Key genes and applications of pachymic acid synthesis

Abstract

This invention discloses a key gene for pamoate synthesis and its application. The nucleotide sequence of the key gene for pamoate synthesis is shown in SEQ ID NO.1. The key gene includes PcCPR and CYP5035. The nucleotide sequence of PcCPR is shown in SEQ ID NO.2, and the nucleotide sequence of CYP5035 is shown in SEQ ID NO.3. Both PcCPR and CYP5035 are located on the pESC-Trp vector plasmid. Both PcCPR and CYP5035 are located after the galactose promoter. PcCPR is located after the GAL10 promoter, and CYP5035 is located after the GAL1 promoter. The nucleotide sequence of the GAL10 promoter is shown in SEQ ID NO.4, and the nucleotide sequence of the GAL1 promoter is shown in SEQ ID NO.5. Through extensive preliminary screening and experiments, this invention identified CYP5035, a key enzyme gene for the synthesis of pachymic acid using lanosterol as a substrate, which involves C-16 hydroxylation and C-20 methyl carboxylation. This invention is expected to construct a complete biosynthetic pathway for pachymic acid, elucidate the intrinsic quality attributes of the synthesis of active substances from Poria cocos, and provide data support for the research and application of pachymic acid biosynthetic cell factories, thereby promoting the innovative development of traditional Chinese medicine resources.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the key genes for the synthesis of pamoate and their applications. Background Technology

[0002] Currently, wild resources of Poria cocos are nearing depletion, and the low content of pachymic acid in Poria cocos severely limits its medicinal development. Therefore, it is of great significance to explore the key enzymes in the biosynthesis of pachymic acid and combine its synthesis with heterologous expression, enabling the synthesis of pachymic acid, which is originally only synthesized in traditional Chinese medicines such as Poria cocos, in microorganisms (such as yeast). Summary of the Invention

[0003] Based on the technical problems existing in the background technology, this invention proposes key genes for the synthesis of pamoate and their applications.

[0004] The key gene for pamoate synthesis proposed in this invention has the nucleotide sequence shown in SEQ ID NO.1.

[0005] The vector proposed in this invention contains the key gene for the synthesis of pamoate.

[0006] The microorganism proposed in this invention includes the above-mentioned carrier.

[0007] The application of the above-mentioned microorganisms proposed in this invention in the synthesis of pamoate.

[0008] Beneficial technical effects of the present invention:

[0009] The near depletion of wild Poria cocos resources and the low content of pachymic acid in Poria cocos have severely limited its medicinal development. This patented invention, for the first time, constructs a complete de novo synthesis pathway for pachymic acid. The key enzymes for the biosynthesis of pachymic acid discovered can not only be used in Poria cocos breeding and metabolic regulation to increase the pachymic acid content in the medicinal material, but also combine pachymic acid synthesis with heterologous expression. This allows pachymic acid, which is originally synthesized only in Poria cocos and other traditional Chinese medicines, to be synthesized in microorganisms such as yeast, significantly increasing its content. After liquid fermentation, the content can reach as high as 17.632 ug / L, while reducing its production cost. Attached Figure Description

[0010] Figure 1 This invention relates to the construction of heterologous expression vectors such as pESC-Trp, pESC-Trp-PcCPR, pESC-Trp-CYP5035, and pESC-Trp-CYP5035-PcCPR.

[0011] Figure 2This is a yeast plate diagram containing the genes pESC-Trp, pESC-Trp-PcCPR, pESC-Trp-CYP5035, and pESC-Trp-CYP5035-PcCPR proposed in this invention.

[0012] Figure 3 This is a PCR verification diagram of the gene inserted into the vector proposed in this invention; M, marker ladder; 1, CYP5035 primer PCR detection of pESC-Trp; 2, CYP5035 primer PCR detection of pESC-Trp-PcCPR; 3, CYP5035 primer PCR detection of pESC-Trp-CYP5035; 4, primer CYP5035 PCR detection of pESC-Trp-CYP5035-PcCPR; 5, primer PcCPR PCR of pESC-Trp; 6, primer PcCPR PCR detection of pESC-Trp-PcCPR; 7, primer PcCPR PCR detection of pESC-Trp-CYP5035; 8, primer PcCPR PCR detection of pESC-Trp-CYP5035-PcCPR.

