Protein from dioscorea zingiberensis as well as coding gene and application thereof

Abstract

The invention provides a protein from dioscorea zingiberensis as well as a coding gene and application thereof. The protein provided by the invention is selected from at least one of a) and b): a) a protein with an amino acid sequence as shown in SEQ ID NO: 2; and b) a fusion protein obtained by connecting a label to the N terminal and / or C terminal of the protein as shown in SEQ ID NO: 2. The protein provided by the invention has P450 enzyme activity, and experiments verify that co-expression of the protein coding gene and other diosgenin synthesis genes in yeast cells can significantly improve the biosynthesis efficiency of diosgenin, and a key gene resource is provided for biosynthesis of diosgenin.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein derived from Dioscorea zingiberensis, a coding gene thereof, and an application thereof. Background Art

[0002] Dioscorea zingiberensis CHWright is currently used industrially to extract diosgenin from D. zingiberensis. This process involves the use of large amounts of inorganic acids, resulting in significant wastewater discharge and severe environmental pollution. The development of synthetic biology promises to revolutionize the synthesis of natural plant products, but this advanced technology requires the complete biosynthetic pathways and isolation of genes responsible for these products from plants. In recent years, several genes involved in diosgenin synthesis have been isolated from D. zingiberensis and their expression cassettes have been integrated into the yeast genome. The resulting engineered yeast strains are capable of synthesizing diosgenin using standard culture media. However, due to the limited availability of diosgenin synthesis genes, diosgenin production by these engineered yeast strains remains low.

[0003] Therefore, it is particularly important to discover new diosgenin synthesis-related genes and improve the biosynthesis efficiency of diosgenin in yeast cells. Summary of the Invention

[0004] The present invention provides a protein derived from Dioscorea zingiberensis, which is used for improving the synthesis efficiency of diosgenin.

[0005] The first aspect of the present invention provides a protein, wherein the protein is selected from at least one of a) and b):

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

[0007] 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: 2.

[0008] Furthermore, the protein is derived from Dioscorea zingiberensis C.H.Wright (scientific name: Dioscorea zingiberensis C.H.Wright).

[0009] Furthermore, the tag is at least one of a 6×His tag, a Flag tag, a MBP tag, a HA tag or a c-Myc tag.

[0010] The second aspect of the present invention provides a nucleic acid molecule encoding the above protein.

[0011] Furthermore, the nucleic acid molecule is at least one of the following d)-f):

[0012] d) a nucleic acid molecule having a nucleotide sequence as set forth in SEQ ID NO: 1;

[0013] e) a nucleic acid molecule that has 75% or more identity with the nucleotide sequence defined in d) and encodes the protein set forth in SEQ ID NO: 2;

[0014] f) A nucleic acid molecule that hybridizes under stringent conditions to the nucleic acid molecule defined in d) or e) and encodes the protein shown in SEQ ID NO: 2.

[0015] Furthermore, the stringent conditions are hybridization in a 6×SSC (containing 0.5% SDS) solution at 68° C., and then washing the membrane once with 2×SSC (containing 0.1% SDS) and once with 1×SSC (containing 0.1% SDS).

[0016] The third aspect of the present invention provides an expression cassette comprising the above nucleic acid molecule.

[0017] The fourth aspect of the present invention provides a recombinant expression vector comprising the above nucleic acid molecule or the above expression cassette.

[0018] A fifth aspect of the present invention provides a recombinant cell, which comprises the aforementioned nucleic acid molecule, the aforementioned expression cassette, or the aforementioned recombinant expression vector.

[0019] Furthermore, the recombinant cell is at least one of an insect cell, a plant cell, a mammalian cell, a fungal cell, or a bacterial cell. Still further, the recombinant cell is a fungal cell, preferably a yeast cell.

[0020] The sixth aspect of the present invention provides the use of the above protein as cytochrome P450.

[0021] The seventh aspect of the present invention provides a protein preparation comprising the above protein.

