CN114134054B

CN114134054B - Aspergillus oryzae chassis strain for high yield of terpenoid and construction of terpene natural product automatic high-flux excavation platform

Info

Publication number: CN114134054B
Application number: CN202111283704.0A
Authority: CN
Inventors: 刘天罡
Original assignee: Wuhan Hesheng Technology Co ltd
Current assignee: Wuhan Hesheng Technology Co ltd
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2024-06-18
Anticipated expiration: 2041-11-01
Also published as: CN114134054A

Abstract

The invention discloses an aspergillus oryzae chassis with high terpenes yield and a method for automatically excavating a terpenes biosynthesis gene cluster FgJ02895 with high flux, which aim at the screening of compounds with high terpenes yield, high throughput heterologous reconstruction and activity as guiding of the terpenes biosynthesis gene cluster. The invention establishes Aspergillus oryzae chassis cells with high terpenes yield based on CRISPR/Cas9 technology mediated gene homologous recombination, and integrates high expression sites in genome to over-express endogenous MVA pathway genes, thereby realizing 41 times and 111 times of improvement of mangicdiene and mangicol J yields. The high throughput mining strategy established in the chassis, including PCR amplification, plasmid library and strain library construction, enables mining of the relevant products of the gene cluster FgJ02895 and the obtaining of the product library of the gene cluster. And meanwhile, the anti-inflammatory activity of the compounds generated in the strain library can be evaluated.

Description

Aspergillus oryzae chassis strain for high yield of terpenoid and construction of terpene natural product automatic high-flux excavation platform

Technical Field

The invention relates to the technical field of natural product biosynthesis, in particular to construction of a chassis with high yield of terpenes by aspergillus oryzae and construction of an automatic high-flux excavation platform of the terpenes natural products based on the chassis strain.

Background

Fungal natural products are one of the important sources of natural medicines, and traditional mining strategies have helped researchers to find many active natural products from microorganisms, such as potent antibiotics penicillin, hypolipidemic lovastatin, immunosuppressant cyclosporine, and the like. However, as more and more natural products are discovered, the difficulty and challenges of being able to mine into novel natural products are increasing. With the development of bioinformatics and sequencing technologies, a large number of fungal natural product biosynthetic gene cluster information was revealed, particularly silent gene clusters, implying great potential for the synthesis of novel natural products. To fully release the synthetic potential of these natural product biosynthetic gene clusters, researchers have developed various strategies such as global or specific pathway regulation, exogenous activator activation, CRISPR/Cas strategy gene editing silencing gene cluster expression, and development of attractive heterologous reconstitution strategies to unlock a range of novel natural product synthesis mechanisms. In addition, in recent years, the development of the field of natural product biosynthesis is further promoted by the genome mining and artificial chromosome technology based on bioinformatics and computer network model predictive analysis technology. Although the development of the above strategies has greatly enriched the natural product libraries, there are still problems to be solved in the implementation process, such as low throughput, low yield, etc.

Terpenoids are a generic term for compounds containing isoprene units. It is widely found in nature, and to date, more than 12 tens of thousands of terpenoids have been found in animals, plants and microorganisms. The compounds have a plurality of physiological activities and are widely applied to the industries of foods, cosmetics and medicines. The invention takes fungus terpenes products as research targets, takes acknowledged strain Aspergillus oryzae (Aspergillus oryzae) with biological safety as a heterologous expression host, creates Aspergillus oryzae chassis with high terpene yield, establishes a filamentous fungus terpenes chassis with high terpene natural product yield, establishes a complete fungus terpenes product genome automatic high-throughput excavation research scheme, improves the throughput of terpene natural product gene cluster research, and solves the problem of low product yield.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an automatic high-throughput excavation platform for high-yield terpenes aspergillus oryzae chassis and terpenes natural products, in particular relates to high-efficiency synthesis of a diterpenoid compound mangicol J with anti-inflammatory activity and high-throughput heterologous reconstruction of a natural product biosynthesis gene cluster FgJ02895, and relates to PCR amplification of functional elements, plasmid library construction, bacterial strain library construction and anti-inflammatory activity-oriented compound screening of the functional elements.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

In a first aspect of the invention, the invention provides a microbial chassis cell comprising a gene that increases the ability of the chassis cell to produce terpenoid compounds, the gene being a gene of an aspergillus oryzae derived MVA pathway gene module; the genes are ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI genes derived from Aspergillus oryzae. Preferably, the nucleotide sequence of the ERG10 gene is set forth in SEQ ID NO:10 is shown in the figure; the nucleotide sequence of the ERG13 gene is shown as SEQ ID NO: 11; the nucleotide sequence of the tHMG1 gene is shown as SEQ ID NO: shown at 12; the nucleotide sequence of the ERG12 gene is shown as SEQ ID NO: 13; the nucleotide sequence of the ERG8 gene is shown as SEQ ID NO: 14; the nucleotide sequence of the MVD1 gene is shown in SEQ ID NO: 15; the nucleotide sequence of the IDI gene is shown as SEQ ID NO: shown at 16.

In one or more embodiments of the invention, the microbial chassis cell is a eukaryotic cell or a prokaryotic cell; preferably, the microbial chassis cell is aspergillus oryzae, escherichia coli, saccharomyces cerevisiae, pichia pastoris, bacillus, aspergillus nidulans, aspergillus niger, neurospora crassa, alternaria alternata or fusarium; more preferably, the chassis cell is aspergillus oryzae.

In one or more embodiments of the present invention, the terpenoid is selected from one or more of a hemiterpenoid, a monoterpenoid, a sesquiterpenoid, a diterpenoid, a triterpene, a tetraterpenoid, and a polyterpenoid. In one or more embodiments of the present invention, the gene hmg1 is a rate-limiting enzyme gene, preferably, in the chassis cell, the rate-limiting enzyme expressed by the gene hmg1 is overexpressed; more preferably, the gene tHMG 1-expressed rate-limiting enzyme is overexpressed in 4 copies.

In a second aspect of the invention, the invention provides the use of a gene from Aspergillus oryzae ERG10, ERG13, HMG1, ERG12, ERG8, MVD1 and IDI for increasing the ability of a chassis cell to produce terpenoid compounds.

In a third aspect of the present invention, the present invention provides a method for preparing a microbial chassis cell according to the first aspect of the present invention, comprising the steps of:

step 1): establishing a promoter library of the microorganism chassis strain: screening promoters from different sources, and characterizing the strength of the promoters suitable for aspergillus oryzae by regulating and controlling the expression of GUS genes;

Step 2): the mevalonate pathway of Saccharomyces cerevisiae is used as a reference, and the mevalonate pathway synthetases ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1 and IDI of the origin of Aspergillus oryzae are screened from a fungal genome database through sequence homology alignment analysis;

Step 3): and (2) respectively constructing 7 mevalonate pathway synthetases of the mevalonate pathway derived from the aspergillus oryzae screened in the step (2) on different plasmids under the regulation of promoters with different intensities represented in the step (1), and increasing the copy number of the rate-limiting enzyme expressed by the rate-limiting enzyme gene tHMG1 to obtain the chassis cell.

In other embodiments of the invention, the preparation method of the chassis cell according to the first aspect of the invention comprises integrating ERG10, ERG13, hmg1, ERG12, ERG8, MVD1 and IDI genes derived from aspergillus oryzae into high expression sites of aspergillus oryzae genome respectively by using CRISPR/Cas9 strategy, increasing the copy number of the rate-limiting enzyme expressed by the rate-limiting enzyme gene hmg1, and obtaining the microbial chassis cell.

In a fourth aspect of the present invention, the present invention provides a strain with high terpene production, wherein the chassis cell of the third aspect of the present invention is used as an initial strain, and the strain with high terpene production is obtained by respectively overexpressing the sesterterpene synthase gene mgcD and coexpression of the sesterterpene synthase gene mgcD and the cytochrome P450 enzyme gene mgcE. Preferably, the nucleotide sequence of gene mgcD is set forth in SEQ ID NO: shown at 17; the nucleotide sequence of the gene mgcE is shown in SEQ ID NO: shown at 18.

In a fifth aspect of the invention, the invention provides a high throughput automated excavation method of a terpenoid biosynthesis gene cluster, comprising the steps of:

1) Performing PCR amplification; 2) Constructing a plasmid library; 3) Constructing a microorganism strain library; 4) Screening for compounds directed to activity.

In one or more embodiments of the present invention, the step 2) includes the steps of:

a. respectively designing 40bp overlapping sequences at the 5 '-end and the 3' -end of each functional gene, promoter and terminator sequence in the terpenoid biosynthesis gene cluster, preparing a DNA polymerase system by an automatic workstation, and carrying out PCR amplification to obtain each fragment;

b. Assembling each fragment by using automated yeast (FIG. 2) to obtain a recombinant plasmid;

c. the recombinant plasmid was transformed into E.coli (FIG. 4), and the plasmid pool was obtained by enrichment.

In one or more embodiments of the present invention, the step 3) includes the steps of: transforming the plasmid library obtained in step 2) into the microorganism chassis cells according to the first aspect of the invention (FIG. 6) based on an automated platform according to a rational combination of the different functional elements involved (FIG. 5), including terpene synthases, cytochrome P450 enzymes, acyltransferases, glycosyltransferases, to obtain a strain library.

In one or more embodiments of the invention, the step 4) includes the steps of: and (3) acting the fermentation product of the microbial strain library obtained in the step (3) on lipopolysaccharide-induced mouse macrophage RAW264.7, and screening out terpenoid (figure 7) with anti-inflammatory activity, such as mangicols-type sesterterpene, by taking the generation of Nitric Oxide (NO) as a detection index.

In a sixth aspect of the present invention, there is provided a novel structure sesquiterpene compound having a structure selected from one of the following structures (FIG. 9):

17 (characterized by the following figures 10-16),

20 (Characterized by the following figures 17-23),

28 (Characterization is as in FIGS. 24-30).

In a seventh aspect of the invention, the invention provides the use of a sesquiterpene compound according to the sixth aspect of the invention for the manufacture of an anti-inflammatory medicament.

In an eighth aspect of the invention, the invention provides the use of the sesquiterpene synthesis gene cluster BGC6-FgJ02895 for the synthesis of a sesquiterpene compound according to the sixth aspect of the invention. The sesquiterpene synthesis gene cluster BGC6-FgJ02895 comprises FgJ02892 gene, fgJ02895 gene, fgJ02896 gene, fgJ02897 gene, fgJ02898 gene, fgJ02899 gene, fgJ02900 gene, fgJ02901 gene, fgJ02902 gene, which respectively encode decarboxylase, terpene synthase, transcription factor, cytochrome P450 enzyme, acetyltransferase, tf, cytochrome P450 enzyme, monooxygenase, copper ion dependent atpase (as shown in fig. 8).

Preferably, the nucleotide sequence of FgJ02892 gene is shown in SEQ ID NO: indicated at 23; the nucleotide sequence of FgJ02895 gene is shown as SEQ ID NO: shown at 24; the nucleotide sequence of FgJ02896 gene is shown as SEQ ID NO: shown at 25; the nucleotide sequence of FgJ02897 gene is shown as SEQ ID NO: 26; the nucleotide sequence of FgJ02898 gene is shown as SEQ ID NO: shown at 27; the nucleotide sequence of FgJ02899 gene is shown as SEQ ID NO: 28; the nucleotide sequence of FgJ02900 gene is shown as SEQ ID NO: 29; the nucleotide sequence of FgJ02901 gene is shown as SEQ ID NO: shown at 30; the nucleotide sequence of FgJ02902 gene is shown as SEQ ID NO: shown at 31.

In a ninth aspect of the invention, the invention provides a vector or vector mixture comprising a gene as described in the first and/or eighth aspect of the invention.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention provides a microorganism chassis cell which is modified to have the capability of producing terpenoid in high yield.

2. The invention provides a preparation method of the microorganism chassis cell, which takes aspergillus oryzae as a dominant expression host, adopts rational design, is based on a genetic transformation strategy of multiple steps and repeated utilization of screening marks in the aspergillus oryzae host mediated by CRISPR/Cas9 technology, over-expresses the whole MVA path in the aspergillus oryzae chassis cell, and establishes the aspergillus oryzae chassis cell with high terpenoid yield by adding 4 copies of tHMG1 genes, thereby providing a new solution for solving the problem of low yield in the natural product biosynthesis process.

3. The invention provides a strain with high terpene yield, which is obtained by modifying the chassis cells.

4. The invention provides a method for producing terpenoid by using the strain with high terpenoid yield.

5. The invention provides application of ERG10, ERG13, HMG1, ERG12, ERG8, MVD1 and IDI genes derived from aspergillus oryzae in improving high-yield terpenoid of chassis cells.

6. In the invention, the constructed rice yeast microorganism chassis cells are preferably used as dominant expression hosts, and the terpene natural product biosynthesis gene cluster BGC-FgJ02895 is efficiently and heterologously reconstructed in the body based on a constructed automatic high-flux platform through rational design, so that the construction from PCR amplification and plasmid library construction to strain library construction can be completed, and a solution is provided for solving the problem of low flux in the natural product biosynthesis process.

7. The invention provides a sesquiterpene compound with a new structure, which is characterized in that: having the structure shown in compounds 17, 20 and 28.

8. The invention provides a sesquiterpene synthesis gene cluster BGC6-FgJ02895, which is characterized in that: encoding and synthesizing the sesquiterpene compounds with the three new structures.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

Fig. 1 is a schematic diagram of a construction flow and a part of experimental results of a terpene high-yield chassis according to an embodiment of the invention.

FIG. 1a is a schematic diagram showing the preparation process of an Aspergillus oryzae mutant strain of the present invention; FIG. 1b is a diagram showing the results of comparison of the yields of the starting strain AO-Y51, the mutant strain AO-S96, and the mutant strain AO-S84 mangicdiene; FIG. 1c is a schematic representation of the engineering process of Aspergillus oryzae mutants AO-S84, AO-S94, AO-S96, AO-S98; FIG. 1d is a schematic diagram showing the results of comparison of the yields of the starting strain AO-Y52, the mutant strain AO-S98 and the mutant strain AO-S94 Mangicol J.

FIG. 2 is a schematic flow chart of a plasmid library constructed by high throughput yeast assembly according to one embodiment of the invention.

FIG. 3 is a schematic of a high throughput plasmid extraction procedure according to one embodiment of the invention.

FIG. 4 is a schematic diagram of a high throughput E.coli transformation scheme according to one embodiment of the invention.

FIG. 5 is a schematic representation of the construction principle of a high throughput Aspergillus oryzae mutant strain according to one embodiment of the present invention.

FIG. 6 is a schematic diagram of a high throughput construction process of a library of Aspergillus oryzae mutants in accordance with one embodiment of the present invention.

FIG. 7 is a schematic diagram of a detection scheme of anti-inflammatory activity in an embodiment of the present invention.

FIG. 8a is a diagram of the results of the bioinformatics analysis of the gene cluster BGC6-FgJ 02895; FIG. 8b is a schematic diagram of heterologous reconstitution of the BGC6-FgJ02895 gene cluster.

FIG. 9 is a GC/MS and HR-LC/MS detection pattern of the gene cluster BGC6-FgJ 02895.

FIG. 10 is a schematic structural view of a compound (17) according to an embodiment of the present invention;

FIG. 11 is a ¹ H NMR spectrum of compound (17) of the present invention;

FIG. 12 is a chart showing the C NMR spectrum of compound (17) ¹³ of the present invention;

FIG. 13 is a chart showing the COSY spectrum of compound (17) ¹H-¹ H of the present invention;

FIG. 14 is a spectrum of HSQC of compound (17) of the present invention;

FIG. 15 is a chart showing the HMBC spectra of compound (17) of the present invention;

FIG. 16 is a ROESY spectrum of compound (17) of the present invention.

FIG. 17 is a schematic structural view of a compound (20) according to an embodiment of the present invention;

FIG. 18 is a ¹ H NMR spectrum of compound (20) of the present invention;

FIG. 19 is a ¹³ C NMR spectrum of the compound (20) of the present invention;

FIG. 20 is a ¹H-¹ H COSY spectrum of compound (20) of the present invention;

FIG. 21 is a HSQC spectrum of the compound (20) of the present invention;

FIG. 22 is a HMBC spectrum of the compound (20) of the present invention;

FIG. 23 is a ROESY spectrum of the compound (20) of the present invention.

FIG. 24 is a schematic structural view of a compound (28) according to one embodiment of the present invention;

FIG. 25 is a ¹ H NMR spectrum of compound (28) of the present invention;

FIG. 26 is a ¹³ C NMR spectrum of compound (28) of the present invention;

FIG. 27 is a ¹H-¹ H COSY spectrum of compound (28) of the present invention;

FIG. 28 is a HSQC spectrum of compound (28) of the present invention;

FIG. 29 is a HMBC spectrum of compound (28) of the present invention;

FIG. 30 is a ROESY spectrum of compound (28) of the present invention.

Detailed Description

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any method or material similar or equivalent to those described may be used in the present invention. Wherein "-" represents a genetic linkage, for example hlyA (nucleotide sequence shown as SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown as SEQ ID NO: 12) NOs (nucleotide sequence shown as SEQ ID NO: 22) represents a nucleotide sequence shown as SEQ ID NO:9, and the nucleotide sequence of the gene hlyA is shown as SEQ ID NO:12, the nucleotide sequence of the gene tHMG1 is shown as SEQ ID NO:22, and the gene nos shown in the above were sequentially linked.

Example 1 determination of mevalonate pathway synthase in Aspergillus oryzae

The fungal genome database was screened for the mevalonate pathway synthetases ERG10, ERG13, HMG1, ERG12, ERG8, MVD1 and IDI of Aspergillus oryzae origin by sequence homology alignment analysis with reference to the Saccharomyces cerevisiae-derived Mevalonate (MVA) pathway.

