CN115247183A - Construction method of recombinant microorganism, related biological material and application thereof - Google Patents
Construction method of recombinant microorganism, related biological material and application thereof Download PDFInfo
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Abstract
The invention discloses a construction method of recombinant saccharomyces cerevisiae, a related biological material and application thereof. The construction method comprises the steps of introducing the coding gene of the recombinant fusion protein into a starting microorganism to obtain a recombinant microorganism; the recombinant fusion protein contains a Pln1 protein and a terpene synthesis-related protein. The construction method repositions a part of key enzymes in a triterpene biosynthesis pathway through the Pln1 protein so as to promote the enzymes to be spatially close to a substrate and promote the conversion of natural product fat-soluble intermediates stored in a lipid drop.
Description
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
Natural compounds and their derivatives have important applications in medical treatment and health care, and the pharmacological actions of more and more natural compound molecules are characterized, and the market demand is increasing. The traditional plant extraction method or chemical synthesis method has many limitations. Plant extraction causes a great waste of plant resources and the obtained active ingredients are extremely low. Chemical synthesis brings environmental pressure on one hand, and on the other hand, when natural compounds with complex structures are processed, the problems of long synthesis route, low yield and the like exist. Synthetic biology is a science of redesigning and engineering construction and application of life systems and processes emerging in recent years, and provides powerful techniques and platform support for large-scale production of natural compounds and design and synthesis of small molecule compounds with new structures. In recent years, reports on the preparation of other active plant natural products by using synthetic biological methods are also continuously reported, the related natural products comprise terpenes, flavonoids, polyphenols, alkaloids and the like, and green and efficient production chains formed by the new technologies are being accepted by science and industry.
Terpenes are one of the most hydrophobic substances with multiple functions and wide application, and can be used as spices and essences, medicines, solvents, cosmetics, food additives and potential advanced biofuels. Yeast is considered to be an ideal host for producing isoprenoid 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 the mitochondria, and β -oxidation of fatty acids is localized in the peroxisomes, which results in the dispersion of cofactors and precursors. This subcellular compartmentalization may present some obstacles to substrate passage in the supply of precursors and cofactors. One of the problems faced in the heterologous synthesis of terpenoid natural products by saccharomyces cerevisiae is that most of its lipid-soluble intermediates are stored in the subcellular space of the yeast cell, inside the lipid droplets.
Despite extensive metabolic engineering, the production of most isoprenoids in yeast has fallen far behind industrial applications, probably due in part to complex metabolic compartmentalization. Thus, systematic studies of cellular sub-compartments and the 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 coding genes of recombinant fusion proteins into starting microorganisms to obtain recombinant microorganisms; the recombinant fusion protein contains a Pln1 protein and a terpene synthesis-related protein.
Optionally, according to the above method, the terpene synthesis-related protein is selected from protopanaxadiol synthase PPDS01 and/or cytochrome P450 reductase ATR1.
The terpene synthesis related protein may include PPDS01 and ATR1, and then PPDS01 and ATR1 may be linked via a linker peptide. ATR1 may be a cytochrome P450 reductase 46tATR1 with a 46 amino acid truncation at the N-terminus, for example the amino acid sequence of 46tATR1 is shown in SEQ ID No.2 from position 780 to position 1425.
Alternatively, according to the above method, in said recombinant fusion protein, said Pln1 protein and said terpene synthesis-related protein are linked via a linker peptide.
Above, the linker peptide may be GGGS or GSTSSG.
Optionally, the recombinant fusion protein is a recombinant protein comprising the Pln1 protein, PPDS01 and ATR1 according to the above method.
Alternatively, the amino acid sequence of the Pln1 protein is as shown in SEQ ID No.2 from position 1 to position 283; the amino acid sequence of the PPDS01 is shown as 288 th to 773 rd positions in SEQ ID No. 2; the amino acid sequence of the ATR1 is shown as 780 th to 1425 th in SEQ ID No. 2.
Alternatively, the amino acid sequence of the recombinant fusion protein is as shown in SEQ ID No.2, according to the method described above. In SEQ ID No.2, the amino acid sequence of the Pln1 protein is shown from position 1 to position 283, the amino acid sequence of the linker peptide GGGS is shown from position 284 to position 287, the amino acid sequence of PPDS01 is shown from position 288 to position 773, the amino acid sequence of the linker peptide GSTSSG is shown from position 774 to position 779, and the amino acid sequence of ATR1 is shown from position 780 to position 1425.
The method may further comprise expressing a gene encoding the above recombinant fusion protein. The specific sequence of the coding gene can be shown as 431 st 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.
Optionally, according to the above method, the encoding 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. The expression cassette sequence for expressing the recombinant fusion protein can be shown as SEQ ID No.1, wherein, the promoter P TEF1 1-430 th site, 431-1279 th site of coding gene of Pln1 protein, 1280-1291 th site of coding gene of connecting peptide GGGS, 1292-2749 th site of coding gene of protein PPDS01, 2750-2767 th site of coding gene of connecting peptide GSTSSG, 2768-4708 th site of coding gene of protein 46tATR1 and terminator T CYC1 From 4709 to 5015.
