CN110305881B - Biosynthetic gene cluster of polyketide neoenterocins and application thereof - Google Patents

Biosynthetic gene cluster of polyketide neoenterocins and application thereof Download PDF

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CN110305881B
CN110305881B CN201910305429.4A CN201910305429A CN110305881B CN 110305881 B CN110305881 B CN 110305881B CN 201910305429 A CN201910305429 A CN 201910305429A CN 110305881 B CN110305881 B CN 110305881B
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polyketide
neoenterocins
streptomyces
neoenterocin
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张长生
郑六眷
蒋晓东
张庆波
朱义广
张海波
张文军
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses a biosynthetic gene cluster of polyketide neoenterocoins and application thereof. The nucleotide sequence of the biosynthetic gene cluster of the polyketide neoenterococcins is shown in SEQ ID NO.1, and the structural formula of the synthesized polyketide neoenterococcins A or B is shown in the formula (I). The invention separates two new compounds neoenterocin A and neoenterocin B synthesized by biosynthetic gene clusters of polyketide neoenterocin from fermentation culture of Streptomyces sp SCSIO11863, wherein neoenterocin B has anti-tumor activity, can be used for preparing anti-tumor active medicaments and has good application prospect.
Figure DDA0002029657890000011

Description

Biosynthetic gene cluster of polyketide neoenterocins and application thereof
The technical field is as follows:
the invention belongs to the field of industrial microorganisms, and particularly relates to a biosynthetic gene cluster of polyketide neoenterocoins and application thereof.
Background art:
cancer is one of the major diseases that endanger human health. Since the second half of the 20 th century, there is an increasing trend in the world toward malignancy and death, and particularly after the 70 s, malignancy has increased at a rate of 3% to 5% per year. Among the three major therapies for cancer, drug therapy plays an important role. In most of the synthetic chemical drugs, natural antitumor active ingredients are used as lead compounds. Therefore, the natural product is an important source of the anti-tumor drug with novel structure and unique action. Neoenterocis is a novel polyketone compound, and the compound of the family has various structures and has various biological activities such as antibiosis, tumor resistance and the like.
The invention content is as follows:
the invention aims to overcome the defects in the prior art and provides a biosynthetic gene cluster of polyketide neoenteroccins and application thereof.
The invention aims to provide a biosynthetic gene cluster of polyketide neoenterococcins, the nucleotide sequence of which is shown in SEQ ID NO.1, and the biosynthetic gene cluster comprises 20 genes, specifically:
(1) genes responsible for the synthesis of the polyketone chain skeleton and modification in the synthesis process, namely, 4 genes of encA, encB, encC and encD:
the encA is positioned at the 12721-14043 base of the sequence shown in SEQ ID NO.1, has the length of 1323 base pairs, encodes beta-polyketide acyl synthetase and has 440 amino acids;
the encB is positioned at the 11501-12724 base of the sequence shown in SEQ ID NO.1, has the length of 1221 base pairs, encodes polyketide chain length factor and has 406 amino acids;
the encC is located at 11205-11453 bases of the sequence shown in SEQ ID NO.1, has the length of 249 base pairs, encodes acyl carrier protein and has 82 amino acids;
encD is positioned at 14981 bases of the sequence shown in SEQ ID NO.1, has the length of 861 base pairs, encodes acetyl coenzyme A reductase and has 266 amino acids;
(2) genes encoding regulators and transporters, namely 3 genes in total, encE, encF and encS:
the encE is positioned at 21825-22277 bases of the sequence shown in SEQ ID NO.1, has the length of 453 base pairs, encodes a reaction regulatory protein and has 150 amino acids;
the encF is positioned at 20216-21067 bases of the sequence shown in SEQ ID NO.1, has the length of 243 base pairs, encodes the StrR family regulatory protein and has 80 amino acids;
the encS is positioned at 2476-3123 bases of the sequence shown in SEQ ID NO.1, has the length of 558 base pairs, and encodes a reaction regulatory protein regX3, 185 amino acids;
other genes involved in the biosynthesis of neoenterococcins:
the encG is positioned at the 19644-20144 bases of the sequence shown in SEQ ID NO.1, has the length of 1212 base pairs, encodes cyclase and has 115 amino acids;
the encH is located at 17870-19477 bases of the sequence shown in SEQ ID NO.1, has the length of 1503 base pairs, codes carbonitriding hydrolase and has 500 amino acids;
the encI is positioned at 17097-17873 bases of the sequence shown in SEQ ID NO.1, has the length of 777 base pairs, encodes the protein of the acetyltransferase family and has 258 amino acids;
encJ is located at 15731-169951 bases of the sequence shown in SEQ ID NO.1, has the length of 1275 base pairs, encodes the enzyme of acyl-CoA hydratase, and has 406 amino acids;
encK is located at the position of 15009-15734 bases of the sequence shown in SEQ ID NO.1, has the length of 726 base pairs, codes for SAM-dependent methyltransferase and has 241 amino acids;
the encL is positioned at the 10129-11151 base of the sequence shown in SEQ ID NO.1, has the length of 915 base pairs, codes glutamine synthetase and has 304 amino acids;
the encM is positioned at 8677-10071 bases of the sequence shown in SEQ ID NO.1, has the length of 1395 base pairs, encodes oxidoreductase and has 464 amino acids;
the encN is positioned at 6997-8565 bases of the sequence shown in SEQ ID NO.1, has the length of 1569 base pairs, encodes unknown functional protein and has 522 amino acids;
the encO is located at 6563-6955 bases of the sequence shown in SEQ ID NO.1, has the length of 393 base pairs, codes for carboxytransferase and has 130 amino acids;
the encP is located at the 4965-6536 th base of the sequence shown in SEQ ID NO.1, has the length of 1572 base pairs, encodes histidine deaminase protein and has 523 amino acids;
the encQ is positioned at the 4415-4660 th base of the sequence shown in SEQ ID NO.1, has the length of 246 base pairs, encodes unknown functional protein and has 81 amino acids;
the encR is positioned at the 3216-4421 th base of the sequence shown in SEQ ID NO.1, has the length of 1203 base pairs, encodes P450 oxidase protein and has 401 amino acids;
the encT is positioned at the 952-1938 base of the sequence shown in SEQ ID NO.1, has the length of 552 base pairs, encodes a cephamycin transporter and has 328 amino acids;
the complementary sequence of the base sequence from position 1 to position 30941 of SEQ ID NO.1 can be obtained at any time according to the principle of DNA base complementarity. The nucleotide sequence or part of the nucleotide sequence from position 1 to position 30941 of SEQ ID No.1 can be obtained by Polymerase Chain Reaction (PCR) or by digestion of the corresponding DNA with suitable restriction endonucleases or by in vitro synthetic techniques of DNA or by other suitable techniques. The present invention provides a means for obtaining a recombinant DNA vector comprising at least part of the DNA sequence in positions 1 to 30941 of SEQ ID NO. 1.
The invention also provides a way to generate a disruption or other genetic modification of neoenterocins biosynthesis genes, at least one of which comprises the nucleotide sequence from position 1 to position 30941 of SEQ ID NO. 1.
The nucleotide sequence or partial nucleotide sequence provided by the invention can obtain genes similar to the biosynthetic gene cluster of neoenterocins from other organisms by using a Polymerase Chain Reaction (PCR) method or a DNA (deoxyribonucleic acid) containing the 1 st site to the 30941 st site of the sequence SEQ ID NO.1 of the invention as a probe by Southern hybridization and other methods.
Cloned DNA comprising the nucleotide sequences provided herein, or at least a portion thereof, can be used to locate further library plasmids from Streptomyces sp. These library plasmids contain at least part of the sequence of the invention and also contain the unclosed DNA of the adjacent region of the Streptomyces sp.
The nucleotide sequences or at least part of the nucleotide sequences provided by the present invention may be modified or mutated in vitro and in vivo, including insertions, substitutions or deletions, polymerase chain reaction, error-mediated polymerase chain reaction, site-specific mutations, re-ligation of different sequences, directed evolution of different parts of the sequence or homologous sequences from other sources, or mutagenesis by ultraviolet light or chemical agents, etc.
The cloned gene comprising the nucleotide sequence provided by the invention or at least part of the nucleotide sequence can be expressed in an exogenous host by a suitable expression system to obtain the corresponding enzyme or other higher bioactive substances or yields. These exogenous hosts include E.coli, Streptomyces, Micromonospora, Pseudomonas, Bacillus, yeast, plants, animals, and the like.
Genes or gene clusters comprising the nucleotide sequences provided by the invention or at least part of the nucleotide sequences can be expressed in heterologous hosts and their function in the metabolism of the host is understood.
Genes or gene clusters comprising the nucleotide sequences or at least part of the nucleotide sequences provided by the present invention can be genetically recombined to construct recombinant vectors to obtain novel biosynthetic pathways, or can be inserted, substituted, deleted or inactivated to obtain other novel biosynthetic pathways or to produce novel compounds.
Cloned genomic DNA fragments comprising the nucleotide sequences provided by the present invention or at least part of the nucleotide sequences may be used to obtain novel structural analogues or precursors of neoenterotoxins by disrupting one or more steps of the biosynthesis of neoenterotoxins. The inclusion of DNA fragments or genes can be used to increase the production of neoenterocins or derivatives thereof and the present invention provides a way to increase production in genetically engineered microorganisms.
The second purpose of the invention is to provide two novel polyketides, namely neoentererocins A or B, which have the structural formula shown in the formula (I):
Figure BDA0002029657870000041
wherein, the compound 1 is neoenterocin A, R is H, and C6 is S configuration; compound 2 is neoenterocin B, R is OH, and C7 is in R configuration.
The third purpose of the invention is to provide the application of the biosynthetic gene cluster of the polyketide neoenterocins in the preparation of the polyketide neoenterocins A and/or B.
The fourth purpose of the invention is to provide the application of Streptomyces sp SCSIO11863 in the preparation of the polyketide compounds neoenterocins A and/or B.
The fifth purpose of the invention is to provide a preparation method of the polyketide neoentericotins A and B, wherein the polyketide neoentericotins A and B are prepared and separated from a fermentation culture of Streptomyces (Streptomyces sp.) SCSIO 11863.
The preparation method of the polyketone compounds neoenterococcins A and B comprises the following specific steps: preparing a fermentation culture of Streptomyces sp SCSIO11863, separating fermentation liquor and mycelium of the fermentation culture, extracting the fermentation liquor by butanone, and distilling and concentrating a butanone layer to obtain an extract A; extracting mycelium with acetone, distilling the extract, and concentrating to obtain extract B; and (3) performing silica gel column chromatography on the crude extract obtained by combining the extract A and the extract B, taking chloroform/methanol as an eluent, and performing column chromatography on the crude extract obtained by mixing the extract A and the extract B according to a volume ratio of 100:0 to 0:100, performing gradient elution, collecting a fraction Fr.2 subjected to gradient elution by a chloroform/methanol volume ratio of 99:1, performing gel sephadex LH-20 column chromatography on the fraction Fr.2, eluting by taking a chloroform/methanol volume ratio of 1:1 as a mobile phase, performing semi-preparative HPLC, using a C-18 reverse phase column and acetonitrile/water volume ratio of 7:3 as a mobile phase, and collecting a fraction with a retention time of 13.8min at a flow rate of 2.5mL/min to obtain a compound neoenterocin A, and collecting a fraction with a retention time of 11.6min to obtain a compound neoenterocin B.
The fermentation culture of the Streptomyces (Streptomyces sp.) SCSIO11863 is prepared by inoculating activated Streptomyces (Streptomyces sp.) SCSIO11863 into a seed culture medium, culturing at 28 ℃ and 200rpm for 2 days to prepare a seed solution, inoculating the seed solution into the fermentation culture medium, and culturing at 28 ℃ and 200rpm for 8 days to prepare the fermentation culture of the Streptomyces (Streptomyces sp.) SCSIO 11863; the seed culture medium and the fermentation culture medium are both prepared from the following components in percentage by weight: the culture medium contains glucose 15g, soybean powder 5g, peptone 10g, CaCO32g, and the balance of seawater or aged seawater with the sea salt mass fraction of 3 percent and the pH value of 7.2.
The sixth purpose of the invention is to provide the application of the polyketide neoenterococcins B or the medicinal salt thereof in preparing the antitumor drugs.
The seventh purpose of the invention is to provide an anti-tumor drug, which comprises an effective amount of the polyketide neoenterococcins B or the pharmaceutically acceptable salts thereof as an active ingredient.
The invention separates two new compounds neoenterocin A and B synthesized by biosynthetic gene clusters of the polyketide neoenterocins from fermentation cultures of Streptomyces sp SCSIO11863, wherein the neoenterocin A and B are novel polyketides with 6/5 furan rings, and the neoenterocin B has antitumor activity, can be used for preparing antitumor active medicaments and has good application prospect.
The Streptomyces sp SCSIO11863 of the present invention is disclosed in the literature: kumar Saurav et al, separation and identification of Enterocins in south China sea marine streptomyces SCSIO11863, natural product research and development 2014,26:1216-1220, and the strain is also held by the applicant and is guaranteed to be provided to the public within 20 years. The Streptomyces coelicolor YF11 disclosed by the invention is disclosed in the literature: zhou, h.; wang, y.; yu, y.; bai, t.; chen, l.; liu, p.; guo, h.; zhu, c.; tao, m.; deng, z. curr. microbiol.2012,64,185-190, the strain the present applicant also holds, warranting provision to the public since 20 years.
