(22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol and preparation method thereof

Abstract

The present invention provides a (22R, 23R)-22,23-dihydroxy-7-dehydrogenated campesterol and a preparation method thereof, and (22R, 23R)-22,23-dihydroxy-7-dehydrogenated campesterol ((22R, 23R)-22,23-diOH-Δ 7 ‑campesterol) as shown in the structural formula (I), (22R, 23R)‑22,23‑dihydroxy‑7‑dehydrocampesterol can be used as a chemical synthesis precursor for brassinosteroids. The present invention also provides a method for preparing the new compound shown in the structural formula (I) and a strain for synthesizing the new compound. The obtained strain can produce a high yield of the compound. #imgabs0#

Description

Technical Field

[0001] The present invention relates to (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol and a preparation method thereof. The present invention obtains a recombinant fungal strain producing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol in fungi by gene editing, and relates to the technical field of synthetic biology and the field of enzyme engineering. Background Art

[0002] Brassinosteroids (BRs) are the sixth class of plant hormones and play a vital role in plant growth processes such as seed germination, root growth, and reproduction. In addition, they can also coordinate with other plant hormones to regulate plant oxidative free radical metabolism, ethylene synthesis, and root gravity response, thereby improving plant resistance to harsh environments such as drought, high salt, and extreme temperatures. As the most biologically active compound among BRs, brassinolide (BL, also known as brassinolide) ( Figure 1 ) has great application potential. However, due to the extremely low content of BL in plants and the lack of precursor substances that can be supplied on a large scale, it has not been mass-produced. So far, two BRs have been widely used in agriculture, namely 24-epi-brassinolide (EBL) and 28-homo-brassinolide (HBL) ( Figure 1 ), and its semi-synthetic precursors are ergosterol (TCMcmorris, et al, J Org Chem, 1993 (58), 2338) ( Figure 2 ) and stigmasterol (TCMcmorris, et al, Phytochemistry, 1994 (36), 585). The applicant previously compared the side chain structures of BL and EBL and proposed, based on retrosynthetic analysis, that 24-epi-ergosterol can be used as a precursor for the semi-synthesis of BL, and constructed recombinant Saccharomyces cerevisiae to achieve large-scale preparation of 24-epi-ergosterol, laying the foundation for the industrial production of BL (patent application number: CN2022115062236; Y. Jiang, et al, Nat Commun, 2023 (14), 437).

[0003] In the semi-synthetic process of BL and its analogs, the most critical step is the double bond dihydroxylation reaction catalyzed by rare platinum group metal (such as osmium and ruthenium) oxides (such as OsO4-catalyzed Sharpless asymmetric dihydroxylation, Figure 3). The study found that in the chemical synthesis of 24-epi-BL and 28-homo-BL, the Δ 2 The dihydroxylation of α-hydroxyl groups usually only forms 2α,3α-dihydroxyl groups, while the side chain Δ 22 Two configurations are formed: 22R, 23R-dihydroxy and 22S, 23S-dihydroxy. The ratio between the two configurations depends mainly on the type of catalyst, the stereostructure of the substrate C-24 and the type of chiral ligand. Among them, the OsO4-catalyzed Sharpless asymmetric dihydroxylation reaction can more efficiently obtain the 22R, 23R-dihydroxy product (JARSalvador, et al, Curr Org Chem, 2008 (12): 492-522). In agricultural applications, compared with BRs with 22R, 23R-dihydroxy, the biological activity of BRs with 22S, 23S-dihydroxy is significantly reduced, or even completely lost (SLAcebedo, et al, Steroids, 2009 (74), 435-440). In order to obtain BRs (such as BL) with a 22R,23R-dihydroxy structure in high yield, highly toxic OsO4 and expensive chiral ligands are required during the chemical synthesis process, which is costly and dangerous, limiting the commercial application of highly active BRs. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a compound (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol represented by structural formula (I) and a preparation method thereof.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] The present invention first provides a compound represented by structural formula (I): (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol (((22R, 23R)-22,23-diOH-Δ 7 -campesterol)):

[0007]

[0008] The present invention further provides a method for constructing a fungus for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, wherein plant-derived C-22 sterol hydroxylase, C-23 sterol hydroxylase and cytochrome P450 reductase genes are recombinantly expressed in 24-epi-ergosterol-producing fungal cells to construct a strain for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0009] As a preferred embodiment of the present invention, the 24-epi-ergosterol-producing fungus is an ergosterol-producing eukaryotic microorganism or an ERG5 gene knockout mutant thereof, preferably a eukaryotic microorganism of the families Saccharomycetaceae, Sclerotiniaceae, Cladosporiaceae, Hypocreaceae, Trichocomaceae, Aspergillaceae, Tricholomataceae or an ERG5 gene knockout mutant of the above eukaryotic microorganisms, particularly preferably Saccharomyces cerevisiae with the ERG5 gene knocked out.

