Transaminase and ethanol dehydrogenase co-expressed genetically engineered bacterium and application thereof in preparation of chiral amine

Abstract

The invention discloses a transaminase and ethanol dehydrogenase co-expression genetically engineered bacterium and application thereof in preparation of chiral amine, and realizes co-expression of double enzymes in the same host on the basis of not influencing high catalytic activity and high optical selectivity of transaminase. According to the method, a sharing mechanism of protein synthesis is utilized, the experimental step of separating and purifying single enzyme is saved, the production cost is saved, and a process for preparing R-FEA by efficiently aminating FTO is established. When a co-expression engineering bacterium BL21 (DE3) / pETDuet-ATA117-ADH is used as a catalyst to catalyze 30mM of a substrate, compared with a free enzyme mixture of ATA117 and ADH, the yield is increased by 8.2%. The co-expression genetically engineered bacterium prepared by the method has the advantages of simple operation steps, mild reaction conditions and the like, and provides reference for later industrial application.

Description

(1) Technical Field

[0001] The present invention relates to the design and construction of a co-expression engineering bacterium of transaminases (TAs) and alcohol dehydrogenase (ADH), and its use in the asymmetric amination reaction of 2-fluoroacetophenone (FTO) to prepare (R)-1-(2-fluorophenyl)ethylamine (R-FEA). (2) Background Art

[0002] Chiral drugs refer to drug molecules containing chiral isomeric elements in their molecular structures. Chiral amines are a class of compounds in which the chiral center is connected to "-NH2". (R)-1-(2-Fluorophenyl)ethylamine (R-FEA), with a Cas number of 185545-90-8, a density of 1.063 ± 0.06 g / cm 3 , a boiling point of 179.8 ± 15.0 °C, and a molecular formula of C8H 10 FN, with a molecular weight of 139.17. R-FEA is a promising chiral amine drug intermediate with important application value. R-FEA can be used as a chiral intermediate for the synthesis of chiral 6-aryl-furo[2,3-d]pyrimidin-4-amine, and chiral 6-aryl-furo[2,3-d]pyrimidin-4-amine is an epidermal growth factor receptor inhibitor, playing an important role in cancer treatment and pain treatment.

[0003] In the synthesis of chiral amines, transaminases (TAs) are one of the most promising enzymes, providing a more concise route for reductive amination than chemical methods. In addition, according to the amino transfer position, they can be divided into α-TAs and ω-TAs. α-TAs achieve amino transfer at the α-position of amino acids, and ω-TAs can catalyze amino transfer at any position other than the α-position. Compared with α-TAs, the substrates of ω-TAs do not require the presence of a carboxylate group, making ω-TAs more suitable for biotransformation than α-TAs.

[0004] Due to the excellent stereoselectivity, broad substrate specificity, and environmental friendliness of ω-TAs, researchers have begun to pay extensive attention to the development and utilization of ω-TAs in the hope of exploring their potential industrial value. The thermodynamic equilibrium of the transamination reaction is one of the biggest problems encountered in catalytic reactions and is also the main bottleneck restricting the scope of application. The transamination reaction is reversible. Therefore, the existing reversible reaction will have a certain impact on the progress of the forward reaction, making it difficult to achieve the ideal 100% conversion. The equilibrium problem of the reaction is an urgent problem to be solved in the reaction of preparing chiral amines catalyzed by ω-TAs. At present, researchers have proposed using physical, chemical, and other methods to solve the reaction equilibrium problem. Physical methods mainly change physical parameters such as pressure and solubility in the reaction system to promote the discharge of reaction by-products, adjust the reaction equilibrium, and thus increase the yield of chiral amines. This method does not completely remove by-products and the effect is not significant. Using chemical methods to adjust the reaction equilibrium usually uses organic compounds, which does not conform to the concept of environmental friendliness.

[0005] When multi-enzyme cascades are used to catalyze coupling reactions, through the synergistic action of multiple enzymes and relying on the differences in the substrates of different enzymes, the reaction process becomes more environmentally friendly and efficient. The transaminase ATA117 belongs to ω-TAs. During the catalytic asymmetric transamination using ATA117, the introduction of alcohol dehydrogenase (ADH) can promote the reaction to move in the direction of synthesizing chiral amines and improve the synthesis efficiency of chiral amines. Compared with separately expressing enzymes in multiple hosts and then mixing the enzymes, due to the sharing of the protein synthesis mechanism, the co-expression of multiple enzymes in the same host can reduce the production cost of the enzymes. However, using pure enzymes for catalysis faces problems such as complex and cumbersome separation and extraction processes and increased corresponding costs. (III) Summary of the Invention

[0006] The object of the present invention is to provide a co-expression genetic engineering bacterium of a transaminase and an alcohol dehydrogenase and its application in the preparation of chiral amines. Using the whole-cell form as a catalyst, there is no need to extract and purify the enzyme from the cells, the loss of enzyme activity is small, the catalytic activity can be exerted to a greater extent, and the cost is reduced.

[0007] The technical solution adopted by the present invention is as follows:

[0008] The present invention provides a co-expression genetic engineering bacterium of a transaminase and an alcohol dehydrogenase. The co-expression genetic engineering bacterium is a recombinant bacterium constructed by transforming a host bacterium with a recombinant plasmid. The recombinant plasmid is obtained by co-transferring the coding genes of a transaminase (ATA117) and an alcohol dehydrogenase (ADH) into the multiple cloning site of a basic plasmid. The recombinant bacterium uses E. coli BL21(DE3) as the host bacterium; the basic plasmid is pETDuet or pACYCDuet.

[0009] Furthermore, the amino acid sequence of the transaminase is as shown in SEQ ID NO:1, and the amino acid sequence of the alcohol dehydrogenase is as shown in SEQ ID NO:2.

[0010] Furthermore, when the basic plasmid is pETDuet, the ATA117 gene is inserted between the EcoRI and SalI sites of the pETDuet plasmid, and the ADH gene is inserted between the NdeI and XhoI of the pETDuet plasmid; the co-expressing genetically engineered bacterium constructed is BL21(DE3) / pETDuet-ATA117-ADH.

[0011] Furthermore, when the basic plasmid is pACYCDuet, the ATA117 gene is inserted between the EcoRI and SalI sites of the pACYCDuet plasmid, and the ADH gene is inserted between the NdeI and XhoI of the pACYCDuet plasmid; the co-expressing genetically engineered bacterium constructed is BL21(DE3) / pACYCDuet-ATA117-ADH.

[0012] Furthermore, the co-expressing genetically engineered bacterium BL21(DE3) / pETDuet-ATA117-ADH is constructed as follows: Design seamless cloning primers, and use PCR amplification to amplify the coding gene fragment of the transaminase ATA117 (JA717225.1) from the strain E. coli BL21(DE3) / pETduet-ATA117; Use double digestion with EcoR I and Sal I to obtain a linearized vector from the pETDuet empty plasmid; Connect the prepared linearized pETDuet vector and the purified and recovered ATA117 insert fragment into pETDuet-ATA117 using seamless cloning technology; Use double digestion with Nde I / Xho I to obtain a linearized vector from the recombinant plasmid pETDuet-ATA117; Design seamless cloning primers, and use PCR amplification to amplify the coding gene fragment of the alcohol dehydrogenase ADH (NP_014555.1) from the strain E. coli BL21(DE3) / pET28a-ADH; Connect the linearized pETDuet-ATA117 vector and the purified and recovered ADH insert fragment into pETDuet-ATA117-ADH using seamless cloning technology; Then transform the competent cells of the cloning host E. coli DH5α, and use a sterile inoculation loop to pick the transformants verified by colony PCR and sequencing verification; Finally, extract the plasmid after culturing the bacteria, and further transform the plasmid into the expression host cell E. coli BL21(DE3) to construct the recombinant engineering bacterium.

