Protein derived from dioscorea zingiberensis, encoding gene and application thereof

Abstract

The present application provides a protein derived from Dioscorea zingiberensis, its encoding gene and application. The protein provided by the present application 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 terminal and / or C terminal of the protein as shown in SEQ ID NO: 2. The protein provided by the present application has P450 enzyme activity, and experiments have verified that the co-expression of the protein encoding gene and other diosgenin synthesis genes in yeast cells can significantly improve the biosynthesis efficiency of diosgenin, thereby providing a key gene resource for the biosynthesis of diosgenin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a protein derived from Dioscorea zingiberensis, its encoding gene and application. BACKGROUND

[0002] Dioscorea zingiberensis C.H.Wright is a medicinal plant for industrial extraction of dioscin. The extraction of dioscin from Dioscorea zingiberensis involves the use of a large amount of inorganic acid, which results in large wastewater discharge and serious environmental pollution. The development of synthetic biology technology will completely revolutionize the synthesis process of plant natural products, but the prerequisite for this advanced technology is to analyze the complete biosynthesis pathway of natural products from plants and isolate natural product synthesis genes. In recent years, some dioscin synthesis-related genes have been isolated from Dioscorea zingiberensis, and the expression cassettes of these genes have been integrated into the genome of yeast to obtain yeast engineering bacteria that can synthesize dioscin using ordinary culture medium. However, due to the limited number of dioscin synthesis genes obtained, the yield of dioscin synthesized by yeast engineering bacteria is still low.

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

[0004] The present application provides a protein derived from Dioscorea zingiberensis for improving the synthesis efficiency of dioscin.

[0005] The first aspect of the present application provides a protein selected from at least one of a), 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 as shown in SEQ ID NO: 2.

[0008] Further, the protein is derived from Dioscorea zingiberensis C.H.Wright.

[0009] Further, the tag is at least one of 6xHis tag, Flag tag, MBP tag, HA tag or c-Myc tag.

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

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

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

[0013] e) a nucleic acid molecule having 75% or more identity to the nucleotide sequence as defined in d), and encoding a protein as set forth in SEQ ID NO: 2;

[0014] f) a nucleic acid molecule hybridizing to the nucleic acid molecule as defined in d) or e) under stringent conditions, and encoding a protein as set forth in SEQ ID NO: 2.

[0015] Further, the stringent conditions are hybridization in a solution of 6xSSC (containing 0.5% SDS) at 68°C, followed by washing the membrane once with 2xSSC (containing 0.1% SDS) and 1xSSC (containing 0.1% SDS), respectively.

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

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

[0018] The fifth aspect of the present application provides a recombinant cell comprising the above nucleic acid molecule or comprising the above expression cassette or comprising the above recombinant expression vector.

[0019] Further, the recombinant cell is at least one of an insect cell, a plant cell, a mammal 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 application provides the above protein for use as a cytochrome P450.

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

[0022] The eighth aspect of the present application provides the above protein or the above protein preparation for use in synthesis of diosgenin.

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

[0024] The present application clones a protein from Dioscorea zingiberensis which can significantly promote the biosynthesis efficiency of diosgenin, the protein has P450 enzyme activity, and co-expression of the protein with other known diosgenin synthesis genes in yeast cells significantly improves the biosynthesis efficiency of diosgenin, thereby providing a key genetic resource for diosgenin biosynthesis. Attached Figure Description

[0025] Figure 1 Mass spectrometry comparison between diosgenin products synthesized by engineered yeast and diosgenin standards;

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

[0027] Figure 2 In the figure, * indicates that p is less than 0.05. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

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

[0030] 1. Extraction of total RNA from Dioscorea zingiberensis tubers: 100 mg of Dioscorea zingiberensis tubers were weighed and rapidly ground in liquid nitrogen. The ground sample powder was then used to extract total RNA using the EASYspin plus plant RNA rapid extraction kit (Adley).

[0031] 2. Reverse transcription of RNA into cDNA: Add the following components to an RNase-free PCR tube: 1.5 μg total RNA extracted in step 1, 1 μL DNase I, 1 μL 10×DNase I buffer, and 0.5 μL RNase inhibitor. Mix the above substances well and incubate at 37°C for 30 min. Then add 1 μL 100 mM EDTA and incubate at 65°C for 10 min to terminate the reaction. Add 1 μL LOligo(dT)18 and 1 μL dNTP MIX (10 mM) to the PCR tube containing the above mixture, pre-denature at 65°C for 5 min, and then immediately place on ice. Then add 4 μL 5× reverse transcriptase buffer, 0.5 μL RNase inhibitor, and 1 μL reverse transcriptase sequentially, mix well, and incubate at 42°C for 1 hour to synthesize first-strand cDNA. Then terminate the reaction at 70°C for 10 min. Store the cDNA at -80°C.