[0010] Furthermore, the C-22 sterol hydroxylase, C-23 sterol hydroxylase and cytochrome P450 reductase genes are derived from plants producing brassinolide compounds, preferably from plants of the Lamiaceae family, the Cruciferae family and the Moraceae family, preferably from plants with a high content of brassinolide compounds.

[0011] Furthermore, the C-22 sterol hydroxylase gene is a gene encoding the amino acid sequence shown in SEQ ID NO.1; the C-23 sterol hydroxylase gene is a gene encoding the amino acid sequence shown in SEQ ID NO.2; and the cytochrome P450 reductase gene is a gene encoding the amino acid sequence shown in SEQ ID NO.3.

[0012] Furthermore, the plant-derived C-22 sterol hydroxylase, C-23 sterol hydroxylase and cytochrome P450 reductase genes are derived from Arabidopsis thaliana (At).

[0013] Furthermore, the fungus producing 24-epi-ergosterol is Saccharomyces cerevisiae.

[0014] The present invention also provides a fungus for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, and the strain is constructed by the above-mentioned construction method.

[0015] The present invention also provides the use of the above-mentioned fungus for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol in the production of (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0016] Furthermore, the fungus constructed by the aforementioned method is inoculated into a seed culture medium to prepare a seed solution; the seed solution is transferred to a fermentation medium, fermented and cultured (20-200 hours), the cultured cells are collected, a saponification reaction solution is added, and after the reaction is completed, petroleum ether is added for extraction to obtain (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The present invention is based on the 24-epi-ergosterol synthesis yeast strain, and the sterol side chain is dihydroxylated to synthesize (22R,23R)-22,23-dihydroxy-7-dehydrogenated campesterol (((22R,23R)-22,23-diOH-Δ 7 -campesterol)) as a precursor for BL chemical synthesis, while RuCl3 and NaIO4 were used to catalyze the Δ 2 The dihydroxylation of OsO4 and chiral ligands can be completely avoided. Figure 4 ), significantly reducing the synthesis cost of BL and effectively ensuring the green, environmentally friendly and safe production process.

[0019] The present invention recombinantly expresses plant-derived C-22 sterol hydroxylase, C-23 sterol hydroxylase and cytochrome P450 reductase genes into 24-epi-ergosterol-producing fungal cells ( Figure 5 ), and a strain synthesizing (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol was constructed. So far, there is no report on (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol in the prior art, nor is there any report on the application of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol as a precursor for the chemical synthesis of BRs.

[0020] The strain YQD630 constructed by the present invention can produce 100.06 mg / L of (22R, 23R)-22, 23-dihydroxy-7-dehydrocampesterol through high-density fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The diagrams are the structures of brassinolide, 24-epi-brassinolide, and 28-homo-brassinolide.

[0022] Figure 2 The present invention is a synthetic route for synthesizing 24-epi-brassinolide using ergosterol as a precursor.

[0023] Figure 3This is a Sharpless asymmetric dihydroxylation reaction in the synthesis of 24-epi-brassinolide using ergosterol as a precursor.

[0024] Figure 4 The invention provides a synthetic route for chemically synthesizing brassinolide using (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol as a precursor.

[0025] Figure 5 This is an artificial synthesis pathway for (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol in Saccharomyces cerevisiae.

[0026] Figure 6 pRS42H-P GAL2 Plasmid maps.

[0027] Figure 7 pRS42H-P GAL2 -CPR1 plasmid map.

[0028] Figure 8 pRS42H-P GAL2 -DWF4 plasmid map.

[0029] Fig. 9 This is the plasmid map of pEB-15-D4D1.

[0030] Fig.10 Liquid phase-UV detection analysis diagram of the starting strain, the recombinant bacteria introduced with C-22 sterol hydroxylase, and the recombinant bacteria introduced with C-22 sterol hydroxylase and C-23 sterol hydroxylase.