Description of the drawings:
FIG. 1 is a high performance liquid chromatogram of a crude extract of Streptomyces (Streptomyces sp.) SCSIO 11863;
high Performance Liquid Chromatography (HPLC) conditions: the chromatographic column is phenomex 150 × 4.6mm (sphereclone SAX), the mobile phase comprises phase A and phase B, and the mobile phase comprises phase A: 10% (volume fraction) acetonitrile + 0.08% (volume fraction) formic acid, the solvent is water, mobile phase B: 90% (volume fraction) acetonitrile, and water as solvent; sample introduction procedure: 0-20min, the mobile phase proportion is A phase/B phase (volume ratio): 95:5-0:100, 20-25min, and the mobile phase ratio is A phase/B phase (volume ratio): 0:100, 25-26min, and the proportion of mobile phase is A phase/B phase (volume ratio): 0:100-95:5, 26-30min, and the mobile phase ratio is A phase/B phase (volume ratio): 95:5, detection wavelength of 190 and 400nm, and flow rate of 1mL/min, wherein 1 represents compound 1 and 2 represents compound 2.
Figure 2 is a key HMBC for compound 1,1H–1h COSY and the crystal diffraction structure of the compound 1.
Figure 3 is the H spectrum of compound 1.
Fig. 4 is a C spectrum of compound 1.
Figure 5 is the H spectrum of compound 2.
Fig. 6 is a C spectrum of compound 2.
FIG. 7 is a schematic diagram showing the overlapping regions of the positive clones, including positive clones pCSG3000, pCSG3001, pCSG3002, pCSG3003 and pCSG3004, the green thick solid lines indicating the positions of amplified fragments of the library screening primers Enc S-F and Enc S-R of the neoenterocin gene cluster, and the boundaries of the neoenterocin biosynthetic gene cluster were determined by end sequencing.
FIG. 8 is a high pressure liquid phase analysis diagram of fermentation products in M-AM3-D medium from heterologous expression strain S.YF11/pCSG3005 and mutant strains S.YF11/pCSG3010, S.YF11/pCSG3011, S.YF11/pCSG3012, S.YF11/pCSG3013 for knocking out genes encD, encK, encM and encR based on heterologous expression: (i) detecting a standard substance of the compound 1, 2, 3, 4 and 5; (ii) detecting products of the heterologous expression strain S.YF11/pCSG3005 fermented for 6 days in an M-AM3-D fermentation culture medium; (iii) product detection of fermentation of heterologous expression strain S.YF11/pSET152AB in M-AM3-D fermentation medium for 6 days; (iv) detecting products of the heterologous expression strain S.YF11/pCSG3010 fermented in an M-AM3-D fermentation medium for 6 days; (v) detecting products of the heterologous expression strain S.YF11/pCSG3011 fermented in an M-AM3-D fermentation medium for 6 days; (vi) product detection of fermentation of heterologous expression strain S.YF11/pCSG3012 in M-AM3-D fermentation medium for 6 days, (vii) product detection of fermentation of heterologous expression strain S.YF11/pCSG3012 in M-AM3-D fermentation medium for 6 days; wherein the numbers 1, 2, 3, 4 and 5 respectively represent neoenterocin A, neoenterocin B, cinnnamic acid, 5-deoxyenterocin and enterocin.
FIG. 9 is a putative biosynthetic pathway for neoenterocins.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1: isolation and preparation of the active metabolite neoenterocin A-B
1. Preparing a culture medium:
preparing a seed culture medium: the seed culture medium contains glucose 15g, soybean powder 5g, peptone 10g, CaCO per liter32g, and the balance of seawater or aged seawater with the sea salt mass fraction of 3%, and the pH value is 7.2, and the preparation method comprises the steps of uniformly mixing the components according to the content, adjusting the pH value, and then sterilizing at 115 ℃ for 30 min.
Fermentation cultureBase preparation: each liter of fermentation medium contains 15g of glucose, 5g of soybean meal, 10g of peptone and CaCO32g, and the balance of seawater or aged seawater with the sea salt mass fraction of 3%, and the pH value is 7.2, and the preparation method comprises the steps of uniformly mixing the components according to the content, adjusting the pH value, and then sterilizing at 115 ℃ for 30 min.
2. Fermentation:
streptomyces sp SCSIO11863 of marine origin activated on a plate was inoculated into a seed medium and cultured at 28 ℃ and 200rpm for 2 days to prepare a seed solution, and 20mL of the seed solution was inoculated into a 1L Erlenmeyer flask containing 200mL of a fermentation medium (14L of the fermentation medium was inoculated thereto in total), and cultured at 28 ℃ and 200rpm for 8 days to prepare a fermentation culture of Streptomyces sp SCSIO 11863.
3. Extracting fermentation liquor:
the fermentation culture is firstly centrifuged (4000 r.min)-110min) to yield a 14L volume of supernatant (broth) and mycelium. Extracting the fermentation liquor with butanone for 3 times, and distilling and concentrating the butanone layer to obtain supernatant extract, namely extract A; the mycelium is leached for 3 times by 2L of acetone at room temperature, the extracting solution is merged, the acetone is recovered under reduced pressure, and the water is evaporated to obtain the mycelium extract, namely extract B. And combining the extract A and the extract B into an extract C (98 g).
Extract A and extract B were analyzed by HPLC and contained compounds 1 and 2, as shown in FIG. 1.
4. Isolation of the Compound
a) And (3) performing silica gel column chromatography (100-: 0 to 0:100, and collecting fraction Fr.2 which is subjected to gradient elution in a chloroform/methanol volume ratio of 99: 1.
b) The fraction fr.2(120mg) was subjected to gel sephadex LH-20 column chromatography (200cm × 250mm), eluted with chloroform-methanol at a volume ratio of 1:1 as a mobile phase to give 1200mL fractions, which were collected as one fraction per 200mL fraction to give fractions fr.2a to fr.4f in this order, and the collected fractions fr.2c to fr.2e were combined and subjected to semi-preparative high performance liquid chromatography (acetonitrile: water 70:30v/v, flow rate 2.5mL/min), prepared using a C-18 reverse phase column (250 × 10.0mm i.d.,5 μm, Phenomenex, USA), collected fractions with retention time of 11.6min were rotary evaporated to dryness to obtain neoenterocin a (37.8mg, compound 1), collected fractions with retention time of 13.8min were rotary evaporated to dryness to obtain neoenterocin B (6.5mg, compound 2).
5. Identification of Compounds
Through structural analysis, 2 compounds 1-2 (corresponding to the compound neoenterocin A-B) (formula (I)) prepared from fermentation culture of Streptomyces sp SCSIO11863 of the invention were identified as follows:
FIG. 2 key HMBC of Compound 1,1H–1h COSY and the crystal diffraction structure of the compound 1.
Figure 3 spectrum H of compound 1.
Figure 4 spectrum C of compound 1.
Figure 5 spectrum H of compound 2.
Figure 6 spectrum C of compound 2.
Compound 1(neoenterocin a) is a pale yellow powder,
Figure BDA0002029657870000071
UV(MeOH)λmax(logε)365nm(2.552),257nm(2.564),205nm(2.755).IRνmax 3510.45cm-1 1670.35cm- 11535.13cm-1 1550.77cm-1 1521.84cm-1 1448.54cm-1 678.94cm-1.1H and 13C NMR data,see Table 1；HRESIMS(m/z 355.1174,[M+H]+,calcd for 355.1182)
the positive source high resolution electrospray mass spectrogram of the compound 1 shows that the quasi-molecular ion peak is M/z 355.1174, [ M + H ]]+The molecular formula is presumed to be C20H18O6(calculated 355.1182, unsaturation 12) and IR spectrum 3510cm-1And 678.94cm-1Strong absorption, suggesting the presence of OH and a benzene ring in the molecule. Analysis of1H,13The C and HSQC (Table 1) data indicate that this compound contains two methyl groups (. delta.) (H 2.16,brs；δH3.72, s), two methylene groups (. delta.))H 3.00；δH 2.70,254), seven methines, 5 of which are monosubstituted CH on the phenyl ring, one SP2Hybrid CH, and an SP3A hybridized CH. In addition, the carbon spectrum also shows eight quaternary carbons. 2D NMR showed that Compound 1 had a benzoyl fragment and a 2-methoxy-6-keto-8-hydroxy-2, 4 nonadiene fragment, and these signals above do not confirm the structure of Compound 1. Finally, the structure of the compound 1 is analyzed through single crystal diffraction analysis, and the structural formula of the compound is shown as 1 in the formula (I), and the compound is named as neoenterocin A.
The NMR spectrum of compound 2 was similar to that of compound 1 (table 1). Compared with compound 1, the molecular weight is 16 more than that of compound 1, and C-7 (delta) in compound 1C47.1) chemical shift of carbon up to 78.0, indicating that C-7 in Compound 1 is methylene and CH-OH in Compound 2. In addition, the split of H-7 in the hydrogen spectrum of Compound 1 was 2.70(dd,16.4, 2.5); 2.54(dd,16.4,6.8), the split of H-7 in the hydrogen spectrum of Compound 2 was 4.46(d,2.5), the larger coupling constant disappeared, and it was therefore presumed that the relative configuration of C-7 was the R configuration. The structural formula of the compound 2 is shown as 2 of the formula (I), and the compound is named as neoenterocin B.
Figure BDA0002029657870000081
TABLE 1 assignment of NMR (500MHz) Nuclear magnetic data for Neoenterocin A (1) and B (2) Compounds
Table1.1H NMR(500MHz)and 13C NMR(125MHz)assignments of compounds 1–2(J in Hz within parentheses).
Figure BDA0002029657870000082
Figure BDA0002029657870000091
Example 2: heterologous expression of biosynthetic gene clusters of polyketides neoenterocins
Streptomyces sp.scsio 11863 genome sequence scanning and biosynthetic gene cluster sequence and functional analysis of neoenterocins:
a 23kb biosynthetic gene cluster of neoenteroccins, containing 20 Open Reading Frames (ORFs), was found by genome-wide scanning and annotation of Streptomyces sp.scsio 11863 (table 2). Analyzing 4 genes of encA, encB, encC and encD according to bioinformatics, wherein the genes are responsible for synthesizing a polyketone chain framework and modifying the polyketone chain framework in the synthesizing process; encE, encF, encS, encG, encH, encI, encJ, encK, encL, encM, encN, encO, encP are responsible for the cyclization and post-modification of the compound neoenterocoins. The biosynthetic pathway of neoenterocins was initially speculated as shown in FIG. 9.
TABLE 2 genes of the neoenterocins biosynthetic gene cluster and functional analysis thereof
Figure BDA0002029657870000092
Figure BDA0002029657870000101
Cloning and analysis of biosynthetic Gene clusters of neoenterocins:
based on genome sequence annotation and bioinformatics analysis, PCR screening primers were designed based on the sequence of Malonyl CoA-acyl carrier protein transferase gene encL located in the middle of the neoenterocins main gene cluster, and screened from 1800 clones of the genomic library of Streptomyces sp.scsio 11863 to obtain 5 positive clones, and then the relative positions of 5 of the cosmids in the gene cluster were determined by end sequencing, and 1 cosmidd (pCSG3000) (the nucleotide sequence of which is shown in SEQ ID No. 1) contained the entire neoenterocins biosynthetic gene cluster, and the other 4 cosmidds (pCSG3001, pCSG3002, pCSG3003, pCSG3004) contained partial gene clusters (fig. 7). Bioinformatics analysis shows that the nucleotide sequence of the biosynthetic gene cluster of the neoenterocins is shown as the base sequence from 952 th site to 22277 th site of SEQ ID NO. 1.
TABLE 3 knockout primers, detection primer names and sequences required for mutant strain construction
Figure BDA0002029657870000102
Figure BDA0002029657870000111
Functional analysis of the genes in the biosynthetic Gene Cluster of neoenterocins
On the basis of cloning and analyzing a complete biosynthetic gene cluster of neoenterocins and researching possible functions of coding proteins of each gene, the invention adopts a heterologous expression method to discuss the biosynthetic mechanism of the neoenterocins. A heterologous expression vector based on pSET152AB was constructed: pCSG3005 (derived from pCSG3000, i.e., a recombinant heterologous expression plasmid pCSG3005 obtained by double digestion of the heterologous expression vector pSET152AB with EcoR I and BamH I by means of homologous recombination to give the fragment pSET152AB sequence which replaces the kanamycin resistance gene in cosmid (pCSG 3000)). Streptomyces coelicolor YF11 is transformed by the empty vectors of pCSG3005 and pSET152AB respectively to obtain corresponding Streptomyces YF11 transformants which are S.YF11/pCSG3005 and S.YF11/pSET152AB respectively.
Compared with S.YF11/pSET152AB of Streptomyces transformant containing empty vector pSET152AB, S.YF11/pCSG3005 successfully expressed 5-deoxyentococin (4), enterocin (5) and neoenterocin A (1), neoenterocin B (2) ( compounds 1 and 2 shown in FIG. 8) which are novel skeletons, and the result shows that the insert of pCSG3005 already contains the complete neoenterocin biosynthesis gene cluster and can express the main product of neoenterocins, and the result is shown in FIG. 8.
The invention fully proves the integrity of the biosynthetic gene cluster of the neoenterocins, successfully realizes the production of the neoenterocins and the structural analogues thereof in heterologous cell factory streptomyces S.YF11 by the biosynthetic gene cluster of the neoenterocins, and provides a new strategy for creating the neoenterocins and the intermediate compounds thereof (figure 8).
The following further provides examples which are useful for understanding the present invention, and are intended to be illustrative only and not limiting as to the scope of the invention.
