CN109097342A - Mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene and application - Google Patents
Mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene and application Download PDFInfo
- Publication number
- CN109097342A CN109097342A CN201810902589.2A CN201810902589A CN109097342A CN 109097342 A CN109097342 A CN 109097342A CN 201810902589 A CN201810902589 A CN 201810902589A CN 109097342 A CN109097342 A CN 109097342A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- protein
- acid molecules
- seq
- complete
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P33/00—Preparation of steroids
- C12P33/06—Hydroxylating
- C12P33/08—Hydroxylating at 11 position
Abstract
The invention discloses a kind of mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene and applications.11 B-hydroxylase of steroid provided by the present invention is 19-527 at least containing SEQ ID No.1, and extends since the 19th of SEQ ID No.1 to the N-terminal of SEQ ID No.1, obtain any one protein (Ac-CYP003 albumen) that length is 509-526 amino acids.In addition the present invention also provides the complete albumen that the protein as shown in SEQ ID No.2 (Ac-CPR albumen) forms by 11 B-hydroxylase of steroid and amino acid sequence.The present invention expresses Ac-CYP003 albumen (or itself and Ac-CPR albumen) in Heterologous Microbial, carries out catalyzing and synthesizing for hydrocortisone (HC), avoids the generation of other by-products, improve the conversion ratio of RSA to HC.
Description
Technical field
The present invention relates to genetic engineering fields, and in particular to mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene
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.
Absidia mould (Absidia coerulea) belongs to Phycomycetes Mucoales Mucoraceae.From Czech's sixties in last century
Started for the first time using the mould catalytic production HC of Absidia, China's recent decades industrially generally use Absidia mould as work
Industry metaplasia produces the production that bacterial strain carries out HC.And recent decades Chinese scholar from strain improvement to zymotechnique on constantly explored
With research, make great efforts the yield for improving fermenting and producing HC.2004 precious congruence [Zhang, B., Effect of
Supercritical Fluids on C11-Hydroxylation Activity of Absidia
Coerulea.Biotechnol Prog, 2004.20:1885-1887.] use supercritical fluid to improve as catalytic media
The yield of substrate RSA solubility and 11 β hydroxylation products.[Fan Zhihua, Sun Aiyou, Hao Jiandong wait to use seaweed to Fan Zhihua etc.
Primary Study [J] the University Of Science and Technology Of Tianjin journal of sour calcium investment immobilization Absidia coerulea bacterium production hydrocortisone,
2003,18 (4): 1-4.] it is significantly improved with the mould cell progress organic solvent RSA synthesis HC of calcium-alginate-immobilized Absidia
HC and the ratio of table HC and the yield of HC in product.
Although the mould hydrocortisone semi-synthesizing technology of Absidia has had the research of decades, presently, there are one
A major issue is that the conversion ratio of RSA to HC is relatively low, and the yield of HC only has 50% or so.Synthetic product is other than HC, also
There is a large amount of table hydrocortisone, this is main, and there are two reasons.First is that there are multiple P450 albumen during Absidia is mould, in addition to
11- β hydroxylating P450 proteins carry synthesizes outside HC, other P450 albumen can catalyze and synthesize by-product.Second is that 11- β hydroxylating
The chiral specificity of P450 proteins carry is not high, and the albumen is in addition to other than 11 progress β hydroxylatings synthesize HC, moreover it is possible to
11 progress α hydroxylatings synthesize table hydrocortisone.Therefore, from Absidia it is mould in excavate 11- β hydroxylating P450 egg
It is white significant.
Summary of the invention
The object of the present invention is to provide a kind of mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene and applications.
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) the 19-527 amino acids sequence at least containing SEQ ID No.1, and from the 19th of SEQ ID No.1 the
Position starts to extend according to the amino acid sequence of SEQ ID No.1 to the N-terminal of SEQ ID No.1, and obtaining length is 509-526 ammonia
Any one protein of base acid；
(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 or amino acid sequence such as SEQ ID
Protein shown in No.1 (wherein, protein shown in SEQ ID No.1 is overall length Ac-CYP003 albumen).
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 overall length Ac-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.
Further, protein shown in described (A1) is specially the 19-527 of amino acid sequence such as SEQ ID No.1
Protein shown in position (Ac-CYP003 albumen --- the Ac-CYP003-T1 albumen after truncating).
Second aspect, claimed nucleic acid molecules or complete nucleic acid molecules.
Nucleic acid molecules provided by the present invention can be nucleic acid molecules 1 or nucleic acid molecules 2.
The nucleic acid molecules 1 be coding above protein described in first aspect nucleic acid molecules (including optimization and it is non-optimum
The nucleic acid molecules of Ac-CYP003 albumen after the coding truncation of change).
The nucleic acid molecules 2 are (the coding Ac-CYP003 overall length egg after optimizing of DNA molecular shown in SEQ ID No.3
White DNA molecular).
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 coding of change truncates and the nucleic acid molecules of non-truncated Ac-CYP003 albumen)；
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 Ac-CPR albumen of change).
Further, the nucleic acid molecules 1 it is concretely any in following (a1)-(a4) shown in DNA molecular:
(a1) the 55-1584 nucleotide sequences at least containing SEQ ID No.3, and from the of SEQ ID No.3
55 start according to SEQ ID No.3 nucleotide sequence to SEQ ID No.3 5 ' end extend, obtain length be 1530 to
Any one DNA molecular of 1583bp；
(a2) the 55-1584 nucleotide sequences at least containing SEQ ID No.5, and from the of SEQ ID No.5
55 start according to SEQ ID No.5 nucleotide sequence to SEQ ID No.5 5 ' end extend, obtain length be 1530 to
Any one DNA molecular of 1583bp；
(a3) hybridize under strict conditions with (a1) or (a2) DNA molecular limited and encode above described in first aspect
(A1) in-(A4) it is any shown in protein DNA molecular；
(a4) with (a1)-(a3) in it is any defined by DNA sequence dna have 99% or more, 95% or more, 90% or more,
Protein shown in 85% or more or 80% or more homology and coding are any in (A1)-(A4) described in first aspect above
DNA molecular.
Further, the nucleic acid molecules A it is concretely as above any in (a1)-(a4) shown in DNA molecular or core
(wherein, SEQ ID No.3 is the overall length of optimization to nucleotide sequence DNA molecular as shown in SEQ ID No.3 or SEQ ID No.5
The encoding gene of Ac-CYP003 albumen, SEQ ID No.5 are the encoding gene of the overall length Ac-CYP003 albumen of unoptimizable).
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 overall length Ac-CPR albumen of optimization, and SEQ ID No.6 is the volume of the overall length Ac-CPR albumen of unoptimizable
Code gene)；
(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.
Further, DNA molecular shown in described (a1) concretely nucleotide sequence such as SEQ ID No.3
DNA molecular shown in 55-1584 (encoding gene of the Ac-CYP003-T1 albumen after optimizing).Shown in (a2)
DNA molecular is specially nucleotide sequence DNA molecular (Ac- being not optimised as shown in 55-1584 of SEQ ID No.5
The encoding gene of CYP003-T1 albumen).
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, a kind of claimed method for preparing the engineering bacteria for producing hydrocortisone.
Method of the preparation provided by the present invention for producing the engineering bacteria of hydrocortisone, it may include following steps: right
Saccharomycete is transformed, and makes its expression protein described in first aspect or complete protein above, improved saccharomycete
As for producing the engineering bacteria of hydrocortisone.
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-CPR-ADHt and expression cassette pTEF-CYP003-
CYC1t composition；The sequence of the expression cassette pPgk-CPR-ADHt is SEQ ID No.7 (or for by the of SEQ ID No.7
Resulting sequence after 813-2864 replaced with SEQ ID No.4)；The sequence of the expression cassette pTEF-CYP003-CYC1t
It is SEQ ID No.8 (or to replace with SEQ ID No.3 or SEQ ID No.3 for 501-2084 of SEQ ID No.8
55-1584 of 55-1584 or SEQ ID No.5 after resulting sequence).Described in being imported into the saccharomycete
While expression cassette pPgk-CPR-ADHt and expression cassette pTEF-CYP003-CYC1t, homology arm marker segment has also been imported
Gal7-URA3-up and homology arm marker segment gal7-URA3-down (realize the Gal7 for being integrated in saccharomyces cerevisiae BY4742
Site)；The sequence of the homology arm marker segment gal7-URA3-up is as shown in SEQ ID No.9；The homology arm
The sequence of marker segment gal7-URA3-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-CPR-ADHt and the expression cassette pTEF-
CYP003-CYC1t composition.When importing the expression cassette pPgk-CPR-ADHt and expression cassette pTEF- into the saccharomycete
While CYP003-CYC1t, homology arm marker segment rDNA-Leu-up and homology arm marker segment rDNA- have also been imported
Leu-down (realizes the site rDNA for being integrated in saccharomyces cerevisiae BY4742)；The homology arm marker segment rDNA-Leu-
The sequence of up is as shown in SEQ ID No.11；The sequence such as SEQ ID of the homology arm marker segment rDNA-Leu-down
Shown in 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) engineering bacteria HC001: by what is obtained after recombinant vector pRS426-CYP003 importing saccharomyces cerevisiae BY4742 bacterial strain
Recombinant bacterium；The recombinant vector pRS426-CYP003 is by the DNA fragmentation (overall length being not optimised shown in SEQ ID No.5
Ac-CYP003 gene) be inserted between the restriction enzyme site SexA1 and Asc1 of pRS426 plasmid after obtained recombinant plasmid.
(2) engineering bacteria HC002: by the expression cassette pPgk-CPR-ADH1t, (Ac-CPR gene therein is unoptimizable base
Cause, as shown in SEQ ID No.6), the expression cassette pTEF-CYP003-CYC1t (Ac-CYP003 gene therein be unoptimizable
Gene, as shown in SEQ ID No.5), the homology arm marker segment gal7-URA3-up and the homology arm marker piece
Section gal7-URA3-down imports the recombinant bacterium obtained after saccharomyces cerevisiae BY4742 bacterial strain and (realizes and be integrated in saccharomyces cerevisiae
The site Gal7 of BY4742).
