Genetically engineered bacteria for improving yield of zeaxanthin and construction method and application thereof

Abstract

The application belongs to the field of genetic engineering and fermentation engineering, and discloses a genetic engineering bacterium for improving the yield of zeaxanthin and a construction method and application thereof. The application provides a cytoskeleton, which comprises (SH3)x-(GBD)y-(PDZ)z, wherein x=1 or 2, y=1 or 2, and z=1 or 2; the sequence of the PDZ is shown as SEQ ID NO. 54, the sequence of the GBD is shown as SEQ ID NO. 53, and the sequence of the SH3 is shown as SEQ ID NO. 52; the cytoskeleton can make the product more easily stored in a lipid droplet, reduce the toxicity of the product to cells, improve the yield of zeaxanthin, and be used for constructing the genetic engineering bacterium for improving the yield of zeaxanthin.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and fermentation engineering, specifically relating to a genetically engineered bacterium that increases maize xanthine yield, its construction method, and its application. Background Technology

[0002] Zeaxanthin is a natural lutein found in many plants and is an important food and feed additive as well as a precursor to pharmaceuticals. Clinically, zeaxanthin is used to treat age-related macular degeneration. In addition, zeaxanthin also has anti-cancer, antioxidant, and anti-radiation effects, and is used to treat cardiovascular aging and Alzheimer's disease.

[0003] The main methods for preparing zeaxanthin include chemical synthesis, plant extraction, and microbial synthesis. Chemical synthesis involves complex processes, high energy consumption, and significant pollution, and chemically synthesized zeaxanthin has low bioactivity, serving only as a pigment. The low content of zeaxanthin in plants leads to low yields for plant extraction. Microbial synthesis of zeaxanthin, however, offers advantages such as low cost and minimal pollution, making it considered the most promising production method. *Yarrowia lipolytica* contains abundant acetyl-CoA, a precursor for synthesizing carotenoid compounds, and its cells accumulate large amounts of lipids, making it suitable for producing fat-soluble compounds. These characteristics give *Yarrowia lipolytica* a natural advantage in synthesizing carotenoids. Figure 1 (Schematic diagram of the pathway for zeaxanthin synthesis in Yersinia lipophila after the introduction of relevant genes).

[0004] Because different enzymes in the synthetic pathway have varying catalytic efficiencies, some metabolic intermediates accumulate, causing toxicity to cells and affecting the yield of the final product. Furthermore, carotenoid products are all lipid-soluble compounds, which can form crystals within cells, damaging them. Carotenoids synthesized by microbial strains are often stored in hydrophobic cell membranes or lipid droplets. Summary of the Invention

[0005] A first aspect of the present invention is to provide a cytoskeleton.

[0006] A second aspect of the present invention is to provide an application of the cytoskeleton of the first aspect of the present invention.

[0007] A third aspect of the present invention is to provide a nucleic acid molecule.

[0008] A fourth aspect of the present invention is to provide an expression system.

[0009] A fifth aspect of the present invention is to provide a cell.

[0010] The sixth aspect of the present invention is to provide a method for constructing cells according to the fifth aspect of the present invention.

[0011] The seventh aspect of the present invention is to provide a method for preparing zeaxanthin.

[0012] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0013] In a first aspect, the present invention provides a cytoskeleton comprising: oleosin and (SH3)x-(GBD)y-(PDZ)z; wherein x = 1 or 2, y = 1 or 2, z = 1 or 2; the sequence of PDZ is shown in SEQ ID NO. 54, the sequence of GBD is shown in SEQ ID NO. 53, and the sequence of SH3 is shown in SEQ ID NO. 52.

[0014] Preferably, the cytoskeleton comprises: oleosin-(SH3)x-(GBD)y-(PDZ)z.

[0015] Preferably, the cytoskeleton comprises any one of (a1) to (a7):

[0016] (a1)oleosin-SH3-GBD-PDZ;

[0017] (a2)oleosin-SH3-GBD-PDZ-PDZ;

[0018] (a3)oleosin-SH3-GBD-GBD-PDZ;

[0019] (a4)oleosin-SH3-SH3-GBD-PDZ;

[0020] (a5)oleosin-SH3-GBD-GBD-PDZ-PDZ;

[0021] (a6)oleosin-SH3-SH3-GBD-PDZ-PDZ;

[0022] (a7)oleosin-SH3-SH3-GBD-GBD-PDZ.

[0023] Preferably, the oleosin is derived from corn.

[0024] Preferably, the accession number of Oleosin is ACG49220.1 (GenBank).

[0025] A second aspect of the present invention provides the use of the cytoskeleton of the first aspect of the present invention in at least one of (d1) to (d3);

[0026] (d1) Increase maize xanthine yield;

[0027] (d2) Increase β-carotene production;

[0028] (d3) Increase astaxanthin production.

[0029] Specifically, when the cytoskeleton of the first aspect of the present invention is used to further improve the yield of zeaxanthin, the cytoskeleton of the first aspect of the present invention localizes the key enzymes of the zeaxanthin synthesis pathway to the surface of cell lipid droplets.

[0030] Specifically, key enzymes (CarRP, CarB, and CrtZ) in the zeaxanthin synthesis pathway are affinity-bound to the PDZ, GBD, and SH3 domains of the cytoskeleton of the first aspect of the present invention via binding ligands linked to their C-termini. The PDZ, GBD, and SH3 domains in the cytoskeleton are covalently linked to the C-terminus of the Oleosin protein in the cytoskeleton located on the outer surface of lipid droplets.

[0031] A third aspect of the present invention provides a nucleic acid molecule comprising the nucleotide sequence of the following gene:

[0032] (b1) The cytoskeleton of the first aspect of the present invention;

[0033] (b2) Key enzyme genes in the maize xanthine synthesis pathway;

[0034] The key enzyme genes in the maize xanthine synthesis pathway include at least one of the following genes:

[0035] (c1) CarRP, a bifunctional enzyme gene that catalyzes the production of phytoene from two molecules of GGPP and the production of β-carotene from phytoene.

[0036] (c2) The gene CarB that catalyzes the production of lycopene from phytoene;

[0037] (c3)β-carotene hydroxylase gene CrtZ.

[0038] Preferably, the key enzyme genes of the zeaxanthin synthesis pathway include at least one of the following genes: CarRP from *Rhizomucor circinelloides*, CarB from *Rhizomucor circinelloides*, and CrtZ from *Erwinia uredovora*; more preferably, the key enzyme genes of the zeaxanthin synthesis pathway include the following genes: CarRP from *Rhizomucor circinelloides*, CarB from *Rhizomucor circinelloides*, and CrtZ from *Erwinia uredovora*.

[0039] Preferably, the sequence of the CarRP is shown as positions 1 to 1842 of SEQ ID NO.49.

[0040] Preferably, the sequence of CarB is shown as positions 1 to 1737 of SEQ ID NO. 50.

[0041] Preferably, the sequence of CrtZ is shown as positions 1 to 525 of SEQ ID NO. 51.

[0042] Preferably, CarRP, CarB, and CrtZ each contain one of the following binding ligands: PDZ binding ligand, GBD binding ligand, and SH3 binding ligand; the binding ligands contained in CarRP, CarB, and CrtZ are all different.

[0043] Preferably, the binding ligand is located at the 3' end of CarRP, CarB, or CrtZ.

[0044] Preferably, the CarRP contains a PDZ binding ligand (sequence shown in positions 1858-1878 of SEQ ID NO.49); the CarB contains a GBD binding ligand (sequence shown in positions 1753-1848 of SEQ ID NO.50); ​​and the CrtZ contains an SH3 binding ligand (sequence shown in positions 541-573 of SEQ ID NO.51).

[0045] Preferably, the 5' ends of the PDZ binding ligand, GBD binding ligand, and SH3 binding ligand further contain binding sequences.

[0046] Preferably, the binding sequence of the PDZ binding ligand is shown in positions 1843 to 1857 of SEQ ID NO.49.

[0047] Preferably, the binding sequence of the GBD binding ligand is shown in positions 1738 to 1852 of SEQ ID NO. 50.

[0048] Preferably, the binding sequence of the SH3 binding ligand is shown in positions 526 to 540 of SEQ ID NO. 51.

[0049] Preferably, the sequence of the CarRP is as shown in SEQ ID NO.49.

[0050] Preferably, the sequence of CarB is as shown in SEQ ID NO.50.

[0051] Preferably, the sequence of CrtZ is as shown in SEQ ID NO.51.

[0052] Preferably, the nucleic acid molecule further comprises the nucleotide sequences of the following genes:

[0053] (b3) Key enzyme genes in the mevalonate pathway;

[0054] (b4) Genes of key enzymes in the farnesyl pyrophosphate pathway;

[0055] The key enzyme genes of the mevalonate pathway include at least one of the following genes: 3-hydroxy-3-methylglutaryl-CoA reductase gene, acetyl-CoA thioesterase gene, and IPP isomerase gene.

[0056] The key enzyme genes of the farnesyl pyrophosphate pathway include the following genes: genes that catalyze the combination of farnesyl diphosphate (FPP) with one molecule of isoprene pyrophosphate to generate digeranyl pyrophosphate (GGPP).

[0057] Preferably, the key enzyme genes of the mevalonate pathway include at least one of the following genes: a 3-hydroxy-3-methylglutaryl-CoA reductase gene from *Saccharomyces cerevisiae* CEN.PK2-1C, an acetyl-CoA thioesterase gene from *Escherichia coli* BL21(DE3), and an IPP isomerase gene from *Yersinia lipolytica* W29; more preferably, the key enzyme genes of the mevalonate pathway include the following genes: a 3-hydroxy-3-methylglutaryl-CoA reductase gene from *Saccharomyces cerevisiae* CEN.PK2-1C (GenBank: YLR450W), an acetyl-CoA thioesterase gene from *Escherichia coli* BL21(DE3) (GenBank: b2224), and an IPP isomerase gene from *Yersinia lipolytica* W29 (GenBank: YALI0F04015p).

