CN118272331A - Alkene reductase mutant and application thereof in (R) -citronellal synthesis - Google Patents

Abstract

The invention discloses an alkene reductase mutant and application thereof in (R) -citronellal synthesis. The invention belongs to the technical field of biocatalysis, and aims to solve the problems of low production efficiency and low optical purity of products existing in the existing technology for synthesizing (R) -citronellal by using alkene reductase. The amino acid sequence of the alkene reductase mutant disclosed by the invention is shown as SEQ ID NO. 4. The alkene reductase mutant body provided by the invention can efficiently catalyze citral to generate (R) -citronellal, the production efficiency is up to 462.3 g L ^‑1 d^‑1, the optical purity (e.e. value) of the product (R) -citronellal is 99.0%, and the method has important industrial application value for green efficient preparation of (R) -citronellal.

Description

Alkene reductase mutant and application thereof in (R) -citronellal synthesis

Technical Field

The invention belongs to the technical field of biocatalysis, and relates to an alkene reductase mutant, a coding nucleic acid molecule, a recombinant vector and a recombinant cell thereof, a preparation method of the alkene reductase mutant and application of the alkene reductase mutant in preparation of (R) -citronellal.

Background

The (R) -citronellal is a key precursor for synthesizing important perfume L-menthol, can also be used for synthesizing perfumes such as hydroxycitronellal, citronellol ester and the like, and is widely applied to foods, beverages and daily fine chemical products. At present, chemical catalytic methods are mainly adopted in industry to synthesize (R) -citronellal, such as chiral BINAP-Rh ⁺ coordination catalyst invented by Japanese high sand (Takasago) company, but the problems of difficult design of metal ligand catalyst, strict reaction condition, low optical purity of (R) -citronellal and the like exist. The technique for synthesizing (R) -citronellal by a biocatalysis method, namely synthesizing (R) -citronellal by using alkene reductase-mediated enantioselective catalysis (E/Z) -citral has the advantages of simple process, mild reaction condition, compliance with atomic economy, environmental protection and great application value.

The invention patent CN113337450B discloses that the conversion rate of catalytic conversion (E/Z) -citral synthesis (R) -citronellal reaction of alkene reductase HP3 from candida tropicalis (Candida tropicalis) reaches more than 99% in 20 hours, but the e.e. value is only 95.4%, and the improvement space is still provided.

In the existing technical route for preparing (R) -citronellal by using a biocatalysis method with the participation of alkene reductase, the problem of low production efficiency of key enzyme-alkene reductase still exists, and the industrial scale application of the key enzyme-alkene reductase is limited. Therefore, the method for developing the alkene reductase with high catalytic efficiency through genome data mining, directed evolution and other methods has important industrial application value in green and high-efficiency synthesis of (R) -citronellal.

Disclosure of Invention

The invention aims to provide the alkene reductase with obviously improved catalytic performance, so that the problems of low production efficiency and low optical purity of products existing in the existing technology for catalyzing and synthesizing (R) -citronellal by using the alkene reductase are solved.

Specifically, the invention takes wild type alkene reductase CvDH (Candida viswanathiidehydrogenase) from candida viscidosa (CANDIDA VISWANATHII) as an original sequence (the amino acid sequence of which is shown as SEQ ID NO: 2, and the nucleotide sequence of which is shown as SEQ ID NO: 1), and performs directed evolution on the original sequence, thereby obtaining alkene reductase mutant CvDH-A181R with remarkable improvement on catalytic performance.

In a first aspect, the invention provides an alkene reductase mutant with improved catalytic activity, compared with wild alkene reductase CvDH shown in SEQ ID NO. 2, the amino acid at the 181 th position is mutated from alanine to arginine, the alkene reductase mutant is named CvDH-A181R, the amino acid sequence of the alkene reductase mutant is shown in SEQ ID NO. 4, and the alkene reductase mutant can efficiently catalyze a substrate citral to generate (R) -citronellal.

The mutant provided by the invention can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing the coding gene, such as from prokaryotes (e.g. escherichia coli) or eukaryotes (e.g. yeast, higher plants) by using recombinant technology.

