CN115247183B - Construction method of recombinant microorganism, related biological material and application thereof - Google Patents

Abstract

The invention discloses a construction method of recombinant saccharomyces cerevisiae, related biological materials and application thereof. The construction method comprises the steps of introducing a coding gene of the recombinant fusion protein into a starting microorganism to obtain a recombinant microorganism; the recombinant fusion protein contains Pln1 protein and terpene synthesis related protein. The construction method relocates part of key enzymes in the triterpene biosynthesis pathway through Pln protein to promote the enzymes to approach substrates spatially, and promote the transformation of the natural product fat-soluble intermediates stored in lipid droplets.

Description

Construction method of recombinant microorganism, related biological material and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a construction method of recombinant microorganisms, related biological materials and application thereof.

Background

The natural compounds and the derivatives thereof have important application in the aspects of medication and health care, and the market demand of the natural compounds is increasing along with the characterization of more and more pharmacological actions of natural compound molecules. Conventional plant extraction methods or chemical synthesis methods have various limitations. Plant extraction results in great waste of plant resources and extremely low active ingredients obtained. On the one hand, chemical synthesis brings about environmental pressure, and on the other hand, when processing natural compounds with complex structures, the problems of long synthetic route, low yield and the like exist. The synthetic biology is the science of redesigning, engineering construction and application of life systems and processes, which is raised in recent years, and provides powerful technology and platform support for the large-scale production of natural compounds and the design and synthesis of small molecular compounds with new structures. There have been reports in recent years that other active plant natural products are prepared by using synthetic biological methods, and the related natural products include terpenes, flavonoids, polyphenols, alkaloids, etc., and the green and efficient production chain formed by these new technologies is being accepted by the science and industry.

Terpenes are one of the most widely used hydrophobic substances with a variety of functions and can be used as fragrances and perfumes, pharmaceuticals, solvents, cosmetics, food additives and potentially higher biofuels. Yeast is considered to be an ideal host for producing isoprenoids due to the characteristics of clear genetic background, simple genetic operation and the like. However, eukaryotic cells are divided into several subcellular organelles, which have complex structures with their own membranes. In particular, cellular metabolism is divided into specialized subcellular organelles. For example, oxidative phosphorylation occurs in mitochondria, and β -oxidation of fatty acids is localized in peroxisomes, which results in the dispersion of cofactors and precursors. This subcellular compartmentalization may present some barriers to substrate passage when supplying precursors and cofactors. One of the problems faced in the heterologous synthesis of terpenoid natural products using Saccharomyces cerevisiae is that its fat-soluble intermediates are mostly stored inside the subcellular space of the yeast cells, the lipid droplets.

Despite extensive metabolic engineering, the production of most isoprenoids in yeast is still far behind industrial applications, which may be due in part to complex metabolic compartmentalization. Thus, systematic studies of cellular subchambers and use of subcellular organelles may provide a viable approach to further enhance isoprenoid biosynthesis in yeast and even other eukaryotic cells.

Disclosure of Invention

The invention provides a method for constructing recombinant microorganisms, which comprises the steps of introducing a coding gene of a recombinant fusion protein into a starting microorganism to obtain the recombinant microorganism; the recombinant fusion protein contains Pln1 protein and terpene synthesis related protein.

Alternatively, according to the above method, the terpene synthesis associated protein is selected from protopanoxadiol synthase PPDS01 and/or cytochrome P450 reductase ATR1.

The terpene synthesis-associated protein may comprise PPDS01 and ATR1, and then PPDS01 and ATR1 may be linked via a linking peptide. ATR1 may be a cytochrome P450 reductase 46 ATR 1N-terminally truncated by 46 amino acids, e.g. 46 ATR1 has the amino acid sequence shown in SEQ ID No.2 at position 780-1425.

Alternatively, according to the above method, in the recombinant fusion protein, the Pln protein and the terpene synthesis-related protein are linked via a connecting peptide.

In the above, the connecting peptide may be GGGS or GSTSSG.

Alternatively, according to the above method, the recombinant fusion protein is a recombinant protein comprising the Pln protein, PPDS01 and ATR1.

Alternatively, according to the above method, the amino acid sequence of the Pln1 protein is shown in positions 1-283 of SEQ ID No. 2; the amino acid sequence of the PPDS01 is shown in 288 th to 773 th positions in SEQ ID No. 2; the amino acid sequence of the ATR1 is shown in 780 th-1425 th positions in SEQ ID No. 2.

Alternatively, according to the above method, the amino acid sequence of the recombinant fusion protein is as shown in SEQ ID No. 2. In SEQ ID No.2, the amino acid sequence of the Pln1 protein is from 1 st to 283 rd, the amino acid sequence of the connecting peptide GGGS is from 284 th to 287 th, the amino acid sequence of the PPDS01 is from 288 th to 773 th, the amino acid sequence of the connecting peptide GSTSSG is from 774 th to 779 th, and the amino acid sequence of the ATR1 is from 780 th to 1425 th.

The method may further comprise expressing a gene encoding the recombinant fusion protein described above. The specific sequence of the coding gene can be shown as 431 th to 4708 th in SEQ ID No. 1.

Alternatively, according to the above method, in the recombinant microorganism, the gene encoding the recombinant fusion protein is integrated into the YPL062W site of the starting microorganism.

Alternatively, according to the above method, the recombinant microorganism is obtained by introducing the gene encoding the recombinant fusion protein into the starting microorganism through an expression cassette expressing the recombinant fusion protein. The sequence of the expression cassette for expressing the recombinant fusion protein can be shown as SEQ ID No1, wherein the promoter P _TEF1 The coding gene of the protein of Pln1 is the 431 th to 1279 th, the coding gene of the connecting peptide GGGS is the 1280 th to 1291 th, the coding gene of the protein PPDS01 is the 1292 th to 2749 th, the coding gene of the connecting peptide GSTSSG is the 2750 th to 2767 th, the coding gene of the protein 46tATR1 is the 2768 th to 4708 th, and the terminator T _CYC1 4709-5015 bits.

Alternatively, according to the above method, the starting microorganism is Saccharomyces cerevisiae, which is a strain obtained by modifying the strain BYT1 as follows A1-A12,

a1, introducing a 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1 gene; a2, introducing a mevalonate kinase gene ERG 12; a3, introducing an alcohol dehydrogenase I gene IDI1 gene; a4, introducing a mevalonate pyrophosphate decarboxylase gene ERG 19; a5, introducing a HMGR gene of a hydroxymethylglutaryl-CoA reductase gene; a6, introducing a hydroxymethyl glutaryl-coenzyme A synthetase gene ERG 13; a7, introducing a phosphomevalonate kinase gene ERG 8; a8, introducing an acetyl coenzyme A acetyltransferase gene ERG 10; a9, introducing a squalene synthase gene AtSQS 2; a10, introducing a squalene monooxygenase gene ERG 1; a11, introducing a farnesyl pyrophosphate synthase gene SmFPS gene; a12, a dammarenediol synthase gene spgDDS gene is introduced.

Alternatively, the sequence of the tHMG1 protein encoded by the tHMG1 gene is genbank accession number: bits 530-1054 of AJS 96703.1; the sequence of ERG12 protein encoded by the ERG12 gene is genbank login number: np_ 013935.1; the sequence of IDI1 protein encoded by the IDI1 gene is genbank accession number: np_ 015208.1; the sequence of ERG19 protein encoded by the ERG19 gene is genbank accession number: np_ 014441.1; the sequence of the HMGR protein coded by the HMGR gene is genbank login number: wp_ 011241944.1; the sequence of ERG13 protein encoded by the ERG13 gene is genbank login number: np_ 013580.1; the sequence of ERG8 protein encoded by the ERG8 gene is genbank login number: np_ 013947.1; the sequence of ERG10 protein encoded by the ERG10 gene is genbank login number: np_ 015297.1; the sequence of the AtSQS2 protein coded by the AtSQS2 gene is genbank login number: np_ 195190.1; the sequence of ERG1 protein encoded by the ERG1 gene is genbank login number: np_ 011691.1; the SmFPS protein coded by the SmFPS gene has the sequence of genbank login number: ABV 08819.1; the sequence of the spgDDS protein coded by the spgDDS gene is a genbank login number: ACZ 71036.1.

Optionally, the sequence of the tHMG1 gene is shown as 757 th to 2340 th positions in SEQ ID No. 3; the sequence of the ERG12 gene is shown in the 801 st position to 2132 nd position in SEQ ID No. 4; the sequence of the IDI1 gene is shown in 1001 st position to 1867 th position in SEQ ID No. 5; the sequence of the ERG19 gene is shown in 1001 st position to 2191 st position in SEQ ID No. 6; the sequence of the HMGR gene is shown in the 563 rd position-1864 th position in SEQ ID No. 7; the sequence of the ERG13 gene is shown in 823 th-2298 th positions in SEQ ID No. 8; the sequence of the ERG8 gene is shown in the 801 st position to 2156 th position in SEQ ID No. 9; the sequence of the ERG10 gene is shown in the 431 th position to 1627 th position of SEQ ID No. 10; the sequence of the AtSQS2 gene is shown in 751 th to 1983 rd positions in SEQ ID No. 11; the sequence of the ERG1 gene is shown in the 801 st position to the 2291 nd position in SEQ ID No. 12; the SmFPS gene has a sequence shown in 431 th to 1480 th positions of SEQ ID No. 13; the sequence of the spgDDS gene is shown in the 431 th position to 2740 th position in SEQ ID No. 15.

The above-mentioned gene may be obtained by introducing the expression cassette for expressing the above-mentioned protein into the strain BYT1 or by expressing the above-mentioned protein into the expression plasmid, and for example, the above-mentioned gene may be integrated into the LEU site and the NDT80 site of the strain BYT1, respectively.

Alternatively, the expression cassette sequence for expressing the tHMG1 protein is shown in SEQ ID No. 3; the sequence of the expression cassette for expressing ERG12 protein is shown as SEQ ID No. 4; the sequence of the expression cassette for expressing IDI1 protein is shown in SEQ ID No. 5; the sequence of the expression cassette for expressing ERG19 protein is shown in SEQ ID No. 6; the sequence of the expression cassette for expressing the HMGR protein is shown in SEQ ID No. 7; the sequence of the expression cassette for expressing ERG13 protein is shown as SEQ D No. 8; the sequence of the expression cassette for expressing ERG8 protein is shown as SEQ ID No. 9; the sequence of the expression cassette for expressing ERG10 protein is shown as SEQ ID No. 10; the sequence of the expression cassette for expressing the AtSQS2 protein is shown as SEQ ID No. 11; the sequence of the expression cassette for expressing ERG1 protein is shown as SEQ ID No. 12; the sequence of the expression cassette for expressing SmFPS protein is shown as SEQ ID No. 13; the sequence of the expression cassette for expressing the spgDDS protein is shown as SEQ ID No. 15.

The invention also provides any one of the following biological materials B1) to B6):

b1 Nucleic acid molecules encoding the recombinant fusion proteins described above; b2 An expression cassette comprising the nucleic acid molecule of B1); b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2); b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3); b5 A recombinant fusion protein as described above; b6 A recombinant microorganism expressing the recombinant fusion protein.

The invention also provides any one of the following applications:

x1, application of the method for constructing recombinant microorganisms in preparing terpene products; x2, the use of the above-described method of constructing a recombinant microorganism in the production of terpenes; x3, application of the biological material in preparing terpene products; x4, application of the biological material in terpene production; application of X5 and Pln1 protein in preparing terpene products; application of X6 and Pln1 proteins in terpene production; application of X7 and Pln1 protein in improving terpene synthesis efficiency (recombination efficiency); application of X8 and the recombinant fusion protein in improving terpene synthesis efficiency (recombination efficiency).

The present invention also provides a method for increasing the efficiency of terpene biosynthesis comprising the step of expressing the recombinant fusion protein described above in a recipient organism to obtain a recombinant organism that synthesizes a terpene more efficiently than the recipient organism.

In the above, the organism may be a microorganism, a plant or a non-human animal.

In the above, the terpene product may be a recombinant bacterium expressing a terpene. The terpene can be protopanaxadiol or cucurbitadienol.

Yeast lipid droplets have the ability to store unwanted hydrophobic substances as a special cell, not only Fatty Acids (FA) in the form of Triacylglycerols (TAG) but also to isolate squalene, sterol esters and retinyl esters in the lipid droplets to avoid damaging the integrity of the membrane. Pln1 protein (also known in the past as Pet10 p) specifically binds to TAG-containing lipid droplets early in lipid droplet formation, thereby maintaining lipid droplet morphology and stability and promoting lipid droplet formation from the endoplasmic reticulum. The present invention repositions a portion of the key enzymes in the triterpene biosynthetic pathway by Pln proteins to facilitate spatial access of the enzymes to the substrate to facilitate conversion of the naturally occurring liposoluble intermediates stored in the lipid droplets.

Drawings

FIG. 1 shows the HPLC detection of DD and PPD contents in strains PTA and LPTA in example 2.

FIG. 2 is an HPLC chart of example 2 strains PTA and LPTA.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Data were processed using SPSS11.5 statistical software and experimental results were expressed as mean.+ -. Standard deviation using One-way ANOVA.

Saccharomyces cerevisiae BY4742 (Saccharomyces cerevisiae BY 4742), described in Zhubo Dai et al Producing aglycons of ginsenosides in bakers' yeast. Sci Rep.2014Jan 15, was used in the examples below; 4:3698..

The gene fragments and protein sequence related information for the following examples are shown in the following table.

Information about Gene fragments

Information about protein sequences

	genbank accession number	Update time
			tHMG1 protein	AJS96703.1, bits 530-1054	2016/5/23
ERG12 proteins	NP_013935.1	2020/10/2
			IDI1 protein	NP_015208.1	2020/10/2
ERG19 proteins	NP_014441.1	2020/10/2
			HMGR protein	WP_011241944.1	2019/6/19
ERG13 proteins	NP_013580.1	2020/10/2
			ERG8 proteins	NP_013947.1	2020/10/2
ERG10 proteins	NP_015297.1	2020/10/2
			AtSQS2 protein	NP_195190.1	2019/2/14
ERG1 proteins	NP_011691.1	2020/10/2
			SmFPS protein	ABV08819.1	2011/4/11
spgDDS protein	ACZ71036.1	2009/12/12

Example 1

1. Cloning of Gene original

1. PCR amplification to obtain Gene fragment

The primer in Table 1 was used to amplify the genomic DNA of Saccharomyces cerevisiae BY4742 as a template to obtain the Pln gene of the complete ORF and the ATR1 gene (46 tATR 1) with an N-terminal truncated of 46 amino acids.

The protopanaxadiol synthase gene PPDS01 was amplified using the primer shown in Table 1 as a template using the plasmid pM13-pgPPDS containing the pgPPDS gene (described in Dai ZB et al, metabolic engineering of Saccharomyces cerevisiae for production of ginoside. Metabolic engineering 2013, 20:145-156, publicly available from Tianjin Industrial Biotechnology research).

The amplification system is as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg ²⁺ plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 1. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, add ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 60℃for 15 seconds, elongation at 68℃for 3 minutes (35 cycles); extension at 72℃for 10 min (1 cycle). The product is recovered and stored by glue.

2. Fusion PCR obtaining fusion fragments

(1) The PPDS01 gene containing 20bp homologous region with 46tATR1 gene was subjected to fusion PCR, and the primers are shown in Table 1.