[0058] Preferably, the gene (GenBank: YALI0D17050p) that catalyzes the combination of farnesyl diphosphate (FPP) with one molecule of isoprene pyrophosphate to generate digeranyl pyrophosphate (GGPP) is derived from Yersinia lipolytica W29.

[0059] Preferably, in order to ensure efficient expression of each gene in the expression system or recombinant cells, a promoter is connected upstream of each gene, and / or a terminator is connected downstream of each gene;

[0060] The promoters include, but are not limited to: pTEF (SEQ ID NO. 58), pEXP (SEQ ID NO. 59), pFBA (SEQ ID NO. 60), and pGPD (SEQ ID NO. 61);

[0061] The terminators include, but are not limited to: tLIP (SEQ ID NO.62), tICL (SEQ ID NO.63), CYC1 (SEQ ID NO.64), and tXPR2 (SEQ ID NO.65).

[0062] More preferably, a promoter is attached upstream of each gene.

[0063] Furthermore, a terminator sequence is added downstream of each gene.

[0064] A fourth aspect of the present invention provides an expression system comprising the nucleic acid molecule of the third aspect of the present invention.

[0065] Preferably, the genes of the nucleic acid molecule are disassembled into several groups and inserted into different expression vectors respectively.

[0066] Preferably, when the nucleic acid molecule does not contain the nucleotide sequences of (b3) and (b4), the gene of the nucleic acid molecule is split into two groups and inserted into two different expression vectors respectively.

[0067] Preferably, an expression system includes plasmid PM1 and plasmid PM2;

[0068] The plasmid PM1 uses pUC19 as a backbone vector and contains genes for key enzymes in the maize xanthine synthesis pathway.

[0069] The plasmid PM2 uses pUC19 as a backbone vector and contains the cytoskeleton of the first aspect of the present invention.

[0070] Preferably, both plasmid PM1 and plasmid PM2 contain the URA3 uracil-deficient screening gene.

[0071] Preferably, when the nucleic acid molecule contains nucleotide sequences (b3) and (b4), the gene of the nucleic acid molecule is split into three groups and inserted into three different expression vectors.

[0072] Preferably, an expression system includes plasmid PM0, plasmid PM1, and plasmid PM2;

[0073] The plasmid PM0 uses pUC19 as a backbone vector and contains key enzyme genes of the mevalonate pathway and the farnesyl pyrophosphate pathway.

[0074] The plasmid PM1 uses pUC19 as a backbone vector and contains genes for key enzymes in the maize xanthine synthesis pathway.

[0075] The plasmid PM2 uses pUC19 as a backbone vector and contains the cytoskeleton of the first aspect of the present invention.

[0076] Preferably, plasmids PM0, PM1, and PM2 all contain the URA3 uracil-deficient screening gene.

[0077] Those skilled in the art will understand that other grouping methods can also achieve the objectives of this invention.

[0078] A fifth aspect of the invention provides a cell comprising a nucleic acid molecule of the third aspect of the invention or an expression system of the fourth aspect of the invention.

[0079] Furthermore, the nucleic acid molecule is inserted into the genome of the cell.

[0080] Furthermore, the nucleic acid molecules are inserted into the genome of the cell via a non-homologous recombinant biological method.

[0081] Those skilled in the art will understand that the insertion into the genome of the cell via non-homologous recombinant biological means is to obtain more strains that are likely to be stably inherited, and the purpose of the present invention can also be achieved by simply transforming the above expression system.

[0082] Preferably, the cell is a yeast cell; more preferably, it is Yeast lipolytica.

[0083] A sixth aspect of the present invention provides a method for constructing cells according to the fifth aspect, wherein a nucleic acid molecule of the third aspect of the present invention or an expression system of the fourth aspect of the present invention is introduced into recipient cells.

[0084] Preferably, the recipient cell is a yeast cell; more preferably, it is Yeast lipolytica.

[0085] Preferably, when the expression system includes plasmid PM1 and plasmid PM2, the cell construction method is as follows: 1) introducing linearized plasmid PM1 into recipient cells to obtain zeaxanthin-producing cells; 2) introducing linearized plasmid PM2 into the zeaxanthin-producing cells obtained in step 1).

[0086] Preferably, the receptor cell is a receptor cell with a uracil synthesis defect.

[0087] Preferably, the plasmid PM1 is prepared as follows: the URA3 gene fragment is cloned between NdeⅠ and KpnⅠ of pUC19 to construct the backbone plasmid pUC19URA3; CarRP, CrtZ, and CarB are ligated into the backbone plasmid pUC19URA3 to obtain plasmid PM1.

[0088] Preferably, the URA3 gene fragment includes a loxP arm.

[0089] Preferably, the sequence of the URA3 gene fragment is shown in SEQ ID NO.55.

[0090] Preferably, the method for linking CarRP, CrtZ, and CarB to the backbone plasmid pUC19URA3 is as follows: pUC19URA3 is double-digested with BamHI and HindIII, and then the digested pUC19URA3 is assembled with CarRP, CrtZ, and CarB by Gibson ligation.

[0091] Preferably, the upstream and downstream of CarRP, CrtZ, and CarB are cloned with promoters and terminators, respectively, to obtain expression frames containing CarRP, CrtZ, and CarB.

[0092] Preferably, the promoters include, but are not limited to: pTEF (SEQ ID NO. 58), pEXP (SEQ ID NO. 59), pFBA (SEQ ID NO. 60), and pGPD (SEQ ID NO. 61);

[0093] The terminators include, but are not limited to: tLIP (SEQ ID NO.62), tICL (SEQ ID NO.63), CYC1 (SEQ ID NO.64), and tXPR2 (SEQ ID NO.65).

[0094] Preferably, the CarRP-containing expression box is pFBA-CarRP-tLIP.

[0095] Preferably, the expression box containing CrtZ is pTEF-CrtZ-CYC1.

[0096] Preferably, the expression box containing CarB is pEXP-CarB-tICL.

[0097] Preferably, the sequence of the CarRP is shown as positions 1 to 1842 of SEQ ID NO.49.

[0098] Preferably, the sequence of CarB is shown as positions 1 to 1737 of SEQ ID NO. 50.

[0099] Preferably, the sequence of CrtZ is shown as positions 1 to 525 of SEQ ID NO. 51.

[0100] Preferably, CarRP, CarB, and CrtZ each contain one of the following binding ligands: PDZ binding ligand, GBD binding ligand, and SH3 binding ligand; the binding ligands contained in CarRP, CarB, and CrtZ are all different.

[0101] Preferably, the binding ligand is located at the 3' end of CarRP, CarB, or CrtZ.

[0102] Preferably, the CarRP contains a PDZ binding ligand (sequence shown in positions 1858-1878 of SEQ ID NO.49); the CarB contains a GBD binding ligand (sequence shown in positions 1753-1848 of SEQ ID NO.50); ​​and the CrtZ contains an SH3 binding ligand (sequence shown in positions 541-573 of SEQ ID NO.51).

[0103] Preferably, the sequence of the CarRP is as shown in SEQ ID NO.49.

[0104] Preferably, the sequence of CarB is as shown in SEQ ID NO.50.

[0105] Preferably, the sequence of CrtZ is as shown in SEQ ID NO.51.

[0106] Preferably, the plasmid PM2 is prepared as follows: the URA3 gene fragment is cloned between NdeⅠ and KpnⅠ of pUC19 to construct the backbone plasmid pUC19URA3; Oleosin-(SH3)x-(GBD)y-(PDZ)z is ligated into the backbone plasmid pUC19URA3 to obtain plasmid PM2.

[0107] Preferably, the URA3 gene fragment includes a loxP arm.

[0108] Preferably, the sequence of the URA3 gene fragment is shown in SEQ ID NO.55.

[0109] Preferably, the method for ligating Oleosin-(SH3)x-(GBD)y-(PDZ)z to the backbone plasmid pUC19URA3 is as follows: pUC19URA3 is double-digested with BamHI and HindIII, and then the digested pUC19URA3 is assembled with Oleosin-(SH3)x-(GBD)y-(PDZ)z by Gibson ligation.

[0110] Preferably, the upstream and downstream of Oleosin-(SH3)x-(GBD)y-(PDZ)z are cloned with promoters and terminators, respectively, to obtain an expression box containing Oleosin-(SH3)x-(GBD)y-(PDZ)z.

[0111] Preferably, the expression box containing Oleosin-(SH3)x-(GBD)y-(PDZ)z includes: promoter-Oleosin and (SH3)x-(GBD)y-(PDZ)z terminator.

[0112] Preferably, the promoters include, but are not limited to: pTEF, pEXP, pFBA, and pGPD;

[0113] The terminators include, but are not limited to: tLIP, tICL, CYC1, and tXPR2.

[0114] Preferably, the expression box containing Oleosin-(SH3)x-(GBD)y-(PDZ)z is pGPD-Oleosin-SH3-GBD-PDZ-tXPR2.

[0115] Preferably, the expression box containing Oleosin-(SH3)x-(GBD)y-(PDZ)z includes pGPD-Oleosin and SH3-GBD-PDZ-tXPR2.

[0116] Preferably, the linearized plasmid PM1 / PM2 is obtained by NotⅠ enzyme digestion.

[0117] Preferably, when the expression system includes plasmid PM0, plasmid PM1, and plasmid PM2, the cell construction method is as follows: 1) linearizing plasmid PM0 is introduced into recipient cells to obtain recipient cells enhanced with genes related to the mevalonate pathway and farnesyl pyrophosphate pathway; 2) linearizing plasmid PM1 is introduced into recipient cells enhanced with genes related to the mevalonate pathway and farnesyl pyrophosphate pathway to obtain zeaxanthin-producing cells; 3) linearizing plasmid PM2 is introduced into the zeaxanthin-producing cells obtained in step 2).

