Carbonyl reductase mutant and application thereof in synthesis of R-lipoic acid chiral intermediate

Abstract

The invention belongs to the technical field of biological catalysis, and particularly relates to a carbonyl reductase mutant and application of the carbonyl reductase mutant in synthesis of an R-lipoic acid chiral intermediate (R)-6-hydroxy-8-chloro ethyl caprylate. According to the method, an unseparated and purified 6-oxy-8-chloro ethyl caprylate stock solution is used as a substrate, carbonyl reductase mutant coupled glucose dehydrogenase BtGDH is used as a catalyst, and the (R)-6-hydroxy-8-chloro ethyl caprylate is asymmetrically synthesized under mild conditions (20-40 DEG C, pH is 6-9). The (R)-6-hydroxy-8-chloro ethyl caprylate enzyme can be used for completely converting 500 g / L of 6-oxy-8-chloro ethyl caprylate stock solution within 8 hours to synthesize optically pure (R)-6-hydroxy-8-chloro ethyl caprylate. According to the method, the substrate does not need to be pretreated (solvent removal), the synthesis yield can reach 87%, and the method has the advantages of being high in space time yield, simple in process, green, environmentally friendly and the like.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to carbonyl reductase mutants and their application in the enzymatic asymmetric synthesis of the chiral intermediate (R)-6-hydroxy-8-chlorooctanoate. Background Technology

[0002] α Alpha-lipoic acid (α-LA) is an important pharmaceutical intermediate and antioxidant with antioxidant, neuroprotective, energy metabolism-promoting, blood glucose-balancing, and cardiovascular-protective effects. It is widely used in the fields of health, biomedicine, and cosmetics. α-LA exists in two configurations, namely (R). α-LA and (S)-α-LA, of which only (R) Only α-LA possesses physiological activity. Because the existing α-LA chemical synthesis processes do not involve the selective synthesis of chiral precursors, (R) α-LA is often prepared by chemically synthesized racemic α-LA through a three-step chemical resolution process, which greatly improves (R). The synthesis cost of α-LA is five times that of its racemic counterpart, therefore most commercially available products are still racemic. However, to reduce the burden on the human body's metabolism of (S)-α-LA, it is necessary to efficiently prepare optically pure (R)-LA. α-LA has significant application value and broad market prospects. The specific configuration of α-LA is determined by the configuration of its chiral precursor, ethyl 6-hydroxy-8-chlorooctanoate (ECHO), therefore, the asymmetric synthesis of (R)-ECHO becomes (R). Cost reduction and efficiency improvement in α-LA synthesis is a research hotspot.

[0003] With the continuous advancement of research on carbonyl reductases, the asymmetric reduction synthesis of (R)-ECHO using ethyl 6-oxo-8-chlorooctanoate (ECCO) has become increasingly mature. CN11004119A utilizes a mutant of the carbonyl reductase CpAR2, with the addition of 0.1 mM NADP... +Under conditions of 5% (v / v) dimethyl sulfoxide, catalysis of 110 g / L (0.5 M) ECCO yielded (R)-ECHO with 85% yield and >99% ee value after 4 h. CN 115948356B discloses a carbonyl reductase mutant SsCRL-211H / V127A / L135I, which, when co-expressed with GDH, can asymmetricly reduce 2 M ECCO to obtain (R)-ECHO with 90% yield and 99% ee value within 3 h. Although the reported substrate loading is sufficient for large-scale production, the carbonyl reductases used are all intolerant to organic solvents, requiring the removal of organic solvents that stabilize the substrate structure before catalysis, resulting in poor compatibility with upstream chemical reactions. However, the substrate after solvent removal is unstable and needs to be immediately introduced into the bioreduction process, which necessitates a strict pretreatment process for the biocatalytic reaction. Summary of the Invention

[0004] To address the aforementioned issues, this invention utilizes protein engineering technology to obtain a carbonyl reductase mutant capable of efficiently and asymmetrically reducing unpurified ethyl 6-oxo-8-chlorooctanoate (ECCO) stock solution (ECCO stock solution is: chemically prepared ECCO dissolved in dichloromethane) to synthesize ethyl (R)-6-hydroxy-8-chlorooctanoate [(R)-ECHO]. When the mutant is applied to the biosynthesis of (R)-ECHO, the ECCO stock solution loading can reach up to 500 g / L, and the conversion rate can reach 99% after 8 hours of reaction, with an ee value >99%.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a carbonyl reductase mutant, which is obtained by mutating amino acid residues based on AxSDR (amino acid sequence as shown in SEQ ID NO. 3, DNA sequence as shown in SEQ ID NO. 4). Specifically, the carbonyl reductase mutant is obtained by mutating any two, three, four, five, or six amino acid residues at any of the following positions on the amino acid sequence shown in SEQ ID NO. 3: G94L, H145C, S153I, Y188C, M198L, L206C.