EXAMPLE 2 construction of expression vectors

All genes are obtained through PCR amplification, and the used primers are shown in a sequence table 1:

table 1 primer sequences used in the examples

The plasmid construction mainly adopts four methods, namely enzyme digestion, goldengate assembly, gibson method and Yeast assembly method. The specific construction method is as follows:

Construction of pSC11 plasmid: using Aspergillus oryzae genome as template, primers 11-2F/11-2R,11-7F/11-7R were used to amplify wA up, wA down (DOI: 10.1038/nature 04300), respectively. pGB123 (DOI: 10.1002/boot. 20160697) was used as template and the promoter alcA (nucleotide sequence shown as SEQ ID NO: 1) was amplified with primer pair 11-3F/11-3R. GUS was amplified using pCAMBIA1301 as template and primer pair 11-4F/11-4R. TamyB and AdeA were amplified with primers 11-5F/11-5R,11-6F/11-6R, respectively, using pAdeA (DOI: 10.1021/ja 3116636) as templates. The vector sequence was amplified using pRS426 (DOI: 10.1016/j. Biortech.2012.08.104) as template and primers 11-1F/11-1R. The 7 amplified fragments were assembled by Yeast assembly method to obtain pSC11 plasmid. Construction of pSC12-pSC19 plasmid: pGB99, pGB98, pGB93, pGB96, pGB92 and rice yeast genome (DOI: 10.1002/boot. 20160697) were used as templates, and primers 12-1F/12-1R,13-1F/13-1R,14-1F/14-1R,15-1F/15-1R,16-1F/16-1R were used to amplify promoter agdA (nucleotide sequence shown as SEQ ID NO: 2), glaA (nucleotide sequence shown as SEQ ID NO: 3), respectively, gpdA (nucleotide sequence shown as SEQ ID NO: 4), oliC (nucleotide sequence shown as SEQ ID NO: 5), trpC (nucleotide sequence shown as SEQ ID NO: 6). The Aspergillus oryzae genome is used as a template, and primers 17-1F/17-1R,18-1F/18-1R,19-1F/19-1R are used for amplifying amyB (the nucleotide sequence is shown as SEQ ID NO: 7), enoA (the nucleotide sequence is shown as SEQ ID NO: 8) and hlyA (the nucleotide sequence is shown as SEQ ID NO: 9). GUS was amplified using pCAMBIA1301 as a template and primer pairs 12-2F/12-2R,13-2F/13-2R,14-2F/14-2R,15-2F/15-2R,16-2F/16-2R,17-2F/17-2R,18-2F/18-2R,19-2F/19-2R (DOI: 10.1016/j.synbio.2016.07.002). The pSC12-pSC19 plasmid was constructed by overlap extension PCR (OE-PCR) ligation of 8 promoter fragments with the corresponding GUS fragment, pacI/MreI cleavage, followed by insertion into the PacI/MreI digested pSC11 plasmid.

Based on the promoter with stronger promoter capability in the fungal host reported in the literature, the promoter strength suitable for aspergillus oryzae is characterized by regulating the expression of GUS reporter gene, and in order to characterize the promoter strength of a series of promoters in the aspergillus oryzae host, the plasmid pSC11-pSC19 is transformed into the aspergillus oryzae to obtain strains AO-S11 to AO-S19 containing corresponding promoters. Finally, the promoter suitable for Aspergillus oryzae is obtained with the sequence of hlyA > oliC > amyB > glaA > enoA > gpdA > agdA > trpC > alcA.

Construction of pSC59 plasmid: the vector fragment was amplified using pSC11 plasmid as template and primers 44-1F/44-1R. The homologous arm fragments ku80-up and ku80-down (DOI: 10.1038/nature 04300) were amplified using Aspergillus oryzae genomic DNA as template and primers 44-2F/44-2R, 44-13F/44-13R. The Aspergillus oryzae cDNA is used as a template, and primers 44-4F/44-4R,44-7F/44-7R,44-10F/44-10R are used to amplify the fragments ERG10 (nucleotide sequence shown as SEQ ID NO: 10), ERG13 (nucleotide sequence shown as SEQ ID NO: 11) and tHMG1 (nucleotide sequence shown as SEQ ID NO: 12), respectively. pGB96 and Aspergillus oryzae genomic DNA were used as templates, and primers 44-3F/44-3R,44-6F/44-6R,44-9F/44-9R were used to amplify the promoter fragments oliC (nucleotide sequence shown as SEQ ID NO: 5), amyB (nucleotide sequence shown as SEQ ID NO: 7) and hlyA (nucleotide sequence shown as SEQ ID NO: 9), respectively. The primers 44-5F/44-5R,44-8F/44-8R,44-11F/44-11R were used to amplify the terminator fragment niaD (nucleotide sequence shown as SEQ ID NO: 20), amyB (nucleotide sequence shown as SEQ ID NO: 19) and NOs (nucleotide sequence shown as SEQ ID NO: 22), respectively, using A. Nidurans FGSC A4, aspergillus oryzae genome and pGB96 as templates. Fragment AdeA was amplified using primers 44-12F/44-12R using plasmid pAdeA (DOI: 10.1021/ja 3116636) as template. The closely sized fragments were subjected to overlap extension PCR (OE-PCR) and finally yeast assembled to give plasmid pSC44. pSC44 was used as a template, and the fragment hlyA (nucleotide sequence shown in SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown in SEQ ID NO: 12) -NOs (nucleotide sequence shown in SEQ ID NO: 22) was amplified with primers 59-1F/59-1R, and subjected to SacI cleavage, and inserted into the SacI-digested pSC44 plasmid to construct a pSC59 plasmid.

Construction of pSC71 plasmid: the vector fragment was amplified using pSC11 plasmid as template and primers 71-1F/71-1R. The homologous arm fragments ku70-up and ku70-down (DOI: 10.1038/nature 04300) were amplified with primers 71-2F/71-2R,71-11F/71-11R, respectively, using Aspergillus oryzae genomic DNA as a template. The Aspergillus oryzae cDNA is used as a template, and the primers 71-5F/71-5R,71-8F/71-8R are used to amplify the fragment MVD1 (the nucleotide sequence is shown as SEQ ID NO: 15) and IDI (the nucleotide sequence is shown as SEQ ID NO: 16). Using pSC44 as a template, the fragment hlyA (nucleotide sequence shown as SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown as SEQ ID NO: 12) -NOs (nucleotide sequence shown as SEQ ID NO: 22) was amplified using primers 71-3F/71-3R. The Aspergillus oryzae genome and pGB98 were used as templates, and primers 71-4F/71-4R,71-7F/71-7R were used to amplify the promoter oliC (nucleotide sequence shown as SEQ ID NO: 5) and glaA (nucleotide sequence shown as SEQ ID NO: 3). The fragment argB-PamyB-mgcD (nucleotide sequence shown as SEQ ID NO: 17) -TamyB was amplified using pYJ152 as a template and primers 71-10F/71-10R. Aspergillus oryzae genomic DNA and A.nidulans FGSC A4 were used as templates, and primers 71-6F/71-6R,71-9F/71-9R were used to amplify terminator agdA (nucleotide sequence shown as SEQ ID NO: 21) and niaD (nucleotide sequence shown as SEQ ID NO: 20). The fragments of similar size were subjected to overlap extension PCR (OE-PCR) and finally yeast assembled to give plasmid pSC71.

Construction of pSC73 plasmid: the vector fragment was amplified using pSC11 plasmid as template and primers 73-1F/73-1R. The homologous arm fragments ku70-up and ku70-down (DOI: 10.1038/nature 04300) were amplified with primers 73-2F/73-2R,73-11F/73-11R, respectively, using Aspergillus oryzae genomic DNA as a template. The Aspergillus oryzae cDNA is used as a template, and primers 73-4F/73-4R,73-7F/73-7R are used to amplify fragments ERG12 (the nucleotide sequence is shown as SEQ ID NO: 13) and ERG8 (the nucleotide sequence is shown as SEQ ID NO: 14). Using pSC44 as a template, the fragment hlyA (nucleotide sequence shown as SEQ ID NO: 9) -tHMG1 (nucleotide sequence shown as SEQ ID NO: 12) -niaD (nucleotide sequence shown as SEQ ID NO: 20) was amplified using primers 73-9F/73-9R. pGB98 and Aspergillus oryzae genomic DNA were used as templates, and primers 73-3F/73-3R and 73-6F/73-6R were used to amplify the promoters glaA (nucleotide sequence shown as SEQ ID NO: 3) and enoA (nucleotide sequence shown as SEQ ID NO: 8). pGB96 and Aspergillus oryzae genomes were used as templates, and terminator NOs (nucleotide sequence shown as SEQ ID NO: 22) and amyB (nucleotide sequence shown as SEQ ID NO: 19) were amplified using primers 73-5F/73-5R and 73-8F/73-8R. Using pUSA as a template, sC was amplified with primers 73-10F/73-10R. The closely sized fragments were subjected to overlap extension PCR (OE-PCR) and finally yeast assembled to give plasmid pSC73.

Construction of pSC111 plasmid: pGB98 was used as template and terminator NOs (nucleotide sequence shown as SEQ ID NO: 22) was amplified using primers 111-1F/111-1R. The pSC11 plasmid was used as a template and the vector fragment was amplified using primers 111-3F/111-3R. The homologous arm fragments koj-down and koj-up (DOI: 10.1038/nature 04300) were amplified with primers 111-2F/111-2R, respectively, using Aspergillus oryzae genomic DNA as a template. ptrA (DOI: 10.1007/s 10529-015-2015-x) was amplified using Aspergillus oryzae genomic DNA as a template and primers 111-5F/111-5R. The Aspergillus oryzae genomic DNA was used as a template, and hlyA was amplified using primers 111-6F/111-6R (nucleotide sequence shown in SEQ ID NO: 9). mgcE (nucleotide sequence shown as SEQ ID NO: 18) was amplified using pYJ153 as a template and primers 111-7F/111-7R. The fragments of similar size were subjected to overlap extension PCR (OE-PCR) and finally yeast assembled to give plasmid pSC111.

Construction of pSC112 plasmid: aspergillus oryzae genomic DNA was used as a template and primer 112-1F/112-1R was used to amplify terminator agdA (nucleotide sequence shown as SEQ ID NO: 21). Using pSC111 as a template, hlyA (nucleotide sequence shown in SEQ ID NO: 9) -mgcE (nucleotide sequence shown in SEQ ID NO: 18) was amplified using primers 112-2F/112-2R. The two fragments were subjected to overlap extension PCR (OE-PCR), digested with NotI/HpaI, and inserted into NotI/HpaI digested pSC111 plasmid to construct pSC112 plasmid.

Construction of pSC253 plasmid: the vector sequence was amplified using pSC11 as template and primers 251-1F/251-1R. PamyB-Cas9-TamyB was amplified with primers 251-2F/251-2R using pAdeA-Cas9 (DOI: 10.1007/s 10529-015-2015-x) as template. hAMA1 (DOI: 10.1006/fgbi.1997.0980) was amplified using the A.nidullasFGSC A4 template with the primers 251-3F/251-3R. ptrA (DOI: 10.1271/bbb.64.1416) was amplified using pSC111 as a template and primers 251-4F/251-4R. Yeast was assembled to give plasmid pSC251. The fragment CEN was amplified using pSC11 as template and primers 78-1F/78-1R. The plasmid pSC78 was constructed by XhoI/BamHI cleavage and insertion into the XhoI/BamHI cleaved pET28 plasmid. P _U6-1,T_U6-1,P_U6-2,T_U6-2,P_U6-3,T_U6 -3 was amplified with primers 251-1F/251-1R,251-2F/251-2R,251-3F/251-3R,251-4F/251-4R,251-5F/251-5R,251-6F/251-6R using Aspergillus oryzae genomic DNA as template. Overlapping extension PCR (OE-PCR) was performed on P _U6 -1 and T _U6-1,P_U6 -2 and T _U6-2,P_U6 -3 and T _U6 -3, and these three fragments were GoldenGate assembled with pSC78 to construct pSC252 plasmid. pSC253 plasmid was constructed by inserting a fragment containing 3 sgRNAs, obtained by cleavage of pSC252 with NotI/PacI, into pSC251 obtained by cleavage with NotI/PacI.

Construction of pSC184 plasmid: the argB and vector sequences were amplified using pSC71 as template and primers 87-1F/87-1R. PamyB-Cas9-TamyB was amplified with primer 87-2F/87-2R using pAdeA (DOI: 10.1021/ja 3116636) as template. hAMA 1A was amplified using primers 87-3F/87-3R using A.nidurans FGSC A4 as template. pyrG was amplified using primers 87-4F/87-4R using A. Fumigatus as template. Four fragment yeasts were assembled to construct pSC87. P _U6-1,T_U6-1,P_U6-2,T_U6-2,P_U6-3,T_U6 -3 was amplified with 98-1F/98-1R,98-2F/98-2R,98-3F/98-3R,98-4F/98-4R,98-5F/98-5R, 98-6F/98-6R. Overlapping extension PCR (OE-PCR) was performed on P _U6 -1 and T _U6-1,P_U6 -2 and T _U6-2,P_U6 -3 and T _U6 -3, and these three fragments were GoldenGate assembled with pSC78 to construct pSC98 plasmid. pSC184 plasmid was constructed by inserting 3 sgRNA-containing fragments of pSC98 digested with NotI/SmaI into pSC87 digested with NotI/SmaI.

Construction of pSC249 plasmid: using A. NidulonsFGSC A4 as a template, the AMA1 sequence split into two fragments was amplified using primers 134-1F/134-1R, 134-2F/134-2R. pyrG was amplified using pSC87 as template and primers 134-3F/134-3R. The vector sequence and PamyB-Cas9-TamyB were amplified with primers 134-4F/134-4R,134-5F/134-5R using pSC251 as template. Four fragment USER Cloning constructed pSC134. P _U6,T_U6 was amplified using the A.oryzae genome as a template and primers 249-1F/249-1R,249-2F/249-2R, overlap extension PCR (OE-PCR), notI/PacI cleavage, and pSC134 digested with NotI/PacI was inserted to construct pSC249 plasmid.

Construction of pSC246 plasmid: the vector sequence was amplified using pSC11 as a template and primers 246-1F/246-1R. HS401 up and HS401 down (DOI: 10.1038/nature 04300) were amplified using the A.oryzae genome as template with primers 246-2F/246-2R, 246-5F/246-5R. PhlyA-tHMG1-Tnos were amplified using pSC44 as a template and primers 246-3F/246-3R. PoliC-MVD-TagdA-PamyB-IDI-TniaD was amplified using pSC71 as template and primers 246-4F/246-4R. The 5 fragments were yeast assembled to construct pSC246 plasmid.

Construction of pSC247 plasmid: using pSC246 as a template, tnos-tHMG1-PhlyA was amplified with primers 247-1F/247-1R, mreI/SalI digested, and pSC246 digested with MreI/SalI was inserted to construct pSC247 plasmid.

Construction of pSC263 plasmid: the pSC263 plasmid was constructed by amplifying P _U6,T_U6 with primers 263-1F/263-1R,263-2F/263-2R using Aspergillus oryzae genome as a template, performing overlap extension PCR (OE-PCR), performing NotI/PacI cleavage, and inserting pSC134 digested with NotI/PacI.

Construction of pSC260 plasmid: the vector sequence was amplified using pSC11 as template and primers 260-1F/260-1R. HS201 up and HS201 down (DOI: 10.1038/nature 04300) were amplified using the A.oryzae genome as template and primers 260-2F/260-2R, 260-4F/260-4R. PoliC-ERG10-TniaD-PamyB-ERG13-TamyB and PhlyA-tHMG1-Tnos were amplified with primers 260-3F/260-3R using pSC44 as template. The 4 fragments were yeast assembled to construct pSC260 plasmid.

Construction of pSC248 plasmid: the pSC248 plasmid was constructed by amplifying P _U6,T_U6 with primers 248-1F/248-1R,248-2F/248-2R using Aspergillus oryzae genome as a template, overlap extension PCR (OE-PCR), notI/PacI cleavage, and insertion of NotI/PacI cleaved pSC 134.

Construction of pSC262 plasmid: the vector sequence was amplified using pSC11 as template and primers 262-1F/262-1R. HS601 up and HS601 down (DOI: 10.1038/nature 04300) were amplified using Aspergillus oryzae genome as a template with primers 262-2F/262-2R,262-4F/262-4R, pSC73 as a template, pglaA-ERG12-Tnos-PenoA-ERG8-TamyB, and PhlyA-tHMG1-TniaD with primers 262-3F/262-3R. The 4 fragments were yeast assembled to construct pSC262 plasmid.

Construction of pSC24 plasmid: the pSC24 plasmid was constructed by amplifying P _U6,T_U6 with primers 24-1F/24-1R,24-2F/24-2R using Aspergillus oryzae genome as a template, overlap extension PCR (OE-PCR), notI/PacI cleavage, and insertion of NotI/PacI cleaved pSC 134.

Construction of pSC162 plasmid: the vector sequence was amplified using pSC11 as template and primers 162-1F/162-1R. HS801 up and HS801 down (DOI: 10.1038/nature 04300) were amplified using the A.oryzae genome as a template with primers 162-2F/162-2R, 162-4F/162-4R. PhlyA-mgcD-Tnos were amplified using pSC71 as a template and primers 162-3F/162-3R. The 4 fragments were yeast assembled to construct pSC162 plasmid.

Construction of pSC27 plasmid: the vector sequence was amplified using pSC11 as template and primers 25-1F/25-1R. HS801 up and HS801 down (DOI: 10.1038/nature 04300) were amplified using the A.oryzae genome as template and primers 25-2F/25-2R, 25-4F/25-4R. PamyB-mgcD-TamyB were amplified using pYJ152 as template and primers 25-3F/25-3R. The 4 fragments were yeast assembled to construct pSC25 plasmid. PhlyA-mgcE-Tnos were amplified using pSC111 as a template and primers 27-1F/27-1R. NotI/PacI cleavage, insertion of NotI/PacI cleaved pSC25 was performed to construct pSC27 plasmid.

Construction of pSC28 plasmid: phlyA-mgcE-Tnos were amplified using pSC111 as a template and primers 28-1F/28-1R. PacI cleavage, insertion of pSC27 digested with PacI was performed to construct pSC28 plasmid.

EXAMPLE 3 construction strategy of Aspergillus oryzae mutant strain highly producing terpenoid

In order to produce target terpenoid at high yield, a series of Aspergillus oryzae mutant strains are rationally designed and constructed. In the construction of the strain, plasmids containing different genes are transformed into Aspergillus oryzae by adopting a protoplast transformation method. After the transformants grow out, 3-4 transformants are selected to extract genome, and PCR verification is performed on the target genes. And carrying out subculture expansion on the strain with positive PCR result, and using the strain for subsequent seed preservation and fermentation experiments.

In order to achieve the yield improvement of the sesterterpene framework compound mangicdiene, the invention co-converts plasmids pSC59, pSC71 and pSC73 into Aspergillus oryzae to obtain a mutant strain AO-S84. To achieve an improved yield of compounds mangicol J with anti-inflammatory activity, the present invention transformed plasmids pSC111 and pSC112 into the A.oryzae mutant strain AO-S84 by increasing the copy number (2 copies) of mgcE to obtain mutants AO-S93 and AO-S94. In order to obtain the free plasmid type aspergillus oryzae chassis strain with high terpenoid yield, the plasmid pSC253 is transformed into an aspergillus oryzae mutant strain AO-S84 to obtain a mutant strain AO-S85.