[0013] Figure 4 This invention proposes a liquid chromatography-mass spectrometry (LC-MS) assay to detect the contents of 16α-hydroxy-tuberculic acid, tomolic acid, 3-O-acetyl-16α-hydroxy-tuberculic acid, and pamoic acid in yeast fermentation broth containing the pESC-Trp, pESC-Trp-PcCPR, pESC-Trp-CYP5035, and pESC-Trp-CYP5035-PcCPR genes. Specifically: (A) is the LC-MS assay of different yeast fermentation broths after 6 hours; (B) is a comparison of the contents of 16α-hydroxy-tuberculic acid, tomolic acid, 3-O-acetyl-16α-hydroxy-tuberculic acid, and pamoic acid in different yeast fermentation broths after 6 hours.

[0014] Figure 5 The present invention proposes a biosynthetic pathway for pachymic acid using CYP5035 as a substrate, through C-16 hydroxylation and C-20 methyl carboxylation. Detailed Implementation

[0015] The present invention will be further explained below with reference to specific embodiments.

[0016] Example 1

[0017] Construction based on pESC-Trp vector plasmid transformation system

[0018] (1) Enzyme cleavage site

[0019] like Figure 1As shown, the PcCPR gene is inserted after the GAL10 promoter of the pESC-Trp vector plasmid via ECORⅠ and ClaⅠ, and the CYP5035 gene is inserted after the GAL1 promoter of the pESC-Trp vector plasmid via BamHⅠ and salⅠ restriction sites.

[0020] The nucleotide sequence of PcCPR is shown in SEQ ID NO.2; the nucleotide sequence of the GAL10 promoter is shown in SEQ ID NO.3; the nucleotide sequence of the GAL1 promoter is shown in SEQ ID NO.4; the nucleotide sequence of the ECORⅠ restriction site is shown in SEQ ID NO.5; the nucleotide sequence of the ClaⅠ restriction site is shown in SEQ ID NO.6; the nucleotide sequence of the BamHI restriction site is shown in SEQ ID NO.7; and the nucleotide sequence of the salⅠ restriction site is shown in SEQ ID NO.8.

[0021] (2) Preservation of microbial strains

[0022] Top 10 bacterial strains were used.

[0023] Lyophilized plasmids -20℃ Glycerin bacteria -80℃

[0024] Since lyophilized plasmids are present in small quantities and easily lost upon opening, centrifugation should be performed before opening to ensure the plasmids settle at the bottom of the tube. After opening, add approximately 40 μL of TE buffer or ddH2O to dissolve the plasmids, mix thoroughly, and then centrifuge again to collect the liquid at the bottom of the tube. Both lyophilized and liquid plasmids should be stored at -20°C. The storage concentration can be adjusted according to the experiment, but should be stored at the highest possible concentration. Glycerol-containing bacteria can be stored long-term at -80°C; repeated freeze-thaw cycles should be avoided.

[0025] (3) Expand training

[0026] It is not recommended to directly inoculate and culture large quantities of glycerol bacteria before use. You can first use an inoculation needle to pick up glycerol bacteria and streak it on a plate with the corresponding antibody, or you can directly spread glycerol bacteria on a plate for recovery, and then pick normally growing colonies the next day for inoculation and expansion culture. Glycerol bacteria are very easy to contaminate after opening, so aseptic operation is required during use to avoid cross-contamination.

[0027] Example 2

[0028] Plasmid transformation and validation

[0029] (1) Preparation of competent states

[0030] (a) Preparation of BT15 competent cells

[0031] Streak BT15 culture (stored at -80℃) on SD-Leu agar plates and incubate at 28℃ for 2-4 days. After 2-4 days, pick a single colony and inoculate it into 3 ml of SD-Leu liquid medium, incubate overnight at 28℃ and 200 rpm; when the OD600 reaches 0.4-0.5, centrifuge at 3000 rpm for 5 min and discard the supernatant. Resuspend the precipitate in 1 ml of Y1 solution (coolaber kit), centrifuge at 3000 rpm for 5 min and discard the supernatant. Resuspend in 100 μl of Y2 (coolaber kit) solution and aliquot into 1.5 ml tubes at 50 μl each.

[0032] (b) Preparation of BT15 competent cells containing pESC-Trp plasmid

[0033] Streak the BT15 bacterial strain containing the pESC-Trp plasmid (stored at -80℃) on SD-Trp-Leu agar plates and incubate at 28℃ for 2-4 days. After 2-4 days, pick a single colony and inoculate it into 3 ml of SD-Trp-Leu liquid medium, incubate overnight at 28℃ and 200 rpm; when the OD600 reaches 0.4-0.5, centrifuge at 3000 rpm for 5 min and discard the supernatant. Resuspend the precipitate in 1 ml of Y1 solution (Coolaber kit), centrifuge at 3000 rpm for 5 min and discard the supernatant. Resuspend in 100 μl of Y2 solution (Coolaber kit) and aliquot into 1.5 ml tubes at 50 μl each.