Example 1: extraction of genomic DNA of neoenterocins and related compound-producing bacteria Streptomyces sp.SCSIO 11863
Inoculating mycelium of Streptomyces sp.SCSIO 11863 in 50mL of 1#Culture medium (1)#The preparation method of the culture medium comprises the following steps: weighing 10g of starch, 4g of yeast powder, 2g of bacteriological peptone and 10g of sea salt, adding water to a constant volume of 1L, and adjusting the pH value to 7.2-7.4), carrying out shaking culture at 28-30 ℃ for about 2-3 days, and centrifuging at 4000rpm for 10 minutes to collect mycelia. The mycelia were washed twice with STE solution (NaCl 75mM, EDTA 25mM, Tris-Cl 20mM), 30mL of STE solution and lysozyme at a final concentration of 3mg/mL were added to the washed mycelia, vortexed uniformly, incubated at 37 ℃ for 3 hours, proteinase K at a final concentration of 0.1-0.2mg/mL was added, mixed well, incubated at 37 ℃ for 10 minutes, SDS at a final concentration of 1-2% was added, mixed well, placed in a 55 ℃ water bath for about 1 hour, and the phases were reversed several times. An equal volume of phenol-chloroform-isoamyl alcohol (V/V25: 24:1) was added, mixed well, and cooled on ice for 30 minutes. Centrifugation was carried out at 12000rpm at 4 ℃ for 10 minutes, and then the supernatant was carefully aspirated into a new centrifuge tube using a cut large-diameter pipette tip, and the treatment was repeated 3 times in the same manner, followed by washing twice with an equal volume of chloroform at 12000rpm and centrifugation at 4 ℃ for 10 minutes. The water phase is sucked out by a cut large-caliber gun head and transferred to a new centrifuge tube, 1/10 volume of 3mol/L NaAc (pH5.2) is added, after mixing, equal volume of isopropanol is added, after mixing, the mixture is placed on ice, and DNA is precipitated. The DNA fiber mass was carefully transferred to a new centrifuge tube with a glass rod, washed twice with 70% ethanol, the liquid was decanted, slightly dried at 37 ℃, dissolved with 5mL of TE, and 3-5U of RNase was added, thereby obtaining Streptomyces sp.SCSIO 11863 genomic DNA.
Example 2: construction of Streptomyces sp.SCSIO 11863 genomic library of neoenterocins producing strain
The amount of restriction endonuclease Sau3A I was first determined by a series of dilution experiments in a 20. mu.L system containing 17. mu.L of Streptomyces sp.SCSIO 11863 genomic DNA, 2. mu.L of 10 × reaction buffer and 1. mu.L of different dilutions of Sau3A I, which stopped the reaction at 4. mu.L of 0.5mol/L EDTA and appropriate loading buffer. The enzyme activity unit of 0.025-0.05U is determined to be more appropriate by groping. On the basis, a large number of parts are digested to obtain 30-42 kb genome DNA fragments, and dephosphorylation treatment is carried out by using dephosphorylation enzyme.
The vector SuperCos l plasmid used for library construction was first cleaved with the restriction endonuclease Xba I from the middle of the two cos sequences, followed by dephosphorylation, and cleavage with the restriction endonuclease Bam HI from the multiple cloning site to obtain two arms. The treated vector was ligated overnight with the previously prepared partially digested 30-42 kb genomic DNA fragment, with a ligation system of 10. mu.L containing 1.25. mu.g of the prepared genomic DNA fragment and 0.5. mu.g of the treated SuperCos 1 plasmid, 1. mu.L of 10 Xbuffer, 0.3U of ligase. The ligation product was treated at 65 ℃ for 15 minutes to inactivate the ligase. A tube of the packaging mixture (50. mu.L) was taken out of the freezer at-80 ℃ on ice, the packaging mixture was rapidly thawed between the fingers, half of the packaging mixture (25. mu.L) was carefully pipetted into a new centrifuge tube, 10. mu.L of the heat-treated ligation product was added, and the remaining packaging mixture was stored at-80 ℃. Carefully mix, incubate for 90 minutes at 30 ℃, add the other half of the packaged mixture (25 μ L), and incubate for 90 minutes at 30 ℃. Add 500. mu.L phage dilution buffer (100mmol/L NaCl, 10mmol/L MgCl)210mmol/L Tris-HCl pH 8.3), followed by addition of 25. mu.L chloroform, gentle mixing, and storage at 4 ℃ to obtain a packaging solution.
The strain E.coli LM392MP frozen at-80 ℃ was plated on LB medium for recovery. One day before the packaging reaction, a single clone was selected and inoculated into LB medium (supplemented with 0.2% maltose and 10mM MgSO4) The cells were cultured overnight with shaking at 37 ℃ and, on the day of the packaging reaction, 5mL of the overnight-cultured broth was added to 50mL of fresh LB medium (supplemented with 0.2% maltose and 10mM MgSO. sub.MgSO.)4) Shaking at 37 ℃ and 200rpm to OD of the culture600When the temperature reaches 0.8-1 ℃, storing at 4 ℃ for later use. Mixing 100 μ L of the above treated host bacterial solution and 100 μ L of appropriately diluted packaging solution, and mixingAfter being incubated at 37 ℃ for 15 minutes, the cells were plated on LB plates containing 100. mu.g/mL ampicillin and 50. mu.g/mL kanamycin, and incubated overnight at 37 ℃. The single colonies grown out were spotted with sterile toothpicks onto 18 96-well plates of LB medium containing 100. mu.g/mL ampicillin and 50. mu.g/mL kanamycin, cultured overnight at 37 ℃, added with glycerol to a final concentration of 20%, mixed well, and stored at-80 ℃. Thus, a genomic library of Streptomyces sp.SCSIO 11863, a strain producing neoenterocins, was obtained.
Example 3: screening of genomic library of Streptomyces sp.SCSIO 11863, a strain producing neoenterocins, for positive clones containing a biological gene synthesized by neoenterocins
Streptomyces sp.SCSIO 11863 genomic DNA was sent to the southern national research center for genome scanning and annotation, primers Enc S-F and Enc S-R were screened by bioinformatics analysis based on the scanning and annotation results by designing PCR with the sequence of gene encL located in the middle of the neoenterocin host gene cluster (primer sequences are shown in Table 3), 5 positive clones were obtained from the genomic library 1800 clones of Streptomyces sp.SCSIO 11863, and the screened positive clones were subjected to end sequencing to determine the relative positions of 5 cosmids in the gene cluster, and 1 cosmidd (pCSG3000) contained the entire neoenterocin biosynthetic gene cluster, and the other 4 cosmidds (pCSG3001, pCSG3002, pCSG3003, pCSG3004) contained partial gene clusters (FIG. 7). The nucleotide sequence of the cosmid (pCSG3000) is shown in SEQ ID NO. 1.
Example 4: obtaining of heterologous expression strain S.YF11/pCSG3005 of neoenterocins biosynthetic gene cluster
A homologous recombination method is used for obtaining a heterologous expression strain, and the specific steps are as follows: (1) the cosmid plasmid pCSG3000 containing the biosynthetic gene cluster gene of neoenterocins is transferred into Escherichia coli E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/pCSG3000, and a lambda/red recombination system is induced to express by 10 mmol/L-arabinose and is prepared into electrotransferase competent cells for later use. (2) The heterologous expression vector pSET152AB was digested with EcoR I and BamH I to give a fragment pSET152AB sequence, which was used to replace the kanamycin resistance gene in cosmid (pCSG3000) to obtain recombinant heterologous expression plasmid pCSG 3005. The strain is transformed into E.coli ET12567/pUZ8002 to construct E.coli ET12567/pUZ8002/pCSG3005 as a donor strain for conjugal transfer.
Streptomyces coelicolor YF11 was streaked on MS (20 g mannitol, 20g soybean meal, 18g agar, water to 1L, pH 7.2) plates for 7-15 days, and the spores were scraped with a sterile cotton swab and placed in a 50mL centrifuge tube as the recipient for conjugal transfer. Coli ET12567/pUZ8002/pCSG3005 in 50mL LB liquid medium containing 50. mu.g/mL kanamycin, 25. mu.g/mL chloramphenicol and 50. mu.g/mL apramycin at 37 ℃ to OD600When the value was about 0.8, the cells were collected by centrifugation (4000rpm, 10min), washed 3 times with LB, suspended in 1mL of LB medium, and used as donor cells for conjugative transfer. And uniformly mixing 400 mu L of the recipient bacterium and 200 mu L of the donor bacterium, coating the mixture on ISP4 solid culture medium without any antibiotic, drying the mixture by blowing, and culturing the mixture for 20 to 24 hours at 28 ℃. The plates were then removed, covered with water containing antibiotics to a final concentration of 50. mu.g/mL apramycin and 50. mu.g/mL trimethoprim, blown dry, placed in a 28 ℃ incubator, incubated for 7-14 days and observed.
After the small bacteria grow on the conjugation transfer plate, the bacteria are transferred to an MS culture medium plate containing 50 mug/mL apramycin and 50 mug/mL trimethoprim by using a sterile toothpick, after the bacteria are cultured for 10 to 14 days at 28 ℃, genome DNA of a conjugant is extracted, and detection primers neo F-F/R and neo R-F/R (the primer sequences are shown in a table 3) of a detection primer of heterologous expression are utilized to obtain positive clone through PCR detection, namely, a heterologous expression strain S.YF11/pCSG3005 of the biosynthetic gene cluster of the neoenterocins is obtained.
Example 5: establishment of a genetic transfer system in the neoenterocins biosynthetic gene cluster and acquisition of gene disruption mutant strains:
obtaining acetyl coenzyme A reductase gene encD frame deletion mutant strain S.YF11/pCSG30010
Obtaining in vivo knockout mutant strains by using a PCR-targeting method. According to the obtained sequence of the biosynthetic gene cluster of neoenterocins, a pair of knock-out primers of encD genes are designed by referring to a PCR-targeting system reported in the literature, and the primer sequences are shown in encD knock-out primers EncD Tar F and EncD Tar R in Table 3. Then constructing an in vitro knockout plasmid by referring to a PCR-targeting method, and then transferring the knockout plasmid into a conjugately transferred donor bacterium. The method comprises the following specific steps: (1) the cosmid plasmid pCSG3005 containing the biosynthetic gene cluster gene of neoenterocins is transferred into Escherichia coli E.coli BW25113/pIJ790 to obtain E.coli BW25113/pIJ790/pCSG3005, and a lambda/red recombination system is induced to express by using 10 mmol/L-arabinose and is prepared into an electrotransferase competent cell for later use. (2) The plasmid pIJ778 was digested with the endonucleases EcoR I and Hind III, and a DNA fragment of about 1.4kb (i.e.: oriT + aadA gene cassette) containing the origin of transfer oriT and spectinomycin resistance gene was recovered, and a 1.4kb PCR product (a fragment containing the origin of transfer and spectinomycin resistance gene at both ends for homologous exchange with the encD gene on pCSG3005) was amplified by PCR using the knock-out primers encD Tar F and encD Tar R of gene encD as primers and the oriT + aadA gene cassette as a PCR template, and a 50. mu.L PCR reaction system: 3U of high-fidelity DNA polymerase, 5 mu L of 10 multiplied by Buffer, 0.5mmol/L of dNTPs, 2.5 mu L of DMSO, 0.5 mu mol/L of each primer and about 1ng of DNA template, and water is added to supplement the volume to 50 mu L. The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; the amplification cycle is 94 ℃ denaturation for 45s, 60 ℃ annealing for 45s, 72 ℃ extension for 90s, and 30 cycles; finally, extension is carried out for 10min at 72 ℃. The 1.4kb PCR product was recovered and purified for use. (3) The recovered 1.4kb PCR product was electroporated into the competent cells prepared in step (1) to be recombined, plated on LB selection plate (containing 50. mu.g/mL spectinomycin), and cultured overnight at 37 ℃. Positive single clones were picked from the plate, and a plasmid in which a partial fragment of the encD gene was replaced with the oriT + aadA gene cassette (i.e., a partial fragment of the encD gene on the pCSG3005 plasmid was replaced with the oriT + aadA gene cassette) was extracted and the recombinant plasmid was designated as pCSG 3006. (4) The constructed recombinant plasmid pCSG3006 is transformed into E.coli DH5 alpha/BT 340 competent cells, and the oriT + aadA gene cassette is deleted in frame to obtain plasmid pCSG3010 (the oriT + aadA gene cassette on the recombinant plasmid pCSG3006 is digested by FLP recombinase expressed by plasmid BT340 to delete the oriT + aadA gene cassette, the obtained recombinant plasmid with part of the gene encD deleted in frame is named as pCSG3010), and the recombinant plasmid is transformed into E.coli ET12567/pUZ8002 to construct E.coli ET12567/pUZ8002/pCSG3010 as a donor bacterium for conjugal transfer.
Streptomyces coelicolor YF11 was streaked on MS (20 g mannitol, 20g soybean meal, 18g agar, water to 1L, pH 7.2) plates for 7-15 days, and the spores were scraped with a sterile cotton swab and placed in a 50mL centrifuge tube as the recipient for conjugal transfer. Coli ET12567/pUZ8002/pCSG3010 Donor bacteria were grown to OD at 37 ℃ in 50mL LB liquid medium containing 50. mu.g/mL kanamycin, 25. mu.g/mL chloramphenicol, and 50. mu.g/mL apramycin600When the value was about 0.8, the cells were collected by centrifugation (4000rpm, 10min), washed 3 times with LB, suspended in 1mL of LB medium, and used as donor cells for conjugative transfer. And uniformly mixing 400 mu L of the recipient bacterium and 200 mu L of the donor bacterium, coating the mixture on ISP4 solid culture medium without any antibiotic, drying the mixture by blowing, and culturing the mixture for 20 to 24 hours at 28 ℃. The plates were then removed, covered with water containing antibiotics to a final concentration of 50. mu.g/mL apramycin and 50. mu.g/mL trimethoprim, blown dry, placed in a 28 ℃ incubator, incubated for 14-21 days and observed.
