Aspergillus versicolor D5 delta stc17 strain of high-yield active Xanthone compound DHST and construction method

文档序号：1932521 发布日期：2021-12-07 浏览：16次中文

阅读说明：本技术 高产活性Xanthone类化合物DHST的杂色曲霉D5Δstc17菌株及构件方法 (Aspergillus versicolor D5 delta stc17 strain of high-yield active Xanthone compound DHST and construction method ) 是由刘京韩小斌马斯琦彭玉龙赵栋霖张成省王小彦芶剑渝温明霞于 2021-10-29 设计创作，主要内容包括：本发明公开了一种高产活性Xanthone类化合物DHST的杂色曲霉D5Δstc17菌株,由敲除片段敲除杂色曲霉D5菌株中细胞色素P450单加氧酶基因获得,敲除片段以杂色曲霉D5菌株的基因组为模板,利用引物扩增细胞色素P450单加氧酶基因上游序列和下游序列得到0.8kb-1.2kb上游片段和0.8kb-1.2kb下游片段,将上游片段和下游片段与潮霉素抗性基因融合得到；细胞色素P450单加氧酶基因序列为序列表中所示的序列；杂色曲霉D5Δstc17菌株现保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC NO.23209,通过对杂色曲霉D5Δstc17菌株代谢产物进行检测发现：其代谢的除草活性Xanthone类化合物DHST的量高出原杂色曲霉D5菌株代谢的除草活性Xanthone类化合物DHST的量三倍；本发明公开了一种高产活性Xanthone类化合物DHST的杂色曲霉D5Δstc17菌株的构件方法。(The invention discloses an aspergillus versicolor D5 Δ stc17 strain of a high-yield active Xanthone compound DHST, which is obtained by knocking out cytochrome P450 monooxygenase gene in an aspergillus versicolor D5 strain by a knock-out fragment, wherein the knock-out fragment is obtained by taking the genome of the aspergillus versicolor D5 strain as a template, amplifying the upstream sequence and the downstream sequence of the cytochrome P450 monooxygenase gene by using a primer to obtain a 0.8kb-1.2kb upstream fragment and a 0.8kb-1.2kb downstream fragment, and fusing the upstream fragment and the downstream fragment with hygromycin resistance genes; the cytochrome P450 monooxygenase gene sequence is a sequence shown in a sequence table; the aspergillus versicolor D5 Δ stc17 strain is now preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.23209, and the detection on the metabolite of the aspergillus versicolor D5 Δ stc17 strain shows that: the amount of the metabolic herbicidal activity Xanthone compound DHST is three times higher than that of the herbicidal activity Xanthone compound DHST metabolized by the original Aspergillus versicolor D5 strain; the invention discloses a building block method of aspergillus versicolor D5 delta stc17 strain for high yield of active Xanthone compounds DHST.)

1. An Aspergillus versicolor D5 delta stc17 strain of high-yield active Xanthone compound DHST, which is characterized in that: the aspergillus versicolor D5 Δ stc17 strain is obtained by knocking out cytochrome P450 monooxygenase gene in aspergillus versicolor D5 strain, the construction of the knock-out segment takes the genome of aspergillus versicolor D5 strain as a template, primers are utilized to amplify the upstream sequence and the downstream sequence of cytochrome P450 monooxygenase gene to obtain 0.8kb-1.2kb upstream segment and 0.8kb-1.2kb downstream segment, and the upstream segment and the downstream segment are fused with hygromycin resistance gene; the cytochrome P450 monooxygenase gene sequence is a sequence shown in a sequence table; the aspergillus versicolor D5 delta stc17 strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 23209.

2. The building block method of aspergillus versicolor D5 Δ stc17 strain of high-yield active Xanthone compound DHST according to claim 1, characterized in that: the method comprises the following steps:

(1) using the genome of the aspergillus versicolor D5 strain as a template, amplifying an upstream sequence and a downstream sequence of cytochrome P450 monooxygenase gene by using primers to obtain a 0.8kb-1.2kb upstream fragment and a 0.8kb-1.2kb downstream fragment, and fusing the upstream fragment and the downstream fragment with a hygromycin resistance gene to obtain a knockout fragment; the cytochrome P450 monooxygenase gene sequence is a sequence shown in a sequence table;

(2) preparing protoplasts of Aspergillus versicolor strain D5;

(3) transforming the knockout fragment into a protoplast to obtain a transformant;

(4) and culturing the transformant, then obtaining a aspergillus versicolor positive transformant by screening, and finally obtaining the aspergillus versicolor D5 Δ stc17 strain by performing gene verification on the aspergillus versicolor positive transformant.

