A carbonyl reductase mutant and a method for catalyzing synthesis of chiral alpha, beta-unsaturated cyclobutanol compounds

Abstract

The present application relates to the technical field of bioengineering, and relates to a carbonyl reductase mutant (named as CR48-M12) and application of the carbonyl reductase mutant in biosynthesis of alpha, beta-unsaturated cyclobutanol compounds. The carbonyl reductase mutant has the function of catalyzing reduction of alpha, beta-unsaturated cyclobutanone C=O bond to synthesize alpha, beta-unsaturated cyclobutanol compounds, has high catalytic activity, is environment-friendly, is simple to operate, and has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a carbonyl reductase mutant and a method for catalytic synthesis of chiral α,β-unsaturated cyclobutanol compounds. Background Technology

[0002] Carbonyl reductase (CR) is a biological enzyme that specifically recognizes and catalyzes the asymmetric reduction of C=O bonds. It uses NAD(H) or NADP(H) as a cofactor and has broad substrate specificity. It can catalyze the reduction of endogenous or exogenous steroidal compounds and other aliphatic aldehydes, ketones, and various exogenous quinones derived from polycyclic aromatic hydrocarbons.

[0003] Chiral α,β-unsaturated cyclobutanols are widely found as molecular building blocks in various natural products and drugs, exhibiting corresponding anti-disease and anti-tumor effects. Furthermore, due to the ease of derivatization of their alkenyl and hydroxyl functional groups, they serve as important intermediates in organic synthesis. Simultaneously, chiral exocyclic allyl alcohols are also key intermediates in many stereooriented reactions, such as the Claisen rearrangement, epoxidation, and Diels-Alder reactions.

[0004] Currently, the synthesis methods for chiral α,β-unsaturated cyclobutanol are mainly divided into two categories: chemical catalysis and biocatalysis. Chemical catalysis primarily involves asymmetric catalytic hydrogenation, which typically relies on noble metal catalysts (such as iridium and ruthenium) and harsh conditions such as high reaction pressure or a hydrogen atmosphere. This not only increases production costs but also limits the feasibility of large-scale industrial applications. In contrast, biosynthetic pathways rely on enzyme catalysis or microbial fermentation systems, offering milder reaction conditions, lower energy consumption, fewer byproducts, and greater environmental friendliness, exhibiting significant green chemistry characteristics. Currently, only two studies have reported the asymmetric reduction of exocyclic α,β-unsaturated cyclic ketones using wild-type yeast, but these studies showed low catalytic activity and poor stereoselectivity.

[0005] Currently, the biosynthesis of important pharmaceutical intermediates such as structurally complex exocyclic α,β-unsaturated chiral cyclic alcohols has not been reported, and the development of chiral biocatalysis theory and methods remains limited. Therefore, based on the understanding of the stereoselective molecular recognition mechanism of asymmetric reduction reactions in biocatalysis, developing synthetic strategies for important chiral molecules such as exocyclic α,β-unsaturated chiral cyclic alcohols, developing precisely regulated chiral biocatalytic systems, and continuously exploring the modification and evolution of bioenzymes and elucidating the stereoselective molecular recognition mechanism are of great significance for promoting the development of green synthesis technologies for drug molecules. Summary of the Invention

[0006] The present invention aims to provide a carbonyl reductase mutant and a method for the enzyme-catalyzed synthesis of chiral α,β-unsaturated cyclobutanol. The present invention solves the technical problems of "low efficiency, poor selectivity and narrow substrate range" in the enzyme-catalyzed synthesis of chiral α,β-unsaturated cyclobutanol through a novel enzyme mutation design.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A carbonyl reductase mutant, named CR48-M12 reductase, has the amino acid sequence shown in SEQ ID No. 1 and the nucleotide sequence shown in SEQ ID No. 2. The amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID No. 3 and the nucleotide sequence is shown in SEQ ID No. 4.

[0008] An isolated nucleic acid molecule encoding the aforementioned carbonyl reductase mutant CR48-M12, the nucleotide sequence of which is shown in SEQ ID No. 2.

[0009] A genetically engineered bacterium containing the encoding gene for the carbonyl reductase mutant CR48-M12.

[0010] A biocatalyst, wherein the biocatalyst is a crude enzyme solution of the carbonyl reductase mutant CR48-M12.

[0011] Furthermore, the biocatalyst is prepared using the aforementioned genetically engineered bacteria.

[0012] If the carbonyl reductase mutant CR48-M12 described above is used as a biocatalyst, or if the crude enzyme solution of CR48-M12 is applied to prepare α,β-unsaturated cyclobutanol compounds of Formula II: Among them, substituent R 1 It is any one of 2-cyclohexyl, phenyl, 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-methoxyphenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 2-furanyl, 2-thienyl, 2-naphthyl, and 2-benzofuranyl.

