Saffron-derived CsCCD2 mutants, genes and applications, and recombinant model organisms for producing crocetin

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a CsCCD2 mutant, gene and application of saffron, and a recombinant model organism for producing crocetin. The protein sequence of the CsCCD2 mutant of the present invention is compared with the protein sequence of the wild-type CsCCD2, and it has a 14-amino acid deletion at the second amino acid. The present invention improves its binding ability and cleavage ability with zeaxanthin by protein modification of the CsCCD2 enzyme derived from saffron, and has the advantages of being more environmentally friendly and low-cost for the biosynthesis of crocetin, and provides a feasible method for the production of crocetin. And the efficiency of the engineering bacteria provided by the present invention in producing crocetin is high, and the concentration of crocetin can reach 1.20mg / g after fermentation for 72h.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to a saffron-derived CsCCD2 mutant, a gene and application thereof, and a recombinant model organism for producing crocetin. Background Art

[0002] Saffron contains 150 volatile and non-volatile components, of which more than 34 are volatile aromatic substances such as terpenes, terpene alcohols and their esters. Among the non-volatile components, crocin, crocetin and picrocrocin are the main secondary metabolites. These three substances are the products of oxidative cleavage of carotenoids based on isoprene compounds. At present, crocetin is mainly found in the stigma of saffron in the Iridaceae family, the flowers of the large-leafed Buddleja in the Scrophulariaceae family, and the fruits of the Gardenia in the Rubiaceae family. The latter two have far less production than saffron and cannot accumulate picrocrocin.

[0003] In 2014, Frusciantea et al. reported a new key enzyme CsCCD2 from saffron, which was proved to be the first key enzyme in the biosynthesis pathway of crocin, which can catalyze the cleavage of zeaxanthin to produce crocin dialdehyde and synthesize crocetin under the action of aldehyde dehydrogenase (ALD). However, when CsCCD2 enzyme is directly used for the synthesis of crocetin, there are problems of low substrate utilization and low synthesis efficiency.

[0004] Therefore, the prior art needs to be further improved and enhanced. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a saffron-derived CsCCD2 mutant, gene and application, and a recombinant model organism for producing crocetin, aiming to solve the problems of low substrate utilization and low synthesis efficiency when the existing CsCCD2 enzyme is used for the synthesis of crocetin.

[0006] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0007] In a first aspect, a saffron-derived CsCCD2 mutant is provided, wherein the protein sequence of the CsCCD2 mutant has a deletion of 14 amino acids at the second amino acid compared with the protein sequence of the wild-type CsCCD2.

[0008] The following are preferred technical solutions of the present invention, but are not intended to limit the technical solutions provided by the present invention. Through the following preferred technical solutions, the objectives and beneficial effects of the present invention can be better achieved and realized.

[0009] As a preferred technical solution, the saffron-derived CsCCD2 mutant, wherein the nucleotide sequence of the CsCCD2 mutant is as shown in SEQ ID NO:1.

[0010] In the second aspect, a gene expressing the CsCCD2 mutant described in the first aspect.

[0011] In a third aspect, a use of the gene described in the second aspect in preparing a recombinant vector expressing the CsCCD2 mutant and / or a recombinant model organism for producing crocetin; the model organism is selected from one of Escherichia coli, yeast, Chlamydomonas reinhardtii and tobacco.

[0012] A fourth aspect is a recombinant vector, wherein the gene described in the third aspect is accessed onto a basic vector.

[0013] As a preferred technical solution, the recombinant vector, wherein the basic vector also contains a gene encoding NcALD.

[0014] A fifth aspect is the use of the recombinant vector in constructing a recombinant Escherichia coli strain for producing crocetin.

[0015] In a sixth aspect, a recombinant Escherichia coli strain for producing crocetin is transformed with the above-mentioned recombinant vector.

[0016] A seventh aspect is the use of the recombinant Escherichia coli strain in the production of crocetin.

[0017] In an eighth aspect, a method for producing crocetin is provided, wherein the recombinant Escherichia coli strain described in the sixth aspect is used for fermentation production.