[0006] In some embodiments, the carbonyl reductase mutant is the six-point mutant AxSDR. mu6 It is obtained by mutating the amino acid residues G94L / H145C / S153I / Y188C / M198L / L206C on the amino acid sequence shown in SEQ ID NO. 3.

[0007] In a second aspect, the present invention provides a recombinant expression vector containing the coding gene for the carbonyl reductase mutant as described in the first aspect and the coding gene for glucose dehydrogenase BtGDH. The DNA sequence of the glucose dehydrogenase BtGDH encoding gene is shown in SEQ ID NO. 2.

[0008] In some embodiments, the encoding genes of glucose dehydrogenase BtGDH and the carbonyl reductase mutant as described in the first aspect are recombined on an expression vector to obtain an expression plasmid; preferably, the expression plasmid is a pET series plasmid; more preferably, it is a pET-30a plasmid.

[0009] In a third aspect, the present invention provides a co-expression engineered bacterium, which is obtained by transforming a recombinant expression vector as described in the second aspect into a host bacterium.

[0010] Preferably, the host bacteria is Escherichia coli; more preferably, it is Escherichia coli BL21(DE3).

[0011] In a third aspect, the present invention provides a method for synthesizing (R)-ECHO, using ethyl 6-oxo-8-chlorooctanoate (ECCO) stock solution as substrate and glucose as co-substrate, and catalyzing the carbonyl reductase mutant coupled with glucose dehydrogenase BtGDH as described in claim 1 or 2 to synthesize (R)-ECHO. The amino acid sequence of the glucose dehydrogenase BtGDH is shown in SEQ ID NO. 1.

[0012] In some embodiments, coenzyme NAD is also added during the enzyme reaction. + .

[0013] In some embodiments, the method for synthesizing (R)-ECHO further includes (R)-ECHO purification: the reaction solution is extracted three times with an equal volume of ethyl acetate, the organic phases are combined, and the solution is concentrated under reduced pressure to obtain an oily liquid, which is (R)-ECHO.

[0014] Furthermore, the carbonyl reductase mutant and glucose dehydrogenase BtGDH are derived from whole cells or cell lysates of recombinant cells co-expressing the carbonyl reductase mutant and glucose dehydrogenase BtGDH using genetic engineering methods, or from whole cells or cell lysates of recombinant cells each expressing the carbonyl reductase mutant and glucose dehydrogenase BtGDH.

[0015] As one possible implementation, further, the carbonyl reductase mutant is used to synthesize (R)-ECHO via whole-cell reduction reaction: the reaction system includes the substrate ECCO stock solution, the co-substrate glucose, and the coenzyme NAD. +Wet cells of the mutant; stir the reaction at 30°C and pH 6.5-7.0 until the reaction is complete.

[0016] (R)-ECHO purification: The reaction solution was extracted three times with equal volumes of ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain an oily liquid, which is (R)-ECHO with a purity and ee value of >99%.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The biocatalyst provided by this invention can tolerate the solvent (dichloromethane) in the ECCO substrate stock solution. After ECCO is prepared by chemical method, the enzyme catalytic reaction can be carried out without separation and purification, eliminating the substrate pretreatment process and avoiding the problem of pure substrate being easily deteriorated. It has the advantage of being compatible with upstream processes. Attached Figure Description

[0018] Figure 1 AxSDR, a carbonyl reductase mutant mu6 HPLC analysis results of the reaction solution.

[0019] Figure 2 The results are HPLC analysis of (R)-ECHO products. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, 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.

[0021] This invention provides a carbonyl reductase mutant, wherein the carbonyl reductase mutant is formed by any two, three, four, five, or six amino acid residue mutations at the following positions in the amino acid sequence shown in SEQ ID NO. 3: G94L, H145C, S153I, Y188C, M198L, L206C.

[0022] Furthermore, this invention also provides a method for synthesizing (R)-ECHO, using ECCO stock solution (ECCO stock solution is: ECCO prepared by chemical method dissolved in dichloromethane) as substrate, glucose as co-substrate, and NAD+ as a substrate. + As a coenzyme, (R)-ECHO was synthesized under the catalysis of the carbonyl reductase mutant coupled with glucose dehydrogenase BtGDH.

