Alkene reductase mutant, engineering bacterium and application thereof

Abstract

The invention discloses an alkene reductase mutant, an engineering bacterium and application of the alkene reductase mutant and the engineering bacterium, and belongs to the technical field of biochemistry. The alkene reductase mutant comprises any one of the following amino acid sequences: SEQ ID NO.3-SEQ ID NO.11. 4-fluoromethyl-2 (5H)-furanone is reduced by expression cells of the alkene reductase mutant to generate (S)-4-(fluoromethyl) dihydrofuran-2-one, so that the problem that a substrate is difficult to catalyze due to fluorine ions is solved.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to an alkene reductase mutant, an engineering bacterium and applications thereof. Background Art

[0002] Dipeptidyl peptidase-IV (DPP-4) inhibitors are a class of drugs used to treat type 2 diabetes. They inhibit the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), increase the levels of endogenous GLP-1 and GIP, promote the release of insulin from pancreatic β cells, and inhibit the secretion of glucagon from pancreatic α cells, thereby lowering blood sugar. This type of drug has the advantage of not easily causing hypoglycemia and weight gain, so it has been widely used in clinical practice. Some common dipeptidyl peptidase-4 inhibitors include sitagliptin, saxagliptin, vildagliptin and linagliptin.

[0003] Roche of Switzerland has developed a new long-acting DPP-4 inhibitor, Carmegliptin. The IC50 value of this drug for DPP-4 is 6.8nmol / L. Oral administration once a day can safely and effectively control blood sugar and has good market expectations. Therefore, the development of its efficient and green synthesis process has important economic and social value.

[0004] From the perspective of retrosynthesis, Carmegliptin molecules can be synthesized by condensation of fluorinated chiral lactone 1 and chiral amine. Chiral lactone 1 can be obtained by tandem fluorination and asymmetric reduction through the Wittig reaction. Enzyme catalysis has the advantages of high efficiency, high selectivity, and green environmental protection. Among them, ene reductase-catalyzed asymmetric reduction is widely used in the preparation of drug intermediates and fine chemicals. Existing ene reductases are difficult to catalyze the asymmetric reduction of 4-fluoromethyl-2(5H)-furanone. One of the difficulties lies in the particularity of the F atom. The F atomic radius is very close to that of the H atom, but it has a very strong electronegativity and may form hydrogen bonds with polar amino acids in the enzyme protein. Therefore, it is necessary to develop a high-yield ene reductase. Summary of the invention

[0005] In view of the above technical problems existing in the prior art, the present invention provides an olefin reductase mutant, an engineered bacterium and applications thereof, which can catalyze asymmetric reduction to prepare a canagliptin intermediate.

[0006] The first aspect of the present invention discloses an olefin reductase mutant, characterized in that it comprises any one of the following amino acid sequences: SEQ ID NO.3-SEQ ID NO.11.

[0007] The second aspect of the present invention provides an engineered bacterium comprising a plasmid or gene sequence expressing the above amino acid sequence.

[0008] Preferably, the method for constructing the engineered bacteria comprises:

[0009] Performing site-directed mutagenesis based on the gene sequence of the wild-type ene reductase to obtain the gene sequence of the ene reductase mutant;

[0010] After the gene sequence is purified and connected to a plasmid, the plasmid is transformed into competent cells to obtain engineered bacteria.

[0011] Preferably, the engineered bacteria is used to reduce the substrate 4-fluoromethyl-2(5H)-furanone to generate (S)-4-(fluoromethyl)dihydrofuran-2-one.

[0012] Preferably, the catalytic reaction conditions are:

[0013] 10-50g / L engineered bacteria wet cells, 1-20g / L substrate, 1.5-155g / L glucose, pH 7-9, reaction temperature 20-40°C, reaction time 10-36h.

[0014] Preferably, the reaction conditions further include 10%, v / v isopropanol.

[0015] Preferably, (S)-4-(fluoromethyl)dihydrofuran-2-one is used to prepare caragliflozin.

[0016] The third aspect of the present invention provides an application of the above-mentioned engineered bacteria: the preparation of (S)-4-(fluoromethyl)dihydrofuran-2-one, the preparation method comprising: catalyzing the substrate 4-fluoromethyl-2(5H)-furanone by the above-mentioned engineered bacteria to obtain (S)-4-(fluoromethyl)dihydrofuran-2-one.