[0118] Preferably, the receptor cell is a receptor cell with a uracil synthesis defect.

[0119] Preferably, the plasmid PMO is prepared as follows: plasmid pUC19 is digested with NdeⅠ and HindⅢ, and then the digested plasmids pUC19, AtoB, HMGR, IDI, GGS1, and URA3 are assembled using Gibson ligation to obtain PMO.

[0120] Preferably, promoters and terminators are cloned upstream and downstream of AtoB, HMGR, IDI, and GGS1, respectively, to obtain expression frames containing AtoB, HMGR, IDI, and GGS1.

[0121] Preferably, the promoters include, but are not limited to: pTEF, pEXP, pFBA, and pGPD;

[0122] The terminators include, but are not limited to: tLIP, tICL, CYC1, and tXPR2.

[0123] Preferably, the upstream and downstream of the URA3 are cloned with promoters and terminators, respectively.

[0124] Preferably, the promoter of URA3 contains the upper part of the 26S rDNA homologous arm.

[0125] Preferably, the terminator of the GGS1 contains the lower part of a 26S rDNA homologous arm downstream.

[0126] Preferably, the sequence of the upper part of the 26S rDNA homologous arm is shown in SEQ ID NO.56.

[0127] Preferably, the sequence of the lower part of the 26S rDNA homologous arm is shown in SEQ ID NO.57.

[0128] Preferably, the plasmid PM1 is prepared as follows: the URA3 gene fragment is cloned between NdeⅠ and KpnⅠ of pUC19 to construct the backbone plasmid pUC19URA3; CarRP, CrtZ, and CarB are ligated into the backbone plasmid pUC19URA3 to obtain plasmid PM1.

[0129] Preferably, the URA3 gene fragment includes a loxP arm.

[0130] Preferably, the sequence of the URA3 gene fragment is shown in SEQ ID NO.55.

[0131] Preferably, the method for linking CarRP, CrtZ, and CarB to the backbone plasmid pUC19URA3 is as follows: pUC19URA3 is double-digested with BamHI and HindIII, and then the digested pUC19URA3 is assembled with CarRP, CrtZ, and CarB by Gibson ligation.

[0132] Preferably, the upstream and downstream of CarRP, CrtZ, and CarB are cloned with promoters and terminators, respectively, to obtain expression frames containing CarRP, CrtZ, and CarB.

[0133] Preferably, the promoters include, but are not limited to: pTEF, pEXP, pFBA, and pGPD;

[0134] The terminators include, but are not limited to: tLIP, tICL, CYC1, and tXPR2.

[0135] Preferably, the CarRP-containing expression box is pFBA-CarRP-tLIP.

[0136] Preferably, the expression box containing CrtZ is pTEF-CrtZ-CYC1.

[0137] Preferably, the expression box containing CarB is pEXP-CarB-tICL.

[0138] Preferably, the sequence of the CarRP is shown as positions 1 to 1842 of SEQ ID NO.49.

[0139] Preferably, the sequence of CarB is shown as positions 1 to 1737 of SEQ ID NO. 50.

[0140] Preferably, the sequence of CrtZ is shown as positions 1 to 525 of SEQ ID NO. 51.

[0141] Preferably, CarRP, CarB, and CrtZ each contain one of the following binding ligands: PDZ binding ligand, GBD binding ligand, and SH3 binding ligand; the binding ligands contained in CarRP, CarB, and CrtZ are all different.

[0142] Preferably, the binding ligand is located at the 3' end of CarRP, CarB, or CrtZ.

[0143] Preferably, the CarRP contains a PDZ binding ligand (sequence shown in positions 1858-1878 of SEQ ID NO.49); the CarB contains a GBD binding ligand (sequence shown in positions 1753-1848 of SEQ ID NO.50); ​​and the CrtZ contains an SH3 binding ligand (sequence shown in positions 541-573 of SEQ ID NO.51).

[0144] Preferably, the sequence of the CarRP is as shown in SEQ ID NO.49.

[0145] Preferably, the sequence of CarB is as shown in SEQ ID NO.50.

[0146] Preferably, the sequence of CrtZ is as shown in SEQ ID NO.51.

[0147] Preferably, the plasmid PM2 is prepared as follows: the URA3 gene fragment is cloned between NdeⅠ and KpnⅠ of pUC19 to construct the backbone plasmid pUC19URA3; Oleosin-(SH3)x-(GBD)y-(PDZ)z is ligated into the backbone plasmid pUC19URA3 to obtain plasmid PM2.

[0148] Preferably, the URA3 gene fragment includes a loxP arm.

[0149] Preferably, the sequence of the URA3 gene fragment is shown in SEQ ID NO.55.

[0150] Preferably, the method for ligating Oleosin-(SH3)x-(GBD)y-(PDZ)z to the backbone plasmid pUC19URA3 is as follows: pUC19URA3 is double-digested with BamHI and HindIII, and then the digested pUC19URA3 is assembled with Oleosin-(SH3)x-(GBD)y-(PDZ)z by Gibson ligation.

[0151] Preferably, the upstream and downstream of Oleosin-(SH3)x-(GBD)y-(PDZ)z are cloned with promoters and terminators, respectively, to obtain an expression box containing Oleosin-(SH3)x-(GBD)y-(PDZ)z.

[0152] Preferably, the promoters include, but are not limited to: pTEF, pEXP, pFBA, and pGPD;

[0153] The terminators include, but are not limited to: tLIP, tICL, CYC1, and tXPR2.

[0154] Preferably, the expression box containing Oleosin-(SH3)x-(GBD)y-(PDZ)z is pGPD-Oleosin-(SH3)x-(GBD)y-(PDZ)z-tXPR2.

[0155] A seventh aspect of the present invention provides a method for preparing zeaxanthin by culturing cells according to the fifth aspect of the present invention.

[0156] A method for preparing zeaxanthin, d1) or d2):

[0157] d1):

[0158] d11) Take the cells from the fifth aspect of this invention, seed them into YPD medium, and culture them at 28-32°C and 180-260 rpm for 20-28 hours to obtain seed culture;

[0159] d12) Inoculate the seed culture into YPD medium at an inoculation rate of 5-15% and incubate at 28-32℃ and 180-260rpm for 70-78 hours;

[0160] d2):

[0161] d21) Take the cells of the fifth aspect of the present invention, seed them in YPD medium, and culture them at 28-32°C and 180-220 rpm for 20-28 hours to obtain the primary seed solution;

[0162] d22) Inoculate the primary seed culture into YPD medium at an inoculation rate of 0.5-1.5% and culture at 28-32℃ and 180-220 rpm until OD600 = 9-11 to obtain the secondary seed culture;

[0163] d23) Inoculate the secondary seed culture into the fermentation medium at an inoculation rate of 5-15% and culture at 28-32℃, 300-800rpm, aeration rate of 1-3L / min, pH 6-8, and dissolved oxygen of 20-30%. After culturing for 10-14 hours, continue fermentation with 50-70% glucose stock solution for 240 hours.

[0164] Preferably, the fermentation medium described in d23) consists of 20–40 g / L yeast extract, 50–70 g / L peptone, and 40–60 g / L glucose.

[0165] The beneficial effects of this invention are:

[0166] This invention provides a cytoskeleton comprising: oleosin and (SH3)x-(GBD)y-(PDZ)z; wherein x = 1 or 2, y = 1 or 2, and z = 1 or 2; the sequence of PDZ is shown in SEQ ID NO. 54, the sequence of GBD is shown in SEQ ID NO. 53, and the sequence of SH3 is shown in SEQ ID NO. 52; this cytoskeleton localizes key enzymes of the zeaxanthin synthesis pathway to the surface of cell lipid droplets (e.g., Figure 2 As shown in the figure, the key enzymes in the zeaxanthin synthesis pathway (CarRP, CarB, and CrtZ) bind to the PDZ, GBD, and SH3 domains of the cytoskeleton via their C-terminal binding ligands. The cytoskeleton is covalently linked to the C-terminus of the oleosin protein on the outer surface of the lipid droplets, which makes it easier for the product to be stored in the lipid droplets, reduces the toxicity of the product to the cells, and increases the yield of zeaxanthin.

[0167] The present invention also provides nucleic acid molecules containing a cytoskeleton and an expression system, which significantly increase the zeaxanthin yield of the obtained cells by introducing nucleic acid molecules or expression systems containing a cytoskeleton into recipient cells, up to 141%. Attached Figure Description

[0168] Figure 1 This is a roadmap for the synthesis of zeaxanthin in zeaxanthin.

[0169] Figure 2 This is a schematic diagram of the protein backbone being localized to the lipid droplet membrane.

[0170] Figure 3 This is a schematic diagram of plasmid pM1.

[0171] Figure 4 This is a schematic diagram of plasmid pM2-1.

[0172] Figure 5 This is a schematic diagram of plasmid pM2-2.

[0173] Figure 6 This is a schematic diagram of plasmid pM2-3.

[0174] Figure 7 This is a schematic diagram of plasmid pM2-4.

[0175] Figure 8 This is a schematic diagram of plasmid pM2-5.

[0176] Figure 9 This is a schematic diagram of plasmid pM2-6.

[0177] Figure 10 This is a schematic diagram of plasmid pM2-7.

[0178] Figure 11 This is a graph showing the results of shake-flask fermentation of zeaxanthin by strains F1, F2-1, F2-2, F2-3, F2-4, F2-5, F2-6 and F2-7.

[0179] Figure 12 This is a diagram showing the results of zeaxanthin production by fermenting strain F2-6 in a fermenter.

[0180] Figure 13 This is a schematic diagram of plasmid pM0. Detailed Implementation

[0181] The present invention will be further described in detail below through specific embodiments.

