CN109097343A - 11 B-hydroxylase of steroid and its encoding gene and application in Curvuluria Iunata - Google Patents
11 B-hydroxylase of steroid and its encoding gene and application in Curvuluria Iunata Download PDFInfo
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Abstract
The invention discloses 11 B-hydroxylase of steroid and its encoding gene and applications in a kind of Curvuluria Iunata.11 B-hydroxylase of steroid provided by the invention is protein (i.e. CL3-CYP021 albumen) shown in SEQ ID No.1.In addition the present invention also provides the complete albumen that protein (i.e. CL3-CPR albumen) forms shown in 11 B-hydroxylase of steroid and SEQ ID No.2.The present invention expresses CL3-CYP021 albumen (or CL3-CYP021 and CL3-CPR albumen) in Heterologous Microbial, carries out catalyzing and synthesizing for hydrocortisone (HC) and 14 α-hydroxylation cortisol.Yeast is substituted original filamentous fungi and carries out biocatalysis fermenting and producing HC by the present invention, with shortening fermentation time, simplify fermentation condition and separation and Extraction step, reduce the advantages such as production cost, has significance for substitution traditional hydrogenation cortisone biocatalysis fermenting and producing mode.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to 11 B-hydroxylase of steroid and its encoding gene in Curvuluria Iunata
With application.
Background technique
Hydrocortisone (Hydrocortisone, HC) chemistry entitled 11 β, 17 α, the pregnant Gona-4-ene-3 of 21- trihydroxy, 20-
Diketone is Adrenal Glucocorticoid drug, accounts in hormone medicine and critical role, structural formula are as shown in fig. 1.HC
Can influence glycometabolism, have the effects that antiviral, anti-inflammatory, antiallergy and Hemorrhagic shock [Dumas, B., et al.,
Hydrocortisone made in yeast:metabolic engineering turns a unicellular
microorganism into a drug-synthesizing factory.Biotechnol J,2006.1(3):299-
307.].It is mainly used for the symptoms such as disease caused by adrenal insufficiency and congenital adrenal function hyperplasia
Treatment, it can also be used to which bronchial asthma, rheumatoid arthritis, gout, rheumatic fever etc. are inflammatory and anaphylactia
Treatment can be also used for seriousness infection and Hemorrhagic shock treatment etc..
The synthetic method of HC is broadly divided into three classes: the method for full chemistry synthetic method, semi-synthesis method and full biosynthesis
[Colingsworth,D.R.,et al."A partial microbiological synthesis of
hydrocortisone."Journal of Biological Chemistry 203.2(1953):807.].Nineteen fifty,
Wendler etc., which is reacted with the fully synthetic method of chemistry through 30 multi-step chemicals, successfully synthesizes HC, but because of its complex process, total recovery is too
It is low, and be worth without industrial production.[Szczebara, F.M., et al., Total the biosynthesis of such as Dumas
hydrocortisone from a simple carbon source in yeast.Nat Biotechnol,2003.21
(2): 143-149.] 11 β hydroxylating P450 (CYP11B1) in user source realize that with glucose be only in saccharomyces cerevisiae
The one fully synthetic hydrocortisone of carbon source bioanalysis, but due to route of synthesis complexity, efficiency of pcr product is lower, and also there is no realize industry
Metaplasia produces.The steroid drugs such as HC are prepared both at home and abroad at present still mainly using semi-synthetic method, i.e., with Chinese yam saponin or plant
The precursors such as sterol obtain the pregnant Gona-4-ene-3 of 17 Alpha-hydroxy of pharmaceutical intermediate, 20- diketone -21- acetate by chemical synthesis
(Cortexolone-21-acetate, RSA) or Compd S 11-deoxycortisol (11-deoxycortisol, RS), then passes through colter
The biological whole-cell catalytic of the microorganisms such as mould (Absidia orchidis) and curvularia lunata (Curvularia lunata)
Reaction obtains HC [Kim, Y.H., et al., Synthesis of α-to its 11 progress β hydroxylatings
ketoglutarate using glutamate dehydrogenase combined with electrochemical
cofactor regeneration system.Journal of Bioscience and Bioengineering,
2009.108:S45-S46.Manosroi,J.,Y.Chisti,and A.Manosroi,Biotransformation of
cortexolone to hydrocortisone by molds using a rapid color-development
assay.Applied Biochemistry and Microbiology,2006.42(5):479-483.].Hydrocortisone closes
Fig. 2 is seen at technique.
Curvularia lunata (Curvularia lunata) belongs to Fungi Imperfecti, Moniliales, An Geng spore section, more spore subfamilies,
Curvularia.The microorganism has 11 β hydroxylating functions, and is used extensively by domestic and foreign scholars.Japanese scholars Kenzi
[Suzuki, K., et al., Purification and properties of cytochrome the P-450 (P- such as Suzuki
450lun)catalyzing steroid 11 beta-hydroxylation in Curvularia lunata.Biochim
Biophys Acta, 1993.1203 (2): 215-223.] it is pure using RS as substrate for induction P450 protein expression in curvularia lunata
The hydroxylated P450 albumen of 11 β has been dissolved, and has thought that the albumen is one and has both 11 β hydroxylatings and 14 α are hydroxylated
Bifunctional enzyme.[Lu, W., et al., Optimisation of Hydrocortisone the Production by such as Lu
Curvularia lunata.Applied Biochemistry and Biotechnology,2007.142(1):17-28.]
The curvularia lunata KA91 obtained by mutagenesis screening ketoconazole resistance mutant strain compares the conversion ratio of HC compared with starting strain CL114
Improve 40%.
Curvularia lunata is better than RSA to the conversion rate of substrate RS.But the bacterium in addition to can specificity introduce C11 β hydroxyl
Base, but can also be generated with C14 α hydroxy by-products.German Schering Corp (Schering AG) by C17 α acetyl compound,
It hinders C14 α hydroxy by-products to generate by stereochemical structure, hydrocortisone yield is made to reach 70% or so.But pass through for many years
Researchers not excavating 11 β hydroxylating P450 of substrate of most critical in hydrocortisone synthesis process successfully,
So the research that the successful excavation of the albumen synthesizes hydrocortisone is significant.
Summary of the invention
The object of the present invention is to provide 11 B-hydroxylase of steroid and its encoding gene and applications in a kind of Curvuluria Iunata.
In a first aspect, claimed following protein or complete protein.
Protein provided by the present invention be it is any in following (A1)-(A4) shown in protein:
(A1) amino acid sequence protein (protein shown in wherein, SEQ ID No.1 as shown in SEQ ID No.1
For CL3-CYP021 albumen)；
(A2) amino acid sequence defined by (A1) is passed through to the substitution and/or missing of one or several amino acid residues
And/or addition and protein with the same function；
(A3) there is 99% or more, 95% or more, 90% or more, 85% with amino acid sequence defined by (A1) or (A2)
Above or 80% or more homology and protein with the same function；
(A4) fusion obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (A1)-(A3)
Albumen.
Complete protein provided by the present invention is made of a-protein and PROTEIN B.
The a-protein be it is any in as above (A1)-(A4) shown in protein.
The PROTEIN B be it is any in following (B1)-(B4) shown in protein:
(B1) amino acid sequence protein (protein shown in wherein, SEQ ID No.2 as shown in SEQ ID No.2
For CL3-CPR albumen)；
(B2) amino acid sequence defined by (B1) is passed through to the substitution and/or missing of one or several amino acid residues
And/or addition and protein with the same function；
(B3) there is 99% or more, 95% or more, 90% or more, 85% with amino acid sequence defined by (B1) or (B2)
Above or 80% or more homology and protein with the same function；
(B4) fusion obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (B1)-(B3)
Albumen.
Second aspect, claimed nucleic acid molecules or complete nucleic acid molecules.
Nucleic acid molecules provided by the present invention can for coding above protein described in first aspect nucleic acid molecules (including
The nucleic acid molecules of the coding CL3-CYP021 albumen of optimization and unoptimizable).
Complete nucleic acid molecules provided by the present invention are made of nucleic acid molecules A and nucleic acid molecules B.
The nucleic acid molecules A be coding above a-protein described in first aspect nucleic acid molecules (including optimization and it is non-optimum
The nucleic acid molecules of the coding CL3-CYP021 albumen of change)；
The nucleic acid molecules B be coding above PROTEIN B described in first aspect nucleic acid molecules (including optimization and it is non-optimum
The nucleic acid molecules of the coding CL3-CPR albumen of change).
Further, the nucleic acid molecules it is concretely any in following (a1)-(a3) shown in DNA molecular:
(a1) nucleotide sequence DNA molecular (wherein, SEQ ID as shown in SEQ ID No.3 or SEQ ID No.5
No.3 is the encoding gene of the CL3-CYP021 albumen of optimization, and SEQ ID No.5 is the coding base of the CL3-CYP021 of unoptimizable
Cause)；
(a2) hybridize under strict conditions with (a1) DNA molecular limited and encode above (A1)-described in first aspect
(A4) in it is any shown in protein DNA molecular；
(a3) with (a1) or (a2) defined by DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% with
The DNA of protein shown in upper or 80% or more homology and coding are any in (A1)-(A4) described in first aspect above
Molecule.
Further, the nucleic acid molecules A it is concretely as above any in (a1)-(a3) shown in DNA molecular.
Further, the nucleic acid molecules B it is concretely any in following (b1)-(b3) shown in DNA molecular:
(b1) nucleotide sequence DNA molecular (wherein, SEQ ID as shown in SEQ ID No.4 or SEQ ID No.6
No.4 is the encoding gene of the CL3-CPR albumen of optimization, and SEQ ID No.6 is the encoding gene of the CL3-CPR of unoptimizable)；
(b2) hybridize under strict conditions with (b1) DNA molecular limited and encode protein described in first aspect above
The DNA molecular of B；
(b3) with (b1) or (b2) defined by DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% with
Upper or 80% or more homology and the DNA molecular for encoding PROTEIN B described in first aspect above.
Wherein, the stringent condition can be as follows: 50 DEG C, in 7% lauryl sodium sulfate (SDS), 0.5M NaPO4With
Hybridize in the mixed solution of 1mM EDTA, is rinsed in 50 DEG C, 2 × SSC, 0.1%SDS；May be used also are as follows: 50 DEG C, 7%SDS,
0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, is rinsed in 50 DEG C, 1 × SSC, 0.1%SDS；May be used also are as follows: 50
DEG C, in 7%SDS, 0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, is floated in 50 DEG C, 0.5 × SSC, 0.1%SDS
It washes；May be used also are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, at 50 DEG C, 0.1 × SSC,
It is rinsed in 0.1%SDS；May be used also are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO4Hybridize in the mixed solution of 1mM EDTA, 65
DEG C, it is rinsed in 0.1 × SSC, 0.1%SDS；It can also are as follows: in 6 × SSC, the solution of 0.5%SDS, hybridize at 65 DEG C, then
With 2 × SSC, 0.1%SDS and 1 × SSC, it is primary that 0.1%SDS respectively washes film.
The third aspect, any one of claimed following biomaterial:
(c1) recombinant vector, for the recombinant vector containing nucleic acid molecules described in second aspect above；
(c2) expression cassette, for the expression cassette containing nucleic acid molecules described in second aspect above；
(c3) transgenic cell line, for the transgenic cell line containing nucleic acid molecules described in second aspect above；
(c4) recombinant bacterium, for the recombinant bacterium containing nucleic acid molecules described in second aspect above；
(c5) complete recombinant vector is made of recombinant vector A and recombinant vector B；The recombinant vector A is containing above the
The recombinant vector of nucleic acid molecules A described in two aspects；The recombinant vector B is to contain nucleic acid molecules described in second aspect above
The recombinant vector of B；
(c6) complete expression cassette is made of expression cassette A and expression cassette B；The expression cassette A is containing in second aspect above
The expression cassette of the nucleic acid molecules A；The expression cassette B is the expression cassette containing nucleic acid molecules B described in second aspect above；
(c7) complete transgenic cell line is made of transgenic cell line A and transgenic cell line B；The transgenosis is thin
Born of the same parents system A is the transgenic cell line containing nucleic acid molecules A described in second aspect above；The transgenic cell line B be containing
The transgenic cell line of nucleic acid molecules B described in second aspect above；
(c8) complete recombinant bacterium is made of recombinant bacterium A and recombinant bacterium B；The recombinant bacterium A is containing in second aspect above
The recombinant bacterium of the nucleic acid molecules A；The recombinant bacterium B is the recombinant bacterium containing nucleic acid molecules B described in second aspect above.
Fourth aspect, it is claimed it is a kind of building for producing hydrocortisone and/or 14 α-hydroxylation cortisol
Engineering bacteria method.
