CN109097343B - Steroid 11 beta-hydroxylase in curvularia lunata as well as coding gene and application thereof - Google Patents

Abstract

The invention discloses a steroid 11 beta-hydroxylase in curvularia lunata, and a coding gene and application thereof. The steroid 11 beta-hydroxylase provided by the invention is a protein shown in SEQ ID No.1 (namely CL3-CYP021 protein). The invention also provides a protein set consisting of steroid 11 beta-hydroxylase and the protein shown in SEQ ID No.2 (namely CL3-CPR protein). The invention expresses CL3-CYP021 protein (or CL3-CYP021 and CL3-CPR protein) in heterologous microorganisms to carry out catalytic synthesis of Hydrocortisone (HC) and 14 alpha-hydrocortisone. The invention produces HC by replacing the original filamentous fungi with yeast for biocatalytic fermentation, has the advantages of shortening the fermentation time, simplifying the fermentation conditions and the separation and extraction steps, reducing the production cost and the like, and has important significance for replacing the traditional biocatalytic fermentation production mode of hydrocortisone.

Description

Steroid 11 beta-hydroxylase in curvularia lunata as well as coding gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to steroid 11 beta-hydroxylase in Curvularia lunata, and a coding gene and application thereof.

Background

Hydrocortisone (HC) has a chemical name of 11 beta, 17 alpha, 21-trihydroxy pregn-4-ene-3, 20-dione, is an adrenoglucocorticoid drug, and plays an important role in hormone drugs, and the structural formula of the Hydrocortisone is shown in figure 1. HC can influence sugar metabolism, and has antiviral, antiinflammatory, antiallergic and antishock effects [ Dumas, B., et al, Hydrocortisone amide in yeast: metabolic engineering wells a non-inflammatory micro-organic factory.Biotechnol J,2006.1(3):299-307 ]. The traditional Chinese medicine composition is mainly used for treating diseases caused by adrenal insufficiency, congenital adrenal cortex function hyperplasia and other symptoms, also can be used for treating inflammatory and allergic diseases such as bronchial asthma, rheumatoid arthritis, gout, rheumatic fever and the like, and also can be used for treating severe infection, antishock and the like.

The synthesis methods of HC are mainly divided into three categories: total chemical synthesis, semi-synthesis and total biosynthesis methods [ Colingsworth, D.R., et al. "A partial microbiological synthesis of Hydrocortisone." Journal of Biological Chemistry 203.2(1953):807 ]. In 1950, Wendler et al successfully synthesized HC by 30 chemical reactions using a total chemical synthesis method, but the process was complicated, the total yield was too low, and the method had no industrial production value. Dumas et al [ Szczebra, F.M., et al, Total biosyntheses of hydrocortisone from a simple carbon source in yeast. Nat Biotechnol,2003.21(2):143-149 ] used 11-site beta-hydroxylated P450(CYP11B1) from human source to achieve Total synthesis of hydrocortisone by a biological method using glucose as a sole carbon source, but due to the complex synthetic route, the product yield is low and industrial production is not achieved. At present, the preparation of steroid drugs such as HC is still mainly a semi-synthetic method, i.e. 17 α -hydroxypregn-4-ene-3, 20-dione-21-acetate (RSA) or 11-deoxycorticol (RS) is obtained by chemical Synthesis from precursors such as diosgenin or phytosterol, and then HC is obtained by β -hydroxylation at position 11 by the whole-cell catalysis reaction of microorganisms such as Absidia (Absidia) and Curvularia lunata (Curvularia lunata) (scaffold Synthesis) and Synthesis of α -ketogenic use bound with biochemical gene Synthesis system. journal of biological and biological engineering,2009.108: S45. S.3532. J.application, biological Synthesis of biological and biological Synthesis of biological engineering, biological engineering and biological engineering of biological, 2006.42(5):479-483.]. The synthesis process of hydrocortisone is shown in figure 2.

Curvularia lunata (Curvularia lunata) belongs to the fungi Imperfecti, Aphyllophorales, Pediobolus viridae, Polysporilidae, Curvularia. The microorganism has 11-position beta hydroxylation function and is widely used by domestic and foreign scholars. The P450 protein, which is purified by the expression of P450 protein using RS as a substrate in Curvularia lunata, is considered to be a bifunctional enzyme having both 11-beta hydroxylation and 14-alpha hydroxylation in Curvularia lunata by Kenzi Suzuki et al [ Suzuki, K., et al, Purification and properties of catalysis P-450(P-450lun) catalysis stereo 11 beta-hydroxylation in Curvularia lunata Biophys Acta 1993.1203(2):215-223 ]. Lu et al [ Lu, W., et al, optimization of Hydrocortisone Production by Curvularia lunata Lunat. applied Biochemistry and Biotechnology,2007.142(1):17-28 ] screening for ketoconazole resistant mutants by mutagenesis gave a 40% increase in HC conversion compared to the starting strain CL 114.

The rate of conversion of the substrate RS by Curvularia lunata is superior to that of RSA. However, the bacterium can specifically introduce C11 beta hydroxyl, but the bacterium is also accompanied by the generation of C14 alpha hydroxyl as a byproduct. Pioneer germany (Schering AG) hindered the formation of C14 α hydroxyl by-product via steric structure with the aid of C17 α acetyl compound, to achieve a cortisol yield of about 70%. However, after years of researches, researchers have not successfully excavated the beta-hydroxylated P450 at the 11 th position of the most key substrate in the synthesis process of the hydrocortisone, so that the successful excavation of the protein has important significance for researching the synthesis of the hydrocortisone.

Disclosure of Invention

The invention aims to provide steroid 11 beta-hydroxylase in curvularia lunata, and a coding gene and application thereof.

In a first aspect, the invention claims the following proteins or protein sets.

The protein provided by the invention is a protein shown in any one of (A1) - (A4) as follows:

(A1) the protein with the amino acid sequence shown as SEQ ID No.1 (wherein, the protein shown as SEQ ID No.1 is CL3-CYP021 protein);

(A2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined in (A1) and having the same function;

(A3) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in (A1) or (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

The protein set provided by the invention consists of protein A and protein B.

The protein A is a protein as shown in any one of (A1) to (A4) above.

The protein B is a protein shown in any one of the following (B1) to (B4):

(B1) a protein with an amino acid sequence shown as SEQ ID No.2 (wherein, the protein shown as SEQ ID No.2 is CL3-CPR protein);

(B2) a protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence defined in (B1) and having the same function;

(B3) a protein having a homology of 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more with the amino acid sequence defined in (B1) or (B2) and having the same function;

(B4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (B1) to (B3).

