CN103627721B - G6PDH gene is improving bread mould to the application in steroidal C11 'alpha '-hydroxylation ability and bacterial strain - Google Patents
G6PDH gene is improving bread mould to the application in steroidal C11 'alpha '-hydroxylation ability and bacterial strain Download PDFInfo
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Abstract
The invention provides G6PDH gene at raising bread mould to the application in steroidal C11 'alpha '-hydroxylation ability, and the bacterial strain that the construction process of G 6 PD gene mutations engineering bacteria obtains with the rear screening of structure.G6PDH is cloned in bread mould by molecular biology method by the present invention, transformation bacterial classification, intend in improved bread mould cell, realize G6PDH catalyzing N ADPH to regenerate, reaction to cytochrome P 450 Enzyme catalysis provides required coenzyme, improves the ability of bread mould to steroidal C11 'alpha '-hydroxylation.Beneficial effect of the present invention is mainly reflected in: the genetic engineering bacterium built according to the inventive method, for transforming 16ALPHA,17ALPHA-epoxyprogesterone C
11'alpha '-hydroxylation produces mold oxide, compares starting strain, and growth is fast, and raw material availability is high, transformation efficiency and transformation efficiency high, there is far-reaching theory significance and higher using value in the development and utilization process of steroid drugs.
Description
(1) technical field
The present invention relates to G6PDH gene at raising bread mould to the application in steroidal C11 'alpha '-hydroxylation ability, and the construction process of G 6 PD gene mutations engineering bacteria, and build and screen higher bacterial strain---bread mould (Rhizopusnigericans) PIe of a strain steroidal C11 'alpha '-hydroxylation obtained.
(2) background technology
The product of Oxidoreductases catalyze synthesis is widely used in the fields such as medicine, food, agricultural chemicals, and in enzyme catalysis process, coenzyme deficiency is the principal element that the most of oxydo-reductase of restriction carries out biocatalysis synthetic product.Therefore, coenzyme cyclic regeneration seems extremely important for enzyme catalysis process, and then embodies great value in the products production that medicine food etc. is relevant.The renovation process of bibliographical information coenzyme comprises the renovation process such as enzyme process, photochemistry, electrochemistry, wherein especially in widespread attention with enzyme process regeneration.The enzyme being usually used in regenerating coenzyme has alcoholdehydrogenase (ADH), hydrogenlyase (FDH), Hexose phosphate dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G6PD) (G6PDH), phosphorous acid desaturase (PTDH) etc., wherein the investigation and application of FDH and GDH is more, such as, Sheldon studies E.coli JM109(pGDA2) overexpression comes from Bacillus megateriumIWG3 glucose dehydrogenase gene, when providing NADP+ and glucose, E.coli JM109(pGDA2) can as the regeneration system rapidly of NADPH.Glucose dehydrogenase gene gdh223 in Bacillus megaterium AS1.223 is built pQE30-gdh223 expression vector by clone by ZhinanXu, and at expression in escherichia coli, realize by the conversion of 4-chloroacetyl acetacetic ester to (R)-CHBE.The report being used for regenerating coenzyme relative to FDH and GDH, G6PDH is relatively less.
Glucose-6-phosphate dehydrogenase (G6PD) (glucose-6-phosphate dehydrogenase, G6PDH, EC1.1.1.49) is a key regulatory enzyme in the enzyme and the regulation and control of PPP pathways metabolism that in pentose-phosphate pathway (PPP) oxidation stage, the catalysis the first step is reacted.G6PDH is extensively present in various biomass cellss such as comprising bacterium, plant, animal, the reaction of its catalysis produces a large amount of reduced coenzyme NADPH, NADPH provides reducing power as negative hydrogen ion donor, meet the needs of many cellular process, as the synthesis of lipid acid steroid, by CO in photosynthesis
2synthesis of glucose, nucleic acid synthesizes, and reduced glutathion level etc. in maintenance red corpuscle, the reaction of thus G6PDH institute catalysis has important effect in reductibility biosynthesizing.In addition, G6PDH also with cell development, haemolytical anaemia, plant stress response, cardiovascular disorder, many human diseasess such as tumour are relevant.