(3) engineering bacteria HC023: by the expression cassette pPgk-CPR-ADH1t (Ac-CPR gene therein be optimization gene,
As shown in SEQ ID No.4), the expression cassette pTEF-CYP003-CYC1t (Ac-CYP003 gene therein be 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 BY4742 bacterial strain and (realizes and be integrated in saccharomyces cerevisiae BY4742
The site Gal7).
(4) engineering bacteria HC030: by the expression cassette pPgk-CPR-ADH1t (Ac-CPR gene therein be optimization gene,
As shown in SEQ ID No.4), the expression cassette pTEF-CYP003-CYC1t (Ac-CYP003 gene therein be optimization Ac-
CYP003-T1 gene, as shown in 55-1584 of SEQ ID No.3), the homology arm marker segment gal7-URA3-
It is (real that the up and homology arm marker segment gal7-URA3-down imports the recombinant bacterium obtained after saccharomyces cerevisiae BY4742 bacterial strain
The site Gal7 for being integrated in saccharomyces cerevisiae BY4742 is showed).
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 is prepared；
(B) catalysis steroid hormone substance carries out 11 β hydroxylatings.
In addition, protein described in first aspect above is also claimed as steroid hormone substance 11 in the present invention
Application in hydroxylase.
Wherein, the steroid hormone substance can be that the object for generating hydrocortisone can be catalyzed by 11 B-hydroxylase of steroid
Matter, it is specific as 7 Alpha-hydroxy pregn-4-ene-3,20-dione -21- acetates (or be deoxidation hydrocortisone 21- acetate,
Cortexolone-21-acetate, RSA) or Compd S 11-deoxycortisol (11-deoxycortisol, RS).
7th aspect, a kind of claimed method for preparing hydrocortisone.
The method provided by the present invention for preparing hydrocortisone can be whole-cell catalysis 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 in reaction product；The substrate
For the substance for generating hydrocortisone can be catalyzed by 11 B-hydroxylase of steroid.
Wherein, the enzyme process may include following steps: extracting in the engineering bacteria described in terms of the above the 5th has steroid
The active substance of 11 B-hydroxylase of class, then catalysis substrate generates hydrogenation in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme
Cortisone；The substrate is that the substance for generating hydrocortisone can be catalyzed by 11 B-hydroxylase of steroid.
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 Ac-CYP003 albumen in Heterologous Microbial carries out catalyzing and synthesizing for HC, it is avoided
The generation of his by-product improves the conversion ratio of RSA to HC.The present invention is can using safer saccharomycete catalytic production hydrogenation
Pine provide possibility, effectively shorten the production cycle compared to traditional filamentous fungi production method, simplify fermentation and set
It is standby, and the expression for being able to carry out single albumen improves product yield, effectively reduces production and extraction cost, for
The innovation of hydrocortisone production technology is of great significance.
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 HC001 catalysate is detected and identified.
Fig. 4 is that saccharomyces cerevisiae engineered yeast HC002 catalysate is detected and identified.
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.
Absidia is mould: 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 embodiment 1, Absidia mould cytochrome reductase and 11 β-hydroxylation enzymes
The clonal expression of gene is divided into following 3 step:
1, the extraction of the mould total serum IgE of Absidia
Firstly, the spore that Absidia is mould to be cultivated a couple of days on plate, and collect certain amount is linked into 50mL Ma Ling
In potato-glucose (PDA) culture medium, it is incubated overnight to a large amount of thallus of synthesis；Then, Absidia mould filament is collected by centrifugation,
It is washed using potassium phosphate buffer (PBS), the bottom of final concentration of 170mg/L is finally resuspended and be added with 50mL buffer
Object deoxygenates hydrocortisone 21- acetate (RSA) and induces 1h, 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 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 Ac-CYP003, Ac-CPR.By the obtained pcr amplification product raw work biology in Shanghai
The PCR product Purification Kit of Engineering Co., Ltd uses SexA1 and Asc1 the digestion DNA piece of Thermo company after purification
Section, glue recovery product are spare.
1 Ac-CYP003 and Ac-CPR gene magnification primer of table
| Primer | Sequence (5 ' -3 ') |
| Ac-CYP003-SexA1-F | gcgaccaggtATGCTTACAGAGTACATTCAT |
| Ac-CYP003-Asc1-R | ggcgcgccTTACTTTCTTGGCACAATCTTGA |
| Ac-CPR-SexA1-F | gcgaccaggtATGGATCTCCCTACAGCAACTGA |
| Ac-CPR-Asc1-R | ggcgcgccCTATGCCCACACGTCTTCCACAT |
Ac-CYP003 gene (wild type) sequence encodes albumen shown in SEQ ID No.1 as shown in SEQ ID No.5
Matter (is named as Ac-CYP003 albumen)；Ac-CPR gene (wild type) sequence encodes SEQ ID as shown in SEQ ID No.6
Protein shown in No.2 (is named as Ac-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
Linked system: 5 μ L 2 × Quick ligation Buffer (NEB company), 0.5 is added in each 50ng of Ac-CYP003 genetic fragment
μ L Quick ligase (NEB company, 400,000cohesive end units/ml) supplements distilled water to 10 μ L, and room temperature is anti-
Answer 10min to obtain connection product, be transferred to ice bath 30 minutes in Trans1-T1 competent cell, 42 DEG C heat shock 30 seconds, immediately as
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 the LB containing ampicillin
On plate, after being incubated overnight, PCR screens 5 positive single colonies, and positive colony is carried out Liquid Culture, extracts positive colony matter
Grain carries out sequence verification, and sequencing result shows to be inserted into target fragment on carrier pRS426, obtains plasmid pRS426-CYP003.
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 Ac-
CPR, Ac-CYP003 genetic fragment are attached conversion, and (the same step 3) of method, and selected clone sequence verification, obtain plasmid M2-
CPR, M3-CYP003.Primer 1-M-pEASY-PGK1-F and 1-M-ADHt- are used as template using the plasmid M2-CPR built
TEF1-R (being shown in Table 2) progress PCR amplification (the same step 2 of method obtains pPgk-Ac-CPR-ADH1t segment (SEQ ID No.7),
The segment includes Pgk promoter (63-812 of SEQ ID No.7), the Ac-CPR gene (SEQ in the mould source of Absidia
813-2864 of ID No.7) and ADH1 terminator (2865-3022 of SEQ ID No.7).With the matter built
Grain M3-CYP003 is that template uses primer 2-M-ADHt-TEF1-F and M-CYC1t-pEASY-R (being shown in Table 2) progress PCR amplification
(the same step 2 of method obtains pTEF-Ac-CYP003-CYC1t segment (SEQ ID No.8), which includes TEF promoter
(51-500 of SEQ ID No.8), the Ac-CYP003 gene (501- of SEQ ID No.8 in the mould source of Absidia
2084) and CYC1 terminator (2085-2391 of SEQ ID No.8).Glue is carried out to the target fragment that amplification obtains
It is recycled spare.
2 Ac-CYP003 and Ac-CPR gene integration fragment amplification primer of table
The building of embodiment 2, saccharomyces cerevisiae engineered yeast HC001
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-CYP003 that embodiment 1 obtains is added, is transferred to after mixing in electric revolving cup, 2.7kv electricity
5.7ms is hit, 1ml 1M sorbierite is added, 30 DEG C of recovery 1h are coated on solid screening and culturing medium (formula: yeast Selective agar 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 HC001.
Embodiment 3, saccharomyces cerevisiae engineered yeast HC001 catalyze and synthesize hydrocortisone
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 (methanol: chloroform=1:9, volume is added
Than), 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 of novel substance for discovery (see Fig. 3).By newly obtained with the comparison confirmation of the chromatograms of standard items both are new
Substance is respectively table hydrocortisone and hydrocortisone (see Fig. 3), it follows that Ac-CYP003 albumen swashs for steroidal
Plain 11 hydroxylase of substance, and the gene can carry out the hydroxylating of 11 α and β simultaneously.
The building of embodiment 4, saccharomyces cerevisiae engineered yeast HC002
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-Ac-CPR-ADH1t, pTEF-Ac-CYP003-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,
URA3 marker gene and the homology region Pgk promoter 400bp), gal7-URA3-down (SEQ ID No.10；This is same
Source arm segment includes the homology region CYC1 terminator 200bp and the site the Gal7 downstream homology region 300bp) it (realizes Ac-
CPR, Ac-CYP003 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, 1ml 1M sorbierite is added in 2.7kv electric shock 5.6ms, and 30 DEG C of recovery 1h are coated on screening and culturing medium plate (formula: 0.8%
Yeast Selective agar medium SD-Ura-Trp, 2% glucose, 0.005%His., 0.01%Leu., 1.5% agar；Each percentage sign
Indicate g/100mL).The condition of screening and culturing are as follows: 30 DEG C, cultivate 36h or more.PCR identifies correct positive colony, name
For bacterial strain HC002.
Embodiment 5, saccharomyces cerevisiae engineered yeast HC002 catalyze and synthesize hydrocortisone (whole-cell catalysis)
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 (methanol: chloroform=1:9, volume is added
Than), 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 HC002 of building can be synthesized with catalysis substrate RSA
Two kinds of substances, confirmation is respectively hydrocortisone and table hydrocortisone, and speculates that its yield is higher than HC001 bacterium from peak area
Strain (Fig. 4), so speculating from the CPR gene of the mould middle clone of Absidia there is side for the production of saccharomyces cerevisiae hydrocortisone
It helps.
Then, quantitative analysis has been carried out to the production result of HC002 bacterial strain, can have been led to by quantitative analysis bacterial strain HC002
The mode for crossing whole-cell catalytic obtains target product hydrocortisone 3.15mg/L, and table hydrocortisone 1.01mg/L is compared to making
3 times (tables 3) are improved with saccharomyces cerevisiae HC001 producing strain, the results showed that the CPR gene of the mould middle clone of Absidia is for making
The production of brewer yeast hydrocortisone is helpful.