The side provided by the present invention constructed for producing the engineering bacteria of hydrocortisone and/or 14 α-hydroxylation cortisol
Method, it may include following steps: being transformed saccharomycete, makes its expression protein described in first aspect or complete egg above
White matter, improved saccharomycete are the engineering bacteria for being used to produce hydrocortisone and/or 14 α-hydroxylation cortisol.
Further, the method may include following steps: will above nucleic acid molecules described in second aspect or it is described at
It covers nucleic acid molecules and imports the saccharomycete, obtain expressing protein described in first aspect above or the complete protein
Recombinant yeast, the as described engineering bacteria.
Further, the nucleic acid molecules are by recombinant vector described in the third aspect above or the recombinant expression
The form of box imports in the saccharomycete；The complete nucleic acid molecules are carried by complete recombination described in the third aspect above
The form of body or the complete expression cassette imports in the saccharomycete.
Further, the nucleic acid molecules or the complete nucleic acid molecules are integrated into the genome of the saccharomycete
Common site, at the site Gal7 or the site rDNA.
Further, the saccharomycete can be saccharomyces cerevisiae (such as BY4742 bacterial strain), Yarrowia lipolytica (such as PO1G bacterium
Strain), schizosaccharomyces pombe (such as CICC1762 bacterial strain) or Pichia pastoris (such as GS115 bacterial strain).
In the specific embodiment of the present invention, the nucleic acid molecules be by way of recombinant vector import described in
In saccharomycete；The recombinant vector be by the nucleic acid molecules be inserted into pRS426 plasmid restriction enzyme site (such as SexA1 and
Asc1 the recombinant plasmid obtained after between).
In another embodiment of the invention, the complete nucleic acid molecules are by way of complete expression cassette
It imports in the saccharomycete；The complete expression cassette is by expression cassette pPgk-CL3-CPR-ADHt and expression cassette pTEF-CL3-
CYP021-CYC1t composition；The sequence of the expression cassette pPgk-CL3-CPR-ADHt is SEQ ID No.7 (or for by SEQ
Resulting sequence after the replaced with SEQ ID No.4 of 813-2903 of ID No.7)；The expression cassette pTEF-CL3-
The sequence of CYP021-CYC1t is SEQ ID No.8 (or to replace with SEQ ID for 501-2021 of SEQ ID No.8
Resulting sequence after No.3).When importing the expression cassette pPgk-CL3-CPR-ADHt and expression cassette into the saccharomycete
While pTEF-CL3-CYP021-CYC1t, homology arm marker segment gal7-URA3-up and homology arm have also been imported
Marker segment gal7-URA3-down (realizes the site Gal7 for being integrated in saccharomyces cerevisiae BY4742)；The homology arm
The sequence of marker segment gal7-URA3-up is as shown in SEQ ID No.9；The homology arm marker segment gal7-URA3-
The sequence of down is as shown in SEQ ID No.10.
In another specific embodiment of the invention, the complete nucleic acid molecules are by way of complete expression cassette
It imports in the saccharomycete；The complete expression cassette is by the expression cassette pPgk-CL3-CPR-ADHt and the expression cassette
PTEF-CL3-CYP021-CYC1t composition.When importing the expression cassette pPgk-CL3-CPR-ADHt and institute into the saccharomycete
While stating expression cassette pTEF-CL3-CYP021-CYC1t, homology arm marker segment rDNA-Leu-up and homologous has also been imported
Arm marker segment rDNA-Leu-down (realizes the site rDNA for being integrated in saccharomyces cerevisiae BY4742)；The homology arm
The sequence of marker segment rDNA-Leu-up is as shown in SEQ ID No.11；The homology arm marker segment rDNA-Leu-
The sequence of down is as shown in SEQ ID No.12.
5th aspect, it is claimed to utilize the engineering bacteria that method described in fourth aspect is prepared above.
In a specific embodiment of the invention, the engineering bacteria is specially following any:
(1) it engineering bacteria HC101: is obtained after recombinant vector pRS426-CL3-CYP021 is imported saccharomyces cerevisiae BY4742 bacterial strain
The recombinant bacterium arrived；The recombinant vector pRS426-CL3-CYP021 is (to be not optimised DNA fragmentation shown in SEQ ID No.5
CL3-CYP021 gene) be inserted between the restriction enzyme site SexA1 and Asc1 of pRS426 plasmid after obtained recombinant plasmid.
(2) engineering bacteria HC102: by the expression cassette pPgk-CL3-CPR-ADHt, (CL3-CPR gene therein is non-optimum
Change gene, as shown in SEQ ID No.6), the expression cassette pTEF-CL3-CYP021-CYC1t (CL3-CYP021 base therein
Because of unoptimizable gene, as shown in SEQ ID No.5), the homology arm marker segment gal7-URA3-up and described homologous
Arm marker segment gal7-URA3-down imports the recombinant bacterium obtained after saccharomyces cerevisiae BY4742 bacterial strain and (realizes and be integrated in wine
The site Gal7 of brewer yeast BY4742).
(3) engineering bacteria HC103: by the expression cassette pPgk-CL3-CPR-ADHt, (CL3-CPR gene therein is optimization
Gene, as shown in SEQ ID No.4), the expression cassette pTEF-CL3-CYP021-CYC1t (CL3-CYP021 gene therein
For optimization gene, as shown in SEQ ID No.3), the homology arm marker segment gal7-URA3-up and the homology arm
Marker segment gal7-URA3-down imports the recombinant bacterium obtained after saccharomyces cerevisiae BY-4741 bacterial strain and (realizes and be integrated in wine
The site Gal7 of brewer yeast BY4742).
6th aspect, protein described in claimed first aspect above or complete protein or above the
Nucleic acid molecules or complete nucleic acid molecules described in two aspects or above biomaterial described in the third aspect or above the 5th
Engineering bacteria described in aspect it is following it is any in application:
(A) hydrocortisone and/or 14 α-hydroxylation cortisol are prepared；
(B) catalysis steroid hormone substance carries out 11 β hydroxylatings and/or 14 α hydroxylatings.
In addition, protein described in first aspect above is also claimed as steroid hormone substance 11 in the present invention
Application in position and/or 14 hydroxylases.
Wherein, the steroid hormone substance can for can by 11 B-hydroxylase of steroid be catalyzed generate hydrocortisone and/
Or the substance of 14 α-hydroxylation cortisol can be generated by 14 α of steroid-hydroxylase catalysis, the specific such as pregnant Gona-4-ene-3 of 7 Alpha-hydroxies,
20- diketone -21- acetate (or being hydrocortisone 21- acetate, Cortexolone-21-acetate, RSA) or 11-
Deoxy-cortisol (11-deoxycortisol, RS).
7th aspect, a kind of claimed method for preparing hydrocortisone and/or 14 α-hydroxylation cortisol.
The method provided by the present invention for preparing hydrocortisone and/or 14 α-hydroxylation cortisol can be whole-cell catalytic
Method or enzyme process.
Wherein, the whole-cell catalysis may include following steps: carry out to engineering bacteria described in the above the 5th aspect
Substrate is added after collecting thallus in fermented and cultured, carries out catalysis reaction, contains hydrocortisone and/or 14 α-in reaction product
It is hydroxylated cortisol；The substrate is that can be catalyzed to generate hydrocortisone and/or can be by steroid 14 by 11 B-hydroxylase of steroid
α-hydroxylase catalysis generates the substance of 14 α-hydroxylation cortisol.
Wherein, the enzyme process may include following steps: extracting in the engineering bacteria described in terms of the above the 5th has steroid
Then the substance of 11 B-hydroxylase of class and/or 14 α-hydroxylase activity is urged in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme
Change substrate and generates hydrocortisone and/or 14 α-hydroxylation cortisol；The substrate is that can be catalyzed to give birth to by 11 B-hydroxylase of steroid
The substance of 14 α-hydroxylation cortisol can be generated at hydrocortisone and/or by 14 α of steroid-hydroxylase catalysis.
Further, concretely 17 Alpha-hydroxy pregn-4-ene-3,20-dione -21- acetates (or are the substrate
Deoxygenate hydrocortisone 21- acetate, Cortexolone-21-acetate, RSA) or Compd S 11-deoxycortisol (11-
Deoxycortisol, RS).
In the specific embodiment of the invention, the whole-cell catalysis that specifically uses.Wherein, the item of the catalysis reaction
Part is specially 30 DEG C of 200-250rpm (such as 250rpm) shaken cultivations 1-4 days (such as 2 days)；Substrate in reaction system is RSA,
Concentration is 10mg-200mg (such as 170mg/L).
It is demonstrated experimentally that the present invention expresses CL3-CYP021 albumen in Heterologous Microbial carries out hydrocortisone and/or 14
α-hydroxylation cortisol catalyzes and synthesizes.The present invention is using the saccharomycete catalytic production hydrocortisone for having more foodsafety
Possibility is provided, the production cycle is effectively shortened compared to traditional filamentous fungi production method, simplifies Zymolysis Equipment, and
And the expression for being able to carry out single albumen improves product yield, effectively reduces production and extraction cost, for hydrogenation
The innovation of cortisone production technology is of great significance, and also catalyzes and synthesizes 14 α-hydroxylation cortisol for biology
Possibility is provided.
Detailed description of the invention
Fig. 1 is the structural formula of hydrocortisone.
Fig. 2 is hydrocortisone production process.
Fig. 3 is that saccharomyces cerevisiae engineered yeast HC101 catalysate is detected and identified.
Fig. 4 is that saccharomyces cerevisiae engineered yeast HC102 catalysate detects and identifies (1).
Fig. 5 is that saccharomyces cerevisiae engineered yeast HC102 catalysate detects and identifies (2).
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Curvuluria Iunata AS3.4381 (ATCC12017): biological product is received in north.
PRS426 plasmid: Shanghai north promise biology Co., Ltd, article No. are addgene 0499.
M2 plasmid: peasy-Blunt-simple carrier (full formula gold biological Co., Ltd) multiple cloning sites in order
Insert SexA1 restriction enzyme site, pPgk promoter, green fluorescent protein (GFP) gene and terminator ADH1t and Asc1 enzyme
Enzyme site.
M3 plasmid: peasy-Blunt-simple carrier (full formula gold biological Co., Ltd) multiple cloning sites in order
Insert SexA1 restriction enzyme site, pTEF promoter, green fluorescent protein (GFP) gene and terminator CYC1t and Asc1 enzyme
Enzyme site.
Saccharomyces cerevisiae (Saccharomyces cerevisiae) BY4742:Thermo Fisher Products.
Yarrowia lipolytica (CICC32520): Chinese industrial Microbiological Culture Collection administrative center (CICC) product.
Schizosaccharomyces pombe (CICC1762): Chinese industrial Microbiological Culture Collection administrative center (CICC) product.
Pichia pastoris (GS115): excellent treasured biological product, product number: ST1030.
The clonal expression of 11 embodiment 1, Curvuluria Iunata β-hydroxylation enzymes
The clonal expression of gene is divided into following 3 step:
1, the extraction of Curvuluria Iunata total serum IgE
Firstly, Curvuluria Iunata AS3.4381 (ATCC12017) is cultivated a couple of days on plate, and collect certain amount
Spore is linked into 50mL Potato-dextrose (PDA) culture medium, is incubated overnight to a large amount of thallus of synthesis；Then, it is collected by centrifugation
Curvuluria Iunata mycelium is washed using potassium phosphate buffer (PBS), is finally resuspended with 50mL buffer and end is added
The substrate hydrocortisone 21- acetate (RSA) that concentration is 170mg/L induces 2h, and sampling carries out RNA extraction.
RNA extraction method:
(1) grinding bead (filling up conical pipe bottom) and 1mL of 0.5mm are added in 2.0mL nut pipe (RNase free)
The mycelia block (about 100mg) that liquid nitrogen flash freezer is crossed is added in Trizol later.
(2) ball mill (BeadBeater) maximum (top) speed 30s is used, is repeated twice.
(3) the mild shake 5min of room temperature, removes the nucleosome of attachment.
(4) chloroform of 0.2mL, maximum (top) speed bead mill 15s is added.
(5) the mild shake 2min of room temperature.
12000g is centrifuged 15min at (6) 4 DEG C, takes supernatant (about 0.5mL) into new EP pipe (RNase free).
(7) isopropanol of isometric (0.5mL) is added, mixes well.
(8) the mild shake 10min of room temperature, visible white precipitates at this time.
12000g is centrifuged 10min at (9) 4 DEG C, removes supernatant.
(10) 75% ethanol wash of 1mL DEPC water configuration precipitates, and 7500g is centrifuged 5min at 4 DEG C, sufficiently removal supernatant.
(11) it is placed at room temperature for about 15-20min and removes ethyl alcohol, indissoluble can be become by hanging RNA too long.