[0013] Furthermore, the co-expression genetically engineered bacterium BL21(DE3) / pACYCDuet-ATA117-ADH was constructed as follows: seamless cloning primers were designed, and the coding gene fragment of transaminase ATA117 (JA717225.1) was amplified from the strain E. coli BL21(DE3) / pETduet-ATA117 by PCR amplification; the pACYCDuet empty plasmid was digested with EcoR I and Sal I to obtain a linearized vector; the prepared linearized pACYCDuet vector and the purified and recovered ATA117 insert fragment were ligated into pACYCDuet-ATA117 by seamless cloning technology; the recombinant plasmid pACYCDuet-ATA117 was digested with Nde I / Xho I to obtain a linearized vector; seamless cloning primers were designed, and the coding gene fragment of alcohol dehydrogenase ADH (NP_014555.1) was amplified from the strain E. coli BL21(DE3) / pET28a-ADH by PCR amplification; the linearized pACYCDuet-ATA117 vector and the purified and recovered ADH insert fragment were ligated into pACYCDuet-ATA117-ADH by seamless cloning technology; then the competent cells of the cloning host E. coli DH5α were transformed, and the transformants verified by colony PCR and sequencing were picked with a sterile inoculation loop; finally, the plasmid was extracted after culturing the bacteria, and the plasmid was further transformed into the expression host cell E. coli BL21(DE3) to construct the recombinant engineering bacterium.

[0014] The present invention also provides a dual-plasmid co-expression genetically engineered bacterium of transaminase and alcohol dehydrogenase. The dual-plasmid co-expression genetically engineered bacterium is a recombinant bacterium obtained by simultaneously transforming a host bacterium with a recombinant plasmid containing transaminase and a recombinant plasmid containing alcohol dehydrogenase. The recombinant bacterium uses E. coli BL21(DE3) as the host bacterium; the recombinant plasmid containing transaminase or the recombinant plasmid containing alcohol dehydrogenase independently uses pETDuet, pACYCDuet or pET28a as the basic vector, and the insertion site is between BamHI and XhoI.

[0015] Furthermore, the recombinant plasmids are pETDuet-ATA117 / pET28a-ADH, pACYCDuet-ATA117 / pETDuet-ADH, pACYCDuet-ATA117 / pET28a-ADH.

[0016] The present invention also provides an application of the co-expression genetically engineered bacterium in the preparation of chiral amines by asymmetric amination. After induced expression, the engineered bacterium produces the transaminase and alcohol dehydrogenase. The application is as follows: using the wet cells obtained by induced expression of the co-expression genetically engineered bacterium containing transaminase and alcohol dehydrogenase or the dual-plasmid co-expression genetically engineered bacterium containing transaminase and alcohol dehydrogenase as a catalyst, using chiral ketone as a substrate, adding a co-solvent, an amino donor isopropylamine hydrochloride (IPA), and a cofactor pyridoxal phosphate (PLP), constructing a reaction system with a pH 6-10 (preferably pH 9) buffer as the reaction medium, and performing a shaking reaction at 25-45 °C and 100-200 rpm (preferably 40 °C and 180 rpm) for 6-36 h (preferably 24 h). The reaction solution is separated and purified to obtain chiral amines; the substrates include acetophenone, 2'-(trifluoromethyl)acetophenone, 3,5-difluoroacetophenone, m-nitroacetophenone, α,α,α-trifluoroacetophenone, 2'-nitroacetophenone (FTO), preferably FTO; the co-solvents include methanol, ethanol, Tween 80, Tween 20, Span 80, glycerol, or dimethyl sulfoxide (DMSO), preferably DMSO.

[0017] Preferably, in the reaction system, the addition amount of the wet cells is 0.05-0.25 g / mL, preferably 0.125 g / mL; the final added concentration of the substrate is 10-50 mM, preferably 30 mM; the final added volume concentration of the co-solvent is 5-25%, preferably 15%; the final added concentration of IPA is 50-250 mM, preferably 150 mM; the final added concentration of PLP is 0.1-10 mM, preferably 1 mM.

[0018] Preferably, the catalyst is prepared by the following method:

[0019] Inoculate the co-expression genetically engineered bacterium containing transaminase and alcohol dehydrogenase (preferably E. coli BL21(DE3) / pETDuet-ATA117-ADH) on an LB solid plate containing 50 μg / mL ampicillin (Amp), incubate it inverted at 37 °C, select single colonies on the plate and inoculate them into an LB liquid medium containing 50 μg / mL Amp, culture them overnight at 37 °C and 180 rpm for 12-16 h (preferably 12 h), inoculate the seed solution into the LB liquid medium at an inoculation amount of 1%-5% (preferably 3%) by volume concentration, culture at 3, 180 rpm until the OD 600 reaches 0.6-0.9 (preferably 0.8), then add IPTG with a final concentration of 0.2-5 mM (preferably 1 mM), induce at 16 °C-25 °C (preferably 18 °C) and 180 rpm for 8 h, centrifuge the fermentation broth at 4 °C (8000 rpm, 10 min), wash it three times with physiological saline, and collect the cell precipitate.

[0020] The present invention constructs a recombinant Escherichia coli strain containing the genes of transaminase (ATA117) and alcohol dehydrogenase (ADH), and successfully asymmetrically amines a series of acetophenone derivatives into the corresponding chiral amines. At the same time, by adjusting the co-expression method of the two enzymes in the dual-enzyme cascade system, the catalytic efficiency and yield in the transamination reaction are improved.

[0021] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0022] On the basis of not affecting the high catalytic activity and high optical selectivity of the transaminase, the present invention realizes the co-expression of the two enzymes in the same host. The present invention utilizes the shared mechanism of protein synthesis, saves the experimental steps of separating and purifying single enzymes, reduces the production cost, and establishes a process for efficiently aminating FTO to prepare R-FEA. When the co-expression engineering strain BL21(DE3) / pETDuet-ATA117-ADH is used as a catalyst to catalyze 30 mM substrate, the yield is increased by 8.2% compared with the mixture of ATA117 and ADH free enzymes. Using the co-expression genetic engineering strain prepared by the method of the present invention as a catalyst to amine FTO to prepare R-FEA has the advantages of simple operation steps and mild reaction conditions, providing a reference for later industrial applications. (IV) Description of the Drawings

[0023] Figure 1 It is a schematic diagram for constructing a single plasmid co-expression recombinant plasmid.

[0024] Figure 2 This is the verification of the construction of the co-expression recombinant plasmid by colony PCR of the present invention; M: standard molecular weight DNA; Lanes 1-7 in A are the colony PCR products of the monoclonal transformants of BL21(DE3) / pETDuet-ATA117-ADH respectively; Lanes 1-7 in B are the colony PCR products of the monoclonal transformants of BL21(DE3) / pACYCDuet-ATA117-ADH respectively.

[0025] Figure 3 It is a schematic diagram of the reaction mechanism for using the co-expression engineering strain of the present invention to catalyze 2-fluoroacetophenone to prepare (R)-1-(2-fluorophenyl)ethylamine.