[0182] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0183] The *Yersinia lipolyticis* strain was Polf (ATCC ID MAY-2613; genotype MATA ura3-302 leu2-270xpr-322axp2-deltaNU49 XPR2::SUC2, purchased from ATCC). The restriction endonucleases used were purchased from Thermo Fisher Scientific. Reagents used for plasmid extraction and gel recovery, PCR and seamless cloning, and *Escherichia coli* (DH5α) for molecular cloning were all purchased from Nanjing Novizan Biotechnology Co., Ltd. Reagents used for genomic DNA extraction from *Saccharomyces cerevisiae*, *Yersinia lipolyticis*, and *Escherichia coli* were purchased from Qiagen.

[0184] Genomic DNA was extracted from Saccharomyces cerevisiae CEN.PK2-1C (ATCC ID MYA-1111; genotype Mata;ura3-52,trp1-289,leu2-3,112,his3Δ1;MAL2-8c;SUC2, purchased from ATCC), Escherichia coli DH5α, and Yersinia lipolyticis Polf using the Qiagen Blood & Cell Culture DNA Mini Kit.

[0185] The chemical and gene names involved in this invention are explained below:

[0186] CarRP gene: a bifunctional enzyme gene that catalyzes the production of phytoene from two molecules of GGPP and the production of β-carotene from lycopene. It is derived from Rhizomucor circinelloides, GenBank: AJ250827.1.

[0187] CarB gene: The gene that catalyzes the production of lycopene from phytoene, derived from Rhizomucor circinelloides, GenBank: AJ238028.1.

[0188] CrtZ gene: β-carotene hydroxylase gene, which adds a hydroxyl group to each end of the β-carotene ring sequentially, and generates zeaxanthin via the intermediate β-cryptoxanthin, which contains a hydroxyl group on only one ring. It is derived from Erwiniauredovora, GenBank: D90087.2.

[0189] Oleosin gene: A gene that expresses a special type of storage protein, mainly expressed in plant seeds, covering the surface of oil bodies, and playing an important biological role in the process of oil body formation and decomposition. It is derived from maize (Zeamays), GenBank: ACG49220.1.

[0190] HMGR gene: 3-hydroxy-3-methylglutaryl-CoA reductase gene, which catalyzes the reduction of HMG-CoA to mevalonic acid, derived from yeast, CP028452.1:571078~572577.

[0191] IDI gene: IPP isomerase gene, which catalyzes the production of dimethylpropene pyrophosphate (DMAPP) from isopentenyl diphosphate (IPP). It is derived from Yersinia lipolytica W29, CP028453.1:585662~584850.

[0192] GGS1 gene: A gene that catalyzes the combination of farnesyl diphosphate (FPP) with one molecule of isoprene pyrophosphate to generate digeranyl pyrophosphate (GGPP), derived from Yersinia lipolytica W29, CP028451.1:2085775~2084792.

[0193] AtoB gene: Acetyl-CoA thioesterase gene, which catalyzes the condensation of acetyl-CoA into acetyl-CoA. It is derived from Escherichia coli BL21(DE3), CP001509.3:2216470~2217654.

[0194] The primers used in the following examples are shown in Table 1.

[0195] Table 1 Primer List

[0196]

[0197]

[0198]

[0199] Example 1: Construction of the backbone plasmid pUC19URA3

[0200] The URA3 gene was amplified using Yersinia lipolyticis genomic DNA as a template with primers loxP-URA3-F / loxP-URA3-R. The URA3 gene fragment was then amplified using primers URA3-F / URA3-R with the PCR product as a template. The plasmid pUC19 was double-digested with restriction enzymes NdeI and KpnI, and the URA3 gene fragment (SEQ ID NO. 55) was cloned into the space between NdeI and KpnI of pUC19 using seamless cloning (Novizan ClonExpress Ultra One Step Cloning Kit) to construct the backbone plasmid pUC19URA3.

[0201] Example 2 Construction of plasmid pM1

[0202] The promoter pFBA, terminator tLIP, promoter pTEF, promoter pEXP, and terminator tICL fragments were amplified using *Yarrowia lipolyticis* Polf genomic DNA as a template, respectively, with primers pFBA-F / pFBA-R, tLIP-F / tLIP-R, pTEF-F / pTEF-R, pEXP-F / pEXP-R, and tICL-F / tICL-R. The terminator CYC1 fragment was amplified using *Saccharomyces cerevisiae* CEN.PK2-1C genomic DNA as a template, with primers CYC1-F / CYC1-R. The CarRP and CarB genes from *Rhizomucor circinelloides* and the CrtZ gene from *Erwinia uredovora*, along with their corresponding affinity ligands, were synthesized by Nanjing GenScript Technology Co., Ltd. after codon optimization. The corresponding base sequences are shown in SEQ ID NO. 49, SEQ ID NO. 50, and SEQ ID NO. 51. The CarRP, CrtZ, and CarB fragments were amplified using the synthesized gene as a template, respectively, with primers CarRP-F / CarRP-R, CrtZ-F / CrtZ-R, and CarB-F / CarB-R.

[0203] The promoter, gene, and terminator were spliced ​​into three expression cassettes: pFBA-CarRP-tLIP, pTEF-CrtZ-CYC1, and pEXP-CarB-tICL, using overlap PCR. The backbone plasmid pUC19URA3 was purified and recovered by double digestion with BamHI and HindIII. Then, the digested plasmid backbone and the three expression cassettes were assembled using Gibson ligation (Novizan ClonExpress Ultra One Step Cloning Kit) to construct plasmid pM1 (see schematic diagram). Figure 3 (As shown).

[0204] Example 3 Construction of plasmid pM2

[0205] The promoter pGPD and terminator tXPR2 fragments were amplified using Yersinia lipolytica Pof genomic DNA as a template, with primers pGPD-F / pGPD-R and tXPR2-F / tXPR2-R. The Oleosin gene and seven cytoskeleton (SH3)x-(GBD)y-(PDZ)z encoding genes from maize (Zea mays) were synthesized by Nanjing Genscript Biotech Co., Ltd. after codon optimization (the optimized cytoskeleton sequences are SH3-GBD-PDZ, SH3-GBD-PDZ-PDZ, SH3-GBD-GBD-PDZ, SH3-SH3-GBD-PDZ, SH3-GBD-GBD-PDZ-PDZ, SH3-SH3-GBD-PDZ, SH3-SH3-GBD-PDZ-PDZ, SH3-SH3-GBD-PDZ-PDZ, SH3-SH3-GBD-GBD-PDZ, SH3-SH3-GBD-GBD-PDZ, SH3-SH3-GBD-GBD-PDZ, where the SH3 sequence is shown in SEQ ID NO. 52, the GBD sequence in SEQ ID NO. 53, and the PDZ sequence in SEQ ID NO. 54. As shown in NO.54, the Oleosin gene fragment and the coding sequence fragments of seven cytoskeleton (SH3)x-(GBD)y-(PDZ)z were amplified using primers Oleosin-F / Oleosin-R and SH3-F / PDZ-R as templates.

[0206] The promoter pGPD and Oleosin were spliced ​​into a pGPD-Oleosin fragment using overlap PCR, and seven cytoskeleton (SH3)x-(GBD)y-(PDZ)z fragments and the terminator tXPR2 fragments were spliced ​​into seven (SH3)x-(GBD)y-(PDZ)z-tXPR2 fragments. The backbone plasmid pUC19URA3 was purified and recovered by double digestion with BamHI and HindIII. Then, using Gibson ligation (Novizan ClonExpress Ultra One Step Cloning Kit), the digested plasmid backbone, pGPD-Oleosin fragment, and seven (SH3)x-(GBD)y-(PDZ)z-tXPR2 fragments were assembled into plasmids (pM2-1, pM2-2, pM2-3, pM2-4, pM2-5, pM2-6, pM2-7). A schematic diagram of plasmids pM2-1, pM2-2, pM2-3, pM2-4, pM2-5, pM2-6, and pM2-7 is shown below. Figures 4-10 As shown.

[0207] Example 4: Construction of engineered bacteria producing zeaxanthin

[0208] 1. Increase the expression of genes related to the mevalonate pathway and farnesyl pyrophosphate pathway.

[0209] Using primers rDNA-up-F / rDNA-up-R, rDNA-dn-F / rDNA-dn-R, pTEF-F / pTEF-R, pEXP-F / pEXP-R, HMGR-F / HMGR-R, tXPR2-F / tXPR2-R1, pGPD-F1 / pGPD-R1, IDI-F / IDI-R, tICL-F / tICL-R1, pFBA-F1 / pFBA-R, GGS1-F / GGS1-R, and tLIP-F / tLIP-R1 as templates, the upper part (SEQ ID NO. 56) and lower part (SEQ ID NO. 57) of the 26S rDNA homologous arm were amplified using *Yersinia lipophila* Polf genomic DNA as a template. The sequence of DNA fragments was as follows: ID NO. 57), promoter pTEF, promoter pEXP, terminator tXPR2, promoter pGPD, terminator tICL, promoter pFBA, and terminator tLIP, as well as genes HMGR, IDI, and GGS1. The terminator CYC1 fragment was amplified using primers CYC1-F / CYC1-R with Saccharomyces cerevisiae CEN.PK2-1C genomic DNA as a template. The AtoB gene was amplified using primers AtoB-F / AtoB-R with Escherichia coli genomic DNA as a template. The URA3 gene was amplified using primers loxP-URA3-F / loxP-R with the URA3 fragment (SEQ ID NO. 55) flanked by loxP as a template.

[0210] The promoter, gene, and terminator were spliced ​​into four expression cassettes: pTEF-AtoB-CYC1, pEXP-HMGR-tXPR2, pGPD-IDI-tICL, and pFBA-GGS1-tLIP using overlap PCR. Then, overlap PCR was used to ligate the upper part of the 26S rDNA homologous arm to the URA3 gene promoter and the lower part of the 26S rDNA homologous arm to the terminator of the pFBA-GGS1-tLIP expression cassette.

