C26 hydroxylase for improving biosynthesis efficiency of diosgenin as well as coding gene, preparation method and application of C26 hydroxylase

Abstract

The invention relates to the technical field of biology, and discloses C26 hydroxylase for improving the biosynthesis efficiency of diosgenin as well as a coding gene, a preparation method and application of the C26 hydroxylase. The protein of the C26 hydroxylase is any one of the following a) or b) or c): a) a protein with an amino acid sequence as shown in SEQ ID NO.2; 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; and c) carrying out substitution and / or deletion and / or addition of one or more amino acid residues on the amino acid sequence as shown in SEQ ID NO.2 to obtain the protein with the same function. The C26 hydroxylase provided by the invention can improve the biosynthesis efficiency of the diosgenin, and provides a key gene resource for producing the diosgenin by using an enzymic method.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a C26 hydroxylase for improving the biosynthesis efficiency of diosgenin, as well as its encoding gene, preparation method and application. Background Art

[0002] Diosgenin, a raw material for various steroidal drugs, is primarily synthesized from plants such as Dioscorea zingiberensis, Fenugreek, and Paris polyphylla. Currently, diosgenin is primarily extracted and purified from plants. However, this method involves the use of large amounts of sulfuric acid or hydrochloric acid, generating significant amounts of acidic wastewater and causing significant environmental pollution. In recent years, genes involved in diosgenin biosynthesis have been isolated from Dioscorea zingiberensis, Fenugreek, and Paris polyphylla. Using synthetic biology techniques, these isolated diosgenin biosynthesis genes have been transformed into cholesterol-producing yeast, and engineered yeast strains capable of synthesizing diosgenin have been constructed. However, due to the limited availability of diosgenin biosynthesis genes, diosgenin production by these engineered yeast strains remains low.

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

[0004] In order to solve the problem of inefficient diosgenin synthesis by yeast engineering bacteria due to limited diosgenin synthesis genes, the present invention provides a C26 hydroxylase that can be used to improve the biosynthesis efficiency of diosgenin in yeast cells, as well as its encoding gene, preparation method and application, providing new genetic elements for promoting the synthetic biology development of diosgenin.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect of the present invention, a C26 hydroxylase is provided that can significantly promote the biosynthesis efficiency of diosgenin. The protein of the C26 hydroxylase is the following protein a) or b) or c):

[0007] a) a protein with an amino acid sequence as shown in SEQ ID NO. 2;

[0008] 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;

[0009] c) The amino acid sequence shown in SEQ ID NO. 2 is substituted and / or deleted and / or added with one or more amino acid residues to obtain a protein with the same function.

[0010] Preferably, the tag is a 6×histidine tag, a Flag tag, an MBP tag, an HA tag or a c-Myc tag.

[0011] The second aspect of the present invention provides a gene encoding the above-mentioned C26 hydroxylase protein, wherein the nucleic acid molecule is the gene of (a), (b), or (c):

[0012] (a) the coding sequence is a cDNA molecule or genomic DNA molecule shown in SEQ ID NO: 1;

[0013] (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: 2;

[0014] (c) A cDNA molecule or genomic DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in (a) or (b) and encodes the protein shown in SEQ ID NO: 2.

[0015] 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).

[0016] Preferably, the gene is derived from the fenugreek plant.

[0017] The third aspect of the present invention provides an expression cassette comprising the gene encoding the C26 hydroxylase protein.

[0018] The fourth aspect of the present invention also provides an expression vector containing the gene encoding the C26 hydroxylase protein.

[0019] Preferably, the expression vector is a yeast expression vector or an insect cell expression vector.

[0020] The fifth aspect of the present invention further provides a transformed cell, which contains the nucleic acid molecule of the gene encoding the C26 hydroxylase protein.

[0021] Preferably, the transformed cell is a yeast cell, an insect cell, a plant cell, a mammalian cell, a fungal cell or a bacterial cell.