[0031] Fig.11 This is the liquid chromatography-mass spectrometry analysis of the YQD630 shake flask fermentation product.

[0032] Fig.12 This is the high-density fermentation diagram of YQD630. DETAILED DESCRIPTION

[0033] The present invention is further described and illustrated below in conjunction with specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0034] The culture medium of the present invention is as follows:

[0035] (1) YPD medium (1% yeast powder, 2% peptone, 2% glucose), solid medium with 2% agar powder added, sterilized at 115°C, for activation and pre-culture of Saccharomyces cerevisiae.

[0036] (2) YPD (HygB / G418) medium (1% yeast powder, 2% peptone, 2% glucose, 100 μg / mL HygB, 200 μg / mL G418), solid medium with 2% agar powder added, sterilized at 115°C, for screening KanMX markers.

[0037] (3) LB medium (1% sodium chloride, 1% tryptone, 0.5% yeast powder), solid medium with 2% agar powder added, sterilized at 121°C, used for activation and pre-culture of E. coli.

[0038] (4) LB (Amp) medium (1% sodium chloride, 1% tryptone, 0.5% yeast powder, 100 μg / mL ampicillin), solid medium with 2% agar powder added, sterilized at 121°C, for culturing Escherichia coli containing plasmids.

[0039] The strain object targeted by the construction method of the present invention can be any fungus that produces 24-epi-ergosterol. Typically, Saccharomyces cerevisiae can be selected. In this embodiment, YQE729 is used as the main experimental object, but other fungi that produce 24-epi-ergosterol can also be selected. Industrial Saccharomyces cerevisiae CICC1746 was purchased from the China Industrial Microbiological Culture Collection Administration Center. YQE729 is based on CICC1746 as the starting strain and is constructed by the method in CN115927436A or the method in the reference (Y. Jiang, et al, Nat Commun, 2023 (14), 437). Strain YQE729 is deposited in the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), and the strain collection number is CCTCC NO: M 2023539, and the collection date is April 17, 2023.

[0040] The plasmid pRS42H-P used in the present invention GAL2 Spectrum Figure 6 Shown

[0041] The plasmid pRS42H-P used in the present invention GAL2 -CPR1 spectrum Figure 7 As shown;

[0042] The plasmid pRS42H-P used in the present invention GAL2 -DWF4 graphs such as Figure 8 As shown;

[0043] The plasmid pEB-15-D4D1 used in the present invention is shown in the following figure: Fig. 9 As shown;

[0044] Example 1: Construction of ERG5 knockout strain YQC512

[0045] The genome of industrial Saccharomyces cerevisiae CICC1746 was used as a template. The primers erg5Δ-UF (SEQ ID NO.4) / erg5Δ-UR (SEQ ID NO.5) were used to perform PCR amplification to obtain the 482 bp homology arm fragment erg5Δ-U upstream of the ERG5 site. The primers erg5Δ-DF (SEQ ID NO.6) / erg5Δ-DR (SEQ ID NO.7) were used to amplify the 491 bp homology arm fragment erg5Δ-D downstream of the ERG5 site. The two amplified fragments were connected by overlap extension PCR to obtain the Kerg5 fragment. The Kerg5 fragment was transformed into YQE729 by lithium acetate / PEG3350 chemical transformation method. The CRISPR-Cas9 gene editing tool was used to perform double-strand break cleavage of DNA at the ERG5 site under the guidance of ERG5-gRNA (SEQ ID NO.8). The DNA fragment was then purified by a PCR product containing 100 μg / mL Hyg B and 200 μg / mL The positive clones were screened in YPD medium containing G418 to obtain YQC512 with ERG5 site knockout.