3 bacterial strain HC001 of table and the quantitative analysis of HC002 catalysate
Note: hydrocortisone/table hydrocortisone unit mg/L be meant that every liter catalysis reaction solution in hydrogenation can
Pine/table hydrocortisone mg number.
Embodiment 6, saccharomyces cerevisiae engineered yeast HC023 building and catalyze and synthesize hydrocortisone
1, the building of saccharomyces cerevisiae engineered yeast HC023
Codon optimization, the optimization and gene chemical synthesis efforts commission south are carried out to Ac-CYP003 and Ac-CPR albumen
Jing Jinsirui Biotechnology Co., Ltd completes, and the unnamed gene after the optimization of acquisition is Ac-CYP003-sy-Sc, Ac-CPR-
sy-Sc.The sequence of Ac-CYP003-sy-Sc gene encodes protein shown in SEQ ID No.1 as shown in SEQ ID No.3；
The sequence of Ac-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
HC023。
2, saccharomyces cerevisiae engineered yeast HC023 catalyzes and synthesizes hydrocortisone (whole-cell catalysis)
The Wine brewing yeast strain HC023 of building is carried out catalyzing and synthesizing hydrocortisone, Catalysis experiments and sample detection side
Method is with embodiment 5, and by catalystic, fermentative, HC023 bacterial strain can synthesize 18.4mg/L hydrocortisone and the hydrogenation of 5.6mg/L table can
Pine, fermentation results are shown in Table 4.
4 bacterial strain HC023 catalysate quantitative analysis of table
Note: hydrocortisone/table hydrocortisone unit mg/L be meant that every liter catalysis reaction solution in hydrogenation can
Pine/table hydrocortisone mg number.
Embodiment 7, using truncated CYP003 albumen saccharomyces cerevisiae engineered yeast strain HC030 building and catalyze and synthesize hydrogen
Change cortisone
1, the building of saccharomyces cerevisiae engineered yeast strain HC030
The Ac-CYP003 gene excavated according to the present invention, since there are more than one startings for part before the gene
Codon, so attempting to truncate the gene, the unnamed gene after the truncation of acquisition is Ac-CYP003-T1.Ac-
The 55- of the sequence such as SEQ ID No.3 of CYP003-T1-sy-Sc gene (the Ac-CYP003 Truncated gene after optimizing)
Shown in 1584, protein (being named as Ac-CYP003-T1 albumen) shown in 19-527 of coding SEQ ID No.1.
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
According to the method in embodiment 4 by above-mentioned Ac-CYP003-T1-sy-Sc gene and original Ac-CPR-sy-Sc gene (SEQ ID
No.4 it) is integrated in the site Gal7 of saccharomyces cerevisiae BY4742, it is final to obtain bacterial strain HC030.
2, saccharomyces cerevisiae engineered yeast HC030 catalyzes and synthesizes hydrocortisone (whole-cell catalysis)
The Wine brewing yeast strain HC030 of building is carried out catalyzing and synthesizing hydrocortisone, Catalysis experiments and sample detection side
Method is with embodiment 5, and by catalystic, fermentative, HC030 bacterial strain can be with synthesizing hydrogenated cortisone and table hydrocortisone, the results showed that cuts
Short Ac-CYP003 albumen, can still be catalyzed C11 β hydroxylatings, the synthesizing hydrogenated cortisone of catalysis substrate RSA, and
Yield is slightly above not truncated Ac-CYP003 albumen, and fermentation results are shown in Table 5.
5 bacterial strain HC030 catalysate quantitative analysis of table
Note: hydrocortisone/table hydrocortisone unit mg/L be meant that every liter catalysis reaction solution in hydrogenation can
Pine/table hydrocortisone mg number.
3, using saccharomyces cerevisiae engineered yeast HC030 with crude enzyme liquid or the synthesizing hydrogenated cortisone of pure enzyme law catalysis
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, OD600nm4.0 left and right；
(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 6) 1-2ml, if resuspension effect is bad, can go
It is ultrasonic in ice-water bath, as pure enzyme solution for subsequent use.
6 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 7)；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 7 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: pass through the above-mentioned albumen crude enzyme liquid extracted in engineering bacteria HC030 or pure enzyme and carries out catalysis reaction,
It successfully equally is detected two kinds of products of hydrocortisone and table hydrocortisone in the product, so proving crude enzyme liquid or pure enzyme
It can also carry out catalytic production HC.
Embodiment 8, the functional verification that source of people CYP11B1 albumen is carried out in saccharomyces cerevisiae
1, the saccharomyces cerevisiae engineering of synthesizing hydrogenated cortisone is used for using source of people CYP11B1 and the building of ADX, ADR albumen
Bacterium HC040
Scholars generally use 11 β hydroxyls in people source in the synthesizing hydrogenated cortisone research of saccharomyces cerevisiae before
Base P450, it has the advantages that product specificity is strong.So in the present invention equally to the CYP11B1 albumen from people into
Row functional verification has synthesized electron transport chain albumin A DX, ADR and source of people P450 of Niu Laiyuan by codon optimization respectively
(gene order is as shown in SEQ ID No.13 after Hm-CYP11B1 optimization for the encoding gene of PROTEIN C YP11B1；After Bo-ADX optimization
Gene order is as shown in SEQ ID No.14；Gene order is as shown in SEQ ID No.15 after Bo-ADR optimization).And according to implementation
Method building saccharomyces cerevisiae polygenes in 1 step 4 of example integrates segment pPgk-Bo-ADX-ADH1t, pTDH3-Bo-ADR-
Said gene is then carried out saccharomyces cerevisiae according to the method in embodiment 4 by TPI1t, pTEF-Hm-CYP11B1-CYC1t
BY4742 chromosomal integration is integrated into the site Gal7, final to obtain bacterial strain HC040.
2, Wine brewing yeast strain HC040 catalyzes and synthesizes hydrocortisone
The Wine brewing yeast strain HC040 of building is carried out catalyzing and synthesizing hydrocortisone, Catalysis experiments and sample detection side
Method is with embodiment 5, and by catalystic, fermentative, HC040 bacterial strain can be with this exclusive product of synthesizing hydrogenated cortisone, but yield is lower
(being shown in Table 8).So compared to the synthesizing hydrogenated cortisone of source of people CYP11B1 proteins carry substrate identified at present, this hair
The Ac-CYP003 albumen in the mould source of the Absidia that explicit example for reference is made has higher catalytic efficiency, but product is not single-minded, explanation
There are problems for albumen stereoselectivity.
The synthesizing hydrogenated cortisone product analysis of the P450 proteins carry of 8 separate sources of table
Note: hydrocortisone/table hydrocortisone unit mg/L be meant that every liter catalysis reaction solution in hydrogenation can
Pine/table hydrocortisone mg number.
Embodiment 9, saccharomyces cerevisiae engineered yeast HC023 catalyze and synthesize the 5L ferment tank of hydrocortisone
The present invention has carried out the fermentation of 5L fermentor to the bacterial strain HC023 of building, by High Density Cultivation, is further promoted
The catalytic capability of bacterial strain, specific fermentation process are as follows:
(1) take out the cryopreservation tube about 1.5ml for saving bacterial strain by -80 DEG C of refrigerators, be placed on ice to melt, be all connected to containing
In the 250ml triangular flask of 30ml seed culture medium, 30 DEG C, 250rpm is cultivated about 20 hours, until OD600nm=5~7, as level-one
Seed.
(2) first order seed is connected in the 1L triangular flask equipped with 100ml seed culture medium, inoculum concentration 3% connects 5 bottles altogether.30
DEG C, 250rpm is cultivated about 20 hours, until OD600nm=5~7, as secondary seed.
(3) secondary seed is connected to culture (inoculum concentration 5% (v/v)) in 7L fermentor by flame inoculation mode.Temperature
30 DEG C of setting, pH setting 5.0 automatically adjust pH, dissolved oxygen setting 30%, by adjusting revolving speed by Feeding ammonia water (25wt.%)
Dissolved oxygen is set to maintain 30% with ventilatory capacity.
When the glucose in bottom material exhausts, dissolved oxygen is increased rapidly, starts flow feeding culture medium 1 at this time, using DO-
The feed profile of Stat.Culture to 59 hours OD=200 or so uses supplemented medium 2 instead and continues culture (to be contained in supplemented medium 2
There is 4.5g/L RSA (RSA adding manner: 80% ethyl alcohol of 30ml to be added in 1g RSA, is heated to being completely dissolved, is then turned rapidly
Enter in fermentor).1g RSA, culture to 143 hours fermentation ends are added when cultivating small to 59,83,100.
Wherein, the medium component used in high density fermentation (trace metal salts and vitamin mother liquor composition as shown in table 9
As shown in table 10).
The medium component used in 9 high density fermentation of table
10 trace metal salts of table and vitamin mother liquor composition
Hydrocortisone yield is 1.23mM (0.44g/L) after fermentation, consumes substrate RSA about 3.68mM, conversion altogether
Rate is about 33.4%.It is shown in Table 11.
11 high density fermentation hydrocortisone yield of table
Note: hydrocortisone/table hydrocortisone unit mmol/L is meant that every liter of fermentation liquid (ferments for 143 hours
At the end of fermentation liquid) in hydrocortisone/table hydrocortisone mmol number.
Embodiment 10, using 11 mould β hydroxylating P450 albumen of Absidia are synthesizing hydrogenated in other saccharomycete can
Pine
Present invention success is in Yarrowia lipolytica (CICC32520), schizosaccharomyces pombe (CICC1762), and finishes red ferment
Success expresses Ac-CPR the and Ac-CYP003 gene mould from Absidia in the form of plasmid expression in female (GS115), right
The bacterial strain (being shown in Table 12) of acquisition is that substrate carries out catalysis reaction (with embodiment 5) with deoxy-cortisol (RS), is also successfully synthesized
Hydrocortisone and its by-product table hydrocortisone.