[0034] (2) Plasmid transformation

[0035] (a) pESC-Trp conversion

[0036] Add 350 μL of Y3 solution (Coolaber kit) and 10 μL of pESC-Trp vector plasmid to 50 μL of BT15 competent cells. Repeatedly pipette the pellet to completely suspend the yeast cells in the premix. Heat shock in a 30°C water bath for 60 min, mixing every 10 min. Centrifuge at 12000 rpm for 15 s and discard the supernatant. Resuspend the cells in 400 μL of sterile deionized water, plate on SD-Trp-Leu selection medium plates, and incubate at 28°C for 2–4 days.

[0037] (b) pESC-Trp-PcCPR conversion

[0038] Add 350 μL of Y3 solution (Coolaber kit) and 10 μL of pESC-Ura vector plasmid carrying the key gene PcCPR to 50 μL of BT15 competent cells containing pESC-Trp plasmid. Repeatedly pipette the pellet to completely suspend the yeast cells in the premix. Heat shock in a 30°C water bath for 60 min, mixing every 10 min. Centrifuge at 12000 rpm for 15 s and discard the supernatant. Resuspend the cells in 400 μL of sterile deionized water, plate on SD-Trp-Ura-Leu selection medium plates, and incubate at 28°C for 2–4 days.

[0039] (c) pESC-Trp-CYP5035 conversion

[0040] Add 350 μL of Y3 solution (Coolaber kit) and 10 μL of pESC-Trp vector plasmid carrying the key gene CYP5035 to 50 μL of BT15 competent cells. Repeatedly pipette the pellet to completely suspend the yeast cells in the premix. Heat shock in a 30°C water bath for 60 min, mixing every 10 min. Centrifuge at 12000 rpm for 15 s and discard the supernatant. Resuspend the cells in 400 μL of sterile deionized water, plate on SD-Trp-Leu selection medium plates, and incubate at 28°C for 2–4 days.

[0041] (d)pESC-Trp-CYP5035-PcCPR conversion

[0042] Add 350 μL of Y3 solution (Coolaber kit) and 10 μL of pESC-Trp vector plasmid carrying the key genes CYP5035 and PcCPR to 50 μL of BT15 competent cells. Repeatedly pipette the pellet to completely suspend the yeast cells in the premix. Heat shock in a 30°C water bath for 60 min, mixing every 10 min. Centrifuge at 12000 rpm for 15 s and discard the supernatant. Resuspend the cells in 400 μL of sterile deionized water, plate on SD-Trp-Leu selection medium plates, and incubate at 28°C for 2–4 days.

[0043] (e) Culture medium

[0044] SD-Leu medium (100ml): SD-Leu 0.8g, glucose 2g, sterilize separately at 121℃ for 20min, cool after sterilization and mix well, store at room temperature, add 2% agar to solid medium.

[0045] SD-Trp-Leu medium (100ml): SD-Trp-Leu 0.8g, glucose 2g, sterilize separately at 121℃ for 20min, cool after sterilization and mix well, store at room temperature, add 2% agar to solid medium.

[0046] SD-Trp-Ura-Leu medium (100ml): SD-Trp-Ura-Leu 0.8g, glucose 2g, sterilize separately at 121℃ for 20min, cool after sterilization and mix well, store at room temperature, add 2% agar to solid medium.

[0047] Figure 2 The image shows a yeast plate containing the genes pESC-Trp, pESC-Trp-PcCPR, pESC-Trp-CYP5035, and pESC-Trp-CYP5035-PcCPR proposed in this invention. The four yeasts are white, round, moist, viscous, easy to pick up, and have a smooth surface.