Example 4 construction strategy of Aspergillus oryzae mutant strain with high terpenoid production

To obtain an A.oryzae strain containing a pyrG defect, the present invention transformed pSC184 plasmid into A.oryzae NSAR1 to obtain mutant AO-S184. In order to obtain the high-expression site integrated Aspergillus oryzae chassis strain with high terpenes yield, pSC249 and pSC246 plasmids are co-transformed into a strain AO-S184. Mutant strains, in which the gene fragment on pSC246 plasmid was integrated at the HS401 site, were cultured for 2-3 generations on plates without selection pressure, spores of the strain (10 ⁴ spores/5. Mu.L) were collected and plated on a medium containing uracil and 5-fluoroorotic acid, and strains, in which homologous recombination had occurred and the pSC249 plasmid had been lost, were selected as starting strains for the next round of gene editing (FIG. 1). Meanwhile, plasmids used for the second and third rounds of gene editing were pSC263 and pSC260, pSC248 and pSC262, respectively, resulting in an integrated Aspergillus oryzae chassis AO-S95 over-expressing the entire MVA pathway and an additional 3 copies of the highly terpenoid of tHMG 1. In order to achieve the yield improvement of the sesterterpene framework compound mangicdiene, the present invention co-converts plasmids pSC24 and pSC162 into A.oryzae to obtain the mutant strain AO-S96. To achieve an improved yield of compound mangicol J with anti-inflammatory activity, plasmids pSC24 and pSC27, pSC24 and pSC28 were co-transformed into the A.oryzae mutant strain AO-S95, respectively, by increasing the copy number of mgcE (2 copies), resulting in mutants AO-S97 and AO-S98.

EXAMPLE 5 enrichment and purification of terpenoids

In order to obtain enough products for compound yield calibration, the invention carries out mass fermentation on Aspergillus oryzae mutant strains AO-S84 and AO-S98. Placing the rice culture medium (5-10 kg) with the thalli in a 30 ℃ incubator, and standing for 20-25d. Crushing the cultured thalli by using a stirrer, adding normal hexane or ethyl acetate with the same volume for extraction for 3-4 times, combining the extracted organic layers, and concentrating by using a rotary evaporator to obtain crude extracts A and B. Purifying the crude extract A by multiple semi-preparative HPLC, eluting with pure acetonitrile and ultrapure water as mobile phases in a volume ratio of 75:25 and 90:10, and collecting fractions to obtain mangicdiene of sesterterpene compounds. Purifying the crude extract B by multiple semi-preparative HPLC, eluting with pure acetonitrile and ultrapure water as mobile phases in a volume ratio of 75:25 and 90:10, and collecting fractions to obtain Mangicol J of sesterterpene compounds.

EXAMPLE 6 Synthesis and detection of terpenoids

In order to detect the target terpenoid, the constructed mutant strain is subjected to amplification culture on a screening plate, 2mL of spore suspension containing 2.5x10 ⁷ is inoculated into 200mL of DPY liquid culture medium, and maltose and glucose are added to 1% of each to induce expression. 140rpm, at 30℃for 7d. Filtering the cultured thallus with nylon cloth, discarding culture medium, pulverizing mycelium with stirrer, adding equal volume of n-hexane or ethyl acetate, extracting for three times, and mixing the three upper organic layers. Spin-drying with rotary evaporator, adding chromatographic grade ethyl acetate or methanol, redissolving the sample, centrifuging at high speed, collecting supernatant, and detecting with GCMS or HR-LCMS.

According to the invention, three mutant strains of AO-S84, AO-S96 and AO-Y51 are subjected to 3 parallel fermentation culture respectively. After redissolving the fermentation product with n-hexane, GCMS was examined. The results showed that the AO-S84 and AO-S96 detected mangicdiene in yields of 87.84mg/L,27.38mg/L, respectively, which were 133-fold and 41-fold higher than the control strain AO-Y51 (0.66 mg/L), respectively. According to the invention, three mutant strains of AO-S93, AO-S94, AO-S97, AO-S98 and AO-Y52 are subjected to 3 parallel fermentation culture respectively. After redissolving the fermentation product with ethyl acetate, GCMS detection was performed. The results showed that the yields of Mangicol J were 9.14mg/L and 7.26mg/L, respectively, as detected by AO-S93 and AO-S97. After the mgcE copy was added, the yields of Mangicol J were detected as 12.09mg/L and 8.93mg/L by AO-S94 and AO-S98, respectively, which were improved by 151 and 111 times as compared with the developed strain AO-Y52 (0.08 mg/L).

EXAMPLE 7 high throughput amplification of recombinant fragments

The DNA fragments required for constructing the gene cluster BGC-FgJ02895 recombinant plasmid library are all obtained through PCR amplification. The Phusion high-fidelity DNA polymerase required for amplification was purchased from NEB company (NEW ENGLAND Biolabs, NEB), and the PRIME STAR GXL DNA polymerase was purchased from TaKaRa company (TaKaRa Bio, inc., shiga, japan). PCR primers were purchased from Kirschner Biotech Inc. (shown in Table 2 below). Magnetic beads for PCR purification of amplified fragments were purchased from Northenan Biotech Inc. (N411-01, vazyme).

Table 2 PCR primers

All functional genes, promoters, terminators and 5 '-and 3' -ends of the vector sequences contain overlapping fragments of 40bp bases for subsequent assembly by utilizing the inherent recombination capacity of Saccharomyces cerevisiae to obtain plasmid libraries. The preparation of the PCR system was done by an automated pipetting station (Biomek FX ^P Laboratory Automation Workstation, beckman Coulter). The PCR amplification adopts an amplification system of 40 mu L, the required primer is synthesized and dissolved and then is split into 96-well plates, a DNA polymerase system (containing buffer, dNTPs, DNA polymerase) is prepared according to the required quantity (589 fragment amplification quantity), and then is split into 96-well plates (34 mu L/well), and the template is diluted and split into 96-well plates (25 ng/mu L). Sequentially taking 2 mu L of the front primer, 2 mu L of the rear primer and 2 mu L of the template respectively, adding the two primers into a DNA polymerase system, blowing and sucking the mixture uniformly, transferring the mixture into a PCR instrument, and setting a program to carry out PCR amplification. The amplification procedure used in this example was as follows:

EXAMPLE 8 construction of recombinant plasmid by Yeast Assembly

The recombinant plasmid related to the gene cluster BGC-FgJ02895 is constructed by a Yeast assembly method. Saccharomyces cerevisiae (Saccharomyces cerevisiae CEN.PK2-1D, EUROSCARF) (genotype MATaura3-52trp1-289leu2-3 112his3Δ1MAL2-8C SUC2) was used as an experimental strain for homologous recombination, and the recombinant plasmid was constructed by the optimized lithium acetate/polyethylene glycol (LiAc/PEG) chemical transformation method. The experimental operations (e.g. "pipetting", "shaking", "pipetting", etc.) involved in the examples were all done by automated pipetting stations (Biomek FX ^P Laboratory Automation Workstation, beckman Coulter), the liquid dispensing operation was done by automated pipetting (Thermo Scientific ^TM Multidrop Combi SMART DISPENSER), the cell monoclonal selection was done by Qpix 460 monoclonal selection system (Molecular Device Qpix 460), the "centrifugation" operation was done by centrifuges configured in the automated stations and the "cell culture" was done in incubators configured in the automated stations. Plasmid extraction magnetic beads were purchased from Magen Bio (MagPure PLASMID LQ KIT) and plasmid miniprep kit was purchased from Axygen (Cat. No. AP-MN-P-250). The specific construction method is described in detail below.

The coding sequences, promoters, terminators and linear vector fragments required for constructing each recombinant plasmid associated with the gene cluster BGC-FgJ02895 obtained in example 7 were each taken at 300ng and mixed for use (total volume 10. Mu.L). The 10. Mu.L of the DNA mixture was transferred to a 96-well deep well plate containing competent cells of Saccharomyces cerevisiae (100. Mu.L/well) using an MP 200-Tip robot arm in an automated pipetting station, followed by sequential addition of boiled fish sperm DNA (20. Mu.L/well) and LiAc/TE/40% PEG4000 solution (700. Mu.L/well), followed by a "aspirate" procedure, pipetting, mixing, and incubation in a 30℃incubator for 30min. Taking out the culture well plate, adding DMSO (88 mu L/hole), blowing and sucking, mixing, transferring the culture plate to a 42 ℃ incubator, heat-shock for 8min, centrifuging at 1500rpm for 5min, discarding supernatant, adding 1mL YPD medium to resuspend thallus, transferring the culture plate to a 30 ℃ incubator, and incubating for 60min. Taking out the culture plate, centrifuging at 1500rpm for 5min, discarding supernatant, adding 1mL of TE solution to wash the thallus, centrifuging at 1500rpm for 5min, discarding part of supernatant, resuspending the thallus with the rest about 100 mu L of TE solution, sucking the thallus, adding the bacterial liquid into a 96-well deep-hole plate containing 1mL of uracil-deficient solid medium (SC-Ura), transferring the culture plate to a shaking module, setting a shake program for 30s, and uniformly coating the bacterial liquid on the solid medium. Transfer the plates to 30℃incubator for 3 days. The specific operation flow is shown in fig. 2.

Example 9 extraction of plasmid by magnetic bead method

Uracil-deficient liquid medium (SC-Ura) (200. Mu.L/well) was added to the cell culture plate, and cells were blown and dissolved to obtain a bacterial liquid. The bacterial solution was transferred to a 96-well deep-well plate containing 1.5mL of SC-Ura liquid medium and incubated overnight at 30 ℃. Centrifuging at 3500rpm for 8min, discarding supernatant, and extracting yeast plasmid by optimized magnetic bead method. Lysozyme (500U/mL, sigma-Aldrich, 20210108) and a plasmid miniprep reagent buffer S1 solution were added sequentially to the well plate, and the well plate was transferred to a 25℃incubator for incubation for 2h. The plate was rotated out, buffer S2 solution was added, the plate was transferred to a "shake" module, 700rpm, and shake for 45S. Buffer S3 was added and the mixture was shaken for 45S at 700 rpm. Subsequently, centrifugation was performed at 3500rpm for 10min, the supernatant was aspirated into a new 96-well deep well plate, 400. Mu.L of the magnetic bead solution was added, mixed by shaking, and allowed to stand for 5min. Transferring the pore plate to a magnetic frame for adsorption for 5min. Discarding the supernatant, sequentially adding buffer W1 and buffer W2, washing, discarding the supernatant, standing for 5min, volatilizing the solution, adding 100 μl of sterile water, transferring the well plate to a magnetic frame, standing for 5min, and dissolving plasmid DNA. And sucking the plasmid solution into a new 96-well PCR plate to obtain the recombinant plasmid library related to the gene cluster BGC-FgJ 02895. The specific operation flow is shown in fig. 3.

EXAMPLE 10 enrichment of recombinant plasmid with E.coli

Competent cells of E.coli (ESCHERICHIA COLI DH B) were prepared using CaCl ₂ chemistry. Competent cells were dispensed by an automated pipetting device into 96-well deep well plates at a rate of 70 μl/well. mu.L of plasmid DNA solution was added to E.coli DH10B competent cells and incubated for 30min at 4 ℃. Subsequently, the plate containing the cells was transferred to a preheated 42℃incubator, and after incubation for 3min, 800. Mu.L of LB medium was added to each well, and incubation was carried out at 37℃for 45min to resuscitate the cells. Subsequently, the plate was transferred to a centrifuge, and centrifuged at 1500g for 8min to collect the cells. A portion of the supernatant was discarded, and the remaining approximately 50. Mu.L of the medium was resuspended, plated onto LA solid medium containing ampicillin (AMPICILLIN, amp) resistance, and cultured overnight at 37 ℃. And picking thalli to LB culture medium by adopting Qpix 460,460 instrument, and performing expansion culture to enrich plasmids. The obtained plasmid related to the gene cluster BGC-FgJ02895 is verified by restriction enzyme digestion. A specific operational flow is shown in fig. 3.

Specifically included is the construction of the following plasmids (FgJ genome, the genome of the filamentous fungus Fusarium graminearum J1-012 (Fusariumgraminearum J-012)):

Construction of pYJ182 plasmid: the fragments pYJ182-F1 and pYJ182-F2 were amplified respectively using FgJ genome and pYJ152 as templates and the primer pairs HQD02895gDNA-Tamy GR (182)/HQD 02895-Pamy GF (182) and Tamy-HQD02895gDNA GR (182)/Pamy-HQD 02895gDNA GF (182) and yeast assembled to obtain plasmid pYJ182.

Construction of pYJ183 plasmid: the fragments pYJ183-F1 and pYJ183-F2 were amplified with the primer pairs HQD2897gDNA-Tamy GR (183)/HQD 2897gDNA-Pamy GF (183) and Tamy-HQD2897gDNA GR (183)/Pamy-HQD 2897gDNA GF (183), respectively, and the above 2 fragments were subjected to yeast assembly to obtain plasmids pYJ183.

Construction of pYJ184 plasmid: the fragments pYJ184-F1 and pYJ184-F2 were amplified with the primer pairs HQD2900gDNA-Tamy GR (184)/HQD 2900gDNA-Pamy GF (184) and Tamy-HQD2900gDNA GR (184)/Pamy-HQD 2900gDNA GF (184), respectively, and the above 2 fragments were subjected to yeast assembly to obtain plasmid pYJ184.

Construction of pYJ185 plasmid: the fragments pYJ185-F1 and pYJ185-F2 were amplified with the primer pairs HQD2901gDNA-Tamy GR (185)/HQD 2901gDNA-Pamy GF (185) and Tamy-HQD2901gDNA GR (185)/Pamy-HQD 2901gDNA GF (185), respectively, and the above 2 fragments were subjected to yeast assembly to obtain plasmids pYJ185.

Construction of pYJ199 plasmid: the fragments pYJ199-F1, pYJ199-F2, pYJ199-F3 and pYJ199-F4 were amplified with the primer pair PglaA-7577gDNA GF(175-176)/PglaA-HQD02901gDNA GR(199)、HQD02901-PglaA GF(199)/HQD02901-TniaD GR(199)、TniaD-HQD02901 GF(199)/TniaD-Tamy GR(175-176)、Tamy-TniaD GF(175-176)/AdeA-PglaAGR(175-176) using pGB98, fgJ genome, pGB127 and pYJ184 as templates, respectively, and the above 4 fragments were subjected to yeast assembly to obtain plasmid pYJ199.

Construction of pYJ200 plasmid: the 7 fragments pYJ200-F1, pYJ200-F2, pYJ200-F3, pYJ200-F4, pYJ200-F5, pYJ200-F6 and pYJ200-F7 were amplified with primer set Tagda-adeAGF(177)/TagdA-HQD02898 GR(200)、HQD02898-TagdA GR(200)/HQD02898-PhlyA GF(200)、PhlyA-HQD02898 GR(200)/PhlyA-PglaAGF(177)、PglaA-PhlyA GF(177)/PglaA-HQD02901gDNA GR(199)、HQD02901-PglaA GF(199)/HQD02901-TniaD GR(199)、TniaD-HQD02901 GF(199)/TniaD-Tamy GR(175-176)、Tamy-TniaD GF(175-176)/AdeA-TagdA GR(177) using pGB127, fgJ genome, pYJ177, pGB98, fgJ genome, pGB127 and pYJ184 as templates, respectively, and yeast assembly was performed to obtain plasmid pYJ200.

Construction of pYJ201 plasmid: the fragments pYJ201-F1, pYJ201-F2, pYJ201-F3 and pYJ201-F4 were amplified with the primer pair PglaA-HQD07575GF(178)/PglaA-HQD02896 GR(201)、HQD02896-PglaA GF(201)/HQD02896-TniaD GR(201)、TniaD-HQD02896GF(201)/TniaD-Tamy GR(175-176)、Tamy-TniaD GF(175-176)/sC-PglaA GR(178) using pGB98, fgJ genome, pGB127 and pYJ183 as templates, respectively, and the above 4 fragments were subjected to yeast assembly to obtain plasmid pYJ201.

Construction of pYJ202 plasmid: the 7 fragments pYJ202-F1, pYJ202-F2, pYJ202-F3, pYJ202-F4, pYJ202-F5, pYJ202-F6 and pYJ202-F7 were amplified with primer set TagdA-sC GR(180)/TagdA-HQD02899 GF(202)、HQD02899-TagdA GR(202)/HQD02899-PhlyA GF(202)、PhlyA-HQD02899 GR(202)/PhlyA-PglaA GF(177)、PglaA-PhlyA GF(177)/PglaA-HQD02896 GR(201)、HQD02896-PglaA GF(201)/HQD02896-TniaD GR(201)、TniaD-HQD02896 GF(201)/TniaD-Tamy GR(175-176)、Tamy-TniaD GF(175-176)/sC-TagdA GR(180) using pGB127, fgJ genome, pYJ177, pGB98, fgJ genome, pGB127 and pYJ183 as templates, respectively, and the above 7 fragments were subjected to yeast assembly to obtain plasmid pYJ202.

Construction of pYJ203 plasmid: the fragments pYJ203-F1, pYJ203-F2, pYJ203-F3 and pYJ203-F4 were amplified with the primer pair PglaA-Pamy GF(203)/PglaA-HQD02902 GR(203)、HQD02902-PglaA GF(203)/HQD02902-TniaD GR(203)、TniaD-HQD02902GF(203)/TniaD-ArgB GR(203)、ArgB-TniaD GF(203)/Pamy-PglaA GR(203) using pGB98, fgJ genome, pGB127 and pYJ182 as templates, respectively, and the above 4 fragments were subjected to yeast assembly to obtain plasmid pYJ203.

Construction of pYJ204 plasmid: the 7 fragments pYJ204-F1, pYJ204-F2, pYJ204-F3, pYJ204-F4, pYJ204-F5, pYJ204-F6 and pYJ204-F7 were amplified with primer set PglaA-Pamy GF(203)/PglaA-HQD02902 GR(203)、HQD02902-PglaA GF(203)/HQD02902-TniaD GR(203)、TniaD-HQD02902 GF(203)/TniaD-TagdA GR(204)、TagdA-TniaD GR(204)/TagdA-HQD02892 GF(204)、HQD02892-TagdA GR(204)/HQD02892-PhlyA GF(204)、PhlyA-HQD02892 GR(204)/Phlya-argB GF(204)、ArgB-PhlyAGF(204)/Pamy-PglaA GR(203) using pGB98, fgJ genome, pGB127, fgJ genome, pYJ177 and pYJ182 as templates, respectively, and yeast-assembled to obtain plasmid pYJ204.