The fusion PCR system was as follows: primeSTARGXLBbuffer (Mg) ²⁺ plus) x 10. Mu.l, dNTPMix 4. Mu.l, primers SexA1-PPDS01-F and Asc1-46tATR 1-R1.5. Mu.l each, DNA template 1.5. Mu.l each of fragment 46tATR1 and fragment PPDS01, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, and ddH was added ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 60℃for 15 seconds, elongation at 68℃for 3 minutes (35 cycles); extension at 72℃for 10 min (1 cycle). Obtaining fusion gene fragment PPDS01-GSTSSG-46tATR1.

A fusion gene fragment 14bp-PPDS01-GSTSSG-46tATR1 containing a 14bp homologous region with the Pln1 gene was obtained in the same manner as described above, and the primers are shown in Table 1. The fragments are subjected to gel recovery treatment.

(2) The 14bp-PPDS01-GSTSSG-46tATR1 gene containing the 14bp homologous region with the Pln gene was subjected to fusion PCR, and the primers are shown in Table 1.

The fusion PCR system was as follows: primeSTAR GXL Buffer (Mg) ²⁺ plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers Pac1-Pln1-F and Asc1-46tATR1-R each 1.5. Mu.l, DNA template was 1.5. Mu.l each fragment Pln and fragment 14bp-PPDS01-GSTSSG-46tATR1, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, supplemented with ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 60℃for 15 seconds, elongation at 68℃for 3 minutes (35 cycles); extension at 72℃for 10 min (1 cycle). The fusion gene fragment Pln-GGGS-PPDS 01-GSTSSG-46tATR1 was obtained.

TABLE 1 primer sequences

2. Construction of recombinant plasmids

1、pM3-PPDS01-GSTSSG-46tATR1

Double digestion was performed with restriction enzymes SexAI and AscI respectively on plasmid pM3-ERG9 (described in Chinese patent application 201210453416. X) and gene PPDS01-GSTSSG-46tATR1, and the target fragment was recovered by tapping: pEASY-Blunt-P _TEF1 -//-T _CYC1 (62 ng) and PPDS01-GSTSSG-46tATR1 (3420 bp,130 ng), the fragment of interest was ligated with the vector as follows: mu.l of 2X Quick Ligation Buffer (NEB Co., ltd.), 0.5. Mu.l of Quick T4 DNA Ligase (NEB Co., 400,000cohesive end units/ml), and ddH were supplemented ₂ O to 10. Mu.l, 25Reacting at the temperature of 13min to obtain a connection product, transferring into a Trans 1T 1 competent cell, and performing sequencing verification to obtain the recombinant vector. Sequencing, the recombinant vector is an expression cassette P of PPDS01-GSTSSG-46tATR1 gene _TEF1 -PPDS01-GSTSSG-46tATR1-T _CYC1 The vector obtained between cloning sites of pEASY-BluntSimple cloning vector (pEASY cloning vector, beijing full gold Biotech (TransGen Biotech) Co., ltd.) was inserted and designated pM3-PPDS01-GSTSSG-46tATR1.

2、pM13-Pln1-GGGS-PPDS01-GSTSSG-46tATR1

Plasmid pM13-pgPPDS (described in literature: dai ZB et al, 2013,Metabolic Engineering 20:145-156, publicly available from Tianjin Industrial Biotechnology institute) and gene fragment Pln1-GGGS-PPDS01-GSTSSG-46tATR1 were digested with restriction enzymes PacI and AscI, respectively, and the desired fragment was recovered by tapping: pEASY-Blunt-P _TEF1 -//-T _CYC1 (50 ng) and pac1-Pln1-GGGS-PPDS01-GSTSSG-46tATR1-Asc1 (4278 bp,104 ng), the desired fragment was ligated with the vector as follows: mu.l of 2X Quick Ligation Buffer (NEB Co., ltd.), 0.5. Mu.l of Quick T4 DNA Ligase (NEB Co., 400,000cohesive end units/ml), and ddH were supplemented ₂ O to 10 μl, reacting at 25deg.C for 13min to obtain a ligation product, transferring into Trans 1T 1 competent cells, and performing sequencing verification to obtain recombinant vector. Sequencing, the recombinant vector is an expression cassette P of Pln1-GGGS-PPDS01-GSTSSG-46tATR1 gene _TEF1 -Pln1-GGGS-PPDS01-GSTSSG-46tATR1-T _CYC1 The vector obtained between cloning sites of pEASY-Blunt Simple cloning vector (pEASY cloning vector, beijing full gold Biotech (TransGen Biotech) Co., ltd.) was inserted and designated pM 13-Pln-GGGS-PPDS 01-GSTSSG-46tATR1.

3. Leu gRNA, NDT80gRNA and YPL062W gRNA

The p426-SNR52p-gRNA.CAN1.Y-SUP4t plasmid from adedge was used as template for amplification with the following primers: gRNA reverse/Leu gRNA forward, gRNA reverse/NDT 80gRNA forward, gRNA reverse/YPL 062W gRNA forward

gRNA reversal: GATCATTTATCTTTCACTGC

LeugRNA forward:

cgcagtgaaagataaatgatcCGATGGTGATGGTGTCGCTTgttttagagctagaaatagcaag

NDT80gRNA forward:

cgcagtgaaagataaatgatcCTGCTTCAGGTGCGGCTTGGgttttagagctagaaatagcaag

YPL062W gRNA forward:

cgcagtgaaagataaatgatcGCACGTCGCCGTGGCTGATGgttttagagctagaaatagcaag

amplifying to obtain three linear fragments Linearized Leu gRNA, linear NDT80gRNA and linear YPL062W gRNA respectively, transferring the three fragments into a Transl T1 competent cell respectively, and carrying out sequencing verification to obtain recombinant plasmids Leu gRNA, NDT80gRNA and YPL062W gRNA.

4. plasmid construction of pM7-HMGR

The genome DNA of Saccharomyces cerevisiae BY4742 is used as a template, primers Pac1-TEF2-F and SexA1-TEF2-R are used for amplification to obtain a promoter pTEF2 (562 bp), and primers Asc1-ENO2-F and Pme1-ENO2-R are used for amplification to obtain a terminator tENO2 (400 bp). The amplification system is as follows: primeSTAR GXL Buffer (Mg) ²⁺ plus). Times.10. Mu.l, dNTPMmix. Times.4. Mu.l, primers Pac1-TEF2-F and SexA1-TEF2-R (Asc 1-ENO2-F and Pme1-ENO 2-R) were each 1.5. Mu.l, and the genomic DNA template was 1.5. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, supplemented with ddH2O to a total volume of 50. Mu.l.

Pac1-TEF2-F：5’-GCTTAATTAAATGGGGCCGTATACTTACATATAGTAGA-3’

SexA1-TEF2-R：5’-GCACCAGGTGTTTAGTTAATTATAGTTCGTTGACCGTATATTCTAAAAAC-3’

Asc1-ENO2-F：5’-GCGGCGCGCCAGTGCTTTTAACTAAGAATTATTAGTCTTTTCTGCT-3’

Pme1-ENO2-R：5’-GCGTTTAAACAGGTATCATCTCCATCTCCCATATGC-3’

The plasmid pUC57-synHMGR (the whole synthesis of the gene was carried out by the company of Kirschner Biotechnology, inc. of the entire synthesis request of the gene) was digested with restriction enzymes SexAI and AscI, and the synHMGR gene was inserted between cloning sites of pUC57 vector (supplied by Kirschner Biotechnology, inc.), to obtain a cloned plasmid pUC57-synHMGR containing the synHMGR gene, and the desired fragment was recovered by tapping to obtain the fragment SexAI-synHMGR-AscI; cutting the section pTEF2 by using restriction endonucleases SexAI and pacI respectively, and recovering a target fragment by tapping to obtain SexAI-pTEF2-pacI; cutting the segment tENO2 by restriction enzymes Ascl and Pme1 respectively, recovering the target segment by tapping to obtain Asc1-tENO2-Pme1, and adding 50ng of each of the three segments into a connecting system: 2ul 10XT4 ligation Buffer (NEB Co.), 1ul T4 ligase (NEB Co., 400,000cohesive end units/ml), distilled water was supplemented to 20ul, and the ligation product was obtained by reacting at room temperature for 2 hours, 1ul ligation product was added, and PCR system: primeSTAR GXL Buffer (Mg2+plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers Pac1-TEF2-F and Pme1-ENO2-R each 1.5. Mu.l, ligation product 1. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, and ddH2O were added to a total volume of 50. Mu.l to give an expression cassette P _TEF2 -HMGR-T _ENO2 . Cloning the expression cassette into a pEASY-Blunt Simple cloning vector (available from Beijing full gold Biotechnology Co., ltd.) to obtain the recombinant vector pM7-HMGR, sequencing, wherein the expression cassette P of the HMGR is obtained _TEF2 -HMGR-T _ENO2 The vector obtained by inserting pEASY-Blunt Simple cloning sites (the sequence is shown as SEQ ID No. 7).

The recombinant plasmids pM3-PPDS01-GSTSSG-46tATR1, pM13-Pln1-GGGS-PPDS01-GSTSSG-46tATR1, leu gRNA, NDT80gRNA and YPL062W gRNA prepared above are shown in Table 2 for relevant information.

TABLE 2 plasmid information

3. Construction of recombinant bacteria

Construction of YSBYT5 Strain

1. Construction of Gene modules

The plasmids described in Table 2 were used as PCR templates (p.delta. -tHMG1, pM9-ERG12, pM16-IDI1, pM5-ERG19, pM8-ERG13, pM11-ERG8, pM3-ERG10, respectively, are described in the literature: creating Saccharomyces cerevisiae)Cell factory fermentation to produce lupeol journal of chinese traditional chinese medicine, lin Tingting, wang Dong, wave-wearing, zhang Xueli, huang Luqi, 2016, 41 (6): 1008-1015) and the corresponding primers of table 3, respectively, to obtain functional module fragments: m1 (comprising P) _PGK1 -tHMG1-T _ADH1 Expression cassette), M2 (comprising P _PDC1 -ERG12-T _ADH2 Expression cassette), M3 (comprising P _ENO2 -IDI1-T- _PDC1 Expression cassette), M4 (comprising P _PYK1 -ERG19-T _PGI1 Expression cassette), M5 (comprising P _TEF2 -HMGR-N-T _ENO2 Expression cassette), M6 (comprising P _FBA1 -ERG13-T _TDH2 Expression cassette) and M7 (comprising P _TDH3 -ERG8-T _TPI1 Expression cassette), M8 (comprising P _TEF1 -ERG10-T _CYC1 An expression cassette).

The amplification system is as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg ²⁺ plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 1. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, add ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 60℃for 15 seconds, elongation at 68℃for 3 minutes (35 cycles); extension at 72℃for 10 min (1 cycle). The product is recovered and stored by glue.

TABLE 3 templates, primers and sequences thereof

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2. Preparation of Yeast competence

Fresh yeast liquid BYT1 (from laboratory save strain, described in Zhubo Dai et al Producing aglycons of ginsenosides in bakers' yeast. Sci Rep.2014Jan 15; 4:3698.) (pre-transferred to P414-P from addgene Corp.) _TEF1 -Cas9-T _CYC1 Plasmid) was prepared (1% inoculum size, 30ul seed liquid was inoculated into 3ml SD-Trp liquid medium (0.8% total synthetic tetra-deficient medium (available from Beijing Pantonoz technologies Co., ltd.) +2% glucose+0.005% His+0.01% Ura+0.01% Leu)).

The operation steps are as follows:

(1) and (3) collecting thalli: 1ml of yeast liquid was dispensed into 1.5ml EP tubes. Centrifuge at 12000rpm for 1min, discard supernatant and suck with a gun. The pellet was washed with 1ml of sterile water, centrifuged by blowing, the supernatant discarded, and washed twice.

(2) And (3) thallus treatment: 1ml of the treatment solution (preparation of the treatment solution: 1M sorbitol+ 10mM LiAc+10mM Tris-HCl (pH 7.5)) (stored in a refrigerator at 4 ℃ C.)) +10ul DTT (available from Bode commercial Co., ltd., product No. 1758-9030-25 g.) (-stored in a refrigerator at 20 ℃ C.), was added, and the metal was heated at 25 ℃ C. For 20 minutes.

(3) After 20min, the supernatant was discarded, sucked out with a gun, 1ml of precooled 1M sob (D-sorbitol, purchased from Beijing Soy technologies Co., ltd.) was added, blown, centrifuged, and the supernatant was discarded. The supernatant was removed by washing twice again with 1M Sob and 50ul of Sob was added for suspension.

(4) Add 2. Mu. l M1, M2, M3, M4, M5, M6, M7 and M8 modules and 2. Mu.l Leu gRNA plasmid were blown and mixed and transferred to a pre-chilled electric rotating cup for 5min in ice bath.

(5) The electric rotating cup is wiped clean, and electric shock is conducted at 2.7 kv. 1ml of the Sob which is sucked in first is added and put into an electric rotating cup, and after uniform mixing, a new 1.5ml EP tube is sucked in. Shaking culture at 30deg.C and 250rpm for 60min.

(6) After 60min, the bacterial liquid is centrifuged to remove part of the supernatant, and the supernatant is evenly coated on a plate of an auxotroph SD-UraTrp (0.8% total synthetic tetra-deficient culture medium+2% glucose+0.005% His+0.01% Leu+2% Ager) and cultured in a 30 ℃ incubator for 36h.

Culturing in an incubator for about two days, picking up a monoclonal to perform PCR colony verification to obtain yeast engineering strain YSBYT5, discarding Leu gRNA plasmid, and performing the next genetic modification.

The construction principle of the yeast engineering bacterium YSBYT5 is that a recombinant plasmid p414-PTEF1-Cas9-TCYC1 capable of expressing Cas9 protein is transferred into a strain BYT1 in advance, then a recombinant plasmid (Leu gRNA) capable of expressing gRNA and a recombinant fragment (M1-M8) are transformed into the strain together, the Leu gRNA recognizes and combines with a Leu site specific PAM region, and simultaneously activates and guides the Cas9 protein to perform a shearing function, so that double-stranded DNA of the Leu site is broken, and at the moment, the recombinant fragments M1-M8 containing the homologous regions are integrated into the strain DNA through homologous recombination repair.

The PCR colony verification method specifically comprises the following steps:

the genome of yeast strain YSBYT5 was extracted using a yeast genomic DNA extraction kit (available from Beijing Tiangen Biochemical technology Co., ltd., cat# DP 307-02). PCR amplification is carried out by using the extracted genome as a template and SacII-PGK1/Asc1-tHMG1-R to obtain a fragment of about 2400bp, which shows that M1 is contained; PCR amplification is carried out on Pac-pPDC1/Asc1-Erg12-R to obtain a fragment with about 2200bp, which shows that M2 is contained; PCR amplification of pac-pENO2/IDI1-Asc1-R to obtain 2200bp fragment, indicating M3; PCR amplification of Pac-PYK1p/Asc1-Erg19-R gave a fragment of about 2200bp, indicating M4; PCR amplification was performed on pac1-pTEF2/Asc1-HMGR-N-R to obtain a fragment of about 1900bp, indicating that M5 was contained; PCR amplification is carried out on pFBA1-YZ-F/Asc1-Erg13-R to obtain a fragment of about 2300bp, which shows that M6 is contained; PCR amplification was performed on Pac-pTDH3/Asc1-Erg8-R to give a fragment of about 2200bp, which indicated that M7 was contained; PCR amplification was performed with SacII-pTEF1/Asc1-Erg10-R to give a fragment of about 1700bp, indicating that M8 was contained. The primers are shown in Table 4.