Steroidal compounds has the effects such as anti-inflammatory, antimycotic, immunosuppression, diuresis, contraception
[76], clinical application is extensive, and demand is large, has become in pharmaceutical industries and has been only second to antibiotic second largest class medicine, C in steroidal hydroxylation
11'alpha '-hydroxylation is one of extremely important steroidal reaction, passes through C
11'alpha '-hydroxylation introduces high physiologically active group, can significantly improve the curative effect of steroid drugs, reduces side effect, the specificity etc. of change effect.Nineteen fifty-two Peterson and Murry etc. utilizes bread mould one step to transform first and realizes progesterone C
11'alpha '-hydroxylation generates C
11alpha-hydroxy progesterone, facilitate cortisone medicine industrialization, subsequently, microbial transformation steroidal more and more comes into one's own in the production of steroid drugs.Through the research of nearly decades, bread mould bio-transformation steroidal C11 'alpha '-hydroxylation technique and technology are relatively ripe, bacterial strain conversion capability is improved by selection by mutation, so be difficult to continue to improve transformation efficiency on existing bacterial strain and Process ba-sis, the throughput how adopting new method to improve bacterial strain further just becomes the problem that this art particularly pays close attention to.
(3) summary of the invention
The present invention seeks to by molecular biology method, G6PDH to be cloned in bread mould, transformation bacterial classification, intend in improved bread mould cell, realize G6PDH catalyzing N ADPH to regenerate, reaction to cytochrome P 450 Enzyme catalysis provides required coenzyme, improves the ability of bread mould to steroidal C11 'alpha '-hydroxylation.
The technical solution used in the present invention is:
Glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) gene is improving bread mould to the application in steroidal C11 'alpha '-hydroxylation ability.
Concrete, described in be applied as: build the expression vector containing G6PDH gene, imported in bread mould, screening positive clone, obtain the genetic engineering bacterium to the raising of steroidal C11 'alpha '-hydroxylation ability.
Concrete, described G6PDH gene order is as shown in SEQ ID No.1:
ATGTCGCATGAGGATTATATCCAACGTATCACTCAATATATCAAGGTGCAAGACCCTGAAAAGTTGGAAGCATTCAAACAGATGACATCTTATGTCTCTGGTCAATATGATGAAGATGCCTCTTTCCAAAAGCTGAACGAGGCCATCGAAGCATCTGAAAAGGAAAGAAAGGCCGAAAAGAAAAATCGCGTGTATTATATGGCCCTGCCTCCTTCCGTCTTTATTCCCGTAGCACAAGGATTGAAACGCAATGTGTACACGCCAGAGGGAAGTAACAGGCTGGTGGTCGAGAAACCGTTCGGGATGGACTCTGAATCCTCTGATCATTTAGGTCGTGAATTGGGTGCTCTCTTTACTGAAAATGAGATTTATCGTATTGATCATTATCTCGGTAAAGAGATGGTGAAGAACATCATGAACCTTCGTTTTGCTAATGTCTTACTTGGACATGCCTGGAGTCGTACTTATGTTGATAACGTTCAGATCACGTTCAAGGAACCTTTTGGCACAGAAGGACGGGGTGGTTATTTTGATGAATTTGGCATCATTCGTGATATCATTCAAAACCATTTACTTCAAGTCCTTTCCTTGATTGCTATGGAAAGACCTATCTCTACTGACTCTGAAGCCATTCGTGATGAAAAAGTCAAGGTGTTGAAGTGTATCTCTCCCATTCGTATCGAAGATACCTTGTTGGGTCAATATGTTGCTGCTGATGGTAAGCCTGGCTATCTTGAAGATGAAACGCTCAAGAACAAGGACAGTTTGACCCCTACTTTTGCTGCTACTGTTTGTTATGTGAATAATGAACGTTGGGAAGGCGTACCCTTTATCTTGAAGGCAGGTAAGGCCTTGAATGAAGCCAAGGTCGAAGTTCGTCTGCAATTCCACCATGTGGCCGGTAATCTGTTTAGCGGGTCCCCTCGTAATGAGCTCGTCATTCGTATTCAACCCAAAGAGGCTGTGTATTTAAAATTCAACAACAAACAACCTGGTTTGTCCTACGAAACCATTCAGACCGATCTCGACTTGACTTATCACGAACGTTATACTGACCTTGCTATCCCTGACGCTTATGAATCTCTCATCTTGGATGTCTTGCGTAATGATCATTCAAACTTTGTAAGAGATGATGAACTTCAGGCTGCCTGGAAGATCTTTACACCTCTGCTTCACAAGATTGACAAGCATGATTCCGATGTGGATATCAAGACATATGCTTATGGTTCTCGTGGTCCAAAGGAATTGGATGAATTCGTAAAGAAGCATGGTTATCACCGTGATACGAATGGTTACACTTGGCCTGTACAAAATGTAAATCCTTCTTCCAACAAGCTTTAA。