Optionally, according to the method, the starting microorganism is Saccharomyces cerevisiae, and the Saccharomyces cerevisiae is obtained by performing A1-A12 transformation on a strain BYT1,
a1, introducing a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene tHMG1 gene; a2, introducing a mevalonate kinase gene ERG12 gene; a3, introducing an IDI1 gene of an alcohol dehydrogenase I gene; a4, introducing a mevalonate decarboxylase pyrophosphate gene ERG19 gene; a5, introducing a hydroxymethyl glutaryl coenzyme A reductase gene HMGR gene; a6, introducing hydroxymethyl glutaryl-coenzyme A synthetase gene ERG 13; a7, introducing a phosphomevalonate kinase gene ERG8 gene; a8, introducing acetyl coenzyme A acetyltransferase gene ERG 10; a9, introducing a squalene synthase gene AtSQS2 gene; a10, introducing a squalene monooxygenase gene ERG1 gene; a11, introducing a farnesyl pyrophosphate synthetase gene SmFPS gene; a12, introducing a dammarenediol synthase gene spgDDS gene.
Optionally, the sequence of tmg 1 protein encoded by the tmg 1 gene is genbank accession number: AJS96703.1, bits 530-1054; the sequence of the ERG12 protein coded by the ERG12 gene is genbank accession number: NP _ 013935.1; the sequence of IDI1 protein coded by the IDI1 gene is genbank accession number: NP _ 015208.1; the sequence of the ERG19 protein coded by the ERG19 gene is genbank accession number: NP-014441.1; the sequence of the HMGR protein coded by the HMGR gene is genbank accession number: WP _ 011241944.1; the sequence of the ERG13 protein coded by the ERG13 gene is genbank accession number: NP-013580.1; the sequence of the ERG8 protein coded by the ERG8 gene is genbank accession number: NP-013947.1; the sequence of the ERG10 protein coded by the ERG10 gene is genbank accession number: NP-015297.1; the sequence of the AtSQS2 protein coded by the AtSQS2 gene is genbank accession number: NP-195190.1; the sequence of the ERG1 protein coded by the ERG1 gene is genbank accession number: NP _ 011691.1; the sequence of SmFPS protein coded by the SmFPS gene is genbank accession number: ABV 08819.1; the sequence of the spgDDS protein coded by the spgDDS gene is genbank accession number: ACZ 71036.1.
Alternatively, the sequence of the tHMG1 gene is shown as 757 th-2340 th positions in SEQ ID No. 3; the sequence of the ERG12 gene is shown as 801 rd to 2132 nd in SEQ ID No. 4; the sequence of the IDI1 gene is shown as 1001 st to 1867 th positions in SEQ ID No. 5; the sequence of the ERG19 gene is shown as 1001 st position to 2191 nd position in SEQ ID No. 6; the sequence of the HMGR gene is shown as 563-1864 in SEQ ID No. 7; the sequence of the ERG13 gene is shown as 823 th-2298 th in SEQ ID No. 8; the sequence of the ERG8 gene is shown as 801 st-2156 th in SEQ ID No. 9; the sequence of the ERG10 gene is shown as 431 th site to 1627 th site in SEQ ID No. 10; the sequence of the AtSQS2 gene is shown as 751 th site to 1983 th site in SEQ ID No. 11; the sequence of the ERG1 gene is shown as 801 th-2291 th in SEQ ID No. 12; the sequence of the SmFPS gene is shown as 431 th site to 1480 th site in SEQ ID No. 13; the sequence of the spgDDS gene is shown as 431 st to 2740 th in SEQ ID No. 15.
The gene can be obtained by introducing an expression cassette for expressing the protein or an expression plasmid for expressing the protein into BYT1, for example, the gene can be integrated into LEU site and NDT80 site of BYT 1.
Alternatively, the sequence of the expression cassette for expressing the tHMG1 protein is shown as SEQ ID No. 3; the sequence of an 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 expression cassette sequence for expressing HMGR protein is shown in SEQ ID No. 7; the sequence of the expression cassette for expressing ERG13 protein is shown in SEQ D No. 8; the sequence of the expression cassette for expressing ERG8 protein is shown in SEQ ID No. 9; the sequence of the expression cassette for expressing ERG10 protein is shown in SEQ ID No. 10; the sequence of an 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 in SEQ ID No. 12; the sequence of an 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 in SEQ ID No. 15.
The invention also provides any one of the following biological materials B1) to B6):
b1 Nucleic acid molecules encoding the above recombinant fusion proteins; b2 An expression cassette comprising the nucleic acid molecule according to B1); b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2); b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3); b5 ) the above recombinant fusion protein; b6 A recombinant microorganism expressing the recombinant fusion protein.
The invention also provides any one of the following applications:
x1, the application of the method for constructing the recombinant microorganism in the preparation of terpene products; x2, the application of the method for constructing the recombinant microorganism in the production of terpene; x3, application of the biological material in preparation of terpene products; x4, the use of the above mentioned biological material for the production of terpenes; the application of the X5 and the Pln1 protein in the preparation of terpene products; the use of X6, the above-mentioned Pln1 protein, for the production of terpenes; the application of X7 and the Pln1 protein in improving terpene synthesis efficiency (recombination efficiency); x8 and the application of 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 above recombinant fusion protein in a recipient organism to obtain a recombinant organism having a higher efficiency of terpene synthesis than the recipient organism.
As above, the organism may be a microorganism, a plant or a non-human animal.
As hereinbefore described, the terpene product may be a recombinant bacterium expressing a terpene. The terpene can be protopanaxadiol or cucurbitadienol.
Yeast lipid droplets, a specialized organelle, have the ability to store unwanted hydrophobic materials, not only Fatty Acids (FA) in the form of Triacylglycerol (TAG), but also squalene, sterol esters, and retinyl esters in the lipid droplet to avoid damaging membrane integrity. Pln1 protein (also known in the past as Pet10 p) that 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 relocates some of the key enzymes in the triterpene biosynthetic pathway through the Pln1 protein to facilitate spatial access of the enzymes to the substrate to facilitate conversion of the natural product lipid soluble intermediates stored in the lipid droplets.
