CN103627721B - G6PDH gene is improving bread mould to the application in steroidal C11 'alpha '-hydroxylation ability and bacterial strain - Google Patents

Abstract

The invention provides the application of the G6PDH gene in improving the ability of Rhizopus niger to steroid C11α-hydroxylation, the construction method of the glucose-6-phosphate dehydrogenase genetically engineered bacteria and the strain obtained by screening after construction. The present invention clones G6PDH into Rhizopus niger by means of molecular biology, transforms the strain, intends to realize the regeneration of NADPH catalyzed by G6PDH in the transformed Rhizopus niger cells, and provides the required coenzyme for the reaction catalyzed by the cytochrome P450 enzyme system , improving the ability of Rhizopus niger to C11α-hydroxylation of steroids. The beneficial effects of the present invention are mainly reflected in: the genetically engineered bacteria constructed according to the method of the present invention are used to transform Woshi oxide C ₁₁ α-hydroxylation to produce mold oxides. Compared with the starting strain, the growth rate is faster and the utilization rate of raw materials is high. The conversion rate and conversion efficiency are high, and it has far-reaching theoretical significance and high application value in the development and utilization of steroid drugs.

Description

Application of G6PDH Gene in Improving the Ability of Rhizopus niger to C11α-Hydroxylation of Steroids and Its Strains

(1) Technical field

The present invention relates to the application of G6PDH gene in improving the ability of Rhizopus niger to steroid C11α-hydroxylation, the construction method of glucose-6-phosphate dehydrogenase genetically engineered bacteria, and a strain of steroid C11α- Higher hydroxylation strain - Rhizopus nigericans PIe.

(2) Background technology

Products catalyzed by oxidoreductases are widely used in medicine, food, pesticides and other fields. The lack of coenzymes in the enzyme catalysis process is the main factor that limits the biocatalytic synthesis of products by most oxidoreductases. Therefore, the recycling of coenzymes is very important for the enzyme-catalyzed process, and it is of great value in the production of related products such as medicine and food. It has been reported in the literature that the regeneration methods of coenzymes include enzymatic, photochemical, electrochemical and other regeneration methods, among which enzymatic regeneration has received extensive attention. Enzymes commonly used for coenzyme regeneration include alcohol dehydrogenase (ADH), formate dehydrogenase (FDH), glucose dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G6PDH), phosphite dehydrogenase (PTDH) ), among which there are many studies and applications of FDH and GDH. For example, Sheldon studied that the overexpression of E.coli JM109 (pGDA2) comes from the Bacillus megaterium IWG3 glucose dehydrogenase gene. When NADP+ and glucose are provided, E.coli JM109 (pGDA2) ) can act as a regeneration system for NADPH. ZhinanXu constructed the pQE30-gdh223 expression vector by cloning the glucose dehydrogenase gene gdh223 in Bacillus megaterium AS1.223, and expressed it in E. - Conversion of ethyl hydroxybutyrate. Compared with FDH and GDH, there are relatively few reports on the use of G6PDH for coenzyme regeneration.

Glucose-6-phosphate dehydrogenase (G6PDH, EC1.1.1.49) is an enzyme that catalyzes the first step in the oxidation stage of the pentose phosphate pathway (PPP) and one of the regulators of the PPP metabolic pathway key regulatory enzymes. G6PDH widely exists in various biological cells including bacteria, plants, animals, etc., and its catalyzed reaction produces a large amount of reduced coenzyme NADPH, which provides reducing power as a negative hydrogen ion donor and meets the needs of many cellular metabolic processes, such as fatty acids The synthesis of sterols, the synthesis of glucose from CO ₂ in photosynthesis, the synthesis of nucleic acids, and the maintenance of reduced glutathione levels in red blood cells, etc. Therefore, the reactions catalyzed by G6PDH play an important role in reductive biosynthesis. In addition, G6PDH is also related to cell growth and development, hemolytic anemia, plant stress response, cardiovascular disease, tumor and many other human diseases.