The invention still further relates to a kind of construction process of G 6 PD gene mutations engineering bacteria, described method comprises:
(1) obtain its G6PDH gene from Rhizopus oryzae clone, it be connected with plasmid PMD19-TSimple and transform, obtaining recombinant plasmid PMD19-TSimple/G6PDH;
(2) recombinant plasmid PMD19-TSimple/G6PDH is after double digestion, is connected and transforms with plasmid PCB1004, obtains expression vector PCB1004-G6PDH;
(3) expression vector PCB1004-G6PDH is dissolved in solution A, makes its concentration reach 1 ~ 5 μ g/ μ l, obtain plasmid solution; Solution A is composed as follows: 50mM CaCl
2, 0.3M N.F,USP MANNITOL, solvent is 10mM MOPS(pH6.3);
(4) every 100 μ L bread mould protoplast solution add 10 μ L plasmid solutions and 10 μ L PEG solution, after placing 30min on ice, then add 1.25ml PEG solution, and room temperature places 30min, obtains conversion fluid; Described PEG solution composition is as follows: 50mM CaCl
2, 40 ~ 60%(w/w) and PEG4000, solvent is 10mM MOPS(pH6.3);
(5) conversion fluid is added to MYG liquid regeneration substratum, after 28 DEG C of quiescent culture 5 ~ 10h, gained nutrient solution is applied to MYG solid plate, cultivate until there is bacterium colony for 28 DEG C, be transferred on the MYG solid plate containing 200 ~ 300 μ g/mL hygromycin B, 28 DEG C of cultivations, screening has the positive colony of hygromycin resistance, extract transformant genome, carry out PCR and determine whether goal gene fragment is incorporated in bread mould genome, preserve after qualification is correct.
Described MYG liquid regeneration substratum is composed as follows: maltose 5g/L, yeast powder 5g/L, glucose 10g/L solvent is water; Described MYG solid plate is composed as follows: maltose 5g/L, yeast powder 5g/L, glucose 10g/L, agar 15g/L, and solvent is water.
Concrete, in above-mentioned steps (1), described G6PDH gene order is as shown in SEQ ID No.1.
The invention still further relates to and build according to the method described above and screen higher bacterial strain---bread mould (Rhizopus nigericans) PIe of a strain steroidal C11 'alpha '-hydroxylation obtained, this bacterial strain is preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number is: CCTCC No:M2013436, and preservation date is: on September 18th, 2013.
Beneficial effect of the present invention is mainly reflected in: the genetic engineering bacterium built according to the inventive method, for transforming 16ALPHA,17ALPHA-epoxyprogesterone C
11'alpha '-hydroxylation produces mold oxide, compares starting strain, and growth is fast, and raw material availability is high, transformation efficiency and transformation efficiency high, there is far-reaching theory significance and higher using value in the development and utilization process of steroid drugs.
