Carotenoid cleavage dioxygenase mutant and application thereof

Abstract

The invention discloses a carotenoid cleavage dioxygenase mutant and application of the carotenoid cleavage dioxygenase mutant. The carotenoid cleavage dioxygenase mutant is subjected to any one or a combination of at least two of the following mutations on the basis of an amino acid sequence SEQ ID NO.1: F181G, F184L or F337M. According to the invention, the substrate beta-carotene is butted to a CCD4 enzyme activity pocket to construct a saturated mutant library, screening is carried out through a bacterial color complementation experiment, and after specific site amino acid mutation, degradation of the substrate beta-carotene is accelerated, thereby laying a certain technical foundation for further cultivation of new varieties of crops.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco genetic engineering and relates to a carotenoid cleavage dioxygenase mutant and application thereof. Background Art

[0002] Carotenoids are important plastid pigments with important physiological functions. They are closely related to plant growth, development and photosynthesis, and affect crop quality traits. Carotenoid cleavage dioxygenases (CCDs) are a family of highly heterogeneous polyene chain oxidases that are active in non-heme iron (Fe) 2+ With the help of 4 histidines, CCDs catalyze the reaction. As a key enzyme, CCDs participate in the production of plant hormones and play an important role in the formation of plant (leaves, flowers, fruits) color and fragrance. CCD1 and CCD4 form easily volatile apocarotenoids through cleavage to produce aroma substances.

[0003] CCD4 is an important enzyme involved in the catalytic cleavage process of carotenoids. Its catalytic carotenoid products contain a variety of flavors and aromatic substances such as β-ionone, which make flowers, fruits and leaves have an aromatic smell and are of great significance to the spread of plant pollen and seeds.

[0004] In existing research, although there have been many studies on the carotenoid cleavage pathway, from the perspective of plant transformation, the use of genetic engineering technology to achieve targeted selection and cultivation is an important technical prerequisite for maximizing the utilization of plants.

[0005] As one of the key technologies in synthetic biology, directed biological evolution technology won the 2018 Nobel Prize in Chemistry. This technology is widely used in the design of enzyme activity. Through directed evolution, it can effectively overcome the shortcomings of natural enzymes in terms of environmental tolerance, stereo / regional selectivity, substrate specificity, catalytic efficiency and product inhibition, so that the evolutionary process of thousands of years in nature can be completed in a relatively short time in the laboratory.

[0006] In the development of enzyme directed evolution technology, traditional directed evolution, such as using error-prone PCR, DNA mixing, sequence saturation mutation, random in vitro recombination and other technologies, has disadvantages such as low mutation efficiency and large screening workload, which restricts the application of in vitro directed evolution of enzyme molecules. Rational design of proteins is a newer directed evolution mutation strategy. This technology is based on the structural information of proteins. First, it uses computer simulation as a basis, and then selects amino acid residues that have direct interactions with the substrate around the catalytic active center of the enzyme. By constructing a "small and precise" mutant library, the screening scale of the mutant library is reduced.

[0007] In general, although directed evolution technology can accelerate the evolution of crop genes and provide high-quality genes for improving crop quality, due to its short development time and other technical difficulties, directed evolution technology has not yet been used for plant gene modification.

[0008] Tobacco is one of the model crops for genetic engineering research, and the degradation efficiency of carotenoids directly affects the quality traits of tobacco. Therefore, if the tobacco terpene metabolism genes can be modified in combination with directed evolution, it will have a very important application significance for accelerating tobacco breeding, and it can also lay a certain technical foundation for the genetic breeding of other crops.

[0009] In summary, the current carotenoid cleavage dioxygenase has problems such as low enzyme activity and poor catalytic effect. It is urgent to provide a carotenoid cleavage dioxygenase mutant with better enzyme activity for the cultivation of new tobacco varieties. Summary of the invention

[0010] In view of the deficiencies of the prior art and actual needs, the present invention provides a carotenoid cleavage dioxygenase mutant and its application, aiming to provide several CCD4 and tobacco CCD4 genes with better enzyme activity, thereby providing a new technical idea for the breeding of new tobacco varieties, and also laying a certain technical foundation for other crop breeding and variety improvement.

[0011] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0012] In a first aspect, the present invention provides a carotenoid cleavage dioxygenase mutant, wherein the carotenoid cleavage dioxygenase mutant undergoes any one or a combination of at least two of the following mutations based on the amino acid sequence SEQ ID NO.1: F181G, F184L or F337M.