[0017] Preferably, the catalytic reaction conditions are:

[0018] 10-50g / L engineered bacteria wet cells, 1-20g / L substrate, 1.5-155g / L glucose, pH 7-9, reaction temperature 20-40°C, reaction time 10-36h.

[0019] Preferably, the reaction conditions further include 10%, v / v isopropanol.

[0020] Compared with the prior art, the present invention has the following beneficial effects: cells expressing the olefin reductase mutant reduce 4-fluoromethyl-2(5H)-furanone to generate (S)-4-(fluoromethyl)dihydrofuran-2-one, thereby overcoming the problem that the substrate is difficult to catalyze due to F ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic diagram of the pET-22b OYE1 plasmid of the olefin reductase of the present invention;

[0022] Figure 2 is the catalytic reaction equation of the substrate;

[0023] Figure 3 This is the reaction formula for preparing caragliflozin. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0026] The ene reductase mutants of the present invention are subjected to site-directed mutagenesis based on the wild-type OYE1, wherein each tyrosine (Y) at position 82 is mutated into alanine (Y82A), leucine (Y82L), and phenylalanine (Y82F), asparagine (N) at position 194 is mutated into alanine (N194A), leucine (N194L), and phenylalanine (N194F), and tyrosine (Y) at position 375 is mutated into alanine (Y375A), leucine (Y375L), and phenylalanine (Y375F). The sequences of the ene reductases are shown in Table 1; the primer sequences of the mutants are shown in Table 2.

[0027] Table 1

[0028] Serial number name Serial Number 1 OYE1 amino acid sequence SEQ ID NO.1 2 OYE1 gene sequence SEQ ID NO.2 3 Y82A amino acid sequence SEQ ID NO.3 4 Y82L amino acid sequence SEQ ID NO.4 5 Y82F amino acid sequence SEQ ID NO.5 6 N194A amino acid sequence SEQ ID NO.6 7 N194L amino acid sequence SEQ ID NO.7 8 N194F amino acid sequence SEQ ID NO.8 9 Y375A amino acid sequence SEQ ID NO.9 10 Y375L amino acid sequence SEQ ID NO.10 11 Y375F amino acid sequence SEQ ID NO.11

[0029] Table 2

[0030]

[0031]

[0032] Construction of mutant strains of the olefin reductase mutant: pET-22b OYE1 plasmid was extracted from the olefin reductase OYE1 seed solution using a plasmid extraction kit. The plasmid structure is as follows: Figure 1 , using the plasmid as a template, PCR site-directed mutagenesis is performed according to the primers in Table 2. The reagents for site-directed mutagenesis are shown in Table 3, and the execution procedure for PCR site-directed mutagenesis is shown in Table 4. After the gene sequence for PCR site-directed mutagenesis is purified and connected to the plasmid, it is transformed into competent cells, and a single colony is picked and identified to obtain a mutant strain. The competent cell can be Escherichia coli.

[0033] Table 3

[0034] Reagents Volume (μL) Final concentration <![CDATA[H2O]]> 30 - dNTP mixture 5 0.16mM DMSO 1 - <![CDATA[MgSO4 solution]]> 3 - Plasmid template 1 1ng / μL Reverse primer 2 0.4μM Forward primer 2 0.4μM 10×Buffer 5 1× KOD-Plus-Neo 1 1.0U / 50μL Total volume 50 -

[0035] Table 4

[0036]

[0037] Screening of mutants: The mutants were subjected to catalytic reaction under the following conditions: 20 g / L wet cells, pH 8, cosolvent isopropanol (5%, v / v), substrate concentration 1 g / L, glucose 5 g / L, 30°C for 10 h. The wet cells can be cultured in a shaker while the substrate is catalyzed. The test results of each mutant are shown in Table 5.

[0038] Among them, the preparation method of wet cells is as follows: take 100 μL of the mutant glycerol bacteria and inoculate it into 5mL LB liquid culture medium (containing 100 μg / mL ampicillin), and shake it at 37°C 200rpm overnight. Inoculate the preculture into a 500mL conical flask (200mL LB liquid culture medium + 100 μg / mL ampicillin) and culture it at 37°C for 4 hours. After cooling at 4°C for 0.5 hours, add 0.2mMIPTG to induce expression. Culture at 18°C, 200rpm for 12 hours. Centrifuge at 4°C for 10 minutes using a refrigerated centrifuge, discard the supernatant, and add PBS buffer (50mM, pH 7-9) as needed.

