Preparation method of all-trans-farnesol by a three-enzyme cascade
By using a two-site mutant of farnesyl pyrophosphate synthase in a three-enzyme cascade reaction with isopentenyl diphosphate isomerase and farnesyl pyrophosphate, the problems of low yield, high cost and environmental pollution in the preparation of farnesol were solved, and a highly efficient and simple all-trans-farnesol preparation was achieved with a product conversion rate of 98.5%.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- WUHAN HUALING BIOTECHNOLOGY CO LTD
- Filing Date
- 2026-03-23
- Publication Date
- 2026-07-31
AI Technical Summary
Existing methods for obtaining farnesol suffer from problems such as low yield, high cost, serious environmental pollution, cumbersome steps, and poor selectivity. Traditional biosynthesis methods also suffer from problems such as numerous byproducts and low efficiency.
All-trans-farnesol was prepared in a one-pot process using a three-enzyme cascade consisting of a two-site mutant of farnesyl pyrophosphate synthase, isopentenyl diphosphate isomerase, and farnesyl pyrophosphate enzyme. The enzyme ratio and reaction conditions were optimized to improve the conversion rate.
A highly efficient and simple preparation of farnesol was achieved, with a product conversion rate of up to 98.5%. This simplified the operation process, reduced intermediate accumulation, and improved selectivity and product concentration.
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Figure CN121896192B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for preparing farnesol, and more particularly to a method for preparing all-trans-farnesol via a three-enzyme cascade reaction, belonging to the field of biocatalytic preparation of farnesol. Background Technology
[0002] Farnesol is an important acyclic sesquiterpene alcohol and a key intermediate in the synthesis of many precious fragrances (such as ambroxol), widely used in high-end perfumes, cosmetics, and daily chemical products. Currently, the acquisition of farnesol mainly relies on plant extraction and chemical synthesis. Plant extraction is limited by factors such as plant growth cycle, local climate, and low content of active ingredients, resulting in low yields and high costs. Chemical synthesis typically involves multi-step reactions, uses heavy metal catalysts, and suffers from poor stereoselectivity (producing a mixture of multiple isomers), severe environmental pollution, and complex post-processing.
[0003] Biocatalysis offers advantages such as mild conditions, high selectivity, and environmental friendliness. Traditional biosynthesis of farnesol often employs whole-cell fermentation, which involves complex metabolic pathways, numerous byproducts, and the target product's toxicity to cells. In vitro multi-step catalysis using whole cells suffers from cumbersome separation steps, unstable intermediates, and low efficiency. Therefore, developing an efficient, concise, and controllable in vitro enzymatic synthesis process is of great significance. Summary of the Invention
[0004] One of the objectives of this invention is to provide a mutant of farnesyl pyrophosphate synthase.
[0005] The second objective of this invention is to apply the mutant of farnesyl pyrophosphate synthase to the catalytic synthesis of farnesol.
[0006] The third objective of this invention is to provide a one-pot method for preparing all-trans-farnesol using a three-enzyme cascade reaction.
[0007] The above-mentioned objective of the present invention is achieved through the following technical solution: One aspect of the present invention is to provide a mutant of farnesyl pyrophosphate synthase, the amino acid sequence of which is shown in SEQ ID No. 3.
[0008] This invention modifies farnesyl pyrophosphate synthase with the amino acid sequence shown in SEQ ID No. 2 by performing a two-site mutation: histidine (H) at position 185 of the amino acid shown in SEQ ID No. 2 is mutated to phenylalanine (F), and asparagine (N) at position 308 is mutated to serine (S). This invention found that, compared with the wild-type farnesyl pyrophosphate synthase before mutation, applying the obtained two-site mutant to the enzymatic synthesis of all-trans-farnesol can significantly improve the conversion rate of all-trans-farnesol.
[0009] Another aspect of the present invention provides a coding gene for a mutant of farnesyl pyrophosphate synthase with the amino acid sequence shown in SEQ ID No. 3, an expression vector containing the coding gene, and a recombinant host cell containing the expression vector.
