Method for synthesizing 16 alpha-hydroxylated steroid compound by using cytochrome P450 enzyme
阅读说明:本技术 一种利用细胞色素P450酶合成16α-羟基化甾体类化合物的方法 (Method for synthesizing 16 alpha-hydroxylated steroid compound by using cytochrome P450 enzyme ) 是由 许莲花 高齐霖 林素素 于 2021-09-13 设计创作,主要内容包括:本发明公开了一种利用细胞色素P450酶合成16α-羟基化甾体化合物的方法,属于生物技术领域。该方法首先构建了来自白酒红链霉菌的P450SabH基因与还原伴侣蛋白RhFRED基因的共表达载体,在大肠杆菌中表达;经过培养后,利用静息细胞法喂养底物甾体化合物进行生物转化,获得16α位羟基化产物。此反应具有严格的区域选择性和立体选择性,产生单一产物。与此同时,此反应对几种特定的甾体化合物例如孕酮、雄烯二酮等的转化率接近100%,在工业生产当中具有极高的利用价值,提高了原料和能源利用率,降低了16α-羟基化甾体化合物的生产成本。(The invention discloses a method for synthesizing a 16 alpha-hydroxylated steroid compound by using cytochrome P450 enzyme, belonging to the technical field of biology. The method firstly constructs a co-expression vector of a P450SabH gene from white spirit streptomyces erythropolis and a reduction chaperonin RhFRED gene, and expresses the co-expression vector in escherichia coli; after culturing, the substrate steroid compound is fed by a resting cell method for biotransformation, and a 16 alpha-position hydroxylation product is obtained. This reaction is strictly regioselective and stereoselective, resulting in a single product. Meanwhile, the conversion rate of the reaction on a plurality of specific steroid compounds such as progesterone, androstenedione and the like is close to 100%, the reaction has extremely high utilization value in industrial production, the utilization rate of raw materials and energy is improved, and the production cost of the 16 alpha-hydroxylated steroid compound is reduced.)
1. A method for synthesizing 16 alpha-hydroxylated steroids by using cytochrome P450 enzyme is characterized by comprising the following steps: the 16 alpha-hydroxylated steroid compound is obtained by carrying out biotransformation on the steroid substrate by utilizing the thallus fermentation broth of the coexpression P450SabH gene and the RhFRED gene. The preparation method of the thallus fermentation liquor for co-expressing the P450SabH gene and the RhFRED gene comprises the following steps: a pET28a vector is connected with a P450SabH gene and an RhFRED gene by utilizing a seamless cloning technology to obtain a recombinant plasmid pET28a-P450SabH-RhFRED, the plasmid is transferred into escherichia coli BL21(DE3) to construct recombinant escherichia coli, and thallus fermentation liquor which co-expresses the P450SabH gene and the RhFRED gene is obtained through screening, pre-culture and expanded culture. The gene sequence of pET28a is shown in SEQ ID NO.1, and the gene sequence of pET28a-P450SabH-RhFRED is shown in SEQ ID NO.2, wherein the gene sequence of P450SabH is shown in SEQ ID NO.3, and the gene sequence of RhFRED is shown in SEQ ID NO. 4.
2. The method according to claim 1, wherein the conditions for bioconversion of steroid substrates by using the microbial fermentation broth co-expressing P450SabH gene and RhFRED gene are as follows: the thallus fermentation liquor is centrifugally resuspended in 50mM phosphate buffer solution, the pH is kept at 7.2, steroid substrates are added for biotransformation, and the reaction is oscillated at 25 ℃. Wherein the volume ratio of the phosphate buffer solution to the thallus fermentation liquor is 5: 1. The concentration of the substrate in phosphate buffer was 100. mu.M.
3. The method according to claim 2, wherein the phosphate buffer consists of phosphate at a final concentration of 50mM (pH 7.2) and glycerol at a volume of 10% of the phosphate buffer.