[0026] Figure 4This is the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the crude enzyme solution induced by the co-expression engineering bacteria of the present invention; M: standard molecular weight protein; In A, lanes 1 and 2 are the broken supernatant and precipitate of BL21(DE3) / pACYCDuet-ATA117-ADH respectively, and lanes 3 and 4 are the broken supernatant and precipitate of BL21(DE3) / pETDuet-ATA117-ADH respectively; In B, lanes 1 and 2 are the broken supernatant and precipitate of BL21(DE3) / pACYCDuet-ATA117 / pETDuet-ADH respectively, lanes 3 and 4 are the broken supernatant and precipitate of BL21(DE3) / pACYCDuet-ATA117 / pET28a-ADH respectively, and lanes 5 and 6 are the broken supernatant and precipitate of BL21(DE3) / pETDuet-ATA117 / pET28a-ADH respectively. (V) Specific Embodiments

[0027] The present invention will be further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0028] In the embodiments of the present invention, the pETDuet plasmid selects ampicillin (Amp) resistance, the pACYCDuet selects chloramphenicol (Cm) resistance, and the pET28a selects kanamycin (Kan) resistance.

[0029] Example 1: Construction of the single plasmid co-expression engineering bacteria BL21(DE3) / pETDuet-ATA117-ADH

[0030] 1. Construction and transformation of the plasmid pETDuet-ATA117

[0031] (1) Construction of the strain E. coli BL21(DE3) / pETduet-ATA117:

[0032] The construction of the E. coli E. coli BL21(DE3) / pETduet-ATA117 engineering bacteria is achieved by inserting the R-ω-transaminase gene (JA717225.1, ATA117, the amino acid sequence is shown in SEQ ID NO: 1) between the EcoR I and Sal I restriction sites of the pETduet expression vector and transforming E. coli BL21(DE3).

[0033] (2) PCR amplification to obtain the ATA117 gene fragment:

[0034] The pETDuet-ATA117 plasmid from the strain E. coli BL21(DE3) / pETduet-ATA117 was used as a template to design the upstream and downstream primers pETDuet-ATA117-F1 and pETDuet-ATA117-R1 (Table 1) required for seamless cloning. PCR Master Mix (purchased from Yisheng) was used as a PCR premix solution, and upstream and downstream primers and DNA template were added to establish a PCR reaction system (Table 2). The most appropriate PCR amplification program (Table 3) was selected for the amplification of ATA117. Afterwards, the PCR product was purified using 0.9% agarose gel electrophoresis, and the target product was recovered using the Sangon SanPrep column-type DNA gel recovery kit to obtain the ATA117 gene fragment (amino acid sequence shown in SEQ ID NO: 1) and stored at -20°C for future use.

[0035] SEQ ID NO: 1:

[0036] MAFSADTPEIVYTHDTGLDYITYSDYELDPANPLAGGAAWIEGAFVPPSEARIPIFDQGFYTSDATYTTFHVWNGNAFRLGDHIERLFSNAESIRLIPPLTQDEVKEIALELVAKTELREAMVTVTITRGYSSTPFERDITKHRPQVYMSASPYQWIVPFDRIRD GVHLMVAQSVRRTPRSSIDPQVKNFQWGDLIRAIQETHARGFELPLLLDCDNLLAEGPGFNVVVIKDGVVRSPGRAALPGITRKTVLEIAESLGHEAILADITPAELYDADEVLGCSTGGGVWPFVSVDGNSISDGVPGPVTQSIIRRYWELNVEPSSLLTPVQY.

[0037] Table 1. PCR primers

[0038]

[0039] Note: The underline represents the restriction site

[0040] Table 2. PCR reaction system

[0041]

[0042] Table 3. PCR amplification program

[0043]

[0044] (3) Linearization of pETDuet vector:

[0045] Extract an appropriate amount of empty pETDuet plasmid from DH5α bacteria storing pETDuet plasmid in the laboratory using a plasmid extraction kit, and verify the plasmid extraction by 0.9% agarose gel electrophoresis. After that, double digest the empty pETDuet plasmid at 37 °C / 180 rpm for 3 h, and the digestion system is shown in Table 4. After the digestion is completed, purify and recover the linearized pETDuet vector by 0.9% agarose gel electrophoresis again.

[0046] Table 4. pETDuet digestion system

[0047]

[0048] (4) Seamless cloning:

[0049] Measure the nucleic acid concentrations of the pETDuet linearized vector prepared in step (3) and the ATA117 gene fragment after purification and recovery in step (2) respectively, mix them evenly according to the molar ratio of 1:3, add 2×In Fusion Cloning Mix, and make up the seamless cloning reaction system with ddH2O, as shown in Table 5. After mixing the seamless cloning system completely evenly, immediately transfer it to an ice-water mixture (0 °C) and let it stand for 10 min to complete the reaction.

[0050] Table 5. Seamless cloning reaction system

[0051]

[0052] (5) Transformation of competent cells:

[0053] Take 10 μL of the seamless cloning reaction product in step (4) and directly add it to the DH5α competent cells (100 μL) melted on ice, gently mix it with a pipette, and incubate it on ice for 30 min. After heat shock at 42 °C for 90 s, incubate it on ice for 2 min. Add 1 mL of antibiotic-free LB medium, and then resuscitate it on a shaker at 37 °C for 1 h. Coat it on an LB plate containing 50 μg / mL ampicillin (Amp), and invert it and culture it overnight in a 37 °C constant temperature incubator. Pick the grown monoclonal colonies from the plate for colony PCR test to screen the positive clone DH5α / pETDuet-ATA117 (EcoR I / Sal I).

[0054] 2. Construction and transformation of pETDuet-ATA117-ADH

[0055] (1) Construction of strain E. coli BL21(DE3) / pET28a-ADH:

[0056] The construction of the engineered E. coli BL21(DE3) / pET28a-ADH strain was achieved by inserting the alcohol dehydrogenase gene (NP_014555.1, ADH, amino acid sequence shown in SEQ ID NO:2) between the BamHI and XhoI restriction sites of the pET-28a(+) expression vector and transforming E. coli BL21(DE3).

[0057] SEQ ID NO:2:

[0058] MSIPETQKGVIFYESHGKLEYKDIPVPKPKANELLINVKYSGVCHTDLHAWHGDWPLPVKLPLVGGHEGAGVVVGMGENVKGWKIGDYAGIKWLNGSCMACEYCELGNESNCPHADLSGYTHDGSFQQYATADAVQAAHIPQGTDLAQVAPILCAGITVYKALKSANLMAGHWVAISGAAGGLGSLAVQYAKAMGYRVLGIDGGEGKEELFRSIGGEVFIDFTKEKDIVGAVLKATDGGAHGVINVSVSEAAIEASTRYVRANGTTVLVGMPAGAKCCSDVFNQVVKSISIVGSYVGNRADTREALDFFARGLVKSPIKVVGLSTLPEIYEKMEKGQIVGRYVVDTSK.

[0059] (2) Obtaining the ADH gene fragment by PCR amplification:

[0060] Using the pET28a-ADH plasmid extracted from E. coli BL21(DE3) / pET28a-ADH as a template, the upstream and downstream primers pETDuet-ATA117-ADH-F1 and pETDuet-ATA117-ADH-R1 (Table 1) required for seamless cloning were designed. Using 2× PCR Master Mix as the PCR premixed solution, and a PCR reaction system was established. The optimal PCR amplification program (Table 3) was selected to amplify the target gene ADH. Subsequently, the PCR product was purified and recovered by 0.9% agarose gel electrophoresis and stored at -20°C for later use.

[0061] (3) Linearization of the pETDuet-ATA117 vector:

[0062] Use a plasmid extraction kit to extract the pETDuet-ATA117 (EcoR I / Sal I) plasmid of the positive clone in Step 1, and double-digest the recombinant plasmid pETDuet-ATA117 with Nde I / Xho I for 3 h. The digestion system is the same as shown in Table 4. After digestion, use 0.9% agarose gel electrophoresis to purify and recover the linearized pETDuet-ATA117 vector again.