[0211] Plasmid pUC19 was purified and recovered by double digestion with NdeI and HindIII, and then ligated using Gibson ligation (Novizan ClonExpress Ultra One Step Cloning Kit) to construct plasmid pM0 (e.g., ...). The digested plasmid and four expression cassettes (pTEF-AtoB-CYC1, pEXP-HMGR-tXPR2, pGPD-IDI-tICL, and pFBA-GGS1-tLIP) along with the URA3 genome were assembled into plasmid pM0. Figure 13 (As shown).

[0212] The plasmid pM0 was linearized by digestion with NotⅠ enzyme, and then the linearized pM0 was transformed into strain Polf using the lithium acetate method to obtain strain F0.

[0213] 2. Zeaxanthin Synthesis

[0214] After culturing strain F0 overnight in 10 mL of YPD medium, it was spread on YPD+5-FOA (100 mg / L) plates and incubated at 28°C for 72 hours. Single colonies were picked to obtain the URA3-deleted strain F0-ΔURA3.

[0215] After linearizing plasmid pM1 with NotI enzyme, strain F0-ΔURA3 was transformed using the lithium acetate method. The transformed strains were then plated on SD-URA deficient medium (PM2040 minimal SD Agar Base, PM2270 DOSupplement-Ura, Beijing Coollab Technology Co., Ltd.). After 2-3 days, 120 transformants with the deepest orange-red color were selected and inoculated into 10 mL of liquid YPD40 medium (1% yeast extract, 2% peptone, 4% glucose). The medium was incubated at 200 rpm and 30℃ for 72 hours. The yield of zeaxanthin was then measured, and strain F1 with high yield was obtained and stored in glycerol tubes at -80℃.

[0216] After culturing strain F1 overnight in 10 mL of YPD medium, it was spread on YPD+5-FOA (100 mg / L) plates and incubated at 28°C for 72 hours. Single colonies were picked to obtain the URA3-deleted strain F1-ΔURA3.

[0217] Plasmids pM2-1 to pM2-7 were linearized by NotI restriction enzyme digestion and transformed into strain F1-ΔURA3 using the lithium acetate method. The transformed strains were plated on SD-URA-deficient medium. After 2-3 days, 120 transformants with the deepest orange-red color were selected from each transformation and inoculated into 10 mL of liquid YPD40 medium. The strains were cultured at 200 rpm and 30℃ for 72 hours. The yield of zeaxanthin was detected, and strains F2-1 to F2-7 with high yields were obtained and stored in glycerol tubes at -80℃.

[0218] Example 5: Production of Zeaxanthin by Shake Flask Fermentation

[0219] F1, F2-1, F2-2, F2-3, F2-4, F2-5, F2-6, and F2-7, stored in glycerol tubes at -80℃, were streaked on YPD plates and incubated at 30℃ for 24 hours. Five single colonies from each plate were inoculated into 5 mL of YPD medium and incubated at 30℃ and 220 rpm for 24 hours. Then, at a 1:10 (v / v) inoculation rate, the culture was transferred to 50 mL of YPD medium and incubated in shake flasks at 30℃ and 220 rpm for 72 hours. After fermentation, 1 mL of the fermentation broth was centrifuged, the supernatant was discarded, and the broth was resuspended in 0.7 mL of DMSO. The culture was then incubated at 55℃ for 10 min, followed by incubation at 45℃ for 15 min with an equal volume of acetone. After incubation, the culture was centrifuged at 12000 rpm for 5 min, and the supernatant was analyzed by HPLC at 445 nm for zeaxanthin content. The results are as follows: Figure 11 As shown, strain F1 produced 53 mg / L of zeaxanthin. After expressing the cytoskeleton, the zeaxanthin production was significantly increased. The highest zeaxanthin production was achieved by strain F2-6, which increased by 141% to 128 mg / L.

[0220] Example 6: Production of Zeaxanthin by Upper Tank Fermentation

[0221] High-density fermentation of genetically engineered bacteria is carried out in a fermenter. The method is as follows:

[0222] 1) Cultivation of primary seed culture: A single colony of recombinant strain F2-6 on a solid YPD plate was inoculated into a 100mL Erlenmeyer flask containing 20mL of LYPD liquid medium and incubated at 30℃ and 200rpm for 48 hours.

[0223] 2) Cultivation of secondary seed culture: The primary seed culture obtained in step 1) was transferred to a 300 mL Erlenmeyer flask containing 100 mL of YPD liquid culture medium at an inoculation rate of 1% (V / V). The flask was cultured at 30°C and 200 rpm for 10 hours until the cell OD600 reached 10.

[0224] 3) The secondary seed culture obtained in step 2) was transferred to a 3L fermenter containing 1L of fermentation medium at an inoculation rate of 10% (V / V) for fermentation. The initial medium composition was 30g / L yeast extract, 60g / L peptone, and 50g / L glucose. Fermentation was carried out at 30℃, stirring speed of 300-800rpm, aeration rate of 2L / min, pH 6.8, and dissolved oxygen above 25%. After 12 hours of culture, the culture was continued with a 60% glucose stock solution for 240 hours of fed-batch fermentation. The results are as follows: Figure 12 As shown, the zeaxanthin yield in zeaxanthin is as high as 1800 mg / L.