In some embodiments, the above-mentioned alkene reductase mutant is obtained by constructing recombinant genetically engineered bacteria by transforming a recombinant expression vector containing its encoding gene into escherichia coli (e.g., e.coli bl21 (DE 3)), then culturing the strain, and adding an inducer to induce expression.

In a second aspect, the present invention provides a nucleic acid molecule encoding a mutant of an alkene reductase as described above.

In some embodiments, the nucleic acid molecule encoding the above alkene reductase mutant is shown in SEQ ID NO. 3.

The above nucleic acid molecules provided by the present invention can be obtained usually by amplification using a PCR instrument or artificial synthesis.

In a third aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule as described in any one of the above;

The recombinant vector comprises a cloning vector for replicating the relevant gene and an expression vector for expressing the relevant gene.

In some embodiments, the recombinant vector is the recombinant expression vector pET-26b (+) -CvDH-A181R.

In a fourth aspect, the invention provides a recombinant cell comprising a nucleic acid molecule and/or recombinant vector as described in any one of the preceding.

In some embodiments, the recombinant cells express the alkene reductase mutants described above after induction.

In some embodiments, the method of constructing a recombinant cell comprises:

the recombinant vector is transformed into an expression host cell, and the expression is induced by culturing and adding an inducer, so that the alkene reductase mutant is obtained.

Further, the recombinant vector is any one of the above recombinant vectors, the expression host cell is a conventional host cell in the art, and the recombinant vector can stably and automatically replicate, and the gene carried by the recombinant vector can be effectively expressed, and the recombinant vector can be a prokaryotic cell or a eukaryotic cell, for example, escherichia coli, yeast and the like, preferably escherichia coli expression host e.colibl21 (DE 3).

In some embodiments, the recombinant cell is E.coli BL21 (DE 3)/pET-26 b (+) -CvDH-A181R, a recombinant genetically engineered bacterium obtained by transforming a recombinant expression vector pET-26b (+) -CvDH-A181R into E.coli BL21 (DE 3). The culture medium used when the recombinant genetically engineered bacterium expresses the alkene reductase mutant may be a culture medium, such as an LB culture medium, which is used in the art to grow the recombinant genetically engineered bacterium and express the alkene reductase mutant of the present invention.

The culture method and the culture conditions have no special requirements, the normal growth of the recombinant genetic engineering strain is ensured, and the expression of the alkene reductase mutant is induced under the condition of proper temperature. The preferred cultivation method is: inoculating recombinant strain E.coliBL21 (DE 3)/pET-26 b (+) -CvDH-A181R into a test tube containing LB liquid medium containing kanamycin, culturing for 12 hours at 37 ℃ and 220 rpm, transferring into a 500mL shake flask containing 100mL LB liquid medium containing kanamycin according to the inoculum size of 1-2% (v/v), culturing at 37 ℃ and 220 rpm, adding isopropyl-beta-D-thiogalactoside (IPTG) with the final concentration of 100-500 mu M as an inducer when the OD ₆₀₀ of the culture solution reaches 0.6-0.8, centrifuging the culture solution after 16 ℃ for 16-24 hours, collecting bacterial sediment, and washing with normal saline to obtain recombinant cells.

In a fifth aspect, the invention provides a method of preparing an alkene reductase comprising:

1) Culturing any one of the recombinant cells described above and inducing expression of the alkene reductase mutant CvDH-a 181R;

2) Isolating the above-mentioned alkene reductase mutant CvDH-A181R from the culture obtained in 1).

Among them, the method of culturing and inducing recombinant cells and the method of separating the alkene reductase mutant CvDH-A181R from the culture are all conventional methods in the art.

In a sixth aspect, the invention provides the use of an alkene reductase mutant as described above, a nucleic acid molecule as described above, a recombinant vector as described above and/or a recombinant cell as described above in the preparation of (R) -citronellal, for example for catalyzing the reduction of the substrate citral to (R) -citronellal.

In a seventh aspect, the present invention provides a process for preparing (R) -citronellal, comprising: the alkene reductase mutant, the recombinant cell and/or the alkene reductase mutant prepared by any one of the methods are used as a catalyst to catalyze citral to perform reduction reaction, so as to obtain (R) -citronellal.