Fermentation catalyzes and synthesizes the yield of hydrocortisone in other saccharomycete of table 12
Embodiment 11, saccharomyces cerevisiae engineered yeast HC061-HC065 building and catalyze and synthesize hydrocortisone
In order to further increase the yield of hydrocortisone, the present invention by the mould middle Ac-CPR excavated of Absidia and
Ac-CYP003 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
Ac-CPR, Ac-CYP003 genetic fragment are integrated in the site rDNA of saccharomyces cerevisiae BY4742) each 1 μ l, it is transferred to electricity after mixing and turns
In 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 HC061, HC062, HC063, HC064, HC065.The wherein highest bacterium of yield
The hydrocortisone yield of strain HC064 can achieve 25.7mg/L.
<110>Tianjin Institute of Industrial Biotechnology, Chinese Accademy of Sciences
<120>mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene and application
<130> GNCLN181612
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 527
<212> PRT
<213> Absidia coerulea
<400> 1
Met Leu Thr Glu Tyr Ile His His Phe Ile Asn Asn Phe Asp Gln Lys
1 5 10 15
Lys Thr Met Asp Gln Leu Gln Thr Met Val Ser Ser Lys Glu Gly Met
20 25 30
Ile Gly Leu Ala Thr Ala Ala Val Leu Met Ser Gly Ala Ala Val Tyr
35 40 45
Lys Ser Thr Arg Ile Glu Arg Gly Cys Pro Gln Val Pro Asn Gln Ser
50 55 60
Tyr Phe Met Gly Ser Thr Lys Glu Tyr Arg Asn Asn Pro Ala Ala Phe
65 70 75 80
Ile Glu Lys Trp Glu Lys Glu Leu Gly Pro Val Tyr Gly Ala Tyr Leu
85 90 95
Phe Gly Gln Tyr Thr Thr Val Val Ser Gly Pro Gln Val Arg Glu Val
100 105 110
Phe Leu Asn Asp Asp Phe Asp Phe Ile Ala Gly Ile Arg Arg Asp Phe
115 120 125
Asp Thr Asn Leu Leu Ser Asn Gly Gly Asp Leu Arg Asp Leu Pro Val
130 135 140
His Lys Phe Ala Gly Ser Ile Lys Lys Asn Leu Ser Pro Lys Leu Pro
145 150 155 160
Phe Tyr Thr Ser Arg Val Ile Glu His Leu Lys Ile Gly Leu Lys Glu
165 170 175
Phe Cys Gly Val Val Pro Asp Glu Gly Lys Glu Phe Asp His Val Tyr
180 185 190
Pro Leu Val Gln His Met Val Ala Lys Ala Ser Ala Ser Val Phe Val
195 200 205
Gly Pro Glu Leu Ala Lys Asn Glu Gln Leu Ile Asp Ser Phe Lys Asn
210 215 220
Met Val Leu Glu Val Gly Ser Glu Leu Ala Pro Lys Pro Tyr Leu Glu
225 230 235 240
Phe Phe Pro Asn Leu Met Arg Leu Arg Met Trp Phe Ile Gly Lys Thr
245 250 255
Ser Gln Lys Val Lys Arg His Arg Asp Gln Leu Arg Ala Ala Leu Ala
260 265 270
Pro Gln Val Glu Tyr Arg Leu Lys Ala Met Lys Glu Asn Asp Ser Asn
275 280 285
Trp Asp Arg Pro Asn Asp Phe Leu Gln Asp Ile Leu Glu Ser Gly Asp
290 295 300
Ile Pro Ala His Val Asp Val Thr Asp His Cys Cys Asp Trp Met Thr
305 310 315 320
Gln Ile Ile Phe Ala Ala Leu His Thr Thr Ser Glu Asn Gly Thr Leu
325 330 335
Ser Phe Tyr Arg Leu Leu Asp Asn Pro Lys Val Leu Glu Asp Leu Leu
340 345 350
Glu Glu Gln Asn Gln Val Leu Glu Asp Ala Gly Tyr Asp Ser Ser Val
355 360 365
Gly Pro Glu Val Phe Thr Arg Glu Ile Leu Asn Lys Phe Val Lys Met
370 375 380
Asp Ser Val Ile Arg Glu Thr Ser Arg Leu Arg Asn Asp Tyr Ile Gly
385 390 395 400
Leu Pro His Lys Asn Ile Ser Ser Lys Thr Ile Thr Leu Ser Gly Gly
405 410 415
Ala Met Ile Arg Pro Gly Glu Arg Ala Tyr Val Asn Ala Tyr Ser Asn
420 425 430
His Arg Asp Gly Thr Ile Gln Lys Val Thr Asp Asn Leu Lys Ser Phe
435 440 445
Glu Pro Tyr Arg Phe Val Asn Gln Asp Arg Asn Ser Thr Lys Ile Gly
450 455 460
Glu Asp Phe Ile Phe Phe Gly Met Gly Lys His Ala Cys Pro Gly Arg
465 470 475 480
Trp Phe Ala Ile Gln Glu Ile Lys Thr Ile Ile Ala Met Met Ile Arg
485 490 495
Ser Tyr Gln Leu Ser Ala Leu Gly Pro Val Thr Phe Pro Thr Asp Asp
500 505 510
Tyr Ser Arg Ile Pro Met Gly Arg Phe Lys Ile Val Pro Arg Lys
515 520 525
<210> 2
<211> 683
<212> PRT
<213> Absidia coerulea
<400> 2
Met Asp Leu Pro Thr Ala Thr Asp Ile Asn Glu Lys Pro Lys Leu Ser
1 5 10 15
Lys Glu Glu Gln Asp Pro Arg Asn Phe Val Lys Leu Met Asn Asp Gln
20 25 30
Asn Arg Asn Glu Leu Ile Ile Phe Tyr Gly Ser Gln Thr Gly Thr Gly
35 40 45
Glu Asp Tyr Ala Gln Arg Leu Gly Lys Glu Cys Lys Lys Arg Phe Asn
50 55 60
Ile Gln Pro Met Val Ala Asp Leu Glu Asn Tyr Asp Leu Gly Tyr Leu
65 70 75 80
Asp Thr Leu Pro Lys Glu Thr Ile Ala Val Phe Val Ile Ser Thr Tyr
85 90 95
Gly Glu Gly Asp Pro Thr Asp Ser Ala Val Asn Phe Trp Glu Leu Leu
100 105 110
Asn Lys Asp Val Pro Thr Phe Ser Lys Gly Cys Ala Val Glu Arg Pro
115 120 125
Leu Lys Asp Leu Arg Tyr Phe Val Phe Gly Leu Gly Asn Arg Thr Tyr
130 135 140
Glu Tyr Phe Asn Gly Ala Ala Ile Gly Val Asp Lys Gln Leu Thr Gln
145 150 155 160
Leu Gly Ala Thr Arg Leu Gly Glu Val Gly Met Gly Asp Asp Asp Asn
165 170 175
Ser Leu Glu Asp Asp Phe Ile Gln Trp Gln Asp Gln Val Trp Pro Leu
180 185 190
Leu Ala Asp Ala Leu Ala Thr Ser Thr Asp Thr Val Asp Glu Gln Ala
195 200 205
Gln Ala Gln His Ala Tyr Lys Val Met Met Gly Gln Glu Lys Glu Asp
210 215 220
Glu Ser Phe Tyr Tyr Met Gly Glu Leu Gly Asp Thr Gln Leu Thr Thr
225 230 235 240
Trp Ser Ala Lys Arg Pro Tyr Pro Ala Pro Val Lys Ile His Asp Leu
245 250 255
Thr Pro Ala Ser Arg Asp Gln Arg His Cys Leu His Leu Asp Val Asp
260 265 270
Leu Ser Asn Ser Asn Ile Ser Tyr Thr Thr Gly Asp His Leu Gly Ile
275 280 285
Trp Pro Thr Asn Asn Glu Asp Glu Val Phe Leu Val Ser Ser Leu Phe
290 295 300
Gly Trp Asn Asp Ala Tyr Leu Asp Gln Val Ile Asn Val Val Pro Thr
305 310 315 320
Asp Ser Thr Asn Lys Pro Pro Phe Pro Gln Pro Thr Thr Leu Arg Ser
325 330 335
Ala Leu Arg His Tyr Leu Asp Ile Ala Gln Leu Pro Ser Arg Ser Thr
340 345 350
Leu Asp Leu Leu Leu Pro Ser Cys Ser Asn Asp Ser Leu Lys Ser Phe
355 360 365
Leu Gln Asn Leu Val Asn Asp Lys Asp Glu His Lys Arg Val Val Leu
370 375 380
Asp Gln Val Arg Asn Leu Gly Gln Leu Leu Ser Phe Ala Leu Glu Thr
385 390 395 400
Ile Gly Ser Thr Thr Thr Asp Gly Ala Leu Lys Asp Ile Pro Val Glu
405 410 415
Val Val Leu Glu Cys Tyr Ser Arg Leu Gln Pro Arg Tyr Tyr Ser Ile
420 425 430
Ser Ser Ser Ser Ser Glu Ser Ala Thr Thr Val Ser Ala Thr Ala Val
435 440 445
Thr Leu Lys Tyr Asn Pro Thr Pro Asp Arg Thr Val Tyr Gly Val Asn
450 455 460
Thr Asn Tyr Leu Trp Ala Ile His Gln Ser Met Ser Ser Thr Pro Ser
465 470 475 480
Ser Asp Val Pro Lys Tyr Val Val Asp Gly Pro Arg Gln Gln Tyr Leu
485 490 495
Ile Thr Lys Glu Ala Asn Ser Asp Ser Ile Lys Ile Lys Ile Pro Val
500 505 510
His Ile Arg Lys Ser Thr Phe Arg Leu Pro Pro Ser Ser Ser Thr Pro
515 520 525
Val Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe
530 535 540
Val Arg Glu Arg Val Tyr Gln Lys Gln Val Leu Gly Glu Asp Val Gly
545 550 555 560
Ala Thr Val Leu Phe Phe Gly Cys Arg Arg Ser Thr Glu Asp Tyr Leu
565 570 575
Tyr Ala Asp Glu Trp Pro Arg Leu Phe Lys Ser Leu Gly Asn Gly Pro
580 585 590
Ser Arg Ile Ile Thr Ala Phe Ser Arg Glu Ser Glu Glu Lys Lys Val
595 600 605
Tyr Val Gln Gln Arg Leu Ala Glu His Gly Gln Glu Met Trp Asp Leu