[0225] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention. SEQUENCE LISTING <110> Guangzhou Zhiteqi Biotechnology Co., Ltd. <120> A genetically engineered bacterium that increases maize xanthine yield, its construction method and application <130> <160> 66 <170> PatentIn version 3.5 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <400> 1 atcgcttcgg ataactcctg ctatacgaag ttatacgaat tcgcgcccag agagccattg 60 <210> 2 <211> 63 <212> DNA <213> Artificial Sequence <400> 2 ataacttcgt atagcataca tcatacgaag ttattctgaa ttccgagaaa cacaacaaca 60 tgc 63 <210> 3 <211> 48 <212> DNA <213> Artificial Sequence <400> 3 attgtactga gagtgcacca gcggccgcat cgcttcggat aactcctg 48 <210> 4 <211> 45 <212> DNA <213> Artificial Sequence <400> 4 aggtcgactc tagaggatcc ccggataact tcgtatagca tacat 45 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <400> 5 acgaagttat ccggggatcc tgcacccaac aataaatggg 40 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <400> 6 ctgggttagt ttgtgtagag 20 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <400> 7 gctatttatc actctttaca ac 22 <210> 8 <211> 37 <212> DNA <213> Artificial sequence <400> 8 ccgccaaccc ggtctctgac ccttcgtggg tctcaat 37 <210> 9 <211> 18 <212> DNA <213> Artificial sequence <400> 9 agagaccggg ttggcggc 18 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <400> 10 ctgcggttag tactgcaaaa 20 <210> 11 <211> 18 <212> DNA <213> Artificial sequence <400> 11 ggagtttggc gcccgttt 18 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <400> 12 tgctgtagat atgtcttgtg 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <400> 13 gcagtttgtt tagcaaaata 20 <210> 14 <211> 41 <212> DNA <213> Artificial sequence <400> 14 gaccatgatt acgccaagct ttgtatgatt gatgttacta c 41 <210> 15 <211> 50 <212> DNA <213> Artificial sequence <400> 15 attgtactga gagtgcacca tatggcggcc gcataactgt cgcgtacggc 50 <210> 16 <211> 41 <212> DNA <213> Artificial sequence <400> 16 caggagttat ccgaagcgat tcttcacttt gacattcaga g 41 <210> 17 <211> 40 <212> DNA <213> Artificial sequence <400> 17 cattgagacc cacgaagggt caattcaacc aagcgcgcgg 40 <210> 18 <211> 51 <212> DNA <213> Artificial sequence <400> 18 ctatgaccat gattacgcca agcttgcggc cgctatgaac gcttgactgc c 51 <210> 19 <211> 39 <212> DNA <213> Artificial sequence <400> 19 ccgccaaccc ggtctctata acttcgtata gcatacatc 39 <210> 20 <211> 38 <212> DNA <213> Artificial sequence <400> 20 ttttgcagta ctaaccgcag atgaagaact gtgtcatc 38 <210> twenty one <211> 42 <212> DNA <213> Artificial sequence <400> twenty one gcgtgacata actaattaca tgattagttc agtcgctcaa tg 42 <210> twenty two <211> 38 <212> DNA <213> Artificial sequence <400> twenty two cacaagacat atctacagca gtgtctaccg acgccaag 38 <210> twenty three <211> 37 <212> DNA <213> Artificial sequence <400> twenty three acaggccatg gaggtactta gccctgacct cggagtc 37 <210> twenty four <211> 19 <212> DNA <213> Artificial sequence <400> twenty four ggttgaaatg aatcggccg 19 <210> 25 <211> 39 <212> DNA <213> Artificial sequence <400> 25 ggccgattca tttcaaccca tctcacttgc gtatgtatg 39 <210> 26 <211> twenty three <212> DNA <213> Artificial sequence <400> 26 tgttgatgtg tgtttaattc aag 23 <210> 27 <211> 42 <212> DNA <213> Artificial sequence <400> 27 cttgaattaa acacacatca acaatgacga cgtcttacag cg 42 <210> 28 <211> 39 <212> DNA <213> Artificial sequence <400> 28 tattttgcta aacaaactgc ctacttgatc caccgccga 39 <210> 29 <211> 40 <212> DNA <213> Artificial sequence <400> 29 cccatttatt gttgggtgca tgtatgattg atgttatactac 40 <210> 30 <211> 20 <212> DNA <213> Artificial sequence <400> 30 tgcacccaac aataaatggg 20 <210> 31 <211> 37 <212> DNA <213> Artificial sequence <400> 31 ctacacaaac taacccagat ggattataac agcgcgg 37 <210> 32 <211> 39 <212> DNA <213> Artificial sequence <400> 32 gtaaagagtg ataaatagct cactgcgcat cctcaaagt 39 <210> 33 <211> 23 <212> DNA <213> Artificial sequence <400> 33 tcatgtaatt agttatgtca cgc 23 <210> 34 <211> 37 <212> DNA <213> Artificial sequence <400> 34 aacgggcgcc aaactccgca aattaaagcc ttcgagc 37 <210> 35 <211> 40 <212> DNA <213> Artificial sequence <400> 35 ctacacaaac taacccagat gctgctgacc tacatggagg 40 <210> 36 <211> 43 <212> DNA <213> Artificial sequence <400> 36 gtaaagagtg ataaatagct taaaccagag attctttaac acc 43 <210> 37 <211> 38 <212> DNA <213> Artificial sequence <400> 37 ttgcagtact aaccgcagat gctgtggatc tggaacgc 38 <210> 38 <211> 37 <212> DNA <213> Artificial sequence <400> 38 gacataacta attacatgat caacgacgac gtttcgg 37 <210> 39 <211> 37 <212> DNA <213> Artificial sequence <400> 39 caagacatat ctacagcaat gtccaagaag cacattg 37 <210> 40 <211> 39 <212> DNA <213> Artificial sequence <400> 40 ttttgctaaa caaactgctt agtcttcgtc ttcgtcacc 39 <210> 41 <211> 43 <212> DNA <213> Artificial sequence <400> 41 atacgaagtt atccggggat ccggttgaaa tgaatcggcc gac 43 <210> 42 <211> 31 <212> DNA <213> Artificial sequence <400> 42 ctatcagcca ttgttgatgt gtgtttaatt c 31 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <400> 43 gtacctccat ggcctgtccc 20 <210> 44 <211> 44 <212> DNA <213> Artificial sequence <400> 44 tgaccatgat tacgccaagc ttcatctcac ttgcgtatgt atgg 44 <210> 45 <211> 27 <212> DNA <213> Artificial sequence <400> 45 caacaatggc tgatagagat aggtctg 27 <210> 46 <211> 37 <212> DNA <213> Artificial sequence <400> 46 gatccggaac cagagccgct agaggctctt ccaccgc 37 <210> 47 <211> 20 <212> DNA <213> Artificial sequence <400> 47 ggctctggtt ccggatctgg 20 <210> 48 <211> 40 <212> DNA <213> Artificial sequence <400> 48 gacaggccat ggaggtactc acttgaagta aggagacacc 40 <210> 49 <211> 1881 <212> DNA <213> Artificial sequence <400> 49 atgctgctga cctacatgga ggtccacctg tactacaccc tgcccgtcct gggcgtcctg 60 tcttggctgt cccgacccta ctacaccgcc accgacgccc tgaagttcaa gttcctgacc 120 ctggtggcct tcaccaccgc ctccgcttgg gacaactaca ttgtctacca caaggcctgg 180 tcctactgcc ccacctgcgt gaccgccgtc attggttacg tgcccctgga ggagtacatg 240 ttcttcatca ttatgaccct gctgaccgtg gccttcacta acctggtcat gcgatggcac 300 ctgcactctt tcttcatccg acccgagacc cccgtgatgc agtctgtcct ggtgcgactg 360 gtccccatca ccgccctgct gatcaccgcc tacaaggcct ggcacctggc cgtccctggt 420 aaacccctgt tctacggctc ttgcattctg tggtacgcct gccccgtgct ggcccttctg 480 tggttcggcg ctggcgagta catgatgcga cgacccctgg ccgtcctggt gtctattgcc 540 ctgcccaccc tgttcctgtg ctgggtcgac gtggtcgcca ttggcgccgg aacctgggac 600 atctccctgg ctacctccac cggcaagttc gtggtgcccc acctgcccgt ggaggagttc 660 atgttcttcg ccctgatcaa caccgtgctg gtgttcggta cctgcgccat cgaccgaacc 720 atggccattc tgcacctgtt caagaacaag tccccctacc agcgacccta ccagcactct 780 aagtccttcc tgcaccagat cctggagatg acctgggcct tctgtctgcc cgaccaggtc 840 ctgcactctg acaccttcca cgacctgtcc gtctcttggg acatcctgcg aaaggcctcc 900 aagtctttct acaccgcctc tgccgtcttc cccggcgacg ttcgacagga gctgggtgtc 960 ctgtacgcct tctgccgagc caccgacgac ctgtgcgaca acgagcaggt gcccgtccag 1020 acccgaaagg agcagctgat cctgacccac cagttcgtct ccgacctgtt cggccagaag 1080 acctccgccc ccaccgctat tgactgggac ttctacaacg accagctgcc cgcctcctgc 1140 atttccgcct tcaagtcctt cacccgactg cgacacgtcc tggaggccgg agctattaag 1200 gagctgctgg acggttacaa gtgggacctg gagcgacgat ccattcgaga ccaggaggac 1260 ctgcgatact actccgcctg cgtggcctcc tctgtcggcg agatgtgcac ccgaatcatt 1320 ctggcccacg ccgacaagcc cgcctcccga cagcagactc agtggatcat ccagcgagcc 1380 cgagagatgg gtctggtcct gcagtacacc aacatcgccc gagacattgt caccgactcc 1440 gaggagctgg gccgatgtta cctgccccag gactggctga ccgagaagga ggtggccctg atccagggcg gtctggctcg agagattggc gaggagcgac tgctgtctct gtctcaccga ctgatctacc aggccgacga gctgatggtc gtcgccaaca agggcattga caagctgccc 1620. tcccactgcc agggtggcgt gcgagctgct tgcaacgtct acgcctccat cggcaccaag 1680 ctgaagtcct acaagcacca ctacccctcc cgagcccacg tgggaaactc tagcgagtg gagatcgccc tgctgtctgt ctacaacctg tacaccgccc ccattgccac ctcttccacc 1800. acccactgtc gacagggtaa aatgcgaaac ctgaacacca tcggtggcgg tggttctggt gttaagaat ctctggttta a <210> 50 <211> 1851 <212> DNA <213> The snowstorm <400> 50 atgtccaaga agcacattgt gatcattggc gccggtgtcg gcggcaccgc tactgctgct cgactggctc gagagggctt caaggtgacc gtggtggaga agaacgactt cggtggtggt cgatgctctc tgattcacca ccagggccac cgattcgacc agggcccttc cctgtacctg atgcccaagt acttcgagga cgcttcgcc gacctggacg agcgaattca ggaccacctg 240 gagctgctgc gatgcgacaa caactacaag gtccacttcg akgacggtga gtccattcag 300 ctgtcctctg acctgacccg aatgaaggcc gagctgggacc gagtcgaggg ccctcttggc 360 ttcggccgat tcctggactt catgaaggag acccacatcc actacgagtc cggtaccctg 420 atcgccctga agaagaactt cgagtctatc tgggacctga tccgaatcaa gtacgcccccc 480 gagattttcc gactgcacct gttcggcaag atctacgacc gagcctccaa gtacttcaag 540 accaagaaga tgcgaatggc cttcaccttc cagaccatgt acatgggtat gtctccctac 600 gacgccccccg ccgtctactc tctgctgcag tacaccgagt tcgccgaggg catctggtac 660 ccccgaggtg gtttcaacat ggtggtccag aagctggagg ccatcgccaa gcagaagtac 720 gacgccgagt tcatctataa cgccccccgtc gccaagatca acaccgacga cgccaccaag 780 caggtcaccg gtgtcaccct ggagaacggt cacattatcg acgccgacgc cgtcgtctgc 840 aacgccgatc tggtctacgc ctaccacaac ctgctgcccc cctgtcgatg gacccagaac 900 accctggcct ccaagaagct gacctcctcc tccatctctt tctactggtc catgtccacc 960 aaggtccccc agctggacgt ccacaacatt ttcctggccg aggcctacca ggagtccttc 1020 gacgagatct tcaaggactt cggtctgccc tccgaggcct ccttctacgt caacgtcccc 1080 tctcgaatcg acccctccgc cgctcctgac ggaaaggact ctgtcatcgt cctggtgccc 1140 attggccaca tgaagtctaa gaccggtgac gcctctaccg agaactaccc cgccatggtc 1200 gacaaggccc gaaagatggt cctggccgtg attgagcgac gactgggcat gtccaacttc 1260 gccgacctca tcgagcacga gcaggtcaac gaccccgccg tgtggcagtc caagttcaac 1320 ctgtggcgag gttctattct gggtctgtct cacgacgtcc tgcaggtgct gtggttccga 1380 ccctccacca aggactccac cggccgatac gacaacctgt tcttcgtggg cgcctccacc 1440 caccccggta ctggtgttcc catcgtcctg gccggctcca agctgacctc tgaccaggtg 1500 gtcaagtctt tcggtaaaac ccccaagccc cgaaagatcg agatggagaa cacccaggcc 1560 cccctggagg agcctgacgc tgagtctacc ttccccgtct ggttctggct gcgagccgcc 1620 ttctgggtca tgttcatgtt cttctacttc ttcccccagt ctaacggtca gacccccgcc 1680 tctttcatca acaacctgct gcctgaggtg ttccgagtcc acaactctaa cgtcatcggt 1740 ggcggtggtt ctctggttgg tgctctgatg cacgttatgc agaaacgttc tcgtgctatc 1800 cactcttctg acgaaggtga agaccaggct ggtgacgaag acgaagacta a 1851 <210> 51 <211> 576 <212> DNA <213> Artificial sequence <400> 51 atgctgtgga tctggaacgc cttgattgtc tttgtgacag tcatcggtat ggaggtgatt 60 gctgctctgg cacacaagta catcatgcat ggatggggct ggggttggca cctcagccac 120 cacgagcccc ggaagggtgc ctttgaagtc aacgacctat atgcggtagt ttttgccgct 180 ctctccattc tgctcatcta cctgggatcg accggcatgt ggcctcttca gtggattgga 240 gccggaatga ccgcctacgg actgttgtac ttcatggtgc atgatggtct ggttcaccaa 300 cgatggccct tccgatacat ccctcgaaag ggctacctca agcgacttta catggcccat 360 cgaatgcacc atgcagtgcg cggcaaggag ggctgcgttt ctttcggctt cctctatgct 420 ccgccacttt ccaagctgca ggccactctg cgggaacgtc acggtgctag agcaggagct 480 