Method for preparing chiral alcohol through asymmetric reduction of large steric hindrance rigid ketone based on aldehyde ketone reductase and mutant thereof

Abstract

The invention belongs to the technical field of biosynthesis, and particularly relates to a method for preparing chiral alcohol through asymmetric reduction of large steric hindrance rigid ketone based on aldehyde ketoreductase and a mutant thereof. Comprising the following steps: adding a recombinant Escherichia coli wet cell for expressing aldehyde ketoreductase, a recombinant Escherichia coli wet cell for expressing formate dehydrogenase, a buffer solution, a substrate, a cosolvent, ammonium formate and coenzyme into a reaction container, reacting at a certain temperature, and after the reaction is completed, extracting, separating and carrying out rotary evaporation to obtain a product alcohol. The biological catalysis method is mild in reaction condition, environmentally friendly and high in regioselectivity and stereoselectivity, chiral resolution and heavy metal residues in products are avoided, and the defects of a chemical method are exactly overcome.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to a method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants. Background Technology

[0002] Chiral alcohols are important intermediates in the synthesis of pharmaceuticals, fragrances, and fine chemicals. The asymmetric reduction of sterically hindered rigid ketones (such as tropinone, quinine, α-thujone, camphor, and fenestrant) presents significant challenges due to their large steric hindrance and rigid structure. Traditional chemical catalysis methods (such as metal complexes or chiral borane reducing agents) can achieve partial substrate conversion, but they typically face problems such as harsh reaction conditions, insufficient stereoselectivity (low ee value), and the need for expensive chiral ligands or metal catalysts. For example, when quinine is used in the reduction of aromatic ketones catalyzed by a cobalt(II) chiral complex, the enantiomeric excess (ee value) of the product is only moderate, and strict anhydrous conditions are required, limiting its industrial application.

[0003] Biocatalysis has become a research hotspot due to its high stereoselectivity, mild reaction conditions, and environmental friendliness. Aldehyde-ketone reductases (AKRs) can efficiently catalyze the asymmetric reduction of prochiral ketones to produce chiral alcohols with high optical purity. In existing technologies, most reported aldehyde-ketone reductases target sterically hindered or flexible ketone substrates (such as acetophenone derivatives), but their catalytic efficiency for sterically hindered rigid ketones is generally low. For example, the aldehyde-ketone reductase BM1-1 from *Bacillus megaterium* can catalyze thiophene ketone substrates, but its activity for other sterically hindered substrates is not mentioned; while *Lekred* from *Rude's yeast* can efficiently reduce ethyl 4-chloroacetoacetate (COBE), its adaptability to sterically hindered substrates has not been verified.

[0004] Furthermore, while progress has been made in the development of quinine reductases (e.g., the enzyme described in Jiangnan University patent CN112941041B can efficiently convert 3-quinine, with an ee value >99%), its substrate spectrum is relatively narrow, and its catalytic ability for other rigid ketones (such as tropine or α-thujone) has not been reported. Similarly, existing aldehyde-ketone reductases often experience significantly reduced reaction rates and conversion rates when catalyzing camphor or fentanyl due to substrate steric hindrance, which makes it difficult for the enzyme's active site to adapt.

[0005] Therefore, developing an aldehyde-ketone reductase with broad substrate adaptability and high stereoselectivity to achieve efficient asymmetric reduction of sterically hindered ketones is of great significance for promoting the synthesis of chiral drugs and green chemistry processes.

[0006] To address the aforementioned issues, this invention has screened an aldehyde-ketone reductase capable of efficiently and asymmetrically reducing sterically hindered rigid substrates. Furthermore, through directed evolutionary modification, the ketone reductase has been rationally modified to significantly enhance its catalytic activity and substrate adaptability for tropinone and 3-quinone. Simultaneously, by combining a coenzyme self-circulation system and optimizing the heterogeneous reaction system, a highly efficient and low-cost biocatalytic process is achieved. This technology not only avoids the use of precious metals and toxic reagents but also overcomes yield bottlenecks through enzyme-reactor synergistic design, providing an innovative solution for the green industrial production of sterically hindered rigid substrates. Summary of the Invention

[0007] To address the problems mentioned in the background art, this invention proposes a method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants. This biocatalytic method not only has mild reaction conditions and is environmentally friendly, but also exhibits high regioselectivity and stereoselectivity. Furthermore, it avoids chiral resolution and heavy metal residues in the products, thus overcoming the shortcomings of chemical methods.

