CN118006474A - Blue Absidia strain for efficiently preparing hydrocortisone and construction method thereof - Google Patents
Blue Absidia strain for efficiently preparing hydrocortisone and construction method thereof Download PDFInfo
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- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 title claims abstract description 36
- 241000235389 Absidia Species 0.000 title claims abstract description 33
- 229960000890 hydrocortisone Drugs 0.000 title claims abstract description 18
- 238000010276 construction Methods 0.000 title abstract description 12
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims abstract description 12
- WHBHBVVOGNECLV-UHFFFAOYSA-N 11-deoxy-17-hydroxy-corticosterone Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)(O)C(=O)CO)C4C3CCC2=C1 WHBHBVVOGNECLV-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000005516 engineering process Methods 0.000 claims abstract description 10
- WHBHBVVOGNECLV-OBQKJFGGSA-N 11-deoxycortisol Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 WHBHBVVOGNECLV-OBQKJFGGSA-N 0.000 claims abstract description 9
- 239000000758 substrate Substances 0.000 claims abstract description 6
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- 108020005004 Guide RNA Proteins 0.000 claims description 4
- 101150078331 ama-1 gene Proteins 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 108091033409 CRISPR Proteins 0.000 claims description 3
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a blue Absidia strain for efficiently preparing hydrocortisone and a construction method thereof, belonging to the technical field of genetic engineering. The invention utilizes CRISPR-Cas9 editing technology to efficiently express a C11 beta-hydroxylase CYP5311B2T329A mutant from blue Absidia in the blue Absidia strain lacking CYP5311B2, and obtains the blue Absidia recombinant strain for efficiently converting substrate 11-deoxycortisol to prepare hydrocortisone. The total dosage of the substrate 11-deoxycortisol is 4g/L, and the yield of the product hydrocortisone reaches 85% after 72h of conversion. The invention provides valuable industrial strains and technical methods for researching and developing the process for producing hydrocortisone by using high-efficiency C11β -hydroxylated 11-deoxycortisol.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a blue Absidia strain for efficiently preparing hydrocortisone and a construction method thereof.
Background
Hydrocortisone is currently the most productive glucocorticoid and is also the core intermediate for the synthesis of other glucocorticoids.
The domestic industry mainly uses blue Absidia (Absidia coerulea) to take 11-deoxycortisol as a substrate and introduces hydroxyl to synthesize hydrocortisone at the C11 beta position (the process is shown as figure 1), but the existing production process has low hydrocortisone feeding amount and more byproducts, the yield is only 42-45%, and the transformation and upgrading of the industry are severely restricted.
The C11 beta-hydroxylation reaction of colestuary is a key reaction technology on which the large-scale production of glucocorticoids depends, whereas the hydroxylation efficiency of steroidal C11 is to a large extent dependent on the c11 beta-hydroxylase activity of the producing species. In order to solve the problems of the current industrial production and application, the enzyme molecules need to be directionally transformed.
At present, saccharomyces cerevisiae (Saccharomyces cerevisiae) is commonly used as an expression host of C11β -hydroxylase, and the method has the advantages of easiness in gene manipulation, easiness in screening and the like in enzyme molecule modification, but has the defects of low material feeding amount, slower conversion rate and the like in the process of preparing hydrocortisone. In contrast, absidia blue has good steroid hydroxylation activity, and is more suitable as a C11 beta-hydroxylase mutant host after being modified by CRISPR-Cas9 technology.
AMA1 sequences isolated from the A.nidulans (Aspergillus nidulans) genomic library are capable of increasing the frequency of transformation and producing phenotypically unstable transformants in filamentous fungi. The invention utilizes a plasmid vector containing an AMA1 sequence to enable a target gene to be efficiently transformed into the blue colpitis protoplast.
Disclosure of Invention
The C11β -hydroxylase gene mutant CYP5311B 2T 329A with high hydroxylation specificity constructed in the early stage of a laboratory provides gene resources for constructing novel recombinant Absidia blue bacteria for efficiently producing hydrocortisone.