[0022] The sixth aspect of the present invention further provides a method for preparing the C26 hydroxylase protein, which comprises at least the following steps:

[0023] (1) amplifying the gene fragment encoding the C26 hydroxylase protein;

[0024] (2) cloning the gene fragment into a yeast expression vector to obtain a yeast expression plasmid;

[0025] (3) The yeast expression plasmid is transferred into Pichia pastoris, Saccharomyces cerevisiae or other yeasts to obtain a strain expressing the C26 hydroxylase, and the expression of the C26 hydroxylase is induced.

[0026] The seventh aspect of the present invention further provides the use of the C26 hydroxylase in any of the following:

[0027] (a1) preparing a product containing diosgenin;

[0028] (a2) preparing a product containing the C26 hydroxylase.

[0029] The present invention also provides a method for improving the biosynthesis efficiency of diosgenin, comprising the following steps:

[0030] (b1) using the gene encoding the C26 hydroxylase protein as the target gene, constructing an expression cassette suitable for expression in yeast cells;

[0031] (b2) co-expressing the expression cassette of the C26 hydroxylase gene and the expression cassettes of other diosgenin synthesis genes in yeast cells to construct an engineered yeast strain;

[0032] (b3) fermenting the engineered yeast to significantly improve the biosynthesis efficiency of diosgenin.

[0033] The present invention has the following advantages:

[0034] The present invention cloned for the first time the full-length sequence of a C26 hydroxylase gene from a fenugreek plant that can significantly promote the biosynthesis efficiency of diosgenin, and identified using biochemical techniques that the C26 hydroxylase can use furanserol, an intermediate in the diosgenin synthesis pathway, as a substrate to perform hydroxylation at the C26 position to form a 26-hydroxylated intermediate. Co-expression of the C26 hydroxylase gene with other known diosgenin synthesis genes in yeast cells significantly improved the biosynthesis efficiency of diosgenin. Therefore, the present invention provides a key gene resource for the biosynthesis of diosgenin.

[0035] In view of the high production cost and severe environmental pollution of directly extracting and purifying diosgenin from plants, the discovery of the gene of the present invention provides the necessary gene resources for the production of diosgenin by synthetic biology technology and has potential application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is an agarose gel electrophoresis diagram of the TfCYP86A164 gene clone according to an embodiment of the present invention;

[0037] Figure 2 The TfCYP86A164 protein expressed by the yeast cells of the embodiment of the present invention catalyzes the formation of a 26-hydroxylated product using furosterol, an intermediate in the diosgenin synthesis pathway, as a substrate;

[0038] Figure 3 is the LC-MS mass spectrum of the 26-hydroxylation product;

[0039] Figure 4 TfCYP86A164 according to the present invention significantly promoted the biosynthesis efficiency of diosgenin in yeast cells. DETAILED DESCRIPTION

[0040] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0041] Example 1

[0042] Obtaining the full-length cDNA sequence of the C26 hydroxylase (TfCYP86A164) encoding gene of the present invention

[0043] 1. Extraction of total RNA from fenugreek plant leaves. The specific method is as follows:

[0044] About 100 mg of young fenugreek leaves 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 (Adelaide).

[0045] 2. Reverse transcribe RNA into cDNA. The specific method is as follows:

[0046] Add the following components to an RNase-free PCR tube: 8 μL (approximately 1.5 μg) of RNA, 1 μL of DNase I, 1 μL of 10× DNase I buffer, and 0.5 μL of RNase inhibitor. Mix thoroughly 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 dNTPMIX to the PCR tube containing the above mixture and pre-denature at 65°C for 5 minutes. Immediately place on ice. Then, add 4 μL of 5× reverse transcriptase buffer, 0.5 μL of RNase inhibitor, and 1 μL of reverse transcriptase. Mix thoroughly 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 the cDNA at -80°C.

[0047] 3. The TfCYP86A614 cDNA sequence was obtained by conventional RT-PCR reaction.

[0048] The C26 hydroxylase gene TfCYP86A164 was amplified by PCR using the following primer pairs:

[0049] TfCYP86A164-F:5'-ATGGATGCATCAAATGCTTTAATG-3';

[0050] TfCYP86A164-R:5'-TTATTTGGCTTTTGAATTGTGC-3'.

