Recombinant yeast engineering bacteria for producing d-borneol

Abstract

The application discloses a recombinant bacterium and a use thereof, the recombinant bacterium is a yeast containing or expressing borneol diphosphate synthetase in vivo, and is used for producing dextro-borneol; experiments prove that the recombinant bacterium after being reformed (such as codon optimization, protein truncation or Kozak increase) can improve the yield of dextro-borneol, and is suitable for industrialized production of dextro-borneol.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and fermentation engineering, specifically to a genetically engineered bacterium for synthesizing borneol, its construction method, and its applications. Background Technology

[0002] Borneol, also known as camphor, is a bicyclic monoterpene compound, classified into dextrorotatory borneol and levorotatory borneol based on its optical rotation. Figure 1 Dextrorotatory borneol is also known as natural camphor, while levorotatory borneol is also known as borneol. Dextrorotatory borneol is currently mainly derived from camphor trees and borneol trees, while levorotatory borneol is found in borneol, lavender, frankincense, pine, fir, citronella oil, and frankincense.

[0003] Borneol is a precious traditional Chinese medicine and high-grade spice, widely used in medicine, food, daily chemicals, pesticides, and other fields. Currently, most pharmaceutical companies and drug manufacturers tend to use inexpensive and readily available synthetic borneol as a raw material and in their finished drug formulations. However, synthetic borneol contains a large amount of toxic isoborneol in addition to borneol. Regarding the plant source of borneol, researchers have adopted plant extraction methods, which have disadvantages such as low yield, long cycle, and difficulty in cultivating high-quality borneol resources. Utilizing fermentation to produce borneol and other monoterpenoid compounds and their derivatives is a feasible approach. Studies have found that the biosynthesis of monoterpenoids all originates from a common precursor (GPP)—in plants, the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway produce isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Under the catalysis of geranyl pyrophosphate monoterpene synthase, IPP and DMAPP condense to form geranyl pyrophosphate (GPP), which is ultimately formed by monoterpene synthases into a wide variety of monoterpenoids (O'BRIEN TE, BERTOLANI SJ, ZHANG Y, et al. Predicting Productive Binding Modes for Substrates and Carbocation Intermediates in Terpene Synthases-BornylDiphosphate Synthase as a Representative Case[J].ACS Catal, 2018, 8:3322.). Zhang Chao et al. (CN110669713A) disclosed a yeast engineered strain that produces high levels of monoterpenoids and reconstructed the limonene synthase biosynthetic pathway in Saccharomyces cerevisiae, enabling heterologous synthesis of D-limonene with a yield of 27.3 mg / L. Zhou Pingping et al. (CN111411101A) constructed a linalool biosynthetic pathway using the cis-linalool synthase t67OMcLIS, by overexpressing the entire MVA pathway and introducing an ERG20 enzyme that reduces FPP formation. F96W / N127W The variant enhanced the supply of precursor GPP, and the final strain produced 53.14 mg / L linalool culture in shake flasks. This demonstrates that the construction of monoterpene engineered bacteria can increase monoterpene production; however, engineered bacteria related to borneol have not yet been reported. Summary of the Invention

[0004] The first aspect of this invention is to provide a yeast engineered strain (recombinant strain) for producing dextrorotatory borneol, said recombinant strain being a yeast strain containing or expressing borneol diphosphate synthase CbTPS1 in vivo. The borneol diphosphate synthase CbTPS1 described in this invention has been disclosed in the applicant's prior patent (CN201911082325.8), which is derived from *Cinnamomum burmanni* and can be used to synthesize or prepare monoterpene synthases of dextrorotatory borneol. The entire contents of that patent are incorporated herein by reference and form part of this application.

[0005] In one aspect, the yeast contains a codon-optimized CbTPS1 gene for borneol diphosphate synthase. This invention is improved based on the codon preference of *Saccharomyces cerevisiae*. The improved method is a conventional technique in the art, such as artificial synthesis based on the sequences of related genes known in the art and referring to the codon preference of *Saccharomyces cerevisiae* (the design of *Saccharomyces cerevisiae* codon preference can be found at http: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=493). Any gene designed based on the codon preference of *Saccharomyces cerevisiae* can be used in this invention.

[0006] Preferably, the codon-optimized CbTPS1-encoded nucleic acid of the present invention is shown in SEQ ID NO:1.

[0007] In a second aspect, the invention further includes protein modification of the key enzyme CbTPS1. The N-terminal amino acid of CbTPS1 is truncated to form three truncated proteins (hereinafter referred to as t10-CbTPS1, t32-CbTPS1, and t37-CbTPS1), and it was found that the yield of dextrorotatory borneol was significantly increased by the truncated proteins. The encoding nucleic acids of t10-CbTPS1, t32-CbTPS1, and t37-CbTPS1 described in this invention are shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0008] In a third aspect of the invention, it was discovered that by adding a Kozak sequence before the start codon ATG of the truncated protein, three new truncated proteins (hereinafter referred to as t10-CbTPS1K, t32-CbTPS1K, and t37-CbTPS1K) were obtained, which can further increase the yield of dextrorotatory borneol.

[0009] In some embodiments, the in vivo presence or expression of borneol diphosphate synthase according to the present invention involves introducing the coding nucleic acid of the borneol diphosphate synthase into the yeast; and / or, the introduction of the coding nucleic acid of the borneol diphosphate synthase into the yeast involves introducing an expression cassette containing the coding nucleic acid of the borneol diphosphate synthase into the yeast; and / or, the expression cassette containing the coding nucleic acid of the borneol diphosphate synthase is introduced into the yeast via a vector expressing the coding nucleic acid expression cassette of the borneol diphosphate synthase; preferably, it is introduced into the yeast in plasmid form.

[0010] In some embodiments, the present invention improves the supply of precursor GPP (hereinafter referred to as M / D) by optimizing the substrate bacteria. For example, the yeast strains described in this invention contain endogenous genes related to the yeast mevalonate pathway, such as the acetyl-CoA transferase gene ERG10 (SEQ ID NO:5), the HMG-CoA synthase gene ERG13 (SEQ ID NO:6), the HMG-CoA reductase gene tHMG1 (SEQ ID NO:7), the mevalonate kinase gene ERG12 (SEQ ID NO:8), the phosphate mevalonate kinase gene ERG8 (SEQ ID NO:9), the mevalonate pyrophosphate decarboxylase gene ERG19 (SEQ ID NO:10), the farnesyl pyrophosphate synthase gene ERG20, and the isopentenyl pyrophosphate isomerase gene IDI1 (SEQ ID NO:11). In some preferred embodiments, the ERG20 used in this invention is a mutant ERG20. F96W-N127W (SEQ ID NO:12).

[0011] In some embodiments, the yeast of the present invention is Saccharomyces cerevisiae; and / or the Saccharomyces cerevisiae is CEN.PK2-1D with genotypes MATα, URA3-52, TRP1-289, LEU2-3112, HIS3Δ1, MAL2-8C, SUC2.