The technical route of the invention is as follows: the C11β -hydroxylase gene CYP5311B2 of Absidia blue is identified in the early stage of a laboratory, and is subjected to directed transformation by semi-rational design and site-directed mutation technology, so that mutant T329A with high hydroxylation specificity and a recombinant saccharomyces cerevisiae strain are obtained. In order to further increase the feeding amount and improve the process efficiency, the blue colpitis with CYP5311B2 function deficiency is obtained through CRISPR-Cas9 editing, and then CYP5311B1 gene in the blue colpitis with C11 beta-hydroxylase gene CYP5311B 2T 329A is replaced through CRISPR-Cas9 editing, so that the blue colpitis recombinant strain for preparing hydrocortisone industrially and efficiently is obtained, and reference is provided for C11 beta-hydroxylation. Wherein, the hydroxylase gene CYP5311B 2T 329A is driven by a 800bp sequence promoter at the upstream of the ORF of the blue Absidia gene CYP5311B 1; the preparation method of the blue Absidia host strain with CYP5311B2 function deletion by using the CRISPR-Cas9 technology comprises the following steps:
(1) Determining a Cas 9-targeted CYP5311B2 gene sequence;
(2) Constructing an expression vector pAMA-gRNA-1-2-Cas 9 containing two gRNA expression cassettes, one Cas9 expression cassette and an AMA1 sequence;
(3) Introducing the recombinant expression vector in the step (2) into wild blue Absidia for gene editing by a protoplast transformation method;
(4) Sequencing and verifying the obtained transformant extracted genome to obtain a C11 beta-hydroxylase gene CYP5311B2 deleted blue colpitis host bacterium;
(5) Transferring the transformant to a non-antibiotic PDA test tube, culturing until spore is produced, washing the spore with sterile water, and diluting the coated plate;
(6) After the colony grows out, the colony is simultaneously and sequentially screened to hygromycin B300 mg/ml and a non-antibiotic PDA flat plate to obtain blue Abelmoschus host bacteria of the C11β -hydroxylase gene CYP5311B2 which is deleted and pAMA-gRNA-1-2-Cas 9 carrier is thrown away.
The nucleotide sequence of the Absidia blue C11 beta-hydroxylase mutant gene CYP5311B 2T 329A is shown in SEQ ID NO.1, and the sequences belong to the protection scope of the invention.
Recombinant expression vectors, recombinant expression plasmids or host cells constructed by using the gene fragment CYP5311B 2T 329A for coding the C11β -hydroxylase also belong to the protection scope of the invention, and the amplification primer sequences used also belong to the protection scope of the invention.
The steroid hydroxylase coded by the mutant gene CYP5311B 2T 329A obtained by adopting the semi-rational design and site-directed mutagenesis technology comprises but is not limited to expression in host cells such as Saccharomyces cerevisiae, pichia pastoris, absidia blue and the like.
The beneficial effects are that: the invention utilizes CRISPR-Cas9 technology to reform wild blue coluba to obtain blue coluba host bacteria inactivated by C11 beta-hydroxylase gene CYP5311B2, and then obtains recombinant blue coluba for efficiently preparing hydrocortisone by CRISPR-Cas9 technology. The hydroxylation specificity of the recombinant Absidia blue is improved, the feeding amount reaches 4g/L, and the research result of the invention has good industrial application value.
Drawings
FIG. 1C 11 beta-hydroxylation of 11-deoxycortisol;
FIG. 2 schematic representation of pAMA1-gRNA-1-2-Cas9 expression vector;
FIG. 3 is a schematic diagram of pCSN44-P B1 -CYP5311B 2T 329A expression vector;
FIG. 4 construction scheme of recombinant mutant Absidia blue
FIG. 5 is a graph showing the yield of recombinant blue Absidia for preparing hydrocortisone at 4 g/L11-deoxycortisol dosage.
Detailed Description
The process according to the invention is described below by way of specific embodiments. The experimental methods in the following examples are conventional methods unless otherwise specified. The embodiments should be considered as illustrative, and not limiting, the scope of the invention being limited only by the claims. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
The invention relates to a strain: coli (ESCHERICHIA COLI JM109,109), colestuary blue (Absidia coerulea AS 3.65), university of Tianjin technical and scientific collection of microorganisms.