(4) accompanying drawing explanation
Fig. 1 is Rhizopus oryzae RNA agarose gel electrophoretogram; M:DL2000DNA Marker; Lane1: Rhizopus oryzae RNA;
Fig. 2 is the G6PDH gene fragment of pcr amplification; M:DNA Marker; Lane1:G6PDH;
Fig. 3 is the transformant colonies PCR of TA clone; M:DNA Marker; Lane1-5:5 transformant colonies PCR;
Fig. 4 is PMD19-TSimple-G6PDH plasmid and digestion verification; M:DNA Marker; Lane1 ~ 5:5 transformant colonies PCR;
Fig. 5 is sequencing result comparison; M:DNA Marker; Lane1:PMD19-TSimple-G6PDH plasmid; Lane2:BamH I single endonuclease digestion; Lane3:Apa I single endonuclease digestion; Lane4:BamH I and Apa I double digestion;
Fig. 6 is PCB1004-G6PDH fungi integrating expression vector building process;
Fig. 7 is transformant colonies PCR in expression vector establishment; M:DNA Marker; Lane1 ~ 5:5 transformant colonies PCR;
Fig. 8 is expression vector and double digestion checking thereof; M:DNA Marker; Lane1:PCB1004 plasmid; Lane2:PCB1004-G6PDH plasmid; Lane3:PCB1004 plasmid BamH I single endonuclease digestion is verified; Lane4:PCB1004 plasmid Apa I single endonuclease digestion is verified; Lane5:PCB1004 plasmid BamH I and the checking of Apa I double digestion; Lane6:PCB1004-G6PDH plasmid BamH I single endonuclease digestion is verified; Lane7:PCB1004-G6PDH plasmid Apa I single endonuclease digestion is verified; Lane8:PCB1004-G6PDH plasmid BamH I and the checking of Apa I double digestion;
Fig. 9 is the transformant of hygromycin resistance screening; A:RG3 transformant; B:RG12 transformant;
Figure 10 is starting strain and transformant genomic dna; M: λ-Hind III digest DNAMarker; Lane1: starting strain genomic dna; Lane2:RG3 transformant genomic dna; Lane3:RG12 transformant genomic dna;
Figure 11 is that PCR identifies transformant; M:DNA Marker; Lane1:PCB1004-G6PDH plasmid (HPH); Lane2:PCB1004-G6PDH plasmid (G6PDH); Lane3:3 transformant genomic dna (HPH); Lane4:12 transformant genomic dna (HPH); Lane5:3 transformant genomic dna (G6PDH); Lane6:12 transformant genomic dna (G6PDH); Lane7: the HPH fragment of purifying; Lane8: starting strain genomic dna (HPH); Lane9: starting strain genomic dna (G6PDH);
Figure 12 is that substrate and product HPLC analyze.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: relevant primer designs
Primers F-g6pdh/R-g6pdh and primers F-hyg/R-hyg(is designed in table 1) according to known Rhizopus oryzae G6DPH gene order (SEQ ID No.1) and E.coli hygromycin gene sequence (SEQ ID No.2), primers F-g6pdh/R-g6pdh adds at two ends BamH I and Apa I restriction enzyme site respectively, and primers F-hyg/R-hyg is used for PCR and identifies bread mould transformant.
Table 1: the primer that the present invention relates to
| Primer | Primer sequence |
| F-g6pdh | 5＇- GGATCCATGTCGCATGAGGATTATATCC-3＇ |
| R-g6pd | 5＇- GGGCCCTTAAAGCTTGTTGGAAGAA-3＇ |
| F-hyg | 5＇-ATGCCTGAACTCACCGCGAC-3＇ |
| R-hyg | 5＇-CTATTCCTTTGCCCTCGGAC-3＇ |
Embodiment 2: the extraction of Rhizopus oryzae total serum IgE
After liquid nitrogen grinding, used by Rhizopus oryzae (CICC40467) mycelium the RNAiso plus Total RNA of Takara company to extract reagent extract total serum IgE, and process removing genomic dna through DNase I, agarose gel electrophoresis detects and spectrophotometric analysis, guarantees that the RNA extracted does not degrade and contaminating genomic dna.
The Rhizopus oryzae total serum IgE that the present invention extracts is through spectrophotometric analysis: OD
260/ OD
280=1.85, RNA concentration is 89 μ g/ml, and agarose gel electrophoresis figure is as Fig. 1, clear according to 28S and 18S RNA band in the total serum IgE that agarose gel electrophoresis interpretation of result is extracted, do not degrade, illustrate that RNA integrity is better, in the RNA extracted in addition, there is no contaminating genomic DNA.
Embodiment 3: Rhizopus oryzae G6PDH gene clone
With the Rhizopus oryzae RNA extracted for template, with primers F-g6pdh/R-g6pdh(in table 1) carry out RT-PCR amplification acquisition not containing the G6PDH gene fragment of intron, PCR primer agarose gel electrophoresis detected result is as Fig. 2, find out that band is between 1200 ~ 1400bp from figure, G6PDH gene (comprising intron is 1439bp, exons 1 346bp) the encoding sequence size reported in this and database meets.