[0022] An eighth aspect of the present invention provides use of the above protein or protein preparation in the synthesis of diosgenin.

[0023] A ninth aspect of the present invention provides a method for synthesizing diosgenin, comprising: constructing a recombinant cell expressing the above-mentioned protein, culturing the recombinant cell, and obtaining the diosgenin from the culture product.

[0024] The present invention cloned a protein from Dioscorea zingiberensis that can significantly promote the biosynthesis efficiency of diosgenin. The protein has P450 enzyme activity, and co-expression with other known diosgenin synthesis genes in yeast cells significantly improves the biosynthesis efficiency of diosgenin, providing a key gene resource for the biosynthesis of diosgenin. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a mass spectrometry comparison diagram between the diosgenin product synthesized by yeast engineering bacteria and the diosgenin standard;

[0026] Figure 2 Comparison of the production of diosgenin synthesized by RH6829-DzCYP86A347(-) and RH6829-DzCYP86A347(+) engineered bacteria.

[0027] Figure 2 In the table, * indicates p less than 0.05. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] Example 1. Obtaining the full-length cDNA sequence of the gene encoding the P450 enzyme (DzCYP86A347)

[0030] 1. Extraction of total RNA from Dioscorea zingiberensis tubers: 100 mg of Dioscorea zingiberensis tubers were weighed and rapidly ground in liquid nitrogen. Total RNA was extracted from the ground sample powder using the EASYspin plus Plant RNA Rapid Extraction Kit (Adler).

[0031] 2. Reverse transcribe RNA into cDNA: Add the following components to an RNase-free PCR tube: 1.5 μg of total RNA extracted in step 1, 1 μL of DNase I, 1 μL of 10× DNase I buffer, and 0.5 μL of RNase inhibitor. Mix well and incubate at 37°C for 30 minutes. Then, add 1 μL of 100 mM EDTA and incubate at 65°C for 10 minutes to terminate the reaction. Add 1 μL of oligo(dT)18 and 1 μL of 10 mM dNTP mix to the PCR tube containing the above mixture and denature at 65°C for 5 minutes. Immediately place on ice. Next, add 4 μL of 5× reverse transcriptase buffer, 0.5 μL of RNase inhibitor, and 1 μL of 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.

[0032] 3. RT-PCR reaction to obtain the DzCYP86A347 cDNA sequence: Based on the coding sequence of the P450 enzyme gene DzCYP86A347, the primer pair DzCYP86A347-F and DzCYP86A347-R were designed:

[0033] DzCYP86A347-F:5'-ATGGCAGTCACTTCTCAGCTTG-3';

[0034] DzCYP86A347-R:5'-TTAAGCAGCAGCGGCAGTAGGC-3'.

[0035] A 20 μL reaction system was prepared by mixing 2 μL of cDNA template, 1 μL each of primers DzCYP86A347-F and DzCYP86A347-R, 10 μL of 2× PrimerSTAR Max Premix (Takara), and 6 μL of ddH₂O. PCR reaction conditions included pre-denaturation at 98°C for 2 min, followed by 30 cycles of denaturation at 95°C for 20 s, annealing at 58°C for 20 s, and extension at 72°C for 1.5 min, with a final extension at 72°C for 5 min. 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 Escherichia coli DH5α competent cells, followed by colony PCR. After completion of the reaction, 5 μL of the reaction product was analyzed by agarose gel electrophoresis to identify positive clones. The plasmids of these positive clones were then extracted and sent to a sequencing company (Beijing Qingke) for sequencing.

[0036] The results showed that the fragment was 1620 bp in length, the nucleotide sequence was shown in SEQ ID NO: 1, and the amino acid sequence encoded by it was shown in SEQ ID NO: 2. It was named DzCYP86A347.