(12) 50 μ L DEPC water, 60 DEG C of water-bath 10min hydrotropies are added.
(13) NanoDrop measures RNA concentration and purity.The usual preferable RNA sample A260/A280 ratio of purity is 1.9
Between~2.0, A260/A230 is typically larger than 2.
2, reverse transcription PCR and gene magnification
First chain Reverse transcription-PCR: no RNA enzyme PCR pipe is taken, expands to obtain cDNA by Thermo reverse transcription reagent box.Specifically
Operating procedure is as follows: template total serum IgE 3 μ L, Oligo (dT)181 μ L, 10mM dNTP Mix of primer, 1 μ l, RNase-free water 10
μl.Moment is centrifuged, 65 DEG C of 5min of PCR, on ice chilling；Reaction solution in following system: 5 × RT Buffer, 4 μ L is added,
1 μ L of Maxima H Minus Enzyme Mix, moment centrifugation carry out reaction 50 DEG C of 50min, 85 DEG C of 5min in PCR instrument, and 4 DEG C
Heat preservation.
PCR amplification: cDNA obtained by reverse transcription is template, and with following primer amplification to target gene, amplification system is
TAKARA5 × Buffer of HS archaeal dna polymerase, 10 μ l, Dntp mix 4 μ l, each 1 μ l of primer (being shown in Table 1),
CDNA, template 0.5 μ l, PrimerSTAR HS polymerase (2.5U/ μ L) 0.5 μ l, adds distilled water to 50 μ l of total volume.Amplification
Condition is 98 DEG C of initial denaturations 2 minutes (1 circulation)；98 DEG C are denaturalized to anneal within 10 seconds, 56 DEG C 15 seconds, 72 DEG C and extend that (30 were followed in 2 minutes
Ring)；72 DEG C extend 8 minutes (1 circulation).
Gained amplified production is named as CL3-CYP021, CL3-CPR.Obtained pcr amplification product is raw with the raw work in Shanghai
The PCR product Purification Kit of object Engineering Co., Ltd uses SexA1 and Asc1 the digestion DNA of Thermo company after purification
Segment, glue recovery product are spare.
1 CL3-CYP021 and CL3-CPR gene magnification primer of table
| Primer | Sequence (5 ' -3 ') |
| CL3-CYP021-SexA1-F | gcgaccaggtATGGATCCCCAGACTGTCGGGCT |
| CL3-CYP021-Asc1-R | ggcgcgccCTACACTACCACTCTCTTGAAA |
| CL3-CPR-SexA1-F | gcgaccaggtATGGCACAACTCGACACGCTCGA |
| CL3-CPR-Asc1-R | ggcgcgccTCATGACCAGACGTCTTCCTGGT |
CL3-CYP021 gene (wild type) sequence encodes albumen shown in SEQ ID No.1 as shown in SEQ ID No.5
Matter (is named as CL3-CYP021 albumen)；CL3-CPR gene (wild type) sequence encodes SEQ ID as shown in SEQ ID No.6
Protein shown in No.2 (is named as CL3-CPR albumen).
3, gene expression plasmid is constructed
With Thermo company SexA1 and Asc1 digested plasmid pRS426, digestion products glue recycles spare.With above-mentioned gained
Each 50ng addition linked system of CL3-CYP021 genetic fragment: 5 μ L 2 × Quick ligation Buffer (NEB company),
0.5 μ L Quick ligase (NEB company, 400,000cohesive end units/ml), supplement distilled water to 10 μ L, room
Temperature reaction 10min obtains connection product, is transferred to ice bath 30 minutes in Trans1-T1 competent cell, 42 DEG C heat shock 30 seconds, immediately
As for 2 minutes on ice.800 μ l LB culture mediums are added, 250rpm, 37 DEG C are incubated for 1 hour, and bacterium solution is coated in containing ampicillin
LB plate on, after being incubated overnight, PCR screens 5 positive single colonies, and positive colony is carried out Liquid Culture, extracts positive gram
Grand plasmid carries out sequence verification, and sequencing result shows to be inserted into target fragment on carrier pRS426, obtains plasmid pRS426-CL3-
CYP021。
4, building yeast polygenes integrates segment
With Thermo company SexA1 and Asc1 digested plasmid M2, M3, digestion products glue recycles spare.With above-mentioned gained
CL3-CPR, CL3-CYP021 genetic fragment are attached conversion, and (the same step 3) of method, and selected clone sequence verification, obtain matter
Grain M2-CL3-CPR, M3-CL3-CYP021.Primer 1-M-pEASY- is used as template using the plasmid M2-CL3-CPR built
PGK1-F and 1-M-ADHt-TEF1-R (being shown in Table 2) carries out PCR amplification, and (the same step 2 of method obtains pPgk-CL3-CPR-ADH1t
Segment (SEQ ID No.7), the segment include Pgk promoter (63-812 of SEQ ID No.7), curvularia lunata source
CL3-CPR gene (813-2903 of SEQ ID No.7) and the ADH1 terminator (2904- of SEQ ID No.7
3061).Primer 2-M-ADHt-TEF1-F and M-CYC1t- is used as template using the plasmid M3-CL3-CYP021 built
PEASY-R (being shown in Table 2) carries out PCR amplification, and (the same step 2 of method obtains pTEF-CL3-CYP021-CYC1t segment (SEQ ID
No.8), which includes TEF promoter (1-450 of SEQ ID No.8), the CL3-CYP021 in curvularia lunata source
Gene (451-1971 of SEQ ID No.8) and CYC1 terminator (1972-2278 of SEQ ID No.8).It is right
It is spare that the target fragment that amplification obtains carries out glue recovery processing.
2 CL3-CYP021 and CL3-CPR gene integration fragment amplification primer of table
The building of embodiment 2, saccharomyces cerevisiae engineered yeast HC101
Bacterium germination saccharomyces cerevisiae (Saccharomyces cerevisiae) BY4742 is incubated overnight in screening and culturing medium out.
Liquid screening medium composition is as follows: SD-Trp (general Jino, Beijing (functional genome) Science and Technology Ltd.), 2% glucose,
0.005%His., 0.01%Leu., 0.01%Ura. (each percentage sign indicates g/100mL).Take 1ml (OD about 0.6-1.0) point
Be attached in 1.5ml EP pipe, 4 DEG C, 10000g be centrifuged 1min, abandon supernatant, precipitating washed with sterile water (4 DEG C), under similarity condition from
The heart abandons supernatant.1ml treatment fluid (10mM LiAc is added in thallus；10mM DTT；0.6M sorbierite；10mM Tris-HCl
(pH7.5), DTT is just added when treatment fluid uses), 20min is placed at 25 DEG C.Centrifugation abandons supernatant, 1ml 1M sorb is added in thallus
Alcohol (0.22 μm of water system film crosses film degerming) is resuspended, centrifugation, abandons supernatant (being resuspended with 1M sorbierite secondary), is about 90 to final volume
μl.The 1.5 μ l of expression plasmid pRS426-CL3-CYP021 that embodiment 1 obtains is added, is transferred to after mixing in electric revolving cup,
2.7kv electric shock 5.7ms, is added 1ml 1M sorbierite, and 30 DEG C of recovery 1h are coated on solid screening and culturing medium (formula: yeast solid
Screening and culturing medium SD-Ura-Trp, 2% glucose, 0.005%His, 0.01%Leu, 1.5% agar；Each percentage sign indicates
g/100mL).The condition of screening and culturing are as follows: 30 DEG C, cultivate 36h or more.PCR identifies correct positive colony, is named as bacterial strain
HC101。
Embodiment 3, saccharomyces cerevisiae engineered yeast HC101 catalyze and synthesize hydrocortisone and 14 α-hydroxylation cortisol
Shake flask fermentation catalysis: in solid selection medium (formula: yeast solid screening and culturing medium SD-Ura-Trp, 2% Portugal
Grape sugar, 0.005%His, 0.01%Leu, 1.5% agar；Each percentage sign indicates g/100mL) in activate HC101 saccharomycete
Strain, in respective liquid Selective agar medium (formula: liquid yeast screening and culturing medium SD-Ura-Trp, 2% glucose, 0.005%
His, 0.01%Leu；Each percentage sign indicates g/100mL) in prepare seed liquor (30 DEG C, 250rpm, 16h), with the inoculation of 1mL
Amount is inoculated in respectively in the 500mL triangular flask of 3 bottles of fluid nutrient mediums of YPD containing 100mL, and 30 DEG C, 250rpm shaken cultivation 2 days,
5000rpm collects yeast cells, and is washed with PBS buffer solution and be finally resuspended in the 250mL triangular flask containing 30mL PBS, adds
The substrate RSA for entering final concentration of 170mg/L, carries out catalysis reaction, and 30 DEG C, 250rpm shaken cultivation 2 days.
It takes the catalysis reaction solution of 5mL in separatory funnel, isometric extractant 1ml (methanol: chloroform=1:9, body is added
Product ratio), lower layer organic phase 4mL is taken, drying in 10mL centrifuge tube is fitted into, is redissolved with 1mL methanol solution, centrifuging and taking supernatant mistake
0.22 μm of organic filter membrane carries out HPLC detection into liquid phase bottle.It is analyzed and is produced by 1260 high performance liquid chromatograph of Agilent (HPLC)
Object, there are two types of the generations (see Fig. 3) of novel substance for discovery, and the product that two kinds of products and curvularia lunata catalystic, fermentative obtain is complete
It is complete consistent, thus it is speculated that both products are respectively hydrocortisone and 14 14 α of α hydroxylation product-hydroxylation cortisols, and are predicted
CL3-CYP021 is that catalysis steroid hormone substance carries out 11 β hydroxylatings and the hydroxylated P450 albumen of 14 α.
The building of embodiment 4, saccharomyces cerevisiae engineered yeast HC102
Bacterium germination saccharomyces cerevisiae (Saccharomyces cerevisiae) BY4742 is incubated overnight in screening and culturing medium out.
Screening and culturing medium composition is as follows: SD-Trp (general Jino, Beijing (functional genome) Science and Technology Ltd.), 2% glucose,
0.005%His., 0.01%Leu., 0.01%Ura. (each percentage sign indicates g/100mL).Take 1ml (OD about 0.6-1.0) point
Be attached in 1.5ml EP pipe, 4 DEG C, 10000g be centrifuged 1min, abandon supernatant, precipitating washed with sterile water (4 DEG C), under similarity condition from
The heart abandons supernatant.1ml treatment fluid (10mM LiAc is added in thallus；10mM DTT；0.6M sorbierite；10mM Tris-HCl
(pH7.5), DTT is just added when treatment fluid uses), 20min is placed at 25 DEG C.Centrifugation abandons supernatant, 1ml 1M sorb is added in thallus
Alcohol (0.22 μm of water system film crosses film degerming) is resuspended, centrifugation, abandons supernatant (being resuspended with 1M sorbierite secondary), is about 90 to final volume
μl.It is separately added into the segment pPgk-CL3-CPR-ADHt, pTEF-CL3-CYP021-CYC1t and homology arm of the acquisition of embodiment 1
Marker segment gal7-URA3-up (SEQ ID No.9；The homology arm segment includes the homology region Gal7 site upstream 400bp,
URA3marker gene and the homology region Pgk promoter 400bp), gal7-URA3-down (SEQ ID No.10；This is homologous
Arm pieces section includes the homology region CYC1 terminator 200bp and the site the Gal7 downstream homology region 300bp) it (realizes CL3-
CPR, CL3-CYP021 genetic fragment are integrated in the site Gal7 of saccharomyces cerevisiae BY4742) each 1 μ l, electric revolving cup is transferred to after mixing
In, 2.7kv shock by electricity 5.6ms, be added 1ml 1M sorbierite, 30 DEG C of recovery 1h, be coated on solid selection medium plate (formula:
Yeast solid screening and culturing medium SD-Ura-Trp, 2% glucose, 0.005%His, 0.01%Leu, 1.5% agar；Each percentage
Number indicate g/100mL).The condition of screening and culturing are as follows: 30 DEG C, cultivate 36h or more.PCR identifies correct positive colony, life
Entitled bacterial strain HC102.
Embodiment 5, saccharomyces cerevisiae engineered yeast HC102 catalyze and synthesize hydrocortisone and 14 α-hydroxylation cortisol (full cell
Catalysis method)
Shake flask fermentation catalysis: HC002 yeast strain is activated in solid selection medium (with embodiment 3), in corresponding liquid
Seed liquor (30 DEG C, 250rpm, 16h) are prepared in body Selective agar medium (with embodiment 3), are inoculated in 3 respectively with the inoculum concentration of 1mL
In the 500ml triangular flask of the bottle fluid nutrient medium of YPD containing 100ml, 30 DEG C, 250rpm shaken cultivation 2 days, 5000rpm collected yeast
Cell, and washed with PBS buffer solution and be finally resuspended in the 250mL triangular flask containing 30mL PBS, make thallus OD600nmReach
60, the substrate RSA of final concentration of 170mg/L is added, carries out catalysis reaction, 30 DEG C, 250rpm shaken cultivation 2 days, must be catalyzed anti-
Answer liquid.
