Carbonyl reductase mutant and application thereof in preparation of (R)-citronellal

Abstract

The invention discloses a carbonyl reductase mutant and application of the carbonyl reductase mutant in preparation of (R)-citronellal. The application comprises the following steps: in a reaction system in the presence of a cosolvent, formate and a cofactor NAD < + >, carrying out catalytic reaction by taking recombinant cells for expressing a carbonyl reductase mutant and formate dehydrogenase as catalysts and (S)-citronellal as a substrate to generate (S)-citronellal, and preparing a reaction solution containing (R)-citronellal and (S)-citronellal; the mutant of the carbonyl reductase comprises an amino acid fragment of which the amino acid sequence is shown as SEQ ID NO: 3. The co-production of the (R)-citronellal and the (S)-citronellal is realized, and the e.e. Value of the obtained (R)-citronellal is greater than 99%.

Description

Technical Field

[0001] The present application relates to the field of biocatalysis technology, specifically to a carbonyl reductase mutant and its use in the preparation of (R)-citronellal, and further to a method for separating and preparing (R)-citronellal and simultaneously co-producing (S)-citronellol. Background Art

[0002] (R)-citronellal is a key precursor for the synthesis of L-menthol and is widely used in related fields such as food, beverages, and daily products. Currently, (R)-citronellal is mainly synthesized industrially using chemical catalysis, using citral as a raw material and produced through a hydrogenation reaction. However, due to the poor chiral selectivity of the chemical catalyst, the synthetic product contains both (R)-citronellal and (S)-citronellal. Simultaneously, a method for synthesizing (R)-citronellal using a biocatalytic method has also been reported, in which alkene reductase catalyzes the synthesis of (R)-citronellal from citral, which has high reaction selectivity but low reaction efficiency. Because the physical and chemical properties of chemically synthesized (R)-citronellal and (S)-citronellal are relatively close, separation is difficult to achieve.

[0003] Therefore, there is an urgent need in this field to develop a method for separating and preparing (R)-citronellal from the product of citral hydrogenation (i.e., a mixture containing both (R)-citronellal and (S)-citronellal) as a raw material, while effectively utilizing (S)-citronellal. Summary of the Invention

[0004] Based on this, in order to address the problem that (R)-citronellal and (S)-citronellal can be obtained by asymmetric hydrogenation of citral but the two cannot be separated, the present application at least provides a carbonyl reductase mutant and its use in the preparation of (R)-citronellal.

[0005] In a first aspect of the present application, there is provided a method for separating (R)-citronellal from a mixture of (R)-citronellal and (S)-citronellal, the method comprising:

[0006] In the presence of cosolvent, formate and cofactor NAD + In a reaction system, a recombinant cell expressing a carbonyl reductase mutant and formate dehydrogenase are used as catalysts, and (S)-citronellal is used as a substrate to carry out a catalytic reaction to generate (S)-citronellal, thereby preparing a reaction solution containing (R)-citronellal and (S)-citronellal;

[0007] The mutant of the carbonyl reductase comprises an amino acid fragment having an amino acid sequence as shown in SEQ ID NO: 3.

[0008] In a second aspect of the present application, a method for preparing (R)-citronellal is provided, comprising:

[0009] Using citral as raw material, (R)-citronellal and (S)-citronellal reactants are prepared through hydrogenation reaction;

[0010] (R)-citronellal was separated from the mixture of (R)-citronellal and (S)-citronellal according to the method described above.

[0011] In a third aspect of the present application, a carbonyl reductase mutant is provided, which comprises mutations Q76K, S189G and N233K relative to the amino acid fragment shown in SEQ ID NO: 1 contained in the wild-type carbonyl reductase.

[0012] In a fourth aspect, the present application provides a nucleic acid encoding the carbonyl reductase mutant as described above.

[0013] In a fifth aspect, the present application provides a gene expression cassette comprising a promoter, the nucleic acid as described above, and a terminator.

[0014] In a sixth aspect of the present application, a recombinant expression vector is provided, comprising a backbone vector and:

[0015] A nucleic acid as described above; and / or,

[0016] A gene expression cassette as described above.

[0017] In a seventh aspect, the present application provides a recombinant cell comprising one or more of the nucleic acid described above, the gene expression cassette described above, and the recombinant expression vector described above.

[0018] In an eighth aspect, the present application provides a method for preparing a whole-cell catalyst comprising the recombinant cell as described above, comprising: performing fermentation culture and induction culture on the recombinant cell as described above;

[0019] a1 fermentation culture: the seed solution of the recombinant cells was inoculated into a fermentation medium for fermentation; the fermentation medium may optionally be a TB liquid medium;

[0020] a2. Induction culture: When the OD value of the fermentation system reaches 25-35, add inducer to induce culture.

[0021] In a ninth aspect, the present application provides use of the recombinant cell described above in the preparation of (R)-citronellal.

[0022] The carbonyl reductase mutant in one embodiment of the present application and the method for preparing (R)-citronellal using the same have at least the following significant advantages:

[0023] 1) This carbonyl reductase mutant has high activity and selectivity, and can selectively convert (S)-citronellal to (S)-citronellol, but has no selectivity for (R)-citronellal, thereby achieving the selective separation of (R)-citronellal.