Transform after the product obtained increasing is connected with PMD19-T Simple carrier (purchased from TaKaDa) after purifying reclaims, from the flat board transformed, picking 5 single bacterium colonies carry out bacterium colony PCR, and result is as Fig. 3.As seen from the figure, 5 transformants selected are the positive.Select No. 2 transformant enlarged culturing, extract plasmid and carry out single double digestion checking, getting 1.0ml bacterium liquid simultaneously and send to order-checking.Plasmid and enzyme cut result as Fig. 4, and as we know from the figure, No. 2 transformants contain G6PDH object fragment, illustrate and are successfully inserted into PMD19-TSimple carrier, construct PMD19-TSimple-G6PDH cloning vector.Cloning vector is checked order, sequence after splicing with DNAMAN software comparison No. 2 transformant gene orders identical with the sequence reported in database (centre is two sections of intron sequences), not sudden change, sequencing result comparison, as Fig. 5, therefore selects No. 2 transformants to preserve G6PDH gene fragment.
Embodiment 4: the structure of fungi integrating expression vector PCB1004-G6PDH
By the object fragment on restructuring PMD19-TSimple-G6PDH carrier with restriction enzyme BamH I and Apa I double digestion and purifying reclaims G6PDH object fragment, then be so kind as to give by Zhejiang Polytechnical University doctor Zhu Tingheng with the expressed in fungi plasmid PCB1004(after BamH I and Apa I double digestion) be connected, build the PCB1004-G6PDH fungus expression vector of restructuring, building process schematic diagram is as Fig. 6.By bacterium colony PCR and double digestion checking screening positive transformant.Bacterium colony PCR and double digestion the result are respectively as Fig. 7, Fig. 8, in 5 transformants of picking as seen from Figure 7, No. 3 transformants are positive, after its enlarged culturing being extracted plasmid, double digestion is verified, digestion verification result shows to carry G6PDH fragment in the plasmid that No. 3 transformants extract, and is positive transformant.In addition, carried out checking order rear (SEQ ID No.1) result than consistent with the G6PDH sequence of report, do not undergone mutation.Therefore, we have successfully constructed PCB1004-G6PDH fungus expression vector.
Embodiment 5: bread mould is tested hygromycin-sensitive seeds
The present invention adopts the method for inoculation bacterium block, observes Totomycin and suppresses bread mould (bacterial strain is provided by the celestial jade pendant medicine company in Taizhou, HG09-11-03) mycelial growth, determine the concentration that hygromycin resistance screens.The hygromycin resistance MYG solid plate of different concns: adopt mixing casting to prepare the Totomycin MYG solid plate of 0 ~ 300 μ g/ml concentration gradient respectively.Eugonic bread mould mycelia is not being obtained in advance containing the method for employing coating spore on the MYG solid plate of Totomycin, punch at colony growth edge with sterilizing rifle head, be inoculated in the dull and stereotyped centre of the hygromycin resistance prepared with the bacterium block after the punching of inoculating needle picking, each gradient does 3 parallel tests.28 DEG C of lucifuges cultivate 36h, and through overtesting, the hygromycin resistance concentration determining screening is 200 ~ 300 μ g/ml.