TABLE 4 colony validation primers and sequences thereof

Primer name	Sequence (5 '-3')
		SacII-PGK1	GCGCCGCGGACGCACAGATATTATAACATC
Asc1-tHMG1-R	GGCGCGCCTTAGGATTTAATGCAGGTGACGGA
		Pac-pPDC1	GCGTTAATTAACATGCGACTGGGTGAGCATATGTTC
Asc1-Erg12-R	GGCGCGCCTTATGAAGTCCATGGTAAATTCGT
		Pac-pENO2	GCGTTAATTAAAATCCTACTCTTGCCGTTGCCATCC
IDI1-Asc1-R	GCGGCGCGCCTTATAGCATTCTATGAATTTGCCTGTCATTTT
		Pac-PYK1p	GCGTTAATTAAAATGCTACTATTTTGGAGATTAATC
Asc1-Erg19-R	GGCGCGCCTTATTCCTTTGGTAGACCAGTCTT
		pac-pTEF2	GCTTAATTAAATGGGGCCGTATACTTACATATAGTAGA
Asc1-HMGR-N-R	GGCGCGCCTTATGTGTTTTCCAAAACTTGCT
		pFBA1-YZ-F	TGGCTTGAACAACAATACCAGCC
Asc1-Erg13-R	GGCGCGCCTTATTTTTTAACATCGTAAGATCTTCTAAA
		Pac-pTDH3	GCGTTAATTAAATACTAGCGTTGAATGTTAGCGTCA
Asc1-Erg8-R	GGCGCGCCTTATTTATCAAGATAAGTTTCCGGATCTTT
		SacII-pTEF1	GCGCCGCGGAGTGATCCCCCACACACCATAGCTT
Asc1-Erg10-R	GGCGCGCCTCATATCTTTTCAATGACAATAGAGGAAGCAC
		SmFPS-Asc1	GCGGCGCGCCTTATTTCTGCCTCTTGTATATCTTGCC
AtSQS2-Asc1	GCGGCGCGCCTCAGTTTGCTCTGAGATATGCAAAGAC
		ERG1-Asc1	GCGGCGCGCCTTAACCAATCAACTCACCAAACAAAAATGG
spgDDS-Asc1-R	GCGGCGCGCCTCATATCTTTAATTGTTGATGCTTAGGTAACCAAAC
		ypl062w-up-256	GGAATTATTCGTAACGTCATACGA
PPDS01-EGPP-R	GTTGTGTGGGTGTAAGTGGATAG
		ATR1-Ce1805-F	TAAGGGCATGGCGAGGGAC
yp1062w-down-249	GTGTAGCTTAGTCATTGTATTCTGAT

Construction of (two) YSBYT30 Strain

1. Construction of Gene modules

The plasmids described in Table 2 were used as PCR templates (pM 3-SmFPS and pM2-AtSQS2 were described in the literature: wang Dong, liu Yi, xu Jiaoyang, wang Jinhe, wearing waves, zhang Xueli, huang Luqi. Creation of a Saccharomyces cerevisiae cell factory to efficiently produce the ginsenoside precursor dammarenediol II [ J ]]Pharmaceutical journal, 2018, 53 (08): 1233-1241, pM11-ERG1 is described in chinese patent application 201210453416.X, publicly available from the institute of biotechnology in the Tianjin industry) and the corresponding primers were subjected to PCR amplification, respectively obtaining functional modules: m9 (comprising P) _PGK1 -AtSQS2-T _ADH1 Expression cassette), M10 (comprising P _TDH3 -ERG1-T _TPI1 Expression cassette), M11 (comprising P _TEF1 -SmFPS-T _CYC1 An expression cassette).

The amplification system is as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg ²⁺ plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 1. Mu.l, primesTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, add ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 60℃for 15 seconds, elongation at 68℃for 3 minutes (35 cycles); extension at 72℃for 10 min (1 cycle). The product is recovered and stored by glue.

2. Preparation of Yeast competence

Fresh yeast YSBYT5 was cultured overnight to make competent (1% inoculum size, 30ul seed liquid was inoculated with 3ml medium) and the procedure was as follows:

(1) and (3) collecting thalli: 1ml of yeast liquid was dispensed into 1.5ml EP tubes. Centrifuge at 12000rpm for 1min, discard supernatant and suck with a gun. The pellet was washed with 1ml of sterile water, centrifuged by blowing, the supernatant discarded, and washed twice.

(2) And (3) thallus treatment: 1ml of the treatment solution (4 ℃ C. Refrigerator preservation) +10ul DTT (-20 ℃ C. Refrigerator preservation) was added, and the metal was heated at 25 ℃ C. For 20min.

(3) After 20min, the supernatant was discarded, sucked out with a gun, 1ml of pre-chilled 1M sob (4 ℃ C. Refrigerator) was added, blown, centrifuged, and the supernatant discarded. The supernatant was removed by washing twice again with 1M Sob and 50ul of Sob was added for suspension.

(4) Add 2. Mu. l M9, M10 and M11 modules and 2. Mu.l NDT80gRNA plasmid, blow mix and transfer to pre-chilled electric rotating cup, ice bath for 5min.

(5) The electric rotating cup is wiped clean, and electric shock is conducted at 2.7 kv. 1ml of the Sob which is sucked in first is added and put into an electric rotating cup, and after uniform mixing, a new 1.5ml EP tube is sucked in. Shaking culture at 30deg.C and 250rpm for 60min.

(6) After 60min, the bacterial liquid is centrifuged to remove part of supernatant, and the supernatant is evenly coated on a plate of an auxotroph Sd-UraTrp (0.8% total synthetic tetra-deficient culture medium+2% glucose+0.005% His+0.01% Leu+2% Ager), and is cultured in a 30 ℃ incubator for 36h.

Culturing in an incubator for about two days, selecting monoclonal for PCR colony verification to obtain yeast engineering strain YSBYT30, discarding NDT80gRNA plasmid, and performing the next genetic transformation

The construction principle of the yeast engineering bacterium YSBYT30 is that a recombinant plasmid p414-PTEF1-Cas9-TCYC1 capable of expressing Cas9 protein is arranged in a strain YSBYT5, a recombinant plasmid (NDT 80 gRNA) expressing NDT80gRNA and a recombinant fragment (M9-M11) are transformed into the strain YSBYT5 together, the gRNA recognizes and combines with a specific PAM region of the NDT80 site, and simultaneously activates and guides the Cas9 protein to perform a shearing function, so that double-stranded DNA of the NDT80 site is broken, and at the moment, the recombinant fragment M9-M11 containing the homologous region is integrated into yeast DNA through homologous recombination repair.

The PCR colony verification method specifically comprises the following steps:

the genome of the yeast strain YSBYT30 was extracted using a yeast genomic DNA extraction kit (available from Beijing Tiangen Biochemical technology Co., ltd., cat# DP 307-02). PCR amplification was performed using the extracted genome as a template and SacII-pTEF1/SmFPS-Asc1 to obtain a fragment of about 1500bp, indicating that M11 was contained; PCR amplification was performed using SacII-PGK1/AtSQS2-Asc1 to give a fragment of about 2000bp, indicating that M9 was contained; PCR amplification was performed using Pac-pTDH3/ERG1-Asc1 to give a fragment of about 2300bp, which was shown to contain M10. The primer sequences are shown in Table 4.

Construction of T30-DD Strain

1. Preparation of Yeast competence

Fresh yeast YSBYT30 was cultured overnight to make competent (1% inoculum size, 30ul seed liquid was inoculated with 3ml medium) and the procedure was as follows:

(1) and (3) collecting thalli: 1ml of yeast liquid was dispensed into 1.5ml EP tubes. Centrifuge at 12000rpm for 1min, discard supernatant and suck with a gun. The pellet was washed with 1ml of sterile water, centrifuged by blowing, the supernatant discarded, and washed twice.

(2) And (3) thallus treatment: 1ml of the treatment solution (4 ℃ C. Refrigerator preservation) +10ul DTT (-20 ℃ C. Refrigerator preservation) was added, and the metal was heated at 25 ℃ C. For 20min.

(3) After 20min, the supernatant was discarded, sucked out with a gun, 1ml of pre-chilled 1M sob (4 ℃ C. Refrigerator) was added, blown, centrifuged, and the supernatant discarded. The supernatant was removed by washing twice again with 1M Sob and 50ul of Sob was added for suspension.

(4) Mu.l pRS425-SpgDDS plasmid (described in documents: wang Dong, liu Yi, xu Jiaoyang, wang Jinhe, wearing waves, zhang Xueli, huang Luqi. Create a plant for efficient production of the ginsenoside precursor dammarenediol II [ J ]. Pharmacology report, 2018, 53 (08): 1233-1241), described in documents as pRS 425-DDS) was blown and mixed and transferred into a pre-cooled electric rotating cup, and ice-bathed for 5min.

(5) The electric rotating cup is wiped clean, and electric shock is conducted at 2.7 kv. 1ml of the Sob which is sucked in first is added and put into an electric rotating cup, and after uniform mixing, a new 1.5ml EP tube is sucked in. Shaking culture at 30deg.C and 250rpm for 60min.

(6) After 60min, the bacterial liquid is centrifuged to remove part of the supernatant, and the supernatant is evenly coated on a plate of an auxotroph SD-TrpLeu (0.8% total synthetic tetra-deficient culture medium+2% glucose+0.005% His+0.01% Ura+2% Ager) and cultured in an incubator at 30 ℃ for 36h.

Culturing in an incubator for about two days, picking monoclonal to perform PCR colony verification to obtain yeast engineering strain T30-DD, and performing the next genetic transformation.

The PCR colony verification method specifically comprises extracting the genome of the yeast strain T30-DD by using a yeast genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical technology Co., ltd., product number: DP 307-02). The extracted genome is used as a template, and the PCR verification is carried out on the strain by using a primer SacII-pTEF1/spgDDS-Asc1-R, so as to obtain a fragment about 2800bp, which indicates that pRS425-SpgDDS plasmid is successfully transferred. The primer sequences are shown in Table 4.

Construction of PTA Strain and LPTA

1. Construction of Gene modules

The plasmids described by the plasmid information of Table 2 (publicly available from Tianjin Industrial Biotechnology research) were used for PCR amplification of the PCR templates and the corresponding primers of Table 3, respectively, to obtain the functional modules: m12 (comprising P) _TEF1 -PPDS01-GSTSSG-46tATR1-T _CYC1 Expression cassette) and M13 (comprising P _TEF1 -Pln1-GGGS-PPDS01-GSTSSG-46tATR1-T _CYC1 An expression cassette).

The amplification system is as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg ²⁺ plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 1. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, add ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 95 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 60℃for 15 seconds, elongation at 68℃for 4 minutes (35 cycles); extension at 72℃for 10 min (1 cycle). The product is recovered and stored by glue.

2. Preparation of Yeast competence

Fresh yeast liquid T30-DD is cultured overnight to prepare competence (1% inoculum size, 30ul seed liquid is connected with 3ml culture medium), and the operation steps are as follows:

(1) and (3) collecting thalli: 1ml of yeast liquid was dispensed into 1.5ml EP tubes. Centrifuge at 12000rpm for 1min, discard supernatant and suck with a gun. The pellet was washed with 1ml of sterile water, centrifuged by blowing, the supernatant discarded, and washed twice.

(2) And (3) thallus treatment: 1ml of the treatment solution (4 ℃ C. Refrigerator preservation) +10ul DTT (-20 ℃ C. Refrigerator preservation) was added, and the metal was heated at 25 ℃ C. For 20min.

(3) After 20min, the supernatant was discarded, sucked out with a gun, 1ml of pre-chilled 1M sob (4 ℃ C. Refrigerator) was added, blown, centrifuged, and the supernatant discarded. The supernatant was removed by washing twice again with 1M Sob and 50ul of Sob was added for suspension.

(4) Two groups were separated, one group was charged with 2. Mu. l M12 module and 2. Mu.l YPL062W gRNA plasmid, and the other group was charged with 2. Mu. l M13 module and 2. Mu.l YPL062W gRNA plasmid, and the mixture was blown and mixed, and transferred to a pre-chilled electric rotating cup, and ice-cooled for 5min.

(5) The electric rotating cup is wiped clean, and electric shock is conducted at 2.7 kv. 1ml of the Sob which is sucked in first is added and put into an electric rotating cup, and after uniform mixing, a new 1.5ml EP tube is sucked in. Shaking culture at 30deg.C and 250rpm for 60min.

(6) After 60min, the bacterial liquid was centrifuged to remove part of the supernatant, and the supernatant was spread on a plate of an auxotroph SD-UraTrpLeu (0.8% total synthetic tetra-deficient medium+2% glucose+0.005% His+2% Ager) and incubated in an incubator at 30℃for 36h. Culturing in an incubator for about two days, and selecting a monoclonal to perform PCR colony verification to obtain yeast engineering bacteria PTA and LPTA.

Verification of PTA strain:

the genome of yeast strain PTA was extracted using a yeast genomic DNA extraction kit. The extracted genome is used as a template, and the primer yp1062w-up-256/yp1062w-down-249 is used for carrying out PCR verification on the strain to obtain a fragment of about 4700bp, which indicates successful transfer into the M12 fragment. The primer sequences are specifically shown in Table 4.

Verification of LPTA strain:

the genome of the yeast strain LPTA was extracted using a yeast genomic DNA extraction kit. PCR verification is carried out by using the extracted genome as a template and using a primer yp1062w-up-256/PPDS01-EGPP-R to obtain about 3000bpThe right fragment was verified by PCR with primer ATR1-Ce1805-F/yp1062w-down-249 to give a fragment of about 1000bp, indicating successful transfer to M13 (P _TEF1 -Pln1-GGGS-PPDS01-GSTSSG-46tATR1-T _CYC1 ) Fragments. The primer sequences are specifically shown in Table 4.

For information on the strains YSBYT5, YSBYT30, T30-DD, PTA, LPTA prepared as described above, see Table 5.

TABLE 5 engineering strain information

Example 2 application of microbial lipid droplet technique in the Production of Protopanaxadiol (PPD)

1. Shaking flask fermentation

(1) Engineering bacteria PTA and LPTA culture

Yeast engineering strains PTA and LPTA were activated in the corresponding solid selection medium SD-UratrpLeu, each genotype engineering strain was transferred to one single clone, seed solutions (30 ℃ C., 250rpm,16 h) were prepared in the corresponding liquid selection medium SD-UratrpLeu, inoculated in 1% inoculum size into 100ml triangular flasks containing 15ml of the corresponding liquid selection medium, and each single clone was transferred to three groups in parallel and cultured for 6 days with shaking at 30 ℃ C., 250 rpm.

