Biosynthetic genes, gene clusters, proteins, and uses thereof for the production of oryzastatin and methods for the production of oryzastatin

Abstract

The present application relates to a biosynthesis gene, gene cluster, protein of a vitamin and its application and a method for preparing vitamin.The gvmA-gvmF combined with gvmT1, gvmT1 and gvmR provided by the present application expand the vitamin biosynthesis gene, gene cluster and enzyme library, which can express enzymes or proteins GvmA-GvmF, GvmT1, GvmT2 and GvmR, can efficiently play the functions of enzyme catalysis, transport and regulation in the biosynthesis of vitamin, ensure the rapid and efficient operation of vitamin biosynthesis, greatly improve the synthesis efficiency of vitamin, reduce the formation of byproduct adenosine, and meet the demand of industrial production of vitamin.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a oryzanol biosynthesis gene, gene cluster, protein, and its application, as well as a method for preparing oryzanol. Background Technology

[0002] In 2013, the inventors isolated a plant growth-promoting bacterium, *Streptomyces caniferus* NEAU6 (CGMCC 16338), from wild *Paris polyphylla*. This bacterium significantly promoted the growth and yield of various crops, including rice, peppers, tomatoes, eggplants, and cucumbers. Subsequently, the active ingredient, oryzanol, was isolated and identified from this strain using an activity-directed assay. Oryzanol is an adenosine analog with a rare 5,6-exenyl allulose glycoside, formerly known as dequamycin or narrowycin A. Its chemical structure is similar to cytokinins, both being adenine derivatives, so it was initially classified as a cytokinin and did not attract much attention. However, compared to zeatin (a cytokinin), it significantly increased the number of tillers or branches in rice and eggplant, which is crucial for their yield. Furthermore, the inventors found that they exhibited opposite regulatory effects on the growth of plant roots and hypocotyls; oryzanol promoted their elongation, while zeatin inhibited their growth. Studies on Arabidopsis thaliana cytokinin receptor mutants further demonstrated that oryzanol does not exert its effects through the cytokinin signal transduction pathway. Field trials conducted for four consecutive years from 2017 to 2020 showed that oryzanol seed soaking treatment increased yields by 6.2-19.6%, higher than treatments with zeatin and brassinolide under the same conditions. In 2021, oryzanol was officially registered as a new type of pesticide in China.

[0003] In 2021, Shiraishi et al. identified a gene cluster involved in the biosynthesis of angustmycins A in *Streptomyces angustmyceticus* NBRC 3934 and *Streptomyces decoyicus* NRRL 2666, and inferred its biosynthetic pathway through bioinformatics analysis (Shiraishi, T.; Xia, J.; Kato, T.; Kuzuyama, T. Biosynthesis of the nucleoside antibiotic angustmycins: identification and characterization of the biosynthetic gene cluster reveal unprecedented dehydratase required for exo-glycal formation. J. Antibiot (Tokyo). 2021, 74, 830-833.). Meanwhile, Yu et al. reconstructed the entire enzymatic pathway in vitro and in E. coli, elucidating the AgmF-catalyzed conversion of angustmycin C to angustmycin A. Researchers introduced six angustmycin A synthesis genes (agmDCAEBF) cloned from *S. angustmyceticus* JCM 4053 into *E. coli* GYJ23 via a 3-plasmid system to construct an angustmycin A-producing strain. After 96 h of fermentation, the yields of angustmycin C and A reached 110 and 370 μg / mL, respectively (Yu, L.; Zhou, W.; She, Y.; Ma, H.; Cai, YS.; Jiang, M.; Deng, Z.; Price, NPJ; Chen, W. Efficient biosynthesis of nucleosidecytokinin angustmycin A containing an unusual sugar system. *Nat. Commun.* 2021, 12, 6633.).

[0004] It is evident that the biosynthetic efficiency of the anthocyanin A-producing strain constructed based on the anthocyanin A synthesis gene cloned from S. angustmyceticus JCM 4053 is low, and the current yield level cannot meet the requirements of industrial production. Therefore, developing genes and proteins for the efficient biosynthesis of oryzanol and applying them to the preparation of oryzanol has become an urgent technical problem to be solved. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the lack of genetically engineered bacteria with high synthesis efficiency for oryzanol production in the prior art, and to provide an oryzanol biosynthesis gene, gene cluster, protein, its application, and a method for preparing oryzanol. The oryzanol biosynthesis gene of this invention can convert fructose-6-phosphate into allulose-6-phosphate, and then efficiently synthesize oryzanol through a series of enzymatic catalytic reactions.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] A first aspect of the present invention provides a gene or combination of genes related to oryzanol biosynthesis, which is selected from one or more of the following genes:

[0008] (1) The gene gvmA encoding phosphoribosyl hydrolase, the nucleotide sequence of which is shown in SEQ ID NO:1;

[0009] (2) The gene gvmB encoding HAD family phosphatases, the nucleotide sequence of which is shown in SEQ ID NO:2;

[0010] (3) The gene gvmC encoding 6-phosphoallosyl-2-pyrophosphate synthase, the nucleotide sequence of which is shown in SEQ ID NO:3;

[0011] (4) The gene gvmD encoding allulose phosphate 3-epimerase, the nucleotide sequence of which is shown in SEQ ID NO:4;

[0012] (5) The gene gvmE encoding a phosphotransferase, the nucleotide sequence of which is shown in SEQ ID NO:5; and

[0013] (6) The gene gvmF encoding a dehydratase, the nucleotide sequence of which is shown in SEQ ID NO:6.

[0014] In some embodiments of the present invention, the genes related to oryzanol biosynthesis include gvmA, gvmB, gvmC, gvmD, gvmE, and gvmF.

[0015] In some embodiments of the present invention, the gene combination related to oryzanol biosynthesis consists of gvmA, gvmB, gvmC, gvmD, gvmE, and gvmF.

[0016] A second aspect of the present invention provides a protein or combination of proteins related to oryzanol biosynthesis, which is selected from one or more of the following proteins:

[0017] (1) Phosphoribose hydrolase GvmA, the amino acid sequence of which is shown in SEQ ID NO:10;

[0018] (2) HAD family phosphatase GvmB, the amino acid sequence of which is shown in SEQ ID NO:11;

[0019] (3) 6-phosphoallosyl-2-pyrophosphate synthase GvmC, wherein the amino acid sequence of GvmC is shown in SEQ ID NO:12;

[0020] (4) Allulose phosphate 3-epimerase GvmD, the amino acid sequence of which is shown in SEQ ID NO:13;

[0021] (5) Phosphotransferase GvmE, the amino acid sequence of which is shown in SEQ ID NO:14; and,

[0022] (6) Dehydratase GvmF, the amino acid sequence of which is shown in SEQ ID NO:15.

[0023] In some embodiments of the present invention, the proteins associated with oryzanol biosynthesis include GvmA, GvmB, GvmC, GvmD, GvmE, and GvmF.

[0024] In some embodiments of the present invention, the protein combination associated with oryzanol biosynthesis consists of GvmA, GvmB, GvmA, GvmB, GvmC, GvmD, GvmE and GvmF.

[0025] A third aspect of the invention provides genes or combinations of genes related to oryzanol transport or regulation, selected from one or more of the following genes:

[0026] (i) The gene encoding transporter 1, namely gvmT1, has the nucleotide sequence shown in SEQ ID NO:7;

[0027] (ii) The gene encoding transporter 2, namely gvmT2, the nucleotide sequence of which is shown in SEQ ID NO:8; and,

[0028] (iii) The gene encoding the regulator is gvmR, and the nucleotide sequence of gvmR is shown in SEQ ID NO:9.

[0029] In some embodiments of the present invention, the genes related to oryzanol transport or regulation include gvmT1, gvmT2, and gvmR.

[0030] In some embodiments of the present invention, the gene combination related to oryzanol transport or regulation consists of gvmT1, gvmT2 and gvmR.

[0031] A fourth aspect of the invention provides a protein or combination of proteins associated with oryzanol transport or regulation, selected from one or more of the following proteins:

[0032] (i) Transporter 1, namely GvmT1, has the amino acid sequence shown in SEQ ID NO:16;

[0033] (ii) Transporter 2, namely GvmT2, the amino acid sequence of which is shown in SEQ ID NO:17; and

[0034] (iii) Regulator GvmR, the amino acid sequence of which is shown in SEQ ID NO:18.

[0035] In some embodiments of the present invention, the proteins associated with oryzanol transport or regulation include GvmT1, GvmT2, and GvmR.

[0036] In some embodiments of the present invention, the protein combination associated with oryzanol transport or regulation consists of GvmT1, GvmT2 and GvmR.

[0037] In this invention, the nucleotide sequence or at least a portion thereof provided by this invention may be modified or mutated. These pathways include insertion, substitution, or deletion; polymerase chain reaction (PCR); error-mediated PCR; site-specific mutation; reconnection of different sequences; directed evolution of different portions of the sequence or homologous sequences from other sources; or mutagenesis induced by ultraviolet light or chemical reagents.

[0038] In this invention, cloned genes containing the nucleotide sequence or at least a portion thereof provided by this invention can be expressed in exogenous hosts through suitable expression systems to obtain the corresponding enzymes or other higher biological activities or yields. These exogenous hosts include Streptomyces, Pseudomonas, Escherichia coli, Bacillus, yeast, plants, and animals, etc.

[0039] In this invention, genes or gene clusters containing the nucleotide sequences or at least a portion thereof provided by this invention can be expressed in a heterologous host and their functions in the host metabolic chain can be understood through DNA microarray technology.