610 615 620
Leu Ala Asn Gln Gly Ala Tyr Phe Tyr Val Cys Gly Asp Ala Lys Tyr
625 630 635 640
Met Ala Lys Asp Val Gln Gln Thr Val Ile Asp Met Ala Lys Ser Phe
645 650 655
Gly Gly Leu Gly Asp Asn Glu Ala Thr Thr Phe Ile Gln Glu Leu Arg
660 665 670
Lys Ser Asn Arg Tyr Val Glu Asp Val Trp Ala
675 680
<210> 3
<211> 1584
<212> DNA
<213> Artificial sequence
<400> 3
atgttgacag aatacatcca tcatttcatc aacaactttg atcaaaagaa aactatggat 60
caattgcaaa ctatggtttc ttcaaaggag ggtatgattg gtttagctac agctgcagtt 120
ttgatgtcag gtgctgcagt ttacaagtct actagaatcg aaagaggttg tccacaagtt 180
ccaaaccaat catacttcat gggttctact aaagaataca gaaacaaccc agctgctttt 240
attgaaaagt gggaaaagga attgggtcca gtttacggtg cttacttatt cggtcaatac 300
acaactgttg tttcaggtcc acaagttaga gaagttttct tgaacgatga tttcgatttc 360
atcgcaggta tcagaagaga tttcgataca aatttgttat ctaatggtgg tgacttgaga 420
gatttgccag ttcataagtt cgctggttct attaagaaaa atttgtctcc aaagttgcca 480
ttctacactt caagagttat tgaacatttg aagatcggtt tgaaggaatt ctgtggtgtt 540
gttccagatg agggtaaaga attcgatcat gtttacccat tggttcaaca tatggttgct 600
aaagcatcag cttctgtttt tgttggtcca gaattagcta aaaatgaaca attgatcgat 660
tcttttaaaa atatggtttt agaagttggt tctgaattgg ctccaaagcc atatttggaa 720
tttttcccaa atttgatgag attgagaatg tggttcatcg gtaaaacatc tcaaaaggtt 780
aagagacata gagatcaatt gagagctgca ttggcaccac aagttgaata cagattgaag 840
gctatgaagg aaaacgattc aaactgggat agaccaaacg atttcttgca agatatcttg 900
gaatctggtg acattccagc acatgttgat gttacagatc attgttgtga ttggatgact 960
caaatcatct tcgctgcatt gcatacaact tcagaaaatg gtactttgtc tttctacaga 1020
ttgttggata acccaaaggt tttggaagat ttgttggaag aacaaaatca agttttagaa 1080
gatgctggtt acgattcttc agttggtcca gaagttttta caagagaaat cttgaataag 1140
ttcgttaaga tggattcagt tattagagaa acttctagat tgagaaacga ttacattggt 1200
ttaccacata agaacatctc ttcaaagaca atcactttgt caggtggtgc aatgattaga 1260
ccaggagaaa gagcatacgt taacgcttac tctaaccata gagatggtac aatccaaaag 1320
gttactgata atttgaaatc attcgaacca tacagattcg ttaaccaaga tagaaattct 1380
acaaaaattg gtgaagattt cattttcttt ggtatgggta aacatgcatg tccaggtaga 1440
tggttcgcta tccaagaaat taaaactatc atcgcaatga tgattagatc atatcaatta 1500
tctgctttgg gtccagttac atttccaact gatgattact ctagaattcc aatgggtaga 1560
ttcaaaattg ttccaagaaa ataa 1584
<210> 4
<211> 2052
<212> DNA
<213> Artificial sequence
<400> 4
atggatttgc caacagctac tgatatcaac gaaaagccaa agttgtctaa ggaagaacaa 60
gatccaagaa acttcgttaa gttgatgaac gatcaaaata gaaatgaatt gattattttc 120
tatggttcac aaacaggtac tggtgaagat tacgcacaaa gattgggtaa agagtgtaag 180
aaaagattca atatccaacc aatggttgct gatttggaaa actacgattt gggttacttg 240
gatactttgc caaaggaaac aatcgcagtt ttcgttatct ctacttatgg tgaaggtgac 300
ccaacagatt cagctgttaa cttctgggaa ttgttgaata aggatgttcc aacattttct 360
aaaggttgtg cagttgaaag accattgaag gatttgagat acttcgtttt cggtttgggt 420
aacagaactt acgaatactt caatggtgct gcaatcggtg ttgataagca attgactcaa 480
ttaggtgcta caagattagg tgaagttggt atgggtgacg atgataactc tttggaagat 540
gatttcatcc aatggcaaga tcaagtttgg ccattgttag ctgatgcatt ggctacatca 600
actgatacag ttgatgaaca agcacaagct caacatgcat acaaagttat gatgggtcaa 660
gaaaaggaag atgaatcttt ctactacatg ggtgaattag gtgacacaca attgactaca 720
tggtcagcaa aaagaccata cccagctcca gttaaaattc atgatttgac tccagcttct 780
agagatcaaa gacattgttt gcatttggat gttgatttgt ctaactcaaa catctcatac 840
actacaggtg accatttggg tatttggcca actaacaacg aagatgaagt tttcttggtt 900
tcttcattgt tcggttggaa cgatgcttac ttagatcaag ttattaatgt tgttccaact 960
gattctacaa ataagccacc atttccacaa ccaactacat tgagatcagc attgagacat 1020
tatttggata tcgctcaatt gccatctaga tcaacattgg atttgttgtt gccatcttgt 1080
tcaaacgatt ctttgaagtc atttttgcaa aatttggtta atgataaaga tgaacataaa 1140
agagttgttt tggatcaagt tagaaatttg ggtcaattgt tgtctttcgc attggaaact 1200
attggttcaa ctacaactga tggtgctttg aaggatatcc cagttgaagt tgttttggaa 1260
tgttactcta gattgcaacc aagatactac tcaatctctt catcttcatc tgaatctgca 1320
acaactgttt cagcaactgc tgttacattg aagtacaacc caactccaga tagaacagtt 1380
tacggtgtta acacaaacta cttgtgggct attcatcaat ctatgtcatc tactccatca 1440
tctgatgttc caaaatatgt tgttgatggt ccaagacaac aatacttaat tactaaagaa 1500
gcaaactctg attcaattaa aattaaaatt ccagttcata ttagaaaatc tacttttaga 1560
ttgccaccat catcttcaac accagttatt atggttggtc caggtactgg tgttgctcct 1620
tttagaggtt tcgttagaga aagagtttac caaaagcaag ttttgggtga agatgttggt 1680
gcaacagttt tgtttttcgg ttgtagaaga tctactgaag attatttgta cgctgatgaa 1740
tggccaagat tgtttaaatc tttgggtaat ggtccatcaa gaatcatcac agcattttct 1800
agagaatcag aagaaaagaa agtttacgtt caacaaagat tggctgaaca tggtcaagaa 1860
atgtgggatt tgttggcaaa tcaaggtgct tatttttacg tttgtggtga cgcaaaatac 1920
atggctaagg atgttcaaca aactgttatt gatatggcaa agtcttttgg tggtttaggt 1980
gacaacgaag ctacaacttt tattcaagaa ttgagaaagt caaatagata tgttgaagat 2040
gtttgggctt aa 2052
<210> 5
<211> 1584
<212> DNA
<213> Absidia coerulea
<400> 5
atgcttacag agtacattca tcattttatc aacaactttg atcaaaagaa gactatggat 60
caattacaaa ccatggtgtc atccaaagaa ggtatgatcg gtttggccac agctgcagtc 120
cttatgtctg gtgcagccgt ttacaaatca accaggatcg aacggggatg ccctcaagta 180
cctaaccagt cctactttat gggatctaca aaagaatatc gcaacaaccc tgctgcattc 240
atcgagaaat gggaaaagga actgggccct gtttatggtg cttatttgtt tggtcagtat 300
actactgttg tctctggtcc tcaagtgcgc gaagtattct tgaacgatga ctttgacttt 360
attgctggca tccgacgaga ctttgatacc aacttgttat caaatggcgg cgatcttcga 420
gacttgcctg tacacaagtt tgcgggtagt atcaagaaga acctcagccc caaactccca 480
ttctacacca gccgcgtgat tgaacatctc aagattggat taaaggaatt ctgtggagta 540
gtgccagatg aaggaaagga attcgatcat gtgtatccct tggtgcaaca tatggtagcc 600
aaagctagtg catccgtctt tgtgggtcct gaattagcca agaatgaaca attgatcgac 660
tcgtttaaga atatggtcct cgaagtggga tctgaattag cccccaagcc ttatttagaa 720
ttcttcccta atctgatgcg tctacgaatg tggtttattg gcaaaacgtc acaaaaggtc 780
aagagacatc gggatcagct tcgtgcggcc ttggcacctc aagtggaata tcgactcaag 840
gctatgaagg aaaacgatag caactgggat cgacctaacg actttttaca agatattctg 900
gaaagcggcg atatcccagc tcatgtggat gtcacggatc attgttgcga ttggatgaca 960
caaattatct ttgcagctct acacacaacc agtgaaaacg gcacattatc cttctatcgc 1020
ttattggata acccaaaggt gttggaagat ttactggaag aacaaaatca agtgttggaa 1080
gatgcaggct atgatagctc cgttggccct gaagtcttta cccgggaaat cctcaacaaa 1140
ttcgtcaaga tggatagtgt gattcgtgaa acgagtcgac tacgaaacga ctatatcggt 1200
ctccctcaca agaatattag ctcaaagaca attacattat cagggggtgc tatgatccgt 1260
ccaggtgaac gtgcttatgt gaatgcttat tccaaccatc gtgatggaac tatccaaaaa 1320
gtgacggaca acttaaaatc atttgaaccc taccgatttg taaaccaaga caggaactct 1380
acaaagattg gtgaagactt tatattcttt ggaatgggta aacatgcctg tcctggtcga 1440
tggttcgcca ttcaagaaat taaaacgatt attgccatga tgatccgaag ctaccaatta 1500
tctgcccttg gtcctgttac cttccccacc gatgactatt ccagaatacc catgggtcga 1560