[0018] Beneficial effects: Compared with the prior art, the present invention functionally transforms the CsCCD2 enzyme, knocks out part of the protein sequence, shortens the distance between the N-terminal hydrophobic substrate binding site and zeaxanthin, improves the binding and cleavage ability of the enzyme and the substrate, and provides an ideal environment for the accumulation and availability of zeaxanthin substrate to CsCCD2. After the mutant is transformed into zeaxanthin-producing Escherichia coli, a strain of Escherichia coli that can efficiently synthesize crocetin in a 20°C environment can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Comparison of amino acid sequences between wild-type CsCCD2 and mutant CsCCD2-E1;

[0020] Figure 2 Figure 1 is a synthetic pathway diagram of crocetin;

[0021] Figure 3 This is the pET-Duet-CsCCD-NcALD plasmid map;

[0022] Figure 4 This is the pET-Duet-CsCCD2cro1-NcALD plasmid map;

[0023] Figure 5 This is the plasmid map of pACCAR-25ΔCrtX;

[0024] Figure 6 is the HPLC chromatogram of crocetin and zeaxanthin standards;

[0025] Figure 7 HPLC chromatogram of the fermentation of recombinant Escherichia coli carrying CsCCD2-E1 in FM medium at 20°C;

[0026] Figure 8 The yields of zeaxanthin and crocetin produced by the recombinant E. coli carrying CsCDD2 of CsCCD2-E1 in FM medium at 20°C. DETAILED DESCRIPTION

[0027] The present invention provides a saffron-derived CsCCD2 mutant, gene and application, and a recombinant model organism for producing crocetin. In order to make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementation methods. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and do not mean a necessary sequence, unless otherwise specified that a certain sequence must be followed. The serial numbers themselves, such as "first", "second", etc., assigned to the components herein are only used to distinguish the objects described and do not have any order or technical meaning.

[0029] The present invention provides a saffron-derived CCD2 mutant and a coding sequence thereof, so that when the CsCCD2 mutant and the coding sequence thereof are used to construct a crocetin-producing recombinant strain, crocetin can be efficiently produced using zeaxanthin as a substrate.

[0030] Specifically, the rate-limiting enzyme CsCCD2 in the crocetin synthesis pathway is mutated and modified, and part of the protein sequence is knocked out (to form a mutant), so that it has a 14-amino acid deletion at the second amino acid. After the deletion of some amino acids, the ability to crack zeaxanthin and generate crocetin is improved, which solves the problem of low substrate utilization in fermentation culture, reduces the cost of industrial production, and improves production efficiency. The CsCCD2 gene deletion mutant is recorded as CsCCD2-E1, and the nucleotide sequence of the CsCCD2-E1 gene is shown in SEQ ID NO: 1.

[0031] It should be noted that in the present invention, the wild-type CsCCD2 gene sequence, CsCCD2-E1 gene sequence and wild-type NcALD gene are all optimized by Escherichia coli codons and appropriately avoid commonly used restriction enzyme sites. The nucleotide sequences of the genes are: the nucleotide sequence of the CsCCD2-E1 gene is shown in SEQ ID NO: 1; the nucleotide sequence of the wild-type CsCCD2 gene is shown in SEQ ID NO: 2; the nucleotide sequence of the wild-type NcALD gene is shown in SEQ ID NO: 3.

[0032] The complete gene sequence of pET-Duet-CsCCD-NcALD plasmid is shown in SEQ ID NO:4, and the plasmid map is shown in Figure 3 As shown;

[0033] The complete gene sequence of pET-Duet-CsCCD2-E1-NcALD plasmid is shown in SEQ ID NO:5, and the plasmid map is shown in Figure 4 As shown;

[0034] The complete gene sequence of the pACCAR-25ΔCrtX plasmid is shown in SEQ ID NO:6, and the plasmid map is shown in Figure 5 shown.

[0035] In the present invention, a recombinant vector expressing a CsCCD2 mutant is provided, so that when the recombinant vector is used to construct a recombinant strain for producing crocetin, crocetin can be efficiently produced using zeaxanthin as a substrate.

