CN115322913B - Recombinant saccharomyces cerevisiae for producing rose essential oil, and construction method and application thereof - Google Patents

Abstract

The invention discloses recombinant saccharomyces cerevisiae, a construction method and application thereof. The construction method comprises the following steps of modifying the original saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae: a1, introducing a geraniol synthase gene ObGES gene; a2, introduction of the double point mutant Gene ERG20 at positions 96 and 127 of the farnesyl pyrophosphate synthetase Gene ^F96W/N127W A gene; a3, introducing A3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1 gene; a4, introducing a geraniol reductase gene OYE 2; a5, introducing a nerol synthase GmNES gene; a6, introducing a neryl diphosphate synthase SINPS 1 gene. The construction method successfully realizes the construction of the rose essential oil microbial cell factory in saccharomyces cerevisiae through heterologous expression, module integration and branch interception regulation and control of farnesyl pyrophosphate synthetase.

Description

Recombinant saccharomyces cerevisiae for producing rose essential oil, and construction method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to recombinant saccharomyces cerevisiae for producing rose essential oil, and a construction method and application thereof.

Background

Rose essential oils are the most expensive essential oils in the world, called "after essential oils" "" liquid gold ". The rose essential oil is yellow brown, has unique fragrance, is widely applied to daily chemicals, medicines, foods and other industries, and has great practical value. About 3000 kg-5000 kg of rose petals are needed to extract 1kg of rose essential oil according to the traditional extraction method, the yield is very low, and the traditional extraction and separation are easy to cause environmental pollution and other problems. At present, as the rose essential oil yield is low and the demand is large, large market gaps exist in China, and how to improve the production technology becomes a problem which needs to be solved urgently.

Since the last 30 th century, microorganisms have been used in large quantities for industrial fermentation production, and fermentation products have been spread over many fields such as chemical industry, foods, and medicines. Metabolic engineering is a science linking cell metabolism to increase metabolite production or to confer new product production capabilities to microbial cells, and has found wide application in the development of cell factories. Saccharomyces cerevisiae (Saccharomyces cerevisiae), which is widely used in the synthetic biotechnology industry because it is generally considered to be in a safe (GRAS) state suitable for large-scale operation, has now been developed as one of the important platform microorganisms for metabolic engineering.

Disclosure of Invention

The invention provides a construction method of recombinant saccharomyces cerevisiae, which comprises the following steps: the method comprises the following steps of modifying the original saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae:

a1, introducing a geraniol synthase gene ObGES gene; a2, introduction of the double point mutant Gene ERG20 at positions 96 and 127 of the farnesyl pyrophosphate synthetase Gene ^F96W/N127W A gene; a3, introducing A3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1 gene; a4, introducing a geraniol reductase gene OYE 2; a5, introducing a nerol synthase GmNES gene; a6, introducing a neryl diphosphate synthase SINPS 1 gene.

Optionally, the construction method comprises the following steps: the following modifications are also carried out on the Saccharomyces cerevisiae:

b1, replacing a promoter of an ERG20 gene of a driving farnesyl pyrophosphate synthetase gene with a promoter of an ERG7 gene named pERG 7; b2, introducing a mevalonate kinase gene ERG 12; b3, introducing an isopentenyl pyrophosphate isomerase gene IDI 1; b4, introducing an MVAPP decarboxylase gene ERG 19; b5, introducing HMG-CoA reductase gene HMGR gene; b6, introducing a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene ERG13 gene; b7, introducing an MVAP kinase gene ERG8 gene; b8, introducing an acetyl-CoA acetyltransferase gene ERG 10.

The construction method of the recombinant saccharomyces cerevisiae can also comprise the step of expressing the introduced genes.

The Saccharomyces cerevisiae may be wild-type Saccharomyces cerevisiae, such as Saccharomyces cerevisiae CEN.PK2-1D, or may be modified Saccharomyces cerevisiae.

Optionally, according to the above construction method, the sequence of the obj gene encoded by the obj gene is genbank accession number: AMK97466.1 sequence 35-569; and/or, the ERG20 ^F96W/N127W Gene encoded ERG20 ^F96W/N127W Protein sequences such as SEQ ID No. 10; and/or, the sequence of the tHMG1 protein coded by the tHMG1 gene is genbank login number: AJS96703.1 sequence is shown at positions 530-1054; and/or, the sequence of the OYE2 protein encoded by the OYE2 gene is genbank login number: the sequence NP 012049.1 is shown; and/or, the sequence of the GmNES protein encoded by the GmNES gene is genbank login number: AEE92791.1 sequence; and/or, the SINPS 1 protein coded by the SINPS 1 gene has the sequence of genbank login number: the QNM36897.1 sequence is shown.

Optionally, according to the above construction method, the sequence of the obj gene is shown in SEQ ID No.8 from position 431 to position 2035; and/or, the ERG20 ^F96W/N127W The sequence of the gene is shown in 2048 th to 3106 th positions in SEQ ID NO. 8; and/or the sequence of the tHMG1 gene is shown in 757 th to 2340 th positions in SEQ ID NO. 2; and/or the sequence of the OYE2 gene is shown in the 801 st position to the 2003 st position in SEQ ID NO. 7; and/or the sequence of the GmNES gene is shown in the 801 st position to 2405 th position in SEQ ID NO. 3; and/or the sequence of the SINPS 1 gene is shown in the 431 th to 1213 rd positions of SEQ ID NO. 4.

The B1 may replace the above-mentioned promoter by CRISPR/CAS9 system, and specifically, CAS9 gene, gRNA gene and DNA fragment containing pERG7 promoter (for example, pERG20-pERG7 fragment used in examples) may be introduced into the Saccharomyces cerevisiae and CAS9 gene and gRNA gene are expressed. The gRNA fragment encoded by the gRNA gene targets the promoter driving the ERG20 gene (pERG 20 promoter). The target sequence may be TTTCTACTTGCCTGTCGCAT, for example.

Alternatively, according to the above construction method, the A1 and A2 are obtained by introducing an ObGES gene and ERG20 into the Saccharomyces cerevisiae ^F96W/N127W Gene expression cassettes (e.g., P _TEF1 -ObGES-ERG20 ^F96W/N127W -T _CYC1 An expression cassette, the sequence of which is shown as SEQ ID No. 8); and/or, the A3 is obtained by introducing into the Saccharomyces cerevisiae a tHMG1 gene expression cassette (e.g., P _PGK1 -tHMG1-T _ADH1 An expression cassette, the sequence of which is shown as SEQ ID No. 2); and/or, the A4 is obtained by introducing an OYE2 gene expression cassette (e.g., P _TDH3 -OYE2-T _TPI1 An expression cassette, the sequence of which is shown as SEQ ID No. 7); and/or, the A5 is obtained by introducing into the Saccharomyces cerevisiae of interest a GmNES gene expression cassette (e.g., P _TDn3 -GmNES-T _TPI1 An expression cassette, the sequence of which is shown as SEQ ID No. 3); and/or, the A6 is obtained by introducing a SINPS 1 gene expression cassette (e.g., P _TEF1 -SINDPS1-T _CYC1 An expression cassette, the sequence of which is shown as SEQ ID No. 4); and/or, the B2 is obtained by introducing an ERG12 gene expression cassette (e.g., P _PDC1 -ERG12-T _ADH2 Expression cassette) implementation; and/or, the B3 is obtained by introducing an IDI1 gene expression cassette (e.g.P _ENO2 -IDI1-T _PDC1 Expression cassette) implementation; and/or, the B4 is obtained by introducing an ERG19 gene expression cassette (e.g., P _PYK1 -ERG19-T _PGI1 Realizing an expression cassette; and/or, the B5 is obtained by introducing an HMGR gene expression cassette (e.g., P _TEF2 -HMGR-T _ENO2 Expression cassette) implementation; and/or, the B6 is obtained by introducing an ERG13 gene expression cassette (e.g., P _FBA1 -ERG13-T _TDH2 Expression cassette) implementation; and/or, the B7 is obtained by introducing an ERG8 gene expression cassette (e.g., P _TDH3 -ERG8-T _TPI1 Expression cassette) implementation; and/or, the B8 is obtained by introducing an ERG10 gene expression cassette (e.g., P _TEF1 -ERG10-T _CYC1 Expression cassette).

Alternatively, according to the above construction method, in the recombinant Saccharomyces cerevisiae, the ObGES gene and ERG20 gene ^F96W/N127W The gene is expressed by introducing an expression plasmid in the saccharomyces cerevisiae; tHMG1 gene, obGES gene and ERG20 ^{F96W /N127W} The gene is integrated into the GAL7 locus of the Saccharomyces cerevisiae; the tHMG1 gene, the GmNES gene and the SINTS 1 gene are integrated into the NDT80 locus of the Saccharomyces cerevisiae; the pERG7 replaces positions 1-248 of the promoter driving the ERG20 gene. The genes B2-B8 described above can be integrated into the YJL064W locus of the Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae constructed by the above method is also within the scope of the present invention.