ttcaagattg tgccaagaaa gtaa 1584
<210> 6
<211> 2052
<212> DNA
<213> Absidia coerulea
<400> 6
atggatctcc ctacagcaac tgatatcaat gaaaaaccca aattgtccaa ggaagaacaa 60
gatccacgca atttcgtaaa gttaatgaac gatcagaatc gaaatgaatt gatcatcttt 120
tatggttctc aaactggtac tggtgaagac tatgcgcaac gcttgggaaa ggaatgcaag 180
aagcgattca acatacaacc aatggtggcc gatctagaaa actatgatct tggctatttg 240
gatacactcc ccaaggaaac gattgccgtg tttgttatct ctacttatgg tgaaggcgac 300
cctacggata gtgcagtcaa cttttgggaa ctcttgaaca aggatgtacc taccttctct 360
aaaggttgcg cggtggaacg acctcttaaa gatttacgct actttgtctt tgggcttggc 420
aatcgaacgt atgaatactt taatggagca gctattggag tggataaaca acttactcag 480
cttggtgcaa cacgattggg cgaagtagga atgggggatg atgataactc tttggaagac 540
gattttattc aatggcaaga tcaagtatgg cctttattag cggatgcttt agcgacaagc 600
acggatacag tggatgaaca agcacaagca caacatgcgt acaaggtgat gatgggccaa 660
gaaaaggaag atgaatcatt ttactatatg ggtgagcttg gcgatactca gcttacaaca 720
tggagtgcga agcgacctta ccctgcacct gtcaagattc atgacctcac acctgcttct 780
cgtgatcaac gtcattgctt acacctggat gtggacttgt ccaacagcaa catctcttat 840
actactggcg atcatctcgg tatttggcct acgaacaacg aagacgaagt gtttttggta 900
tctagtcttt ttggttggaa tgacgcttat ctggatcaag taatcaatgt tgttcccaca 960
gattccacca acaaacctcc attcccccag cctaccacct tacggtctgc tcttcgtcat 1020
tacttggata ttgctcaact tccttctcga tctactcttg atttactgct tccttcttgc 1080
tcaaacgaca gcctaaagtc tttcttacag aacttggtca acgataaaga tgaacacaag 1140
cgggtggtat tggatcaagt tcgtaacctg ggccagcttc tctcttttgc tttggaaact 1200
attggatcca cgactactga tggtgctttg aaggatatac ccgtggaagt tgtattggaa 1260
tgctactctc ggcttcaacc tcgttattac tctatatcat catcatccag cgaatcagca 1320
actacagtta gtgcgaccgc tgtcactttg aaatacaacc caactcctga tcgaactgta 1380
tatggcgtga acaccaatta cctttgggcg atccatcaat caatgtcatc gactccatca 1440
tcggatgtgc caaagtatgt agtggatggg ccgcgtcaac aatatctgat caccaaggaa 1500
gccaacagcg actcgattaa aatcaagatt cctgtacata ttcgcaaatc caccttccgt 1560
ctacctcctt catcaagcac tcctgtcatt atggttggtc ctggtactgg tgttgctcct 1620
ttccgtggat ttgtacgtga acgtgtctac caaaagcaag tcttgggcga agatgttggt 1680
gctactgtcc tcttctttgg ctgccgacga tccaccgaag actatcttta tgctgacgaa 1740
tggccaagat tattcaagtc cctgggaaat ggtccttcta gaatcatcac cgccttctct 1800
agagaatctg aagaaaagaa ggtctacgta cagcaacgac tagccgaaca tggacaggaa 1860
atgtgggact tgttggcaaa tcaaggggct tacttttatg tctgtggtga tgcaaagtat 1920
atggcgaagg atgtgcaaca aaccgtgatc gacatggcaa agtcttttgg tggtcttggc 1980
gataacgaag ctactacctt tattcaagaa ttacggaaat ccaatcgata tgtggaagac 2040
gtgtgggcat ag 2052
<210> 7
<211> 3072
<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 caatggatct ccctacagca actgatatca 840
atgaaaaacc caaattgtcc aaggaagaac aagatccacg caatttcgta aagttaatga 900
acgatcagaa tcgaaatgaa ttgatcatct tttatggttc tcaaactggt actggtgaag 960
actatgcgca acgcttggga aaggaatgca agaagcgatt caacatacaa ccaatggtgg 1020
ccgatctaga aaactatgat cttggctatt tggatacact ccccaaggaa acgattgccg 1080
tgtttgttat ctctacttat ggtgaaggcg accctacgga tagtgcagtc aacttttggg 1140
aactcttgaa caaggatgta cctaccttct ctaaaggttg cgcggtggaa cgacctctta 1200
aagatttacg ctactttgtc tttgggcttg gcaatcgaac gtatgaatac tttaatggag 1260
cagctattgg agtggataaa caacttactc agcttggtgc aacacgattg ggcgaagtag 1320
gaatggggga tgatgataac tctttggaag acgattttat tcaatggcaa gatcaagtat 1380
ggcctttatt agcggatgct ttagcgacaa gcacggatac agtggatgaa caagcacaag 1440
cacaacatgc gtacaaggtg atgatgggcc aagaaaagga agatgaatca ttttactata 1500
tgggtgagct tggcgatact cagcttacaa catggagtgc gaagcgacct taccctgcac 1560
ctgtcaagat tcatgacctc acacctgctt ctcgtgatca acgtcattgc ttacacctgg 1620
atgtggactt gtccaacagc aacatctctt atactactgg cgatcatctc ggtatttggc 1680
ctacgaacaa cgaagacgaa gtgtttttgg tatctagtct ttttggttgg aatgacgctt 1740
atctggatca agtaatcaat gttgttccca cagattccac caacaaacct ccattccccc 1800
agcctaccac cttacggtct gctcttcgtc attacttgga tattgctcaa cttccttctc 1860
gatctactct tgatttactg cttccttctt gctcaaacga cagcctaaag tctttcttac 1920
agaacttggt caacgataaa gatgaacaca agcgggtggt attggatcaa gttcgtaacc 1980
tgggccagct tctctctttt gctttggaaa ctattggatc cacgactact gatggtgctt 2040
tgaaggatat acccgtggaa gttgtattgg aatgctactc tcggcttcaa cctcgttatt 2100
actctatatc atcatcatcc agcgaatcag caactacagt tagtgcgacc gctgtcactt 2160
tgaaatacaa cccaactcct gatcgaactg tatatggcgt gaacaccaat tacctttggg 2220
cgatccatca atcaatgtca tcgactccat catcggatgt gccaaagtat gtagtggatg 2280
ggccgcgtca acaatatctg atcaccaagg aagccaacag cgactcgatt aaaatcaaga 2340
ttcctgtaca tattcgcaaa tccaccttcc gtctacctcc ttcatcaagc actcctgtca 2400
ttatggttgg tcctggtact ggtgttgctc ctttccgtgg atttgtacgt gaacgtgtct 2460
accaaaagca agtcttgggc gaagatgttg gtgctactgt cctcttcttt ggctgccgac 2520
gatccaccga agactatctt tatgctgacg aatggccaag attattcaag tccctgggaa 2580
atggtccttc tagaatcatc accgccttct ctagagaatc tgaagaaaag aaggtctacg 2640
tacagcaacg actagccgaa catggacagg aaatgtggga cttgttggca aatcaagggg 2700
cttactttta tgtctgtggt gatgcaaagt atatggcgaa ggatgtgcaa caaaccgtga 2760
tcgacatggc aaagtctttt ggtggtcttg gcgataacga agctactacc tttattcaag 2820
aattacggaa atccaatcga tatgtggaag acgtgtgggc atagagttat aaaaaaaata 2880
agtgtataca aattttaaag tgactcttag gttttaaaac gaaaattctt attcttgagt 2940
aactctttcc tgtaggtcag gttgctttct caggtatagc atgaggtcgc tcttattgac 3000
cacacctcta ccggcatgcc gattaattaa agtgatcccc cacacaccat agcttcaaaa 3060
tgtttctact cc 3072
<210> 8
<211> 2445
<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 atgcttacag agtacattca tcattttatc aacaactttg 540
atcaaaagaa gactatggat caattacaaa ccatggtgtc atccaaagaa ggtatgatcg 600
gtttggccac agctgcagtc cttatgtctg gtgcagccgt ttacaaatca accaggatcg 660
aacggggatg ccctcaagta cctaaccagt cctactttat gggatctaca aaagaatatc 720
gcaacaaccc tgctgcattc atcgagaaat gggaaaagga actgggccct gtttatggtg 780
cttatttgtt tggtcagtat actactgttg tctctggtcc tcaagtgcgc gaagtattct 840
tgaacgatga ctttgacttt attgctggca tccgacgaga ctttgatacc aacttgttat 900
caaatggcgg cgatcttcga gacttgcctg tacacaagtt tgcgggtagt atcaagaaga 960
acctcagccc caaactccca ttctacacca gccgcgtgat tgaacatctc aagattggat 1020
taaaggaatt ctgtggagta gtgccagatg aaggaaagga attcgatcat gtgtatccct 1080
tggtgcaaca tatggtagcc aaagctagtg catccgtctt tgtgggtcct gaattagcca 1140
agaatgaaca attgatcgac tcgtttaaga atatggtcct cgaagtggga tctgaattag 1200
cccccaagcc ttatttagaa ttcttcccta atctgatgcg tctacgaatg tggtttattg 1260
gcaaaacgtc acaaaaggtc aagagacatc gggatcagct tcgtgcggcc ttggcacctc 1320
aagtggaata tcgactcaag gctatgaagg aaaacgatag caactgggat cgacctaacg 1380
actttttaca agatattctg gaaagcggcg atatcccagc tcatgtggat gtcacggatc 1440
attgttgcga ttggatgaca caaattatct ttgcagctct acacacaacc agtgaaaacg 1500
gcacattatc cttctatcgc ttattggata acccaaaggt gttggaagat ttactggaag 1560