[0013] The present invention docks the substrate beta-carotene to the CCD4 enzyme activity pocket, constructs a saturated mutant library, and screens it through a bacterial color complementation experiment. After the amino acid mutation at a specific site, the degradation of the substrate beta-carotene is accelerated, which lays a certain technical foundation for further breeding of new crop varieties.

[0014] The signal peptide formed by removing the first 28 amino acids from the wild-type CCD4 is composed of 573 amino acids, and the specific sequence is shown in SEQ ID No. 1 (NCBI accession number NP_001312446.1):

[0015] SEQ ID NO.1:

[0016] .

[0017] In a second aspect, the present invention provides a nucleic acid molecule comprising a coding sequence of the carotenoid cleavage dioxygenase mutant described in the first aspect.

[0018] In a third aspect, the present invention provides a recombinant vector comprising the nucleic acid molecule described in the second aspect.

[0019] In a fourth aspect, the present invention provides an expression strain, wherein the expression strain contains the nucleic acid molecule described in the second aspect and / or the recombinant vector described in the third aspect.

[0020] In a fifth aspect, the present invention provides a method for preparing the carotenoid cleavage dioxygenase mutant according to the first aspect, the method comprising the following steps:

[0021] (1) inserting the nucleic acid sequence SEQ ID NO.2 of the carotenoid cleavage dioxygenase into a plasmid to obtain a recombinant plasmid, and using the recombinant plasmid as a template, performing site-directed saturation mutagenesis on any one or a combination of at least two of the 181st, 184th and 337th positions of the carotenoid cleavage dioxygenase, wherein the nucleic acid sequence of the primer for the mutation at the 181st position includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleic acid sequence of the primer for the mutation at the 184th position includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.6, and the nucleic acid sequence of the primer for the mutation at the 337th position includes the sequences shown in SEQ ID NO.7 and SEQ ID NO.8;

[0022] (2) The mutant plasmid obtained in step (1) is transformed into a host bacterium, cultured and purified to obtain the carotenoid cleavage dioxygenase mutant.

[0023] The gene encoding CCD4, minus the first 84 nucleotides, consists of 1719 nucleotides, and the specific sequence is shown in SEQ ID No. 2 (NCBI accession number NM_001325517.1):

[0024] SEQ ID NO.2:

[0025]

[0026] SEQ ID NO. 3: gttatccgaatgtgnnktctggttttaatggt.

[0027] SEQ ID NO. 4: gttcaacgctcagaatattcggtgcaaccataa.

[0028] SEQ ID NO. 5: aatgtgttttctggtnnkaatggtctgaccgca.

[0029] SEQ ID NO. 6: gttcaacgctcagaatattcggtgcaaccataa.

[0030] SEQ ID NO. 7: atgaatccgctggaannkatcaccggtggttcc.

[0031] SEQ ID NO. 8: gttcaacgctcagaatattcggtgcaaccataa.

[0032] Preferably, the plasmid comprises pMAL-c5x.

[0033] Preferably, the host bacteria include Escherichia coli, yeast or Bacillus subtilis.

[0034] In a sixth aspect, the present invention provides use of the carotenoid cleavage dioxygenase mutant described in the first aspect, the nucleic acid molecule described in the second aspect, the recombinant vector described in the third aspect, or the expression strain described in the fourth aspect in pigment degradation.

[0035] Preferably, the pigment comprises beta-carotene.

[0036] In a seventh aspect, the present invention provides a method for preparing β-ionone, the preparation method comprising: culturing the expression strain described in the fourth aspect to obtain a culture containing β-ionone.

[0037] Preferably, the preparation method further comprises extracting the culture to obtain β-ionone.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] The present invention docks the substrate beta-carotene to the CCD4 enzyme activity pocket, constructs a saturated mutant library, and screens it through a bacterial color complementation experiment. After the amino acid mutation at a specific site, the degradation of the substrate beta-carotene is accelerated, which lays a certain technical foundation for further breeding of new crop varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the result of bacterial color experiment of CCD4-181 mutant;

[0041] Figure 2 For CCD4-181 mutants at OD 440 Result graph of absorbance value at ;

[0042] Figure 3 This is a graph showing the results of a bacterial color experiment on the CCD4-184 mutant;

[0043] Figure 4 For CCD4-184 mutants, the OD 440 Result graph of absorbance value at ;

[0044] Figure 5 This is a graph showing the results of a bacterial color experiment on the CCD4-337 mutant;

[0045] Figure 6 For CCD4-337 mutants at OD 440 Result graph of absorbance value at . DETAILED DESCRIPTION

[0046] To further illustrate the technical means and effects of the present invention, the present invention is further described below in conjunction with the embodiments and drawings. It should be understood that the specific implementation methods described herein are only used to explain the present invention, rather than to limit the present invention.