The invention also provides a method for producing terpenes, which comprises the steps of culturing the recombinant saccharomyces cerevisiae to obtain a fermentation product; terpenes are obtained from the fermentation product.

Any of the following applications is also within the scope of the present invention,

x1, application of the method in preparing and producing terpene products; x2, use of the above method for producing terpenes; x3, application of the recombinant saccharomyces cerevisiae in preparing and producing terpene products; x4, application of the recombinant saccharomyces cerevisiae in terpene production.

In the above, the terpene may be at least one selected from geraniol, citronellol, nerol.

In the above, the terpene product may be a recombinant bacterium expressing a terpene.

The main components of the natural rose essential oil are monoterpene compounds citronellol, geraniol and nerol, and in the invention, the construction of a rose essential oil microbial cell factory is successfully realized in saccharomyces cerevisiae through heterologous expression, module integration and branch closure regulation and control of farnesyl pyrophosphate synthetase (ERG 20).

Drawings

FIG. 1 shows the GC-MS analysis results of the Rose year B strain product, wherein A is the peak pattern of the strains NEROL-pERG7, rose year B and the standard of NEROL (Nerol), geraniol (Geraniol) and Citronellol (Citronellol), B (1) is the MS pattern of the database Geraniol, B (2) is the MS pattern of the Geraniol in the Rose year B strain product, C (1) is the MS pattern of the database Citronellol, C (2) is the MS pattern of the Citronellol in the Rose year B strain product, D (1) is the MS pattern of the database NEROL, D (2) is the MS pattern of the NEROL in the Rose year B strain product.

Detailed Description

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

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

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

The following examples used the following media and components in particular:

SD solid selection medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His (available from panno technology limited, beijing), 2% glucose, 0.01% Leu (leucine), 0.005% His (histidine), 0.01% Ura (uracil), 0.01% Trp (tryptophan), and 2% agar powder;

SD liquid selection medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.005% His, 0.01% Ura, 0.01% Trp;

SD-Trp solid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.005% His, 0.01% Ura, 2% agar powder;

SD-Trp liquid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.005% His, 0.01% Ura:

SD-Trp-Ura solid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.005% His,2% agar powder;

SD-Trp-5FOA solid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.005% His, 0.01% Ura, 0.05%5-foa, 2% agar powder;

SD-Trp-His solid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.01% Ura, 2% agar powder;

SD-Trp-His liquid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.01% Leu, 0.01% Ura;

SD-Trp-Leu solid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His (available from pantuno technology limited, beijing), 2% glucose, 0.005% His (histidine), 0.01% Ura (uracil) and 2% agar powder;

SD-Trp-Leu liquid selection Medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 0.005% His, 0.01% Ura;

SD-Trp-Ura-His-Leu solid screening culture medium: 0.8% yeast selection medium SD-Ura-Trp-Leu-His,2% glucose, 2% agar powder.

The information on the gene fragments and protein sequences in the examples described below is given in the following table.

Gene fragment and protein sequence-related information

Protein related information

Example 1

1. Construction of promoter element and acquisition of functional Gene

1. PCR amplification

Saccharomyces cerevisiae CEN.PK2-1D (hereinafter referred to as NK2, purchased from European Saccharomyces cerevisiae collection (EUROSCARF)) genomic DNA was extracted using the Kangji yeast genome extraction kit (cat# CW 0569), and the genome was extracted according to the manufacturer's instructions. The genome DNA of saccharomyces cerevisiae NK2 is used as a template, the primer collocation in the table 1 is adopted respectively, the promoter fragments pERG7 (the pERG7 promoter is the-1 to-774 positions on the upstream of the ERG7 gene) are obtained through respective amplification, and then the promoter fragments pERG20-pERG7 are obtained through amplification by using the primer containing the homologous arm of the pERG20 promoter region.

The amplification system is as follows: TAKARA (Takara Shuzo)Hs DNA polymerase 5 XPS Buffer 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 0.5. Mu.l,/10. Mu.l>HS polymerase (2.5U/. Mu.l) 0.5. Mu.l supplemented with ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 3 minutes (30 cycles); extension at 72℃for 10min (1 cycle). And (5) recovering and storing the product through rubber tapping.

TABLE 1 primer sequences

2. Total synthesis of related genes

The SINSPS 1 gene and the GmNES gene were completely synthesized by the client gold biosciences, and inserted between SexA1 and Asc1 cloning sites of pUC57 vector (supplied by gold biosciences, inc.), to obtain cloning plasmids pUC 57-SINSPS 1 and pUC57-GmNES.

2. Construction of recombinant plasmids

Construction of pEASY-TRP1-URA3 plasmid

The plasmids pRS314 and pGAL7-URA3 are respectively used as templates, and the primers in the primer table 2 are used for amplification to obtain 871bp TRP1 fragment and 1128bp URA3 fragment; then, using TRP1 and URA3 fragments as templates, and using the primers in the primer table 2, a 1999bp TRP1-URA3 fragment was obtained by amplification. (pRS 314) is described in documents Ma Xiaolin, li Weixian, wang Dong, etc. Hederasapogenin biosynthesis analysis and construction of yeast cell factories [ J ]. J.Chinese traditional medicine, 2018, 043 (009): 1844-1850. PGAL7-URA3 is described in documents Lin Tingting, wang Dong, put on waves, etc. A yeast cell factory is created to ferment and produce lupeol [ J ]. J.Chinese traditional Chinese traditional medicine, 2016, 41 (06): 1008-1015. A yeast cell factory is produced.

The amplification system is as follows: TAKARA (Takara Shuzo)HS DNA polymerase 5 XPS Buffer 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 0.5. Mu.l,/o>HS polymerase (2.5U/. Mu.l) 0.5. Mu.l supplemented with ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were as follows: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 3 minutes (30 cycles); extension at 72℃for 10min (1 cycle). And (5) recovering and storing the product through rubber tapping.

Cloning the amplified product TRP1-URA3 into a Trans1-T1 competent cell transformed by a pEASY-Blunt Simple cloning vector, extracting plasmid sequencing and verifying to obtain a plasmid pEASY-TRP-URA.

TABLE 2 primer collocation

2.pRS425-ObGES-ERG20 ^F96W/N127W 、pRS425-LEU-TRP-URA-ObGES-ERG20 ^F96W/N127W Construction of plasmids

With pM3-GES and pM4-ERG20 ^F96W/N127W The plasmid was used as template (pM 3-GES, pM4-ERG 20) ^F96W-N127W The description is in the article: tao W, siwei L, bolin Z, et al engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl acetate [ J]Microbial Cell Factories,2018, 17 (1): 85. in (C), the primers in Table 3 were used to amplify the resulting ObGES-ERG20 ^F96W/N127W A gene fragment.

After purification of the fragments obtained above, the plasmids pRS425-LEU2-TEF1-SynPn3-29 (stored in the laboratory, recorded in the laboratory) were digested with the restriction enzymes SexA1 and Asc1, respectivelyArticles Wang, d., wang, j., shi, y., li, r.,&zhang, x. (2020) Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of panax notoginseng using a synthetic biology plan, metabic Engineering, 61), and fragment oges-ERG 20 ^F96W/N127W Then the rubber tapping is recovered.

Recovering target fragments by tapping: pRS425-LEU2-TEF1-// -CYC1 (7576 bp,120 ng) vector (pRS 425-LEU2-P _TEF1 -Pn3-32-T _CYC1 Rubber cutting recovery product) and ObGES-ERG20 ^F96W/N127W (fragment ObGES-ERG 20) ^F96W/N127W The product was recovered from the tapping) (2763 bp,105 ng) fragment. Respectively connecting the target fragment and the corresponding vector, wherein the connection system is as follows: mu.l 2X Quick Ligation Buffer (NEB Co., ltd.), 0.5. Mu.l Quick T4 DNA Ligase (NEB Co., 400,00 cohesive end units/ml), and ddH were supplemented ₂ O to 10 mul, reacting for 10min at 25 ℃ to obtain a connection product, transferring into a Transl T1 competent cell, and carrying out sequencing verification to obtain a recombinant vector pRS425-LEU-TEF1-ObGES-ERG20 ^{F96W /N127W} (i.e., pRS425-ObGES-ERG20 ^F96W/N127W )。

The TRP-URA fragment was amplified using the primers shown in Table 3, and plasmids pRS425-LEU-TEF1-ObGES-ERG20 were digested with restriction enzymes Xma I and NOT I, respectively ^F96W/N127W And the fragment TRP-URA. P425-LEU-// -TEF1-ObGES-ERG20 ^F96W/N127W CYC1 (50 ng) vector (pRS 425-LEU-TEFi-ObGES-ERG20 ^F96W/N127W Rubber cut recovery product) and TRP-URA (130 ng) (fragment TRP-URA rubber cut recovery product). Respectively connecting the target fragment and the corresponding vector, wherein the connection system is as follows: mu.l of 2X Quick Ligation Buffer (NEB Co., ltd.), 0.5. Mu.l of Quick T4 DNA Ligase (NEB Co., 400,000cohesive end units/ml), and ddH were supplemented ₂ O to 10 μl, reacting at 25deg.C for 10min to obtain ligation product, transferring into Transl T1 competent cells, and performing sequencing verification to obtain pRS425-LEU-TRP-URA-ObGES-ERG20 ^{F96W /N127W} A plasmid.