[0010] Another aspect of the present invention is to apply the mutant of farnesyl pyrophosphate synthase, the coding gene of the mutant, the expression vector containing the coding gene, or the recombinant host cell containing the expression vector to the catalytic synthesis of all-trans-farnesol.
[0011] Another aspect of this invention provides a method for preparing all-trans-farnesol via a three-enzyme cascade reaction, comprising: in the same reaction system, using dimethylallyl diphosphate (DMAPP) as a substrate, and isopentenyl diphosphate isomerase (IDI), farnesyl pyrophosphate synthase (FPPS) or a mutant thereof, and farnesyl pyrophosphate enzyme (FPPase) as catalytic enzymes to prepare all-trans-farnesol. This invention has experimentally demonstrated that replacing farnesyl pyrophosphate synthase (FPPS) with a mutant of farnesyl pyrophosphate synthase (as shown in SEQ ID No. 3) significantly improves the conversion rate of all-trans-farnesol.
[0012] In a preferred embodiment of the present invention, in the method for preparing all-trans-farnesol via a three-enzyme cascade reaction, preferably, during the enzymatic catalytic reaction, isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase or its mutant, and farnesyl pyrophosphate enzyme (FPPase) are added sequentially to carry out a three-stage enzyme-linked catalytic reaction.
[0013] In a preferred embodiment of the present invention, the mass ratio of substrate, isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase or its mutant, and farnesyl pyrophosphate enzyme is (100-200):(10-30):(20-60):(5-30); more preferably, the mass ratio of substrate, isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase or its mutant, and farnesyl pyrophosphate enzyme is (120-150):(15-20):(30-50):(10-20).
[0014] In a preferred embodiment of the present invention, the reaction temperature of the enzyme-catalyzed reaction is controlled at 25-37°C; preferably, the reaction temperature of the enzyme-catalyzed reaction is controlled at 30°C; and the pH value of the reaction process of the enzyme-catalyzed reaction is controlled at 6.0-8.5.
[0015] The reaction mechanism or principle for preparing all-trans-farnesol using the three-enzyme cascade reaction described in this invention is as follows: isoprenyl diphosphate isomerase (IDI) is used as the catalytic enzyme to isoprenyl diphosphate (DMAPP) to form isoprenyl pyrophosphate (IPP). Farnesyl pyrophosphate synthase or its mutant catalyzes the condensation of two molecules of isoprenyl pyrophosphate (IPP) and one molecule of dimethyl allyl pyrophosphate (DMAPP) to generate C15 farnesyl pyrophosphate (FPP). Then, farnesyl pyrophosphatase (FPPase) catalyzes the hydrolysis of C15 farnesyl pyrophosphate (FPP) to remove the pyrophosphate group, thereby obtaining the target product all-trans-farnesol.
[0016] Any isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase, or farnesyl pyrophosphate enzyme, or strains or wet cells of these enzymes, disclosed in the prior art (including literature) or sold commercially (including strains sold in collections), may be applicable to this invention.
[0017] For reference, the amino acid sequence of the isopentenyl diphosphate isomerase described in this invention is shown in SEQ ID No. 1; the amino acid sequence of the farnesyl pyrophosphate synthase is shown in SEQ ID No. 2; and the amino acid sequence of the farnesyl pyrophosphate enzyme is shown in SEQ ID No. 4.
[0018] Compared with the prior art, the main beneficial effects of the present invention include: 1. Simplified process: The preparation method of the present invention adopts a three-enzyme cascade reaction, which eliminates the need to separate and purify intermediates, making the operation simple, the system has few impurities, and is conducive to the purification of the final product.
[0019] 2. Improved product conversion efficiency: The preparation method of this invention adopts a three-enzyme cascade reaction, which reduces the inhibition or degradation that may be caused by the accumulation of intermediate products, and significantly improves the overall conversion efficiency of all-trans-farnesol product.
[0020] 3. High controllability: The reaction process and product yield can be optimized by controlling the ratio, concentration and reaction conditions of enzymes.
