Related protein synthesized by xanthinin and guayule as well as coding gene and application thereof

Abstract

The invention discloses xanthinin and guayule synthesis related protein as well as a coding gene and application thereof. According to the target protein combination provided by the invention, germacene A acid (namely germacene A acid) can be used as a substrate to generate the guayule, the guayule forms 2-hydroxyl guayule under the action of known guayule C2-hydroxylase, and the generated 2-hydroxyl guayule is easy to dehydrate under natural conditions to form the xanthinin. The invention provides a key gene resource for producing xanthinin and guayule by using an enzyme method, and has potential application value.

Description

Technical Field

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

[0002] Xanthin, also known as Xanthate, is a sesquiterpene lactone compound, mainly synthesized from the Asteraceae plant Xanthium sibiricum. 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 control effect reached more than 60% 14 days after field use. Guajulin 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 anticancer drug. In addition, Guajulin has a strong inhibitory effect on fungi such as plant gray mold, and can also be developed as an agricultural fungal inhibitor.

[0003] The main sources of xanthocerin and guayule are extracted and purified from wild plants such as Xanthium sibiricum. However, the content of sesquiterpene lactone compounds (Note: xanthocerin and guayule are both sesquiterpene lactones) in plants is usually only a few ten-thousandths, and the extraction cost is very expensive, which limits the large-scale development and utilization of such compounds. On the other hand, wild plant resources are relatively limited, and excessive harvesting of plant resources by humans will not only damage the ecological environment but also cause unsustainable plant resources. Summary of the invention

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

[0005] In order to achieve the purpose of the present invention, in the first aspect, the present invention provides a protein related to the synthesis of xanthocerin and guayulin, which is the following protein a), b), c) or d): a) a protein having an amino acid sequence as shown in SEQ ID NO: 1-2; 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; c) replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1-2 to obtain a protein with the same function; 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.

[0006] 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): (a) a cDNA molecule or a genomic DNA molecule whose nucleotide sequence is shown in SEQ ID NO:3-4; (b) a cDNA molecule or a 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; (c) a cDNA molecule or a 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.

[0007] Preferably, the stringent conditions are hybridization in a 6×SSC solution (containing 0.5% SDS) 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).

[0008] In a third aspect, the present invention provides biological materials containing the gene, wherein the biological materials include but are not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, transgenic cell lines or plant cells.

[0009] Preferably, the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.

[0010] In a fourth aspect, the present invention provides a recombinant microorganism that expresses the xanthocerin and guayulin synthesis-related proteins or expresses the genes encoding the proteins.

[0011] Further, the microorganism is a yeast or Escherichia coli that produces Germacrene A acid, such as yeast EPY300-GAA (the yeast EPY300-GAA is provided by the laboratory of Professor Dae-Kyun Ro of 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.

[0012] In a fifth aspect, the present invention provides a method for constructing the recombinant microorganism, wherein the coding genes (BfaS and TomS) of the 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 via genetic engineering.

[0013] In a sixth aspect, the present invention provides any of the following applications of the xanthocerin and guayulin synthesis-related proteins, their encoding genes, biological materials containing the genes, or the recombinant microorganisms: (1) Preparation of products containing xanthocerin and guayulin; (2) Preparation of products containing xanthocerasin and guayulin synthase.

[0014] In a seventh aspect, the present invention provides a method for synthesizing guayule, the method comprising: fermenting the recombinant microorganism and separating guayule from the fermentation product.

[0015] In an eighth aspect, the present invention provides an engineered bacterium producing xanthocerin, wherein the engineered bacterium is constructed by introducing the guayule 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.

[0016] The nucleotide sequence of the guayule C2-hydroxylase (TomH2) gene is shown in SEQ ID NO:5.

[0017] 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.