Example 1 obtaining C11β -steroid hydroxylase Gene CYP5311B2 deleted Absidia blue host bacterium Using CRISPR-Cas9
Determination of the Cas 9-targeting CYP5311B2 Gene sequence
Target-1：ACCATGGTGTCATCCAAAGA
Target-2：TGGAACTATCCAAAAAGTGA
Construction of pBlue-PU6-gRNA-1-TU6, pBlue-PU6-gRNA-2-TU6 and pBlue-gRNA-1-2 vector
The plasmid pBlue-PU6-gRNA-TU6 is used as a template, 11 beta-gRNA-1-F/R is used as a primer for carrying out polymerase chain reaction to obtain a target vector fragment pBlue-PU6-gRNA-1-TU6, and 11 beta-gRNA-2-F/R is used as a primer for carrying out polymerase chain reaction to obtain a target vector fragment pBlue-PU6-gRNA-2-TU6. Primers were synthesized by Beijing Hua big Gene company.
11β-gRNA-1-F：ACCATGGTGTCATCCAAAGAGTTTTAGAGCTAGAAATAGCAAG
11β-gRNA-1-R：TCTTTGGATGACACCATGGTACTTGTTCTTCTTTACAATGATT
11β-gRNA-2-F：TGGAACTATCCAAAAAGTGAGTTTTAGAGCTAGAAATAGCAAG
11β-gRNA-2-R：TCACTTTTTGGATAGTTCCAACTTGTTCTTCTTTACAATGATT
PCR reaction System (Total 50. Mu.l):
PCR procedure:
Mu.l of the PCR product was subjected to agarose gel electrophoresis to verify the band size. And (3) purifying and recovering the PCR product after Dpn I digestion, recovering the competence of the product for transforming escherichia coli JM109, picking up transformants to extract plasmids, and sequencing and verifying correct plasmids to obtain pBlue-PU6-gRNA-1-TU6 and pBlue-PU6-gRNA-2-TU6 plasmids.
The pBlue-PU6-gRNA-1-TU6 is subjected to Kpn I single enzyme digestion to prepare a homologous recombination framework, a primer with a homology arm is designed by taking the pBlue-PU6-gRNA-2-TU6 as a template to amplify a PU6-gRNA-2-TU6 target fragment, and 2 mu l of enzyme digestion products and 2 mu l of PCR products are respectively taken for agarose gel electrophoresis to verify the band size. After digestion and purification of the PCR product, homologous recombination is carried out on the target fragment and the skeleton, the recombinant system converts escherichia coli JM109 competence, the transformant is selected to extract plasmids, sequencing verification is correct, the pBlue-gRNA-1-2 plasmids are obtained, and the primers are synthesized by Beijing Hua large gene company.
pBlue-gRNA-2-F：CTCGAGGGGGGGCCCGGTACTAATGCCGGCTCATTCAAACG
pBlue-gRNA-2-R：AGGGAACAAAAGCTGGGTACAGCAGCTCTATATCACGTGAC
PCR reaction System (Total 50. Mu.l):
PCR procedure:
Cleavage reaction System (Total 50. Mu.l):
recombination reaction System (Total 10. Mu.l):
Construction of pAMA1-gRNA-1-2-Cas9 vector
Carrying out BamHI single enzyme digestion on pAMA-Cas 9 plasmid to prepare homologous recombination framework, designing a primer with homology arms by taking pBlue-gRNA-1-2 plasmid as a template to amplify a target fragment containing two gRNA expression cassettes, respectively taking 2 mu l of enzyme digestion products and 2 mu l of PCR products, and carrying out agarose gel electrophoresis to verify the band size. After digestion and purification of the PCR product, homologous recombination is carried out on the target fragment and the skeleton, the recombinant system converts escherichia coli JM109 competent, the transformant is selected to extract plasmids, sequencing verification is correct, and then pAMA-gRNA-1-2-Cas 9 plasmids shown in figure 2 are obtained, and the primers are synthesized by Beijing Hua large gene company.
