Alcohol dehydrogenase mutants and their use in the synthesis of symmetric chiral diols

Abstract

The application discloses an ethanol dehydrogenase mutant and application thereof in synthesis of symmetrical chiral diols, wherein the ethanol dehydrogenase mutant is obtained by single mutation or multiple mutations of amino acids at positions 74, 187, 210, 211 and 352 in the amino acid sequence of ethanol dehydrogenase shown in SEQ ID NO. 2 from Candida orthopsilosis Co. The application utilizes ethanol dehydrogenase to catalyze synthesis of symmetrical chiral diols from prochiral symmetrical diketones, and the method has the advantages of high substrate concentration, high stereoselectivity, mild reaction, simple operation process and environmental friendliness, is more suitable for industrial scale production, and solves the problems in the prior art, such as low substrate concentration, severe reaction condition, relatively complex reaction process difficult to control, many by-products and environmental unfriendliness.

Description

(I)TECHNICAL FIELD

[0001] The present application belongs to the field of biological chemical industry, and particularly relates to an ethanol dehydrogenase mutant, a gene, a recombinant vector containing the gene, and a recombinant genetically engineered bacterium prepared by transformation of the recombinant vector, and an application of the recombinant genetically engineered bacterium in catalyzing synthesis of symmetrical chiral diols (chiral mother nucleus of phosphine ligand) from symmetrical chiral diketones. (II)BACKGROUND

[0002] Chiral phosphine ligand is one of the most widely used ligands in asymmetric catalysis, and the dihedral angle of the biphosphine ligand plays an important role in asymmetric catalysis. A proper dihedral angle is crucial for achieving excellent enantioselectivity. A series of chiral side chain biphosphine ligands are synthesized from central chiral compounds such as methyl sulfonate or p-toluenesulfonate of symmetrical chiral diols (PNAS 2004, 16, 5815-5820. J. Am. Chem. Soc. 2006, 128, 5955-5965.), and the efficient transmission from central chirality to axial chirality is successfully achieved in the synthesis process, and single configuration chiral biphosphine ligands can be obtained without separation, and are successfully applied in asymmetric hydrogenation reaction. Therefore, symmetrical chiral diols have great application prospect in the synthesis of biphosphine ligands. At present, the commonly used process for synthesizing symmetrical chiral diols is to synthesize symmetrical chiral diols from prochiral ketones by metal catalytic hydrogenation, which inevitably increases the complexity and cost of the reaction. In addition, due to the problem of reaction control, many by-products are produced, resulting in low ee value and de value of the product. For example, (2S, 3S)-butanediol and (2R, 3S)-butanediol produced in the process of synthesizing (2R, 3R)-butanediol by metal catalytic hydrogenation cannot be effectively controlled, resulting in too low ee and de values of the product. (III)SUMMARY

[0003] The present application aims to provide an ethanol dehydrogenase mutant and its application in synthesis of symmetrical chiral diols. The present application uses ethanol dehydrogenase to catalyze synthesis of symmetrical chiral diols from prochiral symmetrical diketones, and the method has high substrate concentration, high stereoselectivity, mild reaction, simple operation process, and environmental friendliness, and is more suitable for industrial scale production, and solves the problems of low substrate concentration, severe reaction conditions, complex reaction process, many by-products, and environmental unfriendliness in the prior art.

[0004] The technical scheme adopted by the present application is as follows:

[0005] The present invention provides a highly active and stereoselective alcohol dehydrogenase mutant, which is obtained by subjecting the amino acids at positions 74, 187, 210, 211 and 352 of the alcohol dehydrogenase amino acid sequence shown in SEQ ID NO. 2 from Candida orthopsilosis Co to single or multiple mutations.

[0006] Furthermore, the alcohol dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following: (1) histidine at position 74 is mutated into cysteine ​​(H74C); (2) threonine at position 187 is mutated into leucine (T187L); (3) glycine at position 210 is mutated into lysine (G210K); (4) leucine at position 211 is mutated into alanine (L211A); (5) leucine at position 352 is mutated into valine (L352V); (6) histidine at position 74 is mutated into cysteine, and glycine at position 210 is mutated into lysine (H74C / G210K); (7) histidine at position 74 mutated to cysteine, threonine at position 187 mutated to leucine, glycine at position 210 mutated to lysine, and leucine at position 211 mutated to alanine (H74C / T187L / G210K / L211A); (8) histidine at position 74 mutated to cysteine, glycine at position 210 mutated to lysine, leucine at position 211 mutated to alanine, and leucine at position 352 mutated to valine (H74C / G210K / L211A / L352V).