[0021] 4. High product concentration: Compared with traditional biosynthesis methods, the preparation method of this invention has a short conversion cycle and the highest conversion rate of all-trans-farnesol can reach 98.5%, which has great industrial application value. Attached Figure Description
[0022] Figure 1 The results of the 1H NMR spectrum analysis of trans-farnesol prepared in Example 3 are shown. Detailed Implementation
[0023] The present invention will be further described below with reference to specific preliminary embodiments or examples, and the advantages and features of the present invention will become clearer with the description. However, these preliminary embodiments or examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.
[0024] Preliminary Example 1: Preparation of wet cells containing recombinant expression enzyme The encoding genes for isopentenyl diphosphate isomerase (SEQ ID No. 1), farnesyl pyrophosphate synthase (SEQ ID No. 2), the H185F / N308S double-site mutant of farnesyl pyrophosphate synthase (SEQ ID No. 3), and farnesyl pyrophosphate synthase (SEQ ID No. 4) were synthesized in their entirety and cloned into the pET28a vector. The recombinant vectors containing the target gene sequences were expressed in *E. coli* BL21(DE3). After PCR verification, positive colonies were selected, cultured, and induced to prepare wet bacterial cells expressing isopentenyl diphosphate isomerase (SEQ ID No. 1), farnesyl pyrophosphate synthase (SEQ ID No. 2), the H185F / N308S mutant of farnesyl pyrophosphate synthase (SEQ ID No. 3), and farnesyl pyrophosphate synthase (SEQ ID No. 4).
[0025] Preparation of wet bacterial cells: The recombinant strain E. coli BL21(DE3) glycerol containing the target enzyme was inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin sulfate and cultured at 37℃ for 10 h. Then, it was inoculated into fresh LB liquid medium containing 50 μg / mL kanamycin sulfate at a volume concentration of 1% and cultured at 37℃ and 180 rpm for 2.5 h. Then, IPTG was added to the culture medium to a final concentration of 0.3 mM for induction. After culturing at 25℃ and 180 rpm for 18 h, the culture was centrifuged at 4000 rpm for 15 min. The collected precipitate was the wet bacterial cells containing the target enzyme (namely, wet bacterial cells containing pentenyl diphosphate isomerase, wet bacterial cells containing farnesyl pyrophosphate synthase, wet bacterial cells containing farnesyl pyrophosphate synthase H185F / N308S mutant, or wet bacterial cells containing farnesyl pyrophosphate synthase).
[0026] Example 1: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 120 g / L of dimethyl allyl diphosphate (DMAPP) substrate, 10 mM MgCl2, 15 g / L of isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 30 g / L of farnesyl pyrophosphate synthase wet cells (prepared in Preliminary Example 1), 10 g / L of farnesyl pyrophosphate enzyme wet cells (preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 30℃, and the pH value was controlled at 7.5 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0027] After 26 hours of conversion, the concentration of all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution, detected by external standard method, was 36.7 g / L, and the conversion rate was calculated to be 47%. The product was analyzed by 1H NMR. The NMR parameters were as follows: (400 MHz, CDCl3) δ 5.39–5.43 (m, 1H), 5.06–5.12 (m, 1H), 4.15 (d, 2H), 1.95–2.14 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The structure of the product was identified as all-trans-farnesol by 1H NMR.
[0028] Example 2: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 120 g / L DMAPP substrate, 10 mM MgCl2, 15 g / L isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 45 g / L farnesyl pyrophosphate synthase wet cells (prepared in Preliminary Example 1), 10 g / L farnesyl pyrophosphate enzyme wet cells (prepared in Preliminary Example 1), with the remainder being pure water; the reaction temperature was controlled at 30°C, and the pH was controlled at 7.5 during the reaction process; The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0029] After 23 hours of conversion, the concentration of the product all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution was 56.1 g / L as determined by external standard method, and the conversion rate of all-trans-farnesol was calculated to be 72%.