In some embodiments, in the above methods, the substrate of the reduction reaction is (E/Z) -citral, (E) -citral or (Z) -citral;

Wherein (E/Z) -citral is a mixture of (E) -citral and (Z) -citral, which may be mixed in any ratio, for example a mixture of (E) -citral and (Z) -citral in a molar ratio of 100:1 to 1:100, for example a molar ratio of 100:1, 80:1, 60:1, 40:1, 20:1, 1:1, 1:20, 1:40, 1:60, 1:80, 1:100, or a number or range between any two of these numbers.

In some embodiments, the pH of the reduction reaction is in the range of 5 to 9, e.g., pH5, pH5.5, pH6, pH6.5, pH7, pH7.5, pH8, pH8.5, pH9, or a value or range between any two of these values, preferably pH8.5.

In some embodiments, the temperature of the reduction reaction is 30-50 ℃, such as 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, or a value or range between any two of these values, preferably 35 ℃.

In some embodiments, in any of the above methods, the reduction reaction is performed using NADPH and/or NADH as cofactors; in some embodiments, the cofactor is regenerated enzymatically, e.g., by glucose dehydrogenase; the glucose dehydrogenase may be used as a free or immobilized enzyme or in the form of a free or immobilized cell, and may be prepared alone or may be co-expressed in a recombinant cell expressing the above-mentioned ene reductase.

In some embodiments, in any of the methods described above, the reduction reaction is an aqueous-organic phase biphasic reaction system.

In some embodiments, in any of the above methods, the feeding of the reduction reaction comprises: (E/Z) -citral, glucose dehydrogenase, NADH, alkene reductase;

The final concentration of (E/Z) -citral is 50-500 mM, for example 50 mM, 60mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or a value or range between any two of these values, preferably 500 mM, (E/Z) -citral is added in the form of a substrate solution formulated with n-hexane, n-heptane and/or ethyl acetate;

glucose is added in an amount of 1.0 to 1.10 equivalents of (E/Z) -citral, for example 1.0, 1.02, 1.04, 1.06, 1.08, 1.10 equivalents, preferably 1.02 equivalents;

The final concentration of glucose dehydrogenase is 1-10 g/L, for example 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, or a value or range between any two of these values, preferably 2 g/L;

The final concentration of NADH is in the range of 0.1-0.3 mM, for example 0.1 mM, 0.15mM, 0.2mM, 0.25 mM, 0.3 mM, or a value or range between any two of these values, preferably 0.2mM.

In some embodiments, in the reduction reaction according to any of the above methods, if the volume percentage of n-hexane, n-heptane and/or ethyl acetate is less than 10%, the n-hexane, n-heptane and/or ethyl acetate should be supplemented so that the volume percentage thereof is 10% -50%.

In some embodiments, the buffer used in the reduction reaction according to any of the methods described above is a buffer conventional in the art, such as a phosphate buffer, at a concentration of 50-500 mM, preferably 100 mM.

In some embodiments, any of the above methods comprises catalyzing a reduction reaction of citral with any of the above recombinant cells to yield (R) -citronellal;

Specifically, the lyophilized cells or lyophilized powder of the recombinant cells can be used as a catalyst for the whole cell catalytic production of (R) -citronellal, wherein the amount of the lyophilized cells or lyophilized powder is 0.66-13.16 g/g citral, for example, 0.66, 1.00, 3.00, 5.00, 7.00, 9.00, 11.00, 13.16 g/g citral, or a value or a range between any two of these values can be 0.99 g/g citral. The freeze-dried powder is obtained by crushing any recombinant cell to obtain a cell crushing liquid, and then freeze-drying the cell crushing liquid, wherein the freeze-dried cell is obtained by directly freeze-drying the recombinant cell without crushing.

In some embodiments, in any of the above methods, the reaction system is charged with 50-500 mM final concentration of (E/Z) -citral, (E/Z) -citral 1.0-1.10 times the equivalent of glucose, 1-10 g/L final concentration of glucose dehydrogenase, 0.1-0.3 mM final concentration of NADH and 0.66-13.16 g/g (E/Z) -citral of any of the above recombinant cells lyophilized cells, 10% -50% by volume of n-hexane, n-heptane and/or ethyl acetate, and the reaction is stirred at 30-50℃in phosphate buffer having a pH of 5-9 and a concentration of 50-500 mM.