[0040] In this invention, genes or gene clusters containing the nucleotide sequence or at least a portion of the nucleotide sequence provided by this invention can be used to construct recombinant plasmids through genetic recombination to obtain novel biosynthetic pathways, or they can be used through insertion, substitution, deletion or inactivation to obtain novel biosynthetic pathways.

[0041] In this invention, the provided amino acid sequence can be used to isolate the desired protein and can be used for antibody preparation.

[0042] In this invention, polypeptides containing the amino acid sequence or at least a portion thereof provided by this invention may still have biological activity or even new biological activity after the removal or substitution of certain amino acids, or may have increased yield or optimized protein kinetic characteristics or other properties that are sought to be obtained.

[0043] A fifth aspect of the present invention provides a biosynthetic gene cluster of oryzanol, the biosynthetic gene cluster comprising genes or gene combinations related to oryzanol biosynthesis as described in the first aspect.

[0044] In some embodiments of the present invention, the biosynthetic gene cluster further includes genes or gene combinations related to oryzanol transport or regulation as described in the third aspect.

[0045] A sixth aspect of the present invention provides a recombinant expression vector comprising genes or gene combinations related to oryzanol biosynthesis as described in the first aspect, genes or gene combinations related to oryzanol transport or regulation as described in the third aspect, and biosynthetic gene clusters as described in the fifth aspect.

[0046] In some embodiments of the present invention, each gene or gene combination is located in the same recombinant expression vector or different recombinant expression vectors.

[0047] A seventh aspect of the present invention provides a genetically engineered bacterium that produces oryzanol, the genetically engineered bacterium comprising the recombinant expression vector as described in the sixth aspect.

[0048] In some embodiments of the present invention, the starting strain of the genetically engineered bacteria is Streptomyces coelicolor.

[0049] The eighth aspect of the present invention provides a method for preparing oryzanol, the method comprising fermenting genetically engineered bacteria as described in the seventh aspect in a fermentation medium; or reacting with a protein as described in the second or fourth aspect based on a suitable substrate to obtain oryzanol.

[0050] The ninth aspect of the present invention provides the application of a gene or gene combination related to oryzanol biosynthesis as described in the first aspect, a protein or protein combination related to oryzanol biosynthesis as described in the second aspect, a gene or gene combination related to oryzanol transport or regulation as described in the third aspect, a protein or protein combination related to oryzanol transport or regulation as described in the fourth aspect, a biosynthetic gene cluster as described in the fifth aspect, a recombinant expression vector as described in the sixth aspect, or a genetically engineered bacterium as described in the seventh aspect in the preparation of oryzanol.

[0051] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0052] The reagents and raw materials used in this invention are all commercially available.

[0053] The positive and progressive effects of this invention are as follows:

[0054] The gvmA-gvmF combination of gvmT1, gvmT2, and gvmR provided by this invention expands the genes, gene clusters, and enzyme library for oryzanol biosynthesis. It can express and produce enzymes or proteins GvmA-GvmF, GvmT1, GvmT2, and GvmR, which can efficiently perform enzyme catalysis, transport, and regulation functions in oryzanol biosynthesis, ensuring the rapid and efficient operation of oryzanol biosynthesis, greatly improving the synthesis efficiency of oryzanol, reducing the formation of the byproduct adenosine, and meeting the needs of industrial production of oryzanol. Attached Figure Description

[0055] Figure 1 The chemical formulas of oryzanol and stenomycin C are:

[0056] In the diagram: 1 represents oryzanol, and 2 represents stenomycin C.

[0057] Figure 2 For: HPLC chromatogram of metabolites

[0058] Figure: High performance liquid chromatography (λ = 260 nm) analysis of metabolites from wild-type and mutant strains of S. colicolor M1154 / pJTU2554, S. colicolor M1154 / 10H5, and S. caniferus NEAU6.

[0059] Figure 3 For: Protein SDS-PAGE analysis;

[0060] In the figure: (a) is GvmD, (b) is GvmC, (c) is GvmE, (d) is GvmB, and (e) is GvmA.

[0061] Figure 4For: HPLC analysis of the GvmA and GvmB catalytic reactions and in vitro one-pot reactions (λ = 260 nm)

[0062] In the figure, i-iii represent: cinnamyl ether C(2), adenine, and adenosine standards, respectively;

[0063] iv: HPLC analysis of the GvmB-catalyzed reaction with PMP(5) as substrate;

[0064] v: HPLC analysis of GvmB-catalyzed reaction with PMP(5) as substrate;

[0065] vi: HPLC analysis of the denatured GvmB-catalyzed reaction using AMP as a substrate;

[0066] vii: HPLC analysis of GvmB-catalyzed reactions using AMP as a substrate;

[0067] viii: HPLC analysis of GvmA-catalyzed reaction with AMP as substrate;

[0068] viiii: HPLC analysis of GvmA-catalyzed reactions using AMP as a substrate;

[0069] x: HPLC analysis of GvmB-catalyzed reaction using PMP(5) as substrate;

[0070] xi: HPLC analysis of the in vitro one-pot reaction for biosynthesis using GvmA-GvmE;

[0071] xii: HPLC analysis of the biosynthesis of GvmB-GvmE with added adenine in vitro in a one-pot reaction;

[0072] xiii: HPLC analysis of the in vitro one-pot reaction of GvmB-GvmE.

[0073] Figure 5 For example: the biosynthetic pathway of oryzanol. Detailed Implementation

[0074] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0075] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0076] General Materials and Experimental Procedures

[0077] All chemicals and enzymes are purchased from commercial sources and used directly.

[0078] Primer synthesis and DNA sequencing were performed at Beijing Qingke Biotechnology Co., Ltd. (Beijing).

[0079] PCR amplification was performed using a Bio-Rad T100 thermal cycler (Hercules, CA, USA).

[0080] Semi-preparative high-performance liquid chromatography (HPLC) was performed using a Hitachi Chromaster system with a DAD detector, a YMC-Triart C18 column (250 × 10 mm id, 5 μm, Japan), a flow rate of 3.0 mL / min, and a column temperature of 25 °C. HPLC was performed using a YMC-Triart C18 column (250 mm × 4.6 mm id, 5 μm, Japan), a flow rate of 1.0 mL / min, and a column temperature of 25 °C.

[0081] Column chromatography (CC) was performed using HP-20 macroporous adsorption resin (Mitsubishi Chemical Corp, Japan).

[0082] NMR spectra were recorded in dimethyl sulfoxide (DMSO)-d6 or D2O using a Bruker AVANCE III-600 or 800 spectrometer (Bruker Corp., Switzerland), with tetramethylsilane as an internal standard.

[0083] LC-MS analysis was performed using an Agilent 1290 / 6530 system (Agilent Corp., USA).

[0084] HRESIMS data were obtained using an Agilent G6230 Q-TOF mass instrument (Agilent Technologies, USA).

[0085] Strains culture conditions and plasmids

[0086] The strains and plasmids used in this invention are shown in Table 1. *Escherichia coli* was grown on LB medium at 30°C or 37°C; kanamycin (Kan), chloramphenicol, or apramycin (Apr) were added as needed. *S. caniferus* NEAU6 (CGMCC16338) was cultured on mannitol-soybean flour (MS) medium (2% soybean flour, 2% mannitol, 2% agar, pH 7.2) plates at 30°C. Fermentation was carried out using tryptic soybean broth (TSB) as the seed medium and oryzanol production medium (4% soybean flour, 2% malt extract, 1% sucrose, 0.3% CaCO3, pH 7.2–7.4). Kanamycin, chloramphenicol, and apramycin were added as needed at concentrations of 50 μg / mL, 25 μg / mL, and 50 μg / mL, respectively.

[0087] Table 1. Strains and plasmids used or generated in this invention.

[0088]

[0089]

[0090] Abbreviation: Kan r ,kanamycin resistance; Apr r apramycin resistance

[0091] Example 1: Identification and expression vector construction of the oryzanol biosynthesis gene cluster in Streptomyces caniferus NEAU6

[0092] I. Isolation and Identification of Oryzanol and Narrow-spotted Mycin C

[0093] *S. caniferus* NEAU6 was cultured on MS medium at 30°C for 5 days. Inoculated into 250 mL Erlenmeyer flasks containing 50 mL TSB, it was cultured at 30°C with shaking at 200 rpm for 24 h. Then, equal aliquots (15 mL) of the culture were transferred to 1 L Erlenmeyer flasks containing 250 mL of production medium and cultured at 30°C for 7 days with shaking at 200 rpm. The fermentation broth was centrifuged (4000 rpm, 20 min), and the supernatant was placed on an HP-20 resin column, washed twice with water, and eluted with 100% methanol. The eluent was evaporated under reduced pressure at 40°C, and then separated by semi-preparative high-performance liquid chromatography (HPLC) with a methanol-water solution (15:85, v / v; wavelength 260 nm). The structures of oryzanol and anisodacin C were characterized by HRMS and NMR. The chemical formulas of oryzanol and anisodacin C are shown below. Figure 1 As shown.