[0030] The product was analyzed by 1H NMR. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.39-5.44 (m, 1H), 5.07-5.12 (m, 1H), 4.15 (d, 2H), 1.96-2.15 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The 1H NMR analysis confirmed that the product structure was all-trans-farnesol.
[0031] Example 3: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 120 g / L of DMAPP substrate, 10 mM MgCl2, 15 g / L of isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 30 g / L of farnesyl pyrophosphate synthase H185F / N308S mutant wet cells (prepared in Preliminary Example 1), 10 g / L of farnesyl pyrophosphate enzyme wet cells (preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 30℃, and the pH was controlled at 7.5 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0032] After 19 hours of conversion, the concentration of all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution, determined by external standard method, was 63.3 g / L, indicating a conversion rate of 82%. The product was analyzed by 1H NMR spectroscopy. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.41–5.45 (m, 1H), 5.08–5.14 (m, 1H), 4.15 (d, 2H), 1.97–2.16 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). Figure 1 This is the 1H NMR spectrum of the product prepared in this embodiment; the 1H NMR spectrum identifies the product structure as all-trans-farnesol.
[0033] Example 4: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 120 g / L of DMAPP substrate, 10 mM MgCl2, 15 g / L of isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 45 g / L of farnesyl pyrophosphate synthase H185F / N308S mutant wet cells (prepared in Preliminary Example 1), 10 g / L of farnesyl pyrophosphate enzyme wet cells (prepared in Preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 30℃, and the pH was controlled at 7.5 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0034] After 17 hours of conversion, the concentration of the product all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution was 76.1 g / L as determined by external standard method. The conversion rate of all-trans-farnesol was calculated to be 98.5%.
[0035] The product was analyzed by 1H NMR. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.42-5.47 (m, 1H), 5.09-5.15 (m, 1H), 4.15 (d, 2H), 1.98-2.16 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The 1H NMR analysis confirmed that the product structure was all-trans-farnesol.
[0036] Example 5: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 150 g / L DMAPP substrate, 10 mM MgCl2, 20 g / L isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 40 g / L farnesyl pyrophosphate synthase H185F / N308S mutant wet cells (prepared in Preliminary Example 1), 15 g / L farnesyl pyrophosphate enzyme wet cells (prepared in Preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 25℃, and the pH was controlled at 8.5 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0037] After 24 hours of conversion, the concentration of the product all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution was 75.9 g / L as determined by external standard method. The conversion rate of all-trans-farnesol was calculated to be 88.6%.
[0038] The product was analyzed by 1H NMR. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.40-5.45 (m, 1H), 5.08-5.15 (m, 1H), 4.15 (d, 2H), 1.96-2.15 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The 1H NMR analysis confirmed that the product structure was all-trans-farnesol.
[0039] Example 6: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 150 g / L DMAPP substrate, 10 mM MgCl2, 20 g / L isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 40 g / L farnesyl pyrophosphate synthase H185F / N308S mutant wet cells (prepared in Preliminary Example 1), 15 g / L farnesyl pyrophosphate enzyme wet cells (prepared in Preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 37℃, and the pH was controlled at 6.0 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0040] After 22 hours of conversion, the concentration of the product all-trans-farnesol no longer increased. The concentration of farnesol in the conversion solution was 63.2 g / L as determined by external standard method. The conversion rate of all-trans-farnesol was calculated to be 80.5%.
[0041] The product was analyzed by 1H NMR. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.39-5.45 (m, 1H), 5.06-5.13 (m, 1H), 4.15 (d, 2H), 1.96-2.14 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The 1H NMR analysis confirmed that the product structure was all-trans-farnesol.
[0042] Example 7: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 150 g / L DMAPP substrate, 10 mM MgCl2, 20 g / L isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 40 g / L farnesyl pyrophosphate synthase H185F / N308S mutant wet cells (prepared in Preliminary Example 1), 15 g / L farnesyl pyrophosphate enzyme wet cells (prepared in Preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 37℃, and the pH was controlled at 8.0 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0043] After 24 hours of conversion, the concentration of the product all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution was 82.1 g / L as determined by external standard method. The conversion rate of all-trans-farnesol was calculated to be 85.0%.