In a second aspect, the invention claims a nucleic acid molecule or a set of nucleic acid molecules.

The nucleic acid molecule provided by the present invention may be a nucleic acid molecule encoding a protein as described in the first aspect hereinbefore (including optimized and non-optimized nucleic acid molecules encoding CL3-CYP021 proteins).

The nucleic acid molecule set provided by the invention consists of a nucleic acid molecule A and a nucleic acid molecule B.

The nucleic acid molecule A is a nucleic acid molecule encoding the protein A in the first aspect (including optimized and non-optimized nucleic acid molecules encoding CL3-CYP021 protein);

the nucleic acid molecule B is a nucleic acid molecule encoding the protein B described in the first aspect above (including optimized and non-optimized nucleic acid molecules encoding CL3-CPR protein).

Further, the nucleic acid molecule may specifically be a DNA molecule as shown in any one of (a1) to (a3) below:

(a1) a DNA molecule with a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No.5 (wherein, SEQ ID No.3 is an encoding gene of optimized CL3-CYP021 protein, and SEQ ID No.5 is an encoding gene of non-optimized CL3-CYP 021);

(a2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (a1) and which encodes a protein as set forth in any one of (A1) to (A4) described above in the first aspect;

(a3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology to the DNA sequence defined in (a1) or (a2) and encoding the protein represented by any one of (A1) to (A4) as defined in the first aspect.

Further, the nucleic acid molecule a may specifically be a DNA molecule as shown in any one of (a1) to (a3) above.

Further, the nucleic acid molecule B may specifically be a DNA molecule shown in any one of (B1) to (B3) below:

(b1) a DNA molecule with a nucleotide sequence shown as SEQ ID No.4 or SEQ ID No.6 (wherein, SEQ ID No.4 is an optimized CL3-CPR protein coding gene, and SEQ ID No.6 is a non-optimized CL3-CPR coding gene);

(b2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes the protein B described in the first aspect above;

(b3) a DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more or 80% or more homology to the DNA sequence defined in (B1) or (B2) and encoding the protein B as defined in the first aspect.

Wherein the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in a solution of 6 XSSC, 0.5% SDS,hybridization was carried out at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In a third aspect, the invention claims any of the following biomaterials:

(c1) a recombinant vector comprising a nucleic acid molecule as described in the second aspect above;

(c2) an expression cassette comprising a nucleic acid molecule as described in the second aspect above;

(c3) a transgenic cell line comprising a nucleic acid molecule as described in the second aspect;

(c4) a recombinant bacterium comprising a nucleic acid molecule as described in the second aspect above;

(c5) the complete set of recombinant vector consists of a recombinant vector A and a recombinant vector B; the recombinant vector A is a recombinant vector containing the nucleic acid molecule A described in the second aspect above; the recombinant vector B is a recombinant vector comprising the nucleic acid molecule B as described in the second aspect above;

(c6) the complete set of expression cassette consists of an expression cassette A and an expression cassette B; the expression cassette A is an expression cassette comprising the nucleic acid molecule A as described in the second aspect above; the expression cassette B is an expression cassette comprising the nucleic acid molecule B as described in the second aspect above;

(c7) the complete set of transgenic cell line consists of a transgenic cell line A and a transgenic cell line B; the transgenic cell line A is a transgenic cell line comprising the nucleic acid molecule A as described above in the second aspect; the transgenic cell line B is a transgenic cell line comprising a nucleic acid molecule B as described in the second aspect above;

(c8) the complete set of recombinant bacteria consists of recombinant bacteria A and recombinant bacteria B; the recombinant bacterium A is a recombinant bacterium containing the nucleic acid molecule A in the second aspect; the recombinant bacterium B is a recombinant bacterium containing the nucleic acid molecule B described in the second aspect.

In a fourth aspect, the invention claims a method for constructing engineering bacteria for producing hydrocortisone and/or 14 alpha-hydrocortisone.

The method for constructing the engineering bacteria for producing the hydrocortisone and/or the 14 alpha-hydroxycortinol, which is provided by the invention, can comprise the following steps: the yeast is modified to express the protein or protein set as described in the first aspect, and the modified yeast is an engineering bacterium for producing hydrocortisone and/or 14 alpha-hydroxycortinol.

Further, the method may comprise the steps of: introducing the nucleic acid molecule or the set of nucleic acid molecules of the second aspect into the yeast to obtain a recombinant yeast expressing the protein or the set of proteins of the first aspect, i.e., the engineered yeast.

Still further, the nucleic acid molecule is introduced into the yeast by the form of the recombinant vector or the recombinant expression cassette as described in the third aspect above; the set of nucleic acid molecules is introduced into the yeast by means of the set of recombinant vectors or the set of expression cassettes described in the third aspect above.

Further, the nucleic acid molecule or the set of nucleic acid molecules is integrated into the genome of the yeast at a common site, such as the Gal7 site or the rDNA site.

Further, the yeast can be Saccharomyces cerevisiae (such as BY4742 strain), yarrowia lipolytica (such as PO1G strain), Schizosaccharomyces pombe (such as CICC1762 strain), or Pichia pastoris (such as GS115 strain), etc.

In one embodiment of the invention, the nucleic acid molecule is introduced into the yeast by means of a recombinant vector; the recombinant vector is obtained by inserting the nucleic acid molecule between enzyme cutting sites (such as SexA1 and Asc1) of pRS426 plasmid.

In another embodiment of the invention, the set of nucleic acid molecules is introduced into the yeast in the form of a set of expression cassettes; the complete set of expression cassette consists of an expression cassette pPgk-CL3-CPR-ADHt and an expression cassette pTEF-CL3-CYP021-CYC1 t; the sequence of the expression cassette pPgk-CL3-CPR-ADHt is SEQ ID No.7 (or the sequence obtained after replacing the 813 th and 2903 th positions of SEQ ID No.7 with SEQ ID No. 4); the sequence of the expression cassette pTEF-CL3-CYP021-CYC1t is SEQ ID No.8 (or the sequence obtained after the 501 st and 2021 st positions of the SEQ ID No.8 are replaced by SEQ ID No. 3). When the expression cassette pPgk-CL3-CPR-ADHt and the expression cassette pTEF-CL3-CYP021-CYC1t are introduced into the yeast, a homologous arm marker fragment Gal7-URA3-up and a homologous arm marker fragment Gal7-URA3-down (the Gal7 site integrated in Saccharomyces cerevisiae BY4742 is realized) are also introduced; the sequence of the homologous arm marker fragment gal7-URA3-up is shown as SEQ ID No. 9; the sequence of the homologous arm marker fragment gal7-URA3-down is shown in SEQ ID No. 10.

