A c26 hydroxylase for improving biosynthesis efficiency of diosgenin, and a coding gene, a preparation method and an application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a C26 hydroxylase for improving the biosynthesis efficiency of diosgenin, its encoding gene, preparation method, and application. Background Technology

[0002] Diosgenin is a raw material for various steroidal drugs, mainly synthesized in plants such as yam, fenugreek, and Paris polyphylla. Currently, the primary source of diosgenin is extraction and purification from plants; however, this method involves the use of large amounts of sulfuric acid or hydrochloric acid, generating substantial amounts of acidic wastewater and causing serious environmental pollution. In recent years, several genes involved in diosgenin biosynthesis have been isolated from yam, fenugreek, and Paris polyphylla. Using synthetic biology techniques, these isolated diosgenin synthesis genes have been transformed into cholesterol-synthesizing yeast, and engineered yeast strains capable of synthesizing diosgenin have been constructed. However, due to the limited number of diosgenin synthesis genes currently available, the yield of diosgenin synthesized by engineered yeast 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] To address the problem of inefficient diosgenin synthesis in engineered yeasts due to the limited number of diosgenin synthesis genes, this invention provides a C26 hydroxylase, its encoding gene, preparation method, and application that can be used to improve the biosynthetic efficiency of diosgenin in yeast cells, thus providing a new gene element for promoting the development of synthetic biology of diosgenin.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In a first aspect, the present invention provides a C26 hydroxylase that can significantly promote the biosynthesis efficiency of diosgenin, wherein the protein of the C26 hydroxylase is a protein as follows a) or b) or c):

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

[0008] 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;

[0009] c) The amino acid sequence shown in SEQ ID NO.2 is modified by substitution and / or deletion and / or addition of 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] In a second aspect, the present invention provides a gene encoding the C26 hydroxylase protein described above, wherein the nucleic acid molecule of the gene is one of (a), (b), or (c) as follows:

[0012] (a) Its coding sequence is the 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 nucleotides specified in (a) or (b) and encodes the protein shown in SEQ ID NO: 2.

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

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

[0017] A third aspect of the present invention provides an expression cassette containing the gene encoding the C26 hydroxylase protein described above.

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

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

[0020] In a fifth aspect, the present invention also provides a transformed cell containing a nucleic acid molecule of the gene encoding the C26 hydroxylase protein described above.

[0021] Preferably, the transformed cells are yeast cells, insect cells, plant cells, mammalian cells, fungal cells, or bacterial cells.

[0022] In a sixth aspect, the present invention also provides a method for preparing the C26 hydroxylase protein, which includes at least the following steps:

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

[0024] (2) The gene fragment is cloned 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] A seventh aspect of the invention also provides the use of the C26 hydroxylase in any of the following:

[0027] (a1) Preparation of products containing diosgenin;

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

[0029] This 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 mentioned above as the target gene, an expression cassette suitable for expression in yeast cells was constructed.

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

[0032] (b3) Fermentation of the engineered yeast significantly improved the biosynthesis efficiency of diosgenin.

[0033] The present invention has the following advantages:

[0034] This invention is the first to clone the full-length sequence of a C26 hydroxylase gene from fenugreek that can significantly promote the biosynthesis efficiency of diosgenin. Biochemical techniques were used to identify that this C26 hydroxylase can utilize furanosterol, an intermediate in the diosgenin synthesis pathway, as a substrate, and hydroxylate it at the C26 position to form a 26-hydroxylated intermediate. Co-expression of this C26 hydroxylase gene with other known diosgenin synthesis genes in yeast cells significantly improved the biosynthesis efficiency of diosgenin. Therefore, this invention provides a key gene resource for the biosynthesis of diosgenin.

[0035] Given the high production cost and significant environmental pollution associated with directly extracting and purifying diosgenin from plants, the discovery of the gene in this invention provides the necessary genetic resources for the production of diosgenin using synthetic biology techniques, and has potential application value. Attached Figure Description

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

[0037] Figure 2 In this embodiment of the invention, the TfCYP86A164 protein expressed in yeast cells catalyzes the formation of a 26-hydroxylated product using furan sterol, an intermediate in the diosgenin synthesis pathway, as a substrate.