Steroid compounds have anti-inflammatory, anti-fungal, immunosuppressive, diuretic, contraceptive and other effects ^[76] . They are widely used in clinical practice and are in great demand. They have become the second largest class of drugs in the pharmaceutical industry after antibiotics. Steroid hydroxylation C ₁₁ α-hydroxylation is one of the most important steroid reactions. The introduction of highly physiologically active groups through C ₁₁ α-hydroxylation can significantly improve the efficacy of steroid drugs, reduce side effects, and change the specificity of action. In 1952, Peterson and Murry first used Rhizopus niger to achieve C ₁₁ α-hydroxylation of progesterone to generate C ₁₁ α-hydroxyprogesterone, which promoted the industrialization of cortisone drugs. more and more attention has been paid to the production of body medicines. After decades of research, Rhizopus niger biotransformation steroid C11α-hydroxylation process and technology has been relatively mature, and the transformation ability of strains has been improved through mutation breeding, so it is difficult to continue to improve on the basis of existing strains and processes Transformation rate, how to use new methods to further improve the production capacity of bacterial strains has become a problem of particular concern to personnel in this field.

(3) Contents of the invention

The purpose of the present invention is to clone G6PDH into Rhizopus niger by means of molecular biology methods, transform the bacterial species, intend to realize the regeneration of NADPH catalyzed by G6PDH in the cells of Rhizopus niger after transformation, and provide the required enzyme for the reaction catalyzed by cytochrome P450 enzyme system. A coenzyme that improves the ability of Rhizopus niger to C11α-hydroxylation of steroids.

The technical scheme adopted in the present invention is:

Application of glucose-6-phosphate dehydrogenase (G6PDH) gene in improving the ability of Rhizopus niger to steroid C11α-hydroxylation.

Specifically, the application is: constructing an expression vector containing the G6PDH gene, introducing it into Rhizopus niger, screening positive clones, and obtaining a genetically engineered bacterium with improved steroid C11α-hydroxylation ability.

Specifically, the G6PDH gene sequence is shown in SEQ ID No.1:

ATGTCGCATGAGGATTATATCCAACGTATCACTCAATATATCAAGGTGCAAGACCCTGAAAAGTTGGAAGCATTCAAACAGATGACATCTTATGTCTCTGGTCAATATGATGAAGATGCCTCTTTCCAAAAGCTGAACGAGGCCATCGAAGCATCTGAAAAGGAAAGAAAGGCCGAAAAGAAAAATCGCGTGTATTATATGGCCCTGCCTCCTTCCGTCTTTATTCCCGTAGCACAAGGATTGAAACGCAATGTGTACACGCCAGAGGGAAGTAACAGGCTGGTGGTCGAGAAACCGTTCGGGATGGACTCTGAATCCTCTGATCATTTAGGTCGTGAATTGGGTGCTCTCTTTACTGAAAATGAGATTTATCGTATTGATCATTATCTCGGTAAAGAGATGGTGAAGAACATCATGAACCTTCGTTTTGCTAATGTCTTACTTGGACATGCCTGGAGTCGTACTTATGTTGATAACGTTCAGATCACGTTCAAGGAACCTTTTGGCACAGAAGGACGGGGTGGTTATTTTGATGAATTTGGCATCATTCGTGATATCATTCAAAACCATTTACTTCAAGTCCTTTCCTTGATTGCTATGGAAAGACCTATCTCTACTGACTCTGAAGCCATTCGTGATGAAAAAGTCAAGGTGTTGAAGTGTATCTCTCCCATTCGTATCGAAGATACCTTGTTGGGTCAATATGTTGCTGCTGATGGTAAGCCTGGCTATCTTGAAGATGAAACGCTCAAGAACAAGGACAGTTTGACCCCTACTTTTGCTGCTACTGTTTGTTATGTGAATAATGAACGTTGGGAAGGCGTACCCTTTATCTTGAAGGCAGGTAAGGCCTTGAATGAAGCCAAGGTCGAAGTTCGTCTGCAATTCCACCATGTGGCCGGTAATCTGTTTAGCGGGTCCCCTCGTAATGAGCTCGTCATTCGTATTCAACCCAAAGAGGCTGTGTATTTAAAATTCAACAACAAACAACCTGGTTTGT CCTACGAAACCATTCAGACCGATCTCGACTTGACTTATCACGAACGTTATACTGACCTTGCTATCCCTGACGCTTATGAATCTCTCATCTTGGATGTCTTGCGTAATGATCATTCAAACTTTGTAAGAGATGATGAACTTCAGGCTGCCTGGAAGATCTTTACACCTCTGCTTCACAAGATTGACAAGCATGATTCCGATGTGGATATCAAGACATATGCTTATGGTTCTCGTGGTCCAAAGGAATTGGATGAATTCGTAAAGAAGCATGGTTATCACCGTGATACGAATGGTTACACTTGGCCTGTACAAAATGTAAATCCTTCTTCCAACAAGCTTTAA。