4. The method according to claim 1, wherein the pre-culture and the expansion culture are specifically:
adding the constructed recombinant escherichia coli into an LB culture medium, and culturing for 16h at 37 ℃ to obtain a seed solution of the recombinant escherichia coli. Inoculating the seed solution into a 2 XYT culture medium, wherein the volume ratio of the seed solution to the 2 XYT culture medium is 1:100, and performing amplification culture at 37 ℃ and the rotation speed of a shaking table of 180rpm/min until the OD of a bacterial solution is obtained600And (3) when the value reaches 0.8, adding IPTG and 5-aminolevulinic acid salt to induce the thallus to express protein, reducing the temperature to 20 ℃, and continuously culturing for 20 hours to obtain thallus fermentation liquor. The final concentration of IPTG in 2 XYT medium was 0.1mM and the concentration of 5-aminolevulinic acid salt in 2 XYT medium was 0.5 mM.
5. The method according to claim 1, wherein the substrate is progesterone and the bioconversion provides 16-hydroxysteroid compound is 16 α -hydroxyprogesterone.
6. The method of claim 1, wherein the substrate is androstenedione and the biotransformation yields a 16-hydroxylated steroid compound that is 16 α -hydroxyandrostenedione.
Technical Field
The invention belongs to the technical field of biology, and relates to a method for synthesizing 16 alpha-hydroxylated steroids by using cytochrome P450 enzyme, in particular to a method for obtaining 16 alpha-hydroxylated steroids such as 16 alpha-hydroxyprogesterone and 16 alpha-hydroxyandrostenedione by performing biotransformation by using co-expression recombinant bacteria of novel cytochrome P450 enzyme P450SabH and a reduction partner RhFRED.
Background
The cytochrome P450 enzyme as a biocatalyst has incomparable advantages of chemical catalysts, can catalyze carbon-hydrogen bonds which are not activated in a compound under mild conditions, and has strict local selectivity and stereoselectivity. It has great application potential in the fields of environmental pollutant degradation, fine chemical synthesis and synthetic biology of medicines. With the rapid development of DNA sequencing technology, the accumulation of a large amount of microbial genome data information and the mining of new P450 enzymes with industrial application value are becoming the focus of attention. The successful industrial synthesis of drug intermediates such as artemisinin, pravastatin and the like and drugs by P450 enzyme catalysis has led people to have more confidence in the industrial synthesis of more types of drugs catalyzed by P450.
Steroids are the second largest class of drugs currently in use, second only to antibiotics, and most of them are compounds with novel biological activity obtained by structural modification of natural steroids. The modification reaction of the steroid compound comprises hydroxylation, carbonylation, dehydrogenation, aromatization, bromination and the like. Structural modifications to steroids may cause dramatic changes in physicochemical and pharmaceutical properties. For example, C-15 hydroxylation of 13-methyl-estra-4-ene-3, 17-dione to give hydroxylated products which are important intermediates for contraceptive gestodenes; the 16-position hydroxylation of the glucocorticoid can obviously improve the biological activity and the like.
Disclosure of Invention
The invention aims to disclose a method for synthesizing a 16 alpha-hydroxylated steroid compound by coexpression of recombinant bacteria by using cytochrome P450 enzyme and a reduction partner RhFRED.
The purpose of the invention is realized by the following technical scheme:
a method for synthesizing 16 alpha-hydroxylated steroids by using cytochrome P450 enzyme comprises the following steps:
the 16 alpha-hydroxylated steroid compound is obtained by carrying out biotransformation on the steroid substrate by utilizing the thallus fermentation broth of the coexpression P450SabH gene and the RhFRED gene. The preparation method of the thallus fermentation liquor for co-expressing the P450SabH gene and the RhFRED gene comprises the following steps: a pET28a vector is connected with a P450SabH gene and an RhFRED gene by utilizing a seamless cloning technology to obtain a recombinant plasmid pET28a-P450SabH-RhFRED, the plasmid is transferred into escherichia coli BL21(DE3) to construct recombinant escherichia coli, and thallus fermentation liquor which co-expresses the P450SabH gene and the RhFRED gene is obtained through screening, pre-culture and expanded culture. The gene sequence of pET28a is shown in SEQ ID NO.1, and the gene sequence of pET28a-P450SabH-RhFRED is shown in SEQ ID NO.2, wherein the gene sequence of P450SabH is shown in SEQ ID NO.3, and the gene sequence of RhFRED is shown in SEQ ID NO. 4.