Drawings
FIG. 1 shows the content of DD and PPD in PTA and LPTA detected by HPLC in example 2.
FIG. 2 is an HPLC plot of the strains PTA and LPTA of example 2.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Data were processed using SPSS11.5 statistical software and the results were expressed as mean. + -. Standard deviation using One-way ANOVA test.
Saccharomyces cerevisiae BY4742 (Saccharomyces cerevisiae BY 4742) described in Zhubo Dai et al, production agglutinins of ginsenosides in bakers' yeast. Sci Rep.2014Jan 15;4:3698..
The gene fragments and protein sequence related information referred to in the following examples are shown in the following table.
Information on Gene fragments
Information relating to protein sequences
genbank accession number | Update time | |
tHMG1 protein | AJS96703.1 bits 530-1054 | 2016/5/23 |
ERG12 protein | NP_013935.1 | 2020/10/2 |
IDI1 protein | NP_015208.1 | 2020/10/2 |
ERG19 protein | NP_014441.1 | 2020/10/2 |
HMGR proteins | WP_011241944.1 | 2019/6/19 |
ERG13 protein | NP_013580.1 | 2020/10/2 |
ERG8 protein | NP_013947.1 | 2020/10/2 |
ERG10 protein | NP_015297.1 | 2020/10/2 |
AtSQS2 protein | NP_195190.1 | 2019/2/14 |
ERG1 protein | NP_011691.1 | 2020/10/2 |
SmFPS proteins | ABV08819.1 | 2011/4/11 |
spgDDS proteins | ACZ71036.1 | 2009/12/12 |
Example 1
1. Cloning of Gene elements
1. Obtaining gene fragment by PCR amplification
The Pln1 gene of the complete ORF and the ATR1 gene (46 tATR 1) with 46 amino acids truncated at the N-terminal are obtained BY amplification BY using the genomic DNA of Saccharomyces cerevisiae BY4742 as a template and the primers in Table 1 respectively.
Protopanaxadiol synthase gene PPDS01 was amplified using the primers in Table 1, using plasmid pM13-pgPPDS containing the pgPPDS gene (described in Dai ZB et al, available from Tianjin Industrial Biotechnology institute for production of polysaccharides, methanoic engineering.2013, 20.
The amplification system was as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg 2+ plus) x 10. Mu.l, dNTPmix 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, 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, and extension at 68 ℃ for 3 minutes (35 cycles); extension at 72 ℃ for 10 min (1 cycle). The product is recovered and stored by glue.
2. Obtaining fusion fragment by fusion PCR
(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: primeSTARGXLBuffer (Mg) 2+ plus) x 10. Mu.l, dNTPmix 4. Mu.l, primers SexA1-PPDS01-F and Asc1-46tATR1-R each 1.5. Mu.l, DNA templates of fragment 46tATR1 and fragment PPDS01 each 1.5. Mu.l, 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, and extension at 68 ℃ for 3 minutes (35 cycles); extension at 72 ℃ for 10 min (1 cycle). Obtaining the fusion gene fragment PPDS 01-GSTSG-46 tATR1.
A fusion gene fragment 14bp-PPDS 01-GSTSG-46 tATR1 containing a 14bp homologous region with the Pln1 gene is obtained by the same method, and primers are shown in Table 1. The fragments were recovered by gel.
(2) The 14bp-PPDS 01-GSTSG-46 tATR1 gene containing 14bp homologous region with Pln1 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, dNTPmix 4. Mu.l, primers Pac1-Pln1-F and Asc1-46tATR1-R each 1.5. Mu.l, DNA templates of fragment Pln1 and fragment 14bp-PPDS 01-GSTSG-46 tATR1 each 1.5. Mu.l, 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, and extension at 68 ℃ for 3 minutes (35 cycles); extension at 72 ℃ for 10 min (1 cycle). Obtaining a fusion gene segment Pln1-GGGS-PPDS01-GSTSSG-46tATR1.
TABLE 1 primer sequences
2. Construction of recombinant plasmid
1、pM3-PPDS01-GSTSSG-46tATR1
Carrying out double enzyme digestion by using restriction enzymes SexAI and AscI to respectively double-enzyme digest plasmid pM3-ERG9 (recorded in Chinese patent application 201210453416.X) and gene PPDS 01-GSTSG-46 tATR1, tapping and recovering a target fragment: pEASY-Blunt-P TEF1 -//-T CYC1 (62 ng) and PPDS 01-GSTSG-46 tATR1 (3420bp, 130ng), and connecting the target fragment with a vector in the following system: 5. Mu.l of 2 Xquick Ligation Buffer (NEB), 0.5. Mu.l of Quick T4 DNA Ligase (NEB, 400, 000synergistic end units/ml), supplemented with ddH 2 And reacting at 25 ℃ for 13min until the volume of O is 10 mu l to obtain a ligation product, transferring the ligation product into Trans 1T 1 competent cells, and performing sequencing verification to obtain the recombinant vector. Through sequencing, the recombinant vector is an expression cassette P of PPDS 01-GSTSG-46 tATR1 gene TEF1 -PPDS01-GSTSSG-46tATR1-T CYC1 The vector obtained by being inserted between cloning sites of pEASY-BluntSimple cloning vector (pEASY cloning vector, beijing Quantum Biotechnology (TransGen Biotech) Ltd.) was named pM3-PPDS 01-GSTSG-46 tATR1.