In yet another embodiment of the present invention, the set of nucleic acid molecules is introduced into the yeast in the form of a set of expression cassettes; the set of expression cassette consists of the expression cassette pPgk-CL3-CPR-ADHt and the expression cassette pTEF-CL3-CYP021-CYC1 t. When the expression cassette pPgk-CL3-CPR-ADHt and the expression cassette pTEF-CL3-CYP021-CYC1t are introduced into the yeast, a homologous arm marker fragment rDNA-Leu-up and a homologous arm marker fragment rDNA-Leu-down (realizing the rDNA locus integrated in Saccharomyces cerevisiae BY 4742) are also introduced; the sequence of the homologous arm marker fragment rDNA-Leu-up is shown in SEQ ID No. 11; the sequence of the homologous arm marker fragment rDNA-Leu-down is shown in SEQ ID No. 12.

In a fifth aspect, the invention claims an engineered bacterium prepared by the method described in the fourth aspect.

In a specific embodiment of the present invention, the engineering bacteria is specifically any one of the following:

(1) engineering bacteria HC 101: introducing the recombinant vector pRS426-CL3-CYP021 into a Saccharomyces cerevisiae BY4742 strain to obtain a recombinant strain; the recombinant vector pRS426-CL3-CYP021 is a recombinant plasmid obtained by inserting a DNA fragment (namely, an unoptimized CL3-CYP021 gene) shown in SEQ ID No.5 into a restriction enzyme site SexA1 and Asc1 of a pRS426 plasmid.

(2) Engineering bacteria HC 102: and (2) introducing the expression cassette pPgk-CL3-CPR-ADHt (wherein the CL3-CPR gene is a non-optimized gene and is shown as SEQ ID No. 6), the expression cassette pTEF-CL3-CYP021-CYC1t (wherein the CL3-CYP021 gene is a non-optimized gene and is shown as SEQ ID No. 5), the marker fragment Gal7-URA3-up of the homologous arm and the marker fragment Gal7-URA3-down of the homologous arm into a saccharomyces cerevisiae BY4742 strain to obtain a recombinant bacterium (realizing integration at the Gal7 site of the saccharomyces cerevisiae BY 4742).

(3) Engineering bacteria HC 103: and (3) introducing the expression cassette pPgk-CL3-CPR-ADHt (wherein the CL3-CPR gene is an optimized gene and is shown as SEQ ID No. 4), the expression cassette pTEF-CL3-CYP021-CYC1t (wherein the CL3-CYP021 gene is an optimized gene and is shown as SEQ ID No. 3), the marker fragment Gal7-URA3-up of the homologous arm and the marker fragment Gal7-URA3-down of the homologous arm into a saccharomyces cerevisiae BY-4741 strain to obtain a recombinant bacterium (realizing integration at the Gal7 site of the saccharomyces cerevisiae BY 4742).

In a sixth aspect, the invention claims the use of a protein or a protein set as described in the first aspect hereinbefore or a nucleic acid molecule set as described in the second aspect hereinbefore or a biological material as described in the third aspect hereinbefore or an engineered bacterium as described in the fifth aspect hereinbefore in any one of:

(A) preparing hydrocortisone and/or 14 alpha-hydroxycortinol;

(B) catalyzing the beta hydroxylation at the 11 position and/or the alpha hydroxylation at the 14 position of the steroid hormone substances.

The invention also claims the use of a protein as described in the first aspect as an 11-and/or 14-position hydroxylating enzyme in steroid hormones.

Wherein, the steroid hormone substance can be a substance which can be catalyzed by steroid 11 beta-hydroxylase to generate hydrocortisone and/or can be catalyzed by steroid 14 alpha-hydroxylase to generate 14 alpha-hydroxycorticosterol, such as 7 alpha-hydroxypregna-4-ene-3, 20-dione-21-acetate (or called hydrocortisone 21-acetate, Cortexolone-21-acetate, RSA) or 11-deoxycorticosterol (11-deoxycortisol, RS).

In a seventh aspect, the invention claims a process for the preparation of hydrocortisone and/or 14 α -hydroxycortinol.

The method for preparing the hydrocortisone and/or the 14 alpha-hydrocortisone provided by the invention can be a whole-cell catalysis method or an enzyme method.

Wherein the whole-cell catalysis method may comprise the steps of: carrying out fermentation culture on the engineering bacteria in the fifth aspect, collecting bacteria, adding a substrate, and carrying out catalytic reaction, wherein the reaction product contains hydrocortisone and/or 14 alpha-hydroxycortinol; the substrate is a substance capable of being catalysed by a steroid 11 β -hydroxylase to produce hydrocortisone and/or a steroid 14 α -hydroxylase to produce 14 α -hydroxycortinol.

Wherein, the enzyme method comprises the following steps: extracting a substance with the activity of the steroid 11 beta-hydroxylase and/or 14 alpha-hydroxylase from the engineering bacteria in the fifth aspect, and catalyzing a substrate to generate hydrocortisone and/or 14 alpha-hydrocortisone in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme; the substrate is a substance capable of being catalysed by a steroid 11 β -hydroxylase to produce hydrocortisone and/or a steroid 14 α -hydroxylase to produce 14 α -hydroxycortinol.

Further, the substrate may be 17 α -hydroxypregn-4-ene-3, 20-dione-21-acetate (or called desogestrel-21-acetate, Cortexolone-21-acetate, RSA) or 11-deoxycorticosterol (11-deoxycortisol, RS).

In a particular embodiment of the invention, a whole-cell catalysis method is used. Wherein the conditions of the catalytic reaction are specifically 30 ℃ and 200-; the substrate in the reaction system is RSA, and the concentration of the RSA is 10mg-200mg (such as 170 mg/L).

Experiments prove that the CL3-CYP021 protein is expressed in heterologous microorganisms to carry out catalytic synthesis of hydrocortisone and/or 14 alpha-hydrocortisone. The invention provides possibility for producing hydrocortisone by using yeast with food safety, effectively shortens the production period, simplifies the fermentation equipment, can express single protein, improves the product conversion rate, effectively reduces the production and extraction cost, has important significance for the innovation of hydrocortisone production technology, and also provides possibility for synthesizing 14 alpha-hydrocortisone by biological catalysis.