[0008] The technical solution adopted by the present invention to solve its technical problem is: to provide a method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductase and its mutants, comprising the following steps: adding recombinant Escherichia coli wet cells expressing aldehyde-ketone reductase, recombinant Escherichia coli wet cells expressing formate dehydrogenase, buffer, substrate, solubilizer, ammonium formate and coenzyme to a reaction vessel, reacting at a certain temperature, and after the reaction is completed, obtaining the product alcohol by extraction, separation and rotary evaporation.

[0009] Furthermore, the substrate is at least one selected from tropinone, quinine, α-thujone, camphor, and fentanyl. Its chemical formula is as follows:

[0010] .

[0011] Furthermore, the proportions of each raw material in the 1L reaction system are as follows: 10-200g of recombinant Escherichia coli expressing aldehyde-ketone reductase, 10-200g of recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of solubilizer, 0.2-1mol of ammonium formate, 0.1-2.5mmol of coenzyme, and buffer to a final volume of 1L.

[0012] The aldehyde-ketone reductase is selected from AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, AKR24 or their mutants.

[0013] The NCBI numbers for AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, and AKR24 are EDV10271.1, XP_016271149.1, XP_002491455.1, WP_011237148.1, BAE46987.1, WP_023468191.1, WP_028414523.1, AHC30848.1, WP_263603657.1, and WP_010865121.1.

[0014] Preferably, the proportions of each raw material in the 1L reaction system are as follows: 10-200g of recombinant Escherichia coli expressing aldehyde-ketone reductase, 50-100g of recombinant Escherichia coli expressing formate dehydrogenase, 20-100g of substrate, 50-500mL of solubilizer, 0.2-1mol of ammonium formate, 0.1-2.5 mmol of coenzyme, and the remainder is buffer solution.

[0015] Preferably, the proportions of each raw material in the 1L reaction system are as follows: 10-200g of recombinant Escherichia coli expressing aldehyde-ketone reductase, 25-75g of recombinant Escherichia coli expressing formate dehydrogenase, 20-100g of substrate, 50-500mL of cosolvent, 0.2-1mol of ammonium formate, 0.1-2.5 mmol of coenzyme, and the remainder is buffer solution.

[0016] Furthermore, the reaction temperature is 25℃~35℃, and the reaction time is 2-24h.

[0017] Furthermore, the mutant of AKR19 is selected from AKR19-K42C / P99Q / Y167F / E182A or AKR19-K42Y / P99A / Y167A;

[0018] Among them, AKR19-K42C / P99Q / Y167F / E182A is a mutation where K at position 42 of the AKR19 amino acid sequence is changed to C, P at position 99 is changed to Q, Y at position 167 is changed to F, and E at position 182 is changed to A.

[0019] AKR19-K42Y / P99A / Y167A is an AKR19 amino acid sequence in which K is mutated to Y at position 42, P is mutated to A at position 99, and Y is mutated to A at position 167.

[0020] Furthermore, in a 1L reaction system, the amount of recombinant Escherichia coli wet cells expressing formate dehydrogenase is 50–100g.

[0021] Furthermore, the buffer solution is selected from one of TEA buffer, PB buffer, and Tris-HCl buffer.

[0022] Further, the concentration of the TEA buffer is 0.05–0.20 M, and the pH value is 6–8; the concentration of the PB buffer is 0.05–0.10 M, and the pH value is 6–7; the concentration of the Tris-HCl buffer is 0.05–0.10 M, and the pH value is 7–8.

[0023] Furthermore, the coenzyme is NADP+ or NAD+.