[0024] 2) The co-production of (R)-citronellal and (S)-citronellol was achieved, with the resulting (R)-citronellal exhibiting an ee value exceeding 99%. Both can be used in fragrances with high atom economy. Furthermore, the resulting citronellol possesses a more elegant rose aroma, making it an indispensable ingredient in the formulation of various rose-based floral fragrances, thus expanding the product's application areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the implementation methods and examples of this application and to more completely understand the application and its beneficial effects, the following briefly introduces the drawings required for the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0026] Figure 1 Schematic diagram of a reaction formula in one embodiment of the present application. DETAILED DESCRIPTION

[0027] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] In this application, unless otherwise specified, "one or more" refers to any one of the listed items or any combination of the listed items. Similarly, "one or more" and other similar expressions that refer to "one or more" are also understood in the same way unless otherwise specified.

[0030] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0031] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0032] In this application, the terms "further," "further," "particularly," "for example," "such as," "example," and "for example" are used for descriptive purposes to indicate that the preceding and following technical solutions are related in terms of the content covered, but should not be construed as limiting the preceding technical solution or the scope of protection of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it is understood that A is not limited to B.

[0033] In this application, the terms "optionally," "optional," and "optional" mean optional, that is, they refer to either option selected from the two parallel options of "yes" or "no." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "optional" is independent. Unless otherwise specified, the descriptions "optionally include," "optionally include," etc. in this application, taking "optionally include" as an example, mean "may include or not include."

[0034] As used herein, the terms "comprising," "including," and "include" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0035] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0036] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, the terms "first," "second," "third," "fourth," etc. in "the first aspect," "the second aspect," "the third aspect," "the fourth aspect," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor should they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc. serve only for the purpose of non-exhaustive enumeration and description, and should be understood not to constitute a closed-ended limitation on quantity.

[0038] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval only refers to an integer in the numerical interval, including the two endpoint integers of the numerical range and each integer between the two endpoints, is equivalent to directly enumerating each integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0039] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0040] One aspect of the present application provides a method for preparing (R)-citronellal and simultaneously producing (S)-citronellal. The method uses a mixture of (R)-citronellal and (S)-citronellal as a raw material and utilizes carbonyl reductase to convert (S)-citronellal into (S)-citronellal, while leaving the (R)-citronellal unreacted. Due to the significant differences in the physical and chemical properties of (R)-citronellal and (S)-citronellal, separation is possible with high atom economy.

[0041] In the present application, the carbonyl reductase and its mutants have the following characteristics: Specifically, the corresponding carbonyl reductase CRase-1 was obtained from Yarrowia lipolytica, whose amino acid sequence is shown in SEQ ID NO: 1 and nucleotide sequence is shown in SEQ ID NO: 2. To screen for the highly active mutant CRase-1-mut, error-prone PCR technology was used to further mutate it. Throughput screening using (R / S)-citronellal as the reaction substrate, a mutant with high conversion activity was obtained, whose amino acid sequence is shown in SEQ ID NO: 3 and nucleotide sequence is shown in SEQ ID NO: 4.

[0042] In one aspect of the present application, a parent carbonyl reductase is provided, which comprises an amino acid fragment having an amino acid sequence as shown in SEQ ID NO: 1.

[0043] In another aspect of the present application, a nucleic acid molecule encoding the above-mentioned parent carbonyl reductase is provided. In some embodiments, the nucleic acid sequence of the nucleic acid molecule is shown in SEQ ID NO: 2.

[0044] The nucleic acid molecules can be obtained by, but are not limited to, PCR amplification or artificial synthesis.

[0045] In another aspect of the present application, a mutant of the above-mentioned parent carbonyl reductase is provided, which comprises mutations Q76K, S189G and N233K relative to the amino acid fragment shown in SEQ ID NO: 1 contained in the wild-type carbonyl reductase.

[0046] In some embodiments, the mutant of the parent carbonyl reductase has an amino acid sequence such as an amino acid fragment shown in SEQ ID NO: 3, and has higher catalytic activity and selectivity than the above-mentioned parent carbonyl reductase.

[0047] The parent carbonyl reductase or its mutant can be obtained by conventional methods in the art. For example, the encoding gene of the parent carbonyl reductase or its mutant is first synthesized and then expressed in a biological manner, such as by expressing it in a prokaryotic organism (Escherichia coli) using recombinant technology.

[0048] In some embodiments, the above-mentioned parent carbonyl reductase or its mutant is obtained by transforming a recombinant vector containing its encoding gene (nucleic acid sequence is shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively) into an Escherichia coli expression host (e.g., E. coli BL21 (DE3)), thereby constructing a recombinant genetic engineering strain, and then culturing the recombinant strain and inducing expression by adding an inducer.

[0049] In another aspect of the present application, a nucleic acid molecule encoding the above carbonyl reductase mutant is provided.

[0050] The nucleic acid molecule has one of the following characteristics:

[0051] (1) comprising a nucleic acid sequence as shown in SEQ ID NO: 4;

[0052] (2) A nucleic acid sequence that encodes the same protein as the nucleic acid sequence shown in (1), but is different from the nucleic acid sequence shown in C1 due to the degeneracy of the genetic code;

[0053] (3) A nucleic acid sequence that hybridizes with the nucleic acid sequence defined in (1) or (2) under stringent conditions and encodes the carbonyl reductase mutant according to claim 1 or 2;

[0054] (4) A nucleic acid sequence obtained by replacing, deleting or adding one or more nucleotides to the nucleic acid sequence shown in any one of (1) to (3), and a nucleic acid sequence having the same or similar function as the nucleic acid sequence shown in any one of (1) to (3). It should be understood that the above-mentioned nucleic acid molecules provided in this application are usually obtained by PCR amplification or artificial synthesis.