Drawings

FIG. 1 is the structural formula of hydrocortisone.

FIG. 2 shows the production process of hydrocortisone.

FIG. 3 shows the detection and identification of catalytic products of Saccharomyces cerevisiae engineering bacteria HC 101.

FIG. 4 shows the detection and identification (1) of catalytic products of Saccharomyces cerevisiae engineering bacteria HC 102.

FIG. 5 shows the detection and identification (2) of catalytic products of Saccharomyces cerevisiae engineering bacteria HC 102.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Crescent moon fungus AS3.4381(ATCC 12017): a Beina biological product.

(ii) pRS426 plasmid: shanghai North Nuo Biometrics, Inc., under the addgene 0499.

M2 plasmid: a SexA1 cleavage site, a pPgk promoter, a Green Fluorescent Protein (GFP) gene, and terminators ADH1t and Asc1 cleavage sites were inserted in this order into the multiple cloning site of the peasy-Blunt-simple vector (all-alone King Bio Inc.).

M3 plasmid: a SexA1 cleavage site, a pTEF promoter, a Green Fluorescent Protein (GFP) gene, and terminators CYC1t and Asc1 cleavage sites were inserted in this order into the multiple cloning site of the peasy-Blunt-simple vector (all-alone gold organism Co., Ltd.).

Saccharomyces cerevisiae (Saccharomyces cerevisiae) BY 4742: products of Thermo Fisher corporation.

Yarrowia lipolytica (CICC 32520): china center for Industrial microbial culture Collection (CICC) product.

Schizosaccharomyces pombe (CICC 1762): china center for Industrial microbial culture Collection (CICC) product.

Pichia pastoris (GS 115): youbao biological product, product number: ST 1030.

Example 1 cloning and expression of beta-hydroxylase at position 11 of Curvularia lunata

The cloning and expression of the gene are divided into the following 3 steps:

1. extraction of total RNA of Curvularia lunata

Firstly, culturing Curvularia lunata AS3.4381(ATCC12017) on a plate for several days, collecting a certain number of spores, inoculating into 50mL of potato-glucose (PDA) culture medium, and culturing overnight until a large amount of thallus is synthesized; subsequently, curvularia lunata mycelia were collected by centrifugation, washed with potassium Phosphate Buffer (PBS), finally resuspended in 50mL buffer and induced for 2h with the addition of the substrate hydrocortisone 21-acetate (RSA) to a final concentration of 170mg/L, and sampled for RNA extraction.

The RNA extraction method comprises the following steps:

(1) to 2.0mL of nutated tube (RNase free) were added 0.5mm ground beads (filling the bottom of the cone) and 1mL of Trizol, followed by the addition of liquid nitrogen quick-frozen pieces of mycelium (ca. 100 mg).

(2) This was repeated twice using a bead mill (BeadBeater) at maximum speed of 30 s.

(3) Remove the attached nucleosomes by gentle shaking at room temperature for 5 min.

(4) 0.2mL of chloroform was added and the beads were milled at maximum speed for 15 s.

(5) Gently shaken at room temperature for 2 min.

(6) Centrifugation at 12000g for 15min at 4 ℃ took the supernatant (ca. 0.5mL) into a fresh EP tube (RNase free).

(7) An equal volume (0.5mL) of isopropanol was added and mixed well.

(8) After gentle shaking at room temperature for 10min, a white precipitate was visible.

(9) Centrifugation was carried out at 12000g for 10min at 4 ℃ to remove the supernatant.

(10) The precipitate was washed with 1mL of 75% ethanol in DEPC water, centrifuged at 7500g at 4 ℃ for 5min, and the supernatant was removed completely.

(11) The mixture is left at room temperature for about 15-20min to remove ethanol, and the RNA becomes insoluble after being left for a long time.

(12) Adding 50 μ L DEPC water, and performing water bath at 60 deg.C for 10min to help dissolve.

(13) The concentration and purity of RNA were determined by NanoDrop. The ratio of A260/A280 of the RNA sample with better purity is 1.9-2.0, and A260/A230 is usually more than 2.

2. Reverse transcription PCR and gene amplification

First strand reverse transcription-PCR: taking a PCR tube without RNase, and amplifying according to a Thermo reverse transcription kit to obtain cDNA. The specific operation steps are as follows: template Total RNA 3. mu.L, oligo (dT)₁₈mu.L of primer, 1. mu.L of 10mM dNTP Mix, and 10. mu.L of RNase-free water. Performing instant centrifugation, performing PCR at 65 ℃ for 5min, and performing rapid cooling on ice; then adding reaction liquid in the following system: 5 × RT Buffer 4 μ L, Maxima H Minus Enzyme Mix 1 μ L, instant centrifugation, reaction in PCR instrument 50 deg.C 50min, 85 deg.C 5min, 4 deg.C heat preservation.

And (3) PCR amplification: the cDNA obtained by reverse transcription is used as a template, and the target gene is amplified by using the following primers, wherein the amplification system is TAKARA

10. mu.l of HS DNA polymerase, 10. mu.l of Dntp mix, 1. mu.l each of primers (see Table 1), cDNA, 0.5. mu.l of template, 0.5. mu.l of PrimerSTAR HS polymerase (2.5U/. mu.L), and distilled water were added to a total volume of 50. mu.l. Amplification conditions were 98 ℃ pre-denaturation for 2min (1 cycle); denaturation at 98 ℃ for 10 seconds, annealing at 56 ℃ for 15 seconds, and extension at 72 ℃ for 2 minutes (30 cycles); extension at 72 ℃ for 8 min (1 cycle).

The resulting amplification product was named CL3-CYP021, CL 3-CPR. And purifying the obtained PCR amplification product by using a PCR product purification kit of Shanghai biological engineering Co., Ltd, carrying out enzyme digestion on the DNA fragment by using SexA1 and Asc1 of Thermo after purification, and recovering the product for later use.