[0007] The present invention also relates to the alcohol dehydrogenase encoding gene, a recombinant expression vector containing the encoding gene, and a recombinant genetically engineered bacterium constructed with the recombinant expression vector; the recombinant vector of the present invention is not limited, as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and eukaryotic cells, and the vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., preferably using the pET-28a(+) plasmid as the expression vector and Escherichia coli as the expression host (Escherichia coli BL21 cells or Escherichia coli DH5α).

[0008] The present invention provides an application of the alcohol dehydrogenase mutant in catalyzing the synthesis of symmetrical chiral diols from prochiral diketones. The application method comprises the following steps: using wet bacteria obtained by fermentation and culturing recombinant genetically engineered bacteria expressing the alcohol dehydrogenase mutant as a catalyst, using the prochiral diketone as a substrate, and using an organic alcohol as a reaction medium to form a reaction system; carrying out the reaction at 200 rpm and 30-37° C.; obtaining a reaction solution containing the symmetrical chiral diol after the reaction is completed; and separating and purifying the reaction solution to obtain the symmetrical chiral diol.

[0009] Furthermore, the organic alcohol includes isopropyl alcohol.

[0010] Further, the substrate includes 2,3-butanedione, 2,4-pentanedione, 2,5-hexanedione.

[0011] Further, the catalyst is used in an amount of 10-200 g / L (preferably 50 g / L) based on the weight of the wet bacteria, and the substrate is added in a concentration of 200-500 g / L (preferably 250-350 g / L).

[0012] Further, the wet bacteria are prepared as follows: the recombinant engineering bacteria expressing the ethanol dehydrogenase mutant are inoculated into LB culture solution containing kanamycin at a final concentration of 50 mg / L, and cultured at 37°C for 8 h to obtain a seed solution; then the seed solution is inoculated into sterile fermentation medium containing kanamycin at a final concentration of 50 mg / L at a volume concentration of 2%, and cultured at 37°C for about 1.5-2.5 h to make the bacteria concentration OD600 be 0.4-0.8; then isopropyl thiogalactoside (IPTG) at a final concentration of 0.1-1.0 mM (preferably 0.1 mM) is added to the culture solution, and expression is induced at 26°C for 12 h; and then the wet bacteria are collected by centrifugation at 4°C and 4000 rpm for 30 min; the LB liquid medium is as follows: proteose peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, solvent is deionized water, and pH is 7.0.

[0013] The ethanol dehydrogenase mutant of the present application can be catalyzed in the form of whole cells, or by the crude enzyme solution of cell disruption or the pure enzyme of complete disruption. In addition, the above two enzymes can be prepared into immobilized enzymes or enzymes in the form of immobilized cells by using specific immobilization technology.

[0014] Compared with the prior art cells, the present application has the following advantages:

[0015] (1) The present application mutates the 74th, 187th, 210th, 211th and 352nd amino acids of the wild-type ethanol dehydrogenase respectively by single-point or multi-point superimposed combination mutation to obtain corresponding mutants, and selects the mutants producing activity or improving the activity of diketones. The advantage mutants H74C / G210K, H74C / T187L / G210K / L211A, H74C / G210K / L211A / L352V can basically convert the prochiral diketones (2,3-butanedione, 2,4-pentanedione, 2,5-hexanedione) with high substrate concentration into corresponding high stereoselective symmetrical chiral diols, the substrate concentration reaches 200-350 g / L, the conversion rate is as high as 98.2%, and the ee% value is more than 99%, thereby solving the problems of low yield of chiral alcohol, low substrate loading capacity and low optical purity of product in the prior art.

[0016] (2) The recombinant vector constructed by the alcohol dehydrogenase mutant coding gene and the recombinant genetically engineered bacteria prepared by transforming the recombinant vector realize the expression of the alcohol dehydrogenase in the E. coli engineering bacteria.

