Method for synthesizing dehydroepiandrosterone through chemical-multienzyme coupling catalysis
By utilizing a chemical-multi-enzyme coupled catalytic synthesis route and optimizing reaction conditions with specific enzyme systems, the problems of lengthy and environmentally polluting dehydroepiandrosterone (DHEA) synthesis routes have been solved, achieving efficient and green DHEA synthesis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGXI BAISIKANGRUI PHARMA
- Filing Date
- 2026-01-15
- Publication Date
- 2026-05-05
AI Technical Summary
Existing technologies for the synthesis of dehydroepiandrosterone (DHEA) involve lengthy routes, highly corrosive reagents, and severe environmental pollution, lacking efficient and green synthetic methods.
A chemical-multi-enzyme coupled catalytic synthesis route was adopted, which involved esterification, hydrolysis by lipases, and synergistic catalysis by ketone reductase and glucose dehydrogenase. Specific enzyme systems such as lipase Pp-lipase, ketone reductase Ss-SDR mutant, and glucose dehydrogenase Es-GDH were used to optimize reaction conditions in order to improve the synthesis efficiency of DHEA.
This method achieves high-yield synthesis of DHEA, reduces synthesis steps, avoids the use of toxic reagents, improves reaction efficiency and product optical purity, and constructs a more environmentally friendly synthesis process.
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Figure CN121975899A_ABST
Abstract
Description
Technical Field
[0001] This application relates to a method for the chemical-multi-enzyme coupled catalytic synthesis of the raw material dehydroepiandrosterone (DHEA), belonging to the field of pharmaceutical formulation technology with specific therapeutic activity. Background Technology
[0002] Dehydroepiandrosterone (DHEA), a raw material for the synthesis of abiraterone, mainly relies on semi-synthesis from plant-based sources such as diosgenin. This route is lengthy, involves highly corrosive reagents, and causes significant environmental pollution. With the increasing emphasis on green synthesis, the chemical-enzymatic synthesis route has emerged and become a current research hotspot. The core of this route lies in integrating the advantages of chemical synthesis and biocatalysis: typically, a core is constructed first through esterification, and then enzymes with high regioselectivity and stereoselectivity (lipases, ketone reductases, glucose dehydrogenases, etc.) are used to catalyze key reactions such as hydroxylation, dehydrogenation, or side-chain modification at specific sites. This method aims to significantly reduce synthetic steps, avoid the use of toxic reagents, and improve reaction efficiency and product optical purity, thereby constructing a more atom-economical and environmentally friendly synthetic process. In-depth research on this route not only has significant industrial value for achieving the green manufacturing of DHEA but also provides technical guidance for the biosynthesis of other complex drugs. However, current research mainly focuses on the synthetic routes or applications of abiraterone, with relatively little research on the synthesis of DHEA. Summary of the Invention
[0003] In view of this, this application provides a method for the chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone (DHEA), which directly synthesizes DHEA through chemical reaction-multi-enzyme coupling.
[0004] Specifically, this application is implemented through the following scheme: A method for the chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone (DHEA) comprises the following steps: Step 1: Using 4-androstenedione (4-AD) as the initial substrate, 3-acetyloxy-androsta-3,5-dien-17-one was synthesized by esterification. Step 2: Add lipase to hydrolyze and catalyze the synthesis of 5-androstenedione (5-AD) from 3-acetoxyandrost-3,5-dien-17-one. Step 3: Add ketone reductase and glucose dehydrogenase, and the two enzymes are coupled and synergistically catalyze the synthesis of dehydroepiandrosterone (DHEA) from 5-androstenedione.
[0005] Furthermore, as a preferred option: In step one, In the esterification synthesis conversion system, the concentration of 4-androstenedione is 10~100 g / L.
[0006] The esterification synthesis conversion system incorporates p-toluenesulfonic acid and acetic anhydride for hydrolysis and esterification. The concentration of toluenesulfonic acid in the conversion system is 10–100 g / L, and the concentration of acetic anhydride is 43.2–432 g / L. Both toluenesulfonic acid and acetic anhydride participate in the reaction and regulate the system polarity. The resulting composite catalytic system achieves highly efficient activation of the substrate hydroxyl site through significant synergistic effects: p-toluenesulfonic acid promotes protonation to form an oxonium ion intermediate, while acetic anhydride provides an acetyl group and captures the generated water molecule, shifting the reaction equilibrium towards esterification. This catalytic strategy significantly improves the selective acylation efficiency of the 3-hydroxyl group and suppresses other competing side reactions, such as rearrangement or over-acylation. Under optimized conditions, the reaction proceeds directionally, ultimately achieving a high yield of the key intermediate 3-acetoxyandrost-3,5-dien-17-one, which has important applications in steroid drug synthesis. This method is not only mild and easy to operate, but also has a significant catalytic synergistic effect, providing an efficient and specific synthetic route for the esterification modification of steroids with similar structures.
[0007] Step one can also be specifically set as follows: Under nitrogen protection, in a dry reaction flask, add 4-AD, acetic anhydride, and p-toluenesulfonic acid. Heat the reaction mixture to a certain temperature (e.g., 40~50℃) and reflux with stirring at this temperature for several hours. The reaction time needs to be monitored by TLC until the 4-AD starting material is essentially eliminated. After the reaction is complete, cool the reaction solution to room temperature or cool in an ice bath. Slowly pour the reaction solution into ice water or a saturated sodium bicarbonate (NaHCO3) solution for quenching. Perform multiple extractions using the organic solvent ethyl acetate, and combine the organic phases. Wash the organic phase successively with a saturated sodium bicarbonate solution (to neutralize residual acid), water, and saturated brine. After drying with anhydrous sodium sulfate or magnesium sulfate, filter, concentrate under reduced pressure to obtain the crude product. The crude product is usually purified by recrystallization from ethanol to obtain high-purity 3-acetoxyandrost-3,5-dien-17-one.