[0018] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention clones the full-length sequence of the gene related to the synthesis of xanthin or guayulin as shown in SEQ ID NO:3-4 from the Xanthium plant for the first time. When the gene shown in SEQ ID NO:3-4 is expressed in a yeast producing geranyl 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 A acid, the recombinant yeast can automatically synthesize xanthin by fermentation in a simple culture medium. The present invention provides a key gene resource for biological production of xanthin or guayulin. The production cost of xanthin or guayulin by fermentation of the microorganism provided by the present invention is greatly reduced compared with the direct extraction and purification of xanthin or guayulin from plants, and the present invention is environmentally friendly. The present invention provides the necessary gene resources for producing xanthin or guayulin using synthetic biology technology, and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the LC-MS detection spectrum of the expression of the gene shown in SEQ ID NO:3-4 in the germacrene A acid yeast in the preferred embodiment of the present invention, and the recombinant yeast can synthesize guayule by fermentation using a simple culture medium. Among them, a is the peak line diagram of the extracted ion 249; b is the ion fragment diagram of the guayule product and the guayule standard.

[0020] Figure 2 This is the LC-MS detection spectrum of the co-expression of the gene shown in SEQ ID NO:3-4 and the known guayule C2-hydroxylase (TomH2) encoding in the preferred embodiment of the present invention, and the recombinant yeast can synthesize xanthocyanin by fermentation using a simple culture medium. Among them, a is the peak line diagram of the extracted ion 247; b is the ion fragment diagram of the xanthocyanin product and the xanthocyanin standard. DETAILED DESCRIPTION

[0021] The present invention provides xanthocerin and guayulein synthesis-related proteins and their encoding genes and applications, and provides a new method and approach for the synthesis and preparation of xanthocerin (guayulein is the precursor of xanthocerin) and guayulein.

[0022] The present invention adopts the following technical solution: The present invention provides xanthocerin or guayulin synthesis-related proteins (BfaS and TomS), which are proteins shown in any of the following a) or b) or c) or d): a) a protein with an amino acid sequence as shown in SEQ ID NO: 1-2; 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; c) replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1-2 to obtain a protein with the same function; d) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 1-2 and having the same function.

[0023] Preferably, the tag is a 6×histidine tag, a Flag tag, an MBP tag, an HA tag or a c-Myc tag.

[0024] The present invention also provides a gene encoding the above-mentioned xanthocerin and guayulin synthesis-related protein, wherein the nucleic acid molecule is the following gene (a) or (b) or (c): (a) Its coding sequence is a cDNA molecule or genomic DNA molecule shown in SEQ ID NO:3-4; (b) a cDNA molecule or a genomic DNA molecule having 75% or more identity with the nucleotide sequence defined in (a) and encoding the protein shown in SEQ ID NO: 1-2; (c) a cDNA molecule or a genomic DNA molecule that hybridizes with the nucleotide sequence defined in (a) or (b) under stringent conditions and encodes the protein shown in SEQ ID NO: 1-2.

[0025] Preferably, the stringent conditions are hybridization in a 6×SSC solution (containing 0.5% SDS) 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).

[0026] Preferably, the gene is derived from the Xanthium sibiricum plant.

[0027] The present invention also provides an expression box, which contains the nucleic acid molecule of the gene encoding the xanthocerin and guayulin synthesis-related proteins.

[0028] The present invention also provides an expression vector, which contains the nucleic acid molecule of the gene encoding the xanthocerin and guayulein synthesis-related proteins.

[0029] Preferably, the expression vector is a yeast expression vector or an E. coli expression vector.

[0030] The present invention also provides a transformed cell, which contains the nucleic acid molecule of the gene encoding the xanthocerin and guayule synthesis-related proteins.

[0031] Preferably, the transformed cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insect cell or a plant cell.

[0032] The present invention also provides a recombinant microorganism, which expresses the xanthocerin and guayulein synthesis-related proteins or expresses the coding genes thereof.

[0033] Preferably, the microorganism is yeast or Escherichia coli that synthesizes Germacrene A acid.

[0034] 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 the recombinant microorganism expressing the xanthocerin or guayulin synthesis-related proteins.

[0035] The present invention also provides the use of the protein encoding xanthocerin and guayule synthesis-related protein in any of the following: (a1) preparing products containing guayule; (a2) preparing products containing xanthocerin; (a3) A product of preparing proteins related to the synthesis of xanthocerasin or guayulin.

