Xanthocerin and guayulin synthesis-related proteins, their encoding genes and applications

Abstract

The present invention discloses proteins related to the synthesis of xanthin and guayulin, their encoding genes and applications. The target protein combination provided by the present invention can use germacrene A acid (i.e., germacrene A acid) as a substrate to generate guayulin. Guayulin forms 2-hydroxyguayulin under the action of known guayulin C2-hydroxylase. The generated 2-hydroxyguayulin is easily dehydrated to form xanthin under natural conditions. The present invention provides a key gene resource for the enzymatic production of xanthin and guayulin, and has potential application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to xanthocerin and guayulin synthesis-related proteins, and their encoding genes and applications. Background Art

[0002] Xanthin, also known as Xanthin, is a sesquiterpene lactone compound that is mainly synthesized from the Asteraceae plant Xanthin. Xanthin has a strong inhibitory effect on tumors such as lung cancer, liver cancer, and colon cancer. In agriculture, xanthin is expected to be developed into a new type of botanical fungicide. For example, the 30% xanthin emulsifiable concentrate developed by Gansu Agricultural University has a strong antibacterial activity against tomato gray mold and cucumber black spot fungus. The prevention and control effect reached more than 60% 14 days after field application. Guaijuliin can significantly induce apoptosis of pancreatic cancer cells and inhibit their proliferation, migration and invasion, and has the potential to be developed into a new anti-cancer drug. In addition, guayjuliin has a strong inhibitory effect on fungi such as plant gray mold, and can also be developed as an agricultural fungus inhibitor.

[0003] The primary sources of xanthocyanin and guayulin are extraction and purification from wild plants such as Xanthium sibiricum. However, the concentration of sesquiterpene lactones (note: xanthocyanin and guayulin are both sesquiterpene lactones) in plants is typically only a few parts per million, making extraction very expensive, thus limiting the large-scale development and utilization of these compounds. Furthermore, wild plant resources are relatively limited, and excessive harvesting of these resources not only damages the ecological environment but also threatens to make plant resources unsustainable. Summary of the Invention

[0004] The purpose of the present invention is to provide xanthocerin and guayulin synthesis-related proteins, their encoding genes and applications.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a protein related to the synthesis of xanthocerin and guayulin, which is the protein of a), b), c) or d) below:

[0006] a) a protein having an amino acid sequence as shown in SEQ ID NO: 1-2;

[0007] b) fusion proteins obtained by connecting tags to the N-terminus and / or C-terminus of the proteins shown in SEQ ID NOs: 1-2;

[0008] c) replacing, deleting, and / or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1-2 to obtain a protein having the same function;

[0009] d) A protein having 75% or more homology to the amino acid sequence shown in SEQ ID NO: 1-2 and having the same function.

[0010] In a second aspect, the present invention provides a gene encoding a protein related to the synthesis of xanthocerin and guayulin, which is the gene of (a), (b) or (c):

[0011] (a) a cDNA molecule or genomic DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 3-4;

[0012] (b) a cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence described in (a) and encodes the protein shown in SEQ ID NO: 1-2;

[0013] (c) a cDNA molecule or genomic DNA molecule that hybridizes with the nucleotide sequence described in (a) or (b) under stringent conditions and encodes the protein shown in SEQ ID NO: 1-2.

[0014] Preferably, the stringent conditions are hybridization in a 6×SSC (containing 0.5% SDS) solution at 68° C., followed by washing the membrane once with 2×SSC (containing 0.1% SDS) and once with 1×SSC (containing 0.1% SDS).

[0015] In a third aspect, the present invention provides biological materials containing the gene, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, transgenic cell lines or plant cells.

[0016] Preferably, the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.

[0017] In a fourth aspect, the present invention provides a recombinant microorganism that expresses the xanthosporin and guayulin synthesis-related proteins or expresses the genes encoding the same.