[0012] A fourth aspect of the present invention provides a method for producing dextrorotatory borneol, the method comprising:

[0013] The recombinant yeast strain that produces dextrorotatory borneol is inoculated into a culture medium and fermented. The fermented bacterial broth is then extracted and separated to obtain the target product, dextrorotatory borneol. The recombinant yeast strain can ferment to produce GPP. Attached Figure Description

[0014] Figure 1 A schematic diagram of the biosynthetic pathways of GPP and dextrorotatory borneol in Saccharomyces cerevisiae;

[0015] Figure 2The graph shows the geraniol production in the modified *Bacillus subtilis* strain, where A is the growth curve of strain M / D; and B is the geraniol production of strain M / D.

[0016] Figure 3 The image shows the GC-MS qualitative detection results of dextrorotatory borneol, where A represents the gas chromatography detection results of dextrorotatory borneol; and B represents the mass spectrometry identification results of dextrorotatory borneol.

[0017] Figure 4 The effect of different N-terminal truncation methods of borneol diphosphate synthase CbTPS1 on the yield of dextrorotatory borneol was investigated. In this study, A represents the qualitative analysis of the product of the protein truncated strain by GC-MS, and B represents the comparison of dextrorotatory borneol yield among different strains. Detailed Implementation

[0018] Experimental materials

[0019] Table 1. Vector and strain information

[0020]

[0021]

[0022] Table 2 Primer Information

[0023]

[0024]

[0025]

[0026] Main reagents:

[0027] Gene JET Gel Extraction Kit (Thermo Scientific, USA); EZNATMplasmid mini kit I (Omega Bio-Tek, USA); pEASY-Uni Seamless Cloning and Assembly Kit (Beijing Quanjin Biotechnology Co., Ltd.); 2×EasyTaq PCR SuperMix(+dye) (Beijing Quanjin Biotechnology Co., Ltd.); EasyPure Genomic DNA Kit (Beijing Quanjin Biotechnology Co., Ltd.); Phusion high-fidelity Master Mix (NEB, USA); BsaI (NEB, USA); BamHI-HF (NEB, USA); T4 DNA Ligase (NEB, USA); SD-Ura yeast culture medium (Beijing Fanjinuo Technology Co., Ltd.); SD-Leu-Ura yeast culture medium (Beijing Fanjinuo Technology Co., Ltd.); Frozen-EZ Yeast Transformation II Kit TM Zymo Research, a biotechnology company;

[0028] YPD solid plates: 1% yeast extract + 2% peptone + 2% glucose + 1.5% agar; without agar, it becomes the corresponding liquid medium (YPD liquid medium);

[0029] YPL induction medium: 1% yeast extract + 2% peptone + 2% galactose;

[0030] SD-Ura solid plates: SD-Ura + 2% glucose + 2% agar; without agar, it becomes the corresponding liquid culture medium (SD-Ura liquid culture medium);

[0031] SD-Ura-Leu solid plates: SD-Ura-Leu + 2% glucose + 2% agar; without agar, it becomes the corresponding liquid culture medium (SD-Ura-Leu liquid culture medium).

[0032] Example 1: Construction of recombinant yeast chassis

[0033] Using the increase of GPP, a precursor of dextrorotatory borneol, as an indicator, the *Saccharomyces cerevisiae* CEN.PK2-1D was modified. The modification method is referenced in the following literature: (Jianga GZ, Yaoa MD, Wanga Y, et al. Manipulation of GES and ERG20 for geraniol overproduction in *Saccharomyces cerevisiae*[J]. Metabolic Engineering, 2017, 41:57-66.)(Xiao-Jing G, Wen-Hai X, Ying W, et al. Metabolic engineering of *Saccharomyces cerevisiae* for 7-dehydrocholesterol overproduction[J]. Biotechnology for biofuels, 2018, 11(1):192.). (Information on the recombinant yeast chassis strain of this invention is shown in Table 1.)

[0034] 1. Construction of yeast strains

[0035] The initial strain used in this study was CEN.PK2-1D from *Saccharomyces cerevisiae* (Table 1). All endogenous genes in the MVA pathway, ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19, IDI1, and ERG20, were derived from the genomic DNA of CEN.PK2-1D. The ERG20 mutant used in this study was ERG20. F96W-N127WIt has been reported to have higher monoterpene production efficiency. The gene expression cassette was integrated into the yeast chromosome using the M2S integration method (Li S, Ding W, Zhang X, et al. Development of amodularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae[J]. Biotechnology for Biofuels,2016,30(1):232-243.). Simply put, ERG10 and ERG13 were amplified by adding BsaI restriction sites, and the head-to-head promoter (pGAL1-pGAL10) was ligated into the termination vector T1-(TPI1-PGI1) to generate plasmid T1-(ERG10-ERG13). Two terminators were inserted into the plasmid, and homologous arms L1 and L2 were designed in the terminators, respectively. Similarly, T2-(tHMG1-tHMG1), T3-(tHMG1-ERG12), T4-(ERG8-ERG19), and T5-(IDI1-ERG20) F96W-N127W They each have specific homologous arms L2 and L3, L3 and L4, L4 and L5, and L5 and L6, respectively.

[0036] Each expression module with a homologous arm was amplified separately. In this study, the exogenous gene was constructed at locus 15 (YPRCΔ15) in the *Saccharomyces cerevisiae* genome, using the Ura selection marker. The upstream homologous arm YPRCΔ15-UP was amplified from CEN.PK2-1D genomic DNA; the URA3 module containing the promoter was amplified from the pESC-URA vector; and L1 was amplified from the T1 vector. The three modules were assembled using overlap PCR to form the selection marker module YPRCΔ15up-ura3-L1. The downstream homologous arm YPRCΔ15DOWN was amplified from CEN.PK2-1D genomic DNA, and L6 was amplified from the T5 vector. These were then combined to generate the downstream homologous arm module L6-YPRCΔ15.

[0037] All modules were electroporated into CEN.PK2-1D for assembly and integration. The transformation product was dropped into the center of a defective SD-Ura solid plate and spread evenly using a spreader until the entire bacterial culture was absorbed. The plate was then incubated upside down at 30°C for 2-3 days. Single colonies were picked for sequencing verification, yielding a positive strain MD (see attached image). Figure 1 ).

[0038] 2. Fermentation

[0039] To determine the GPP production capacity of the MD strain, fermentation assays were performed. The detailed procedure is as follows:

[0040] (1) Pick a single colony of MD that has grown on the SD-Ura solid plate and place it in 10 mL of SD-Ura liquid culture medium, 30℃, 200 rpm for 48 h;

[0041] (2) Collect the cells by centrifugation at 5000g for 5 min at room temperature, transfer them to 10mL YPL induction medium, and induce culture at 30℃ and 200rpm for 48h to obtain the fermentation product.