[0046] Example 2: Construction of Saccharomyces cerevisiae strain YQC433 expressing cytochrome P450 reductase gene

[0047] Using the plasmid containing the cytochrome P450 reductase gene AtCPR1 from Arabidopsis thaliana synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. as a template, the CPR1 fragment was amplified by PCR using primers VG2-CPR1-F (SEQ ID NO.9) / VG2-CPR1-R (SEQ ID NO.10); the CPR1 fragment was ligated to the linearized (SalI and BamHI double-digested) pRS42H-P by seamless cloning. GAL2 Plasmid pRS42H-P GAL2 -CPR1; plasmid pRS42H-P GAL2 -CPR1 was used as template and primer Dor.-16Ty1-P with 50 bp homology arm was used. TEF1 (SEQ ID NO.11) / Dor.-16Ty1-P ADH2 (SEQ ID NO.12) was amplified by PCR to obtain the CPR1 expression cassette; the CPR1 expression cassette was transformed into YQC512 using a lithium acetate / PEG3350 chemical transformation method; using the CRISPR-Cas9 gene editing tool, the 16Ty1 site was double-stranded broken and cut DNA under the guidance of 16Ty1-gRNA (SEQ ID NO.13), and the cerevisiae yeast strain YQC433 expressing the cytochrome P450 reductase gene was constructed.

[0048] Example 4: Construction of recombinant bacteria YQD610 with C-22 sterol hydroxylase

[0049] The plasmid AtDWF4 containing the C-22 sterol hydroxylase gene from Arabidopsis thaliana synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. was used as a template to amplify the DWF4 fragment by PCR using primers VG2-DWF4-F (SEQ ID NO.14) / VG2-DWF4-R (SEQ ID NO.15); the DWF4 fragment was ligated to the linearized (double-digested with SalI and BamHI) pRS42H-P by seamless cloning. GAL2 Plasmid pRS42H-P GAL2 -DWF4; plasmid pRS42H-P GAL2 -DWF4 was used as template and primer Dor.-15Ty2-P with 50 bp homology arm was used. TEF1 (SEQ ID NO.16) / Dor.-15Ty2-T ADH2 (SEQ ID NO.17) was amplified by PCR to obtain the DWF4 expression cassette; the DWF4 expression cassette was transformed into YQC433 using the lithium acetate / PEG3350 chemical transformation method; the CRISPR-Cas9 gene editing tool was used to perform double-strand break cleavage of the 15Ty2 site under the guidance of 15Ty2-gRNA (SEQ ID NO.18), and the DNA was integrated into the Saccharomyces cerevisiae genome, coated on HygB / G418 plates, and positive clone strains were screened to construct the recombinant bacteria YQD610 introduced with C-22 sterol hydroxylase.

[0050] Example 4: Construction of recombinant bacteria YQD630 with C-22 sterol hydroxylase and C-23 sterol hydroxylase

[0051] The plasmid pEB-15-D4D1 used to construct a strain co-expressing C-22 sterol hydroxylase gene and C-23 sterol hydroxylase gene is constructed as follows:

[0052] The plasmid AtDWF4 containing the C-22 sterol hydroxylase gene from Arabidopsis thaliana synthesized by Shanghai Biotechnology Co., Ltd. was used as a template. GAL7 -DWF4 (SEQ ID NO. 19) / PT MFA1-DWF4 (SEQ ID NO.20) was amplified by PCR to obtain the DWF4 fragment; the plasmid At90D1 containing the C-23 sterol hydroxylase gene from Arabidopsis thaliana synthesized by Sangon Biotechnology (Shanghai) Co., Ltd. was used as a template, and the primers PP GAL2 -90D1 (SEQ ID NO. 21) / PT PGK1 -90D1 (SEQ ID NO.22) was amplified by PCR to obtain the 90D1 fragment; the genome of Saccharomyces cerevisiae CICC1746 was used as a template and primers P-pEB-T MFA1 (SEQ ID NO.23) and P-DWF4-T MFA1 (SEQ ID NO.24) was amplified to obtain T MFA1 fragment, using primers P-DWF4-P GAL7 (SEQ ID NO.25) and PP GAL2 -P GAL7 (SEQ ID NO.26) was amplified to obtain P GAL7 fragment, through primer PP GAL7 -P GAL2 (SEQ ID NO. 27) and P-90D1-P GAL2 (SEQ ID NO.28) was amplified to obtain P GAL2 fragment, using primer P-90D1-T PGK1 (SEQ ID NO.29) and P-pEB-T PGK1 (SEQ ID NO.30) was amplified to obtain T PGK1 Fragments with homologous sequences were fused using overlap extension PCR and Simple Cloning Vectors Kit was used to construct pEB-15-D4D1.