EXAMPLE 11 construction of Aspergillus oryzae mutant library for terpene Synthesis

By bioinformatics analysis, the terpene biosynthesis gene cluster can be divided into 3 synthesis modules, which are respectively: 1) An upstream terpene synthase module: contains terpene synthases (TERPENE SYNTHASE, TS), and catalyzes precursor (such as FPP, GGPP, GFPP) to synthesize terpene skeleton compounds; 2) Midstream oxidation module: contains cytochrome P450 enzymes (cytochrome protein P, CYP 450), which catalyze the synthesis of double bonds, carbonyl or hydroxyl functions from the core backbone; 3) Downstream post-modification module: contains optional post-modification catalytic enzymes such as acylase (ACYLTRANSFERASE, ACT) and glycosylase (glycosylation enzyme, GE) which catalyze the intermediate compounds synthesized in the step 2) so as to synthesize terpenoid compounds with various structures and potential activities. Therefore, in order to reconstruct the terpenoid gene cluster with high throughput, obtain intermediates and final terpenoid compounds and analyze the biosynthesis path, the invention designs the combined transformation of recombinant plasmids rationally according to the inherent sequential catalytic characteristics of the terpenoid gene cluster synthesis, and constructs the Aspergillus oryzae mutant strain screening library related to the gene cluster BGC-FgJ02895 (shown in figure 5). The strain is constructed by adopting a protoplast transformation method, and plasmids containing different genes are transformed into Aspergillus oryzae based on an automatic pipetting workstation. The method comprises the following specific steps:

Before the experiment, an automated pipetting workstation was UV sterilized. Aspergillus oryzae strain is cultured on DPY solid plate for about 3 days, spore and mycelium are scraped and transferred to 100mL DPY liquid culture medium, and cultured at 30deg.C for 2 days, and the thallus is collected. And preparing Aspergillus oryzae protoplast by adopting an optimized enzymatic hydrolysis method. Protoplast solutions were dispensed into 96-well deep well plates by an automated pipetting apparatus in an amount of 100. Mu.L/well. The plasmid DNA mixture to be transformed was taken and added to the protoplast solution, the plate was transferred to an incubator and incubated at 30℃for 1 hour. Subsequently, 1.25ml of a solution of EG6000 was added and incubated for 30min at room temperature. The plate was transferred to a centrifuge and centrifuged at 420g for 25min, and part of the solution was discarded. To the remaining 200. Mu.L of solution, 800. Mu.L of STC solution was added, and 420g was centrifuged for 25min, and part of the solution was discarded. Cells were resuspended in approximately 100 μl of solution and transferred from a 96-well deep-well plate to a 24-well deep-well plate containing a triple-defect (sC ^-,ΔargB,adeA^-) solid screening medium using a flexible 8-channel robotic arm. The 24-well deep-well plate was then transferred to a shaking module, and after shaking the coated cells, the plate was transferred to an incubator and incubated at 30℃for 2-5 days (as shown in FIG. 6). After the transformant grows, extracting the genome, and carrying out PCR verification on the target gene. In the invention, BGC-FgJ02895 is taken as an example, and a strain library containing 11 mutant strains is finally constructed.

Specifically comprises the following construction of Aspergillus oryzae mutant strains:

The AO-Y23 cotransformation plasmid pGB366/pUSA/pAdeA, comprising terpene synthases FgJ02895; the AO-Y24 cotransformation plasmid pGB366/pYJ183/pAdeA comprises terpene synthase FgJ02895 and cytochrome P450 enzyme FgJ02897; the AO-Y25 cotransformation plasmid pGB366/pUSA/pYJ184 contains terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02900; the AO-Y26 cotransformation plasmid pGB366/pUSA/pYJ185 comprises terpene synthases FgJ02895 and monooxygenases FgJ02901; the AO-Y27 cotransformation plasmid pGB366/pYJ183/pYJ184 comprises terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897 and cytochrome P450 enzyme FgJ02900; the AO-Y28 cotransformation plasmid pGB366/pYJ183/pYJ199 comprises terpene synthases FgJ02895, cytochrome P450 enzymes FgJ02897, cytochrome P450 enzymes FgJ02900 and monooxygenases FgJ02901; the AO-Y29 cotransformation plasmid pGB366/pYJ183/pYJ200 comprises terpene synthases FgJ02895, cytochrome P450 enzymes FgJ02897, cytochrome P450 enzymes FgJ02900, monooxygenases FgJ02901 and acetyltransferases FgJ02898; the AO-Y30 cotransformation plasmid pGB366/pYJ201/pYJ200 comprises terpene synthase FgJ02895, cytochrome P450 enzyme FgJ02897, transcription factor FgJ02896, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ02901 and acetyltransferase FgJ02898; the AO-Y31 cotransformation plasmid pGB366/pYJ202/pYJ200 comprises terpene synthases FgJ02895, cytochrome P450 enzymes FgJ02897, transcription factors FgJ02896, tfFgJ02899, cytochrome P450 enzymes FgJ02900, monooxygenases FgJ02901 and acetyltransferases FgJ02898; the AO-Y32 cotransformation plasmid pYJ203/pYJ202/pYJ200 comprises terpene synthase FgJ02895, cu < 2+ > -ATPaseFgJ02902, cytochrome P450 enzyme FgJ02897, transcription factors FgJ02896, tfFgJ02899, cytochrome P450 enzyme FgJ02900, monooxygenase FgJ02901 and acetyl transferase FgJ02898; the AO-Y cotransformation plasmid pYJ204/pYJ202/pYJ200 comprises terpene synthase FgJ02895, cu < 2+ > -ATPaseFgJ02902, decarboxylase FgJ02892 (the nucleotide sequence is shown as SEQ ID NO: 25), cytochrome P450 enzyme FgJ02897 (the nucleotide sequence is shown as SEQ ID NO: 30), transcription factor FgJ02896 (the nucleotide sequence is shown as SEQ ID NO: 29), tf FgJ02899 (the nucleotide sequence is shown as SEQ ID NO: 32), Cytochrome P450 enzyme FgJ02900 (nucleotide sequence shown as SEQ ID NO: 33), monooxygenase FgJ02901 (nucleotide sequence shown as SEQ ID NO: 34), and acetyltransferase FgJ02898 (nucleotide sequence shown as SEQ ID NO: 31).

Example 12 Synthesis and detection of terpenoids

Aspergillus oryzae strains positive in PCR verification result are transferred to 24-well deep-hole plate culture containing rice solid culture medium, each strain is transferred to 6 compound wells (each well contains 1g of rice and 1.5mL of deionized water), and fermentation culture is carried out for 2 weeks at 30 ℃. Acetone (5 mL/well) was added to the cultured 24-well plate, and the plate was immersed at room temperature for 2 hours. The supernatant was transferred to a new 24-well deep well plate, ethyl acetate (5 mL/well) was added to the cells, and the cells were immersed for 2 hours at room temperature. The acetone and ethyl acetate extracts were combined, transferred to a fume hood from a 24-well plate, and naturally volatilized to obtain a crude extract. The production of terpenoid products in the mutants was detected by GC/MS and HR-LC/MS.

In the invention, 3 terpene compounds (17, 20 and 28) with new structures are separated and purified, and the structures are analyzed by nuclear magnetic resonance spectroscopy, and the results are as follows:

The data of C ₁₅H₂₄,¹ H-NMR and ¹³ C-NMR of the compound 17 are shown in tables 3 and 4 and FIGS. 10 to 16; HR-ESI-MS [ M+H ] ⁺ M/z 205.1948 in positive ion mode (calculated value 205.1951).

Compound 20 was taken up in C ₁₅H₂₄ O,-80.5(c 0.1,MeOH),UV(MeOH)λ_max(logε):196(3.81);IR(KBr)v_max3362,2955,2921,1710,1637,1460,1378,1270,1114,1064,907cm^–1;¹H-NMR And ¹³ C-NMR data are shown in tables 1 and 2 and FIGS. 17 to 23; HR-ESI-MS [ M+H ] ⁺ M/z 221.1908 in positive ion mode (calculated value 221.1900).

Compound 28 was taken up in the presence of C ₁₇H₂₈O₃,-38.5(c 0.12,MeOH),UV(MeOH)λ_max(logε):196(3.45);IR(KBr)v_max3432,2963,2928,1737,1632,1454,1384,1243,1045cm^–1;¹H-NMR And ¹³ C-NMR data are shown in tables 1 and 2 and FIGS. 24 to 30; HR-ESI-MS [ M+H ] ⁺ M/z 281.2117 in positive ion mode (calculated value 281.2111).

Table 3. ¹ H-NMR data for compounds 17, 20 and 28.

^a Recorded at 500MHz,and the assignments were based on DEPT,HSQC,COSY,HMBC,and ROESY experiments.

Table 4. ¹³ C-NMR data for compounds 17, 20 and 28.

Example 13 high throughput screening of terpenoid anti-inflammatory Activity

1) In vitro cytotoxicity assessment

In order to exclude the effect of cytotoxicity caused by the compounds on anti-inflammatory activity screening, the toxic effect of the compounds to be screened on the cells is first evaluated prior to the activity screening experiment. In this example, mouse macrophage RAW264.7 was used as a subject. Cells were inoculated at 2.0X10 ⁴ cells/well into 24-well plates, incubated at 37℃in a 5% CO ₂ incubator for 24 hours, and then the crude extract to be screened (final concentration 500. Mu.g/mL) was added thereto and incubated at 37℃in a 5% CO ₂ incubator for 24 hours. To each well 100. Mu.L of CCK-8 dilution (10. Mu.L CCK-8 in 90. Mu.L medium) was added, and after incubation in a 5% CO ₂ incubator at 37℃for 1h, the plate was transferred to a microplate reader and absorbance was measured at 450nm (as shown in FIG. 7).

2) In vitro anti-inflammatory Activity Screen

In this example, lipopolysaccharide (LPS) -induced mouse macrophage RAW264.7 was used as an in vitro inflammatory cell model. RAW264.7 mouse macrophages were inoculated into 24-well plates at 3.0X10 ⁵ cells/well, incubated in a 5% CO ₂ incubator at 37℃for 24 hours, and LPS (1. Mu.g/mL) was applied to the RAW264.7 mouse macrophages to perform modeling, thereby obtaining inflammatory cells. Crude extracts (final concentration 500. Mu.g/mL) were applied to model cells, respectively, after 16 hours, the supernatant was taken, NO levels in the supernatant were detected by Griess reagent chromogenic method, and the anti-inflammatory activity of the compounds was evaluated using the ability of the test drug to inhibit NO release as a screening index. After drug treatment of the cells, the supernatant was transferred from 24 well plates to 96 well plates using a flexible 8-channel robotic arm, followed by sequential addition of Griess I and Griess II reagents. The plate was transferred to a microplate reader and UV absorbance was measured at 540nm (as shown in FIG. 7).

While embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are illustrative and not to be construed as limiting the present invention, and that variations, modifications, alternatives and variations of the above embodiments may be made by those skilled in the art within the scope of the present invention and are intended to be included within the scope of the present invention.

Sequence listing

<110> University of Wuhan

<120> Construction of an Aspergillus oryzae Chassis strain for high yield of terpenoid and an automated high throughput excavation platform for terpenoid natural products