[0094] II. Chemical Synthesis of Alloxanone Adenosine (PMP)

[0095] Pyrophosphoryl chloride (77.9 mg, 309.48 μM) was added to 1 mL of triethyl phosphate containing stenomycin C (23 mg, 77.37 μM) under constant stirring at -78 °C. The mixture was stirred at -78 °C for 5 h. The reaction mixture was then poured into ice water and stirred at 0 °C for 0.5 h. The mixture was then washed with dichloromethane (5 mL, 3 times), and the aqueous phase was slowly titrated with 1 M NaOH to approximately pH 7.0, followed by lyophilization. The remaining white solid was dissolved in 0.5 mL of ddH2O, and then subjected to high-performance liquid chromatography (HPLC) using a Dionex Carbopac™ PA10 carbohydrate column with gradient elution of water as solvent A and 1.0 M ammonium acetate as solvent B at a flow rate of 1 mL / min and a UV detection wavelength of 260 nm. The following conditions were used: 0–5 min, 5% solvent B; 5.1–10 min, 20% solvent B; 10.1–15 min, 40% solvent B; 15.1–18 min, 100% solvent B. The eluent was collected at 12.5 min and lyophilized to obtain allulose adenosine (2.1 mg, 5.57 μM, yield 7.2%) as a white solid.

[0096] III. Genome sequencing and genome library construction

[0097] The genome of *S. caniferus* NEAU6 was sequenced using PacBio RS II single-molecule real-time (SMRT) technology. Low-quality reads were filtered using SMRT 2.3.018,19, and the filtered reads were assembled into a gapless contig. Genomic DNA was partially digested with MboI according to standard procedures. DNA fragments of 30–45 kb were isolated and ligated to cosmid SuperCos1 or pJTU2554. Extracts were packaged using MaxPlax Lambda packaging buffer. Approximately 2000 *E. coli* clones were selected and stored in 20 96-well microplates at 80°C.

[0098] IV. Identification of the oryzanol biosynthesis gene cluster in Streptomyces caniferus NEAU6

[0099] Based on the whole genome sequencing results of Streptomyces caniferus NEAU6, the nucleotide and amino acid sequences of the gvmT1 to gvmF genes were obtained. The amino acid sequences were then compared with the amino acid sequences of the homologous genes AgmT1 to AgmF of S. angustmyceticus. The results are shown in Table 2.

[0100] Table 2. Functional predictions of genes in the oryzanol gene cluster.

[0101]

[0102] a Amino acids b :Identity / Positive.

[0103] Results: This gene cluster spanned approximately 8.9 kbp and contained 9 open reading frames. It shared 85-92% homology with 9 genes (agmT1, T2, R, A, B, C, D, E, and F) responsible for oryzanol biosynthesis in *S. angustmyceticus* NBRC3934. However, the amino acid sequence identity of gvmA was only 92%, gvmB only 85%, and gvmR only 87%. Furthermore, the enzyme length encoded by the gvmR gene in *S. caniferus* NEAU6 (259 aa) was significantly shorter than that of AgmR (290 aa in *S. angustmyceticus* NBRC 3934).

[0104] V. Functional Identification of the Gene for Oryzanol Biosynthesis

[0105] To further identify the function of this gene cluster, the plasmid pJTU2554-10H5 containing the gvmT1 to gvmF gene from *S. caniferus* NEAU6 was transferred via conjugation into the heterologous host *Streptomyces coelicolor* M1154, which does not produce the gvmT1 and gvmF genes. An empty pJTU2554 vector was used as a negative control, and the same conjugation was performed in parallel. The fermentation medium and conditions for the recombinant strain M1154 / 10H5 were the same as those for the wild-type strain (see Example 1, "I. Isolation and Identification of Oryzanol and Narrow-spotted Mycin C"). The fermentation extract was then analyzed by HPLC.

[0106] Results: HPLC analysis of the fermentation extract showed that *S. colicolor* M1154 / 10H5 produced oryzanol, while the control group did not. Figure 2 The gene clusters gvmT1 to gvmF contain all the genes required for oryzanol biosynthesis. The genetically engineered bacteria containing plasmid pJTU2554-10H5 were fermented, and the fermentation extract was analyzed. The results showed that the oryzanol content in the fermentation broth was 1567 μg / mL.

[0107] Example 2: Oryzanol Biosynthetic Enzyme

[0108] I. Preparation of Recombinant Bacteria

[0109] Genes encoding GvmA, GvmB, GvmC, GvmD, and GvmE were amplified by PCR from cosmid SuperCos1-3E3 (primers are shown in Table 3). The gvmA and gvmC genes were cloned into the pET26b vector, and the gvmB, gvmD, and gvmE genes were cloned into the pET28a vector using NdeI and HindIII restriction sites. The resulting plasmids were used to transform *E. coli* BL21(DE3) cells. Recombinant colonies were selected on LB agar plates containing kanamycin (50 μg / mL). Positive clones were identified by colony PCR, and the corresponding plasmids were isolated and sequenced for confirmation.

[0110] II. Protein Expression and Measurement

[0111] E. coli BL21(DE3) cells containing pET26b-gvmA, pET26b-gvmC, pET28a-gvmB, pET28a-gvmD, and pET28a-gvmE were cultured in LB liquid medium (50 μg / mL) containing kanamycin at 37°C, with an OD600 of 0.6. The cultures were cooled on ice for 10 min and induced with 0.25 mM IPTG at 16°C for 18 h. After centrifugation at 4000 rpm for 15 min at 4°C, the cells were resuspended in 50 mL of buffer A (Tris 50 mM, NaCl 300 mM, imidazole 15 mM, glycerol 10%, pH 8.0) and homogenized by sonication on ice. After centrifugation at 24000 rpm for 1 h, the supernatant was filtered and purified using the AKTA purification system and then added to a 5 mL Hisstrap™ FF column (GE Healthcare). Samples were eluted using a linear imidazole gradient of buffer A and buffer B (imidazole 500 mM, Tris 50 mM, NaCl 100 mM, pH 8.0). The target protein was concentrated by Amicon Ultra-4 (10K, Millipore) ultrafiltration and stored in a buffer of NaH2PO4 100 mM, glycerol 10%, pH 8.0 at -80℃. Protein concentration was determined using the Bradford method. The molecular weight of the protein was determined by SDS-PAGE (see [link to SDS-PAGE]). Figure 3 Ultimately, the following proteases were obtained: GvmA encoded by the gvmA gene, GvmB encoded by the gvmB gene, GvmC encoded by the gvmC gene, GvmD encoded by the gvmD gene, and GvmE encoded by the gvmE gene.

[0112] Table 3. Primer pairs used for PCR amplification of target genes

[0113]

[0114]

[0115] Example 3: Determination of the biochemical activity of oryzanol biosynthetic enzyme

[0116] I. In vitro biochemical activity assay of GvmB

[0117] The dephosphorylation of AMP and PMP catalyzed by GvmB was carried out in 50 μL of a reaction mixture containing 1 μM GvmB, 1 mM AMP, 1 mM PMP, and 5 mM MgCl2 at 30 °C under 50 mM Tris·HCl (pH 8.0). The reaction was quenched by adding 50 μL of methanol. After centrifugation to remove protein, the supernatant was analyzed by HPLC.

[0118] Results: GvmB catalyzes the dephosphorylation of PMP. Incubation of PMP with GvmB yielded 2 (…) by HPLC analysis. Figure 4 In addition, GvmB can catalyze the dephosphorylation of AMP to form adenosine (v). Figure 4 (vii) indicates that GvmB is a phosphatase with significant substrate heterogeneity because it can process both PMP and AMP.

[0119] II. In vitro biochemical activity assay of GvmA

[0120] The enzyme activity of GvmA was determined by reacting the sample in 50 μL of 50 mM Tris·HCl (pH 8.0) solution at 30 °C for 1 h. The reaction was quenched by adding 50 μL of methanol. After centrifugation to remove protein, the supernatant was analyzed by HPLC.

[0121] Results: The function of GvmA is to provide the substrate adenine and coordinate primary and secondary metabolism. HPLC analysis showed that incubation of GvmA with AMP produces an adenine peak ( Figure 4 (viiii), corresponding to the true standard. Considering that PMP and AMP have similar structures, the enzymatic activity of GvmA against PMP was also tested under the same conditions. HPLC analysis showed that no detectable product was produced by the enzymatic hydrolysis mixture. Figure 4This result indicates that GvmA can specifically recognize the substrate AMP, but not the substrate PMP. GvmA plays a key role in mitigating substrate competition for GvmB and reducing the formation of the byproduct adenosine. AMP catalyzed by GvmA releases adenine, which is a substrate of GvmE, thus eliminating the need for additional adenine in the biosynthetic pathway. To further confirm the role of GvmA in oryzanol biosynthesis, we co-incubated GvmA-E with D-fructose 6-phosphate and ATP, but without adenine, thus allowing the observation of adenine and the final product stenomycin C, while adenosine was not detected. Figure 4 The production of ximycin C requires the addition of adenine, and at the same time, a large amount of adenosine is produced. Figure 4 (xii and xiii). The results showed that GvmA prevented competition between adenine and primary metabolism in the biosynthetic pathway, while reducing the formation of the byproduct adenosine. This resulted in a faster and more efficient biosynthesis of oryzanol. Finally, the biosynthetic pathway of oryzanol was determined as follows: Figure 5 As shown.

[0122] III. In vitro biochemical activities of GvmC, GvmD and GvmE

[0123] Enzyme activity was achieved for 2 h at 30 °C using 50 μL of a solution containing 1 μM GvmD, 1 μM GvmC, 1 mM d-fructose 6-phosphate, 1 mM ATP, 5 mM MgCl2, 5 mM KPi (pH 8.0), and 50 mM Tris·HCl (pH 8.0). The reaction was then quenched by adding 50 μL of chloroform. After centrifugation to remove protein, the supernatant was analyzed by HRMS.

[0124] The GvmC / GvmE coupled enzyme activity was determined by reacting the enzyme in a 50 μL solution containing 1 μM GvmD, 1 μM GvmC, 1 μM GvmE, 1 mM ATP, 1 mM adenine, 5 mM MgCl2, 5 mM KPi (pH 8.0), and 50 mM Tris·HCl (pH 8.0) at 30 °C for 2 h. The reaction was quenched by adding 50 μL of chloroform. After centrifugation to remove protein, the supernatant was used for HPLC analysis.