[0008] In step two, The hydrolysis catalysis system has a pH of 5-10 (preferably 6-9) and a temperature of 15-60℃ (preferably 45-55℃). Preferably, the hydrolysis catalysis conversion system contains any one of the following as a reaction medium: phosphate-citric acid buffer (pH approximately 5-6), phosphate buffer (pH approximately 6-8), Tris / HCl buffer (pH approximately 7.5-9), or glycine-NaOH buffer (pH approximately 9-10), with phosphate buffer and Tris / HCl buffer being preferred.
[0009] The concentration of the 3-acetoxyandrost-3,5-diene-17-one in the conversion system is 10~50 g / L.
[0010] The lipolytic enzyme is a lipolytic enzyme. Pa -lipase or lipolytic enzyme Ps lipase, lipolytic enzyme Pl lipase, lipolytic enzyme Pp lipase, lipolytic enzyme Pg lipase and its corresponding expression plasmid or genetically engineered bacteria, lipohydrolase Pa -lipase or lipolytic enzyme Ps lipase, lipolytic enzyme Pl lipase, lipolytic enzyme Pp lipase, lipolytic enzyme Pg The nucleotide sequences of -lipase are SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively. It is a lipolytic enzyme. Pp -lipase (from) Pseudomonas paraeruginosa () is the better option.
[0011] The above-mentioned lipohydrolases Pp -lipase can also be replaced with expressing the lipolytic enzyme. Pp -lipase-mediated genetically engineered bacterial wet cells expressing lipolytic enzymes Pp -lipase, cell lysate of genetically engineered bacterial wet cells, or the lipohydrolase Pp -lipase is a pure enzyme solution separated from the wet cells of genetically engineered bacteria after cell disruption and protein purification.
[0012] When the lipolytic enzyme is expressed Pp When adding lipase-in the form of wet bacterial cells, the dosage is 6 g DCW / L based on the dry weight of the wet bacterial cells (DCW: dry weight of cells, L: volume of the transformation system).
[0013] Step two can also be specifically set as follows: using the product 3-acetoxyandros-3,5-dien-17-one as a substrate, add a lipohydrolase. Pp Engineered bacterial cells containing the -lipase gene were used as a catalyst for the hydrolytic synthesis of 5-AD. The reaction solution was analyzed by HPLC after the reaction was terminated to determine the amount of 5-AD produced. The obtained product was extracted with ethyl acetate at 50°C, and the concentrate was dissolved in methanol and then cooled for crystallization to obtain crude 5-AD.
[0014] In step three, The pH of the dual-enzyme coupled synergistic catalytic transformation system is 6-7, and the temperature is 30-40℃. Preferably, PBS buffer is added as the reaction medium in the dual-enzyme coupled synergistic catalytic transformation system.
[0015] The solvent of the dual-enzyme coupled synergistic catalytic conversion system is any one of methanol, ethanol, isopropanol, n-hexane, dimethyl sulfoxide (DMSO), and 2-methyltetrahydrofuran, with methanol, dimethyl sulfoxide, and 2-methyltetrahydrofuran being preferred.
[0016] In the dual-enzyme coupled synergistic catalytic conversion system, the concentration of 5-androstenedione in the conversion system is 10~100g / L.
[0017] The dual-enzyme coupled synergistic catalytic transformation system also contains NADP. + (Nicotinamide Adenine Dinucleotide Phosphate) and glucose, NADP + As a co-substrate, glucose is NADP. + NADP, a recycled substrate + The concentration of NADP in the conversion system is 0.5–3.5 mM (mmol / L), with 2.0–3.5 mM being the preferred concentration. The concentration of glucose in the conversion system is 50–200 g / L, and its molar ratio to 5-AD in the reaction is 0.5–2:1. During the reaction, NADP… + It is converted into NADPH, while glucose is converted into gluconic acid.
[0018] The ketone reductase is an SDR family NAD(P)-dependent oxidoreductase. Rh (recorded as) Rh -SDR), SDR family NAD(P)-dependent oxidoreductases Sj (recorded as) Sj -SDR), SDR family NAD(P)-dependent oxidoreductases Ss (recorded as) Ss -SDR) or SDR family NAD(P)-dependent oxidoreductases Sw (recorded as) Sw -SDR), the corresponding nucleotide sequences are SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, and SEQ ID NO.19, respectively.
[0019] The ketone reductase is Ss -SDR mutant, this mutant is Ss -SDR is obtained by mutation at at least one of the following sites in its amino acid sequence: (1) The phenylalanine at position 26 is replaced with alanine (denoted as ) Ss -SDR-F26A), (2) Arginine at position 106 is replaced with serine (denoted as...) Ss -SDR-R106S), (3) the tryptophan at position 157 is replaced with glycine (denoted as...). Ss -SDR-W157G), (4) Tyrosine at position 249 is replaced with serine (denoted as...) Ss -SDR-Y249S). The optimal mutant is the combination of mutations at positions 26 and 249, denoted as . Ss -SDR-F26A / Y249S (named) Ss -SDR-M2).
[0020] The glucose dehydrogenase is glucose dehydrogenase. Es -GDH and its corresponding expression plasmids or genetically engineered bacteria, glucose dehydrogenase Es The nucleotide sequence of -GDH is SEQ ID NO.11.