[0063] (4) Seamless cloning:

[0064] Mix the linearized pETDuet-ATA117 vector and the purified and recovered ADH gene fragment evenly at a molar ratio of 1:3, add 2×In Fusion Cloning Mix and make up the reaction system to 10 μL with dd H2O. Transfer the reaction system to an ice-water mixture (0 °C) and let it stand for 10 min to complete the reaction.

[0065] (5) Transformation of competent cells:

[0066] Take 10 μL of the seamless cloning reaction product that has reacted in the ice-water mixture for 10 min and add it to DH5α competent cells (100 μL). Culture by the method in Step 1, and pick the grown monoclonal colonies from the plate for colony PCR test ( Figure 2 ) to screen the positive clone DH5α / pETDuet-ATA117-ADH.

[0067] 3. Co-expression engineering bacteria

[0068] Carefully add 10 μL of the recombinant plasmid pETDuet-ATA117-ADH to Escherichia coli BL21(DE3) competent cells, mix well, and place on ice for 20 min. Take it out from the ice, then transfer it to a 42 °C water bath for heat shock for 90 s, quickly put it on ice, and let it stand in the ice bath for 2 min. In the laminar flow hood, continue to add 1 mL of LB liquid medium without antibiotics to the centrifuge tube containing the cells at room temperature, mix well, then incubate at 37 °C with constant shaking for 1 h. Centrifuge the centrifuge tube at a speed of 8000×g for 30 s to remove 800 μL of the supernatant. Gently mix the remaining supernatant and the cells with a pipette, pipette 100 μL and evenly spread it on an LB solid plate containing 50 μg / mL Amp antibiotic, invert the plate and culture it overnight in a 37 °C incubator to prepare the BL21(DE3) / pETDuet-ATA117-ADH genetic engineering bacteria.

[0069] Example 2: Construction of the single-plasmid co-expression engineering bacteria BL21(DE3) / pACYCDuet-ATA117-ADH

[0070] Referring to the construction method of Example 1, first, seamless cloning primers pACYCDuet-ATA117-F2 and pACYCDuet-ATA117-R2 (Table 1) were designed. Using the pETDuet-ATA117 plasmid as a template, a PCR reaction system was established with 2× PCR Master Mix as the PCR premixed solution to obtain the PCR amplification product of ATA117. The pACYCDuet linearized vector was obtained by digesting with the restriction enzymes EcoR I and Sal I at 37 °C for 3 h. Then, the pACYCDuet linearized vector and the purified and recovered ATA117 insert fragment were mixed evenly at a molar ratio of 1:3, 2×In Fusion CloningMix was added, and the reaction system was made up with dd H2O. The reaction system was placed in an ice-water mixture (0 °C) for 10 min. The reaction product was directly transformed into Escherichia coli DH5α competent cells, and monoclonal colonies that grew out were picked from the plate for colony PCR experiments to screen for DH5α / pACYCDuet-ATA117 (EcoR I / Sal I) positive clones.

[0071] Similarly, using pET28a-ADH as the PCR template, pACYCDuet-ATA117-ADH-F2 and pACYCDuet-ATA117-ADH-R2 (Table 1) as the upstream and downstream primers for PCR, 2× PCR Master Mix was used as the PCR premixed solution to establish a PCR reaction system to obtain the PCR amplification product of the ADH gene. The pACYCDuet-ATA117 (EcoR I / Sal I) linearized vector was obtained by digesting the pACYCDuet-ATA117 (EcoR I / Sal I) plasmid with the restriction enzymes Nde I / Xho I at 37 °C for 3 h. Then, a seamless cloning reaction system was formed with the linearized vector and the purified and recovered ADH insert fragment. The reaction product was then transformed into Escherichia coli BL21(DE3) competent cells, and monoclonal colonies were picked from the plate for colony PCR experiments ( Figure 2 ) to screen for BL21(DE3) / pACYCDuet-ATA117-ADH positive clones.

[0072] Example 3: Cultivation and expression of a single-plasmid co-expression genetically engineered bacterium

[0073] (1) Cultivation: Use a clean and sterile inoculation loop to pick single colonies of the correctly constructed genetic engineering bacteria BL21(DE3) / pETDuet-ATA117-ADH and BL21(DE3) / pACYCDuet-ATA117-ADH from the plate respectively, and inoculate them into 50 mL of LB liquid medium containing the corresponding antibiotics (Amp, Cm), where the final concentrations of Amp and Cm are 50 μg / mL and 34 μg / mL respectively. Incubate with constant shaking at 37 °C / 180 rpm overnight to obtain the seed solutions of the above two single-plasmid co-expression genetic engineering bacteria respectively.

[0074] (2) Induced expression: According to an inoculation amount of 3% by volume, pipette the seed solution into 150 mL of LB liquid medium containing the corresponding antibiotics (Amp, Cm), where the final concentrations of Amp and Cm are 50 μg / mL and 34 μg / mL respectively. Transfer to a constant temperature shaker at 37 °C / 180 rpm for cultivation. When the OD600 measured by a spectrophotometer reaches 0.8, add IPTG (1 mM) to the culture medium respectively. Then, transfer the two genetic engineering bacteria to a shaker (18 °C / 180 rpm) for induced expression for 8 h. After induced expression, centrifuge the fermentation broth at 8000 rpm and 4 °C for 10 min to remove the culture medium components. Then wash the bacterial cells three times with an appropriate amount of physiological saline to ensure complete removal of the culture medium components, and collect the wet bacterial cells for standby.

[0075] Example 4: Construction of a two-plasmid co-expression genetic engineering bacterium

[0076] 1. Construction of the engineering bacterium containing the plasmid

[0077] The construction of E.coli BL21(DE3) / pETduet-ATA117 is the same as that in Example 1.

[0078] The construction of E.coli BL21(DE3) / pET28a-ATA117 is achieved by inserting the R-ω-transaminase gene (JA717225.1, ATA117, the amino acid sequence is shown in SEQ ID NO:1) between the BamHI and XhoI restriction sites of the pET28a expression vector and transforming Escherichia coli BL21(DE3).

[0079] The construction of E.coli BL21(DE3) / pACYCDuet-ATA117 is achieved by inserting the R-ω-transaminase gene (JA717225.1, ATA117, the amino acid sequence is shown in SEQ ID NO:1) between the BamHI and XhoI restriction sites of the pACYCDuet expression vector and transforming Escherichia coli BL21(DE3).

[0080] The construction of E. coli BL21(DE3) / pETDuet-ADH engineering bacteria was achieved by inserting the alcohol dehydrogenase gene (NP_014555.1, ADH, amino acid sequence shown in SEQ ID NO:2) between the BamHI and XhoI restriction sites of the pETDuet expression vector and then transforming E. coli BL21(DE3).

[0081] The construction of E. coli BL21(DE3) / pET28a-ADH was achieved by inserting the alcohol dehydrogenase gene (NP_014555.1, ADH, amino acid sequence shown in SEQ ID NO:2) between the BamHI and XhoI restriction sites of the pET28a expression vector and then transforming E. coli BL21(DE3).

[0082] The construction of E. coli BL21(DE3) / pACYCDuet-ADH was achieved by inserting the alcohol dehydrogenase gene (NP_014555.1, ADH, amino acid sequence shown in SEQ ID NO:2) between the BamHI and XhoI restriction sites of the pACYCDuet expression vector and then transforming E. coli BL21(DE3).

[0083] 2. Plasmid extraction:

[0084] Recombinant plasmids pETDuet-ATA117, pET28a-ATA117, pACYCDuet-ATA117, pETDuet-ADH, pET28a-ADH, and pACYCDuet-ADH were extracted from the above-mentioned engineering bacteria respectively.

