Carbonyl reductase mutants and their applications in the synthesis of posaconazole intermediates

Abstract

The present invention discloses a carbonyl reductase mutant and its application in the synthesis of posaconazole intermediates. The mutant protein is a non-natural protein and has significantly improved catalysis ( S )-2-benzyloxy-3-pentanone is reduced to form R The carbonyl reductase mutant strain with enhanced activity in the present invention is applied to the production process of posaconazole intermediates, and 100-200 g / L substrate can be completely converted to produce (2 S ,3 R )-2-benzyloxy-3-pentanol.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and enzyme engineering, and relates to a carbonyl reductase mutant and an application thereof in synthesizing a posaconazole intermediate. Background Art

[0002] Carbonyl reductases, also known as alcohol dehydrogenases, are a class of biocatalysts that reversibly catalyze the reduction of carbonyl groups to alcohols. To achieve catalytic activity, carbonyl reductases require a coenzyme to transfer hydrogen ions to the carbonyl substrate. During carbonyl reductase catalysis, the coenzyme is used in equal molar amounts to the substrate. To optimize the reaction economy, a corresponding coenzyme regeneration system is often incorporated into the carbonyl reductase catalysis. With the development of directed evolution techniques, carbonyl reductases are increasingly used in the preparation of chiral alcohols.

[0003] Posaconazole is a broad-spectrum triazole antifungal drug developed by Merck and first approved for marketing in 2005. It is a derivative of itraconazole and exerts a potent antifungal effect by inhibiting the biosynthesis of ergosterol in fungal cell membranes (targeting 14α-demethylase, CYP51). (2 S ,3 R )-2-benzyloxy-3-pentanol is a key intermediate in the synthesis of chiral modules ( Figure 1 ).

[0004] Currently, there are synthesized (2 S ,3 R )-2-benzyloxy-3-pentanol, such as using ( S )-2-benzyloxy-3-pentanone as substrate, using chiral catalyst ( S )-CBS and the reducing agent BH3 achieve the preparation of chiral alcohols (CN 103936564 A); the use of chemical catalysis inevitably involves the use of hazardous reagents, and the development of green and efficient catalytic technology is imperative. The present invention obtains the conversion ( S )-2-benzyloxy-3-pentanone mutant with significantly improved activity, which is (2 S ,3 R )-2-benzyloxy-3-pentanol lays the foundation for green biomanufacturing. Summary of the Invention

[0005] The present invention provides a carbonyl reductase mutant, its encoding gene and application, which improves the enzyme activity and substrate structure diversity, etc., so as to meet the needs of industrial use. Limosilactobacillus fastidiosusThe enzyme (LfCR, GenBank: WP_182581089.1) was functionally determined and directed evolution was performed to obtain mutants with significantly improved enzyme activity. S )-2-benzyloxy-3-pentanone to produce (2 S ,3 R )-2-benzyloxy-3-pentanol.

[0006] The present invention provides a mutant of carbonyl reductase, whose protein sequence has at least 90% identity with SEQ ID NO: 1, and the mutant is obtained by substitution mutation at one or more of positions V90, F93, I141, E142, L150, P185, Y187, T190, P191, L192, V193, Q196, L210, and H211 in the amino acid sequence of SEQ ID NO: 1 from positions 1 to 250.

[0007] Specifically, it includes one or more mutations in the following positions corresponding to the amino acid sequence shown in SEQ ID NO: 1:

[0008] The 90th valine (V) mutated to tryptophan (W), phenylalanine (F), and arginine (R); the 93rd phenylalanine (F) mutated to alanine (A), glycine (G), isoleucine (I), lysine (K), leucine (L), methionine (M), proline (P), arginine (R), and threonine (T); the 141st isoleucine (I) mutated to alanine (A), serine (S), threonine (T), valine (V), and asparagine (N); the 142nd glutamic acid (E) mutated to serine (S), threonine (T), and isoleucine (I); the 150th leucine (L) mutated to alanine (A), cysteine ​​(C), glycine (G), valine (V), serine (S), and threonine (T); the 185th proline (P) mutated to threonine (T), valine (V), and leucine (L); Tyrosine (Y) at position 7 mutated to alanine (A), glycine (G), and valine (V); threonine (T) at position 190 mutated to cysteine ​​(C), glycine (G), and leucine (L); proline at position 191 mutated to valine (V), threonine (T), and glycine (G); leucine (L) at position 192 mutated to cysteine ​​(C), glycine (G), phenylalanine (F), methionine (M), threonine (T), tryptophan (W), and proline (P); valine (V) at position 193 mutated to phenylalanine (F), tryptophan (W), and tyrosine (Y); and glutamine (Q) at position 196 mutated to asparagine (N) and serine (S); leucine (L) at position 210 mutated to alanine (A), valine (V), and isoleucine (I); and histidine (H) at position 211 mutated to tyrosine (Y) and isoleucine (I).