Construction of YJL064W-gRNA, pERG20gRNA plasmid

The pLPPgRNA is used as a template, and the primers in the table 3 are respectively matched, so that gRNA fragments YJL064W-gRNA and pERG20-gRNA are respectively obtained through amplification. The amplified gRNA fragment PCR product is treated by Dpnl enzyme for more than three hours, then 5-10 mu l is transferred into Trans 1T 1 competent cells and is subjected to sequencing verification, and YJL064W-gRNA plasmid and pERG20gRNA plasmid are obtained, and the related information of the plasmids is shown in Table 5. ( pLPPgRNA is described in: wang, d., wang, j, shi, y, li, r, & Zhang, x (2020) & Elucidation of the complete biosynthetic pathway of the main triterpene glycosylation products of panax notoginseng using a synthetic biology platform.metabic Engineering,61 )

TABLE 3 primer sequences

4. Construction of pRS 425-SINPS 1, pM4-OYE2, pM4-GmNES recombinant plasmid

(1) PCR amplification

The functional gene fragment of geraniol reductase OYE2 is amplified by taking genomic DNA of saccharomyces cerevisiae NK2 as a template and adopting primers in a table 3.

(2) Construction of recombinant plasmid by enzyme digestion and ligation

After purification of the fragments obtained above, plasmids pRS425-LEU2-TEF1-SynPn3-29, pUC57-GmNES, pUC 57-SINSPS 1 and plasmid pM4-tHMG1 (stored in the laboratory, described in Tao W, siwei L, bolin Z, et al engineering Saccharomyces cerevisiae for the production of the valuable monoterpene ester geranyl acetate [ J ]. Microbial Cell Factories,2018, 17 (1): 85) and fragment OYE2 were digested with restriction enzymes, respectively, and then the resulting pM4 and p425 vectors with the cohesive ends of the sites of SexA1, ASC1 and fragments of OYE2, SINSS 1, gmNES were recovered by tapping.

Recovering target fragments by tapping: pEASY-Blunt-TDH3-// -TPI1 (5749 bp,100 ng) vector (plasmid pM4-tHMG1 tapping recovery product) and GmNES (plasmid pUC57-GmNES tapping recovery product) (1605 bp,20 ng), OYE2 (fragment OYE2 tapping recovery product) (1203 bp,30 ng) fragment, p425-LEU2-TEF 1-/-CYC 1 (120 ng) vector (plasmid pRS425-LEU2-TEF1-SynPn3-29 rubber cut recovery product) and SINPS 1 (783 bp,20 ng) (fragment SINPS 1 rubber cut recovery product). Respectively connecting the target fragment and the corresponding vector, wherein the connection system is as follows: mu.l of 2X Quick Ligation Buffer (NEB Co., ltd.), 0.5. Mu.l of Quick T4 DNA Ligase (NEB Co., 400,000cohesive end units/ml), and ddH were supplemented ₂ O to 10 μl, reacting at 25deg.C for 10min to obtain a ligation product, transferring into Trans 1T 1 competent cells, and performing sequencing verification to obtain recombinant vector.

The recombinant vector has been sequenced to insert the expression cassette of the gene between cloning sites of the cloning vector and was designated pRS 425-SINTS 1, pM4-OYE2, pM4-GmNES.

5. plasmid construction of pM7-HMGR

The genomic DNA of Saccharomyces cerevisiae NK2 was used as a template, the primers Pac1-TEF2-F and SexA1-TEF2-R were used for amplification to obtain the promoter pTEF2 (562 bp), and the primers Asc1-ENO2-F and Pme1-ENO2-R were used for amplification to obtain the terminator tENO2 (400 bp). The amplification system is as follows: primeSTAR GXL Buffer (Mg2+plus). Times.10. Mu.l, dNTPMmix. Times.4. Mu.l, primers Pac1-TEF2-F and SexA1-TEF2-R (Asc 1-ENO2-F and Pme1-ENO 2-R) were each 1.5. Mu.l, genomic DNA template was 1.5. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, and ddH2O was added to a total volume of 50. Mu.l.

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

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

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

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

The plasmid pUC57-synHMGR is digested with restriction enzymes SexA I and Asc I respectively, and the target fragment is recovered by tapping to obtain a fragment SexA I-synHMGR-Asc I; cutting the section pTEF2 by using restriction endonucleases SexA I and pac I respectively, and recovering a target fragment by tapping to obtain SexA I-pTEF2-pac I; cutting the segment tENO2 by restriction enzymes Asc1 and Pme1 respectively, recovering the target segment by tapping to obtain Asc1-tENO2-Pme1, and adding 50ng of each of the three segments into a connecting system: 2ul 10XT4 ligationBuffer (NEB Co.) 1ul T4 ligase(NEB Co., 400,000cohesive end units/ml), distilled water was supplemented to 20ul, and the reaction was carried out at room temperature for 2 hours to give a ligation product, 1ul of the ligation product was added, and the PCR system: primeSTAR GXL Buffer (Mg2+plus) x 10. Mu.l, dNTPMmix 4. Mu.l, primers Pac1-TEF2-F and Pme1-ENO2-R each 1.5. Mu.l, ligation product 1. Mu.l, primeSTAR GXL DNA Polymerase (1.25U/. Mu.l) 1. Mu.l, and ddH2O were added to a total volume of 50. Mu.l to give an expression cassette P _TEF2 -HMGR-T _ENO2 . The expression cassette was cloned into pEASY-Blunt Simple cloning vector (purchased from Beijing full gold Biotechnology Co., ltd.) to obtain recombinant vector pM7-HMGR.

The information on the recombinant vector prepared above is shown in Table 4.

TABLE 4 recombinant vector information

3. Construction of recombinant bacteria

Construction of Chaetomium HP001-pERG7-ERG20

The amplification system is as follows: TAKARA (Takara Shuzo)HS DNA polymerase 5 XPS Buffer 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, DNA template 0.5. Mu.l,/o>HS polymerase (2.5U/. Mu.l) 0.5. Mu.l supplemented with ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 3 minutes (30 cycles); extension at 72℃for 10min (1 cycle). And (5) recovering and storing the product through rubber tapping.

TABLE 5 primer information

Activating yeast bottom in SD liquid mediumDisc cells NK2, competent cells were prepared by lithium acetate method. Mu.l of p414-TEF1p-Cas9-CYC1t plasmid (stored in laboratory, purchased from addgene.) was added and transformants were streaked on SD-Trp solid selection medium after incubation for 48 hours at 30 ℃. Activating the strain obtained in the above manner in SD-Trp liquid selection medium, and preparing competent cells by using a lithium acetate method. 2. Mu.l of YJL064W-gRNA plasmid was added to the competence, and N1, N2, N3, N4, N5, N6 (P) amplified as described above was added _FBA1 -ERG13-T _TDH2 ) And 8 modular segments of N7 and N8 (total 8. Mu.l) were added to the yeast competent cells in a molar ratio of 1:1:1:1:1:1:1. Transformants were streaked after culturing in SD-Trp-Ura solid selection medium at 30℃for 48 hours in an incubator, and PCR was performed to verify that the correct strain was cultured in SD-Trp-5FOA solid selection medium for 24 hours to eliminate the gRNA plasmid, designated HP001.

The construction principle of HP001 is that recombinant plasmid p414-PTEF1-Cas9-TCYC1 capable of expressing Cas9 protein is transferred into strain NK2 in advance, then recombinant plasmid (YJL 064W-gRNA plasmid) capable of expressing gRNA is transformed into strain together with recombinant fragment (N1-N8), YJL064W-gRNA plasmid recognizes and binds specific PAM region of YJL064W site, at the same time, cas9 protein is activated and guided to exert cleavage function, double-stranded DNA of YJL064W site is broken, at the moment, recombinant fragment N1-N8 containing homologous region is integrated into strain DNA by homologous recombination repair.