2、pM13-Pln1-GGGS-PPDS01-GSTSSG-46tATR1
Plasmid pM13-pgPPDS (described in Dai ZB et al, 2013, metabolic Engineering 20, publicly available from the institute for biotechnology of Tianjin) and gene fragment Pln1-GGGS-PPDS01-GSTSSG-46tATR1, were double-digested with restriction enzymes PacI and AscI, respectively, and the fragment of interest was recovered by tapping: pEASY-Blunt-P TEF1 -//-T CYC1 (50 ng) and pac1-Pln1-GGGS-PPDS01-GSTSSG-46tATR1-Asc1 (4278bp, 104ng), and the target fragment is connected with the vector by the following connection system: 5 ul 2 XQuick Ligation Buffer (NEB), 0.5 ul Quick T4 DNA Ligase (NEB, 400, 000genetic end units/ml), supplemented with ddH 2 And reacting at 25 ℃ for 13min until the volume of O is 10 mu l to obtain a ligation product, transferring the ligation product into Trans 1T 1 competent cells, and performing sequencing verification to obtain the recombinant vector. After sequencing, the recombinant vector is an expression cassette P of Pln1-GGGS-PPDS 01-GSTSG-46 tATR1 gene TEF1 -Pln1-GGGS-PPDS01-GSTSSG-46tATR1-T CYC1 Insert pEASY-Blunt Simple cloning vector (pEASY clone)Vector, a vector obtained between cloning sites of Beijing Quanyu Biotechnology (TransGen Biotech) Ltd.), and named pM13-Pln1-GGGS-PPDS 01-GSTSG-46 tATR1.
3. Leu gRNA, NDT80gRNA and YPL062W gRNA
The p426-SNR52p-gRNA. CAN1.Y-SUP4t plasmid purchased from addge company is taken as a template, and the following primers are respectively used for amplification: gRNA antisense/Leu gRNA forward, gRNA antisense/NDT 80gRNA forward, gRNA antisense/YPL 062W gRNA forward
gRNA reverse: GATCATTTATCTTTCACTGC
LeugRNA forward:
cgcagtgaaagataaatgatcCGATGGTGATGGTGTCGCTTgttttagagctagaaatagcaag
NDT80gRNA forward:
cgcagtgaaagataaatgatcCTGCTTCAGGTGCGGCTTGGgttttagagctagaaatagcaag
YPL062W gRNA forward:
cgcagtgaaagataaatgatcGCACGTCGCCGTGGCTGATGgttttagagctagaaatagcaag
amplifying to obtain three linear segments of Linear Leu gRNA, linear NDT80gRNA and Linear YPL062W gRNA, transferring the three segments into Transl T1 competent cells respectively, and performing 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, a promoter pTEF2 (562 bp) is obtained BY adopting primers Pac1-TEF2-F and SexA1-TEF2-R for amplification, and a terminator tENO2 (400 bp) is obtained BY adopting primers Asc1-ENO2-F and Pme1-ENO2-R for amplification. The amplification system was as follows: primeSTAR GXL Buffer (Mg) 2+ plus). Times.10. Mu.l, dNTPmix. Times.4. Mu.l, 1.5. Mu.l each of the primers Pac1-TEF2-F and SexA1-TEF2-R (Asc 1-ENO2-F and Pme1-ENO 2-R), 1.5. Mu.l of genomic DNA template, 1. Mu.l of PrimeSTAR GXL DNA Polymerase (1.25U/. Mu.l), and 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’
Carrying out double digestion on plasmid pUC57-synHMGR (the synHMGR gene is totally synthesized by totally-synthesized consignjinsry biotechnology limited of the gene and is inserted into the cloning sites of a pUC57 vector (provided by the kingsry biotechnology limited) by using restriction enzymes SexAI and AscI respectively to obtain a cloning type plasmid pUC57-synHMGR containing the synHMGR gene, and carrying out tapping to recover a target fragment to obtain a SexAI-synHMGR-AscI fragment; respectively double-digesting the fragment pTEF2 by using restriction enzymes SexAI and pacI, and tapping and recovering the target fragment to obtain SexAI-pTEF2-pacI; respectively double-digesting the fragment tENO2 by using restriction endonucleases Ascl and Pme1, tapping and recovering a target fragment to obtain Asc1-tENO2-Pme1, and adding 50ng of each of the three fragments into a connection system: 2ul 10XT4 ligation Buffer (NEB), 1ul T4 ligation Buffer (NEB, 400, 000covalent end units/ml), distilled water was added to 20ul, and the reaction was carried out at room temperature for 2 hours to obtain a ligation product, 1ul of the ligation product was added, and the PCR system: primeSTAR GXL Buffer (Mg 2+ plus) x 10. Mu.l, dNTPmix 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, supplemented with ddH2O to a total volume of 50. Mu.l, to obtain expression cassette P TEF2 -HMGR-T ENO2 . Cloning the expression cassette into pEASY-Blunt Simple cloning vector (purchased from Beijing Quanyujin Biotechnology Co., ltd.) to obtain recombinant vector pM7-HMGR, and sequencing TEF2 -HMGR-T ENO2 (the sequence is shown as SEQ ID No. 7) is inserted between cloning sites of pEASY-Blunt Simple to obtain a vector.
The recombinant plasmids pM3-PPDS 01-GSTSG-46 tATR1, pM13-Pln1-GGGS-PPDS 01-GSTSG-46 tATR1, leu gRNA, NDT80gRNA, YPL062W gRNA and pM7-HMGR are prepared and related information is shown in Table 2.