[0024] Furthermore, the extractant used for extraction is at least one of ethyl acetate, methyl tert-butyl ether, methanol, dichloromethane, and n-butanol.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] (1) The biocatalytic method of the present invention is not only mild and environmentally friendly, but also highly regioselective and stereoselective. It also avoids chiral resolution and heavy metal residues in the products, thus making up for the shortcomings of chemical methods.

[0027] (2) The method of the present invention can obtain the target product in only one step, and uses fewer reagents, has mild reaction conditions, high catalytic activity, and reduces production costs. It has broad application prospects and considerable market value. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0030] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0031] In the method of the present invention, the aldehyde-ketone reductase can be a free enzyme (e.g., used in the form of enzyme powder), or in the form of cells expressing the aldehyde-ketone reductase (e.g., wet bacterial cells). Or other forms, such as cell lysate supernatant or whole-cell immobilization expressing the aldehyde-ketone reductase, or immobilization of free enzyme powder.

[0032] In the preparation method of aldehyde-ketone reductase of the present invention, the host microorganism for enzyme protein expression can be *Escherichia coli* BL21(DE3), and the recombinant plasmid can be pET28 plasmid. The molecular biological operations involved in the steps are all well-known and routine experimental procedures in the biological field, including gene acquisition (PCR), splicing of plasmids and target genes (i.e., vector construction), introduction of plasmids containing target gene fragments into bacterial cells (i.e., transformation), bacterial culture in culture medium, and enzyme production (i.e., fermentation).

[0033] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 g / L agar.

[0034] Example 1:

[0035] Obtaining recombinant bacterial cells expressing aldehyde-ketone reductase:

[0036] (1) Expression vectors for AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, and AKR24 were synthesized externally (Shanghai Sangon Biotech). The target gene fragment was inserted into the commercial plasmid pET28 to complete the construction of the expression vector. Gene sequences: EDV10271.1, XP_016271149.1, XP_002491455.1, WP_011237148.1, BAE46987.1, WP_023468191.1, WP_028414523.1, AHC30848.1, WP_263603657.1, WP_010865121.1.

[0037] (2) Expression of enzyme proteins using *Escherichia coli* BL21(DE3). The constructed plasmids were sequentially transformed into the same *E. coli* strain and plated on LB agar plates (containing 50 mg / L kanamycin and 20 mg / L chloramphenicol, respectively). The specific implementation is as follows:

[0038] First, the bacterial cells transformed with plasmid pET28 were spread on LB agar plates containing kanamycin resistance (50 mg / L kanamycin) and cultured at 37°C for 12 hours. Then, single colonies were selected and transferred to LB liquid medium (with additional kanamycin added to a final concentration of 50 mg / L) and cultured at 37°C for 12 hours to prepare competent cells. Then, the plasmid pET28 containing the target gene was transformed into competent cells and spread on LB agar plates containing chloramphenicol resistance (20 mg / L) and cultured at 37°C for 12 hours to obtain recombinant bacterial cells.

[0039] Obtaining recombinant bacterial cells expressing formate dehydrogenase:

[0040] (1) The formate dehydrogenase expression vector was synthesized externally (Shanghai Sangon Biotech). The commercial plasmid pET28 was used to insert the target gene fragment into pET28 to complete the construction of the expression vector. The formate dehydrogenase gene sequence is located in GenBank as AXT18256.1.

[0041] (2) Expression of enzyme proteins using *Escherichia coli* BL21(DE3). The constructed plasmids were sequentially transformed into the same *E. coli* strain and plated on LB agar plates (containing 50 mg / L kanamycin and 20 mg / L chloramphenicol, respectively). The specific implementation is as follows:

[0042] First, the bacterial cells transformed with plasmid pET28 were spread on LB agar plates containing kanamycin resistance (50 mg / L kanamycin) and cultured at 37°C for 12 hours. Then, single colonies were transferred to LB liquid medium (with additional kanamycin added to a final concentration of 50 mg / L) and cultured at 37°C for 12 hours to prepare competent cells. Then, the plasmid pET28 containing the target gene was transformed into competent cells and spread on LB agar plates containing chloramphenicol resistance (20 mg / L) and cultured at 37°C for 12 hours to obtain recombinant bacterial cells.