[0042] 3. Extraction of fermentation products

[0043] The target component is a terpenoid compound, which is lipid-soluble and readily soluble in ethyl acetate. Therefore, ethyl acetate was selected as the solvent to extract the fermentation product and obtain the target compound. The extraction steps are as follows:

[0044] (1) After fermentation, take 5 ml of fermentation liquid, add an equal volume of ethyl acetate and sonicate for 1 h. During this time, mix by inverting the mixture every 10 minutes and add ice to keep the sonication temperature at 10°C to prevent product volatilization.

[0045] (2) Centrifuge at 13,000g for 10 minutes, remove the upper organic phase, add an appropriate amount of anhydrous sodium sulfate (dried at 120℃ for 30 minutes), and shake while adding to remove the water from the extract;

[0046] (3) Transfer to a liquid chromatography vial, seal, and use for GC-MS detection.

[0047] 4. GC-MS detection of fermentation products

[0048] GC-MS analysis conditions were as follows: column: TR-5ms (30m × 0.25mm); injection: 1 μL sample; splitless mode; incubation: 50℃ for 2 min; 5℃·min -1 Raise to 230℃, hold for 5 minutes, then increase by 10℃·min. -1 The temperature was raised to 300℃ and held for 2 minutes; the injection port temperature and transfer line temperature were both 280℃, and the electron energy was 70 eV. The sample was scanned in the range of 50-300 m / z. A standard curve was constructed using geraniol as a control, and the geraniol content in the sample was determined.

[0049] In the MD strain, the geraniol yield was detected to be 12.52 mg·L⁻¹. -1 (Appendix) Figure 2 ).

[0050] Example 2 Construction of CbTPS1 eukaryotic expression vector

[0051] The monoterpene synthase CbTPS1 cloned from *Cinnamomum camphora* (see prior Chinese Patent 201911082325.8, all contents of which are incorporated herein by reference) was improved according to the codon preference of *Saccharomyces cerevisiae*. The improvement method is a conventional technique in the art. An exemplary improved codon sequence of the monoterpene synthase gene is shown in SEQ ID NO: 1. The improved monoterpene synthase gene was then used to design homologous arm primers and constructed into the BamHI site of the eukaryotic expression vector pESC-LEU via homologous recombination. The specific operation is as follows:

[0052] (1) Primers CbTPS1-F' and CbTPS1-R' with homologous arms containing the BamHI site were designed to amplify the monoterpene synthase CbTPS1 by PCR from the pET32a::CbTPS1 plasmid (Chinese Patent 201911082325.8). The amplified product was then recovered and purified. The primer sequences are shown in Table 2 (underlined sequences indicate vector homologous regions):

[0053] (2) Take the pESC-LEU vector (Agilent Technologies), digest it with the restriction endonuclease BamHI, and recover the linearized vector backbone.

[0054] (3) Take the purified PCR product obtained in step 1) and clone it into the linearized vector backbone of step 2) according to the instructions of Beijing TransGen Biotech Co., Ltd. pEASY-UniSeamless Cloning and Assembly Kit to obtain the recombinant plasmid pESC-LEU::CbTPS1.

[0055] The recombinant plasmid pESC-LEU::CbTPS1 was transformed into Escherichia coli Trans5α competent cells, plated on LB agar plates, and identified by positive clone PCR (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain the pESC-LEU::CbTPS1 recombinant bacteria. The pESC-LEU::CbTPS1 recombinant plasmid was extracted using the EZNATM plasmid mini kit I (Omega Bio-Tek).

[0056] Example 3: Fermentation of dextrorotatory borneol

[0057] The constructed pESC-LEU::CbTPS1 recombinant plasmid was transformed into MD strain, and the yield of dextrorotatory borneol was detected by fermentation. The specific operation is as follows:

[0058] 1. Preparation of MD yeast competent cells

[0059] Yeast competent cells were prepared using the ZYMO RESEARCH Frozen-EZ Yeast Transformation II kit:

[0060] (1) Pick a single colony of newly activated MD yeast from the SD-Ura solid plate, inoculate it into 10 mL of SD-Ura liquid medium, and culture it with shaking at 30 °C until the OD600 = 0.8-1.0;

[0061] (2) Centrifuge at 500g for 4 minutes at room temperature, and discard the supernatant;

[0062] (3) Add 10 mL of Frozen-EZ Solution 1 to suspend the bacterial cells, centrifuge at 500 g for 4 min at room temperature, and discard the supernatant;

[0063] (4) Add 1 mL of Frozen-EZ Solution 2 to suspend the bacterial cells to obtain BY-Mono yeast competent cells, and dispense 50 μL into sterile 1.5 mL EP tubes;

[0064] (5) Slowly cool to -70℃ (4℃, 1h; -20℃, 1h; -40℃, 1h; -70℃ for storage), and do not use liquid nitrogen to quickly freeze competent cells.

[0065] 2. Recombinant plasmid pESC-Leu::CbTPS1 was transformed into MD yeast competent cells.

[0066] (1) Take 0.2-1 μg of recombinant plasmid pESC-Leu::CbTPS1 (less than 5 μL) and mix it with 50 μL of BY-Mono yeast competent cells;

[0067] (2) Add 500 μL of Frozen-EZ Solution 3 and mix vigorously;

[0068] (3) Incubate at 30℃ for 1-2 hours, mixing 2-3 times during the process;

[0069] (4) Take 50-150 μL of the incubated bacterial solution, spread it on an SD-Ura-Leu solid plate, air-dry it, and then incubate it upside down at 30℃ for 48 h to obtain recombinant yeast transformed with the recombinant plasmid pESC-Leu::CbTPS1, which is named MD-1. Fermentation and detection of dextrorotatory borneol products are described in Example 1. The initial strain obtained from fermentation contained 30 μg·L⁻¹. -1 Right-handed borneol (with) Figure 3 ).

[0070] Example 4: Protein structure optimization of the high-yield module for dextrorotatory borneol

[0071] In this invention, the obtained yeast codon-optimized CbTPS1 was further modified. The chloroplast transport peptide (C37) at the N-terminus of protein CbTPS1 was truncated. Simultaneously, gene expression increased when the start codon ATG directly followed by UCU. Since S10 (TCC) and S32 (TCA) are both serine residues, consistent with the amino acid encoded by UCU, truncation experiments were also performed at S10 and S32, and the amino acid was mutated to TCT, thereby obtaining three truncated proteins: t10-CbTPS1, t32-CbTPS1, and t37-CbTPS1. The nucleic acid sequences encoding the truncated proteins t10-CbTPS1, t32-CbTPS1, and t37-CbTPS1 are shown in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, respectively. The truncated proteins were introduced into MD strains to obtain new strains (Table 1). On the other hand, based on the truncated protein, the yeast-specific Kozak sequence (AAAAAA) was incorporated before the truncated ATG. The specific procedure is as follows:

[0072] (1) Shortened primers were designed (see Table 2). The monoterpene synthase CbTPS1 was amplified by PCR from the pESU-LEU::CbTPS1 plasmid, and the purified PCR product was recovered and purified. The primer sequences are shown in Table 2 (underlined sequences represent vector homologous regions):

[0073] (2) Take the pESC-LEU vector (Agilent Technologies), digest it with the restriction endonuclease BamHI, and recover the linearized vector backbone.