[0021] The glucose dehydrogenase Es -GDH to express the glucose dehydrogenase Es - Add the genetically engineered GDH bacteria in the form of wet cells, at a rate of 1–5 g DCW / L (transformation system) based on the dry weight of the wet cells.
[0022] The SDR family of NAD(P)-dependent oxidoreductases SsMutants to express the SDR family NAD(P)-dependent oxidoreductases Ss The mutant genetically engineered bacteria are added in the form of wet bacterial cells, at a rate of 5–10 g DCW / L (transformation system) based on the dry weight of the wet bacterial cells.
[0023] glucose dehydrogenase Es -GDH can express the glucose dehydrogenase. Es -GDH genetically engineered bacterial wet cells expressing glucose dehydrogenase Es -GDH genetically engineered bacterial cell lysate or glucose dehydrogenase Es -Pure enzyme solution isolated from the wet cells of GDH genetically engineered bacteria after cell disruption and protein purification; SDR family NAD(P)-dependent oxidoreductases Ss The mutant is a wet cell of a genetically engineered bacterium expressing the mutant, a cell lysate of a wet cell of a genetically engineered bacterium expressing the mutant, or a pure enzyme solution separated from a wet cell of a genetically engineered bacterium expressing the mutant after cell lysis and protein purification.
[0024] Step three can also be specifically set as follows: separately culturing and obtaining ketone reductases. Ss -SDR mutant and glucose dehydrogenase Es Engineered bacterial cells containing the GDH genes were used, and then a mixture of the two bacteria was used as a catalyst and 5-AD as a substrate to synthesize DHEA via dual-enzyme catalysis. The amount of DHEA produced was detected by liquid chromatography after the reaction was terminated.
[0025] This application uses 4-androstenedione as the initial substrate and further catalyzes the process through the coupling of three enzymes: lipase, ketone reductase, and glucose dehydrogenase. First, the three enzyme pathways were screened and applied from NCBI, and the optimal catalyst for the lipase was determined to be derived from... Pseudomonas paraeruginosa Lip hydrolase ( Pp -lipase), through sequence screening and enzyme compatibility analysis, was selected to obtain the optimal catalytic performance, derived from Sphingopyxis sp. DBS4 ketone reductase ( Ss -SDR) and the corresponding glucose dehydrogenase required for catalytic function ( Es -GDH). Analysis using a halved enzyme dosage experiment determined that the key enzyme in the third step of the multi-enzyme cascade was ketone reductase. Then, through rational design, the enzyme was molecularly modified by site-directed mutagenesis of amino acid residues near the enzyme-substrate pocket. Since 5-androstenedione is a large side-chain substrate, the enzyme was modified according to the principle of increasing the substrate pocket size. Mutant strains with 325.3–427.6% higher enzyme activity than the original strain were screened. Ss -SDR-F26A Ss -SDR-R106S Ss-SDR-W157G and Ss -SDR-Y249S. A mutant strain with 503.1% increased enzyme activity was obtained through combined mutations. Ss -SDR-F26A / Y249S, the optimal mutant was applied to the synthesis of abiraterone intermediate (DHEA) by multi-enzyme cascade catalysis. The yield of DHEA was further improved by optimizing reaction conditions, ensuring constant reaction pH through pH titration, and using multi-enzyme synergistic immobilization. Attached Figure Description
[0026] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application.
[0027] Figure 1 This is a schematic diagram of the synthesis process of this application.
[0028] Figure 2 This is an SDS-PAGE electrophoresis image of the supernatant of the three enzymes in this application. Figure 2 The A in the formula is lipase. Pp SDS-PAGE electrophoresis image of lipase supernatant, lane M: standard protein molecule marker; lane 1: Pa -lipase; Lane 2: Pl -lipase; Lane 3: Pg -lipase; Lane 4: Pp -lipase.
[0029] Figure 2 B in the formula stands for glucose dehydrogenase. Es -GDH and ketone reductase Ss - SDS-PAGE electrophoresis image of SDR supernatant, lane 5: Ps -lipase; Lane 6: Es -GDH; Lane 7: Ss -SDR; Lane 8: Rh -SDR; Lane 9: Sj -SDR; Lane 10: Sw -SDR.
[0030] Figure 3 The effect of lipohydrolase on the synthesis of 5-AD from the substrate 3-acetoxyandrost-3,5-dien-17-one. Figure 3In the diagram, A represents the TLC chromatogram, where 1 represents the reaction sample without lipase, 2 represents the reaction sample containing lipase, and the control refers to the standards containing three compounds (3-acetoxyandrosten-3,5-dien-17-one, 4-AD, 5-AD). Figure 3 In the figure, B represents the hydrolysis yield of lipases from different sources, and the horizontal axis represents: Pa -lipase refers to lipolytic enzymes Pa -lipase, Ps -lipase refers to lipolytic enzymes Ps -lipase, Pl -lipase refers to lipolytic enzymes Pl -lipase, Pp -lipase refers to lipolytic enzymes Pp -lipase, Pg -lipase refers to lipolytic enzymes Pg -lipase.
[0031] Figure 4 To illustrate the synthesis effects of 5-AD under different reaction conditions, Figure 4 In this diagram, A represents the effect of different pH values on the yield of 5-AD catalytic synthesis. Figure 4 B in the figure represents the effect of different temperatures on the yield of 5-AD catalytic synthesis.
[0032] Figure 5 The relative enzyme activities of ketone reductases from different sources. In the x-axis, Rh -SDR finger ketone reductase Rh , Sj -SDR finger ketone reductase Sj , Ss -SDR finger ketone reductase Ss , Sw -SDR finger ketone reductase Sw .