Further, the conditions for bioconversion of steroid substrates by using the thallus fermentation broth co-expressing the P450SabH gene and the RhFRED gene are as follows: the thallus fermentation liquor is centrifugally resuspended in 50mM phosphate buffer solution, the pH is kept at 7.2, steroid substrates are added for biotransformation, and the reaction is oscillated at 25 ℃. Wherein the volume ratio of the phosphate buffer solution to the thallus fermentation liquor is 5: 1. The concentration of the substrate in phosphate buffer was 100. mu.M.
Further, the phosphate buffer consisted of phosphate (pH 7.2) at a final concentration of 50mM and glycerol at a volume of 10% of the phosphate buffer.
Further, the pre-culture and the expanded culture specifically comprise:
adding the constructed recombinant escherichia coli into an LB culture medium, and culturing for 16h at 37 ℃ to obtain a seed solution of the recombinant escherichia coli. Inoculating the seed solution into a 2 XYT culture medium, wherein the volume ratio of the seed solution to the 2 XYT culture medium is 1:100, and performing amplification culture at 37 ℃ and the rotation speed of a shaking table of 180rpm/min until the OD of a bacterial solution is obtained600And (3) when the value reaches 0.8, adding IPTG and 5-aminolevulinic acid salt to induce the thallus to express protein, reducing the temperature to 20 ℃, and continuously culturing for 20 hours to obtain thallus fermentation liquor. The final concentration of IPTG in 2 XYT medium was 0.1mM and the concentration of 5-aminolevulinic acid salt in 2 XYT medium was 0.5 mM.
Further, the substrate is progesterone, and the 16-hydroxylated steroid compound obtained through biotransformation is 16 alpha-hydroxyprogesterone.
Further, the substrate is androstenedione, and the 16-hydroxylated steroid compound obtained through biotransformation is 16 alpha-hydroxyandrostenedione.
The invention has the beneficial effects that: the P450SabH related to the invention is a novel enzyme which is not disclosed, the recombinant bacteria of the invention can convert steroids compounds such as progesterone, androstenedione and the like to generate 16 alpha-position hydroxylation products, and the products are mainly obtained from a chemical synthesis method in the industry at present; the P450SabH enzyme has strict regioselectivity and stereoselectivity to the progesterone and the androstenedione, generates unique products, namely 16 alpha-hydroxyprogesterone and 16 alpha-hydroxyandrostenedione, and can improve the separation and extraction efficiency; the catalytic system constructed by the invention can selectively carry out oxidation reaction under mild conditions, and has great application value in the field of biological pharmacy compared with the complex process flow of the traditional chemical reaction; and fourthly, by utilizing the biotransformation of the recombinant bacteria, side reaction products can be greatly reduced, and the desired product is easy to separate, thereby achieving high efficiency. The catalytic system constructed by the invention has extremely high conversion rate to specific steroid compounds, the conversion rate to progesterone and androstenedione is close to 100%, the utilization efficiency of raw materials and energy is improved, and the catalytic system has huge market prospect.
Drawings
FIG. 1 is a scheme of bioconversion of P450SabH with a substrate;
fig. 2 is an HPLC profile of the experimental sample, wherein fig. 2A is progesterone and fig. 2B is androstenedione.
Detailed Description
The present invention will be further described in detail with reference to examples. The embodiments are provided to facilitate a better understanding of the invention and are not intended to limit the invention.
Example 1
FIG. 1 is a flow chart of bioconversion of P450SabH with a substrate, specifically comprising the steps of:
1. preparation of co-expressed recombinant E.coli:
the pET28a plasmid (SEQ ID NO.1) was linearized with restriction enzymes NdeI and XhoI to give a linearized vector.
The whole genome of the white spirit streptomyces erythreus is taken as a template, and the sequence (SEQ ID NO.3) of the coding gene sab3040 of the P450SabH is obtained through PCR amplification. Wherein, the forward primer sequence used for PCR amplification is SEQ ID NO.5, which specifically comprises: 5'-CGCGCGGCAGCCATATGAACTGCCCGCACGCCG-3', the sequence of the reverse primer is SEQ ID NO.6, which is specifically: 5'-GCAGCACGCCCAGGAGGACGGGGAAGG-3' are provided.