[0009] Preferably, it includes any one of the following mutations in the amino acid sequence corresponding to SEQ ID NO: 1:

[0010] The 90th valine (V) mutated to tryptophan (W),

[0011] The phenylalanine (F) at position 93 was mutated to lysine (K).

[0012] Leucine (L) at position 150 was mutated to alanine (A).

[0013] Tyrosine (Y) at position 187 mutated to alanine (A),

[0014] Leucine (L) at position 192 mutated to tryptophan (W),

[0015] The 90th valine (V) mutated to tryptophan (W), and the 93rd phenylalanine (F) mutated to lysine (K).

[0016] The 90th valine (V) mutated to tryptophan (W), and the 187th tyrosine (Y) mutated to alanine (A).

[0017] The 90th valine (V) mutated to tryptophan (W), and the 192nd leucine (L) mutated to tryptophan (W),

[0018] The 93rd phenylalanine (F) mutated to lysine (K), and the 187th tyrosine (Y) mutated to alanine (A).

[0019] The 93rd phenylalanine (F) mutated to lysine (K), and the 192nd leucine (L) mutated to tryptophan (W).

[0020] Tyrosine (Y) at position 187 mutated to alanine (A), and leucine (L) at position 192 mutated to tryptophan (W).

[0021] The 93rd phenylalanine (F) mutated to lysine (K), the 93rd phenylalanine (F) mutated to lysine (K), and the 187th tyrosine (Y) mutated to alanine (A).

[0022] The 93rd phenylalanine (F) mutated to lysine (K), the 93rd phenylalanine (F) mutated to lysine (K), and the 192nd leucine (L) mutated to tryptophan (W).

[0023] The phenylalanine (F) at position 93 was mutated to lysine (K), the tyrosine (Y) at position 187 was mutated to alanine (A), and the leucine (L) at position 192 was mutated to tryptophan (W).

[0024] The present invention also provides a nucleic acid encoding the mutant of the carbonyl reductase.

[0025] The present invention further provides a recombinant expression vector encoding the nucleic acid.

[0026] The present invention also provides a recombinant bacterium encoding the nucleic acid.

[0027] The present invention also provides a synthetic posaconazole intermediate (2 S ,3 R )-2-benzyloxy-3-pentanol, comprising the steps of:

[0028] The carbonyl reductase mutant protein is contacted with a reaction substrate, and a coenzyme circulation system is added to perform a catalytic reaction, thereby obtaining the (2 S ,3 R )-2-benzyloxy-3-pentanol.

[0029] Specifically, the pH of the reaction system is 6.0-8.0, the reaction time is 12-48 hours, and the reaction substrate concentration is 80-260 g / L.

[0030] Preferably, the pH of the reaction system is pH 6.5; and the reaction time is 15-36 hours.

[0031] Optionally, further comprising separating and purifying the (2 S ,3 R )-2-benzyloxy-3-pentanol step.

[0032] The carbonyl reductase mutant with enhanced activity in the present invention is applied to the production process of posaconazole intermediates, and 100-200 g / L substrate can be completely converted to produce (2 S ,3 R )-2-benzyloxy-3-pentanol, and therefore has more practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Posaconazole and (2 S ,3 R )-2-benzyloxy-3-pentanol structure.

[0034] Figure 2 Shows ( S )-2-benzyloxy-3-pentanone sodium borohydride reduction liquid phase spectrum.

[0035] Figure 3 The liquid phase spectrum of the LfCR-catalyzed conversion reaction is shown. DETAILED DESCRIPTION

[0036] The experimental techniques and methods used in this example are conventional unless otherwise specified. For example, in the following examples, where specific conditions are not specified, conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions recommended by the manufacturer are generally followed. Materials and reagents used in the examples were obtained through commercial channels unless otherwise specified.

[0037] Example 1: Construction of a carbonyl reductase mutant library and screening of mutants

[0038] 1. Screening of carbonyl reductase library

[0039] The conversion reaction was constructed by using a library of laboratory-preserved enzymes, phosphate buffered at pH 7.5): 20 mM (2.5 g / L) substrate, 5% DMSO, 50 mM (10 g / L) glucose, 5 U / mL glucose dehydrogenase (GDH), 10 g / L Escherichia coli expressing the carbonyl reductase gene, and the coenzyme NAD(P) + 0.3 g / L, react at 30℃ for 24 hours. Extract with equal volume of ethyl acetate and detect by chiral GC. Figure 2 The comparison shown in FIG. 1 shows that the carbonyl reductase LfCR, whose amino acid sequence is shown in SEQ ID NO: 1, has good stereoselectivity, such as Figure 3 shown.