TABLE 2 plasmid information
3. Construction of recombinant bacterium
(I) construction of YSBYT5 Strain
1. Construction of Gene modules
Respectively taking the plasmids described in the table 2 as PCR templates (p delta-tHMG 1, pM9-ERG12, pM16-IDI1, pM5-ERG19, pM8-ERG13, pM11-ERG8 and pM3-ERG10 are described in the literature: creating Saccharomyces cerevisiae cell factory for fermentation production of lupeol, china journal of Chinese traditional medicine, lingting, wangDong, wearing Bo, zhang Xuanli, huangluqin, 2016, 41 (6): 1008-1015) and corresponding primers in the table 3 for PCR amplification to respectively obtain functional module fragments: m1 (containing 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 was as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg 2+ plus) x 10. Mu.l, dNTPmix 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, 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, and extension 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
2. Preparation of Yeast competence
Fresh yeast strain liquid BYT1 (derived from a laboratory-maintained strain, described in Zhubo Dai et al, producing agglomerans of ginsenosides in bakers' yeast. Sci Rep.2014Jan 15, ex vivo) was cultured overnight (P414-P purchased from Addge company TEF1 -Cas9-T CYC1 Plasmid) competent (1% inoculum size, 30ul seed inoculum inoculated into 3ml SD-Trp broth (0.8% total synthesis four-deficiency medium (purchased from Beijing Pankeno technology Co., ltd.) +2% glucose +0.005% His +0.01% by weight 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 the supernatant, and pipette clean. The precipitate was washed with 1ml of sterile water, centrifuged by pipetting, the supernatant discarded and washed twice.
(2) And (3) thallus treatment: 1ml of a treatment solution (preparation of treatment solution: 1M sorbitol +10mM LiAc +10mM Tris-HCl (pH 7.5)) (preservation in a refrigerator at 4 ℃) +10ul DTT (purchased from Beijing Lanbobedrid commercial and trade Co., ltd., product number: 1758-9030-25 g) (-preservation in a refrigerator at 20 ℃), and metal heating at 25 ℃ for 20min.
(3) After 20min, centrifugation was carried out, the supernatant was discarded, the mixture was aspirated off by a gun, 1ml of pre-cooled 1M sob (D-sorbitol, available from Beijing Soilebao Tech. Co., ltd.) (4 ℃ freezer) was added, whipped, centrifuged, and the supernatant was discarded. The column was washed twice with 1M Sob, the supernatant was aspirated off, and 50ul of Sob was added for suspension.
(4) Adding 2 μ l of M1, M2, M3, M4, M5, M6, M7 and M8 modules and 2 μ l of Leu gRNA plasmid, blowing, mixing, transferring into pre-cooled electric rotary cup, and ice-cooling for 5min.
(5) The electric revolving cup is wiped clean with 2.7kv electric shock. Add the first 1ml of sob to the electric rotor, mix well and suck a new 1.5ml EP tube. Shaking-culturing at 30 deg.C and 250rpm for 60min.
(6) After 60min, the supernatant was centrifuged and partially removed from the broth, applied to a plate with auxotrophy SD-UraTrp (0.8% total synthetic medium +2% glucose +0.005% His +0.01% Leu +2% Ager), and cultured in an incubator at 30 ℃ for 36h.
After about two days of culture box culture, selecting a single clone to carry out PCR colony verification to obtain a yeast engineering bacterium YSBYT5, discarding the Leu gRNA plasmid, and carrying out the next step of genetic modification.
The construction principle of the yeast engineering bacteria YSBYT5 is specifically 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) for expressing gRNA and a recombinant fragment (M1-M8) are transformed into the strain together, leu gRNA identifies and combines a specific PAM region of a Leu site, and simultaneously, the Leu gRNA is activated 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 fragment M1-M8 containing a homologous region is integrated into the DNA of the strain 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 genome DNA extraction kit (purchased from Beijing Tiangen Biochemical technology Co., ltd., cat # DP 307-02). Performing PCR amplification by using the extracted genome as a template and SacII-PGK1/Asc1-tHMG1-R to obtain a fragment of about 2400bp, which indicates that the fragment contains M1; PCR amplification is carried out on Pac-pPDC1/Asc1-Erg12-R to obtain a fragment of about 2200bp, which indicates that the fragment contains M2; PCR amplification is carried out on pac-pENO2/IDI1-Asc1-R, and a fragment of about 2200bp is obtained, which indicates that the fragment contains M3; PCR amplification is carried out on Pac-PYK1p/Asc1-Erg19-R to obtain a fragment about 2200bp, which indicates that the fragment contains M4; PCR amplification is carried out on pac1-pTEF2/Asc1-HMGR-N-R, and a fragment of about 1900bp is obtained, which indicates that M5 is contained; carrying out PCR amplification on pFBA1-YZ-F/Asc1-Erg13-R to obtain a fragment of about 2300bp, which indicates that the fragment contains M6; PCR amplification is carried out on Pac-pTDH3/Asc1-Erg8-R, and a fragment of about 2200bp is obtained, which indicates that the fragment contains M7; PCR amplification of SacII-pTEF1/Asc1-Erg10-R yielded a fragment of about 1700bp, indicating M8. The primers are specifically 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 YSBYT30 Strain
1. Construction of Gene modules
The plasmids described in Table 2 were used as PCR templates (pM 3-SmFPS and pM2-AtSQS2 are described in: wang Dong, liu Yi, xu jiao Yang, wang jin He, zhang Bo, zhang Xun, huang Qi, creating Saccharomyces cerevisiae cell factory for efficient production of dammaraenediol II [ J ] which is a ginsenoside precursor]Pharmaceutical bulletin, 2018, 53 (08): 1233-1241, pM11-ERG1 is described in chinese patent application 201210453416.X, publicly available from tianjin industrial biotechnology research institute) and corresponding primers for PCR amplification to obtain functional modules: m9 (containing 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 was as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg 2+ plus) x 10. Mu.l, dNTPmix 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, 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, and extension at 68 ℃ for 3 minutes (35 cycles); extension for 10 min at 72 ℃ (1 cycle). The product is recovered and stored by glue.