[0017] (3) By optimizing the reaction system, the pure isopropyl alcohol reaction system is adopted in the application, which effectively prevents the subsequent extraction emulsification and other problems, facilitates the extraction and separation of the subsequent product, the by-product acetone generated in the reaction has certain economic value and can be used as another product. The solvent isopropyl alcohol generated at the same time can be recycled and reused, which meets the concept of green chemistry.

[0018] (4) The application can realize the reaction cycle without additional addition of coenzyme, which simplifies the operation of production and reduces the production cost.

[0019] (5) The application reduces the production cost and avoids the environmental problems generated by the traditional method, has certain significance for green production of high stereoselectivity chiral diols and provides basic guarantee for the synthesis of chiral phosphine ligands. (Four)DETAILED DESCRIPTION

[0020] Figure 1 The liquid chromatograms of (2,3)-butanediol (A) and (2S,3S)-butanediol (B) in Example 2.

[0021] Figure 2 The liquid chromatograms of (2,4)-pentanediol (A) and (2S,4S)-pentanediol (B) in Example 3.

[0022] Figure 3 The liquid chromatograms of (2,5)-hexanediol (A) and (2S,5S)-hexanediol (B) in Example 4. (Five)SPECIFIC EMBODIMENTS

[0023] The application will be further described below in combination with specific embodiments, but the protection scope of the application is not limited to this:

[0024] LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of sodium chloride, and deionized water as a solvent, pH 7.0. The LB plate is prepared by adding 20 g / L of agar to the LB liquid medium.

[0025] Example 1: Screening of ethanol dehydrogenase mutant and preparation of recombinant engineering bacteria

[0026] 1. Wild-type ethanol dehydrogenase

[0027] The alcohol dehydrogenase gene CoC from Candida orthopsilosis Co, NCBI accession number: XP_003865936.1 (nucleotide sequence shown in SEQ ID NO. 1, amino acid sequence shown in SEQ ID NO. 2), was integrated into the pET-28a(+) commercial plasmid by a gene synthesis company to directly obtain the recombinant plasmid pET-28a(+)-CoC and the recombinant engineered bacteria E. coli BL21(DE3) / pET-28a(+)-COC.

[0028] 2. Selection of mutation sites

[0029] Through molecular docking, key sites 74, 187, 210, 211, and 352 were rationally analyzed and selected, and primers were designed. Some primers are shown in Table 1.

[0030] Table 1. Key site primers (partial)

[0031]

[0032] 3. Site-directed saturation mutagenesis

[0033] PCR amplification was performed using the pET-28a(+)-COC plasmid as a template and a high-fidelity DNA polymerase. The PCR system is shown in Table 2.

[0034] Table 2. PCR system

[0035]

[0036] The PCR reaction program was as follows: pre-denaturation at 98°C for 3 min, followed by the following cycle: denaturation at 98°C for 1 min, annealing at 63°C for 10 s, extension at 72°C for 1 min 30 s, for a total of 30 cycles, and final extension at 72°C for 10 min, followed by storage at 4°C.

[0037] Mix 3 μL of the PCR product with 2 μL of Loding Buffer and verify PCR success by gel electrophoresis by measuring band size. After successful PCR, add 1 μL of Dpn I to the product and digest it at 37°C for 1 hour to eliminate the original DNA template left over from PCR. Place the product in a 65°C oven for 10 minutes to inactivate Dpn I. Purify the digested product using the Clean Up Kit and store it in a refrigerator at 4°C.

[0038] 4. Expression of transformants

[0039] The E. coli BL21 (DE3) (Invitrogen) competent cells stored at -80°C were ice-bathed at 0°C for 10 min, 5 μL of the purified PCR product was added in a clean bench, ice-bathed at 0°C for 30 min, heat-shocked at 42°C for 90 s, ice-bathed at 0°C for 2 min, 600 μL of LB liquid medium was added, cultured at 37°C, 200 rpm for 1 h, finally spread on LB plates containing 50 μg / mL kanamycin resistance, cultured at 37°C for 8-12 h, and sequencing was performed to determine whether the mutation was successful. The recombinant E. coli carrying the mutant was obtained.

[0040] The recombinant E. coli carrying the mutant was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C overnight. The culture was inoculated into a 250 mL flask containing 100 mL of LB liquid medium at a 1% (v / v) inoculation amount, and cultured at 37°C, 180 rpm. When the OD600 of the culture reached 0.6, IPTG was added as an inducer at a final concentration of 0.1 mM, and the culture was induced at 26°C for 14 h. The culture was centrifuged, the precipitate was resuspended with 3 mL of 100 mM phosphate buffer (pH 6.0), transferred to an EP tube, and stored at -20°C after centrifugation.