[0040] 2. The First Round of Vitality Transformation

[0041] The relationship between LfCR and coenzyme NADP was obtained by RoseTTAFold All-Atom simulation (Science 384, 291 (2024)). + and substrate ( S )-2-benzyloxy-3-pentanone docking results.

[0042] An 8-Å range of amino acid positions centered on the substrate was analyzed, with S140 / Y153 / K157 representing the catalytic triad. Structural analysis was performed, and the following sites were selected for the construction of a mutation library: V90, G91, F93, I141, E142, L150, P185, Y187, T190, P191, L192, V193, Q196, L210, and H211. Mutation primers were designed using the degenerate codon NNK, and pET21a-LfCR was used as a template. Two-step PCR was performed using the high-fidelity polymerase Pfx.

[0043] 1. Construction of pET21a-LfCR plasmid

[0044] The wild-type amino acid sequence of carbonyl reductase is shown in SEQ ID NO: 1. The corresponding nucleotide sequence was fully synthesized and cloned into the restriction endonuclease sites NdeI and XhoI of the pET-21a vector to obtain the recombinant plasmid pET21a-LfCR, which was further transformed into the expression host. E. coli BL21 (DE3), pick the positive clones, and obtain the recombinant expression transformants E. coli BL21(DE3) / pET21a-LfCR.

[0045] 2. Construction of a carbonyl reductase mutant library

[0046] Using pET21a-LfCR as a template, amplification was performed using the primers in Table 1. Forward primer 20F was combined with reverse primers 90-93-R for PCR, and forward primer 100-F was combined with reverse primer 141-211-R for PCR. The PCR reaction system and reaction conditions were as follows:

[0047] 1) First step PCR reaction system and reaction conditions

[0048] Round 1: To a 25 μL PCR reaction system, add 15 ng of template, 12.5 μL of 2× PfxMix, and 0.5 μL of each of a pair of mutation primers (10 μM). Add sterile distilled water to 25 μL.

[0049] The first step of PCR reaction procedure: ① pre-denaturation at 98℃ for 2 min, ② denaturation at 98℃ for 30 sec, ③ annealing at Tm-5℃ for 30 sec, ④ extension at 72℃ for 30 sec, ⑤ final extension at 72℃ for 5 min. Steps ② to ④ were performed for a total of 30 cycles.

[0050] 2) Second-step PCR reaction system and reaction conditions

[0051] Round 2: To a 50 μL PCR reaction system, add 30 ng of template, 25 μL of 2× Pfx Mix, 1 μL of the first-round PCR product fragment and primer, and add sterilized distilled water to 50 μL.

[0052] The second step PCR reaction procedure was as follows: ① pre-denaturation at 98°C for 2 min, ② denaturation at 98°C for 30 sec, ③ annealing at 55°C for 30 sec, ④ extension at 72°C for 3 min, ⑤ final extension at 72°C for 10 min. Steps ② to ④ were repeated for a total of 25 cycles.

[0053] The PCR products obtained in the above steps were verified by agarose gel electrophoresis analysis and then digested with restriction endonuclease DpnI at 37°C for 2h. E. coli BL21 (DE3) competent cells were plated on plates containing 10 μg / mL ampicillin antibiotics and placed in a 37°C incubator for about 12 hours to grow single colonies and obtain a carbonyl reductase mutant library.

[0054] At the same time, the pET21a-LfCR mutation library plasmid was transferred into E. coli BL21 (DE3) competent cells were spread on plates containing 100 mg / mL ampicillin antibiotics and placed in a 37°C incubator for static culture for about 12 hours to grow single colonies and obtain a strain expressing the carbonyl reductase mutant.

[0055] Table 1. Primer sequences for the first round of modification

[0056]

[0057] 3. Inducible expression of carbonyl reductase mutants

[0058] The monoclonal colony obtained after the above step 2 was picked into 4 mL of LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) containing ampicillin (100 mg / L) and cultured overnight at 37°C and 200 rpm to obtain a culture medium. The culture medium was inoculated into the fermentation medium (LB liquid medium) at a 1% (v / v) inoculum and cultured in a shaker at 37°C and 200 rpm until the OD 600 The pH value was 0.6-0.8, IPTG was added to a final concentration of 0.1 mM, and the cells were induced at 25°C, 200 rpm on a shaker for 8-12 hours. The culture medium was centrifuged at 6000 g to collect the cells and crushed under high pressure to obtain a crude carbonyl reductase enzyme solution for subsequent enzyme activity assays.