[0125] The enzyme activity of GvmC was determined by reacting the sample in a 50 μL volume containing 1 μM GvmC, 1 mM d-ribose-5-phosphate, 1 mM ATP, 5 mM MgCl2, 5 mM KPi (pH 8.0), and 50 mM Tris·HCl (pH 8.0) at 30 °C for 2 h. The enzyme activity of GvmE against PRPP was determined by reacting the sample in a 50 μL volume containing 1 μM GvmE, 1 mM PRPP, 1 mM adenine, and 5 mM MgCl2, and 50 mM Tris·HCl (pH 8.0) at 30 °C for 2 h. The reaction was quenched by adding 50 μL of chloroform. After centrifugation to remove protein, the supernatant was analyzed by HPLC.

[0126] Results: GvmC / GvmE also function as a prase / APRTase (PPPP synthesis / phosphotransferase) in the adenine rescue pathway. Incubation with ATP bound to GvmC using d-ribose 5-phosphate or incubation with adenine bound to GvmE using PRPP can generate AMP (e.g., AMP). Figure 5 As shown in the figure, AMP is one of the sources of adenine, a precursor for the synthesis of oryzanol.

[0127] Example 4 Kinetic analysis of oryzanol biosynthetic enzyme

[0128] Kinetic analysis of GvmA and GvmB. The kinetics of GvmA with respect to AMP were analyzed in a reaction mixture containing 50 mM Tris·HCl (pH 8.0), 10 nM GvmA, and AMP concentrations ranging from 25 to 1000 μM. The kinetics of GvmB with respect to PMP were investigated in a reaction mixture containing 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2, 20 nM GvmB, and PMP concentrations ranging from 100 to 2000 μM. The reaction kinetics of GvmB with AMP were analyzed in a mixed reaction of 50 mM Tris·HCl (pH 8.0), 5 mM MgCl2, 2.92 μM GvmB, and AMP concentrations ranging from 250 to 5000 μM. In addition, using the AvmA and AvmB genes disclosed by Yu et al. as references, the corresponding enzymes AgmA and AgmB were obtained by obtaining the enzymes GvmA and GvmB, respectively, and the differences in their catalytic efficiencies were compared. High-performance liquid chromatography (HPLC) was used to determine the kinetics. All assays were performed in triplicate, according to the Michaelis-Menten equation V0 = (V max ×[S]) / (K m +[S]) performs curve fitting and calculates K m and V max Value. According to the formula k cat =Vmax / [E] calculate k cat value.

[0129] Table 4. Kinetic analysis of GvmA and GvmB

[0130]

[0131]

[0132] Results: Kinetic experiments showed that the catalytic efficiency of GvmA for AMP (k) cat / K m =2.83×10 4 M -1 s -1 The catalytic efficiency (kb) of GvmB for PMP is significantly higher than that of AgmA. cat / K m =6.92×10 3 M -1 s -1 The activity of GvmB is significantly higher than that of AgmB. Furthermore, GvmB's PMP dephosphorylation is more efficient: the catalytic efficiency of GvmB's PMP dephosphorylation is 96 times that of AMP dephosphorylation, while AgmB's PMP dephosphorylation efficiency is only 17 times that of AMP dephosphorylation. This indicates that the enzymes GvmA and GvmB encoded by the gvmA gene and gvmB gene in *S. caniferus* NEAU6 exhibit higher catalytic activity and a certain degree of substrate selectivity or preference. Because of the high catalytic activity and specific substrate selectivity or preference of GvmA and GvmB, the biosynthetic pathway avoids or reduces competition with the adenosine metabolic pathway, while also reducing the formation of the byproduct adenosine, making the biosynthesis of oryzanol faster, more efficient, and more productive.

[0133] 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.

[0134] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0135] References:

[0136] 1. Shiraishi, T.;