After the growth of the microspores on the conjugative transfer plate, the microspores are transferred to a Gao's first plate containing 50 mug/mL of apramycin and 50 mug/mL of trimethoprim by using a sterile toothpick, after the culture is carried out for 10 to 14 days at 28 ℃, genome DNA of each mutant strain is extracted, and positive clones are obtained by PCR detection by using detection primers EncD test F and EncD test R (primer sequences are shown in Table 3) of encD, namely, a mutant strain S.YF11/pCSG3010 with the gene encD deleted in frame is obtained (namely, a plasmid pCSG3010 with the gene encD deleted in frame is transformed into Streptomyces coelicolor YF11, and the obtained Streptomyces YF11 containing the plasmid pCSG3010 is named as S.YF11/pCSG 3010).
The inactivation primers and detection primers of other genes are shown in Table 3, and the PCR-targeting technology is used to obtain mutant strains with knocked-out genes by referring to the method.
The method comprises the following specific steps:
the gene encK (encoding SAM-dependent methyltransferase) is deleted in the same frame, the used knockout primers are EncK Tar F and EncK Tar R, the detection primers are EncK test F and EncK test R, and a mutant strain S.F11/pCSG3011 is obtained; the oxidoreductase gene encM is deleted in the same frame, primers EncM Tar F and EncM Tar R are knocked out, detection primers are EncM test F and EncM test R, and a mutant strain S.F11/pCSG3012 is obtained; the P450 oxidase gene encR is deleted in the same frame, primers EncR Tar F and EncR Tar R are knocked out, detection primers are EncR test F and EncR test R, and a mutant strain S.YF11/pCSG3013 is obtained.
Example 6: biological fermentation and detection of neoenterocoins and derivatives thereof
After S.YF11 and heterologous expression strain S.YF11/pCSG3005 and mutant strain thereof are activated, inoculating the activated strain into 50mL fermentation medium M-AM3-D (glucose 10g, soybean powder 5g, peptone 10g, CaCO) according to the inoculum size of 5 percent32g of sea salt, adding water to a constant volume of 1L, and adjusting the pH value to 7.2-7.4), culturing at 28 ℃ for 6-8 days, adding butanone with the same volume, carrying out ultrasonic treatment for 30min to break cells, stirring for 30min, and then standing for layering. Separating butanone extract from water phase, evaporating butanone to dryness by using a rotary evaporator, dissolving crude extract in methanol to form a sample, and performing High Performance Liquid Chromatography (HPLC) detection under the following detection conditions: phenomex C184.6X 150mm reversed-phase column, wherein the mobile phase A is 10% acetonitrile (containing 0.1% formic acid), and the mobile phase B is 90% acetonitrile; the flow rate was 1mL/min and the detection wavelength was 258 nm. HPLC procedure: 0-20min, 5% -100% of phase B; 21-25min, 100% phase B; 26-30min, 100% of phase B.
Example 7: use of biosynthetic gene clusters of neoenterocins-genetic modification of biosynthetic genes to obtain structural analogs:
in-frame deletions of some of the genes in the biosynthetic gene cluster of neoenterocins were performed by the methods described in examples 4 and 5, and intermediate neoenterocin a (1), compound 1 and 5-deoxyenterocin (4), compound 4, was obtained in the mutant strain s.yf11/pCSG3013 (fig. 8), with the following results:
(1) heterologous expression strain s.yf11/pCSG3005 contains the complete neoenterocins biosynthetic gene cluster, obtained via fermentation, neoenterocins a and neoenterocins B (fig. 8 (II)); whereas the Streptomyces transformant S.YF11/pSET152AB, which contained the empty vector pSET152AB, was unable to produce neoenterococcins A and neoenterococcins B after fermentation (FIG. 8 (III));
(2) knocking off acetyl coenzyme A reductase gene encD, obtaining a mutant strain S.YF11/pCSG3010 which can not produce neoenterocins A and B and related compounds thereof after fermentation (figure 8 (IV));
(3) knocking out SAM-dependent methyltransferase gene encK, obtaining a mutant strain S.YF11/pCSG3011, and fermenting to be incapable of producing neoenterocins A and B and related compounds (figure 8 (V));
(4) knocking out oxidoreductase gene encM, obtaining a mutant strain S.YF11/pCSG3012, and fermenting to generate a related compound wailupenmycins (figure 8 (VI));
(5) the P450 oxidase gene encR was knocked out to obtain a mutant strain S.YF11/pCSG3013 which, after fermentation, could produce the intermediates neoenterocin A (1) -Compound 1 and 5-deoxyenterocin (4) -Compound 4, but could not produce neoenterocin B (2) and enterocin (5) (FIG. 8 (VII)).
Example 8: determination of the antitumor Activity of Neoenterocin A, B
Neoenterocin a-B was assayed for anti-tumor activity against 4 tumor cell lines, experimental method reference [ Liu, h; tan, h.; chen, y.; guo, x.; wang, w.; guo, h.; liu, z.; zhang, w.org.lett.2019,21,1063]Cissplatin was used as a control. The results show the IC of neoenterocin B on four tumor cell lines5018.88-73.78 muM (Table 4), and can be developed into new antitumor drugs by biological engineering or chemical modification.
TABLE 4 determination of antitumor Activity of Compounds Neoenterocin A and B
Figure BDA0002029657870000171
aCisplatin,positive control.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> biosynthetic gene cluster of polyketide neoenterocins and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30941
<212> DNA
<213> Streptomyces SCSIO11863 (Streptomyces sp. SCSIO 11863)
<400> 1
gggggtgtcc atgccggtcc actacgggtg tggaagtgca ggtcagggcc ttctgatcca 60
gcagacttca tccagttggt gatcgctgcg tgacagacga gtgatacggc gaggtcccgc 120
agggcgcgtc cgggccggac tttgcgtggc gtcgcgcgtt gacgaagccg cgacggagca 180
ttccgtcgcg gcttcgtcgt gagaccctgt cggctgccgc gcacgaagag gttggcactc 240
cctgctgtcg gagccttatg acgcttcgcg ggtgcgccca cttcgcgatc gcactcattg 300
ccgggagcgg atttcatgca tcccggaatg cgcgcacaca tgcaggtgcc ctccgcattg 360
aggccgtgag cgtcaggcgt ggcgcaggca gagggagccc cactgtctgt acggcaccgt 420
gctcacctcc ggcgccgcga gcctggtcgg cctcacctat gccaagctca ccgcccacct 480
tggctatgcc gcgttgatgc ggatcgcggc cggagcgtgg acggcggcgc tgctggtctt 540
tgccgtcgcc gggcagtggg ccgtgctgct gatcgtgccg gtgctcaccg ggatcggcag 600
cggcatcacg atgcccaccc tgaccgtcct ggtcgaccgc ccgcccccgc cgaacagcgc 660
ggcaccgcca cctcgctgca ggcgaccgcg ctcttcggcg gccagttcgc ctcgcctctg 720
ctctccgggc cgctgatcga ctcgacctcg atcgccacgg gtgcactgtt cgccgccagc 780
ggcaccgcgg gcatcctcgc cgcgctgttc cggctgaggg aaccggacca gtcggccgac 840
atcgcaggcc ggaacgagaa gacccgcgcc ggactcccgt cggtgaatga tcacagcgtg 900
acacctgacg agggcagccc agcacctcct taccggaggc cggcacggaa agtgaccgcc 960
gcatcgcgac ggactcgtcc cggccctctt gtggcgcagg cagcgccaag gccatcaccg 1020
caacgcccgc gcacacagcc gccgtcgcca cgaacaccgc cctgtacgac aggtccggac 1080
cgccgtcggc ggtagcgagg gtgacgaggg cagcgagccc gacgacaccg ccgagctggc 1140
gggtggtgtt catcagcccg gaggctgccc ccgcatcctg ctcgcggatg ccggaggtga 1200
cggtggtagt gatcggggtg atgagcagtc ccatgccggc ggacatgacg atcgcgggcc 1260
cgagcagtcc gtccaggtag ccgctgtccg tgctgatgct gctctgccag aggaagccga 1320
cggcgctgag gacggcgctg agaacgatga ggccgcgggc gccggtgcgc tccatgacgg 1380
ccggggcgag gcgggcagcc gcgataccga caagcgtgtg gggcaggaaa ccgacgcccg 1440
tggccaaggc cccgtagtgg aggacctcct gcatgtacag cgacaggaag taccacatag 1500
ggatgaagca gccgcccgcc agcagcatgg tgacgttgcc ggcccagatc gggcgcagcc 1560
gcaccaggcg gggcggcatc aggggggccg ccgtgtaccg gctctccgtc accgcgaacg 1620
cggtcagcgc cacggcgctg accgccaggc ccgtcagggt ggtcgcccct gtccagccct 1680
gtgttccggc ctggttgacg ccgtagacga gtgcggtgac gccgagggtg gccagcaccg 1740
ccccggggac gtcgagcctg ccggataccg cgcgggagcg gtcggacggc agcagacgca 1800
gggccgccag gacggccacg gtgccgatcg gcacgttgat cagcaggatc caccgccagg 1860
acagtgcgtc ggtcaggacg ccgccgatca ggttgcccgc cgcgcctccg gcggagctga 1920
ctgcagtcca gatcgtgacg ctggtgcggc ccggcccttc ggggaaggtg gtggtcagga 1980
tggtcagcgt ggccggggcc agcaccgcgg cgcccaggcc ctgcaccgcc cgcatggcga 2040
tcagcaggcc ggggccggtg gcgagtccgc ccaccaggct ggagacggag aacagcgcca 2100
gcccccacac gaacgcccga cgtcggccgt acaggtccgc gagccgaccg ccgagaagca 2160
ggaaacccgc gaagaccagc gcgtagctgc ccacgaccca ctgcagtccc gccgcgtcga 2220
agccgagcgc ggtctggatc gacggcagcg cgacgttcac caccgacacg tcgagcacca 2280
ccatgaactg tgccgcgcag gccagccaca acagcgccgc ctgacggggc accagtgcgc 2340
ctgtctcttt ctcggtgtcg actgatccgt cggcggcacg ggcttcgcgt tgtcccatgt 2400
ctcttcccca tcgtcgcggc cagcttttat agtacagcgg tactatgaaa aagggtcgaa 2460
cgggaagggg tcacggtgcc gaaacgtgtt gaccatgccc agcgccgggc gcacattgct 2520
gacgccctgg tccgggtggc cgcgcgggac ggactgcatg cggtgaccat gcgcgcggtc 2580
gctgccgagg cgggcatgtc cttgaacctg gtgcagtact acttcgacac caaggcccag 2640
ttgatgcacg ccgctctgca gcaccttgag cagcagagcc atgagcggtg gtcggcgcgg 2700
ctggagagcc tggaggatcc ggggtcggca cgggcctgtc tggaagcgtt cgtggatgaa 2760
gccctgccgg tcgacgcgga cagccacacc ttccggctgg tgtggacgtc ctacgcggta 2820
ctggccatga ccgaccccga gctggcggag cagcccttcg tcgaggggcc gaaccggctt 2880
gagcgacagc tcacccacat cctggccacg gcgcaggcgg cgggagagat cgcggcttat 2940
ctggatgtct ccgccgaggc cgcgcgtctg ctgagcctca gtcacggtgt cgggaccagc 3000
gtgctggtcg ggcagcggtc ggcggcacaa gcccggtacg tgatgctcta ccacctctcc 3060