TABLE 1 CL3-CYP021 and CL3-CPR gene amplification primers

Primer name	Sequence (5 '-3')
		CL3-CYP021-SexA1-F	gcgaccaggtATGGATCCCCAGACTGTCGGGCT
CL3-CYP021-Asc1-R	ggcgcgccCTACACTACCACTCTCTTGAAA
		CL3-CPR-SexA1-F	gcgaccaggtATGGCACAACTCGACACGCTCGA
CL3-CPR-Asc1-R	ggcgcgccTCATGACCAGACGTCTTCCTGGT

The CL3-CYP021 gene (wild type) has a sequence shown in SEQ ID No.5, and codes a protein shown in SEQ ID No.1 (named as CL3-CYP021 protein); the CL3-CPR gene (wild type) has the sequence shown in SEQ ID No.6 and codes the protein shown in SEQ ID No.2 (named CL3-CPR protein).

3. Construction of Gene expression plasmids

Plasmid pRS426 was digested with SexA1 and Asc1 from Thermo, and the digested product gel was recovered for use. Adding a connecting system into each 50ng of the obtained CL3-CYP021 gene fragment: mu.L of 2 Xquick ligation Buffer (NEB), 0.5. mu.L of Quick ligation Buffer (NEB, 400,000 covalent end units/ml), distilled water was added to 10. mu.L, the mixture was reacted at room temperature for 10min to obtain a ligation product, which was transferred to Trans1-T1 competent cells and subjected to ice-bath for 30 min, heat-shock at 42 ℃ for 30 sec, and immediately placed on ice for 2 min. Adding 800 mu l LB culture medium, incubating at 250rpm and 37 ℃ for 1 hour, coating the bacterial liquid on LB plate containing ampicillin, after overnight culture, PCR screening 5 positive single colonies, carrying out liquid culture on positive clones, extracting positive clone plasmids for sequencing verification, wherein sequencing results show that a target fragment is inserted on the vector pRS426 to obtain the plasmid pRS426-CL3-CYP 021.

4. Construction of Yeast PolyGene integration fragments

The plasmids M2 and M3 are digested by SexA1 and Asc1 of Thermo company, and the digested product gel is recovered for later use. And (3) performing connection transformation with the obtained CL3-CPR and CL3-CYP021 gene fragment (the method is the same as the step 3), and selecting clone sequencing verification to obtain plasmids M2-CL3-CPR and M3-CL3-CYP 021. PCR was performed using the constructed plasmid M2-CL3-CPR as a template and using primers 1-M-pEASY-PGK1-F and 1-M-ADHt-TEF1-R (see Table 2) (the same procedure as in step 2), to obtain pPgk-CL3-CPR-ADH1t fragment (SEQ ID No.7) comprising Pgk promoter (positions 63-812 of SEQ ID No.7), Curvularia lunata-derived CL3-CPR gene (position 813-2903 of SEQ ID No.7) and ADH1 terminator (position 2904-3061 of SEQ ID No. 7). PCR was performed using the constructed plasmid M3-CL3-CYP021 as a template and using primers 2-M-ADHt-TEF1-F and M-CYC1t-pEASY-R (see Table 2) (the same procedure as in step 2), to obtain pTEF-CL3-CYP021-CYC1t fragment (SEQ ID No.8) comprising a TEF promoter (1-450 th position of SEQ ID No.8), a C3-CYP 021 gene derived from curvularia lunata (451-1971 th position of SEQ ID No.8), and a CYC1 terminator (1972-2278 th position of SEQ ID No. 8). And performing gel recovery treatment on the target fragment obtained by amplification for later use.

TABLE 2 CL3-CYP021 and CL3-CPR Gene integration fragment amplification primers

Example 2 construction of Saccharomyces cerevisiae engineering bacterium HC101

The starting strain Saccharomyces cerevisiae BY4742 was cultured overnight in the screening medium. The liquid screening medium consisted of: SD-Trp (beijing pan kino (functional genome) science and technology ltd), 2% glucose, 0.005% his, 0.01% leu, 0.01% Ura (each percentage number indicates g/100 mL). 1ml (OD about 0.6-1.0) was taken and dispensed into 1.5ml EP tubes, centrifuged at 4 ℃ at 10000g for 1min, the supernatant was discarded, the precipitate was washed with sterile water (4 ℃), centrifuged under the same conditions, and the supernatant was discarded. The cells were incubated at 25 ℃ for 20min with 1ml of a treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5) added thereto, and the treatment solution was used. After centrifugation, the supernatant was discarded, 1ml of 1M sorbitol (0.22 μ M aqueous membrane filtration sterilization) was added to the cells for resuspension, and the cells were centrifuged, and the supernatant was discarded (resuspended twice with 1M sorbitol) to a final volume of about 90 μ l. The expression plasmid pRS426-CL3-CYP 0211.5. mu.l obtained in example 1 was added thereto, mixed, transferred to an electric cuvette, shocked at 2.7kv for 5.7ms, added with 1mL of 1M sorbitol, thawed at 30 ℃ for 1h, and spread on a solid screening medium (formulation: solid yeast screening medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage indicates g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. The correct positive clone was identified by PCR and was designated strain HC 101.

Example 3 catalytic Synthesis of hydrocortisone and 14 alpha-Hydrocortole by Saccharomyces cerevisiae engineering bacterium HC101

And (3) flask fermentation catalysis: activating HC101 yeast strain in solid selective culture medium (formula: solid yeast screening culture medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage number represents g/100mL), preparing seed solution (30 ℃, 250rpm, 16h) in corresponding liquid selective culture medium (formula: liquid yeast screening culture medium SD-Ura-Trp, 2% glucose, 0.005% His, 0.01% Leu; each percentage number represents g/100mL), inoculating 1mL into 3 bottles of 500mL triangular flasks containing 100mL YPD liquid culture medium, shaking and culturing at 30 ℃, 250rpm for 2 days, collecting yeast cells at 5000rpm, washing with PBS buffer solution, finally suspending in 250mL triangular flasks containing 30mL, adding substrate with final concentration of 170mg/L, performing catalytic reaction, performing 30 RSA, shaking at 250rpm for 2 days.

5mL of the catalyzed reaction solution was placed in a separatory funnel, an equal volume of 1mL of an extractant (methanol: chloroform: 1:9, volume ratio) was added, 4mL of the lower organic phase was taken, dried in a 10mL centrifuge tube, redissolved in 1mL of methanol solution, centrifuged, and the supernatant was filtered through a 0.22 μm organic filter membrane and placed in a liquid phase bottle for HPLC detection. The products were analyzed by an agilent 1260 High Performance Liquid Chromatography (HPLC) method, and it was found that two new substances were produced (see fig. 3), and the two products were completely identical to the product obtained by catalytic fermentation of curvularia lunata, and it was presumed that the two products were hydrocortisone and 14 α -hydroxycortinol, respectively, and CL3-CYP021 was predicted to be P450 protein catalyzing the β -hydroxylation at position 11 and the α -hydroxylation at position 14 of steroid hormones.