Using pET28b-RhFRED plasmid as a template, obtaining a coding gene RhFRED sequence (SEQ ID NO.4) of RhFRED through PCR amplification, wherein the forward primer sequence used for PCR amplification is SEQ ID NO.7, and specifically comprises the following steps: 5'-TCCTGGGCGTGCTGCACCGCCATCAACCG-3', the sequence of the reverse primer is SEQ ID NO.8, which is specifically: 5'-GGTGGTGGTGCTCGATCAGAGGCGCAGGGCCAG-3' are provided.
Mixing PCR product (P450SabH coding gene and RhFRED coding gene) and linearization carrier according to kit specification in a certain proportion, and addingII, reacting for 30min at 37 ℃ under the catalysis of recombinase to obtain a recombinant plasmid pET28a-P450SabH-RhFRED, wherein the pET28a-P450SabH-RhFRED gene sequence is shown in SEQ ID NO. 2. Transferring the recombinant plasmid pET28b-CYP154C2-RhFRED into escherichia coli BL21(DE3) competent cells, and screening out a monoclonal by using an LB solid culture medium containing kanamycin to obtain the recombinant escherichia coli with multi-enzyme co-expression.
2. Pre-culture and scale-up culture of recombinant E.coli:
inoculating the coexpression recombinant Escherichia coli into LB liquid culture medium, culturing at 37 deg.C and 220rpm under pH of 7.4-7.6 for 16 hr to obtain seed solution of recombinant Escherichia coli. Inoculating the seed liquid into a 2 XYT culture medium for amplification culture and fermentation, wherein the volume ratio of the seed liquid to the 2 XYT culture medium is 1: 100; after inoculation, the recombinant Escherichia coli is cultured for 3-4h at 37 deg.C and shaking table rotation speed of 180rpm until the OD of bacterial liquid600The value reaches 0.8, then IPTG and 5-aminolevulinic acid are added to induce the thalli to express a large amount of protein, the temperature is reduced to 20 ℃, and the process is continuedCulturing for 20h to obtain fermentation liquor. The concentration of IPTG in 2 XYT medium is 0.1mM and the concentration of 5-aminolevulinic acid salt in 2 XYT medium is 0.5 mM.
3. Biotransformation to synthesize 16 α -hydroxyprogesterone:
the fermentation broth was centrifuged at low speed to collect the cells, and the collected cells were resuspended in phosphate buffer consisting of phosphate at a final concentration of 50mM and 10 vol% glycerol, maintaining pH at 7.2. Adding progesterone, carrying out oscillation reaction at 25 ℃ for 24h, adding equal volume of ethyl acetate for extraction, collecting and combining ethyl acetate layers, and obtaining the 16 alpha-hydroxyprogesterone after the ethyl acetate is completely volatilized. The volume ratio of the fermentation liquid before centrifugation to the phosphate buffer is 5:1 (for example, 100ml of phosphate buffer is added to 500ml of the centrifuged thallus of the fermentation liquid), and the concentration of the progesterone in the phosphate buffer is 100 mu M.
Example 2
And (3) replacing the substrate in the step 3 of the example 1 with androstenedione, wherein the concentration of the androstenedione in a phosphate buffer solution is 100 mu M, and the rest conditions are unchanged to finally obtain the 16 alpha-hydroxyandrostenedione.
To 16 α -hydroxyprogesterone obtained in example 1 or 16 α -hydroxyandrostenedione obtained in example 2, 100 μ L of methanol was added for dissolution and HPLC analysis was performed. In the invention, the HPLC analysis adopts a Waters E2695 high performance liquid chromatography system of Singapore and a COSMOSIL packed column, the mobile phase adopts 35 vt-100 vt% methanol-water gradient elution for 20min, the detection wavelength is 240nm, and the flow rate is 1 ml/min.