Yeast chassis cells HP001 were activated in SD-Trp medium and competent cells were prepared by lithium acetate method. The pERG20gRNA plasmid obtained above, together with the fragment pERG20-pERG7, was added to yeast competent cells at a molar ratio of 1:1. Transformants were incubated in SD-Trp-Ura solid screening medium at 30℃for 48 hours in an incubator. Extracting yeast genome, using the extracted strain genome as a template, adopting the following primers to amplify fragments by PCR, and verifying by sequencing, wherein the correct strain is named HP001-pERG7-ERG20.

ERG20-OUT-F：CGGCTCTTACGCCATAACCTTTATG

ERG20-R：CTTCAGGTGCAGTGATTAAGTCCATC

Construction of recombinant Strain

1. Construction of high-yield nerol strain

(1) Construction of Gene Module

The PCR templates described in Table 6 (pNDT 80-HIS3, described in articles Zhang Lili, ma Xiaolin, wang Dong, yu Peng, huang Luqi,&zhang Xueli et al (2017) Yeast cell factory creation of high-yield nerolidol journal of Chinese traditional medicine, 42 (015), 2962-2968, p delta-tHMG 1 is described in literature: journal of chinese traditional medicine, lin Tingting, wang Dong, wear waves, zhang Xueli, huang Luqi, 2016, 41 (6): 1008-1015) and corresponding primers to obtain functional modules respectively: m1 (comprising the NDT80-HIS3-up fragment), M2 (comprising P) _PGK1 -tHMG1-T _ADH1 Expression cassette), M3 (comprising P _TDH3 -GmNES-T _TPI1 Expression cassette), M4 (comprising P _TEF1 -SINDPS1-T _CYC1 Expression cassette), M5 (comprising NDT80-HIS3-down fragment).

The amplification system is as follows: TAKARA (Takara Shuzo)HS DNA polymerase 5 XPS Buffer 10. Mu.l, dNTPMmix 4. Mu.l, primers 1.5. Mu.l each, RNA template 0.5. Mu.l,/o>HS polymerase (2.5U/. Mu.l) 0.5. Mu.l supplemented with ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 3 minutes (30 cycles); extension at 72℃for 10min (1 cycle). And (5) recovering and storing the product through rubber tapping.

TABLE 6 primer sequences

(2) Construction of recombinant strains

The resulting chassis strain HP001-pERG7-ERG20 was subjected to elimination of the gRNA plasmid with SD-5FOA-Trp plates. Activating yeast chassis cells HP001-pERG7-ERG20 in SD-TRP culture medium, and preparing competent cells by a lithium acetate method. M1, M2, M3, M4, M5 were each added to the yeast competent cells in a molar ratio of 1:1:1:1:1 (total 5. Mu.l). Transformants were streaked after culturing in SD-Trp-His solid screening medium in an incubator at 30℃for 48 hours. Activating the strain in SD-Trp-His liquid culture medium, extracting yeast genome with the method, taking the extracted strain genome as a template, verifying functional genes according to primer collocation in Table 7, verifying that the correct strain is named NEROL-pERG7, shaking seed liquid, and performing shake flask fermentation.

TABLE 7 primer sequences

2. Construction of Rose essential Yeast Strain

(1) Construction of Gene Module

PCR amplification was performed using the PCR templates and the corresponding primers described in Table 8, respectively, to obtain functional modules: m6 (containing GAL7-URA3-up fragment), M7 (containing P) _PGK1 -tHMG1-T _ADH1 Expression cassette), M8 (comprising P _TDH3 -OYE2-T _TPI1 Expression cassette), M9 (comprising P _TEF1 -ObGES-ERG20 ^F96W/N127W -T _CYC1 Expression cassette), M10 (comprising GAL7-URA3-down fragment).

The amplification system is as follows: TAKARA (Takara Shuzo)HS DNA polymerase 5 XPS Buffer 10 μl, dNTPMmix 4 μl, primers 1.5 μl each, RNA template 0.5 μl,/10 μl>HS polymerase (2.5U/. Mu.l) 0.5. Mu.l supplemented with ddH ₂ O to a total volume of 50. Mu.l.

The amplification conditions were: pre-denaturation at 98 ℃ for 3min (1 cycle); denaturation at 98℃for 10 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 3 minutes (30 cycles); extension at 72℃for 10min (1 cycle). And (5) recovering and storing the product through rubber tapping.

TABLE 8 primer sequences

(2) Construction of recombinant strains

Activating the engineering yeast cell NEROL-pERG7 obtained in the above way in SD-Trp-His culture medium, and preparing competent cells by a lithium acetate method. M6, M7, M8, M9, M10 were collocated and added to yeast competent cells in a molar ratio of 1:1:1:1:1 (total 5. Mu.L). Transformants were streaked after culturing in SD-Trp-Ura-His solid screening medium in an incubator at 30℃for 48 hours. Activating the strain in SD-Trp-Ura-His liquid culture medium, extracting yeast genome by the method, taking the extracted strain genome as a template, verifying functional genes according to the primer collocation PCR in table 9, verifying that the correct strain is named as Rose year A, shaking seed liquid, and performing shake flask fermentation.

TABLE 9 primer sequences

3. Optimization of rose essential oil yeast strains

After eliminating the plasmid containing the TRP1 selection marker in the Rose yeast A strain by reverse screening in YPD medium, the engineering yeast cell Rose yeast A obtained above was activated in SD-His-Ura medium, and competent cells were obtained by lithium acetate method. Mu.l pRS425-LEU-TRP-URA-ObGES-ERG20 ^F96W/N127 The plasmid was added to yeast competent cells. Transformants were streaked after 48 hours of incubation in SD-Trp-Ura-His-Leu solid screening medium at 30 ℃. Activating strain in SD-Trp-Ura-His-Leu liquid culture medium, extracting strain fermentThe master plasmid (yeast plasmid was extracted according to manufacturer's instructions using the Tiangen yeast plasmid extraction kit (product number: DP 112-02)) and the yeast plasmid extracted was used as a template to verify the functional plasmid according to the primer set in Table 10, and the correct strain was named Rose yeast B and the seed solution was shaken for shake flask fermentation.

TABLE 10 primer sequences

The information on the recombinant bacteria prepared above is shown in Table 11.

TABLE 11 engineering strain information

4. Shake flask fermentation and detection

1. Engineering bacteria culture and product extraction

According to Table 12, the yeast engineering strain prepared as described above was activated in the corresponding solid selection medium, seed solution (30 ℃,250rpm,16 h) was prepared in the corresponding liquid selection medium, inoculated in a 100ml Erlenmeyer flask containing 15ml of the corresponding liquid selection medium at an inoculum size of 1%, shake-cultured at 30 ℃,250rpm for 1 day, then 1.5ml of methyl oleate was added, and shake-cultured was continued for 5 days. Finally, the liquid in the triangular flask was transferred to a 50ml centrifuge tube, centrifuged at 5000rpm for 5min, and the organic phase was collected for use.

TABLE 12 Medium used for culturing strains

Strain name	Solid selection medium	Liquid selection medium
			NEROL-pERG7	SD-Trp-His	SD-Trp-His
Rose yeast A	SD-Trp-Ura-His	SD-Trp-Ura-His
			Rose yeast B	SD-Trp-Ura-His-Leu	SD-Trp-Ura-His-Leu

2. Qualitative and quantitative analysis

GC-MS detection

The converted material was diluted 10-fold with n-hexane, passed through an organic nylon membrane (0.22 μm) and detected by GC-MS. Detection instrument: agilent gas chromatograph-mass spectrometer Agilent 7890A/5975C.

NEROL-pERG7 strain fermentation product GC-MS assay conditions: the temperature of the sample inlet is 250 ℃, the sample inlet volume is 1 mu L, no flow division is performed, and the solvent is delayed for 3min; chromatographic column: HP-5ms (30 m.0.25 mM); chromatographic conditions: preserving heat at 45 ℃ for 1min at 10 ℃/min to 300 ℃ for 5min; MS conditions: full Scan:50-750amu. Qualitative and quantitative determination was performed using a standard of nerol, which was purchased from Zhejiang river Biotechnology Co., ltd.