cgcttgttca acgaccccac tccacccgag gcccctggcc agcacttagc ggcagcttcc 3120
tgactggtgc cggctcggaa acgggacgcc ccgccgtgcg atgtgcggca gggctcgcga 3180
gtggactgcc ccgcttgacg gacaggagaa gctcagtgac cacccatacc cagcagttgt 3240
gggactttcc cttcgcgccc cccgccgagc tgcacatgga accggccttc gcgcaattgc 3300
gcgaagagga gcccatcagt cgcgtccgcc tcccgtacgg gggtgaggca tggctggtca 3360
cgcgttatca ggacatcaag accgtgctgg gcgacccgcg gtttagtcgt gccgccacgc 3420
agcacgccca ggcgccgcgg atccagcctg acccggcggg tgaaggcgtg ctgatgtcgc 3480
tggatccccc ggaccacacg cggctgcgta aaacggtcgc gggtgtcttc accaagcgca 3540
gggtggaaga tctgcggccc gccacgcaga ggatcgccga ggagttgctg gaggcgatgg 3600
aagcctcggg cgcaccggcc gatctggtcg catcctatgc actgccgctg cccgtcacgg 3660
tgatctgcga cctgctgggt gtacccgggg acgaccgtga acagttgcgg ggctggtccg 3720
atgcgctgct gtccaccacg gcctgcactc cagctgaatc ggcggcggcc gcgcaggcca 3780
tggccgacca cttcgccgcc ctggtgagcc agcggcgctg ccagccgacc gatgacctgc 3840
tcggggctct ggtgcagacg tgggaccggg aggaggggct gcttcgcgac gaggagctgg 3900
tccttctcac ccgtgatctg ctcatcgccg gccatgagac cacggcgagc cagatcgcca 3960
actgcaccta cctgctgctc cagcgcccgc atgacatgga ccgcttgcgc acggatcccg 4020
cggcgatggc gtccgcagtg gaggagctgc tgcggttcat cccgctgggc tcgggcagtt 4080
tccgggccag ggtcgccacc gagccggtgg aactgtgcgg ggtgaggatc cagcccggtg 4140
acaccgtgtt cgcccccact gtggcggcca actgggatcc ggacgtcttt gctgagccgg 4200
gccgcctcga tatcgacagg tcccccaacc cgcacgtcgc cttcgggcac ggtgtgcacc 4260
actgcctggg tgcgcagttg gccaggctgg agctccaggt ggcgctgggc gtcctgttgc 4320
gccgtctgcc gcggctgcga ctggctgtcg acgaagcgga gatcgtgtgg aagacgggca 4380
tgcaggtacg tgggccgaag accctgccgg tgaagtggtg accggggagt ctgcggctgc 4440
aggccgctgg cggctggcgg tggacgcgac gttgtgcatc ggatcgggtg cgtgtgccgg 4500
ttcctctgcc catttccgga tgacgcagga cggccacgcc gagccgcgcg aggacgtcat 4560
cgtcgccgat gacacggcac gggacgccgc ggagtgttgc ccagtggagg ccatcacggt 4620
gtccgacacc agtaccggcg agttgatcgc accgacgtga gccgcggggc atgtcaccac 4680
ttcagcatca catcccctgt ggccatggag gaaccgcctg tcgactacgg gccgcctcgc 4740
gggggagcgc cgaggggctc agggggtgac cttgggcggg ccatgaggct gccttgcttg 4800
ggcgtggagt gacgggcgct tcagtgaatt cggtgcgtca ctcagccgac ctgcccggtc 4860
gcgatccgga tggccgtcgg cgtgccgcgc ctgtccgctg gcatgggcag acggtctgta 4920
caacccgtcg agtccaccga ttctccggcc gcaaagggaa cttcatgacc ctcgtcatag 4980
acctcgacat gaacgtcacg ctcgaccaac ttgaggacgc ggcgcgacag cgcacgcccg 5040
tggagctgtc cgcacccgtc cgctcccgcg tccgcgcctc gcgcgacgta ttggagaagt 5100
tcgtgcagga cgaacgtgtc atctacgggg tcaacaccag catggggggc ttcgtcgacc 5160
acctcgtccc ggtgtcccag gcccggcagc tccaggagaa cctgatcaac gcggtcgcca 5220
ccaacgtggg ggcgtatctg gacgacacga ccgcccggac catcatgctg tcccgcatcg 5280
tgtcgctggc gcgcgggaac tccgcgatca ccccggcgaa tctggacaag ctggtggccg 5340
tactcaacgc cgggatcgtg ccgtgcatcc cggagaaggg ctctttgggc accagcggtg 5400
acctcggccc gctggccgcg atcgccctgg tgtgcgcggg gcagtggaag gcccgctaca 5460
acggtcagat catgcccggg cggcaggccc tgtccgaggc tggcgtcgag ccgatggagc 5520
tgagctacaa ggatggcctg gccctgatca acggcacgtc gggcatggtc ggcctgggca 5580
ccatggccct ccaggccgcg cgccggctcg tggaccgcta cctgcaggtg tccgcgttgt 5640
cggtcgaggg cctggcaggc atgacgaaac cgttcgaccc tcgcgtgcac ggcgtcaagc 5700
cgcaccgcgg gcagcgtcag gtggcctcgc ggttgtggga ggggcttgcc gactcgcacc 5760
tggcggtcaa cgaactggac actgagcaga ccctggccgg agagatgggc acggtcgcca 5820
aggccggttc gctggcgatc gaggacgcct actccatccg gtgcacgccg cagatcctcg 5880
gtcccgtggt cgatgtgctg gaccggatcg gggcgaccct gcaggacgag ctgaactcct 5940
ccaacgacaa cccgatcgtc ctgccggagg aggcggagct gttccacaac gggcacttcc 6000
acggccagta cgtggccatg gccatggacc acctgaacat ggccctggcc accgtgacca 6060
atctcgccaa ccggcgcgtg gaccgcttcc tggacaagag caacagcaac gggctgcccg 6120
ccttcctgtg ccgggaagat ccgggactgc gcctgggcct gatgggcggc cagttcatga 6180
ccgcgtcgat caccgcggag acccgcaccc tgaccattcc gatgtcggtg cagtccctca 6240
cgagtacggc ggacttccag gacatcgtgt ccttcggatt cgtcgccgcc cgccgcgccc 6300
gggaggtact caccaacgcc gcctacgtgg tggccttcga gctgctgtgc gcctgccagg 6360
ccgccgacat ccgcggcgcg gacaaactgt cctctttcac ccgcccgctc tatgagcgca 6420
cccgcacgat cgtgccgttc ctcgaccggg acgagaccat caccgactac gtcgagaagc 6480
tggcggccga cctgatcgcg ggcgagcctg tcgacgctgc cgtggcggcg cactgaagcc 6540
gcacctggaa gggagacaca ccatgacgac cacactggcc gagcaggcgg ttacggactt 6600
cgtccgggaa tggagcacgg ccgaagagcg gggagagcac agggcgctgg acgacctgct 6660
cgcgaaggac ttcgtcggcg tcgggcccca gggcttcgtg cgcagccgcg aggagtggct 6720
ggcccgctac agcacgggca ccgtgcgcaa cacgtcgttt gaggtgagcg acctgcgtat 6780
ccgctcctac ggggagacgg cggtcgtggt ggcggcccag acccagcaga gtgtgaacgg 6840
tgatgccgac gccagtggtg cattccgctt cacgctcatc gtcgtccgcc aggacgggcg 6900
gctgcggctc gccggcctgc acctgagccc gaacgccgtc ccggcaggcg gctgagcccc 6960
tgccgggacg gcgtgggctc cggccgcccg cgctggtcac acggcgtggg cggccggagc 7020
atccgccttt cccgtctcct gtgagcgcag ggcgaaccgc tgaatcttgc ccgtaggcgt 7080
cttgggcagg tccgcggcga actcgaccag gtgcggaaac tgatgactgg ccagacggtc 7140
cttgcaccac tgccgcagct gccgggccag tgcgtcgctg ccctggcagc cgtcgcgcag 7200
cacgatgaac gccttgatcc gggtgaggcg gtcggtgtcc acgccgacga ccgctgcctc 7260
ggcaacatcc gggtgctgct gaagggcggt ctcgatcgcg gcgggagcga cccacagccc 7320
ggagaccttc atcatgtcgt cggcacgtcc ctcgtaccag tagaagccgt cctcgtcgcg 7380
gcggcagcgg tcgccggtgc ggtaccagct ccccttcaac accgagcggg tctggtcggc 7440
acggtgccag tactgggcca gcatgctgcc gccccgcacg aacatctctc cgctgcccgc 7500
gccttccaca ttgccgccgt cggggtcgac gagcttgatc tggtagccgg gcacggccct 7560
cccggaggag ccggggcgaa gcgcccgctc ggtgttgctg cagtagatgt gcagcatttc 7620
ggtggacccc acaccgtcca ggatcgtcag cccgtaaagg tcgcgccatc gttcccagac 7680
gtgggcgggc aggctctcgg cggccgagac gcacagccgg atggacgtca ggtcccgaga 7740
gccagcgccg ggcgagcggg gcatggcgtt gtacaaggcc gggacggaga acagcagcgt 7800
gggacgtact cgttcgatgg tctccagcac gaggtgcggt gccggaaacc cgggcagcag 7860
gacgcatgtc ccgccgaacc acagcggaaa cagcaggctg ttgcccaggc cgtaggcgtg 7920
gaagagcttg gtggtggaca ggtgcacgtc gtcctgagtg acacccagga catcgcccgc 7980
gtagtgccgg caggcgggtg cgatgtcccg ctgcaggtgg accacccctt tgggccgtcc 8040
ggtggacccg gagctgtaca gccacagcgc gggatcctcg gcgtgtgtgg acgcggcgcc 8100
cagctcgccc tcgtggccgc ggatcaggtc gcgcatgtgg aacctgccgt cgtccggccc 8160
tgcgggctcg gtgctgatca ccatcacgcc ggcctcctgc agagcgggcg cgagctcctc 8220
gtaccggtcc gcctcgatca cagccgcccg ggcgtaactg tcctgggcgt agaaacgcac 8280
ctgttcaggg tcggtttggt agttgatcgg gaccgggacg gcgccgatgc ggccggcgcc 8340
gaggaaggcg gtgaagaacg ccggggtgtc gtccaggatg agcaggacgc gctgttcgcg 8400
ctggatgcca agctcacgca gagcccggcc catcgcacac atgctccggt acagctcgcc 8460
gtagctgacg tggccgtggg ggctgtagag ggcggccttg tcgctcaggc cgctgtcgag 8520
atggtggtcg accagggtgg agaggttgaa gaggtcggtg gtcatgtcgg ctcctgttcg 8580
agaggcatgg agctgatcgg cccccgggtg tgaccctgcc ccggtcgcgg cattggccgg 8640
gagccggggc agagctgggt gggggcgtgg tgtgggtcag gggctgctcg ggggtatgtt 8700
ctggttgagg cggaacaggt tcgtcgggtc gtacttggcc ttgacgccct gcaaccgttc 8760
gaacttggcg gccccgtaag cctcccgcgt gcggtccgcc tcgcccgggt tcatgaagtt 8820
gacgtagccg ccggacaagt ggccggccag ggcccggtag ccctcgcggg cccaggcggt 8880
gtggcgggcg tcctcggtgg ggtccatcca ggcggcggcc aggttggtca cgaacggcga 8940
ctggcggttg gggtaggcgg tggcgtcgtc gggcacgcgc gccacggccc cgcccaggta 9000
gagcagttcg agctgggtga acggggaggc gatgtcggcg gcgtgttcca ggaccgtgtc 9060
ggtggcttca tcgctcagtt cgttcagata gccgctcttg gtgtagatcc ggtcggggac 9120
gacggcgccg ggaaaggagt acgcctgcag ggcccggtag ggcaaggtcg ccttggtcag 9180
accgtggggc ttgccggcgt gaaggatgga ctcaagctgc cgctcaccct cgtgcgggtc 9240
cccgatccag cagctcatgg cgcagatcac cggcttgccg tgcatgtcgg cgggcagttc 9300
gggcagcggc ggtgccaggc gcagatacaa cgcccaggtc agttcgtcgg gggcggtggc 9360
catgtggtcg cgccaggccc ggatgacctg gggtccttcg tccagcgagt agtaggtgct 9420
ggcgaaacgg acgggcccga cccggtggag gccgaactcg aatgcggtca ccacaccgaa 9480
gttgccgccg ccgccgcgga cggcccagaa caggtcgggg ttctcggtgt cgctcgcagt 9540
gaggacgccg ccgtccgcgg taacgatctc cacggaggtg aggttgtcga tggacaggcc 9600
gtacttgcgg ctcagccagc cgaatccgcc gccgaggacg agtcccccca atccggtgtg 9660
ggagaccacg ccggcggggg tggcgagcat gtgggcctgt gtggcggtgt cgaacgcccc 9720
cagcaggcag ccgccctggg ctcgcgcgcg ccgcaggcgg cggctgacct tgatgctgtt 9780
catcaaggag agatcgatga cgatcccgcc gtcgcatacc gagtgcccgg ccatgctgtg 9840
tccgccgccg cggaccgcga ccagcaggcc gctcttgcgg gcgaagctca ccgcggcgac 9900
cacgtccggg gtgctggtgc agcgggcgat cagcgcgggg cgccgatcga tcgtgccgtt 9960
ccagatgcgg cgcgcctcgt cgtagtcggc gtcgctcggc cagatcaact ccccccggaa 10020
cgccgcgctg aacgcggcca gggcggcagg gtcgagctgt gggaactgca tgctggtccc 10080
tttctgcggc gccacgacgg gcgcttgacg gggctggcgg ctggggggtc agaagtccgg 10140
agacagcggg tcgcatccgc aggtggcgag ctgcttcagg gcggcccgta cggcttcgat 10200
atcggcgtcc ggctcgcccg cgcgggccgg cagcagggcg agggccttcg cgtcaccgcg 10260
ggcaaccgcg ggcgtgcgcc gtgccagggc ggtcaggctc tgtccggggc ctgcctcgac 10320
caagaggtga gggccactgc tcagcagatg ctccagggtg gggccgaaca ggaccggctc 10380
ggcgggctga ccggtccaga actgccagtc ggtggccagc tcgttggcaa gttcctgccc 10440
ggtgtaggcg gagtagaggg tggtgtgagg cgggtggagg ggcggacggg tgagcaagcc 10500
cgcgtgggcc gtgatgacgt cgcgcatcac cgggtggtgg aagggctgca gggcgcgggc 10560
gcgggaacag gtgataccgg caccgcggag ggcggcttcc acgtcggcca gggcctcttc 10620
cggggcggag agcagaacct ggcgcggtgc gttgacggcg ccgatgacca cgcccggcac 10680
caggtgggct tcgacctccc tgggactggc ggccacggca agcatgccgc cgcgcggggc 10740