[0059] 4. Screening of carbonyl reductase mutants

[0060] The screening method involves detecting the decrease in NADPH at 340 nm. The reaction consists of 10 mM substrate, 0.25 mg / mL NADPH, 10 μL crude enzyme solution, and potassium phosphate buffer to 200 μL. NADPH has a characteristic absorbance at 340 nm, and the decrease in absorbance at 340 nm is detected using a microplate reader. High enzyme activity indicates rapid NADPH consumption, resulting in a steeper slope of the decrease curve. Beneficial mutations identified through screening for increased enzyme activity include V90, F93, I141, E142, L150, P185, Y187, T190, P191, L192, V193, Q196, L210, and H211.

[0061] 3. The Second Round of Vitality Transformation

[0062] Based on the saturation mutagenesis results, sites with more obvious activity improvements were selected to construct combination mutants of single mutation sites. The resulting mutants were picked and cultured in a test tube containing 4 mL of LB medium. The activity of the expressed proteins was tested. The results of significant activity improvement are shown in Table 2. Mutants 8 and 10 had the highest activity, which was 75.9 and 85 times higher than that of the wild type.

[0063] Table 2. Relative activities of KaCR and its mutants towards substrates

[0064]

[0065] Example 2: Whole-cell catalytic synthesis of mutant 8 (2S ,3 R )-2-Benzyloxy-3-pentanol

[0066] Mutant 8 was obtained by inducing expression according to the method of Example 1, and the cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 100 mL potassium phosphate buffer (pH 7.0, 100 mM), 80 g / L ( S )-2-benzyloxy-3-pentanone, 10% / v DMSO, 100 g / L glucose, 0.3 g / L NADP + The reaction was stirred at 30°C for 12 hours with 30 U / mL GDH and 10 g / L wet cells. The pH was maintained at 7.0. HPLC analysis showed a conversion rate of 98% and a product desaturase value of 99.28%.

[0067] Example 3: Catalytic synthesis of (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0068] Mutant 9 was induced to express according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and the crude enzyme solution after disruption was used as a biocatalyst. Other conditions were the same as those of Example 2. The reaction time was 10 hours, the conversion rate was 97%, and the product de value was 99.59%.

[0069] Example 4: Whole-cell catalytic synthesis of mutant 10 (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0070] Mutant 10 was obtained by inducing expression according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 100 mL potassium phosphate buffer (pH 7.0, 100 mM), 160 g / L ( S )-2-benzyloxy-3-pentanone, 200 g / L glucose, 0.2 g / L NADP + The reaction was performed with 30 U / mL GDH and 10 mg / mL whole cells at 30°C for 15 hours, with the pH maintained at 7.0. HPLC analysis revealed a 97% conversion rate and a product desaturase of 99.67%.

[0071] Example 5. Whole-cell catalytic synthesis of mutant 10 (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0072] Mutant 10 was obtained by inducing expression according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 300 mL potassium phosphate buffer (pH 7.0, 100 mM), 140 g / L ( S )-2-benzyloxy-3-pentanone, 180 g / L glucose, 0.2 g / L NADP + The reaction was continued at 25°C for 15 hours with 30 U / mL GDH and 5 mg / mL wet cells. The pH was maintained at 7.0. HPLC analysis showed a conversion rate of 97% and a product desaturase of 99.78%.

[0073] Example 6: Whole-cell catalytic synthesis of mutant 10 (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0074] Mutant 10 was obtained by inducing expression according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 300 mL potassium phosphate buffer (pH 7.0, 100 mM), 140 g / L ( S )-2-benzyloxy-3-pentanone, 180 g / L glucose, 0.2 g / L NADP + The reaction was continued at 35°C for 10 hours with 30 U / mL GDH and 10 mg / mL wet cells. The pH was maintained at 7.0. HPLC analysis showed a conversion rate of 98% and a product desaturase of 99.77%.

[0075] Example 7: Whole-cell catalytic synthesis of mutant 8 (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0076] Mutant 8 was obtained by inducing expression according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 300 mL potassium phosphate buffer (pH 7.0, 100 mM), 120 g / L ( S )-2-benzyloxy-3-pentanone, 160 g / L glucose, 0.2 g / L NADP + The reaction was continued at 30°C for 10 hours with 30 U / mL GDH and 10 mg / mL wet cells. The pH was maintained at 7.0. HPLC analysis showed a conversion rate of 98% and a product desaturase of 99.79%.