Conditions for GC-MS measurement of the fermentation products of the Rose eye A and Rose eye B strains: the temperature of the sample inlet is 250 ℃, the sample inlet volume is 1 mu L, no flow division is performed, and the solvent is delayed for 3min; chromatographic column: HP-5ms (30 m.0.25 mM); chromatographic conditions: preserving heat at 70 ℃,35min,3 ℃/min to 115 ℃,20 ℃/min to 300 ℃ for 5min; MS conditions: full Scan:50-750amu. The quality and quantity of geraniol, citronellol and nerol standard products are respectively purchased from Allatin company, shanghai Yuan Ye Biotechnology Co., ltd, zhejiang Ling Biotechnology Co., ltd, geraniol (product number: G107515), citronellol (product number: G107515) and nerol (product number: N0077).

The experiment was repeated three times, and as a result, the yield of each engineering bacterium at 6 days of fermentation was as follows:

NEROL yield of NEROL-pERG7 strain was 12.46mg/L.

The yield of nerol of Rose yeast A reaches 3.37mg/L, the yield of citronellol reaches 35.51mg/L, and the yield of geraniol reaches 22.70mg/L.

The yield of nerol of Rose yeast B reaches 12.78mg/L, the yield of citronellol reaches 77.24mg/L, the yield of geraniol reaches 33.96mg/L, and the GC-MS analysis result of the fermentation product is shown in FIG. 1.

3. Bioreactor fermentation culture

1) Culture medium configuration

Calcium chloride mother liquor: 19.2g/L calcium chloride dihydrate.

Trace metal salt mother liquor: 19.1g/L disodium ethylenediamine tetraacetate; 10.2g/L zinc sulfate heptahydrate; 0.5g/L manganese chloride tetrahydrate; 0.86g/L cobalt chloride hexahydrate; 0.78g/L copper sulfate pentahydrate; 0.56g/L sodium molybdate dihydrate; 5.12g/L of seven Shui Ya ferric sulfate.

Vitamin mother liquor: 0.05g/L biotin; 0.2g/L sodium p-aminobenzoate; 1g/L nicotinic acid; 1g/L calcium pantothenate; 1g/L pyridoxine hydrochloride; 1g/L thiamine hydrochloride; 25g/L inositol.

Seed medium and fermentation medium: 25g/L glucose, 15g/L ammonium sulfate, 6.15g/L magnesium sulfate heptahydrate, 0.72g/L zinc sulfate heptahydrate, 8g/L potassium dihydrogen phosphate, 2ml/L calcium chloride mother liquor and 10ml/L trace metal salt mother liquor; 12ml/L vitamin mother liquor, 1g/L tryptophan, and the balance of water.

Feed medium: 800g/L glucose, 5.125g/L magnesium sulfate heptahydrate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 9g/L potassium dihydrogen phosphate, 1g/L tryptophan and the balance being water.

2) Engineering bacterium Rose yeast B fermentation

Activating engineering bacteria Rose yeast B by the method of '1, engineering bacteria culture and product extraction'. After the amino acid screening mark is supplemented, the monoclonal on the flat plate is selected to a test tube filled with SD-Trp-Ura-His-Leu culture medium, and the culture is carried out at 30 ℃ with shaking at 250rpm for overnight; sucking 500 mu L of bacterial liquid into a 250ml triangular flask filled with 50ml of SD-Trp-Ura-His-Leu culture medium, and carrying out shaking culture at 30 ℃ for 24 hours at 250 rpm;

picking up the monoclonal on the plate to a test tube filled with sD-TRP-Ura-His-Leu medium, and culturing at 30 ℃ with shaking at 250rpm for overnight; sucking 500 mu L of bacterial liquid into a 250ml triangular flask filled with 50ml of SD-Ura-His-Leu culture medium, and carrying out shaking culture at 30 ℃ for 24 hours at 250 rpm;

respectively sucking 2ml of bacterial liquid into 3 1L triangular flasks filled with 100ml of seed culture medium, and carrying out shaking culture at 30 ℃ and 250rpm for 48 hours; finally, the seed solution was added to a 7L fermenter containing 3L of fermentation medium via a flame inoculation loop (Eppendorf company, germany, model:) Is a kind of medium.

The parameter set values in the fermentation process are respectively as follows: the temperature is 30 ℃, the pH is 5.0, the dissolved oxygen is 30%, the air flow is 3-20L/min, the stirring rotating speed is 300-1000rpm, and the dissolved oxygen, the stirring rotating speed and the ventilation are cascaded. When the dissolved oxygen value is more than 60%, feeding a feeding culture medium into the fermentation tank until the glucose concentration in the fermentation liquid is 5g/L.

3 hours before the end of fermentation, 10% (relative to the volume of the broth) of olive oil was added, and the organic phase was separated after the end of fermentation.

The method is processed according to a conversion method and a detection method of '2 and qualitative and quantitative analysis', and the engineering bacterium Rose year B is subjected to high-density fermentation for 96 hours to obtain 0.32g/L (relative to the culture solution) of nerol, 3.89g/L (relative to the culture solution) of citronellol and 0.66g/L (relative to the culture solution) of geraniol.

Recombinant bacteria meeting the purposes of the present invention, including but not limited to the specific experimental examples described in the table, can be fermented and cultured according to the fermentation method described in "3, bioreactor fermentation and culture" to obtain rose essential oil.

Specific information on the expression cassette in the above N2-N8 fragment and the pERG20-pERG7 fragment are given in the following table.

Expression cassette in N2-N8 fragment and pERG20-pERG7 fragment related information table

Expression cassette in N2-N8 fragment and pERG20-pERG7 fragment sequence table

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

Sequence listing

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

<120> recombinant Saccharomyces cerevisiae for producing rose essential oil, construction method and application thereof

<130> 210858

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 726

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga 60

ccatgattac gccaagctct gttccattga tttcttctct attgttatat cataattttc 120

atgaggtaat tcattgattt ttttgaatgc taatgagcct actttgaaaa tattctcatg 180

ttcaaaagag cgtgccggtt tcagttcgat ttgcttcata ttaacagaaa aactacttga 240

gtaaactaaa tcctcctttt ctaaacaccc ttccatacct cttgaagatg aactcggata 300

tttcagagag gaatctggac attgattttt caaagcaccc agtttgttct ggatatgttc 360

aagagtctcc acccttttat ttggcctgaa gaacggatcc tcattttcct tcgaagcttc 420

cattggtgtg gattgacgag atttgatagg cgagatggag gccccagagt tcggtgcacc 480

tagcgccact ttggatcgcc ttttcgaagg tcttgcattt aagcaacttt catttaacgg 540

ctgcgccatt gatggcattt ttctttttgt gctgttttgt gaggaattga cactcgacgg 600

tgttctcaca gttattcgct gagatgacgc atagttagaa ggtgatcttc ctctaataat 660

aagaggaggc gttttcattt cttgcaaata cacaaacatc ccttttgatc cattttttaa 720

agccag 726

<210> 2

<211> 2498

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

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 2498

<210> 3

<211> 2807

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

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 atggataata tctatattaa acaagcattg gttttgaagg 840