[0043] Example 2:

[0044] Preparation of wet bacterial cells of S1, AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, and AKR24: The recombinant engineered bacteria AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, and AKR24 prepared in Example 1 were inoculated (1% inoculum) into 10 mL of LB liquid medium and cultured at 37°C and 180 rpm for 8-10 h. Then, 1% of the bacterial culture was inoculated into 100 mL of LB medium and cultured at 37°C and 180 rpm for 2-2.5 h (equivalent to an OD600 value of 0.6-0.8). Finally, IPTG was added to a final concentration of 0.1 mM, and the culture was induced at 24°C and 180 rpm for 12 h. Pour the cultured bacterial solution into a 500ml centrifuge cup, centrifuge at 8000rpm and 4℃ for 10min, discard the supernatant, collect the bacterial cells and weigh them for subsequent use.

[0045] Preparation of wet formate dehydrogenase cells: The recombinant formate dehydrogenase engineered bacteria prepared in Example 1 were inoculated (1% inoculum) into 10 mL of LB liquid medium and cultured at 37°C and 180 rpm for 8-10 h. Then, 1% of the bacterial culture was inoculated into 100 mL of LB medium and cultured at 37°C and 180 rpm for 2-2.5 h (equivalent to an OD600 value of 0.6-0.8). Next, 0.1 mM IPTG was added, and the culture was induced at 24°C and 180 rpm for 12 h. The cultured bacterial culture was poured into a 500 mL centrifuge cup and centrifuged at 8000 rpm and 4°C for 10 min. The supernatant was discarded, and the bacterial cells were collected and weighed for subsequent use.

[0046] S2. Take 0.15g*5 portions of wet bacterial cells of AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, and AKR24 respectively and add them to 2mL EP tubes, and set up a blank control group.

[0047] S3. Add 0.15 g of wet formate dehydrogenase cells to each EP tube. Add 700 µL of PB buffer (0.1 M, pH 7), 100 µL of 7 M ammonium formate solution, 100 µL of 25 m M NADP+ solution, and 100 µL of substrate solution (the substrates are tropinone, quinineone, α-thujone, camphor, and fenestrant, with a final substrate concentration of 50 g / L). Place the EP tubes in a constant temperature mixer and react at 30 °C and 1200 rpm for 16 h.

[0048] S4. After the reaction is complete, take 200µL of the reaction solution, add 200µL of 4M NaOH and 200µL of n-butanol or dichloromethane, shake to mix for 2 min, centrifuge at 12000 rpm for 1 min, take the supernatant and send it for GC analysis. The product is obtained after rotary evaporation.

[0049] The conversion rates and ee values ​​of each aldehyde-ketone reductase were determined by GC analysis, as shown in Table 1.

[0050] Table 1 shows the conversion rate and ee value of each aldehyde-ketone reductase.

[0051]

[0052] The results showed that AKR19 had good catalytic effects on five sterically hindered rigid substrates. Among them, it had the best effect on tropinone and quinine, with a conversion rate of >99% and good stereoselectivity, and high modifiability.

[0053] Example 3: Construction and screening of a mutant library of aldehyde-ketone reductases catalyzing quinone.

[0054] 1. Starting strain:

[0055] Using AKR19, selected in Example 2, as the original strain, the plasmid pET28b(+)-AKR-19 was activated and extracted.

[0056] 2. Single mutation:

[0057] (1) Construction of mutant libraries

[0058] The AKR-19 mutant library was prepared by site-directed mutagenesis. Using the vector pET28b(+)-AKR-19 from the original strain as a template, primers were designed (examples of primers are shown in Table 2), and polymerase chain reaction (PCR) was performed. The recombinant plasmid digested with DpnI was transferred into Escherichia coli BL21(DE3) competent cells, and the clones were inoculated into 10 mL LB agar plates and cultured at 37°C for 12–16 h.

[0059] Table 2 is the primer table.