It will be appreciated that the alkene reductase mutants CvDH-A181R of the present invention may be used in the form of whole cells of the engineering bacterium, as crude enzymes without purification, or as partially or fully purified enzymes. The alkene reductase mutants CvDH-A181R of the present invention can also be prepared as immobilized enzymes or catalysts in the form of immobilized cells using immobilization techniques known in the art.

In some embodiments, in any of the above methods, the reduction reaction is performed under stirring or shaking, e.g., with stirring of 100-500 rpm.

The beneficial effects of the invention are as follows:

the alkene reductase mutant CvDH-A181R provided by the invention can efficiently catalyze citral to generate (R) -citronellal, the production efficiency is up to 462.3 g L ^-1d^-1, the optical purity (e.e. value) of the product (R) -citronellal is 99.0%, and the method has important industrial application value for green efficient preparation of (R) -citronellal.

Detailed Description

The invention will be further illustrated with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Unless otherwise indicated, all technical means used in the examples are routine procedures in the art or experimental methods suggested by manufacturers of kits and instruments. Reagents and biological materials used in the examples were obtained commercially unless otherwise specified.

PET-26b (+) is a product of Novagen company under the product catalog number 69862-3CN.

The standard of neral ((Z) -citral) and geranial standard ((E) -citral) are products of Ruo Zhou Ruifeng chemical industry Co.

The (S) -citronellal standard is manufactured by Shanghai Yuan leaf Biotechnology Co., ltd, and the product catalog number is B29239.

The (R) -citronellal standard is a Shanghai Yuan Yes Biotechnology Co., ltd, and the product catalog number is S28339.

The (E/Z) -citral used in the examples was a mixture of (E) -citral and (Z) -citral, and was an Ala-dine product, the molar ratio of E-citral to Z-citral being 1:1.

The glucose dehydrogenase used in the examples is derived from Bacillus megaterium (Bacillus megaterium), and has the amino acid sequence shown in SEQ ID NO. 6 and the gene sequence shown in SEQ ID NO. 5.

Example 1: construction of pET-26b (+) -CvDH-A181R

PCR was performed using CvDH gene sequence shown in SEQ ID NO. 1 as a template and CvDH-FP and CvDH-RP as primers to obtain a fragment containing CvDH gene; the BamHI and NotI double enzyme cuts the fragment containing CvDH genes to obtain gene fragment; double-enzyme cutting pET-26b (+) by BamHI and NotI to obtain a vector fragment; the gene fragment and the vector fragment are connected to obtain a recombinant expression plasmid pET-26b (+) -CvDH, and the result is consistent with the expected result through whole plasmid sequencing. The recombinant expression plasmid pET-26b (+) -CvDH contains a nucleotide Sequence of encoding wild-type alkene reductase CvDH (NCBI Sequence ID: RCK 57628.1) derived from candida viscidosa (CANDIDA VISWANATHII), which is shown in SEQ ID NO. 1, and the amino acid Sequence of encoding wild-type alkene reductase CvDH is shown in SEQ ID NO. 2.

CvDH-FP：5’-cagtGGATCCATGACTATCGACGCTGAAAAATTCC-3’(SEQ ID NO: 7)；

CvDH-RP：5’-cagtGCGGCCGCCTAAACCAAAGCTGTAGGGAAG-3’(SEQ ID NO: 8)。

And (3) carrying out full-plasmid PCR by taking recombinant expression plasmid pET-26b (+) -CvDH as a template and taking CvDH-A181R-FP and CvDH-A181R-RP as primers, introducing A181R mutation sites to obtain recombinant expression plasmid pET-26b (+) -CvDH-A181R, and sequencing the plasmid, wherein the result is consistent with the expected result. The nucleotide sequence of the alkene reductase mutant CvDH-A181R contained in the recombinant expression plasmid pET-26b (+) -CvDH-A181R is shown as SEQ ID NO. 3, and the amino acid sequence of the coded alkene reductase mutant CvDH-A181R is shown as SEQ ID NO. 4.