gtgccggtag acgccgatgt attcggacag gtagcgcagg ccgcagtcga aggagaacac 10800
gccggccagg gtggcggcgg ccagttcccc cacactgtgg cccagcagga cgtcggggcg 10860
cagaccggcc tcggtgacca tggcgcccat cgcgcagttg accgcgtaca gcaggggctg 10920
ggcgtaggag atgtcgtcga aggccgcggg tggtgtggcc gcgagccatc cttcgcgcag 10980
tgtgcgtccc gccggtccga aggcatcgaa ggcgtggtcc atcgtggagg tgaaggactt 11040
ggagacgccg tagaggccgg cggccatgcg ggcgtgctgt gccccttggc cggggaaggt 11100
gagaacgact ttggggcgcg gcggacgggg gtgtggggtg ggggcgtcca tcagctggct 11160
cctgcggtgc aaaaaaaggg gcgggaggcg ggcggacggg tgcctcagtt cctgctggcg 11220
gggacgttga cgtactccag gacggcgcgc ggtgtcttca ggtcccggat gacgtcctcc 11280
gggatgtcga tgtcgtagcg gtgcttgagc tgggcggagg tctccagcag gaccagggag 11340
tcgtagccga ggacctcgaa ctcgcggtcg aggatgtcgt cgccctgggc gagggcctgc 11400
tcgtcgccgg agcactcgct gaggatctgc aggagctggt cgctgctgag catgcggaac 11460
ggtcccttct caatgggtgg gaacacaggg agtggtggtt tcagggggtg tgctcgacga 11520
tgacggccga gttgaagccg ccggcgccgc gcgcgaggac gagggccctc gacagggagc 11580
acgtgcgtgg ctcgtcacgg acgaggtcga tctggtagtc gtcgacgacg tcggtgacgt 11640
tgacggccgg tgggatgacg ttgtcgcgca gcgcgagcag ggcgcacacc acatcgagtg 11700
gccctccgcc ggccatcagg cgaccgacca tggtcttggg cgcacagacg gggacgccgt 11760
acgggccgaa gacggcccgc agagcatcgg cttcctgggc gtcgaggtcg gcgacgcctg 11820
ccgcgtcggc gacgacgagg tcgatggcgt cgggcttaag ccccgcatcc gccagagcca 11880
ggtccacggc gcgccgcaga ccggagggcc gctgcgctcc gggggcgggg tcgaaggtcg 11940
ccgcgtagcc ggtgatcttt ccgtagggac gccggtcgat gcgtccgcgc gccgattggg 12000
cgtcctccat gacgaggagg gcaccgcctt ctcccggcac atagccgcag gcccgctggt 12060
cgaacggcag gtaggcggtg gcggggtcgg tggccgtgct gacggtgccg gaggccaggt 12120
gggacaccca gccccatggg tcgaaggagg actcgacgcc gccggtgacc atcaggcggc 12180
tgcggccgcg ggtgatcgtg cggcgggcgt gcccgatcgc atcgaggccg cctgcctgtt 12240
cggcgacgac cacgccgccc gggccacgca tcttgtgccg gatggagatc tggccggtgt 12300
tgacggcgta gaaccaggcg aagcactggt agacgctgac acgatcgggg ccctgcgtcc 12360
acagccgctg catctcgcgg tgggagaact cgaagccgcc ggtcgcgttc gaggtgacga 12420
caccgcactc gtaggtgccg agttcgccga cgtcgacctg ggcgtcctcc agcgcgcggt 12480
cggcggcgac cagggcgagc cgggtgacgc ggtcggtctg cggcagcagc cgcccgggca 12540
ggtgcctctt ggcctcgaag ccggtgatct gcccggccag ccgcgccgtg tacggcgtgg 12600
gatcgaactc ctgcagcggg gcgatgccct gggtgccggc cagggtggcg ttccagtact 12660
ccttgacgct caggccggta ggcgcggtca cacccatccc ggtgatcacg gcatcggtgc 12720
tcatacgtgg cctcctcgcc acttctccac gaccatggcg ctctggaacc ccccgaaccc 12780
gctgcccacg gtcaggacga cgtcaaggtc cacatcacgc gcctcaaggg ggacgtagtc 12840
caggtcgcag tccgggtcgg gctcatggag gttcgcggtg gggggcacca ctccgttgcg 12900
gatggccagc gcgcaggttg caacctcgat cgcgccgatc gcgcccaggg aatgcccgat 12960
catcgacttg acggagctga ccgggacgtt gtaagcgtgc tcgcccaggg ccttcttgaa 13020
cgcctcggtt tcatgcaggt cgttctgttt cgtgccagag ccgtgggcgt tgatgtaccc 13080
gacggccgtg gggtcggtgc gggcctcgtc cagggcggtc gtgatggcgc gggccatctc 13140
attgccgtcc cgggacaggc ccgtcatgtg gtaagcgttc gagtgcgcgg cgtaaccggt 13200
gatggtgccg taaatgtcgg ctccgcgccg ccgggcgtgt tcgctgtcct ccaggaccag 13260
catggccgcc ccctcggcga tggcgaatcc gtcccgggtg cggtcgaagg gccgcggcgc 13320
gtgttcggga tcgtcgttac ggcgcgtggt ggccttgatc gcatcgaagc aggccaccgc 13380
gatcggggtg acgggagcat cggtggcgcc ggcgaccatc acgtcggcac tgccctcctg 13440
gatgagctgg caggcatggc cgagggcgtc gatgccagag gtgcagccgg ccgagatgac 13500
cgacaccggt ccttcggcac cggccgccca ggcggtctcg cgggcgaggg aacccgggac 13560
gaagtagtcg aacaggtggg gggactggta cttctggtcg accagccact ggcgtccctc 13620
gtcggacagc acgacgtact cgttctcgat acttgtggcg gatcccaccg cgttgcccac 13680
gctcacaccg atgcggccag ggtcgacact cgtaccggag gtgatcccgg cttccgccac 13740
cgcccgtctg gcgcaggcca cggccagcag gctggcccgg tccagacgcc ggacctcgcg 13800
gcggctgaac ccacaggcca gcggatcgag atcgacctct cccgccacct gggaacggaa 13860
cggcgtgggg tcgaatgccg tgatcctgcg gatcgcgctg cgccccgctg tgatcatctt 13920
ccagaagccc tcggtatccg tcgaaccggg tgcgaggacg ctcaggccag ttatgactac 13980
gtgtcggctc acaccttgag accttgcggg gtgctgctcc agcggcagtc caccgccgct 14040
catcctctgc tccagagcgc cttgcttggg cggcagagag cgccttgccc gcgcgtggac 14100
cggccctgca ccgccagcag ggaccctcgt cgtcgggccg gcccgttccg gccgccccga 14160
cgaagggaga aagtctcacc atgcccgcac acaccgagtc cccaagcgtc gctctggtca 14220
caggcggcac cagcggcatt ggtctgtcga tcgcccaggc cctgctgcag gatgggctga 14280
aggtcatcat caccggccgc gaccccggga agctggagac ggtcgtcagg tcgctgaaga 14340
tggaggacga ccagttcgac gtggccggtt acacctgcga cgtacgcgac cgcgacgcgg 14400
tgcacgacat ggtggaacgg tgcgccaagg agcacggcga gccgaaggtc ctggtcaaca 14460
acgccggccg cagcggcggc ggcgtgaccg ccgcgatccc cgacgagttg tggctggacg 14520
tcatcaacac caacctcaac agcgtcttct gggtgacccg cgaggtcatc cggagcagcg 14580
acgcgatcaa gcagccagcc ggccggatca tcaacatcgc gtccaccggc ggcaagcagg 14640
gcgtcccgta cggggccccg tactcggcgg ccaaggccgg tgtcatcggc ttcaccaagg 14700
cactcgccaa ggaattcgcc gcccacggtg gccccaccgt gaacgcggta tgccccggct 14760
acgtggaaac gcccatggcc caggacatcc gcgccagtta cgccaagctc aacaacgtca 14820
ccgaggagca ggtcctcgcc gacttcgagg ccaaaatccc cctcggccgc tactgcaccc 14880
ccgaggaggt ggcgggcatg gtgcgctacc tggtcagccc ggcagccgcg tcggtgaccg 14940
cccaggcact caacgtctgc ggcggactgg gcagttactg acgcttcgag tggaagaggg 15000
acacgcgcat gaccatcgat gcatcccccg agtcctccgc cttcggcttc gagggccagt 15060
cctactacga acaccggcgg ctgcgcctgt atgacgccgt ggtcgtccat gcgtcgaacc 15120
ggctgctgtg gcgctgcccg aagtcacggc tgctcgagca ctacaacgac tggatcacct 15180
cccgccacct ggaggtcggc cccggctcgg gctactacct ggaccactgc cacttccccg 15240
caggcacacc cgagctgacc ctgctcgacc tcaaccccga cccgctcaag ttcgccgcgc 15300
accgcctgcg ccgctacacc ccgcgctgcg tgcagtcgga catcctggaa cccttgcccg 15360
acttcgtcgg cagcggattc acatccatcg cctacaacta cgtcatgcac tgcctgcccg 15420
agccaccggg cggcaagcac atcgtcttca agcacctgcg cgaggcactc gcgcccgacg 15480
gcgtcctctt cggcagcacc gtcctgtccc agggcgtgaa acacaccgcc ctctcccgcc 15540
gcttcaacat cctctaccag cgccagggct ccttccacaa ccagcatgac agcgtccacg 15600
gactgcacca ggccctggag gaacacttcg ccagccactg gatcgaggta cgcggcagcg 15660
tcgcactctt cgcggcccgc gcatcgcaca ccaccgcgcc ccgccgcccc ggagcatccc 15720
tggaccagcg atgagtgagg agaagcgcat gacgtccggg gccacctacc gtgatgcggc 15780
cattcccctg acggcgctgt ggacctcgcc cttcgcgcgg tggggaggcg ccctgtccca 15840
cctctccagc cttgacctgg ccgtggccgc caccggccgg gcactgtcgg agcgcggcat 15900
cgaccgcgac accatcgagg gcatggtgct gggctggacc gtgccgcagc ccgccatctt 15960
ctacggagcc accacgctcg cggcccgact gggcatgccc caggtctccg gaccgatgct 16020
caaccaagcc tgcgccacct ccgtcgcggc cctgcaccag gccgccgcgg cagtaggaac 16080
aggagtcggc ccacaactga tcgtcaccac cgaccgcacc agcaacggcc cggacctctc 16140
ctggccctca cccgccggtt ccgggcaggg accggtgcgc gagaactggg tacgcgacag 16200
cttctcccgg gaccccgtca ccggacagag catgctcgcc acagccgaag ccgtcgcggc 16260
cgagtgcggc ttcacccgcg acgacctgga tgccctcgcc gtcctgcgct ccgagcagta 16320
caccgccgcg ctcagcaacg accgcacctt ccagcgtgac ttcatggtcg gcatcgagat 16380
cgaacaggac gggctcggca ccgtacggct cttgtccgac gaaggcgtgc ggccggtcac 16440
ggccgacagc gccggtcaac tcccgacggt ccgccccggc ggcgtccaca ccgccgccac 16500
gcaaacgcat cccgccgacg gcacagcggg ggccctggtg accacgacac cgtgcgcccg 16560
ggaattcgcc gaccgcggcc cgatcgtgcg cctggtcgca tccggattcg cccgcgtgga 16620
gccggcccgc atgcccaggg cactcgttcc ggccgcgcag gcggcgctgc acgccgccgg 16680
gctgaagatc gacacgatgg atgccatcag cacccacaac cccttcgcgg tgaacgacct 16740
gtacttcgcc cgggaaaccg gggtgacgct ggaggccatg aacgcgtccg gctgcagcct 16800
tctcttcgga catccccaag gcccgaccgg gctgcggatg gtcacggaac tcgcacacgt 16860
actgaaggca cgaggcggag ggctgggact gttcaccggg tgcgccgcgg gggacaccgc 16920
ggcagcactg atcatcgaag tgaccgactg acccggcctc ctgctgccca gtgctgggca 16980
ggcagggaca cccagcgggc tgtggcgaag gaggctttcg gccttcacca cagcccgttc 17040
ggtttgtgcc tcggcggccc ccttcggggt acggccccgg cccgcagcag caaaggtcac 17100
ttcccgtggc cagccagcac cgtggcgatg ccggcctggg caatgccgct ctcctggacc 17160
tgccgtacgg ccgcgccctc gcgtgccatg gccgcttcta ccgcacgcag cgagggccgc 17220
atcagcctca gatgtgcggc cgtggacgaa cccggcggcg tccactggtg gatcagccgg 17280
atggcggcgg ggatgagatc ctccggctcg acgatgtcgt cgaggagacc gaaggccagt 17340
gcctgggagg cgctgagccg ggcactgttc atgatcaggt gcagggctcg gggtgcacca 17400
gcgaggcggg gcaggaacag gctcgcggca ttggacacgg tgagcccgac actggtctcc 17460
ggccatgaga tcgcggcctg agcggtggcg atgcgggcgt cgaagctcag ggccatctca 17520
ccggccccgc cgacggcgat gccgttgagg gcggccacga ccgggacttc cgtgatgaga 17580
gcggcgcggg tgatgtcgtt gaacagctcc atctccgcca gcaagtcgac ttccgggccg 17640
gcgacttcgc gcaggtccat tccggcagag aaggcttttg cctggccggt gatcaccact 17700
ccgcgagcct ggctgccgtc gccgtagtgg cgcagcagct cggccaggtc ccggcgcatg 17760
ccgcgggtca gggcgttgag tttcgccgga cggctcatcg cgatcacagc aacatcgctg 17820
tcgaccgtga cggtcaggtc ggcggagctc agcaactgct gtacgtcgct catgcccatg 17880
catccgctgc ggcgtcgaac gcaccggtgc gctggtcgtc tccgggacga agcagctggc 17940
gcttctggat acgggcggag ggggtcatgg ggaaggagtc gacgaattcg acataggcag 18000
ggaccttgaa tttcgcgagg tggcggcggg cgtgcgcgag gacgctgcga gccgttgccg 18060
tggtgggccg gtagccgggg cgcagctgca ggaaggcttt gggcagctca ccgaagagct 18120
cgtccgggat gccggcgagc gcggcggcca ggaccgccgg gtgggcttcg aggacgctct 18180
cgacctcggc cgctgcgatg ttctccccac cgcgtcggat catgtccttc agccgcccgg 18240
cgtgcaccac gctcccggcc gcctcggcac gtccgaggtc accggtgtgg taccagccgc 18300
cgcggaacgc ccgctcggta ctgtccgggt tgttccagta gccattcatc atgggccttc 18360