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 246

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 1

cgggatagtt ccgacctagg attggatgca tgcggaaccg cacgagggcg gggcggaaat 60

tgacacacca ctcctctcca cgcaccgttc aagaggtacg cgtatagagc cgtatagagc 120

agagacggag cactttctgg tactgtccgc acgggatgtc cgcacggaga gccacaaacg 180

agcggggccc cgtacgtgct ctcctacccc aggatcgcat ccccgcatag ctgaacatct 240

atataa 246

<210> 2

<211> 720

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 2

gccatcggat gctcccgtca tggcaccact agagatggcg tggaaaaccc tcaccggcac 60

accggagggg tttaggcacc ttggaatatg aggtggggaa cgatgtattt gccagtattg 120

actctggtga atggatctct cgagaaatac taccttttca gggctcaagc gtcgtgtcgg 180

gcatttatcg ggggatggac caatcagcgt agggatatca gatgatcgcc agcattggtc 240

aggaacgttt ccaatttccg gacacggaag tactgtaact gctcccaaga atcaacacac 300

tcttttccgg tctcgtcctt tgctcggcag agattcatct cccatcgtcg gcttaaccgg 360

tactctttcg tcacgttcca aaaggcttga tcatgctgtc cccactccgt gcgggtgaag 420

ccacctcatt gctgcgtagg acctataccc ttcaactagc gtgacttctt cccctctcat 480

ggtcgagaga ttgcaggcaa tgcccctcgg acgtttgacg gggaatgttt tgccttcacg 540

gcaggtagca caaatcgatg ggaacgggac gggccatcaa ttgtgaggga tttcccgtgg 600

acacctggtt cgtcaagaca tatacatcta gctacaattc cggttcggag acggcagagg 660

ggtccgtttc ttaaaagaac aactacaaca cggtccggaa tcaacttggc ggaccacgac 720

<210> 3

<211> 745

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 3

ggatccgaac tccaaccggg gggagtatat tgagtggccg cagtggaagg aatcgcggca 60

gttgatgaat ttcggagcga acgacgccag tctccttacg gatgatttcc gcaacgggac 120

atatgagttc atcctgcaga ataccgcggc gttccacatc tgatgccatt ggcggagggg 180

tccggacggt caggaactta gccttatgag attaatgatg gacgtgtctg gcctcggaaa 240

aggatatatg gggatcataa tagtactagc catattaatg aagggtatat accacgcgtt 300

ggacctgcgt tatagcttcc cgttagttat agtaccatcg ttataccagc caatcaagtc 360

accacgcacg accggggacg gcgaatcccc gggaattgaa agaaattgca tcccaggcca 420

gtgaggccag cgattggcca catctccaag gcacagggcc attctgcagc gctggtggat 480

tcatcgcaat ttcccccggc ccggcccgac accgctatag gctggttctc ccacaccatc 540

ggagattcgt cgcctaatgt ctcgtccgtt cacaagctga agagcttgaa gtggcgagat 600

gcctctgcag gaattcaagc tagatgctaa gcgatattgc atggcaattt gtgttgatgc 660

atgtgcttct tccttcagct tcccctcgtg cagatgaggt ttggctataa attgaagtgg 720

ttggtcgggg ttccgtgagg ggctg 745

<210> 4

<211> 708

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 4

acagtgaccg gtgactcttt ctggcatgcg gagagacgga cggacgcaga gagaagggct 60

gagtaataag cgccactgcg ccagacagct ctggcggctc tgaggtgcag tggatgatta 120

ttaatccggg accggccgcc cctccgcccc gaagtggaaa ggctggtgtg cccctcgttg 180

accaagaatc tattgcatca tcggagaata tggagcttca tcgaatcacc ggcagtaagc 240

gaaggagaat gtgaagccag gggtgtatag ccgtcggcga aatagcatgc cattaaccta 300

ggtacagaag tccaattgct tccgatctgg taaaagattc acgagatagt accttctccg 360

aagtaggtag agcgagtacc cggcgcgtaa gctccctaat tggcccatcc ggcatctgta 420

gggcgtccaa atatcgtgcc tctcctgctt tgcccggtgt atgaaaccgg aaaggccgct 480

caggagctgg ccagcggcgc agaccgggaa cacaagctgg cagtcgaccc atccggtgct 540

ctgcactcga cctgctgagg tccctcagtc cctggtaggc agctttgccc cgtctgtccg 600

cccggtgtgt cggcggggtt gacaaggtcg ttgcgtcagt ccaacatttg ttgccatatt 660

ttcctgctct ccccaccagc tgctcttttc ttttctcttt cttttccc 708

<210> 5

<211> 844

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 5

ctgcagctgt ggagccgcat tcccgattcg ggccggattg gtcaagattt gcgtccgagg 60

tgccgtctat cattctagct tgcggtcctg ggcttgtgac tggtcgcgag ctgccactaa 120

gtggggcagt accattttat cggacccatc cagctatggg acccactcgc aaatttttac 180

atcattttct ttttgctcag taacggccac cttttgtaaa gcgtaaccag caaacaaatt 240

gcaattggcc cgtagcaagg tagtcagggc ttatcgtgat ggaggagaag gctatatcag 300

cctcaaaaat atgttgccag ctggcggaag cccggaaggt aagtggattc ttcgccgtgg 360

ctggagcaac cggtggattc cagcgtctcc gacttggact gagcaattca gcgtcacgga 420

ttcacgatag acagctcaga ccgctccacg gctggcggca ttattggtta acccggaaac 480

tcagtctcct tggccccgtc ccgaagggac ccgacttacc aggctgggaa agccagggat 540

agaatacact gtacgggctt cgtacgggag gttcggcgta gggttgttcc caagttttac 600

acacccccca agacagctag cgcacgaaag acgcggaggg tttggtgaaa aaagggcgaa 660

aattaagcgg gagacgtatt taggtgctag ggccggtttc ctccccattt ttcttcggtt 720

ccctttctct cctggaagac tttctctctc tctcttcttc tcttcttcca tcctcagtcc 780

atcttccttt cccatcatcc atctcctcac ctccatctca actccatcac atcacaatcg 840

atcc 844

<210> 6

<211> 359

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 6

cagaagatga tattgaagga gcactttttg ggcttggctg gagctagtgg aggtcaacaa 60

tgaatgccta ttttggttta gtcgtccagg cggtgagcac aaaatttgtg tcgtttgaca 120

agatggttca tttaggcaac tggtcagatc agccccactt gtagcagtag cggcggcgct 180

cgaagtgtga ctcttattag cagacaggaa cgaggacatt attatcatct gctgcttggt 240

gcacgataac ttggtgcgtt tgtcaagcaa ggtaagtgaa cgacccggtc ataccttctt 300

aagttcgccc ttcctccctt tatttcagat tcaatctgac ttacctattc tacccaagc 359

<210> 7

<211> 604

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 7

tcatggtgtt ttgatcattt taaattttta tatggcgggt ggtgggcaac tcgcttgcgc 60

gggcaactcg cttaccgatt acgttagggc tgatatttac gtaaaaatcg tcaagggatg 120

caagaccaaa gtagtaaaac cccggagtca acagcatcca agcccaagtc cttcacggag 180

aaaccccagc gtccacatca cgagcgaagg accacytcta ggcatcggac gcaccatcca 240

attagaagca gcaaagcgaa acagcccaag aaaaaggtcg gcccgtcggc cttttctgca 300

acgctgatca cgggcagcga tccaaccaac accctccaga gtgactaggg gcggaaattt 360

aaagggatta atttccactc aaccacaaat cacagtcgtc cccggtattg tcctgcagaa 420

tgcaatttaa actcttctgc gaatcgcttg gattccccgc ccctggccgt agagcttaaa 480

gtatgtccct tgtcgatgcg atgtatcaca acatataaat actagcaagg gatgccatgc 540

ttggaggata gcaaccgaca acatcacatc aagctctccc ttctctgaac aataaacccc 600

acag 604

<210> 8

<211> 737

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 8

cagccagcga gagtcacaga agactgatga gccccaccat ttcattggaa agattcggga 60

ggacgaggtc gagagctttt gccggggtag aggacgagga tggtacaaga actagacctt 120

tccaacttta attgttgaca cctatttaat tctctccttc ttctttattt tatttttcat 180

ttctccaacg acgactgtct cattactagt ctactagtaa ctctgtctta tcgtcatctc 240

ccataggtga gtttggttgt tttgtttcca ctgagatcat gacctcctcc taccccacca 300

tcccactatt tttgttacgg tagccatgac ccctccatgg caaagagaga ggaggacgag 360

gacgatcagg aaactgtgtc tcgccgtcat accacaatcg tgttatcctg attgacatct 420

tcttaaatat cgttgtaact gttcctgact ctcggtcaac tgaaattgga tctccccacc 480

actgcctcta ccttgtactc cgtgactgaa ccatccgatc attctttttg ggtcgtcggt 540

gaacacaacc cccgctgcta gtctccttcc aacaccgatc cagaattgtt ttgattttcc 600

attcccttcg tttatatctg tcgtctctcc tccctttccg tctcttttct tccgtcctcc 660

aagttagtcg actgaccaat tccgcagctc gtcaaaatgc ctatcaccaa gatccacgcc 720

cgctctgttt acgactc 737

<210> 9

<211> 1499

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 9

tgagtgaagt tccggtcgga gcaatcaggg tcggttggag tatatcagaa atgtatatct 60

ttggaaaggg tagttataat atgtcgaata tgaatttagg taagagtatt tgaaacaaca 120

agtatgagga tggttcactg gatggcttgc cctaacatcc acaccatcgc atccggccac 180

ctcaggaaca atcactcctc ctcacaaggt atatcccgcc attgaccaac gacaatccag 240

agtatgagat acttgcagaa cgccgagtgg cgttatcaac cgcgtgacct attcagtacc 300

tttgcctcac gccaagcaaa tttccccaca caaaagcaac cataaatgtc gttggccacc 360

gaagctcaaa ctttcggagt cgctctcagt ggttacttta ctgctggtgc ccacgggatc 420

gtaaatggac cttgaccaat tcgagtagtt ccgcagacga tcgctcatcg ctcgatttgg 480

tcgcgtgtcc agagtgttgc tacagcatgg tctggattcc aatccacgca gcagatatct 540

cttttacgca gtaactattc gatagccatt tccgttcaga tcagccgtcg gatccgagga 600

gcgacgacat caatgcgtgt tattagtcaa attttggagg ggttgcgtgc ccactgcagg 660

cagatgtagc cgtggcacca caacaccggc cagccctgga ttgggtggtg gaaccaagat 720

atgagaacca gtatctattg gcacggaggc gtttccggag cctgccgcgt gtgatacgtg 780

cagagtcaat tcctacggac tgtctggggt aggatcacaa ctaatcagtt gcagcgatgg 840

tttgacaaca ggggtcgatc aaagtttccg aagaatagga agcgaggaca gcaccaaggc 900

cgctgaacca caggaaacaa aggaaggaaa aacacaacaa aaccaaagac agacacatag 960

gaaatgacat acttaccaga gataagatga aaagcaccat ggggagggag ggtatggatg 1020

gggaagttga tcaacagtct gaaacccgcc cgaaatgaat gcatgacgcg acgattccat 1080

ctccacctaa gcttcatccc gtcaacctct aacaacgcgc tcggaggaga aaaagagggg 1140

gatcaacagc aatctaggtt gttgttcccc caggatttct ccgcaaagaa tgtttagggt 1200

tccgtggatc gggtgaccga atcggccaac cgcattgtct gatcgtctcg tcatatgatc 1260

cagtgcatga cgtcttccat caatccatca ccccagactg aatcctcaca ttaatttaac 1320

aattgtcgct cggggaaatc aataaatacc cgctcgtctc ctccctccct cgtgctggtt 1380

cagttgattg ttcactcatc gactttatca atcttccatt gaccattcca ggcttgtcgc 1440

ccactcatat aatcttcttt ccccggtctc acatcaatca cccttcacac cacaacacc 1499

<210> 10

<211> 1200

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 10

atgtcttctc ttccgccggt ctatattgtt tcctctgccc gcaccccagt cggctctttc 60

ttggggtcgc tctcaagtct cactgccccg cagttaggct ctcatgctat taaagctgcg 120

ctcagcaaag cggatggaat caagccgtct gatatccagg aggtcttctt tggcaatgtc 180

atctccgcaa acgttggaca aaatcctgct agacagtgtg ctctcggcgc tggtctcaat 240

gaatcaactg tctgtactac ggttaataag gtgtgcgcgt ctggcttgaa agcggttatt 300

ctcggtgcac agaccatcat gactggcaat gcggatattg tcgtagcagg cggtgctgaa 360

tccatgtcta acgcccctca ttaccttcca aaccttcgcg tcggtgcgaa atacggcaac 420

cagagtctgg tggacggtat tatgaaggat ggcttgacag acgcaggaaa gcaggaactc 480

atgggcttgc aagccgagga gtgtgctcag gatcatggct ttagcaggga acaacaggat 540

gattatgcca ttcgcactta cgaaaaagca caggcggctc aaaaggctgg cctttttgac 600

gaagaaattg cgcctattga acttcctggc tttaggggca agccaggtgt gactgtgtca 660

caagacgaag aaccaaagaa tcttaacccg gataagcttc gagctatcaa gcctgcattt 720

atccccggat ccggcacggt cacagccccg aattcctcac ctcttaacga cggtgctgct 780

gctgttatcc tcgtctcaga agctaaactg aaagagctta acctaaagcc tgttgcaaag 840

attcttggct ggggagatgc cgcccagcag ccaagcaaat tcacaactgc cccagctcta 900

gcaattccca aggccctcag ccatgcaggt gtggctcagg atgctgttga tgcgttcgag 960

attaacgaag cgttcagcgt agttgctctg gccaatatga aactcctggg gttggctgaa 1020

gataaagtca acatccatgg tggtgcagtg gctatcggtc atcctatcgg cgccagcggt 1080

gctcgtatct tgactacatt gctcggtgta ttgaaagcga gaaagggtaa gattggttgt 1140

gccgggattt gtaatggagg aggtggtgct agcgctattg ttgtcgaatc tctcgtctga 1200

<210> 11

<211> 1383

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 11

atgtctgctc gtcctcagaa cattggtgtc aaggccattg aggtctattt tcctaagcaa 60

tgtgtcgaac aaaccgagct ggagaagttc gacggtgtca gtgagggcaa gtacacaatc 120

ggtctgggac agacaaaaat gagcttctgt gatgaccgtg aggatatcta ctctgtcgcc 180

ctgaccactc tctcctccct ctttcgcaaa tacaacgtcg accccaagtc cgttggtcgt 240

ctcgaagtcg gtactgagac tctcctggac aaatccaagt ccgtcaagtc cgttctgatg 300

cagctctttg ccgagagcgg aaacttcaac gttgagggtg ttgataacgt caacgcttgc 360

tatggaggta ccaacgctgt cttcaacagc atcaactggc ttgagtcttc cgcctgggat 420

ggaagagatg ccgttgttgt ctgcggtgac attgctctgt atgccgaggg acctgctcgc 480

cctactggtg gtgctggctg tgttgccctc ctcattggtc ctgatgcccc tattgtcttt 540

gagcccggtc ttcgtggctc ttacgtcacc cacacctacg atttctacaa gcctgatctc 600

accagcgaat accccgttgt tgacggtcag cactcccttc agtgctacac tgaggctgtt 660

gatgcttgct acaaggccta cgccgctcgc gagaagacgc tgaaggaaaa gactcagaac 720

ggaaccaacg gtgtggccca tgatgaatcc aagactcctt tggaccgctt tgactatatc 780

cttttccact cccctacctg caagttggtc cagaagtcgt acggccgtat gctttacaac 840

gatttcctcg agaaccccac ccaccccgct ttcgctgaag tcgctcctga gctgcgcgat 900

ctggactaca gcaagtctct cactgacaag aacgtcgaga agactttcat gggtctgacc 960

aagaagcgct tcgctgagcg tgtgaagccc agccttgatg ttgccactct ctgtggtaac 1020

atgtacaccg ccaccgtcta cgccggcctg gccagcttgc tcagcaacgt caccttcgac 1080

cccagccagc ctaagcgcat tggccttttc tcctacggca gtggtctcgc tgcttccatg 1140

ttcagcgcga agattgttgg tgacgtgtct tacatggctg agaagcttga tcttcacaac 1200

cgcctcaatg ctagggatgt cttggccccc caggcctatg ttgagatgtg tgctctgcgt 1260

aagcaagctc acttgaagaa gaacttcaag ccctccggta acacggagac gcttttcccc 1320

aacacctact acctcactga ggtggacgac atgttccgcc gcaagtacga ggtcaaggca 1380

tga 1383

<210> 12

<211> 2497

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 12

atggaaaagc cgattatcct tactcgggct gtgctcaatg cttcggctga caacaagcgc 60

cgaggtggtg ctggtccttc cagccaatcg agcccgacca ctgcgaagtc catccaagac 120

tcgatccaga ccgcaatcag ggaacagggc tttgagatcg tccgggatta ctgtattgag 180

atcgctatcc ttgttgctgg cgcggcttcg ggggtgcaag gcggcctgaa acagttctgt 240

ttcctggctg cttggattct gttcttcgac tgcctcctgc tatttacctt ctacacgact 300

attctttgca tcaagcttga aatcactcgc attaagcgtc acattgcgct ccgtaaagct 360

ttggaagagg atggtattac gcaccgtgtc gccgaaaatg tggcctcaaa caatgattgg 420

cctcaggatg gctcggaaaa cagtgacacc agcatcttcg gaaggaaaat caagtcaagc 480

aatgtgcgtc gtttcaagat tctcatggtt ggaggtttcg tcttgattaa tgtggtgaac 540

ttgtctgcca ttcctttccg gaattccgct ctgggccctg ttccgttact ctcccgggta 600

tccaatgtgc tcgcgcctac tccaattgac cctttcaagg tcgctgaaaa cggtcttgat 660

tcgatctacg tgactgcgaa gagccagatg accgaaactg ttgtgactgt cattccacca 720

atcaagtaca agctcgagta tccttcagtc cattatgctg cgccaggaga tagccaatcc 780

tttgacattg aatacactga tcaactcttg gatgctgttg gtggacgcgt gattgaaagt 840

ttgctgaaga gcgtcgagga cccggtcatc agcaaatgga ttattgctgc gctcaccctg 900

agcatcgtat tgaatggtta ccttttcaac gcggcacggt ggagtatcaa ggaacccgag 960

gctgcccctg cacccaaggc cgtcgagccg aaggtttacc ccaaggtgga tttgaatgca 1020

gacagctcga agagaagtgc agaggaatgt gaagtattcc tgaaggaaaa gcgggcgccc 1080

tatttgtcgg acgaggatct gattgagctt tgcttgcgag gcaagattcc agggtatgct 1140

ttggagaaga ccatggaaaa tgaagacctt atgagccgcg ttgatgcctt cacgagggca 1200

gtcaagatca gaagggctgt ggtatctagg accaaggcta cgtctgccgt tacaagctct 1260

ttggaggcct cgaaactccc ttacaaggac tacaactata cgttggttca cggtgcatgc 1320

tgtgagaacg ttattggata tttgcctctg ccccttggag ttgctggacc tcttactatc 1380

gatggccaaa gctactttat tcccatggct accactgagg gtgtattggt agcaagtgcc 1440

agccgtggtg ccaaggctat caatgcaggt ggtggtgcag tgactgtcct caccggcgat 1500

ggtatgactc gtggtccctg tgttggtttc cctaccctgg cacgcgctgc cgctgcaaaa 1560

gtttggattg actctgagga gggtcagagt atcatgaagg ccgcgttcaa ctctaccagc 1620

cgctttgctc gtctccagac tatgaaaacc gctcttgctg gtacatacct gtatattcgt 1680

ttcaagacaa cgaccggcga tgctatgggt atgaacatga tctccaaggg cgttgagaag 1740

gcgcttcatg tgatgtctac agaatgtgga tttgatgaca tggccaccat caccatctct 1800

ggtaatttct gtacagacaa gaagtctgct gctcttaact ggatcgatgg acgtggcaag 1860