2gRNA-F-BamHI：CTGTTTCCGCTAGCAGATCACTATAGGGCGAATTGGAGCTC
2gRNA-R-BamHI：CCAGCTCACATCCTGGATCCAGCTATGACCATGATTACGC
PCR reaction System (Total 50. Mu.l):
PCR procedure:
Cleavage reaction System (Total 50. Mu.l):
recombination reaction System (Total 10. Mu.l):
Recombinant Absidia blue host bacteria constructed by protoplast transformation of pAMA1-gRNA-1-2-Cas9 vector
3-5 Μg of recombinant plasmid pAMA-gRNA-1-2-Cas 9 was added to wild Absidia blue protoplast with a concentration > 10 8/ml and 680 μl of PEG6000 solution was added, ice-bath for 20min;
2ml PEG6000 buffer solution is added and the mixture is left at room temperature for 10min;
Adding 4ml STC and 4ml upper medium which are preheated and mixed uniformly in advance at 48 ℃, plating on a lower medium with hygromycin B concentration of 300mg/ml, placing a flat plate in a 28 ℃ incubator, growing transformants after 5-7d, transferring the transformants into a non-antibiotic PDA test tube for culturing until spores are produced, extracting genome for sequencing verification, obtaining blue Abelmoschus host bacteria with C11 beta-hydroxylase gene CYP5311B2 deletion, and confirming CYP5311B2 function deletion through 11-deoxycortisol conversion experiments;
Washing the blue Absidia spores with sterile water, diluting the coated plate, and after colonies grow out, simultaneously adding the colonies to a hygromycin B300 mg/ml and a non-antibiotic PDA plate, screening to obtain blue Absidia host bacteria of which the C11 beta-hydroxylase gene CYP5311B2 is deleted and pAMA-gRNA-1-2-Cas 9 vectors are discarded.
Example 2 determination of C11 alpha-steroid hydroxylase Gene CYP5311B1 deleted blue Absidia host Using CRISPR-Cas9 determination of C11 beta-steroid hydroxylase Gene CYP5311B1 deleted blue Absidia host 1.Cas9 targeting CYP5311B1 Gene sequence Using CRISPR-Cas9
Target-3：CTTTGATCAAAAAAAGACCA
Target-4：AGACAAGAACTCTACAAAGG
Construction of pBlue-PU6-B1-gRNA-1-TU6, pBlue-PU6-B1-gRNA-2-TU6 and pBlue-B1-gRNA-1-2 vector
The plasmid pBlue-PU6-gRNA-TU6 is used as a template, B1-gRNA-1-F/R is used as a primer for carrying out polymerase chain reaction to obtain a target vector fragment pBlue-B1-PU6-gRNA-1-TU6, and B1-gRNA-2-F/R is used as a primer for carrying out polymerase chain reaction to obtain a target vector fragment pBlue-PU6-B1-gRNA-2-TU6. Primers were synthesized by Beijing Hua big Gene company.
B1-gRNA-1-F：CTTTGATCAAAAAAAGACCAGTTTTAGAGCTAGAAATAGCAAG
B1-gRNA-1-R：GGGTAAATCTATTTCTATTTTTACTTTCTTGGCACAATC
B1-gRNA-2-F：AGACAAGAACTCTACAAAGGGTTTTAGAGCTAGAAATAGCAAG
B1-gRNA-2-R：CCTTTGTAGAGTTCTTGTCTACTTGTTCTTCTTTACAATGATT
PCR reaction System (Total 50. Mu.l):
PCR procedure:
Mu.l of the PCR product was subjected to agarose gel electrophoresis to verify the band size. And (3) carrying out purification recovery on the PCR product after Dpn I digestion, recovering the competence of the product for transforming escherichia coli JM109, picking up transformants to extract plasmids, and carrying out sequencing verification to verify correctness to obtain pBlue-PU6-B1-gRNA-1-TU6 and pBlue-PU6-B1-gRNA-2-TU6 plasmids.