Example 4 construction of Saccharomyces cerevisiae engineering bacterium HC102

The starting strain Saccharomyces cerevisiae BY4742 was cultured overnight in the screening medium. The screening medium consisted of: SD-Trp (beijing pan kino (functional genome) science and technology ltd), 2% glucose, 0.005% his, 0.01% leu, 0.01% Ura (each percentage number indicates g/100 mL). 1ml (OD about 0.6-1.0) was taken and dispensed into 1.5ml EP tubes, centrifuged at 4 ℃ at 10000g for 1min, the supernatant was discarded, the precipitate was washed with sterile water (4 ℃), centrifuged under the same conditions, and the supernatant was discarded. The cells were incubated at 25 ℃ for 20min with 1ml of a treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5) added thereto, and the treatment solution was used. After centrifugation, the supernatant was discarded, 1ml of 1M sorbitol (0.22 μ M aqueous membrane filtration sterilization) was added to the cells for resuspension, and the cells were centrifuged, and the supernatant was discarded (resuspended twice with 1M sorbitol) to a final volume of about 90 μ l. The fragment pPgk-CL3-CPR-ADHt, pTEF-CL3-CYP021-CYC1t and the homologous arm marker fragment Gal7-URA3-up (SEQ ID No. 9; the homologous arm fragment comprising 400bp homologous region upstream of Gal7 site, URA3marker gene, and 400bp homologous region downstream of Pgk promoter), Gal7-URA3-down (SEQ ID No. 10; the homologous arm fragment comprising 200bp homologous region of CYC1 terminator and 300bp homologous region downstream of Gal7 site) (CL 3-CPR, CL3-CYP021 gene fragment is integrated into Gal7 site of Saccharomyces cerevisiae BY 4742) were added, respectively, after mixing, the mixture was transferred to a cuvette, 2.7kv shock 5.6ms, 1ml of 1M sorbitol was added, 1h at 30 ℃, and a solid culture medium selection (SD-Ura. RTM. medium: Ura screening medium), 2% glucose, 0.005% His, 0.01% Leu, 1.5% agar; each percentage number represents g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. PCR identified the correct positive clone, designated strain HC 102.

Example 5 catalytic Synthesis of hydrocortisone and 14 α -Hydrocortole by engineering bacteria of Saccharomyces cerevisiae HC102 (Whole cell catalysis)

And (3) flask fermentation catalysis: HC002 yeast strain was activated in solid selection medium (same as in example 3), seed solutions (30 ℃, 250rpm, 16h) were prepared in corresponding liquid selection medium (same as in example 3), and inoculated in 1mL portions into 3 flasks each containing 100mL of YPD liquid culture mediumThe cells were cultured in a 500mL Erlenmeyer flask at 30 ℃ under shaking at 250rpm for 2 days, yeast cells were collected at 5000rpm, washed with PBS buffer solution and finally resuspended in a 250mL Erlenmeyer flask containing 30mL PBS to obtain the OD of the cells_600nmWhen the concentration reaches 60 ℃, adding a substrate RSA with the final concentration of 170mg/L for catalytic reaction, and carrying out shaking culture at 30 ℃ and 250rpm for 2 days to obtain a catalytic reaction solution.

5mL of the catalyzed reaction solution was placed in a separatory funnel, an equal volume of 1mL of an extractant (methanol: chloroform: 1:9, volume ratio) was added, 4mL of the lower organic phase was taken, dried in a 10mL centrifuge tube, redissolved in 1mL of methanol solution, centrifuged, and the supernatant was filtered through a 0.22 μm organic filter membrane and placed in a liquid phase bottle for HPLC detection. The product was analyzed by agilent 1260 High Performance Liquid Chromatography (HPLC).

By comparison with the standard through HPLC identification, it was confirmed that the constructed s.cerevisiae strain HC102 can catalyze the substrate RSA to synthesize two substances, one can be confirmed as hydrocortisone (confirmed by standard comparison), the other can be confirmed as 14-alpha hydroxylation product 14 alpha-hydrocortisone (see FIGS. 4 and 5), and the yield thereof is calculated from the peak area to be higher than that of the strain HC101, so it is presumed that the CPR gene identified and cloned from Curvularia lunata is helpful for the production of the s.cerevisiae hydrocortisone.

Subsequently, the production result of HC102 strain was analyzed quantitatively, and by analyzing the strain HC102, the target product hydrocortisone 1.97mg/L can be obtained by whole cell catalysis. Compared with 2-fold increase of the yield of the strain HC101 by using the saccharomyces cerevisiae, the result shows that the CPR gene cloned in the Curvularia lunata is helpful for the production of the hydrocortisone of the saccharomyces cerevisiae.

TABLE 3 quantitative analysis of catalytic products of strains HC101 and HC102

Name of Strain	Genotype(s)	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 meaning of the unit mg/L of hydrocortisone is the mg of hydrocortisone per liter of catalytic reaction solution.

Example 6 construction and catalytic Synthesis of hydrocortisone and 14 α -Hydroxycortisol by Saccharomyces cerevisiae engineering bacterium HC103

1. Construction of saccharomyces cerevisiae engineering bacteria HC103

The codon optimization is carried out on CL3-CYP021 and CL3-CPR proteins, the optimization and gene synthesis work is finished by Nanjing Kingsler Biotechnology Co., Ltd, and the obtained optimized genes are named as CL3-CYP021-sy-Sc and CL 3-CPR-sy-Sc. The sequence of the CL3-CYP021-sy-Sc gene is shown in SEQ ID No.3, and the CL3-CYP021-sy-Sc gene codes protein shown in SEQ ID No. 1; the CL3-CPR-sy-Sc gene has the sequence shown in SEQ ID No.4 and codes the protein shown in SEQ ID No. 2.

The obtained genes were subjected to construction of a Saccharomyces cerevisiae polygene integration fragment according to the method in step 4 of example 1, and then the two genes were integrated at the Gal7 locus of Saccharomyces cerevisiae BY4742 according to the method in example 4, to finally obtain strain HC 103.