[0074] (3) Take the purified PCR product obtained in step 1) and clone it into the linearized vector backbone of step 2) according to the instructions of Beijing TransGen Biotech Co., Ltd. pEASY-UniSeamless Cloning and Assembly Kit to obtain the recombinant plasmid pESC-LEU::CbTPS1.

[0075] The recombinant plasmids pESC-LEU::t10-CbTPS1, pESC-LEU::t32-CbTPS1, pESC-LEU::t37-CbTPS1, pESC-LEU::CbTPS1K, pESC-LEU::t10-CbTPS1K, pESC-LEU::t32-CbTPS1K, and pESC-LEU::t37-CbTPS1K were transformed into Escherichia coli Trans5α competent cells, plated on LB agar plates, and identified by positive clone PCR (purchased from Beijing TransGen Biotech Co., Ltd.) to obtain recombinant bacteria. The recombinant plasmids were then extracted using the EZNATM plasmid mini kit I (Omega Bio-Tek).

[0076] The recombinant plasmid was transformed into MD strains according to the method in Example 3, resulting in new strains MD-2, MD-3, MD-4, MD-5, MD-6, MD-7, and MD-8. Fermentation and extraction were then performed, and the yields are shown in the attached figure. Figure 4 As shown in the figure. The results showed that the production of dextrorotatory borneol was significantly increased in all strains of the truncated fusion protein, with strains MD-3, MD-5, and MD-7 producing 1.48 mg·L⁻¹. -1 2.16 mg·L -1 and 1.53 mg·L -1 Furthermore, the yields of strains MD-4, MD-5, and MD-8, optimized using the Kozak sequence, were also increased, reaching 1.67 mg·L⁻¹. -1 2.89 mg·L -1 and 1.75 mg·L -1 .