[0023] The amino acid sequence and DNA sequence of glucose dehydrogenase BtGDH are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0024] (R)-ECHO purification: The reaction solution was extracted three times with equal volumes of ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain an oily liquid, which is (R)-ECHO with a purity and ee value of >99%.

[0025] Example 1: Construction of co-expressed engineered bacteria Using the synthesized AxSDR encoding gene (sequence shown in SEQ ID NO.4) as a template, and F-F1 and F-R1 as primers, PCR amplification was performed using KOD one DNA polymerase to obtain... axsdr A gene fragment (804 bp) was obtained. Simultaneously, using the synthesized BtGDH encoding gene (sequence shown in SEQ ID NO.2) as a template, and with F-F2 and F-R2 as primers, PCR amplification was performed using KOD one DNA polymerase to obtain... btgdh Gene fragment (825 bp). axsdr Gene fragments and btgdh Using the gene fragment as a template, and F-F1 and F-R2 as primers, PCR amplification was performed using KOD one DNA polymerase to obtain... axsdr-btgdh Gene fragment (1610 bp). Using plasmid pET30a as a template and VF and VR as primers, PCR amplification was performed using KOD one DNA polymerase to obtain the backbone fragment (5368 bp). Homologous recombination enzyme was then used to... axsdr-btgdh Homologous recombination of gene fragments and backbone fragments was performed, followed by incubation at 37°C for 30 min, and then transferred into... E. coli BL21(DE3) competent cells were plated on LB agar plates containing 50 mg / L kanamycin. Single colonies were picked and colony PCR was performed using primers F-F1 and F-R2. Transformants that amplified a fragment of approximately 1600 bp were considered positive clones. Positive clones were sequenced, and those confirmed to be positive were identified as co-expression engineered bacteria. E. coli BL21(DE3)(pET30a-AxSDR-BtGDH). The primer information is shown in Table 1 below.

[0026] Table 1. Primer Information

[0027] Example 2: Construction of mutants Using recombinant plasmid pET30a-AxSDR-BtGDH or its mutant as a template, circular plasmid expression plasmids were amplified by KOD one DNA polymerase. The primers used are shown in Table 1. The amplification program was: 98℃, 3 min; 98℃, 10 s, 60℃, 20 s, 68℃, 40 s, 30 cycles; 68℃, 2 min. After PCR, 0.3 U DMT was added to the reaction system, and the reaction was carried out at 37℃ for 1 h to digest the template. After digestion, 3 µL of the digestion product was transferred into… E. coli BL21(DE3) competent cells were spread on LB agar plates containing 50 mg / L kanamycin resistance.

[0028] Example 3: Mutant Screening Single colonies from the resistance plates in the site-directed mutagenesis experiment were picked and inoculated into 1 mL of LB liquid medium containing 50 mg / L kanamycin. The culture was incubated at 200 rpm and 37°C for 8 h to obtain the primary seed culture. 0.5 mL of the primary seed culture was inoculated into 25 mL of fresh resistant LB liquid medium and incubated at 200 rpm and 37°C for 2–3 h to obtain the secondary culture. IPTG was added to the secondary culture to a final concentration of 0.1 mM, and the culture was transferred to 25°C and 200 rpm for another 12 h. After incubation, the fermentation broth was centrifuged (12000 rpm, 3 min), the supernatant was discarded, and the bacterial resuspended in phosphate buffer (100 mM, pH 7.0) to a concentration of 100 mg / mL.

[0029] Add 50% (v / v) of the above whole-cell resuspension, 75 mM glucose, and 3% (v / v) ECCO stock solution to an EP tube, and bring the volume to 500 μL with water. Incubate the mixture at 37°C and 200 rpm for 2 h, then add an equal volume of ethyl acetate and invert the tube 8-9 times. Centrifuge (12000 rpm, 1 min), collect the upper organic phase, add an appropriate amount of anhydrous sodium sulfate to remove water, and then perform GC detection. The chromatographic column was a CP-Chirasil Dex CB (25 m, 0.32 mm, 0.25 μm, Agilent, USA), with nitrogen 0.1 MPa, hydrogen 0.1 MPa, air 0.1 MPa, injector temperature 240°C, column oven temperature 150°C, detector temperature 240°C, and injection volume 1 μL. Calculate the conversion rate of the substrate ECCO based on the peak areas of the substrate and product, and calculate the enantiomeric excess value ee based on the peak areas of the two configurations of the product. The conversion rates of ECCO and the optical purity of the products for each mutant are shown in Table 2. The optimal six-point mutant, G94L / H145C / S153I / Y188C / M198L / L206C (Mu6), was finally obtained. It completely converted 3% ECCO stock solution within two hours, producing (R)-ECHO with an ee value >99% (see attached table). Figure 1 (As shown).