[0077] Example 8: Whole-cell catalytic synthesis of mutant 10 (2 S,3 R )-2-Benzyloxy-3-pentanol

[0078] Mutant 10 was obtained by inducing expression according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 300 mL potassium phosphate buffer (pH 7.0, 100 mM), 180 g / L ( S )-2-benzyloxy-3-pentanone, 250 g / L glucose, 0.2 g / L NADP + The reaction was carried out at 30°C for 14 hours with 30 U / mL GDH and 10 mg / mL wet cells. The pH was maintained at 7.0. HPLC analysis showed a conversion rate of 99% and a product desaturase of 99.74%.

[0079] Example 9: Whole-cell catalytic synthesis of mutant 10 (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0080] Mutant 10 and glucose dehydrogenase were engineered and expressed according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 300 mL potassium phosphate buffer (pH 7.0, 100 mM), 200 g / L ( S )-2-benzyloxy-3-pentanone, 300 g / L glucose, 0.2 g / L NADP + The mixture was stirred at 30°C for 20 hours with 10 mg / mL wet cells. The pH was maintained between 6.0 and 7.0. HPLC analysis revealed a 99% conversion rate and a product desaturase (DE) of 99.78%.

[0081] Example 10: Whole-cell catalytic synthesis of mutant 10 (2 S ,3 R )-2-Benzyloxy-3-pentanol

[0082] Mutant 10 and glucose dehydrogenase were engineered and expressed according to the method of Example 1. The cells were collected by centrifugation (6000 rpm) and used as biocatalysts. The cells were resuspended in 300 mL potassium phosphate buffer (pH 7.0, 100 mM), 260 g / L ( S )-2-benzyloxy-3-pentanone, 380 g / L glucose, 0.2 g / L NADP +The mixture was stirred at 30°C for 22 hours with 15 mg / mL wet cells. The pH was maintained between 6.0 and 7.0. HPLC analysis revealed a conversion rate of 98.6% and a product dex value of 99.79%.

Claims

1. A mutant of carbonyl reductase, characterized in that The protein sequence of the mutant corresponds to the carbonyl reductase represented by GenBank accession number WP_182581089.1, and only the following mutations are present: Valine at position 90 was mutated to tryptophan; Phenylalanine at position 93 was mutated to lysine; Leucine at position 150 was mutated to alanine; Tyrosine at position 187 was mutated to alanine; Leucine at position 192 was mutated to tryptophan; The valine at position 90 was mutated to tryptophan, and the phenylalanine at position 93 was mutated to lysine; The valine at position 90 was mutated to tryptophan, and the tyrosine at position 187 was mutated to alanine; The valine at position 90 was mutated to tryptophan, and the leucine at position 192 was mutated to tryptophan; Phenylalanine at position 93 mutated to lysine, and tyrosine at position 187 mutated to alanine; Phenylalanine at position 93 was mutated to lysine, and leucine at position 192 was mutated to tryptophan; Tyrosine at position 187 was mutated to alanine, and leucine at position 192 was mutated to tryptophan; The valine at position 90 was mutated to tryptophan, the phenylalanine at position 93 was mutated to lysine, and the tyrosine at position 187 was mutated to alanine; Valine at position 90 is mutated to tryptophan, and phenylalanine at position 93 is mutated to lysine and leucine at position 192 is mutated to tryptophan; or, The valine at position 90 was mutated to tryptophan, and the tyrosine at position 187 was mutated to alanine and the leucine at position 192 was mutated to tryptophan.

2. A nucleic acid encoding the mutant of carbonyl reductase according to claim 1.

3. A recombinant expression vector containing the encoding nucleic acid according to claim 2.

4. A recombinant bacterium containing the encoding nucleic acid according to claim 2.

5. A synthetic intermediate of posaconazole (2 S ,3 R )-2-benzyloxy-3-pentanol, characterized in that Including steps: The carbonyl reductase mutant of claim 1 is contacted with a reaction substrate (S)-2-benzyloxy-3-pentanone, and a coenzyme circulation system is added to carry out a catalytic reaction, thereby obtaining the (2 S ,3 R )-2-benzyloxy-3-pentanol.

6. The method according to claim 5, wherein The pH of the reaction system is 6.0-8.0, the reaction time is 12-48 hours, and the reaction substrate concentration is 80-260 g / L.

7. The method according to claim 6, wherein The pH of the reaction system is 6.5; the reaction time is 15-36 hours.

8. The method according to any one of claims 5 to 7, wherein: Also includes separating and purifying the (2 S ,3 R )-2-benzyloxy-3-pentanol step.

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