3. The building block method of aspergillus versicolor D5 Δ stc17 strain of high-yielding active Xanthone compound DHST according to claim 2, characterized in that: the amplification conditions in the step (1) are as follows: denaturation at 94 deg.C for 5 min; denaturation at 95 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 60s, 32 cycles; extension at 72 ℃ for 7 min.

4. The building block method of aspergillus versicolor D5 Δ stc17 strain of high-yielding active Xanthone compound DHST according to claim 2, characterized in that: the protoplast for preparing the aspergillus versicolor D5 strain in the step (2) is specifically as follows: inoculating Aspergillus versicolor D5 strain to potato solid culture medium plate, and culturing at 30 deg.C for 5-7 days; selecting a fungus block with a proper size, smashing, inoculating into 100ml of liquid potato culture medium, culturing at 30 ℃ for 24h, filtering and collecting mycelia; cleaning mycelium with 0.8M magnesium chloride, adding mycelium into enzymolysis solution of 1% lysozyme, and performing enzymolysis at 30 deg.C and 100rpm for 1-2 hr; the mycelia were removed by filtration, and centrifuged at 3000rpm for 30min to obtain protoplasts of Aspergillus versicolor D5 strain.

5. The building block method of aspergillus versicolor D5 Δ stc17 strain of high-yielding active Xanthone compound DHST according to claim 2, characterized in that: the step (3) of transforming the knockout fragment into the protoplast to obtain a transformant specifically comprises the following steps: washing protoplasts of the Aspergillus versicolor D5 strain with 1.2M sorbitol solution, and suspending the protoplasts with an appropriate amount of sorbitol solution to a concentration of 108/ml; adding 10ul of knockout fragment into every 150ul of protoplast, adding 50ul of 40% PEG4000, gently mixing, and ice-cooling for 30 min; adding 1ml of 40% PEG4000, mixing uniformly, standing at room temperature for 10min, mixing with 15ml of upper agar culture medium, pouring onto 5 regenerated hygromycin screening culture medium plates, and performing dark culture at 30 ℃ for 5d to obtain a transformant.

6. The building block method of aspergillus versicolor D5 Δ stc17 strain of high-yielding active Xanthone compound DHST according to claim 2, characterized in that: the transformant is cultured in the step (4), then a aspergillus versicolor positive transformant is obtained through screening, and finally the aspergillus versicolor D5 Δ stc17 strain is obtained through gene verification of the aspergillus versicolor positive transformant, wherein the strain specifically comprises: transferring the transformant obtained in the step (3) to a screening plate, carrying out passage purification for 5 days at 30 ℃, repeating twice, then randomly selecting 3 cultured transformants, collecting mycelia to extract genome DNA, carrying out genome PCR verification to obtain a aspergillus versicolor positive transformant, finally carrying out single spore separation purification on the aspergillus versicolor positive transformant, and selecting 3 single colonies from the transformants to carry out genome PCR verification again; the Aspergillus versicolor D5 Δ stc17 strain was finally obtained.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a aspergillus versicolor D5 delta stc17 strain for producing active Xanthone compounds DHST at high yield and a construction method thereof.

Background

According to incomplete statistics, there are over 3 million weeds worldwide, of which over 1800 species are harmful to the growth of crop plants. Weeds reduce the yield or quality of crops by competing with the crops for nutrients and space, even cause dead harvest in severe cases, and if the weeds are not controlled, the yield of grains can be reduced by 10% -15% all over the world, and even the yield of the crops can be reduced by more than 50% in severe cases. Over the past several decades, chemical herbicides have played a major role in weed control. The action targets of most of the current herbicides are mainly the following: synthesis and metabolism of photosynthetic pigments and related components, ammonia metabolism and amino acid biosynthesis, lipid biosynthesis, and photosynthetic electron transport system. Less action targets enable weeds to easily generate drug resistance, and reports show that 272 drug-resistant weed biotypes appear at present. Meanwhile, the long-term, continuous and large-area use of chemical herbicides also causes a series of problems such as pesticide residue, food safety and environmental pollution, and thus, the research of novel environment-friendly biological herbicides is urgently needed.