[0033] Figure 6 The synthesis process of DHEA, Figure 6 In this equation, A represents the reaction of DHEA synthesized via two-cell coupling catalysis of ketone reductase and glucose dehydrogenase. Figure 6 B in the formula is ketone reductase. Ss -SDR and its mutants Ss Yield of DHEA synthesized by SDR-M2 catalysis.
[0034] Figure 7 To test the effects of different organic solvents on ketone reductase SsThe effect of -SDR-M2 enzyme activity.
[0035] Figure 8 The effect of adjuvants on DHEA yield, Figure 8 A in this context stands for NADP. + Effect of concentration on DHEA yield Figure 8 In this context, B represents the effect of the molar ratio of glucose to 5-AD on the yield of DHEA.
[0036] Figure 9 The effect of two enzymes on DHEA synthesis. Figure 9 In this context, A represents the reaction process catalyzed by the two-cell immobilization of ketone reductase and glucose dehydrogenase to synthesize DHEA. Figure 9 In this context, B represents the yield of DHEA in different batches of immobilized cells. Detailed Implementation
[0037] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit the technical solutions of this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without creative effort are within the scope of protection of this application.
[0038] Example 1
[0039] In this embodiment, an esterification reaction was performed to synthesize 3-acetoxyandrost-3,5-diene-17-one.
[0040] In exploring esterification methods for 4-androstenediene-3,17-dione (4-AD), we compared three different esterification reaction systems: conventional sulfuric acid-catalyzed esterification, organic base-catalyzed esterification, and p-benzenesulfonic acid / acetic anhydride esterification. Experimental results showed that the p-benzenesulfonic acid / acetic anhydride esterification method yielded the highest yield (details are shown in Table 1).
[0041] Under nitrogen protection, the esterification reaction of 4-AD proceeds consisted of 100 g / L 4-AD, 100 g / L p-toluenesulfonic acid, and 400 mL acetic anhydride, mixed thoroughly in a reactor and stirred at 40°C to synthesize 3-acetoxyandrost-3,5-dien-17-one. During the reaction, small samples of the reaction solution were periodically taken for identification using thin-layer chromatography (TLC). By comparing the chromatographic behavior with that of a standard sample under the same developing conditions, the esterification process of the reactants was precisely tracked, and the reaction termination time was determined accordingly to ensure optimal reaction outcome.
[0042] After the reaction was completed, the reaction products were post-processed and then analyzed by high-performance liquid chromatography (HPLC). HPLC results showed that this step had good reaction efficiency, with a final catalytic yield as high as 94.6%, fully demonstrating the superiority of the p-benzenesulfonic acid / acetic anhydride esterification method in the 4-AD esterification reaction.
[0043] Table 1: Effect of different esterification conditions on esterification yield .
[0044] Example 2
[0045] In this embodiment, 5-AD is synthesized by hydrolysis catalysis catalyzed by lipohydrolase.
[0046] Sequences of lipases, a superfamily of lipases known to hydrolyze large steroid acetate substrates, were used as probes for homology searches in the National Center for Biotechnology Information (NCBI) protein database. The search strategy focused on identifying homologous enzymes with sequence similarity within a specific range (30–80%) to ensure that the obtained enzymes possessed both functional relevance and structural novelty. After screening the preliminary results, a focus was placed on candidate enzymes whose substrate-binding pockets might accommodate large steroid side chains, and five representative ketone reductases were selected from these candidates. Pa -lipase ( Pseudomonas aeruginosa (GenBank: OHQ70763.1, amino acid sequence see SEQ ID NO.2) Ps -lipase ( Pseudomonas simiae (GenBank: AJP51387.1, amino acid sequence see SEQ ID NO.4) Pl -lipase ( Pseudomonas lurida (GenBank: PRC23824.1, amino acid sequence see SEQ ID NO.6) Pp -lipase ( Pseudomonas paraeruginosa (GenBank: PHJ32035.1, amino acid sequence see SEQ ID NO.8) and Pg -lipase ( Pseudomonas gelidaquae (GenBank: OZO05934.1, amino acid sequence shown in SEQ ID NO.10). Codon optimization was performed using *E. coli* as the host (optimized nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9), and the nucleotides were synthesized by Qingke Biotechnology Co., Ltd. Pa -lipase, Ps-lipase, Pl -lipase, Pp -lipase and Pg The cDNA fragments of the lipase were ligated into the pET-28a(+) plasmid after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site), respectively, to obtain the plasmid pET28a(+)- Pa -lipase, pET28a (+) - Ps -lipase, pET28a (+) - Pl -lipase, pET28a (+) - Pp -lipase and pET28a (+) - Pg -lipase. The five plasmids were respectively transformed into... E. coli In BL21(DE3), strains were obtained E. coli BL21(DE3) / pET28a(+)- Pa -lipase, E. coli BL21(DE3) / pET28a(+)- Ps -lipase, E. coli BL21(DE3) / pET28a(+)- Pl -lipase, E. coli BL21(DE3) / pET28a(+)- Pp -lipase and E. coli BL21(DE3) / pET28a(+)- Pg -lipase. The above-mentioned ketone reductase-containing strains were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h; 1% (v / v) was inoculated into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. Then, IPTG was added to the culture medium to a final concentration of 0.1 mM, and the culture was incubated at 28°C for 12 h. After centrifugation at 4°C and 9000 xg for 10 min, the corresponding wet bacterial cells were obtained. The expression of the five lipase proteins is as follows: Figure 2 As shown in A in the diagram.
