A carbonyl reductase mutant and its application in producing (R)-(+)-3-chloro-1-phenyl-1-propanol

Abstract

The invention discloses a carbonyl reductase mutant and its application in producing (R)-(+)-3-chloro-1-phenyl-1-propanol, mainly relates to a recombinant vector constructed by a carbonyl reductase mutant encoding gene and a recombinant genetic engineering bacterium prepared by transformation of the recombinant vector, and the application of the carbonyl reductase mutant in asymmetric catalysis of 3-chlorophenylacetone to synthesize (R)-(+)-3-chloro-1-phenyl-1-propanol. The present invention realizes the expression of carbonyl reductase in Escherichia coli engineering bacteria, solves the problems of low conversion rate, low yield and low ee value; adopts a pure isopropanol reaction system, effectively prevents the problems of subsequent extraction emulsification, facilitates the extraction and separation of subsequent products, and the solvent isopropanol produced can be recycled and reused, greatly reducing the production cost and the environmental protection problems generated; realizes the addition and regeneration without the need for coenzymes, simplifies the production operation and reduces the production cost.

Description

Technical Field

[0001] The invention relates to the field of biochemical engineering, and relates to the application of a carbonyl reductase mutant and a recombinant Escherichia coli thereof in catalyzing chiral pure (R)-(+)-3-chloro-1-phenyl-1-propanol. Background Art

[0002] ((R)-(+)-3-chloro-1-phenyl-1-propanol is an important chiral intermediate that can be used to prepare atomoxetine hydrochloride and dapoxetine hydrochloride. CN101012147A mentions a method of producing (R)-(+)-3-chloro-1-phenyl-1-propanol by reacting 3-chlorophenylacetone with NaBH4 or KBH4 under the catalysis of the left-handed isomer of a chiral amino acid derivative, wherein the left-handed isomer of the chiral amino acid derivative includes a left-handed tryptophan derivative, a left-handed isoleucine derivative, etc., which is relatively expensive, and the total yield of the method is only 25%. The high production cost greatly limits the application of the method in actual production. CN104496830A uses 3-chlorophenylacetone and (-) diisopinocampheyl chloroborane in an organic phase solvent to react at 10 to 80°C to generate (R)-(+)-3-chloro-1-phenyl-1-propanol. This method uses a large amount of organic solvent, which pollutes the environment to a certain extent, and requires high reaction conditions, which is difficult to operate. CN112980895A provides a method for enzymatically synthesizing (R)-(+)-3-chloro-1-phenyl-1-propanol. This method uses 3-chlorophenylacetone as a substrate and carbonyl reductase as a catalyst. In the coenzyme / coenzyme cycle hydrogen donor NADP + In the presence of NADPH, coenzyme cycle enzymes, organic cosolvents and PB buffer, a biocatalytic reaction is carried out to produce (R)-(+)-3-chloro-1-phenyl-1-propanol. This method has a low substrate concentration, the product extraction is difficult, and additional coenzymes and coenzyme cycle enzymes need to be added. There is much room for improvement in cost and operation. Summary of the invention

[0003] The object of the present invention is to provide a carbonyl reductase mutant and its application in the production of (R)-(+)-3-chloro-1-phenyl-1-propanol in view of the above problems, and to provide a novel enzymatic synthesis method of (R)-(+)-3-chloro-1-phenyl-1-propanol in view of the high cost, high difficulty in operation, low yield and large environmental pollution in the current existing process. The method mines and obtains carbonyl reductase mutants through directed evolution, and prepares (R)-(+)-3-chloro-1-phenyl-1-propanol by constructing a recombinant carbonyl reductase Escherichia coli to catalyze the asymmetric reduction of 3-chlorophenylacetone, which is low in cost, simple and easy to operate, and environmentally friendly and suitable for industrial production.

[0004] The present invention is achieved through the following technical solutions.

[0005] Technical solution 1 of the present invention provides a carbonyl reductase mutant, characterized in that: the carbonyl reductase mutant is a mutant in which the isoleucine at position 93 in the amino acid sequence shown in SEQ ID NO.1 is mutated to tryptophan (I93W), the proline at position 186 is mutated to alanine (P186M), and the glutamic acid at position 196 is mutated to proline (E196P).

[0006] The nucleotide sequence is shown in SEQ ID NO.1, and the sequence of the carbonyl reductase mutant is shown in SEQ ID NO.2.