[0037] Example 2: Protein significantly improves the biosynthesis efficiency of diosgenin in yeast cells

[0038] The coding gene of the above-mentioned protein DzCYP86A347 was cloned into the yeast expression vector pESC-leu2d-CPR-CYP94N8 through SpeI (this vector has been disclosed in the literature Zhou et al., 22R-but not 22S-hydroxycholesterolis recruited for diosgenin biosynthesis. the Plant Journal. 109(4): 940-951(2022) and can be provided by the research group of Zhang Yansheng of Shanghai University) to obtain the expression vector pESC-leu2d-CPR-CYP94N8-DzCYP86A347.

[0039] The expression vectors pESC-leu2d-CPR-CYP94N8-DzCYP86A347 and pESC-leu2d-CPR-CYP94N8 were respectively combined with another yeast expression vector pESC-URA-DzCYP90B71-DzCYP90G6 (this vector has been disclosed in the literature Zhou et al., 22R-but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis. the Plant Journal. 109(4): 940-951(2022) and can be provided by the research group of Zhang Yansheng of Shanghai University) in the cholesterol-synthesizing yeast RH6829 strain (this strain has been disclosed in the literature Souza, CM et al. A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic 13, 555-569 (2011), the strain was provided by Professor Howard Riezman of the University of Geneva, Switzerland) and co-expressed to obtain engineered bacteria RH6829-DzCYP86A347(-) and RH6829-DzCYP86A347(+).

[0040] Multiple transgenic yeast colonies were selected and placed in 5 ml of SD-URA-LEU-deficient medium (containing 2% glucose) and cultured at 30°C, 250 rpm, for 48 hours. The cells were harvested by centrifugation at 5000 rpm, washed three times with double-distilled water, and resuspended in 30 ml of SD-URA-LEU medium containing 2% galactose (OD600 was controlled between 0.4 and 0.6 after resuspension). Induction culture was then performed at 30°C for 48 hours. The resulting cells were harvested by centrifugation, resuspended in methanol containing 2% KOH, and disrupted by shaking with 0.45 mm acid-washed glass beads. The disrupted yeast cells were incubated at 22°C for 1 hour for saponification, and then extracted with n-hexane. The n-hexane extract was evaporated to dryness and dissolved in methanol for LC-MS analysis.

[0041] like Figure 1 As shown in Figure 2, mass spectrometry comparative analysis showed that the engineered bacteria could successfully synthesize diosgenin. The content of diosgenin in RH6829-DzCYP86A347(-) and RH6829-DzCYP86A347(+) was detected, and the results were as follows: Figure 2 As shown, the content of diosgenin synthesized by RH6829-DzCYP86A347(-) was 0.28 μg / mg, and the content of diosgenin synthesized by RH6829-DzCYP86A347(+) was 0.59 μg / mg, and the biosynthesis efficiency was increased by 1.13 times.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein, characterized in that The protein is selected from at least one of a) and b) a) a protein with an amino acid sequence as shown in SEQ ID NO: 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:

2.

2. A nucleic acid molecule encoding the protein according to claim 1.

3. An expression cassette comprising the nucleic acid molecule of claim 2.

4. A recombinant expression vector comprising the nucleic acid molecule of claim 2 or the expression cassette of claim 3.

5. A recombinant cell, characterized in that The recombinant cell comprises the nucleic acid molecule of claim 2 or the expression cassette of claim 3 or the recombinant expression vector of claim 4.

6. The recombinant cell according to claim 5, characterized in that The recombinant cell is at least one of an insect cell, a plant cell, a mammalian cell, a fungal cell or a bacterial cell.

7. Use of the protein according to claim 1 as cytochrome P450.

8. A protein preparation, characterized in that The invention comprises the protein according to claim 1.

9. Use of the protein according to claim 1 or the protein preparation according to claim 8 in the synthesis of diosgenin.

10. A method for synthesizing diosgenin, characterized in that: Constructing a recombinant cell that expresses the protein according to claim 1, culturing the recombinant cell, and obtaining the diosgenin from the culture product.

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