aagttaagca tgttttccaa aaattgatcg gtgaagatcc aatggaatct atgtacatgg 900

ttgatacaat ccaaagattg ggtatcgaac atcatttcga agaagaaatc gaagctgcat 960

tgcaaaagca acatttgatt ttctcttctc atttgtctga tttcgcaaac aaccataaat 1020

tgtgtgaagt tgctttgcct tttagattgt tgagacaaag aggtcattac gttttggcag 1080

atgttttcga taatttgaag tcaaataaga aagaattcag agaaaaacat ggtgaagatg 1140

ttaagggttt gatttcttta tacgaagcta ctcaattggg tattgaaggt gaagattcat 1200

tggatgatgc aggttactta tgtcatcaat tgttacatgc ttggttaact agacatgaag 1260

aacataacga agcaatgtac gttgctaaga cattgcaaca tccattacat tacgatttgt 1320

ctagattcag agatgatact tcaatcttgt tgaacgattt caagacaaaa agagaatggg 1380

aatgtttgga agaattagca gaaattaatt cttcaatcgt tagattcgtt aatcaaaatg 1440

aaattacaca agtttacaag tggtggaagg atttgggttt aaataacgag gttaagttcg 1500

caagatacca accattgaag tggtacatgt ggccaatggc ttgttttact gatccaagat 1560

tttcagaaca aagaatcgaa ttgacaaaac caatttcttt agtctacatc atcgatgata 1620

tttttgatgt ttacggtact ttggatcaat tgactttgtt tactgatgca attaaaagat 1680

gggaattggc ttctactgaa caattgccag atttcatgaa gatgtgtttg agagttttgt 1740

acgaaattac aaatgatttt gctgaaaaga tttgtaagaa acatggtttt aatccaattg 1800

aaactttgaa gagatcttgg gttagattgt tgaacgcatt tttagaagaa gctcattggt 1860

tgaactctgg tcatttgcca agatcagcag aatatttgaa caacggtatc gtttcaacag 1920

gtgttcatgt tgttttggtt cattctttct ttttgatgga ttactcaatt aataatgaaa 1980

ttgttgctat cgttgataac gttccacaaa tcatccattc tgttgcaaag atcttgagat 2040

tgtcagatga tttggaaggt gctaaatctg aagatcaaaa cggtttggat ggttcataca 2100

tcgattgtta catgaacgaa catcaagatg tttctgcagg tgacgctcaa agacatgttg 2160

ctcatttgat ttcatgtgaa tggaagagat taaatagaga aatcttgact caaaaccaat 2220

tgccatcttc ttttactaac ttttgtttga atgctgcaag aatggttcca ttgatgtacc 2280

attacagatc taacccaggt ttgtcaactt tgcaagaaca tgttaaattg ttgtctaata 2340

atgctgttgc aggtgctgaa agacatgttg ttcatatttt gtgtttacaa tttgttattg 2400

aataagatta atataattat ataaaaatat tatcttcttt tctttatatc tagtgttatg 2460

taaaataaat tgatgactac ggaaagcttt tttatattgt ttctttttca ttctgagcca 2520

cttaaatttc gtgaatgttc ttgtaaggga cggtagattt acaagtgata caacaaaaag 2580

caaggcgctt tttctaataa aaagaagaaa agcatttaac aattgaacac ctctatatca 2640

acgaagaata ttactttgtc tctaaatcct tgtaaaatgt gtacgatctc tatatgggtt 2700

actcataagt gtaccgaaga ctgcattgaa agtttatgtt ttttcactgg aggcgtcatt 2760

ttcgcgttga gaagatgttc ttatccaaat ttcaactgtt atataga 2807

<210> 4

<211> 1520

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

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 atgtctgcta gaggtttgaa taagatctct tgttctttga atttgcaaac 480

tgaaaaattg tgttacgaag ataacgataa cgatttggat gaagaattga tgccaaagca 540

tatcgctttg atcatggatg gtaacagaag atgggcaaaa gataaaggtt tagaagttta 600

cgaaggtcat aagcatatca tcccaaaatt gaaggaaatt tgtgatattt cttcaaaatt 660

gggtattcaa atcatcactg ctttcgcatt ttctacagaa aactggaaga gatcaaagga 720

agaagttgat ttcttgttac aaatgttcga agaaatctat gatgaatttt ctagatcagg 780

tgttagagtt tctatcatcg gttgtaagtc agatttgcca atgactttgc aaaagtgtat 840

cgctttgaca gaagaaacta caaagggtaa taagggtttg catttggtta ttgcattgaa 900

ctacggtggt tactacgata tcttgcaagc tactaagtca atcgttaata aggcaatgaa 960

cggtttgtta gatgttgaag atatcaataa gaatttgttc gatcaagaat tagaatctaa 1020

gtgtccaaac ccagatttgt tgattagaac tggtggtgaa caaagagttt caaatttctt 1080

gttgtggcaa ttggcttaca cagaattcta cttcactaac acattgttcc cagatttcgg 1140

tgaagaagat ttgaaggaag caatcatgaa cttccaacaa agacatagaa gattcggtgg 1200

tcatacatat taaccgctga tcctagaggg ccgcatcatg taattagtta tgtcacgctt 1260

acattcacgc cctcccccca catccgctct aaccgaaaag gaaggagtta gacaacctga 1320

agtctaggtc cctatttatt tttttatagt tatgttagta ttaagaacgt tatttatatt 1380

tcaaattttt cttttttttc tgtacagacg cgtgtacgca tgtaacatta tactgaaaac 1440

cttgcttgag aaggttttgg gacgctcgaa ggctttaatt tgcaagctgc ggccctgcat 1500

taatgaatcg gccaacgcgc 1520

<210> 5

<211> 1993

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

catcataagg aattccggga ttctccccat gaaaggtatc tggatctacc actgcaccta 60

aaattacatg caagctaaag tgtttatttt gttgggatgg tttcttcacg ctaattgatg 120

aagcaaattg aactctttca taacgggcaa ctttctgaat agaatcttct ggataggaaa 180

acaataaaga gtccactcca tattcttcat aattaacgtg gtctctgtgc aaataaaaag 240

tggaatcgta ttttttcatt ttagtgatat ttcgaacatt tgaagcttct cttatgattt 300

gatgttttgg caaaggggaa ggcaccaacg gacatactga aggacaaaat tgaggacctt 360

tgtcgcgttt cgctgtatgc tggacgagat tgatttccgt gtcgtcgtcg tcattcttag 420

cttttatttt gatagcgaaa tattgcactc ttaatctgct ttctactgaa gagtcttcaa 480

cgagaagatc aaaactgctc tttaaaaaag tatccaaatc acaatttgcc gtttcaaacg 540

ttgatactaa ggtaaaataa tttcttttat aacctaccca ctcttcatca atatggtcga 600

acgtttaaag cggtttaaac gtgtcactac ataagaacac ctttggtgga gggaacatcg 660

ttggtaccat tgggcgaggt ggcttctctt atggcaaccg caagagcctt gaacgcactc 720

tcactacggt gatgatcatt cttgcctcgc agacaatcaa cgtggagggt aattctgcta 780

gcctctgcaa agctttcaag aaaatgcggg atcatctcgc aagagagatc tcctactttc 840

tccctttgca aaccaagttc gacaactgcg tacggcctgt tcgaaagatc taccaccgct 900

ctggaaagtg cctcatccaa aggcgcaaat cctgatccaa acctttttac tccacgcgcc 960

agtagggcct ctttaaaagc ttgaccgaga gcaatcccgc agtcttcagt ggtgtgatgg 1020

tcgtctatgt gtaagtcacc aatgcactca acgattagcg accagccgga atgcttggcc 1080

agagcatgta tcatatggtc cagaaaccct atacctgtgt ggacgttaat cacttgcgat 1140

tgtgtggcct gttctgctac tgcttctgcc tctttttctg ggaagatcga gtgctctatc 1200

gctaggggac caccctttaa agagatcgca atctgaatct tggtttcatt tgtaatacgc 1260

tttactaggg ctttctgctc tgtcatcttt gccttcgttt atcttgcctg ctcatttttt 1320

agtatattct tcgaagaaat cacattactt tatataatgt ataattcatt atgtgataat 1380

gccaatcgct aagaaaaaaa aagagtcatc cgctaggtgg aaaaaaaaaa atgaaaatca 1440

ttaccgaggc ataaaaaaat atagagtgta ctagaggagg ccaagagtaa tagaaaaaga 1500

aaattgcggg aaaggactgt gttatgactt ccctgactaa tgccgtgttc aaacgatacc 1560

tggcagtgac tcctagcgct caccaagctc ttaaaacgga attatggtgc actctcagta 1620

caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 1680

cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg 1740

ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgatc cggacgccac 1800

cgctggtacc gagctcggat ccactagtaa cggccgccag tgtgctggaa ttgcccttaa 1860

gggcaattct gcagatatcc atcacactgg cggccgctcg agcatgcatc tagagggccc 1920

aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac 1980

tgggaaaacc ctg 1993

<210> 6

<211> 1746

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg 60

caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat 120

aacaatttca cacaggaaac agctatgacc atgattacgc caagctgggc tggcttaact 180

atgcggcatc agagcagatt gtactgagag tgcaccatac caccttttca attcatcatt 240

ttttttttat tctttttttt gatttcggtt tccttgaaat ttttttgatt cggtaatctc 300

cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata tacgcatatg 360

tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca gaacaaaaac 420

ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg ctgctactca 480

tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa caaacttgtg 540

tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag cattaggtcc 600

caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca tggagggcac 660

agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg aagacagaaa 720

atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat acagaatagc 780

agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg ttagcggttt 840

gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt tagcagaatt 900

gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg acattgcgaa 960

gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg gaagagatga 1020

aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg gagacgcatt 1080

gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca ttattattgt 1140

tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac gttacagaaa 1200

agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaagcgg tttaaacatg 1260

actgctgaag aatttgattt ttctagccat tcccatagac gttacaatcc actaaccgat 1320

tcatggatct tagtttctcc acacagagct aaaagacctt ggttaggtca acaggaggct 1380

gcttacaagc ccacagctcc attgtatgat ccaaaatgct atctatgtcc tggtaacaaa 1440

agagctactg gtaacctaaa cccaagatat gaatcaacgt atattttccc caatgattat 1500

gctgccgtta ggctcgatca acctatttta ccacagaatg attccaatga ggataatctt 1560

aaaaataggc tgcttaaagt gcaatctgtg agaggcaatt gtttcgtcat atgttttagc 1620

cccaatcata atctaaccat tccacaaatg aaacaatcag atctggttca tattgttaat 1680

tcttggcaag cattgactga cgatctctcc agagaagcaa gagaaaatca taagcctttc 1740

aaatat 1746

<210> 7

<211> 2405

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

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 atgccatttg ttaaggactt taagccacaa gctttgggtg 840