[0085] 3. Transformation of BL21(DE3) competent cells:

[0086] E. coli BL21(DE3) competent cells were taken out from the -80°C ultra-low temperature freezer and placed on ice to melt. 10 μL of pETDuet-ATA117 and 10 μL of pET28a-ADH recombinant plasmids were taken, and the two recombinant plasmids were gently added to the BL21(DE3) competent cells simultaneously using a pipette. After mixing evenly, it was ice-bathed for 30 min, heat-shocked at 42°C for 90 s, left standing in the ice bath for 2 min, and then 1 mL of antibiotic-free LB medium was added and placed in a 37°C shaker for activation for 1 h.

[0087] 4. Pouring plates:

[0088] After cooling the autoclaved LB solid medium to about 50 °C, add Kan and Amp with a final concentration of 50 μg / mL each, and gently shake well. Pour the medium into a 90-mm diameter Petri dish in a laminar flow hood, about 25 mL of LB solid medium. After pouring the medium into the Petri dish, open the lid and irradiate with ultraviolet light for 10 - 15 min. Let the agar solidify at room temperature.

[0089] 5. Plating:

[0090] Use a sterile spreader to evenly spread the activated mixture on the surface of the solid LB plate. Place the plate upright at 37 °C for half an hour to allow the moisture on the surface of the plate to be fully absorbed, and then incubate it overnight in a 37 °C constant temperature incubator after inverting.

[0091] 6. Colony picking: After incubating overnight at 37 °C, the single colonies grown on the plate containing the two antibiotics (Kan and Amp) are the successfully transformed BL21(DE3) with the pETDuet-ATA117 and pET28a-ADH double recombinant plasmids. Use a sterile inoculation loop to pick a single colony growing on the plate and inoculate it into an LB liquid medium containing both 50 μg / mL of Kan and Amp, and culture it overnight in a 37 °C constant temperature shaker to obtain the E. coli BL21(DE3) / pETDuet-ATA117 / pET28a-ADH double recombinant plasmid co-expressing genetically engineered bacteria.

[0092] The construction methods of the other five co-expressing genetically engineered bacteria containing the pETDuet-ATA117 / pACYCDuet-ADH, pACYCDuet-ATA117 / pET28a-ADH, pACYCDuet-ATA117 / pETDuet-ADH, pET28a-ATA117 / pACYCDuet-ADH, and pET28a-ATA117 / pETDuet-ADH double recombinant plasmids are similar to that of the E. coli BL21(DE3) / pETDuet-ATA117 / pET28a-ADH double recombinant plasmid co-expressing genetically engineered bacteria. Among them, the final concentration of Kan and Amp is 50 μg / mL, and the final concentration of Cm is 34 μg / mL.

[0093] Example 5: Cultivation and expression of genetically engineered bacteria with double plasmids co-expressing

[0094] Cultivation: Use a clean and sterile inoculation loop to pick single colonies of the dual-plasmid co-expression genetically engineered bacteria constructed in Example 4 from the plate, and inoculate them into 50 mL of LB liquid medium containing two corresponding antibiotics. The final concentrations of Kan, Amp, and Cm are 50 μg / mL, 50 μg / mL, and 34 μg / mL, respectively. Place the culture solution inoculated with the genetically engineered bacteria in a constant temperature shaker at 37°C / 180 rpm and incubate overnight to obtain the seed solutions of the above six dual-plasmid co-expression genetically engineered bacteria respectively.

[0095] Induced expression: According to an inoculation amount of 3% by volume, pipette the seed solution and transfer it to 150 mL of LB liquid medium containing two corresponding antibiotics (Kan, Amp, Cm). The final concentrations of Kan, Amp, and Cm are 50 μg / mL, 50 μg / mL, and 34 μg / mL, respectively. Transfer the culture solutions of the six dual-plasmid co-expression genetically engineered bacteria to a constant temperature shaker at 37°C / 180 rpm for incubation. When measuring OD 600 reaches 0.8 using a spectrophotometer, add IPTG (1 mM) to the culture solutions respectively. Then, transfer the genetically engineered bacteria containing the dual plasmids of pACYCDuet-ATA117 / pETDuet-ADH, pETDuet-ATA117 / pACYCDuet-ADH, pET28a-ATA117 / pACYCDuet-ADH, pET28a-ATA117 / pETDuet-ADH to a constant temperature shaker at 18°C / 180 rpm for induced expression for 15 h. For the genetically engineered bacteria containing the dual plasmids of pETDuet-ATA117 / pET28a-ADH and pACYCDuet-ATA117 / pET28a-ADH, they are induced at 23°C / 180 rpm for 14 h. After induced expression, centrifuge the fermentation broth at 8000 rpm and 4°C for 10 min to remove the culture medium components. Then wash the bacterial cells with an appropriate amount of physiological saline three times to ensure complete removal of the culture medium components, and reserve for use.

[0096] Example 6: Analysis of the protein expression of the co-expression genetically engineered bacteria

[0097] In order to investigate the expression of the co-expression genetically engineered bacteria, the Bradford method was used to measure the protein concentrations of the single-plasmid co-expression and dual-plasmid co-expression genetically engineered bacteria respectively (Table 6).

[0098] Table 6. Protein concentrations of the co-expression genetically engineered bacteria

[0099]

[0100] Both the pACYCDuet and pETDuet plasmids are E. coli protein dual-expression vectors with two multiple cloning sites and can be used to simultaneously express two target genes. Therefore, the ATA117 and ADH genes were simultaneously ligated onto the pACYCDuet or pETDuet plasmid to construct the pACYCDuet-ATA117-ADH or pETDuet-ATA117-ADH recombinant plasmid to achieve the co-expression of the two genes. The results showed that compared with the BL21(DE3) / pACYCDuet-ATA117-ADH genetically engineered bacteria, the protein soluble expression of the BL21(DE3) / pETDuet-ATA117-ADH genetically engineered bacteria was higher. In addition, it was found that the content of the target protein in the broken supernatant of BL21(DE3) / pETDuet-ATA117-ADH was higher, while the content of the target protein in the cell precipitate was lower, which was more in line with the requirements. This indicated that the genes located in the pET plasmid had better expression levels than those in the pACYC plasmid.

[0101] Among the six dual-plasmid co-expression genetically engineered bacteria, the protein concentrations of three dual-plasmid engineered bacteria, namely BL21(DE3) / pETDuet-ATA117 / pET28a-ADH, BL21(DE3) / pACYCDuet-ATA117 / pETDuet-ADH, and BL21(DE3) / pACYCDuet-ATA117 / pET28a-ADH, were higher.

[0102] Therefore, the wet cell bodies of the co-expression genetically engineered bacteria BL21(DE3) / pACYCDuet-ATA117-ADH, BL21(DE3) / pETDuet-ATA117-ADH, BL21(DE3) / pACYCDuet-ATA117 / pET28a-ADH, BL21(DE3) / pACYCDuet-ATA117 / pETDuet-ADH, and BL21(DE3) / pETDuet-ATA117 / pET28a-ADH after induced expression were suspended with physiological saline and sonicated at 400 W for 5 min with a 3 s sonication interval and a 7 s interval, and the supernatant and precipitate were collected respectively for SDS-PAGE experiments to verify the protein expression situation( Figure 4 ).