[0137] 2.Yu,L.,et al;.Efficient biosynthesis of nucleoside cytokininangustmycin A containing an unusual sugar system.Nat.Commun.2021,12,6633. SEQUENCE LISTING <110> Yuanda Industrial Holdings Co., Ltd. <120> Oryzanol biosynthesis genes, gene clusters, proteins and their applications, and methods for preparing oryzanol. <130> P22012966C <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> gvmA <400> 1 atggagacct caatggaatc ggccgtcacc gtgtattgcg gagcctcact cggcgagcac 60 cccgcctacg ccgacgcagc gcgtcaatta ggtcaggcgt tggcccggtc cggaatgaac 120 ttggtctacg gcggggcgtc caccggcctg atggggtgcg tggcggacgc ggcactcctc 180 ggcggcagcc gcgtcaccgg cgtcatcccg cagagcctcg tgcggtacga ggtcgcgcac 240 accgccctca ccgacctgcg cgtcgttgcg accatgcatg aacgcaaggc actgatggcc 300 gagttgggag acgccttcgt cgcgatgccg ggcggcttcg gcaccgcgga ggaactgttc 360 gaggtcctga catgggccca gctcggactg cactccaagc cgtgcgccct gctcaacacc 420 aacggctact acaccccgct cctgcggttc ctggagcacg ccgccgacga agggttcgtg 480 caccggcgct acctcggcaa tgtgatcgtc gaagacgatc cgcacgtcct gctctcccgg 540 ctcacgagcc accgcgcgct gccgcacctg ttcgaagagg tggccgaggc atga 594 <210> 2 <211> 675 <212> DNA <213> Artificial Sequence <220> <223> gvmB <400> 2 atgaccacca caccccgccc ggccgccacg ggtgcccgct cccgcccctc cggcgccact 60 ccccccgccc cggcactcgc cgtgttcgac ctcgacggca ccttgcttcc ggagacgacc 120 tcctgccggg agatcgcccg ggtggtcggc aggcacatgg acatccacga cctggaagcg 180 gcgtacggcc ggggcgagat cgacagccgc gccttcgcgg tcagcgcgct gcggtgctgg 240 agcgcgggcg gcaaggccgt ctaccgccag gcgttcgacg ccgcacgctt cctccacggc 300 atccaggact gtctgacgtg gctgcgcacg cacgggacga caaccatcct ggtcacgatg 360 tcgcaccggg agttcgccga gtgcctcgac ggcttcgacc acatcttcgc gtccgactac 420 cccgacgaca tcctcaaccc cgaggacaag ccgctgatcg tcgagaaact ccgcaccgac 480 ctgggcatca cggccgacga catcatcgcc ttcggcgact cgacatccga cctgccgctc 540 ttccgcaccc tgcggcgcac ggtcgccgtc aatgcctccc ccgccctcac cgcgctggcc 600 gcgcaccact accagggcga cgatctgcgc gaggcgctgc gcctggtctg ccctgacgcg 660 gagttcacct catga 675 <210> 3 <211> 1077 <212> DNA <213> Artificial Sequence <220> <223> gvmC <400> 3 atgagcgcgc ccggccgcgc cgtgccctcc gccaccacgg ccgcctcacc cgctcccggt 60 gccggcaccg tcaccgccgt ctgccggtcc ttcgccgagg acttccccgg cgggctcgac 120 gccctccacg acgccctggg ccggcgtggc ctgaccgccg ggacacacgg acaggtcgac 180 gggcggttcc ccgacggaga gctgtgtgtg gacctgccag gggaccccac cggggcgcat 240 gtgctcatcc accagtccct gaccgggccc gccgaacagg tcgaccagtc ggtgatggca 300 ctgctggccg tggcccgtgc ctaccgggag cacggggccc ggaaggtgac cgccgtcgta 360 ccgcacctcg cctatgcgcg ccacgaccgg cacgtacccg gcgaacgccg cccggtcatg 420 gccggcttgc tggcacagct cgccgcgacg gccggtatcg accagctcat caccctggcc 480 tcgggcgccc aggaccgtct cggacacctc agcgagccga tggatctggc gcagctcccc 540 gcggacgcgg cactgatgtc cctggtgcgc ccctgggtcg tgcccggccg tacggtgctc 600 gtggcccctg atgcgggggc cgcccaccag gtccgcagga tcggtgaccg gctacggctt 660 ccggtgatca ccgccacgaa acgccgcacc ggaccggact ccgtcaccgc ggccctcacc 720 tcgtccgcac ccggccctgc cgaccgcgtc atcatcgtgg acgacctgat caccagtgcg 780 ggaacgatgc tggcggtggc ccggctggca cgggcggagt tcgacgggtg ccaggtgctc 840 tgcgcggcga cccatctgcg ccccaccccg accggcgaac agcggctggc cgctgccctg 900 cgggacggga cgctgcaccg cgtgctcacc accgacgcgg ccggccaccg cccccgtatc 960 cccggcatcg acgtcaccgc cgtcctcccg ctgatcgccg acgccgtcgc cgaacacgtc 1020 ctccctgccc cctccccctt agcggaaagg cccaccgcac atgcaggctc ccactga 1077 <210> 4 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> gvmD <400> 4 atgcaggctc ccactgagct gctcgaaccc gttcccccga ccacgctgag cccggccttg 60 tccgtgtccg tggtctgcct cgaccagacc cggctcggcg aacacgtcgc acagttacgg 120 acgttggggg tcagccgact gcacgtcgac ctcatcgacc cggccttcgc ccggaacgtc 180 ggactgcccc tggagaccat cagcgacctg gcggccacct gcggtctgcc gatcgacgtc 240 cacgccatgc tggcgcgccc cggtgacctg ctggagcagg ccgcggcacg cggggcggcc 300 accatctgcg tccaccagcg caccgtcacc ccccaggtcc tggcttgctt acagcgcatc 360 gccgccgggc ccgccgaggt gggcctggtc atcgacccgg gtgagggact ggaccgtgcg 420 ctgatcgagc agctggcacc cgcccagctg acggtgatga gcgtcgaacc gggcggcgtg 480 gggcggccgt tccgcgacga ggccctgctc accctcgccg aggccgccga gctgcgcgcc 540 cgcggcatcg tccggcgggt cgaagccgac ggggcggtcg gtcctgccac cgtcgcgcgg 600 ctggtgtccg ccggggccgg ccaactcgtc ctgggcagca cggtcttccc gcgccgcggc 660 ccggaaggca accgcctgga cgaactggcc ctggcactca ccgccctctc gcccgcctga 720 <210> 5 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> gvmE <400> 5 atgacgctca cgcacaccat caccatcggc gacgtccgcc gcgagctgcc gatcgtacgg 60 gtggcggacg acgcccggat cgccttcctc aagctgtacg gcgacgtgga actcaccgtc 120 gcctgcgcac gcgcactggc cgggcggatg ccggcggacg tcgatgtcat cgtgggcccg 180 gagaccgggg gcatcctgct cgcccacgaa ctcgccgagc actccgggcg tccctacgtg 240 atcgcccgca agaagctgcg ccccaacatg gtcaagccgc tccgcgtccc ggtacagagc 300 atcggcaccc cgggacagca ggaactcttc ctcggtgagg acgacgcggc gctcatcaag 360 ggccgccggg tcgccgtcgt cgacgaggtc atcagcagcg gcggcaccct caaggcgctc 420 catgaactcg tcgccgcggc cggcggcacc gtgcagcagg tgctgaccgt ggccaccgaa 480 ggggagcgca ggccggacgt ggagtcgctg ctgcatctgc ccgtctacac ggactag 537 <210> 6 <211> 1272 <212> DNA <213> Artificial Sequence <220> <223> gvmF <400> 6 atgacgccca ccatccatgc aagcgaggtc gctgacatcg gcctcgccgg ccatggcgaa 60 ggactgtgcg actggtcggc ccaatcgatg cccatcctgg cgcggatacg cgagcgttac 120 gccgtcgaga agcccttcca gggcaagcgc atcgccatcg ccgtgcacat caccaccgag 180 accgccaacc tcgctctgac cctccgggcc ggcggcgccg aggtcttcct gtgcgcctcc 240 aacgtacgga cgacgaacga cgcggtggcc gcctacgcag cccagcaggg cgtgtacgtc 300 tacggcatcc agggcgagga ccgcgagacc tactccacgc actgccaccg tgtcctggac 360 gcccgcccgc acctggtcat cgacgacggt gcggatctga ccggtctgct gcacggcgcg 420 cgcaccgacg ccgcgtccga cgtcatcgcg ggcctggagg agaccacggc cggcatccag 480 cgcattcgca gcctggaggc cgacggactg ctgcggtacc ccgtcgtcgc ggtcaacgag 540 gcacgcacca agcatctgtt cgacaaccgc tacggcaccg ggcagagcac catggacgcc 600 atcctgcggc tgacgaacac cctggtcgcc ggctcacggg tggtcgtggc cggttacggc 660 atgtgcggaa agggcgtcgc ggcacgggcc cgcggtatgg gcgccgatgt cctggtgacc 720 gaggtggacc atgtccgggc cctggaggcc gtcatggacg ggtaccgggt catggagatg 780 gaccgtgccg cggaactggg cgacatcttc atcacggtca ccagcaacaa gaacgtcctc 840 ggggcggagc acttcgcccg tatgcccgac ggcgccctgg tgtgcaactc cggccacttc 900 gacgtggaga tcgatgtgcc ggcactgcgc gccgcggcgc tcaagcacac ccggccccgc 960 cccggcgtcg accagtacac gctcgccgac ggccggcgga tcaacctgct ctccgcgggg 1020 gaggtggtga atctcgccgc ggccgagggc aacccggcga cgttgatgga catgtccttc 1080 accaaccagg tcctggggtg tgagtacctc ctcaagaacg ccgcggacct ggaccgccgc 1140 gtccacaccc tgccctcgga catcgacgac caggtggcac acctgaaact ccagacgctg 1200 ggcgtcggca tcgacaccct cagcgaagag cagaccgcgt acctgcgctc ctgggatcag 1260 ggcagcgcct ga 1272 <210> 7 <211> 1311 <212> DNA <213> Artificial Sequence <220> <223> gvmT1 <400> 7 gtggaaccga gtgacgagtt gacgccgtcg cacgaacggc cgcatccgcc cggtcaggga 60 ccggccgcct gggcccggcg ggcggcacgc agcgttcctc cggggcgggc ggcccgcacc 120 ttcctgctgg tcgccttcgt cgacgccgcg ggccgtggca tcttcctcgc gggctccgcg 180 ctcttctaca cgcaggtcat cgggctcacc aacgcgcagg tcggactggg gctgtcgatc 240 gccggcctgg tggggctggt gtgcgccatt cccatcggct ggctcgcgga ccggttcggt 300 gacgggcccg tcctggtggc gctgcaactg tggcgggccg cggcgttcct ggtctatccc 360 ctggtcgacg acttcgggct gttccttgtg gtggcctgtc tggtgggcgc ggtggagtgg 420 gccgcgatgc ccatcatcca gtcggtcgcc ggagccacca gcgaggaggg ctccatggtg 480 gaggccatgg ccgtggtcgc ggtggcccgc aactccgcgt acgcgctctc cgccctggcc 540 gccaccgccg tgatcacctg ggcgtccccc cgcgcctatg tcgtcttcgt gctgttcaac 600 gcgctgggat tcctcgtctc ggggatgctg ctgctgagac tgcggctgcc gcgccgggcg 660 gtggaccaca gcgcgaagga gccggccgga tcgaagctgc tgccgttcag ggatgtgcgg 720 ttcctgctgc tgtccgtcac gaacgggatc ctgtacgccc atatgccgat cctgtccgtg 780 gccttcccgc tgtgggtcgt cacgcgtacc gatgcgccgc gcgccctcgt cggtgtggcg 840 ctgaccatca acaccgtgat ggcggtgatg ttgcagatcc ggctcagcaa gggcggggac 900 gacctcgtgc gcgccggccg caagcagcgg ctcgccggac tggcgctggc cgcgttctgt 960 gtgctcgtcg cgttcacccg gtcggtggac ggactcaccg ccacggtcct gttgctgctc gccgcggtcg cgctgaccct gggcgagctg tggcagtcgg cggggcggctg gggcatctcc 1080 tacggcctcg ctcctgaggc ccagcgcacc tactacctga gcgtctacca actcggggcc 1140 accgggtgca cggttgcggg gccggcggtg ctggtgatgg ccgtcgtgga cgtgggtccg gcaggctggg cggggctggc ggccctgctc gcgctcacgg gcctggtgac ccccgcgctc 1260 acccggccgc cgcgccacaa ggcagcggtc gccccggcgc cggccgactg a <210> 8 <211> 1200 <212> DNA <213> Artificial Sequence <220> <223> gvmT2 <400> 8 atgcctctgg caatcctcgc cctggccatc agcgccttcg cggtcggcac gaccgagttc gtgatcatgg gcttgctctc ggacgtggca gccgacctgt ccgtctccgt gcccgtggcc 120 ggctacctgg tctccggcta cgccctcggc gtcgtggccg gtgcgctgcc gctggccgcc 180 