It takes the catalysis reaction solution of 5mL in separatory funnel, isometric extractant 1ml (methanol: chloroform=1:9, body is added
Product ratio), lower layer organic phase 4mL is taken, drying in 10mL centrifuge tube is fitted into, is redissolved with 1mL methanol solution, centrifuging and taking supernatant mistake
0.22 μm of organic filter membrane carries out HPLC detection into liquid phase bottle.It is analyzed and is produced by 1260 high performance liquid chromatograph of Agilent (HPLC)
Object.
It is identified by HPLC and is compared with the product of marking, confirm that the Wine brewing yeast strain HC102 of building can be synthesized with catalysis substrate RSA
Two kinds of substances, one kind, which can be confirmed, (to be compared through standard items and confirms) for hydrocortisone, and another kind can be confirmed as 14 α hydroxyls
Change 14 α of product-hydroxylation cortisol (see Fig. 4 and Fig. 5), and be higher than HC101 bacterial strain from its yield of calculated by peak area, so speculating
The CPR gene identified and cloned from curvularia lunata is helpful for the production of saccharomyces cerevisiae hydrocortisone.
Then, quantitative analysis has been carried out to the production result of HC102 bacterial strain, can have been led to by quantitative analysis bacterial strain HC102
The mode for crossing whole-cell catalytic obtains target product hydrocortisone 1.97mg/L.Compared to using saccharomyces cerevisiae HC101 bacterial strain
2 times of output increased, the results showed that the CPR gene cloned in curvularia lunata has side for the production of saccharomyces cerevisiae hydrocortisone
It helps.
3 bacterial strain HC101 of table and the quantitative analysis of HC102 catalysate
| Bacterial strain name | Genotype | Hydrocortisone (mg/L) |
| HC101 | BY4742,pRS426-CL3-CYP021 | 0.602±0.08 |
| HC102 | BY4742,Gal7::URA3-PPGK-CL3-CPR+PTEF-CL3-CYP021 | 1.97±0.21 |
Note: the unit mg/L of hydrocortisone is meant that the mg number of hydrocortisone in every liter of catalysis reaction solution.
Embodiment 6, saccharomyces cerevisiae engineered yeast HC103 building and catalyze and synthesize hydrocortisone and 14 α-hydroxylation cortisol
1, the building of saccharomyces cerevisiae engineered yeast HC103
Codon optimization is carried out to CL3-CYP021 and CL3-CPR albumen, the optimization and gene chemical synthesis efforts are entrusted
Nanjing Genscript Biotechnology Co., Ltd. completes, and the unnamed gene after the optimization of acquisition is CL3-CYP021-sy-Sc, CL3-
CPR-sy-Sc.The sequence of CL3-CYP021-sy-Sc gene encodes egg shown in SEQ ID No.1 as shown in SEQ ID No.3
White matter；The sequence of CL3-CPR-sy-Sc gene encodes protein shown in SEQ ID No.2 as shown in SEQ ID No.4.
Segment is integrated according to the method building saccharomyces cerevisiae polygenes in 1 step 4 of embodiment to the gene of acquisition, is then pressed
Above-mentioned two gene integration is finally obtained into bacterial strain in the site Gal7 of saccharomyces cerevisiae BY4742 according to the method in embodiment 4
HC103。
2, saccharomyces cerevisiae engineered yeast HC103 catalyzes and synthesizes hydrocortisone and 14 α-hydroxylation cortisol (whole-cell catalysis)
The Wine brewing yeast strain HC103 of building is carried out catalyzing and synthesizing hydrocortisone, Catalysis experiments and sample detection side
Method is with embodiment 5, by catalystic, fermentative, HC103 bacterial strain can synthesize 3.87mg/L hydrocortisone and 2.43mg/L 14
α-hydroxylation cortisol, fermentation results are shown in Table 4, by codon optimization, further improve saccharomyces cerevisiae and utilize curvularia lunata
11 β hydroxylating P450 albumen carry out catalystic, fermentative production hydrocortisone yield.
4 bacterial strain HC103 catalysate quantitative analysis of table
Note: the unit mg/L of hydrocortisone and 14 α-hydroxylation cortisol is meant that in every liter of catalysis reaction solution that hydrogenation can
Pine or 14 α-hydroxylation cortisol mg number.
3, using saccharomyces cerevisiae engineered yeast HC103 with crude enzyme liquid or the synthesizing hydrogenated cortisone of pure enzyme law catalysis and 14 α-hydroxylation
Cortisol
Step 1: the extraction of albumen (following reactions are all carried out at 4 DEG C on ice)
(1) fermented and cultured, the corresponding Selective agar medium of 200ml, ferment 48h after collect thallus, OD600nmIt is 4.0 or so；
(2) microorganism collection (5000rpm is centrifuged 5min, abandons supernatant), clasmatosis is (thin using liquid nitrogen grinding bacterial cell disruption
Born of the same parents, mill three times, the ground crude protein phosphate buffer of 1mL pH7.25 are resuspended, is placed on ice) it is used as thick enzyme
Liquid is for subsequent use；
(3) then to increase microsome extraction process if you need to purifying protein, process is as follows:
A, the yeast crude enzyme liquid being resuspended is centrifuged 10min with 7000g, collects supernatant, then precipitating 5ml TES-B
Buffer (table 6) is washed once, is repeated the above steps primary；
B, the supernatant of collection is divided in ultracentrifugation pipe, ultracentrifugation 25000g/10min；
C, the supernatant after taking centrifugation, 100000g is centrifuged 1h in being divided in ultracentrifugation pipe；
D, exhaust supernatant, and the precipitating of acquisition is resuspended with TEG buffer (table 5) 1-2ml, if resuspension effect is bad, can go
It is ultrasonic in ice-water bath, as pure enzyme solution for subsequent use.
5 TES-B buffer of table and TEG buffer formulation:
| TES-B | Final concentration | TEG | Final concentration |
| Tris-HCL pH 7.4 | 50mmol | Tris-HCL pH 7.4 | 50mmol |
| EDTA | 1mmol | EDTA | 1mmol |
| Sorbitol | 600mmol | Glycerol | 20/30% |
| BSA | 10g/l | Water | |
| Beta -mercaptoethanol | 1.5mmol | ||
| Water |
Step 2: catalysis reaction synthesis HC
Crude enzyme liquid or pure Enzyme catalyzed synthesis HC: catalyst system total volume 2mL (table 6)；Reaction is carried out at 30 DEG C, on shaking table
70rpm slowly shakes, and originates reaction with the addition of NADPH, the addition of corresponding extractant methanol chloroform terminates reaction, it is anti-to obtain catalysis
Answer liquid.
The catalyst system of 6 crude enzyme liquid of table or pure Enzyme catalyzed synthesis HC
| Catalyst system | Final concentration |
| RSA | 0.2μmol |
| Phosphate buffer pH7.25 | 100mmol |
| NADPH | 1mmol |
| Crude enzyme liquid or pure enzyme |
Step 3: catalysate extraction and detection
It takes the catalysis reaction solution of 2mL in separatory funnel, isometric extractant (methanol: chloroform=1:9, volume is added
Than), lower layer organic phase 2mL is taken, drying in 5mL centrifuge tube is fitted into, is redissolved with 1mL methanol solution, centrifuging and taking supernatant mistake
0.22 μm of organic filter membrane carries out HPLC detection into liquid phase bottle.It is analyzed and is produced by 1260 high performance liquid chromatograph of Agilent (HPLC)
Object.
As the result is shown: by above-mentioned extraction albumen crude enzyme liquid or pure enzyme in engineering bacteria HC103 and catalysis reaction is carried out,
It successfully equally is detected two kinds of products of hydrocortisone and 14 α-hydroxylation cortisol in the product, so proving crude enzyme liquid or pure
Enzyme can also carry out catalytic production HC and 14 α-hydroxylation cortisol.
Embodiment 7, using 11 β hydroxylating P450 albumen of curvularia lunata in other saccharomycete it is synthesizing hydrogenated can
Pine and 14 α-hydroxylation cortisol
Present invention success is in Yarrowia lipolytica (PO1G), schizosaccharomyces pombe (CICC1762) and Pichia pastoris
(GS115) CL3-CPR from curvularia lunata is expressed in, CL3-CYP021 gene, to the bacterial strain (being shown in Table 7) of acquisition with de-
Oxygen cortisol (RS) is that substrate carries out catalysis reaction (with embodiment 5), has also successfully synthesized hydrocortisone and its by-product
14 α-hydroxylation cortisol.
Fermentation catalyzes and synthesizes the yield of hydrocortisone in other saccharomycete of table 7
| Bacterial strain name | Genotype | Hydrocortisone (mg/L) | 14 α-hydroxylation cortisol (mg/L) |
| HC151 | CICC32520, pINA1269-CL3-CPR+CL3-CYP021 | 1.03±0.11 | 0.61±0.06 |
| HC152 | CICC1702, pCAD1-CL3-CPR+CL3-CYP021 | 1.06±0.18 | 0.64±0.08 |
| HC153 | GS115, pPIC3.5K-CL3-CPR+CL3-CYP021 | 1.26±0.14 | 0.75±0.06 |
Embodiment 8, saccharomyces cerevisiae engineered yeast HC111-HC115 building and catalyze and synthesize hydrocortisone and 14- α and be hydroxylated
Cortisol
In order to further increase the yield of hydrocortisone, we by the CL3-CPR excavated in curvularia lunata and
CL3-CYP021 is integrated in the multicopy site site rDNA of saccharomyces cerevisiae BY4742.
Bacterium germination saccharomyces cerevisiae (Saccharomyces cerevisiae) BY4742 is incubated overnight in screening and culturing medium out.
Screening and culturing medium composition is as follows: SD-Trp (general Jino, Beijing (functional genome) Science and Technology Ltd.), 2% glucose,
0.005%His., 0.01%Leu., 0.01%Ura. (each percentage sign indicates g/100mL).Take 1ml (OD about 0.6-1.0) point
Be attached in 1.5ml EP pipe, 4 DEG C, 10000g be centrifuged 1min, abandon supernatant, precipitating washed with sterile water (4 DEG C), under similarity condition from
The heart abandons supernatant.1ml treatment fluid (10mM LiAc is added in thallus；10mM DTT；0.6M sorbierite；10mM Tris-HCl
(pH7.5), DTT is just added when treatment fluid uses), 20min is placed at 25 DEG C.Centrifugation abandons supernatant, 1ml 1M sorb is added in thallus
Alcohol (0.22 μm of water system film crosses film degerming) is resuspended, centrifugation, abandons supernatant (being resuspended with 1M sorbierite secondary), is about 90 to final volume
μl.It is separately added into the segment pPgk-CL3-CPR-ADHt, pTEF-CL3-CYP021-CYC1t and homology arm of the acquisition of embodiment 1
Marker segment rDNA-Leu-up (SEQ ID No.11；The homology arm segment includes rDNA site upstream about 500bp homologous region
Domain, Leu marker gene and the homology region Pgk promoter 400bp), rDNA-Leu-down (SEQ ID No.12；This is same
Source arm segment includes the homology region CYC1 terminator 200bp and the site the rDNA downstream homology region about 500bp) (realizing will
CL3-CPR, CL3-CYP021 genetic fragment are integrated in the site rDNA of saccharomyces cerevisiae BY4742) each 1 μ l, electricity is transferred to after mixing
In revolving cup, 1ml 1M sorbierite is added, and 30 DEG C of recovery 1h are coated on solid selection medium plate and (match in 2.7kv electric shock 5.6ms
Side: yeast solid screening and culturing medium SD-Leu-Trp, 2% glucose, 0.005%His, 0.01%Ura, 1.5% agar；Each hundred
Branch indicates g/100mL).The condition of screening and culturing are as follows: 30 DEG C, cultivate 36h or more.PCR identifies correct positive colony,
Five clones are selected at random, are respectively designated as bacterial strain HC111, HC112, HC113, HC114, HC115.The wherein highest bacterium of yield
The hydrocortisone yield of strain HC113, which can achieve 6.42mg/L, 14- α hydroxylation cortisol yield, can achieve 4.06mg/L.