[0047] The buffer system and reaction temperature for the catalytic reaction were optimized, and the results are as follows: Figure 4As shown, after optimizing the catalytic reaction conditions, phosphate-citric acid buffer, phosphate buffer, Tris / HCl buffer, and glycine-NaOH buffer were added as reaction media to screen the catalytic activity of the bacteria under different pH conditions. Then, the catalytic activity of the bacteria was screened at different temperatures: 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 °C. 50 g / L of the substrate 3-acetoxyandro-3,5-dien-17-one was added, and the catalyst dosage was 6 g DCW / L based on the total dry weight of the lipase cells. A conversion system was constructed using lipase as a catalyst, 3-acetoxyandrost-3,5-dien-17-one as a substrate, and a 100 mM Tris / HCl buffer solution at pH 8.0 as the reaction medium. The reaction was carried out at 50 °C and 800 rpm. After the reaction was terminated, liquid chromatography was performed to detect the amount of 5-AD produced to calculate the product yield. Figure 3 As shown. The results indicate that: originating from Pseudomonas paraeruginosa Lip hydrolase Pp -lipase produced the highest yield of 5-AD among four different ketone reductases, with a yield of 93.6% for 5-AD synthesis.
[0048] Example 3
[0049] This embodiment focuses on the discovery and screening of genetically engineered bacteria containing ketone reductase.
[0050] Using sequences of known short-chain dehydrogenases / reductases (SDRs) superfamily members capable of catalyzing large ketone substrates such as steroids as probes, a homology search was performed in the National Center for Biotechnology Information (NCBI) protein database. The search strategy focused on discovering homologous enzymes with sequence similarity within a specific range (30–80%) to ensure that the obtained enzymes possess both functional relevance and structural novelty. After screening the preliminary results, a focus was placed on candidate enzymes whose substrate-binding pockets might accommodate large steroid side chains, and four representative ketone reductases were selected from these candidates. Rh -SDR ( Rhizorhabdus histidinilytica (GenBank: WP_209046370.1, amino acid sequence see SEQ ID NO.14) Sj -SDR ( Sphingomonas jatrophae (GenBank: WP_093313358.1, amino acid sequence see SEQ ID NO.16) Ss -SDR ( Sphingopyxis sp.DBS4 (GenBank: WP_257549357.1, amino acid sequence see SEQ ID NO.18) and Sw -SDR ( Sphingopyxis witflariensis (GenBank: WP_088473921.1, amino acid sequence shown in SEQ ID NO. 20). Codon optimization was performed using *E. coli* as the host (optimized nucleotide sequences are shown in SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 17, and SEQ ID NO. 19), and the nucleotides were synthesized by Qingke Biotechnology Co., Ltd. Rh -SDR、 Sj -SDR、 Ss -SDR and Sw The -SDR cDNA fragments were ligated into the pET-28a(+) plasmid after TATACCAT (before the NcoI restriction site) and before CTCGAG (XhoI restriction site), respectively, to obtain the plasmid pET28a(+)- Rh -SDR, pET28a(+)- Sj -SDR, pET28a(+)- Ss -SDR and pET28a (+)- Sw -SDR. The four plasmids were transferred into... E. coli In BL21(DE3), strains were obtained E. coli BL21(DE3) / pET28a(+)- Rh -SDR、 E. coli BL21(DE3) / pET28a(+)- Sj -SDR、 E. coli BL21(DE3) / pET28a(+)- Ss -SDR and E. coli BL21(DE3) / pET28a(+)- Sw -SDR. The above-mentioned ketone reductase-containing strains were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h. They were then inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. IPTG was then added to the culture medium to a final concentration of 0.1 mM and cultured at 28°C for 12 h. Finally, the cultures were centrifuged at 4°C and 9000 xg for 10 min to obtain the corresponding wet bacterial cells.
[0051] 1) Screening of bacterial catalytic activity: 10 g / L of substrate 5-AD was added, and the catalyst dosage was 4 g DCW / L based on the total dry weight of the mixed bacterial cells. Ss -SDR: Es -GDH ratio is 5:1. Es The nucleotide sequence of -GDH is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12. (Source: [Original Source Name]) Exiguobacterium sp. UBA6282, GenBank: WP_290773133.1). Using ketone reductase and glucose dehydrogenase as catalysts, and 5-AD as substrate, NADP... + As a co-substrate, glucose is NADP. + The recovered substrate (glucose to gluconic acid) was converted using a transformation system constructed with pH 7.0, 50 mM PBS buffer as the reaction medium. The reaction was carried out at 30°C and 800 rpm. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to detect the amount of DHEA produced, and the amount of product was calculated to determine the enzyme activity of SDR. Figure 5 As shown.
[0052] The results show that: originating from Sphingopyxis sp. DBS4 ketone reductase Ss -SDR exhibits the highest enzyme activity for DHEA production among four different ketone reductases, making it the preferred choice. Ss -SDR further modifies the chassis cells to improve the efficiency of DHEA synthesis.
[0053] 2) Liquid Chromatography (HPLC) Conditions: Samples were analyzed using a Kromasil ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, Waters) and a UVD170U detector at 260 nm. The mobile phase consisted of 0.1% (v / v) formic acid aqueous solution (A) and methanol (B), using isocratic elution mode, A:B = 1:9. Column temperature: 35℃; flow rate: 0.6 mL / min; injection volume: 10 μL. The retention time of 4-AD was 10.5 min, and the standard curve was y = 2248.52x - 180.41. The retention time of DHEA was 11.8 min, and the standard curve was y = 1767.63x - 167.52. The retention time of 5-AD was 14.8 min, and the standard curve was y = 1235.58x - 127.43.