[0013] A method for biosynthesizing α,β-unsaturated cyclobutanol compounds, using compound I as a substrate, in the presence of a coenzyme, a coenzyme cycling hydrogen donor, a solubilizer, a buffer solution, and a crude enzyme solution of the carbonyl reductase mutant according to claim 1, to generate α,β-unsaturated cyclobutanol of formula II via a biocatalytic reaction; wherein the coenzyme is NADP. +The coenzyme cycling hydrogen donor and solubilizer are isopropanol, and the buffer solution is PB buffer (100 mM, pH = 8.0). , where R 1 It is any one of 2-cyclohexyl, phenyl, 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-methoxyphenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 2-furanyl, 2-thienyl, 2-naphthyl, and 2-benzofuranyl.

[0014] Furthermore, the volume ratio of compound I to the crude enzyme solution of the carbonyl reductase mutant is 1:5.

[0015] Compared with existing technologies that use purely chemical routes to synthesize chiral α,β-unsaturated cyclobutanol compounds (Formula II), the method of this invention does not require harsh catalytic environments such as high temperature, high pressure, and H2, while reducing the use of complex and hazardous catalysts, thus reducing waste generation and being environmentally friendly. Furthermore, because the carbonyl reductase mutant of this invention exhibits excellent stereoselectivity, it can effectively improve the yield and optical purity of the product, demonstrating excellent industrialization potential. Attached Figure Description

[0016] Figure 1 , Figure 2 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳa; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 10.074 min (major). Figure 3 , Figure 4 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳb; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 16.075 min (major). Figure 5 , Figure 6The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳc; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 80 / 20, flow 0.8mL / min, λ = 254 nm, retention time = 20.359 min(major). Figure 7 , Figure 8 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳd; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 9.922 min (major). Figure 9 , Figure 10 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳe; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 13.640 min (major). Figure 11 , Figure 12 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳf; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 15.591 min (major). Figure 13 , Figure 14 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳg; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 11.383 min (major). Figure 15 , Figure 16 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳh; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 20.785 min (major). Figure 17 , Figure 18 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳi; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 10.994 min (major). Figure 19 , Figure 20 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳj; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 12.128 min (major). Figure 21 , Figure 22 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳk; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 8.246 min (major). Figure 23 , Figure 24 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳl; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 95 / 5, flow 0.8mL / min, λ = 254 nm, retention time = 19.844 min (major). Figure 25 , Figure 26 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳm; Chiral HPLC analysis: Chiralcel AD-H (Hexane / i-PrOH = 95 / 5, flow 0.8mL / min, λ = 254 nm, retention time = 20.814 min (major). Figure 27 , Figure 28 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-Ⅳn; Chiral HPLC analysis: Chiralcel AD-H (Hexane / i-PrOH = 90 / 10, flow 0.8mL / min, λ = 254 nm, retention time = 13.111 min (major). Figure 29 , Figure 30 The standard and the compound prepared by the reaction were respectively R Chromatogram of optical purity analysis of )-Ⅳo; Chiral HPLC analysis: Chiralcel OJ-H (Hexane / i-PrOH = 98 / 2, flow 0.8mL / min, λ = 254 nm, retention time = 32.384 min (major). Figure 31 , Figure 32 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-IIp; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 95 / 5, flow 0.8mL / min, λ = 254 nm, retention time = 15.940 min (major). Figure 33 , Figure 34 The standard and the compound prepared by the reaction were respectively S Chromatogram of optical purity analysis of )-IIq; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 95 / 5, flow 0.8mL / min, λ = 254 nm, retention time = 22.805 min (major). Figure 35 , Figure 36 The standard and the compound prepared by the reaction were respectively R Chromatogram of optical purity analysis of )-IIs; Chiral HPLC analysis: Chiralcel OD-H (Hexane / i-PrOH = 95 / 5, flow 0.8mL / min, λ = 254 nm, retention time = 17.106 min (major). Detailed Implementation

[0017] The following detailed description illustrates the specific implementation method: Example 1: In this implementation, homology analysis of TbSADH (from Thermoanaerobacterbrockii) was performed from the NCBI database, and the gene information of strain (CR48) with 70.66% homology to TbSADH was obtained. The gene information was synthesized by Jiangsu Saisofe Biotechnology Co., Ltd.

[0018] CR48 was engineered using site-directed saturation mutagenesis, and the mutagenic primers are shown in Table 1 below.

[0019] Table 1 Primer design for CR48-M12 single-point mutation

[0020] A plasmid containing the parental carbonyl reductase (CR48) gene was extracted and used as a DNA template (the amino acid sequence of the wild-type carbonyl reductase is shown in SEQ ID No. 3, and the nucleotide sequence is shown in SEQ ID No. 4). PCR amplification was performed using the primers described above, and the amplification system is shown in Table 2 below.

[0021] Table 2 PCR amplification system

[0022] PCR amplification procedure: Step 1, pre-denaturation at 95℃ for 5 min; Step 2, denaturation at 98℃ for 10 s, annealing at 60℃ for 5 s, for 30 cycles; Step 3, extension at 72℃ for 10 min, cool to 4℃, and end the reaction.