CvDH-A181R-FP：5’-CAAGAAACCGTCTTGCTGCGGGATTCG-3’ (SEQ ID NO: 9)；

CvDH-A181R-RP：5’-CAGCAAGACGGTTTCTTGCAGCGTTAGG-3’ (SEQ ID NO: 10)。

Compared with the wild-type alkene reductase CvDH shown in SEQ ID NO. 2, the amino acid at 181 th position of alkene reductase mutant CvDH-A181R is mutated from alanine to arginine.

Example 2: construction of wild-type CvDH expression Strain and mutant CvDH-A181R expression Strain

The mutant recombinant expression plasmid pET-26b (+) -CvDH-A181R obtained in example 1 was transferred into an expression host E. coliBL21 (DE 3) by means of chemical transformation, and screened by coating LB solid medium containing 50 μg/mL kanamycin to obtain recombinant strain E.coliBL21 (DE 3)/pET-26 b (+) -CvDH-A181R expressing mutant CvDH-A181R.

Recombinant E.coli BL21 (DE 3)/pET-26 b (+) -CvDH expressing wild-type CvDH was obtained in the same manner, except that the recombinant expression plasmid pET-26b (+) -CvDH-A181R was replaced with pET-26b (+) -CvDH.

Example 3: preparation of enzymes

Recombinant E.coli BL21 (DE 3)/pET-26 b (+) -CvDH-A181R obtained in example 2 was inoculated into a test tube containing LB liquid medium containing 50. Mu.g/mL kanamycin, cultured at 37℃for 12 hours, then transferred to a 500 mL shake flask containing 100mL LB liquid medium containing 50. Mu.g/mL kanamycin at 1% (v/v), cultured at 37℃and 220 rpm until OD ₆₀₀ = 0.6-0.8, added with an inducer IPTG having a final concentration of 300. Mu.M, induced and cultured at 16℃for 20 hours, the induced and cultured bacterial liquid was centrifuged at 9000 rpm for 10 minutes, and bacterial pellet was collected and washed with physiological saline to obtain resting cells. The resting cells are directly freeze-dried to obtain freeze-dried cells of mutant CvDH-A181R, or freeze-dried after ultrasonic disruption to obtain freeze-dried powder, and the freeze-dried powder is preserved at 4 ℃.

Lyophilized cells and lyophilized powder of wild-type CvDH were obtained in the same manner, except that recombinant E.coli BL21 (DE 3)/pET-26 b (+) -CvDH-A181R was replaced with E.coli BL21 (DE 3)/pET-26 b (+) -CvDH.

Example 4: enzymatic preparation of (R) -citronellal

The mutant enzyme CvDH-A181R prepared in example 3 was used as a biocatalyst in the preparation reaction of (R) -citronellal.

100ML reaction system: 500 mM (E/Z) -citral ((E/Z) -citral was added as a substrate solution formulated with n-hexane to a concentration of 3M), 510 mM glucose (added as an aqueous glucose solution), 2 g/L glucose dehydrogenase, 0.2 mM NADH (added as an aqueous NADH solution), n-hexane was fed so that the volume percentage of n-hexane in the system was 20%, 7.5 g (equivalent to 0.99 g/g (E/Z) -citral) of the lyophilized cells of mutant CvDH-A181R obtained in example 3), 100mM phosphate buffer (pH 8.5) was fed to 100mL, and the reaction was stirred for 4 hours under 200 rpm at 35 ℃.

The reaction formula is as follows:

After the reaction, adding 1mL reaction liquid into an equal volume of ethyl acetate, repeatedly reversing and mixing, centrifuging 10 min at 10000 rpm, taking out supernatant, adding the equal volume of ethyl acetate again, extracting according to the method, mixing the supernatant solutions collected twice, adding anhydrous sodium sulfate for drying, centrifuging 10 min at 10000 rpm, taking a proper amount of supernatant extract, and detecting the content of (E/Z) -citral and citronellal through gas chromatography analysis.