ttgaccaccc gtttcccccg tcgggccgtc ctcgtgctcc tcatggccgt cttcgtcgtg 240 ggcaacctcc tcgcgggtgt cgcccccggg tacggcatgc tgatgtccgc ccgcgtgctg 300 gtgtccctcg cccacggggc gttcttcggc gtcgccgcgg tcaccgccgc ggacctggtc 360 gccccgtccc ggcgggcgag cgccatctcc ctgattttca gcggcatgac cgtctcgacg 420 attctcgggg taccgctggg cacgcttatc ggccagcact ttggctggcg ggccaccttt 480 ctgagcgtgg cactgatcgg tttcgtcgga ctcctcagca ttcttgtgct gctgcccgcg 540 caaccgcgcc ccacgggtgg tgggctccgc gcggaattgg gcgctttccg ccggaaacag 600 gtgtgggtca ctctcaccgt caccatgctg gtttccgcag cgattttcgc gtccttcacc 660 tacatcgagc cgatgctcgt caaggtgagc ggtttcgcgc cgagttccgt cacggtgatt 720 ctcatgctct atggcatcgg gatgtccgtc ggaaatcttc tgggcggaca ccttgccggt 780 aaattcccgg tcgcgacgct gcacggcacg ctcatcgtgc tggcggtgct gctcgcggtc 840 ttcgcctaca gcattcattt ccggacacct gccgtgatca ccctgacgct gctggggctg 900 tgcgggaccg ccgcgattcc cacgttgcag caccgcatca tgcaggtggc ctccgaagcg 960 cgcacgctcg cctcggccgc gaatgtcgcc gctctcaacg tcggcaatgc actcgccgcc 1020 tgggtcggcg gggccgccat cggcgccgga ttcggctacg cctcgcccaa cctggtcggt 1080 gccgcgctcg ccgtggccgc caccctcatc gtcgtcgtgg ccgacctcac cgcccgcggg 1140 cgggaccgga acgggacggc gcgacgcgcc gacgagcagg tacgcacccc cgcttcatga 1200 <210> 9 <211> 780 <212> DNA <213> Artificial Sequence <220> <223> gvmR <400> 9 atgctggtcg tgccgtccct gacgaacccc catttcgccg atgtgcacag cggagcggcc 60 cgcgtcggcg ccgaccacgg gatcggggtg atcgtctacc ccctccaggg cgaggacggg 120 aagggcccgt tcccctctcc ccgccaggcc atcgacggcg ttctcgcctg tgccgtgtcc 180 gccgggagca tcgacgtgct gcgggacggc gtcgccatgg tcgccctcga cagcgacccg 240 gacgccggca tctgttcggt caccatggac gtcggcgccg ggatgcgctc cgccgtggac 300 cacctcgtcc ggctggggca ccgagacatc ctgcatgtcc gtgcggagcg cgacgcctgg 360 acattcgcgc agcgcaccga ggccttcgac cgccggctcg gggcgcaccg ccgcgtgcgg 420 gggcggagcc tggccgttcc cttcgacggc gcgcgggcgc gggaacggat actggccagg 480 ctgcgcgacg acaactcggt gaccgcggtc atctgcgacg acgacaatct ggccgcctgt 540 gcctactccg ccgcgcacgg cctcgggctg tccatccccg gcgatctgtc cgtcatcgga 600 ttcgacgacc tgcccctcgc ctccttgatg gtgcccgccc tgacgacggt gcgccttccc 660 gcccatgagc tggggcgcct gggcatggag cagttcctgc gcaacgacac ggcgacggcc 720 aaaacggtca tgccggtcga gttcgtcgag cgggcctccg tggggccgcc acggcggtga 780 <210> 10 <211> 197 <212> PRT <213> Artificial Sequence <220> <223> gvmA <400> 10 Met Glu Thr Ser Met Glu Ser Ala Val Thr Val Tyr Cys Gly Ala Ser 1 5 10 15 Leu Gly Glu His Pro Ala Tyr Ala Asp Ala Ala Arg Gln Leu Gly Gln 20 25 30 Ala Leu Ala Arg Ser Gly Met Asn Leu Val Tyr Gly Gly Ala Ser Thr 35 40 45 Gly Leu Met Gly Cys Val Ala Asp Ala Ala Leu Leu Gly Gly Ser Arg 50 55 60 Val Thr Gly Val Ile Pro Gln Ser Leu Val Arg Tyr Glu Val Ala His 65 70 75 80 Thr Ala Leu Thr Asp Leu Arg Val Val Ala Thr Met His Glu Arg Lys 85 90 95 Ala Leu Met Ala Glu Leu Gly Asp Ala Phe Val Ala Met Pro Gly Gly 100 105 110 Phe Gly Thr Ala Glu Glu Leu Phe Glu Val Leu Thr Trp Ala Gln Leu 115 120 125 Gly Leu His Ser Lys Pro Cys Ala Leu Leu Asn Thr Asn Gly Tyr Tyr 130 135 140 Thr Pro Leu Leu Arg Phe Leu Glu His Ala Ala Asp Glu Gly Phe Val 145 150 155 160 His Arg Arg Tyr Leu Gly Asn Val Ile Val Glu Asp Asp Pro His Val 165 170 175 Leu Leu Ser Arg Leu Thr Ser His Arg Ala Leu Pro His Leu Phe Glu 180 185 190 Glu Val Ala Glu Ala 195 <210> 11 <211> 224 <212> PRT <213> Artificial Sequence <220> <223> gvmB <400> 11 Met Thr Thr Thr Pro Arg Pro Ala Ala Thr Gly Ala Arg Ser Arg Pro 1 5 10 15 Ser Gly Ala Thr Pro Pro Ala Pro Ala Leu Ala Val Phe Asp Leu Asp 20 25 30 Gly Thr Leu Leu Pro Glu Thr Thr Ser Cys Arg Glu Ile Ala Arg Val 35 40 45 Val Gly Arg His Met Asp Ile His Asp Leu Glu Ala Ala Tyr Gly Arg 50 55 60 Gly Glu Ile Asp Ser Arg Ala Phe Ala Val Ser Ala Leu Arg Cys Trp 65 70 75 80 Ser Ala Gly Gly Lys Ala Val Tyr Arg Gln Ala Phe Asp Ala Ala Arg 85 90 95 Phe Leu His Gly Ile Gln Asp Cys Leu Thr Trp Leu Arg Thr His Gly 100 105 110 Thr Thr Thr Ile Leu Val Thr Met Ser His Arg Glu Phe Ala Glu Cys 115 120 125 Leu Asp Gly Phe Asp His Ile Phe Ala Ser Asp Tyr Pro Asp Asp Ile 130 135 140 Leu Asn Pro Glu Asp Lys Pro Leu Ile Val Glu Lys Leu Arg Thr Asp 145 150 155 160 Leu Gly Ile Thr Ala Asp Asp Ile Ile Ala Phe Gly Asp Ser Thr Ser 165 170 175 Asp Leu Pro Leu Phe Arg Thr Leu Arg Arg Thr Val Ala Val Asn Ala 180 185 190 Ser Pro Ala Leu Thr Ala Leu Ala Ala His His Tyr Gln Gly Asp Asp 195 200 205 Leu Arg Glu Ala Leu Arg Leu Val Cys Pro Asp Ala Glu Phe Thr Ser 210 215 220 <210> 12 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> gvmC <400> 12 Met Ser Ala Pro Gly Arg Ala Val Pro Ser Ala Thr Thr Ala Ala Ser 1 5 10 15 Pro Ala Pro Gly Ala Gly Thr Val Thr Ala Val Cys Arg Ser Phe Ala 20 25 30 Glu Asp Phe Pro Gly Gly Leu Asp Ala Leu His Asp Ala Leu Gly Arg 35 40 45 Arg Gly Leu Thr Ala Gly Thr His Gly Gln Val Asp Gly Arg Phe Pro 50 55 60 Asp Gly Glu Leu Cys Val Asp Leu Pro Gly Asp Pro Thr Gly Ala His 65 70 75 80 Val Leu Ile His Gln Ser Leu Thr Gly Pro Ala Glu Gln Val Asp Gln 85 90 95 Ser Val Met Ala Leu Leu Ala Val Ala Arg Ala Tyr Arg Glu His Gly 100 105 110 Ala Arg Lys Val Thr Ala Val Val Pro His Leu Ala Tyr Ala Arg His 115 120 125 Asp Arg His Val Pro Gly Glu Arg Arg Pro Val Met Ala Gly Leu Leu 130 135 140 Ala Gln Leu Ala Ala Thr Ala Gly Ile Asp Gln Leu Ile Thr Leu Ala 145 150 155 160 Ser Gly Ala Gln Asp Arg Leu Gly His Leu Ser Glu Pro Met Asp Leu 165 170 175 Ala Gln Leu Pro Ala Asp Ala Ala Leu Met Ser Leu Val Arg Pro Trp 180 185 190 Val Val Pro Gly Arg Thr Val Leu Val Ala Pro Asp Ala Gly Ala Ala 195 200 205 His Gln Val Arg Arg Ile Gly Asp Arg Leu Arg Leu Pro Val Ile Thr 210 215 220 Ala Thr Lys Arg Arg Thr Gly Pro Asp Ser Val Thr Ala Ala Leu Thr 225 230 235 240 Ser Ser Ala Pro Gly Pro Ala Asp Arg Val Ile Ile Val Asp Asp Leu 245 250 255 Ile Thr Ser Ala Gly Thr Met Leu Ala Val Ala Arg Leu Ala Arg Ala 260 265 270 Glu Phe Asp Gly Cys Gln Val Leu Cys Ala Ala Thr His Leu Arg Pro 275 280 285 Thr Pro Thr Gly Glu Gln Arg Leu Ala Ala Ala Leu Arg Asp Gly Thr 290 295 300 Leu His Arg Val Leu Thr Thr Asp Ala Ala Gly His Arg Pro Arg Ile 305 310 315 320 Pro Gly Ile Asp Val Thr Ala Val Leu Pro Leu Ile Ala Asp Ala Val 325 330 335 Ala Glu His Val Leu Pro Ala Pro Ser Pro Leu Ala Glu Arg Pro Thr 340 345 350 Ala His Ala Gly Ser His 355 <210> 13 <211> 239 <212> PRT <213> Artificial Sequence <220> <223> gvmD <400> 13 Met Gln Ala Pro Thr Glu Leu Leu Glu Pro Val Pro Pro Thr Thr Leu 1 5 10 15 Ser Pro Ala Leu Ser Val Ser Val Val Cys Leu Asp Gln Thr Arg Leu 20 25 30 Gly Glu His Val Ala Gln Leu Arg Thr Leu Gly Val Ser Arg Leu His 35 40 45 Val Asp Leu Ile Asp Pro Ala Phe Ala Arg Asn Val Gly Leu Pro Leu 50 55 60 Glu Thr Ile Ser Asp Leu Ala Ala Thr Cys Gly Leu Pro Ile Asp Val 65 70 75 80 His Ala Met Leu Ala Arg Pro Gly Asp Leu Leu Glu Gln Ala Ala Ala 85 90 95 Arg Gly Ala Ala Thr Ile Cys Val His Gln Arg Thr Val Thr Pro Gln 100 105 110 Val Leu Ala Cys Leu Gln Arg Ile Ala Ala Gly Pro Ala Glu Val Gly 115 120 125 Leu Val Ile Asp Pro Gly Glu Gly Leu Asp Arg Ala Leu Ile Glu Gln 130 135 140 Leu Ala Pro Ala Gln Leu Thr Val Met Ser Val Glu Pro Gly Gly Val 145 150 155 160 Gly Arg Pro Phe Arg Asp Glu Ala Leu Leu Thr Leu Ala Glu Ala Ala 165 170 175 Glu Leu Arg Ala Arg Gly Ile Val Arg Arg Val Glu Ala Asp Gly Ala 180 185 190 Val Gly Pro Ala Thr Val Ala Arg Leu Val Ser Ala Gly Ala Gly Gln 195 200 205 Leu Val Leu Gly Ser Thr Val Phe Pro Arg Arg Gly Pro Glu Gly Asn 210 215 220 Arg Leu Asp Glu Leu Ala Leu Ala Leu Thr Ala Leu Ser Pro Ala 225 230 235 <210> 14 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> gvmE <400> 14 Met Thr Leu Thr His Thr Ile Thr Ile Gly Asp Val Arg Arg Glu Leu 1 5 10 15 Pro Ile Val Arg Val Ala Asp Asp Ala Arg Ile Ala Phe Leu Lys Leu 20 25 30 Tyr Gly Asp Val Glu Leu Thr Val Ala Cys Ala Arg Ala Leu Ala Gly 35 40 45 Arg Met Pro Ala Asp Val Asp Val Ile Val Gly Pro Glu Thr Gly Gly 50 55 60 Ile Leu Leu Ala His Glu Leu Ala Glu His Ser Gly Arg Pro Tyr Val 65 70 75 80 Ile Ala Arg Lys Lys Leu Arg Pro Asn Met Val Lys Pro Leu Arg Val 85 90 95 Pro Val Gln Ser Ile Gly Thr Pro Gly Gln Gln Glu Leu Phe Leu Gly 100 105 110 Glu Asp Asp Ala Ala Leu Ile Lys Gly Arg Arg Val Ala Val Val Asp 115 120 125 Glu Val Ile Ser Ser Gly Gly Thr Leu Lys Ala Leu His Glu Leu Val 130 135 140 Ala Ala Ala Gly Gly Thr Val Gln Gln Val Leu Thr Val Ala Thr Glu 145 150 155 160 Gly Glu Arg Arg Pro Asp Val Glu Ser Leu Leu His Leu Pro Val Tyr 165 170 175 Thr Asp <210> 15 <211> 423 <212> PRT <213> Artificial Sequence <220> <223> gvmF <400> 15 Met Thr Pro Thr Ile His Ala Ser Glu Val Ala Asp Ile Gly Leu Ala 1 5 10 15 Gly His Gly Glu