[0018] Furthermore, the microorganism is a yeast or Escherichia coli that produces Germacrene A acid, such as yeast EPY300-GAA (Yeast EPY300-GAA was provided by Professor Dae-Kyun Ro's laboratory at the University of Calgary, Canada, reference Nguyen, DT, Go¨pfert, JC, Ikezawa, N., Macnevin, G., Kathiresan, M., Conrad, J., Spring, O., and Ro, DK (2010) J. Biol. Chem. 285, 16588–16598.

[0019] In a fifth aspect, the present invention provides a method for constructing the recombinant microorganism, wherein the coding genes (BfaS and TomS) for proteins related to the synthesis of xanthocerin and guayulin are introduced into the microorganism via a plasmid or integrated into the chromosome of the microorganism by genetic engineering.

[0020] In a sixth aspect, the present invention provides any of the following applications of the xanthosporin and guayulin synthesis-related proteins, their encoding genes, biological materials containing the genes, or the recombinant microorganisms:

[0021] (1) Preparation of products containing xanthocerin and guayulin;

[0022] (2) Preparation of products containing xanthocerin and guayulin synthase.

[0023] In a seventh aspect, the present invention provides a method for synthesizing guayule, which comprises: fermenting the recombinant microorganism and separating guayule from the fermentation product.

[0024] In an eighth aspect, the present invention provides an engineered bacterium that produces xanthosporin, wherein the engineered bacterium is constructed by introducing the guayulin C2-hydroxylase gene (TomH2) into the recombinant microorganism via a plasmid or integrating it into the chromosome of the recombinant microorganism via genetic engineering means.

[0025] The nucleotide sequence of the guayulin C2-hydroxylase (TomH2) gene is shown in SEQ ID NO: 5.

[0026] In a ninth aspect, the present invention provides a method for synthesizing xanthocerin, the method comprising: fermenting the engineered bacteria, and separating xanthocerin from the fermentation product.

[0027] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0028] The present invention clones the full-length sequence of the gene related to the synthesis of xanthium sibiricum or guayulin as shown in SEQ ID NO: 3-4 from the Xanthium sibiricum plant for the first time. When the gene shown in SEQ ID NO: 3-4 is expressed in a yeast producing geranyl ester A acid, the recombinant yeast can automatically synthesize guayulin by fermentation in a simple culture medium; when the gene shown in SEQ ID NO: 3-4 is co-expressed with the gene encoding guayulin C2 hydroxylase (TomH2) in a yeast producing geranyl ester A acid, the recombinant yeast can automatically synthesize xanthium sibiricum by fermentation in a simple culture medium. The present invention provides a key gene resource for the biological production of xanthium sibiricum or guayulin. The production of xanthium sibiricum or guayulin by fermentation of the microorganisms provided by the present invention greatly reduces the production cost compared to directly extracting and purifying xanthium sibiricum or guayulin from plants, and is environmentally friendly. The present invention provides the necessary gene resources for the production of xanthium sibiricum or guayulin using synthetic biology technology, and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the LC-MS analysis of the expression of the genes represented by SEQ ID NOs: 3-4 in a yeast strain producing geraniol in a preferred embodiment of the present invention. The recombinant yeast was fermented in a simple culture medium to produce guayulin. a is the peak line plot of the extracted ion 249; b is the ion fragmentation plot of the guayulin product and a guayulin standard.

[0030] Figure 2 This is the LC-MS analysis of the co-expression of the gene represented by SEQ ID NO: 3-4 with the known enzyme encoding guayulin C2-hydroxylase (TomH2) in a preferred embodiment of the present invention, resulting in the synthesis of xanthocyanin by recombinant yeast fermented in a simplified culture medium. Figure a is the peak line plot of the extracted ion 247; figure b is the ion fragmentation plot of the xanthocyanin product and the xanthocyanin standard. DETAILED DESCRIPTION

[0031] The present invention provides xanthocyanin and guayulein synthesis-related proteins and their encoding genes and applications, providing a new method and approach for the synthesis and preparation of xanthocyanin (guayulein is the precursor of xanthocyanin) and guayulein.