The present invention also relates to a method for constructing a glucose-6-phosphate dehydrogenase genetically engineered bacterium, the method comprising:

(1) Obtain the G6PDH gene from Rhizopus oryzae, connect it to the plasmid PMD19-TSimple and transform it to obtain the recombinant plasmid PMD19-TSimple/G6PDH;

(2) After the recombinant plasmid PMD19-TSimple/G6PDH was digested with double enzymes, it was ligated with the plasmid PCB1004 and transformed to obtain the expression vector PCB1004-G6PDH;

(3) Dissolve the expression vector PCB1004-G6PDH in solution A to a concentration of 1-5 μg/μl to obtain a plasmid solution; the composition of solution A is as follows: 50 mM CaCl ₂ , 0.3 M mannitol, and the solvent is 10 mM MOPS (pH6. 3);

(4) Add 10 μL plasmid solution and 10 μL PEG solution to every 100 μL Rhizopus niger protoplast solution, place it on ice for 30 minutes, then add 1.25ml PEG solution, and place it at room temperature for 30 minutes to obtain a transformation solution; the composition of the PEG solution is as follows: 50 mM CaCl ₂ , 40~60% (w/w) PEG4000, the solvent is 10mM MOPS (pH6.3);

(5) The transformation solution was added to the MYG liquid regeneration medium, and after static culture at 28°C for 5-10 hours, the resulting culture solution was spread on the MYG solid plate, cultured at 28°C until colonies appeared, and then transferred to a medium containing 200-300 μg/mL tide On the MYG solid plate of mycin B, cultivate at 28°C, screen the positive clones with hygromycin resistance, extract the genome of the transformant, and perform PCR to determine whether the target gene fragment is integrated into the Rhizopus niger genome, and save it after identification.

The composition of the MYG liquid regeneration medium is as follows: maltose 5g/L, yeast powder 5g/L, glucose 10g/L solvent is water; the composition of the MYG solid plate is as follows: maltose 5g/L, yeast powder 5g/L, glucose 10g /L, agar 15g/L, solvent is water.

Specifically, in the above step (1), the G6PDH gene sequence is shown in SEQ ID No.1.

The present invention also relates to a strain of Rhizopus nigericans (Rhizopus nigericans) PIe, which is constructed and screened according to the above-mentioned method, and is obtained from a higher steroidal C11α-hydroxylation. The strain is preserved in the China Type Culture Collection Center, address: China, Wuhan, Wuhan University, postcode: 430072, deposit number: CCTCC No: M2013436, deposit date: September 18, 2013.

The beneficial effects of the present invention are mainly reflected in: the genetically engineered bacteria constructed according to the method of the present invention are used to transform Woshi oxide C ₁₁ α-hydroxylation to produce mold oxides. Compared with the starting strain, the growth rate is faster and the utilization rate of raw materials is high. The conversion rate and conversion efficiency are high, and it has far-reaching theoretical significance and high application value in the development and utilization of steroid drugs.

(4) Description of drawings

Figure 1 is the pattern of Rhizopus oryzae RNA agarose gel electrophoresis; M: DL2000DNA Marker; Lane1: RNA of Rhizopus oryzae;

Figure 2 is the G6PDH gene fragment amplified by PCR; M: DNA Marker; Lane1: G6PDH;

Figure 3 is the colony PCR of transformants cloned by TA; M: DNA Marker; Lane1-5: PCR of 5 transformant colonies;

Figure 4 shows the PMD19-TSimple-G6PDH plasmid and enzyme digestion verification; M: DNA Marker; Lane1-5: PCR of 5 transformant colonies;

Figure 5 shows the comparison of sequencing results; M: DNA Marker; Lane1: PMD19-TSimple-G6PDH plasmid; Lane2: single digestion with BamHI; Lane3: single digestion with ApaⅠ; Lane4: double digestion with BamHI and ApaⅠ;

Figure 6 is the construction process of PCB1004-G6PDH fungal integrated expression vector;

Figure 7 is the PCR of transformant colonies in the construction of expression vector; M: DNA Marker; Lane1-5: PCR of 5 transformant colonies;

Figure 8 shows the expression vector and its double-digestion verification; M: DNA Marker; Lane1: PCB1004 plasmid; Lane2: PCB1004-G6PDH plasmid; Lane3: PCB1004 plasmid BamHⅠ single-digestion verification; Lane4: PCB1004 plasmid ApaⅠ single-digestion verification; Lane5 Lane6: PCB1004-G6PDH plasmid BamHⅠ single enzyme digestion verification; Lane7: PCB1004-G6PDH plasmid ApaⅠ single enzyme digestion verification; Lane8: PCB1004-G6PDH plasmid BamHI and ApaⅠ double enzyme digestion verification;