[0055] In another aspect of the present application, a gene expression cassette is provided, which contains a promoter, the nucleic acid molecule as described above, and a terminator.

[0056] In another aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid molecule as described above.

[0057] In another aspect of the present application, a recombinant expression vector is provided, comprising the gene expression cassette as described above.

[0058] In some embodiments, the recombinant expression vector is pETDuet-1-CRase or pETDuet-1-ΔCRase-mut, wherein the nucleic acid molecule encoding the carbonyl reductase and its mutant replaces the sequence between the BamHI and EcoRI restriction sites of pETDuet-1, and the rest of the sequence remains unchanged.

[0059] In another aspect of the present application, a recombinant cell is provided, comprising one or more of the nucleic acid molecule, gene expression cassette and recombinant expression vector as described above.

[0060] In some embodiments, the recombinant cell is induced to express the parent carbonyl reductase or a mutant thereof.

[0061] In some embodiments, the method for constructing the recombinant cell comprises the following:

[0062] The recombinant vector is transformed into an expression host cell, cultured and induced to express by adding an inducer to obtain the parent carbonyl reductase or a mutant thereof.

[0063] Furthermore, the recombinant vector is any of the above-mentioned recombinant vectors, and the expression host cell is a prokaryotic cell or a eukaryotic cell, such as Escherichia coli, yeast, etc., preferably an Escherichia coli expression host E. coli BL21 (DE3), Rosetta (DE3), BL21 (DE3) plysS, M15, W3110 or Top10f'.

[0064] The culture medium used when the recombinant genetically engineered bacteria express the parent carbonyl reductase or its mutant is a conventional culture medium in the art that can allow the recombinant genetically engineered bacteria to grow and express, preferably LB culture medium.

[0065] There are no special requirements for the culture method and culture conditions. It is sufficient to ensure that the recombinant genetic engineering strain grows normally and induces the expression of the parent carbonyl reductase and its mutants at 20°C.

[0066] In another aspect of the present application, a method for preparing a recombinant cell of a carbonyl reductase or a mutant thereof is provided, comprising:

[0067] Cultivating any of the above-mentioned recombinant cells and adding an inducer to induce the cells to obtain a culture; and separating the cells from the culture by centrifugation;

[0068] Among them, the methods of culturing and inducing recombinant cells and the methods of isolating carbonyl reductase and its mutants from the culture are conventional methods in the art.

[0069] In another aspect of the present application, a method for preparing a whole-cell catalyst comprising the recombinant cell as described above is provided, comprising: subjecting the recombinant cell as described above to fermentation culture and induction culture;

[0070] a1 fermentation culture: the seed solution of the recombinant cells was inoculated into a fermentation medium for fermentation; the fermentation medium may optionally be a TB liquid medium;

[0071] a2. Induction culture: When the OD value of the fermentation system reaches 25-35, add inducer to induce culture.

[0072] In some embodiments, in step a1, the initial concentration of glucose in the fermentation system is 30 g / L to 50 g / L. For example, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L, 41 g / L, 42 g / L, 43 g / L, 44 g / L, 45 g / L, 46 g / L, 47 g / L, 48 g / L, 49 g / L, 50 g / L, or a range or value between any two values.

[0073] In some embodiments, in step a1, the initial OD of the recombinant cells in the fermentation system is 600 It is 0.3~0.5, for example, 0.3, 0.4 or 0.5.

[0074] In some embodiments, in step a1, the glucose concentration in the fermentation system is controlled to be 5 g / L to 10 g / L by feeding glucose.

[0075] In some embodiments, in step a1, the fermentation culture conditions include: controlling the dissolved oxygen to 25% to 35%, culturing at 37° C. and 200 rpm to 950 rpm. Exemplary conditions include 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm, 800 rpm, 850 rpm, 900 rpm, 950 rpm, or any range or value between any two values.

[0076] In some embodiments, in step a2, the inducer comprises IPTG (Isopropyl β-D-thiogalactopyranoside).

[0077] In some embodiments, in step a2, the concentration of the inducer in the fermentation system is 0.2 mM to 0.5 mM, for example, 0.2 mM, 0.3 mM, 0.4 mM or 0.5 mM.

[0078] In some embodiments, in step a2, the induction culture conditions include: induction culture at 15°C to 25°C for 30 to 35 hours. Exemplarily, the induction culture temperature is 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or a range or value between any two values; and the induction culture time is, for example, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, or a range or value between any two values.

[0079] In another aspect of the present application, a method for separating and preparing (R)-citronellal and simultaneously co-producing (S)-citronellol is provided, which uses a raw material containing (R)-citronellal and (S)-citronellal as a substrate, carbonyl reductase or a mutant thereof and formate dehydrogenase as a catalyst, and the reaction system also contains a buffer salt, a cosolvent, formate and a cofactor for catalytic reaction.

[0080] The cosolvent is used to dissolve the substrates (R)-citronellal and (S)-citronellal, and is introduced as a solvent for dissolving the substrates when the substrates are added into the reaction system.