tcagttgtag cagaggccat cattcctggt gatgttgtca agagtgtgct caagagtaac 1920

gttgacgcgc tggtcgaatt gaacaccagc aagaacttga tcggaagtgc aatggctgga 1980

agcttgggtg gcttcaacgc gcacgcatcg aacattgtta ctgccatttt cttggcaact 2040

ggtcaggacc ctgcgcagaa tgtggaaagc agtagctgca tcactactat gagaaagtaa 2100

gttacaggtt gccaatgttc acgattcaca tcagtgtact aattgtcatg ctcccagtct 2160

caacggtgac cttcagatct ccgtgtcaat gccctcaatc gaggtcggaa ccattggtgg 2220

tggtacgatt cttgaaggac agtccgctat gcttgaccta ctcggtgtgc gcggttccca 2280

ccccaccaac cctggcgaca acgcccgtca acttgcacgg attgttgccg ccgctacgct 2340

tgcaggcgaa ctgagcttgt gctctgcact tgctgccggc catcttgtcc gtgcgcacat 2400

ggcgcacaat cgcagtagcg ctcctacacg gtcatcaact cccgtctcgg ccgctgttgg 2460

cgctgctcgg ggattgacta tgacgagttc gaaatga 2497

<210> 13

<211> 1605

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 13

atgcacaacg gccgtggacg ccgaaagaac ggcagtgtta aagctccaaa gaatcgtcag 60

cgccctacta tgtcacactt aatctcggaa caatcccttc ccacccgctc aaaatcctcg 120

gtcgctagcg acgatagcgt agacacctcc gacgacacct cagccgctcc ctccttcagc 180

agtagtccgc cgagcacgaa gtctatcacc aacggtgttc acagaccagc tatggctcga 240

aaggcttctt ctccaatggc acccgccttc atggtgtccg ccccgggcaa ggtcattgtc 300

tttggtgagc atgccgtcgt gcatggtaaa gccgccatgg cggcagccat ctccctacga 360

tcctacctcc ttgttacaac cttgaccaaa tcgcaacgca ccatcacctt aaacttcaga 420

gatattggat tgaatcacac ctggagcatc gacgagctgc cgtgggactt gtttcaccaa 480

ccgaccaaga agaagtacta ttacgacctg gtcacctcga ttgaccccga acttctggac 540

gcgatcttgc ctctcgtgga gcgcatctcc ccagacctac ccgaagacaa gcgaaaacat 600

cagcgtggcg ctgcgactgc gttcctctat cttttctgtg cgttgggttc cccgcaacac 660

cctggagcga tctataccct tcggtcgacg atcccaactg gcgcggggtt gggcagcagt 720

gctagtatat gcgtttgtat cagtgctgca ctccttcttc agattcgtac tctagctgga 780

ccgcaccccg accaaccgcc cgacgaggcg gaggtgcaga tcgagcgcat caaccgatgg 840

gcattcgttg gtgagatgtg cattcacgga aatcccagcg gagtggataa cacggtcgcc 900

gcaggcggca aggccgtgat tttcagaaga ggcgattatt ccaaaccacc ggccgttagc 960

tcactcccca atttccccga gctacctcta ctgctcgtgg acactcgaca atcccgttcc 1020

acggcggttg aagtagcaaa ggtcggtcag ctgaaagaag aacaaccact agtgacggag 1080

gcgatccttg ataccatcga gaaggtgaat gcttccgctc aggagattat acgggaaacg 1140

gattcgtcag gtatttccaa ggatacgctc gagcgcattg gagcgcttat ccgcatcaac 1200

cacggcttgt tggtctcgct gggagtctct caccctcgac tcgagcgcat tcgtgagctt 1260

gtagattttg cgaacattgg ttggacgaaa ctcaccggcg ctggtggagg aggatgcgcg 1320

atcaccctcc tacgtccgga tgccgacccg agtgctatcc gccaattgga ggaaaagctg 1380

gacgaagaag gattcgcgaa gtacgagact actctgggag gagatggtgt cggtgtcctg 1440

tggcccgccg tggtccgcaa tggaaccgac gaagaaggtg gtgaggagat tgaccagcag 1500

aagttcgaaa atgcagatgg ccccgaaggc atcgagcgtc tcgtcggtgt gggcacacag 1560

gaaaagagag agggctggaa gttttggaaa cgagcaatgc attaa 1605

<210> 14

<211> 1452

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 14

atgtcttatc cgccatccgg gagcaccgcc ttgtccgccc cgggcaaggt ccttcttacc 60

ggtggttacc tcgttttaga ccgcaattac acggggacag tgatcgcact cgatgctagg 120

atccacgtta tcgttcaaca attgaaaaga ggccatcgac gtggtgcatc attcagctca 180

gtgaaggggg gtcccgatac ggagacagtc gaggacggaa gtgctgtgga cgacaaggaa 240

aaagaagacg tcgtggttgt acgctcgccg caatttgtca atgcaatttg ggagtacggt 300

atacagcgtt gtgagaatgg aggtggaatc aaagtgattc aaaggaacga cgggcgttcc 360

aatccgttcg tcgaaacttc cctcaactac gctctcacct atatcagcta tgtggccgac 420

tcgaaggact ttgggtccct ctccgtgacc atcctggccg acactgatta ctactctgag 480

actgccttct ctagggtttc tgagtcccct ggaagattcg tgaacttcgg tgttcctctt 540

cacgaggccc acaagacagg actaggttcc tctgcggctc tagtaactgc cctagtatca 600

tccctcgtta ttcaccgtac cctgcagcct gacgaccttg gagcttctcg tgacaagctt 660

cataacttgg cacaggctgc ccactgtgct gctcaaggta aagtgggatc cgggtttgat 720

gtggctgctg ctatctacgg ctcttgccta tatcgccgat tctccccaag cattttggaa 780

tccgtcggcg acgccggttc acctgggttt gaagagcggc tgtttgcagt cgtggaggac 840

gccgacccta agcatccgtg ggatacagag tgcttggatt tcggcatgcg acttcctcga 900

ggcatgcaga tggtcctgtg cgatgttgag tgcggctcaa attccccttc gatggtcaag 960

aaagtgctcg aatggcgaaa acaaaaccag caggaagccg atcttctttg ggctgccctc 1020

cagtcaaaca atgaaaggct ctgtcttcag ctcaagcagc tagcccagag ccctgaccaa 1080

gaatcgcccg aagatttcaa tgatgtccgc aacctcatcc agcgctcacg caatcacctt 1140

cgcagcatga cccgcaaggc gggagtcccc attgagccgc gggtacagac agaactgctt 1200

gatgccgtgt cagccgttga cggagtgatt ggtggtgtgg ttcctggtgc gggagggtat 1260

gatgcgattg ctgtcttgat ccgcgatgac caggaggtgc ttaaaaagtt aactgagctc 1320

tttaagaact gggaaagtaa ggtggaggac gatttcggtg gcaagatcgg aactgtccgg 1380

ctcctcggtg tgcgtcacgg ctcagatgga gtcaaaaatg aggttctcga ccaatatgcc 1440

ggttggcttt aa 1452

<210> 15

<211> 1215

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 15

atggctgctc cttctgacag tacggtcttt cgggccacca ccacagcccc agtcaatatc 60

gctgttatca aatactgggg taaaagggac gccactctga acttgcctac gaactcctcc 120

ctctctgtaa ccctctcgca gcgttctttg cgcaccctca ccactgcctc atgctctgcc 180

aagtacccga ctgccgacga gcttatcctc aatggcaaac ctcaagatat ccagtcctcc 240

aagcgcaccc ttgcttgcct ttcgaatctg cgctccctcc gccaggaact tgaagctgcc 300

gactcttctc tgccgagact gtctaccctt cccctacgga tcgtttccga gaacaacttc 360

cccaccgccg ctggcctcgc cagttccgcc gccggtttcg cagcgctcgt ccgtgccgtc 420

gccgaccttt accagcttcc ccagtccccc cgagacctca gccgcatcgc tcgtcaggga 480

tctggttccg cttgtcggtc tctgatgggc ggatatgtgg cctggcgcgc cggaaatctt 540

gccgatggta gcgacagctt ggctgaggag gttgctcccg agtcacactg gcctgagatg 600

cgtgcgctca tcctggtcgt cagcgctgaa aagaaggatg tgcccagtac ggagggtatg 660

caaaccaccg ttgctacttc caacctcttc gcgacccgcg cggaatctgt tgtacccgag 720

cggatggcgg ctattgagac tgccattcag aaccgagatt tccctgcctt cgccgagatt 780

accatgcggg actccaatgg cttccacgcc acctgtctcg actcctggcc tcccatcttc 840

tatatgaacg atgtctctcg cgccgctgtt aggctcgtcc acgatattaa ccgtgccgtc 900

ggtcgtacgg tttgcgctta cacttttgat gctggtccga acgcggtcat ctactacctt 960

gagaaggatt ctgaactcgt tgccggtacc gtcaaggcta tcctgggcgc cagcagtgag 1020

ggctgggacg gtccgttcta cgaacctctt aagagcttca ccgctccggg tgtggcattg 1080

gataaggtgg actctagggc tgttgatgtg ctcaaggatg gtgtcagtcg tgtgatctta 1140

accggcgtcg gtgagggtcc tgtcagtgtc aacgaccacc tcgtcagtga gacaggtgac 1200

attctctcca actaa 1215

<210> 16

<211> 819

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 16

atgagcgtaa ctactacaac caccgagccc cctagaatca cggcggagaa tgtcgccact 60

ctcttcccag aggtcgatac ttctttggct cgtgaagtcc tcccaaaagc cgatggcaat 120

ccctccgcag ctagcagcaa cgagctggcg ggttatgacg acgaacaggt ccgtctaatg 180

gatgaagtat gcatcgttct ggatgatgat gacaagccta ttggaagtgc cagcaagaag 240

acctgccacc tcatgaccaa cattgaccgc ggccttctcc accgtgcctt ttccgtgttc 300

ctcttcgatt ccaacaagcg cttgcttctc caacagcgcg ccactgagaa gattacattc 360

ccagatatgt ggacaaacac ttgctgctct caccctcttg gaattgctgg cgagaccggt 420

tctgagctgg atgccgctat cttgggcgtg aagcgggctg cgcagcggaa gttggaacat 480

gagcttggaa ttaagccgga gcaagtaccc ctggataagt tcgatttctt cacgagaata 540

cattacaagg ctcctagtga tgggaagtgg ggagagcatg agatcgacta tattctcttc 600

atccaggcag atgtagagct gaagcctagc ccgaatgagg ttcgagacac gaagtacgtc 660

tcggctgacg aattgaagac gatgtttgag cagccggggt tgaaattcac gccttggttc 720

aaacttatct gcaattcgat gttgttcgaa tggtggagcc atctcggctc tccaaccctg 780

gagaagtaca agggcgagaa aggtatccgg cgtatgtga 819

<210> 17

<211> 2268

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 17

atggacttca cataccgcta ttcgttcgag cctacggact atgacactga cggtctctgt 60

gatggtgttc cggtccgtat gcacaagggt gcagacttgg acgaggttgc catcttcaaa 120

gctcagtatg actgggagaa gcatgttggt cctaagctgc cctttcgggg tgcattgggg 180

ccaagacaca acttcatctg tcttactctg ccagagtgct tgcctgagag actagagatt 240

gtgtcttacg ccaatgagtt tgccttcctt cacgatgata ttactgatgt cgagtcagct 300

gagacggttg ccgctgagaa cgatgagttc cttgatgccc ttcaacaagg tgttagagaa 360

ggtgacatcc agagccgtga gtccggaaag cgtcatctcc aggcttggat cttcaagtcc 420

atggtggcca ttgaccgtga tagagctgtg gccgctatga acgcttgggc cacctttatc 480

aacacaggtg caggatgcgc tcacgataca aacttcaagt cacttgatga gtatcttcac 540

tacagggcta cagatgtcgg ctacatgttc tggcacgctc ttatcatctt cggatgcgcc 600

atcaccattc ctgaacatga gattgagcta tgccatcaac tcgctcttcc agccatcatg 660

tccgtgactt tgacaaacga catctggtca tatggcaaag aagcagaggc agctgagaaa 720

tccggcaagc ccggagattt tgtcaacgct ctcgttgttc tgatgagaga gcacaactgc 780

tccattgaag aagccgagcg tctctgcaga gcgcgaaaca aaatcgaggt agccaagtgt 840

cttcaagtca caaaagagac acgagagcga aaagatgttt cacaagatct caaagattac 900

ctctaccata tgctgtttgg tgtcagtgga aatgcgatct ggagcactca gtgccgaaga 960

tatgacatga cagcgcctta caacgaaaga cagcaggcca gactcaagca gaccaagggt 1020

gagcttactt ccacatatga tcctgttcag gctgccaagg aggccatgat ggagtctact 1080

cgtcctgaga tccacagact gcctactccc gatagtccca ggaaggagag ctttgctgtt 1140

cgtcctttgg tgaatggcag tggacaatac aatggcaaca atcacatcaa tggagtctcc 1200

aatgaagttg acgtgcgtcc ttctattgag agacatgcct caaccaagcg agctacttca 1260

gctgatgaca tcgactggac ggcacataag aaggttgata gtggggctga ccacaagaag 1320

accctgtccg atatcatgct gcaagagttg cctcctatgg aagacgatgt cgtcatggaa 1380

ccataccgat atctgtgttc tcttccctca aagggagtta gaaacaagac tattgacgct 1440

cttaacttct ggctcaaggt tcctattgaa aatgcaaaca ccatcaaggc catcactgaa 1500

agccttcacg gatcatcact catgcttgat gatatcgagg accattcaca actgcgacgt 1560

ggcaagcctt cggcccacgc tgtttttggt gaggcacaga ccatcaactc tgcaacattc 1620

cagtacattc agtctgttag cctgattagc cagcttagaa gccctaaggc tttgaacatc 1680

tttgttgatg agattcgaca acttttcatc ggtcaggctt acgagctcca gtggacctct 1740

aacatgattt gcccaccttt ggaggagtat ttgcgaatgg ttgacggaaa aactggcggg 1800

ttattccgcc ttctcactcg tctcatggct gctgagtcca ctactgaggt agatgttgac 1860

tttagccgtc tgtgccagct ttttggtcgc tacttccaga tccgagacga ttacgccaac 1920

ctcaagctcg cagactacac cgaacaaaag ggtttctgtg aagacctcga cgagggcaag 1980

ttctcactcc ctctcatcat tgccttcaac gagaacaaca aggcccccaa agccgtagct 2040

caactgcgcg gcctcatgat gcagcgctgt gtcaacggcg gcctcacctt tgaacagaag 2100

gtgctagcac tgaatctcat tgaggaggct ggtggaattt cgggcacgga gaaggtgctg 2160

cactcacttt atggtgagat ggaggctgag ctggaaaggt tggctggtgt ctttggggcg 2220

gagaatcatc agcttgagct tattctggag atgctgcgta tagattag 2268

<210> 18

<211> 1858

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 18

atggaaaggc tcaagatgga tagcctcaac atcaacatca atgactggtt cgagaaagac 60

ctgccagcca ctgcagactg gaagctcttt gctctagcca gtgttgtctt tgttgttcta 120

cgatttactt gcattgtcat ctatcgtatc tacttttctc ccctttccaa gttccctggt 180

cccaagctcg ctgctgcgac gcatctttat gagtcttact atgacttttg gaagaaaggg 240

cagtactaca aagtgattca gcgtatgcac gaggtctacg gaccgcttgt tcgtgtcacg 300

cctgatgaac tttcgatcaa cgaccctgac tattacgaca ctgtctatgt caacggtaat 360

gttcgacgta ctgagtcctt cggccattcc tttggtggcg gacttggtat tgaagacacc 420

ttcttcgcct ctcaggacca tgacctccac cgcaagagaa gaaaacccat cgagccttac 480

ttctctcgca atggtgtctt gaagctcgag aatcttatcg gtgaacgtgt tgagaaactg 540

ttccacaagt tccacgagct gtctggtact ggtgttgttg cccgtcttga ctatgccttt 600

gaggccttca ctggcgatgt catgcagcat atttgcattg agaagcctga atcactactc 660

aacagcgatg acttttcttc tgagtggttt gagatgcttc gcaatgtctc cttgtccgta 720

cctcttatgg gaatgatccc ttggcttgtc cacgtactga agttcatccc cgagagtgtc 780

atcatgtggc tcgcgccctc agctgcccac ttccagacct tccgtgttca agctggtcgt 840

cagattgagc aagccaagca cgagaaagtg gagaatgatc gcaaaggtat cactactgtc 900

ggcggcaagc ccaccctctt ccgcttcctt gtccacgaga gtggtctcgc accggaagac 960

ctgagcaccg agagactcca gaaggaggca atggttctac ttggcggtgg cactacaact 1020

actgcgcgta ctgcgaccat gacttgcttc tggatgctca gcatgcctga gaagggccaa 1080

cgtcttcgcg acgagctcaa ggacatcatg gccgagtacc ccaagaagaa gccttctttg 1140

accgagcttg agaagctgcc ttatttggga gctgtgatcc aagagagttt gagaatggcg 1200

tacggatcca tgcgtcgact tcctcgcact tctcccgatg tagctctgca gttcaaagat 1260

tgggtgatcc ctcctgggac tcctgttggc atgaatgctt attatcttca cactgatcct 1320

aatgcgttcc ccgagccatt tgagtacaag cccgaacgat ggcttggaaa tgtcacaccc 1380

gcgatgaagc gtagttttgt gcccttttcg cgcggttcgc gccgatgccc cggatctagc 1440

ttggctcttg ccgatctcca tttcgttctt gcagcattgt tcggaccaac tggacccaaa 1500

tttgagttgt tcgaatcgga caggtctgac gtggatgcca ttcatgacta cctgatgccg 1560

ctgcctcggt tggattccaa gggtgttcgc gtcactgtca agtaggatcg ttcaaacatt 1620

tggcaataaa gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa 1680

tttctgttga attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg 1740

agatgggttt ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa 1800

atatagcgcg caaactagga taaattatcg cgcgcggtgt catctatgtt actagatc 1858

<210> 19

<211> 182

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 19

gatctgtagt agctcgtgaa gggtggagag tatatgatgg tactgctatt caatctggca 60

ttggacagtg agtttgagtt tgatgtacag ttggagtcgt tactgctgtc atccccttat 120

actcttcgat tgtttttcga accctaacgc caagcacgct agtctattat aggaaaggat 180

cc 182

<210> 20

<211> 269

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 20

actagacggg ttcgcatagg tttggggttg tatcttggcg ttgggacgga ctgggtatgg 60

tgtttctttt ggatatatga catgatatgt acacggccgt gaatctttaa ctttatatca 120

ttatagaaat gcacttgcac atttcaacac gctgcgagca gaatctcgaa gattgttccg 180

caagtattag atcatgagag cattttcatt tcctttcagg cagtgggagt aggccatcct 240

gaaaacaagg cggccactgt agactagag 269

<210> 21

<211> 567

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 21

ttgtattggt attaccagaa cgtcacgaga ccgcctgccg caatgttctc atcctttctt 60

tgctactagc aagttttttc gcaatttctc gaagctttag atggtctttt cttttccagt 120

cgaaagctac tagccggtct tttctgtcct attcagctta ggcagttata ggatatcttc 180

aagattcagg tactctttga ctacacacaa tgccctatga tatgaagggt aaatgtgtgg 240

gtatcattca ttggatctta agtaaggcac agcccgcgga gcagaaatga agctccttgt 300

atgacatcac caaatggtca cttaatgcaa tttcgaaccc tttccatccg agctcaagtt 360

cgagagctca gttcccattt tactcatctt ttttacttag aagagggata taatctaata 420

acaaactgat ttaaatgaac acagagctat tactttcaaa tttggcttaa tgtccacttc 480

tcgccaccca acggtcctat ggggtagtgg ttatcccacc ggattttgat gaacaatata 540

catgtcctgt atatgtgtag gaagatc 567

<210> 22

<211> 253

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 22

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210> 23

<211> 741

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 23

atggttaacg gaaccttctc catccaagac gacaaggccc ttcctgtgtt tgcaccccca 60