The natural product herbicide mainly refers to a herbicide developed by secondary metabolites with herbicidal activity generated by plants, animals, microorganisms and the like, and has the advantages of abundant resources, no damage to ecological environment, easy biodegradation, low toxicity, less residue, strong selectivity, safety to non-target organisms and mammals, good environmental compatibility, low development cost, novel chemical structure, unique action mode, high target selectivity and the like which are incomparable with synthetic herbicides. Natural product herbicides can be divided into plant-derived herbicides, animal-derived herbicides and microbial-derived herbicides according to their origin. At present, animal-derived herbicides are not reported, plant-derived herbicides are rarely reported, and main research and application objects are focused on microbial-derived herbicides.

The microbial herbicide has the advantages of rich resources, small environmental pollution and the like, meets the development requirement of sustainable agriculture, and has attracted extensive attention of people in recent years. Microbial herbicides are divided into two types, live microorganisms and microbial secondary metabolites. Compared with living microorganisms and chemical herbicides, microbial secondary metabolites have the following advantages: firstly, the chemical structure is novel, and the novel phytotoxic compound is a potential novel phytotoxic compound which is difficult to discover by chemical synthesis; secondly, compared with active microbial herbicides, the herbicide is easier to store, beneficial to formulation processing, convenient to use and less interfered by the environment; thirdly, the multi-target action site and mode are generally adopted, so that the weed resistance is not easy to generate; fourthly, the biological antibacterial agent is easy to degrade in the environment, is low in toxicity to mammals mostly, and is safe to non-target organisms; finally, development and registration costs are low.

Aspergillus versicolor from marine origin (Aspergillus versicolor) D5 strain is separated and identified that polyketide Dihydrosterigmatocystin (DHST) has obvious herbicidal activity, the minimum weed suppression concentration of Amaranthus retroflexus reaches 8.0 mug/mL, namely, weeds have disease symptoms and stop growing at the concentration; the minimum weed control concentration reaches 31 mug/mL. The herbicidal activity of the compound is twice that of the positive drug glyphosate, the acting time is fast, the weeds have diseases after two days of application, and the weeds completely die after 4 days. Meanwhile, the compound is subjected to weed resistance spectrum screening aiming at amaranth weeds, and the compound is found to have obvious weeding effect on amaranth weeds including amaranthus spinosus, amaranthus viridis, purslane and allium alternantherum. However, DHST is currently produced in low yields and difficult to extract.

Disclosure of Invention

In view of the above, the present invention provides a aspergillus versicolor D5 Δ stc17 strain for producing a xanthene compound DHST with high activity in high yield, and provides a building block method of the aspergillus versicolor D5 Δ stc17 strain for producing the xanthene compound DHST with high activity in high yield.

One of the purposes of the invention is realized by the following technical scheme:

a aspergillus versicolor D5 Δ stc17 strain of a high-yield active Xanthone compound DHST is obtained by knocking out a cytochrome P450 monooxygenase gene in an aspergillus versicolor D5 strain by a knock-out fragment, wherein the knock-out fragment is obtained by taking a genome of the aspergillus versicolor D5 strain as a template, an upstream sequence and a downstream sequence of the cytochrome P450 monooxygenase gene are amplified by using a primer to obtain a 0.8kb-1.2kb upstream fragment and a 0.8kb-1.2kb downstream fragment, and the upstream fragment and the downstream fragment are fused with a hygromycin resistance gene to obtain the aspergillus versicolor D5 Δ stc17 strain; the cytochrome P450 monooxygenase gene sequence is a sequence shown in a sequence table; the aspergillus versicolor D5 delta stc17 strain is preserved in China general microbiological culture Collection center, the preservation address is No. 3 of Xilu No. 1 of Beijing Korean district, the preservation number is CGMCC NO.23209, and the preservation date is 30 years from 8 months and 12 days in 2021.

The second purpose of the invention is realized by the following technical scheme:

the construction method of aspergillus versicolor D5 Δ stc17 strain of high-yield active Xanthone compound DHST comprises the following steps:

(2) preparing protoplasts of Aspergillus versicolor strain D5;

(3) transforming the knockout fragment into a protoplast to obtain a transformant;

Further, the amplification conditions in the step (1) are as follows: denaturation at 94 deg.C for 5 min; denaturation at 95 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 60s, 32 cycles; extension at 72 ℃ for 7 min.