Fig. 2 is an HPLC profile of an experimental sample, wherein fig. 2A is a standard substance of progesterone and a 16 α -hydroxyprogesterone profile obtained after 24 hours of reaction, retention times of progesterone and 16 α -hydroxyprogesterone are 19.486min and 15.069min, respectively, and the conversion rate of the biocatalysis is 99.7% calculated according to the integrated peak area of 16 α -hydroxyprogesterone/(integrated peak area of 16 α -hydroxyprogesterone + integrated peak area of progesterone) in the experimental sample; fig. 2B shows the standard of androstenedione and the 16 α -hydroxyandrostenedione spectra obtained after 24 hours of reaction, the retention times of androstenedione and 16 α -hydroxyandrostenedione are 16.268min and 13.111min, respectively, and the conversion rate of the biocatalysis is calculated to be 98.7% according to the peak integral area of 16 α -hydroxyandrostenedione/(peak integral area of 16 α -hydroxyandrostenedione + peak integral area of androstenedione) in the experimental sample. The results all show that the product obtained is the only product and the conversion efficiency is extremely high, indicating the stricter substrate selectivity and the high efficiency of catalysis.
The 16 alpha-hydroxylated steroid compound obtained by the catalytic system is expected to have better application value in the field of biological pharmacy, and compared with the disadvantages of complex process flow and multiple byproducts of traditional chemical synthesis, the product obtained by the catalytic system is single, the catalytic efficiency is extremely high, and better economic benefit and environmental benefit are achieved.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should all embodiments be exhaustive. And obvious variations or modifications of the invention may be made without departing from the scope of the invention.
Sequence listing
<110> Zhejiang university of science and engineering
<120> method for synthesizing 16 alpha-hydroxylated steroid compound by using cytochrome P450 enzyme
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gccgcagagg ccttcctcgc agtcgctggg cacgtcgatg ttgttggcgc gcaacgcgtc 360
gaggacggtc tgggtgggtt cgacccgcac ggtcagcccc gagtcacgca gttcgaggtc 420
gaaggcgtgc tcgacgtccg gatcgagcgc cgcgagggac gaggtgaagt gctcgacgtg 480
cagcgccccg tcgggccagt tccggctcgc gtcctcgagt ccggcgagca gccgcccggg 540
cccgcacgcg tagatctgga cgccgggggc gggctcggcg aggagggcgg cgaggtcgat 600
ccgggtgcct tcctcggaca cgtgcagggc ggcccggtca ccgtgcccgg cgacacgctc 660
gagataggcc atgccggaac ggtttcggcc gcagtagtgc agttcgtagc tccacccccg 720
ggcgcgggcg tggtcggcca tggccaggac tggggtgatg ccgatgccgc cggcgacgaa 780
cacgtagtgc tcggcgccgg ggtcgagcgc gaaatggttc cgagggccgc gcatccggag 840
cgggcttccc acctcgagct gttcgtggat gtagcgcgaa ccgccgcggc tctcgggatc 900
caggtgcacg gcaatctcgt agctcggcgc atcgggcgcg ccgcacaggg agtactggcg 960
cgacagcgcg ccgaggtcga ggtcgatatg ggcgccggga gtccacgtgg gcaatgtctt 1020
tccgccggcg tcgcgcagga cgaggcgcag cacgtcgtcg gcgatccggt ccaggcgctc 1080
gacggtgacg gtgcgggaca ccgcccgggc ggcgggttct ccgatggtga ccggttgatg 1140
gcggtgcagc acgcccagga ggacggggaa ggccgccatg tcgttctgcg tcatcacggg 1200
aagcctggtg atctcctcgt ccgggacggc gagacggagt tcggggaagc gggcgaacag 1260
cgcgggcagc gcgatgcccg cctcgacgcg cgacagagcg gcgccggggc agatgtgcgg 1320
gccgtgaccg aaggtcatgt ggcggttacg ggtgggccgg gtgatgtcga aggtgtcggc 1380