[0038] Figure 3 The LC-MS mass spectrum of the 26-hydroxylated product is shown below.

[0039] Figure 4 The TfCYP86A164 of this invention significantly promotes the biosynthesis efficiency of diosgenin in yeast cells. Detailed Implementation

[0040] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are 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 described in this invention

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

[0044] Weigh approximately 100 mg of fenugreek plant tender leaves and rapidly grind them in liquid nitrogen. Extract total RNA from the ground sample powder using the EASYspin plus Plant RNA Rapid Extraction Kit (Adley).

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

[0046] Add the following components to an RNase-free PCR tube: RNA: 8 μL (approximately 1.5 μg), DNase I: 1 μL, 10×DNase I buffer: 1 μL, RNase inhibitor: 0.5 μL. Mix the above substances thoroughly and incubate at 37°C for 30 min. Then add 1 μL of 100 mM EDTA and incubate at 65°C for 10 min to terminate the reaction. Add 1 μL of Oligo(dT)18 and 1 μL of dNTPMIX (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 of 5× reverse transcriptase buffer, 0.5 μL of RNase inhibitor, and 1 μL of reverse transcriptase sequentially, mix well, and incubate at 42°C for 1 hour to synthesize first-strand cDNA. Then incubate at 70°C for 10 min 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 pair:

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

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

[0051] The 20 μL PCR reaction system consisted of: 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: 98℃ pre-denaturation for 2 min; cycling program: 95℃ denaturation for 20 s, 58℃ annealing for 20 s, 72℃ extension for 1.5 min, 30 cycles, with a final extension at 72℃ for 5 min. The reaction product was recovered using a standard agarose gel DNA recovery kit (purchased from Beijing Adley) and ligated into the pEASY-Blunt Simple vector (Beijing TransGen). The ligation product was transformed into *E. coli* DH5α competent cells, followed by colony PCR. After the reaction, 5 μL of the product was subjected to agarose gel electrophoresis to identify positive clones. Plasmids from positive clones were extracted and sent to a sequencing company (Beijing Qingke) for sequencing. Positive clone PCR identification is as follows: Figure 1 As shown, Figure 1The red arrow in the middle points to the band of the target gene being amplified.

[0053] The results showed that the fragment was 1554 bp in length, and its deoxyribonucleotide sequence was shown in SEQ ID NO: 1, while the amino acid sequence it encoded 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, whose sequencing results were correct in Example 1, was cloned into the yeast expression vector pESC-HIS via BamHI and SalI sites, thus constructing the yeast expression plasmid pESC-HIS-CYP86A164 containing the C26 hydroxylase gene. The vector pESC-HIS-CYP86A164, along with other diosgenin synthesis gene vectors pESC-URA-CYP90B71-CYP90G6 and pESC-Leu-CPR-CYP82J17 (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 Zhang Yansheng research group of Shanghai University), were co-transformed into the cholesterol-producing Saccharomyces cerevisiae strain RH6829 (this strain has been disclosed in the literature Souza, CM et 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 Professor Zhang Yansheng's research group at Shanghai University. Yeast transformants were obtained and plated on SD-URA-HIS-LEU auxotrophic solid medium for screening to obtain positive clones. Single colonies of positive yeast were picked and placed in 5 ml of SD-URA-HIS-LEU auxotrophic medium (containing 2% glucose), and cultured at 30°C and 250 rpm for 48 hours. 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 was controlled between 0.4 and 0.6 after resuspending). The cells were induced and cultured at 30°C for 48 hours. The bacterial cells obtained from the above culture were collected by centrifugation, resuspended in methanol solvent containing 2% KOH, and subjected to oscillation by filling with 0.45 mm acid-washed glass beads. The lysate was cultured 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.

