Carotenoid dioxygenase mutant and its application

Abstract

The present invention relates to the field of genetic engineering, and more specifically to a carotenoid dioxygenase mutant, its encoding gene, expression vector, expression strain, application, and method for preparing β-ionone. The mutant of the present invention has good enzymatic activity and can be used in a biosynthetic method to produce β-ionone, thereby achieving a high β-ionone yield. The expression strain of the present invention also has stable and sustainable production capacity, a simple and pollution-free extraction method, and few by-products.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a carotenoid dioxygenase mutant, its encoding gene, expression vector, expression strain, application and a method for preparing beta-ionone. Background Art

[0002] β-ionone has a violet-like aroma, a blend of fruity and woody notes. Its threshold is extremely low, at 0.007 nL / L in water and 0.12 ng / L in air. β-ionone is widely found in osmanthus, roses, tomatoes, citrus fruits, strawberries, and grapes. Its unique aroma has made it highly sought after in the flavor and fragrance market. β-ionone has also been shown to inhibit the proliferation of breast and gastric cancer cells, suggesting potential applications in medicine.

[0003] At present, compared with β-ionone obtained by chemical synthesis, natural β-ionone is safer and more environmentally friendly. At present, natural β-ionone is mainly obtained by extraction from plants. Due to the low content of β-ionone in plants (19.8-31.9μg / kg in grapes and 1.72mg / kg in raspberries are the highest content in plants), the yield of natural β-ionone is low and the price is very high. In addition, due to natural conditions, the yield of β-ionone extracted from plants fluctuates greatly, making it difficult to meet sustained and stable commercial demand. With the rapid development of biotechnology, it has become possible to produce β-ionone through biosynthetic pathways, and the biosynthesis of β-ionone has become a research hotspot.

[0004] Carotenoid cleavage dioxygenase (CCD) is the rate-limiting protein in the biosynthesis of β-ionone. This family of proteins possesses a seven-lobed β-helical structure with ferrous ion as a cofactor. The ferrous ion is coordinated by four conserved histidine residues, forming the active site of the enzyme. The α1 and α3 helices form the dome of the CCD protein, while two helices composed primarily of hydrophobic amino acids constitute the membrane-binding region. Studies have shown that improving the membrane binding capacity of CCDs is a potential effective approach to increase β-ionone production. However, there have been no studies investigating the potential for enhancing β-ionone production by increasing the membrane binding capacity of CCDs through site-directed mutagenesis.

[0005] Tobacco (Nicotiana tabacum) is an important economic crop and a model species for biological research. As an aromatic plant, tobacco contains a large number of carotenoid degradation products, such as β-ionone, with concentrations as high as 1.08 mg / 100 g in its essential oil. This may be due to the presence of highly catalytically active CCDs in tobacco. However, there are currently no published reports on the catalytic activity of tobacco carotenoid dioxygenases (NtCCDs). Summary of the Invention

[0006] The present invention aims to overcome the problems of the prior art by providing a carotenoid dioxygenase mutant, its encoding gene, expression vector, expression strain, application, and method for preparing β-ionone. The mutant exhibits good enzymatic activity and can be used in a biosynthetic method to produce β-ionone, resulting in a high β-ionone yield. Furthermore, the expression strain of the present invention has stable and sustainable production capacity, a simple and pollution-free extraction method, and minimal byproducts.

[0007] To achieve the above objectives, the present invention provides a carotenoid dioxygenase mutant in a first aspect, characterized in that the mutant is a mutant produced by mutating at least one amino acid in the carotenoid dioxygenase with an amino acid sequence as shown in SEQ ID NO.1, wherein, based on the carotenoid dioxygenase with an amino acid sequence as shown in SEQ ID NO.1, the mutation site of the mutant is located at at least one of positions 25, 31, 38, 42, 128, 140, 155, 157, and 164 of SEQ ID NO.1.

[0008] The second aspect of the present invention provides a gene encoding the mutant as described above.

[0009] The third aspect of the present invention provides an expression vector, which contains the gene as described above.

[0010] The fourth aspect of the present invention provides an expression strain, which contains the expression vector as described above or the gene encoding the mutant as described above.

[0011] The fifth aspect of the present invention provides the use of the aforementioned mutant, the aforementioned gene, the aforementioned expression vector, and the aforementioned expression strain in the preparation of β-ionone.