[0007] Technical solution 2 of the present invention: Provide a recombinant vector containing the carbonyl reductase mutant encoding gene construction and a recombinant genetically engineered bacterium prepared by transformation of the recombinant 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 / or eukaryotic cells. The vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., preferably using Pet28b plasmid as an expression vector and Escherichia coli as an expression host (Escherichia coli BL21 cells or Escherichia coli DH5α). The recombinant genetically engineered bacteria can be constructed by selecting various conventional engineering strains as the chassis, such as Escherichia coli, Saccharomyces cerevisiae, etc., preferably using Escherichia coli BL21 cells as the chassis microorganism to construct the recombinant genetically engineered bacteria.

[0008] Technical solution three of the present invention: provides an application of the carbonyl reductase mutant in asymmetric catalysis of 3-chloropropiophenone to synthesize (R)-(+)-3-chloro-1-phenyl-1-propanol, wherein the application method is as follows: using wet bacteria obtained by fermentation culture of recombinant genetically engineered bacteria containing a carbonyl reductase encoding gene as a catalyst, using 3-chloropropiophenone as a substrate, and using isopropanol as a reaction medium to form a reaction system, reacting at 200 rpm and 37° C. After the reaction is completed, a reaction solution containing (R)-(+)-3-chloro-1-phenyl-1-propanol is obtained, and the reaction solution is separated and purified to obtain (R)-(+)-3-chloro-1-phenyl-1-propanol.

[0009] Furthermore, the amount of the catalyst is 60-200 g / L (preferably 100 g / L) based on the weight of the wet bacteria, and the initial concentration of the substrate is 0.5-3 M (preferably 1 M).

[0010] Furthermore, the wet cell was prepared as follows: the recombinant engineered bacteria containing the carbonyl reductase mutant were inoculated into an LB culture solution 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 was inoculated into a sterile fermentation medium containing kanamycin with a final concentration of 50 mg / L at an inoculum concentration of 2% by volume, and cultured at 37°C for about 1.5-2.5 hours to make the cell concentration OD 600 is 0.4-0.8, and then isopropylthio-β-D-galactoside (IPTG) is added to the culture solution at a final concentration of 0.1-1.0mM (preferably 0.1mM), after inducing expression at 26°C for 12h, centrifuge at 4°C and 4000rpm for 30min to collect wet bacteria; LB liquid culture medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, solvent is deionized water, pH 7.0.

[0011] The carbonyl reductase mutant of the present invention can perform catalysis in the form of whole cells, or in the form of crude enzyme solution of cell crushing or pure enzyme crushing. In addition, it can also be prepared into an enzyme in the form of immobilized enzyme or immobilized cells by using a specific immobilization technology.

[0012] The beneficial effects of the present invention are as follows: 1. The recombinant vector constructed by the gene encoding the carbonyl reductase mutant of the present invention and the recombinant genetic engineering bacteria prepared by transformation of the recombinant vector realize the expression of carbonyl reductase in Escherichia coli engineering bacteria. Compared with the prior art, the superposition mutation of the mutation sites of 93-position Ile to Trp, 186-position Pro to Met and 196-position Glu to Pro increases the enzyme activity of the mutant by 6.2 times, and the optical purity (ee value) is increased to 99%. Finally, when the bacterial concentration is 20g (dcw) / L, the conversion rate of 168g / L substrate can reach more than 95% within 12h and the optical purity (ee value) is more than 99%, solving the problems of low conversion rate, low yield and low ee value. 2. Through the exploration and optimization of the reaction system, the present invention adopts a pure isopropanol reaction system, effectively preventing the problems of subsequent extraction emulsification, facilitating the extraction and separation of subsequent products, and the solvent isopropanol produced can be recycled and reused, greatly reducing the production cost and the environmental protection problems generated. 3. At the same time, the present invention realizes the elimination of the need for the addition and regeneration of coenzymes, simplifies the production operation and reduces the production cost.

[0013] Instruction Manual

[0014] Figure 1 This is the gas phase detection spectrum of (R)-(+)-3-chloro-1-phenyl-1-propanol according to an embodiment of the present invention. DETAILED DESCRIPTION

[0015] The technical solution of the present invention is further described in detail below through embodiments and accompanying drawings.