Further, the protoplast for preparing the aspergillus versicolor D5 strain in the step (2) is specifically: inoculating Aspergillus versicolor D5 strain to potato solid culture medium plate, and culturing at 30 deg.C for 5-7 days; selecting a fungus block with a proper size, smashing, inoculating into 100ml of liquid potato culture medium, culturing at 30 ℃ for 24h, filtering and collecting mycelia; cleaning mycelium with 0.8M magnesium chloride, adding mycelium into enzymolysis solution of 1% lysozyme, and performing enzymolysis at 30 deg.C and 100rpm for 1-2 hr; the mycelia were removed by filtration, and centrifuged at 3000rpm for 30min to obtain protoplasts of Aspergillus versicolor D5 strain.

Further, the step (3) of transforming the knock-out fragment into protoplast to obtain a transformant is specifically as follows: washing protoplasts of the Aspergillus versicolor D5 strain with 1.2M sorbitol solution, and suspending the protoplasts with an appropriate amount of sorbitol solution to a concentration of 108/ml; adding 10ul of knockout fragment into every 150ul of protoplast, adding 50ul of 40% PEG4000, gently mixing, and ice-cooling for 30 min; adding 1ml of 40% PEG4000, mixing uniformly, standing at room temperature for 10min, mixing with 15ml of upper agar culture medium, pouring onto 5 regenerated hygromycin screening culture medium plates, and performing dark culture at 30 ℃ for 5d to obtain a transformant.

Still further, the step (4) of culturing the transformants, then obtaining aspergillus versicolor positive transformants by screening, and finally obtaining the aspergillus versicolor D5 Δ stc17 strain by performing gene verification on the aspergillus versicolor positive transformants specifically comprises the following steps: transferring the transformant obtained in the step (3) to a screening plate, carrying out passage purification for 5 days at 30 ℃, repeating twice, then randomly selecting 3 cultured transformants, collecting mycelia to extract genome DNA, carrying out genome PCR verification to obtain a aspergillus versicolor positive transformant, finally carrying out single spore separation purification on the aspergillus versicolor positive transformant, and selecting 3 single colonies from the transformants to carry out genome PCR verification again; the Aspergillus versicolor D5 Δ stc17 strain was finally obtained.

The invention has the beneficial effects that:

the aspergillus versicolor D5 delta stc17 strain of the high-yield active Xanthone compound DHST has the following beneficial effects:

(1) through detection of metabolites of aspergillus versicolor D5 Δ stc17 strain, the following results are found: the amount of the metabolic herbicidal activity Xanthone compound DHST is three times higher than that of the herbicidal activity Xanthone compound DHST metabolized by the original Aspergillus versicolor D5 strain;

(2) the growth speed of the aspergillus versicolor D5 Δ stc17 strain is obviously higher than that of the aspergillus versicolor D5 strain;

(3) the metabolite abundance of the D5 Δ stc17 strain was significantly improved.

The method for constructing the aspergillus versicolor D5 delta stc17 strain of the high-yield active Xanthone compound DHST has simple steps and easy realization.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof.

Drawings

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings, in which:

FIG. 1 is a liquid chromatogram comparison of a metabolite of Aspergillus versicolor D5 Δ stc17 strain with a metabolite of Aspergillus versicolor D5 strain;

FIG. 2 is a graph showing the comparison of Aspergillus versicolor D5. DELTA. stc17 strain and Aspergillus versicolor D5 strain obtained by inoculating the same mass of mycelium of Aspergillus versicolor D5. DELTA. stc17 strain and mycelium of Aspergillus versicolor D5 strain to obtain different bacterial cell amounts.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be understood that the preferred embodiments are illustrative of the invention only and are not limiting upon the scope of the invention.

A aspergillus versicolor D5 Δ stc17 strain of a high-yield active Xanthone compound DHST is obtained by knocking out cytochrome P450 monooxygenase gene in an aspergillus versicolor D5 strain by a knock-out fragment, wherein the knock-out fragment is obtained by taking the genome of the aspergillus versicolor D5 strain as a template, amplifying the upstream sequence and the downstream sequence of the cytochrome P450 monooxygenase gene by using a primer to obtain a 0.8kb-1.2kb upstream fragment and a 0.8kb-1.2kb downstream fragment, and fusing the upstream fragment and the downstream fragment with hygromycin resistance gene; the cytochrome P450 monooxygenase gene sequence is a sequence shown in a sequence table; the aspergillus versicolor D5 delta stc17 strain is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 23209.