[0030] Table 2. Mutant screening

[0031] Example 4: High-density fermentation of Mu6 engineered bacteria mutant engineered strains E. coli BL21(DE3)(pET30a-AxSDR mu6-BtGDH was activated on LB agar plates containing 50 mg / L kanamycin. After single colonies grew, single colonies of the engineered bacteria were picked and inoculated into 150 mL of LB medium containing kanamycin. The culture was carried out at 37°C and 200 rpm for 12 h to obtain the seed culture. The seed culture was then inoculated into a 5 L fermenter (containing 3 L of liquid, 10 g / L glycerol, 15 g / L yeast extract, 4 g / L K2HPO4·3H2O, 2.24 g / L NaH2PO4·2H2O, 3 g / L NaCl, 2.5 g / L (NH4)2SO4, 2.1 g / L citric acid, 2 g / L glucose, 0.49 g / L MgSO4·7H2O, and 0.3 g / L FeSO4) to start fermentation. The stirring speed was set to 300 rpm, and the temperature to 37℃. During fermentation, the pH was controlled at around 7.0 by automatically adding NH4OH (25%, v / v). In the early stages of fed-batch fermentation, dissolved oxygen (DO) slowly decreased as the cells proliferated. At this point, the stirring speed was increased to maintain DO at around 30%, with an upper limit of 600 rpm. When nutrients were depleted, dissolved oxygen and pH spiked. At this point, fed-batch fermentation (a mixture of 110 g / L yeast extract and 600 g / L glycerol) was initiated, and the DO was maintained at around 15% by adjusting the feeding speed. When OD... 600 When the concentration reaches approximately 60%, IPTG at a final concentration of 0.3 mM is added to induce the expression of the target gene. After fermentation for 25 h, the fermentation broth is centrifuged (6000 rpm, 10 min) to obtain 550 g of wet cells.

[0032] Example 5: Asymmetric synthesis of (R)-ECHO (10% v / v substrate stock solution) Add 10 mL of ECCO stock solution, 3.2 g of glucose, and 6.6 mg of NAD to a 250 mL beaker. + Add 5 g of wet Mu6 cells and bring the volume to 100 mL with water. Initiate the reaction by magnetic stirring at 30°C. During the reaction, control the pH of the reaction solution to approximately 6.5-7.0 by adding 1 M NaOH solution dropwise. Perform GC analysis periodically to monitor the reaction progress. After 2 h of reaction, the substrate was completely converted to (R)-ECHO (>99% ee).

[0033] Example 6: Asymmetric synthesis of (R)-ECHO (20% v / v substrate stock solution) Add 20 mL of ECCO stock solution, 6.4 g of glucose, and 6.6 mg of NAD to a 250 mL beaker. +Add 5 g of wet Mu6 cells and bring the volume to 100 mL with water. Initiate the reaction by magnetic stirring at 30°C. During the reaction, control the pH of the reaction solution to approximately 6.5-7.0 by adding 1 M NaOH solution dropwise. Perform GC analysis periodically to monitor the reaction progress. After 3 h of reaction, the substrate was completely converted to (R)-ECHO (>99% ee).

[0034] Example 7: Asymmetric synthesis of (R)-ECHO (30% v / v substrate stock solution) Add 30 mL of ECCO stock solution, 9.6 g of glucose, and 6.6 mg of NAD to a 250 mL beaker. + Add 5 g of wet Mu6 cells and bring the volume to 100 mL with water. Initiate the reaction by magnetic stirring at 30°C. During the reaction, control the pH of the reaction solution to approximately 6.5-7.0 by adding 1 M NaOH solution dropwise. Perform GC analysis periodically to monitor the reaction progress. After 5 h of reaction, the substrate was completely converted to (R)-ECHO (>99% ee).

[0035] Example 8: Asymmetric synthesis of (R)-ECHO (40% v / v substrate stock solution) Add 40 mL of ECCO stock solution, 12.8 g of glucose, and 6.6 mg of NAD to a 250 mL beaker. + Add 5 g of wet Mu6 cells and bring the volume to 100 mL with water. Initiate the reaction by magnetic stirring at 30°C. During the reaction, control the pH of the reaction solution to approximately 6.5-7.0 by adding 1 M NaOH solution dropwise. Perform GC analysis periodically to monitor the reaction progress. After 8 h of reaction, the substrate was completely converted to (R)-ECHO (>99% ee).