2. Preparation of Yeast competence
Culturing fresh yeast liquid YSBYT5 at night to prepare competence (1% inoculation amount, 30ul seed liquid inoculated to 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 the supernatant, and suck clean with a gun. The precipitate was washed with 1ml of sterile water, centrifuged by pipetting, the supernatant discarded and washed twice.
(2) And (3) thallus treatment: adding 1ml of the treatment solution (4 deg.C storage in refrigerator), 10ul DTT (-20 deg.C storage in refrigerator), and heating at 25 deg.C for 20min.
(3) After 20min centrifugation was carried out, the supernatant was discarded, the mixture was aspirated off with a gun, 1ml of precooled 1M sob (in a4 ℃ freezer) was added, whipped, centrifuged and the supernatant discarded. The column was washed twice with 1M Sob, the supernatant was aspirated off, 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 well, transfer to pre-cooled electric rotor, ice-bath 5min.
(5) The electric revolving cup is wiped clean with 2.7kv electric shock. Add the first 1ml of sob into the electric rotating cup, mix well and suck the new 1.5ml EP tube. Shaking-culturing at 30 deg.C and 250rpm for 60min.
(6) After 60min, the supernatant from the centrifugation of the bacterial suspension was spread evenly on a plate of auxotroph Sd-UraTrp (0.8% total synthetic four-deficiency medium +2% glucose +0.005% His +0.01% Leu +2% Ager), and cultured at 30 ℃ in an incubator for 36h.
Culturing in an incubator for about two days, selecting monoclonal for PCR colony verification to obtain yeast engineering bacteria YSBYT30, discarding NDT80gRNA plasmid, and performing genetic modification
The construction principle of the yeast engineering bacteria YSBYT30 is specifically that a recombinant plasmid p414-PTEF1-Cas9-TCYC1 capable of expressing Cas9 protein exists in a strain YSBYT5, the 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 a specific PAM region of an 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 the recombinant fragment M9-M11 containing a homologous region is integrated into yeast DNA through homologous recombination repair.
The PCR colony verification method specifically comprises the following steps:
the genome of yeast strain YSBYT30 was extracted using a yeast genome DNA extraction kit (purchased from Beijing Tiangen Biochemical technology Co., ltd., cat # DP 307-02). Performing PCR amplification by using the extracted genome as a template and SacII-pTEF1/SmFPS-Asc1 to obtain a fragment of about 1500bp, which indicates that the fragment contains M11; performing PCR amplification by using SacII-PGK1/AtSQS2-Asc1 to obtain a fragment of about 2000bp, which indicates that the fragment contains M9; PCR amplification was performed using Pac-pTDH3/ERG1-Asc1 to obtain a fragment of about 2300bp, indicating that the fragment contains M10. Primer sequences are shown in Table 4.
(III) construction of T30-DD Strain
1. Preparation of Yeast competence
Fresh yeast liquid YSBYT30 is cultured overnight to prepare competence (1 percent of inoculum size, 30ul of seed liquid is inoculated to 3ml of culture medium), and the operation steps are as follows:
(1) and (3) collecting thalli: 1ml of yeast solution was taken and dispensed into 1.5ml of EP tube. Centrifuge at 12000rpm for 1min, discard the supernatant, and pipette clean. The precipitate was washed with 1ml of sterile water, centrifuged by pipetting, the supernatant discarded and washed twice.
(2) And (3) thallus treatment: adding 1ml of the treatment solution (4 deg.C storage in refrigerator), 10ul DTT (-20 deg.C storage in refrigerator), and heating at 25 deg.C for 20min.
(3) After 20min centrifugation was carried out, the supernatant was discarded, the mixture was aspirated off with a gun, 1ml of precooled 1M sob (in a4 ℃ freezer) was added, whipped, centrifuged and the supernatant discarded. The column was washed twice with 1M Sob, the supernatant was aspirated off, and 50ul of Sob was added for suspension.
(4) Adding 2 μ l pRS425-SpgDDS plasmid (described in the literature: radix asparagi, liuyi, xue Yang, gold crane, zhangzhu, zhangli, huangqi. Creating Saccharomyces cerevisiae cell factory for high-efficiency production of ginsenoside precursor dammarenediol II [ J ]. Pharmaceutical science, 2018, 53 (08): 1233-1241, the plasmid described in the literature as pRS 425-DDS), blowing, mixing, transferring into a precooled electric rotary cup, and ice-cooling for 5min.
(5) The electric revolving cup is wiped clean, and the electric shock is carried out at 2.7 kv. Add the first 1ml of sob to the electric rotor, mix well and suck a new 1.5ml EP tube. Shaking-culturing at 30 deg.C and 250rpm for 60min.
(6) After 60min, the supernatant was centrifuged off part of the bacterial suspension and spread evenly on a plate of auxotroph SD-TrpLeu (0.8% total synthesis medium +2% glucose +0.005% His +0.01% Ura +2% Ager) and cultured for 36h at 30 ℃ in an incubator.
After about two days of incubator culture, selecting the single clone to carry out PCR colony verification, obtaining the engineered yeast T30-DD, and carrying out the next step of genetic modification.
The PCR colony verification method specifically comprises the step of extracting the genome of the yeast strain T30-DD by using a yeast genome 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 a strain is subjected to PCR verification by using a primer SacII-pTEF1/spgDDS-Asc1-R to obtain a fragment of about 2800bp, which indicates that the pRS425-spgDDS plasmid is successfully transferred. The primer sequences are shown in Table 4.