[0039] Table 5

[0040] Serial number Enzymes Yield / % ee value / % 1 WT 94 73 2 Y82A 26 70 3 Y82 23 80 4 Y82F 34 75 5 N194A 75 86 6 N194L 86 89 7 N194F 98 97 8 Y375A 92 64 9 Y375L 91 82 10 Y375F 95 91

[0041] Among them, the ee value is expressed as enantiomeric excess and is used for chiral GC detection. From Table 5, it can be seen that the yield and ee value of N194F are both ideal.

[0042] Optimization of catalytic reaction conditions: According to the conditions in Table 6, the N194F mutant strain was subjected to catalytic reaction.

[0043] Table 6

[0044]

[0045]

[0046] It can be seen from Table 6 that when the wet cell concentration is 20-50 g / L, the yield and ee value are relatively ideal.

[0047] The mutant strain of the present invention can reduce the substrate 4-fluoromethyl-2(5H)-furanone to generate (S)-4-(fluoromethyl)dihydrofuran-2-one while being cultured. Glucose dehydrogenase (GDH) contained in E. coli cells can directly recycle the coenzyme without adding additional reagents, thus reducing the cost. The catalytic reaction formula is shown in Figure 2 , wherein compound 2 is a substrate and compound 1 is a product, which is an important intermediate of carmegliptin. The reaction formula for preparing carmegliptin by compound 1 is shown in Figure 3 The preparation method is prior art and will not be described in detail in the present invention. It can be seen that the whole-cell catalyst of the present application can overcome the problem that the substrate is difficult to catalyze due to F atoms.

[0048] The application method of the ketone reductase mutant of the present invention is to ferment the engineered strain E. coli containing the gene of OYE1 or its mutant to obtain the fermentation liquid of OYE1 or its mutant, collect the whole cells as a catalyst, and use it for catalytic reaction. The whole cell catalyst can effectively perform reduction catalysis and can meet the production needs. However, it is theoretically feasible to express and purify the corresponding ketone reductase mutant from the cell and then perform the catalytic reaction.

[0049] In the asymmetric reduction reaction system, the amount of cells containing the enone reductase mutant is 10-50 g / L of wet cells, the concentration of the raw material 4-fluoromethyl-2(5H)-furanone is 1-20 g / L, the amount of glucose is 1-5 molar equivalents of the raw material, the reaction pH is controlled at 7-9, the temperature is 20-40°C, and the reaction time is 10-36 hours.

[0050] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An olefin reductase mutant, characterized in that Includes any of the following amino acid sequences: SEQ ID NO.3-SEQ ID NO.

11.

2. An engineered bacterium, characterized in that: Contains a plasmid or gene sequence expressing the amino acid sequence as claimed in claim 1.

3. The engineered bacteria according to claim 2, characterized in that Methods for constructing engineered bacteria include: Performing site-directed mutagenesis based on the gene sequence of the wild-type ene reductase to obtain the gene sequence of the ene reductase mutant; After the gene sequence is purified and connected to a plasmid, the plasmid is transformed into competent cells to obtain engineered bacteria.

4. The engineered bacteria according to claim 3, characterized in that The engineered bacteria is used to reduce the substrate 4-fluoromethyl-2(5H)-furanone to generate (S)-4-(fluoromethyl)dihydrofuran-2-one.

5. The engineered bacteria according to claim 4, characterized in that The catalytic reaction conditions are: 10-50g / L engineered bacteria wet cells, 1-20g / L substrate, 1.5-155g / L glucose, pH 7-9, reaction temperature 20-40°C, reaction time 10-36h.

6. The engineered bacteria according to claim 5, characterized in that The reaction conditions also included 10% v / v isopropanol.

7. The engineered bacteria according to claim 5, characterized in that (S)-4-(Fluoromethyl)dihydrofuran-2-one is used to prepare cargliptin.

8. A method for preparing (S)-4-(fluoromethyl)dihydrofuran-2-one, characterized in that: The substrate 4-fluoromethyl-2(5H)-furanone is catalyzed by the engineered bacteria as claimed in claim 2 or 3 to obtain (S)-4-(fluoromethyl)dihydrofuran-2-one.

9. The preparation method according to claim 8, characterized in that: The catalytic reaction conditions are: 10-50g / L engineered bacteria wet cells, 1-20g / L substrate, 1.5-155g / L glucose, pH 7-9, reaction temperature 20-40°C, reaction time 10-36h.

10. The preparation method according to claim 9, characterized in that: The reaction conditions also included 10% v / v isopropanol.

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