[0081] In some embodiments, there is provided a method for separating (R)-citronellal from a mixture of (R)-citronellal and (S)-citronellal, the method comprising:

[0082] In the presence of cosolvent, formate and cofactor NAD + In a reaction system, a recombinant cell expressing a carbonyl reductase mutant and formate dehydrogenase are used as catalysts, and (S)-citronellal is used as a substrate to carry out a catalytic reaction to generate (S)-citronellal, thereby preparing a reaction solution containing (R)-citronellal and (S)-citronellal;

[0083] The mutant of the carbonyl reductase comprises an amino acid fragment having an amino acid sequence as shown in SEQ ID NO: 3.

[0084] In some embodiments, in the above method, the formate dehydrogenase is derived from a commercial enzyme preparation and is mainly used in coenzyme circulation.

[0085] In some embodiments, the formate dehydrogenase is derived from a product of Shangke Biopharmaceuticals (Shanghai) Co., Ltd., with the product number being ES-FDH-101.

[0086] In some embodiments, in any of the methods described above, the dosage (initial concentration) of formate dehydrogenase in the reaction system is 1 U / mL to 5 U / mL, for example, 1 U / mL, 2 U / mL, 3 U / mL, 4 U / mL, 5 U / mL, or any value and range between these values, for example, 3 U / mL to 5 U / mL.

[0087] In some embodiments, in any of the methods described above, in the reaction system, the initial concentration of (R)-citronellal and (S)-citronellal is 70 g / L to 100 g / L, for example, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, or any value and range therebetween, for example, 85 g / L to 100 g / L.

[0088] In the mixture of (R)-citronellal and (S)-citronellal, (R)-citronellal and (S)-citronellal are present in any mass ratio, for example, a mass ratio of 60:40 to 95:5, such as 60:40, 65:35, 70:30, 75:25, 80:20, 90:10, 95:5, or any ratio and range between these ratios, such as 70:30 to 95:5.

[0089] In some embodiments, in any of the above methods, the amount of recombinant cells added is 0.25-0.75 g / g (R / S)-citronellal, for example, 0.25 g / g (R / S)-citronellal, 0.35 g / g (R / S)-citronellal, 0.45 g / g (R / S)-citronellal, 0.55 g / g (R / S)-citronellal, 0.65 g / g (R / S)-citronellal, 0.75 g / g (R / S)-citronellal, or any value and range therebetween, for example, 0.5 g / g (R / S)-citronellal to 0.75 g / g (R / S)-citronellal.

[0090] In some embodiments, in any of the above methods, the co-solvent is one or more of ethyl acetate, n-heptane and n-octane, and the volume fraction in the reaction system is 30% to 45%, for example, 30%, 35%, 40% and 45%, or any value and range between these values, for example, 35% to 45%.

[0091] In some embodiments, in any of the above methods, the formate is one or more of sodium formate and potassium formate, such as sodium formate, and the molar ratio of the formate to (R / S)-citronellal is 1 to 1.5:1, such as 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, or any ratio and range between these ratios, such as (1 to 1.2):1.

[0092] In some embodiments, in any of the above methods, the cofactor is NAD +, the concentration in the reaction system is 0.05 mM~0.15 mM, for example, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.10 mM, 0.11 mM, 0.12 mM, 0.13 mM, 0.14 mM, 0.15 mM, or any value and range therebetween, for example, 0.05 mM~0.1 mM.

[0093] In some embodiments, in any of the methods described above, the buffer of the reaction system is a phosphate buffer or a Tris-HCl buffer, and the pH of the catalytic reaction is controlled to be 6.0-7.5, for example, pH 6.0, 6.5, 7.0, 07.5, or any value and range between these values, for example, pH 7.0-7.5.

[0094] In some embodiments, in any of the above methods, the reaction temperature of the catalytic reaction is 30°C to 45°C, for example, 30°C, 35°C, 40°C, 45°C, or any value and range therebetween, for example, 35°C to 45°C;

[0095] The reaction time can be 15 h to 25 h, for example, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h, or any value and range therebetween, preferably 20 h to 25 h.

[0096] In some embodiments, in any of the above methods, after the reaction is completed, the reaction solution is filtered to obtain a filtrate, the bacterial cells are extracted with the cosolvent to obtain a supernatant, the filtrate and the supernatant are combined and allowed to stand for phase separation to obtain an upper organic phase, and the organic phase is distilled to prepare (R)-citronellal and (S)-citronellol, and the ee value of the obtained citronellal is greater than 99%.

[0097] In some embodiments, the method further comprises a separation step to separate (R)-citronellal and (S)-citronellol in the reaction solution;

[0098] The separation step includes the following sub-steps:

[0099] Filtration: filtering the reaction solution to obtain a filtrate and recombinant cells;

[0100] Extraction: extracting the recombinant cells to obtain a supernatant;

[0101] Standing and distilling: the filtrate and the supernatant are combined and then standing for phase separation to obtain an upper organic phase, and the organic phase is distilled to prepare (R)-citronellal and (S)-citronellol.

[0102] In some embodiments, in the above method, the distillation conditions are: intermittent distillation, the number of distillation plates is 35-45, the bottom temperature is 40°C-50°C, the top pressure is 50 Pa-70 Pa, and the reflux ratio is 1-1.6:1.

[0103] In another aspect of the present application, a method for preparing (R)-citronellal is provided, comprising:

[0104] Using citral as raw material, (R)-citronellal and (S)-citronellal reactants are prepared through hydrogenation reaction;

[0105] (R)-citronellal was separated from the mixture of (R)-citronellal and (S)-citronellal according to the method described above.