The pBlue-PU6-B1-gRNA-1-TU6 is subjected to Kpn I single enzyme digestion to prepare a homologous recombination framework, primers with homology arms are designed by taking the pBlue-PU6-B1-gRNA-2-TU6 as templates to amplify PU6-B1-gRNA-2-TU6 target fragments, 2 mu l of enzyme digestion products and 2 mu l of PCR products are respectively taken for agarose gel electrophoresis to verify the band sizes. After digestion and purification of the PCR product, homologous recombination is carried out on the target fragment and the skeleton, the recombinant system converts escherichia coli JM109 competence, the transformant is selected to extract plasmids, sequencing verification is correct, the pBlue-gRNA-1-2 plasmids are obtained, and the primers are synthesized by Beijing Hua large gene company.
pBlue-gRNA-2-F：CTCGAGGGGGGGCCCGGTACTAATGCCGGCTCATTCAAACG
pBlue-gRNA-2-R：AGGGAACAAAAGCTGGGTACAGCAGCTCTATATCACGTGAC
PCR reaction System (Total 50. Mu.l):
PCR procedure:
Cleavage reaction System (Total 50. Mu.l):
recombination reaction System (Total 10. Mu.l):
construction of pAMA1-B1-gRNA-1-2-Cas9 vector
Carrying out BamHI single enzyme digestion on pAMA-Cas 9 plasmid to prepare homologous recombination framework, designing a primer with homology arms by taking pBlue-B1-gRNA-1-2 plasmid as a template, amplifying a target fragment containing two gRNA expression cassettes, respectively taking 2 mu l of enzyme digestion products and 2 mu l of PCR products, and carrying out agarose gel electrophoresis to verify the band size. After digestion and purification of the PCR product, homologous recombination is carried out on the target fragment and the skeleton, the recombinant system converts escherichia coli JM109 competence, the transformant is selected to extract plasmids, sequencing verification is correct, then pAMA-B1-gRNA-1-2-Cas 9 plasmids are obtained, and the primers are synthesized by Beijing Hua large gene company.
2gRNA-F-BamHI：CTGTTTCCGCTAGCAGATCACTATAGGGCGAATTGGAGCTC
2gRNA-R-BamHI：CCAGCTCACATCCTGGATCCAGCTATGACCATGATTACGC
PCR reaction System (Total 50. Mu.l):
PCR procedure:
Cleavage reaction System (Total 50. Mu.l):
recombination reaction System (Total 10. Mu.l):
Construction of pCSN44-P B1 -CYP5311B 2T 329A recombinant expression vector and acquisition of target fragment
The method comprises the steps of amplifying a homologous recombination skeleton containing a CYP5311B1 promoter and a terminator part by inverse PCR by taking a pCSN44-G418-CYP5311B2 recombinant plasmid constructed in the earlier stage of a laboratory as a template, designing a primer with a homology arm by taking a pYes2-CYP5311B 2T 329A plasmid as the template to amplify a target fragment containing a CYP5311B 2T 329A gene, carrying out homologous recombination on the skeleton and the target fragment after digestion and purification, recombining and systemizing escherichia coli JM109 competence, picking a transformant to extract plasmids, sequencing and verifying correctness, thus obtaining the pCSN44-P B1 -CYP5311B 2T 329A recombinant expression vector shown in figure 3, amplifying by using a primer C1-F/R to obtain the target fragment, and synthesizing the primer by Beijing Hua large gene company.
PCSN44-P B1 backbone-F: GTTTTGTTAGAATGGGATCAG A
PCSN44-P B1 backbone-R: AAATAGAAATAGATTTACCCTCGC A
F58-CYP5311B2(PB1)：GATCCCATTCTAACAAAACATGCTTACAGAGTACATTCATC
R56-CYP5311B2(PB1)：GGGTAAATCTATTTCTATTTTTACTTTCTTGGCACAATC
C1-F：GCATTGACATCGAGTCCCATT
C1-R：AGGTAACACTAACTCTTCTAG
PCR reaction System (Total 50. Mu.l):
PCR procedure:
recombination reaction System (Total 10. Mu.l):
EXAMPLE 3 construction of C11.beta. -hydroxylase mutant recombinant Absidia blue
Adding 3-5 mug recombinant plasmid pAMA-gRNA-1-2-Cas 9 and 30-50 mug target fragment CYP5311B 2T 329A into wild Absidia blue protoplast with concentration more than 10 8/ml and adding 680 mug PEG6000 solution, ice-bath for 20min;
2ml PEG6000 buffer solution is added and the mixture is left at room temperature for 10min;
Adding 4ml STC and 4ml upper medium at 48 ℃ which are preheated and mixed uniformly in advance, plating on a lower medium with hygromycin B concentration of 300mg/ml, placing a flat plate in a 28 ℃ incubator, growing transformants after 5-7d, transferring the transformants into a non-antibiotic PDA test tube for culturing until sporulation and extracting genome for sequencing verification, and performing fixed-point CYP5311B 2T 329A replacement on CYP5311B1 to obtain the recombinant blue Abelmoschus with a C11 beta-hydroxylase mutant, wherein the construction schematic diagram is shown in figure 4.