gtcggggccg tgctgctcgg tgtcccggcc gatggcccgg taggagatga cgacgccttc 1440
ccccgtgcgg atcaccccgt cgccgacggc tatgtcctcg gtggcgaacc gcatcagcag 1500
atgggtggtg ggggtgtccc agcgcagcgt ctcctcgatc acggcgtccc agccgatctc 1560
cccgtcgagc accttgcgca gctggcccgg gtgggagagg agggcgcgca cggcgttgag 1620
gacgagtccg atggtggtct cgtgcccggc ggcgaccatg gccttgaggt tgcccaccac 1680
ctcctcctcg gtgagcggtt cgccgccctc ctcggccagg atcagcgcac tggtgaggtc 1740
gtccgtgggc cgggcggtct tctcccgtac gaggccggcg tagaagacgt ccagctccgc 1800
cagcagggcg aggcgttcgt cctgcggggt gagcatcgag aagaacgcct tgtactgccg 1860
ggtgagcatg gcgtgctggg acgcgtcgac gcccatcagc atgccgacga ccttcatcgg 1920
cagcggctgg gcgaagaccg ccttcaggtc gacgaccccg tcctcgccct gggcggcgtc 1980
gaggtcgtcc agcagctctt cggtgaactt ctcgatgtcc gggcggatcg cctccaggcg 2040
ccggggagtc agcgcctgcg aggtcttggt gcgcagccgc cggtgttcgg caccgtcgac 2100
ggtgaacatg gaacgcccgg cgtcgatcat gccgatcagc ggccaggcac gtgtcaccgc 2160
tccgctccgc cacgcacccc aggcgtcgat gtccttcacc aggcgtgggt cgacgagcag 2220
ttggcgcgcc tcggcgtgcc gggtgaccgc ccaggccggg acgccgagca ggtcgatccg 2280
ggcgaggacc ccggcgtcgc gcagtcgcag ggtctcgccg tccaggtcct ggaccatggg 2340
gtcgatggtg atgacctcgg cgtcccggcc ggctcggtcg gtttcggcgt gcgggcagtt 2400
catatggctg ccgcgcggca ccaggccgct gctgtgatga tgatgatgat ggctgctgcc 2460
catggtatat ctccttctta aagttaaaca aaattatttc tagaggggaa ttgttatccg 2520
ctcacaattc ccctatagtg agtcgtatta atttcgcggg atcgagatct cgatcctcta 2580
cgccggacgc atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat 2640
cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt 2700
cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgca 2760
tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg gctgcttcct 2820
aatgcaggag tcgcataagg gagagcgtcg agatcccgga caccatcgaa tggcgcaaaa 2880
cctttcgcgg tatggcatga tagcgcccgg aagagagtca attcagggtg gtgaatgtga 2940
aaccagtaac gttatacgat gtcgcagagt atgccggtgt ctcttatcag accgtttccc 3000
gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg ggaaaaagtg gaagcggcga 3060
tggcggagct gaattacatt cccaaccgcg tggcacaaca actggcgggc aaacagtcgt 3120
tgctgattgg cgttgccacc tccagtctgg ccctgcacgc gccgtcgcaa attgtcgcgg 3180
cgattaaatc tcgcgccgat caactgggtg ccagcgtggt ggtgtcgatg gtagaacgaa 3240
gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct cgcgcaacgc gtcagtgggc 3300
tgatcattaa ctatccgctg gatgaccagg atgccattgc tgtggaagct gcctgcacta 3360
atgttccggc gttatttctt gatgtctctg accagacacc catcaacagt attattttct 3420
cccatgaaga cggtacgcga ctgggcgtgg agcatctggt cgcattgggt caccagcaaa 3480
tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg tctgcgtctg gctggctggc 3540
ataaatatct cactcgcaat caaattcagc cgatagcgga acgggaaggc gactggagtg 3600
ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga gggcatcgtt cccactgcga 3660
tgctggttgc caacgatcag atggcgctgg gcgcaatgcg cgccattacc gagtccgggc 3720
tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga taccgaagac agctcatgtt 3780
atatcccgcc gttaaccacc atcaaacagg attttcgcct gctggggcaa accagcgtgg 3840
accgcttgct gcaactctct cagggccagg cggtgaaggg caatcagctg ttgcccgtct 3900
cactggtgaa aagaaaaacc accctggcgc ccaatacgca aaccgcctct ccccgcgcgt 3960
tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 4020
cgcaacgcaa ttaatgtaag ttagctcact cattaggcac cgggatctcg accgatgccc 4080
ttgagagcct tcaacccagt cagctccttc cggtgggcgc ggggcatgac tatcgtcgcc 4140
gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc agcgctctgg 4200
gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga tgatcggcct gtcgcttgcg 4260
gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca ctggtcccgc caccaaacgt 4320
ttcggcgaga agcaggccat tatcgccggc atggcggccc cacgggtgcg catgatcgtg 4380
ctcctgtcgt tgaggacccg gctaggctgg cggggttgcc ttactggtta gcagaatgaa 4440
tcaccgatac gcgagcgaac gtgaagcgac tgctgctgca aaacgtctgc gacctgagca 4500
acaacatgaa tggtcttcgg tttccgtgtt tcgtaaagtc tggaaacgcg gaagtcagcg 4560
ccctgcacca ttatgttccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca 4620
cctacatctg tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc 4680
cgcatccata ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca 4740
tcagtaaccc gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac 4800
agaaatcccc cttacacgga ggcatcagtg accaaacagg aaaaaaccgc ccttaacatg 4860
gcccgcttta tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg 4920
gatgaacagg cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc 4980
tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg 5040
gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg 5100
ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat 5160
actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atatgcggtg 5220
tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc 5280
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 5340
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 5400
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 5460
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 5520
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 5580
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 5640
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 5700
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 5760
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 5820
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5880
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 5940
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 6000
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 6060
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgaacaataa 6120
aactgtctgc ttacataaac agtaatacaa ggggtgttat gagccatatt caacgggaaa 6180
cgtcttgctc taggccgcga ttaaattcca acatggatgc tgatttatat gggtataaat 6240
gggctcgcga taatgtcggg caatcaggtg cgacaatcta tcgattgtat gggaagcccg 6300
atgcgccaga gttgtttctg aaacatggca aaggtagcgt tgccaatgat gttacagatg 6360
agatggtcag actaaactgg ctgacggaat ttatgcctct tccgaccatc aagcatttta 6420
tccgtactcc tgatgatgca tggttactca ccactgcgat ccccgggaaa acagcattcc 6480
aggtattaga agaatatcct gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc 6540
tgcgccggtt gcattcgatt cctgtttgta attgtccttt taacagcgat cgcgtatttc 6600
gtctcgctca ggcgcaatca cgaatgaata acggtttggt tgatgcgagt gattttgatg 6660
acgagcgtaa tggctggcct gttgaacaag tctggaaaga aatgcataaa cttttgccat 6720
tctcaccgga ttcagtcgtc actcatggtg atttctcact tgataacctt atttttgacg 6780
aggggaaatt aataggttgt attgatgttg gacgagtcgg aatcgcagac cgataccagg 6840
atcttgccat cctatggaac tgcctcggtg agttttctcc ttcattacag aaacggcttt 6900
ttcaaaaata tggtattgat aatcctgata tgaataaatt gcagtttcat ttgatgctcg 6960
atgagttttt ctaagaatta attcatgagc ggatacatat ttgaatgtat ttagaaaaat 7020
aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgaaat tgtaaacgtt 7080
aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 7140
gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt 7200
gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga 7260
aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg 7320
gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct 7380
tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc 7440
gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt 7500
aatgcgccgc tacagggcgc gtcccattcg cca 7533
<210> 3
<211> 1254
<212> DNA
<213> white spirit Streptomyces erythropolis (Streptomyces)
<400> 3
gtgaactgcc cgcacgccga aaccgaccga gccggccggg acgccgaggt catcaccatc 60
gaccccatgg tccaggacct ggacggcgag accctgcgac tgcgcgacgc cggggtcctc 120
gcccggatcg acctgctcgg cgtcccggcc tgggcggtca cccggcacgc cgaggcgcgc 180
caactgctcg tcgacccacg cctggtgaag gacatcgacg cctggggtgc gtggcggagc 240
ggagcggtga cacgtgcctg gccgctgatc ggcatgatcg acgccgggcg ttccatgttc 300
accgtcgacg gtgccgaaca ccggcggctg cgcaccaaga cctcgcaggc gctgactccc 360
cggcgcctgg aggcgatccg cccggacatc gagaagttca ccgaagagct gctggacgac 420
ctcgacgccg cccagggcga ggacggggtc gtcgacctga aggcggtctt cgcccagccg 480
ctgccgatga aggtcgtcgg catgctgatg ggcgtcgacg cgtcccagca cgccatgctc 540
acccggcagt acaaggcgtt cttctcgatg ctcaccccgc aggacgaacg cctcgccctg 600
ctggcggagc tggacgtctt ctacgccggc ctcgtacggg agaagaccgc ccggcccacg 660
gacgacctca ccagtgcgct gatcctggcc gaggagggcg gcgaaccgct caccgaggag 720
gaggtggtgg gcaacctcaa ggccatggtc gccgccgggc acgagaccac catcggactc 780
gtcctcaacg ccgtgcgcgc cctcctctcc cacccgggcc agctgcgcaa ggtgctcgac 840
ggggagatcg gctgggacgc cgtgatcgag gagacgctgc gctgggacac ccccaccacc 900
catctgctga tgcggttcgc caccgaggac atagccgtcg gcgacggggt gatccgcacg 960
ggggaaggcg tcgtcatctc ctaccgggcc atcggccggg acaccgagca gcacggcccc 1020
gacgccgaca ccttcgacat cacccggccc acccgtaacc gccacatgac cttcggtcac 1080
ggcccgcaca tctgccccgg cgccgctctg tcgcgcgtcg aggcgggcat cgcgctgccc 1140
gcgctgttcg cccgcttccc cgaactccgt ctcgccgtcc cggacgagga gatcaccagg 1200
cttcccgtga tgacgcagaa cgacatggcg gccttccccg tcctcctggg ctga 1254
<210> 4
<211> 990
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gtgctgcacc gccatcaacc ggtcaccatc ggagaacccg ccgcccgggc ggtgtcccgc 60
accgtcaccg tcgagcgcct ggaccggatc gccgacgacg tgctgcgcct cgtcctgcgc 120
gacgccggcg gaaagacatt gcccacgtgg actcccggcg cccatatcga cctcgacctc 180
ggcgcgctgt cgcgccagta ctccctgtgc ggcgcgcccg atgcgccgag ctacgagatt 240
gccgtgcacc tggatcccga gagccgcggc ggttcgcgct acatccacga acagctcgag 300
gtgggaagcc cgctccggat gcgcggccct cggaaccatt tcgcgctcga ccccggcgcc 360
gagcactacg tgttcgtcgc cggcggcatc ggcatcaccc cagtcctggc catggccgac 420
cacgcccgcg cccgggggtg gagctacgaa ctgcactact gcggccgaaa ccgttccggc 480
atggcctatc tcgagcgtgt cgccgggcac ggtgaccggg ccgccctgca cgtgtccgag 540
gaaggcaccc ggatcgacct cgccgccctc ctcgccgagc ccgcccccgg cgtccagatc 600
tacgcgtgcg ggcccgggcg gctgctcgcc ggactcgagg acgcgagccg gaactggccc 660
gacggggcgc tgcacgtcga gcacttcacc tcgtccctcg cggcgctcga tccggacgtc 720
gagcacgcct tcgacctcga actgcgtgac tcggggctga ccgtgcgggt cgaacccacc 780
cagaccgtcc tcgacgcgtt gcgcgccaac aacatcgacg tgcccagcga ctgcgaggaa 840
ggcctctgcg gctcgtgcga ggtcgccgtc ctcgacggcg aggtcgacca tcgcgacacg 900
gtgctgacca aggccgagcg ggcggcgaac cggcagatga tgacctgctg ctcgcgtgcc 960
tgtggcgacc ggctggccct gcgcctctga 990
<210> 5
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
cgcgcggcag ccatatgaac tgcccgcacg ccg 33
<210> 6
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gcagcacgcc caggaggacg gggaagg 27
<210> 7
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tcctgggcgt gctgcaccgc catcaaccg 29
<210> 8
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ggtggtggtg ctcgatcaga ggcgcagggc cag 33