Alcohol dehydrogenase mutant and application thereof in synthesis of symmetric chiral diol

Abstract

The invention discloses an ethanol dehydrogenase mutant and an application of the ethanol dehydrogenase mutant in synthesis of symmetric chiral diol. The ethanol dehydrogenase mutant is obtained by carrying out single mutation or multiple mutations on amino acids at 74th, 187th, 210th, 211th and 352th sites of an ethanol dehydrogenase amino acid sequence as shown in SEQ ID NO.2 from Candida orbiculata Co. The invention further discloses a preparation method of the ethanol dehydrogenase mutant, and the preparation method comprises the following steps: carrying out single mutation or multiple mutations on the amino acids at the 74th, 187th, 210th, 211th and 352th sites of the ethanol dehydrogenase amino acid sequence as shown in SEQ ID NO.2. Alcohol dehydrogenase is utilized to catalyze prochiral symmetric diketone to synthesize symmetric chiral diol, the method is high in substrate concentration, high in stereoselectivity, mild in reaction, simple in operation process, environment-friendly and more suitable for industrial amplification production, and the problems that in the prior art, the substrate concentration is not high, the reaction condition is violent, the reaction process is complex and difficult to control, and the production cost is low are solved. And the method is not environment-friendly enough, and the like.

Description

(I) Technical field

[0001] The invention belongs to the field of biochemical engineering, and particularly relates to an alcohol dehydrogenase mutant, a gene, a recombinant vector containing the gene, and a recombinant genetic engineering bacterium prepared by transforming the recombinant vector, as well as an application in catalyzing the synthesis of symmetrical chiral diols (phosphine ligand chiral mother nucleus) from symmetrical diketones. (II) Background technology

[0002] Chiral phosphine ligand is one of several types of ligands that are most widely used in asymmetric catalysis, and the dihedral angle of bisphosphine ligand plays an important role in asymmetric catalysis, and suitable dihedral angle is crucial to obtaining excellent enantioselectivity. Utilize the bisphosphine ligand (PNAS 2004,16,5815-5820.J.Am.Chem.Soc.2006,128,5955-5965.) of a series of chiral side chains synthesized by central chiral compounds such as methyl sulfonate or p-toluene sulfonate of symmetrical chiral diols, successfully realize the efficient transfer from central chirality to axial chirality in the synthesis process, do not need to split and obtain the chiral bisphosphine ligand of single configuration, and be successfully applied in asymmetric hydrogenation. Therefore symmetrical chiral diols have great application prospects in the synthesis of bisphosphine ligands. At present, the relatively common process of synthesizing symmetrical chiral alcohols is basically obtained by metal catalytic hydrogenation with chiral ketone, which inevitably increases the complexity and manufacturing cost of reaction. In addition, due to reaction control problems, a large number of by-products are produced, resulting in low ee and de values ​​of the products. For example, the (2S,3S)-butanediol and (2R,3S)-butanediol produced during the metal-catalyzed hydrogenation synthesis of (2R,3R)-butanediol cannot be effectively controlled, resulting in too low ee and de values ​​of the products. (III) Summary of the invention

[0003] The invention aims to provide an alcohol dehydrogenase mutant and application thereof in synthesizing symmetrical chiral diols. The invention utilizes alcohol dehydrogenase to catalyze prochiral symmetrical diketones to synthesize symmetrical chiral diols. The method has high substrate concentration, high stereoselectivity, mild reaction, simple operation process, and is environmentally friendly. The method is more suitable for industrial scale-up production, and solves the problems in the prior art of low substrate concentration, drastic reaction conditions, complex reaction process that is difficult to control, many by-products, and environmentally unfriendly.