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>11 B-hydroxylase of steroid and its encoding gene and application in Curvuluria Iunata
<130> GNCLN181613
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 506
<212> PRT
<213> Curvularia lunata
<400> 1
Met Asp Pro Gln Thr Val Gly Leu Val Val Arg Ala Leu Gln Thr Thr
1 5 10 15
Ala Ile Ala Ala Val Leu Leu Ala Val Trp Thr Tyr Val Pro Lys Leu
20 25 30
Gln Leu Asn Ala Gln Leu Arg Lys Leu Pro Ser Leu Thr Pro Glu Gly
35 40 45
Thr Thr Lys Ala Arg Asp Lys Phe Met Ala Ser Ala Arg Lys Leu Tyr
50 55 60
Gln Asp Gly Tyr His Lys Phe Arg Asp Ser Ala Tyr Thr Leu Ile Asn
65 70 75 80
Glu Asn Gly Asn Ala Asn Val Ile Val Pro Pro Gln Phe Leu Pro Glu
85 90 95
Leu Arg Gln Leu Pro Asp Asp Val Leu Ser Phe Pro Glu Ala Leu Thr
100 105 110
Glu Asp Leu Glu Ile Lys Tyr Thr His Leu Ser Ile Glu Asn Pro Thr
115 120 125
Ala Ala Gly Leu Ile Lys Lys Lys Leu Thr Pro Ala Leu Pro Arg Leu
130 135 140
Asn Pro Ser Ile Cys Gln Asp Val Asp Arg Ala Val Lys Thr Tyr Met
145 150 155 160
Pro Pro Cys Asp Asp Trp Thr Glu Val Asn Ile Asn Glu Lys Leu Leu
165 170 175
Arg Ile Val Ala Lys Val Ser Gly Thr Ile Phe Val Gly Pro Glu Leu
180 185 190
Ala Ser Asp Ser Asp Tyr Leu Asp Ala Ala Cys Phe Tyr Thr Val Asp
195 200 205
Leu Met Asn Ala Val Thr Ala Met Lys Lys Ile Arg Pro Trp Leu Lys
210 215 220
Pro Phe Leu Ala Ser Arg Thr Pro Glu Ile Ile Ala Leu Arg Ala Arg
225 230 235 240
Glu Lys His Thr Glu Arg Val Leu Ile Pro Ile Val Glu Gln Arg Ile
245 250 255
Ala Ala Lys Ala Asn Asp Pro Asn Trp Gln Glu Pro Asp Asp Phe Leu
260 265 270
Gln Trp Met Leu Asp Met Arg Glu Gly Thr Glu Ser Ile Gln Glu Leu
275 280 285
Ala Lys Thr Gln Leu Ser Leu Ile Phe Ala Ala Ile His Thr Thr Thr
290 295 300
Met Thr Val Thr Asn Thr Met Tyr Thr Leu Ala Ala Met Pro Glu Tyr
305 310 315 320
Leu Glu Pro Leu Arg Glu Glu Ile Arg Asn Val Met Leu Asp Glu Gly
325 330 335
Gly Val Ile Thr Ser Arg Ala Leu Gln Arg Met Glu Lys Leu Asp Ser
340 345 350
Tyr Met Lys Glu Val Leu Arg Phe Thr Gly Pro Thr Met Thr Ser Phe
355 360 365
Thr Arg Arg Ala Arg Lys Gly Ile Thr Leu Ser Asn Gly Gln Tyr Ile
370 375 380
Pro Ala Gly Val Ile Ile Glu Val Pro Ser Ala Ala Ile Tyr Gln Asp
385 390 395 400
Asn Ala Phe Tyr Pro Ser Ser Asp Ser Phe Asp Gly Phe Arg Ala Tyr
405 410 415
Lys Ala Arg Ser Thr Gly Lys Ala Ala Asp Ile Ala Arg Asn Gln Phe
420 425 430
Val Thr Ser Asn Glu Glu Asn Leu Thr Phe Gly Tyr Gly Arg His Ala
435 440 445
Cys Pro Gly Arg Phe Phe Ala Ala Asn Glu Ile Lys Met Met Ile Thr
450 455 460
Arg Leu Ile Leu Asp Tyr Asp Ile Lys Met Pro Asn Gly Glu Lys Glu
465 470 475 480
Arg Tyr Pro Gln Ile Glu Ile Gly Lys Met Ser Ile Pro Asp Pro Thr
485 490 495
Lys Thr Leu Ala Phe Lys Arg Val Val Val
500 505
<210> 2
<211> 696
<212> PRT
<213> Curvularia lunata
<400> 2
Met Ala Gln Leu Asp Thr Leu Asp Ile Ile Val Leu Ala Val Leu Leu
1 5 10 15
Val Gly Thr Val Ala Tyr Phe Thr Lys Gly Thr Tyr Trp Ala Val Ser
20 25 30
Ala Asp Pro Tyr Gly Ser Ser Leu Ala Thr Ala Asn Gly Ala Ala Lys
35 40 45
Ala Gly Lys Ser Arg Asn Ile Ile Glu Lys Met Asp Glu Thr Asp Lys
50 55 60
Asn Cys Val Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Asp Tyr
65 70 75 80
Ala Ser Arg Ile Ser Lys Glu Gly His Ser Arg Phe Gly Leu Lys Thr
85 90 95
Met Val Ala Asp Leu Glu Glu Tyr Asp Tyr Asp Asn Leu Asp Ala Phe
100 105 110
Pro Glu Asp Lys Leu Ala Val Phe Val Leu Ala Thr Tyr Gly Glu Gly
115 120 125
Glu Pro Thr Asp Asn Ala Val Glu Phe Tyr Glu Phe Ile Gly Ser Glu
130 135 140
Asp Ile Ser Phe Ser Gln Gly Gly Gly Ile Asp Asp Lys Pro Leu Ser
145 150 155 160
Asn Leu Asn Tyr Val Thr Phe Gly Leu Gly Asn Asn Thr Tyr Glu His
165 170 175
Tyr Asn Ser Met Val Arg Asn Val Asp Lys Tyr Leu Thr Arg Leu Gly
180 185 190
Ala Lys Arg Leu Gly Ala Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr
195 200 205
Met Glu Glu Asp Phe Leu Ala Trp Lys Glu Pro Met Trp Ala Ala Val
210 215 220
Ala Glu Lys Met Gly Leu Glu Glu Arg Glu Ala Met Tyr Glu Pro Val
225 230 235 240
Phe Glu Val Thr Glu Lys Pro Glu Leu Ser Pro Glu Asp Asp Thr Val
245 250 255
Tyr Leu Gly Glu Pro Asn Lys Asn His Leu Glu Gly Asn Gln Lys Gly
260 265 270
Pro Phe Asn Ala Asn Asn Pro Phe Ile Ala Pro Ile Val Glu Ser Ala
275 280 285
Glu Leu Phe Lys Asp Ser Asp Arg Asn Cys Leu His Met Glu Ile Ser
290 295 300
Ile Ala Gly Ser Asn Leu Ser Tyr Thr Thr Gly Asp His Ile Ala Ile
305 310 315 320
Trp Pro Thr Asn Ala Gly Lys Glu Val Asp Arg Leu Phe Lys Val Leu
325 330 335
Gly Lys Glu Asp Lys Arg His Thr Val Ile Ser Val Arg Gly Leu Asp
340 345 350
Pro Thr Ala Lys Val Pro Phe Pro Ser Pro Thr Thr Tyr Asp Ala Ala
355 360 365
Leu Arg Tyr His Ile Glu Ile Asn Ala Ala Val Ser Arg Gln Leu Val
370 375 380
Ser Val Val Ala Gln Phe Ala Pro Asn Glu Asp Ile Lys Ala Glu Ile
385 390 395 400
Val Lys Leu Gly Gly Asp Lys Asp Tyr Phe Lys Glu Gln Val Thr Asp
405 410 415
Arg Asn Leu Asn Leu Gly Gln Leu Leu Glu Ile Thr Gly Lys Gly Ala
420 425 430
Thr Trp Asp Lys Ile Pro Phe Ser Phe Leu Phe Glu Thr Met Val Lys
435 440 445
Ile Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu Val Gln Lys
450 455 460
Asp Lys Ile Ser Ile Thr Ala Val Val Glu Ser Ile Glu Lys Pro Gly
465 470 475 480
Ala Pro Tyr Ala Leu Lys Gly Val Thr Thr Asn Tyr Leu Leu Ala Leu
485 490 495
Lys Gln Lys Gln His Gly Asp Pro Asn Pro Asp Pro His Gly Leu Ser
500 505 510
Tyr Ser Ile Thr Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Val Pro
515 520 525
Val His Val Arg His Ser Asn Phe Lys Leu Pro Ser Asp Pro Ser Lys
530 535 540
Pro Ile Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly
545 550 555 560
Phe Val Gln Glu Arg Ala Ala Gln Ala Lys Ala Gly Gln Asn Val Gly
565 570 575
Lys Thr Val Leu Phe Phe Gly Cys Arg Lys Gln Ser Glu Asp Phe Met
580 585 590
Tyr Ala Asp Glu Trp Lys Gln Tyr Gln Gln Asp Leu Gly Asp Lys Phe
595 600 605
Glu Met His Thr Ala Phe Ser Arg Asp Gly Pro Gln Lys Val Tyr Val
610 615 620
Gln His Lys Leu Glu Glu Asn Gly Glu Glu Val Asn Arg Leu Leu Glu
625 630 635 640
Gln Lys Ala Tyr Phe Tyr Val Cys Gly Asp Ala Ala His Met Ala Arg
645 650 655
Glu Val Asn Thr Leu Leu Gly Lys Ile Ile Ala Lys Tyr Arg Asn Val
660 665 670
Ser Glu Thr Lys Gly Glu Glu Ile Val Lys Ala Met Arg Ala Ser Asn
675 680 685
Gln Tyr Gln Glu Asp Val Trp Ser
690 695
<210> 3
<211> 1521
<212> DNA
<213> Artificial sequence
<400> 3
atggatccac aaactgttgg tttggttgtt agagctttac aaactacagc aattgctgca 60
gttttgttag ctgtttggac ttacgttcca aagttgcaat tgaacgcaca attgagaaag 120
ttgccatctt tgacaccaga aggtactaca aaagctcgtg ataagtttat ggcttctgca 180
agaaagttgt accaagatgg ttaccataag tttagagatt cagcttatac tttgattaat 240
gaaaacggta acgcaaacgt tatcgttcca ccacaatttt taccagaatt gagacaatta 300
ccagatgatg ttttgtcttt tccagaagct ttgactgaag atttggaaat taaatacaca 360
catttgtcaa tcgaaaatcc aactgctgca ggtttgatta aaaagaaatt gactccagct 420
ttgccaagat tgaacccatc tatctgtcaa gatgttgata gagcagttaa aacttacatg 480
ccaccatgtg atgattggac agaagttaac atcaacgaaa agttgttgag aatcgttgct 540
aaagtttcag gtacaatctt tgttggtcca gaattggcat ctgattcaga ttatttggat 600
gctgcatgtt tttacacagt tgatttgatg aatgctgtta ctgcaatgaa gaaaattaga 660
ccatggttga agccattttt agcttcaaga actccagaaa tcattgcttt gagagcaaga 720
gaaaagcata cagaaagagt tttgatccca atcgttgaac aaagaattgc tgcaaaagct 780
aatgatccaa attggcaaga accagatgat ttcttgcaat ggatgttaga tatgagagaa 840
ggtactgaat ctatccaaga attggcaaag acacaattgt cattgatctt cgctgcaatc 900
catactacaa ctatgacagt tactaacaca atgtacactt tggctgcaat gccagaatac 960
ttggaaccat tgagagaaga aatcagaaac gttatgttag atgaaggtgg tgttattaca 1020
tctagagctt tgcaaagaat ggaaaagttg gattcataca tgaaggaagt tttgagattc 1080
actggtccaa ctatgacatc ttttactaga agagctagaa agggtatcac attgtcaaac 1140
ggtcaataca ttccagcagg tgttattatt gaagttccat ctgctgcaat ctatcaagat 1200
aatgcttttt acccatcttc agattctttc gatggtttta gagcttacaa agcaagatca 1260
actggtaaag ctgcagatat tgctagaaac caattcgtta cttcaaacga agaaaatttg 1320
acatttggtt acggtagaca tgcatgtcca ggtagatttt tcgctgcaaa cgaaattaaa 1380
atgatgatta caagattgat cttggattac gatatcaaga tgccaaatgg tgaaaaggaa 1440
agatacccac aaatcgaaat cggtaaaatg tctatcccag atccaactaa aacattagct 1500
tttaaaagag ttgttgttta a 1521
<210> 4
<211> 2091
<212> DNA
<213> Artificial sequence
<400> 4
atggctcaat tggatacttt ggatatcatc gttttagcag ttttgttggt tggtacagtt 60
gcttacttca ctaagggtac atactgggct gtttctgcag atccatatgg ttcttcattg 120
gctactgcaa atggtgctgc aaaagctggt aaatcaagaa acatcatcga aaagatggat 180
gaaacagata agaactgtgt tgttttctat ggttctcaaa ctggtacagc tgaagattac 240
gcatctagaa tctcaaagga aggtcattca agatttggtt tgaaaactat ggttgcagat 300
ttggaagaat acgattacga taatttggat gctttcccag aggataagtt ggctgttttc 360
gttttggcaa cttatggtga aggtgaacca acagataacg ctgttgaatt ctacgaattc 420
attggttctg aagatatctc tttttcacaa ggtggtggta tcgatgataa gccattgtca 480
aatttgaatt atgttacttt tggtttgggt aacaacacat acgaacatta caactctatg 540
gttagaaacg ttgataagta cttgactaga ttaggtgcta agagattggg tgctgcaggt 600
gaaggtgacg atggtgcagg tacaatggaa gaagatttct tggcttggaa agaaccaatg 660
tgggctgcag ttgcagaaaa gatgggttta gaagaaagag aagctatgta cgaaccagtt 720
ttcgaagtta ctgaaaagcc agaattgtct ccagaagatg atacagttta cttgggtgaa 780
ccaaataaga accatttgga aggtaaccaa aagggtcctt ttaatgctaa caaccctttt 840
attgcaccaa tcgttgaatc tgctgaattg tttaaagatt cagatagaaa ctgtttacat 900
atggaaatct caatcgctgg ttctaatttg tcatatacta caggtgacca tattgcaatt 960
tggccaacta atgctggtaa agaagttgat agattgttta aagttttggg taaagaagat 1020
aaaagacata ctgttatttc tgttagaggt ttggatccaa cagctaaagt tccatttcca 1080
tcaccaacta catatgatgc tgcattgaga taccatattg aaattaatgc tgcagtttct 1140
agacaattag tttcagttgt tgctcaattt gcaccaaacg aagatatcaa ggctgaaatc 1200
gttaaattgg gtggtgacaa ggattacttc aaggaacaag ttacagatag aaatttgaat 1260
ttgggtcaat tgttggaaat tactggtaaa ggtgcaacat gggataagat cccattttca 1320
tttttgttcg aaactatggt taagatccaa ccaagatact actcaatctc ttcatcttca 1380