(IV) construction of PTA Strain and LPTA
1. Construction of Gene modules
The plasmids described by the plasmid information in Table 2 (publicly available from the institute of biotechnology in Tianjin industry) were used as PCR templates and the corresponding primers in Table 3 for PCR amplification, respectively to obtain functional modules: m12 (including 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 was as follows:GXL DNA Polymerase PrimeSTAR GXL Buffer(Mg 2+ plus) x 10. Mu.l, dNTPmix 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, 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, and extension 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
The method for preparing competence (1% inoculation amount, 30ul seed solution inoculated to 3ml culture medium) by culturing fresh yeast liquid T30-DD overnight comprises the following operation steps:
(1) and (3) collecting thalli: 1ml of yeast liquid was dispensed into 1.5ml EP tubes. Centrifuge at 12000rpm for 1min, discard the supernatant, and pipette clean. The precipitate was washed with 1ml sterile water, blown down and centrifuged, the supernatant was discarded and washed twice.
(2) And (3) thallus treatment: adding 1ml of the treatment solution (4 deg.C storage in refrigerator), 10ul DTT (-20 deg.C storage in refrigerator), and heating at 25 deg.C for 20min.
(3) After 20min centrifugation was carried out, the supernatant was discarded, the pipette was used up, 1ml of pre-cooled 1M sob (4 ℃ freezer) was added, whipped, centrifuged and the supernatant discarded. Wash twice more with 1M Sob, aspirate the supernatant and add 50ul of Sob to suspend.
(4) Divided into two groups, one group is added with 2 mul M12 module and 2 mul YPL062W gRNA plasmid, the other group is added with 2 mul M13 module and 2 mul YPL062W gRNA plasmid, and the mixture is blown, beaten, evenly mixed and transferred into a precooled electric rotating cup and ice-bathed for 5min.
(5) The electric revolving cup is wiped clean, and the electric shock is carried out at 2.7 kv. Add the first 1ml of sob to the electric rotor, mix well and suck a new 1.5ml EP tube. Shaking-culturing at 30 deg.C and 250rpm for 60min.
(6) After 60min, the supernatant was centrifuged and partially removed from the broth, applied to a plate of auxotrophy SD-UraTrpLeu (0.8% total synthetic medium +2% glucose +0.005% His +2% Ager), and cultured in an incubator at 30 ℃ for 36h. After about two days of culture box culture, picking single clone to carry out PCR colony verification, and obtaining yeast engineering bacteria PTA and LPTA.
Verification of PTA strain:
and (3) extracting the genome of the yeast strain PTA by using a yeast genome DNA extraction kit. PCR verification is carried out on the strain by taking the extracted genome as a template and using a primer yp1062w-up-256/yp1062w-down-249 to obtain a fragment of about 4700bp, which indicates that the M12 fragment is successfully transferred. See table 4 for primer sequences.
Validation of LPTA strain:
the yeast strain LPTA genome is extracted by using a yeast genome DNA extraction kit. The extracted genome is taken as a template, a primer yp1062w-up-256/PPDS01-EGPP-R is used for carrying out PCR verification to obtain a fragment of about 3000bp, a primer ATR1-Ce1805-F/yp1062w-down-249 is used for carrying out PCR verification on the strain to obtain a fragment of about 1000bp, and the result shows that M13 (P13) is successfully transferred (the P gene is expressed by the DNA sequence) TEF1 -Pln1-GGGS-PPDS01-GSTSSG-46tATR1-T CYC1 ) And (3) fragment. See table 4 for primer sequences.
The related information of the prepared strains YSBYT5, YSBYT30, T30-DD, PTA and LPTA is shown in Table 5.
TABLE 5 engineering strain information
EXAMPLE 2 use of microbial lipid droplet technology for Production of Protopanaxadiol (PPD)
1. Shake flask fermentation
(1) Culturing engineering bacteria PTA and LPTA
Yeast engineered strains PTA and LPTA were activated in the corresponding solid selection medium SD-UraTrpLeu, one monoclonal was inoculated to each of the genotype engineered strains, seed solutions (30 ℃,250rpm, 169h) were prepared in the corresponding liquid selection medium SD-UraTrpLeu, inoculated in a 100ml Erlenmeyer flask containing 15ml of the corresponding liquid selection medium at an inoculum size of 1%, three sets of each monoclonal were inoculated in parallel, and cultured for 6 days at 30 ℃,250rpm with shaking.
(2) Extraction of engineering bacteria PTA and LPTA products
And (4) sucking 2ml of bacterial liquid 6 days after shaking the flask for fermentation, centrifuging at 12000rpm for 1min, discarding the supernatant, and sucking the supernatant completely by using a gun. Washing the precipitate twice with ddH2O, transferring to a crushing tube, centrifuging at 12000rpm for 1min, and removing the supernatant; adding glass beads (diameter 0.5 mm) and lml extract (the extract is composed of methanol and acetone, volume ratio of methanol to acetone is 1: 1), vibrating and crushing for 5min,2 times, and ultrasonically crushing for 30min; centrifuging at 12000rpm for 2min, removing precipitate, and filtering the supernatant with 0.22 μm organic filter membrane to obtain solutions, which are named PTA solution and LPTA solution, respectively.
2. Qualitative and quantitative analysis by HPLC
(1) Qualitative analysis by HPLC
The standard products are protopanaxadiol PPD and dacarbazine DD, and are purchased from Shanghai-derived leaf Biotechnology Co., ltd. The samples were PTA solution and LPTA solution.