A building block method of Aspergillus versicolor D5 Δ stc17 strain of high-yield active Xanthone compound DHST, comprising the following steps:

(1) the genome of a strain of aspergillus versicolor D5 is used as a template, primers stc17-uF (AGTTCGTCAATGTGGCAGG) and stc17-u (hpn) R (ctttacgcttgcgatccagCCgaCCGCCCATCATGGGTGTAGAAAA) are used for amplifying an upstream sequence of a cytochrome P450 single oxygenase gene, primers (hpn) stc17D-F (cctgggttcgcaaagagatagggGCGCAGGCAGGCTATACGACGAAG) and stc17D-R CCCGATATCCCAGGCCTGAGGCTCG) are used for amplifying a downstream sequence of the P450 single oxygenase gene, the cytochrome P450 single gene sequence is a sequence shown in a sequence table, and the amplification conditions are as follows: denaturation at 94 deg.C for 5 min; denaturation at 95 ℃ for 30s, renaturation at 58 ℃ for 30s, extension at 72 ℃ for 60s, 32 cycles; extending for 7min at 72 ℃ to obtain a 1kb upstream fragment and a 1kb downstream fragment, and fusing the upstream fragment and the downstream fragment with the hygromycin resistance gene to obtain a knockout fragment, wherein the upstream fragment and the downstream fragment are respectively positioned at two sides of the hygromycin resistance gene;

(2) inoculating Aspergillus versicolor D5 strain to potato solid culture medium plate, and culturing at 30 deg.C for 6 days; selecting a fungus block with a proper size, smashing, inoculating into 100ml of liquid potato culture medium, culturing at 30 ℃ for 24h, filtering and collecting mycelia; cleaning mycelium with 0.8M magnesium chloride, adding mycelium into enzymolysis solution of 1% lysozyme, and performing enzymolysis at 30 deg.C and 100rpm for 1.5 hr; the mycelia were removed by filtration, and centrifuged at 3000rpm for 30min to obtain protoplasts of Aspergillus versicolor D5 strain.

(3) Washing protoplasts of the Aspergillus versicolor D5 strain with 1.2M sorbitol solution, and suspending the protoplasts with an appropriate amount of sorbitol solution to a concentration of 108/ml; adding 10ul of knockout fragment into every 150ul of protoplast, adding 50ul of 40% PEG4000, gently mixing, and ice-cooling for 30 min; adding 1ml of 40% PEG4000, mixing uniformly, standing at room temperature for 10min, mixing with 15ml of upper agar culture medium, pouring onto 5 regenerated hygromycin screening culture medium plates, and performing dark culture at 30 ℃ for 5d to obtain a transformant.

(4) Transferring the transformant obtained in the step (3) to a screening plate, carrying out passage purification for 5 days at 30 ℃, repeating twice, then randomly selecting 3 cultured transformants, collecting mycelia to extract genome DNA, carrying out genome PCR verification to obtain a aspergillus versicolor positive transformant, finally carrying out single spore separation purification on the aspergillus versicolor positive transformant, and selecting 3 single colonies from the transformants to carry out genome PCR verification again; the Aspergillus versicolor D5 Δ stc17 strain was finally obtained.

In order to verify the metabolite changes of the aspergillus versicolor D5 Δ stc17 strain, a test group and a comparison group are established;

test group

Fermenting Aspergillus versicolor D5 Δ stc17 strain, detecting the metabolite change of the mutant strain by LC-MS, which comprises the following steps:

a fermentation culture of Aspergillus versicolor D5 Δ stc17 strain: a potato dextrose liquid culture medium is adopted, 5.5g/L potato extract powder, 18g/L glucose, 28 g/L crude sea salt and 380mL water are added into a 1000 mL conical flask, and after aspergillus versicolor D5 delta stc17 strain is inoculated, fermentation culture is carried out for 35 days at 28 ℃.

b, extracting the fermentation liquor obtained in the step a by using ethyl acetate, and concentrating; the fermentation bacteria are fermented with methanol: soaking in 1:1 dichloromethane solution, concentrating under reduced pressure, extracting the residual water phase with ethyl acetate, concentrating under reduced pressure, mixing with the fermentation broth extract, and drying to obtain extract.