[0023] After the PCR reaction was completed, 10×DpnI buffer (5.2 μL) and DpnI enzyme (1.8 μL) were added to the product and digested at 37°C for 3-5 h. The digested PCR product was then purified and recovered according to the steps of a commercially available purification and recovery kit, and transformed into *E. coli* BL21 (DE3) competent cells by a 90-second heat shock at 42°C. After recovery and culture at 37°C and 150 rpm for 1 h, the cells were evenly spread on LB agar containing kanamycin sulfate and cultured overnight at 37°C. Single colonies were then picked, fermented, and induced to obtain cells containing the carbonyl reductase CR48 mutant. These cells were then used as a biocatalyst to investigate the conversion rate and optical purity of α,β-unsaturated cyclobutanol compounds, yielding the mutant CR48-M12 (conversion rate >95%, ee >99%).

[0024] Example 2: This example provides a method for obtaining CR48-M12 reductase, specifically including the following steps: CR48-M12 glycerol bacteria stored at -80℃ were activated on LB agar plates (incubated at 37℃ for 14-16 h). Single colonies were picked and placed in 5 mL of liquid LB medium containing the corresponding antibiotic (50 mg / mL Kan, final concentration 50 μg / mL). The medium was incubated at 37℃ with shaking for 8-10 h. The next day, a 1% inoculum was transferred to 50 mL of fresh LB liquid medium containing antibiotics and cultured at 37℃ with shaking at 250 rpm.600 Add IPTG to a final concentration of 0.2 mM and incubate at 17℃ and 250 rpm for 12 h. After induction, centrifuge 50 mL of bacterial culture at 9000 rpm for 3 min to collect the bacterial cells, and resuspend the cells in PB buffer (100 mM, pH = 8.0).

[0025] Example 3: The wet cells of the strain cultured in Example 2 were suspended in 5 mL of PB buffer (100 mM, pH = 8.0) at a cell concentration of 10 g / L and sonicated under ice bath conditions (60% power, continuous for 2 s, intermittent for 3 s, continuous for 3 min) to obtain the required crude enzyme solution.

[0026] Substrate I (compound I) was dissolved in isopropanol to prepare a 125 mM substrate stock solution. In a 5 mL reaction system, 3.6 mL of PB buffer solution (100 mM, pH = 8.0), 1 mL of crude enzyme solution, and 0.2 mL of NADPH were added. + 0.2 mL of substrate stock solution (125 mM, 50 mM) and 0.2 mL of substrate stock solution (125 mM) were added to make a final substrate concentration of 5 mM. The reaction was carried out in a constant temperature shaker at 30 °C and 250 rpm for 12 h. After the reaction was completed, an equal volume of ethyl acetate was added for extraction (12000 rpm, 3 min). The results were analyzed by HPLC and are shown in Tables 3 and 4.

[0027] Table 3:

[0028] Table 4:

[0029] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A carbonyl reductase mutant, named CR48-M12, characterized in that: Its amino acid sequence is shown in SEQ ID No.

1.

2. An isolated nucleic acid molecule, characterized in that: The carbonyl reductase mutant of claim 1 is encoded, wherein the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No.

2.

3. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contain the encoding gene of the carbonyl reductase mutant as described in claim 1.

4. A biocatalyst, characterized in that: The biocatalyst is the crude enzyme solution of the carbonyl reductase mutant as described in claim 1.

5. The biocatalyst according to claim 4, characterized in that: It is prepared using the genetically engineered bacteria described in claim 3.

6. The hydroxyl reductase mutant of claim 1, or the biocatalyst of claim 4, can be used as a biocatalyst to prepare α,β-unsaturated cyclobutanol compounds of formula II: ,in, Substituent R 1 It is any one of 2-cyclohexyl, phenyl, 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-methoxyphenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 2-furanyl, 2-thienyl, 2-naphthyl, and 2-benzofuranyl.

7. A method for biosynthesizing α,β-unsaturated cyclobutanol compounds, characterized in that: Using compound I as a substrate, in the presence of a coenzyme, a coenzyme cycling hydrogen donor, a solubilizer, a buffer solution, and a crude enzyme solution of the carbonyl reductase mutant according to claim 1, a biocatalytic reaction is carried out to produce α,β-unsaturated cyclobutanol as shown in formula II; the coenzyme is NADP. + The coenzyme cycle hydrogen donor and cosolvent are isopropanol, and the buffer solution is PB buffer. , where R 1 It is any one of 2-cyclohexyl, phenyl, 2-methylphenyl, 2-methoxyphenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 3-methylphenyl, 3-methoxyphenyl, 3-fluorophenyl, 3-chlorophenyl, 3-bromophenyl, 4-methoxyphenyl, 4-chlorophenyl, 4-bromophenyl, 4-trifluoromethylphenyl, 2-furanyl, 2-thienyl, 2-naphthyl, and 2-benzofuranyl.

8. The method for biosynthesizing α,β-unsaturated cyclobutanol compounds according to claim 7, characterized in that: The volume ratio of compound I to the crude enzyme solution of the carbonyl reductase mutant is 1:5.

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