(2) Engineering bacterium PTA and LPTA product extraction

After 6 days of fermentation in a shaking flask, 2ml of the bacterial liquid is sucked up, the bacterial liquid is centrifuged at 12000rpm for 1min, the supernatant is discarded, and the bacterial liquid is sucked up by a gun. The precipitate was washed twice with ddH2O and transferred to a disruption tube, centrifuged at 12000rpm for 1min, and the supernatant was discarded; adding glass beads (diameter of 0.5 mm) and lml extract (the extract consists of methanol and acetone with volume ratio of 1:1) into the precipitate, vibrating and crushing for 5min,2 times, and ultrasonically crushing for 30min; centrifuge at 12000rpm for 2min, discard the precipitate, pass the supernatant through 0.22 μm organic filter membrane into liquid phase bottle to obtain solution, named PTA solution and LPTA solution respectively.

2. Qualitative and quantitative HPLC analysis

(1) HPLC qualitative analysis

The standard is protopanoxadiol PPD and dactylenediol DD, which are all purchased from Shanghai Yuan Yes Biotechnology Co. The samples were PTA solution and LPTA solution.

Instrument: agilent high performance liquid chromatography 1260

HPLC detection conditions: DAD monitor, monitoring wavelength 203nm, watersChromatographic column (250 mm. Times.4.6 mm,5 μm), mobile phase A10% methanol, mobile phase B acetonitrile, isocratic 20min,10% A+90% B

(2) Quantitative analysis by HPLC

The yield of each engineering bacterium after fermentation for 6 days is as follows:

on the basis of T30-DD, a YPL062W locus is knocked out from a yeast genome, a PPD module without yeast fat drip compartmentalization positioning and a PPD module with yeast fat drip compartmentalization positioning are respectively integrated, and the HPLC detection result shows that the PPD yield of the PTA engineering bacteria is 5.39mg/L/OD, the DD yield is 13.88mg/L/OD, the DD-to-PPD conversion rate is 27.97%, the PPD yield of the corresponding LPTA engineering bacteria is 19.30mg/L/OD, the DD yield is 3.03mg/L/OD, and the DD-to-PPD conversion rate is 86.43%.

The results are shown in FIG. 1 and FIG. 2, FIG. 1 shows the DD and PPD contents of the strains PTA and LPTA by HPLC, and FIG. 2 shows the HPLC patterns of the strains PTA and LPTA.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of Tianjin Industrial biotechnology, national academy of sciences

<120> construction method of recombinant microorganism, related biological material and application thereof

<130> 210689

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5015

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa atgtctgaat catctatttc ttcttctaaa ccatctgtgg aattgccaca 480

agcaacctgg tcgcatctgc aaagataccc agctttatcc aagtttatta aatatgcgga 540

atctctgcca cctgtggaga gattgatttc cttcaacctc gttgttttgg gatctgtgaa 600

ccagtgggtt tccgaatcgt ccagctctcc tcgtctggtg aagcaagttg ttgctgctgg 660

gaaggaaggg gccttcaagt tggacgagtt agttaacctc ttggtgttca aggagggtgt 720

cgacggcttg ctgtacaatt ggaaatcaca ttccaacacg ccagggatct ggctggtgtg 780

gttcttcgtc gactacgtcg ccaacatttc taatactctg ttgagggagt tcctgatcaa 840

gccattgcac ttgcaaggtt ctaccgcatc gaaggagatc ggctcttccg gtgaggagaa 900

caaggtcact gatgcttctt ctttgcccca cgtggcagag ttgtcttcaa cgaccagagg 960

tatgtcgcag gagatccagt ccaaggtcaa gtcgaactat atcgacccaa ccaaggacct 1020

ggctaaagaa aagtacgacg ccatagtgaa gcccacaact gacaagttgc agtctgtgta 1080

catcgaccca acaaagacta agcttaacga aacctaccaa cgcttcacca ctgtctatga 1140

aaacaatcta agtaaatctg aaagcgtccc taaagccatt gtatccaccg ggttggactt 1200

gggcaatgcc accattgaga agctaaaggc ctcaagagaa gaccaaacca attctaagcc 1260

cgcggctgtg tcgaccaatg gtggtggttc tatggcagcc gctatggttt tgttcttttc 1320

attgtcctta ttgttgttac ctttgttatt gttgtttgct tatttctctt acactaaaag 1380

aataccacaa aaagaaaatg attccaaggc tcctttacct ccaggtcaaa ccggttggcc 1440

attgatcggt gaaactttga actatttgtc atgtgttaag tccggtgtca gtgaaaactt 1500

cgtaaagtac agaaaggaaa agtactctcc aaaggttttc agaacttcat tgttaggtga 1560

accaatggcc attttatgcg gtcctgaagg taataagttc ttgtactcta cagaaaagaa 1620

attggtacaa gtttggtttc catcttcagt tgaaaagatg ttccctagat ctcatggtga 1680

atcaaacgca gataacttct ctaaagttag aggtaaaatg atgttcttgt taaaggtcga 1740

tggtatgaaa aagtatgtag gtttgatgga cagagttatg aagcaattct tggaaacaga 1800

ttggaacaga caacaacaaa ttaatgtaca caacaccgtt aaaaagtaca ccgtcactat 1860

gtcctgtaga gtattcatga gtatagatga cgaagaacaa gttaccagat tgggttccag 1920

tattcaaaac atagaagctg gtttgttagc agtcccaatc aatattcctg gtacagccat 1980

gaacagagct atcaaaacag taaagttgtt aaccagagaa gtcgaagccg taattaaaca 2040

aagaaaggtt gacttgttgg aaaataagca agcatctcaa ccacaagatt tgttgagtca 2100

tttgttgttg actgctaacc aagatggtca atttttatct gaatcagaca tcgcatcaca 2160

cttaattggt ttgatgcaag gtggttacac tacattgaac ggtacaatca ccttcgtctt 2220

gaactatttg gcagaattcc ctgacgtcta caatcaagta ttgaaggaac aagttgaaat 2280

cgccaactct aagcatccaa aggaattgtt gaactgggaa gatttgagaa agatgaagta 2340

ctcatggaac gttgctcaag aagtcttgag aattatacct ccaggtgttg gtacttttag 2400

agaagcaatt accgatttca cttatgccgg ttacttaatt cctaaaggtt ggaagatgca 2460

cttgatacca catgacactc acaagaatcc tacatacttc ccatctcctg aaaagttcga 2520

tcctactaga ttcgagggta acggtccagc tccttatact tttacaccat tcggtggtgg 2580

tccaagaatg tgccctggta tcgaatacgc aagattagtt atattgatct ttatgcataa 2640

tgttgtcaca aacttcagat gggaaaaatt gatcccaaac gaaaagatct tgactgaccc 2700

tatcccaaga ttcgcccacg gtttacctat ccacttacac ccacacaacg gttctacttc 2760

ttcaggttgg aagaaaacga cggcggatcg gagcggggag ctgaagcctt tgatgatccc 2820

taagtctctt atggctaagg acgaggatga tgatttggat ttgggatccg ggaagactag 2880

agtctctatc ttcttcggta cgcagactgg aacagctgag ggatttgcta aggcattatc 2940

cgaagaaatc aaagcgagat atgaaaaagc agcagtcaaa gtcattgact tggatgacta 3000

tgctgccgat gatgaccagt atgaagagaa attgaagaag gaaactttgg catttttctg 3060

tgttgctact tatggagatg gagagcctac tgacaatgct gccagatttt acaaatggtt 3120

tacggaggaa aatgaacggg atataaagct tcaacaacta gcatatggtg tgtttgctct 3180

tggtaatcgc caatatgaac attttaataa gatcgggata gttcttgatg aagagttatg 3240

taagaaaggt gcaaagcgtc ttattgaagt cggtctagga gatgatgatc agagcattga 3300

ggatgatttt aatgcctgga aagaatcact atggtctgag ctagacaagc tcctcaaaga 3360

cgaggatgat aaaagtgtgg caactcctta tacagctgtt attcctgaat accgggtggt 3420

gactcatgat cctcggttta caactcaaaa atcaatggaa tcaaatgtgg ccaatggaaa 3480

tactactatt gacattcatc atccctgcag agttgatgtt gctgtgcaga aggagcttca 3540

cacacatgaa tctgatcggt cttgcattca tctcgagttc gacatatcca ggacgggtat 3600

tacatatgaa acaggtgacc atgtaggtgt atatgctgaa aatcatgttg aaatagttga 3660

agaagctgga aaattgcttg gccactcttt agatttagta ttttccatac atgctgacaa 3720

ggaagatggc tccccattgg aaagcgcagt gccgcctcct ttccctggtc catgcacact 3780

tgggactggt ttggcaagat acgcagacct tttgaaccct cctcgaaagt ctgcgttagt 3840

tgccttggcg gcctatgcca ctgaaccaag tgaagccgag aaacttaagc acctgacatc 3900

acctgatgga aaggatgagt actcacaatg gattgttgca agtcagagaa gtcttttaga 3960

ggtgatggct gcttttccat ctgcaaaacc cccactaggt gtattttttg ctgcaatagc 4020

tcctcgtcta caacctcgtt actactccat ctcatcctcg ccaagattgg cgccaagtag 4080

agttcatgtt acatccgcac tagtatatgg tccaactcct actggtagaa tccacaaggg 4140

tgtgtgttct acgtggatga agaatgcagt tcctgcggag aaaagtcatg aatgtagtgg 4200

agccccaatc tttattcgag catctaattt caagttacca tccaaccctt caactccaat 4260

cgttatggtg ggacctggga ctgggctggc accttttaga ggttttctgc aggaaaggat 4320

ggcactaaaa gaagatggag aagaactagg ttcatctttg ctcttctttg ggtgtagaaa 4380

tcgacagatg gactttatat acgaggatga gctcaataat tttgttgatc aaggcgtaat 4440

atctgagctc atcatggcat tctcccgtga aggagctcag aaggagtatg ttcaacataa 4500

gatgatggag aaggcagcac aagtttggga tctaataaag gaagaaggat atctctatgt 4560

atgcggtgat gctaagggca tggcgaggga cgtccaccga actctacaca ccattgttca 4620

ggagcaggaa ggtgtgagtt cgtcagaggc agaggctata gttaagaaac ttcaaaccga 4680

aggaagatac ctcagagatg tctggtgacc gctgatccta gagggccgca tcatgtaatt 4740

agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg 4800

agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag 4860

aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa 4920

cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcaa 4980

gctgcggccc tgcattaatg aatcggccaa cgcgc 5181

<210> 2

<211> 1425

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Ser Glu Ser Ser Ile Ser Ser Ser Lys Pro Ser Val Glu Leu Pro