Figure 9 shows the transformants screened for resistance to hygromycin; A: RG3 transformants; B: RG12 transformants;

Fig. 10 is starting bacterial strain and transformant genomic DNA; M: λ-HindⅢ digest DNAMarker; Lane1: starting bacterial strain genomic DNA; Lane2: RG3 transformant genomic DNA; Lane3: RG12 transformant genomic DNA;

Figure 11 is PCR identification of transformants; M: DNA Marker; Lane1: PCB1004-G6PDH plasmid (HPH); Lane2: PCB1004-G6PDH plasmid (G6PDH); Lane3: No. 3 transformant genomic DNA (HPH); Lane4: No. 12 transformation Subgenome DNA (HPH); Lane5: No. 3 transformant genomic DNA (G6PDH); Lane6: No. 12 transformant genomic DNA (G6PDH); Lane7: purified HPH fragment; Lane8: starting strain genomic DNA (HPH); Lane9: Starting strain genomic DNA (G6PDH);

Figure 12 is the substrate and product HPLC analysis.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1: Related primer design

Primer F-g6pdh/R-g6pdh and primer F-hyg/ R-hyg (see Table 1), BamHI and ApaI restriction sites were added to both ends of the primer F-g6pdh/R-g6pdh, respectively, and the primer F-hyg/R-hyg was used for PCR identification of Rhizopus niger transformants.

Table 1: Primers involved in the present invention

Primer name Primer sequence F-g6pdh 5'- GGATCC ATGTCGCATGAGGATTATATCC-3' R-g6pd 5'- GGGCCC TTAAAGCTTGTTGGAAGAA -3' F-hyg 5'-ATGCCTGAACTCACCGCGAC-3' R-hyg 5'-CTATTCCTTTGCCCTCGGAC-3'

Embodiment 2: the extraction of Rhizopus oryzae total RNA

The Rhizopus oryzae (CICC40467) mycelium was ground with liquid nitrogen, and the total RNA was extracted with RNAiso plus Total RNA extraction reagent from Takara Company, and the genomic DNA was removed by DNase I treatment, and agarose gel electrophoresis detection and absorbance analysis were performed to ensure that the extracted RNA does not degrade and contaminate genomic DNA.

The Rhizopus oryzae total RNA extracted by the present invention is analyzed by absorbance: OD ₂₆₀ /OD ₂₈₀ =1.85, the RNA concentration is 89 μ g/ml, and the agarose gel electrophoresis figure is as shown in Figure 1, and the total RNA extracted is analyzed according to the agarose gel electrophoresis result The 28S and 18S RNA bands are clear and not degraded, indicating that the RNA integrity is good, and there is no genomic DNA contamination in the extracted RNA.

Embodiment 3: Rhizopus oryzae G6PDH gene clone

Using the extracted Rhizopus oryzae RNA as a template and primers F-g6pdh/R-g6pdh (see Table 1) for RT-PCR amplification to obtain the G6PDH gene fragment without introns, the PCR product agarose gel electrophoresis detection results As shown in Figure 2, it can be seen from the figure that the band is between 1200 and 1400bp, which is consistent with the size of the coding sequence of the G6PDH gene (including introns 1439bp and exons 1346bp) reported in the database.

After purification and recovery, the amplified product was ligated with the PMD19-T Simple vector (purchased from TaKaDa) and transformed. Five single colonies were picked from the transformed plate for colony PCR. The results are shown in Figure 3. As can be seen from the figure, the selected 5 transformants were all positive. The No. 2 transformant was selected for expansion culture, the plasmid was extracted and verified by single and double enzyme digestion, and 1.0ml bacterial liquid was taken and sent for sequencing. The results of plasmid and enzyme digestion are shown in Figure 4. It can be seen from the figure that the No. 2 transformant contains the G6PDH target fragment, indicating that it was successfully inserted into the PMD19-TSimple vector, and the PMD19-TSimple-G6PDH cloning vector was constructed. The cloning vector was sequenced, and after splicing, the DNAMAN software was used to compare the gene sequence of the No. 2 transformant with the sequence reported in the database (two intron sequences in the middle), without mutation, and the sequencing results were compared as shown in Figure 5, therefore The No. 2 transformant was selected to preserve the G6PDH gene fragment.