Embodiment 6: the protoplast transformation of the preparation of bread mould protoplastis and PEG mediation and screening method thereof
1, the preparation of bread mould protoplastis:
1) on PDA culture medium flat plate, actication of culture is carried out, culture temperature 28 ~ 30 DEG C to bread mould (the celestial jade pendant medicine company in Taizhou, HG09-11-03); PDA substratum final concentration consists of: potato 200g/L, glucose 20g/L, agar powder 15g/L, pH nature;
2) cultivate after 3 ~ 5 days on PDA inclined-plane, with bread mould spore under aseptic washing, granulated glass sphere break up and with sterile gauze filter make spore suspension after be seeded to MYG liquid nutrient medium, as quiescent culture 14 ~ 16h at 28 ~ 30 DEG C; Described MYG liquid nutrient medium final concentration is: maltose 5g/L, yeast powder 5g/L, glucose 10g/L, and solvent is water;
3) step 2 is collected) mycelia of gained, after washing 2 times with aseptic deionized water, then use enzymolysis solution (0.5mol/L MgSO
4, 50mmol/L toxilic acid, solvent is water, pH5.0) wash 2 times;
4) lywallzyme(is purchased from Guangdong institute of microbiology) and Yatalase(purchased from Takara) mass ratio is that the mixed enzyme of 5:7 is dissolved in aseptic 0.5mol/L MgSO
4, in 50mmol/L DL-Maleateaicd solution, make the cell wall degrading enzyme liquid that final concentration is 50mg/mL; Every 1ml cell wall degrading enzyme liquid adds about 1.0g wet thallus, and put 30 DEG C of water-baths, every 20min shakes up gently, and enzymolysis dissociates protoplastis 3 ~ 4h, obtains protoplastis enzymolysis solution;
5) by the 1mol/L Sorbitol Solution USP dilution of protoplastis enzymolysis solution, then cross with double-layer sterile lens wiping paper and filter mycelia relic, the centrifugal 10min of 5000r/min, obtains protoplast pellet, and regulates protoplast concentration to be 10 with the suspension of 1mol/L Sorbitol Solution USP
7~ 10
8individual/mL, obtains protoplast solution; Described Sorbitol Solution USP is composed as follows: 1mol/L sorbyl alcohol, 10mmol/L Tris-Cl, 50mmol/L CaCl
2, solvent is water, pH7.0;
2, the protoplast transformation method of PEG mediation:
1. with solution A (10mM MOPS(pH6.3), 50mM CaCl
2, 0.3M N.F,USP MANNITOL) dissolve embodiment 4 build expression vector PCB1004-G6PDH, make its concentration reach 5 μ g/ μ L, obtain plasmid solution;
2. get 10 μ L plasmid solutions to add in protoplast solution prepared by 100 μ L, place 30min on ice, add 10 μ l PEG solution (10mM MOPS(pH6.3), 50mM CaCl
2, 50%PEG4000), place 30min on ice;
3. add 1.25ml PEG solution (10mM MOPS(pH6.3), 50mM CaCl
2, 50%PEG4000), room temperature places 30min, then conversion fluid is all added in 10ml YPG liquid regeneration substratum, 28 DEG C of quiescent culture 5 ~ 10h, each MYG solid plate are coated with the nutrient solution of 500 μ l quiescent culture, and 28 DEG C of incubators are cultivated until there is bacterium colony.Punch with aseptic rifle head when colony diameter grows to 0.5 ~ 1.0cm and be transferred on the flat board of hygromycin resistance, 28 DEG C of cultivations, arrange the negative control group of bread mould starting strain simultaneously.
4. observe each transformant growing state, the transformant that can grow in resistant panel is gone down to posterity after 10 generations by punching inoculation bacterium block method and observes its growing state on hygromycin resistance flat board.
Transformant RG3 and RG12 that 2 strains have hygromycin resistance has been screened, as shown in Figure 9 by aforesaid method.
Embodiment 7: transformant extracting genome DNA and PCR qualification
Primers F-g6pdh/R-g6pdh and primers F-hyg/R-hyg(table 1 according to design), carry out PCR with the transformant genomic dna extracted for template, extract the strain gene group DNA that sets out simultaneously and test as a control group PCR qualification is carried out to RG3 and RG12 screened in embodiment 6.The genome dna electrophoresis figure extracted and PCR qualification result are as Figure 10,11.PCR primer order-checking is confirmed with known sequence completely the same simultaneously, so RG3 and the RG12 transformant genomic dna of screening all incorporates hygromycin B resistant gene and G6PDH gene fragment, illustrate that the present invention constructs two strain bread mould genetic engineering bacterium RG3 and RG12, by RG12 called after PIe higher for activity, carried out culture presevation, its deposit number is CCTCC No:M2013436.
Embodiment 8: substrate and product HPLC analyzing and testing condition and transformation efficiency calculate
Draw 0.5ml nutrient solution, the centrifugal 2min of 12000rpm/min, discards supernatant liquor, adds 2ml methyl alcohol, and concussion makes mycelia fully suspend, and 60 DEG C are soaked 3h, the then centrifugal 2 ~ 5min of 12000rpm/min, and after getting supernatant dilution, HPLC detects, chromatographic condition: chromatographic column C
18post, moving phase: acetonitrile: water (60:40, v/v), flow velocity: 1.0ml/min, sample size: 5 μ l, determined wavelength λ: 240nm, column temperature: room temperature.
