycobacterium, carry out the analysis of amino acid conserved sequence, the result shows that both have the conservative region in conjunction with cofactor flavin adenine two nucleic acids (FAD) simultaneously, and choline dehydrogenase (BetA) conservative region that carries out cholesterol C3 position dehydrogenation, therefore the cho-2 that assert the NwIB-00 that is comprised of these 538 amino acid may possess the ability of carrying out steroidal C3 position dehydrogenation reaction, may be one section rCO.

1.2, cho-2, the heterogenous expression of cho-1 gene in intestinal bacteria

The clone of cho-2 full-length gene order is as example in the NwIB-00, design and synthesize amplimer NwIB-00.CHO2-F1 ' (shown in SEQ ID NO:9) and NwIB-00.CHO2-R1 ' (shown in SEQ ID NO:10), wherein, introduce the EcoRI restriction enzyme site at primer NwIB-00.CHO2-F1 ', introduce the HindIII restriction enzyme site at primer NwIB-00.CHO2-R1 '.

Take the total DNA of NwIB-00 as template, carry out pcr amplification with primer NwIB-00.CHO2-F1 ' and NwIB-00.CHO2-R1 ', actual conditions is as follows:

Reaction system: ddH ₂O 5.4 μ l, 2 * GC BufferI, 10 μ L, 2.5mM dNTP 3.2 μ L, each 0.4 μ L of primer (20 μ mol/L), template DNA 0.4 μ l, taq enzyme 0.2 μ l.

Reaction process: 95 ℃ of 5min; 95 ℃ of 30s, 69 ℃ of 30s, 72 ℃ of 120s, 30 circulations; 72 ℃ of 10min.

The PCR product is carried out agarose electrophoresis detect, the result according to Fig. 3 result, reclaims the fragment of the about 1600bp of size as shown in Figure 3.

Reclaim fragment through EcoRI, HindIII double digestion, after target DNA fragment reclaims purifying, with equally through plasmid pET-28a (+) that digestion with restriction enzyme is crossed (5369bp) under the effect of T4DNA ligase enzyme, 16 ℃ of connections, screening obtains recombinant plasmid pET28a-NwIB-00.CHO2.The recombinant plasmid of double digestion checking gained is seen Fig. 4.With its order-checking, sequencing result shows that the cho-2 sequence among the NwIB-00 that is cloned into is shown in SEQ ID NO:3.The escherichia coli expression host selects BL21 (DE3), and 42 ℃ of thermal shocks obtain genetic engineering bacterium BL21/NwIB-00.CHO2 with the recombinant plasmid transformed e. coli bl21.

Similar, by above amplification, the method that transforms etc. equally also can obtain to express the genetic engineering bacterium BL21/NwIB-00.CHO1 of rCO.

From transforming dull and stereotyped any picking BL21/NwIB-00.CHO1 and BL21/NwIB-00.CHO2 bacterium colony, access 5mL contains in the LB substratum of 100 μ g/mL penbritins, is cultured to OD in 37 ℃ _600nmAfter reaching 0.4～0.8, add IPTG to final concentration 1mmol/L, at the different time of inducing, centrifugal collection thalline, carrying out ultrasonic bacteria breaking, centrifugal collection supernatant, the SDS-PAGE electrophoresis, the result is as shown in Figure 5.According to Fig. 5 result, recombination bacillus coli BL21/NwIB-00.CHO1 and BL21/NwIB-00.CHO2 expressing quantity are all higher, near the 60kDa place, present obvious clear band, coincide with the enzyme molecular weight size of expection, all successful expression in escherichia expression system of success of two kinds of enzyme genes are described.

Embodiment 2, the structure of cho-2, cho-1 missing gene engineering strain among the mycobacterium NwIB-00

Present embodiment knocks out as example take cho-2's, the technical way and the method that knock out so as to homologous recombination double exchange commonly used in the explanation mycobacterium, and finished knocking out of cho-1 with this method.

Cho-2 knocks out plasmid among the structure mycobacterium NwIB-00, and this plasmid transforms through electricity and enters the mycobacterium competent cell.Utilize card to receive penicillin and hygromycin B and carry out the recon preliminary screening, then utilize the sucrose flat board to carry out multiple sieve, obtain gene knockout recon NwIB-00.CHO2-405, utilize simultaneously PCR method that above-mentioned recon is verified.Genetically deficient double exchange operation can be referring to prior art (gene replacement using pretreated DNABhavna G.Gordhan and Tanya Parish).