tacacttccc aggagccctt ccactttagc ggcatgactt ctgtctctat catgtaccgc 120

gtccgtgcct cccagatcca gcaccttgtg ccgcgtgaac tagaaatgga ggacgaaccg 180

atcatgactt ccttcttcgt tcattacggc acctctaccg tcggcgagta caacgaatac 240

ggcaacgctg tccaggtcaa gtataatggc aagacgttcg actattactt ggttcttgtc 300

ctcgacaacg acggcgccat ctttgctggt agagaaatat ttggatatcc caaaatcttc 360

ggcaaaacca acttccaccc gtcagctggg tccaaggtca tgacgggaaa cgtggagcgt 420

cccgcaggac gatctctgat cgagtttgag ttcgctccca aggcgcctct tgaattgtcg 480

gcagaaccca cgatttccaa ggcgctgaac ttgcgtgtta ttcccagtac ggacccgaca 540

aagcctgcca ttaaggagtt tgtcgctgta gatatgcaag tcgaattcag cgagaaatgg 600

agcggagaag gaagtctcaa gttcaacaaa ggattcgcat cggttccatg ggccaacata 660

gacgtagtct cgtacgtcgg gtctttcatg ggaaagagca aggccattct caccaatgag 720

gtagagcgtt tccctctcta a 741

<210> 24

<211> 1273

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 24

tcacagcgcc gccaaactct ttggtatcaa caactctcgc gttttcctaa cccgagcacc 60

atcctcctta ctcttgaaat accgacccgt cttgtagctc cagagcagac taccaagagc 120

atggttcttt tcaacttcaa gaaagccaag gagtgcacgg tcagcctctt caccgaagca 180

tgggagcttc gatagtgctc tgtaatagcg tctatagcag ttgttgatca taactccgac 240

ctcgtttatg gcctgctggg ttgagtgccc cttggctttg agaagtcgga ccatgttgtg 300

gtctaccccg agacgaaggt ctttctcgta agacaggatg tcgttgacta ataggatctg 360

gtcgctgatg atgaccatga tttcgaatac ggaaggatga tcagctacct cctctggcaa 420

gtcaattccg taacaccact cgttattaac aagggctgga taagcaccga gagaacctcg 480

acgcatagcc atgtattcct ctgggcgacg tgtgtaggac cggccctcca cattagtccg 540

aacctggtcg accagtccct gccagtaaag ctcatgtgcc cacatccagc gcttatagaa 600

acgatctgat gatggctttc caatgaagaa accctctggg ctttgcttta tgcgatcaca 660

caatgtctgg aacacatagc gaatggggtg ttctgagtcg gcggtatacc gaggggcggt 720

accctccatg atctcgcgcg tcctggcaat ctcgctgatg gcgccttcta gatcattgca 780

caaatggccc tcgtcaaact ggtcatcgaa gagaaaggcc cacgagttcc aatcagctga 840

cgtacgtaag gcaaaggcac tgcagtcagg tgcccagatg ttggcaagat aggtaaagtc 900

aacccgcttg tttctgttgg tccagtctgc atctgctttg atcacgcttt ataagttgtt 960

agtagacgat gattcttttg gtctgtgtca tacgtagggg gttaactcac gaggaaatcc 1020

aatcatctgc ttctgctttg acgctagcat agtttgggtt ctcacgggct gggaccgaca 1080

tgagtgagct gaagaggtca ggaagtatga gaacctccat ctcatcacgg ccctggaact 1140

gatcatgttt gggttcgttg tagactccgt taccgttctt catcttgagt ttgacctcat 1200

gtttggtgtc gatatagacc tcatccccgg ttgtcatctc tgactcggaa ctactgctgt 1260

cgaatttgac cat 1273

<210> 25

<211> 1692

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 25

atgtttagta ctctacggca aacgtctggc gatggagacc gtctcctcag cgtggagatt 60

gctccaccct ttgacaagga caggatgtca atgcggaccg cgtgtgagag atgccgcata 120

cagaaggtag gtagtatcga agccattcac attgtgtcac gtcggagagt ctttctagat 180

aaaccctaga cgtcgttcct attctcactt gataattatg aaactaattg actgcattag 240

tccaagtgtg tcagtggcga gaatggctgt atgctgtgtg ttgccaagaa ccagaaatgc 300

gagtacctgg ttgtgtcgag gccacgacgg cgcaagtcca acattgctgg cggtggccca 360

aagtcccgcg acgataacga tgatgaggac ggcgacaaca cagtcgtcaa tcagcaaaag 420

cagtcaaatc aggctaggat gcaaaaacga tggtctcgaa tgcagacaat acgacagtca 480

tcgccattat cagggccatc atctccaccc actgatcaag tgagggtttc agcgaagccc 540

tcgattcaag atgacaacag taacataata gggccagacc tgggcctaga agatgcacat 600

caaataatat tcaacggtgg tctcttcccg gactcccttg gacaaatccc ggctctcttt 660

ccacacctcg agcccgcagc cattaaagac ttctttagca atatgcctat agctgcggag 720

aatacccatg caggccgtgg tgttaccgca gaagcattga ctgctcgagg agacagtggc 780

gggctaaaca gtcacacctc tgtatactca acctcatcag cagatagcct gtttgacttt 840

ggagttgatc agatggactt tctgatggac gagtcacaca tcactgctat agctcccaga 900

agtaccagta gcagcaataa agttgcaaga gacgttcgtg ctcctgccac tcattcttca 960

acaccatcag aatcaaactc tacatccatt tctacatctt cttcttgcag ttgtatgatg 1020

acagctgtgg gtatctatga ggctttgcaa gttgagctga actggggcga tccaattgct 1080

ggcccatcgg ctacatcaag tcccaaatcc tcatcggcgg gctcttcata ctcgtctggg 1140

ccgccatcgt ggtcagactc aggttcgata acaaaccatt caacaacact gatgacccag 1200

caaacgatct tgaaacgcca aaagacggtg ctactccgtt gtgactccct cacccggtgt 1260

ggcacctgct ggtcccgccc ggactttgtt atgctaatca ttaccatatg tgatcgcata 1320

ttgactagtc tggaggcggt tgagcgtttt gtctgtaaca agaaagacga tgacataaat 1380

agaatcagtt ccaacggctc taccactgcc gtagatatcc aggccgcacg cacggagcta 1440

gactcatccc tgtccaccac ttcccaggga ctacaatctg gggttggtgc atggcagatt 1500

gatgacgagg atgagcagga aatggttatt agcttgatca agtcccgcgt tacaagactc 1560

ggcaatctga tcaacatagc tgaggggaca attagcgcaa acgggtggcc ttggcatgag 1620

aggctggctc aggctctacg gaggaggtct aacaagcttg ccatatcctt gtcatttcgc 1680

ggttttccgt aa 1692

<210> 26

<211> 1934

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 26

atggcatatc ctatcatcct catcggcgtt attggccttc ccattacaat cctgctccgc 60

tggctcctga catacacgcg caccgtcctc aaggtccgcc gtaatggcca tcctgcaatt 120

cacagtggtg tcatgatctt tgagtccgtc gtgcgttcat ggtatcctcg aatccccttt 180

ctggtaccta tggagaagtt caccctgaag gatcccttca agaagttcgc cgatgcgcgc 240

tctgacatga tcgttataac tgaggctgta agctacccta ccctttttgc aaaaagaatg 300

accactgacg tgtgagaagt cgtcacctaa tggtatcgcc tacctcttcg gatcaccaaa 360

gatcttccgt gaaattggcc gcaacggcga catcttcctc aagccgcttg agaagattcg 420

ctatcgcatg ctcaatacct ttggactcca gctcgcctca acacagaatg gcacccagca 480

tgagagacac aagcgcgttg tcaaagcagt attcaataat gagcttatgg agaacgggtg 540

gcaaaacatg cgcaacatgt ggcgaagcct cctccgcgaa gagggcgtct atccaacagc 600

tgccaactca gacgctgctc ccattgtacg agacatgaag tcgacgatgc tgaaagtcac 660

acttggtgcc attggtgcct cgtggtttga cattgacatt ccctggaatc cggctaagga 720

gacgcaacgc caaaatgacg agttgatgcc attcgccgag acactgaaag ttgtttggga 780

ttcaccattt gtgcagacta ttctaccact gtggttcatg gaatggagcc cttcgttgca 840

tcttcgtcgc gctgcttggg cacagcgatc ccttgttacc cacatcaaga atgcgcaggc 900

tgagacaaga cgaagaatcg aggatagcaa ggataaagct caggttcaag gccggatgcg 960

aaagtacagg aacctcatcg atgcgctggt ggattctcag aatgacgtgg agatggctga 1020

gaaggccgag aagggatatt tggcccccaa tgtcggcctg tcagacaaag aagtccaagg 1080

caacattttc tctttcatgg tgtaagttca aaggcacttg atgttgaact acagaatact 1140

aataaatggt taatttagaa ctggtcatga aacatcctca cacaccctga cttgggtctt 1200

gtcactgctc gccaagaaca ccgactggca agaaaggctc tatgctgaag tcagcaaggt 1260

caacactctc cccttgaacg aggccgaaag cgcgaacggc gcaaagccac tgaagtgcct 1320

tggttatgaa gaaatggcaa acttccctct gatccttgcg gctactgtcg aaacactccg 1380

catgcgtgat ttggctatgc agatgacgcg tgtggcctct cgcaacacca cgctcagtta 1440

cacaacatgg gatggtgacg caactaaccc atccgaggcc aaggttcagc aacacacgat 1500

tacaattcct gctggcacac gtgtgcacct cgatacagcg gcctttgggg ttaacccgtt 1560

caagtgggaa gacccagaga catataaccc agaacgtcac cttcgcgaga ctgaggatat 1620

gaatggcaac aaaaaggtga cggtacgttt atctcatctt tctatttaaa ggaataaccc 1680

tgtactaaca ttcgatacag atctcatatg aggatttcat cggctactca tccggttccc 1740

gtcagtgcat tggtaaacgt tttgccgagg tgacaatggt gtgtttcctg gcacatatga 1800

ttctgaacta tcgatgggag gtcgtgcctg aggcgggcga gacacaggag caagctaaag 1860

tgagggcgtc tactggttcg gaacagttca tgttgacacc accggcgtac gatctgcgct 1920

tcatcagacg ctaa 1934

<210> 27

<211> 3949

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 27

atggctatcg gtagaaccaa gcgaggaaag atcggaaaca ggttgattcc acagattttg 60

gacgatctcg ccgctacgga gcctgatcgc atcatttact cctgggccaa gtcctcggac 120

ttgtcacagg gtttccgcca tgtatctgct cgtgccttta cgagagcagt cgataagacg 180

gcgtggttac ttcagcgaga actaggagag acctcagaga tccgcgctgt gggttatatt 240

gggcctcgtt agttaagctg tccaagagcg tcgcactcgg gccatttgac taacgacacg 300

gcatctagac gatcttcgac agatcttgtt gacattcgcc tgtatcaaag ccaactacac 360

cgtgagtttc tccttaacct caacgtgtac ctaacgactt gactctgttt gtttaacctc 420

tttttacaca ggctttattc ctctctccaa agaacagtat tgagggagct ttagccgttc 480

tcgatgcggc agattgtaac atctgggtca accccgctgg cgagaggcca gcaccactcg 540

tggatgactt cgtgcagcaa cgtgccatgc acgtgatgca cctgccgctg ttgagcgagc 600

ttcttcctga agacgagagc gagatggagg acgtgaagcc gtttccttac acaaagtgct 660

gggaagatgc gataaacgac acattctgca ttcttcatac ctcgggctcg actggcctgt 720

caaagcccat caagtggaca cacgggttga ttggtactgt ggatgctgtc cgactgcttc 780

cccctcatga aggtatggag ccctgggcga aagggtggga tgatggtgat acgctgtact 840

cgacattccc gatgagccat gtaagcattc cagtcccagt ctcaatgaaa aaatcagcat 900

gatgctaacc atgtatcaag ggagcgggga tcttgatgga cgtcgttata gcgccactat 960

ttggcctgca ctgcgtcctt gggcctcgag acgtgatacc caatttggaa ctcatatcgt 1020

ctctggcaga ccacatcgaa attgacatct ggagcatgat tccttcgctc agtgacgaac 1080

ttggcgaagc acctgacatc ttgccaaaac ttagccggtc gaaatttatt tgtgcttcag 1140

gaggtaagac agttgtcctc atgccttcca ctcccagacg tttgctgata tatataatta 1200

ccttacaggg cccgtgagca gtgttttggg ctccaaggtg aacgagttca ttcgtgtctt 1260

gaatctgacc ggcacatcag aaggtctatt catcggcaac ctgtgggtgg acagaaaaga 1320

ctggcactgg tttgccttcc acccttggtc tggctttgac tttaagatgg tcgagcctgg 1380

tctttatgaa cagtggatcc atcgtaacga acatgcagac cttttccagg gtctctttca 1440

aacctttcaa gatgtggaga gtttcaactt caaggacctg tatgttccgc acccgactaa 1500

gccgggcctc tgggcatctc acggtcgcag cgatgatgtt gtggtccttt cgaacggata 1560

caagatctcg cctcttgata cagaggccct tgtcgcatct cacccggccg ttgatgggtg 1620

tttgatggta agtccagaat ccctcaagag gttgaagaag aatgctgatt gaagtgtacc 1680

acagatcgga tcaggtaagc cacaagctgg tctactcatc gagctgaaag atccaacaat 1740

aaagaaggac gacgacaacg ctgaagcgct cttcaacagc atctgggccg tggttgagag 1800

ggccaactcc ctgtctctgc acaagaacca gctgcaccgg gactatgtcg ccttttctga 1860

agcggacaag ccatttatcc gtaccgataa gcgcaccatc aaacgtcggg ctactatggc 1920

actgtatgaa gattacatac agcgcttcta ccagtcaaga acagaggatg atagcggaga 1980

tggagctgcg gctatcgggt tcatcacagt cgacacatca tccttggact cgactactcg 2040

cgcagttcga catgtgctgg cttcgattgt gcctgtggta aaagattccc ccgcggacgc 2100

tgatatgttt actcttgggt tcgactcact tctcgtcttc cgcgctacca agacgattgc 2160

ggcagttaca gatcttggtg ggaaattctc accgaggaac ttctatgctg gcccgacgat 2220

tgaagcgatc gtcgcaactg tcatgcgact agcttctgag cgcagagcca tgataataga 2280

tggcaccgtt gcctcatcgc cgaccgaaca gcatcaacaa gacccaaagg aagtaatgat 2340

gagtacactc ataaaacgac acaaggctgt tctatcctcc aagctgggcc caatggacct 2400

ttttggaggg aacatgtacg agggtattaa cgtcttcata cctctctgtc cagatgtgcc 2460

gtttaagcag gcgtataaag tgctccagcg aggtcttgtt cgtgcgatgg agatcgtgcc 2520

ggatctcgca ggtaaagtta taccctgttc ggagcacgag attggataca agaagggaga 2580

tcttcgtctc agtcttcctc cactgccctc tactgtcttg gggatgactg ccccggagga 2640

gccacgacaa ctgcgcttca atgacctgtc gtccgtcctg ccatcctacg ctgagcagaa 2700

agtttctgga ttcttgacgt cagcttaccc cgacgagctt ctgactacgt gtccggcttt 2760

tccttcactg cctgcggacg tgtgtaatat ccaggccaat ttcatcgagg gtggttgtgt 2820

gctggccttc aatgttcatc atcacgctct tgatggtgtc ggattgttga tagcacttac 2880

ggtttgggca gaatgctgtc gattcgtcca gggtgatcag tctgcgactt gcacgtggct 2940

gcacccagag agcctcaatc gtgatatgct atctgtcttg tacgagttgg agggcttcgc 3000

gaagccggca agtgaaatcg accccaaggt ttggggtttc ctaccatacg ccgatccggc 3060

gctgaacgcc aaggacgcaa ctgcagccaa cggacatggg actgagccaa gaaccgagaa 3120

ggctcctttg tctcgcaatc tgcctgaacc gcctcgtcta cctccatgcg agcactggcc 3180

acccaaagct cgattggatg gtcgcacact ggcagcgtcg accttcctta tctcggccga 3240

gaagctgaag agactccaag agagtgtaga gcttgctgaa gctactgatc cagaacgtca 3300

aagcctcagc aacgagtcag tgtcactatc cctcggcgat gtactccaag ccttcttctg 3360

gcgcgccgcc gtccgggctc gtcgtcgccc tgagaacacc tcggccgatg acacatccat 3420

catcgagatg cccaccgacg tccgacccta cttcagcgcg cacctaccgc caacgtacat 3480

ggccaacagc gtcatcatga atcgacagca cgtgtccgtc tcgaagctct gctcatccga 3540

gacaaccatt tacgaaattg ctcaaatctg tcgcgaggcg cgcactcgaa ttgatcaaga 3600

gcttgtccac gatgcatttg gtctgcttca tacgatccaa gacaacagtc caggaaacca 3660

caccacagct ttcctgggcc agggtatcca ggacggtcca cactcgctct tcaacaacat 3720

gatgctattc cacgcaaagg atattggggc atttggcgga aacatctttg atgcgcctga 3780

tgctgtgagg gtccaaatgg attggctaaa caaagccttc aggagtctgt ttattctgcc 3840

gataagagat gatggcggtg tcgagttgct tctcgggacg tttcctgagg aactggatgc 3900

tatgagaaat gatgaggagt ttatgcagtt tgccgagttt ctgggatag 3949

<210> 28

<211> 1495

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 28

atggatatcg tggacgacgt caagataatg accgacacta tcgttctaac ctaccccgaa 60

cctactacca tcatgaacaa tacctcaaca gaccatggca tcgtctttga tgacggagtc 120

tcgaagctca agacaccggc tatcaccata gcttcatcag acgtctcgtt tgacgacaca 180

atgtcaacca gatcctccaa ctccagtctc ggtactgaga tgacgggtcc tccagccaca 240

gatgacaaaa tggaagtctc caacgaaacc aaagtctctg agatatccga gagaatttta 300

gacgtcattc tgaaatactc tctcaacaag ttcgaaagca cctccgagct ccacagcaaa 360

ggcagaccca agtttcttgc cgtcatctca cgctttgtcc aagagcaaca gaaagtggtt 420

atgtgtctgc cagcattccc attcaagtct gcgaacaagg ttgaaaaggt gctgggcagt 480

cttcccgaca aggccgagga agtatcactc gccagattaa actccatgtg taccaccatc 540

ggacagtttt atgagccggg agctcagttg accatcatct cggacggtct ggtttacaat 600

ggttcgttca cctcttttat ttctgcctca aattaatgaa gtatttcatg gctgctggca 660

catcacttac atttcttctt cgtcatatag acttgctagg catttcagac ctcgagacct 720

ggcgttacgg ctccgcgcta cgagccatgg ccgagcgcaa ggcctttact aacctatcat 780

tttcccgtct tcaagacttg gtcgcagcca agggcttgcc caatgacctc aatgagctga 840

catatgttgc caacgccacc aacttccgtc gaaccctgtt caacaagtac ggacgggacg 900

acgatctcga cattgatcac gaaatcgcca caaacgcaga cacactcggg acgtataagg 960

ggtactgccg tttcctcaag tcagatctgc aacacatcta cggcccagcc aagagttctg 1020

ccaagtacag gaaggacgtc aagtatcttg ctaagcaact gctgatccgg ggatatgtaa 1080

gtttcgagct cactagtcca accagttatt gaatactgta tcgactcctt acagctaacg 1140

caatctcaag gcctttgctg gagctgtcaa agcgcgcttc ccagaccatt tgcgtctcag 1200

tatccaccaa tcgaccgggg agcacaagat ttcaattagc cttctcaaca ccaaaagcgg 1260

cttcacaacg ccatggcatt gcagcgtggc attgatggaa gatggcgaat ggcttagcgg 1320

ccttacgatc gacttcaaag ccgatcggtt gctggaactt gtccaagagg agggacggcc 1380

gagttacttc aaagaggtcg cccgtcagcg gccatacctg acggagagcg ctaagccacg 1440

cgttgttgtt aagcaggaac cacaggccca tagacctagg atacgcgcat cttaa 1495

<210> 29

<211> 1875

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 29

tcaagatatt ttgatgcgag tcgaaatatg tcccttcttc gtctgtagca caaccagatc 60

gtggtcatac gtgaggtctt caatcgtagt ctcgaagagt tccgatttag gaatgactcg 120

aaacgccatc agggcagcca tgatgtagat ctcacacagc gcaagactac aagatacaac 180

tgttagaatg aaaacttcaa cttgaacatg taccgtagtg gacagtagga tgtaagtaag 240

actcaccttt caccaacgca aactctagat ccctttgaaa acgagataag gaatttctga 300