[0060]

[0061] (2) GC screening

[0062] Randomly select positive clones and the original bacterial strain from the plate and inoculate them into 10 mL of LB liquid medium (with kanamycin, 50 mg / L added to the test tube), and incubate at 37°C and 200 rpm in a temperature-controlled shaker for 8-10 h. Inoculate 1% of the culture solution into 100 mL of LB liquid medium (with kanamycin, 50 mg / L added to the shake flask), and incubate at 37°C and 180 rpm in a temperature-controlled shaker for 2-2.5 h (equivalent to an OD600 value of 0.6-0.8). Add IPTG inducer to a final concentration of 0.1 mM, and continue incubation at 24°C and 180 rpm in a temperature-controlled shaker for 12 h. Pour the cultured solution into an 800 mL centrifuge cup, centrifuge at 8000 rpm and 4°C for 10 min using a low-temperature high-speed centrifuge, discard the supernatant, collect the bacterial cells and weigh them for subsequent use.

[0063] The concentrations and chirality of quinone and R-3-quinol were detected by GC, and the conversion rate was calculated. The dominant strains were screened based on the conversion rate and chirality.

[0064] The dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored at -80℃. The final dominant mutants were AKR19-K42C, AKR19-P99Q, AKR19-Y114I, AKR19-Y167F, AKR19-E182A, and AKR19-T193Y.

[0065] 3. Iterative mutation

[0066] Using vectors pET28b(+)-AKR19-K42C, AKR19-P99Q, AKR19-Y114I, AKR19-Y167F, AKR19-E182A, and AKR19-T193Y as templates, primers were designed (primers are the same as in Table 2) for polymerase chain reaction (PCR). Based on the GC screening procedure, dominant mutations were further screened from single-point mutants.

[0067] PCR reaction system (25µL): 1µL forward primer (100µM), 1µL reverse primer (100µM), 12.5µL 2×Phanta buffer (Novizan, China), 0.5µL dNTP mixture (10mM each), 1µL plasmid template, 0.5µL DNA polymerase Phanta (Novizan, China) and 8.5µL ultrapure water.

[0068] The PCR program set according to the Phanta Super-Fidelity DNA Polymerase Manual (Novizan, China) is as follows: 95℃ pre-denaturation for 5 min, then 30 cycles (95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 4 min), 72℃ final extension for 10 min, and incubation at 16℃.

[0069] The iterative mutant (two-site mutant) was obtained:

[0070] AKR19-K42C / P99Q, AKR19-K42C / Y114I, AKR19-K42C / Y167F, AKR19-K42C / E182 A. AKR19-K42C / T193Y, AKR19-P99Q / Y114I, AKR19-P99Q / Y167F, AKR19-P99Q / E 182A, AKR19-P99Q / T193Y, AKR19-Y114I / Y167F, AKR19-Y114I / E182A, AKR19-Y 114I / T193Y, AKR19-Y167F / E182A, AKR19-Y167F / T193Y, AKR19-E182A / T193Y.

[0071] 4. Catalytic activity detection

[0072] Single mutant strains, iterative mutant strains, and control strains (AKR19-WT as a control) were used as catalysts, with quinine as the substrate, to compare the catalytic activity of each mutant. The reaction system consisted of 1 mL of culture medium, with a catalyst concentration of 100 g / L (wet weight of cells), a final substrate concentration of 50 g / L, and a pH 7.0, 100 mM MPa buffer solution. The reaction medium included 0.7 M ammonium formate, 100 g / L formate dehydrogenase, and 2.5 mM NADPH. The reaction was carried out at 30°C and 1200 rpm for 2 h. 200 μL of the reaction solution was then added to 200 μL of 4 M NaOH (for alkali adjustment), followed by extraction once with 200 μL of n-butanol. The concentrations and chirality of quinine and R-3-quinine were determined using GC as described in Example 1, and the conversion rate was calculated.

[0073] The screening results are shown in Table 3:

[0074] Table 3 shows the screening results of various mutants of quinone aldehyde reductase.

[0075]

[0076] Finally, further screening yielded the dominant strain K42C / P99Q / Y167F / E182A, which is an AKR19 mutant.

[0077] Amino acid sequence: K42C / P99Q / Y167F / E182A (see SEQ ID NO. 13).