Example 4 11-deoxycortisol transformation experiments with C11.beta.mutant recombinant Absidia blue
The 11-deoxycortisol steroid transformation experiment of the C11β mutant recombinant blue Absidia is carried out, the spore suspension is added with a substrate after shaking culture for 24 hours at 28 ℃, the addition amount of the substrate 11-deoxycortisol is 4g/L, the fermentation transformation is carried out for 24 hours/48 hours/72 hours, sampling is carried out, and extraction is carried out by adopting ethyl acetate, wherein the transformation condition is shown in figure 5.
The above examples merely illustrate several embodiments of the present invention, but are not intended to limit the present invention. It should be noted that modifications to the embodiments described above may be made by any person skilled in the art without departing from the spirit and scope of the present invention. Accordingly, the scope of the invention should be determined by the following claims.
Claims (5)
1. A recombinant blue-plowshare mold is characterized in that a CRISPR-Cas9 technology is utilized to obtain blue-plowshare mold with inactivated C11β -hydroxylase gene CYP5311B2, and a CYP5103B 2T 329A mutant nucleotide sequence shown in SEQ ID NO.1 is integrated into a blue-plowshare mold genome with a missing function, so that the blue-plowshare mold recombinant strain for preparing hydrocortisone industrially and efficiently is obtained.
2. The C11 β -hydroxylase gene CYP5311B2 inactivated blue Absidia according to claim 1, wherein the steps of constructing are as follows:
(1) Determining a Cas 9-targeted CYP5311B2 gene sequence;
(2) Constructing an expression vector pAMA-gRNA-1-2-Cas 9 containing two gRNA expression cassettes, one Cas9 expression cassette and an AMA1 sequence;
(3) Introducing the recombinant expression vector in the step (2) into wild blue Absidia through a protoplast transformation mode for gene editing;
(4) Sequencing and verifying the obtained transformant extracted genome to obtain 11 alpha-hydroxylase gene CYP5311B2 deleted blue Absidia;
(5) Transferring the transformant to a non-antibiotic PDA test tube, culturing until spore is produced, washing the spore with sterile water, and diluting the coated plate;
(6) After the colony grows out, the colony is simultaneously screened to hygromycin B300 mg/ml and a non-antibiotic PDA plate to obtain 11 alpha-hydroxylase gene CYP5311B2 which is deleted and the pAMA1-gRNA-1-2-Cas9 carrier blue Abelmoschus host bacteria is lost.
3. The CYP5311B 2T 329A mutant nucleotide sequence of claim 1, wherein the mutant nucleotide sequence is driven by a 800bp sequence promoter upstream of the ORF of the blue coulter gene CYP5311B 1.
4. The integration of the CYP5103B 2T 329A mutant nucleotide sequence into the genome of a deletional blue-colpitis mold according to claim 1, wherein the C11 β -hydroxylation deletional blue-colpitis mold CYP5311B1 gene is replaced by CYP5103B 2T 329A using CRISPR-Cas9 technology and the fragment of interest for replacement contains homology arms upstream and downstream.
5. The recombinant blue-coluba according to claim 1, wherein the recombinant blue-coluba catalyzes the production of hydrocortisone from 11-deoxycortisol, the total amount of substrate 11-deoxycortisol fed is 4g/L, the conversion is carried out for 72 hours, and the yield of the product hydrocortisone is up to 85%.
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