2. Saccharomyces cerevisiae engineering bacterium HC103 catalyzed synthesis of hydrocortisone and 14 alpha-hydrocortisone (whole cell catalysis method)

The constructed saccharomyces cerevisiae strain HC103 is catalyzed to synthesize hydrocortisone, the catalytic experiment and the sample detection method are the same as those in example 5, the HC103 strain can synthesize hydrocortisone at 3.87mg/L and 14 alpha-hydrocortisone at 2.43mg/L through catalytic fermentation, the fermentation result is shown in table 4, and the yield of the hydrocortisone produced by the saccharomyces cerevisiae through catalytic fermentation by utilizing the 11-site beta hydroxylated P450 protein of the Curvularia lunata is further improved through codon optimization.

TABLE 4 quantitative analysis of HC103 catalytic products by Strain

Note: the unit mg/L of hydrocortisone and 14 alpha-hydrocortisone means the mg of hydrocortisone or 14 alpha-hydrocortisone per liter of the catalytic reaction solution.

3. Catalytic synthesis of hydrocortisone and 14 alpha-hydrocortisone by using saccharomyces cerevisiae engineering bacteria HC103 through crude enzyme solution or pure enzyme method

The first step is as follows: extraction of protein (the following reactions were all carried out on ice at 4 ℃ C.)

(1) Fermenting and culturing, 200ml of corresponding selective medium, fermenting for 48h, collecting thallus, OD_600nmAbout 4.0;

(2) collecting thallus (centrifuging at 5000rpm for 5min, discarding supernatant), and breaking cells (grinding thallus with liquid nitrogen for breaking cells, grinding for three times, resuspending the ground crude protein with 1mL phosphate buffer solution with pH7.25, and placing on ice) as crude enzyme solution for subsequent use;

(3) if protein purification is needed, a microsome extraction process is added, and the process is as follows:

A. centrifuging the resuspended yeast crude enzyme solution at 7000g for 10min, collecting the supernatant, washing the precipitate with 5ml TES-B buffer (Table 6) once, and repeating the above steps once;

B. the collected supernatant is respectively loaded in an ultracentrifuge tube, and ultracentrifugation is carried out for 25000g/10 min;

C. taking the centrifuged supernatant, and subpackaging in an ultracentrifuge tube at 100000g for centrifugation for 1 h;

D. the supernatant was aspirated off, and the pellet was resuspended in 1-2ml TEG buffer (Table 5), and if the resuspension effect was poor, it was sonicated in an ice-water bath and used as a pure enzyme solution for subsequent use.

Table 5 TES-B buffer and TEG buffer formulations:

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 (W)
				Beta-mercaptoethanol	1.5mmol
Water (W)

The second step is that: catalytic reaction for synthesizing HC

And (3) catalyzing and synthesizing HC by crude enzyme liquid or pure enzyme: total volume of the catalyst system was 2mL (table 6); the reaction was carried out at 30 ℃ with slow shaking at 70rpm on a shaker, starting with the addition of NADPH and terminating with the addition of the corresponding extractant methanol chloroform to obtain a catalyzed reaction solution.

TABLE 6 catalytic system for HC synthesis catalyzed by crude enzyme solution or pure enzyme

Catalytic system	Final concentration
		RSA	0.2μmol
Phosphate buffer pH7.25	100mmol
		NADPH	1mmol
Crude enzyme solution or pure enzyme

The third step: catalytic product extraction and detection

2mL of the catalyzed reaction solution was placed in a separatory funnel, an equal volume of an extractant (methanol: chloroform: 1:9, volume ratio) was added, 2mL of the lower organic phase was taken, the mixture was put into a 5mL centrifuge tube and dried, 1mL of a methanol solution was used for redissolution, and the supernatant was centrifuged and filtered through a 0.22 μm organic filter to a liquid phase bottle for HPLC detection. The product was analyzed by agilent 1260 High Performance Liquid Chromatography (HPLC).

The results show that: by extracting crude enzyme liquid or pure enzyme from the engineering bacteria HC103 and carrying out catalytic reaction, two products of hydrocortisone and 14 alpha-hydrocortisone are successfully detected in the products, so that the crude enzyme liquid or the pure enzyme can be used for catalytically producing HC and 14 alpha-hydrocortisone.

Example 7 Synthesis of hydrocortisone and 14 α -Hydroxycortisol in other yeasts Using β -hydroxylated P450 protein in position 11 of Curvularia lunata

The invention successfully expresses CL3-CPR and CL3-CYP021 genes from curvularia lunata in yarrowia lipolytica (PO1G), schizosaccharomyces pombe (CICC1762) and pichia pastoris (GS115), and carries out catalytic reaction on the obtained strain (shown in Table 7) by taking deoxycorticosterol (RS) as a substrate (the same as example 5), and also successfully synthesizes hydrocortisone and a byproduct 14 alpha-hydroxycorticosterol thereof.

TABLE 7 yield of fermentation catalyzed Synthesis of hydrocortisone in other yeasts

Name of Strain	Genotype(s)	Hydrocortisone (mg/L)	14 alpha-Hydroxycortisol (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

Example 8 construction and catalytic Synthesis of hydrocortisone and 14-alpha Hydrocortol by Saccharomyces cerevisiae engineering bacteria HC111-HC115

In order to further improve the yield of hydrocortisone, CL3-CPR and CL3-CYP021 excavated in curvularia lunata are integrated into a multicopy site rDNA site of Saccharomyces cerevisiae BY 4742.