[0047] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0048] Example 1

[0049] This example is to obtain the carotenoid cleavage dioxygenase (CCD4) gene.

[0050] The tobacco NtCCD4 gene was obtained from common tobacco by molecular cloning and sequencing, which is completely consistent with the sequence published in the NCBI database (NCBI accession number NM_001325517.1), as shown in SEQ ID NO. 2. In order to avoid differences in protein expression caused by codon preference, the tobacco NtCCD4 codon was optimized to make it more conducive to expression in Escherichia coli. The codon optimization and vector pMAL-CCD4 construction were commissioned to Beijing Qingke Biotechnology Co., Ltd. The protein expression vector is pMAL-c5x, and the homologous cloning site is BamHI.

[0051] Example 2

[0052] In this example, a bacterial color complementation experiment was used to verify the recombinant engineering strain of carotenoid cleavage dioxygenase (CCD4).

[0053] (1) The recombinant vector pMAL-CCD4 constructed in Example 1 was co-transformed with the pAC-β plasmid into the expression strain E. coli BL21 (DE3), and the empty vector pMAL-c5x was co-transformed with the pAC-β plasmid as a negative control strain.

[0054] (2) After overnight culture, positive clones were selected for identification, and the β-carotene content of the positive clone strains with correct sequencing was further tested.

[0055] The principle of β-carotene content detection is: pAC-β contains carotenoid synthase, which can synthesize β-carotene in E. coli, causing E. coli to transform from white to yellow. Therefore, compared with the control, the color of the recombinant plasmid co-transformed bacteria is significantly lighter, indicating that CCD4 has catalytic activity, the substrate β-carotene can be oxidized and degraded, and the reduction in the amount of substrate causes the color to become lighter. Further, the OD is measured by a spectrophotometer. 440 The absorbance value of the final product can be used to determine the β-carotene content.

[0056] Test results such as Figure 1 , Figure 3 , Figure 5 As shown, the vector pAC-β contains β-carotene synthase, which can synthesize β-carotene in Escherichia coli, turning Escherichia coli from white to yellow; after the saturated mutations at the 181st, 184th and 337th sites of CCD4 were co-transformed with the pAC-β double plasmid into Escherichia coli BL21 (DE3), the color of the bacterial solution changed significantly after IPTG induction. Using the pMAL-c5x empty vector as a negative control, it can be seen that the colors of the F181G mutant, F184L mutant and F337M mutant were significantly lighter than those of the wild type and other mutants. Figure 2 , Figure 4 , Figure 6 For OD 440 The absorbance values ​​at OD were 0.04 and 0.06, respectively. Empty pMAL-c5x was used as negative control. It can be seen that mutants F181G, F184L, and F337M showed significantly lower absorbance than wild-type CCD4 at OD 440 The absorbance values ​​decreased significantly.

[0057] Example 3

[0058] In this example, saturation mutations were performed at positions 181, 184 and 337 of the carotenoid cleavage dioxygenase CCD4 (the specific amino acid sequence is shown in SEQ ID NO. 1).

[0059] Construction of a CCD4 saturation mutant library for site 181: Using the pMAL-CCD4 constructed in Example 1 as a template, SEQ ID NO.3 as a forward primer, and SEQ ID NO.4 as a reverse primer, a whole plasmid PCR was performed. Construction of a CCD4 saturation mutant library for site 184: Using the pMAL-CCD4 constructed in Example 1 as a template, SEQ ID NO.5 as a forward primer, and SEQ ID NO.6 as a reverse primer, a whole plasmid PCR was performed. Construction of a CCD4 saturation mutant library for site 337: Using the pMAL-CCD4 constructed in Example 1 as a template, SEQ ID NO.7 as a forward primer, and SEQ ID NO.8 as a reverse primer, a whole plasmid PCR was performed.