[0078] SEQ ID NO. 13:

[0079] MTFTVVTGANGYIAKHILKSLLEDGHRVIGTVRNSKKAEELCRTVNDENLIVELVPDMLVENAFDELFKKYNTQIKYVFHTASPVLETSKDYEKSLIEQAITGAKSMVEAIRKYSLTSVEHIVYTSSIAASSLESEFTDPTLVVSEDSWNPQGLEEAKTEFFTAYSFSKKIA EKTMWDFVEAYKGTEHEIKLTTVNPCFNIGPQAYEADVTETMNFTAELINHVVKSKVGDPLPPTRIVPYVDVRDTARAHVDALKNEKLAFQRLLVVGPFLSSQQIYDIVNERFPQLRGKIARGEPGSDKLDPAKLAKFDHARTTQALGWEFTPIEKAIADEVAQILRVGAYRG

[0080] Example 4: Construction and screening of a mutant library of aldehyde-ketone reductases that catalyze tropinone.

[0081] 1. Starting strain:

[0082] Using AKR19, selected in Example 2, as the original strain, the plasmid pET28b(+)-AKR-19 was activated and extracted.

[0083] 2. Single mutation:

[0084] (1) Construction of mutant libraries

[0085] Same as Example 3.

[0086] (2) GC screening

[0087] Same as Example 3.

[0088] The concentrations and chirality of tropinone and R-tropinol were detected by GC, and the conversion rate was calculated. The dominant strains were screened based on the conversion rate and chirality.

[0089] The dominant strains were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing and stored at -80℃. The final dominant mutants were AKR19-K42C, AKR19-P99Q, AKR19-Y114I, AKR19-Y167F, and AKR19-E182A.

[0090] 3. Iterative mutation

[0091] Using vectors pET28b(+)-AKR19-K42Y, AKR19-P99A, AKR19-Y167A, AKR19-E182A, and AKR19-T193A as templates, primers were designed (as shown in Table 2) for polymerase chain reaction (PCR). Following the GC screening procedure, dominant mutations were further screened based on single-point mutants.

[0092] PCR reaction system (25µL): 1µL forward primer (100µM), 1µL reverse primer (100µM), 12.5µL 2×Phanta buffer (Novizan, China), 0.5µL dNTP mixture (10mM each), 1µL plasmid template, 0.5µL DNA polymerase Phanta (Novizan, China) and 8.5µL ultrapure water.

[0093] The PCR program set according to the Phanta Super-Fidelity DNA Polymerase Manual (Novizan, China) is as follows: 95℃ pre-denaturation for 5 min, then 30 cycles (95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 4 min), 72℃ final extension for 10 min, and incubation at 16℃.

[0094] The iterative mutant (two-site mutant) was obtained:

[0095] AKR19-K42Y / P99A, AKR19-K42Y / Y167A, AKR19-K42Y / E182A, AKR19-K42Y / T193A, AKR19-P99A / Y167A, AK R19-P99A / E182A, AKR19-P99Q / T193A, AKR19-Y167A / E182A, AKR19-Y167A / T193A, AKR19-E182A / T193A.

[0096] 4. Catalytic activity detection

[0097] Single mutant strains, iterative mutant strains, and control strains (AKR19-WT as a control) were used as catalysts, with tropidine as the substrate, to compare the catalytic activity of each mutant. The reaction system consisted of 1 mL of tropidine, with a catalyst concentration of 100 g / L (wet weight of bacterial cells), a final substrate concentration of 50 g / L, and a pH 7.0, 100 mM MPa buffer solution as the reaction medium. The reaction mixture contained 0.7 M ammonium formate, 100 g / L formate dehydrogenase, and 2.5 mM NADPH. The reaction was carried out at 30°C and 1200 rpm for 2 h. 200 μL of the reaction solution was then added to 200 μL of 4 M NaOH (to adjust the alkali), followed by extraction once with 200 μL of dichloromethane. The concentrations and chirality of tropidine and R-tropidine alcohol in the dichloromethane sample were determined using GC as described in Example 1, and the conversion rate was calculated.

[0098] The screening results are shown in Table 4.

[0099] Table 4 shows the screening results of various mutants of tropinone-catalyzing aldehyde-ketone reductase.