Gly Leu Cys Asp Trp Ser Ala Gln Ser Met Pro Ile 20 25 30 Leu Ala Arg Ile Arg Glu Arg Tyr Ala Val Glu Lys Pro Phe Gln Gly 35 40 45 Lys Arg Ile Ala Ile Ala Val His Ile Thr Thr Glu Thr Ala Asn Leu 50 55 60 Ala Leu Thr Leu Arg Ala Gly Gly Ala Glu Val Phe Leu Cys Ala Ser 65 70 75 80 Asn Val Arg Thr Thr Asn Asp Ala Val Ala Ala Tyr Ala Ala Gln Gln 85 90 95 Gly Val Tyr Val Tyr Gly Ile Gln Gly Glu Asp Arg Glu Thr Tyr Ser 100 105 110 Thr His Cys His Arg Val Leu Asp Ala Arg Pro His Leu Val Ile Asp 115 120 125 Asp Gly Ala Asp Leu Thr Gly Leu Leu His Gly Ala Arg Thr Asp Ala 130 135 140 Ala Ser Asp Val Ile Ala Gly Leu Glu Glu Thr Thr Ala Gly Ile Gln 145 150 155 160 Arg Ile Arg Ser Leu Glu Ala Asp Gly Leu Leu Arg Tyr Pro Val Val 165 170 175 Ala Val Asn Glu Ala Arg Thr Lys His Leu Phe Asp Asn Arg Tyr Gly 180 185 190 Thr Gly Gln Ser Thr Met Asp Ala Ile Leu Arg Leu Thr Asn Thr Leu 195 200 205 Val Ala Gly Ser Arg Val Val Val Ala Gly Tyr Gly Met Cys Gly Lys 210 215 220 Gly Val Ala Ala Arg Ala Arg Gly Met Gly Ala Asp Val Leu Val Thr 225 230 235 240 Glu Val Asp His Val Arg Ala Leu Glu Ala Val Met Asp Gly Tyr Arg 245 250 255 Val Met Glu Met Asp Arg Ala Ala Glu Leu Gly Asp Ile Phe Ile Thr 260 265 270 Val Thr Ser Asn Lys Asn Val Leu Gly Ala Glu His Phe Ala Arg Met 275 280 285 Pro Asp Gly Ala Leu Val Cys Asn Ser Gly His Phe Asp Val Glu Ile 290 295 300 Asp Val Pro Ala Leu Arg Ala Ala Ala Leu Lys His Thr Arg Pro Arg 305 310 315 320 Pro Gly Val Asp Gln Tyr Thr Leu Ala Asp Gly Arg Arg Ile Asn Leu 325 330 335 Leu Ser Ala Gly Glu Val Val Asn Leu Ala Ala Ala Glu Gly Asn Pro 340 345 350 Ala Thr Leu Met Asp Met Ser Phe Thr Asn Gln Val Leu Gly Cys Glu 355 360 365 Tyr Leu Leu Lys Asn Ala Ala Asp Leu Asp Arg Arg Val His Thr Leu 370 375 380 Pro Ser Asp Ile Asp Asp Gln Val Ala His Leu Lys Leu Gln Thr Leu 385 390 395 400 Gly Val Gly Ile Asp Thr Leu Ser Glu Glu Gln Thr Ala Tyr Leu Arg 405 410 415 Ser Trp Asp Gln Gly Ser Ala 420 <210> 16 <211> 436 <212> PRT <213> Artificial Sequence <220> <223> gvmT1 <400> 16 Met Glu Pro Ser Asp Glu Leu Thr Pro Ser His Glu Arg Pro His Pro 1 5 10 15 Pro Gly Gln Gly Pro Ala Ala Trp Ala Arg Arg Ala Ala Arg Ser Val 20 25 30 Pro Pro Gly Arg Ala Ala Arg Thr Phe Leu Leu Val Ala Phe Val Asp 35 40 45 Ala Ala Gly Arg Gly Ile Phe Leu Ala Gly Ser Ala Leu Phe Tyr Thr 50 55 60 Gln Val Ile Gly Leu Thr Asn Ala Gln Val Gly Leu Gly Leu Ser Ile 65 70 75 80 Ala Gly Leu Val Gly Leu Val Cys Ala Ile Pro Ile Gly Trp Leu Ala 85 90 95 Asp Arg Phe Gly Asp Gly Pro Val Leu Val Ala Leu Gln Leu Trp Arg 100 105 110 Ala Ala Ala Phe Leu Val Tyr Pro Leu Val Asp Asp Phe Gly Leu Phe 115 120 125 Leu Val Val Ala Cys Leu Val Gly Ala Val Glu Trp Ala Ala Met Pro 130 135 140 Ile Ile Gln Ser Val Ala Gly Ala Thr Ser Glu Glu Gly Ser Met Val 145 150 155 160 Glu Ala Met Ala Val Val Ala Val Ala Arg Asn Ser Ala Tyr Ala Leu 165 170 175 Ser Ala Leu Ala Ala Thr Ala Val Ile Thr Trp Ala Ser Pro Arg Ala 180 185 190 Tyr Val Val Phe Val Leu Phe Asn Ala Leu Gly Phe Leu Val Ser Gly 195 200 205 Met Leu Leu Leu Arg Leu Arg Leu Pro Arg Arg Ala Val Asp His Ser 210 215 220 Ala Lys Glu Pro Ala Gly Ser Lys Leu Leu Pro Phe Arg Asp Val Arg 225 230 235 240 Phe Leu Leu Leu Ser Val Thr Asn Gly Ile Leu Tyr Ala His Met Pro 245 250 255 Ile Leu Ser Val Ala Phe Pro Leu Trp Val Val Thr Arg Thr Asp Ala 260 265 270 Pro Arg Ala Leu Val Gly Val Ala Leu Thr Ile Asn Thr Val Met Ala 275 280 285 Val Met Leu Gln Ile Arg Leu Ser Lys Gly Gly Asp Asp Leu Val Arg 290 295 300 Ala Gly Arg Lys Gln Arg Leu Ala Gly Leu Ala Leu Ala Ala Phe Cys 305 310 315 320 Val Leu Val Ala Phe Thr Arg Ser Val Asp Gly Leu Thr Ala Thr Val 325 330 335 Leu Leu Leu Leu Ala Ala Val Ala Leu Thr Leu Gly Glu Leu Trp Gln 340 345 350 Ser Ala Gly Gly Trp Gly Ile Ser Tyr Gly Leu Ala Pro Glu Ala Gln 355 360 365 Arg Thr Tyr Tyr Leu Ser Val Tyr Gln Leu Gly Ala Thr Gly Cys Thr 370 375 380 Val Ala Gly Pro Ala Val Leu Val Met Ala Val Asp Val Gly Pro 385 390 395 400 Ala Gly Trp Ala Gly Leu Ala Ala Leu Leu Ala Leu Thr Gly Leu Val 405 410 415 Thr Pro Ala Leu Thr Arg Pro Pro Arg His Lys Ala Ala Val Ala Pro 420 425 430 Wing Pro Wing Asp 435 <210> 17 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> gvmT2 <400> 17 Met Pro Leu Ala Ile Leu Ala Leu Ala Ile Ser Ala Phe Ala Val Gly 1 5 10 15 Thr Thr Glu Phe Val Ile Met Gly Leu Leu Ser Asp Val Ala Ala Asp 20 25 30 Leu Ser Val Ser Val Pro Val Ala Gly Tyr Leu Val Ser Gly Tyr Ala 35 40 45 Leu Gly Val Val Ala Gly Ala Leu Pro Leu Ala Ala Leu Thr Thr Arg 50 55 60 Phe Pro Arg Arg Ala Val Leu Val Leu Leu Met Ala Val Phe Val Val 65 70 75 80 Gly Asn Leu Leu Ala Gly Val Ala Pro Gly Tyr Gly Met Leu Met Ser 85 90 95 Ala Arg Val Leu Val Ser Leu Ala His Gly Ala Phe Phe Gly Val Ala 100 105 110 Ala Val Thr Ala Ala Asp Leu Val Ala Pro Ser Arg Arg Ala Ser Ala 115 120 125 Ile Ser Leu Ile Phe Ser Gly Met Thr Val Ser Thr Ile Leu Gly Val 130 135 140 Pro Leu Gly Thr Leu Ile Gly Gln His Phe Gly Trp Arg Ala Thr Phe 145 150 155 160 Leu Ser Val Ala Leu Ile Gly Phe Val Gly Leu Leu Ser Ile Leu Val 165 170 175 Leu Leu Pro Ala Gln Pro Arg Pro Thr Gly Gly Gly Leu Arg Ala Glu 180 185 190 Leu Gly Ala Phe Arg Arg Lys Gln Val Trp Val Thr Leu Thr Val Thr 195 200 205 Met Leu Val Ser Ala Ala Ile Phe Ala Ser Phe Thr Tyr Ile Glu Pro 210 215 220 Met Leu Val Lys Val Ser Gly Phe Ala Pro Ser Ser Val Thr Val Ile 225 230 235 240 Leu Met Leu Tyr Gly Ile Gly Met Ser Val Gly Asn Leu Leu Gly Gly 245 250 255 His Leu Ala Gly Lys Phe Pro Val Ala Thr Leu His Gly Thr Leu Ile 260 265 270 Val Leu Ala Val Leu Leu Ala Val Phe Ala Tyr Ser Ile His Phe Arg 275 280 285 Thr Pro Ala Val Ile Thr Leu Thr Leu Leu Gly Leu Cys Gly Thr Ala 290 295 300 Ala Ile Pro Thr Leu Gln His Arg Ile Met Gln Val Ala Ser Glu Ala 305 310 315 320 Arg Thr Leu Ala Ser Ala Ala Asn Val Ala Ala Leu Asn Val Gly Asn 325 330 335 Ala Leu Ala Ala Trp Val Gly Gly Ala Ala Ile Gly Ala Gly Phe Gly 340 345 350 Tyr Ala Ser Pro Asn Leu Val Gly Ala Ala Leu Ala Val Ala Ala Thr 355 360 365 Leu Ile Val Val Val Ala Asp Leu Thr Ala Arg Gly Arg Asp Arg Asn 370 375 380 Gly Thr Ala Arg Arg Ala Asp Glu Gln Val Arg Thr Pro Ala Ser 385 390 395 <210> 18 <211> 259 <212> PRT <213> Artificial Sequence <220> <223> gvmR <400> 18 Met Leu Val Val Pro Ser Leu Thr Asn Pro His Phe Ala Asp Val His 1 5 10 15 Ser Gly Ala Ala Arg Val Gly Ala Asp His Gly Ile Gly Val Ile Val 20 25 30 Tyr Pro Leu Gln Gly Glu Asp Gly Lys Gly Pro Phe Pro Ser Pro Arg 35 40 45 Gln Ala Ile Asp Gly Val Leu Ala Cys Ala Val Ser Ala Gly Ser Ile 50 55 60 Asp Val Leu Arg Asp Gly Val Ala Met Val Ala Leu Asp Ser Asp Pro 65 70 75 80 Asp Ala Gly Ile Cys Ser Val Thr Met Asp Val Gly Ala Gly Met Arg 85 90 95 Ser Ala Val Asp His Leu Val Arg Leu Gly His Arg Asp Ile Leu His 100 105 110 Val Arg Ala Glu Arg Asp Ala Trp Thr Phe Ala Gln Arg Thr Glu Ala 115 120 125 Phe Asp Arg Arg Leu Gly Ala His Arg Arg Val Arg Gly Arg Ser Leu 130 135 140 Ala Val Pro Phe Asp Gly Ala Arg Ala Arg Glu Arg Ile Leu Ala Arg 145 150 155 160 Leu Arg Asp Asp Asn Ser Val Thr Ala Val Ile Cys Asp Asp Asp Asn 165 170 175 Leu Ala Ala Cys Ala Tyr Ser Ala Ala His Gly Leu Gly Leu Ser Ile 180 185 190 Pro Gly Asp Leu Ser Val Ile Gly Phe Asp Asp Leu Pro Leu Ala Ser 195 200 205 Leu Met Val Pro Wing Leu Thr Thr Val Arg Leu Pro Wing His Glu Leu 210 215 220 Gly Arg Leu Gly Met Glu Gln Phe Leu Arg Asn Asp Thr Ala Thr Ala 225 230 235 240 Lys Thr Val Met Pro Val Glu Phe Val Glu Arg Ala Ser Val Gly Pro 245 250 255 Pro Arg Arg <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GaF3 <400> 19 cgccatatgg agacctcaat ggaatcg 27 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GaR3 <400> 20 cccaagcttt gcctcggcca cctcttc 27 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GbF3 <400> 21 ggaattccat atgaccacca caccccgc 28 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GbR3 <400> 22 cccaagcttt catgaggtga actccgc 27 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GcF3 <400> 23 ggaattccat atgagcgcgc ccggccgc 28 <210> 24 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GcR3 <400> 24 cccaagcttg tgggagcctg catgtgc 27 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GdF3 <400> 25 ggaattccat atgcaggctc ccactgag 28 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GdR3 <400> 26 cccaagcttt caggcgggcg agagggc 27 <210> 27 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> GeF3 <400> 27 ggaattccat atgacgctca cgcacacc 28 <210> 28 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GeR3 <400> 28 cccaagcttc tagtccgtgt agacggg 27