[0012] A sixth aspect of the present invention provides a method for preparing β-ionone, comprising: culturing the expression strain according to claim 6 or 7 to obtain a culture containing β-ionone.

[0013] Through the above technical solution, the present invention has the following beneficial effects:

[0014] (1) The mutants of the present invention have good enzymatic activity, and the enzymatic activity of the preferred mutants can be increased by about 3 times compared with the wild type;

[0015] (2) An engineered strain containing the mutant of the present invention is prepared, which can obtain a higher yield of β-ionone when used to prepare β-ionone. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the β-ionone production of the wild type and mutant NtCCD1-3 in Example 2.

[0017] Figure 2 This is the plasmid map of the tpLADH1 plasmid.

[0018] Figure 3 This is the plasmid map of the pColdTF-rox1-NtCCD1-3-LEU2 plasmid. DETAILED DESCRIPTION

[0019] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0020] In the present invention, unless otherwise specified, the mutant refers to a mutant of tobacco carotenoid dioxygenase (NtCCD1-3) (i.e., wild type) (NP_001312918.1, amino acid sequence shown in SEQ ID NO.1, nucleotide sequence of its encoding gene shown in SEQ ID NO.2).

[0021] In the present invention, unless otherwise specified, the various expression elements or sequences described in the present invention are shown and connected in the order of 5' to 3'.

[0022] A first aspect of the present invention provides a carotenoid dioxygenase mutant, wherein the mutant is a carotenoid dioxygenase mutant having an amino acid sequence as shown in SEQ ID NO.1, wherein the mutation site of the mutant is located at at least one of positions 25, 31, 38, 42, 128, 140, 155, 157, and 164 of SEQ ID NO.1.

[0023] SEQ ID NO.1:

[0024] MGRKEEDDTVERTEGGVVVVNPKPKKGVIGKAIDLEKVIIKLMHDSTKPLHYLSGNFAPTDETPPLKDLSVTGHLPECLNGEFVRVGPNPKFAPVAGYHWFDGDGMIHGLRIKDGKATYVSRYVRTSRLKQEEFFGGAKFMKIGDLKGLFGLFTVYMQVLRAKLKVLDITYGNGTANTALVYHHGKLLALSEADPYALKVMEDGDLQTLGMLDYDKRLAHSFTAHPKVDPVTGEMFTFGYSQNPPYITYRVISKGGIMQDPVPPITIPEPIM MHDFAITENYAIMMDLPLCFRPKEMVKNNQLAFFFDATKNARFGVLPRYAQSEALIKWFELPNCFIFHNANAWEEGDEVVLITCRVQNPNLDMVNGVVKEKLENFSNELYEMRFNMKSGAASQKKLSESAVDFPRI NENYTGRKQRYVYGTTLDSIAKVTGIIKFDLHAEPETGKAQLEVGGNVQGIFDLGPGRFGSEAVFVPRQPGTECEEDGGYLIFFVHDENTGKSAVNVIDAKTMSAEPVAVVELPKRVPYGFHAFFVTEEQIQEQAKL

[0025] SEQ ID NO.2:

[0026]

[0027] In some embodiments of the present invention, the mutant is a single-point mutation mutant.

[0028] In some embodiments of the present invention, the mutant is a mutation in which at least one amino acid at positions 25, 31, 38, 42, 128, 140, 155, 157, or 164 of SEQ ID NO. 1 is mutated to a hydrophobic amino acid. The hydrophobic amino acid is at least one of tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline, and methionine.

[0029] In some embodiments of the present invention, the mutation site of the mutant is position 25 of SEQ ID NO.1, and preferably the mutant is mutant K25A in which lysine at position 25 of SEQ ID NO.1 is mutated to alanine.

[0030] In some embodiments of the present invention, the mutation site of the mutant is position 31 of SEQ ID NO.1, and preferably the mutant is mutant K31A in which lysine at position 31 of SEQ ID NO.1 is mutated to alanine.

[0031] In some embodiments of the present invention, the mutation site of the mutant is position 38 of SEQ ID NO.1, and preferably the mutant is mutant K38A in which lysine at position 38 of SEQ ID NO.1 is mutated to alanine.

[0032] In some embodiments of the present invention, the mutation site of the mutant is position 42 of SEQ ID NO.1, and preferably the mutant is mutant K42A in which lysine at position 42 of SEQ ID NO.1 is mutated to alanine.

[0033] In some embodiments of the present invention, the mutation site of the mutant is position 128 of SEQ ID NO.1, and preferably the mutant is mutant S128A in which the serine at position 128 of SEQ ID NO.1 is mutated to alanine.

[0034] In some embodiments of the present invention, the mutation site of the mutant is position 140 of SEQ ID NO.1, and preferably the mutant is mutant K140M in which lysine at position 140 of SEQ ID NO.1 is mutated to methionine.

[0035] In some embodiments of the present invention, the mutation site of the mutant is position 155 of SEQ ID NO.1, and preferably the mutant is mutant T155M in which the threonine at position 155 of SEQ ID NO.1 is mutated to methionine.

[0036] In some embodiments of the present invention, the mutation site of the mutant is position 157 of SEQ ID NO.1, preferably the mutant is mutant Y157A in which the tyrosine at position 157 of SEQ ID NO.1 is mutated to alanine, or mutant Y157L in which the tyrosine at position 157 of SEQ ID NO.1 is mutated to leucine.

[0037] In some embodiments of the present invention, the mutation site of the mutant is position 164 of SEQ ID NO.1, and preferably the mutant is mutant K164L in which lysine at position 164 of SEQ ID NO.1 is mutated to leucine.

[0038] The inventors discovered that mutants K31A, K38A, Y157A, and Y157L exhibited higher enzymatic activity than the wild type and produced more β-ionone. Among them, mutant K38A exhibited the highest enzymatic activity, approximately three times higher than the wild type. Therefore, preferably, the mutants are K31A, K38A, Y157A, and Y157L, with K38A being even more preferred.

[0039] In the present invention, the mutant can be obtained by artificial synthesis, or the encoding gene can be synthesized first and then obtained by biological expression.

[0040] The second aspect of the present invention provides a gene encoding the mutant as described above.

[0041] It is well known in the art that, of the 20 different amino acids that make up proteins, with the exception of Met (ATG) and Trp (TGG), which are each encoded by a single codon, the other 18 amino acids are encoded by 2-6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989, see Appendix D on page 950). Due to the degeneracy of the genetic code, there is often more than one codon that determines an amino acid, and substitution of the third nucleotide in a triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different. Based on the known codon table, those skilled in the art can fully deduce the nucleotide sequences of genes encoding these amino acids from the amino acid sequences disclosed herein, and obtain the nucleotide sequences by biological methods (such as PCR methods, mutagenesis methods) or chemical synthesis methods. Therefore, such partial nucleotide sequences should be included within the scope of the present invention.

[0042] The nucleotide sequences provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis.

[0043] The third aspect of the present invention provides an expression vector, which contains the gene as described above.

[0044] In the present invention, the expression vector is a recombinant vector capable of expressing the mutant as described above, which can be obtained by integrating the gene encoding the mutant into the starting vector using any genetic engineering means.

[0045] It should be understood that after obtaining the expression vector, the expression vector can be transferred into a host cell to express the mutant, or a fragment of the gene contained in the expression vector can be transferred into a host cell (e.g., an expression strain) by enzyme cutting or the like to express the mutant.

[0046] In the present invention, the starting vector used in the expression vector can be selected from various vectors for protein expression known in the art, such as various commercially available plasmids, etc. The type of vector can be selected by those skilled in the art according to actual conditions.

[0047] In the present invention, it should be understood that the expression vector contains the promoter and terminator of the gene encoding the mutant, and those skilled in the art can routinely select the promoter and terminator. Preferably, in order to further improve the expression level of the mutant, the expression vector contains at least one of the promoters ADH1, TEF, or GAP.

[0048] In some embodiments of the present invention, when the expression strain is Saccharomyces cerevisiae, in order to integrate the coding gene of the mutant into a specific position in the genome of Saccharomyces cerevisiae, the expression vector also contains an upstream homology arm and a downstream homology arm of a single copy site (i.e., integration site) in the Saccharomyces cerevisiae genome, the upstream homology arm of the Saccharomyces cerevisiae single copy site is located at the 5' end of the gene encoding the mutant, and the downstream homology arm of the Saccharomyces cerevisiae single copy site is located at the 3' end of the gene encoding the mutant. It should be understood that the upstream homology arm refers to a fragment complementary to the 5' end sequence of the integration site (i.e., single copy site) in the Saccharomyces cerevisiae genome, and the downstream homology arm refers to a fragment complementary to the 3' end sequence of the integration site (i.e., single copy site) in the Saccharomyces cerevisiae genome. The length, sequence, and preparation method of the upstream homology arm and the downstream homology arm are conventional techniques in the art, and those skilled in the art can select and prepare them according to the specific Saccharomyces cerevisiae single copy site. Preferably, the Saccharomyces cerevisiae single copy site can be at least one of rox1, XI-5, XI-4, XI-3, XI-2, and XI-1.

[0049] In some embodiments of the present invention, in order to facilitate the screening of transformants, the expression vector may further contain a gene expression cassette encoding a screening tag, wherein the screening tag is selected by conventional techniques in the art, for example, it may be at least one of leucine (Leu), histidine (His) or methionine (Met) screening tags.

[0050] In some preferred embodiments of the present invention, the expression vector comprises the following elements, from the 5' end to the 3' end: an upstream homology arm of the Saccharomyces cerevisiae single-copy locus, a promoter of the gene encoding the mutant, a gene encoding the mutant, a terminator of the gene encoding the mutant, an expression cassette encoding a gene for a selection tag, and a downstream homology arm of the Saccharomyces cerevisiae single-copy locus. It should be understood that to prevent elements from being too close together, each element is separated by a base sequence. The selection of such base sequences is well known to those skilled in the art and will not be elaborated upon here.

[0051] The fourth aspect of the present invention provides an expression strain, which contains the expression vector as described above or the gene encoding the mutant as described above.

[0052] It should be understood that when the expression strain is used to produce β-ionone, the expression strain is capable of expressing β-carotene. The expression strain capable of expressing β-carotene can be commercially available or prepared independently. The preparation method is a conventional technique in the art. Those skilled in the art can prepare an expression strain capable of expressing β-carotene according to actual conditions, and the details will not be repeated here.

[0053] In the present invention, the expression vector can be transformed into the strain by conventional methods in the art, such as chemical transformation using the calcium chloride method or high-voltage electroporation, preferably electroporation.

[0054] In the present invention, the host strain may be at least one of yeasts such as Saccharomyces cerevisiae and Yarrowia lipolytica, more preferably Saccharomyces cerevisiae.

[0055] In the present invention, the gene of the mutant can be transferred into the strain using conventional techniques in the art. For example, the expression vector described above can be digested with enzymes and then a fragment containing the gene encoding the mutant can be transferred into the strain. Preferably, the fragment of the gene encoding the mutant also contains the promoter and terminator of the gene encoding the mutant. Preferably, when the strain is Saccharomyces cerevisiae, the fragment also contains the upstream homology arm and downstream homology arm of the Saccharomyces cerevisiae single copy site. The upstream homology arm of the Saccharomyces cerevisiae single copy site is located at the 5' end of the gene encoding the mutant, and the downstream homology arm of the Saccharomyces cerevisiae single copy site is located at the 3' end of the gene encoding the mutant to ensure that the fragment containing the mutant gene and the Saccharomyces cerevisiae genome undergo homologous recombination so that the gene encoding the mutant is integrated into a specific location in the Saccharomyces cerevisiae genome. Preferably, the Saccharomyces cerevisiae single copy site can be at least one of rox1, XI-5, XI-4, XI-3, XI-2, and XI-1. Preferably, in order to facilitate the screening of transformants that have successfully integrated the mutant gene, the fragment containing the gene encoding the mutant also contains a gene expression cassette encoding a screening tag, and the gene expression cassette encoding the screening tag is located between the gene encoding the mutant and the downstream homology arm of the single copy site. Preferably, the screening tag can be at least one of leucine (Leu), histidine (His) or methionine (Met). Preferably, the fragment containing the gene encoding the mutant contains the following elements from the 5' end to the 3' end in sequence: the upstream homology arm of the Saccharomyces cerevisiae single copy site, the promoter of the gene encoding the mutant, the gene encoding the mutant, the terminator of the gene encoding the mutant, the gene expression cassette encoding the screening tag, and the downstream homology arm of the Saccharomyces cerevisiae single copy site. It should be understood that in order to prevent the elements from being too close to each other, each element is spaced by some base sequences. The selection of the base sequences is well known to those skilled in the art and will not be repeated here.

[0056] The fifth aspect of the present invention provides the use of the aforementioned mutant, the aforementioned gene, the aforementioned expression vector, and the aforementioned expression strain in the preparation of β-ionone.

[0057] A sixth aspect of the present invention provides a method for preparing β-ionone, the method comprising: culturing the expression strain as described above to obtain a culture containing β-ionone.

[0058] In the present invention, the culture method is selected according to conventional techniques in the art, for example, the expression strain can be inoculated into a culture medium.

[0059] In the present invention, the culture medium is selected by conventional techniques in the art, and those skilled in the art can select an appropriate culture medium according to the strain of the expression strain. For example, when the expression strain is Saccharomyces cerevisiae, the culture medium can be YPD medium.

[0060] In the present invention, the culture conditions are selected by conventional techniques in the art, and those skilled in the art can select appropriate culture conditions according to the strain of the expression strain. For example, when the expression strain is Saccharomyces cerevisiae, the culture conditions are: 28-30°C, 180-200rpm, 9-10d. Preferably, the expression strain can be activated before being cultured to obtain seed liquid, and then inoculated into the culture medium in the form of seed liquid for culture. The inoculation amount of the seed liquid can be selected within a wide range, for example, it can be 1%-4% (V / V). Among them, the activation method and conditions are selected by conventional techniques in the art. For example, when the expression strain is Saccharomyces cerevisiae, the expression strain can be inoculated into YPD culture medium, 28-30°C, 180-200rpm, 48-72h to obtain seed liquid, and the OD of the seed liquid is 600nm The value can be selected within a wide range and may be 0.6-0.8.

[0061] In some preferred embodiments of the present invention, the method comprises: inoculating the expression strain into YPD medium at 28-30°C, 180-200 rpm, for 48-72 hours to obtain a seed solution, and then inoculating the seed solution into YPD medium at an inoculum amount of 1%-4% (V / V), at 28-30°C, 180-200 rpm, for 9-10 days to obtain a culture containing β-ionone.

[0062] In some embodiments of the present invention, the method further comprises extracting the culture to obtain β-ionone. The extraction method comprises adding an extractant to extract the β-ionone, and performing solid-liquid separation after the culture is completed to obtain a supernatant containing β-ionone. The extractant is dodecane and / or isopropyl myristate.

[0063] In the present invention, the timing of adding the extractant is selected according to conventional techniques in the art, and can be added when the strain is activated or when it is cultured.

[0064] In the present invention, the amount of the extractant added is selected according to conventional techniques in the art. Relative to 100 mL of culture, the amount of the extractant added can be 4-10 mL.

[0065] The present invention will be described in detail below by way of examples. In the following examples, unless otherwise specified, all methods used are conventional methods in the art, and all reagents used are conventional reagents that can be purchased commercially.

[0066] Strain A10 was transformed from Saccharomyces cerevisiae BY4741 (purchased from the National Center for Type Culture Collection, NTCC). Plasmids containing crtE, crtYB, and crtI genes from Phaffia rhodozyma were constructed using pRS406 as a backbone (the construction method can be found in Table 2 of René Verwaal, Wang J, Meijnen JP, et al. High-Level Production of Beta-Carotene in Saccharomyces cerevisiae by Successive Transformation with Carotenogenic Genes from Xanthophyllomyces dendrorhous [J]. Applied and Environmental Microbiology, 2007, 73(13): 4342-4350. The preparation method of YEplac195YB / I / E is different in that the present application uses pRS406 as the backbone), and a plasmid containing the crtE, crtYB, and crtI genes from Phaffia rhodozyma is transferred into Saccharomyces cerevisiae BY4741, so that the crtE, crtYB, and crtI genes from Phaffia rhodozyma are inserted into the uracil-deficient gene position of the BY4741 genome. Strain A10 is obtained by screening, and strain A10 can successfully synthesize β-carotene.

[0067] The vector tpLADH1 plasmid contains The ampicillin resistance gene on the vector and the Escherichia coli self-replicator ori, 2μori on the p426GPD vector, and the leucine selection tag gene (LEU2) expression cassette are shown in the plasmid map. Figure 2 shown.

[0068] Unless otherwise specified, primers and sequences to be synthesized were commissioned to Qingke Biotechnology Co., Ltd.

[0069] Sequencing was commissioned to Qingke Biotechnology Co., Ltd.

[0070] The cloning vector was purchased from Quanshijin Biotechnology Co., Ltd.

[0071] The vector pColdTF-rox1-NtCCD1-3-LEU2 was prepared by the following method:

[0072] (1) pColdTF plasmid (purchased from Quanshijin Biotechnology Co., Ltd.) was digested with EcoRI enzyme to obtain a linear plasmid;

[0073] (2) Using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, PCR amplification was performed with upstream homology arm primers (forward primer sequence is SEQ ID NO. 27: CGGTACCCTCGAGGGATCCGATGAATCCTAAATCCTCTACACCTAAG, reverse primer sequence is SEQ ID NO. 28: GAGGTTGCCTATTCCTTCTGTTAAATCCTTTTGTTGTTTCCGGGTGTAC) to obtain the upstream homology arm sequence of rox1 (rox1-up);

[0074] (2) Using the genomic DNA of Saccharomyces cerevisiae BY4741 as a template, PCR amplification was performed with downstream homology arm primers (forward primer sequence is SEQ ID NO. 29: GAAACGGCCTTAACGACGTAGTCGATTACTTCTAACTATATGGTCTCCAG, reverse primer sequence is SEQ ID NO. 30: CTGGAGACCATATAGTTAGAAGTAATCGACTACGTCGTTAAGGCCGTTTC) to obtain the downstream homology arm sequence of rox1 (rox1-down);

[0075] (3) Synthesize a sequence containing the promoter, NtCCD1-3 encoding gene sequence (SEQ ID NO. 2), and terminator (SEQ ID NO. 31);

[0076] (4) Synthesize the leucine selection tag gene (LEU2) expression cassette;

[0077] (5) The sequence obtained in steps (1)-(4) is passed through The Seamless Cloning and Assembly Kit was assembled according to the instructions in the kit to obtain the vector pColdTF-rox1-NtCCD1-3-LEU2, the map of which is shown in FIG. Figure 3 shown.

[0078] SEQ ID NO.31:

[0079]

[0080] Example 1

[0081] This example is used to illustrate the construction of mutants and engineered strains of NtCCD1-3.

[0082] Lys-25, Lys-31, Lys-38, Lys-42, Ser-128, Lys-140, Thr-155, Tyr-157, and Lys-164 on the amino acid sequence of NtCCD1-3 (SEQ ID NO. 1) were selected as site-directed mutagenesis sites and mutated to obtain the corresponding mutants. The specific mutations were: K25A, K31A, K38A, K42A, S128A, K140M, T155M, Y157A, Y157L, and K164L. The coding gene sequence of NtCCD1-3 is shown in SEQ ID NO. 2.

[0083] The specific method is as follows:

[0084] 1. Preparation of vector T1 containing mutant gene sequence

[0085] 1-1. Preparation of vector T1-K25A

[0086] Using the tpLADH1-NtCCD1-3 plasmid (commissioned to GenScript Biotech for synthesis, wherein the plasmid tpLADH1 was digested with Xho I and BamH I, and the NtCCD1-3 encoding gene sequence was ligated to the digested plasmid) as a template, PCR reaction 1 was performed with primers N3-F (SEQ ID NO. 3) and K25A-R1 (SEQ ID NO. 4) to amplify the first half of NtCCD1-3 (PCR product 1); using the tpLADH1-NtCCD1-3 plasmid as a template, PCR reaction 2 was performed with primers K25A-F2 (SEQ ID NO. 5) and N3-R (SEQ ID NO. 6) to amplify the second half of NtCCD1-3 (PCR product 2). The PCR program was as follows: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 50 seconds, annealing at 55-60°C for 30 seconds, and extension at 72°C for 2 minutes; 35 cycles, and finally extension at 72°C for 10 minutes.

[0087] After the PCR reaction, PCR products 1 and 2 were recovered and purified. Then, an overlapping PCR reaction (PCR reaction 3) was performed using PCR products 1 and 2 as templates and primers N3-F and N3-R. The overlapping PCR reaction system and overlapping PCR procedure are shown in Table 1.

[0088] Table 1

[0089]

[0090]

[0091] The obtained overlapping PCR product (PCR amplification product 3) was recovered and purified, and the overlapping PCR product and The cloning vectors were connected to obtain vector T1-K25A, which contained a mutant gene capable of encoding mutant K25A, and the mutant gene was referred to as K25A mutant gene for short.

[0092] 1-2. Preparation of vectors T1-K31A, T1-K38A, T1-K42A, T1-S128A, T1-K140M, T1-T155M, T1-Y157A, T1-Y157L, and T1-K164L: Follow step 1-1, except that different primers are used in PCR reaction 1 and PCR reaction 2 (the primer sequences for PCR reaction 1, PCR reaction 2, and overlapping PCR reaction are shown in Table 2).

[0093] Table 2

[0094]

[0095]

[0096]

[0097] Note: The underlined part is the mutated base sequence

[0098] 2. Construction of recombinant vectors and mutant Saccharomyces cerevisiae engineering strains

[0099] 2-1. Construction of recombinant vector containing K25A mutant gene and strain A10-rox1-K25A

[0100] Using vector T1-K25A as a template, PCR amplification was performed with the N3mut-F primer (SEQ ID NO. 25: 5'-CATGCCATGGATGGGTAGAAA-3') and the N3mut-R primer (SEQ ID NO. 26: 5'-GGACTAGTTCATAACTTGGCTTGTT-3') (PCR reaction 4) to obtain PCR product 4 having Nco I and Spe I restriction sites and the K25A mutant gene sequence.

[0101] PCR product 4 was digested with Nco I and Spe I. The vector pColdTF-rox1-NtCCD1-3-LEU2 was also digested with Nco I and Spe I, and the portion containing the NtCCD1-3 gene encoding sequence was discarded. The remaining half of the vector fragment was recovered as a backbone. This backbone was ligated to the digested PCR product 4 using T4 DNA ligase and transformed into competent E. coli TransT1 cells. Transformants were selected on solid LB medium supplemented with 50 μg / mL ampicillin. The plasmid was extracted and verified by digestion with Nco I and Spe I. Once sequencing confirmed, a recombinant vector containing the K25A mutant gene was constructed.

[0102] The recombinant vector containing the K25A mutant gene was digested with Pst I and Sma I to obtain a rox1-up-K25A-LEU2-rox1-down fragment, which was then transformed into strain A10. Transformants on the screening plate were selected and verified by PCR amplification using N3mut-F and N3mut-R primers. The correct one was the mutant Saccharomyces cerevisiae engineered strain (i.e., strain A10-rox1-K25A).

[0103] 2-2. The recombinant vectors and mutant Saccharomyces cerevisiae engineering strains (strains A10-rox1-K31A, A10-rox1-K38A, A10-rox1-K42A, A10-rox1-S128A, A10-rox1-K140M, A10-rox1-T155M, A10-rox1-Y157A, A10-rox1-Y157L and A10-rox1-K164L) were constructed according to the method of 2-1. The difference was that in PCR reaction 4, vectors T1-K31A, T1-K38A, T1-K42A, T1-S128A, T1-K140M, T1-T155M, T1-Y157A, T1-Y157L and T1-K164L were used as templates, respectively.

[0104] The 10 mutant Saccharomyces cerevisiae engineered strains obtained in this example are collectively referred to as A10-rox1-NtCCD1-3mut.

[0105] Example 3

[0106] This example is used to illustrate the fermentation of a mutant Saccharomyces cerevisiae engineered strain and the detection of its products.

[0107] Construction of strain A10-rox-NtCCD1-3: Digest the vector pColdTF-rox1-NtCCD1-3-LEU2 with Pst I and Sma I to obtain the rox1-up-NtCCD1-3-LEU2-rox1-down fragment, which is then transformed into strain A10. Select transformants on the screening plate and verify the transformation by PCR amplification using primers N3-F and N3-R. The correct transformants are strain A10-rox-NtCCD1-3.

[0108] Strain A10, the 10 strains obtained in Example 2 (A10-rox1-NtCCD1-3mut), and strain A10-rox-NtCCD1-3 were inoculated into 20 mL of YPD medium and incubated at 30°C with shaking at 200 rpm for 48 hours to obtain seed solution (OD 600 = about 0.8). Subsequently, 0.4 mL of the seed solution was inoculated into 20 mL of fresh YPD medium, and 1 mL of dodecane was added. Finally, the culture was shaken at 30°C and 200 rpm for 9 days for fermentation. After fermentation, the supernatant organic phase and precipitated cells were collected by centrifugation at 12,000 rpm. Anhydrous sodium sulfate was added to the supernatant organic phase for dehydration, and then the peak area and peak height of β-ionone were determined by gas chromatography-mass spectrometry (GC-MS). The mass of β-ionone was calculated. The operating conditions of the gas chromatography-mass spectrometry were as follows: capillary column HP-5MS (non-polar, 30 m×0.25 mm×0.25 μm); inlet temperature was 250° C.; carrier gas was high-purity helium (purity ≥99.999%), flow rate was 1 mL / min; injection mode was split injection, injection volume was 1 μL, split ratio was 30:1; programmed temperature conditions were: initial temperature was 100° C., maintained for 2 min, then increased to 210° C. at 10° C. / min, then increased to 290° C. at 20° C. / min, and maintained for 5 min. Mass spectrometry conditions: ionization mode: EI+; ionization voltage: 70 eV; scanning range: 40-400 amu; ion source temperature: 200°C; transfer line temperature: 290°C; spectrum search: WILEY and NIST08 spectral libraries were searched.

[0109] The collected cells were lyophilized and used for dry weight measurement to calculate the yield of β-ionone.

[0110] β-ionone titer = [β-ionone mass (μg) / medium volume

[0111] β-ionone yield = β-ionone mass (μg) / fermentation cell weight (g)

[0112] Three biological replicates were performed for each sample. The results are shown in Table 3 and Figure 1 shown.

[0113] Table 3

[0114] strain β-ionone titer β-ionone production A10 0mg / L 0mg / g A10-rox1-NtCCD1-3 0.353mg / L 0.048mg / g A10-rox1-K25A 0.203mg / L 0.031mg / g A10-rox1-K31A 0.498mg / L 0.078mg / g A10-rox1-K38A 1.081mg / L 0.175mg / g A10-rox1-K42A 0.269mg / L 0.045mg / g A10-rox1-S128A 0.277mg / L 0.055mg / g A10-rox1-K140M 0.388mg / L 0.057mg / g A10-rox1-T155M 0.298mg / L 0.058mg / g A10-rox1-Y157A 0.298mg / L 0.058mg / g A10-rox1-Y157L 0.550mg / L 0.077mg / g A10-rox1-Y164L 0.304mg / L 0.049mg / g

[0115] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A carotenoid dioxygenase mutant, characterized in that: The mutants are mutant K31A in which the lysine at position 31 of SEQ ID NO. 1 is mutated to alanine, mutant K38A in which the lysine at position 38 of SEQ ID NO. 1 is mutated to alanine, mutant Y157A in which the tyrosine at position 157 of SEQ ID NO. 1 is mutated to alanine, or mutant Y157L in which the tyrosine at position 157 of SEQ ID NO. 1 is mutated to leucine.

2. A gene encoding the mutant according to claim 1.

3. An expression vector, characterized in that The expression vector contains the gene according to claim 2.

4. An expression strain, characterized in that The strain contains the expression vector according to claim 3 or the gene encoding the mutant according to claim 2.

5. The expression strain according to claim 4, wherein The expression strain is yeast.

6. The expression strain according to claim 5, wherein The expression strain is Saccharomyces cerevisiae and / or Yarrowia lipolytica.

7. Use of the mutant according to claim 1, the gene according to claim 2, the expression vector according to claim 3, or the expression strain according to any one of claims 4 to 6 in the preparation of β-ionone.

8. A method for preparing β-ionone, characterized in that: The method comprises: culturing the expression strain according to any one of claims 4 to 6 to obtain a culture containing β-ionone.

9. The method according to claim 8, wherein The method further comprises extracting the culture to obtain β-ionone.

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