1 5 10 15

Gln Ala Thr Trp Ser His Leu Gln Arg Tyr Pro Ala Leu Ser Lys Phe

20 25 30

Ile Lys Tyr Ala Glu Ser Leu Pro Pro Val Glu Arg Leu Ile Ser Phe

35 40 45

Asn Leu Val Val Leu Gly Ser Val Asn Gln Trp Val Ser Glu Ser Ser

50 55 60

Ser Ser Pro Arg Leu Val Lys Gln Val Val Ala Ala Gly Lys Glu Gly

65 70 75 80

Ala Phe Lys Leu Asp Glu Leu Val Asn Leu Leu Val Phe Lys Glu Gly

85 90 95

Val Asp Gly Leu Leu Tyr Asn Trp Lys Ser His Ser Asn Thr Pro Gly

100 105 110

Ile Trp Leu Val Trp Phe Phe Val Asp Tyr Val Ala Asn Ile Ser Asn

115 120 125

Thr Leu Leu Arg Glu Phe Leu Ile Lys Pro Leu His Leu Gln Gly Ser

130 135 140

Thr Ala Ser Lys Glu Ile Gly Ser Ser Gly Glu Glu Asn Lys Val Thr

145 150 155 160

Asp Ala Ser Ser Leu Pro His Val Ala Glu Leu Ser Ser Thr Thr Arg

165 170 175

Gly Met Ser Gln Glu Ile Gln Ser Lys Val Lys Ser Asn Tyr Ile Asp

180 185 190

Pro Thr Lys Asp Leu Ala Lys Glu Lys Tyr Asp Ala Ile Val Lys Pro

195 200 205

Thr Thr Asp Lys Leu Gln Ser Val Tyr Ile Asp Pro Thr Lys Thr Lys

210 215 220

Leu Asn Glu Thr Tyr Gln Arg Phe Thr Thr Val Tyr Glu Asn Asn Leu

225 230 235 240

Ser Lys Ser Glu Ser Val Pro Lys Ala Ile Val Ser Thr Gly Leu Asp

245 250 255

Leu Gly Asn Ala Thr Ile Glu Lys Leu Lys Ala Ser Arg Glu Asp Gln

260 265 270

Thr Asn Ser Lys Pro Ala Ala Val Ser Thr Asn Gly Gly Gly Ser Met

275 280 285

Ala Ala Ala Met Val Leu Phe Phe Ser Leu Ser Leu Leu Leu Leu Pro

290 295 300

Leu Leu Leu Leu Phe Ala Tyr Phe Ser Tyr Thr Lys Arg Ile Pro Gln

305 310 315 320

Lys Glu Asn Asp Ser Lys Ala Pro Leu Pro Pro Gly Gln Thr Gly Trp

325 330 335

Pro Leu Ile Gly Glu Thr Leu Asn Tyr Leu Ser Cys Val Lys Ser Gly

340 345 350

Val Ser Glu Asn Phe Val Lys Tyr Arg Lys Glu Lys Tyr Ser Pro Lys

355 360 365

Val Phe Arg Thr Ser Leu Leu Gly Glu Pro Met Ala Ile Leu Cys Gly

370 375 380

Pro Glu Gly Asn Lys Phe Leu Tyr Ser Thr Glu Lys Lys Leu Val Gln

385 390 395 400

Val Trp Phe Pro Ser Ser Val Glu Lys Met Phe Pro Arg Ser His Gly

405 410 415

Glu Ser Asn Ala Asp Asn Phe Ser Lys Val Arg Gly Lys Met Met Phe

420 425 430

Leu Leu Lys Val Asp Gly Met Lys Lys Tyr Val Gly Leu Met Asp Arg

435 440 445

Val Met Lys Gln Phe Leu Glu Thr Asp Trp Asn Arg Gln Gln Gln Ile

450 455 460

Asn Val His Asn Thr Val Lys Lys Tyr Thr Val Thr Met Ser Cys Arg

465 470 475 480

Val Phe Met Ser Ile Asp Asp Glu Glu Gln Val Thr Arg Leu Gly Ser

485 490 495

Ser Ile Gln Asn Ile Glu Ala Gly Leu Leu Ala Val Pro Ile Asn Ile

500 505 510

Pro Gly Thr Ala Met Asn Arg Ala Ile Lys Thr Val Lys Leu Leu Thr

515 520 525

Arg Glu Val Glu Ala Val Ile Lys Gln Arg Lys Val Asp Leu Leu Glu

530 535 540

Asn Lys Gln Ala Ser Gln Pro Gln Asp Leu Leu Ser His Leu Leu Leu

545 550 555 560

Thr Ala Asn Gln Asp Gly Gln Phe Leu Ser Glu Ser Asp Ile Ala Ser

565 570 575

His Leu Ile Gly Leu Met Gln Gly Gly Tyr Thr Thr Leu Asn Gly Thr

580 585 590

Ile Thr Phe Val Leu Asn Tyr Leu Ala Glu Phe Pro Asp Val Tyr Asn

595 600 605

Gln Val Leu Lys Glu Gln Val Glu Ile Ala Asn Ser Lys His Pro Lys

610 615 620

Glu Leu Leu Asn Trp Glu Asp Leu Arg Lys Met Lys Tyr Ser Trp Asn

625 630 635 640

Val Ala Gln Glu Val Leu Arg Ile Ile Pro Pro Gly Val Gly Thr Phe

645 650 655

Arg Glu Ala Ile Thr Asp Phe Thr Tyr Ala Gly Tyr Leu Ile Pro Lys

660 665 670

Gly Trp Lys Met His Leu Ile Pro His Asp Thr His Lys Asn Pro Thr

675 680 685

Tyr Phe Pro Ser Pro Glu Lys Phe Asp Pro Thr Arg Phe Glu Gly Asn

690 695 700

Gly Pro Ala Pro Tyr Thr Phe Thr Pro Phe Gly Gly Gly Pro Arg Met

705 710 715 720

Cys Pro Gly Ile Glu Tyr Ala Arg Leu Val Ile Leu Ile Phe Met His

725 730 735

Asn Val Val Thr Asn Phe Arg Trp Glu Lys Leu Ile Pro Asn Glu Lys

740 745 750

Ile Leu Thr Asp Pro Ile Pro Arg Phe Ala His Gly Leu Pro Ile His

755 760 765

Leu His Pro His Asn Gly Ser Thr Ser Ser Gly Trp Lys Lys Thr Thr

770 775 780

Ala Asp Arg Ser Gly Glu Leu Lys Pro Leu Met Ile Pro Lys Ser Leu

785 790 795 800

Met Ala Lys Asp Glu Asp Asp Asp Leu Asp Leu Gly Ser Gly Lys Thr

805 810 815

Arg Val Ser Ile Phe Phe Gly Thr Gln Thr Gly Thr Ala Glu Gly Phe

820 825 830

Ala Lys Ala Leu Ser Glu Glu Ile Lys Ala Arg Tyr Glu Lys Ala Ala

835 840 845

Val Lys Val Ile Asp Leu Asp Asp Tyr Ala Ala Asp Asp Asp Gln Tyr

850 855 860

Glu Glu Lys Leu Lys Lys Glu Thr Leu Ala Phe Phe Cys Val Ala Thr

865 870 875 880

Tyr Gly Asp Gly Glu Pro Thr Asp Asn Ala Ala Arg Phe Tyr Lys Trp

885 890 895

Phe Thr Glu Glu Asn Glu Arg Asp Ile Lys Leu Gln Gln Leu Ala Tyr

900 905 910

Gly Val Phe Ala Leu Gly Asn Arg Gln Tyr Glu His Phe Asn Lys Ile

915 920 925

Gly Ile Val Leu Asp Glu Glu Leu Cys Lys Lys Gly Ala Lys Arg Leu

930 935 940

Ile Glu Val Gly Leu Gly Asp Asp Asp Gln Ser Ile Glu Asp Asp Phe

945 950 955 960

Asn Ala Trp Lys Glu Ser Leu Trp Ser Glu Leu Asp Lys Leu Leu Lys

965 970 975

Asp Glu Asp Asp Lys Ser Val Ala Thr Pro Tyr Thr Ala Val Ile Pro

980 985 990

Glu Tyr Arg Val Val Thr His Asp Pro Arg Phe Thr Thr Gln Lys Ser

995 1000 1005

Met Glu Ser Asn Val Ala Asn Gly Asn Thr Thr Ile Asp Ile His His

1010 1015 1020

Pro Cys Arg Val Asp Val Ala Val Gln Lys Glu Leu His Thr His Glu

1025 1030 1035 1040

Ser Asp Arg Ser Cys Ile His Leu Glu Phe Asp Ile Ser Arg Thr Gly

1045 1050 1055

Ile Thr Tyr Glu Thr Gly Asp His Val Gly Val Tyr Ala Glu Asn His

1060 1065 1070

Val Glu Ile Val Glu Glu Ala Gly Lys Leu Leu Gly His Ser Leu Asp

1075 1080 1085

Leu Val Phe Ser Ile His Ala Asp Lys Glu Asp Gly Ser Pro Leu Glu

1090 1095 1100

Ser Ala Val Pro Pro Pro Phe Pro Gly Pro Cys Thr Leu Gly Thr Gly

1105 1110 1115 1120

Leu Ala Arg Tyr Ala Asp Leu Leu Asn Pro Pro Arg Lys Ser Ala Leu

1125 1130 1135

Val Ala Leu Ala Ala Tyr Ala Thr Glu Pro Ser Glu Ala Glu Lys Leu

1140 1145 1150

Lys His Leu Thr Ser Pro Asp Gly Lys Asp Glu Tyr Ser Gln Trp Ile

1155 1160 1165

Val Ala Ser Gln Arg Ser Leu Leu Glu Val Met Ala Ala Phe Pro Ser

1170 1175 1180

Ala Lys Pro Pro Leu Gly Val Phe Phe Ala Ala Ile Ala Pro Arg Leu

1185 1190 1195 1200

Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Pro Arg Leu Ala Pro Ser

1205 1210 1215

Arg Val His Val Thr Ser Ala Leu Val Tyr Gly Pro Thr Pro Thr Gly

1220 1225 1230

Arg Ile His Lys Gly Val Cys Ser Thr Trp Met Lys Asn Ala Val Pro

1235 1240 1245

Ala Glu Lys Ser His Glu Cys Ser Gly Ala Pro Ile Phe Ile Arg Ala

1250 1255 1260

Ser Asn Phe Lys Leu Pro Ser Asn Pro Ser Thr Pro Ile Val Met Val

1265 1270 1275 1280

Gly Pro Gly Thr Gly Leu Ala Pro Phe Arg Gly Phe Leu Gln Glu Arg

1285 1290 1295

Met Ala Leu Lys Glu Asp Gly Glu Glu Leu Gly Ser Ser Leu Leu Phe

1300 1305 1310

Phe Gly Cys Arg Asn Arg Gln Met Asp Phe Ile Tyr Glu Asp Glu Leu

1315 1320 1325

Asn Asn Phe Val Asp Gln Gly Val Ile Ser Glu Leu Ile Met Ala Phe

1330 1335 1340

Ser Arg Glu Gly Ala Gln Lys Glu Tyr Val Gln His Lys Met Met Glu

1345 1350 1355 1360

Lys Ala Ala Gln Val Trp Asp Leu Ile Lys Glu Glu Gly Tyr Leu Tyr

1365 1370 1375

Val Cys Gly Asp Ala Lys Gly Met Ala Arg Asp Val His Arg Thr Leu

1380 1385 1390

His Thr Ile Val Gln Glu Gln Glu Gly Val Ser Ser Ser Glu Ala Glu

1395 1400 1405

Ala Ile Val Lys Lys Leu Gln Thr Glu Gly Arg Tyr Leu Arg Asp Val

1410 1415 1420

Trp

1425

<210> 3

<211> 2498

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

acgcacagat attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg 60

aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120

tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180

cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240

gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300

gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360

caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420

gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480

tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540

tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600

atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660

agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720

ttatctactt tttacaacaa atataaaaca aaaacaatgg ctgcagacca attggtgaaa 780

actgaagtca ccaagaagtc ttttactgct cctgtacaaa aggcttctac accagtttta 840

accaataaaa cagtcatttc tggatcgaaa gtcaaaagtt tatcatctgc gcaatcgagc 900

tcatcaggac cttcatcatc tagtgaggaa gatgattccc gcgatattga aagcttggat 960

aagaaaatac gtcctttaga agaattagaa gcattattaa gtagtggaaa tacaaaacaa 1020

ttgaagaaca aagaggtcgc tgccttggtt attcacggta agttaccttt gtacgctttg 1080

gagaaaaaat taggtgatac tacgagagcg gttgcggtac gtaggaaggc tctttcaatt 1140

ttggcagaag ctcctgtatt agcatctgat cgtttaccat ataaaaatta tgactacgac 1200

cgcgtatttg gcgcttgttg tgaaaatgtt ataggttaca tgcctttgcc cgttggtgtt 1260

ataggcccct tggttatcga tggtacatct tatcatatac caatggcaac tacagagggt 1320

tgtttggtag cttctgccat gcgtggctgt aaggcaatca atgctggcgg tggtgcaaca 1380

actgttttaa ctaaggatgg tatgacaaga ggcccagtag tccgtttccc aactttgaaa 1440

agatctggtg cctgtaagat atggttagac tcagaagagg gacaaaacgc aattaaaaaa 1500

gcttttaact ctacatcaag atttgcacgt ctgcaacata ttcaaacttg tctagcagga 1560

gatttactct tcatgagatt tagaacaact actggtgacg caatgggtat gaatatgatt 1620

tctaaaggtg tcgaatactc attaaagcaa atggtagaag agtatggctg ggaagatatg 1680

gaggttgtct ccgtttctgg taactactgt accgacaaaa aaccagctgc catcaactgg 1740

atcgaaggtc gtggtaagag tgtcgtcgca gaagctacta ttcctggtga tgttgtcaga 1800

aaagtgttaa aaagtgatgt ttccgcattg gttgagttga acattgctaa gaatttggtt 1860

ggatctgcaa tggctgggtc tgttggtgga tttaacgcac atgcagctaa tttagtgaca 1920

gctgttttct tggcattagg acaagatcct gcacaaaatg ttgaaagttc caactgtata 1980

acattgatga aagaagtgga cggtgatttg agaatttccg tatccatgcc atccatcgaa 2040

gtaggtacca tcggtggtgg tactgttcta gaaccacaag gtgccatgtt ggacttatta 2100

ggtgtaagag gcccgcatgc taccgctcct ggtaccaacg cacgtcaatt agcaagaata 2160

gttgcctgtg ccgtcttggc aggtgaatta tccttatgtg ctgccctagc agccggccat 2220

ttggttcaaa gtcatatgac ccacaacagg aaacctgctg aaccaacaaa acctaacaat 2280

ttggacgcca ctgatataaa tcgtttgaaa gatgggtccg tcacctgcat taaatcctaa 2340

agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt taaaacgaaa 2400

attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg tatagcatga 2460

ggtcgctctt attgaccaca cctctaccgg catgccga 2580

<210> 4

<211> 2532

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

catgcgactg ggtgagcata tgttccgctg atgtgatgtg caagataaac aagcaaggca 60

gaaactaact tcttcttcat gtaataaaca caccccgcgt ttatttacct atctctaaac 120

ttcaacacct tatatcataa ctaatatttc ttgagataag cacactgcac ccataccttc 180

cttaaaaacg tagcttccag tttttggtgg ttccggcttc cttcccgatt ccgcccgcta 240

aacgcatatt tttgttgcct ggtggcattt gcaaaatgca taacctatgc atttaaaaga 300

ttatgtatgc tcttctgact tttcgtgtga tgaggctcgt ggaaaaaatg aataatttat 360

gaatttgaga acaattttgt gttgttacgg tattttacta tggaataatc aatcaattga 420

ggattttatg caaatatcgt ttgaatattt ttccgaccct ttgagtactt ttcttcataa 480

ttgcataata ttgtccgctg cccctttttc tgttagacgg tgtcttgatc tacttgctat 540

cgttcaacac caccttattt tctaactatt ttttttttag ctcatttgaa tcagcttatg 600

gtgatggcac atttttgcat aaacctagct gtcctcgttg aacataggaa aaaaaaatat 660

ataaacaagg ctctttcact ctccttgcaa tcagatttgg gtttgttccc tttattttca 720

tatttcttgt catattcctt tctcaattat tattttctac tcataacctc acgcaaaata 780

acacagtcaa atcaatcaaa atgtcattac cgttcttaac ttctgcaccg ggaaaggtta 840

ttatttttgg tgaacactct gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg 900

cgttgagaac ctacctgcta ataagcgagt catctgcacc agatactatt gaattggact 960

tcccggacat tagctttaat cataagtggt ccatcaatga tttcaatgcc atcaccgagg 1020

atcaagtaaa ctcccaaaaa ttggccaagg ctcaacaagc caccgatggc ttgtctcagg 1080

aactcgttag tcttttggat ccgttgttag ctcaactatc cgaatccttc cactaccatg 1140

cagcgttttg tttcctgtat atgtttgttt gcctatgccc ccatgccaag aatattaagt 1200

tttctttaaa gtctacttta cccatcggtg ctgggttggg ctcaagcgcc tctatttctg 1260

tatcactggc cttagctatg gcctacttgg gggggttaat aggatctaat gacttggaaa 1320

agctgtcaga aaacgataag catatagtga atcaatgggc cttcataggt gaaaagtgta 1380

ttcacggtac cccttcagga atagataacg ctgtggccac ttatggtaat gccctgctat 1440

ttgaaaaaga ctcacataat ggaacaataa acacaaacaa ttttaagttc ttagatgatt 1500

tcccagccat tccaatgatc ctaacctata ctagaattcc aaggtctaca aaagatcttg 1560

ttgctcgcgt tcgtgtgttg gtcaccgaga aatttcctga agttatgaag ccaattctag 1620

atgccatggg tgaatgtgcc ctacaaggct tagagatcat gactaagtta agtaaatgta 1680

aaggcaccga tgacgaggct gtagaaacta ataatgaact gtatgaacaa ctattggaat 1740

tgataagaat aaatcatgga ctgcttgtct caatcggtgt ttctcatcct ggattagaac 1800

ttattaaaaa tctgagcgat gatttgagaa ttggctccac aaaacttacc ggtgctggtg 1860

gcggcggttg ctctttgact ttgttacgaa gagacattac tcaagagcaa attgacagct 1920

tcaaaaagaa attgcaagat gattttagtt acgagacatt tgaaacagac ttgggtggga 1980

ctggctgctg tttgttaagc gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag 2040

tattccaatt atttgaaaat aaaactacca caaagcaaca aattgacgat ctattattgc 2100

caggaaacac gaatttacca tggacttcat aagcggatct cttatgtctt tacgatttat 2160

agttttcatt atcaagtatg cctatattag tatatagcat ctttagatga cagtgttcga 2220

agtttcacga ataaaagata atattctact ttttgctccc accgcgtttg ctagcacgag 2280

tgaacaccat ccctcgcctg tgagttgtac ccattcctct aaactgtaga catggtagct 2340

tcagcagtgt tcgttatgta cggcatcctc caacaaacag tcggttatag tttgtcctgc 2400

tcctctgaat cgtctccctc gatatttctc attttccttc gcatgccagc attgaaatga 2460

tcgaagttca atgatgaaac ggtaattctt ctgtcattta ctcatctcat ctcatcaagt 2520

tatataattc ta 2616

<210> 5

<211> 2267

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aatcctactc ttgccgttgc catccaaaat gagctagaag gtggattaac aaatataatg 60