acaccaactt attcaaacca atcaaaattg gtaacaatga acttctacac cgtgctgtca 900

ttcctccatt gactagaatg agagcccaac atccaggtaa tattccaaac agagactggg 960

ccgttgaata ctacgctcaa cgtgctcaaa gaccaggaac cttgattatc actgaaggta 1020

cctttccctc tccacaatct gggggttacg acaatgctcc aggtatctgg tccgaagaac 1080

aaattaaaga atggaccaag attttcaagg ctattcatga gaataaatcg ttcgcatggg 1140

tccaattatg ggttctaggt tgggctgctt tcccagacac ccttgctagg gatggtttgc 1200

gttacgactc cgcttctgac aacgtgtata tgaatgcaga acaagaagaa aaggctaaga 1260

aggctaacaa cccacaacac agtataacaa aggatgaaat taagcaatac gtcaaagaat 1320

acgtccaagc tgccaaaaac tccattgctg ctggtgccga tggtgttgaa atccacagcg 1380

ctaacggtta cttgttgaac cagttcttgg acccacactc caataacaga accgatgagt 1440

atggtggatc catcgaaaac agagcccgtt tcaccttgga agtggttgat gcagttgtcg 1500

atgctattgg ccctgaaaaa gtcggtttga gattgtctcc atatggtgtc ttcaacagta 1560

tgtctggtgg tgctgaaacc ggtattgttg ctcaatatgc ttatgtctta ggtgaactag 1620

aaagaagagc taaagctggc aagcgtttgg ctttcgtcca tctagttgaa cctcgtgtca 1680

ccaacccatt tttaactgaa ggtgaaggtg aatacaatgg aggtagcaac aaatttgctt 1740

attctatctg gaagggccca attattagag ctggtaactt tgctctgcac ccagaagttg 1800

tcagagaaga ggtgaaggat cctagaacat tgatcggtta cggtagattt tttatctcta 1860

atccagattt ggttgatcgt ttggaaaaag ggttaccatt aaacaaatat gacagagaca 1920

ctttctacaa aatgtcagct gagggataca ttgactaccc tacgtacgaa gaagctctaa 1980

aactcggttg ggacaaaaat taagattaat ataattatat aaaaatatta tcttcttttc 2040

tttatatcta gtgttatgta aaataaattg atgactacgg aaagcttttt tatattgttt 2100

ctttttcatt ctgagccact taaatttcgt gaatgttctt gtaagggacg gtagatttac 2160

aagtgataca acaaaaagca aggcgctttt tctaataaaa agaagaaaag catttaacaa 2220

ttgaacacct ctatatcaac gaagaatatt actttgtctc taaatccttg taaaatgtgt 2280

acgatctcta tatgggttac tcataagtgt accgaagact gcattgaaag tttatgtttt 2340

ttcactggag gcgtcatttt cgcgttgaga agatgttctt atccaaattt caactgttat 2400

ataga 2405

<210> 8

<211> 3413

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

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 atgtctgctt gtactccttt ggcatctgct atgccattgt cttctactcc 480

attgattaac ggtgataatt ctcagagaaa aaatacaaga caacatatgg aagagtcttc 540

atctaaaaga agagaatatt tgttggaaga aacaacaaga aaattgcaaa gaaacgatac 600

tgaatctgtt gagaaattaa aattgataga taacattcaa caattaggta ttggatacta 660

ttttgaagat gctataaatg ctgttttacg ttctcctttt tctacaggtg aagaagattt 720

gtttacagct gcattgagat ttaggttgtt gaggcataat ggtattgaaa tttctcctga 780

aattttcttg aagttcaaag atgaaagggg aaagttcgat gaatctgata ctttgggttt 840

attgtcttta tacgaggctt caaacttggg tgttgctggt gaagagattt tggaggaggc 900

tatggagttc gctgaagcta ggttgaggag gtctttgtct gagccagcag ctccattgca 960

cggtgaagtt gcacaggctt tagacgtccc aaggcacttg agaatggcta gattggaagc 1020

tagaagattt attgaacaat acggtaagca gtctgaccat gacggtgact tgttggaatt 1080

ggcaatttta gattataacc aagtccaggc tcagcaccag tctgaattga cagaaattat 1140

taggtggtgg aaagaattgg gattggttga taaattatct tttggtagag atagaccatt 1200

ggaatgcttt ttgtggactg tcggtttgtt gccagagcca aagtattctt ctgttagaat 1260

tgagttggct aaagctattt ctattttgtt ggttattgat gatattttcg atacatacgg 1320

tgaaatggat gatttaattt tgttcactga cgctattaga aggtgggact tagaggctat 1380

ggaaggttta cctgaatata tgaagatttg ctacatggct ttgtacaata ctacaaacga 1440

agtttgttat aaagttttaa gagatactgg tagaattgtt ttgttaaatt tgaaatcaac 1500

ttggattgat atgattgaag gttttatgga agaggctaaa tggttcaacg gaggttctgc 1560

tcctaagttg gaggaatata ttgaaaatgg tgtttcaact gctggtgctt acatggcttt 1620

cgctcatatt ttctttttaa ttggagaagg tgttactcat caaaattctc aattgttcac 1680

tcaaaagcca tatccaaaag tcttttctgc tgcaggtaga attttgagat tgtgggacga 1740

cttgggtaca gctaaggagg agcaagagag gggtgattta gcttcttgtg ttcaattatt 1800

tatgaaggaa aaatcattga ctgaagagga ggctagatct agaattttgg aagaaattaa 1860

gggtttatgg agagatttga atggtgagtt ggtctataat aagaatttac cattatcaat 1920

tattaaagtc gctttgaaca tggctagggc ttctcaggtc gtctataaac acgatcaaga 1980

tacttacttt tcttctgttg acaattacgt tgatgcttta tttttcactc aataaggtgg 2040

tggttctatg gcttcagaaa aagaaattag gagagagaga ttcttgaacg ttttccctaa 2100

attagtagag gaattgaacg catcgctttt ggcttacggt atgcctaagg aagcatgtga 2160

ctggtatgcc cactcattga actacaacac tccaggcggt aagctaaata gaggtttgtc 2220

cgttgtggac acgtatgcta ttctctccaa caagaccgtt gaacaattgg ggcaagaaga 2280

atacgaaaag gttgccattc taggttggtg cattgagttg ttgcaggctt actggttggt 2340

cgccgatgat atgatggaca agtccattac cagaagaggc caaccatgtt ggtacaaggt 2400

tcctgaagtt ggggaaattg ccatctggga cgcattcatg ttagaggctg ctatctacaa 2460

gcttttgaaa tctcacttca gaaacgaaaa atactacata gatatcaccg aattgttcca 2520

tgaggtcacc ttccaaaccg aattgggcca attgatggac ttaatcactg cacctgaaga 2580

caaagtcgac ttgagtaagt tctccctaaa gaagcactcc ttcatagtta ctttcaagac 2640

tgcttactat tctttctact tgcctgtcgc attggccatg tacgttgccg gtatcacgga 2700

tgaaaaggat ttgaaacaag ccagagatgt cttgattcca ttgggtgaat acttccaaat 2760

tcaagatgac tacttagact gcttcggtac cccagaacag atcggtaaga tcggtacaga 2820

tatccaagat aacaaatgtt cttgggtaat caacaaggca ttggaacttg cttccgcaga 2880

acaaagaaag actttagacg aaaattacgg taagaaggac tcagtcgcag aagccaaatg 2940

caaaaagatt ttcaatgact tgaaaattga acagctatac cacgaatatg aagagtctat 3000

tgccaaggat ttgaaggcca aaatttctca ggtcgatgag tctcgtggct tcaaagctga 3060

tgtcttaact gcgttcttga acaaagttta caagagaagc aaatagccgc tgatcctaga 3120

gggccgcatc atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc 3180

tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat 3240

agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt ttctgtacag 3300

acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt tgggacgctc 3360

gaaggcttta atttgcaagc tgcggccctg cattaatgaa tcggccaacg cgc 3413

<210> 9

<211> 911

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gtccaaatat ttgaaaacaa aggtacagcc atgggttgtt ccaacttaca tccacatggc 60