ttggttcaaa aggataagat ctctatcaca gctgttgttg aatcaatcga aaagccaggt 1440
gctccatacg cattgaaggg tgttactaca aactacttgt tggcattgaa gcaaaagcaa 1500
catggtgacc caaatccaga tccacatggt ttgtcttact caatcactgg tccaagaaat 1560
aagtacgatg gtattcatgt tccagttcat gttagacatt ctaacttcaa gttgccatct 1620
gatccatcaa aaccaattat tatggttggt ccaggtactg gtgttgctcc ttttagaggt 1680
tttgttcaag aaagagctgc acaagctaaa gcaggtcaaa acgttggtaa aacagttttg 1740
tttttcggtt gtagaaagca atctgaagat ttcatgtacg ctgatgaatg gaagcaatac 1800
caacaagatt tgggtgacaa gttcgaaatg catacagcat tttcaagaga tggtccacaa 1860
aaggtttacg ttcaacataa gttggaagaa aacggtgaag aagttaacag attgttggaa 1920
caaaaggcat acttttacgt ttgtggtgac gctgcacata tggctagaga agttaatact 1980
ttgttgggta aaatcatcgc aaagtacaga aacgtttctg aaacaaaggg tgaagaaatc 2040
gttaaggcta tgagagcatc taatcaatac caagaagatg tttggtcata a 2091
<210> 5
<211> 1521
<212> DNA
<213> Curvularia lunata
<400> 5
atggatcccc agactgtcgg gctggtagtg cgtgcgctgc agacgacagc catagccgct 60
gtcctccttg cggtatggac ctatgtcccc aaactacaac tcaatgccca gcttcgaaaa 120
ctgccttctt tgactcctga aggaactaca aaagcaagag acaagttcat ggcttcggcc 180
aggaagttgt atcaagacgg gtaccacaag ttcagagata gtgcctacac attgatcaat 240
gagaacggca acgcaaacgt cattgtccct ccgcaatttc tacccgaact acgacaacta 300
cccgacgatg ttctcagttt tccagaggct cttaccgagg acttggaaat caaatacaca 360
catctatcga ttgaaaaccc cacagcggcc ggactgatta aaaaaaagct tacgcccgcg 420
cttcctcgct tgaacccttc catctgccag gatgtcgaca gggcagtcaa gacgtatatg 480
ccgccatgtg acgactggac cgaagtcaac atcaacgaga aactcctccg tatcgttgcc 540
aaggtctctg gtaccatctt cgtcggcccc gaactagcca gcgattccga ctacctcgat 600
gccgcgtgct tctacacggt tgatctcatg aatgccgtca ctgccatgaa gaagattcgt 660
ccctggctga aacccttcct tgcgtcacgc accccggaaa tcattgccct tcgtgcacgc 720
gagaagcaca ctgagcgtgt cctgatcccc attgttgaac aacgcattgc ggccaaggcc 780
aacgacccca actggcagga gccagatgac tttctgcaat ggatgctcga tatgcgcgaa 840
ggcacggagt ctattcaaga gctcgccaaa acccagcttt ctctgatctt cgccgccatt 900
cacacgacta ctatgactgt cacaaatacg atgtacacgc ttgctgccat gccagagtac 960
ctggagccgc ttcgcgagga gatccgcaat gtcatgctcg atgaaggtgg tgttattacg 1020
tcccgtgcct tgcaaagaat ggagaagctg gatagttata tgaaggaggt tttgcgattc 1080
accggaccta ccatgacatc ctttacccgc cgtgcccgta aaggcatcac cctcagcaat 1140
ggtcaataca ttcctgctgg cgtcatcatt gaagtcccct ccgccgccat ctaccaagac 1200
aacgcgttct acccttcgtc cgactccttc gacggcttcc gcgcctacaa ggcgcgctcc 1260
accggaaaag ccgcagacat tgcccgcaac caattcgtca cttccaacga ggagaacctc 1320
acgtttggct acggacgaca cgcctgccca ggacgcttct ttgctgccaa cgaaatcaag 1380
atgatgatca ccaggcttat ccttgattac gacatcaaga tgccaaatgg tgagaaggag 1440
agataccctc agattgaaat cggcaagatg tccattccgg atccgacaaa gacgttggct 1500
ttcaagagag tggtagtgta g 1521
<210> 6
<211> 2091
<212> DNA
<213> Curvularia lunata
<400> 6
atggcacaac tcgacacgct cgacatcatc gtgctggccg tcctcctggt cggcacggtc 60
gcctacttca caaagggtac ctactgggcc gtctcagctg acccgtacgg cagctcgctg 120
gccacggcca atggcgctgc taaggcgggc aagtcccgca acatcataga gaagatggac 180
gagacggaca agaactgcgt cgtcttctac ggcagtcaga cgggcacggc cgaggactat 240
gcctcccgca tctccaagga gggtcattcg cgatttggtc tcaagacaat ggttgccgat 300
ctcgaggagt acgactacga caatctcgac gccttccccg aagacaagct tgccgtcttt 360
gtcctggcca cctatggcga gggcgaaccc accgacaacg ccgtagagtt ctacgagttc 420
attggctccg aagacatctc cttctcccag ggcggtggca tcgacgacaa gcccctcagc 480
aacctcaact acgtcacttt cggtctcggc aacaacacct acgaacacta caactccatg 540
gtccgcaacg tcgacaagta cctgacaagg ctcggtgcga agcgactggg tgctgctggc 600
gaaggtgacg acggtgcggg aacaatggaa gaagacttcc tcgcctggaa ggagcctatg 660
tgggccgccg tggcagagaa gatgggcttg gaagagcgtg aggccatgta cgagcctgtc 720
ttcgaagtca ccgagaagcc agaactctct cctgaggatg acaccgtgta tctgggcgag 780
cccaacaaga accatctcga gggaaaccaa aagggcccct tcaacgccaa caaccccttc 840
attgccccca ttgtcgaatc cgccgagctc ttcaaggact ctgaccgaaa ctgcctgcac 900
atggaaatca gcattgctgg ctccaacctg tcctatacca ctggtgatca cattgctatc 960
tggcctacca acgctggcaa ggaagtcgat cgtttgttca aggtcctcgg caaagaggac 1020
aagcgtcaca cagtcatctc cgtccgaggc ctcgacccta ctgccaaggt tcccttccct 1080
tcgccaacta cctacgatgc cgccctccgc taccacattg agatcaacgc cgccgtgtcc 1140
cgccaacttg tatctgtcgt tgctcagttt gctccaaatg aagacatcaa ggctgaaatc 1200
gtaaagctcg gtggtgataa ggactacttc aaggaacagg tcaccgatcg caacctcaac 1260
ctgggacaac tccttgaaat caccggaaag ggcgcgactt gggacaagat tcctttctca 1320
ttcctgtttg agaccatggt caagatccag ccccgttact actcaatctc ctcgtcgtcc 1380
ctggttcaga aagacaagat ctccatcact gccgtcgtcg agtccattga gaaacctggc 1440
gcgccatatg ctctcaaagg tgtcactacc aactatctgc tcgccctgaa gcagaagcag 1500
cacggagacc ccaacccaga tcctcatggt ttgagctact ctatcactgg tccacgcaac 1560
aagtacgatg gtatccacgt tcccgttcat gtccgccact ccaacttcaa acttccttcg 1620
gatccatcaa agcccatcat catggttggc cctggtaccg gtgttgcacc cttccgtggc 1680
tttgtccaag agcgagctgc gcaagccaag gctggtcaaa acgttggcaa gactgtcttg 1740
ttcttcggat gcaggaagca gtcggaagac ttcatgtacg ccgacgaatg gaagcaatac 1800
caacaagacc tgggtgacaa attcgaaatg cataccgctt tctcccgaga cggcccccaa 1860
aaggtctacg ttcagcacaa gctcgaagag aacggcgaag aggtcaaccg acttctcgag 1920
caaaaggctt acttctacgt ctgcggtgac gccgctcaca tggcccgcga ggtaaacact 1980
ttgctcggca agattatcgc caagtaccga aacgtgtcgg aaaccaaggg cgaggaaatt 2040
gtaaaggcaa tgagagcttc gaaccaatac caggaagacg tctggtcatg a 2091
<210> 7
<211> 3111
<212> DNA
<213> Artificial sequence
<400> 7
ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccttaatt 60
aaacgcacag atattataac atctgcacaa taggcatttg caagaattac tcgtgagtaa 120
ggaaagagtg aggaactatc gcatacctgc atttaaagat gccgatttgg gcgcgaatcc 180
tttattttgg cttcaccctc atactattat cagggccaga aaaaggaagt gtttccctcc 240
ttcttgaatt gatgttaccc tcataaagca cgtggcctct tatcgagaaa gaaattaccg 300
tcgctcgtga tttgtttgca aaaagaacaa aactgaaaaa acccagacac gctcgacttc 360
ctgtcttcct attgattgca gcttccaatt tcgtcacaca acaaggtcct agcgacggct 420
cacaggtttt gtaacaagca atcgaaggtt ctggaatggc gggaaagggt ttagtaccac 480
atgctatgat gcccactgtg atctccagag caaagttcgt tcgatcgtac tgttactctc 540
tctctttcaa acagaattgt ccgaatcgtg tgacaacaac agcctgttct cacacactct 600
tttcttctaa ccaagggggt ggtttagttt agtagaacct cgtgaaactt acatttacat 660
atatataaac ttgcataaat tggtcaatgc aagaaataca tatttggtct tttctaattc 720
gtagtttttc aagttcttag atgctttctt tttctctttt ttacagatca tcaaggaagt 780
aattatctac tttttacaac aaatataaaa caatggcaca actcgacacg ctcgacatca 840
tcgtgctggc cgtcctcctg gtcggcacgg tcgcctactt cacaaagggt acctactggg 900
ccgtctcagc tgacccgtac ggcagctcgc tggccacggc caatggcgct gctaaggcgg 960
gcaagtcccg caacatcata gagaagatgg acgagacgga caagaactgc gtcgtcttct 1020
acggcagtca gacgggcacg gccgaggact atgcctcccg catctccaag gagggtcatt 1080
cgcgatttgg tctcaagaca atggttgccg atctcgagga gtacgactac gacaatctcg 1140
acgccttccc cgaagacaag cttgccgtct ttgtcctggc cacctatggc gagggcgaac 1200
ccaccgacaa cgccgtagag ttctacgagt tcattggctc cgaagacatc tccttctccc 1260
agggcggtgg catcgacgac aagcccctca gcaacctcaa ctacgtcact ttcggtctcg 1320
gcaacaacac ctacgaacac tacaactcca tggtccgcaa cgtcgacaag tacctgacaa 1380
ggctcggtgc gaagcgactg ggtgctgctg gcgaaggtga cgacggtgcg ggaacaatgg 1440
aagaagactt cctcgcctgg aaggagccta tgtgggccgc cgtggcagag aagatgggct 1500
tggaagagcg tgaggccatg tacgagcctg tcttcgaagt caccgagaag ccagaactct 1560
ctcctgagga tgacaccgtg tatctgggcg agcccaacaa gaaccatctc gagggaaacc 1620
aaaagggccc cttcaacgcc aacaacccct tcattgcccc cattgtcgaa tccgccgagc 1680
tcttcaagga ctctgaccga aactgcctgc acatggaaat cagcattgct ggctccaacc 1740
tgtcctatac cactggtgat cacattgcta tctggcctac caacgctggc aaggaagtcg 1800
atcgtttgtt caaggtcctc ggcaaagagg acaagcgtca cacagtcatc tccgtccgag 1860
gcctcgaccc tactgccaag gttcccttcc cttcgccaac tacctacgat gccgccctcc 1920
gctaccacat tgagatcaac gccgccgtgt cccgccaact tgtatctgtc gttgctcagt 1980
ttgctccaaa tgaagacatc aaggctgaaa tcgtaaagct cggtggtgat aaggactact 2040
tcaaggaaca ggtcaccgat cgcaacctca acctgggaca actccttgaa atcaccggaa 2100
agggcgcgac ttgggacaag attcctttct cattcctgtt tgagaccatg gtcaagatcc 2160
agccccgtta ctactcaatc tcctcgtcgt ccctggttca gaaagacaag atctccatca 2220
ctgccgtcgt cgagtccatt gagaaacctg gcgcgccata tgctctcaaa ggtgtcacta 2280
ccaactatct gctcgccctg aagcagaagc agcacggaga ccccaaccca gatcctcatg 2340
gtttgagcta ctctatcact ggtccacgca acaagtacga tggtatccac gttcccgttc 2400
atgtccgcca ctccaacttc aaacttcctt cggatccatc aaagcccatc atcatggttg 2460
gccctggtac cggtgttgca cccttccgtg gctttgtcca agagcgagct gcgcaagcca 2520
aggctggtca aaacgttggc aagactgtct tgttcttcgg atgcaggaag cagtcggaag 2580
acttcatgta cgccgacgaa tggaagcaat accaacaaga cctgggtgac aaattcgaaa 2640
tgcataccgc tttctcccga gacggccccc aaaaggtcta cgttcagcac aagctcgaag 2700
agaacggcga agaggtcaac cgacttctcg agcaaaaggc ttacttctac gtctgcggtg 2760
acgccgctca catggcccgc gaggtaaaca ctttgctcgg caagattatc gccaagtacc 2820
gaaacgtgtc ggaaaccaag ggcgaggaaa ttgtaaaggc aatgagagct tcgaaccaat 2880
accaggaaga cgtctggtca tgaagttata aaaaaaataa gtgtatacaa attttaaagt 2940
gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg 3000
ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cggcatgccg 3060
attaattaaa gtgatccccc acacaccata gcttcaaaat gtttctactc c 3111
<210> 8
<211> 2382
<212> DNA
<213> Artificial sequence
<400> 8
ggtatagcat gaggtcgctc ttattgacca cacctctacc ggcatgccga ttaattaaag 60
tgatccccca agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac 120
tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag 180
catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt 240
tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat 300
ttttatcacg tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac 360
ctcccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt 420
tcttgttcta ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta 480
atctaagttt taattacaaa atggatcccc agactgtcgg gctggtagtg cgtgcgctgc 540