gccagagatg cccagggtgg cgaggacgac cccgcgtctg ggaaaggcgg tggcggttct 540 ccgccgccgg ctctgccgcc gaaacgtcgt cgttga 576 <210> 52 <211> 171 <212> DNA <213> Artificial sequence <400> 52 gcagagtatg tgcgggccct ctttgacttt aatgggaatg atgaagaaga tcttcccttt 60 aagaaaggag acatcctgag aatccgggat aagcctgaag agcagtggtg gaatgcagag 120 gacagcgaag gaaagagggg gatgattcct gtcccttacg tggagaagta t 171 <210> 53 <211> 237 <212> DNA <213> Artificial sequence <400> 53 accaaggcag atattggaac accaagtaat ttccagcaca ttggacatgt tggttgggat 60 ccaaatacag gttttgatct aaataatttg gatccagaat tgaagaatct ttttgatatg 120 tgtgggatct ctgaggccca gcttaaagac agagaaacat caaaagttat ttatgacttt 180 attgaaaaaa caggaggtgt agaagctgtt aaaaatgaac tccgaaggca agcacca 237 <210> 54 <211> 285 <212> DNA <213> Artificial sequence <400> 54 ctccagcggc gccgcgtgac ggtgcgcaag gccgacgccg gcgggctggg catcagcatc 60 aaggggggcc gggaaaacaa gatgcctatt ctcatttcca agatcttcaa gggactggca 120 gcagaccaga cggaggccct ttttgttggg gatgccatcc tgtctgtgaa tggtgaagat 180 ttgtcctctg ccacccacga tgaagcggta caggccctca agaagacagg caaggaggtt 240 gtgttggagg ttaagtacat gaaggaggtg tctccttact tcaag 285 <210> 55 <211> 1297 <212> DNA <213> Artificial sequence <400> 55 atcgcttcgg ataactcctg ctatacgaag ttatacgaat tcgcgcccag agagccattg 60 acgttctttc taatttggac cgatagccgt atagtccagt ctatctataa gttcaactaa 120 ctcgtaacta ttaccataac atatacttca ctgccccaga taaggttccg ataaaaagtt 180 ctgcagacta aatttatttc agtctcctct tcaccaccaa aatgccctcc tacgaagctc 240 gagctaacgt ccacaagtcc gcctttgccg ctcgagtgct caagctcgtg gcagccaaga 300 aaaccaacct gtgtgcttct ctggatgtta ccaccaccaa ggagctcatt gagcttgccg 360 ataaggtcgg accttatgtg tgcatgatca agacccatat cgacatcatt gacgacttca 420 cctacgccag cactgtgctc cccctcaagg aacttgctct taagcacggt ttcttcctgt 480 tcgaggacag aaagttcgca gatattggca acactgtcaa gcaccagtac aagaacggtg 540 tctaccgaat cgccgagtgg tccgatatca ccaacgccca cggtgtaccc ggaaccggaa 600 tcattgctgg cctgcgagct ggtgccgagg aaactgtctc tgaacagaag aaggaggacg 660 tctctgacta cgagaactcc footcaagg agttcctggt cccctctccc aacgagaagc 720 tggccagagg tctgctcatg ctggccgagc tgtcttgcaa gggctctctg gccactggcg 780 agtactccaa gcacaccatt gagcttgccc gatccgaccc cgagtttgtg gttggcttcg 840 ttgcccagaa ccgacctaag ggcgactctg aggactggct tattctgacc cccggggtgg 900 960 ctaccggaac ggatatcata attgtcggcc gaggtctgta cggccagaac cgagatccta 1020 tcgaggaggc caagcgatac cagaaggctg gctgggaggc ttaccagaag attaactgtt 1080 agaggttaga ctatggatat gtaatttaac tgtgtatata gagagcgtgc aagtatggag 1140 cgcttgttca gcttgtatga tggtcagacg acctgtctga tcgagtatgt atgatactgc 1200 acaacctgtg tatccgcatg atctgtccaa tggggcatgt tgttgtgttt ctcggaattc 1260 agaataactt cgtatgatgt atgctatacg aagttat 1297 <210> 56 <211> 627 <212> DNA <213> Artificial Sequence <400> 56 gcataactgt cgcgtacggc ccgataaggg ccttctccaa aagggaagcc ggttgaaatt 60 ccggcacttg gatgtggatt ctccacggca acgtaactga atgtggggac ggtggcacaa 120 gtcttggaag gagttatctt ttctttttaa cggagtcaac accctggaat tagtttgtct 180 agagataggg tatcgttccg gaagaggggg gcagctttgt cccctccgat gcacttgtga 240 cgccccttga aaacccgcag gaaggaatag ttttcacgcc aagtcgtact gataaccgca 300 gcaggtctcc aaggtgaaca gcctctagtt gatagaataa tgtagataag ggaagtcggc 360 aaaatagatc cgtaacttcg ggataaggat tggctctggg ggttggtgga tggaagcgtg 420 ggagacccca agggactggc agctgggcaa ctggcagccg gacccgcggc agacactgcg 480 tcgctccgtc cacatcatca accgccccag aactggtacg gacaagggga atctgactgt 540 ctaattaaaa catagctttg cgatggttgt aaaacaatgt tgacgcaaag tgatttctgc 600 ccagtgctct gaatgtcaaa gtgaaga 627 <210> 57 <211> 608 <212> DNA <213> Artificial sequence <400> 57 aattcaacca agcgcgcggg taaacggcgg gagtaactat gctctcttaa ggtagccaaa 60 tgcctcgtca tctaattagt gacgcgcatg aatggattaa cgagattccc actgtcccta 120 tctactatgt agcgaaacca cagccaaggg aacgggcttg gcagaatcag cggggaaaga 180 agaccctgtt gagcttgact ctagtttgac attgtgaaga gacatagggg gtgtagaata 240 agtgggagct tcggcgccgg tgaaatacca ctacccttat cgtttcttta cttatttagt 300 aagtggaagt ggtttaacaa ccattttcta gcattccttt ccaggctgaa gacattgtca 360 ggtggggagt ttggctgggg cggcacatct gttaaaagat aacgcagatg tcctaagggg 420 gactcaatga gaacagaaat ctcatgtaga acaaaagggt aaaagtcccc ttgattttga 480 ttttcagtgt gaatacaaac catgaaagtg tggcctatcg atcctttagt tgttcggagt 540 ttgaacctag aggtgccaga aaagttacca cagggataac tggcttgtgg cagtcaagcg 600 ttcatagc 608 <210> 58 <211> 531 <212> DNA <213> Artificial sequence <400> 58 agagaccggg ttggcggcgt atttgtgtcc caaaaaacag ccccaattgc cccaattgac 60 cccaaattga cccagtagcg ggcccaaccc cggcgagagc ccccttcacc ccacatatca 120 aacctccccc ggttcccaca cttgccgtta agggcgtagg gtactgcagt ctggaatcta 180 cgcttgttca gactttgtac tagtttcttt gtctggccat ccgggtaacc catgccggac 240 gcaaaataga ctactgaaaa tttttttgct ttgtggttgg gactttagcc aagggtataa 300 aagaccaccg tccccgaatt acctttcctc ttcttttctc tctctccttg tcaactcaca 360 cccgaaatcg ttaagcattt ccttctgagt ataagaatca ttcaaaatgg tgagtttcag 420 aggcagcagc aattgccacg ggctttgagc acacggccgg gtgtggtccc attcccatcg 480 acacaagacg ccacgtcatc cgaccagcac tttttgcagt actaaccgca g 531 <210> 59 <211> 1000 <212> DNA <213> Artificial sequence <400> 59 ggagtttggc gcccgttttt tcgagcccca cacgtttcgg tgagtatgag cggcggcaga 60 ttcgagcgtt tccggtttcc gcggctggac gagagcccat gatgggggct cccaccacca 120 gcaatcaggg ccctgattac acacccacct gtaatgtcat gctgttcatc gtggttaatg 180 ctgctgtgtg ctgtgtgtgt gtgttgtttg gcgctcattg ttgcgttatg cagcgtacac 240 cacaatattg gaagcttatt agcctttcta ttttttcgtt tgcaaggctt aacaacattg 300 ctgtggagag ggatggggat atggaggccg ctggagggag tcggagaggc gttttggagc 360 ggcttggcct ggcgcccagc tcgcgaaacg cacctaggac cctttggcac gccgaaatgt 420 gccacttttc agtctagtaa cgccttacct acgtcattcc atgcatgcat gtttgcgcct 480 tttttccctt gcccttgatc gccacacagt acagtgcact gtacagtgga ggttttgggg 540 gggtcttaga tgggagctaa aagcggccta gcggtacact agtgggattg tatggagtgg 600 catggagcct aggtggagcc tgacaggacg cacgaccggc tagcccgtga cagacgatgg 660 gtggctcctg ttgtccaccg cgtacaaatg tttgggccaa agtcttgtca gccttgcttg 720 cgaacctaat tcccaatttt gtcacttcgc acccccattg atcgagccct aacccctgcc 780 catcaggcaa tccaattaag ctcgcattgt ctgccttgtt tagtttggct cctgcccgtt 840 tcggcgtcca cttgcacaaa cacaaacaag cattatatat aaggctcgtc tctccctccc 900 aaccacactc acttttttgc ccgtcttccc ttgctaacac aaaagtcaag aacacaaaca 960 accaccccaa cccccttaca cacaagacat atctacagca 1000 <210> 60 <211> 838 <212> DNA <213> Artificial Sequence <400> 60 ctgcacccaa caataaatgg gtagggttgc accaacaaag ggatgggatg gggggtagaa 60 gatacgagga taacggggct caatggcaca aataagaacg aatactgcca ttaagactcg 120 tgatccagcg actgacacca ttgcatcatc taagggcctc aaaactacct cggaactgct 180 gcgctgatct ggacaccaca gaggttccga gcactttagg ttgcaccaaa tgtcccacca 240 ggtgcaggca gaaaacgctg gaacagcgtg tacagtttgt cttagcaaaa agtgaaggcg 300 ctgaggtcga gcagggtggt gtgacttgtt atagccttta gagctgcgaa agcgcgtatg 360 gatttggctc atcaggccag attgagggtc tgtggacaca tgtcatgtta gtgtacttca 420 atcgccccct ggatatagcc ccgacaatag gccgtggcct catttttttg ccttccgcac 480 atttccattg ctcggtaccc acaccttgct tctcctgcac ttgccaacct taatactggt 540 ttacattgac caacatctta caagcggggg gcttgtctag ggtatatata aacagtggct 600 ctcccaatcg gttgccagtc tcttttttcc tttctttccc cacagattcg aaatctaaac 660 tacacatcac acaatgcctg ttactgacgt ccttaagcga aagtccggtg tcatcgtcgg 720 cgacgatgtc cgagccgtga gtatccacga caagatcagt gtcgagacga cgcgttttgt 780 gtaatgacac aatccgaaag tcgctagcaa cacacactct ctacacaaac taacccag 838 <210> 61 <211> 1000 <212> DNA <213> Artificial Sequence <400> 61 ggttgaaatg aatcggccga cgctcggtag tcggaaagag ccgggaccgg ccggcgagca 60 taaaccggac gcagtaggat gtcctgcacg ggtctttttg tggggtgtgg agaaaggggt 120 gcttggagat ggaagccggt agaaccgggc tgcttggggg gatttggggc cgctgggctc 180 caaagagggg taggcatttc gttggggtta cgtaattgcg gcatttgggt cctgcgcgca 240 tgtcccattg gtcagaatta gtccggatag gagacttatc agccaatcac agcgccggat 300 ccacctgtag gttgggttgg gtgggagcac ccctccacag agtagagtca aacagcagca 360 gcaacatgat agttgggggt gtgcgtgtta aaggaaaaaa aaagaagctt gggttatatt 420 cccgctctat ttagaggttg cgggatagac gccgacggag ggcaatggcg ccatggaacc 480 ttgcggatat cgatacgccg cggcggactg cgtccgaacc agctccagca gcgttttttc 540 cgggccattg agccgactgc gaccccgcca acgtgtcttg gcccacgcac tcatgtcatg 600 ttggtgttgg gaggccactt tttaagtagc acaaggcacc tagctcgcag caaggtgtcc 660 gaaccaaaga agcggctgca gtggtgcaaa cggggcggaa acggcgggaa aaagccacgg 720 gggcacgaat tgaggcacgc cctcgaattt gagacgagtc acggccccat tcgcccgcgc 780 aatggctcgc caacgcccgg tcttttgcac cacatcaggt taccccaagc caaacctttg 840 tgttaaaaag cttaacatat tataccgaac gtaggtttgg gcgggcttgc tccgtctgtc 900 caaggcaaca tttatataag ggtctgcatc gccggctcaa ttgaatcttt tttcttcttc 960 tcttctctat attcattctt gaattaaaca cacatcaaca 1000 <210> 62 <211> 500 <212> DNA <213> Artificial Sequence <400> 62 gctatttatc actctttaca acttctacct caactatcta ctttaataaa tgaatatcgt 60 ttattctcta tgattactgt atatgcgttc ctctaagaca aatcgaaacc agcatgcgat 120 cgaatggcat acaaaagttt cttccgaagt tgatcaatgt cctgatagtc aggcagcttg 180 agaagattga cacaggtgga ggccgtaggg aaccgatcaa cctgtctacc agcgttacga 240 atggcaaatg acgggttcaa agccttgaat ccttgcaatg gtgccttgga tactgatgtc acaaacttaa gaagcagccg cttgtcctct tcctcgaaac tctcaaacac agtccagagg tcctttatag cttgatctgt atccagatag cctccgtaat tggtgtgtgt cttcaaatcc cagacgtcca cattggcatg tcctccactg ataagcattt gaagttcatc tgcgttgaac attgagaccc acgaagggtc <210> 63 <211> 512 <212> DNA <213> The snowstorm <400> 63 gcagtttgtt tagcaaaata tatttaacga gtttgataga ggcgctggac tacataatta ctgaatcacg cgtacatgtt tcagctcaaa ttgtatcacg gtttctttgt agcaatggag ggggagagtt gacaaggcat tagagaagag agcgagagga gaagacaagt ggatagga ctgcaatcat atgatctgca caaactgcga tgttttcctg tcagatcatg ttcttttgct catagttaag ctatcgtgac tttacggatc cgccgagcat cttagtagcg aggtttgcgg tctgggctga tcggcttttg ttgatcgggt tcggaacgaa tgaagtgg gtcgatagca 360. atgatcaatt cggggtgagt gagtgcggtt agtgggaaac cggggagata ctgtggggat 420 ttaggggaca gtgttaagaa gaggaagggg tgagggaggt gaggtgaggg agagggaagt 480 atagaggttt atgtagtaac atcaatcata ca 512 <210> 64 <211> 248 <212> DNA <213> Artificial sequence <400> 64 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ctccacatcc gctctaaccg 60 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240 taatttgc 248 <210> 65 <211> 412 <212> DNA <213> Artificial sequence <400> 65 gtacctccat ggcctgtccc cacgttgccg gtcttgcctc ctactacctg tccatcaatg 60 acgaggttct cacccctgcc caggtcgagg ctcttattac tgagtccaac accggtgttc 120 ttcccaccac caacctcaag ggctctccca acgctgttgc ctacaacggt gttggcattt 180 aggcaattaa cagatagttt gccggtgata attctcttaa cctcccacac tcctttgaca 240 taacgattta tgtaacgaaa ctgaaatttg accagatatt gttgtaaata gaaaatctgg 300 cttgtaggtg gcaaaatccc gtctttgttc atcaattccc tctgtgacta ctcgtcatcc 360 ctttatgttc gactgtcgta tttttatttt ccatacatac gcaagtgaga tg 412 <210> 66 <211> 21 <212> DNA <213> Artificial Sequence <400> 66 gacccttcgt gggtctcaat g 21