[0057] LC-MS detection results are as follows Figure 2 As shown, where Figure 2The color symbols in a, from left to right, represent 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 indicates that diosgenin and its intermediates in the synthesis pathway are... Figure 2 The composition of each combination is shown in figure a. For example... Figure 2 As shown in b: Expression of TfCYP86A164 alone does not produce diosgenin or any intermediate components in its synthetic pathway; combined expression of DzCYP90B71 / DzCYP90G6 synthesizes intermediate compounds 1 and 2 in the diosgenin synthetic pathway; combined expression of DzCYP90B71 / DzCYP90G6 / TfCYP86A164 leads to a significant decrease in intermediate component 2 and a significant increase in intermediate 3; combined expression of DzCYP90B71 / DzCYP90G6 / TfCYP82J17 produces intermediates 1 and 2, as well as 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 spectrometry identification chromatogram of the 26-hydroxylated product is shown below. Figure 3 As shown.

[0058] Example 3

[0059] The C26 hydroxylase significantly enhances the biosynthetic efficiency of diosgenin in yeast cells.

[0060] The gene for the C26 hydroxylase TfCYP86A164 was cloned into the vector pESC-URA-CYP90B50, a gene for diosgenin synthesis derived from fenugreek, using SpeI / SacI. (This vector has been described in the literature by Zhou et al., 22R-but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.) The expression vector pESC-URA-CYP90B50-TfCYP86A164 was obtained by publishing the gene for diosgenin synthesis from fenugreek in Plant Journal. 109(4):940-951 (2022), which can be provided by Professor Zhang Yansheng's research group at Shanghai University. The vectors pESC-URA-CYP90B50 and pESC-URA-CYP90B50-TfCYP86A164 were then combined with the vector pESC-Leu-CPR-CYP72A613 (a vector already described in the literature Zhou et al., 22R-but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis). (Published in Journal.109(4):940-951(2022), available from Professor Zhang Yansheng's research group at Shanghai University) Co-expressed in cholesterol-synthesizing yeast RH6829 to obtain engineered strains RH6829-CYP86A164(-) and RH6829-CYP86A164(+); Following the method in Example 2, the engineered strains were induced to ferment, and the fermentation products were extracted from the yeast cells to compare the synthesis efficiency of diosgenin.

[0061] like Figure 4 As shown, when the engineered bacteria were co-expressed with the gene of the C26 hydroxylase TfCYP86A164, 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 protein of this C26 hydroxylase is either a) or b) of the following: 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 gene encoding the C26 hydroxylase as described in claim 1, characterized in that, Its nucleic acid molecule is a gene that is one of the following (a), (b), or (c): (a) Its coding sequence is the cDNA molecule or genomic DNA molecule shown in SEQ ID NO: 1; (b) has more than 75% identity with the nucleotide sequence defined in (a) and encodes a cDNA molecule or genomic DNA molecule that encodes the protein shown in SEQ ID NO: 2; (c) A cDNA molecule or genomic DNA molecule that hybridizes under stringent conditions with the nucleotides 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 box, characterized in that, It contains the gene as described in claim 2.

5. An expression carrier, characterized in that, It contains the gene as described in claim 2.

6. A transformed cell, characterized in that, It contains the gene as described in claim 2.

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

8. The method for preparing C26 hydroxylase as described in claim 1, characterized in that, It should include at least the following steps: (1) Amplify the gene fragment encoding the amino acid sequence of the C26 hydroxylase; (2) The gene fragment is cloned 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 be expressed.

9. The application of the C26 hydroxylase as described in claim 1, characterized in that, For (a1) Preparing products containing diosgenin; and / or (a2) Prepare a product containing the C26 hydroxylase.

10. A method for improving the biosynthesis efficiency of diosgenin, characterized in that, Includes the following steps: (b1) The gene encoding the C26 hydroxylase amino acid sequence as described in claim 1 is co-transformed with other genes involved in diosgenin synthesis into cholesterol synthesis chassis yeast to construct an engineered yeast strain; (b2) Fermentation culture of engineered yeast significantly improved the biosynthesis efficiency of diosgenin.

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