[0100]

[0101] Finally, further screening yielded the dominant strain K42Y / P99A / Y167A, which is an AKR19 mutant.

[0102] Amino acid sequence: K42Y / P99A / Y167A (see SEQ ID NO. 14).

[0103] SEQ ID NO. 14:

[0104] MTFTVVTGANGYIAKHILKSLLEDGHRVIGTVRNSKKAEELYRTVNDENLIVELVPDMLVENAFDELFKKYNTQIKYVFHTASPVLETSKDYEKSLIEAAITGAKSMVEAIRKYSLTSVEHIVYTSSIAASSLESEFTDPTLVVSEDSWNPQGLEEAKTEFFTAYSASKKIA EKTMWDFVEAYKGTEHEIKLTTVNPCFNIGPQAYEADVTETMNFTAELINHVVKSKVGDPLPPTRIVPYVDVRDTARAHVDALKNEKLAFQRLLVVGPFLSSQQIYDIVNERFPQLRGKIARGEPGSDKLDPAKLAKFDHARTTQALGWEFTPIEKAIADEVAQILRVGAYRG

[0105] Example 5: Extractant screening.

[0106] The steps are as follows:

[0107] S1. Prepare a 100g / L stock solution by mixing quinine / quinine alcohol and tropine / tropine alcohol standards.

[0108] S2. Take 0.5 mL of the mother liquor and add 0.5 mL of 4M NaOH and 0.5 mL of organic solvent to 2 mL EP tubes respectively. Mix thoroughly and centrifuge at 12000 rpm for 1 min. Collect the supernatant and perform TLC detection (developing solvent: dichloromethane:methanol = 5:1). Develop the solution in an iodine bath. Ethyl acetate, methyl tert-butyl ether, methanol, dichloromethane, and n-butanol are used.

[0109] The results are shown in Table 5 (more plus signs indicate better extraction results):

[0110] Table 5 shows the extraction effects of various extractants.

[0111]

[0112] The results showed that n-butanol and methanol had better extraction effects for quinine and quinol, but methanol easily extracted other impurities and was too soluble in water, making it difficult to extract. Therefore, n-butanol was chosen as the extractant. For tropidine and tropidine alcohol, dichloromethane had better extraction effects, and dichloromethane was chosen as the extractant.

[0113] Example 6: Scale-up verification and preparation of kilogram-scale R-3-quinol

[0114] S1. In a 50L reaction flask (30L reaction system), add 900g each of AKR19-K42C / P99Q / Y167F / E182A wet bacterial cells and formate dehydrogenase wet bacterial cells, 18L of PB buffer (0.1M pH 6.5), 3L of 15M ammonium formate solution, 3L of 3mM NADP+ solution, and 6L (7.6kg) of 1267g / L quinine hydrochloride solution. The reaction is mechanically stirred at 30℃ and 200rpm.

[0115] S2. After 2 hours of reaction, take 0.5 mL of the reaction solution, add 0.5 mL of 4M NaOH (to adjust the alkali), add 0.5 mL of n-butanol to extract once, centrifuge and take the upper organic phase. TLC detection showed no substrate residue, and then send it for GC detection.

[0116] S3. The reaction solution was adjusted to pH 13 with NaOH, extracted twice with an equal volume of n-butanol, the organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure, slurried with n-heptane, filtered and dried to obtain 5.7 kg of product (content of 106.5% of the purchased reference standard), with a molar yield of 95%, product purity >99%, and product ee value of 100%.

[0117] Example 7: Scale-up verification and preparation of kilogram-scale R-tropine alcohol

[0118] S1. In a 50L reaction flask (30L reaction system), add 800g each of AKR19-K42Y / P99A / Y167A wet bacterial cells and formate dehydrogenase wet bacterial cells, 18L of PB buffer (0.1M pH 6.5), 3L of 15M ammonium formate solution, 3L of 3mM NADP+ solution, and 6L (7.5kg) of 1250g / L tropidine solution. The reaction is mechanically stirred at 30℃ and 200rpm.