agagtgtaat caggctttcc gtcgggtagc agccatcgat cagggttaaa ggaatcaggg 360

tcgggaaaca gccgcgtgtc ccagtggtta atcatggctg tcatgctcac aggcgtgcct 420

cgcgggatca tgaattgtgt cttgccatct tgactcttat agaagagatc ttcctcacgg 480

gcgatacgag cagatcgacc ggctgcgcca ggttggtgcc gcagagactc ttgaacaagg 540

gcccagaggt agggtctctg ttcgagttgc gcccatttga gatttttagg atcaattcct 600

tcaaggtctt tcataagacg agcgtagacc ttgggttgat gaagcatttg gaacgtcatg 660

acagtaagga tagcctgtgc cgggttagct gtgaaaatta taattgtctg tcatggatcg 720

cgaactaaca gcagtggttt ccgttccagc gaggagaaag acaaatccct cacccgaaag 780

acgaaacatg gttttctcct cttcaggtag gacgttggag tttagaatct cgttaaagac 840

acggccgccg tccgggttgg ccaaggctgt cttgatgtag gctggaatga catgattcat 900

ctggtgcatg atcctcttga catcctcccc catatagtca gcgaagatgg gtaggatatc 960

agctagccgc ctagccaaag cattgtggcg cactggttgc tatccgttag caaaattgtc 1020

aagtcaaagt atttgaaagt tcaatgcaaa aacgtactca tgtaagtggt tttcaagaag 1080

gatgacgtcc aggtgccaaa gttgggctcc cagccctctt gctcaataaa gcccatgggc 1140

tctccaaatg catactgcga gaagacgtcg gcagtgtagc aattgaaggc gcccttgact 1200

tcaaaggctt cttttccagt ccagcgcagc attttctgga tgaagagctc ggcgaatttt 1260

agtacttcat cttcgagctt aagaacttgt ccacgtgaga agaagcgtgc atgggcagcc 1320

ctgcgcttgc gatgcaactc atggggccca gctgtgccgg ttgcctgtga ggctgggcca 1380

gcacctgcta tcttgaggtg gtgctgccat ttgtcgcgga ctagaagatg atagacgtga 1440

gcgacagagt cccatcgaga caataatgtt gcgactaaat aaacttactt cgacctggct 1500

ttcctgtgta gatctcgtcg gcaaagtagg gatcgctgca gtggagttca tcagggctca 1560

cgcgcacgat gggaccttga aaggctgtta gtggtcactg acgatccatc cggaatcatt 1620

caagcactta ccatactgct catgcatctt ttctatcctc ctgccatagc ggccttgaag 1680

gatccagtca tagtaagcct catagacata cgaggcagcg gcaatcttcg gtccagggaa 1740

cttggagagt gggtggaaag gagagatgtt gtaaagcgcg agagcgacac ggtaaccaag 1800

ccaaagacca gcgagtccaa gcagcgctga tggactcaca aaacgcttga gggcgatgat 1860

attgaagtca tccat 1875

<210> 30

<211> 1771

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 30

ctatgacctg cgcttgacct ccaattggac tcccggatcg gcaaacagct caaaaccaaa 60

cggcatcagc ttgggtgtat aaccctcagc caaccgcaca tcgtaatcga gcaagatggt 120

agcaatagct gtcttcacct ctgcgacagc gaagaatctt cctgggcaga taggtttgcc 180

aataccaaat acaaaatgat cctcacttgt agaagtagca gaactagcgt acgtccactt 240

gccgccctga gcacgaagct tcgcatatcg atatgcgtca aaggtgtctg ggttctcgta 300

cacctcaggg tttcggagac gagacatgta aattgagaga gcggttcctt tggggagata 360

ctggccgttg ggaagggtga cgggtgatag gacctggcgg tcgagattga ctttaatcta 420

ttagtacatt gttgtataat gatgaagtaa tcgtcttatt gacttaccaa taacagcgga 480

attcatcctc tgtgtctctt tgatgacgct atcgagcaat cgcatatttg taagcgcagc 540

gggtgaccat ccatgctcgt gaaccacggc ttcaatctct tctcggagag gtgtgatcag 600

atcatgcgag caaatctcaa tgaccgtctg cttcagcaac tccgaagtag tcacaacagc 660

agccatagac attccaagct gagcggcgac aggatcatag ctcttgcccg ccgcaatttc 720

actgaaccaa gtgatagagt catcgtgctc tttgccttca gccgctctct tggccaaatt 780

cttttccaag acttcgcgtg cacgcttaac ttctgctctg caactcttac aagcggggag 840

gaaccattga ataagaggtc tcagccatcg cggccattgt cgcaacgcgc gagccgcgag 900

gaacacagtc attccgtagt tggtactgac ttgttgccat gtctcgtcgt gtgacagctc 960

ctcgccaaca aagacggatg cggacattcg gctgatgagg ttaagaccgt ccttggccca 1020

gtcgactgct ttccaagcta ttctgggtgt cagtcaatac tcacgatggg agaatgctca 1080

atacgtaccg ccacatcctt ccggccaagc tgcaaggatg tctctctgca catttttgtg 1140

cagtttgctg acatctggaa agaaactagt cagtgggatg acgccaaggg tatggtggcg 1200

gtggacttac gagggttttg aacaagcttc ttcttgataa cattggccaa cagtttggaa 1260

gggtcagaga tggctgccgt gccattgaaa ccagggtagc ccgcaaagaa atcctaaaga 1320

gacaatttag taagttattg ataggactat ctagaaagca agggttttac cttgcgaaca 1380

agcggctggt gatcaagttg aggaccatga cgcctgatcc attcaaaatg gtcttcaggg 1440

aggatcaatc tatctcccac caatgtgatc atgcggaaag ggttttggaa ctgatttagt 1500

tattagtgtc tagttgtctg gataactatc attcgcgtac cttggcaaac ccctgtttga 1560

tgaggccatc ggcattagtg acgaaggcta tttgggcctt gatggagaac caatcattgg 1620

gatatttgtt gatgactggg attttttctt ttggcgagac aagtactaga agtgataaaa 1680

cgaggatgat ggcaccggca gcgacgaggc cagcaccctc gggagtatag tcccccacac 1740

ctagcgtagt ggtgttttga ctgaaatcca t 1771

<210> 31

<211> 3320

<212> DNA

<213> Artificial sequence (ARTIFICIAL SEQUENCE)

<400> 31

atgcgcctgc cagaagatgg cgatggtaca gctggcagac tttccgtgtc tggtagcgca 60

catcttgcaa caaccacgct gcaggtcggc ggcatgacgt gagtcaccac ctgttaacat 120

tttgctcttt tagtactcat aactaacgtt gtagcaataa tagatgcggt gcttgcacat 180

cagccgttga gtccggtttc aaaggcgttg acggtatcgg aacagtctct atcagtctcg 240

taatggagcg cgccgtagtc acacatgacc cacggatcat acccgccgag aagatacatg 300

agatcatcga ggatcgcggg tttgatgcag aggttctctc gacagatatt cccaacgctg 360

gggctactcg gaccaacaac cacttcaacg agagcactgc catcaatggc gaaactacaa 420

ccactgcgac aacaactttc gccatcgagg gaatgacatg tggtgcctgc acatcagcag 480

ttgagggcag ctttaagggc gtggacagta tcctcaagtt taacattagc cttttggctg 540

agcgcgccgt tattacgtac gatgaaacca agatttcccc cgaggagata gccgagatta 600

tcgaggatcg cggattcgat gctaccattc tatccacaca acgcgacatg gcctgccagg 660

gacgagacac cacatctgcc caattcaaag tctttgggtg caaagatgca acgacggccc 720

aagctctgga ggaaggcctc attgcgatcc aaggcatcca atcagtatct ctcagcctca 780

gtacggatcg tcttacagtt gtttatcaac ccatgaccat aggacttcgt ggcattgtcg 840

aagccataga ggcgcagggc ttgaatgccc tagttgcaag cggagaagat aacaacgcgc 900

aacttgaatc gttggcaaaa acgcgtgaaa tcactgagtg gaggagagca ttcaagatct 960

cactcgcctt tgcgattcct gtccttctaa ttggaatgat cattcctatg gccttccctg 1020

cgatagacat tgggagtttc gaactcattc ctggtctatt cttgggtgac attgtgtgtc 1080

tcattatcac attacctgtc cagttcggca ttggcaagag attctatatt tctggttaca 1140

agtctctcaa gcatggatca ccgacaatgg atgttctggt cgttcttggc acaacgtgtg 1200

cttttctctt tagcgtcttc tcgatgctgg tctcagttct tcttgagccg cattccaaac 1260

cttccaccat cttcgataca agtactatgc tcattacttt cataacacta tctcgatggc 1320

tcgaaaatcg cgccaagggc aagacctcca aggcattatc tcgccttatg tccctagctc 1380

cgtcaacagc agccatctat gctgatccga tcgccgtgga aaaagcagcc gagaactggg 1440

caaagtcttt tgacgagccg tcaacgccaa ggacacctgg taaccaaact ggcggatccg 1500

cttgggagga aaaggtcatc ccaacagagc tacttgaggt tgacgatatc gtggtcatcc 1560

ggccaggtga caagattcca gcagatggtg tcctggtccg gggtacaaca ttcgttgatg 1620

aaagcatggt tacaggagaa gctatgcctg tccacaagcg tataggtgat aacatgatcg 1680

ccggtactat caatggtgac ggacgtgttg atcttcgtgt tactcgagct ggccatgcta 1740

cccaattaag ccaaatcgtc aagttggttc aagatgcgca aacggcccgc gccccaatcc 1800

aggagctcgc tgataagctg gccggctact ttgttcccat gattctcatt cttggtctta 1860

gcacattcct tgtatggatg gtcctttgtc acgttttatc tcaccctcct gagatcttcc 1920

ttgaagacaa cagcggtggt aaaatcgtgg tatgtgtcaa gttgtgcatt tccgtcatcg 1980

tctttgcctg tccatgtgcc cttgggcttg ctacgcccac ggcagtcatg gtcggtacag 2040

gagttggggc cgagaacgga attctcatca aaggaggcgc tgccttggag cgtataacca 2100

aggtcacgca tatcatcctt gataaaactg gcacaattac gtacggaaaa atgagcgttg 2160

ccagcacaga tctcatctcg cagtgggcca gaagcgatgt caacaaacga ctgtggtggt 2220

ccatcgtggg tctggccgag atggggagcg aacaccccgt tggcaaggct atcctgggcg 2280

ctgcaaagga agaactgggc atggatcccg agggaaccat tgatggcact gtcggtgact 2340

tcaaagctgt tgtaggcaag ggtgtcagtg tgactgtgga gccagctacc tcgagccgta 2400

cacgatacct cgttcaagtt ggaaatctcg tctttttgca agataatggt gttgatgtcc 2460

ccgaggatgc tgtccaggct gcagagaaga tcaacttgtc ggctgacgta ggtaaatcga 2520

cggtcaagag cagcggcgct ggaaccacca acatctttgt ggccatcgat ggcgtttaca 2580

caggctatgt atgtctgtct gataagatca aggaggacgc tgctgcggct atctcggttc 2640

tgcaccgcat gggcatcaaa acctcgatag taacaggcga tcaacggtct accgcactcg 2700

ctgtcgcttc tgtcgtgggt attgatgccg ataatgtcta cgctggcgtt agtcccgatc 2760

aaaagcaagc catcgtacaa gagatccaac agtctggtga agttgtcggc atggttggtg 2820

acggcattaa tgactctcca gcccttgcga cagcagacgt tggcatcgcc atggcaagcg 2880

gcacggatgt agcgatggaa gcagcagatg tcgttcttat gagaccgaca gagctcatga 2940

ttatacctgc tgctttgact cttacacaca ctattttccg tcgaatcaag ttaaaccttg 3000

gatgggcttg tctatataac gccattggtc tcccgatcgc aatgggcttt tttcttccgc 3060

tgggtctgag cgtacaccct atcatggcga gtcttgcgat ggcgtttagc agtgtcacgg 3120

tggtggttag tagtctcatg cttaactcat ggaaaaggcc tacttggatg aatgaaatag 3180

ctatgaacga tgacaagacg cccaaggcgg agaggtgggc atttggaagg ggcatcgttg 3240

gctgggtgag ggaaatgatg ggacgtaggg gaaaggtgga ggaaattggg tatttgccgt 3300

tacagaacat ggagggctga 3320

Claims

1. A method for improving mangicdiene yield is characterized by comprising fermenting a strain AO-S84 or AO-S96, wherein the strain is an Aspergillus oryzae NSAR1 as a starting strain,

The strain AO-S84 comprises 3×PhlyA-tHMG1-Tnos、PoliC-MVD1-TagdA、PglaA-IDI-TniaD、PamyB-mgcD-TamyB、PoliC-ERG10-TniaD、PamyB-ERG13-TamyB、PglaA-ERG12-Tnos、PenoA-ERG8-TamyB、PhlyA-tHMG1-TniaD;

The strain AO-S96 is obtained by taking aspergillus oryzae NSAR1 as a starting strain to obtain pyrG-deficient aspergillus oryzae NSAR1, integrating 2X PhlyA-tHMG1-Tnos, poliC-MVD1-tagdA and PamyB-IDI-TniaD at the HS401 site of the pyrG-deficient aspergillus oryzae NSAR1, integrating PoliC-ERG10-TniaD and PamyB-ERG13-TamyB at the HS201 site of the pyrG-deficient aspergillus oryzae NSAR1, integrating PglaA-ERG12-Tnos, penoa-ERG8-TamyB and PhlyA-tHMG1-TniaD at the HS601 site of the pyrG-deficient aspergillus oryzae NSAR1, and integrating PhlyA-mgcD-TniaD at the HS201 site of the pyrG-deficient aspergillus oryzae NSAR 1;

Wherein, the nucleotide sequence of PglaA is shown as SEQ ID NO:3 is shown in the figure; poliC has the nucleotide sequence shown in SEQ ID NO:5 is shown in the figure; pamyB has the nucleotide sequence shown in SEQ ID NO:7, penoA has the nucleotide sequence shown as SEQ ID NO:8, phlyA has the nucleotide sequence shown as SEQ ID NO: shown as 9; the nucleotide sequence of the ERG10 gene is shown as SEQ ID NO:10 is shown in the figure; the nucleotide sequence of the ERG13 gene is shown as SEQ ID NO: 11; the nucleotide sequence of the tHMG1 gene is shown as SEQ ID NO: shown at 12; the nucleotide sequence of the ERG12 gene is shown as SEQ ID NO: 13; the nucleotide sequence of the ERG8 gene is shown as SEQ ID NO: 14; the nucleotide sequence of the MVD1 gene is shown in SEQ ID NO: 15; the nucleotide sequence of the IDI gene is shown as SEQ ID NO: shown at 16; the nucleotide sequence of the amplified fragment mgcD is shown in SEQ ID NO: shown at 17; tamyB has the nucleotide sequence shown in SEQ ID NO:19, tniaD has the nucleotide sequence shown in SEQ ID NO:20, tagdA has the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of Tnos is shown in SEQ ID NO: shown at 22.

2. A method for improving mangicol J yield is characterized by comprising the steps of fermenting a strain AO-S93, AO-S94, AO-S97 or AO-S98, wherein a starting strain of the strain is Aspergillus oryzae NSAR1,

The strain AO-S93 comprises 3×PhlyA-tHMG1-Tnos、PoliC-MVD1-TagdA、PglaA-IDI-TniaD、PamyB-mgcD-TamyB、PoliC-ERG10-TniaD、PamyB-ERG13-TamyB、PglaA-ERG12-Tnos、PenoA-ERG8-TamyB、PhlyA-tHMG1-TniaD、PhlyA-mgcE-Tnos;

The strain AO-S94 comprises 3×PhlyA-tHMG1-Tnos、PoliC-MVD1-TagdA、PglaA-IDI-TniaD、PamyB-mgcD-TamyB、PoliC-ERG10-TniaD、PamyB-ERG13-TamyB、PglaA-ERG12-Tnos、PenoA-ERG8-TamyB、PhlyA-tHMG1-TniaD、2×PhlyA-mgcE-Tnos;

The strain AO-S97 is constructed by taking aspergillus oryzae NSAR1 as a starting strain to obtain pyrG-deficient aspergillus oryzae NSAR1, integrating 2X PhlyA-tHMG1-Tnos, poliC-MVD1-tagdA and PamyB-IDI-TniaD at the HS401 site of the pyrG-deficient aspergillus oryzae NSAR1, integrating PoliC-ERG10-TniaD, pamyB-ERG13-TamyB and PhoyA-tHMG 1-Tnos at the HS601 site of the pyrG-deficient aspergillus oryzae NSAR1, integrating PglaA-ERG12-Tnos, penoa-ERG8-TamyB and PhoyA-tHMG 1-TniaD at the HS201 site of the pyrG-deficient aspergillus oryzae NSAR1, and integrating PhlyA-mgcD-TnD and PhoyA-mgcE-Tnos at the HS601 site of the pyrG-deficient aspergillus oryzae NSAR 1;

The strain AO-S98 is prepared by taking aspergillus oryzae NSAR1 as a starting strain to obtain pyrG-deficient aspergillus oryzae NSAR1, integrating 2X PhlyA-tHMG1-Tnos, poliC-MVD1-TagdA and PamyB-IDI-TniaD at the HS401 site of the pyrG-deficient aspergillus oryzae NSAR1, integrating PoliC-ERG10-TniaD, pamyB-ERG13-TamyB and PhoyA-tHMG 1-Tnos at the HS601 site of the pyrG-deficient aspergillus oryzae NSAR1, integrating PglaA-ERG12-Tnos, penoa-ERG8-TamyB and PhoyA-tHMG 1-TniaD at the HS201 site of the pyrG-deficient aspergillus oryzae NSAR1, and integrating PhlyA-mgcD-TniaD and 2X PhlyA-mgcE-Tnos at the HS601 site of the pyrG-deficient aspergillus oryzae NSAR 1;

Wherein, the nucleotide sequence of PglaA is shown as SEQ ID NO:3 is shown in the figure; poliC has the nucleotide sequence shown in SEQ ID NO:5 is shown in the figure; pamyB has the nucleotide sequence shown in SEQ ID NO:7, penoA has the nucleotide sequence shown as SEQ ID NO:8, phlyA has the nucleotide sequence shown as SEQ ID NO: shown as 9; the nucleotide sequence of the ERG10 gene is shown as SEQ ID NO:10 is shown in the figure; the nucleotide sequence of the ERG13 gene is shown as SEQ ID NO: 11; the nucleotide sequence of the tHMG1 gene is shown as SEQ ID NO: shown at 12; the nucleotide sequence of the ERG12 gene is shown as SEQ ID NO: 13; the nucleotide sequence of the ERG8 gene is shown as SEQ ID NO: 14; the nucleotide sequence of the MVD1 gene is shown in SEQ ID NO: 15; the nucleotide sequence of the IDI gene is shown as SEQ ID NO: shown at 16; the nucleotide sequence of the amplified fragment mgcD is shown in SEQ ID NO: shown at 17; mgcE has the nucleotide sequence shown in SEQ ID NO: shown at 18; tamyB has the nucleotide sequence shown in SEQ ID NO:19, tniaD has the nucleotide sequence shown in SEQ ID NO:20, tagdA has the nucleotide sequence shown in SEQ ID NO: 21; the nucleotide sequence of Tnos is shown in SEQ ID NO: shown at 22.

3. A strain for improving mangicdiene yield is characterized by comprising a strain AO-S84 or AO-S96, wherein a starting strain of the strain is Aspergillus oryzae NSAR1,

4. A strain for improving mangicol J yield is characterized by comprising a strain AO-S93 or AO-S94 or AO-S97 or AO-S98, wherein the original strain of the strain is Aspergillus oryzae NSAR1,