[0041] 5. Screening of transformants

[0042] The wet bacteria of step 4 were used as catalysts for the following substrate conversion reactions, and the substrate conversion rates were detected to screen the superior mutants.

[0043] 2,3-Butanedione reaction system 10 mL: 2,3-butanedione 2.58 g, wet bacteria 0.5 g, isopropyl alcohol was added to 10 mL, and the reaction was carried out at 30°C, 200 rpm overnight. The conversion rate was detected by GC.

[0044] 2,4-Pentanedione reaction system 10 mL: 2,4-pentanedione 3 g, wet bacteria 0.5 g, isopropyl alcohol was added to 10 mL, and the reaction was carried out at 37°C, 200 rpm overnight. The conversion rate was detected by GC.

[0045] 2,5-Hexanedione reaction system 10 mL: 2,5-hexanedione 3.42 g, wet bacteria 0.5 g, isopropyl alcohol was added to 10 mL, and the reaction was carried out at 37°C, 200 rpm overnight. The conversion rate was detected by GC.

[0046] The GC detection conditions were as follows: Rt-βDEXsa chiral column, injection port and detector temperature 230°C, carrier gas N2, flow rate 1 m / s. The column oven temperature was 100°C for 13 min, then increased to 140°C at 10°C / min, and maintained for 2 min.

[0047] 6. Screening of mutants

[0048] (1) Single point mutation

[0049] Taking the mutation site 74 screened in step 2 as an example, mutation was performed using the method in step 3, expression was performed using the method in step 4, and screening was performed using the method in step 5. The results are shown in Table 3.

[0050] Table 3. Results of 74-site single mutation screening

[0051]

[0052] The same method was used to screen single-site mutations at sites 187, 210, 211, and 352 screened in step 2. The results showed that at site 74, by mutating H to C, mutating threonine at position 187 to leucine (T187L), mutating glycine at position 210 to lysine (G210K), mutating leucine at position 211 to alanine (L211A), and mutating leucine at position 352 to valine (L352V), the enzyme activity towards the three substrates was greatly improved, while having little effect on enantioselectivity.

[0053] (2) Superimposed mutation

[0054] Using pET-28a(+)-COC-H74C as a new template, other mutation sites were superimposed on this basis. Some of the results are shown in Table 4. The dominant mutants finally screened were H74C / G210K, H74C / T187L / G210K / L211A, and H74C / G210K / L211A / L352V.

[0055] Table 4. Catalytic performance of mutants

[0056]

[0057] Example 2: Synthesis of (2S,3S)-butanediol from 2,3-butanedione catalyzed by mutant COC-H74C / G210K

[0058] The following catalytic reactions were carried out using E. coli BL21(DE3) / pET-28a(+)-COC-H74C / G210K wet cells as catalyst:

[0059] 10mL reaction system: 2,3-butanedione 2.58g, wet bacteria 0.5g, add isopropanol to 10mL, react at 30℃, 200rpm overnight, GC detection ( Figure 1 ) conversion rate 98.1%, ee 99.6%, de 97.1%. As shown in Table 5.

[0060] 1 L reaction system: 2,3-butanedione 258 g, wet cell 50 g, add isopropyl alcohol to 1 L, 30 °C, 200 rpm, overnight reaction, GC detection conversion rate 98.3%, ee 99.7%, de 96.8%.

[0061] Example 3: Mutant COC-H74C / T187L / G210K / L211A catalyzes the synthesis of (2S,4S)-pentanediol from 2,4-pentanedione

[0062] The following catalytic reaction was carried out with E. coli BL21(DE3) / pET-28a(+)-COC-H74C / T187L / G210K / L211A wet cell as catalyst:

[0063] 10 mL reaction system: 2,4-pentanedione 3 g, wet cell 0.5 g, add isopropyl alcohol to 10 mL, 37 °C, 200 rpm, overnight reaction, GC detection conversion rate 84.1%, ee 99.9%, de 99%. As shown in Table 5. Figure 2 ) conversion rate 84.1%, ee 99.9%, de 99%. As shown in Table 5.