acaaatcgtt gcttgtctga cttgattcca ctacagttac aaatatttga cattgtatat 120

aagttttgca agttcatcaa atctatgaga gcaaaattat gtcaactgga ccccgtacta 180

tatgagaaac acaaaagcgg gatgatgaaa acactaaacg aaggctatcg tacaaacaat 240

ggcggtcagg aagatgttgg ttaccaagaa gatgccgccc tggaattaat tcagaagctg 300

attgaataca ttagcaacgc gtccagcatt tttcggaagt gtctcataaa ctttactcaa 360

gagttaagta ctgaaaaatt cgacttttat gatagttcaa gtgtcgacgc tgcgggtata 420

gaaagggttc tttactctat agtacctcct cgctcagcat ctgcttcttc ccaaagatga 480

acgcggcgtt atgtcactaa cgacgtgcac caacttgcgg aaagtggaat cccgttccaa 540

aactggcatc cactaattga tacatctaca caccgcacgc cttttttctg aagcccactt 600

tcgtggactt tgccatatgc aaaattcatg aagtgtgata ccaagtcagc atacacctca 660

ctagggtagt ttctttggtt gtattgatca tttggttcat cgtggttcat taattttttt 720

tctccattgc tttctggctt tgatcttact atcatttgga tttttgtcga aggttgtaga 780

attgtatgtg acaagtggca ccaagcatat ataaaaaaaa aaagcattat cttcctacca 840

gagttgattg ttaaaaacgt atttatagca aacgcaattg taattaattc ttattttgta 900

tcttttcttc ccttgtctca atcttttatt tttattttat ttttcttttc ttagtttctt 960

tcataacacc aagcaactaa tactataaca tacaataata atgactgccg acaacaatag 1020

tatgccccat ggtgcagtat ctagttacgc caaattagtg caaaaccaaa cacctgaaga 1080

cattttggaa gagtttcctg aaattattcc attacaacaa agacctaata cccgatctag 1140

tgagacgtca aatgacgaaa gcggagaaac atgtttttct ggtcatgatg aggagcaaat 1200

taagttaatg aatgaaaatt gtattgtttt ggattgggac gataatgcta ttggtgccgg 1260

taccaagaaa gtttgtcatt taatggaaaa tattgaaaag ggtttactac atcgtgcatt 1320

ctccgtcttt attttcaatg aacaaggtga attactttta caacaaagag ccactgaaaa 1380

aataactttc cctgatcttt ggactaacac atgctgctct catccactat gtattgatga 1440

cgaattaggt ttgaagggta agctagacga taagattaag ggcgctatta ctgcggcggt 1500

gagaaaacta gatcatgaat taggtattcc agaagatgaa actaagacaa ggggtaagtt 1560

tcacttttta aacagaatcc attacatggc accaagcaat gaaccatggg gtgaacatga 1620

aattgattac atcctatttt ataagatcaa cgctaaagaa aacttgactg tcaacccaaa 1680

cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat gatttgaaaa ctatgtttgc 1740

tgacccaagt tacaagttta cgccttggtt taagattatt tgcgagaatt acttattcaa 1800

ctggtgggag caattagatg acctttctga agtggaaaat gacaggcaaa ttcatagaat 1860

gctataagcg atttaatctc taattattag ttaaagtttt ataagcattt ttatgtaacg 1920

aaaaataaat tggttcatat tattactgca ctgtcactta ccatggaaag accagacaag 1980

aagttgccga cagtctgttg aattggcctg gttaggctta agtctgggtc cgcttcttta 2040

caaatttgga gaatttctct taaacgatat gtatattctt ttcgttggaa aagatgtctt 2100

ccaaaaaaaa aaccgatgaa ttagtggaac caaggaaaaa aaaagaggta tccttgatta 2160

aggaacactg tttaaacagt gtggtttcca aaaccctgaa actgcattag tgtaatagaa 2220

gactagacac ctcgatacaa ataatggtta ctcaattcaa aactgcc 2341

<210> 6

<211> 2591

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

aatgctacta ttttggagat taatctcagt acaaaacaat attaaaaaga ggtgaattat 60

ttttcccccc ttattttttt tttgttaaaa ttgatccaaa tgtaaataaa caatcacaag 120

gaaaaaaaaa aaaaaaaaaa aaatagccgc catgaccccg gatcgtcggt tgtgatacgg 180

tcagggtagc gccctggtca aacttcagaa ctaaaaaaat aataaggaag aaaaaaatag 240

ctaatttttc cggcagaaag attttcgcta cccgaaagtt tttccggcaa gctaaatgga 300

aaaaggaaag attattgaaa gagaaagaaa gaaaaaaaaa aaatgtacac ccagacatcg 360

ggcttccaca atttcggctc tattgttttc catctctcgc aacggcggga ttcctctatg 420

gcgtgtgatg tctgtatctg ttacttaatc cagaaactgg cacttgaccc aactctgcca 480

cgtgggtcgt tttgccatcg acagattggg agattttcat agtagaattc agcatgatag 540

ctacgtaaat gtgttccgca ccgtcacaaa gtgttttcta ctgttctttc ttctttcgtt 600

cattcagttg agttgagtga gtgctttgtt caatggatct tagctaaaat gcatattttt 660

tctcttggta aatgaatgct tgtgatgtct tccaagtgat ttcctttcct tcccatatga 720

tgctaggtac ctttagtgtc ttcctaaaaa aaaaaaaagg ctcgccatca aaacgatatt 780

cgttggcttt tttttctgaa ttataaatac tctttggtaa cttttcattt ccaagaacct 840

cttttttcca gttatatcat ggtccccttt caaagttatt ctctactctt tttcatattc 900

attctttttc atcctttggt tttttattct taacttgttt attattctct cttgtttcta 960

tttacaagac accaatcaaa acaaataaaa catcatcaca atgaccgttt acacagcatc 1020

cgttaccgca cccgtcaaca tcgcaaccct taagtattgg gggaaaaggg acacgaagtt 1080

gaatctgccc accaattcgt ccatatcagt gactttatcg caagatgacc tcagaacgtt 1140

gacctctgcg gctactgcac ctgagtttga acgcgacact ttgtggttaa atggagaacc 1200

acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc gacctacgcc aattaagaaa 1260

ggaaatggaa tcgaaggacg cctcattgcc cacattatct caatggaaac tccacattgt 1320

ctccgaaaat aactttccta cagcagctgg tttagcttcc tccgctgctg gctttgctgc 1380

attggtctct gcaattgcta agttatacca attaccacag tcaacttcag aaatatctag 1440

aatagcaaga aaggggtctg gttcagcttg tagatcgttg tttggcggat acgtggcctg 1500

ggaaatggga aaagctgaag atggtcatga ttccatggca gtacaaatcg cagacagctc 1560

tgactggcct cagatgaaag cttgtgtcct agttgtcagc gatattaaaa aggatgtgag 1620

ttccactcag ggtatgcaat tgaccgtggc aacctccgaa ctatttaaag aaagaattga 1680

acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc attgttgaaa aagatttcgc 1740

cacctttgca aaggaaacaa tgatggattc caactctttc catgccacat gtttggactc 1800

tttccctcca atattctaca tgaatgacac ttccaagcgt atcatcagtt ggtgccacac 1860

cattaatcag ttttacggag aaacaatcgt tgcatacacg tttgatgcag gtccaaatgc 1920

tgtgttgtac tacttagctg aaaatgagtc gaaactcttt gcatttatct ataaattgtt 1980

tggctctgtt cctggatggg acaagaaatt tactactgag cagcttgagg ctttcaacca 2040

tcaatttgaa tcatctaact ttactgcacg tgaattggat cttgagttgc aaaaggatgt 2100

tgccagagtg attttaactc aagtcggttc aggcccacaa gaaacaaacg aatctttgat 2160

tgacgcaaag actggtctac caaaggaata aacaaatcgc tcttaaatat atacctaaag 2220

aacattaaag ctatattata agcaaagata cgtaaatttt gcttatatta ttatacacat 2280

atcatatttc tatattttta agatttggtt atataatgta cgtaatgcaa aggaaataaa 2340

ttttatacat tattgaacag cgtccaagta actacattat gtgcactaat agtttagcgt 2400

cgtgaagact ttattgtgtc gcgaaaagta aaaattttaa aaattagagc accttgaact 2460

tgcgaaaaag gttctcatca actgtttaaa aggaggatat caggtcctat ttctgacaaa 2520

caatatacaa atttagtttc aaagatgaat cagtgcgcga aggacataac tcatgaagcc 2580

tccagtatac c 2677

<210> 7

<211> 2264

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

atggggccgt atacttacat atagtagatg tcaagcgtag gcgcttcccc tgccggctgt 60

gagggcgcca taaccaaggt atctatagac cgccaatcag caaactacct ccgtacattc 120

atgttgcacc cacacattta tacacccaga ccgcgacaaa ttacccataa ggttgtttgt 180

gacggcgtcg tacaagagaa cgtgggaact ttttaggctc accaaaaaag aaagaaaaaa 240

tacgagttgc tgacagaagc ctcaagaaaa aaaaaattct tcttcgacta tgctggaggc 300

agagatgatc gagccggtag ttaactatat atagctaaat tggttccatc accttctttt 360

ctggtgtcgc tccttctagt gctatttctg gcttttccta tttttttttt tccatttttc 420

tttctctctt tctaatatat aaattctctt gcattttcta tttttctctc tatctattct 480

acttgtttat tcccttcaag gttttttttt aaggagtact tgtttttaga atatacggtc 540

aacgaactat aattaactaa acatgactgg taaaacaggt catatcgatg gtttgaattc 600

tagaatcgaa aagatgagag atttggaccc agcacaaaga ttagttagag ttgctgaagc 660

tgcaggtttg gaaccagaag ctatttctgc attagctggt aatggtgcat tgccattatc 720

attggctaac ggtatgatcg aaaacgttat cggtaaattc gaattgccat tgggtgttgc 780

tactaacttc acagttaacg gtagagatta tttgatccca atggctgttg aagaaccatc 840

tgttgttgct gcagcttcat acatggcaag aattgctaga gaaaatggtg gttttactgc 900

acatggtaca gctccattga tgagagctca aattcaagtt gttggtttag gtgacccaga 960

aggtgcaaga caaagattgt tagctcataa agcagctttt atggaagcag ctgatgctgt 1020

tgatccagtt ttagttggtt tgggtggtgg ttgtagagat atcgaagttc atgtttttag 1080

agatactcca gttggtgcta tggttgtttt gcatttgatc gttgatgtta gagatgcaat 1140

gggtgctaac actgttaaca caatggcaga aagattggct ccagaagttg aaagaattgc 1200

aggtggtact gttagattga gaattttgtc taatttggct gatttgagat tggttagagc 1260

aagagttgaa ttggctccag aaacattaac tacacaaggt tatgatggtg cagatgttgc 1320

tcgtggtatg gttgaagcat gtgctttagc aattgttgat ccatacagag cagctactca 1380

taataagggt atcatgaacg gtatcgatcc agttgttgtt gcaactggta atgattggag 1440

agctattgaa gctggtgcac atgcttatgc agctagaact ggtcattaca cttcattgac 1500

aagatgggaa ttagctaatg atggtagatt ggttggtact attgaattac cattggcatt 1560

aggtttggtt ggtggtgcta ctaaaacaca tccaacagca agagcagctt tagctttgat 1620

gcaagttgaa actgcaacag aattggctca agttacagca gctgttggtt tagctcaaaa 1680

tatggcagct attagagcat tggctactga aggtattcaa agaggtcata tgacattgca 1740

tgcaagaaac atcgctatta tggcaggtgc tactggtgca gatatcgata gagttacaag 1800

agttattgtt gaagctggtg acgtttcagt tgcaagagct aagcaagttt tggaaaacac 1860

ataaagtgct tttaactaag aattattagt cttttctgct tattttttca tcatagttta 1920

gaacacttta tattaacgaa tagtttatga atctatttag gtttaaaaat tgatacagtt 1980

ttataagtta ctttttcaaa gactcgtgct gtctattgca taatgcactg gaaggggaaa 2040

aaaaaggtgc acacgcgtgg ctttttcttg aatttgcagt ttgaaaaata actacatgga 2100

tgataagaaa acatggagta cagtcacttt gagaaccttc aatcagctgg taacgtcttc 2160

gttaattgga tactcaaaaa agatggatag catgaatcac aagatggaag gaaatgcggg 2220

ccacgaccac agtgatatgc atatgggaga tggagatgat acct 2338

<210> 8

<211> 2699

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gatccaactg gcaccgctgg cttgaacaac aataccagcc ttccaacttc tgtaaataac 60