[0077] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. sequence list <110> Sichuan Hongda Pharmaceutical Co., Ltd. <120> A recombinant yeast strain for producing dextrorotatory borneol <130> KH20201127 <160> 12 <170> SIPOSequenceList 1.0 <210> 1 <211> 1812 <212> DNA <213> Artificial Sequence <400> 1 atggccttgc aaatgaccgt cccattcttg tcctccttct tgccaaaccc aagacacaga 120. ccaacagctc acggtttcat tccacagga agggtttcca agcacatctc ttgctctacc 180. accactc cctactctac accgttc aggagatccg gcaactac gccatccatt tgggactacg atttcgtcca atccttggtg tccgactaca aggttgaagc tcacggtact 240 agggtcgaaa agttgaagga ggtcgtcaag aacttgttga aggaaactga ttcctcccta gctcaaatgg agttgatcga cagcttgcac aggttggggag ttaggtggtt gttcgagaac gagatcaagc aggtgttgta caccgtctct tccgacaaca cctccatcga gatgaagaag gatttgcacg ccgtctctac cagattcaga ttgttgagac agcacggctt caaagtttct 480 acagacgtgt tcaacgactt cgaggacgaa aagggttgct tcaagccatc cttgtccatg 540 gcatcaagg gcatgttgag cttgtacgag gcttctcatt tggccttcca aggcgaaact atcttggacg aagccagagc tttcactcac gctcacttga tgggcatcaa ggagaacatc 660 gacccaatca tccacaagaa ggtcgaacac gctttggaca tgccattgca ttggaggttg 720 gagaagttgg aagccaggtg gtacatggac atgtacatga gggaggaggg tatgaacagc 780 agcttgttgg agttggccat gttgcacttc aacatcgtcc aagccacctt ccaaaccaac 840 ttgaagtccc taagcaggtg gtggaaggat ttgggtttgg gcgaacagtt gtccttctct 900 agagacaggc tagtcgagtg cttcttttgg gctgcagcta tgacttccga accacaattc 960 ggtaggtgtc aagaagccgt tgctaaggtt gtccaattga ccactaccat cgacgacatc 1020 tacgacgttt acggtaccgt tgacgagttg gaattgttca ccaacgccgt tgataggtgg 1080 gatttggaag ctatggagca gttgccagag tacatgaaga cttgcttctt ggccttgtac 1140 aactccatca acgagatcgg ttacggcatc ttgaaggagc aaggtaggaa cgtcatccca 1200 tacttgagga acgcttggac cgaactttgc aaggcctacc tagtcgaagc taagtggtac 1260 tcttccggtt acaccccaat gttggaagag ttcttgcaga cctcttggat ctcagtcggt 1320 tctttgccaa tgcagaccta cgctttcgct ttgttgggcc aaaacttggc tccagagtct tgcgatttcg ccgaacaaat ctccgacatc ttgccattgg ccggtatgtt gatcaggttc ccagacgatt tgggtacttc cttggacgaa ttgaagagag gcgacgttcc aaagtccatc cagtgttaca tgcacgaagc aggcgttaca gaagacgttg ctagagatca tatcatgggt ttgttcaggg agacttgga gagttgac gagtacttgg tcgaatcctc tatcccacac gctttcatcg accaagctat gaacttgggc aggtctcct attgcaccta caaacacggc 1680. gacggtttct cagacggttt tggagatcca ggttcccagg agaagagat gtacatgtcc 1740. ttgttcgtcg agccaatcca agttgacgag gctaaaggca tctccttttg cgttgacggt ggttacgctt of 1812. <210> 2 <211> 1785 <212> DNA <213> Artificial Sequence <400> 2 atgtcttcct tcttgccaaa cccaagacac agaccaacag ctcacggttt cattccacag gaaagggttt ccaagcacat ctcttgctct accaccactc caacctactc tactaccgtt ccaaggagat ccggcaacta caagccatcc atttgggact acgatttcgt ccaatccttg 180 gtgtccgact acaaggttga agctcacggt actagggtcg aaaagttgaa ggaggtcgtc 240 aagaacttgt tgaaggaaac tgattcctcc ctagctcaaa tggagttgat cgacagcttg 300 cacaggttgg gagttaggtg gttgttcgag aacgagatca agcaggtgtt gtacaccgtc 360 tcttccgaca acacctccat cgagatgaag areatttgc acgccgtctc taccagattc 420 480 gaaaagggtt gcttcaagcc atccttgtcc atggacatca agggcatgtt gagcttgtac 540 gaggctttc atttggcctt ccaaggcgaa actatcttgg acgaagccag agctttcact 600 cacgctcact tgatgggcat caaggaaac atcgacccaa tcatccacaa gaaggtcgaa 660 cacgctttgg acatgccatt gcattggagg ttggagaagt tggaagccag gtggtacatg 720 gacatgtaca tgagggaga gggtatgaac agcagcttgt tggagttggc catgttgcac 780 ttcaacatcg tccaagccac cttccaaacc aacttgaagt ccctaagcag gtggtggaag 840 gatttgggtt tgggcgaaca gttgtccttt tctagagaca ggctagttcga gtgcttcttt 900 tgggctgcag ctatgacttc cgaaccacaa ttcggtaggt gtcaagaagc cgttgctaag 960 gttgtccaat tgaccactac catcgacgac atctacgacg tttacggtac cgttgacgag 1020 ttggaattgt tcaccaacgc cgttgatagg tgggatttgg aagctatgga gcagttgcca 1080 gagtacatga agacttgctt cttggccttg tacaactcca tcaacgagat cggttacggc 1140 atcttgaagg agcaagtag gaacgtcatc ccatacttga ggaacgcttg gaccgaactt 1200 tgcaaggcct acctagtcga agctaagtgg tactcttccg gttacacccc aatgttggaa 1260 gagttcttgc agacctcttg gatctcagtc ggttctttgc caatgcagac ctacgctttc 1320 gctttgttgg gccaaaactt ggctccagag tcttgcgatt tcgccgaaca aatctccgac 1380 atcttgccat tggccggtat gttgatcagg ttcccagacg atttgggtac ttccttggac 1440 Gaattgaga gaggcgacgt tccaaagtcc atccagtgtt acatgcacga agcaggcgtt 1500 acagaagcg ttgctagaga tcatatcatg ggtttgttca gggagacttg gaagaagttg 1560 aacgagtact tggtcgaatc ctctatccca cacgctttca tcgaccaagc tatgaacttg 1620 ggcagagtct cctattgcac ctacaaacac ggcgacggtt tctcagacgg ttttggagat 1680 ccaggttccc aggagaagaa gatgtacatg tccttgttcg tcgagccaat ccaagttgac 1740 gaggctaaag gcatctcctt ttgcgttgac ggtggttacg cttga 1785 <210> 3 <211> 1719 <212> DNA <213> Artificial Sequence <400> 3 atgtctaagc acatctcttg ctctaccacc actccaacct actctactac cgttccaagg 60 agatccggca actacaagcc atccatttgg gactacgatt tcgtccaatc cttggtgtcc 120 gactacaagg ttgaagctca cggtactagg gtcgaaaagt tgaaggaggt cgtcaagaac 180 ttgttgaagg aaactgattc ctccctagct caaatggagt tgatcgacag cttgcacagg 240 ttgggagtta ggtggttgtt cgagaacgag atcaagcagg tgttgtacac cgtctcttcc 300 gacaacacct ccatcgagat gaagaaggat ttgcacgccg tctctaccag attcagattg 360 ttgagacagc acggcttcaa agtttctaca gacgtgttca acgacttcga ggacgaaaag 420 ggttgcttca agccatcctt gtccatggac atcaagggca tgttgagctt gtacgaggct 480 tctcatttgg ccttccaagg cgaaactatc ttggacgaag ccagagcttt cactcacgct 540 cacttgatgg gcatcaagga gaacatcgac ccaatcatcc acaagaaggt cgaacacgct 600 ttggacatgc cattgcattg gaggttggag aagttggaag ccaggtggta catggacatg 660 tacatgaggg aggagggtat gaacagcagc ttgttggagt tggccatgtt gcacttcaac 720 atcgtccaag ccaccttcca aaccaacttg aagtccctaa gcaggtggtg gaaggatttg 780 ggtttgggcg aacagttgtc cttctctaga gacaggctag tcgagtgctt cttttgggct 840 gcagctatga cttccgaacc acaattcggt aggtgtcaag aagccgttgc taaggttgtc 900 caattgacca ctaccatcga cgacatctac gacgtttacg gtaccgttga cgagttggaa 960 ttgttcacca acgccgttga taggtgggat ttggaagcta tggagcagtt gccagagtac 1020 atgaagactt gcttcttggc cttgtacaac tccatcaacg agatcggtta cggcatcttg 1080 aaggagcaag gtaggaacgt catcccatac ttgaggaacg cttggaccga actttgcaag 1140 gcctacctag tcgaagctaa gtggtactct tccggttaca ccccaatgtt ggaagagttc 1200 ttgcagacct cttggatctc agtcggttct ttgccaatgc agacctacgc tttcgctttg 1260 ttgggccaaa acttggctcc agagtcttgc gatttcgccg aacaaatctc cgacatcttg 1320 ccattggccg gtatgttgat caggttccca gacgatttgg gtacttcctt ggacgaattg 1380 aagagaggcg acgttccaaa gtccatccag tgttacatgc acgaagcagg cgttacagaa 1440 gacgttgcta gagatcatat catgggtttg ttcagggaga cttggaagaa gttgaacgag 1500 tacttggtcg aatcctctat cccacacgct ttcatcgacc aagctatgaa cttgggcaga 1560 gtctcctatt gcacctacaa acacggcgac ggtttctcag acggttttgg agatccaggt 1620 tcccaggaga agaagatgta catgtccttg ttcgtcgagc caatccaagt tgacgaggct 1680 aaaggcatct ccttttgcgt tgacggtggt tacgcttga 1719 <210> 4 <211> 1704 <212> DNA <213> Artificial Sequence <400> 4 atgtgctcta ccaccactcc aacctactct actaccgttc caaggagatc cggcaactac 60 aagccatcca tttgggacta cgatttcgtc caatccttgg tgtccgacta caaggttgaa 120 180 gattcctccc tagctcaaat ggagttgatc