[0064] 100 L reaction system: 2,4-pentanedione 30 kg, wet cell 5 kg, add isopropyl alcohol to 100 L, 37 °C, 200 rpm, overnight reaction, GC detection conversion rate 81.7%, ee 99.9%, de 99%.

[0065] Example 4: Mutant COC-H74C / G210K / L211A / L352V catalyzes the synthesis of (2S,5S)-hexanediol from 2,5-hexanedione

[0066] The following catalytic reaction was carried out with E. coli BL21(DE3) / pET-28a(+)-COC-H74C / G210K / L211A / L352V wet cell as catalyst:

[0067] 10 mL reaction system: 2,5-hexanedione 3.42 g, wet cell 0.5 g, add isopropyl alcohol to 10 mL, 37 °C, 200 rpm, overnight reaction, GC detection conversion rate 77.4%, ee 99.9%, de 99%. As shown in Table 5. Figure 3 ) conversion rate 77.4%, ee 99.9%, de 99%. As shown in Table 5.

[0068] 5 L reaction system: 2,5-hexanedione 1.7 kg, wet cell 250 g, add isopropyl alcohol to 5 mL, 37 °C, 200 rpm, overnight reaction, GC detection conversion rate 77.2%, ee 99.9%, de 99%.

[0069] Table 5, catalytic performance of superior mutants

[0070]

Claims

1. A highly active and stereoselective alcohol dehydrogenase mutant, characterized in that: The alcohol dehydrogenase mutant is a mutant in which the amino acid sequence shown in SEQ ID NO. 2 is mutated into one of the following: (1) histidine at position 74 is mutated into cysteine, H74C; (2) threonine at position 187 is mutated into leucine, T187L; (3) glycine at position 210 is mutated into lysine, G210K; (4) leucine at position 211 is mutated into alanine, L211A; (5) leucine at position 352 is mutated into valine, L352V; (6) histidine at position 74 is mutated into cysteine, and glycine at position 210 is mutated into lysine, H74C / G 210K; (7) Histidine at position 74 mutated to cysteine, Threonine at position 187 mutated to Leucine, Glycine at position 210 mutated to Lysine, and Leucine at position 211 mutated to Alanine, H74C / T187L / G210K / L211A; (8) Histidine at position 74 mutated to cysteine, Glycine at position 210 mutated to Lysine, Leucine at position 211 mutated to Alanine, and Leucine at position 352 mutated to Valine, H74C / G210K / L211A / L352V.

2. Use of the alcohol dehydrogenase mutant according to claim 1 in catalyzing the synthesis of symmetrical chiral diols from prochiral diketones.

3. The use according to claim 2, characterized in that The application method comprises the following steps: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria expressing an alcohol dehydrogenase mutant as a catalyst, using a potentially chiral diketone as a substrate, and using an organic alcohol as a reaction medium to form a reaction system; carrying out the reaction at 200 rpm and 30-37° C.; obtaining a reaction solution containing a symmetrical chiral diol after the reaction is completed; and separating and purifying the reaction solution to obtain the symmetrical chiral diol.

4. The use according to claim 3, characterized in that The organic alcohol includes isopropyl alcohol.

5. The use according to claim 3, characterized in that The substrates include 2,3-butanedione, 2,4-pentanedione, and 2,5-hexanedione.

6. The use according to claim 3, characterized in that The amount of the catalyst used is 10-200 g / L based on the weight of the wet bacteria, and the concentration of the substrate added is 200-500 g / L.

7. The use according to claim 3, characterized in that The wet bacteria were prepared as follows: recombinant engineered bacteria expressing an alcohol dehydrogenase mutant were inoculated into LB culture medium containing kanamycin at a final concentration of 50 mg / L, and cultured at 37° C. for 8 h to obtain seed solution; The seed liquid was then inoculated into a sterile fermentation medium containing a final concentration of 50 mg / L kanamycin at an inoculum concentration of 2% by volume, and cultured at 37°C for 1.5-2.5 hours to make the bacterial concentration OD600 of 0.4-0.

8. Isopropylthio-β-D-galactoside was then added to the culture medium at a final concentration of 0.1-1.0 mM. After inducing expression at 26°C for 12 hours, the culture was centrifuged at 4°C and 4000 rpm for 30 minutes to collect the wet bacteria.

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