ggcggtacgc cagtgccacc agtaccgtta cctttcggta tacctccttt ccccatgttt 120

ccaatgccct tcatgcctcc aacggctact atcacaaatc ctcatcaagc tgacgcaagc 180

cctaagaaat gaataacaat actgacagta ctaaataatt gcctacttgg cttcacatac 240

gttgcatacg tcgatataga taataatgat aatgacagca ggattatcgt aatacgtaat 300

agttgaaaat ctcaaaaatg tgtgggtcat tacgtaaata atgataggaa tgggattctt 360

ctatttttcc tttttccatt ctagcagccg tcgggaaaac gtggcatcct ctctttcggg 420

ctcaattgga gtcacgctgc cgtgagcatc ctctctttcc atatctaaca actgagcacg 480

taaccaatgg aaaagcatga gcttagcgtt gctccaaaaa agtattggat ggttaatacc 540

atttgtctgt tctcttctga ctttgactcc tcaaaaaaaa aaaatctaca atcaacagat 600

cgcttcaatt acgccctcac aaaaactttt ttccttcttc ttcgcccacg ttaaatttta 660

tccctcatgt tgtctaacgg atttctgcac ttgatttatt ataaaaagac aaagacataa 720

tacttctcta tcaatttcag ttattgttct tccttgcgtt attcttctgt tcttcttttt 780

cttttgtcat atataaccat aaccaagtaa tacatattca aaatgaaact ctcaactaaa 840

ctttgttggt gtggtattaa aggaagactt aggccgcaaa agcaacaaca attacacaat 900

acaaacttgc aaatgactga actaaaaaaa caaaagaccg ctgaacaaaa aaccagacct 960

caaaatgtcg gtattaaagg tatccaaatt tacatcccaa ctcaatgtgt caaccaatct 1020

gagctagaga aatttgatgg cgtttctcaa ggtaaataca caattggtct gggccaaacc 1080

aacatgtctt ttgtcaatga cagagaagat atctactcga tgtccctaac tgttttgtct 1140

aagttgatca agagttacaa catcgacacc aacaaaattg gtagattaga agtcggtact 1200

gaaactctga ttgacaagtc caagtctgtc aagtctgtct tgatgcaatt gtttggtgaa 1260

aacactgacg tcgaaggtat tgacacgctt aatgcctgtt acggtggtac caacgcgttg 1320

ttcaactctt tgaactggat tgaatctaac gcatgggatg gtagagacgc cattgtagtt 1380

tgcggtgata ttgccatcta cgataagggt gccgcaagac caaccggtgg tgccggtact 1440

gttgctatgt ggatcggtcc tgatgctcca attgtatttg actctgtaag agcttcttac 1500

atggaacacg cctacgattt ttacaagcca gatttcacca gcgaatatcc ttacgtcgat 1560

ggtcattttt cattaacttg ttacgtcaag gctcttgatc aagtttacaa gagttattcc 1620

aagaaggcta tttctaaagg gttggttagc gatcccgctg gttcggatgc tttgaacgtt 1680

ttgaaatatt tcgactacaa cgttttccat gttccaacct gtaaattggt cacaaaatca 1740

tacggtagat tactatataa cgatttcaga gccaatcctc aattgttccc agaagttgac 1800

gccgaattag ctactcgcga ttatgacgaa tctttaaccg ataagaacat tgaaaaaact 1860

tttgttaatg ttgctaagcc attccacaaa gagagagttg cccaatcttt gattgttcca 1920

acaaacacag gtaacatgta caccgcatct gtttatgccg cctttgcatc tctattaaac 1980

tatgttggat ctgacgactt acaaggcaag cgtgttggtt tattttctta cggttccggt 2040

ttagctgcat ctctatattc ttgcaaaatt gttggtgacg tccaacatat tatcaaggaa 2100

ttagatatta ctaacaaatt agccaagaga atcaccgaaa ctccaaagga ttacgaagct 2160

gccatcgaat tgagagaaaa tgcccatttg aagaagaact tcaaacctca aggttccatt 2220

gagcatttgc aaagtggtgt ttactacttg accaacatcg atgacaaatt tagaagatct 2280

tacgatgtta aaaaataaat ttaactcctt aagttacttt aatgatttag tttttattat 2340

taataattca tgctcatgac atctcatata cacgtttata aaacttaaat agattgaaaa 2400

tgtattaaag attcctcagg gattcgattt ttttggaagt ttttgttttt ttttccttga 2460

gatgctgtag tatttgggaa caattataca atcgaaagat atatgcttac attcgaccgt 2520

tttagccgtg atcattatcc tatagtaaca taacctgaag cataactgac actactatca 2580

tcaatacttg tcacatgaga actctgtgaa taattaggcc actgaaattt gatgcctgaa 2640

ggaccggcat cacggatttt cgataaagca cttagtatca cactaattgg cttttcgcc 2787

<210> 9

<211> 2558

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

atactagcgt tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca 60

aaaagattcc ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt 120

cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 180

agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 240

acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 300

acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 360

aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 420

ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 480

ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 540

atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 600

agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 660

atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 720

tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 780

aacacacata aacaaacaaa atgtcagagt tgagagcctt cagtgcccca gggaaagcgt 840

tactagctgg tggatattta gttttagata caaaatatga agcatttgta gtcggattat 900

cggcaagaat gcatgctgta gcccatcctt acggttcatt gcaagggtct gataagtttg 960

aagtgcgtgt gaaaagtaaa caatttaaag atggggagtg gctgtaccat ataagtccta 1020

aaagtggctt cattcctgtt tcgataggcg gatctaagaa ccctttcatt gaaaaagtta 1080

tcgctaacgt atttagctac tttaaaccta acatggacga ctactgcaat agaaacttgt 1140

tcgttattga tattttctct gatgatgcct accattctca ggaggatagc gttaccgaac 1200

atcgtggcaa cagaagattg agttttcatt cgcacagaat tgaagaagtt cccaaaacag 1260

ggctgggctc ctcggcaggt ttagtcacag ttttaactac agctttggcc tccttttttg 1320

tatcggacct ggaaaataat gtagacaaat atagagaagt tattcataat ttagcacaag 1380

ttgctcattg tcaagctcag ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat 1440

atggatctat cagatataga agattcccac ccgcattaat ctctaatttg ccagatattg 1500

gaagtgctac ttacggcagt aaactggcgc atttggttga tgaagaagac tggaatatta 1560

cgattaaaag taaccattta ccttcgggat taactttatg gatgggcgat attaagaatg 1620

gttcagaaac agtaaaactg gtccagaagg taaaaaattg gtatgattcg catatgccag 1680

aaagcttgaa aatatataca gaactcgatc atgcaaattc tagatttatg gatggactat 1740

ctaaactaga tcgcttacac gagactcatg acgattacag cgatcagata tttgagtctc 1800

ttgagaggaa tgactgtacc tgtcaaaagt atcctgaaat cacagaagtt agagatgcag 1860

ttgccacaat tagacgttcc tttagaaaaa taactaaaga atctggtgcc gatatcgaac 1920

ctcccgtaca aactagctta ttggatgatt gccagacctt aaaaggagtt cttacttgct 1980

taatacctgg tgctggtggt tatgacgcca ttgcagtgat tactaagcaa gatgttgatc 2040

ttagggctca aaccgctaat gacaaaagat tttctaaggt tcaatggctg gatgtaactc 2100

aggctgactg gggtgttagg aaagaaaaag atccggaaac ttatcttgat aaataagatt 2160

aatataatta tataaaaata ttatcttctt ttctttatat ctagtgttat gtaaaataaa 2220

ttgatgacta cggaaagctt ttttatattg tttctttttc attctgagcc acttaaattt 2280

cgtgaatgtt cttgtaaggg acggtagatt tacaagtgat acaacaaaaa gcaaggcgct 2340

ttttctaata aaaagaagaa aagcatttaa caattgaaca cctctatatc aacgaagaat 2400

attactttgt ctctaaatcc ttgtaaaatg tgtacgatct ctatatgggt tactcataag 2460

tgtaccgaag actgcattga aagtttatgt tttttcactg gaggcgtcat tttcgcgttg 2520

agaagatgtt cttatccaaa tttcaactgt tatataga 2642

<210> 10

<211> 1934

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc 480

attccagggt tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg 540

cgccttggct aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg 600

taacgttctt tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg 660

tttgagtaat catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc 720

aatcattttg ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg 780

ttgtgaatct atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt 840

tggccaaact gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg 900

tctagccatg ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca 960

acaagacaat tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa 1020

attcgacaat gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca 1080

agtcacgaag gacgaggaac ctgctagatt acacgttgaa aaattgagat ctgcaaggac 1140

tgttttccaa aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg 1200

tgctgcagcc gtcatcttgg tttccgaaaa agttttgaag gaaaagaatt tgaagccttt 1260

ggctattatc aaaggttggg gtgaggccgc tcatcaacca gctgatttta catgggctcc 1320

atctcttgca gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga 1380

ttactttgaa ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa 1440

gctagaccca tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg 1500

ttgttctggt gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa 1560

gatcggtgtt gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa 1620

gatatgaccg ctgatcctag agggccgcat catgtaatta gttatgtcac gcttacattc 1680

acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta 1740

ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat 1800

ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct 1860

tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctgcggccct gcattaatga 1920

atcggccaac gcgc 1998

<210> 11

<211> 2141

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

acgcacagat attataacat ctgcacaata ggcatttgca agaattactc gtgagtaagg 60

aaagagtgag gaactatcgc atacctgcat ttaaagatgc cgatttgggc gcgaatcctt 120

tattttggct tcaccctcat actattatca gggccagaaa aaggaagtgt ttccctcctt 180

cttgaattga tgttaccctc ataaagcacg tggcctctta tcgagaaaga aattaccgtc 240

gctcgtgatt tgtttgcaaa aagaacaaaa ctgaaaaaac ccagacacgc tcgacttcct 300

gtcttcctat tgattgcagc ttccaatttc gtcacacaac aaggtcctag cgacggctca 360

caggttttgt aacaagcaat cgaaggttct ggaatggcgg gaaagggttt agtaccacat 420

gctatgatgc ccactgtgat ctccagagca aagttcgttc gatcgtactg ttactctctc 480

tctttcaaac agaattgtcc gaatcgtgtg acaacaacag cctgttctca cacactcttt 540

tcttctaacc aagggggtgg tttagtttag tagaacctcg tgaaacttac atttacatat 600

atataaactt gcataaattg gtcaatgcaa gaaatacata tttggtcttt tctaattcgt 660

agtttttcaa gttcttagat gctttctttt tctctttttt acagatcatc aaggaagtaa 720

ttatctactt tttacaacaa atataaaaca atggggagct tggggacgat gctgagatat 780

ccggatgaca tatatccgct cctgaagatg aaacgagcga ttgagaaagc ggagaagcag 840

atccctcctg agccacactg gggtttctgc tattcgatgc tccacaaggt ttctcgaagc 900

ttttctctcg ttattcagca actcaacacc gagctccgta acgccgtgtg tgtgttctac 960

ttggttctcc gagctcttga tactgttgag gatgatacta gcataccaac tgatgaaaag 1020

gttcccatcc tgatagcttt tcaccggcac atatacgata ctgattggca ttattcatgt 1080

ggtacgaagg agtacaagat tctaatggac caatttcacc atgtttctgc agcttttttg 1140

gaacttgaaa aagggtatca agaggctatc gaggaaatta ctagaagaat gggtgcaggg 1200

atggccaagt ttatctgcca agaggtagaa actgttgatg actacgatga atactgccac 1260

tatgttgctg ggcttgttgg tttaggtttg tcgaaactct tcctcgctgc aggatcagag 1320

gttttgacac cagattggga ggcgatttcc aattcaatgg gtttatttct gcagaaaaca 1380

aacattatca gagattatct tgaggacatt aatgagatac caaaatcccg catgttttgg 1440

cctcgcgaga tttggggcaa atatgctgac aagcttgagg atttaaaata cgaggagaac 1500

acaaacaaat ccgtacagtg cttaaatgaa atggttacca atgcgttgat gcatattgaa 1560

gattgcctga aatacatggt ttccttgcgt gatccttcca tatttcggtt ctgtgccatc 1620

cctcagatca tggcgattgg aacacttgca ttatgctata acaatgaaca agtattcaga 1680

ggcgttgtga aactgaggcg aggtcttact gctaaagtca ttgatcgtac aaagacaatg 1740

gctgatgtct atggtgcttt ctatgatttt tcctgcatgc tgaagacaaa ggttgacaag 1800

aacgatccaa atgccagtaa gacactaaac cgacttgaag ccgttcagaa actctgcaga 1860

gacgctggag ttcttcaaaa cagaaaatct tatgttaatg acaaaggaca accaaacagt 1920

gtctttatta taatggttgt gattctactg gccatagtct ttgcatatct cagagcaaac 1980

tgaagttata aaaaaaataa gtgtatacaa attttaaagt gactcttagg ttttaaaacg 2040

aaaattctta ttcttgagta actctttcct gtaggtcagg ttgctttctc aggtatagca 2100

tgaggtcgct cttattgacc acacctctac cggcatgccg a 2211

<210> 12

<211> 2693

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

atactagcgt tgaatgttag cgtcaacaac aagaagttta atgacgcgga ggccaaggca 60

aaaagattcc ttgattacgt aagggagtta gaatcatttt gaataaaaaa cacgcttttt 120

cagttcgagt ttatcattat caatactgcc atttcaaaga atacgtaaat aattaatagt 180

agtgattttc ctaactttat ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt 240

acatgcccaa aatagggggc gggttacaca gaatatataa catcgtaggt gtctgggtga 300

acagtttatt cctggcatcc actaaatata atggagcccg ctttttaagc tggcatccag 360

aaaaaaaaag aatcccagca ccaaaatatt gttttcttca ccaaccatca gttcataggt 420

ccattctctt agcgcaacta cagagaacag gggcacaaac aggcaaaaaa cgggcacaac 480

ctcaatggag tgatgcaacc tgcctggagt aaatgatgac acaaggcaat tgacccacgc 540

atgtatctat ctcattttct tacaccttct attaccttct gctctctctg atttggaaaa 600

agctgaaaaa aaaggttgaa accagttccc tgaaattatt cccctacttg actaataagt 660

atataaagac ggtaggtatt gattgtaatt ctgtaaatct atttcttaaa cttcttaaat 720

tctactttta tagttagtct tttttttagt tttaaaacac caagaactta gtttcgaata 780

aacacacata aacaaacaaa atgtctgctg ttaacgttgc acctgaattg attaatgccg 840

acaacacaat tacctacgat gcgattgtca tcggtgctgg tgttatcggt ccatgtgttg 900

ctactggtct agcaagaaag ggtaagaaag ttcttatcgt agaacgtgac tgggctatgc 960

ctgatagaat tgttggtgaa ttgatgcaac caggtggtgt tagagcattg agaagtctgg 1020

gtatgattca atctatcaac aacatcgaag catatcctgt taccggttat accgtctttt 1080

tcaacggcga acaagttgat attccatacc cttacaaggc cgatatccct aaagttgaaa 1140

aattgaagga cttggtcaaa gatggtaatg acaaggtctt ggaagacagc actattcaca 1200

tcaaggatta cgaagatgat gaaagagaaa ggggtgttgc ttttgttcat ggtagattct 1260

tgaacaactt gagaaacatt actgctcaag agccaaatgt tactagagtg caaggtaact 1320

gtattgagat attgaaggat gaaaagaatg aggttgttgg tgccaaggtt gacattgatg 1380

gccgtggcaa ggtggaattc aaagcccact tgacatttat ctgtgacggt atcttttcac 1440

gtttcagaaa ggaattgcac ccagaccatg ttccaactgt cggttcttcg tttgtcggta 1500

tgtctttgtt caatgctaag aatcctgctc ctatgcacgg tcacgttatt cttggtagtg 1560

atcatatgcc aatcttggtt taccaaatca gtccagaaga aacaagaatc ctttgtgctt 1620

acaactctcc aaaggtccca gctgatatca agagttggat gattaaggat gtccaacctt 1680

tcattccaaa gagtctacgt ccttcatttg atgaagccgt cagccaaggt aaatttagag 1740

ctatgccaaa ctcctacttg ccagctagac aaaacgacgt cactggtatg tgtgttatcg 1800

gtgacgctct aaatatgaga catccattga ctggtggtgg tatgactgtc ggtttgcatg 1860

atgttgtctt gttgattaag aaaataggtg acctagactt cagcgaccgt gaaaaggttt 1920

tggatgaatt actagactac catttcgaaa gaaagagtta cgattccgtt attaacgttt 1980

tgtcagtggc tttgtattct ttgttcgctg ctgacagcga taacttgaag gcattacaaa 2040

aaggttgttt caaatatttc caaagaggtg gcgattgtgt caacaaaccc gttgaatttc 2100

tgtctggtgt cttgccaaag cctttgcaat tgaccagggt tttcttcgct gtcgcttttt 2160

acaccattta cttgaacatg gaagaacgtg gtttcttggg attaccaatg gctttattgg 2220

aaggtattat gattttgatc acagctatta gagtattcac cccatttttg tttggtgagt 2280

tgattggtta agattaatat aattatataa aaatattatc ttcttttctt tatatctagt 2340

gttatgtaaa ataaattgat gactacggaa agctttttta tattgtttct ttttcattct 2400

gagccactta aatttcgtga atgttcttgt aagggacggt agatttacaa gtgatacaac 2460

aaaaagcaag gcgctttttc taataaaaag aagaaaagca tttaacaatt gaacacctct 2520

atatcaacga agaatattac tttgtctcta aatccttgta aaatgtgtac gatctctata 2580

tgggttactc ataagtgtac cgaagactgc attgaaagtt tatgtttttt cactggaggc 2640

gtcattttcg cgttgagaag atgttcttat ccaaatttca actgttatat aga 2781

<210> 13

<211> 1787

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa atggctaatt tgaatggtga atctgctgat ttgagagcaa catttttggg 480