cccgcaccag gatctcgccg atctgccctg gctggacggg gcgttggcgc tcgtcggcga 18420
cgaggacctc cttgccggac ggcgggtacc ccatcgcgcc gctgcccacg gtggcctcct 18480
caccaggcgg catgatcaag tccagtccgc tctcggtgga gccgtacacc tcccgccagg 18540
ccgcacccca gcggtcctcg aacgcatggt gcaagtcgcg cgggatgccc gagcacaaga 18600
ccagccgcat cgcatgatcg cggtcgcccg agtggggcgg ctgcttgtac agcagcatgg 18660
gcatgctgcc cagcacgtag gtcagcgtgg cccggtgctg gcggacggag tgccagaagc 18720
cggaggcgga gaaccgcggg agcaccacca gcggaacgcc gcccatcagg cacatcacgg 18780
ctttccactg gggatccatg taggagaacg cctgggccgt cagcacgacg tcgtcgggac 18840
ggaggccgga gtggacagcg atcacccacg ccgtgcgcag ccagtagtcg tggctgagca 18900
tgcaggcttt cggaaagcct gtggtgccgg aggtgtactg gaagttgacg gtgtcgtccg 18960
cgtgcgccga caggcccgga ccatccaccg gccggtcgcc gaagccggcc ttcagttcgc 19020
gtagcgtccg gaccgcacag gggtgcccga tgctgccgct cacgtcacgg gccaacggca 19080
cgcagcctgg ggaggccagg accagcacgg cctgagagtc cctcaggacg tgcgcgaggt 19140
ccgtagcgcc gaagcgggtg ttgaccggca ccgtgatggc accggccttg atggccgcga 19200
accagctcaa cggccagtcc gccacattgt ccatcatgac cgcgacacgg tcacccggtt 19260
tgacaccttg ggcgatcagc gcctgcgcca accggtcggt gcgggtgtcg gtctcggaga 19320
agctgagcgt cttgtcgccg catcggagga attccttgtc gccgtgcagc aacgcggcat 19380
ctttgatgag ctcgccgatg gtctcccaac gcggtcccgc ttcggggtgc cgagcgtcga 19440
tcacaggttg tgtgggggcc tgaggggccg gttccatgct tgtggcctcc gtcgcatcag 19500
gcggtggccc ttgccaccgg tctccactgg acagcgcacc accggcacct tgagggggaa 19560
gcaagacagc gtcacgggct gctctcgcga cttgccctgg ccgagcttgt ctctttaccg 19620
ggcatgaaga agcagccctc accgtgacta cggcaggagc tgacccgacc caagcagagg 19680
agagcgcctt gacacaggcc ccgcacaagc cggaggagac cgcggtgtcc gcggcgttgt 19740
tcgcagagat tcagcagttc tatgggcgcc acatgcgggc catggacgaa ggccgagtgg 19800
aggactggac cggcgacttc atgcccgacg cggtcttcgc gaccaacgcc cgacccgagc 19860
cccagcgcgg gcgggccgag atcgcccgaa acgcggaggc cgcagcgcgg cagctgcagg 19920
agaagggcat cctgcgccgc cactgcgtca ccaccctgga actccagcag gcgagtggac 19980
tgacgctcct ggccaagacc tacgccctga tcaccaagac ggtggtggga ggccggtcgg 20040
agctggagtt cgtgtgtacc tgcgaggacg tgctcgtccg gaacgagggc cgatggttca 20100
tccggcaccg ccaggtcttc cgggacgatc tccccaagag gtgagacgac aacgtcccaa 20160
caaccctcgg ctggcacaac gcagcggctc cgcactcagc caccgctgcg ccgcctcaca 20220
ccttccaggg gctggccgtc ggccacggga cctgcagagg agccggatca gcggtcgtgc 20280
gcaggaacgg ggtccgctca gcggtgtcca gccgagggtc cgcggacaga accgcctgct 20340
gcaacatctg cagctcccgg gagggctcca gccccaactc ggtcacgagg gactgccgca 20400
gctggttgaa caccttcagc gcctgcggac gtctgccgca acggtagagt gcgagcatca 20460
gttgacggtg gaatccttca tgctgcggac actgcgacgc cagtccgctc agctcgctca 20520
gcacctcatg atgacgcccc agttcgagtt ccgcttcgat tctggtttcc tggaccccga 20580
tacggctctc ttccagcctt acgatctcag cctcaagatg catcccacgc cggacattcg 20640
ccagaggcgg cgccgcaaag atgtgcagag cggcgcggaa ctgctcgaca gcagccatgt 20700
tctcgcccgc ttgaaaggct tgctggccgg ccaggcgatg ccgttcgtag cgttgcaggt 20760
ccacgttctc cggcgggacg cggaattcat agccgttcgg cgtggtcacc aggagctgct 20820
tagggcattc acctggacgg atcttcctca acagctgccg cagctggaac acgtacgtct 20880
gcaacgcact gagcgcgctc cgaggaggct gctcggccca cagctcttcg atcagtacat 20940
cggtgctgac cattctgttt gcgttgacca gcaggagggc aaggacctgc ctggccttgg 21000
gggcgctcgg cgtccggcgc tcatcgccga ccttcagcgt cacatgcccg agaacttcga 21060
cgtccacagt tatttcaccc cgtcgactgt ttgcatttac cgcacagcgg gcagcgggca 21120
gcgagacgat ttcgcatgca attccctgag tcagttgacg cgtgtgggtc gcgcggctgt 21180
gcgcgacaca tgaaaactcg cttgcattac cgaccactga ggcgccattg ccgcagcacg 21240
acacaaaagc atggcccctg catgtgactg aacgtatccg cacggtaatt acagggtcaa 21300
ggactcagga agcgaaaccg gccacaccgc gccccttcgt tccgcttctc tgccgacacc 21360
ccacccggga gcccttccgc acctgcgtgc gagagtgtga catcgcaccg cttcctcatc 21420
tcttccctct cgtaagggta gagtccagca tcaacgtgaa ccccttcctg cgctgcgcgc 21480
gcgactgcat gtgatcgcgg gtgtgcgccg cgcaccgacg gcgcacaccc gccgagtctc 21540
gcgcgggccc tccggacgcc ctcgatcggg gaaaggcccg cttttggctc cgctcccagc 21600
aagcggcgtc gggccaggtc agcgccttac tgcgtatgcc gcacctgcgc acttgccggc 21660
aaggagagcc gcgacggcgg gttgggtcag gagtatggcg cagcttgtct gattacagcc 21720
agatgcaccg tgtccacatc gcgcggattg cgcgctgtga ccgcgaatcg cacaccttct 21780
gccgcgtcga gcgcctgcat ggctacccgg caagaccggg tgcatcactc gtgagccaga 21840
gcctccgcag agggccctcc ggaagtagcc cgctcacctg ccgaccgagc tcgggcgtcc 21900
gcattcatcc ggtcacgcaa cgtggcggcg aagtcctcgg ccatcgccag tcgcgcgcgc 21960
agtgtctcgc accgcgcgtc ggccaccttg cggtacgcgt caagtcgctc acggagtcgc 22020
tcgcgctcca caggcgcggg cggatcatcc tgggcgccga gcctgtcagt gatctccagc 22080
aggtcccgca tctcctcaag ggggaaagcc agcggcttaa aatgccgaat caccatgagc 22140
cgactgacgt cggcctgagt gtagagacgg aagccgccct tgctgcgggc ggagggaacg 22200
atgaggccga cctcttcgta ggcgaggacg tcccagccgc gcagcacctg cacgaaggga 22260
accagtggag cgttcacagc gcaccccctt cctgcaaccg gctcccttgt tgcgtatgta 22320
ttgcaacaag gagccggtgt tgtcgcaagg ggccccacca gtgccgtgcg gtcaggagcc 22380
cgcggcaggc cggccttcac gcctccggtg cagcagcccc agcagggcgt cgaggaccag 22440
gaaggtcagt aggggccagc ccagcagggc gccactccag ggcagcggcc gacggctgcc 22500
gaccttcctg tagcctctcg ggcctctcca gtgacgggcg tcacacttgc cctgaggaac 22560
tggacagcct gcggccccga cttctgtacc ggtacggctc ggtccagtgg aggccgtgtg 22620
gcgtgcgacg cgatcgggag agtcgacaga tggcccagac acggcaaccc gcttccggta 22680
cgccggccgg cctgctgcgc cgtgtcctgc cccctcctgc cctgtgcggg ttcctcgtgc 22740
tgctggtgct ggtgttcgcc gtctcctacg ccgtcggccg gagcgtcggc cccgtcgcgc 22800
ccgggatgca cggtcccggg atcaccgagg acggccatga gggcggcggc acggacgtgg 22860
aggacgacga catgggcggc atgaaccacg ggagcggaca ctgatggccg gcgaaccggc 22920
gtccgtggtg gaggtgaccg acctcgcggt cggcggcatg acctgcgcgg cctgcgtgaa 22980
gcgggtggag aagaagttgg ccaagctgga cggcgttacc gcgagcgtga acctggccac 23040
cggacgggcc cgcgtgcacc atccgcccga cgtgctcccg gaacagctcg tcgccaccgt 23100
cgagcaggcc ggatacaccg caacgctgcc cgagccgccc gaagcgcggc gccgcgagga 23160
cggtgacgac aaggatacgg agacggagca ggaacgccac cggctgacgg tcaccgtcct 23220
gctcgccgtc cccgtgctcg ttctgtcgat ggtgcccgcc tggcagttcc gcaactggca 23280
atggctgtgc ttcgtactcg ccgcgcccgt cgtggtctgg ggcgcctggc ccttccacca 23340
gcgggcggcg cgggcactgc ggcactccac ctcgaccatg gacaccctgg tgtcgcttgg 23400
cgttgtggcc tccttcgcct ggtcctccta cgccctgttc ctcggcggcg ccggcgatcc 23460
gggcatgcgg atgcccttca gcctggtgcc gacggcatcg gacggcgtcg cccacatcta 23520
cctggaggcc gccgtcggcg tccccctgtt cgtcctggcg ggccggtacc tggaggcgag 23580
ggcccggcgc ggcaccggag cggccctgcg ctcaccggcg gaactggccg ccaaggaggt 23640
cgccgtacgt gacgacacgg gcgagaggct catcgccata gaggacctcc gggtcggcca 23700
gatcttcgtc gtacggcccg gagaacgcgc agccaccgac ggcaccgtcg tcgaaggcgg 23760
ttcggccgtc gacctctccc tggtcaccgg ggagagcgaa ccggccgagg tcgctcccgg 23820
cacggccgtg atcggcggct ccctgaacgt cggcggcctg ctggcggtgc gggccaccgc 23880
ggtcggcgcc gacacccgac tggcccgcat cacccacctg gtcaccgagg cgcaggccgg 23940
caaggcgcgg gcacagagcc tcgccgacaa ggtcgccgga gtcttcgtac cggtcgtgct 24000
caccctggcc gccaccgtcc tcggcttctg gttcggcgcg ggcgccgacc cgcaggccgc 24060
ggtcaccgcc tccgtggccg tcctggtcgt cgcctgcccc tgcgccctcg gccttgccac 24120
cccgaccgct ctgatggccg ccaccggccg tggcgcccaa ctgggcgtcc tggtcagcgg 24180
cccgcaggcc ctggagggac tgcagcacat cgacgccgtg gtcctcgaca agaccggcac 24240
cctgacctcc gggcacatga gcgtcgcccg ggtcacggcc gcacccggcg gcatcggcga 24300
ggaacaggcg atcaggctcg cgggcgcggt cgaacagggc tccgagcacc cgctgggcca 24360
ggccgtcacg gcctacgccc gacgcaccgc accggacggc acgctgccgg acgtgaccgg 24420
cttcgcggca ctaccgggac gcggcgtgcg cgggaatgtc gacggacggc tggtggaggt 24480
gctggccccg gacgacgagc tgcccgcacc cctggacgac gcactgtccg cagccgagac 24540
ggccgcccac acgcccgtcg tggtccgcgt cgacggtgtg accgaggcgc tcgtcgaggt 24600
cggagacgta ctgcggccgg gcagctaccg ggcagtggac cggctgcggc gtctgggcgt 24660
acggccggtg ctggccaccg gagaccgtga ggcacccgcc cgcgccgtcg ccaccgccct 24720
gcgcatcgat gacgtacacg cccgctgcac ccccgaggac aaggcccggc tcgtccgcga 24780
actacgggaa gagggctacc gggtcgccgt cgtcggcgac ggcgtcaacg atgccgcggc 24840
cctggccgtg gtcgtccggc ccggaaccga atgacaacgg cacgcgaaga cgtacgacag 24900
ccgagacgac caggaaaggg cgcgtgggct gcggcgggac atcggcaccg gccctgtacc 24960
tgctggaacg ccactggcct tgcggacatg gcgaggtgtg cgacacggaa accctggacg 25020
ggaacgacga ggtctgacaa gcgcctgagc tcccggtcca ccggctgtcg ctacgccgcg 25080
tcagctcgga cgccggagcg cggagcagcg ggtgcgaagc cggaggtggc gggtggacca 25140
caccgggccc accgggaggg cccgcacccg cacagcatcg atgggatgcc cactctcatg 25200
agggtactga ggcccggtcc tggttggtga cgttggtgac gagcgaaaca taggccgtgg 25260
tcgccggttg gccaaaccgg cgatcacggc aatccttgga ggagttcgtc agcccccggg 25320
cttggcgttc gacatgcaca cagaaggctg cgcggaaggc ccacgcggga gcgatcgccc 25380
ttcgacttcc gcaggtctga tgaccgcagt gtccgtcgct gagccccatc ggctcctctg 25440
ccctgcgcag aagggctccc acactcccaa ggaaagcgag tagtcctgtc ctatccgatc 25500
aagaagctcg cccccaattc caggggaaag gtgcggtacc ggttcgtggt cgacgcaggc 25560
cctgacccgg tcaccgggaa gcgtaagcag gtgacccgta ccttcggcac gctgcgggag 25620
gcgaaggcag agtacgcgag catcatgcat cgtcgctacg aggcggcgcg tgcaccgctc 25680
agtcaggtca cggtggacga gtggctcgat cagtggctgt ccacgaaagc ggaggatctg 25740
gaagagagca ctgccaacag ctacaccatg acgttggccc gcgtccgcgg aaggctcggg 25800
cacatccgac ttcaggacct caccgaggag cacgtcgagg catggatgcg atgggcgctc 25860
caggaagggc gtgtgcgggg aggcaagacg ggcaccggcc tggcgacgac ctcggtcgag 25920
atgtcgctgg cccggctgaa agacgcgctc ggccgggcgg tgacacgggg cctggtcgag 25980