The starting strain Saccharomyces cerevisiae BY4742 was cultured overnight in the screening medium. The screening medium consisted of: SD-Trp (beijing pan kino (functional genome) science and technology ltd), 2% glucose, 0.005% his, 0.01% leu, 0.01% Ura (each percentage number indicates g/100 mL). 1ml (OD about 0.6-1.0) was taken and dispensed into 1.5ml EP tubes, centrifuged at 4 ℃ at 10000g for 1min, the supernatant was discarded, the precipitate was washed with sterile water (4 ℃), centrifuged under the same conditions, and the supernatant was discarded. The cells were incubated at 25 ℃ for 20min with 1ml of a treatment solution (10mM LiAc; 10mM DTT; 0.6M sorbitol; 10mM Tris-HCl (pH7.5) added thereto, and the treatment solution was used. After centrifugation, the supernatant was discarded, 1ml of 1M sorbitol (0.22 μ M aqueous membrane filtration sterilization) was added to the cells for resuspension, and the cells were centrifuged, and the supernatant was discarded (resuspended twice with 1M sorbitol) to a final volume of about 90 μ l. The fragment pPgk-CL3-CPR-ADHt, pTEF-CL3-CYP021-CYC1t and the homologous arm marker fragment rDNA-Leu-up (SEQ ID No. 11; the homologous arm fragment comprises a homologous region of about 500bp upstream of the rDNA site, a Leu marker gene and a homologous region of 400bp of a Pgk promoter), rDNA-Leu-down (SEQ ID No. 12; the homologous arm fragment comprises a homologous region of 200bp of a CYC1 terminator and a homologous region of about 500bp downstream of the rDNA site) (realizing that 1 mul of the rDNA 3-CPR and the CL3-CYP021 gene fragment are integrated into the rDNA site of the Saccharomyces cerevisiae BY 4742), after being mixed uniformly, the mixed solution is transferred into an electrotransfer, 2.7kv shock is carried out for 5.6ms, 1ml of 1M sorbitol is added, the mixed solution is recovered for 1h at 30 ℃, the mixed solution is coated on a solid selection medium plate (formula: solid yeast screening Trp-Try, 2% glucose, 0.005% His, 0.01% Ura, 1.5% agar; each percentage number represents g/100 mL). The conditions of the screening culture are as follows: culturing at 30 deg.C for 36 hr or more. Correct positive clones were identified by PCR and five clones were randomly selected and named strains HC111, HC112, HC113, HC114, HC115, respectively. Wherein the hydrocortisone yield of the strain HC113 with the highest yield can reach 6.42mg/L, and the 14-alpha hydroxycorticol yield can reach 4.06 mg/L.

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> steroid 11 beta-hydroxylase in curvularia lunata, and coding gene and application thereof

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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 (11)

1. A set of proteins consisting of protein a and protein B;

the protein A is shown as the following (A1) or (A2):

(A1) protein with amino acid sequence shown as SEQ ID No. 1;

(A2) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in (A1);

the protein B is shown as the following (B1) or (B2):

(B1) protein with amino acid sequence shown as SEQ ID No. 2;

(B2) and (B1) attaching a tag to the N-terminus and/or C-terminus of the protein defined in (B1).

2. A set of nucleic acid molecules consisting of a nucleic acid molecule A and a nucleic acid molecule B;

the nucleic acid molecule A is a nucleic acid molecule encoding the protein A of claim 1;

the nucleic acid molecule B is a nucleic acid molecule encoding the protein B according to claim 1.

3. The kit of nucleic acid molecules according to claim 2, wherein:

the nucleic acid molecule A is a DNA molecule with a nucleotide sequence shown as SEQ ID No.3 or SEQ ID No. 5;

the nucleic acid molecule B is a DNA molecule with a nucleotide sequence shown as SEQ ID No.4 or SEQ ID No. 6.

4. Any of the following biological materials:

(c1) the complete set of recombinant vector consists of a recombinant vector A and a recombinant vector B; the recombinant vector A is a recombinant vector containing the nucleic acid molecule A in claim 2 or 3; the recombinant vector B is a recombinant vector containing the nucleic acid molecule B of claim 2 or 3;

(c3) the complete set of expression cassette consists of an expression cassette A and an expression cassette B; the expression cassette A is an expression cassette comprising the nucleic acid molecule A of claim 2 or 3; the expression cassette B is an expression cassette comprising the nucleic acid molecule B according to claim 2 or 3;

(c4) the complete set of transgenic cell line consists of a transgenic cell line A and a transgenic cell line B; the transgenic cell line A is a transgenic cell line containing the nucleic acid molecule A according to claim 2 or 3; the transgenic cell line B is a transgenic cell line containing the nucleic acid molecule B according to claim 2 or 3;

(c5) the complete set of recombinant bacteria consists of recombinant bacteria A and recombinant bacteria B; the recombinant bacterium A is a recombinant bacterium containing the nucleic acid molecule A in claim 2 or 3; the recombinant bacterium B is a recombinant bacterium containing the nucleic acid molecule B according to claim 2 or 3.

5. A method for constructing engineering bacteria for producing hydrocortisone and/or 14 alpha-hydroxycortinol, which comprises the following steps: modifying yeast to express the protein set of claim 1, wherein the modified yeast is an engineering bacterium for producing hydrocortisone and/or 14 alpha-hydrocortisone.

6. The method of claim 5, wherein: the method comprises the following steps: the recombinant yeast expressing the protein set of claim 1 is obtained by introducing the nucleic acid molecule set of claim 2 or 3 into the yeast, namely the engineering bacteria.

7. The method of claim 6, wherein: the set of nucleic acid molecules is introduced into the yeast in the form of the set of recombinant vectors or the set of expression cassettes of claim 4.

8. The method of claim 6, wherein: the set of nucleic acid molecules is integrated into the genome of the yeast at the Gal7 site or at the rDNA site.

9. An engineered bacterium produced by the method of any one of claims 5 to 8.

10. Use of the set of proteins of claim 1 or the set of nucleic acid molecules of claim 2 or 3 or the biological material of claim 4 or the engineered bacterium of claim 9 in any one of:

(A) preparing hydrocortisone and/or 14 alpha-hydroxycortinol;

(B) catalyzing the beta hydroxylation at the 11 position and/or the alpha hydroxylation at the 14 position of the steroid hormone substances.

11. A method for preparing hydrocortisone and/or 14 alpha-hydrocortisone by a whole-cell catalysis method or an enzyme method;

the whole-cell catalysis method comprises the following steps: carrying out fermentation culture on the engineering bacteria of claim 9, collecting bacteria, adding a substrate, and carrying out catalytic reaction, wherein the reaction product contains hydrocortisone and/or 14 alpha-hydroxycortinol; the substrate is a substance capable of being catalysed by a steroid 11 β -hydroxylase to produce hydrocortisone and/or a steroid 14 α -hydroxylase to produce 14 α -hydroxycortinol;

the enzyme method comprises the following steps: extracting a substance with steroid 11 beta-hydroxylase and/or 14 alpha-hydroxylase activity from the engineering bacteria as claimed in claim 9, and then catalyzing a substrate to generate hydrocortisone and/or 14 alpha-hydroxycortinol in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme; the substrate is a substance capable of being catalysed by a steroid 11 β -hydroxylase to produce hydrocortisone and/or a steroid 14 α -hydroxylase to produce 14 α -hydroxycortinol.

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