[0106] Some examples are provided below.

[0107] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0108] pETDuet-1 is a product of EMD Biosciences (Novagen).

[0109] Formate dehydrogenase is a product of Shangke Biopharmaceuticals (Shanghai) Co., Ltd., product number: ES-FDH-101.

[0110] (R)-Citronellal, (S)-citronellal, and (S)-citronellol were all products of Sigma-Aldrich.

[0111] Gas chromatography detection of (R)-citronellal, (S)-citronellal, and (S)-citronellol was performed using a β-DEX225 capillary column (30 m × 0.25 mm × 0.25 μm) with a split ratio of 100:1; injection temperature: 220°C; FID detector temperature: 250°C; carrier gas: nitrogen, 34 mL / min; air flow: 400 mL / min; hydrogen flow: 40 mL / min; column flow rate: 1 mL / min; injection volume: 1 μL. The column temperature was initially set at 95°C for 35 min, then increased at a rate of 5°C / min to 160°C for 2 min, and then increased at a rate of 10°C / min to 200°C for 5 min. Quantitative analysis was performed using an external standard method. The retention time of (R)-citronellal standard was 25.145 min, the retention time of (S)-citronellal standard was 25.058 min, and the retention time of (S)-citronellol was 23.052 min.

[0112] Example 1: Obtaining the maternal carbonyl reductase gene sequence

[0113] 1) A carbonyl reductase gene from Yarrowia lipolytica was synthesized (the nucleotide sequence of the parent carbonyl reductase is shown in SEQ ID NO: 2), and BamHI and EcoRI restriction sites were added upstream and downstream, respectively.

[0114] 2) Using the obtained gene sequence as a template, PCR was performed with primer 1 (5'-ttctcgaacccaaggctgac-3', SEQ ID NO: 5) and primer 2 (5'-gatttcgtggcccataacgc-3', SEQ ID NO: 6) to obtain an amplified fragment containing the maternal carbonyl reductase gene.

[0115] Example 2: Obtaining the gene sequence of the carbonyl reductase mutant using error-prone PCR technology

[0116] The PCR amplified fragment obtained in Example 1 was used as a template, and Primer 1 and Primer 2 in Example 1 were used as a primer pair to perform the following error-prone PCR, obtaining a total of 96 mutants.

[0117] Error-prone PCR reaction system: 5 μl of 10× amplification buffer, 4 μl of each of the four dNTP mixtures (2.5 mmol / L), 50 pmol of each primer, 1.5 μg of template DNA, 0.5 μl of Taq DNA polymerase, MgCl2 2+ 7 mmol / L, add double-distilled water to 50 μl.

[0118] PCR reaction procedure: (1) Pre-denaturation: 96℃-3.5 min; (2) Denaturation: 96℃-30 s; Annealing: 60℃-30 s; Extension: 72℃-2 min; 40 cycles in total; (3) Post-extension: 75℃-10 min; (4) Incubation at 4℃.

[0119] Example 3: Cloning of the parent carbonyl reductase gene and construction of the expression strain

[0120] 1) The PCR amplified fragment containing the parent carbonyl reductase gene obtained in Example 1 was double-digested with BamHI and EcoRI to obtain a gene fragment; pETDuet-1 was double-digested with BamHI and EcoRI to obtain a vector fragment; the gene fragment and the vector fragment were ligated to obtain a recombinant expression plasmid, which was named pETDuet-1-CRase. This plasmid was sequenced, and the results were consistent with expectations.

[0121] 2) The recombinant expression plasmid pETDuet-1-CRase was transformed into competent E. coli DH5α cells using the heat shock method. The cells were then plated with LB solid medium containing 35 μg / mL kanamycin to generate a single clone. The strain was amplified and the plasmid was extracted to obtain the pETDuet-1-CRase plasmid. This plasmid was then chemically transformed into the expression host E. coli BL21(DE3). Screening was performed by plating with LB solid medium containing 35 μg / mL kanamycin to obtain recombinant strains expressing the parent invertase, CRase.

[0122] Example 4: Cloning of carbonyl reductase mutant genes and construction of expression strains

[0123] According to the method of Example 3, the 96 mutants of the carbonyl reductase gene obtained in Example 2 were respectively constructed into recombinant plasmids pETDuet-1-ΔCRase-mut-1 to pETDuet-1-ΔCRase-mut-96, and recombinant bacteria were numbered 1 to 96.

[0124] Example 5: High-throughput screening of carbonyl reductase mutants with high catalytic and high selectivity

[0125] 1.5 mL of LB culture medium was added to a deep-well 96-well plate, and the recombinant bacteria verified by PCR in Examples 3 and 4 were cultured therein. After shaking and culturing for 4 hours in a 37°C environment, IPTG was added at a final concentration of 0.5 mM at 20°C to induce protein expression. After 20 hours of induction expression, the deep-well plate was centrifuged at 6000 rpm at 4°C for 10 minutes. The supernatant of the culture solution was removed, and 1 mL of 100 mM pH 7.0 phosphate buffer was added to the resulting bacteria, and the bacteria were resuspended. The resuspended bacteria solution was placed in a -80°C refrigerator and frozen for 12 hours, then taken out and thawed in a 4°C environment; the operation was repeated twice to achieve freeze-thaw fragmentation of the bacteria. The deep-well plate was centrifuged at 6000 rpm at 4°C for 10 minutes to obtain a supernatant containing carbonyl reductase.