[0044] The product was analyzed by 1H NMR. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.40-5.43 (m, 1H), 5.06-5.15 (m, 1H), 4.15 (d, 2H), 1.96-2.17 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The 1H NMR analysis confirmed that the product structure was all-trans-farnesol.
[0045] Example 8: Preparation of all-trans-farnesol using a one-pot method based on a three-enzyme cascade reaction The reaction system consisted of 50 ml containing 150 g / L DMAPP substrate, 10 mM MgCl2, 20 g / L isopentenyl diphosphate isomerase wet cells (prepared in Preliminary Example 1), 50 g / L farnesyl pyrophosphate synthase H185F / N308S mutant wet cells (prepared in Preliminary Example 1), 20 g / L farnesyl pyrophosphate enzyme wet cells (prepared in Preliminary Example 1), and the remainder being pure water. The reaction temperature was controlled at 28℃, and the pH was controlled at 6.0 during the reaction process. The concentration of the product all-trans-farnesol was monitored by GC during the reaction process until the product concentration no longer changed, at which point the reaction was stopped.
[0046] After 26 hours of conversion, the concentration of the product all-trans-farnesol no longer increased. The concentration of all-trans-farnesol in the conversion solution was 78.5 g / L as determined by external standard method. The conversion rate of all-trans-farnesol was calculated to be 81.3%.
[0047] The product was analyzed by 1H NMR. The 1H NMR parameters were as follows: (400 MHz, CDCl3) δ 5.42-5.47 (m, 1H), 5.09-5.16 (m, 1H), 4.15 (d, 2H), 1.98-2.16 (m, 8H), 1.68 (s, 6H), 1.60 (s, 6H). The 1H NMR analysis confirmed that the product structure was all-trans-farnesol.
Claims
1. A mutant of farnesyl pyrophosphate synthase, characterized in that, Its amino acid sequence is shown in SEQ ID No.
3.
2. The coding gene of the mutant according to claim 1.
3. An expression vector containing the encoding gene as described in claim 2.
4. A recombinant host cell containing the expression vector of claim 3.
5. The use of the mutant of claim 1, the encoding gene of claim 2, the expression vector of claim 3, or the recombinant host cell of claim 4 in the biocatalytic preparation of all-trans-farnesol.
6. A method for preparing all-trans-farnesol via a three-enzyme cascade reaction, characterized in that, include: In the same reaction system, all-trans-farnesol was prepared by enzymatic catalysis using dimethylallyl diphosphate as substrate and isopentenyl diphosphate isomerase, a mutant of farnesyl pyrophosphate synthase, and farnesyl pyrophosphate as catalysts; the amino acid sequence of the mutant of farnesyl pyrophosphate synthase is shown in SEQ ID No.
3.
7. The preparation method according to claim 6, characterized in that, The amino acid sequence of the isopentenyl diphosphate isomerase is shown in SEQ ID No. 1; the amino acid sequence of the farnesyl pyrophosphatase is shown in SEQ ID No.
4.
8. The preparation method according to claim 6, characterized in that, During the enzymatic catalytic reaction, isopentenyl diphosphate isomerase, a mutant of farnesyl pyrophosphate synthase, and farnesyl pyrophosphate are added sequentially to carry out a three-stage enzyme-linked catalytic reaction.
9. The preparation method according to claim 6, characterized in that, The mass ratio of substrate, isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase mutant, and farnesyl pyrophosphate enzyme is (100-200):(10-30):(20-60):(5-30); the reaction temperature of the enzyme-catalyzed reaction is controlled at 25-37℃; the pH value of the enzyme-catalyzed reaction process is controlled at 6.0-8.
5.
10. The preparation method according to claim 9, characterized in that, The mass ratio of substrate, isopentenyl diphosphate isomerase, farnesyl pyrophosphate synthase mutant, and farnesyl pyrophosphate synthase was (120-150): (15-20): (30-50): (10-20). The reaction temperature of the enzyme-catalyzed reaction is controlled at 30°C.