agacgacagc catagccgct gtcctccttg cggtatggac ctatgtcccc aaactacaac 600
tcaatgccca gcttcgaaaa ctgccttctt tgactcctga aggaactaca aaagcaagag 660
acaagttcat ggcttcggcc aggaagttgt atcaagacgg gtaccacaag ttcagagata 720
gtgcctacac attgatcaat gagaacggca acgcaaacgt cattgtccct ccgcaatttc 780
tacccgaact acgacaacta cccgacgatg ttctcagttt tccagaggct cttaccgagg 840
acttggaaat caaatacaca catctatcga ttgaaaaccc cacagcggcc ggactgatta 900
aaaaaaagct tacgcccgcg cttcctcgct tgaacccttc catctgccag gatgtcgaca 960
gggcagtcaa gacgtatatg ccgccatgtg acgactggac cgaagtcaac atcaacgaga 1020
aactcctccg tatcgttgcc aaggtctctg gtaccatctt cgtcggcccc gaactagcca 1080
gcgattccga ctacctcgat gccgcgtgct tctacacggt tgatctcatg aatgccgtca 1140
ctgccatgaa gaagattcgt ccctggctga aacccttcct tgcgtcacgc accccggaaa 1200
tcattgccct tcgtgcacgc gagaagcaca ctgagcgtgt cctgatcccc attgttgaac 1260
aacgcattgc ggccaaggcc aacgacccca actggcagga gccagatgac tttctgcaat 1320
ggatgctcga tatgcgcgaa ggcacggagt ctattcaaga gctcgccaaa acccagcttt 1380
ctctgatctt cgccgccatt cacacgacta ctatgactgt cacaaatacg atgtacacgc 1440
ttgctgccat gccagagtac ctggagccgc ttcgcgagga gatccgcaat gtcatgctcg 1500
atgaaggtgg tgttattacg tcccgtgcct tgcaaagaat ggagaagctg gatagttata 1560
tgaaggaggt tttgcgattc accggaccta ccatgacatc ctttacccgc cgtgcccgta 1620
aaggcatcac cctcagcaat ggtcaataca ttcctgctgg cgtcatcatt gaagtcccct 1680
ccgccgccat ctaccaagac aacgcgttct acccttcgtc cgactccttc gacggcttcc 1740
gcgcctacaa ggcgcgctcc accggaaaag ccgcagacat tgcccgcaac caattcgtca 1800
cttccaacga ggagaacctc acgtttggct acggacgaca cgcctgccca ggacgcttct 1860
ttgctgccaa cgaaatcaag atgatgatca ccaggcttat ccttgattac gacatcaaga 1920
tgccaaatgg tgagaaggag agataccctc agattgaaat cggcaagatg tccattccgg 1980
atccgacaaa gacgttggct ttcaagagag tggtagtgta gccgctgatc ctagagggcc 2040
gcatcatgta attagttatg tcacgcttac attcacgccc tccccccaca tccgctctaa 2100
ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt tttatagtta 2160
tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg tacagacgcg 2220
tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg 2280
ctttaatttg caagctgcgg ccctgcatta atgaatcggc caacgcgcca gggttttccc 2340
agtcacgacg ttgtaaaacg acggccagtg aattgtaata cg 2382
<210> 9
<211> 1604
<212> DNA
<213> Artificial sequence
<400> 9
ggaaaagttg taaatattat tggtagtatt cgtttggtaa agtagagggg gtaatttttc 60
ccctttattt tgttcataca ttcttaaatt gctttgcctc tccttttgga aagctatact 120
tcggagcact gttgagcgaa ggctcattag atatattttc tgtcattttc cttaacccaa 180
aaataaggga aagggtccaa aaagcgctcg gacaactgtt gaccgtgatc cgaaggactg 240
gctatacagt gttcacaaaa tagccaagct gaaaataatg tgtagctatg ttcagttagt 300
ttggctagca aagatataaa agcaggtcgg aaatatttat gggcattatt atgcagagca 360
tcaacatgat aaaaaaaaac agttgaatat tccctcaaaa atgtcgaaag ctacatataa 420
ggaacgtgct gctactcatc ctagtcctgt tgctgccaag ctatttaata tcatgcacga 480
aaagcaaaca aacttgtgtg cttcattgga tgttcgtacc accaaggaat tactggagtt 540
agttgaagca ttaggtccca aaatttgttt actaaaaaca catgtggata tcttgactga 600
tttttccatg gagggcacag ttaagccgct aaaggcatta tccgccaagt acaatttttt 660
actcttcgaa gacagaaaat ttgctgacat tggtaataca gtcaaattgc agtactctgc 720
gggtgtatac agaatagcag aatgggcaga cattacgaat gcacacggtg tggtgggccc 780
aggtattgtt agcggtttga agcaggcggc agaagaagta acaaaggaac ctagaggcct 840
tttgatgtta gcagaattgt catgcaaggg ctccctatct actggagaat atactaaggg 900
tactgttgac attgcgaaga gcgacaaaga ttttgttatc ggctttattg ctcaaagaga 960
catgggtgga agagatgaag gttacgattg gttgattatg acacccggtg tgggtttaga 1020
tgacaaggga gacgcattgg gtcaacagta tagaaccgtg gatgatgtgg tctctacagg 1080
atctgacatt attattgttg gaagaggact atttgcaaag ggaagggatg ctaaggtaga 1140
gggtgaacgt tacagaaaag caggctggga agcatatttg agaagatgcg gccagcaaaa 1200
ctaaacgcac agatattata acatctgcac aataggcatt tgcaagaatt actcgtgagt 1260
aaggaaagag tgaggaacta tcgcatacct gcatttaaag atgccgattt gggcgcgaat 1320
cctttatttt ggcttcaccc tcatactatt atcagggcca gaaaaaggaa gtgtttccct 1380
ccttcttgaa ttgatgttac cctcataaag cacgtggcct cttatcgaga aagaaattac 1440
cgtcgctcgt gatttgtttg caaaaagaac aaaactgaaa aaacccagac acgctcgact 1500
tcctgtcttc ctattgattg cagcttccaa tttcgtcaca caacaaggtc ctagcgacgg 1560
ctcacaggtt ttgtaacaag caatcgaagg ttctggaatg gcgg 1604
<210> 10
<211> 499
<212> DNA
<213> Artificial sequence
<400> 10
agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt 60
tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac 120
cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagctgc ggccctgcat 180
taatgaatcg gccaacgcgc aaagaaagtg gaatattcat tcatatcata ttttttctat 240
taactgcctg gtttctttta aattttttat tggttgtcga cttgaacgga gtgacaatat 300
atatatatat atatttaata atgacatcat tatctgtaaa tctgattctt aatgctattc 360
tagttatgta agagtggtcc tttccataaa aaaaaaaaaa aagaaaaaag aattttagga 420
atacaatgca gcttgtaagt aaaatctgga atattcatat cgccacaact tcttatgctt 480
ataaaagcac taatgcctg 499
<210> 11
<211> 2181
<212> DNA
<213> Artificial sequence
<400> 11
tcctctaatc aggttccacc aaacagatac cccggtgttt cacggaatgg tacgtttgat 60
atcgctgatt tgagaggagg ttacacttga agaatcacag tcttgcgacc ggctattcaa 120
caaggcattc ccccaagttt gaattctttg aaatagattg ctattagcta gtaatccacc 180
aaatccttcg ctgctcacca atggaatcgc aagatgccca cgatgagact gttcaggtta 240
aacgcaaaag aaacacactc tgggaatttc ttcccaaatt gtatctctca atacgcatca 300
acccatgtca attaaacacg ctgtatagag actaggcaga tctgacgatc acctagcgac 360
tctctccacc gtttgacgag gccatttaca aaaacataac gaacgacaag cctactcgaa 420
ttcgtttcca aactcttttc gaacttgtct tcaactgctt tcgcatgaag tacctcccaa 480
ctacttttcc tcacacttgt actccatgac taaacccccc ctcccattac aaactaaaat 540
cttactttta ttttcttttg ccctctctgt cgctctgcct taactacgta tttctcgccg 600
agaaaaactt caatttaagc tattctccaa aaatcttagc gtatattttt tttccaaagt 660
gacaggtgcc ccgggtaacc cagttcatgt ctgcccctaa gaagatcgtc gttttgccag 720
gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa gctatttctg 780
atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt gctgctatcg 840
atgctacagg tgttccactt ccagatgagg cgctggaagc ctccaagaag gctgatgccg 900
ttttgttagg tgctgtgggt ggtcctaaat ggggtaccgg tagtgttaga cctgaacaag 960
gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca tgtaactttg 1020
catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa ggtactgact 1080
tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag gaagacgatg 1140
gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa agaatcacaa 1200
gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg tccttggata 1260
aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa accatcaaga 1320
acgaattccc tacattgaag gttcaacatc aattgattga ttctgccgcc atgatcctag 1380
ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt ggtgatatca 1440
tctccgatga agcctccgtt atcccaggtt ccttgggttt gttgccatct gcgtccttgg 1500
cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac ggttctgctc 1560
cagatttgcc aaagaataag gtcaacccta tcgccactat cttgtctgct gcaatgatgt 1620
tgaaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca gttaaaaagg 1680
ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc accgaagtcg 1740
gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta aacgcacaga tattataaca 1800
tctgcacaat aggcatttgc aagaattact cgtgagtaag gaaagagtga ggaactatcg 1860
catacctgca tttaaagatg ccgatttggg cgcgaatcct ttattttggc ttcaccctca 1920
tactattatc agggccagaa aaaggaagtg tttccctcct tcttgaattg atgttaccct 1980
cataaagcac gtggcctctt atcgagaaag aaattaccgt cgctcgtgat ttgtttgcaa 2040
aaagaacaaa actgaaaaaa cccagacacg ctcgacttcc tgtcttccta ttgattgcag 2100
cttccaattt cgtcacacaa caaggtccta gcgacggctc acaggttttg taacaagcaa 2160
tcgaaggttc tggaatggcg g 2181
<210> 12
<211> 705
<212> DNA
<213> Artificial sequence
<400> 12
agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt 60
tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac 120
cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagctgc ggccctgcat 180
taatgaatcg gccaacgcgc ctcactattt tttactgcgg aagcggaagc ggaaaatacg 240
gaaacgcgcg ggaacataca aaacatacaa aatatacctt tctcacacaa gaaatatatg 300
ctacttgcaa aatatcatac caaaaaactt ttcacaaccg aaaccaaaac caacggatat 360
catacattac actaccacca ttcaaacttt actactatcc tcccttcagt ttcccttttt 420
ctgccttttt cggtgacgga aatacgcttc agagacccta aagggaaatc catgccataa 480
caggaaagta acatcccaat gcggactata ccaccccacc acactcctac caataacggt 540
aactattcta tgttttctta ctcctatgtc tattcatctt tcatctgact acctaatact 600
atgcaaaaat gtaaaatcat cacacaaaac ataaacaatc aaaatcagcc atttccgcac 660
cttttcctct gtccactttc aaccgtccct ccaaatgtaa aatgg 705
Claims (10)
1. protein or complete protein, it is characterised in that:
The protein be it is any in following (A1)-(A4) shown in protein:
(A1) amino acid sequence protein as shown in SEQ ID No.1；
(A2) by amino acid sequence defined by (A1) by one or several amino acid residues substitution and/or missing and/or
Addition and protein with the same function；
(A3) have 99% or more, 95% or more, 90% or more, 85% or more with amino acid sequence defined by (A1) or (A2)
Or 80% or more homology and protein with the same function；
(A4) fusion protein obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (A1)-(A3)；
The complete protein is made of a-protein and PROTEIN B；
The a-protein be it is any in as above (A1)-(A4) shown in protein；
The PROTEIN B be it is any in following (B1)-(B4) shown in protein:
(B1) amino acid sequence protein as shown in SEQ ID No.2；
(B2) by amino acid sequence defined by (B1) by one or several amino acid residues substitution and/or missing and/or
Addition and protein with the same function；
(B3) have 99% or more, 95% or more, 90% or more, 85% or more with amino acid sequence defined by (B1) or (B2)
Or 80% or more homology and protein with the same function；
(B4) fusion protein obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (B1)-(B3).