[0051] The 20 μL PCR reaction system was as follows: 2 μL cDNA template, 1 μL each of primers TfCYP86A164-F and TfCYP86A164-R, 10 μL 2× PrimerSTAR Max Premix (Takara), and 6 μL ddH2O.

[0052] PCR reaction conditions: pre-denaturation at 98°C for 2 minutes; cycle program: denaturation at 95°C for 20 seconds, annealing at 58°C for 20 seconds, extension at 72°C for 1.5 minutes, 30 cycles, and final extension at 72°C for 5 minutes. The reaction product was recovered using a common agarose gel DNA recovery kit (purchased from Beijing Aidelai) and then 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, 5 μL of the reaction product was taken for agarose gel electrophoresis detection to determine the positive clone. The plasmid of the positive clone was extracted and sent to a sequencing company (Beijing Qingke) for sequencing. Positive clone PCR identification as follows Figure 1 As shown, Figure 1The red arrow in the middle points to the band of the amplified target gene.

[0053] The results showed that the length of the fragment was 1554 bp, the deoxyribonucleotide sequence thereof was shown in SEQ ID NO: 1, and the amino acid sequence encoded by the fragment was shown in SEQ ID NO: 2.

[0054] Example 2

[0055] Identification of the catalytic activity of the C26 hydroxylase (TfCYP86A164)

[0056] The C26 hydroxylase (CYP86A164) gene with the correct sequencing results in Example 1 was cloned into the yeast expression vector pESC-HIS through the BamHI and SalI sites to form the yeast expression plasmid pESC-HIS-CYP86A164 containing the C26 hydroxylase gene; the vector pESC-HIS-CYP86A164 and other diosgenin synthesis gene vectors pESC-URA-CYP90B71-CYP90G6 and pESC-Leu-CPR-CYP82J17 (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), and can be provided by the research group of Zhang Yansheng of Shanghai University) were jointly transformed into the cholesterol-producing Saccharomyces cerevisiae RH6829 strain (the strain has been disclosed in the literature Souza, CMet al. Astable 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 can be provided by the research group of Zhang Yansheng at Shanghai University) to obtain yeast transformants, which were plated on SD-URA-HIS-LEU nutrient-deficient solid medium for screening to obtain positive clones; a single positive yeast colony was picked and incubated in 5 ml of SD-URA-HIS-LEU deficiency medium (containing 2% glucose) at 30°C and 250 rpm for 48 hours, and the cells were collected by centrifugation at 5000 rpm, washed three times with double-distilled water, and resuspended in 30 ml of SD-URA-HIS-LEU medium containing 2% galactose (OD600 after resuspension was controlled between 0.4 and 0.6), and induced at 30°C for 48 hours. The yeast cells were collected by centrifugation and resuspended in a methanol solvent containing 2% KOH. 0.45 mm acid-washed glass beads were added and the yeast cells were shaken and disrupted. The disrupted liquid was incubated at 22°C for 1 hour for saponification reaction and then extracted with n-hexane. The n-hexane extract was evaporated to dryness and dissolved in methanol for LC-MS analysis.

[0057] LC-MS test results are as follows Figure 2 As shown, Figure 2The color legend in a represents, from left to right, the expression of TfCYP86A164 alone, the combined expression of DzCYP90B71 / DzCYP90G6, the combined expression of DzCYP90B71 / DzCYP90G6 / TfCYP86A164, and the combined expression of DzCYP90B71 / DzCYP90G6 / TfCYP82J17 in the Saccharomyces cerevisiae RH6829 strain, respectively; Figure 2 b represents the intermediates in diosgenin and its synthesis pathway. Figure 2 a shows the synthesis of each combination expression. Figure 2 As shown in b: Single expression of TfCYP86A164 did not produce diosgenin and any intermediate components in its synthesis pathway; combined expression of DzCYP90B71 / DzCYP90G6 synthesized intermediate compounds 1 and 2 in the diosgenin synthesis pathway; combined expression of DzCYP90B71 / DzCYP90G6 / TfCYP86A164 led to a significant decrease in intermediate component 2 and a significant increase in intermediate 3; combined expression of DzCYP90B71 / DzCYP90G6 / TfCYP82J17 produced intermediates 1, 2, and the final product diosgenin. These results indicate that TfCYP86A164 can use intermediate 2 as a substrate and hydroxylate it at its C26 position to form a 26-hydroxylated product (i.e., intermediate compound 3); the LC-MS mass spectrometric identification of the 26-hydroxylated product is shown in Figure 2. Figure 3 shown.