gacagcttgc acaggttggg agttaggtgg 240 ttgttcgaga acgagatcaa gcaggtgttg tacaccgtct cttccgacaa cacctccatc 300 your gagat aggatttgca cgccgtctct accagattca gattgttgag acagcacggc 360 ttcaaagttt ctacagacgt gttcaacgac ttcgaggacg aaaagggttg cttcaagcca 420 tccttgtcca tggacatcaa gggcatgttg agcttgtacg aggcttctca tttggccttc 480 540 aaggaaca tcgacccaat catccacaag aaggtcgaac acgctttgga catgccattg 600 cattggagagt tggagaagtt ggaagccagg tggtacatgg acatgtacat gaggaggag 660 ggtatgaaca gcagcttgtt ggagttggcc atgttgcact tcaacatcgt ccaagccacc 720 ttccaaacca acttgaagtc cctaagcagg tggtggaagg atttgggttt gggcgaacag 780 ttgtccttct ctagagacag gctagtcgag tgcttctttt gggctgcagc tatgacttcc 840 gaaccacaat tcggtaggtg tcaagaagcc gttgctaagg ttgtccaatt gaccactacc 900 atcgacgaca tctacgacgt ttacggtacc gttgacgagt tggaattgtt caccaacgcc 960 gttgataggt gggatttgga agctatggag cagttgccag agtacatgaa gacttgcttc 1020 ttggccttgt acaactccat caacgagatc ggttacggca tcttgaagga gcaaggtagg 1080 aacgtcatcc catacttgag gaacgcttgg accgaacttt gcaaggccta cctagtcgaa 1140 gctaagtggt actcttccgg ttacacccca atgttggaag agttcttgca gacctcttgg 1200 atctcagtcg gttctttgcc aatgcagacc tacgctttcg ctttgttggg ccaaaacttg 1260 gctccagagt cttgcgattt cgccgaacaa atctccgaca tcttgccatt ggccggtatg 1320 ttgatcaggt tcccagacga tttgggtact tccttggacg aattgaagag aggcgacgtt 1380 ccaaagtcca tccagtgtta catgcacgaa gcaggcgtta cagaagacgt tgctagagat 1440 catatcatgg gtttgttcag ggagacttgg aagaagttga acgagtactt ggtcgaatcc 1500 tctatcccac acgctttcat cgaccaagct atgaacttgg gcagagtctc ctattgcacc 1560 tacaaacacg gcgacggttt ctcagacggt tttggagatc caggttccca ggagaagaag 1620 atgtacatgt ccttgttcgt cgagccaatc caagttgacg aggctaaagg catctccttt 1680 tgcgttgacg gtggttacgc ttga 1704 <210> 5 <211> 1197 <212> DNA <213> Artificial Sequence <400> 5 atgtctcaga acgtttacat tgtatcgact gccagaaccc caattggttc attccagggt 60 tctctatcct ccaagacagc agtggaattg ggtgctgttg ctttaaaagg cgccttggct 120 aaggttccag aattggatgc atccaaggat tttgacgaaa ttatttttgg taacgttctt 180 tctgccaatt tgggccaagc tccggccaga caagttgctt tggctgccgg tttgagtaat 240 catatcgttg caagcacagt taacaaggtc tgtgcatccg ctatgaaggc aatcattttg 300 ggtgctcaat ccatcaaatg tggtaatgct gatgttgtcg tagctggtgg ttgtgaatct 360 atgactaacg caccatacta catgccagca gcccgtgcgg gtgccaaatt tggccaaact 420 gttcttgttg atggtgtcga aagagatggg ttgaacgatg cgtacgatgg tctagccatg 480 ggtgtacacg cagaaaagtg tgcccgtgat tgggatatta ctagagaaca acaagacaat 540 tttgccatcg aatcctacca aaaatctcaa aaatctcaaa aggaaggtaa attcgacaat 600 gaaattgtac ctgttaccat taagggattt agaggtaagc ctgatactca agtcacgaag 660 gacgaggac ctgctagatt acacgttgaa aaattgagat ctgcaaggac tgttttccaa 720 aaagaaaacg gtactgttac tgccgctaac gcttctccaa tcaacgatgg tgctgcagcc 780 gtcatcttgg tttccgaaaa agttttgaag gaaagaatt tgaagccttt ggctattatc 840 aaaggttgg gtgaggccgc tcatcaacca gctgatttta catgggctcc atctcttgca 900 gttccaaagg ctttgaaaca tgctggcatc gaagacatca attctgttga ttactttgaa 960 ttcaatgaag ccttttcggt tgtcggtttg gtgaacacta agattttgaa gctagaccca 1020 tctaaggtta atgtatatgg tggtgctgtt gctctaggtc acccattggg ttgttctggt 1080 gctagagtgg ttgttacact gctatccatc ttacagcaag aaggaggtaa gatcggtgtt 1140 gccgccattt gtaatggtgg tggtggtgct tcctctattg tcattgaaaa gatatga 1197 <210> 6 <211> 1476 <212> DNA <213> Artificial Sequence <400> 6 atgaaactct caactaaact ttgttggtgt ggtattaaag gaagacttag gccgcaaaag 60 caacaacaat tacacaatac aaacttgcaa atgactgaac taaaaaaaca aaagaccgct 120 gaacaaaaaa ccagacctca aaatgtcggt attaaaggta tccaaattta catcccaact 180 caatgtgtca accaatctga gctagagaaa tttgatggcg tttctcaagg taaatacaca 240 attggtctgg gccaaaccaa catgtctttt gtcaatgaca gagaagatat ctactcgatg 300 tccctaactg ttttgtctaa gttgatcaag agttacaaca tcgacaccaa caaaattggt 360 agattagaag tcggtactga aactctgatt gacaagtcca agtctgtcaa gtctgtcttg 420 atgcaattgt ttggtgaaaa cactgacgtc gaaggtattg acacgcttaa tgcctgttac 480 ggtggtacca acgcgttgtt caactctttg aactggattg aatctaacgc atgggatggt 540 agagacgcca ttgtagtttg cggtgatatt gccatctacg ataagggtgc cgcaagacca accggtggtg ccggtactgt tgctatgtgg atcggtcctg atgctccaat tgtatttgac 660 tctgtaagag cttcttacat ggaacacgcc tacgattttt acaagccaga tttcaccagc gaatcctt acgtcgatgg tcatttttca ttaacttgtt acgtcaaggc tcttgatcaa gtttacaaga gttattccaa gaaggctatt tctaaagggt tggttagcga tcccgctggt tcggatgctt tgaacgtttt gaaatatttc gactacaacg ttttccatgt tccaacctgt aaattggtca caaaatcata cggtagatta ctataacg atttcagagc caatcctcaa ttgttcccag aagttgacgc cgaattagct actcgcgatt atgacgaatc tttaaccgat aagaacattg aaaaaacttt tgttaatgtt gctaagccat tccacaaaga gagagttgcc caatctttga ttgttccaac aaacacaggt aacatgtaca ccgcatctgt ttatgccgcc tttgcatctc father tgttggatct gacgacttac aaggcaagcg tgttggttta ttttcttacg gttccggttt agctgcatct ctatattctt gcaaaattgt tggtgacgtc 1260 caacatatta tcaaggaatt agatattact aacaaattag ccaagagaat caccgaaact 1320 ccaaaggatt acgaagctgc catcgaattg agagaaaatg cccatttgaa gaagaacttc 1380 aaacctcaag gttccattga gcatttgcaa agtggtgttt actacttgac caacatcgat 1440 gacaaattta gaagatctta cgatgttaaa aaataa 1476 <210> 7 <211> 1584 <212> DNA <213> Artificial Sequence <400> 7 atggctgcag accaattggt gaaaactgaa gtcaccaaga agtcttttac tgctcctgta 60 caaaaggctt ctacaccagt tttaaccaat aaaacagtca tttctggatc gaaagtcaaa 120 agtttatcat ctgcgcaatc gagctcatca ggaccttcat catctagtga ggaagatgat 180 tcccgcgata ttgaaagctt ggataagaaa atacgtcctt tagaagaatt agaagcatta 240 ttaagtagtg gaaatacaaa acaattgaag aacaaagagg tcgctgcctt ggttattcac 300 ggtaagttac ctttgtacgc tttggagaaa aaattaggtg atactacgag agcggttgcg 360 gtacgtagga aggctctttc aattttggca gaagctcctg tattagcatc tgatcgttta 420 ccatataaaa attatgacta cgaccgcgta tttggcgctt gttgtgaaaa tgttataggt 480 tacatgcctt tgcccgttgg tgttataggc cccttggtta tcgatggtac atcttatcat 540 ataccaatgg caactacaga gggttgtttg gtagcttctg ccatgcgtgg ctgtaaggca 600 atcaatgctg gcggtggtgc aaactgtt ttaactaagg atggtatgac aagaggccca 660 gtagtccgtt tcccaacttt gaaaagatct ggtgcctgta agatatggtt agactcagaa 720 gagggacaaa acgcaattaa aaaagctttt aactctacat caagatttgc acgtctgcaa 780 catattcaaa cttgtctagc aggagattta ctcttcatga gatttagaac aactactggt 840 gacgcaatgg gtatgaatat gatttctaaa ggtgtcgaat actcattaaa gcaaatggta 900 gaagagtatg gctgggaaga tatggaggtt gtctccgttt ctggtaacta ctgtaccgac 960 aaaaaaccag ctgccatcaa ctggatcgaa ggtcgtggta agagtgtcgt cgcagaagct 1020 actattcctg gtgatgttgt cagaaaagtg ttaaaaagtg atgtttccgc attggttgag 1080 ttgaacattg ctaagaattt ggttggatct gcaatggctg ggtctgttgg tggatttaac 1140 gcacatgcag ctaatttagt gacagctgtt ttcttggcat taggacaaga tcctgcacaa 1200 aatgttgaaa gttccaactg tataacattg atgaaagaag tggacggtga tttgagaatt 1260 tccgtatcca tgccatccat cgaagtaggt accatcggtg gtggtactgt tctagaacca 1320 caaggtgcca tgttggactt attaggtgta agaggcccgc atgctaccgc tcctggtacc 1380 aacgcacgtc aattagcaag aatagttgcc tgtgccgtct tggcaggtga attatcctta 1440 tgtgctgccc tagcagccgg ccatttggtt caaagtcata tgacccacaa caggaaacct 1500 gctgaaccaa caaaacctaa caatttggac gccactgata taaatcgttt gaaagatggg 1560 