[0036] The present invention also provides a method for synthesizing guayule and xanthocerin, comprising the following steps: (b1) constructing a yeast expression vector using the gene encoding the above-mentioned xanthocerin and guayulin synthesis-related protein as the target gene; (b2) expressing the yeast expression vector in a yeast that produces geranyl A acid, and synthesizing guayule by fermentation using only a simple culture medium; (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 geranyl A acid, and xanthocyanin can be synthesized by fermentation using only a simple culture medium.

[0037] 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.

[0038] 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.

[0039] Example 1 Obtaining the full-length cDNA sequence of the gene encoding the protein related to the synthesis of xanthocerin or guayulin (BfaS and TomS)

[0040] 1. Extraction of total RNA from leaves of Xanthium sibiricum. The specific method is as follows: About 100 mg of young leaves of Xanthium sibiricum were weighed and quickly ground in liquid nitrogen, and total RNA was extracted from the ground sample powder using EASYspin plus Plant RNA Rapid Extraction Kit (Adlai).

[0041] 2. Reverse transcribe RNA into cDNA. The specific method is as follows: Add the following components to an RNase-free PCR tube: RNA: 8µL (about 1.5µg), DNase I: 1µL, 10×DNaseI buffer: 1µL, RNase inhibitor: 0.5µL. Mix the above substances and incubate at 37℃ for 30min. Then add 1µL 100mM EDTA and incubate at 65℃ for 10 min to terminate the reaction. Add 1µL Oligo(dT)18 and 1µL dNTP MIX (10 mM) to the PCR tube containing the above mixture, pre-denature at 65℃ for 5min, and then immediately place on ice. Then add 4µL 5× reverse transcriptase buffer, 0.5µL RNase inhibitor and 1µL reverse transcriptase in sequence, mix well, and incubate at 42℃ for 1 hour to synthesize the first-strand cDNA. Then incubate at 70℃ for 10 min to terminate the reaction. Store cDNA in a -80℃ refrigerator.

[0042] 3. Conventional RT-PCR reaction BfS and TomS cDNA sequence.

[0043] The cDNA encoding the BfaS protein was amplified by PCR using the following primer pairs: BfaS-F:5'-ATGTTAACCTTCTTTCCTCCA-3'; BfaS-R:5'-TTATTTGGCTTTTGAATTGTG-3'.

[0044] The cDNA encoding TomS protein was amplified by PCR using the following primer pairs: TomS-F:5'-ATGGAGCTTTCACAATCTT-3'; TomS-R:5'-CTAGTTTGATTTTGACAGGG-3'.

[0045] The 20µL PCR reaction system was as follows: 2µL cDNA template, 1µL each primer, 10µL 2× PrimerSTAR MaxPremix (Takara), and 6µL ddH2O.

[0046] PCR reaction conditions: 98℃ pre-denaturation for 2min; cycle program: 95℃ denaturation for 20s, 58℃ annealing for 20s, 72℃ extension for 1.5min, 30 cycles, and finally 72℃ extension for 5min. The reaction product was recovered by a common agarose gel DNA recovery kit (purchased from Beijing Aidelai) and connected to the pEASY-Blunt Simple vector (Beijing Quanshijin). The ligation product was transformed into Escherichia coli DH5α competent cells, and then colony PCR was performed. After the reaction was completed, 5µL of the reaction product was taken for agarose gel electrophoresis detection to determine the positive clones. The plasmid of the positive clone was extracted and sent to a sequencing company (Beijing Qingke) for sequencing. The results showed that BfS The length of the fragment is 1473 bp, 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 length of the fragment is 1482 bp, its deoxyribonucleotide sequence is shown in SEQ ID NO:4, and its encoded amino acid sequence is shown in SEQ ID NO:2.