[0054] Example 4
[0055] This embodiment describes the construction and screening of a ketone reductase mutant library.
[0056] Based on the substrate pocket reduction strategy of dual-substrate molecules, the preparation of the ketone reductase mutant library was achieved through site-directed mutagenesis, and the primer design is shown in Table 2. E. coliBL21(DE3) / pET28a(+)- Ss -SDR carrier pET28a(+)- Ss Using -SDR as a template, and F26A-F and F26A-R as the 26th amino acid mutation primers in Table 2, site-directed mutagenesis PCR was performed to induce ketoreductase... Ss The phenylalanine at position 26 of the -SDR amino acid sequence was mutated to alanine, and the resulting monoclonal mutant was obtained. Ss -SDR-F26A. Using the R106S-F and R106S-R primers from Table 2 as the 106th amino acid mutants, site-directed mutagenesis PCR was performed to remove the ketoreductase. Ss The arginine at position 106 of the SDR amino acid sequence is mutated to serine, and the resulting monoclonal mutant ketone reductase is obtained. 、Ss -SDR-R106S. Using W157G-F and W157G-R as the mutant primers for amino acid 157 in Table 2, site-directed mutagenesis PCR was performed to remove the ketoreductase. Ss The tryptophan at position 157 of the SDR amino acid sequence was mutated to glycine, and the resulting monoclonal mutant was transformed. Ss -SDR-W157G. Using Y249S-F and Y249S-R as the mutant primers for amino acid 249 in Table 2, site-directed mutagenesis PCR was performed to remove ketoreductase. Ss The tyrosine residue at position 249 of the SDR amino acid sequence was mutated to serine, and the resulting monoclonal mutant was transformed. Ss -SDR-Y249S.
[0057] PCR reaction system (50 µL): 2 µL forward primer (10 μM), 2 µL reverse primer (10 μM), 25 µL 2×Phanta buffer, 1 µL dNTP mixture (10 mM each), 1 µL plasmid template, 1 µL DNA polymerase, and 18 µL ultrapure water. The PCR program set according to the Phanta Super-Fidelity DNA polymerase instructions was as follows: 95℃ pre-denaturation for 5 min, followed by 29 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 5 min), final extension at 72℃ for 10 min, and incubation at 16℃. The resulting recombinant plasmid was transformed into DNA using a 42℃ heat shock method. E. coliBL21(DE3) competent cells were evenly spread on LB agar plates containing 50 µg / µL kanamycin and incubated at 37°C for 14 h. Single colonies were picked and transferred to 1 mL of LB liquid medium containing 50 µg / mL kanamycin. After incubation at 37°C and 180 rpm for 10 h in 100 mL shake flasks, the cells were induced and then collected by centrifugation after incubation at 28°C for 12 h. The crude enzyme was prepared as follows: the cells were resuspended in 50 mM phosphate buffer (pH 7.5) at a concentration of 100 g / L and sonicated on an ice-water mixture for 6 min. The sonication conditions were: 400 W power, 1 s of sonication followed by a 2 s pause. The resulting mixture was collected to obtain the crude enzyme solution. The crude enzyme solution was purified by ammonium sulfate fractionation: 100 mL of culture supernatant was placed in an ice bath, and 50% saturated ammonium sulfate powder was added while stirring until the ammonium sulfate was completely precipitated. The mixture was then incubated on ice for 1 h, followed by centrifugation at 12000 rpm at 4°C for 10 min to obtain the primary precipitate of crude ketoreductase. To the obtained supernatant, 80% saturated ammonium sulfate powder was added while stirring in an ice bath until the ammonium sulfate was completely dissolved. The mixture was then incubated on ice for 2 h, followed by centrifugation at 12000 rpm at 4°C for 10 min. The resulting precipitate was dissolved in phosphate buffer (pH 7.0, 20 mM) to obtain purified ketoreductase. The collected purified enzyme was dialyzed overnight with 20 mM phosphate buffer (pH 7.0). All purification steps were performed at 4°C.
[0058] The yields of DHEA synthesized by each mutant were determined using the catalytic system described in Example 3. Ketoreductase Ss The specific enzyme activity assay system for the -SDR mutant was the same as in Example 3. The mutant enzyme catalyst was catalyzed with purified enzyme at a final concentration of 1 mg / mL. A transformation system was constructed using 100 mM PBS buffer at pH 7.0 as the reaction medium. The reaction was carried out at 30°C and 800 rpm for 3 h. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to detect the amount of DHEA produced, thus calculating the specific enzyme activity of the mutant.
[0059] Table 2: Ketoreductases Ss -SDR site-directed mutagenesis primer design .
[0060] Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 micromole of DHEA per minute at 30°C and pH 7.0. Specific enzyme activity is defined as the number of activity units per milligram of enzyme protein, U / mg.
[0061] Protein concentration was determined using a diquinoline carboxylic acid protein assay kit (Nanjing Kaiji Biotechnology Development Co., Ltd., Nanjing).
[0062] Ss The specific enzyme activity of -SDR was 89.52 U / mg. Through rational design and screening, four single mutants with increased enzyme activity were obtained, and the results are shown in Table 3. The strains with increased enzyme activity were... E. coli BL21(DE3) / pET28a(+)- Ss -SDR-F26A E. coli BL21(DE3) / pET28a(+)- Ss -SDR-R106S E. coli BL21(DE3) / pET28a(+)- Ss -SDR-W157G and E. coli BL21(DE3) / pET28a(+) -Ss -SDR-Y249S. During the catalytic synthesis of DHEA, Ss The specific enzyme activity of -SDR-F26A is 319.05 U / mg; Ss The specific enzyme activity of -SDR-R106S is 291.21 U / mg; Ss The specific enzyme activity of -SDR-W157G is 363.63 U / mg; Ss The specific enzyme activity of -SDR-Y249S is 382.79 U / mg (Table 3).