[0048] (3) Validation of key gene transformation

[0049] (a) Extraction of yeast plasmids

[0050] Select a single colony from the selection medium and inoculate it into 3 ml of the corresponding liquid medium. Incubate overnight at 28°C and 200 rpm. Centrifuge at 5000 rpm for 5 min to collect the cells; discard the supernatant, add 480 μL Buffer SE, 4.8 μL β-mercaptoethanol, and 20 μL lyticase solution (OMEGA kit), vortex to resuspend the cells, and incubate at 30°C for at least 30 min; centrifuge at 5000 rpm for 5 min, discard the supernatant, add 250 μL Buffer YPⅠ / RNase A (OMEGA kit) and 50 mg glass beads, vortex at maximum speed for 5 min, and after the glass beads settle, transfer the supernatant; add 250 μL YPⅡ (OMEGA kit), gently invert and mix 4-6 times until the lysis buffer becomes clear, incubate at room temperature for 20 min, then add 350 μL YPⅢ (OMEGA kit), gently invert and mix several times until a white flocculent precipitate appears, centrifuge at 10000 rpm for 1 min at room temperature; transfer the supernatant to HiBindDNAmini. Centrifuge the HiBind DNA mini column (pre-inserted into a 2ml collection tube) at 10,000 rpm for 1 min at room temperature and discard the filtrate. Insert the HiBind DNA mini column back into the 2ml collection tube, add 500 μL of Buffer HBC (OMEGA kit), centrifuge at 10,000 rpm for 1 min, and discard the filtrate. Insert the HiBind DNA mini column back into the 2ml collection tube, add 700 μL of DNAWash Buffer (OMEGA kit), centrifuge at 10,000 rpm for 1 min, and discard the filtrate. Repeat twice. Centrifuge the empty column at 13,000 rpm for 2 min to dry the column matrix. Insert the HiBind DNA mini column into a new 1.5ml centrifuge tube, add 50 μL of sterile deionized water to the column, and centrifuge at 10,000 rpm for 1 min to elute the DNA.

[0051] (b) PCR verification of key genes

[0052] The extracted yeast plasmids were amplified by PCR using T5colony.

[0053] The PCR reaction system is as follows:

[0054]

[0055] PCR conditions for CYP5035 and PcCPR gene amplification and verification:

[0056]

[0057]

[0058] The PCR primers for amplification and verification are as follows:

[0059]

[0060] Figure 3 This is a PCR verification diagram of the gene inserted into the vector proposed in this invention; M, marker ladder; 1, CYP5035 primer PCR detection of pESC-Trp; 2, CYP5035 primer PCR detection of pESC-Trp-PcCPR; 3, CYP5035 primer PCR detection of pESC-Trp-CYP5035; 4, primer CYP5035 PCR detection of pESC-Trp-CYP5035-PcCPR; 5, primer PcCPR PCR of pESC-Trp; 6, primer PcCPR PCR detection of pESC-Trp-PcCPR; 7, primer PcCPR PCR detection of pESC-Trp-CYP5035; 8, primer PcCPR PCR detection of pESC-Trp-CYP5035-PcCPR showed that the CYP5035 band was 1683 bp and the PcCPR band was 2211 bp. Results indicated that pESC-Trp did not contain the genes CYP5035 and PcCPR, pESC-Trp-PcCPR contained the gene PcCPR but not CYP5035, pESC-Trp-CYP5035 contained the gene CYP5035 but not PcCPR, and pESC-Trp-CYP5035-PcCPR contained both the genes CYP5035 and PcCPR. The bands for each gene were of consistent size and bright.

[0061] Example 3

[0062] Fermentation produces pachymic acid

[0063] (1) Making fermentation liquid

[0064] (a) pESC-Trp, pESC-Trp-CYP5035, pESC-Trp-CYP5035-PcCPR

[0065] A single colony was picked from the SD-Trp-Leu selection medium and inoculated into 3 ml of SD-Trp-Leu liquid medium (2% glucose added). The colonies were incubated overnight at 28°C and 200 rpm on a shaker. The colonies were then transferred to 100 ml of SD-Trp-Leu liquid medium (2% glucose added) for further culture and incubated overnight. The colonies were then aliquoted into 50 ml centrifuge tubes, centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of sterile water. The precipitate was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the colonies were washed twice with water. The centrifuged colonies were then inoculated into 100 ml of SD-Trp-Leu liquid medium (2% raffinose and galactose added) for fermentation. Samples were taken at 6 h and 24 h.

[0066] (b)pESC-Trp-PcCPR

[0067] A single colony was picked from the SD-Trp-Ura-Leu selection medium and inoculated into 3 ml of SD-Trp-Ura-Leu liquid medium (2% glucose added). The culture was incubated overnight at 28°C and 200 rpm on a shaker. The colony was then expanded by inoculating into 100 ml of SD-Trp-Ura-Leu liquid medium (2% glucose added) and incubated overnight. The colony was aliquoted into 50 ml centrifuge tubes, centrifuged at 5000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in sterile water. The colony was centrifuged at 5000 rpm for 5 min, the supernatant was discarded, and the colony was washed twice with water. The centrifuged colony was then inoculated into 100 ml of SD-Trp-Ura-Leu liquid medium (2% raffinose and galactose added) for fermentation. Samples were taken at 6 h and 24 h.