[0053] The recombinant bacteria YQD630 introduced with C-22 sterol hydroxylase and C-23 sterol hydroxylase was constructed, and the construction steps were as follows:

[0054] Plasmid pEB-15-D4D1 was used as template and primer Dor.-erg5site-T with 50 bp homology arm was used. MFA1 (SEQ ID NO.31) / Dor.-erg5site-T CYC1(SEQ ID NO.32) was amplified by PCR to obtain the DWF4 and 90D1 expression cassettes; the DWF4 and 90D1 expression cassettes were transformed into the cerevisiae strain YQC433 using the lithium acetate / PEG3350 chemical transformation method; the CRISPR-Cas9 gene editing tool was used to perform double-strand break cleavage of the ERG5 knockout site under the guidance of erg5site-gRNA (SEQ ID NO.33), and the DNA was integrated into the cerevisiae genome, coated on HygB / G418 plates, and positive clone strains were screened to obtain YQD630.

[0055] Example 5: Fermentation, post-treatment and liquid phase detection

[0056] The successfully edited transformants YQC433, YQD610 and YQD630 were streaked on YPD plates, cultured at 30°C for 48 h, and single clones were picked to inoculate 5 mL of YPD liquid culture medium and cultured at 30°C and 220 rpm for 96 h. After the culture was completed, 500 μL of fermentation broth was collected, centrifuged to remove the supernatant, and 600 μL of ethanol-KOH solution (25% [w / v] KOH in 50% ethanol) was added. After sufficient vortexing, the solution was saponified in a boiling water bath for 1 h, cooled to room temperature, and 400 μL of water and 800 μL of petroleum ether were added. After sufficient vortexing, 500 μL of the upper petroleum ether extract was vacuum-dry, 500 μL of anhydrous ethanol was added to re-dissolve, and filtered through a 0.22 μm PVDF filter membrane into the insert tube in the liquid phase detection bottle as the detection sample.

[0057] The metabolites in the samples were analyzed by Agilent1160 high performance liquid chromatography to analyze the changes in the sterol composition of YQC512, YQD610 and YQD630 samples. The chromatographic column model was Thermo C-18 column (ODS Hypersil, 4.6×250 mm, 5μm), the detection wavelength was 280nm, the column temperature was 30℃, and the gradient mobile phase was: 90% methanol, 20min; 100% methanol, 10min; 90% methanol, 6min.

[0058] The YQD630 sample was analyzed by fast high performance liquid chromatography-quadrupole time of flight (Q-TOF) mass spectrometry (LC-MS), the chromatographic column was Thermo C-18column (ODS Hypersil, 4.6×250mm, 5μm) reverse phase column, the ion source was atmospheric pressure chemical ion source (APCI), and the mobile phase was 90% methanol. The mobile phase flow rate was 0.8mL / min, the column temperature was 30℃, the positive ion mode, and the m / z range was 100-1500.

[0059] The results of liquid phase detection showed that ( Fig.10), compared with YQC512 (no exogenous hydroxylase was introduced), YQD610 (introduced C-22 sterol hydroxylase) and YQD630 (introduced C-22 sterol hydroxylase and C-23 sterol hydroxylase) both produced new substances, and compared with YQD610, YQD630 had a new peak at around 6.7min, which was speculated to be the target substance (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol. The YQD630 sample was then analyzed by LC-MS ( Fig.11 ), and the product was determined to be the target product (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol ((22R,23R)-22,23-diOH-Δ 7 -campesterol, 22,23-diOH-Δ 7 -CR).

[0060] Example 6: Fermentation verification of recombinant strain YQD630 in a fermenter

[0061] A single colony was inoculated into 5 mL YPD medium, cultured at 30°C and 220 rpm for 24 h, and inoculated into two 50 mL YPD shake flasks at a 2% inoculum (v / v). After culturing for 20 h, the culture broth in the two shake flasks was inoculated into a 2 L bioreactor (the fermentation tank medium formula was 10 g / L D-glucose, 10 g / L (NH4)2SO4, 8 g / L KH2PO4, 3 g / L MgSO4, 0.72 g / L ZnSO4·7H2O, 10 mL / L trace metal solution and 12 mL / L vitamin solution, and the initial fermentation volume after inoculation was 1 L).