Claims

1. A nucleic acid molecule comprising the nucleotide sequence of the following gene: (b1) Cytoskeleton; and (b2) Key enzyme genes in the maize xanthine synthesis pathway; The cytoskeleton comprises: oleosin-SH3-SH3-GBD-PDZ-PDZ; the sequence of PDZ is shown in SEQ ID NO.54, the sequence of GBD is shown in SEQ ID NO.53, and the sequence of SH3 is shown in SEQ ID NO.

52. The key enzyme genes in the zeaxanthin synthesis pathway include: (c1) CarRP, a bifunctional enzyme gene that catalyzes the production of phytoene from two molecules of GGPP and the production of β-carotene from phytoene. (c2) The gene CarB that catalyzes the production of lycopene from phytoene; and (c3) β-carotene hydroxylase gene CrtZ; The CarRP, CarB, and CrtZ each contain one of the following binding ligands: PDZ binding ligand, GBD binding ligand, and SH3 binding ligand; the binding ligands contained in CarRP, CarB, and CrtZ are all different. The nucleic acid molecule also contains the nucleotide sequences of the following genes: (b4) Key enzyme genes in the mevalonate pathway; and (b5) Genes of key enzymes in the farnesyl pyrophosphate pathway; The key enzyme genes of the mevalonate pathway include the following genes: 3-hydroxy-3-methylglutaryl-CoA reductase gene HMGR, acetyl-CoA thioesterase gene AtoB, and IPP isomerase gene IDI; The key enzyme gene in the farnesyl pyrophosphate pathway is GGS1, which catalyzes the combination of farnesyl diphosphate with one molecule of isoprene pyrophosphate to generate digeranyl pyrophosphate.

2. The nucleic acid molecule according to claim 1, characterized in that, The gene has an upstream promoter and / or a downstream terminator.

3. An expression system comprising the nucleic acid molecule according to any one of claims 1 to 2.

4. The expression system according to claim 3, characterized in that: The expression system comprises the nucleic acid molecule of claim 1; The nucleic acid molecule's gene is broken down into three groups and inserted into three different expression vectors: plasmid PM0, plasmid PM1, and plasmid PM2. The plasmid PM0 uses pUC19 as a backbone vector and contains the key enzyme genes of the mevalonate pathway and the key enzyme genes of the farnesyl pyrophosphate pathway. The plasmid PM1 uses pUC19 as a backbone vector and contains the key enzyme genes of the maize xanthine synthesis pathway. The plasmid PM2 uses pUC19 as a backbone vector and contains the cytoskeleton.

5. A cell comprising the nucleic acid molecule of any one of claims 1 to 2 and / or the expression system of any one of claims 3 to 4.

6. The method for constructing cells according to claim 5, characterized in that: The nucleic acid molecule of any one of claims 1 to 2 and / or the expression system of any one of claims 3 to 4 are introduced into recipient cells.

7. The construction method according to claim 6, characterized in that, The recipient cells are yeast cells.

8. A method for preparing zeaxanthin, obtained by culturing the cells described in claim 5.

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