Example 4: Construction of Fungal Integrated Expression Vector PCB1004-G6PDH

The target fragment on the recombinant PMD19-TSimple-G6PDH vector was double-digested with restriction endonucleases BamHI and ApaI and purified to recover the G6PDH target fragment, and then combined with the fungal expression plasmid PCB1004 after double-digestion with BamHI and ApaI (provided by Zhejiang University of Technology Dr. Zhu Tingheng) was connected to construct the recombinant PCB1004-G6PDH fungal expression vector. The schematic diagram of the construction process is shown in Figure 6. Positive transformants were screened by colony PCR and double enzyme digestion. The results of colony PCR and double enzyme digestion verification are shown in Figure 7 and Figure 8, respectively. From Figure 7, it can be seen that No. 3 transformant among the 5 picked transformants was positive. The verification results showed that the plasmid extracted from No. 3 transformant carried the G6PDH fragment, which was a positive transformant. In addition, after it was sequenced (SEQ ID No.1), the result was consistent with the reported G6PDH sequence, and no mutation occurred. Therefore, we successfully constructed the PCB1004-G6PDH fungal expression vector.

Embodiment 5: Rhizopus niger is sensitive to hygromycin test

The present invention adopts the method of inoculating bacteria block, observes hygromycin inhibition of rhizopus niger (strain provided by Taizhou Xianju Pharmaceutical Co., Ltd., HG09-11-03) mycelia growth, and determines the concentration of hygromycin resistance screening. Hygromycin-resistant MYG solid plates with different concentrations: Hygromycin MYG solid plates with a concentration gradient of 0-300 μg/ml were prepared by the mixed pouring method. Use the method of coating spores on the MYG solid plate without hygromycin in advance to obtain vigorously growing Rhizopus niger mycelium, use a sterilized gun tip to punch holes at the edge of the colony growth, and use an inoculation needle to pick out the punched bacterial blocks Inoculate in the center of the prepared hygromycin resistance plate, and do 3 parallel experiments for each gradient. Incubate at 28°C in the dark for 36 hours. After testing, it is determined that the concentration of hygromycin resistance used for screening is 200-300 μg/ml.

Embodiment 6: Rhizopus niger protoplast preparation and PEG-mediated protoplast transformation and screening method thereof

1, the preparation of Rhizopus niger protoplast:

1) Activate Rhizopus niger (Taizhou Xianju Pharmaceutical Co., Ltd., HG09-11-03) on a PDA medium plate at a culture temperature of 28-30°C; the final concentration of the PDA medium is: potato 200g/L, glucose 20g /L, agar powder 15g/L, pH natural;

2) After culturing on a PDA slant for 3 to 5 days, wash the Rhizopus niger spores with sterile water, break up the glass beads and filter them with sterile gauze to make a spore suspension, then inoculate them into MYG liquid medium, and place them at 28-30°C Static culture for 14-16 hours; the final concentration of the MYG liquid medium is: maltose 5g/L, yeast powder 5g/L, glucose 10g/L, solvent is water;

3) Collect the hyphae obtained in step 2), wash them twice with sterile deionized water, and then use the enzymatic hydrolysis solution (0.5mol/L MgSO ₄ , 50mmol/L maleic acid, the solvent is water, pH5.0) Just wash 2 times;

4) The mixed enzyme of lywallzyme (purchased from Guangdong Institute of Microbiology) and Yatalase (purchased from Takara) with a mass ratio of 5:7 was dissolved in sterile 0.5mol/L MgSO ₄ , 50mmol/L DL-Maleateaicd solution to prepare Cell wall degrading enzyme solution with a final concentration of 50 mg/mL; add about 1.0 g of wet bacteria per 1 ml of cell wall degrading enzyme solution, place in a 30°C water bath, shake gently every 20 minutes, and enzymatically hydrolyze free protoplasts for 3 to 4 hours to obtain protoplasts body enzyme hydrolyzate;

5) Dilute the protoplast enzymatic solution with 1mol/L sorbitol solution, then filter with double-layer sterile lens paper to remove mycelium fragments, centrifuge at 5000r/min for 10min to obtain protoplast precipitation, and use 1mol/L sorbitol solution Suspend to adjust the protoplast concentration to 10 ⁷ -10 ⁸ /mL to obtain a protoplast solution; the composition of the sorbitol solution is as follows: 1mol/L sorbitol, 10mmol/L Tris-Cl, 50mmol/L CaCl ₂ , and the solvent is water , pH7.0;