[0004] The technical solution adopted by the present invention is:

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

[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) the histidine at position 74 is mutated into cysteine ​​(H74C); (2) the threonine at position 187 is mutated into leucine (T187L); (3) the glycine at position 210 is mutated into lysine (G210K); (4) the leucine at position 211 is mutated into alanine (L211A); (5) the leucine at position 352 is mutated into valine (L352V); (6) the histidine at position 74 is mutated into cysteine, and the 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 by 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 pET-28a (+) plasmid as an expression vector and Escherichia coli as an expression host (Escherichia coli BL21 cells or Escherichia coli DH5α).

[0008] The 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 culture of recombinant genetic engineering bacteria expressing the alcohol dehydrogenase mutant as a catalyst, using prochiral diketones as substrates, and using organic alcohols as reaction media to form a reaction system, reacting at 200 rpm and 30-37° C., obtaining a reaction solution containing symmetrical chiral diols after the reaction is completed, and separating and purifying the reaction solution to obtain the symmetrical chiral diols.

[0009] Furthermore, the organic alcohol includes isopropanol.

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

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

[0012] Further, the wet bacteria are prepared as follows: the recombinant engineering bacteria expressing the alcohol dehydrogenase mutant are inoculated into an LB culture medium containing kanamycin with a final concentration of 50 mg / L, and cultured at 37°C for 8 hours to obtain a seed solution; then the seed solution is inoculated into a sterile fermentation medium containing kanamycin with a final concentration of 50 mg / L at an inoculum amount of 2% by volume, and cultured at 37°C for about 1.5-2.5 hours to make the bacterial concentration OD600 0.4-0.8, and then isopropylthio-β-D-galactoside (IPTG) with a final concentration of 0.1-1.0 mM (preferably 0.1 mM) is added to the culture solution, after inducing expression at 26°C for 12 hours, centrifuged at 4°C and 4000 rpm for 30 minutes, and the wet bacteria are collected; LB liquid culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, pH 7.0.

[0013] The alcohol dehydrogenase mutant of the present invention can perform catalysis in the form of whole cells, or in the form of crude enzyme solution obtained by cell crushing or pure enzyme crushed completely. In addition, the 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 beneficial effects of the present invention are mainly reflected in:

[0015] (1) The present invention performs single-point or multi-point superposition combination mutations on the amino acids at positions 74, 187, 210, 211, and 352 of the wild-type alcohol dehydrogenase to obtain corresponding mutants, and screens mutants that produce activity or have improved diketone activity. The dominant mutants H74C / G210K, H74C / T187L / G210K / L211A, and H74C / G210K / L211A / L352V can basically convert potential chiral diketones (2,3-butanedione, 2,4-pentanedione, and 2,5-hexanedione) with high substrate concentrations into corresponding symmetrical chiral diols with high stereoselectivity, with a substrate concentration of 200-350 g / L, a conversion rate of up to 98.2%, and an ee% value of more than 99%, solving the problems of low chiral alcohol yield, low substrate loading, and low product optical purity.

[0016] (2) The recombinant vector constructed by the gene encoding the alcohol dehydrogenase mutant of the present invention and the recombinant genetic engineering bacteria prepared by transformation of the recombinant vector achieve the expression of alcohol dehydrogenase in the Escherichia coli engineering bacteria.

[0017] (3) Through the exploration and optimization of the reaction system, the present invention adopts a pure isopropanol reaction system, which effectively prevents the problems of subsequent extraction emulsification and facilitates the extraction and separation of subsequent products. The by-product acetone produced by the reaction has certain economic value and can be used as another product. At the same time, the solvent isopropanol produced can be recycled and reused, which is in line with the concept of green chemistry.

[0018] (4) The present invention can realize the reaction cycle without adding additional coenzymes, which simplifies the production operation and reduces the production cost.

[0019] (5) The present invention reduces the production cost while avoiding the environmental problems caused by the traditional method, which is of certain significance for the green production of high stereoselective chiral diols and provides a basic guarantee for the synthesis of chiral phosphine ligands. (IV) Description of the drawings

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

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

[0022] Figure 3 , liquid chromatograms of (2,5)-hexanediol (A) and (2S,5S)-hexanediol (B) in Example 4. (V) Specific implementation methods

[0023] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0024] LB liquid medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, solvent is deionized water, pH 7.0. LB plate is LB liquid medium with 20g / L agar added.