[0047] Example 2 Application of proteins related to xanthocerin or guayulin synthesis (BfaS and TomS)

[0048] 1. Synthesis of guayule by yeast fermentation

[0049] The correct sequencing results in Example 1 BfS and TomS Genes are separated by SpeI as well as BamHI / SalI The 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 yeast EPY300-GAA producing geranyl A acid 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 was plated on SD-Leu nutrient-deficient solid medium for progressive culture at a temperature of 30°C. Several positive yeast colonies identified by colony PCR were picked and placed in 5 ml 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 SD-Ura medium containing 2% galactose (OD after resuspension was 0.05). 600 The concentration of 1 μg / mL was controlled between 0.4 and 0.6), and the culture was induced at 30°C for 48 hours. The culture solution was collected by centrifugation at 5000 rpm, and an equal volume of ethyl acetate was added for extraction. The ethyl acetate extract was evaporated to dryness and dissolved in 200 μL methanol solution for LC-MS detection. The LC-MS detection results are shown in Figure 1As shown, the empty vector control group did not produce guayule, while the expression BfS and TomS Yeast with the gene synthesized guayule.

[0050] 2. Synthesis of xanthocerin by yeast fermentation

[0051] The recombinant plasmid pESC-Leu2d-BfaS-TomS constructed above and the pESC-URA-TomH2 plasmid constructed earlier in the laboratory were co-transformed into EPY300-GAA yeast (TomH2 encodes guayule C2 hydroxylase), and the transgenic yeast was screened and cultured on SD-Leu-Ura nutritional deficiency medium. The other culture conditions were the same as above. After galactose induction culture, part of the culture medium was collected for ethyl acetate extraction, and the ethyl acetate extract was evaporated to dryness and resuspended in methanol solvent for LC-MS detection. The LC-MS test results are shown in 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.

[0052] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A protein related to the synthesis of xanthocerin and guayulin, characterized in that: It is a protein of a), b), c) or d) as follows: a) a protein having an amino acid sequence as shown in SEQ ID NO: 1-2; 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; c) replacing, deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1-2 to obtain a protein with the same function; 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.

2. The gene encoding the xanthocerin and guayulin synthesis-related protein according to claim 1, characterized in that: It is a gene of (a), (b) or (c) below: (a) a cDNA molecule or a genomic DNA molecule whose nucleotide sequence is shown in SEQ ID NO:3-4; (b) a cDNA molecule or a 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; (c) a cDNA molecule or a 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.

3. The biological material containing the gene according to claim 2, characterized in that: The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line; Preferably, the cell is a bacterial cell, a mammalian cell, a fungal cell, a yeast cell or an insect cell.

4. A recombinant microorganism, characterized in that It expresses the xanthocerin and guayulin synthesis-related protein described in claim 1 or expresses the gene described in claim 2.

5. The recombinant microorganism according to claim 4, characterized in that The microorganism is yeast or Escherichia coli that produces geranyl acid.

6. The method for constructing a recombinant microorganism according to claim 4 or 5, characterized in that: The gene according to claim 2 is introduced into the microorganism via a plasmid or integrated into the chromosome of the microorganism via genetic engineering.

7. Any of the following uses of the xanthocerin and guayulin synthesis-related protein of claim 1, the gene of claim 2, the biomaterial of claim 3, or the recombinant microorganism of claim 4 or 5: (1) Preparation of products containing xanthocerin and guayulin; (2) Preparation of products containing xanthocerasin and guayulin synthase.

8. A method for synthesizing guayule, characterized in that: The method comprises: fermenting the recombinant microorganism according to claim 5, and separating guayule from the fermentation product.

9. An engineered bacterium producing xanthocerin, characterized in that: The engineered bacteria is constructed by introducing the guayule C2-hydroxylase gene into the recombinant microorganism of claim 5 via a plasmid or integrating it into the chromosome of the recombinant microorganism by genetic engineering means; Among them, the nucleotide sequence of the guayule C2-hydroxylase gene is shown in SEQ ID NO:

5.

10. A method for synthesizing xanthocerin, characterized in that: The method comprises: fermenting the engineered bacteria according to claim 9, and separating xanthocerin from the fermentation product.

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