Claims

1. The application of a biosynthetic gene cluster of oryzanol, a protein combination encoded by the biosynthetic gene cluster, a recombinant expression vector containing the biosynthetic gene cluster, or a genetically engineered bacterium containing the biosynthetic gene cluster in the preparation of oryzanol, characterized in that, The biosynthetic gene cluster includes the following genes: (1) The gene gvmA encoding phosphoribosyl hydrolase, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) The gene gvmB encoding HAD family phosphatases, the nucleotide sequence of which is shown in SEQ ID NO: 2; (3) The gene gvmC encoding allosyl-2-phosphate synthase 6-phosphate, the nucleotide sequence of which is shown in SEQ ID NO: 3; (4) The gene gvmD encoding allulose phosphate 3-epimerase, the nucleotide sequence of which is shown in SEQ ID NO: 4; (5) The gene gvmE encoding phosphotransferase, the nucleotide sequence of which is shown in SEQ ID NO: 5; (6) The gene gvmF encoding a dehydratase, the nucleotide sequence of which is shown in SEQ ID NO: 6; (7) The gene encoding transporter 1 is gvmT1, and the nucleotide sequence of gvmT1 is shown in SEQ ID NO:7; (8) The gene encoding transporter 2, namely gvmT2, has the nucleotide sequence shown in SEQ ID NO:8; and, (9) The gene encoding the regulator is gvmR, and the nucleotide sequence of gvmR is shown in SEQ ID NO:

9.

2. The application as described in claim 1, characterized in that, The starting strain of the genetically engineered bacteria is *Streptomyces azureense* ( Streptomyces coelicolor ).

3. A method for preparing oryzanol, characterized in that, The method includes fermenting genetically engineered bacteria containing a biosynthetic gene cluster of oryzanol in a fermentation medium; or obtaining oryzanol by reacting with a combination of proteins encoded by the biosynthetic gene cluster based on a suitable substrate. The biosynthetic gene cluster Including the following genes: (1) The gene gvmA encoding phosphoribosylhydrolase, the nucleotide sequence of which is shown in SEQ ID NO: 1; (2) The gene gvmB encoding HAD family phosphatases, the nucleotide sequence of which is shown in SEQ ID NO: 2; (3) The gene gvmC encoding 6-phosphoallosyl-2-pyrophosphate synthase, the nucleotide sequence of which is shown in SEQ ID NO: 3; (4) The gene gvmD encoding allulose phosphate 3-epimerase, the nucleotide sequence of which is shown in SEQ ID NO: 4; (5) The gene gvmE encoding phosphotransferase, the nucleotide sequence of which is shown in SEQ ID NO: 5; (6) The gene gvmF encoding a dehydratase, the nucleotide sequence of which is shown in SEQ ID NO: 6; (7) The gene encoding transporter 1 is gvmT1, and the nucleotide sequence of gvmT1 is shown in SEQ ID NO:7; (8) The gene encoding transporter 2, namely gvmT2, has the nucleotide sequence shown in SEQ ID NO:8; and, (9) The gene encoding the regulator is gvmR, and the nucleotide sequence of gvmR is shown in SEQ ID NO:

9.

4. The method as described in claim 3, characterized in that, The starting strain of the genetically engineered bacteria is *Streptomyces azureense* ( Streptomyces coelicolor ).

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