2. nucleic acid molecules or complete nucleic acid molecules, it is characterised in that: the nucleic acid molecules are to encode egg as claimed in claim 1 or 2
The nucleic acid molecules of white matter；
The complete nucleic acid molecules are made of nucleic acid molecules A and nucleic acid molecules B；
The nucleic acid molecules A is the nucleic acid molecules for encoding a-protein described in claims 1 or 2；
The nucleic acid molecules B is the nucleic acid molecules for encoding PROTEIN B described in claims 1 or 2.
3. nucleic acid molecules according to claim 2 or complete nucleic acid molecules, it is characterised in that:
The nucleic acid molecules be it is any in following (a1)-(a3) shown in DNA molecular:
(a1) nucleotide sequence DNA molecular as shown in SEQ ID No.3 or SEQ ID No.5；
(a2) hybridize and encode in (A1)-(A4) described in claim 1 with (a1) DNA molecular limited under strict conditions and appoint
The DNA molecular of protein shown in one；
(a3) with (a1) or (a2) defined by DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or
80% or more homology of person and encode it is any in (A1)-(A4) described in claim 1 shown in protein DNA molecular；
The nucleic acid molecules A be it is any in as above (a1)-(a3) shown in DNA molecular；
The nucleic acid molecules B be it is any in following (b1)-(b3) shown in DNA molecular:
(b1) nucleotide sequence DNA molecular as shown in SEQ ID No.4 or SEQ ID No.6；
(b2) hybridize under strict conditions with (b1) DNA molecular limited and encode the DNA of PROTEIN B described in claim 1
Molecule；
(b3) with (b1) or (b2) defined by DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or
80% or more homology of person and the DNA molecular for encoding PROTEIN B described in claim 1.
4. any one of following biomaterial:
(c1) recombinant vector, for the recombinant vector containing nucleic acid molecules described in Claims 2 or 3；
(c2) expression cassette, for the expression cassette containing nucleic acid molecules described in Claims 2 or 3；
(c3) transgenic cell line, for the transgenic cell line containing nucleic acid molecules described in Claims 2 or 3；
(c4) recombinant bacterium, for the recombinant bacterium containing nucleic acid molecules described in Claims 2 or 3；
(c5) complete recombinant vector is made of recombinant vector A and recombinant vector B；The recombinant vector A is to contain claim 2
Or the recombinant vector of nucleic acid molecules A described in 3；The recombinant vector B is to contain nucleic acid molecules B described in Claims 2 or 3
Recombinant vector；
(c6) complete expression cassette is made of expression cassette A and expression cassette B；The expression cassette A is containing described in Claims 2 or 3
The expression cassette of nucleic acid molecules A；The expression cassette B is the expression cassette containing nucleic acid molecules B described in Claims 2 or 3；
(c7) complete transgenic cell line is made of transgenic cell line A and transgenic cell line B；The transgenic cell line A
For the transgenic cell line containing nucleic acid molecules A described in Claims 2 or 3；The transgenic cell line B is to want containing having the right
Seek the transgenic cell line of nucleic acid molecules B described in 2 or 3；
(c8) complete recombinant bacterium is made of recombinant bacterium A and recombinant bacterium B；The recombinant bacterium A is containing described in Claims 2 or 3
The recombinant bacterium of nucleic acid molecules A；The recombinant bacterium B is the recombinant bacterium containing nucleic acid molecules B described in Claims 2 or 3.
5. a kind of method of building for producing the engineering bacteria of hydrocortisone and/or 14 α-hydroxylation cortisol, including walk as follows
It is rapid: saccharomycete being transformed, it is made to express protein of any of claims 1 or 2 or complete protein, improved yeast
Bacterium is the engineering bacteria for being used to produce hydrocortisone and/or 14 α-hydroxylation cortisol.
6. according to the method described in claim 5, it is characterized by: described method includes following steps: by Claims 2 or 3
The nucleic acid molecules or complete nucleic acid molecules import the saccharomycete, obtain expressing protein described in claim 1 or complete
The recombinant yeast of protein, the as described engineering bacteria.
7. according to the method described in claim 6, it is characterized by: the nucleic acid molecules are by weight described in claim 4
The form of group carrier or the expression cassette imports in the saccharomycete；The complete nucleic acid molecules are by claim 4
The form of the complete recombinant vector or the complete expression cassette imports in the saccharomycete；
And/or
The nucleic acid molecules or the complete nucleic acid molecules be integrated into the genome of the saccharomycete the site Gal7 or
At the site rDNA.
8. the engineering bacteria being prepared using any the method in claim 5-7.
9. protein described in claim 1 or complete protein or nucleic acid molecules described in claim 2 or 3 or complete nucleic acid
Molecule or biomaterial as claimed in claim 4 or engineering bacteria according to any one of claims 8 it is following it is any in application:
(A) hydrocortisone and/or 14 α-hydroxylation cortisol are prepared；
(B) catalysis steroid hormone substance carries out 11 β hydroxylatings and/or 14 α hydroxylatings；
Or
Protein described in claim 1 is as the application in steroid hormone substance 11 and/or 14 hydroxylases.
10. a kind of method for preparing hydrocortisone and/or 14 α-hydroxylation cortisol is whole-cell catalysis or enzyme process；
The whole-cell catalysis includes the following steps: to collect thallus to engineering bacteria according to any one of claims 8 progress fermented and cultured
After substrate is added, carry out catalysis reaction, hydrocortisone and/or 14 α-hydroxylation cortisol contained in reaction product；The bottom
Object is that can be catalyzed to generate hydrocortisone and/or can generate 14 by 14 α of steroid-hydroxylase catalysis by 11 B-hydroxylase of steroid
α-hydroxylation cortisol substance；
The enzyme process include the following steps: from engineering bacteria according to any one of claims 8 extract have 11 B-hydroxylase of steroid and/or
The substance of 14 α-hydroxylase activity, then catalysis substrate generation hydrogenation can in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme
Pine and/or 14 α-hydroxylation cortisol；The substrate be can by 11 B-hydroxylase of steroid be catalyzed generate hydrocortisone and/or
The substance of 14 α-hydroxylation cortisol can be generated by 14 α of steroid-hydroxylase catalysis.
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| RU2732611C1 (en) * | 2019-10-14 | 2020-09-21 | Федеральное государственное бюджетное учреждение науки Федеральный исследовательский центр Тюменский научный центр Сибирского отделения Российской академии наук | Cortisol producer |
| CN111748022A (en) * | 2019-03-29 | 2020-10-09 | 中国科学院天津工业生物技术研究所 | Curvularia lunata-derived steroid substance transport protein and coding gene and application thereof |
| CN112608909A (en) * | 2020-11-16 | 2021-04-06 | 天津科技大学 | Construction and application of curvularia lunata steroid 11 beta-hydroxylase CYP5103B6 mutant |
| CN112708602A (en) * | 2019-10-25 | 2021-04-27 | 中国科学院天津工业生物技术研究所 | Dioscorea zingiberensis-derived diosgenin synthesis related protein, coding gene and application |
| CN112852651A (en) * | 2020-11-25 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Method for increasing yield of hydrocortisone produced by saccharomyces cerevisiae biotransformation |
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| CN114907997A (en) * | 2021-02-07 | 2022-08-16 | 中国科学院天津工业生物技术研究所 | Construction and application of diosgenin synthesis strain |
| CN117778342A (en) * | 2024-02-27 | 2024-03-29 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in synthesis of 11 beta-hydroxy steroid compounds |
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| CN111748022B (en) * | 2019-03-29 | 2022-05-31 | 中国科学院天津工业生物技术研究所 | Curvularia lunata-derived steroid substance transport protein and coding gene and application thereof |
| CN111748022A (en) * | 2019-03-29 | 2020-10-09 | 中国科学院天津工业生物技术研究所 | Curvularia lunata-derived steroid substance transport protein and coding gene and application thereof |
| RU2732611C1 (en) * | 2019-10-14 | 2020-09-21 | Федеральное государственное бюджетное учреждение науки Федеральный исследовательский центр Тюменский научный центр Сибирского отделения Российской академии наук | Cortisol producer |
| CN112708602A (en) * | 2019-10-25 | 2021-04-27 | 中国科学院天津工业生物技术研究所 | Dioscorea zingiberensis-derived diosgenin synthesis related protein, coding gene and application |
| CN112708602B (en) * | 2019-10-25 | 2022-04-05 | 中国科学院天津工业生物技术研究所 | Dioscorea zingiberensis-derived diosgenin synthesis related protein, coding gene and application |
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| CN112852651A (en) * | 2020-11-25 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Method for increasing yield of hydrocortisone produced by saccharomyces cerevisiae biotransformation |
| CN112852651B (en) * | 2020-11-25 | 2022-02-18 | 中国科学院天津工业生物技术研究所 | Method for increasing yield of hydrocortisone produced by saccharomyces cerevisiae biotransformation |
| CN114907997A (en) * | 2021-02-07 | 2022-08-16 | 中国科学院天津工业生物技术研究所 | Construction and application of diosgenin synthesis strain |
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| CN114196641B (en) * | 2021-12-14 | 2023-10-03 | 上海交通大学 | Steroid C14 alpha hydroxylase, expression vector, engineering bacteria and application thereof |
| CN117778342A (en) * | 2024-02-27 | 2024-03-29 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in synthesis of 11 beta-hydroxy steroid compounds |
| CN117778342B (en) * | 2024-02-27 | 2024-05-28 | 中国科学院天津工业生物技术研究所 | Carbonyl reductase mutant and application thereof in synthesis of 11 beta-hydroxy steroid compounds |
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