The enforcement concrete steps are as follows:

2.1, the acquisition of cho-2 gene upstream and downstream sequence and knock out the structure of plasmid

According to the method for embodiment 1.1, knock out primer for mycobacterium NwIB-01 known array design upstream and downstream: NwIB-00.CHO2-F2 (shown in SEQ ID NO:11), NwIB-00.CHO2-R2 (shown in SEQ ID NO:12), NwIB-00.CHO2-F3 (shown in SEQ ID NO:13) and NwIB-00.CHO2-R3 (shown in SEQ IDNO:14).Specific as follows:

Carry out pcr amplification take the NwIB-00 genome as template, obtain the upstream and downstream gene fragment of cho-2, again respectively with the gene clone of target gene upstream and downstream to the pMD19-T carrier, then use respectively HindIII, SalI, SalI, PacI enzyme are cut, and enzyme is cut product and is connected on the mycobacterium gene knockout plasmid pNL that corresponding enzyme is cut.Cut with the PacI enzyme respectively and carry out non-directional behind above-mentioned plasmid and the pG19 plasmid and connect, make up gene knockout plasmid NwIB-00-QC-CHO2.

2.2, knock out Plasmid Transformation mycobacterium competent cell

The preparation of mycobacterium competent cell: first order seed OD is long to about 0.5～1.5,1%～10% switching secondary seed; Add 2%～5% glycine behind 14～24h and continue to cultivate 1～4d.Centrifugal collection thalline, minute four usefulness 10% glycerine flushing suspension thalline and centrifugal add at last 1ml glycerine suspension thalline, and packing are preserved.

Electricity transforms: 10 μ l plasmids add in the 300 μ l competence thalline places 10min, the following 12.5kv/cm of electric shock condition, 25 μ F, 20ms.

2.3, recombinant screen and checking:

Electricity is changed the line of production behind thing adding substratum recovery cultivation 3～24h, coat on the substratum (medium component is as follows: hyg50 μ g/ml, Kn 20 μ g/ml, X-gal 50 μ g/ml), and leave standstill in 30 ℃ and to cultivate 3～15d, until blue colonies appears in flat board, choose to liquid nutrient medium and cultivate, then PCR verifies the correct generation of single recon (SCO).The sucrose of checking SCO bacterium coating 2%～10% well is dull and stereotyped, continue to cultivate until blueness occurs simultaneously with white colony, the picking white colony, and carry out simultaneously the PCR checking.

The checking of recon: comprise the PCR checking of single recon (SCO) and double exchange (DCO), the checking principle is as described in the front citing document, and the checking primer uses NwIB-00.CHO2-F1 and NwIB-00.CHO2-R1.

The result the band of two sizes about 500bp and 1600bp occur for single recon (SCO) theory as shown in Figure 6; For the theoretical band that the 500bp size only occurs of double exchange (DCO), sign originally reached the complete cholesterol oxidase gene from rhodococcus equi of 1617bp and was successfully lacked as size only is the fragment of 500bp, had destroyed the function of original enzyme.

With above-mentioned method, the experimenter has finished at mycobacterium NwIB-00 rCO cho-1 has been knocked out, and has finished the structure of the relevant bacterial strain of NwIB-00.CHO1-405, and its PCR the result as shown in Figure 7.

Embodiment 3, mycobacterium NwIB-00 and NwIB-01 be to conversion and the interpretation of result method of steroidal compounds

Utilize 1%～10% materials such as tensio-active agent, polymkeric substance or organic solvent (routine Tween80, ethanol, silicone oil, soya-bean oil etc.) that steroidal compounds is carried out hydrotropy.Adopt two-stage or three grades of cultivations, with 1%～20% the final conversion substratum of seed inoculation, steroidal compounds can add the time in office.The condition of Steroid Transformation is: 25～37 ℃ of culture temperature, and high oxygen dissolving value, pH is controlled between 5.0～8.0, with concluding time and the conversion results of thin-layer chromatography (TLC) or the conversion reaction of gas-chromatography (GC) Analysis deterrmination.After reaction finished, the Steroid Transformation thing can be used the organic solvent extractions such as ethyl acetate with volume, chloroform three times, merges reaction solution, vacuum-drying, and then analyze with product and prepare.

Shake-flask culture mycobacterium NwIB-00 and NwIB-01 transformation phytosterin, Tween80 or the silicone oil of employing 1%～5% are solubility promoter, be to adopt two-stage to cultivate in 30 milliliters 250 ml shake flasks at sample-loading amount, wherein inoculate the secondary medium of plant sterol concentration 0.4g/L～4.0g/L with 1～20% grain weight, at 26～35 ℃, 200～300rpm, cultivate under the condition between the pH 5.0～8.0, sampling after 3～7 days, the ethyl acetate oscillation extraction, organic phase is by the growing amount of the check of the methods such as TLC and GC and detection sterol transformation efficiency and product A DD.