The substrate that the present invention relates to is 16ALPHA,17ALPHA-epoxyprogesterone, and product is mold oxide, and transformation efficiency calculates as follows:
As shown in figure 12, product mold oxide comparatively early goes out peak to the HPLC collection of illustrative plates that substrate and product detect, substrate more late go out peak.
Embodiment 10: the fermentation test of engineering strain
Adopt bread mould genetic engineering bacterium RG3, R.nigericans PIe(and RG12 of qualification in embodiment 7) and starting strain, respectively at fermention medium (glucose 30g/L, corn steep liquor 20g/L, dried silkworm chrysalis meal 10g/L, ammonium sulfate 1g/L, dipotassium hydrogen phosphate 5g/L, solvent is water) in carry out microbe conversion test, fermentation 18h drops into 1.5%(W/V, and namely every 100mL fermented liquid drops into 1.5g) 16ALPHA,17ALPHA-epoxyprogesterone, continues to transform 48h.Detect transformation efficiency by embodiment 9, experimental result is starting strain, RG3, R.nigericans PIe transformation efficiency is respectively 31.2 ± 0.5%, 36.1 ± 0.5% and 53.2 ± 0.5%.
Therefore, the engineering bacteria built according to the inventive method has huge potentiality to the hydroxylated transformation efficiency of raising 16ALPHA,17ALPHA-epoxyprogesterone, has far-reaching theory significance and higher using value in the development and utilization process of steroid drugs.
Claims (4)
1. glucose-6-phosphate dehydrogenase (G6PD) (G6PDH) gene is at raising bread mould to the application in steroidal C11 'alpha '-hydroxylation ability, and described G6PDH gene order is as shown in SEQ ID No.1.
2. apply as claimed in claim 1, be applied as described in it is characterized in that: build the expression vector containing G6PDH gene, imported in bread mould, screening positive clone, obtain the genetic engineering bacterium that steroidal C11 'alpha '-hydroxylation ability is improved.
3. a construction process for G 6 PD gene mutations engineering bacteria, described method comprises:
(1) obtain its G6PDH gene from Rhizopus oryzae clone, it be connected with plasmid PMD19-TSimple and transform, obtaining recombinant plasmid PMD19-TSimple/G6PDH; Described G6PDH gene order is as shown in SEQ ID No.1;
(2) recombinant plasmid PMD19-TSimple/G6PDH is after double digestion, is connected and transforms with plasmid PCB1004, obtains expression vector PCB1004-G6PDH;
(3) expression vector PCB1004-G6PDH is dissolved in solution A, makes its concentration reach 1 ~ 5 μ g/ μ l, obtain plasmid solution; Solution A is composed as follows: 50mM CaCl
2, 0.3M N.F,USP MANNITOL, solvent is 10mM MOPS, pH 6.3;
(4) every 100 μ L bread mould protoplast solution add 10 μ L plasmid solutions and 10 μ L PEG solution, after placing 30min on ice, then add 1.25ml PEG solution, and room temperature places 30min, obtains conversion fluid; Described PEG solution composition is as follows: 50mM CaCl
2, 40 ~ 60%PEG 4000, solvent is 10mM MOPS, pH 6.3;
(5) conversion fluid is added to MYG liquid regeneration substratum, after 28 DEG C of quiescent culture 5 ~ 10h, gained nutrient solution is applied to MYG solid plate, cultivate until there is bacterium colony for 28 DEG C, be transferred on the MYG solid plate containing 200 ~ 300 μ g/mL hygromycin B, 28 DEG C of cultivations, screening has the positive colony of hygromycin resistance, extract transformant genome, carry out PCR and determine whether goal gene fragment is incorporated in bread mould genome, preserve after qualification is correct.
4. bread mould engineering bacteria---bread mould (Rhizopusnigericans) PIe of a strain steroidal C11 'alpha '-hydroxylation ability raising, be preserved in China typical culture collection center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number is: CCTCC No:M 2013436, and preservation date is: on September 18th, 2013.
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