[0126] Mutant screening was performed in a 96-well plate. The reaction system consisted of 200 μL of 12 mg of (R)-citronellal, 8 mg of (S)-citronellal, 50 μL of supernatant containing carbonyl reductase, NADH (final concentration: 0.2 mM), and 50 μL of 500 g / L glucose in water. The volume was made up with DMSO. The 96-well plate was incubated at 35°C with a shaker at 120 rpm for 2 hours. After the reaction, the absorbance of the samples was measured at 340 nm using a microplate reader.

[0127] Five reaction solutions with lower absorbance were extracted with ethyl acetate, and after removing water, the conversion rates of (R)-citronellal and (S)-citronellal were detected by gas chromatography. It was found that only (S)-citronellal reacted in the reaction solution No. 85#, while (R)-citronellal had no selectivity. The corresponding 85# mutant bacteria were subjected to plasmid extraction, and the extracted plasmids were sequenced to obtain a gene sequence encoding a carbonyl reductase mutant as shown in SEQ ID NO: 4, and the amino acid sequence of the carbonyl reductase mutant encoded by it is shown in SEQ ID NO: 3. Compared with the amino acid sequence of the parent carbonyl reductase shown in SEQ ID NO: 1, the mutant has the following mutations: Q at position 76 is mutated to K, S at position 189 is mutated to G, and N at position 233 is mutated to K.

[0128] Example 6: Preparation of whole-cell catalyst

[0129] The strain CRase containing the parent carbonyl reductase constructed in Example 3 was inoculated into 10 mL of LB liquid medium (containing 35 μg / mL chloramphenicol) and cultured at 37°C and 200 rpm until the OD 600=0.6-0.8, then transferred to a 500 mL shake flask containing 100 mL LB liquid medium (containing 35 μg / mL chloramphenicol) at a 1% (v / v) inoculation volume, and cultured at 37°C and 200 rpm for 24 h as the fermentation seed liquid. 1.9 L TB liquid medium was added to a 3 L fermentor, and glucose was added to make the initial glucose content 50 g / L. The seed liquid was added to make the initial OD 600 =0.3-0.5, glucose concentration was controlled to be ≥5 g / L and ≤10 g / L by feeding glucose during the fermentation process, aeration volume was 4 vvm, dissolved oxygen was controlled to be 35%, and culture was carried out at 37°C and 200 rpm~950 rpm. 600 After reaching 25, the inducer IPTG was added with a final concentration of 0.2 mM, and the culture was induced at 18°C ​​for 35 h. The induced culture solution was centrifuged at 8000 rpm for 10 minutes, the bacterial pellet was collected, washed with 0.9% saline, resuspended and centrifuged again at 8000 rpm for 10 minutes, the bacterial pellet was collected and weighed, and a total of 125.6 g of wet cells containing the parent carbonyl reductase were harvested.

[0130] The same method as above was used to obtain 126.8 g of wet cells of the mutant strain pETDuet-1-ΔCRase-mut-85, with the only difference being that the mutant strain pETDuet-1-ΔCRase-mut-85 was used to replace the strain CRase containing the parent carbonyl reductase.

[0131] Example 7: (S)-Citronellal Selectivity Verification

[0132] 50 mL reaction system: 42 g / L (R)-citronellal, 28 g / L (S)-citronellal ((R)-citronellal and (S)-citronellal were prepared as a mother solution with ethyl acetate, with ethyl acetate accounting for 35% of the reaction system), 52.5 g / L mutant pETDuet-1-ΔCRase-mut-85 wet cells (equivalent to 0.75 g / g (R / S)-citronellal), 0.68 mol / L potassium formate (the molar ratio of potassium formate to (R / S)-citronellal was 1.5:1), and 0.15 mM NAD + (Prepare the stock solution with water) Commercial formate dehydrogenase 5 U / mL was added to the reaction system with 100 mM PBS (pH 7.5) to make up to 50 mL. During the reaction, 1 M hydrochloric acid was used to control the pH to 7.5. The reaction was carried out at 45°C and 700 rpm for 25 h.

[0133] The reaction is as follows Figure 1 shown.

[0134] After the reaction, about 1 mL of the reaction solution was added to 2 mL of ethyl acetate and mixed evenly for extraction. The mixture was centrifuged at 8000 rpm for 5 min, and the supernatant was removed. 2 mL of ethyl acetate was further added to the lower layer solution, mixed evenly for extraction, and centrifuged at 8000 rpm for 5 min. The supernatant was removed. The supernatants of the two extractions were combined for gas chromatography detection.

[0135] The retention time of (R)-citronellal in the reaction solution was 25.145 min, and the retention time of (S)-citronellal was 25.058 min.

[0136] The conversion rate of (R)-citronellal (((R)-citronellal initial amount - (R)-citronellal remaining amount) / (R)-citronellal initial amount × 100%) was measured to be 0.03%, and the conversion rate of (S)-citronellal (((S)-citronellal initial amount - (S)-citronellal remaining amount) / (S)-citronellal initial amount × 100%) was measured to be 99.3%.