[0036] Example 9: Asymmetric synthesis of (R)-ECHO (50% v / v substrate stock solution) Add 50 mL of ECCO stock solution, 16.0 g of glucose, and 6.6 mg of NAD to a 250 mL beaker. + Add 5 g of wet Mu6 cells and bring the volume to 100 mL with water. Initiate the reaction by magnetic stirring at 30°C. During the reaction, control the pH of the reaction solution to approximately 6.5-7.0 by adding 1 M NaOH solution dropwise. Perform GC analysis periodically to monitor the reaction progress. After 11 h of reaction, the substrate was completely converted to (R)-ECHO (>99% ee).

[0037] Example 10: Preparation of (R)-ECHO (50% v / v substrate stock solution, 2 L system) Add 1 L of ECCO stock solution, 320 g of glucose, and 132 mg of NAD to a 3 L beaker. + Add 100 g of wet Mu6 cells and bring the volume up to 2 L with water. Initiate the reaction by mechanical stirring at 30°C. During the reaction, maintain the pH of the reaction solution at approximately 6.5-7.0 by adding 1 M NaOH solution dropwise. Perform GC analysis periodically to monitor the reaction progress. After 8 h of reaction, the substrate was completely converted to (R)-ECHO (>99% ee, ≥99% conv.). Extract three times with 2 L of ethyl acetate, collect the upper organic phase, and concentrate under reduced pressure to remove the solvent, finally obtaining 132 g of (R)-ECHO, with a yield of 87% and an ee value >99% (see attached figure). Figure 2 (As shown).

[0038] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is formed by any two, three, four, five, or six amino acid residue mutations at the following positions in the amino acid sequence shown in SEQ ID NO. 3: G94L, H145C, S153I, Y188C, M198L, L206C.

2. The carbonyl reductase mutant according to claim 1, characterized in that, The carbonyl reductase mutant is the six-point mutant AxSDR. mu6 It is obtained by mutating the amino acid residues G94L / H145C / S153I / Y188C / M198L / L206C on the amino acid sequence shown in SEQ ID NO.

3.

3. A recombinant expression vector, characterized in that, Contains the coding gene for the carbonyl reductase mutant as described in claim 1 or 2 and the coding gene for glucose dehydrogenase BtGDH. The DNA sequence of the glucose dehydrogenase BtGDH encoding gene is shown in SEQ ID NO.

2.

4. The recombinant expression vector according to claim 3, characterized in that, The encoding genes for glucose dehydrogenase BtGDH and the carbonyl reductase mutant as described in claim 1 were recombined on the pET-30a plasmid.

5. A co-expression engineered bacterium, characterized in that, The co-expression engineered bacteria are obtained by converting the recombinant expression vector as described in claim 3 or 4 into host bacteria.

6. The co-expression engineered bacteria according to claim 5, characterized in that, The host bacterium is Escherichia coli BL21(DE3).

7. A method for synthesizing (R)-ECHO, characterized in that, (R)-ECHO was synthesized using ethyl 6-oxo-8-chlorooctanoate stock solution as substrate and glucose as co-substrate, under the catalysis of carbonyl reductase mutant coupled with glucose dehydrogenase BtGDH as described in claim 1 or 2. The amino acid sequence of the glucose dehydrogenase BtGDH is shown in SEQ ID NO.

1.

8. The method for synthesizing (R)-ECHO according to claim 7, characterized in that, During the enzyme reaction, coenzyme NAD is also added. + .

9. The method for synthesizing (R)-ECHO according to claim 7, characterized in that, It also includes (R)-ECHO purification: the reaction solution is extracted three times with equal volumes of ethyl acetate, the organic phases are combined, and the solution is concentrated under reduced pressure to obtain an oily liquid, which is (R)-ECHO.

10. The method for synthesizing (R)-ECHO according to claim 7, wherein the carbonyl reductase mutant and glucose dehydrogenase BtGDH are derived from whole cells or cell lysates of recombinant cells co-expressing the carbonyl reductase mutant and glucose dehydrogenase BtGDH using genetic engineering methods, or are whole cells or cell lysates of recombinant cells each expressing the carbonyl reductase mutant and glucose dehydrogenase BtGDH.

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