And c, performing primary treatment on the extract obtained in the step b by adopting a normal-phase silica gel column, eluting by using 10% ethyl acetate-petroleum ether and 10% methanol-ethyl acetate respectively, concentrating the 10% methanol-ethyl acetate elution part until the part is dried, and dissolving by using methanol to prepare 10.0 mg/mL. Performing HPLC analysis by using Agilent 1290 system, wherein the system is provided with a DAD detector, and the detection wavelength range is 210-700 nm; the analytical column was Waters SunAire C18 (5 μm, 4.6X 250 mm), eluting with A pump, methanol, B pump, water (0.1% trifluoroacetic acid added); the flow rate was 1.0 mL/min, and the composition and ratio of the mobile phase were selected according to the polarity of the sample. The mass spectrum detection device is a Saimer fly LTQ Orbitrap XL, the spraying voltage is 4.0 kv, the tube voltage is 16 v, the mirror voltage is 35 v, the capillary temperature is 300 ℃, the sheath gas flow rate is 40L/min, the auxiliary gas flow rate is 10L/min, and the mass precision generated by external calibration of the mass spectrum is better than 3 ppm; mass spectrum data are respectively obtained in a positive ionization mode and a negative ionization mode, the resolution is 30000, the mass range is 100-1500 Da, and then data correlation scanning is carried out in a CID mode; data acquisition and analysis were performed using the software of model 4.1, model f.

Comparison group

The comparative group was different from the test group only in that the strain of the comparative group was the A.versicolor D5 strain.

The liquid chromatogram of the test group and the comparison group are shown in figure 1, and as can be seen from figure 1, the yield of the herbicidal active Xanthone compound DHST (dihydrostermatostatin) is greatly improved, namely the DHST peak area corresponding to the Aspergillus versicolor D5 delta stc17 strain is 11841613 mv · s, the DHST peak area corresponding to the Aspergillus versicolor D5 strain is 4200800 mv · s, the yield of the Aspergillus versicolor D5 delta stc17 strain DHST is 3 times higher, and meanwhile, the abundance of metabolites of the Aspergillus versicolor D5 delta stc17 strain is also obviously improved;

the growth rate of aspergillus versicolor strain D5 Δ stc17 was increased: that is, the same quality of mycelia was inoculated, and the amount of mycelia was observed by photographing at 14D of fermentation, as shown in FIG. 2, the growth rate of Aspergillus versicolor D5 Δ stc17 strain was significantly higher than that of Aspergillus versicolor D5 strain.

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Sequence listing

SEQUENCE LISTING

<110> Guizhou province tobacco company Zunyi City company

Tobacco institute of Chinese academy of agricultural sciences (Qingzhou tobacco institute of Chinese tobacco Co., Ltd.)

<120> Aspergillus versicolor D5 delta stc17 strain with high-yield activity Xanthone compound DHST and component method

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Thr Asn Ile Asp Val Thr Ser Ala Gln Phe Ala Tyr Gly Leu Ile Asn

180 185 190

Met Gly Lys Ser Pro Ala Ala Gly Arg Arg Leu Tyr Asp Glu Val Ser

195 200 205

Thr Val Ala Val Glu Glu Asp Glu Ile Asp Phe Tyr Ala Arg Lys Asp

210 215 220

Asp Thr Phe Leu His Lys Met Tyr Leu Glu Ile Leu Arg Ser Asn Pro

225 230 235 240

Pro Ile Trp Phe Thr Phe Pro Glu Thr Thr Ala Val Glu Lys Arg Ile

245 250 255

Asp Gly Phe Leu Ile Pro Ala Gly Thr Asn Val Val Ile Asp Thr Leu

260 265 270

Arg Leu Asn Lys Ser Ser Pro Ile Trp Gly Asn Thr Gly His Glu Phe

275 280 285

His Pro Glu Arg Trp Asp His Ile Thr Thr Gly Gln Ala Arg Tyr Ser

290 295 300

Trp Leu Gly Tyr Gly Met Gly Pro Arg Lys Cys Leu Gly Lys Asn Phe

305 310 315 320

Ala Asn Ile Ile Leu Lys Leu Phe Leu Ile Ser Asn Thr Leu Leu Asn

325 330 335