[0103] Different plasmids using the same replication system cannot coexist in the same host cell. When two plasmids are introduced into the same cell simultaneously, they compete with each other during replication and subsequent distribution to daughter cells. In some cells, one plasmid dominates, while in others, the other plasmid prevails. After several generations of cell growth, the minority plasmid will be lost, so only one of the two plasmids remains in the cell progeny. Therefore, this fact is considered to be the reason why the pETDuet-ATA117 and pET28a-ADH combination grows more slowly than other combinations. When the OD 600 of other combinations reaches 0.8 at 3 h, the pETDuet-ATA117 / pET28a-ADH combination takes 4 h to reach the same OD 600 . In addition, the three double-plasmid co-expression engineering bacteria showed a certain degree of expression after induction at their respective optimal temperatures, and there was no significant difference in the amount of the target protein. The ADH protein (39.5 kDa) is 0.5 kDa larger than the ATA117 protein, so there is a higher possibility that their bands partially overlap in the SDS-PAGE diagram. Considering the plasmid stability and specific activity comprehensively, pETDuet-ATA117-ADH is considered to be the best choice.

[0104] Example 7: Screening of the substrate spectrum of co-expression engineering bacteria

[0105] In a 2 mL reaction system, different prochiral substrate ketones (Table 7) were added. 0.3 mL of DMSO was used as a co-solvent to dissolve 7 substrate ketones separately (each with a final concentration of 30 mM), 150 mM IPA, 1 mM PLP, and 0.25 g of the wet cells of the E. coli BL21(DE3) / pETDuet-ATA117-ADH co-expression genetically engineered bacteria after centrifugation and washing in Example 1. The reaction bottle was made up to 2 mL with 0.1 mM Tris-HCl buffer at pH 9.0. The reaction bottle was placed at 40 °C and reacted at 180 rpm for 24 h. After the reaction, it was centrifuged at 8000 rpm for 10 min at 4 °C, the supernatant was collected and extracted with an equal volume of ethyl acetate. After repeating the extraction three times, the collected extract was dried with an appropriate amount of anhydrous sodium sulfate, and the peak areas of the substrate and product were detected by gas chromatography (GC). The internal standard method (adding dodecane as the internal standard) was used to calculate the content, and the yield and enantiomeric excess value (ee) were calculated by formula (1) and formula (2). The results are shown in

[0106] Table 7.

[0107]

[0108] In formula (1), Ms: molecular weight of the substrate; Mp: molecular weight of the (R)-type product; Q: mass of the substrate at the beginning of the reaction; P: mass of the (R)-type product at the end of the reaction.

[0109]

[0110] In formula (2), C R : Concentration of (R)-type product; Cs: Concentration of (S)-type product.

[0111] Table 7. Screening of substrate spectra of co-expressing engineered bacteria

[0112]

[0113] In this example, the asymmetric amination ability of the E. coli BL21(DE3) / pETDuet-ATA117-ADH co-expressing genetically engineered bacterium towards different prochiral ketones was investigated. It can be concluded from the above table that the presence of an electron-withdrawing group is beneficial to the catalysis of acetophenone derivatives by the catalyst, and the yield has a certain degree of increase compared to acetophenone. When substituted at the ortho position, the effect of the F group is better than that of trifluoromethyl and better than that of nitro. When using two F substitutions on the benzene ring simultaneously, the effect is not as good as that of single F substitution at the ortho position. In addition, when nitro is used as a substituent, meta substitution is slightly better than ortho substitution. Considering the yield and enantiomeric excess value comprehensively, 2-fluoroacetophenone was selected as the substrate for subsequent research.

[0114] Example 8: Effect of temperature on the transamination reaction catalyzed by co-expressing engineered bacteria

[0115] The catalytic activity of the enzyme is affected by the reaction temperature. Therefore, the effect of temperature on the transamination reaction catalyzed by co-expressing engineered bacteria was investigated at 30 °C - 50 °C. When the temperature is too high, protein denaturation leads to the loss of part or all of the enzyme activity, and thus the yield of the reaction decreases.

[0116] Add the substrate FTO solution (substrate with a final concentration of 30 mM dissolved in 0.3 mL of cosolvent DMSO), 150 mM amino donor IPA, 1 mM cofactor PLP, and 0.25 g of the wet cells of the E. coli

[0117] BL21(DE3) / pETDuet-ATA117-ADH genetically engineered bacteria obtained by centrifuging and washing in Example 1 into the reaction system, and use 0.1 M Tris-HCl buffer (pH 9) to supplement the 2 mL system. The reaction system was reacted in a shaker at 30, 35, 40, 45, and 50 °C at a rotation speed of 180 rpm for 24 h. After the reaction, centrifuge at 8000 rpm for 10 min at 4 °C, collect the supernatant and add an equal volume of ethyl acetate for extraction. After repeating the extraction three times, the collected extract was dried with an appropriate amount of anhydrous sodium sulfate, and the yield and enantiomeric excess value of the product were detected by the GC method mentioned in Example 7. The results are shown in Table 8.

[0118] Table 8. Effect of temperature on the transamination reaction catalyzed by co-expressing engineered bacteria

[0119]

[0120] As can be seen from Table 8, for the co-expressing engineering bacteria, within the temperature range of 30°C - 50°C, the productivity shows a trend of first increasing and then decreasing. The catalytic activity reaches the optimum at 40°C, manifested as the highest productivity. At relatively low temperatures of 30°C and relatively high temperatures of 45°C, the productivity drops sharply. Therefore, it is recommended to control the temperature reaction conditions within the range of 35°C - 45°C to ensure the optimal conditions for the reaction temperature.

[0121] Example 9: Influence of Substrate Concentration on the Transamination Reaction Catalyzed by Co-expressing Engineering Bacteria

[0122] When a biocatalyst catalyzes an organic reaction, unnatural substrates and products can have a significant impact on its catalytic activity. Under the condition of higher substrate concentration, the inhibitory effect on the enzymatic reaction is more intense. This example examines the influence of different substrate concentrations on the asymmetric amination reaction. As the substrate concentration increases, the productivity of the reaction catalyzed by the co-expressing genetically engineered bacteria shows an obvious downward trend.

[0123] Take 0.25 g of the wet cells of the co-expressing genetically engineered bacteria E. coli BL21(DE3) / pETDuet-ATA117-ADH after centrifugation and washing in Example 1, resuspend them in 0.1 M Tris-HCl buffer (pH 9), and make up the volume to 1.4 mL to form a Tris-HCl buffer system. Dissolve FTO with different final concentrations (10, 20, 30, 40, 50 mM) in 0.3 mL of DMSO respectively, and add 5 equivalents of IPA and 1 mM PLP to the above Tris-HCl buffer system to form a 2 mL reaction system. Keep the reaction system at 40°C and 180 rpm for 24 h. After the reaction is completed, centrifuge at 8000 rpm for 10 min at 4°C, collect the supernatant, add an equal volume of ethyl acetate for extraction, repeat the extraction three times, dry the collected extract with an appropriate amount of anhydrous sodium sulfate, and detect the productivity and enantiomeric excess value of the product by the GC method mentioned in Example 7. The results are shown in Table 9.

[0124] Table 9. Influence of Substrate Concentration on the Transamination Reaction Catalyzed by Co-expressing Engineering Bacteria

[0125]

[0126] When the substrate concentration is within the range of 10 - 30 mM, the productivity corresponding to the BL21(DE3) / pETDuet-ATA117-ADH genetically engineered bacteria is at a relatively high value (≥85%). Further increasing the substrate concentration, the productivity drops significantly. Considering the considerable productivity and better enantiomeric excess value, 30 mM is determined as the optimal substrate concentration for the system.

[0127] Example 10: Influence of pH on the transamination reaction catalyzed by the co-expressing engineered bacteria

[0128] In the range of pH 6 - pH 10, the influence of buffer pH on the transamination reaction catalyzed by BL21(DE3) / pETDuet-ATA117-ADH was studied using FTO as the substrate.