[0032] 3. Obtaining DzCYP86A347 cDNA sequence by RT-PCR reaction: According to the coding sequence of P450 enzyme gene DzCYP86A347, primer pair DzCYP86A347-F and DzCYP86A347-R was designed:

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

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

[0035] 2 μL of cDNA template, 1 μL of primer DzCYP86A347-F and DzCYP86A347-R, 10 μL of 2 × PrimerSTAR Max Premix (Takara) and 6 μL of ddH2O were mixed to obtain a 20 μL reaction system. The PCR reaction conditions were as follows: 98 °C pre-denaturation for 2 min; the cycle program was 95 °C denaturation for 20 s, 58 °C annealing for 20 s, 72 °C extension for 1.5 min, 30 cycles, and finally 72 °C extension for 5 min. The reaction product was recovered by a general agarose gel DNA recovery kit (purchased from Beijing Aidley) and then connected to a pEASY-Blunt Simple vector (Beijing Quanshijin). The connection product was transformed into E. coli DH5α competent cells, and then colony PCR was performed. After the reaction, 5 μL of the reaction product was detected by agarose gel electrophoresis to determine the positive clone. The plasmid of the positive clone was extracted and sent to a sequencing company (Beijing Qikexin) for sequencing.

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

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

[0038] The coding gene of the above protein DzCYP86A347 was cloned into the yeast expression vector vector pESC-leu2d-CPR-CYP94N8 (the 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), which can be provided by Professor Zhang Yan-sheng's research group of Shanghai University) through Spel, obtaining the expression vector pESC-leu2d-CPR-CYP94N8-DzCYP86A347.

[0039] The expression vectors pESC-leu2d-CPR-CYP94N8-DzCYP86A347 and pESC-leu2d-CPR-CYP94N8 were respectively co-expressed with another yeast expression vector pESC-URA-DzCYP90B71-DzCYP90G6 (the 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), which can be provided by Professor Zhang Yan-sheng's research group of Shanghai University) in the cholesterol synthesis yeast RH6829 strain (the strain has been disclosed in the literature Souza, C. M. et al. A stable yeast strain efficiently producing cholesterol instead of ergosterol is functional for tryptophan uptake, but not weak organic acid resistance. Metab. Eng. 13, 555-569 (2011), which was provided by Professor Howard Riezman of the University of Geneva, Switzerland), obtaining the engineering bacteria RH6829-DzCYP86A347(-) and RH6829-DzCYP86A347(+).

[0040] Several transgenic yeast single colonies were picked and cultured in 5 ml SD-URA-LEU deficient medium (containing 2% glucose) at 30°C, 250 rpm for 48 hours. The cells were collected by centrifugation at 5000 rpm, washed with double distilled water for 3 times, and resuspended in 30 ml SD-URA-LEU medium containing 2% galactose (the OD600 was controlled between 0.4-0.6 after resuspension), and induced at 30°C for 48 hours. The cells obtained from the above culture were collected by centrifugation, resuspended in 2% KOH-methanol solution, and filled with 0.45 mm acid-washed glass beads for yeast cell shaking disruption. The disruption solution was incubated at 22°C for 1 hour for saponification reaction, then extracted with n-hexane. The n-hexane extract was evaporated to dryness and dissolved in methanol for LC-MS analysis.

[0041] As shown in Figure 1 , the mass spectrum comparison analysis showed that the engineered bacteria could successfully synthesize dioscin. The content of dioscin in RH6829-DzCYP86A347(-) and RH6829-DzCYP86A347(+) was detected, and the results are shown in Figure 2 . The content of dioscin synthesized by RH6829-DzCYP86A347(-) was 0.28 μg / mg, and the content of dioscin 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 examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. The application of protein in the synthesis of diosgenin, characterized in that, The protein is selected from at least one of a) and b): a) A protein with the amino acid sequence shown in SEQ ID NO:2; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:

2.

2. A method for synthesizing diosgenin, characterized in that, Recombinant cells expressing the protein were constructed, the recombinant cells were cultured, and the diosgenin was obtained from the culture product. The protein is selected from at least one of a) and b): a) A protein with the amino acid sequence shown in SEQ ID NO:2; b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID NO:2; The recombinant cells are cholesterol-synthesizing yeast.

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