[0025] Example 1: Screening of alcohol dehydrogenase mutants and preparation of recombinant engineered bacteria

[0026] 1. Wild-type alcohol dehydrogenase

[0027] Based on the alcohol dehydrogenase gene CoC from Candida orthopsilosis Co, NCBI accession number: XP_003865936.1 (nucleotide sequence is shown in SEQ ID NO.1, amino acid sequence is shown in SEQ ID NO.2), it 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 of the primers are shown in Table 1.

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

[0031]

[0032] 3. Site-directed saturation mutagenesis

[0033] Using pET-28a(+)-COC plasmid as template, PCR amplification was performed using 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] Take 3 μL of PCR product and mix it with 2 μL of Loding buffer, and use gel electrophoresis to verify whether the PCR is successful. Add 1 μL of Dpn I to the successfully verified PCR product and place it at 37℃ for 1 hour to eliminate the original DNA template left during PCR, and place it in a 65℃ oven for 10 minutes to inactivate Dpn I. Purify the digested product according to the Clean up kit and store the purified product in a 4℃ refrigerator.

[0038] 4. Expression of transformants

[0039] The competent Escherichia coli BL21 (DE3) (Invitrogen) cells stored at -80°C were placed in an ice bath at 0°C for 10 min, 5 μL of the purified PCR product was added in a clean bench, the cells were placed in an ice bath at 0°C for 30 min, heat-shocked in a 42°C water bath for 90 s, and placed in an ice bath at 0°C for 2 min. 600 μL of LB liquid culture medium was added, and the cells were cultured at 37°C, 200 rpm, on a shaking table, for 1 h. Finally, the cells were spread on an LB plate containing 50 μg / mL kanamycin resistance and cultured at 37°C for 8-12 h. After sequencing confirmed that the mutation was successful, the recombinant Escherichia coli carrying the mutant was obtained.

[0040] The recombinant Escherichia coli carrying the mutant was inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C overnight with shaking. The inoculum was inoculated into a 250mL Erlenmeyer flask containing 100mL LB liquid medium at a 1% (v / v) inoculation amount, and cultured in a shaker at 37°C and 180rpm with shaking. When the OD600 of the culture reached 0.6, IPTG was added as an inducer with a final concentration of 0.1mM, and induced at 26°C for 14h. The culture was centrifuged, and the precipitated cells were resuspended in 3mL100mM phosphate buffer solution (pH 6.0), transferred to an EP tube, and the wet bacteria were stored at -20°C after centrifugation.

[0041] 5. Screening of transformants

[0042] Use the wet bacteria in step 4 as a catalyst to carry out the following substrate conversion reaction, detect the substrate conversion rate, and screen for dominant mutants.

[0043] 2,3-Butanedione reaction system 10mL: 2,3-Butanedione 2.58g, wet bacteria 0.5g, add isopropanol to 10mL, react at 30℃, 200rpm overnight, and detect the conversion rate by GC.

[0044] 2,4-Pentanedione reaction system 10mL: 2,4-Pentanedione 3g, wet bacteria 0.5g, add isopropanol to 10mL, react at 37℃, 200rpm overnight, and detect the conversion rate by GC.

[0045] 2,5-hexanedione reaction system 10mL: 2,5-hexanedione 3.42g, wet bacteria 0.5g, add isopropanol to 10mL, react at 37℃, 200rpm overnight, and detect the conversion rate by GC.

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

[0047] 6. Screening of mutants

[0048] (1) Single point mutation

[0049] Taking the mutation site No. 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 single mutation screening at 74 sites

[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, the activity of the enzyme towards the three substrates was greatly improved by mutating H to C, threonine at position 187 to leucine (T187L), glycine at position 210 to lysine (G210K), leucine at position 211 to alanine (L211A), and leucine at position 352 to valine (L352V), while having little effect on enantioselectivity.