[0016] Example 1: Preparation of ketocarbonyl reductase recombinant vector

[0017] The carbonyl reductase gene (SDRA) from Chryseobacterium was obtained by screening, and the NCBI number is WP_048502227.1 (the nucleotide sequence is shown in SEQ ID NO.1)

[0018] The above genes were connected by genetic engineering using pET28b plasmid as a vector to construct a carbonyl reductase expression vector pET28b-SDRA, and E. coli BL21 (DE3) / pET28b-SDRA was constructed.

[0019] Upstream primer: 5'-AAGGAGATATACCATGGGAATTTTAG-3'

[0020] Downstream primer: 5'-CTAAAATTCCCATGGTATATCTCCTT-3'

[0021] Construction of the expression plasmid Using the above primers and the SDRA gene as a template, high-fidelity DNA polymerase was used for PCR amplification, and the amplified gene fragment was connected to pET28b by one-step cloning to construct the plasmid pET28b-SDRA.

[0022] Example 2: Construction of carbonyl reductase mutants and recombinant engineered bacteria

[0023] Using pET28b-SDRA gene as template, the triple mutant pET28b-SDRA-I93W-P186M-E196 (nucleotide sequence is shown in SEQ ID NO. 2) was obtained by site-directed mutagenesis. The recombinant expression plasmid was transformed into E. coli BL21 (DE3) competent cells.

[0024] Construction of recombinant E. coli: First, the competent cells of E. coli BL21 (DE3) (Invitrogen) stored at -80°C were placed in an ice bath at 0°C for 10 minutes, then 5 μL of plasmid pET28b-SDRA was added in a clean bench, ice bathed at 0°C for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, ice bathed at 0°C for 2 minutes, and 600 μL of LB medium was added, and cultured at 37°C and 200 rpm on a shaker for 1 hour; spread on an LB plate containing 50 μg / ml kanamycin resistance, cultured at 37°C for 8-12 hours, centrifuged at 8000 rpm to collect the cells, and stored at -20°C for later use. The recombinant E. coli BL21 (DE3) / pET28b-SDRA-I93W-P186M-E196 containing the expression recombinant plasmid was obtained by screening by enzyme activity detection method. The results of enzyme activity detection were: 455U / g wet bacteria for pET28b-SDRA, and 9193U / g wet bacteria for pET28b-SDRA-I93W-P186M-E196. The enzyme activity was increased by 20.2 times after triple mutation, providing a basis for industrial production.

[0025] Standard enzyme activity detection method: accurately weigh 0.84g 3-chloropropiophenone and dissolve it in 7.85g isopropanol to prepare a 0.5M mother solution. After keeping warm at 37°C, add 1mL of the mother solution to 0.1g of wet bacteria, react at 37°C and 200rpm for 30min, take a sample, add 2 volumes of ethyl acetate and centrifuge at 12000rpm to terminate the reaction, dry over anhydrous sodium sulfate, and then detect the conversion rate of (R)-(+)-3-chloro-1-phenyl-1-propanol by GC, and calculate the amount of (R)-(+)-3-chloro-1-phenyl-1-propanol.

[0026] Enzyme activity definition: Under standard conditions, the amount of enzyme required to generate 1 μmol of (R)-(+)-3-chloro-1-phenyl-1-propanol per second is defined as one enzyme activity unit U.

[0027] The positive transformants were selected and sequenced to identify the nucleotide sequence as shown in SEQ ID No. 2, thereby obtaining the ketocarbonyl reductase expression engineering bacteria: T3.

[0028] Example 3: Application of carbonyl reductase 3 mutant recombinant Escherichia coli T3 in asymmetric catalytic synthesis of (R)-(+)-3-chloro-1-phenyl-1-propanol

[0029] Glucose dehydrogenase (GDH) coenzyme circulation system: 3-chloropropiophenone 84g (0.5M), glucose monohydrate 120g (0.6M), T3 wet bacteria 120g, GDH 10g, NADP + 0.1mM, add water to make up the volume to 1L, control the reaction pH to 6.5-7 with sodium bicarbonate at 37℃, and the substrate conversion rate is 96.7% and ee99% after 6h reaction.