2. PEG-mediated protoplast transformation method:

1. Dissolve the expression vector PCB1004-G6PDH constructed in Example 4 with solution A (10mM MOPS (pH6.3), 50mM CaCl ₂ , 0.3M mannitol) to a concentration of 5 μg/μL to obtain a plasmid solution;

2. Add 10 μL plasmid solution to 100 μL prepared protoplast solution, place on ice for 30 minutes, add 10 μl PEG solution (10 mM MOPS (pH6.3), 50 mM CaCl ₂ , 50% PEG4000), and place on ice for 30 minutes;

3. Add 1.25ml PEG solution (10mM MOPS (pH6.3), 50mM CaCl ₂ , 50%PEG4000), let stand at room temperature for 30min, then add all the transformation solution into 10ml YPG liquid regeneration medium, and culture at 28℃ for 5～ After 10 hours, 500 μl of the static culture solution was coated on each MYG solid plate, and cultured in a 28° C. incubator until colonies appeared. When the diameter of the colony grows to 0.5-1.0 cm, punch a hole with a sterile pipette tip and transfer to a hygromycin-resistant plate, culture at 28°C, and set a negative control group for the Rhizopus niger origin strain.

4. Observe the growth of each transformant, and observe the growth of the transformant that can grow on the resistance plate after 10 generations on the hygromycin resistance plate by punching and inoculating the bacterial block method.

Two hygromycin-resistant transformants RG3 and RG12 were screened by the above method, as shown in FIG. 9 .

Embodiment 7: Transformant genomic DNA extraction and PCR identification

According to the designed primers F-g6pdh/R-g6pdh and primers F-hyg/R-hyg (Table 1), PCR was carried out using the extracted transformant genomic DNA as a template, and the genomic DNA of the starting strain was extracted as a control group. RG3 and RG12 screened in 6 were identified by PCR. The electropherograms and PCR identification results of the extracted genomic DNA are shown in Figures 10 and 11. Simultaneously, the sequencing of the PCR product was confirmed to be completely consistent with the known sequence, so the hygromycin B resistance gene and the G6PDH gene fragment were integrated on the genomic DNA of the RG3 and RG12 transformants screened, indicating that the present invention has constructed two strains of Rhizopus niger Genetically engineered bacteria RG3 and RG12, named RG12 with higher activity as PIe, and carried out strain preservation, and its preservation number is CCTCC No: M2013436.

Embodiment 8: Substrate and product HPLC analysis detection condition and conversion ratio calculation

Take 0.5ml of culture solution, centrifuge at 12000rpm/min for 2min, discard the supernatant, add 2ml of methanol, oscillate to suspend the mycelia fully, soak at 60°C for 3h, then centrifuge at 12000rpm/min for 2-5min, take the supernatant and dilute it for HPLC detection , Chromatographic conditions: chromatographic column C ₁₈ column, mobile phase: acetonitrile: water (60:40, v/v), flow rate: 1.0ml/min, injection volume: 5μl, detection wavelength λ: 240nm, column temperature: room temperature.

The substrate involved in the present invention is Woshi oxide, and the product is mold oxide, and the conversion rate is calculated by the following formula:

The HPLC chromatograms of substrate and product detection are shown in Figure 12, the product mold oxide peaks out earlier, and the substrate peaks out later.

Embodiment 10: the fermentation test of engineering strain

Adopt Rhizopus niger genetically engineered bacteria RG3, R.nigericans PIe (i.e. RG12) identified in Example 7 and the starting bacterial strain, respectively in fermentation medium (glucose 30g/L, corn steep liquor 20g/L, silkworm chrysalis powder 10g/L, Ammonium sulfate 1g/L, dipotassium hydrogen phosphate 5g/L, solvent is water) for fermentation conversion test, 1.5% (W/V, that is, 1.5g per 100mL fermentation liquid) of Woshi oxide was added to the fermentation for 18 hours, and the conversion was continued 48h. The conversion rate was detected according to Example 9. The experimental results showed that the conversion rates of the starting strain, RG3, and R. nigericans PIe were 31.2 ± 0.5%, 36.1 ± 0.5% and 53.2 ± 0.5%, respectively.

Therefore, the engineering bacterium constructed according to the method of the present invention has great potential for improving the conversion rate of the hydroxylation of the Wormton oxide, and has far-reaching theoretical significance and high application value in the development and utilization 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.