[0032] The present invention adopts the following technical solutions:

[0033] The present invention provides xanthophyll or guayulin synthesis-related proteins (BfaS and TomS), which are proteins shown in any one of the following a) or b) or c) or d):

[0034] a) a protein with an amino acid sequence as shown in SEQ ID NO: 1-2;

[0035] b) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO: 1-2;

[0036] c) replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence of SEQ ID NO: 1-2 to obtain a protein having the same function;

[0037] d) A protein having 75% or more homology to the amino acid sequence shown in SEQ ID NO: 1-2 and having the same function.

[0038] Preferably, the tag is a 6×histidine tag, a Flag tag, an MBP tag, an HA tag or a c-Myc tag.

[0039] The present invention also provides a gene encoding the aforementioned xanthocerin and guayulin synthesis-related protein, wherein the nucleic acid molecule is the following gene (a) or (b) or (c):

[0040] (a) The coding sequence is a cDNA molecule or genomic DNA molecule shown in SEQ ID NO: 3-4;

[0041] (b) a cDNA molecule or genomic DNA molecule that has 75% or more identity with the nucleotide sequence defined in (a) and encodes the protein shown in SEQ ID NO: 1-2;

[0042] (c) a cDNA molecule or genomic DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in (a) or (b) and encodes the protein shown in SEQ ID NO: 1-2.

[0043] Preferably, the stringent conditions are hybridization in a 6×SSC (containing 0.5% SDS) solution at 68° C., followed by washing the membrane once with 2×SSC (containing 0.1% SDS) and once with 1×SSC (containing 0.1% SDS).

[0044] Preferably, the gene is derived from the plant Xanthium sibiricum.

[0045] The present invention also provides an expression cassette containing the nucleic acid molecule of the gene encoding xanthocerin and guayulin synthesis-related proteins.

[0046] The present invention also provides an expression vector containing the nucleic acid molecule of the gene encoding xanthocerin and guayulin synthesis-related proteins.

[0047] Preferably, the expression vector is a yeast expression vector or an Escherichia coli expression vector.

[0048] The present invention also provides a transformed cell, which contains the nucleic acid molecule of the gene encoding xanthocerin and guayulin synthesis-related proteins.

[0049] Preferably, the transformed cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.

[0050] The present invention also provides a recombinant microorganism, which expresses the xanthocerin and guayulin synthesis-related proteins or expresses the coding genes thereof.

[0051] Preferably, the microorganism is yeast or Escherichia coli that synthesizes Germacrene A acid.

[0052] The present invention also provides a method for constructing the recombinant microorganism, wherein the coding genes of the xanthocerin and guayulin synthesis-related proteins are introduced into the microorganism to obtain a recombinant microorganism expressing the xanthocerin or guayulin synthesis-related proteins.

[0053] The present invention also provides the use of the protein encoding xanthocerin and guayulin synthesis-related protein in any of the following:

[0054] (a1) preparing products containing guayule;

[0055] (a2) preparing products containing xanthocerin;

[0056] (a3) Preparation of products of proteins related to the synthesis of xanthocerin or guayulin.

[0057] The present invention also provides a method for synthesizing guayulin and xanthocerin, comprising the following steps:

[0058] (b1) constructing a yeast expression vector using the gene encoding the xanthocerin and guayulin synthesis-related protein as the target gene;

[0059] (b2) expressing the yeast expression vector in a yeast strain that produces germacrene A acid, and synthesizing guayulein by fermentation using only a simple culture medium;

[0060] (b3) The above yeast expression vector is co-expressed with a vector containing the known guayule C2-hydroxylase (TomH2) gene in a chassis organism (such as yeast) that produces geraniol, and xanthocyanin can be synthesized by fermentation using only a simple culture medium.

[0061] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0062] The yeast EPY300-GAA and vector pESC-Leu2d-CPR used in the following examples were provided by Professor Dae-Kyun Ro's laboratory at the University of Calgary, Canada. The vector pESC-URA-TomH2 was constructed by the present invention. The main construction method is as follows: TomH2 The gene's reading frame sequence was cloned into the commercial yeast expression vector pESC-URA (Stratagene, USA) through the BamHI and SalI restriction sites.