[0058] Example 3

[0059] The C26 hydroxylase significantly improves the biosynthesis efficiency of diosgenin in yeast cells

[0060] The C26 hydroxylase TfCYP86A164 gene was cloned into the fenugreek-derived diosgenin biosynthesis gene vector pESC-URA-CYP90B50 (the vector has been reported in the literature Zhou et al., 22R-but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis. Plant Journal. 109 (4): 940-951 (2022) disclosed, can be provided by the Zhang Yansheng research group of Shanghai University), to obtain the expression vector pESC-URA-CYP90B50-TfCYP86A164; the vectors pESC-URA-CYP90B50 and pESC-URA-CYP90B50-TfCYP86A164 were respectively combined with the fenugreek-derived diosgenin synthesis gene vector pESC-Leu-CPR-CYP72A613 (the vector has been reported 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 the research group of Zhang Yansheng of Shanghai University) was co-expressed in the cholesterol-synthesizing yeast RH6829 to obtain engineered bacteria RH6829-CYP86A164(-) and RH6829-CYP86A164(+); according to the method in Example 2, the engineered bacteria were induced to ferment, and the fermentation products were extracted from the yeast cells for comparison of the synthesis efficiency of diosgenin.

[0061] like Figure 4 As shown, when the C26 hydroxylase TfCYP86A164 gene was co-expressed in the engineered bacteria, the biosynthesis efficiency of diosgenin was increased by 6.86 times.

Claims

1. A C26 hydroxylase for improving the biosynthesis efficiency of diosgenin, characterized in that: The C26 hydroxylase protein is the protein of a) or b) or c) below: 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; c) The amino acid sequence shown in SEQ ID NO. 2 is substituted and / or deleted and / or added with one or more amino acid residues to obtain a protein with the same function.

2. A gene encoding a protein having C26 hydroxylase activity according to claim 1, characterized in that: The nucleic acid molecule is the gene of (a), (b), or (c): (a) the coding sequence is a cDNA molecule or genomic DNA molecule shown in SEQ ID NO: 1; (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: 2; (c) A cDNA molecule or genomic DNA molecule that hybridizes under stringent conditions with the nucleotide sequence defined in (a) or (b) and encodes the protein shown in SEQ ID NO:

2.

3. The gene according to claim 2, characterized in that The gene is derived from the fenugreek plant.

4. An expression cassette, characterized in that It contains the gene according to claim 2.

5. An expression vector, characterized in that It contains the gene according to claim 2.

6. A transformed cell, characterized in that It contains the gene according to claim 2.

7. A transformed cell according to claim 6, characterized in that The transformed cells are yeast cells, bacterial cells, mammalian cells, fungal cells, insect cells or plant cells.

8. The method for preparing C26 hydroxylase according to claim 1, wherein At least the following steps are included: (1) amplifying a gene fragment encoding the amino acid sequence of the C26 hydroxylase; (2) cloning the gene fragment into a yeast expression vector to obtain a yeast expression plasmid; (3) The yeast expression plasmid is transferred into yeast cells to obtain a strain expressing the C26 hydroxylase, and the C26 hydroxylase is induced to express.

9. The use of C26 hydroxylase according to claim 1, characterized in that Used for (a1) preparing a product containing diosgenin; and / or (a2) preparing a product containing the C26 hydroxylase.

10. A method for improving the biosynthesis efficiency of diosgenin, characterized in that: The steps include: (b1) transforming the gene encoding the amino acid sequence of C26 hydroxylase as described in claim 1 together with other genes involved in diosgenin synthesis into "cholesterol synthesis chassis biological yeast" to construct a yeast engineering strain; (b2) The biosynthesis efficiency of diosgenin was significantly improved by fermentation and cultivation of engineered yeast.

Images