[0129] 0.3 mL of DMSO, 150 mM of IPA, and 1 mM of PLP, in which FTO (with a final concentration of 30 mM) was dissolved, were added to a reaction flask. Then, 0.25 g of the wet cells of the co-expressing engineered bacteria BL21(DE3) / pETDuet-ATA117-ADH after centrifugation and washing in Example 1 were added to the above system, and a 2 mL reaction system was constituted with 0.1 M buffers of different pH values. Phosphate buffer (pH 6 - 8), Tris-HCl buffer (pH 8 - 9), and glycine-NaOH buffer (pH 9 - 10) were used as the buffers. After reacting at 40 °C and 180 rpm for 24 h, the reaction supernatant was obtained by centrifugation (8000 rpm, 10 min). After repeating the extraction three times, the extracts were collected together and dehydrated with anhydrous sodium sulfate. The yield and enantiomeric excess value of the product were detected by the GC method mentioned in Example 7, and the results are shown in Table 10.

[0130] Table 10. Influence of pH on the transamination reaction catalyzed by the co-expressing engineered bacteria

[0131]

[0132] The experimental results showed that the yield gradually increased with the increase of the pH value in the range of pH 6 - pH 9, and when the pH was further increased, the yield began to decrease (Table 10). Therefore, the pH value of the reaction medium would affect the catalytic performance of the co-expressing engineered bacteria to a certain extent, which might be due to the change of the pH value of the buffer causing the change of the three-dimensional conformation of the enzyme active center. When the buffer pH for the catalytic reaction of the engineered bacteria BL21(DE3) / pETDuet-ATA117-ADH was alkaline (pH > 7), the yield was controlled above 83%, and the yield corresponding to pH 9 was the highest. Therefore, pH 9 was the optimal pH for it.

[0133] Example 11: Comparison of the catalytic abilities between the co-expressing engineered bacteria and free dual enzymes

[0134] (1) ATA117 enzyme powder:

[0135] The R-ω-transaminase gene (JA717225.1) was inserted between the BamHI and XhoI restriction sites of the pETduet-1 expression vector and transferred into DH5α bacteria. The recombinant plasmid pETduet-ATA117 was extracted from DH5α bacteria and transformed into Escherichia coli BL21(DE3). The obtained transformants were cultured overnight at 37 °C and 180 rpm in 50 mL of LB medium containing 50 μg / mL Amp to obtain the seed culture of recombinant Escherichia coli. The seed culture was transferred to 50 mL of fresh LB medium containing 50 μg / mL Amp at an inoculation amount of 3% and fermented at 37 °C and 180 rpm until the optical density (OD 600 ) at 600 nm reached 0.8, and isopropyl-β-thiogalactoside (IPTG) with a final concentration of 1 mM was added to induce gene expression. Then the culture was transferred to a shaker at 18 °C and 180 rpm for 15 h of induced expression. The culture solution was aliquoted into 50 mL centrifuge tubes and centrifuged at 4 °C and 8000 rpm for 10 min to collect the overexpressed cell precipitate after 15 h of induction, and washed 3 times with 0.9% (v / w) physiological saline. The harvested wet cells were resuspended in 100 mM sodium phosphate buffer (pH 7.0) at 50 g / L, and the mixture was sonicated for 5 minutes (power 400 W, working for 3 seconds and breaking for 7 seconds). The lysate was collected in a 50 mL centrifuge tube and centrifuged at 8000 rpm and 4 °C for 10 min, and then the supernatant was taken to obtain the crude ATA117 enzyme solution. The BeyoGold TM His-tag purification column was pre-equilibrated with non-denaturing lysis buffer (pH 8.0, 50 mM sodium phosphate buffer and 300 mM NaCl), centrifuged at 8000 rpm and 4 °C for 1 min to discard the liquid, and the equilibration was repeated twice, and the supernatant was discarded. Then the crude enzyme solution of the sample (mixed in a ratio of 1:8) was added to the gel from which the liquid had been discarded above, shaken and mixed well, placed on ice, and slowly shaken in a shaker at 40 rpm for 1 h. The sample and the mixture of chelating gel resistant to reduction were loaded into an empty chromatography column, and 1-2 column volumes of non-denaturing washing buffer (50 mM sodium phosphate buffer, 300 mM NaCl and 2 mM imidazole) were added each time to remove the impurity proteins, and the column was washed 5 times. Then the target protein ATA117 was eluted and purified by adding one column volume of non-denaturing elution buffer (50 mM sodium phosphate buffer, 300 mM NaCl and 50 mM imidazole) each time. After pre-freezing in a -80 °C refrigerator for 8 h, it was placed in a freeze dryer and vacuum dried at -65 °C for 12 h to obtain the ATA117 enzyme powder with an enzyme activity of 0.18 U / mg.

[0136] (2) ADH enzyme powder:

[0137] The ethanol dehydrogenase ADH gene (NP_014555.1) was inserted between the BamHI and XhoI restriction sites of the pET-28a(+) expression vector and transferred into Escherichia coli DH5α. The pET28a-ADH recombinant plasmid was extracted from Escherichia coli DH5α and introduced into competent BL21(DE3). After activation in a shaker at 37°C and 180 rpm for 1 h, it was spread on a plate containing 50 μg / mL Kan antibiotic and incubated overnight at 37°C in an inverted position. A single colony was picked and inoculated into 50 mL of LB liquid medium containing 50 μg / mL Kan antibiotic and cultured overnight in a shaker at 37°C and 180 rpm to obtain a seed culture. It was transferred to 0.15 L of fermentation medium (5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, pH 7) at an inoculation amount of 3% and continued to be cultured at 37°C and 180 rpm until the OD 600 reached 0.7, then IPTG with a final concentration of 1 mM was added, and induction expression was carried out at 23°C and 180 rpm for 15 h. After the induction was completed, the culture solution was aliquoted into centrifuge tubes and placed at 4°C, and centrifuged at 8000 rpm for 10 min in a centrifuge. The collected cell precipitate was washed with 10 mL of 0.9% (v / w) normal saline each time, and washed a total of 3 times. Centrifuged at 4°C and 8000 rpm for 10 min to collect wet cells. The harvested wet cells were resuspended in 100 mM sodium phosphate buffer (pH 7.0) at 50 g / L, and the mixture was sonicated in an ice bath for 5 minutes (power 400 W, working for 3 seconds, breaking for 7 seconds). The sonicated lysate was collected, centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was taken to obtain the crude ADH enzyme solution, which was then purified by the above ATA117 purification method. After pre-freezing in a -80°C refrigerator for 8 h, it was placed in a freeze dryer and vacuum dried at -65°C for 12 h to obtain ADH enzyme powder with an enzyme activity of 0.15 U / mg.

[0138] (3) Co-expression catalysis

[0139] In a 2 mL reaction system, 0.3 mL of DMSO dissolving FTO (final concentration added 30 mM), 150 mM IPA, and 1 mM PLP were added, and 0.25 g of wet cells of the co-expression engineered bacteria BL21(DE3) / pETDuet-ATA117-ADH after centrifugation and washing in Example 1 were added, and made up to 2 mL with 0.1 M Tris-HCl buffer at pH 9. The reaction flask was placed at 40°C and 180 rpm for reaction for 24 h. After the reaction was completed, it was centrifuged at 4°C and 8000 rpm for 10 min, the supernatant was collected and an equal volume of ethyl acetate was added for extraction. After repeating the extraction three times, the collected extract was dried with an appropriate amount of anhydrous sodium sulfate, and the yield and enantiomeric excess value of the product were detected by the GC method in Example 7. The results are shown in Table 11.

[0140] (4) Catalysis by free enzyme ATA117 / ADH

[0141] Change the wet cells of the co-expressing engineering bacteria in step (3) to 0.125 g of ATA117 enzyme powder and 0.125 g of ADH enzyme powder, and then add NADH with a final concentration of 10 mM. Other operations are the same.