Thin plate preparation (TLC) condition is: developping agent adopts sherwood oil: ethyl acetate (6: 4 to 7: 3); Thin plate adopts the precoated plate of 5 * 10cm of Yantai silica gel factory product; Colour developing, the sulphuric acid soln of employing 20% evenly sprays, and baking 5min observes after showing spot in 105 ℃ of baking ovens.

Embodiment 4, mycobacterium NwIB-00 cholesterol oxidase gene from rhodococcus equi crosses the construction process of expression strain in NwIB-01

Utilize pAL5000 (to take from (the Howard Hughes MedicalInstitute of U.S. Howard Hughes Institute for Medical Research, USA) Jacobs Jr. professor) and derivative vector pFZ2 mycobacterium-bacillus coli shuttle expression carrier (take from (the Howard Hughes Medical Institute of U.S. Howard Hughes Institute for Medical Research, USA) Jacobs Jr. professor) cholesterol oxidase gene from rhodococcus equi among the NwIB-00 is imported mycobacterium NwIB-01, so that the rCO of NwIB-01 was realized expressing, successfully make up NwIB-01.CHO1-405 and NwIB-01.CHO2-405.The building process of expression strain hereinafter still was described as an example of cho-2 example.

The primer sequence is NwIB-00.CHO2-F4 (shown in SEQ ID NO:15) and NwIB-00.CHO2-R4 (shown in SEQ ID NO:16).

Utilize above primer, NwIB-00 clones the CHO2 gene of 1.6kb from mycobacterium, is connected with carrier pMD19-T through reclaiming, and makes up the recombinant clone plasmid, sequence verification.With restriction enzyme EcoRI, SalI double digestion recombinant vectors, recovery is inserted in the mycobacterium that same enzyme is cut-bacillus coli shuttle expression plasmid pAL5000 or pFZ2, construction recombination plasmid pAL5000-NwIB-00.CHO2 or pFZ2-NwIB-00.CHO2, and process double digestion checking fragment is inserted successfully.

Electricity turns recombinant plasmid pAL5000-NwIB-00.CHO2 or pFZ2-NwIB-00.CHO2 and enters mycobacterium NwIB-01 and screen with the kantlex flat board.What need to emphatically point out is, shuttle expression plasmid pAL5000 or pFZ2 be in low copy type plasmid, it is at the very large copy number height that is decided by its plasmid of the expression level of original strain.Electricity at the dull and stereotyped enterprising row filter that antibiotic concentration increases progressively, obtains the engineering bacteria of anti-high antibiotic concentration after transforming the mycobacterium competent cell.Because the said gene engineering bacteria only carrier of recombinant plasmid there are differences, and the difference of bearer type is not brought substantial impact to genetic engineering bacterium, in fact, to those skilled in the art, adopt as required other carriers also to be fine, therefore the genetic engineering bacterium of above-mentioned acquisition is unified called after NwIB-01.CHO2-405.

Adopt above-mentioned identical method, made up genetic engineering bacterium NwIB-01.CHO1-405.

Embodiment 5, rCO crosses the expressing gene engineering bacteria and prepares application among the ADD at sterol degradation

The culture condition of engineering strain and the conversion condition of steroidal can be with reference to embodiment 3.Genetic engineering bacterium NwIB-01.CHO1-405 and NwIB-01.CHO2-405 finish under the operational condition of embodiment 4.In shaking flask (30ml fills liquid/250ml shaking flask), the plant sterol charging capacity is under the condition of 0.5-5%, and transformation time is 5-10 days, and the result of engineering strain transformation phytosterin as shown in Figure 8.In addition, it must be noted that two strains are carried out engineering strain after rCO is strengthened when low concentration of substrate, continue to keep high conversion, and have greatly shortened transformation time; Meanwhile, when concentration of substrate in the shake flask fermentation raise, engineering bacteria then embodied obvious high transformation efficiency advantage than original NwIB-01.Along with the raising of concentration of substrate in the fermention medium, when reaching 3g/l, transformation efficiency is brought up to about 80% by 60%; When concentration reached 4g/l, transformation efficiency brought up to 60% by 40%, and the accumulation volume of product A DD improves nearly 1 times than original NwIB-01, the highest level near 2g/l simultaneously.In addition, Figure 9 shows that NwIB-01.CHO2-405 transforms gas-chromatography (GC) analytical results that the 2.0g/L plant sterol generates product A DD, finally, substrate plant sterol transformation efficiency is higher, and product A DD peak type is intact.