[0030] Grade g pure isopropanol system: 3-chloropropiophenone 101g (0.6M), T3 wet bacteria 160g, isopropanol added to 1L volume, 37℃ 200rpm reaction for 4h, then 3-chloropropiophenone 101g (0.6M), continued reaction for 10h, final conversion rate 95%, ee 99%. After concentration, filtration, decolorization and crystallization, the reaction solution obtained 128g of product with purity 99.5%, ee 99%, and final yield 63.3%.

[0031] kg-level pure isopropanol system: 3-chloropropiophenone 250kg, T3 wet bacteria 225kg, isopropanol supplemented to about 1850L volume, 37℃ 200rpm reaction for 8h, final conversion rate 97%, ee 99%. The reaction solution was concentrated, filtered, decolorized, and crystallized to obtain 187kg of product with a purity of 99.5%, ee 99%, and a final yield of 74.8%.

[0032] The gas phase detection spectrum of (R)-(+)-3-chloro-1-phenyl-1-propanol is as follows Figure 1 .

[0033] The sequence table involved in the present invention is as follows:

[0034] SEQ ID NO.1:

[0035] ATGGGAATTTTAGAAAACAAAGTTGCACTGGTAACAGGTGCAGGTTCAGGAATCGGTTTAGCAGTGGCTCAGCTGTATGCTAAAGAAGGTGCAAAAGTTATCGTTCGATATCAATGAAGAGCATGGTAAAAAAGCTGTAGAAAAGATTAAGTCGGAAGGAGGGGAAGCTTTTTAAAGCTTTAAAGCTTAAAGAGCATGCTAAAAGGTGCAAAAGTTATCGTTCGAATCAATGAAGAGCATGGTAAAAAAGCT ACACTGCCAGCGCAGAAGAAGTAGAAAACCTTGTCAGAAACTGCCGAGATTTGGAAAGCTTGATATTGCATGCAATAATGCAGGAATTGGCGGCGAAGCAGCTTTAACCGGAGATTATAGCCTGGATAGCTGGAGAAAAGTCTTAAGTTAATCTGATGGCCGTATTATGGCTGCTGTATTAT GAATCCTGGAAATGGAGAAACGGAGGTGGAGTGATTGTCAATATGGCTTCTATCCATGGAAGCGTAGCTGCACCACTTTCTTCGGCATATACAACGGCAAAACATGCGGTGGTAGGCTTACCAAATATAGGAGCAGAGTATGGACAGAAAGGAATCCGTTGCAATGCCATGCGTAGGGAT ATATTGATACCCCGCTTTTACAGGAAATGGATGAAGAAAAAAAGGAATTATTGGTAGGGAAACATCCTATAGCCGTCTCGGTACTGCTCAGGAAGTTGCAGAATTAGTTCTATTCCTAAGTTCAGATAAATCTTCATTTATAACAGGAGGATATTCTTATAGATGGTGGTATACTGCTGTTTAATT

[0036] SEQ ID NO.1:

[0037] MGILENKVALVTGAGSGIGLAVAQLYAKEGAKVIVSDINEEHGKKAVEKIKSEGGEASFIKADTASAEEVENLVRKTAEIYGKLDIACNNAGIGGEAALTGDYSLDSWRKVLSINLDGVFYGCKYEILEMEKNGGGVIVNMASIHGSVAAPLSSAYTTAKHAVVGLTKNIGAEYGQKGIRCNAVGPGYIDTPLLQEMDEEKKELLVGKHPIGRLGTAQEVAELVLFLSSDKSSFITGGYYLIDGGYTAV

[0038] SEQ ID NO.2:

[0039] ATGGGAATTTTAGAAAACAAAGTTGCACTGGTAACAGGTGCAGGTTCAGGAATCGGTTTAGCAGTGGCTCAGCTGTATGCTAAAGAAGGTGCAAAAGTTATCGTTCGATATCAATGAAGAGCATGGTAAAAAAGCTGTAGAAAAGATTAAGTCGGAAGGAGGGGAAGCTTTTTAAAGCTTTAAAGCTTAAAGAGCATGCTAAAAGGTGCAAAAGTTATCGTTCGAATCAATGAAGAGCATGGTAAAAAAGCT ACACTGCCAGCGCAGAAGAAGTAGAAAACCTTGTCAGAAAAACTGCCGAGATTTGGAAAGCTTGATATTGCATGCAATAATGCAGGATGGGGCGGCGAAGCAGCTTTAACCGGAGATTATAGCCTGGATAGCTGGAGAAAAGTCTTAAGTTAATCTTGATGGCGTATTATGGCTTATTATGGCTTATTAT GAATCCTGGAAATGGAGAAGAACGGAGGTGGAGTGATTGTCAATATGGCTTCTATCCATGGAAGCGTAGCTGCACCACTTTCTTCGGCATATACAACGGCAAAACATGCGGTGGTAGGCTTACCAAAAATAGGAGCAGAGTATGGACAGAAAGGAATCCGTTGCAATGGGAGTGGGGAT ATATTGATACCCCGCTTTTACAGCCAATGGATGAAGAAAAAAAGGAATTATTGGTAGGGAAACATCCTATAGCCGTCTCGGTACTGCTCAGGAAGTTGCAGAATTAGTTCTATTCCTAAGTTCAGATAAATCTTCATTTATAACAGGAGGATATTCTTATAGATGGTGGTATACTGCTGTTTAATT

[0040] SEQ ID NO.2:

[0041] MGILENKVALVTGAGSGIGLAVAQLYAKEGAKVIVSDINEEHGKKAVEKIKSEGGEASFIKADTASAEEVENLVRKTAEIYGKLDIACNNAGWGGEAALTGDYSLDSWRKVLSINLDGVFYGCK YEILEMEKNGGGVIVNMASIHGSVAAPLSSAYTTAKHAVVGLTKNIGAEYGQKGIRCNAVGMGYIDTPLLQPMDEEKKELLVGKHPIGRLGTAQEVAELVLFLSSDKSSFITGGYYLIDGGYTAV

[0042] The above are preferred embodiments of the present invention. It should be pointed out that a person skilled in the art may make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A carbonyl reductase mutant, characterized in that: The carbonyl reductase mutant is a mutant in which the isoleucine at position 93 is mutated to tryptophan (I93W), the proline at position 186 is mutated to methionine (P186M), and the glutamic acid at position 196 is mutated to proline (E196P) in the amino acid sequence shown in SEQ ID NO.

1.

2. A recombinant vector comprising the gene encoding the carbonyl reductase mutant according to claim 1.

3. A recombinant genetically engineered bacterium prepared by transformation with the recombinant vector as claimed in claim 2.

4. The recombinant vector according to claim 2, characterized in that The Pet28b plasmid was used as the expression vector and Escherichia coli was used as the expression host.

5. The recombinant genetically engineered bacterium according to claim 3, characterized in that: Escherichia coli BL21 cells were used as chassis microorganisms to construct recombinant genetically engineered bacteria.

6. Use of the carbonyl reductase mutant according to claim 1 in asymmetric catalysis of 3-chloropropiophenone to synthesize (R)-(+)-3-chloro-1-phenyl-1-propanol.

7. The use according to claim 6, characterized in that: The application method is as follows: wet bacteria obtained by fermentation culture of recombinant genetic engineering bacteria containing a carbonyl reductase encoding gene are used as catalysts, 3-chloropropiophenone is used as a substrate, and isopropanol is used as a reaction medium to form a reaction system, and the reaction is carried out at 200 rpm and 37° C. After the reaction is completed, a reaction liquid containing (R)-(+)-3-chloro-1-phenyl-1-propanol is obtained, and the reaction liquid is separated and purified to obtain (R)-(+)-3-chloro-1-phenyl-1-propanol.

8. The use according to claim 7, characterized in that: The catalyst dosage is 60-200 g / L based on the weight of wet bacteria, and the initial addition concentration of the substrate is 0.5-3 M.

9. The use according to claim 7, characterized in that: The wet bacterial cells are prepared as follows: the recombinant engineered bacteria containing the carbonyl reductase mutant are inoculated into LB culture solution containing kanamycin with a final concentration of 50 mg / L, and cultured at 37° C. for 8 hours to obtain seed solution; Then the seed solution was inoculated into a 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 hours until the bacterial concentration reached OD 600 The pH value was 0.4-0.8, and then isopropylthio-β-D-galactoside (IPTG) was added to the culture medium with a final concentration of 0.1-1.0 mM. After inducing expression at 26°C for 12 hours, the wet bacteria were collected by centrifugation at 4°C and 4000 rpm for 30 minutes. LB liquid culture medium: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, the solvent was deionized water, pH 7.0.

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