[0119] S2. After 2 hours of reaction, take 0.5 mL of the reaction solution, add 0.5 mL of 4M NaOH (to adjust the alkali), add 0.5 mL of dichloromethane to extract once, centrifuge and take the upper organic phase. TLC detection showed no substrate residue, and then send it for GC detection.

[0120] S3. The reaction solution was adjusted to pH 13 with NaOH, extracted twice with an equal volume of dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, evaporated under reduced pressure, slurryed with methanol, filtered and dried to obtain 7.1 kg of product (content of 101.4% of the purchased reference standard), with a molar yield of 94.5%, product purity >99%, and product ee value of 99.48%.

[0121] This invention provides 10 aldehyde-ketone reductases AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, and AKR24, as well as mutants of preferred enzymes, capable of catalyzing the conversion of sterically hindered rigid ketone substrates into alcohol products. Specifically, the mutant AKR19-K42Y / P99A / Y167A for the conversion of tropinone to R-tropinol achieves a conversion rate of 99% and an optical purity greater than 99%. Similarly, the mutant AKR19-K42C / P99Q / Y167F / E182A for the conversion of quinineone to R-3-quinineol achieves a conversion rate of 99% and an optical purity greater than 99%. This biocatalytic method not only features mild reaction conditions and is environmentally friendly, exhibiting high regioselectivity and stereoselectivity, but also avoids chiral resolution and heavy metal residues in the products, effectively compensating for the shortcomings of chemical methods.

[0122] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants, characterized in that, The process includes the following steps: adding recombinant Escherichia coli wet cells expressing aldehyde-ketone reductase, recombinant Escherichia coli wet cells expressing formate dehydrogenase, buffer, substrate, solubilizer, ammonium formate, and coenzyme to a reaction vessel, reacting at a certain temperature, and after the reaction is completed, extracting, separating, and rotary evaporating to obtain the product alcohol.

2. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, The substrate is at least one of tropinone, quinineone, α-thujone, camphor, and fentanyl.

3. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, The proportions of each raw material in the 1L reaction system are as follows: 10-200g of recombinant Escherichia coli expressing aldehyde-ketone reductase, 10-200g of recombinant Escherichia coli expressing formate dehydrogenase, 20g-100g of substrate, 50-500mL of solubilizer, 0.2-1mol of ammonium formate, 0.1-2.5mmol of coenzyme, and buffer to a final volume of 1L. The aldehyde-ketone reductase is selected from AKR15, AKR16, AKR17, AKR18, AKR19, AKR20, AKR21, AKR22, AKR23, AKR24 or their mutants.

4. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, The reaction temperature is 25℃~35℃, and the reaction time is 2-24h.

5. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 3, characterized in that, The mutant of AKR19 is selected from AKR19-K42C / P99Q / Y167F / E182A or AKR19-K42Y / P99A / Y167A; Among them, AKR19-K42C / P99Q / Y167F / E182A is a mutation where K at position 42 of the AKR19 amino acid sequence is changed to C, P at position 99 is changed to Q, Y at position 167 is changed to F, and E at position 182 is changed to A. AKR19-K42Y / P99A / Y167A is an AKR19 amino acid sequence in which K is mutated to Y at position 42, P is mutated to A at position 99, and Y is mutated to A at position 167.

6. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, In a 1L reaction system, the amount of recombinant Escherichia coli wet cells expressing formate dehydrogenase is 50–100g.

7. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, The buffer solution is selected from one of TEA buffer, PB buffer, and Tris-HCl buffer.

8. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 7, characterized in that, The concentration of the TEA buffer is 0.05–0.20 M, and the pH value is 6–8; the concentration of the PB buffer is 0.05–0.10 M, and the pH value is 6–7; the concentration of the Tris-HCl buffer is 0.05–0.10 M, and the pH value is 7–8.

9. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, The coenzyme is NADP+ or NAD+.

10. The method for preparing chiral alcohols based on the sterically hindered asymmetric reduction of rigid ketones using aldehyde-ketone reductases and their mutants according to claim 1, characterized in that, The extractant used for extraction is at least one of ethyl acetate, methyl tert-butyl ether, methanol, dichloromethane, and n-butanol.

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