The instrument comprises the following steps: agilent high performance liquid chromatography 1260
HPLC detection conditions: DAD monitor, monitoring wavelength 203nm, watersChromatography column (250 mm. Times.4.6 mm,5 μm), mobile phase A10% methanol, mobile phase B acetonitrile, isocratic elution 20min,10% A +90%
(2) Quantitative analysis by HPLC
The yield of each engineering bacterium after being fermented for 6 days is as follows:
on the basis of T30-DD, a PPD module without yeast lipid droplet compartmentalization localization and a PPD module with yeast lipid droplet compartmentalization localization are respectively integrated at a yeast genome knockout YPL062W locus, HPLC detection results show that the PPD yield of PTA engineering bacteria is 5.39mg/L/OD, the DD yield is 13.88mg/L/OD, the conversion rate from DD to PPD is 27.97%, the PPD yield of corresponding LPTA process bacteria is 19.30mg/L/OD, the DD yield is 3.03mg/L/OD, and the conversion rate from DD to PPD is 86.43%.
The results are shown in fig. 1 and fig. 2, fig. 1 is a graph of HPLC detection of DD and PPD content in strains PTA and LPTA, and fig. 2 is a graph of HPLC of strains PTA and LPTA.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the 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 reference to specific examples, 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 use of some of the essential features is made possible within the scope of the claims attached below.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese 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 (10)
1. A method of constructing a recombinant microorganism, comprising: comprises introducing the coding gene of the recombinant fusion protein into a starting microorganism to obtain a recombinant microorganism; the recombinant fusion protein contains a Pln1 protein and a terpene synthesis-related protein.
2. The method of claim 1, wherein: the terpene synthesis related protein is selected from protopanaxadiol synthase PPDS01 and/or cytochrome P450 reductase ATR1.
3. The method according to claim 1 or 2, characterized in that: in the recombinant fusion protein, the Pln1 protein and the terpene synthesis-related protein are linked via a linker peptide.
4. The method according to any one of claims 1-3, wherein: the recombinant fusion protein is a recombinant protein containing the Pln1 protein, PPDS01 and ATR1.
5. The construction method according to claim 4, wherein:
the amino acid sequence of the Pln1 protein is shown as 1 st to 283 th in SEQ ID No. 2;
the amino acid sequence of the PPDS01 is shown as 288 th to 773 rd positions in SEQ ID No. 2;
the amino acid sequence of the ATR1 is shown as 780 st to 1425 th in SEQ ID No. 2.
6. The method according to claim 4 or 5, characterized in that:
the amino acid sequence of the recombinant fusion protein is shown in SEQ ID No. 2.
7. The method according to any one of claims 1-6, wherein: in the recombinant microorganism, the coding gene of the recombinant fusion protein is integrated into YJL062W locus of the starting microorganism.
8. The method according to any one of claims 1-7, wherein: and introducing the coding gene of the recombinant fusion protein into the starting microorganism through an expression cassette for expressing the recombinant fusion protein to obtain the recombinant microorganism.
9. The method according to any one of claims 1-8, wherein: the starting microorganism is saccharomyces cerevisiae, the saccharomyces cerevisiae is a strain obtained by carrying out the following A1-A12 transformation on a strain BYT1,
a1, introducing a 3-hydroxy-3-methylglutaryl coenzyme A reductase gene tHMG1 gene;
a2, introducing a mevalonate kinase gene ERG12 gene;
a3, introducing an IDI1 gene of an alcohol dehydrogenase I gene;
a4, introducing a mevalonate pyrophosphate decarboxylase gene ERG19 gene;
a5, introducing a hydroxymethyl glutaryl coenzyme A reductase gene HMGR gene;
a6, introducing hydroxymethyl glutaryl-coenzyme A synthetase gene ERG 13;
a7, introducing a phosphomevalonate kinase gene ERG8 gene;
a8, introducing acetyl coenzyme A acetyltransferase gene ERG 10;
a9, introducing a squalene synthase gene AtSQS2 gene;
a10, introducing a squalene monooxygenase gene ERG1 gene;
a11, introducing a farnesyl pyrophosphate synthetase gene SmFPS gene;
a12, introduction of dammarenediol synthase gene spgDDS gene.
10. Biomaterial, use or method, characterized in that: the biological material is any one of the following B1) -B6),
b1 A nucleic acid molecule encoding the recombinant fusion protein of claim 1;
b2 An expression cassette comprising the nucleic acid molecule according to B1);
b3 A recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);
b4 A recombinant microorganism containing the nucleic acid molecule according to B1), or a recombinant microorganism containing the expression cassette according to B2), or a recombinant microorganism containing the recombinant vector according to B3);
b5 A recombinant fusion protein according to claim 1;
b6 A recombinant microorganism expressing the recombinant fusion protein of claim 1;
the application is any one of the following applications,
use of X1, a process according to any one of claims 1 to 9, for the manufacture of a terpene product;
use of X2, the method of any one of claims 1-9, for the production of a terpene;
x3, the application of the biological material in the preparation of terpene products;
x4, the use of said biomaterial in the production of terpenes;
use of X5, the Pln1 protein of claim 1, in the manufacture of a terpene product;
use of X6, the Pln1 protein of claim 1, for the production of a terpene;
use of X7, the Pln1 protein of claim 1, for increasing the efficiency of terpene synthesis;
use of X8, the recombinant fusion protein of claim 1 for increasing efficiency of terpene synthesis;
the method is a method for increasing efficiency of terpene biosynthesis, comprising the step of expressing the recombinant fusion protein of claim 1 in a recipient organism to obtain a recombinant organism having a higher efficiency of terpene synthesis than the recipient organism.
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