[0137] The remaining reaction liquid was filtered through a 1000-mesh filter cloth to obtain a filtrate. The filtered bacterial cells were washed and extracted with 50 mL of ethyl acetate, and the mixture was centrifuged at 8000 rpm for 5 minutes. The supernatant was removed. The filtrate and supernatant were combined and allowed to stand for phase separation. The upper organic phase was distilled. The distillation conditions were batch distillation, 40 plates, a bottom temperature of 45°C, a top pressure of 50 Pa, and a reflux ratio of 1.6:1. 1.401 g of (S)-citronellol and 2.09 g of (R)-citronellal were produced (the overall process yield was 99.5%, and the ee value of (R)-citronellal was 99.1%).

[0138] Gas chromatography detection showed that the retention time of (S)-citronellol was 23.052 min.

[0139] Example 8: (S)-Citronellal Selectivity Verification

[0140] 59.5 g / L (R)-citronellal, 25.5 g / L (S)-citronellal ((R)-citronellal and (S)-citronellal were prepared into a mother solution with n-heptane, and the volume fraction of n-heptane in the reaction system was 45%), 42.5 g / L mutant bacteria pETDuet-1-ΔCRase-mut-85 wet cells (equivalent to 0.5 g / g (R / S)-citronellal), 0.66 mol / L sodium formate (the molar ratio of sodium formate to (R / S)-citronellal was 1.2:1), 0.1 mM NAD +Commercial formate dehydrogenase (3 U / mL) was added as a stock solution prepared with water. 100 mM Tris-HCl buffer (pH 7.1) was used to make up the reaction system to 50 mL. 1 M hydrochloric acid was used to control the pH at 7.1 during the reaction. The reaction was carried out at 35°C and 650 rpm for 24 h.

[0141] The reaction solution sample testing and distillation process were the same as in Example 7, except that the solvent was replaced with n-heptane instead of ethyl acetate. Gas chromatography detection of the reaction solution revealed a retention time of 25.145 min for (R)-citronellal and 25.058 min for (S)-citronellal.

[0142] The conversion rate of (R)-citronellal was measured to be 0.001%, while the conversion rate of (S)-citronellal was 99.93%.

[0143] 2.96 g of (R)-citronellal (the overall process yield was 99.5%, and the ee value of (R)-citronellal was 99.9%) and 1.284 g of (S)-citronellol were prepared by distillation.

[0144] Gas chromatography detection showed that the retention time of (S)-citronellol was 23.052 min.

[0145] Example 9: (S)-Citronellal Selectivity Verification

[0146] A 50 mL reaction system included 95 g / L (R)-citronellal, 5 g / L (S)-citronellal ((R)-citronellal and (S)-citronellal were prepared as stock solutions in n-octane, with n-octane accounting for 30% of the reaction volume), 25 g / L wet cells of the mutant strain pETDuet-1-ΔCRase-mut-85 (equivalent to 0.25 g / g (R / S)-citronellal), 0.65 mol / L sodium formate (sodium formate: (R / S)-citronellal) at a 1:1 molar ratio), 0.05 mM NAD+ (added from a stock solution prepared in water), and 1 U / mL of commercial formate dehydrogenase. The reaction system was made up to 50 mL with 100 mM Tris-HCl buffer (pH 6.0). The pH was maintained at 6.0 with 1 M HCl. The reaction was incubated at 30°C and 650 rpm for 15 h.

[0147] The reaction solution sample testing and distillation process were the same as in Example 7, except that the solvent was replaced with n-octane instead of ethyl acetate. Gas chromatography detection of the reaction solution revealed a retention time of 25.145 min for (R)-citronellal and 25.058 min for (S)-citronellal.

[0148] The conversion of (R)-citronellal was measured to be 0.003%, while the conversion of (S)-citronellal was 99.5%.

[0149] 4.726 g of (R)-citronellal (the overall process yield was 99.5%, and the ee value of (R)-citronellal was 99.9%) and 0.25 g of (S)-citronellol were prepared by distillation.

[0150] Gas chromatography detection showed that the retention time of (S)-citronellol was 23.052 min.

[0151] Comparative Example 1: (S)-citronellal selectivity verification using parent carbonyl reductase as catalyst

[0152] The steps of Example 7 were repeated, except that the wet cells of the mutant strain pETDuet-1-ΔCRase-mut-85 were replaced with wet cells of the parent carbonyl reductase.

[0153] The conversion of (R)-citronellal was measured to be 35.8%, while the conversion of (S)-citronellal was 55.9%.

[0154] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0155] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be determined by the appended claims, and the specification and drawings shall serve to interpret the claims.

Claims

1. A method for separating (R)-citronellal from a mixture of (R)-citronellal and (S)-citronellal, characterized in that: The method comprises: In the presence of cosolvent, formate and cofactor NAD + In a reaction system, a recombinant cell expressing a carbonyl reductase mutant and formate dehydrogenase are used as catalysts, and (S)-citronellal is used as a substrate to carry out a catalytic reaction to generate (S)-citronellal, thereby preparing a reaction solution containing (R)-citronellal and (S)-citronellal; The mutant of the carbonyl reductase comprises an amino acid fragment having an amino acid sequence as shown in SEQ ID NO:

3.