[0068] (c) Culture medium

[0069] SD-Trp-Leu medium (100ml, glucose 2%): SD-Trp-Leu 0.8g, glucose 2g, sterilize separately at 121℃ for 20min, cool after sterilization, mix well, and store at room temperature.

[0070] SD-Trp-Leu medium (100ml, raffinose and galactose are both 2%): SD-Trp-Leu 0.8g, raffinose and galactose 2g each, SD-Trp-Leu (with 80ml sterile water) and 20ml sterile water are sterilized at 121℃ for 20min. After sterilization and cooling, raffinose and galactose are directly added to sterile water to dissolve, filtered to remove bacteria, mixed with SD-Trp-Leu, and stored at room temperature.

[0071] SD-Trp-Ura-Leu medium (100ml, 2% glucose): SD-Trp-Ura-Leu 0.8g, glucose 2g, sterilize separately at 121℃ for 20min, cool after sterilization, mix well, and store at room temperature.

[0072] SD-Trp-Ura-Leu medium (100ml, raffinose and galactose are both 2%): SD-Trp-Ura-Leu 0.8g, raffinose and galactose 2g each, SD-Trp-Ura-Leu (with 80ml sterile water) and 20ml sterile water are sterilized at 121℃ for 20min. After sterilization and cooling, raffinose and galactose are directly added to sterile water to dissolve, filtered to remove bacteria, and mixed with SD-Trp-Ura-Leu. Store at room temperature.

[0073] (2) Sample processing

[0074] Fermentation lasted 6 hours, with sampling every 24 hours. 50 ml samples were taken each time, with 3 ml reserved for OD (600 nm) value measurement. The remaining sample was centrifuged at 5000 rpm for 10 minutes, and the supernatant was transferred to a 50 ml flask. 20 ml of ethyl acetate was added to the supernatant for extraction. After separation, the upper layer was transferred to an evaporating dish and evaporated to dryness in a 100°C water bath. After drying, it was allowed to cool slightly, and 1 ml of anhydrous ethanol was added to dissolve the precipitate. The precipitate was then transferred to a LC-MS vial using a 1 ml syringe. 4 ml of anhydrous ethanol was added to the precipitate, and the mixture was sonicated for 20 minutes. After standing for a while, a portion of the supernatant was transferred to a LC-MS vial using a 1 ml syringe.

[0075] (3) Liquid mass

[0076] The liquid chromatography-mass spectrometry (LC-MS) conditions are as follows:

[0077]

[0078]

[0079] Figure 4 This invention proposes a liquid chromatography-mass spectrometry (LC-MS) assay for the content of 16α-hydroxy- ... Comparison of the contents of 16α-hydroxy- ...

[0080] Example 4

[0081] BT15 fermentation scale-up culture increases pachymic acid yield

[0082] As shown in Example 3, the yeast fermentation broth containing the pESC-Trp-CYP5035-PcCPR gene exhibited the highest pamoic acid yield at 6 hours. Therefore, the fermentation broth was expanded to 100 L for an increased pamoic acid yield. All other operations were performed as in Example 3. Pamoic acid was then separated using liquid chromatography, yielding a pamoic acid content as high as 17.632 ug / L.

[0083] Figure 5 The proposed pathway for the biosynthesis of pachymic acid using CYP5035 as a substrate involves C-16 hydroxylation and C-20 methyl carboxylation. Under the regulation of the CYP5035 gene, lanosterol is converted into 16-Hydroxytrametenolic acid, which is then converted into pachymic acid under the regulation of the SMT1 (ERG6) gene.

[0084] The above embodiments are merely examples of several implementations of the present invention and should not be construed as limiting the scope of the invention patent. Sequences and applications that have the same function based on this gene sequence are also protected by this patent.

Claims

1. A key gene for the synthesis of pachymic acid, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

1.

2. A carrier, characterized in that, It contains the key gene for pamoate synthesis as described in claim 1.

3. A microorganism, characterized in that, It includes the carrier as described in claim 2.

4. The application of the microorganisms described in claim 3 in the synthesis of pachymic acid.

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