[0063] Example 1 Obtaining the full-length cDNA sequence of the gene encoding the protein related to xanthocerin or guayulin synthesis (BfaS and TomS)

[0064] 1. Extraction of total RNA from Xanthium sibiricum leaves. The specific method is as follows:

[0065] About 100 mg of young leaves of Xanthium sibiricum were weighed and quickly ground in liquid nitrogen. Total RNA was extracted from the ground sample powder using the EASYspin plus Plant RNA Rapid Extraction Kit (Adela).

[0066] 2. Reverse transcribe RNA into cDNA. The specific method is as follows:

[0067] Add the following components to an RNase-free PCR tube: 8µL RNA (approximately 1.5µg), 1µL DNase I, 1µL 10× DNase I buffer, and 0.5µL RNase inhibitor. Mix well and incubate at 37°C for 30 minutes. Then, add 1µL 100mM EDTA and incubate at 65°C for 10 minutes to terminate the reaction. Add 1µL Oligo(dT)18 and 1µL dNTP mix (10 mM) to the PCR tube containing the above mixture and denature at 65°C for 5 minutes. Immediately place on ice. Then, add 4µL 5× reverse transcriptase buffer, 0.5µL RNase inhibitor, and 1µL reverse transcriptase. Mix well and incubate at 42°C for 1 hour to synthesize first-strand cDNA. Then, incubate at 70°C for 10 minutes to terminate the reaction. Store cDNA at -80°C.

[0068] 3. Conventional RT-PCR reaction BfS and TomS cDNA sequence.

[0069] The cDNA encoding the BfaS protein was amplified by PCR using the following primer pairs:

[0070] BfaS-F:5'-ATGTTAACCTTCTTTCCTCCA-3';

[0071] BfaS-R:5'-TTATTTGGCTTTTGAATTGTG-3'.

[0072] The cDNA encoding TomS protein was amplified by PCR using the following primer pairs:

[0073] TomS-F:5'-ATGGAGCTTTCACAATCTT-3';

[0074] TomS-R:5'-CTAGTTTGATTTTGACAGGG-3'.

[0075] The 20µL PCR reaction system consisted of: 2µL cDNA template, 1µL each primer, 10µL 2× PrimerSTAR MaxPremix (Takara), and 6µL ddH2O.

[0076] PCR reaction conditions included pre-denaturation at 98°C for 2 minutes, followed by 30 cycles of denaturation at 95°C for 20 seconds, annealing at 58°C for 20 seconds, and extension at 72°C for 1.5 minutes, with a final extension at 72°C for 5 minutes. The reaction product was recovered using a standard agarose gel DNA recovery kit (purchased from Beijing Adlai) and ligated into the pEASY-Blunt Simple vector (Beijing Quanshijin). The ligation product was then transformed into competent E. coli DH5α cells, followed by colony PCR. After the reaction, 5 µL of the reaction product was analyzed by agarose gel electrophoresis to identify positive clones. The plasmids from these clones were then extracted and sent to a sequencing company (Beijing Qingke) for sequencing. The results showed that BfS The fragment is 1473 bp in length, its deoxyribonucleotide sequence is shown in SEQ ID NO: 3, and its encoded amino acid sequence is shown in SEQ ID NO: 1; TomS The fragment is 1482 bp in length, its deoxyribonucleotide sequence is shown in SEQ ID NO: 4, and its encoded amino acid sequence is shown in SEQ ID NO: 2.