[0063] Table 3: Ss-SDR Its catalytic properties and specific enzyme activity of single mutants .
[0064] Example 5
[0065] This embodiment involves ketone reductase. Ss Construction and screening of combined mutant libraries of -SDR mutants.
[0066] Ketone reductase Ss The preparation of the -SDR mutant combinatorial mutant library was achieved through batch site-directed saturation mutagenesis. Primer design is shown in Table 3. E. coli BL21(DE3) / pET28a(+)- Ss -SDR carrier pET28a(+)- Ss Using SDR-F26A as a template, primers for sites 106, 157, and 249 in Table 2 were used for combined mutations. The PCR system and procedure were the same as in Example 4.
[0067] Ketone reductase was obtained through screening Ss-SDR combined mutant Ss -SDR-F26A / Y249S. It was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C for 10 h. Then, it was inoculated at a 1% (v / v) inoculation rate into fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180 rpm for 2 h. Next, 0.1 mM IPTG was added to the culture medium, and the culture was incubated at 28°C for 12 h. Finally, it was centrifuged at 4°C and 8000 xg for 10 min to obtain the corresponding wet bacterial cells. The specific enzyme activity and the yield of DHEA synthesized by each mutant were determined using the catalytic system described in Example 3. The results showed that the combined mutants... Ss -SDR-F26A / Y249S exhibits the highest specific enzyme activity and catalytic performance, with a specific enzyme activity of 455.38 U / mg and a catalytic synthesis yield of DHEA of 85.63% (Table 4).
[0068] Table 4: Ax Catalytic performance and specific enzyme activity of PGA and its combined mutants .
[0069] Example 6
[0070] This embodiment performs Ss -SDR's optimal mutant Ss -SDR-F26A / Y249S is used for screening optimal organic solvents for the catalytic synthesis of DHEA.
[0071] The ketone reductase prepared in Example 5 E. coli BL21(DE3) / pET28a(+)- Ss The SDR-F26A / Y249S mutant cells were used as catalysts. The calculation method for the catalytic performance of the mutant under different organic solvents was the same as in Example 3. Six different organic solvents were added: methanol, ethanol, isopropanol, n-hexane, dimethyl sulfoxide (DMSO), and 2-methyltetrahydrofuran, with a control group (no solvent added). Under the above co-solvents, 100 g / L of 5-AD was added to test the catalytic ability of the mutant cells. The catalyst dosage was 8 g DCW / L based on the total dry weight of the mixed cells. Ss -SDR: EsThe reaction was carried out at 30°C and 800 rpm for 24 h (GDH ratio 5:1). After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to detect the amount of DHEA produced in order to calculate the product yield and compare the effects of different co-solvents. Ss -SDR's optimal mutant Ss Catalytic performance of SDR-F26A / Y249S. Under different co-solvent conditions, Ss The yield of DHEA synthesized by SDR-F26A / Y249S catalysis is as follows: Figure 7 As shown.
[0072] The results showed that adding the co-solvent DMSO... Ss The highest yield was obtained by synthesizing DHEA using the SDR-F26A / Y249S catalysis.
[0073] Example 7
[0074] This embodiment describes a mutant. Ss -Optimization of the reaction substrate ratio for the synthesis of DHEA catalyzed by SDR-F26A / Y249S.
[0075] The ketone reductase mutant was obtained using the same method as in Example 5. Ss -SDR-F26A / Y249S engineered bacteria cells. Different NADP levels were investigated. + The yield of DHEA synthesized by mutants at different concentrations and substrate concentration ratios.
[0076] 1) Different NADPs + Effect of concentration on the reaction: Using 50 mM PBS buffer at pH 8.0 as the buffer system, 100 g / L of substrate 5-AD was added. The catalyst dosage was 8 g DCW / L based on the total dry weight of the mixed bacterial cells. Ss -SDR: Es (GDH ratio 5:1), add 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5 mM (i.e., mmol / L) NADP respectively. + The reaction was carried out at 50℃ and 800 rpm for 3 h. After the reaction was terminated, the reaction solution was analyzed by liquid phase detection to detect the amount of product generated in order to calculate the yield.
[0077] The results are as follows Figure 8 As shown in A: NADP + At a concentration of 3.0 mM, the mutant Ss The highest yield was obtained by synthesizing DHEA using the SDR-F26A / Y249S catalysis.
[0078] 2) Effect of different substrate concentration ratios on the reaction: Five different substrate gradients were set up (glucose / 5-AD molar ratios of 0.5:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, and 2:1). Using 50 mM PBS buffer at pH 8.0 as the buffer system, 100 g / L of substrate 5-AD and 3.0 mM NADP were added. + The catalyst dosage, based on the total dry weight of the mixed bacterial cells, is 8 gDCW / L. Ss -SDR: Es The reaction was carried out at 50°C and 800 rpm for 3 h (GDH ratio 5:1). After the reaction was terminated, the reaction solution was analyzed by liquid phase detection to determine the amount of product generated and to calculate the product yield.
[0079] The results are as follows Figure 8 As shown in B: When the glucose / 5-AD molar ratio is 1.4:1, the mutant... Ss The highest yield can be achieved by using SDR-F26A / Y249S catalytic synthesis of DHEA.