aacaaaatca agtgttggaa gatgcaggct atgatagctc cgttggccct gaagtcttta 1620
cccgggaaat cctcaacaaa ttcgtcaaga tggatagtgt gattcgtgaa acgagtcgac 1680
tacgaaacga ctatatcggt ctccctcaca agaatattag ctcaaagaca attacattat 1740
cagggggtgc tatgatccgt ccaggtgaac gtgcttatgt gaatgcttat tccaaccatc 1800
gtgatggaac tatccaaaaa gtgacggaca acttaaaatc atttgaaccc taccgatttg 1860
taaaccaaga caggaactct acaaagattg gtgaagactt tatattcttt ggaatgggta 1920
aacatgcctg tcctggtcga tggttcgcca ttcaagaaat taaaacgatt attgccatga 1980
tgatccgaag ctaccaatta tctgcccttg gtcctgttac cttccccacc gatgactatt 2040
ccagaatacc catgggtcga ttcaagattg tgccaagaaa gtaaccgctg atcctagagg 2100
gccgcatcat gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc 2160
taaccgaaaa ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag 2220
ttatgttagt attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac 2280
gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga 2340
aggctttaat ttgcaagctg cggccctgca ttaatgaatc ggccaacgcg ccagggtttt 2400
cccagtcacg acgttgtaaa acgacggcca gtgaattgta atacg 2445
<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
<210> 13
<211> 1512
<212> DNA
<213> Artificial sequence
<400> 13
atggctttga gagctaaagc tgaagtttgt atggctgttc catggttatc tttgcaaaga 60
gctcaagctt tgggtactag agctgctaga gttccaagaa ctgttttgcc atttgaagct 120
atgccaagaa gaccaggaaa tagatggttg agattattac aaatctggag agaacaaggt 180
tatgaagatt tgcatttgga agttcatcaa acttttcaag aattgggtcc aatttttaga 240
tacgatttgg gtggtgctgg tatggtttgt gttatgttac cagaagatgt tgaaaaattg 300
caacaagttg attctttgca tccacataga atgtctttgg aaccatgggt tgcttataga 360
caacatagag gtcataaatg tggtgttttt ctattgaatg gtccagaatg gagattcaat 420
agattaagat tgaatccaga ggttttgtct ccaaatgctg ttcaaagatt tttaccaatg 480
gttgatgctg ttgctagaga tttttctcaa gctttaaaaa agaaggtctt gcaaaatgct 540
agaggttctt tgactttgga tgttcaacca tctatttttc attacactat cgaagcttct 600
aacttggctt tgtttggtga aagattaggt ttggttggtc attctccatc ttctgcttct 660
ttgaattttc tacatgcttt agaagtcatg ttcaaatcta ctgttcaatt gatgttcatg 720
ccaagatctt tgtctagatg gacatctcca aaagtttgga aagaacattt tgaagcttgg 780
gattgtattt ttcaatacgg tgacaattgt atccaaaaaa tatatcagga gctggctttt 840
tctagaccac aacaatatac atctatcgtt gctgaattgt tattgaatgc tgaattatct 900
ccagatgcta ttaaggctaa ttctatggaa ttaactgctg gttctgttga tactactgtt 960
tttccattgt tgatgacatt attcgaattg gctagaaatc caaatgttca acaagctttg 1020
agacaagaat ctttagctgc tgctgcttct atttctgaac atccacaaaa agctactact 1080
gaattaccat tgttaagagc tgctttgaaa gaaactttaa gattgtatcc agtcggtttg 1140
tttttggaaa gagttgcttc ttctgatttg gttttgcaaa attatcacat cccagctggt 1200
acattggtta gagtttttct atattctctg ggtagaaatc cagctttgtt tccaagacca 1260
gaaagatata atccacaaag atggttggat attagaggtt ctggtagaaa tttttaccat 1320
gttccatttg gtttcggtat gagacaatgt ttgggtagaa gattggctga agctgaaatg 1380
ttgttgttat tgcatcatgt tttgaagcat ttgcaagttg aaactttaac acaagaagat 1440
atcaagatgg tttactcttt tatcttgaga ccatctatgt ttccattatt aacattcaga 1500
gctatcaact aa 1512
<210> 14
<211> 561
<212> DNA
<213> Artificial sequence
<400> 14
atggctgcaa gattgttaag agtcgcttct gccgctttag gtgacactgc aggtagatgg 60
agattgttag ttagaccaag agcaggtgcc ggtggtttaa gaggttctag aggtcctggt 120
ttgggtggtg gtgctgtagc aaccagaact ttgtcagttt ccggtagagc tcaatcttca 180
tccgaagata agataaccgt tcatttcatc aacagagacg gtgaaacttt gactacaaag 240
ggtaaaattg gtgactcatt gttagacgtt gtcgtacaaa ataacttgga tatagacggt 300
ttcggtgcat gtgaaggtac attagcctgt tccacctgcc atttgatctt cgaacaacac 360
atcttcgaaa agttagaagc cattacagat gaagaaaacg atatgttgga cttagcatat 420
ggtttgaccg acagatcaag attaggttgt caaatatgct tgactaaggc tatggataac 480
atgacagtta gagtcccaga tgccgttagt gacgctagag aatctataga tatgggtatg 540
aacagttcta agatcgaata a 561
<210> 15
<211> 1479
<212> DNA
<213> Artificial sequence
<400> 15
atggctccta gatgttggag atggtggcca tggtcttcat ggaccagaac tagattacca 60
ccttctagat caatccaaaa cttcggtcaa catttctcca cccaagaaca aactcctcaa 120
atatgcgttg tcggtagtgg tccagcaggt ttctacactg cccaacattt gttgaagcat 180
cactctagag ctcacgtcga tatatacgaa aagcaattgg tacctttcag attggttaga 240
gtttggttag cattgactac accaagatct agaatgttgt tgaacacttt tactcaaact 300
gcaagatcag atagatgtgc cttctatggt aacgtcgaag taggtagaga cgttactgtc 360
caagaattga gagtttacag attgacagca gtagttttgt cttacggtgc tgaagatcat 420
caagcattgg acattcctgg tgaagaattg ccaggtgttt tctctgctag agcattcgtc 480
ggttggtaca atggtttacc tgaaaacaga gaattagctc cagatttgtc atgcgacact 540
gcagttatat tgggtcaggg taacgtcgca ttagatgtag ccagaatctt gttaacacca 600
cctgatcact tggaaaagac tgacattaca gaagctgcat taggtgcttt gagacaatct 660
agagtaaaga cagtttggat cgtcggtaga agaggtccat tgcaagttgc ttttactatt 720
aaagaattga gagaaatgat acaattgcca ggtacaagac ctatgttaga tccagctgac 780
ttcttaggtt tgcaagatag aattagagaa gccgctagac ctagaaagag attgatggaa 840
ttgttgttga gaacagcaac cgaaaagcca ggtgttgaag aagcagccag aagagcctcc 900
gctagtagag cctggggttt aagatttttc agatcccctc aacaagtttt aagattgcca 960
gatggtagag ctagaagatc cgcatggcaa agtcctgaat tggaaggtat tggtgaagcc 1020
catccaggtt ctgctcactg gggttgtggt ggtccacctt gcggtttagt tttgtccagt 1080
atcggttata aatccagacc aattgatcct agtgtcccat ttgaccctaa gttgggtgtc 1140
gtacctaata tggaaggtag agttgtcgat gttccaggtt tatactgttc cggttgggta 1200
aaaagaggtc caactggtgt tataaccact acaatgacag attcattttt gaccggtcaa 1260
atcttattgc aagacttgaa agctggtcat ttgccatctg gtccaagacc aggatctgcc 1320
tttattaagg ctttattgga ttcaagaggt gtatggccag tttctttctc agattgggaa 1380
aagttagacg ccgaagaagt ttctagaggt caagcatctg gtaaacctag agaaaagtta 1440
ttggacccac aagaaatgtt gagattattg ggtcactaa 1479
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) the 19-527 amino acids sequence at least containing SEQ ID No.1, and opened from the 19th of SEQ ID No.1
Begin to extend according to the amino acid sequence of SEQ ID No.1 to the N-terminal of SEQ ID No.1, obtaining length is 509-526 amino acids
Any one protein；
(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 is protein or amino acid sequence such as SEQ ID No.1 institute shown in any in as above (A1)-(A4)
The protein shown；
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. protein according to claim 1 or complete protein, it is characterised in that: protein shown in (A1) is
Amino acid sequence protein as shown in 19-527 of SEQ ID No.1.
3. nucleic acid molecules or complete nucleic acid molecules, it is characterised in that: the nucleic acid molecules are nucleic acid molecules 1 or nucleic acid molecules 2；
The nucleic acid molecules 1 are the nucleic acid molecules for encoding protein as claimed in claim 1 or 2；
The nucleic acid molecules 2 are DNA molecular shown in SEQ ID No.3；
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.