tccgtcacct gcattaaatc ctaa 1584 <210> 8 <211> 1332 <212> DNA <213> Artificial Sequence <400> 8 atgtcattac cgttcttaac ttctgcaccg ggaaaggtta ttatttttgg tgaacactct 60 gctgtgtaca acaagcctgc cgtcgctgct agtgtgtctg cgttgagaac ctacctgcta 120 ataagcgagt catctgcacc agatactatt gaattggact tcccggacat tagctttaat 180 cataagtggt ccatcaatga tttcaatgcc atcaccgagg atcaagtaaa ctcccaaaaa 240 ttggccaagg ctcaacaagc caccgatggc ttgtctcagg aactcgttag tctttttggat 300 ccgttgttag ctcaactatc cgaatccttc cactaccatg cagcgttttg tttcctgtat 360 atgtttgttt gcctatgccc ccatgccaag aatattaagt tttctttaaa gtctacttta 420 cccatcggtg ctgggttggg ctcaagcgcc tctatttctg tatcactggc cttagctatg 480 gcctacttgg gggggttaat aggatctaat gacttggaaa agctgtcaga aaacgataag 540 catatagtga atcaatgggc cttcataggt gaaaagtgta ttcacggtac cccttcagga 600 atagataacg ctgtggccac ttatggtaat gccctgctat ttgaaaaaga ctcacataat 660 ggaacaataa acacaaacaa ttttaagttc ttagatgatt tcccagccat tccaatgatc 720 ctaacctata ctagaattcc aaggtctaca aaagatcttg ttgctcgcgt tcgtgtgttg 780 gtcaccgaga aatttcctga agttatgaag ccaattctag atgccatggg tgaatgtgcc 840 ctacaaggct tagagatcat gactaagtta agtaaatgta aaggcaccga tgacgaggct 900 gtagaaacta ataatgaact gtatgaacaa ctattggaat tgataagaat aaatcatgga 960 ctgcttgtct caatcggtgt ttctcatcct ggattagaac ttattaaaaa tctgagcgat 1020 gatttgagaa ttggctccac aaaacttacc ggtgctggtg gcggcggttg ctctttgact 1080 ttgttacgaa gagacattac tcaagagcaa attgacagct tcaaaaagaa attgcaagat 1140 gattttagtt acgagacatt tgaaacagac ttgggtggga ctggctgctg tttgttaagc 1200 gcaaaaaatt tgaataaaga tcttaaaatc aaatccctag tattccaatt atttgaaaat 1260 aaaactacca caaagcaaca aattgacgat ctattattgc caggaaacac gaatttacca 1320 tggacttcat aa 1332 <210> 9 <211> 1356 <212> DNA <213> Artificial Sequence <400> 9 atgtcagagt tgagagcctt cagtgcccca gggaaagcgt tactagctgg tggatattta 60 gttttagata caaaatatga agcatttgta gtcggattat cggcaagaat gcatgctgta 120 gcccatcctt acggttcatt gcaagggtct gataagttttg aagtgcgtgt gaaaagtaaa 180 caatttaaag atggggagtg gctgtaccat ataagtccta aaagtggctt cattcctgtt 240 tcgataggcg gatctaagaa ccctttcatt gaaaaagtta tcgctaacgt atttagctac 300 tttaaaccta acatggacga ctactgcaat agaaacttgt tcgttattga tattttctct 360 gatgatgcct accattctca ggaggatagc gttaccgaac atcgtggcaa cagaagattg 420 agttttcatt cgcacagaat tgaagaagtt cccaaaacag ggctgggctc ctcggcaggt 480 ttagtcacag ttttaactac agctttggcc tccttttttg tatcggacct ggaaaataat 540 gtagacaaat atagagaagt tattcataat ttagcacaag ttgctcattg tcaagctcag 600 ggtaaaattg gaagcgggtt tgatgtagcg gcggcagcat atggatctat cagatataga 660 agattcccac ccgcattaat ctctaatttg ccagatattg gaagtgctac ttacggcagt 720 aaactggcgc atttggttga tgaagaagac tggaatatta cgattaaaag taaccattta 780 ccttcgggat taactttatg gatgggcgat attaagaatg gttcagaaac agtaaaactg 840 gtccagaagg taaaaaattg gtatgattcg catatgccag aaagcttgaa aatatataca 900 gaactcgatc atgcaaattc tagatttatg gatggactat ctaaactaga tcgcttacac 960 gagactcatg acgattacag cgatcagata tttgagtctc ttgagaggaa tgactgtacc 1020 tgtcaaaagt atcctgaaat cacagaagtt agagatgcag ttgccacaat tagacgttcc 1080 tttagaaaaa taactaaaga atctggtgcc gatatcgaac ctcccgtaca aactagctta 1140 ttggatgatt gccagacctt aaaaggagtt cttacttgct taatacctgg tgctggtggt 1200 tatgacgcca ttgcagtgat tactaagcaa gatgttgatc ttagggctca aaccgctaat 1260 gacaaaagat tttctaaggt tcaatggctg gatgtaactc aggctgactg gggtgttagg 1320 aaagaaaaag atccggaaac ttatcttgat aaataa 1356 <210> 10 <211> 1191 <212> DNA <213> Artificial Sequence <400> 10 atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60 gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120 caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact 180 ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240 gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300 caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360 tccgctgctg gctttgctgc attggtttct gcaattgcta agttatacca attaccacag 420 tcaacttcag aaattctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480 tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540 gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc 600 gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660 ctatttaaag aaagaattga acatgcgta ccaaagagat ttgaagtcat gcgtaaagcc 720 attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780 catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt 840 atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt gcatttatct ataattgtt tggctctgtt cctggatggg acaagaaatt tactactgag cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a <210> 11 <211> 867 <212> DNA <213> Artificial Sequence <400> 11 atgactgccg acaacaatag tatgccccat ggtgcagtat ctagttacgc caaattagtg snow snow snow snow snow snow snow cover snow cover snow cover agcctaata cccgatctag tgagacgtca aatgacgaaa gcggagaaac atgtttttct ggtcatgatg aggagcaaat tagttaatg aatgaaaatt gtattgtttt ggattgggac gataatgcta ttggtgccgg taccaagaaa gtttgtcatt taatggaaaa tattgaaaag 300 ggtttactac atcgtgcatt ctccgtcttt attttcaatg aacaaggtga attactttta 360 caacaaagag ccactgaaaa aataactttc cctgatcttt ggactaacac atgctgctct 420 catccactat gtattgatga cgaattaggt ttgaagggta agctagacga taagattaag 480 ggcgctatta ctgcggcggt gagaaaacta gatcatgaat taggtattcc agaagatgaa 540 actaagacaa ggggtaagtt tcacttttta aacagaatcc attacatggc accaagcaat 600 gaaccatggg gtgaacatga aattgattac atcctatttt ataagatcaa cgctaaagaa 660 aacttgactg tcaacccaaa cgtcaatgaa gttagagact tcaaatgggt ttcaccaaat 720 gatttgaaaa ctatgtttgc tgacccaagt tacaagttta cgccttggtt taagattatt 780 tgcgagaatt acttattcaa ctggtgggag caattagatg acctttctga agtggaaaat 840 gacaggcaaa ttcatagaat gctataa 867 <210> 12 <211> 1059 <212> DNA <213> Artificial Sequence <400> 12 atggcttcag aaaaagaaat taggagagag agattcttga acgttttccc taaattagta 60 gaggaattga acgcatcgct tttggcttac ggtatgccta aggaagcatg tgactggtat 120 gcccactcat tgaactacaa cactccaggc ggtaagctaa atagaggttt gtccgttgtg 180 gacacgtatg ctattctctc caacaagacc gttgaacaat tggggcaaga agaatacgaa 240 aaggttgcca ttctaggttg gtgcattgag ttgttgcagg cttactggtt ggtcgccgat 300 gatatgatgg acaagtccat taccagaaga ggccaaccat gttggtacaa ggttcctgaa 360 gttggggaaa ttgccatctg ggacgcattc atgttagagg ctgctatcta caagcttttg 420 aaatctcact tcagaaacga aaaatactac atagatatca ccgaattgtt ccatgaggtc 480 accttccaaa ccgaattggg ccaattgatg gacttaatca ctgcacctga agacaaagtc 540 gacttgagta agttctccct aaagaagcac tccttcatag ttactttcaa gactgcttac 600 tattctttct acttgcctgt cgcattggcc atgtacgttg ccggtatcac ggatgaaaag 660 gatttgaaac aagccagaga tgtcttgatt ccattgggtg aatacttcca aattcaagat 720 gactacttag actgcttcgg taccccagaa cagatcggta agatcggtac agatatccaa 780 gataacaaat gttcttgggt aatcaacaag gcattggaac ttgcttccgc agaacaaaga 840 aagactttag acgaaaatta cggtaagaag gactcagtcg cagaagccaa atgcaaaaag 900 attttcaatg acttgaaaat tgaacagcta taccacgaat atgaagagtc tattgccaag 960 gatttgaagg ccaaaatttc tcaggtcgat gagtctcgtg gcttcaaagc tgatgtctta 1020 actgcgttct tgaacaaagt ttacaagaga agcaaatag 1059