[0060] F181-F (SEQ ID NO.3): 5'-gttattccgaatgtgnnktctggttttaatggt-3';

[0061] general-R (SEQ ID NO.4): 5'-gttcaacgctcagaatattcggtgcaaccataa-3';

[0062] F184-F (SEQ ID NO.5): 5'-aatgtgttttctggtnnkaatggtctgaccgca-3';

[0063] general-R (SEQ ID NO.6): 5'-gttcaacgctcagaatattcggtgcaaccataa-3';

[0064] F337-F (SEQ ID NO.7): 5'-atgaatccgctggaankatcaccggtggttcc-3',

[0065] general-R (SEQ ID NO. 8): 5'-gttcaacgctcagaatattcggtgcaaccataa-3'.

[0066] The PCR amplification product was digested with DpnI, and then the digested product and the pAC-β double plasmid were co-transformed into E. coli BL21 (DE3) competent cells. After the transformation, the bacterial solution was spread on a plate containing 100 μg / L ampicillin and 34 μg / L chloramphenicol to finally obtain a saturated mutation library.

[0067] For further screening, positive monoclones of F181, F184 and F337 were selected and cultured overnight in 20 mL LB liquid medium containing ampicillin (concentration, 100 μg / L) and chloramphenicol (concentration, 34 μg / L), and then induced with IPTG at a final concentration of 0.1 mM at 18°C ​​for 18 h. After the culture was completed, the supernatant was discarded by centrifugation, photographed, and resuspended in 3 mL of acetone to measure OD 440 value.

[0068] The above high-throughput screening method was used to finally screen and identify mutants with significantly reduced absorbance and further sequence them. The results showed that when the 181st Phe amino acid (F) mutated to the amino acid Gly (G), the 184th Phe amino acid (F) mutated to the amino acid Leu (L), and the 337th Phe amino acid (F) mutated to the amino acid Met (M), the carotenoid cleavage dioxygenase CCD4 all had significant changes.

[0069] In summary, the directed mutation evolution method of the present application targeting specific sites 181, 184 and 337 can effectively improve the CCD4 enzyme activity, thereby laying a good foundation for further affecting the β-carotene content in crops.

[0070] The applicant declares that the present invention illustrates the detailed method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed method, that is, it does not mean that the present invention must rely on the above-mentioned detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A carotenoid cleavage dioxygenase mutant, characterized in that: The carotenoid cleavage dioxygenase mutant has any one or a combination of at least two of the following mutations based on the amino acid sequence SEQ ID NO.1: F181G, F184L or F337M.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule contains the coding sequence of the carotenoid cleavage dioxygenase mutant according to claim 1.

3. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule according to claim 2.

4. An expression strain, characterized in that The expression strain contains the nucleic acid molecule according to claim 2 and / or the recombinant vector according to claim 3.

5. A method for preparing the carotenoid cleavage dioxygenase mutant according to claim 1, characterized in that: The method comprises the following steps: (1) inserting the nucleic acid sequence SEQ ID NO.2 of the carotenoid cleavage dioxygenase into a plasmid to obtain a recombinant plasmid, and using the recombinant plasmid as a template, performing site-directed saturation mutagenesis on any one or a combination of at least two of the 181st, 184th and 337th positions of the carotenoid cleavage dioxygenase, wherein the nucleic acid sequence of the primer for the mutation at the 181st position includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4, the nucleic acid sequence of the primer for the mutation at the 184th position includes the sequences shown in SEQ ID NO.5 and SEQ ID NO.6, and the nucleic acid sequence of the primer for the mutation at the 337th position includes the sequences shown in SEQ ID NO.7 and SEQ ID NO.8; (2) The mutant plasmid obtained in step (1) is transformed into a host bacterium, cultured and purified to obtain the carotenoid cleavage dioxygenase mutant.

6. The method for preparing a carotenoid cleavage dioxygenase mutant according to claim 5, characterized in that: The plasmids include pMAL-c5x; Preferably, the host bacteria include Escherichia coli, yeast or Bacillus subtilis.

7. Use of the carotenoid cleavage dioxygenase mutant according to claim 1, the nucleic acid molecule according to claim 2, the recombinant vector according to claim 3 or the expression strain according to claim 4 in pigment degradation.

8. The use according to claim 7, characterized in that: The pigment includes beta-carotene.

9. A method for preparing β-ionone, characterized in that: The preparation method comprises: culturing the expression strain according to claim 4 to obtain a culture containing β-ionone.

10. The method for preparing β-ionone according to claim 9, characterized in that: The preparation method further comprises extracting the culture to obtain β-ionone.

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