4. nucleic acid molecules according to claim 3 or complete nucleic acid molecules, it is characterised in that:
The nucleic acid molecules 1 be it is any in following (a1)-(a4) shown in DNA molecular:
(a1) the 55-1584 nucleotide sequences at least containing SEQ ID No.3, and from the 55th of SEQ ID No.3
Start to extend according to the nucleotide sequence of SEQ ID No.3 to the 5 ' ends of SEQ ID No.3, obtaining length is 1530 to 1583bp
Any one DNA molecular；
(a2) the 55-1584 nucleotide sequences at least containing SEQ ID No.5, and from the 55th of SEQ ID No.5
Start to extend according to the nucleotide sequence of SEQ ID No.5 to the 5 ' ends of SEQ ID No.5, obtaining length is 1530 to 1583bp
Any one DNA molecular；
(a3) hybridize under strict conditions with (a1) or (a2) DNA molecular limited and encode (A1)-described in claim 1
(A4) in it is any shown in protein DNA molecular；
(a4) with (a1)-(a3) in it is any defined by DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% with
Upper or 80% or more homology and encode it is any in (A1)-(A4) described in claim 1 shown in DNA points of protein
Son；
The nucleic acid molecules A be it is any in as above (a1)-(a4) shown in DNA molecular or nucleotide sequence such as SEQ ID
DNA molecular shown in No.3 or SEQ ID No.5；
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.
5. nucleic acid molecules according to claim 4 or complete nucleic acid molecules, it is characterised in that: DNA shown in (a1)
Molecule is nucleotide sequence DNA molecular as shown in 55-1584 of SEQ ID No.3；
DNA molecular shown in (a2) is nucleotide sequence DNA molecular as shown in 55-1584 of SEQ ID No.5.
6. any one of following biomaterial:
(c1) recombinant vector, for the recombinant vector containing the nucleic acid molecules any in claim 3-5；
(c2) expression cassette, for the expression cassette containing the nucleic acid molecules any in claim 3-5；
(c3) transgenic cell line, for the transgenic cell line containing the nucleic acid molecules any in claim 3-5；
(c4) recombinant bacterium, for the recombinant bacterium containing the nucleic acid molecules any in claim 3-5；
(c5) complete recombinant vector is made of recombinant vector A and recombinant vector B；The recombinant vector A is to contain claim 3-
5 it is any described in nucleic acid molecules A recombinant vector；The recombinant vector B be containing claim 3-5 it is any described in nucleic acid point
The recombinant vector of sub- B；
(c6) complete expression cassette is made of expression cassette A and expression cassette B；The expression cassette A be containing claim 3-5 it is any in
The expression cassette of the nucleic acid molecules A；The expression cassette B be containing claim 3-5 it is any described in nucleic acid molecules B expression
Box；
(c7) complete transgenic cell line is made of transgenic cell line A and transgenic cell line B；The transgenic cell line A
For containing claim 3-5 it is any described in nucleic acid molecules A transgenic cell line；The transgenic cell line B is containing having the right
Benefit require 3-5 it is any described in nucleic acid molecules B transgenic cell line；
(c8) complete recombinant bacterium is made of recombinant bacterium A and recombinant bacterium B；The recombinant bacterium A be containing claim 3-5 it is any in
The recombinant bacterium of the nucleic acid molecules A；The recombinant bacterium B be containing claim 3-5 it is any described in nucleic acid molecules B recombination
Bacterium.
7. a kind of method for preparing the engineering bacteria for producing hydrocortisone, includes the following steps: to be transformed saccharomycete,
It is set to express protein of any of claims 1 or 2 or complete protein, improved saccharomycete is as used to produce hydrogenation can
Pine engineering bacteria；
Further, described method includes following steps: by the nucleic acid molecules any in claim 3-5 or complete nucleic acid point
Son imports the saccharomycete, obtains the recombinant yeast for expressing protein of any of claims 1 or 2 or complete protein, i.e.,
For the engineering bacteria；
Further, the nucleic acid molecules are led by way of being the recombinant vector described in the claim 6 or the expression cassette
Enter in the saccharomycete；The complete nucleic acid molecules are by complete recombinant vector described in claim 6 or described complete
The form of 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 claim 7 the method.
9. in protein of any of claims 1 or 2 or complete protein or claim 3-5 any nucleic acid molecules or
Complete nucleic acid molecules or biomaterial as claimed in claim 6 or engineering bacteria according to any one of claims 8 it is following it is any in answer
With:
(A) hydrocortisone is prepared；
(B) catalysis steroid hormone substance carries out 11 β hydroxylatings；
Or
Protein of any of claims 1 or 2 is as the application in 11 hydroxylase of steroid hormone substance.
10. a kind of method for preparing hydrocortisone 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 contained in reaction product；The substrate is can be by 11 β of steroid-hydroxyl
Change the substance that enzymatic generates hydrocortisone；
The enzyme process, which includes the following steps: to extract from engineering bacteria according to any one of claims 8, has 11 B-hydroxylase of steroid activity
Substance, then in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder or pure enzyme catalysis substrate generate hydrocortisone；The substrate is
The substance for generating hydrocortisone can be catalyzed by 11 B-hydroxylase of steroid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810902589.2A CN109097342B (en) | 2018-08-09 | 2018-08-09 | Steroid 11 beta-hydroxylase in Absidia coerulea, coding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810902589.2A CN109097342B (en) | 2018-08-09 | 2018-08-09 | Steroid 11 beta-hydroxylase in Absidia coerulea, coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109097342A true CN109097342A (en) | 2018-12-28 |
| CN109097342B CN109097342B (en) | 2021-09-28 |
Family
ID=64849270
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810902589.2A Active CN109097342B (en) | 2018-08-09 | 2018-08-09 | Steroid 11 beta-hydroxylase in Absidia coerulea, coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109097342B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885869A (en) * | 2019-12-24 | 2020-03-17 | 天津科技大学 | Method for producing 7 α -hydroxy-dehydroepiandrosterone by microbial transformation |
| 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 |
-
2018
- 2018-08-09 CN CN201810902589.2A patent/CN109097342B/en active Active
Non-Patent Citations (5)
| Title |
|---|
| FELPETO-SANTERO ET AL.: "Identification and expression of the 11 beta-steroid hydroxylase from Cochliobolus lunatus in Corynebacterium glutamicum.", 《MICROBIAL BIOTECHNOLOGY》 * |
| J. MANOSROI ET AL.: "Biotransformation of Cortexolone to Hydrocortisone by Molds Using a Rapid Color-Development Assay.", 《APPLIED BIOCHEMISTRY AND MICROBIOLOGY》 * |
| KIM ET AL.: "Synthesis of α-ketoglutarate using glutamate dehydrogenase combined with electrochemical cofactor regeneration system.", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》 * |
| WANG ET AL.: "Cloning and identification of a novel steroid 11α-hydroxylase gene from Absidia coerulea.", 《JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY》 * |
| WANG,R.ET AL.: "steroid 11 beta hydroxylase [Absidia caerulea]", 《GENBANK: ATF27934.1》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885869A (en) * | 2019-12-24 | 2020-03-17 | 天津科技大学 | Method for producing 7 α -hydroxy-dehydroepiandrosterone by microbial transformation |
| 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 |
| CN114907997B (en) * | 2021-02-07 | 2024-02-06 | 中国科学院天津工业生物技术研究所 | Construction and application of diosgenin synthetic 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109097342B (en) | 2021-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109097343B (en) | Steroid 11 beta-hydroxylase in curvularia lunata as well as coding gene and application thereof | |
| CN110438099B (en) | Application of glycosyltransferase and related materials thereof in construction of engineering bacteria for producing ginsenosides Rb1 and Rg1 | |
| CN108060092B (en) | Recombinant bacterium and application thereof | |
| CN115011616B (en) | Acetaldehyde dehydrogenase gene RKALDH and application thereof | |
| CN107099516A (en) | 7 β hydroxy sterols dehydrogenase mutants and its application in ursodesoxycholic acid synthesis | |
| CN109097342A (en) | Mould middle 11 B-hydroxylase of steroid of Absidia and its encoding gene and application | |
| CN110283805B (en) | Monascus purpureus ester synthetase LIP05, encoding gene and application thereof | |
| CN109055327A (en) | Aldehyde Ketoreductase mutant and its application | |
| CN102382805B (en) | Polypeptide containing oxidoreductase and its use | |
| CN114107255B (en) | Panax japonicus saponin glycoside hydrolase and application thereof in production of ginger-like notoginsenoside R1 | |
| CN116987603A (en) | Recombinant saccharomyces cerevisiae strain for high yield of cannabigerolic acid as well as construction method and application thereof | |
| JP6633535B2 (en) | Method for producing 7-dehydrocholesterol and vitamin D3 | |
| CN111334522B (en) | Recombinant saccharomyces cerevisiae for producing ambergris alcohol and construction method | |
| CN107488638B (en) | 15 α -hydroxylase and preparation method and application thereof | |
| CN109385377A (en) | For producing the engineering bacteria and the preparation method and application thereof of cucurbit dienol | |
| CN111690549A (en) | Recombinant yarrowia lipolytica strain for producing protopanoxadiol by using xylose and construction method and application thereof | |
| CN107523582A (en) | A kind of engineering bacteria for producing coniferyl alcohol, construction method and the purposes for producing coniferyl alcohol | |
| CN112708602B (en) | Dioscorea zingiberensis-derived diosgenin synthesis related protein, coding gene and application | |
| CN112812983B (en) | Saccharomyces cerevisiae engineering bacterium for producing campesterol and construction method thereof | |
| CN109971730A (en) | A kind of monoamine oxidase from aspergillus niger is used for the preparation of chiral amine intermediates | |
| CN111808830A (en) | Method for producing androstadienedione by microbial degradation of phytosterol | |
| CN113430186A (en) | Fructokinase from fungus traditional Chinese medicine and coding gene and application thereof | |
| CN107903227B (en) | Succinic anhydride compound, gene and protein related to succinic anhydride compound and preparation method of succinic anhydride compound | |
| CN112646834A (en) | Lupeol derivative and synthesis method and application thereof | |
| CN113684191A (en) | Pear head mould steroid 11 beta-hydroxylase CYP5311B2 mutant construction and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |