An olefin bond reductase and its mutant and application

Abstract

The application provides an olefin bond reductase and a mutant thereof and application, and belongs to the technical field of catalysis and modification of functional enzyme molecules. The amino acid sequence of the olefin bond reductase disclosed by the application is shown as SEQ ID NO:1, and the amino acid sequence of the mutant thereof is shown as SEQ ID NO:3. The olefin bond reductase and the mutant thereof provided by the application are used in combination with glucose dehydrogenase to catalyze citral, without adding coenzyme NADH in a reaction system, while glucose salt is produced as a by-product, the economic benefit of the reaction is improved, the cost of the reaction is greatly reduced, and the olefin bond reductase and the mutant thereof have more potential and value in industrial production in the preparation of (R)-citronellal.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of catalysis and modification of functional enzyme molecules, and particularly relates to an ene reductase derived from Candida sake and a mutant thereof, a nucleic acid molecule encoding the ene reductase or the mutant thereof, a recombinant vector and a recombinant cell containing the nucleic acid molecule, a preparation method of the ene reductase and the mutant thereof, and application of the ene reductase and the mutant thereof, the nucleic acid molecule, the recombinant vector or the recombinant cell in preparation of (R)-citronellal. BACKGROUND

[0002] The molecular formula of citronellal is C 10 H 18 O, also known as 3,7-dimethyl-6-octenal, is an acyclic monoterpene aldehyde, which naturally exists in essential oils such as citronella oil and lemon eucalyptus oil, and is mostly present in the form of (R / S)-citronellal mixture under natural conditions.

[0003] L-menthol has a unique minty aroma and excellent cooling effect, and is widely used in fine chemicals, food, medical treatment, health and other fields, and is one of the top three fragrances in the world in terms of sales. (R)-citronellal is an important chiral intermediate in the reaction process of synthesizing L-menthol, and can also be used to synthesize important products such as natural vitamin E and male giant waxworm sex pheromone. Since the quality and yield of menthol naturally extracted from mint oil are greatly affected by natural environmental factors, and (R) configuration citronellal is mainly used, the chemical synthesis method is currently the mainstream method for industrial preparation of (R)-citronellal. However, when relying on chemical asymmetric hydrogenation to prepare products, the metal catalyst used faces a series of problems such as high cost, high requirement for reaction environment, difficulty in catalyst recovery and use, and high reaction energy consumption.

[0004] Biological enzyme catalysis of citral to synthesize (R)-citronellal can better solve the predicament faced by chemical catalysis. However, the existing biological enzyme catalysis of citral to synthesize (R)-citronellal has problems such as low conversion rate and low ee value of the product. SUMMARY

[0005] To solve the above problems in the prior art, the present application provides an ene reductase derived from Candida sake and a mutant thereof, which is combined with glucose dehydrogenase to obtain a catalytic reaction system with coenzyme cycle, so that only NAD + is needed to be added in the reaction system, and citral can be catalytically converted into (R)-citronellal efficiently.

[0006] In a first aspect, the present application provides an ene reductase, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] Amino acid sequence of the enoyl reductase (SEQ ID NO: 1)

[0008] MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVE YYAQRAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAW VQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAKKANNPQHSITKDEIKQ YVKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEW DAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAFV HLVEPRVTNPFLTEGEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTL IGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKK

[0009] The enoyl reductase provided by the present application can be a natural, recombinant or synthetic active polypeptide, which can be a natural purified product, a chemically synthesized product, or a product produced using recombinant technology from a prokaryotic host (such as E. coli) or a eukaryotic host (such as yeast, higher plants).

[0010] In some embodiments, the enoyl reductase is obtained by introducing a recombinant vector containing a gene encoding the same into an expression host (such as E. coli BL21 (DE3)) to obtain a recombinant genetically engineered strain, then culturing the recombinant genetically engineered strain and inducing expression to obtain the enoyl reductase.

[0011] In a second aspect, the present application provides a nucleic acid molecule encoding the above-mentioned enoyl reductase, the nucleotide sequence of which is shown in SEQ ID NO: 2.

[0012] Nucleotide sequence encoding the enoyl reductase (SEQ ID NO: 2)

[0013]

[0014] The nucleic acid molecule provided by the present application can be obtained by PCR amplification or artificial synthesis.

[0015] In a third aspect, the present application provides an ene reductase mutant, the amino acid sequence of which is shown as SEQ ID NO: 3.

[0016] Amino acid sequence of the ene reductase mutant (SEQ ID NO: 3)

[0017] MSFVKDFKPQALGDTNLFKPIKIGNNELLHRAVIPPLTRMRAQHPGNIPNRDWAVEYYAQRAQRPGTLIITEGTFPSPQSGGYDNAPGIWSEEQIKEWTKIFKAIHENKSFAWVQLWVLGWAAFPDTLARDGLRYDSASDNVYMNAEQEEKAK T ANNPQHSITKDEIKQYVKEYVQAAKNSIAAGADGVEIHSANGYLLNQFLDPHSNNRTDEYGGSIENRARFTLEVVDAVVDAIGPEKVGLRLSPYGVFNSMSGGAETGIVAQYAYVLGELERRAKAGKRLAF H HLVEPRVTNPFLTEGEGEYNGGSNKFAYSIWKGPIIRAGNFALHPEVVREEVKDPRTLIGYGRFFISNPDLVDRLEKGLPLNKYDRDTFYKMSAEGYIDYPTYEEALKLGWDKK

[0018] The ene reductase mutant provided by the present application is obtained by modifying the above-mentioned ene reductase. Specifically, the present application mainly uses a semi-rational design method to modify it. First, 96 enzyme mutant molecule libraries are obtained by error-prone PCR technology, and then after high-throughput reaction screening, an intermediate mutant of the ene reductase with improved catalytic conversion of citral is obtained, which has the following site mutations: the amino acid Lys at position 154 is mutated to Asn, and the amino acid Val at position 286 is mutated to Asp; continue to perform site saturation mutation at the above-mentioned positions 154 and 286, respectively, and after screening, a mutant of the ene reductase with better catalytic conversion of citral is obtained, which has the following site mutations: the amino acid Lys at position 154 is mutated to Thr, and the amino acid Val at position 286 is mutated to His. Compared with the ene reductase, the mutant has higher citral conversion activity.

[0019] The above-mentioned enoyl reductase mutant can be artificially synthesized, or the coding gene thereof can be synthesized first and then expressed biologically, such as using a recombination technology to express in a prokaryotic host (E. coli) or a eukaryotic host (such as yeast, higher plants).

[0020] In some embodiments, the above-mentioned enoyl reductase mutant is obtained by introducing a recombinant vector containing the coding gene thereof into an expression host (such as E. coli BL21 (DE3)) to obtain a recombinant genetically engineered strain, then culturing the recombinant genetically engineered strain and inducing expression to obtain the enoyl reductase mutant.

[0021] In a fourth aspect, the present application provides a nucleic acid molecule encoding the above-mentioned enoyl reductase mutant, and the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO: 4.

[0022] Nucleotide sequence (SEQ ID NO: 4) encoding enoyl reductase mutant

[0023] atgtcatttgttaaggatttcaagccacaagctttgggtgacaccaacttattcaaaccaatcaaaattggtaacaatgaacttctacaccgtgctgtcattcctccattgactagaatgagagcccaacatccaggtaatattccaaacagagactgggccgttgaatactacgctcaacgtgctcaaagaccaggaaccttgattatcactgaaggtacctttccctctccacaatctgggggttacgacaatgctccaggtatctggtccgaagaacaaattaaagaatggaccaagattttcaaggctattcatgagaataaatcgttcgcatgggtccaattatgggttctaggttgggctgctttcccagacacccttgctagggatggtttgcgttacgactccgcttctgacaacgtgtatatgaatgcagaacaagaagaaaaggctaag acggctaacaacccacaacacagtataacaaaggatgaaattaagcaatacgtcaaagaatacgtccaagctgccaaaaactccattgctgctggtgccgatggtgttgaaatccacagcgctaacggttacttgttgaaccagttcttggacccacactccaataacagaaccgatgagtatggtggatccatcgaaaacagagcccgtttcaccttggaagtggttgatgcagttgtcgatgctattggccctgaaaaagtcggtttgagattgtctccatatggtgtcttcaacagtatgtctggtggtgctgaaaccggtattgttgctcaatatgcttatgtcttaggtgaactagaaagaagagctaaagctggcaagcgtttggctttc cac catctagttgaacctcgtgtcaccaacccatttttaactgaaggtgaaggtgaatacaatggaggtagcaacaaatttgcttattctatctggaagggcccaattattagagctggtaactttgctctgcacccagaagttgtcagagaagaggtgaaggatcctagaacattgatcggttacggtagattttttatctctaatccagatttggttgatcgtttggaaaaagggttaccattaaacaaatatgacagagacactttctacaaaatgtcagctgagggatacattgactaccctacgtacgaagaagctctaaaactcggctgggacaagaag

[0024] The nucleic acid molecule of the above-mentioned olefin bond reductase mutant provided by the present application can be obtained by PCR amplification or artificial synthesis.

[0025] In a fifth aspect, the present application provides a recombinant vector comprising the nucleic acid molecule according to any one of the above-mentioned aspects.

[0026] The recombinant vector provided by the present application includes a cloning vector for replication of the relevant sequence and an expression vector for expression of the relevant enzyme gene.

[0027] In some embodiments, the expression vector comprises pET-28a(+) for expression of the enoyl reductase and mutant genes thereof.

[0028] In a sixth aspect, the present application provides a recombinant cell comprising the recombinant vector of any one of the above.

[0029] In some embodiments, the method for preparing the recombinant cell comprises the step of transforming the recombinant vector into an expression host cell.

[0030] In some preferred embodiments, the expression host cell is a prokaryotic cell or a eukaryotic cell, such as E. coli, yeast, etc., and more preferably, an E. coli expression host E. coli BL21(DE3) is used.

[0031] More specifically, the method for constructing the recombinant cell provided by the present application comprises the following steps:

[0032] (i) obtaining the enoyl reductase gene and the enoyl reductase positive mutant gene;

[0033] (ii) constructing the enoyl reductase and enoyl reductase mutant expression vectors, respectively;

[0034] (iii) transforming the above recombinant expression vectors into host strains, respectively;

[0035] (iv) screening for positive clone strains on plate resistance medium.

[0036] In a seventh aspect, the present application provides a method for preparing an enoyl reductase or mutant thereof, comprising:

[0037] culturing the recombinant cell of any one of the above, inducing expression to obtain a culture;

[0038] a step of isolating the above enoyl reductase or the above enoyl reductase mutant from the culture.

[0039] In the present application, the culturing method and culturing conditions are not particularly required, as long as the recombinant cell can grow normally. The method for isolating the above enoyl reductase or the above mutant from the culture is also a conventional method in the art.

[0040] In some embodiments, the culture medium used in the method for preparing the enoyl reductase or mutant thereof is a culture medium that can express proteins in the art, and preferably TB culture medium.

[0041] In some embodiments, the induction temperature is 16-25℃, for example 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, or any value or range between any two of these values, preferably the induction temperature is 22℃; the final concentration of IPTG used is preferably 0.5mM.

[0042] In some embodiments, the separation method is as follows: 8000rpm, 4℃, 10min, centrifugal collection of bacterial cells, 5-10 times mass volume of pH 8.5 PBS solution is added to wash and resuspend the bacterial cells, ultrasonic crushing or high-pressure homogenizer is used to crush the resuspended bacterial cells, 10000rpm, 4℃, 20min, centrifugal collection of supernatant, Ni ion column is used to purify the ene reductase or mutant, ultrafiltration tube is used to concentrate and exchange the purified protein into pH 8.5 PBS buffer, and store at -80℃ for use.

[0043] In an eighth aspect, the present application provides a use of the above-mentioned ene reductase, the above-mentioned nucleic acid molecule, the above-mentioned ene reductase mutant, the above-mentioned recombinant vector, the above-mentioned recombinant cell, or the above-mentioned preparation method for preparing (R)-citronellal.

[0044] In a ninth aspect, the present application provides a preparation method of (R)-citronellal, comprising the following steps: using the above-mentioned ene reductase, the above-mentioned ene reductase mutant, the above-mentioned recombinant cell, or the above-mentioned preparation method for preparing ene reductase or its mutant as a catalyst to catalyze the reaction of citral to obtain (R)-citronellal.

[0045] In some preferred embodiments, the catalyst further comprises a glucose dehydrogenase, which is preferably derived from Cedecea neteri; in some more preferred embodiments, the amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO: 5.

[0046] Amino acid sequence of glucose dehydrogenase (SEQ ID NO: 5)

[0047] VNTNKTGSQLFVTLSALFAVLCGLYLLFGGIWLVAIGGSWYYPIAGLVMLGVAWLLWKSKASALWLYA ALLLCTMAWGVWEVGFDFWALTPRCHVLVFFGVWLLLPFVSRRLVIPASGAAASLVVSLLITAVVLVWAGF NDPQEINGTLQLDNTAAAAGSSIPDADWPAYGRNQEGQRFSPLKQITTQNVGQLKEAWRFQTGDVKRATDP GEITNEVTPIKIRDMLYLCTAHQQLFALDASTGKEKWKFDPQLNTDPSFQHVTCRGVTYHEATADNASADVV ADCPRRIILPVNDGRLFALNADTGKLCESFANKGILNLQTNQPVTTPGMYEPTSPPIVTDKVIVIAGAVTDN FSTREPSGVIRGFDVNTGKLLWAFDPGAKDPNAIPDDGHHYTLNSPNSWAPAAYDAKLDLVYLPMGVTTPDI WGGNRTAEQERYASSIVALNATTGKLAWSYQTVHHDLWDMDMPAQPTLADITDKSGNKVPVVYAPAKTGN IFVLDRRDGKLVVPAPEQPVPQGAAKGDHVSPTQPFSELTFRPKKDLSGADMWGATMFDQLVCRVMFHSM RYEGIFTPPSEKGTLVFPGNLGMFEWGGISVDPNNQVAMTNPMARPFVSKLIPRGPGNPMEPPKDAKGSGT ES GVQPQYGVPYGVTLNPFLSPLGLPCKQPAWGYISGVDLKTNEIVWKKRIGTVEDSMPFPTGIPMKFRMG MPMLGGPISTAGNVMFIGATADNYLRAFDMRDGKQLWEARLPAGGQATPMTYEVNGKQYVVISAGGHGSF GTKMGDYIVAYALPDDAK

[0048] The above-mentioned glucose dehydrogenase provided by the present application can be a natural, recombinant or synthetic active polypeptide, which can be a naturally purified product, a chemically synthesized product, or a product produced from a prokaryotic host (e.g., E. coli) or a eukaryotic host (e.g., yeast, higher plants) using a recombinant technique.

[0049] In some embodiments, the above-mentioned glucose dehydrogenase is obtained by introducing a recombinant vector containing the gene encoding the same into an expression host (e.g., E. coli BL21(DE3)), then culturing the recombinant genetically engineered strain, and inducing expression to obtain the glucose dehydrogenase.

[0050] In some embodiments, the present application provides a nucleic acid molecule encoding the above-mentioned glucose dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO: 6.

[0051] Nucleotide sequence encoding glucose dehydrogenase (SEQ ID NO: 6)

[0052]

[0053] The nucleic acid molecules provided by this invention can usually be obtained by PCR amplification or artificial synthesis.

[0054] In some embodiments, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding the aforementioned glucose dehydrogenase. The recombinant vector provided by the present invention includes a cloning vector and an expression vector, wherein the expression vector includes pET-21a(+), the pET-21a(+) being used for the expression of the glucose dehydrogenase gene.

[0055] In some embodiments, the present invention provides a recombinant cell comprising a recombinant vector encoding a nucleic acid molecule encoding the aforementioned glucose dehydrogenase. The method for preparing the recombinant cell includes the step of transforming the recombinant vector into an expression host cell.

[0056] In some embodiments, the present invention provides a method for preparing glucose dehydrogenase, comprising culturing recombinant cells containing a recombinant vector encoding a nucleic acid molecule of the above-mentioned glucose dehydrogenase, inducing the expression of glucose dehydrogenase to obtain a culture; and isolating the above-mentioned glucose dehydrogenase from the culture.

[0057] In this invention, there are no special requirements for the culture method and conditions; it is sufficient to ensure the normal growth of the recombinant cells. Furthermore, the methods for isolating the aforementioned glucose dehydrogenase from the culture are all conventional methods in the art.

[0058] In some embodiments, the culture medium used in the preparation method of the glucose dehydrogenase is a protein-expressing culture medium in the art, preferably TB medium.

[0059] In some embodiments, the induction temperature is 16-25°C, for example, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or any two of these values ​​or a range thereof, preferably 18°C; the final concentration of the inducing agent IPTG is preferably 0.5 mM.

[0060] In some implementations, the separation method is as follows: centrifuge at 8000 rpm, 4°C, for 10 min to collect bacterial cells; add 5-10 times the mass volume of pH 8.5 PBS solution to wash and resuspend the bacterial cells; use ultrasonic disruption or high-pressure homogenizer to disrupt the resuspended bacterial cells; centrifuge at 10000 rpm, 4°C, for 20 min to collect the supernatant; purify glucose dehydrogenase using a Ni ion column; concentrate the purified protein using an ultrafiltration tube, change the buffer to pH 8.5 PBS buffer, and freeze at -80°C for later use.

[0061] The ene reductase or mutant thereof and glucose dehydrogenase provided by the present application can be added into the reaction system in the form of whole cells, or in the form of crude enzyme solution without purification, or in the form of semi-pure or pure enzyme after partial or complete purification. The ene reductase or mutant thereof and glucose dehydrogenase described in the present application can also be prepared into catalysts in the form of immobilized enzyme or immobilized cells by using conventional immobilization means in the art, and then added into the reaction system.

[0062] In some preferred embodiments, the ene reductase or mutant thereof and glucose dehydrogenase in the present application are added into the reaction system in the form of whole cells. Whole cells are more conducive to the maintenance of enzyme activity, and have the advantage of lower cost.

[0063] In some preferred embodiments, the ene reductase or mutant thereof and glucose dehydrogenase in the present application are separately expressed in different cells, and then added into the reaction system in the form of whole cells, so as to avoid the problem that the expression rhythm is not matched when the two enzymes are co-expressed in a single cell, and the proportion of the two enzymes in the catalytic cascade reaction cannot be reconciled, thereby causing insufficient reaction, poor reaction effect, and difficulty in separation of the product.

[0064] In some embodiments, the bacteria (containing the ene reductase or mutant thereof and containing the glucose dehydrogenase) after induction culture are collected by centrifugation, washed, and then the wet bacteria obtained are used as catalysts. The concentration of the wet bacteria of the ene reductase or mutant thereof is 40-110 g / L, for example, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, or any two values or a range between any two values in these values, preferably 80 g / L. The concentration of the wet bacteria of the glucose dehydrogenase is 64-176 g / L, for example, 64 g / L, 80 g / L, 95 g / L, 110 g / L, 128 g / L, 140 g / L, 155 g / L, 176 g / L, or any two values or a range between any two values in these values, preferably 128 g / L. The mass ratio of the wet bacteria of the ene reductase or mutant thereof to the wet bacteria of the glucose dehydrogenase is 1:0.6-1:2.0, for example, 1:0.6, 1:0.8, 1:1.0, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2.0, or any two ratios or a ratio range between any two ratios in these ratios, preferably 1:1.6.

[0065] In some embodiments, the recombinant cells containing the ene reductase or its mutants and the recombinant cells containing the glucose dehydrogenase can be lyophilized into bacterial powder. And the method for preparing the lyophilized bacterial powder can be the conventional method in the art, for example, prepared by the following method: centrifugal collection of bacterial bodies at 8000 rpm, 4°C, 10 min, washing of the bacterial bodies with 5-10 times mass volume of PBS solution at pH 8.5, centrifugal collection, freezing at -20°C overnight, running of a vacuum freeze dryer, freeze drying at -20°C, vacuum degree 15 Pa for 48 h, and obtaining of the lyophilized bacterial powder.

[0066] In some preferred embodiments, the lyophilized bacterial powder of the ene reductase or its mutants has a catalyst concentration of 12-32 g / L, for example 12 g / L, 14 g / L, 16 g / L, 18 g / L, 20 g / L, 22 g / L, 24 g / L, 26 g / L, 28 g / L, 30 g / L, 32 g / L, or a value or a range between any two of these values, preferably 24 g / L; the lyophilized bacterial powder of the glucose dehydrogenase has a concentration of 24-64 g / L, for example 24 g / L, 34 g / L, 48 g / L, 54 g / L, 64 g / L, or a value or a range between any two of these values, preferably 48 g / L; and the mass ratio of the lyophilized bacterial powder of the ene reductase or its mutants to the lyophilized bacterial powder of the glucose dehydrogenase is 1:1.6-1:2.4, for example 1:1.6, 1:1.8, 1:2.0, 1:2.2, 1:2.4, or a ratio or a ratio range between any two of these ratios, preferably 1:2.0.

[0067] In some embodiments, as the reaction involving the redox enzyme known in the art, it can be understood that DMSO, glucose and NAD + .

[0068] wherein the DMSO is used as a cosolvent, and has a volume concentration of 15-35%, for example 15%, 17%, 19%, 21%, 23%, 25%, 27%, 29%, 31%, 33%, 35%, or a value or a range between any two of these values, preferably 25%; the glucose has a concentration of 100-800 mM, for example 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, or a value or a range between any two of these values, preferably 700 mM; and the NAD +the concentration of the citral is 20-850 mM, for example 20 mM, 80 mM, 140 mM, 200 mM, 260 mM, 320 mM, 380 mM, 460 mM, 520 mM, 580 mM, 640 mM, 650 mM, 700 mM, 760 mM, 820 mM, 850 mM, or a value or a range between any two of these values, preferably 650 mM; the temperature of the reaction is 20-45℃, for example 20℃, 23℃, 26℃, 29℃, 32℃, 35℃, 38℃, 41℃, 44℃, 45℃, or a value or a range between any two of these values, preferably 35℃; the pH of the reaction is 7.5-9.0, for example 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or a value or a range between any two of these values, preferably 8.5; in some preferred embodiments, 2M sodium hydroxide can be used to adjust the pH of the reaction system; the time of the reaction is 22-40h, for example 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, or a value or a range between any two of these values, preferably 28h.

[0069] In the present application, in the reaction system for catalyzing citral to prepare (R)-citronellal, coenzyme NADH does not need to be added, and citral can be catalyzed to convert to (R)-citronellal. At the same time, glucose salt is produced as a byproduct, and additional economic benefits can be obtained by separating and purifying the glucose salt.

[0070] In some embodiments, the concentration of the citral is 20-850 mM, for example 20 mM, 80 mM, 140 mM, 200 mM, 260 mM, 320 mM, 380 mM, 460 mM, 520 mM, 580 mM, 640 mM, 650 mM, 700 mM, 760 mM, 820 mM, 850 mM, or a value or a range between any two of these values, preferably 650 mM; the temperature of the reaction is 20-45℃, for example 20℃, 23℃, 26℃, 29℃, 32℃, 35℃, 38℃, 41℃, 44℃, 45℃, or a value or a range between any two of these values, preferably 35℃; the pH of the reaction is 7.5-9.0, for example 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, or a value or a range between any two of these values, preferably 8.5; in some preferred embodiments, 2M sodium hydroxide can be used to adjust the pH of the reaction system; the time of the reaction is 22-40h, for example 22h, 24h, 26h, 28h, 30h, 32h, 34h, 36h, 38h, 40h, or a value or a range between any two of these values, preferably 28h.

[0071] Compared with the prior art, the present application has the following beneficial effects:

[0072] The present application provides an ene reductase derived from Candida sake, and further mutates the ene reductase to obtain an ene reductase mutant, wherein, compared with the wild-type ene reductase, the ene reductase mutant has a 99.9% conversion rate of citral 28h, an ee value of 98% of the product (R)-citronellal, a yield of 92%, and a space-time yield of 3.29 g / (L*h) when catalyzing the conversion of citral into (R)-citronellal, and the conversion rate of the ene reductase mutant is 2.64 times that of the original wild-type ene reductase. In addition, by combining with glucose dehydrogenase, the catalytic efficiency of the ene reductase mutant is further improved, and without adding coenzyme NADH in the reaction system, the economic benefit of the reaction is improved while by-product gluconate is produced, the cost of the reaction is greatly reduced, and the ene reductase mutant has great potential and value in the industrial production of (R)-citronellal. BRIEF DESCRIPTION OF DRAWINGS

[0073] Figure 1 Reaction schematic diagram for the catalytic synthesis of (R)-citronellal by the ene reductase and glucose dehydrogenase of the present application;

[0074] Figure 2 Gas chromatogram of the citral standard in Example 5 of the present application;

[0075] Figure 3 Gas chromatogram of the (R)-citronellal standard in Example 5 of the present application;

[0076] Figure 4 Gas chromatogram of the conversion of citral into (R)-citronellal in Example 5 of the present application;

[0077] Figure 5 Determination chromatogram of the ee value of the reaction product (R)-citronellal in Example 5 of the present application. DETAILED DESCRIPTION

[0078] The technical solutions of the present application will be described in detail below in combination with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0079] The experimental methods not specified in the embodiments are usually carried out according to the conventional conditions and the conditions described in the manual, or according to the conditions suggested by the manufacturers, and the general equipment, materials, reagents, etc. used are commercially available, unless otherwise specified.

[0080] Candida sake has been disclosed in the literature (Gueguen, Y. "Purification and characterization of an intracellular beta-glucosidase from a candida sake strain isolated from fruit juices." Applied Biochemistry & Biotechnology, 95(3), 151.) and is publicly available from Wanhua Chemical Group Co., Ltd.

[0081] Cedecea neteri has been disclosed in the literature (Chan K G. Complete Genome Sequence of Cedecea neteri Strain SSMD04, a Bacterium Isolated from Pickled Mackerel Sashimi." American Society for Microbiology, 2014(6).01339-14) and is publicly available from Wanhua Chemical Group Co., Ltd.

[0082] In the present application, the conversion rate of citral is determined by gas chromatography, and the specific method is as follows:

[0083] About 1 mL of the sample to be tested is extracted with two volumes of ethyl acetate, and after centrifugation, the supernatant is taken and an appropriate amount of anhydrous sodium sulfate is added to remove water, and after centrifugation, the supernatant is used for gas chromatography detection.

[0084] The conditions for gas chromatography detection include: using a SH-RTX-5 capillary column, nitrogen as the carrier gas, FID detector temperature: 300℃, injection port temperature: 280℃, column oven temperature: 100℃, injection volume: 1 μL, column flow rate: 0.86 mL / min, and area normalization is used to calculate the conversion rate of citral.

[0085] Example 1: Obtaining of the ene reductase gene

[0086] 1) Extract the genomic DNA of Candida sake;

[0087] 2) Using the genomic DNA obtained in step 1) as the template, PCR amplification is performed using upstream primer 1 and downstream primer 2 to obtain a DNA fragment containing the wild-type ene reductase gene.

[0088] The primer sequences are as follows:

[0089] Upstream primer 1: 5'-cgcggatccatgtcatttgttaagg-3' (SEQ ID NO: 7)

[0090] Downstream primer 2: 5'-ctcgaattcttacttcttgtcccagcc-3' (SEQ ID NO: 8)

[0091] wherein the nucleotide sequence of the enoyl reductase is shown as SEQ ID NO: 2; the amino acid sequence of the enoyl reductase is shown as SEQ ID NO: 1.

[0092] Example 2: Establishing enoyl reductase gene mutant library by error-prone PCR technology

[0093] Using the DNA fragment of wild-type enoyl reductase gene obtained in Example 1 as a template, and continuing to use upstream primer 1 and downstream primer 2 as primers, an error-prone PCR reaction was performed according to the following reaction system to obtain 96 enoyl reductase gene mutants.

[0094] Error-prone PCR reaction system: 10x amplification buffer 5 μl, 4 kinds of dNTP mixture 4 μl (2.5 mM), upstream primer 1 and downstream primer 2 each 1.5 μl, template DNA 1.5 μl, Taq DNA polymerase 1 μl, Mn 2+ 5 mM, and double-distilled water to 50 μl.

[0095] PCR reaction program: (1) pre-denaturation: 94°C, 3 min; (2) denaturation: 94°C, 30 s; annealing: 60°C, 30 s; extension: 72°C, 3 min, 30 cycles; (3) post-extension: 72°C, 10 min; (4) 4°C incubation.

[0096] Example 3: High-throughput screening to obtain potential modification points of enoyl reductase and performing site-directed saturation mutation

[0097] 1) Using BamH I and EcoR I double enzyme digestion of the DNA segment of the 96 enoyl reductase gene mutants obtained in Example 2, obtain the gene segment, use BamH I and EcoR I double enzyme digestion of the pET-28a(+) vector to obtain the vector segment, and link the gene segment and the vector segment to obtain the recombinant expression plasmid. After sequencing, the recombinant expression plasmid is respectively transferred into the expression host E. coli BL21(DE3) by heat shock. The positive clone strains selected are cultured in 8 ml TB medium for 4-6 h, then 0.5 mM IPTG is added, and the strains are induced at 22°C for 16 h. 6 mL of the bacterial solution is centrifuged, resuspended with 0.2 mL of pH 8.5 PBS, and 110 μL is transferred to a 96-well plate. The enoyl reductase catalyzes the formation of (R)-citronellal and NAD + from citral in the presence of NADH, which causes a change in the absorbance value at a wavelength of 340 nm, to determine the catalytic activity of the enoyl reductase.

[0098] 2) Continue to add 90 μL of substrate solution (0.5 mM NADH; 56.5 μL of 2.3 M citral DMSO solution; 19 μL of DMSO solution, 14.5 μL of pH 8.5 PBS solution; can be configured as a mother liquor), incubate at 35°C, 120 rpm for 4 h, and measure the absorbance at a wavelength of 340 nm.

[0099] 3) Extract the plasmid of the strain corresponding to the reaction solution with a lower absorbance value, and perform sequencing to confirm the mutation site: the amino acid Lys at position 154 is mutated to Asn, and the amino acid Val at position 286 is mutated to Asp. For these two amino acid sites, a single-point site-directed saturation mutation is designed using the codon NNK, 48 mutants are obtained, and the screening method in steps 1) and 2) is continued to screen. Two positive single mutants with good catalytic effect are screened: positive single mutant 1: the amino acid Lys at position 154 is mutated to Thr, and positive single mutant 2: the amino acid Val at position 286 is mutated to His. According to the mutation information of the above two positive single mutants, primers are designed for site-directed saturation mutation.

[0100] The primer sequence for site-directed saturation mutation is as follows:

[0101] Upstream primer 3 at position 154: 5'-gaacaagaagaaaaggctaagnnkgctaac-3' (SEQ ID NO: 9)

[0102] Downstream primer 4 at position 154: 5'-tgtgggttgttagcnnkcttagcc-3' (SEQ ID NO: 10)

[0103] 286 upstream primer 5: 5'-cgtttggctttcnnkcatctagtt-3' (SEQ ID NO: 11)

[0104] 286 downstream primer 6: 5'-cacgaggttcaactagatgnnkgaaa-3' (SEQ ID NO: 12)

[0105] Site-directed saturation mutation whole plasmid PCR reaction system: DNA polymerase mixture 10 μl, primer and template (plasmid of the strain corresponding to the above lower absorbance value reaction solution) each 1 μl, add double distilled water to 20 μl.

[0106] PCR reaction program: (1) pre-denaturation: 96℃, 3min (2) denaturation: 96℃, 30s; annealing: 60℃, 30s; extension: 72℃, 2.0min, cycle 30 times; (3) extension: 72℃, 5min; (4) 4℃ continuous incubation.

[0107] Among them, using 154 upstream primer 3, 154 downstream primer 4, 286 upstream primer 5 and 286 downstream primer 6, the combination mutant of the 154th amino acid Lys mutated to Thr and the 286th amino acid Val mutated to His is obtained.

[0108] Example 4 Comparison of catalytic efficiency of combination mutant and single mutation point

[0109] 1) The DNA fragment of the combination mutant (or positive single mutant 1, positive single mutant 2) obtained in Example 3 is double enzyme cut using BamH I and EcoR I, a gene fragment is obtained, the vector fragment is obtained by double enzyme cutting pET-28a(+) vector using BamH I and EcoR I, the gene fragment and the vector fragment are connected to obtain a recombinant expression plasmid, the plasmid with correct sequence is transformed into the expression host E. coli BL21(DE3) by heat shock, and the single colony is picked and inoculated into 5ml LB medium for activation, then inoculated into 100ml TB medium at 1% inoculation amount, and cultured at 37℃ for 4-6h, then 0.5mM IPTG is added, and induced at 22℃ for 12-16h, then 40ml of bacterial solution is centrifuged, and the bacterial body is washed with pH 8.5 PBS, the wet bacterial body concentration is adjusted to 80g / L, 0.2mM NADH, 100mM citral, 25% DMSO (volume fraction) are added, and the reaction is carried out at 35℃ for 1h, then the citral conversion rate is determined by gas chromatography.

[0110] The results show that the transformation rate of positive single mutant 1 (K154T) is 43.18%, the transformation rate of positive single mutant 2 (V286H) is 40.86%, and the transformation rate of the combined mutant (K154T and V286H) is 57%. It can be determined that the combined mutant has the best reaction effect, and therefore, the mutant of the ene reductase with higher citral conversion activity is obtained.

[0111] The nucleotide sequence of the mutant is shown as SEQ ID NO: 4; and the amino acid sequence of the mutant is shown as SEQ ID NO: 3.

[0112] Example 5 Efficiency determination of the combination of ene reductase or its mutant and glucose dehydrogenase in catalyzing the conversion of citral to (R)-citronellal

[0113] 1) Extract the genomic DNA of Cedecea neteri;

[0114] 2) Using the genomic DNA obtained in step 1) as a template, perform PCR amplification with the upstream primer 1 and the downstream primer 2 to obtain a DNA fragment containing the glucose dehydrogenase gene.

[0115] The primer sequences are as follows:

[0116] Upstream primer 7: 5'-atccgaattcgtgaatacaaataaaaccgg-3' (SEQ ID NO: 13)

[0117] Downstream primer 8: 5'-gcaagcttttacttcgcgtcgtc-3' (SEQ ID NO: 14)

[0118] The nucleotide sequence of the glucose dehydrogenase is shown as SEQ ID NO: 6; and the amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO: 5:

[0119] 3) using EcoRI and Hind III double enzyme cleavage of the DNA fragment of the glucose dehydrogenase gene obtained in step 2) and using BamH I and EcoR I double enzyme cleavage of the DNA fragment of the wild type ene bond reductase gene obtained in example 1, respectively obtaining a first gene fragment and a second gene fragment, using EcoR I and Hind III double enzyme cleavage of the pET-21a(+) vector and using BamH I and EcoR I double enzyme cleavage of the pET-28a(+) vector, respectively obtaining a first carrier fragment and a second carrier fragment, connecting the first gene fragment and the first carrier fragment to obtain a first recombinant expression plasmid, connecting the second gene fragment and the second carrier fragment to obtain a second recombinant expression plasmid, after sequencing, the first recombinant expression plasmid and the second recombinant expression plasmid are respectively transformed into the expression host E. coli BL21(DE3) by heat shock, and positive clone strains containing glucose dehydrogenase and positive clone strains containing ene bond reductase are screened. Using TB medium, the strains containing ene bond reductase mutants obtained in example 4, the above-mentioned strains containing glucose dehydrogenase, and the above-mentioned strains containing ene bond reductase are subjected to shake flask fermentation, and after induction culture (the induction conditions for the strains containing ene bond reductase mutants and the strains containing ene bond reductase are as follows: adding IPTG with a final concentration of 0.5 mM, and inducing at 22°C for 16 h; the induction conditions for the strains containing glucose dehydrogenase are as follows: adding IPTG with a final concentration of 0.5 mM, and inducing at 18°C for 16 h), the wet bacteria after induction culture are respectively collected by centrifugation, and the bacteria are washed with PBS solution with pH 8.5 and then collected by centrifugation for use.

[0120] 4) Select 100 mL three-necked reaction flask for catalytic reaction, and the fixed reaction system is 20 mL. Two systems are respectively carried out: in system 1, wet bacteria are added, and the specific conditions are as follows: the final concentration of ene bond reductase mutant wet bacteria is 80 g / L, and the concentration of glucose dehydrogenase wet bacteria is 128 g / L; the mass ratio of ene bond reductase mutant wet bacteria to glucose dehydrogenase wet bacteria is 1:0.6, 1:0.8, 1:1.0, 1:1.2, 1:1.4, 1:1.6, 1:1.8, and 1:2.0, respectively, which are added to different batches of reaction systems to screen the proportion of bacteria. The reaction system also includes 25% volume concentration of DMSO solution, 700 mM final concentration of glucose solution, 650 mM of citral, 0.15 mM final concentration of NAD + , 2M sodium hydroxide solution is used to control the pH of the reaction system to 8.5, the reaction is carried out for 28 h, and the conversion rate of citral is determined by gas chromatography. The results are shown in the following table 1:

[0121] Table 1 Conversion rate of citral

[0122]

[0123] As can be seen from Table 1, when the mass ratio of the wet mycelium of the ene reductase mutant and the wet mycelium of the glucose dehydrogenase is 1:1.6, the conversion rate of citral is the highest.

[0124] In system 2, the freeze-dried bacterial powder is added as follows: the concentration of the freeze-dried bacterial powder of the ene reductase mutant is 24 g / L, and the concentration of the freeze-dried bacterial powder of the glucose dehydrogenase is 48 g / L; the freeze-dried bacterial powder of the ene reductase mutant and the freeze-dried bacterial powder of the glucose dehydrogenase are added to different batches of reaction systems in a mass ratio of 1:1.6, 1:1.8, 1:2.0, 1:2.2 and 1:2.4, respectively, to screen the proportion of the bacterial powder, and the reaction system further includes a 25% volume concentration DMSO solution, a glucose solution with a final concentration of 700 mM, 650 mM citral, 0.15 mM NAD+ with a final concentration, 2M sodium hydroxide solution is used to control the pH of the reaction system to be 8.5, and if the volume is insufficient, PBS solution with pH 8.5 is used to make up, the reaction is carried out for 28 h, and the conversion rate of citral is determined by gas chromatography, and the results are shown in Table 2 as follows:

[0125] Table 2 Conversion rate of citral

[0126]

[0127] As can be seen from Table 2, when the mass ratio of the freeze-dried bacterial powder of the ene reductase mutant and the freeze-dried bacterial powder of the glucose dehydrogenase is 1:2.0, the conversion rate of citral is the highest.

[0128] In the present application, Figure 1 The reaction schematic diagram of the ene reductase and the glucose dehydrogenase catalyzing the synthesis of (R)-citronellal is shown in the figure.

[0129] Further, the ee value and yield of the reaction product of the freeze-dried bacterial powder of the ene reductase mutant and the freeze-dried bacterial powder of the glucose dehydrogenase in system 2 in a mass ratio of 1:2.0 are determined by gas chromatography, and the freeze-dried bacterial powder of the ene reductase mutant is replaced by the freeze-dried bacterial powder of the ene reductase to carry out the reaction, and the conversion rate of citral is determined by gas chromatography.

[0130] It is found that when the mass ratio of the obtained freeze-dried bacterial powder of the ene reductase mutant and the freeze-dried bacterial powder of the glucose dehydrogenase is 1:2.0, the catalytic reaction can obtain a higher conversion rate of citral: the conversion rate is 99.9% after 28 h, the ee value of the product (R)-citronellal is 98%, the yield is 92%, and the space-time yield is 3.29 g / (L*h), which is 2.64 times of the conversion rate (37.8%) of the wild ene reductase freeze-dried bacterial powder and the glucose dehydrogenase freeze-dried bacterial powder.

[0131] The (R)-citronellal space-time yield calculation formula is as follows:

[0132] (R)-citronellal space-time yield = molar concentration of citral * (R)-citronellal yield * molar mass of (R)-citronellal * 10 -3 / reaction time

[0133] Figure 2 is a citral standard gas chromatogram; wherein the citral standard contains two standards, which are neral with a peak time of 7.557 min and geranial with a peak time of 7.907 min; Figure 3 is a (R)-citronellal standard gas chromatogram; wherein the (R)-citronellal has a peak time of 5.964 min; Figure 4 is a gas chromatogram of a sample of citral converted into (R)-citronellal catalyzed by the alkenyl-reductase and glucose dehydrogenase; Figure 5 is an ee value determination chromatogram of the reaction product (R)-citronellal.

[0134] Comparative Example

[0135] The system 2 in Example 5 was used for the reaction, except that the glucose dehydrogenase was replaced by a commercial glucose dehydrogenase from Pseudomonas (purchased from Aladdin Biochem Technology, product number G139687), and the mass ratio of the lyophilized bacteria powder of the alkenyl-reductase mutant to the lyophilized bacteria powder of the glucose dehydrogenase was 1:2.0 for the citral conversion reaction. The conversion rate of citral was determined by gas chromatography, and the conversion rate was 72.1%.

[0136] The results show that the alkenyl-reductase mutant in the present application further improves the conversion rate of citral by using a glucose dehydrogenase that is specifically matched thereto.

[0137] The above examples are only specific embodiments of the present application, which are used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the foregoing examples, it should be understood by those skilled in the art that any person skilled in the art can modify or easily think of changes to the technical solutions recorded in the foregoing examples, or make equivalent replacements to some technical features therein, without departing from the technical range disclosed by the present application. Such modifications, changes or replacements do not make the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application, and should be covered within the protection scope of the present application.

Claims

1. An enoyl reductase mutant, characterized in that, The amino acid sequence of the ene-reductase mutant is shown as SEQ ID NO:

3.

2. A nucleic acid molecule encoding the enone reductase mutant of claim 1, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO:

4.

3. A recombinant vector, characterized in that, The nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that, The recombinant cell of claim 4 is E. coli or yeast.

5. A method of producing an enoyl reductase mutant, characterized by, Comprising: The recombinant cell of claim 4 is induced to express, to obtain a culture; The ene-reductase mutant of claim 1 is isolated from the culture.

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

7. A process for the preparation of (R)-citronellal, characterized in that, The mutant of claim 1 or the recombinant cell of claim 4 is used as a catalyst to catalyze the reaction of citral to obtain (R)-citronellal.

8. The preparation method according to claim 7, characterized in that, The catalyst further comprises glucose dehydrogenase.

9. The preparation method according to claim 8, characterized in that, The amino acid sequence of the glucose dehydrogenase is shown as SEQ ID NO:

5.

10. The production method according to claim 8 or 9, characterized by, When the ene-reductase mutant is in the form of wet cells, its concentration is 40-110 g / L; when the glucose dehydrogenase is in the form of wet cells, its concentration is 64-176 g / L; the mass ratio of the wet cells of the ene-reductase mutant to the wet cells of the glucose dehydrogenase is 1:0.6-1:2.0; when the ene-reductase mutant is in the form of freeze-dried bacterial powder, its concentration is 12-32 g / L; when the glucose dehydrogenase is in the form of freeze-dried bacterial powder, its concentration is 24-64 g / L; the mass ratio of the freeze-dried bacterial powder of the ene-reductase mutant to the freeze-dried bacterial powder of the glucose dehydrogenase is 1:1.6-1:2.

4.

11. The method of claim 10, wherein, When the ene-reductase mutant is in the form of wet cells, its concentration is 80 g / L; when the glucose dehydrogenase is in the form of wet cells, its concentration is 128 g / L; the mass ratio of the wet cells of the ene-reductase mutant to the wet cells of the glucose dehydrogenase is 1:1.6; when the ene-reductase mutant is in the form of freeze-dried bacterial powder, its concentration is 24 g / L; when the glucose dehydrogenase is in the form of freeze-dried bacterial powder, its concentration is 48 g / L; the mass ratio of the freeze-dried bacterial powder of the ene-reductase mutant to the freeze-dried bacterial powder of the glucose dehydrogenase is 1:2.

0.

12. The preparation method according to claim 7, characterized in that, NAD is also added to the reaction + ; The concentration of said NAD + is 0.05-0.2 mM; The temperature of the reaction is 20-45℃; The pH of the reaction is 7.5-9.0; The time of the reaction is 22-40 h; The concentration of the citral is 20-850 mM.

13. The method of claim 12, wherein, The concentration of said NAD + 0.15 mM; The temperature of the reaction is 35℃; The pH of the reaction is 8.5; The time of the reaction is 28 h; The concentration of the citral is 650 mM.

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