[0062] Fermentation tank parameter setting: temperature was set to 30°C, ammonia was added to control pH to maintain at 5.0, and dissolved oxygen was maintained at >25% saturation by adjusting the stirring rate (300 rpm to 950 rpm) and airflow rate (1 vvm to 3 vvm). After the carbon source in the initial culture medium was used up, a feed solution containing 500 g / L glucose and 12 mL / L vitamin solution was fed into the fermentation tank according to the pseudo-exponential feeding model; when the bacterial OD in the tank reached 600 When the fermentation temperature reaches about 200, anhydrous ethanol is added at a rate of 6 mL / h until the fermentation ends. The feed rate F in the pseudo-exponential feeding stage S Determined by the following equation:

[0063]

[0064] Where X0, V0 and S are the initial biomass density (gDCW / L), initial culture volume (L) and glucose concentration in the culture medium (g / L); Y X / Sis the productivity of cell biomass to glucose (gDCW / g glucose); μ is the specific growth rate (h -1 ); m is the maintenance factor (g glucose / gDCW / h), and t is the time after the start of feeding (h). The specific growth rate was set to 0.12h -1 , Y X / S Set to 0.5 and m to 0.05.

[0065] After fermentation, HPLC-UV was used to detect the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol in YQD630 strain. Since there was no standard substance for this product, and the detection wavelength was set at 280nm, the main detection was the sterol with Δ 5,7 The side chain groups of sterols basically do not affect the response value, so the solution prepared with ergosterol standard was used to prepare the standard curve for quantitative analysis. The test results showed that the yield of (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol was 100.60mg / L ( Fig.12 ).

[0066] Example 7: Synthesis of brassinolide using (22R,23R)-22,23-dihydroxy-7-dehydrocampesterol as substrate

[0067] The overall reaction route is Figure 4 As shown, (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol (486 mg, 1.13 mmol) was dissolved in 14 mL tetrahydrofuran, 2,2-dimethoxypropane (1.55 mL, 12.6 mmol) and p-toluenesulfonic acid monohydrate (43.7 mg, 0.23 mmol) were added, and the mixture was reacted at 25°C for 2 h. Water (25 mL) and ethyl acetate (10 mL) were added, and the organic phase was separated. The aqueous phase was extracted with ethyl acetate (10 mL × 2). The organic phases were combined and rotary evaporated to obtain a crude product with side chain dihydroxy protection. The crude product was dissolved in 5 mL pyridine, cooled to 0°C, p-toluenesulfonyl chloride (430 mg, 2.26 mmol) was slowly added, and the reaction was stirred at below 15°C for 3 to 4 h. After the reaction was completed, ice water (20 mL) was slowly added to precipitate a large amount of solids, which were filtered, washed with water, and dried to obtain the sulfonylated product.

[0068] Potassium acetate (490 mg, 4.99 mmol) was dissolved in 3.5 mL of water, and acetone (15 mL) was added. The temperature was raised to reflux, and then the sulfonylated product was added to the above reflux reaction in batches. After the addition was completed, the reflux reaction was continued for 8 to 10 hours. After the reaction was completed, the acetone was evaporated, and then the reaction solution was cooled to room temperature. Ice brine was added to the reaction solution under stirring to precipitate solids. After filtration, washing and drying, the solids were dissolved in acetone (10 mL), cooled to 0°C, and Jones reagent (0.5 mL) was slowly added dropwise. When the reaction solution turned slightly red, isopropanol (0.5 mL) was added to terminate the reaction. Chloroform (10 mL) and water (10 mL) were added, the organic phase was separated, and the aqueous phase was extracted with chloroform (5 mL×2). The organic phases were combined and rotary evaporated to obtain a crude oxidation product.

[0069] Place the reaction bottle in a -70℃ container (dry ice plus acetone), pass ammonia gas, liquefy into liquid ammonia, collect about 5mL of liquid ammonia. Add metal lithium sheet (0.175mg, 25mmol), dissolve, drop the crude oxidation product dissolved in tetrahydrofuran into the reaction bottle, react for 0.5h, add anhydrous ammonium chloride solid until the blue disappears, stop the reaction, take it out from the low temperature device and place it, evaporate the liquid ammonia until it is completely, add water, extract with ethyl acetate, wash the organic solvent layer with water twice, dry it with anhydrous sodium sulfate, rotary evaporate it, dissolve it in 6mL N,N-dimethylacetamide, add pyridine hydrobromide (0.06g, 0.375mmol) and lithium bromide (0.064g, 0.734mmol), heat it to 170℃, and react for 10-12h. After the reaction was completed, when the reaction temperature dropped to about 80°C, most of the solvent was removed by rotary evaporation, ice water (20 mL) and petroleum ether (10 mL) were added, the organic phase was separated, the aqueous phase was extracted twice with petroleum ether, the organic phases were combined, and after rotary evaporation, column chromatography separation and purification were performed to obtain the isomerized product (291 mg, 0.62 mmol).