[0142] Table 11. Comparison of the catalytic abilities of co-expressing engineering bacteria and free dual enzymes

[0143]

[0144] In this example, the catalytic activities of the co-expressing engineering bacteria and free dual enzymes under the optimal conditions were investigated. By co-expressing the genes of ATA117 and ADH in the same host cell, the steps of enzyme separation and purification can be reduced. Compared with free dual enzymes as catalysts, the fermentation cost of the genetically engineered bacteria with dual enzyme co-expression is lower and the steps are more concise. In addition, without the addition of coenzyme factors, the yield is 8.2% higher than that of the free dual enzyme catalysis group, making the catalysis of the engineering bacteria more economical.

Claims

1. A genetically engineered bacterium co-expressing transaminase and alcohol dehydrogenase, characterized in that, The co-expressing genetically engineered bacterium is a recombinant bacterium obtained by transforming a host bacterium with a recombinant plasmid. The recombinant plasmid is obtained by co-transferring the coding genes of transaminase ATA117 and alcohol dehydrogenase ADH into the multiple cloning site of a basic plasmid. The recombinant bacterium uses E. coli BL21(DE3) as the host bacterium; the basic plasmid is pETDuet or pACYCDuet.

2. The co-expressing genetically engineered bacterium of transaminase and alcohol dehydrogenase according to claim 1, wherein The amino acid sequence of the transaminase is shown in SEQ ID NO:1, and the amino acid sequence of the alcohol dehydrogenase is shown in SEQ ID NO:

2.

3. The co-expressing genetically engineered bacterium of transaminase and alcohol dehydrogenase according to claim 1, characterized in that, When the basic plasmid is pETDuet, the ATA117 gene is inserted between the EcoRI and SalI sites of the pETDuet plasmid, and the ADH gene is inserted between the NdeI and XhoI of the pETDuet plasmid; the constructed co-expressing genetically engineered bacterium is BL21(DE3) / pETDuet-ATA117-ADH.

4. The co-expressing genetically engineered bacterium of transaminase and alcohol dehydrogenase according to claim 1, characterized in that, When the basic plasmid is pACYCDuet, the ATA117 gene is inserted between the EcoRI and SalI sites of the pACYCDuet plasmid, and the ADH gene is inserted between the NdeI and XhoI of the pACYCDuet plasmid; the constructed co-expressing genetically engineered bacterium is BL21(DE3) / pACYCDuet-ATA117-ADH.

5. The co-expressing genetically engineered bacterium of transaminase and alcohol dehydrogenase according to claim 3, characterized in that, The co-expressing genetically engineered bacterium BL21(DE3) / pETDuet-ATA117-ADH is constructed as follows: Design seamless cloning primers, and use PCR amplification to amplify the coding gene fragment of transaminase ATA117 from the strain E. coli BL21(DE3) / pETduet-ATA117; Use double digestion with EcoR I and Sal I to obtain a linearized vector from the pETDuet empty plasmid; Connect the prepared pETDuet linearized vector and the purified and recovered ATA117 insert fragment into a recombinant plasmid pETDuet-ATA117 using seamless cloning technology; Use double digestion with Nde I / Xho I to obtain a linearized vector from the recombinant plasmid pETDuet-ATA117; Design seamless cloning primers, and use PCR amplification to amplify the coding gene fragment of alcohol dehydrogenase ADH from the strain E. coli BL21(DE3) / pET28a-ADH; Connect the pETDuet-ATA117 linearized vector and the purified and recovered ADH insert fragment into pETDuet-ATA117-ADH using seamless cloning technology; Then transform the competent cells of the cloning host E. coli DH5α, and use a sterile inoculation loop to pick the transformants verified by colony PCR and sequencing verification; Finally, extract the plasmid after culturing the bacteria, and further transform the plasmid into the expression host cell E. coli BL21(DE3) to construct a recombinant engineering bacterium.

6. The co-expressing genetically engineered bacterium of transaminase and alcohol dehydrogenase according to claim 4, wherein The co-expressing genetically engineered bacterium BL21(DE3) / pACYCDuet-ATA117-ADH was constructed as follows: seamless cloning primers were designed, and the coding gene fragment of transaminase ATA117 was amplified from the strain E. coli BL21(DE3) / pETduet-ATA117 by PCR amplification; the pACYCDuet empty plasmid was digested with EcoR I and Sal I to obtain a linearized vector; the prepared linearized pACYCDuet vector and the purified and recovered ATA117 insert fragment were ligated into the recombinant plasmid pACYCDuet-ATA117 by seamless cloning technology; the recombinant plasmid pACYCDuet-ATA117 was digested with Nde I / Xho I to obtain a linearized vector; seamless cloning primers were designed, and the coding gene fragment of alcohol dehydrogenase ADH was amplified from the strain E. coli BL21(DE3) / pET28a-ADH by PCR amplification; the linearized pACYCDuet-ATA117 vector and the purified and recovered ADH insert fragment were ligated into pACYCDuet-ATA117-ADH by seamless cloning technology; then the competent cells of the cloning host E. coli DH5α were transformed, and the transformants verified by colony PCR and sequencing were picked with a sterile inoculation loop; finally, the plasmid was extracted after culturing the bacteria, and the plasmid was further transformed into the expression host cell E. coli BL21(DE3) to construct the recombinant engineering bacterium.

7. A dual-plasmid co-expression genetically engineered bacterium of transaminase and alcohol dehydrogenase, characterized in that, The dual-plasmid co-expressing genetically engineered bacterium is a recombinant bacterium obtained by simultaneously transforming the host bacterium with a recombinant plasmid containing transaminase and a recombinant plasmid containing alcohol dehydrogenase. The recombinant bacterium uses E. coli BL21(DE3) as the host bacterium; the recombinant plasmid containing transaminase or the recombinant plasmid containing alcohol dehydrogenase independently uses pETDuet, pACYCDuet or pET28a as the basic vector, and the insertion site is between BamHI and XhoI.

8. The dual-plasmid co-expression genetically engineered bacterium according to claim 7, characterized in that, The recombinant plasmids are pETDuet-ATA117 / pET28a-ADH, pACYCDuet-ATA117 / pETDuet-ADH, pACYCDuet-ATA117 / pET28a-ADH.

9. Use of the co-expressing genetically engineered bacterium according to claim 1 or 7 in the preparation of chiral amines by asymmetric amination, characterized in that, The application is as follows: Using the wet cells obtained by induced expression of a co-expressing genetically engineered bacterium containing transaminase and alcohol dehydrogenase or a dual-plasmid co-expressing genetically engineered bacterium containing transaminase and alcohol dehydrogenase as a catalyst, using chiral ketone as a substrate, adding a cosolvent, an amino donor isopropylamine hydrochloride, and a cofactor pyridoxal phosphate, constructing a reaction system with a pH 6-10 buffer as the reaction medium, and performing a shaking reaction at 25-45 °C and 100-200 rpm for 6-36 h. The reaction solution is separated and purified to obtain chiral amine; the substrates include acetophenone, 2'-(trifluoromethyl)acetophenone, 3,5-difluoroacetophenone, m-nitroacetophenone, a,a,a-trifluoroacetophenone, 2'-nitroacetophenone; the cosolvents include methanol, ethanol, Tween 80, Tween 20, Span 80, glycerol or dimethyl sulfoxide.

10. The application according to claim 9, characterized in that In the said reaction system, the addition amount of the wet cells is 0.05-0.25 g / mL; the final added concentration of the substrate is 10-50 mM; the final added concentration of the cosolvent volume is 5-25%; the final added concentration of isopropylamine hydrochloride is 50-250 mM; the final added concentration of pyridoxal phosphate is 0.1-10 mM.

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