[0053] (2) Superposition 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] 1L reaction system: 258g 2,3-butanedione, 50g wet bacteria, add isopropanol to 1L, react overnight at 30℃ and 200rpm. GC detection conversion rate is 98.3%, ee is 99.7%, and de is 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 reactions were carried out using E.coli BL21(DE3) / pET-28a(+)-COC-H74C / T187L / G210K / L211A wet cells as catalyst:

[0063] 10mL reaction system: 2,4-pentanedione 3g, wet bacteria 0.5g, add isopropanol to 10mL, react at 37℃, 200rpm overnight, GC detection ( Figure 2 ) conversion rate 84.1%, ee 99.9%, de 99%. As shown in Table 5.

[0064] 100L reaction system: 2,4-pentanedione 30kg, wet bacteria 5kg, add isopropanol to 100L, react overnight at 37℃, 200rpm, GC detection conversion rate 81.7%, ee99.9%, de99%.

[0065] Example 4: Synthesis of (2S,5S)-hexanediol from 2,5-hexanedione catalyzed by mutant COC-H74C / G210K / L211A / L352V

[0066] The following catalytic reactions were carried out using E.coli BL21(DE3) / pET-28a(+)-COC-H74C / G210K / L211A / L352V wet cells as catalyst:

[0067] 10mL reaction system: 2,5-hexanedione 3.42g, wet bacteria 0.5g, add isopropanol to 10mL, react at 37℃, 200rpm overnight, GC detection ( Figure 3 ) conversion rate 77.4%, ee 99.9%, de 99%. As shown in Table 5.

[0068] 5L reaction system: 1.7kg 2,5-hexanedione, 250g wet bacteria, add isopropanol to 5mL, react overnight at 37℃ and 200rpm. GC detection conversion rate is 77.2%, ee99.9%, de99%.

[0069] Table 5. Catalytic performance of dominant mutants

[0070]

Claims

1. A highly active and stereoselective alcohol dehydrogenase mutant, characterized in that: The alcohol dehydrogenase mutant is obtained by subjecting the amino acids at positions 74, 187, 210, 211 and 352 of the amino acid sequence shown in SEQ ID NO.2 to single mutation or multiple mutation.

2. The alcohol dehydrogenase mutant according to claim 1, 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) the histidine at position 74 is mutated into cysteine, H74C; (2) the threonine at position 187 is mutated into leucine, T187L; (3) the glycine at position 210 is mutated into lysine, G210K; (4) the leucine at position 211 is mutated into alanine, L211A; (5) the leucine at position 352 is mutated into valine, L352V; (6) the histidine at position 74 is mutated into cysteine, and the glycine at position 210 is mutated into lysine, H74C / G 210K; (7) the histidine at position 74 mutated to cysteine, the threonine at position 187 mutated to leucine, the glycine at position 210 mutated to lysine, and the leucine at position 211 mutated to alanine, H74C / T187L / G210K / L211A; (8) the histidine at position 74 mutated to cysteine, the glycine at position 210 mutated to lysine, the leucine at position 211 mutated to alanine, and the leucine at position 352 mutated to valine, H74C / G210K / L211A / L352V.

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

4. The use according to claim 3, 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 potential chiral diketone as a substrate, and using an organic alcohol as a reaction medium to form a reaction system, reacting at 200 rpm and 30-37° C. After the reaction is completed, a reaction liquid containing a symmetrical chiral diol is obtained, and the reaction liquid is separated and purified to obtain the symmetrical chiral diol.

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

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

7. The use according to claim 4, 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.

8. The use according to claim 4, characterized in that The wet bacterial cells are prepared as follows: the recombinant engineering bacteria expressing the alcohol 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 hours 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 h to make the bacterial concentration OD600 of 0.4-0.

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

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