Embodiment 6, NwIB-01.CHO2-405 transforms the situation of 15g/l plant sterol in 10 liters of fermentor tanks

By to embodiment 5 interpretations of result, determine that NwIB-01.CHO2-405 is the production bacterial strain of comparatively ideal ADD.Throughput and present situation for further research engineering bacterial strain, excavate its inherent industrial application potentiality, the scale of now will fermenting is amplified to the 10L fermentor tank from the 250ml shaking flask, initial fermentating liquid volume is 7.0L, 2～20% (v/v) inoculum size access seed liquor, air flow is controlled at 0.2～10vvm, and leavening temperature is 20～40 ℃, rotating speed 200～1000rpm, initial pH are 5.0～9.0 and by 4M NaOH and 4M H ₃PO ₄All the time keep pH in controlled range, observation OD after the inoculation _600nmVariation, work as OD _600nmReach 1.0～5.0 rear addings through the plant sterol of emulsification and continue fermentation culture 100h～300h.The check of tunning and detect can be with reference to the mode of embodiment 3, and after fermentation ends, measure the transformation efficiency of substrate and the actual concentrations of product A DD by gas-chromatography (GC).Figure 10 shows that in the 10L fermentor tank add the fermentation situation of 15g/L substrate plant sterol: the transformation efficiency of plant sterol can reach 95%, improves about 20% than original strain; Simultaneously, product A DD accumulates in a large number, but because to the toxicity of thalline, therefore there is inevitably degraded in high density product itself in the fermentation middle and later periods, however, the fermentation level of product A DD is also brought up to 5.57g/L from original 4.23g/L, increases obviously.

In sum, adopt the bacterial strain of hypercholesterolemia oxydase enzymic activity of the present invention can greatly improve utilising efficiency and the speed of substrate plant sterol, compare with original bacterium, realized the stable raising of transformation efficiency when high concentration of substrate feeds intake, thereby improve product A DD real attenuation level.If therefore improved strain excellent is put into production, can greatly improve production efficiency and the product quality of steroid medicine, help to reduce the energy consumption in the steroid medicine production process, the utilization ratio that improves prodrug, simplification production stage, reduce production costs, and reaction conditions is gentle, environmental friendliness, be suitable for wideling popularize application, have higher economic benefit and social benefit.

In this specification sheets, the present invention is described with reference to its specific embodiment.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention, the present invention is only take the engineering strain that produces ADD as example, all the other engineering strains of producing AD and 9 α-derivatives such as OHAD are relating to rCO related fields of the present invention without any difference with the engineering strain that produces ADD, therefore, though the present invention is take the engineering strain that produces ADD as example, but be not limited to the engineering strain that produces ADD, also should be believed to comprise the engineering strain of AD and 9 α-derivatives such as OHAD.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.

Claims

1. the gene of a rCO is one of following nucleotide sequences:

1) it is the base sequence shown in the SEQ ID NO:1 or 3 in the sequence table;

2) protein that the aminoacid sequence shown in the SEQ ID NO:2 or 4 forms in the coding sequence table.

2. rCO, it is the protein that aminoacid sequence shown in the SEQ ID NO:2 or 4 forms in the sequence table.

3. the expression vector that comprises the nucleotide sequence of cholesterol oxidase gene from rhodococcus equi claimed in claim 1.

4. a rCO mycobacterium engineering strain is characterized in that, described mycobacterium engineering strain is expressed the aminoacid sequence of rCO shown in SEQ ID NO:2.

5. mycobacterium engineering strain as claimed in claim 4 is characterized in that, the nucleotide sequence of the described rCO of encoding is shown in SEQ ID NO:1.

6. mycobacterium engineering strain as claimed in claim 4 is characterized in that, the gene of the described rCO of encoding is positioned on the recombinant plasmid.

7. mycobacterium engineering strain as claimed in claim 4, it is that expression vector claimed in claim 3 is transformed into the engineering strain that obtains in the host microorganism.

8. be used for 3-hydroxy kind steroidal compounds is carried out the application of 3 hydroxyl dehydrogenations such as each described rCO mycobacterium engineering strain among the claim 4-7.

9. be used for 3-hydroxy kind steroidal compounds is carried out the application of sterol metabolism such as each described rCO mycobacterium engineering strain among the claim 4-7.

10. be used for the application of degraded sterol such as each described rCO mycobacterium engineering strain among the claim 4-7.