2. The method according to claim 1, wherein The method meets one or more of the following conditions A1 to A8: A1. The initial concentration of formate dehydrogenase in the reaction system is 1 U / mL ~ 5 U / mL; A2. The initial concentration of the (R)- citronellal and (S)- citronellal mixture in the reaction system is 70 g / L ~ 100 g / L, optionally, the mass ratio of the (R)- citronellal and the (S)- citronellal is 60:40 ~ 95:5; A3. The recombinant cells were used in an amount of 0.25 g~0.75 g / g (R)-citronellal and (S)-citronellal mixture; A4. The cofactor NAD + The initial concentration in the reaction system is 0.05 mM to 0.15 mM; A5. The reaction system further comprises a buffer salt, the buffer salt optionally comprising a phosphate buffer or a Tris-HCl buffer; and, A6. The molar ratio of the formate to the (R)-citronellal and (S)-citronellal mixture is (1 to 1.5): 1; A7. The host cell of the recombinant cell is Escherichia coli; and A8. The cosolvent comprises one or more of ethyl acetate, n-heptane and n-octane, and the volume fraction of the cosolvent in the reaction system is 30% to 45%; and / or, The catalytic reaction satisfies one or more of the following conditions B1 to B3: B1. The catalytic reaction temperature is 30 ℃ ~ 45 ℃, optionally 35 ℃ ~ 45 ℃; B2. The catalytic reaction is carried out for 15 h~25 h, optionally 20 h~25 h; and, B3. The pH of the catalytic reaction is 6.0-7.5, optionally 7.0-7.

5.

3. The method according to claim 1 or 2, wherein: The method further comprises a separation step to separate (R)-citronellal and (S)-citronellol in the reaction solution; The separation step includes the following sub-steps: Filtration: filtering the reaction solution to obtain a filtrate and recombinant cells; Extraction: extracting the recombinant cells to obtain a supernatant; Standing and distilling: combining the filtrate and the supernatant, standing and separating the phases to obtain an upper organic phase, and distilling the organic phase to prepare (R)-citronellal and (S)-citronellol; in: The distillation optionally satisfies the following conditions: Batch distillation, the number of distillation plates is 35~45, the bottom temperature is 40℃~50℃, the top pressure is 50 Pa~70 Pa, and the reflux ratio is (1~1.6):

1.

4. A method for preparing (R)-citronellal, characterized in that: It includes: Using citral as raw material, (R)-citronellal and (S)-citronellal reactants are prepared through hydrogenation reaction; (R)-citronellal is separated from the mixture of (R)-citronellal and (S)-citronellal according to the method according to any one of claims 1 to 3.

5. A carbonyl reductase mutant, characterized in that The amino acid fragment relative to the wild-type carbonyl reductase as shown in SEQ ID NO: 1 comprises mutations Q76K, S189G and N233K; Optionally, the carbonyl reductase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO:

3.

6. Nucleic acid, characterized in that It encodes the carbonyl reductase mutant according to claim 5; Optionally, the nucleic acid has one of the following characteristics: C1. comprising a nucleic acid sequence as shown in SEQ ID NO: 4; C2. A nucleic acid sequence that encodes the same protein as the nucleic acid sequence shown in C1 but differs from the nucleic acid sequence shown in C1 due to the degeneracy of the genetic code; C3. A nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence defined by C1 or C2 and encodes a carbonyl reductase mutant according to claim 1 or 2; C4. A nucleic acid sequence obtained by substituting, deleting or adding one or more nucleotides to the nucleic acid sequence shown in any one of C1-C3, and having the same or similar functions as the nucleic acid sequence shown in any one of C1-C3.

7. A gene expression cassette, characterized in that It contains a promoter, the nucleic acid according to claim 6, and a terminator.

8. A recombinant expression vector, characterized in that: It contains a backbone vector and one or more of the following items: D1. The nucleic acid according to claim 6; D2. The gene expression cassette of claim 7.

9. A recombinant cell, characterized in that It comprises one or more of the nucleic acid according to claim 6, the gene expression cassette according to claim 7, and the recombinant expression vector according to claim 8; Optionally, the host cell of the recombinant cell is a prokaryotic cell or a eukaryotic cell; Further optionally, the host cell is Escherichia coli BL21 (DE3), Rosetta (DE3), BL21 (DE3) plysS, M15, W3110 or Top10f'.

10. A method for preparing a whole-cell catalyst comprising the recombinant cell according to claim 9, characterized in that: It includes: Performing fermentation and induction culture on the recombinant cell according to claim 9; a1 fermentation culture: the seed solution of the recombinant cells was inoculated into a fermentation medium for fermentation; the fermentation medium may optionally be a TB liquid medium; a2. Induction culture: When the OD of the fermentation system reaches 25~35, an inducer is added thereto for induction culture; Wherein, the fermentation culture may optionally satisfy one or more of the following E1 to E4: E1. The initial glucose concentration in the fermentation system is 30 g / L–50 g / L. E2. Initial OD of recombinant cells in fermentation system 600 0.3~0.5; E3. During the fermentation process, the glucose concentration in the fermentation system was controlled to be 5 g / L~10 g / L by feeding glucose; and E4. Fermentation conditions include: dissolved oxygen maintained at 25%–35%, culture at 37°C, and a speed of 200–950 rpm. The induced culture may optionally satisfy one or more of the following F1 to F3: F1. The inducer includes IPTG; F2. The concentration of the inducer in the fermentation system is 0.2 mM~0.5 mM; and, F3. Induction culture conditions include: 15℃~25℃ for 30 h~35 h.

11. Use of the recombinant cell according to claim 9 in the preparation of (R)-citronellal.

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