[0070] The isomerized product (291 mg, 0.62 mmol) was dissolved in ethyl acetate (9 mL), acetone (4 mL), and acetonitrile (4 mL), and cooled to 3-5°C. A solution of RuCl3·3H2O (9.78 mg, 0.045 mmol) and NaIO4 (203 mg, 0.95 mmol) dissolved in water (0.8 mL) was added, and the reaction was stirred at 3-5°C for 3 min. A 20% sodium thiosulfate solution (10 mL) was added and stirred for 5 min to terminate the reaction. After all the organic solvents were removed by rotary evaporation, ethyl acetate was used for extraction (3×30 mL). The organic phases were combined, the organic solvent layer was washed twice with water, dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain a crude ring dihydroxylation product. The crude ring dihydroxylation product was dissolved in a mixture of acetic acid (10 mL) and water (3 mL), reacted at 60 ° C for 1 h, and then cooled to room temperature, the solvent was removed by rotary evaporation, and purified by column chromatography (chloroform / isopropanol = 10 / 1-4 / 1) to obtain brassinosteroid ketone (182 mg, 0.392 mmol).

[0071] Chloroform (4 mL) and trifluoroacetic anhydride (2.77 mL, 1.96 mol) were added to a reaction flask, cooled to below 0°C, 30% hydrogen peroxide solution (0.45 mL, 3.92 mmol) was slowly added dropwise, stirred for 0.5 h after the addition, and brassinolide (182 mg, 0.392 mmol) dissolved in chloroform (11 mL) was added dropwise, the temperature was controlled to be below 15°C and reacted for 2 h, and then neutralized with potassium carbonate aqueous solution, washed with water, layered, extracted with chloroform, dried, and the solvent was removed by rotary evaporation to obtain a crude product of brassinolide. The crude product was recrystallized from ethyl acetate to obtain brassinolide (121 mg, 0.252 mmol).

[0072] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and cannot be construed as limitations on the present invention. A person skilled in the art may change and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol, characterized in that Its structural formula is shown in formula (I):

2. Use of the (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol according to claim 1 in the preparation of brassinosteroid compounds.

3. A method for preparing the compound of structural formula (I) according to claim 1, characterized in that: Inoculating a fungus that synthesizes (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol into a seed culture medium to prepare a seed solution; The seed liquid is transferred to a fermentation medium for fermentation culture; the cultured cells are collected and added to a saponification reaction solution for saponification reaction; after the reaction, petroleum ether is added for extraction to obtain (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol; The fungus for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol is constructed by the following method: the plant-derived C-22 sterol hydroxylase gene, the C-23 sterol hydroxylase gene and the cytochrome P450 reductase gene are recombinantly expressed into the strain YQC512 to construct a fungal strain for synthesizing (22R, 23R)-22,23-dihydroxy-7-dehydrocampesterol; the strain YQC512 is the strain YQE729 with the ERG5 site knocked out; wherein, the strain YQE729 is deposited in the China Center for Type Culture Collection, the strain deposition number is CCTCC M2023539, and the deposition date is April 17, 2023.

4. The preparation method according to claim 3, characterized in that: The C-22 sterol hydroxylase, C-23 sterol hydroxylase and cytochrome P450 reductase genes are derived from plants producing brassinolide compounds.

5. The preparation method according to claim 3, characterized in that: The C-22 sterol hydroxylase, C-23 sterol hydroxylase and cytochrome P450 reductase genes are derived from plants of the Lamiaceae, Cruciferae and Moraceae families.

6. The preparation method according to claim 3, characterized in that: The C-22 sterol hydroxylase gene is a gene encoding the amino acid sequence shown in SEQ ID NO.1; the C-23 sterol hydroxylase gene is a gene encoding the amino acid sequence shown in SEQ ID NO.2; and the cytochrome P450 reductase gene is a gene encoding the amino acid sequence shown in SEQ ID NO.3.

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