tgtttactct gttttgaagt cagaattgtt gaatgatcca gcatttgaat ggacagatgg 540

ttcaagacaa tgggttgaaa gaatgttgga ttacaacgtt ccaggtggta aattgaacag 600

aggtttgtct gttattgatt catacaaatt gttgaagggt ggtaaagatt tgactgatga 660

tgaagttttc ttggcttctg cattaggttg gtgtgttgaa tggttacaag catacttttt 720

ggttttggat gatatcatgg ataactcaca tacaagaaga ggtcaaccat gttggtttag 780

agttccaaaa gttggtatga tcgcaattaa tgatggtatc atcttgagaa atcatattcc 840

aagaattttg aagaaacatt ttagaactaa accatactac gttgatttgt tggatttgtt 900

taatgaagtt gaattccaaa cagcttctgg tcaaatgatc gatttgatca ctacaatcga 960

aggtgaaaag gatttgtcta agtactcatt gccattgcat agaagaatcg ttcaatacaa 1020

gactgcttat tactcatttt acttgccagt tgcttgtgca ttgttaatgg caggtgaaga 1080

tttggaaaaa catccaacag ttaaggatgt tttgattaat atgggtatct atttccaagt 1140

tcaagatgat tacttagatt gttttggtga accagaaaag attggtaaaa tcggtactga 1200

tatcgaagat ttcaagtgtt cttggttggt tgttaaagca ttggaattgt gtaacgaaga 1260

acaaaagaaa actttatttg aacattatgg taaagaagat ccagctgatg ttgcaaagat 1320

taaagttttg tacaacgaaa ttaatttgca aggtgttttc gcagaattcg aatctaagtc 1380

atacgaaaaa ttgaattctt caattgaagc tcatccatct aagtcagttc aagcagtttt 1440

gaaatcattt ttgggtaaaa tctataaaag acaaaaatga ccgctgatcc tagagggccg 1500

catcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac 1560

cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat 1620

gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt 1680

gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc 1740

tttaatttgc aagctgcggc cctgcattaa tgaatcggcc aacgcgc 1845

<210> 14

<211> 4154

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa atggcagccg ctatggtttt gttcttttca ttgtccttat tgttgttacc 480

tttgttattg ttgtttgctt atttctctta cactaaaaga ataccacaaa aagaaaatga 540

ttccaaggct cctttacctc caggtcaaac cggttggcca ttgatcggtg aaactttgaa 600

ctatttgtca tgtgttaagt ccggtgtcag tgaaaacttc gtaaagtaca gaaaggaaaa 660

gtactctcca aaggttttca gaacttcatt gttaggtgaa ccaatggcca ttttatgcgg 720

tcctgaaggt aataagttct tgtactctac agaaaagaaa ttggtacaag tttggtttcc 780

atcttcagtt gaaaagatgt tccctagatc tcatggtgaa tcaaacgcag ataacttctc 840

taaagttaga ggtaaaatga tgttcttgtt aaaggtcgat ggtatgaaaa agtatgtagg 900

tttgatggac agagttatga agcaattctt ggaaacagat tggaacagac aacaacaaat 960

taatgtacac aacaccgtta aaaagtacac cgtcactatg tcctgtagag tattcatgag 1020

tatagatgac gaagaacaag ttaccagatt gggttccagt attcaaaaca tagaagctgg 1080

tttgttagca gtcccaatca atattcctgg tacagccatg aacagagcta tcaaaacagt 1140

aaagttgtta accagagaag tcgaagccgt aattaaacaa agaaaggttg acttgttgga 1200

aaataagcaa gcatctcaac cacaagattt gttgagtcat ttgttgttga ctgctaacca 1260

agatggtcaa tttttatctg aatcagacat cgcatcacac ttaattggtt tgatgcaagg 1320

tggttacact acattgaacg gtacaatcac cttcgtcttg aactatttgg cagaattccc 1380

tgacgtctac aatcaagtat tgaaggaaca agttgaaatc gccaactcta agcatccaaa 1440

ggaattgttg aactgggaag atttgagaaa gatgaagtac tcatggaacg ttgctcaaga 1500

agtcttgaga attatacctc caggtgttgg tacttttaga gaagcaatta ccgatttcac 1560

ttatgccggt tacttaattc ctaaaggttg gaagatgcac ttgataccac atgacactca 1620

caagaatcct acatacttcc catctcctga aaagttcgat cctactagat tcgagggtaa 1680

cggtccagct ccttatactt ttacaccatt cggtggtggt ccaagaatgt gccctggtat 1740

cgaatacgca agattagtta tattgatctt tatgcataat gttgtcacaa acttcagatg 1800

ggaaaaattg atcccaaacg aaaagatctt gactgaccct atcccaagat tcgcccacgg 1860

tttacctatc cacttacacc cacacaacgg ttctacttct tcaggttgga agaaaacgac 1920

ggcggatcgg agcggggagc tgaagccttt gatgatccct aagtctctta tggctaagga 1980

cgaggatgat gatttggatt tgggatccgg gaagactaga gtctctatct tcttcggtac 2040

gcagactgga acagctgagg gatttgctaa ggcattatcc gaagaaatca aagcgagata 2100

tgaaaaagca gcagtcaaag tcattgactt ggatgactat gctgccgatg atgaccagta 2160

tgaagagaaa ttgaagaagg aaactttggc atttttctgt gttgctactt atggagatgg 2220

agagcctact gacaatgctg ccagatttta caaatggttt acggaggaaa atgaacggga 2280

tataaagctt caacaactag catatggtgt gtttgctctt ggtaatcgcc aatatgaaca 2340

ttttaataag atcgggatag ttcttgatga agagttatgt aagaaaggtg caaagcgtct 2400

tattgaagtc ggtctaggag atgatgatca gagcattgag gatgatttta atgcctggaa 2460

agaatcacta tggtctgagc tagacaagct cctcaaagac gaggatgata aaagtgtggc 2520

aactccttat acagctgtta ttcctgaata ccgggtggtg actcatgatc ctcggtttac 2580

aactcaaaaa tcaatggaat caaatgtggc caatggaaat actactattg acattcatca 2640

tccctgcaga gttgatgttg ctgtgcagaa ggagcttcac acacatgaat ctgatcggtc 2700

ttgcattcat ctcgagttcg acatatccag gacgggtatt acatatgaaa caggtgacca 2760

tgtaggtgta tatgctgaaa atcatgttga aatagttgaa gaagctggaa aattgcttgg 2820

ccactcttta gatttagtat tttccataca tgctgacaag gaagatggct ccccattgga 2880

aagcgcagtg ccgcctcctt tccctggtcc atgcacactt gggactggtt tggcaagata 2940

cgcagacctt ttgaaccctc ctcgaaagtc tgcgttagtt gccttggcgg cctatgccac 3000

tgaaccaagt gaagccgaga aacttaagca cctgacatca cctgatggaa aggatgagta 3060

ctcacaatgg attgttgcaa gtcagagaag tcttttagag gtgatggctg cttttccatc 3120

tgcaaaaccc ccactaggtg tattttttgc tgcaatagct cctcgtctac aacctcgtta 3180

ctactccatc tcatcctcgc caagattggc gccaagtaga gttcatgtta catccgcact 3240

agtatatggt ccaactccta ctggtagaat ccacaagggt gtgtgttcta cgtggatgaa 3300

gaatgcagtt cctgcggaga aaagtcatga atgtagtgga gccccaatct ttattcgagc 3360

atctaatttc aagttaccat ccaacccttc aactccaatc gttatggtgg gacctgggac 3420

tgggctggca ccttttagag gttttctgca ggaaaggatg gcactaaaag aagatggaga 3480

agaactaggt tcatctttgc tcttctttgg gtgtagaaat cgacagatgg actttatata 3540

cgaggatgag ctcaataatt ttgttgatca aggcgtaata tctgagctca tcatggcatt 3600

ctcccgtgaa ggagctcaga aggagtatgt tcaacataag atgatggaga aggcagcaca 3660

agtttgggat ctaataaagg aagaaggata tctctatgta tgcggtgatg ctaagggcat 3720

ggcgagggac gtccaccgaa ctctacacac cattgttcag gagcaggaag gtgtgagttc 3780

gtcagaggca gaggctatag ttaagaaact tcaaaccgaa ggaagatacc tcagagatgt 3840

ctggtgaccg ctgatcctag agggccgcat catgtaatta gttatgtcac gcttacattc 3900

acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta 3960

ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat 4020

ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct 4080

tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctgcggccct gcattaatga 4140

atcggccaac gcgc 4292

<210> 15

<211> 3047

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

agtgatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat 60

tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat 120

ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa 180

aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg 240

tttctttttc ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga 300

tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta 360

ttacaacttt ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt 420

taattacaaa atgtggaagt taaaggtagc tcaaggtaat gacccttact tatactcaac 480

caacaatttc gtcggtagac aatactggga atttcaacca gatgctggta cacctgaaga 540

aagagaagaa gtcgaaaagg caagaaagga ctacgtaaac aacaaaaagt tacatggtat 600

tcacccatgt tcagatatgt tgatgagaag acaattgata aaagaatcag gtatcgactt 660

gttatccatt ccacctttga gattggatga aaacgaacaa gttaactacg acgccgtcac 720

tacagctgtt aaaaaggctt tgagattaaa tagagcaatt caagcccatg atggtcactg 780

gccagctgaa aacgcaggta gtttgttgta caccccacct ttgataatag ctttgtacat 840

ctctggtact atagatacaa tcttaaccaa gcaacataaa aaggaattga tcagattcgt 900

ctacaaccac caaaacgaag atggtggttg gggtagttac atcgaaggtc attctactat 960

gattggttcc gttttgagtt acgtcatgtt gagattgttg ggtgaaggtt tagccgaatc 1020

agatgacggt aatggtgctg ttgaaagagg tagaaaatgg atcttggatc atggtggtgc 1080

tgcaggtatt ccatcttggg gtaaaacata tttggctgta ttgggtgttt acgaatggga 1140

aggttgtaat ccattaccac ctgaattttg gttgttccct tcttcatttc cattccatcc 1200

tgcaaaaatg tggatctatt gtagatgcac ctacatgcca atgtcatatt tgtacggtaa 1260

aagataccac ggtcctataa ctgatttggt tttatccttg agacaagaaa tctataacat 1320

cccatacgaa caaattaaat ggaaccaaca aagacacaac tgttgcaagg aagatttgta 1380

ttaccctcac actttagtac aagatttggt ttgggacggt ttgcattact tctctgaacc 1440

attcttgaag agatggcctt ttaataagtt gagaaagaga ggtttgaaga gagttgtcga 1500

attaatgaga tacggtgcta cagaaactag attcattacc actggtaatg gtgaaaaagc 1560

attgcaaatc atgtcatggt gggccgaaga tccaaacggt gacgaattca agcatcactt 1620

agccagaatt cctgatttct tgtggatagc tgaagacggt atgacagttc aatcttttgg 1680

ttcacaattg tgggattgta tattggccac tcaagctatc attgcaacaa atatggtcga 1740

agaatatggt gacagtttga agaaagctca tttctttatc aaggaatctc aaatcaagga 1800

aaacccacgt ggtgactttt tgaaaatgtg tagacaattc accaagggtg catggacttt 1860

ttcagatcaa gaccacggtt gtgtagtttc cgattgcacc gcagaagcct tgaagtgctt 1920

gttgttgttg tctcaaatgc cacaagacat tgtaggtgaa aagcctgaag ttgaaagatt 1980

gtacgaagcc gttaacgtct tgttgtactt gcaatccaga gttagtggtg gtttcgctgt 2040

ttgggaacca cctgtcccaa aaccttattt ggaaatgttg aacccatcag aaatctttgc 2100

tgatatagtc gtagaaagag aacatatcga atgtacagct tccgtaatca aaggtttgat 2160

ggcttttaaa tgcttgcatc caggtcacag acaaaaggaa atagaagata gtgttgctaa 2220

ggcaatcaga tatttggaaa gaaaccaaat gcctgacggt tcttggtatg gtttttgggg 2280

tatatgtttc ttatacggta ctttctttac attgagtggt tttgcctctg ctggtagaac 2340

atacgataat tcagaagcag tcagaaaagg tgtaaagttt ttcttatcca cccaaaacga 2400

agaaggtggt tggggtgaat ctttggaatc atgcccatcc gaaaaattca ctcctttgaa 2460

gggtaacaga acaaacttgg ttcaaacctc ttgggcaatg ttaggtttga tgtttggtgg 2520

tcaagccgaa agagatccaa ctcctttgca tagagccgct aaattgttga ttaatgcaca 2580

aatggataac ggtgacttcc cacaacaaga aatcacaggt gtttactgta agaactctat 2640

gttgcactac gccgaataca gaaacatttt tcctttgtgg gccttgggtg aatacagaaa 2700

aagagtttgg ttacctaagc atcaacaatt aaagatatga ccgctgatcc tagagggccg 2760

catcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac 2820

cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat 2880

gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt 2940

gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc 3000

tttaatttgc aagctgcggc cctgcattaa tgaatcggcc aacgcgc 3047

Claims (7)

1. A method of constructing a recombinant microorganism, characterized by: comprises the steps of introducing a coding gene of a recombinant fusion protein into a starting microorganism to obtain a recombinant microorganism;

the starting microorganism is Saccharomyces cerevisiae, which is a strain obtained by modifying the strain BYT1 by A1-A12,

a1, introducing a 3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1 gene;

a2, introducing a mevalonate kinase gene ERG 12;

a3, introducing an alcohol dehydrogenase I gene IDI1 gene;

a4, introducing a mevalonate pyrophosphate decarboxylase gene ERG 19;

a5, introducing a HMGR gene of a hydroxymethylglutaryl-CoA reductase gene;

a6, introducing a hydroxymethyl glutaryl-coenzyme A synthetase gene ERG 13;

a7, introducing a phosphomevalonate kinase gene ERG 8;

a8, introducing an acetyl coenzyme A acetyltransferase gene ERG 10;

a9, introducing a squalene synthase gene AtSQS 2;

a10, introducing a squalene monooxygenase gene ERG 1;

a11, introducing a farnesyl pyrophosphate synthase gene SmFPS gene;

a12, introducing a dammarenediol synthase gene spgDDS gene;

the terpene synthesis related proteins are protopanoxadiol synthase PPDS and cytochrome P450 reductase ATR1;

the ATR1 is cytochrome P450 reductase 46tATR1 with N-terminal truncated by 46 amino acids;

the recombinant fusion protein is a recombinant protein containing the Pln protein, PPDS and 46tATR1 or a recombinant protein containing the PPDS and 46tATR1.

2. The method according to claim 1, characterized in that: in the recombinant fusion protein, the Pln1 protein and the terpene synthesis related protein are connected through a connecting peptide.

3. The construction method according to claim 1 or 2, characterized in that:

the amino acid sequence of the Pln protein is shown in the 1 st position to 283 rd position in SEQ ID No. 2;

the amino acid sequence of the PPDS is shown as 288 th to 773 th positions in SEQ ID No. 2;

the amino acid sequence of the ATR1 is shown in 780 th-1425 th positions in SEQ ID No. 2.

4. The method according to claim 1 or 2, characterized in that: the amino acid sequence of the recombinant fusion protein is shown in SEQ ID No. 2.

5. The method according to claim 1 or 2, characterized in that: in the recombinant microorganism, the coding gene of the recombinant fusion protein is integrated into the YJL062W site of the starting microorganism.

6. The method according to claim 1 or 2, characterized in that: the coding gene of the recombinant fusion protein is introduced into the starting microorganism through an expression cassette for expressing the recombinant fusion protein, so as to obtain the recombinant microorganism.

7. The application is any one of the following applications,

use of X1, the method of any one of claims 1-6 for the preparation of a terpene product;

use of X2, the method of any one of claims 1-6 for the production of terpenes;

the terpene is protopanoxadiol.