The gas chromatography detection method comprises the following steps: HP-5 column (30 m X0.32 mm X0.25 μm), split ratio 10:1, sample injection temperature: 220 ℃; FID detector temperature: 250 ℃; carrier gas: nitrogen, 54 mL/min; air flow rate: 400 mL/min; hydrogen flow rate: 30 mL/min; column flow rate: 1 mL/min; sample injection volume: 1. mu L; the initial column temperature was 100 ℃, maintained at2 min, and raised to 150 ℃ at a rate of 10 ℃ per minute maintained at 4 min. The content of citronellal and citral ((E) -citral and (Z) -citral was obtained by the external standard method.

Citronellal standards (misconvergence citronellal, available from Allatin under the product catalog number C118628-1 ml), neral standards, geranial standards retention times were 3.687 min, 4.609 min, 4.920 min, respectively.

The retention time of citronellal in the reaction solution was 3.687 min.

The enantioselective assay of product citronellal uses a beta-DEX 225 vapor capillary column (30 m X0.25 mm X0.25 μm) with a split ratio of 100:1; sample introduction temperature: 220 ℃; FID detector temperature: 250 ℃; carrier gas: nitrogen, 34 mL/min; air flow rate: 400 mL/min; hydrogen flow rate: 40 mL/min; column flow rate: 1 mL/min; sample injection volume: 1. mu L; the initial column temperature was 95 ℃ and maintained at 35 min, elevated to 160 ℃ at 5 ℃ per minute and maintained at 2 min, and elevated to 200 ℃ at 10 ℃ per minute and maintained at 5 min. The content of (S) -citronellal and (R) -citronellal is obtained by an external standard method.

The retention times of the (S) -citronellal standard and the (R) -citronellal standard were 25.058 min and 25.145 min, respectively.

The retention times of (S) -citronellal and (R) -citronellal in the reaction solutions were 25.058 min and 25.145 min, respectively.

Calculated, the substrate (E/Z) -citral conversion (conversion= (initial amount of reactant-amount of reactant remaining)/initial amount of reactant x 100%) was 99.9%, the production efficiency of product (R) -citronellal (production efficiency = amount of product formed by reaction/(volume of reaction) reaction time)) was 462.3 g L ^-1d^-1, and the optical purity (e.e. value) of product (R) -citronellal was 99.0%.

The (R) -citronellal preparation reaction was performed in the same manner using wild-type CvDH lyophilized cells, except that mutant CvDH-A181R lyophilized cells were replaced with wild-type CvDH lyophilized cells. Calculated, the conversion of substrate (E/Z) -citral was 53%, the production efficiency of product (R) -citronellal was 245.3 g L ^-1d^-1, and the optical purity (e.e. value) of product (R) -citronellal was 99.0%.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical solution and the concept of the present invention, and should be covered by the scope of the present invention.

Claims (10)

1. An alkene reductase mutant has an amino acid sequence shown in SEQ ID NO. 4.

2. A nucleic acid molecule encoding the alkene reductase mutant of claim 1.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. A recombinant cell comprising the nucleic acid molecule of claim 2 and/or the recombinant vector of claim 3.

5. A method of preparing an alkene reductase: comprising the following steps:

1) Culturing the recombinant cell of claim 4 and inducing expression of the alkene reductase mutant of claim 1;

2) Isolating the alkene reductase mutant of claim 1 from the culture obtained in 1).

6. Use of the alkene reductase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3 and/or the recombinant cell of claim 4 for the preparation of (R) -citronellal.

7. A process for preparing (R) -citronellal, comprising: the (R) -citronellal is obtained by catalyzing citral to perform a reduction reaction with the alkene reductase mutant as claimed in claim 1, the recombinant cell as claimed in claim 4 and/or the alkene reductase prepared by the method as claimed in claim 5 as a catalyst.

8. The method according to claim 7, wherein: the substrate of the reduction reaction is (E/Z) -citral, (E) -citral or (Z) -citral.

9. The method according to claim 7, wherein: the pH of the system of the reduction reaction is 5-9.

10. The method according to any one of claims 7-9, wherein: the temperature of the reduction reaction is 30-50 ℃.