[0077] Example 2 Application of proteins related to xanthocerin or guayulin synthesis (BfaS and TomS)

[0078] 1. Synthesis of guayule by yeast fermentation

[0079] The correct sequencing results in Example 1 BfS and TomS Genes are respectively SpeI as well as BamHI / SalIThe site was cloned into the yeast expression vector pESC-Leu2d-CPR to construct the recombinant plasmid pESC-Leu2d-BfaS-TomS. The recombinant plasmid pESC-Leu2d-BfaS-TomS was transformed into the geranyl A acid-producing yeast EPY300-GAA by the conventional lithium acetate transformation method. The yeast cells transformed with the empty vector (pESC-Leu2d-CPR) were used as the control group. The transgenic yeast were plated on SD-Leu nutrient-deficient solid medium and cultured at 30°C. Several positive yeast colonies identified by colony PCR were picked and placed in 5 ml of SD-Leu nutrient-deficient liquid medium, cultured at 30°C and 250 rpm for 48 hours, and the bacterial solution was collected by centrifugation at 5000 rpm, washed once with double-distilled water, and resuspended in 500 ml of SD-Ura medium containing 2% galactose (OD after resuspension was 0.05). 600 The pH was controlled between 0.4 and 0.6), and the culture was induced at 30°C for 48 hours. The culture medium was collected by centrifugation at 5000 rpm and extracted with an equal volume of ethyl acetate. The ethyl acetate extract was evaporated to dryness and dissolved in 200 μL of methanol solution for LC-MS analysis. The LC-MS test results are shown in the figure below. Figure 1 As shown, the empty vector control group did not produce guayule, while the expression BfS and TomS Yeast with the gene synthesized guayule.

[0080] 2. Synthesis of xanthocerasin by yeast fermentation

[0081] The recombinant plasmid pESC-Leu2d-BfaS-TomS constructed above was co-transformed with the pESC-URA-TomH2 plasmid constructed earlier in the laboratory into EPY300-GAA yeast (TomH2 encodes guayulin C2 hydroxylase). The transgenic yeast was screened and cultured on SD-Leu-Ura nutrient-deficient medium. The other culture conditions were the same as above. After galactose induction, the culture medium was collected and used for ethyl acetate extraction. The ethyl acetate extract was evaporated to dryness and resuspended in methanol solvent for LC-MS detection. The LC-MS detection results are shown as follows: Figure 2 As shown, the empty vector control group did not produce xanthocerin, while the expression BfS 、 TomS as well as TomH2 Yeast with the gene synthesized xanthocerasin.

[0082] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. Application of xanthocerin and guayulin synthesis-related proteins, their encoding genes, and biomaterials containing said genes in the preparation of guayulin products using germacrene A acid as a substrate; The amino acid sequences of the xanthocerin and guayulin synthesis-related proteins are shown in SEQ ID NOs: 1 and 2.

2. Use of recombinant microorganisms expressing xanthocerin and guayulin synthesis-related proteins or their encoding genes in the preparation of guayulin products; The amino acid sequences of the xanthocerin and guayulin synthesis-related proteins are shown in SEQ ID NOs: 1 and 2; The microorganism is yeast or Escherichia coli that produces germacreous acid.

3. The use according to claim 2, characterized in that The method for constructing the recombinant microorganism comprises: introducing the coding genes of the xanthosibiricum and guayulin synthesis-related proteins described in claim 1 into the microorganism via a plasmid or integrating them into the chromosome of the microorganism via genetic engineering means.

4. The use according to claim 1, characterized in that The biological material is an expression cassette, a transposon, a plasmid vector, a virus vector, an engineered bacterium or a transgenic cell line.

5. The use according to claim 4, characterized in that The engineered bacteria are yeast; the transgenic cell line is mammalian cells or insect cells.

6. A method for synthesizing guayule, characterized in that: The method comprises: fermenting the recombinant microorganism according to claim 2 or 3, and separating guayule from the fermentation product.

7. An engineered bacterium producing xanthocerin, characterized in that: The engineered bacteria are constructed by introducing the guayule C2-hydroxylase gene into the recombinant microorganism described in claim 2 or 3 via a plasmid or integrating it into the chromosome of the recombinant microorganism by genetic engineering means; The nucleotide sequence of the guayule C2-hydroxylase gene is shown in SEQ ID NO:

5.

8. A method for synthesizing xanthocerin, characterized in that: The method comprises: fermenting the engineered bacteria according to claim 7, and separating xanthocerin from the fermentation product.

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