gcgaacgtcg ctcgcgaggt gaacattccc cgtaaggcac gcaaggcaga gcgcagggcc 26040
agggcggcag tgccgccgtg gagcgtcgcg gaagtgcgtg cctttgttcg tgcagtcgca 26100
gacgaccgcc tgcaggcacc attccttctg gctctgatgg gtcttcggcc ggcggagatc 26160
tgtggcatgc gctgggcgga cgtcgacctg gacgaagtga cggtgtccat catcaacacc 26220
cgaacagtga tgggaaacaa gtccgtggtg gagaaggacg ccaagtcgct tgccggtgag 26280
cgacagcttc ctctgccagg tccggttgcg gctgccttga ccgcgttccg ggctgctcag 26340
gcagacgaga agacgacggc cgggcagggg tacgaagaca gcggctacgt tctcgtagac 26400
gcccggggca gggcgctcaa cggacgtcag ctgcgtgagc gggcctacaa ggtcatggac 26460
cgcagcgggc tgcgtcgggt ccgtctgtac gacgcccctg cgagctgcct gacgtacctg 26520
gcgaaccacg gggtgccgga ccaccttctc gcccggtggg ccggccacgc cgacgtccgg 26580
accacgaagc gctggtacgt gaagccggat gtggaggacc tgcggccggc ggcggacacg 26640
tgttgaggag gtctggcgag tggctccgcc cccgttcacg gagagaacgt gagatgtggg 26700
agcgtgaacg ggtgagtgag aagaacgtcg acagcatgca ataccgcttt gacgggccgg 26760
aacaggcccc ggtcctcgtc atggggccgt ccctcggtgc tacatggcac aggttgtaga 26820
gaccgtccag gggcgtctat gccagtccac tatgggcatg aaattgcagg tcggagcctc 26880
ttggcccgag agcgtctgcc cagttggtga tggctgcgtg atacgcgggt gatatggcga 26940
ggccccgccg aggtccgtcc tggcggggtc tctgcccgga tccgtgcgac gtcgaagccg 27000
cacccgagtg ctccggtgcg gcttcgacgt gaggtcgaac gcgccggcgc gcacgatgcg 27060
gtcggtgccg ctaaggagtt cgtgtgcctg taactctctc gggtgcctga tttgcgatcg 27120
cacccgctca tccgtccgta ccggggtgcg ttttccaccc agcgcgaccg ggacgcgtga 27180
gtgcgggagc ggttacggct ggatcgacgg gctggccacc ggcagctgaa ctggccatct 27240
cggcctgtgg ccaggccacg gctgcatcgc tgttgcgtgt gacgtccatg tgggcgtcga 27300
tccgtactcc cgtaactact acgaacgccg catcggaccc gcgcgagaca gtcacatgcg 27360
tctcgattgc tactttgccc gagaggctgg ccacttggtc aggctcacac ggtcatgatg 27420
cccactagaa gcgcagtcgt aggctatgaa gcatgaagat cagggtccgt gacgctcatc 27480
cagccgacgt caaatggatg agcaacaaca ccgagggctg gacggtcacc gtcgagaagc 27540
aggagggggc cacctccctg gcggaggcgc tgcacgccct gatcgcagag gatcccgacc 27600
aaggcgtgcg tatggggtgg cttcactcca gtctgcagcg tgcggcaggc ggcgagcccg 27660
gctacgtcag gctttatgag ctccacgttt ctcccgagtt ccgacaaatg ggagtggcac 27720
gagctttggt cgatgagttg tttgctcggg tgcccgatca agagatcatc ttgtctgcgt 27780
gggaccggga gctgtacgga gtgtggctta agctcggatt tacatacgtc cctgaacccg 27840
acgaaatgtc aggggatcac agttaccccg gtgacatggt gcgcccaccg gtagaagctt 27900
catcctgaag aatcggcatc gtttgtcggc atagtcgtcc aggatcgttt cctgcgggaa 27960
cgaatgtcca gcctgggcaa tgccgagctg ttccgcacct gcgcgaccgt cagcccgccc 28020
gatggtgggg gagccgagga cgcggtggcc caggccaccc acctgacgtt gcgcatgctc 28080
gccgagcgca tcgagcagct caccgggcag atcgatgcgc tgaaccagcg actgacccgg 28140
ctcgtcgagc accacgcccg cagctgctcg taccggtggg catcggcccg gatagcgccg 28200
tcaccctgct gatcaccatg ggggacaatc ccgagcggct gaacaccgag gcgtcctttg 28260
ctgcactttg tggagtcagc cccatcgagt actcctccgg ccgacggcgc acgcgccggc 28320
tcaaccacgg cggcgaccgc caggccaacg ccgccctgca ccgcatcgtc ttcacccggc 28380
tgcgccacga cccgcgcacc caggcgtact tcgaacgccg cacccaggaa ggcaagaccc 28440
gacgcgaaat catccgatgc ctcaagcgat acgcagcccg cgaggtcttc aacctggtca 28500
gaccggtttc ccgcaccctt gcgttgtagg ggcgtcggtg atacggcgag ggccgccggg 28560
gtccctcctg accgaacctc tgcgctgaac cgtgagacga cgaagccgca ccggagcgct 28620
ccggtgcggc ttcgtcgtga gatcagctgc gctggcgcca cgatgcggtc ggtgcctcat 28680
gcgatccgtg tttcctgtca cgcttccagg gtgcgcctac tttgcgattg cactcgccgg 28740
gaggagtccg tgcactccgg gagtgcgctg atgctgaacg gaaaatcgac cttcgggagg 28800
gcaggcggca gcattcgggc cgcttgcgga ccggatgtcg gggtggggct ggtcgggtgg 28860
tcacgggttc gagggtgacc tggtggccga gggcctggag ctggtggacg aggtcgcggg 28920
tcgtgcgggc ggggttgagg tgacgctggt gccagtcggc gccgagttcc tggtaggagg 28980
tgccggggtc gttgatcagg tgccaggcga tgacgagtat cgagcggtga cggcgacgag 29040
ggctttggcg tgaccgcggc gcttgacgat gcgccggtaa cgggcgccga ggaaggtgtt 29100
ggtgcgggcg gcggcgttgg cagcctcccc gagcgctccc ttgagcccgg ggttgccctg 29160
cccggccggg cccgcgccgt tcttcgttcc ggactggatg gttctgggac acagcttcgc 29220
ccacgagacg aggtgttcgg gggtggggaa gcctcatgtc cgtgcggatc tcggcgagga 29280
tgatctgtgc ggtagccggt ccgatgtcgg gggcggcgtc cagccgctcg gccaggaccc 29340
gggcggtcgc tccgtcagcg gccggttcgg tgccggttct cgcttggtcg ttgggtgggg 29400
tggtgacggt caggccggtc atacacgagc cggaatccga agagccatcg tcaccggagg 29460
cggtggctgg gcctgccgcg acgcgccgac tcccctcaga aaagaccgtg tggaactggc 29520
tgcacggaaa gtcgcggctg tcggcgggcg gccccaccgg cgactctgta cagaacagcg 29580
gacaacaatc cgggaaaaag tccatacctg atcggcagcc gcgacgagct gtaccgcatc 29640
cgtaagggcc actgcctcgg tcagagccag gcggcgagat caccgaggcc gttcagccag 29700
cgcagcccac cgccctcacg ccagggcgca gtcgccaagg cgtcggccgc cgggggccta 29760
cccaccgtcg acaacggcgg ctaacgtcag gtcttctgag cgccgcagtg ccgacacgag 29820
gcaggtcgcc ctccgacgtc gtcgggctct tcttcatacg gatccggaca ggcggttcga 29880
atgaacaaga tcaaaatgat cctcatccag gccgatgaca gcgagaatca cggaacctac 29940
ttcaattcag cacccctcgg tattcaccgc atcagaaaat ggctggagag tgaattcagg 30000
gacggagttg aagtttcctg cttcgatccc aacctctaca cggacgcccg cgagacactc 30060
aatggtctcc tcgaggcagt gcgattcgat atcgtgggtt acagccctct tcatgacacc 30120
ctctcgcgag atatcggcca catgctggac gccggtgaca tactcccgca tgcattacat 30180
atagcgggag gccagcaagc gggactctcg agggagctgc ttttccggca cgtcccgcat 30240
ctcagtctga tcgctcgcgg ggaaggcgaa aagcctatca ctgaactgct ggaaaaagtt 30300
cacgggtacg ggatcgatgc gatcggaagg gacccccggc ggtatttgtc tcgagtccga 30360
ggattttata tcagagacgg tgaaaagtca ttcttcaccg gataccccga accgttcacg 30420
gtggaagaat tcacgcgcgc cacgctcgca gtggacttca cggatgtggg actggatatg 30480
tattgggacc agttgaaggc ccactattcc gcggagcaac tcggtgaccc ggtcctgctg 30540
gggaagatcc tcaccatcaa gccgtacacg agtaactact gccccatggg ctgcgcattc 30600
tgcagcacaa ccacctttca ccgcgacggc aacggccgga ccgccaaggt catcgccatc 30660
cgcaaacacg agctggccgc atacgtgcgg aatctgctcc tgaagaatcc gcaggcccgg 30720
aaggtgatgt tcaaggacga cctgttcttc ctcagaggtg gttcgcggag cgatctcctg 30780
gaagatctcc aagcccttga ggacgttcga aacgagattc acgccctcga tggcagggac 30840
gtcacctatc acggaaaggc ccgggttgac acctgggtcg acccgaggac cctcacggtc 30900
gacacccgcc tcctcgaagc cacacggcgc gcaggattca c 30941

Claims (8)

1. A biosynthetic gene cluster of polyketides is characterized in that the nucleotide sequence of the biosynthetic gene cluster is shown as SEQ ID NO. 1.
2. Polyketide neoenterocins A or B, the structural formula of which is shown in formula (I):
Figure FDA0002941062130000011
wherein, the compound 1 is neoenterocin A, R is H, and C6 is S configuration; compound 2 is neoenterocin B, R is OH, and C7 is in R configuration.
3. Use of a cluster of biosynthetic genes of a polyketide according to claim 1 for the preparation of a polyketide neoenterococcins a and/or B according to claim 2.
4. Use of Streptomyces sp SCSIO11863 for the preparation of polyketide compounds neoenterocins A and/or B according to claim 2.
5. A preparation method of polyketides neoenterocins A and B as claimed in claim 2, characterized by comprising the following specific steps: preparing a fermentation culture of Streptomyces sp SCSIO11863, separating fermentation liquor and mycelium of the fermentation culture, extracting the fermentation liquor by butanone, and distilling and concentrating a butanone layer to obtain an extract A; extracting mycelium with acetone, distilling the extract, and concentrating to obtain extract B; and (3) subjecting the crude extract obtained by combining the extract A and the extract B to silica gel column chromatography, performing gradient elution from a volume ratio of 100: 0-0: 100 by using chloroform/methanol as an eluent, collecting a fraction Fr.2 subjected to gradient elution from the chloroform/methanol volume ratio of 99:1, subjecting the fraction Fr.2 to gel sephadex LH-20 column chromatography, eluting by using a chloroform/methanol volume ratio of 1:1 as a mobile phase, performing semi-preparative HPLC, using a C-18 reverse phase column, using an acetonitrile/water volume ratio of 7:3 as a mobile phase, carrying out a flow rate of 2.5mL/min, collecting a fraction with a retention time of 13.8min to obtain a compound neoenterocin A, and collecting a fraction with a retention time of 11.6min to obtain a compound neoenterocin B.
6. The preparation method according to claim 5, wherein the fermentation culture of Streptomyces sp SCSIO11863 is prepared by inoculating activated Streptomyces sp SCSIO11863 into a seed culture medium, culturing at 28 ℃ and 200rpm for 2 days to obtain a seed solution, inoculating the seed solution into the fermentation culture medium, and culturing at 28 ℃ and 200rpm for 8 days to obtain the fermentation culture of Streptomyces sp SCSIO 11863; the seed culture medium and the fermentation culture medium are both prepared from the following components in percentage by weight: the culture medium contains glucose 15g, soybean powder 5g, peptone 10g, CaCO32g, and the balance of seawater or aged seawater with the sea salt mass fraction of 3 percent and the pH value of 7.2.
7. The use of the polyketide neoenterococcins B of claim 2 in the preparation of an anti-tumor medicament.
8. An antitumor agent comprising an effective amount of the polyketide neoenterocins B according to claim 2 as an active ingredient.
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