caagcttggt gcttagaatc catccctagt gaagtttcgc aagaattgaa atcttttgat 120

aaatataaac gtgaacacaa tactgatttg tttgccgatt acgtcaaatt agaatcaaga 180

gagaagtcaa gagtcgtagt ggagaatgaa tcctttattg ttgttgttcc atactgggcc 240

atctggccat ttgagacctt ggtcatttca aagaagaagc ttgcctcaat tagccaattt 300

aaccaaatgg tgaaggagga cctcgcctcg attttaaagc aactaactat taagtatgat 360

aatttatttg aaacgagttt cccatactca atgggtatcc atcaggctcc tttgaatgcg 420

actggtgatg aattgagtaa tagttggttt cacatgcatt tctacccacc tttactgaga 480

tcagctactg ttcggaaatt cttggttggt tttgaattgt taggtgagcc tcaaagagat 540

ttaacttcgg aacaagctgc tgaaaaacta agaaatttag atggtcagat tcattatcta 600

caaagactgt aatggtaccg agctcggatc cactagtaac ggccgccagt gtgctggaat 660

tgcccttaag ggcaattctg cagatatcca tcacactggc ggccgctcga gcatgcatct 720

agagggccca attcgcccta tagtgagtcg tattacaatt cactggccgt cgttttacaa 780

cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 840

ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 900

agcctgaatg g 911

<210> 10

<211> 352

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 10

Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe

1 5 10 15

Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met

20 25 30

Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr

35 40 45

Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala

50 55 60

Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu

65 70 75 80

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

85 90 95

Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln

100 105 110

Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Trp Asp

115 120 125

Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe

130 135 140

Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val

145 150 155 160

Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro

165 170 175

Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe

180 185 190

Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala

195 200 205

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

210 215 220

Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp

225 230 235 240

Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly

245 250 255

Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu

260 265 270

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

275 280 285

Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp

290 295 300

Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys

305 310 315 320

Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys

325 330 335

Ala Asp Val Leu Thr Ala Phe Leu Asn Lys Val Tyr Lys Arg Ser Lys

340 345 350

Claims (8)

1. A construction method of recombinant saccharomyces cerevisiae is characterized in that: comprising the following steps: and (3) modifying the original saccharomyces cerevisiae 1), 2) or 3) to obtain the recombinant saccharomyces cerevisiae:

the 1) modifications are the following A3, A5 and A6 modifications;

said 2) being adapted to further perform A1, A2, A3 and A4 modifications on the recombinant Saccharomyces cerevisiae obtained by said 1) modification;

said 3) engineering to further perform A1 and A2 engineering on the recombinant Saccharomyces cerevisiae obtained by said 2) engineering;

a1, introducing a geraniol synthase gene ObGES gene;

a2, introduction of the double point mutant Gene ERG20 at positions 96 and 127 of the farnesyl pyrophosphate synthetase Gene ^F96W/N127W Genes of ERG20 ^F96W/N127W Gene encoded ERG20 ^F96W/N127W The sequence of the protein is shown as SEQ ID No. 10;

a3, introducing A3-hydroxy-3-methylglutaryl-CoA reductase gene tHMG1 gene;

a4, introducing a geraniol reductase gene OYE 2;

a5, introducing a nerol synthase GmNES gene;

a6, introducing a neryl diphosphate synthase SINPS 1 gene;

the Saccharomyces cerevisiae is also modified as follows B1-B8:

b1, replacing a promoter of an ERG20 gene of a driving farnesyl pyrophosphate synthetase gene with a promoter of an ERG7 gene named pERG 7;

b2, introducing a mevalonate kinase gene ERG 12;

b3, introducing an isopentenyl pyrophosphate isomerase gene IDI 1;

b4, introducing an MVAPP decarboxylase gene ERG 19;

b5, introducing HMG-CoA reductase gene HMGR gene;

b6, introducing a 3-hydroxy-3-methylglutaryl-CoA synthase gene ERG 13;

b7, introducing an MVAP kinase gene ERG8 gene;

b8, introducing an acetyl-CoA acetyltransferase gene ERG 10.

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

the sequence of the ObGES protein encoded by the ObGES gene is genbank login number: AMK97466.1 sequence 35-569;

and/or, the sequence of the tHMG1 protein coded by the tHMG1 gene is genbank login number: AJS96703.1 sequence is shown at positions 530-1054;

and/or, the sequence of the OYE2 protein encoded by the OYE2 gene is genbank login number: the sequence NP 012049.1 is shown;

and/or, the sequence of the GmNES protein encoded by the GmNES gene is genbank login number: AEE92791.1 sequence;

and/or, the SINPS 1 protein coded by the SINPS 1 gene has the sequence of genbank login number: the QNM36897.1 sequence is shown.

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

the sequence of the ObGES gene is shown in the 431 th to 2035 th positions of SEQ ID NO. 8;

and/or, the ERG20 ^F96W/N127W The sequence of the gene is shown in 2048 th to 3106 th positions in SEQ ID NO. 8;

and/or the sequence of the tHMG1 gene is shown in 757 th to 2340 th positions in SEQ ID NO. 2;

and/or the sequence of the OYE2 gene is shown in the 801 st position to the 2003 st position in SEQ ID NO. 7;

and/or the sequence of the GmNES gene is shown in the 801 st position to 2405 th position in SEQ ID NO. 3;

and/or the sequence of the SINPS 1 gene is shown in the 431 th to 1213 rd positions of SEQ ID NO. 4.

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

the A1 and A2 are obtained by introducing an ObGES gene and ERG20 into the Saccharomyces cerevisiae ^F96W/N127W Realizing a gene expression cassette;

and/or, said A3 is achieved by introducing a hmg1 gene expression cassette into said s.cerevisiae;

and/or, the A4 is realized by introducing an OYE2 gene expression cassette into the saccharomyces cerevisiae;

and/or, the A5 is realized by introducing a GmNES gene expression cassette into the saccharomyces cerevisiae;

and/or, said A6 is achieved by introducing a SINDPS1 gene expression cassette into said s.cerevisiae;

and/or, said B2 is achieved by introducing an ERG12 gene expression cassette into said s.cerevisiae;

and/or, said B3 is achieved by introducing an IDI1 gene expression cassette into said s.cerevisiae;

and/or, said B4 is achieved by introducing an ERG19 gene expression cassette into said s.cerevisiae;

and/or, said B5 is achieved by introducing an HMGR gene expression cassette into said s.cerevisiae;

and/or, said B6 is achieved by introducing an ERG13 gene expression cassette into said s.cerevisiae;

and/or, said B7 is achieved by introducing an ERG8 gene expression cassette into said s.cerevisiae;

and/or, the B8 is realized by introducing an ERG10 gene expression cassette into the saccharomyces cerevisiae.

5. The method according to claim 1 or 2, characterized in that: in the recombinant Saccharomyces cerevisiae, the ObGES gene and ERG20 ^F96W/N127W The gene is expressed by introducing an expression plasmid in the saccharomyces cerevisiae; tHMG1 gene, obGES gene and ERG20 ^F96W/N127W The gene is integrated into the Gal70 locus of the Saccharomyces cerevisiae; the tHMG1 gene, the GmNES gene and the SINTS 1 gene are integrated into the NDT80 locus of the Saccharomyces cerevisiae; the pERG7 replaces positions 1-248 of the promoter driving the ERG20 gene.

6. Recombinant s.cerevisiae constructed by the method of any one of claims 1-5.

7. A process for producing a terpene, characterized by either:

(1) Culturing the recombinant s.cerevisiae obtained by the modification of 1) in the method of any one of claims 1 to 5 to obtain a fermentation product; obtaining nerol from the fermentation product;

(2) Culturing the recombinant s.cerevisiae obtained by the modification of 2) in the method of any one of claims 1 to 5 to obtain a fermentation product; obtaining geraniol, citronellol, and/or nerol from the fermentation product;

(3) Culturing the recombinant s.cerevisiae obtained by the modification of 3) in the method of any one of claims 1 to 5 to obtain a fermentation product; geraniol, citronellol, and/or nerol are obtained from the fermentation product.

8. In any of the applications described in the following,

use of X1a, the method of any one of claims 1-5 for the production of nerol when 1) retrofitting is performed;

use of X1b, the method of any one of claims 1-5 for the production of geraniol, citronellol and/or nerol when 2) retrofitting is performed;

use of X1c, the method of any one of claims 1-5 for the production of geraniol, citronellol and/or nerol when carrying out the modification of 3);

use of the recombinant saccharomyces cerevisiae obtained by 1) modification of X2a, the method according to any one of claims 1-5, for the production of nerol;

use of the recombinant saccharomyces cerevisiae obtained by 2) modification of the method according to any of the claims 1-5 for the production of geraniol, citronellol and/or nerol;

use of the recombinant saccharomyces cerevisiae obtained by 3) modification of the method according to any of the claims 1-5 for the production of geraniol, citronellol and/or nerol.