[0080] Example 8
[0081] This embodiment involves immobilization. Ss Catalytic reusability performance of SDR-F26A / Y249S was determined.
[0082] The ketone reductase mutant was obtained using the same method as in Example 5. Ss -SDR-F26A / Y249S engineered bacteria cells. The purified... Ss -SDR-F26A / Y249S and GDH were mixed at a mass ratio of 5:1 and co-immobilized on an epoxy resin support using an adsorption-crosslinking method to prepare a co-immobilized dual-enzyme preparation. Figure 9 (A) Using 50 mM PBS buffer (pH 8.0) as the buffer system, add 100 g / L of substrate 5-AD and 3.0 mM NADP. + The glucose / 5-AD molar ratio was 1.4:1, and the catalyst dosage was 15 g DCW / L. The reaction was carried out at 50℃ and 800 rpm for 3 h. After the reaction was terminated, the reaction solution was analyzed by liquid chromatography to detect the product formation. To investigate its reusability for industrial applications, after each batch of reaction, the immobilized enzyme was recovered by simple filtration, washed with phosphate buffer, and directly added to the next batch of fresh reaction solution for continuous batch reactions. A total of 10 batches were conducted. The results showed that even after the 7th batch of reaction, the co-immobilized dual-enzyme preparation could still maintain more than 90% of the initial catalytic efficiency. Figure 9 The B in the enzyme exhibits excellent operational stability and reusability, significantly reducing the cost of using the enzyme.
[0083] The above-described embodiments are merely illustrative of several feasible implementations of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the present invention, nor are the embodiments intended to limit the scope of protection in the claims of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the concept of the present invention. All equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.
Claims
1. A method for the chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone, characterized in that, The steps are as follows: Step 1: Using 4-androstenedione as a substrate, p-toluenesulfonic acid and acetic anhydride were added to synthesize 3-acetoxyandrost-3,5-dien-17-one by esterification. Step two: Add lipohydrolase to hydrolyze and catalyze the synthesis of 5-androstenedione from 3-acetoxyandrost-3,5-dien-17-one. The lipohydrolase is a lipohydrolase with the nucleotide sequence SEQ ID NO.
1. Pa -lipase, whose nucleotide sequence is SEQ ID NO.3, is a lipohydrolase. Ps -lipase, whose nucleotide sequence is SEQ ID NO.5, is a lipolytic enzyme. Pl -lipase, whose nucleotide sequence is SEQ ID NO.7, is a lipolytic enzyme. Pp -lipase, whose nucleotide sequence is SEQ ID NO.9, is a lipohydrolase. Pg -lipase and its corresponding expression plasmid or genetically engineered bacterial wet cells, expressing lipohydrolase Pp -lipase, cell lysate of genetically engineered bacterial wet cells, or the lipohydrolase Pp -lipase is a pure enzyme solution separated from the wet cells of genetically engineered bacteria after cell disruption and protein purification. Step 3: Add ketone reductase and glucose dehydrogenase; the two enzymes are coupled to catalyze the synthesis of dehydroepiandrosterone from 5-androstenedione. The glucose dehydrogenase is the glucose dehydrogenase with the nucleotide sequence SEQ ID NO.
11. Es -GDH, its corresponding expression plasmid, cell lysate of its genetically engineered bacteria, or the glucose dehydrogenase. Es -Pure enzyme solution isolated from the wet cells of GDH genetically engineered bacteria after cell disruption and protein purification; The ketone reductase is the one with the nucleotide sequence SEQ ID NO.
13. Rh -SDR, SEQ ID NO.15 Sj -SDR, SEQ ID NO.17 Ss -SDR, SEQ ID NO.19 Sw -SDR and its mutants, expression plasmids of mutants or wet cells of genetically engineered bacteria, cell lysate of wet cells of genetically engineered bacteria expressing ketone reductase, or pure enzyme solution separated from wet cells of genetically engineered bacteria expressing ketone reductase after cell disruption and protein purification.
2. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that: In step one, in the esterification synthesis conversion system, the final concentration of 4-androstenedione is 10~100 g / L; the final concentration of toluenesulfonic acid is 10~100 g / L; and the concentration of acetic anhydride is 43.2~432 g / L.
3. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that: In step two, the pH of the hydrolysis-catalyzed conversion system is 5-10, and the temperature is 15-60℃.
4. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that: In step two, one of the following is added to the hydrolysis-catalyzed conversion system as a reaction medium: phosphate citrate buffer, phosphate buffer, Tris / HCl buffer, or glycine-NaOH buffer.
5. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that: In step three, the pH of the dual-enzyme coupled synergistic catalytic conversion system is 6-7, and the temperature is 30-40℃.
6. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that: The dual-enzyme coupled synergistic catalytic transformation system also contains NADP. + And glucose, NADP + The concentration in the conversion system is 0.5~3.5 mM, and the molar ratio of glucose to 5-androstenedione is 0.5~2:
1.
7. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that: The solvent of the dual-enzyme coupled synergistic catalytic conversion system is any one of methanol, ethanol, isopropanol, n-hexane, dimethyl sulfoxide, and 2-methyltetrahydrofuran.
8. The method for chemical-multi-enzyme coupled catalytic synthesis of dehydroepiandrosterone according to claim 1, characterized in that, The Ss -SDR mutants are mutants obtained by mutating at least one of the following sites in their amino acid sequence: (1) Phenylalanine at position 26 is replaced with alanine; (2) Arginine at position 106 is replaced with serine; (3) Tryptophan at position 157 is replaced with glycine; (4) Tyrosine at position 249 is replaced with serine.