Claims

1. A recombinant bacterium, characterized in that... The recombinant bacteria are yeast strains that contain or express borneol diphosphate synthase in vivo. The gene containing or expressing borneol diphosphate synthase in vivo has undergone codon optimization. The nucleic acid encoding the borneol diphosphate synthase in vivo is the nucleic acid described in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

2. The recombinant bacteria according to claim 1, characterized in that... The recombinant bacteria further includes the Kozak sequence.

3. The recombinant bacteria according to any one of claims 1-2, characterized in that... The yeast contains or expresses borneol diphosphate synthase, which is obtained by introducing the encoding nucleic acid of borneol diphosphate synthase into the yeast. And / or introducing the nucleic acid encoding borneol diphosphate synthase into the yeast is equivalent to introducing a cassette containing a nucleic acid encoding borneol diphosphate synthase into the yeast. And / or a nucleic acid expression cassette encoding borneol diphosphate synthase is introduced into the yeast via a vector expressing the nucleic acid expression cassette encoding borneol diphosphate synthase.

4. The recombinant bacteria according to claim 3, characterized in that... The yeast was introduced in plasmid form.

5. The recombinant bacteria according to any one of claims 1-2, characterized in that... The yeast further comprises the following genes: acetyl-CoA transferase gene ERG10, HMG-CoA synthase gene ERG13, HMG-CoA reductase gene tHMG1, mevalonate kinase gene ERG12, mevalonate phosphate kinase gene ERG8, mevalonate pyrophosphate decarboxylase gene ERG19, farnesyl pyrophosphate synthase gene ERG20, and isopentenyl pyrophosphate isomerase gene IDI1.

6. The recombinant bacteria according to claim 5, characterized in that... The ERG20 is a mutant ERG20. F96W-N127W .

7. The recombinant bacteria according to any one of claims 1-2, characterized in that... The yeast mentioned is brewer's yeast.

8. The recombinant bacteria according to claim 7, characterized in that... The yeast is CEN.PK2-1D.

9. The use of the recombinant bacteria according to any one of claims 1-8 in the production of dextrorotatory borneol.

10. A method for preparing dextrorotatory borneol, characterized in that the recombinant bacteria as described in any one of claims 1-8 are inoculated into a culture medium, fermented, and the fermented bacterial broth is extracted and separated to obtain the target product, dextrorotatory borneol.

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