[0036] Specifically, a plasmid with CsCCD2 and NcALD genes was constructed. The pETDuet-1 vector is designed to express two foreign genes simultaneously in E. coli. The vector contains two multiple cloning sites, each of which is composed of a T7 promoter / Lac operator and a ribosome binding site. pETDuet-1 carries the Co lE1 replicon, the la c I gene and the ampicillin resistance gene derived from the pBR322 plasmid. The wild-type CsCCD2 gene sequence and the CsCCD2-E1 gene sequence were inserted into the pETDuet-1 vector together with the wild-type NcALD gene, respectively, to construct the following plasmids:

[0037] pET-Duet-CsCCD-NcALD and pET-Duet-CsCCD2-E1-NcALD.

[0038] The present invention provides a recombinant Escherichia coli strain for producing crocetin, so that the recombinant Escherichia coli strain can efficiently produce crocetin using zeaxanthin as a substrate.

[0039] Specifically, since E. coli does not synthesize zeaxanthin in the background, the pACCAR-25ΔCrtX plasmid is designed to produce zeaxanthin in BL21 (DE3) E. coli. The plasmid contains the Crt E gene, Crt X gene, Crt Y gene, Crt I gene, Crt B gene and Crt Z gene required for constructing a pathway for heterologous synthesis of zeaxanthin in E. coli, as well as a chloramphenicol (CPL) resistance gene for resistance screening. The plasmid pACCAR-25ΔCrtX is transformed into BL21 (DE3) competent E. coli to construct a pathway for heterologous synthesis of zeaxanthin, and E. coli with high zeaxanthin production is preferably used as the starting strain. The construction method of the plasmid pACCAR-25ΔCrtX refers to the paper DO I: 10.1128 / jb.172.12.6704-6712.1990.

[0040] E. coli strains with high zeaxanthin production:

[0041] BL21(DE3), pACCAR-25ΔCrtX

[0042] The above two plasmids were transformed into Escherichia coli strains with high zeaxanthin production, and high-yield crocetin-producing Escherichia coli were constructed and screened.

[0043] The genotype of the saffron-producing engineered bacteria constructed by the present invention is:

[0044] BL21(DE3), pACCAR-25ΔCrtX, pET-Duet-CsCCD-NcALD;

[0045] BL21(DE3), pACCAR-25ΔCrtX, pET-Duet-CsCCD2-E1-

[0046] NcALD.

[0047] The recombinant Escherichia coli provided by the present invention can efficiently synthesize crocetin in FM medium at 20°C, and the engineered bacteria of the present invention have the advantages of being more environmentally friendly and low-cost compared to the biosynthesis of crocetin by fermentation culture and other methods in the prior art, and provide a feasible method for heterologous production of crocetin in Escherichia coli. The engineered bacteria provided by the present invention have a high efficiency in producing crocetin, and the yield of crocetin can reach 1.20 mg / g after fermentation for 72 hours in a culture environment of FM medium at 20°C.

[0048] A fermentation medium suitable for crocetin production provided by the present invention adopts FM medium, so that the high-yield zeaxanthin Escherichia coli containing wild-type CsCCD2 gene / CsCCD2-E1 gene and NcALD gene can normally produce crocetin. Isopropyl-β-D-thiogalactoside (IPTG) is used as an inducer in the fermentation medium, which is responsible for opening the transcription of the Lac operon and initiating the transcription and expression of the downstream CsCCD2 gene / CsCCD2-E1 gene and NcALD gene. In the initial culture medium, due to the presence of glucose, the transcription of the Lac operon is inhibited by glucose; as the fermentation proceeds, glucose is rapidly consumed until the glucose inhibition effect is relieved, IPTG opens the transcription of the Lac operon, induces the synthesis of CsCCD enzyme / CsCCD2-E1 enzyme and NcALD enzyme, thereby cutting zeaxanthin and generating crocetin.

[0049] The culture medium includes:

[0050] Table 1 Basic components of FM culture medium

[0051]

[0052] Table 2 Basic components of Trace meta l in FM medium

[0053]

[0054]

[0055] The preparation method of crocetin provided by the invention further comprises an extraction step after fermentation.

[0056] The extraction method is as follows: grind the freeze-dried bacteria into powder, weigh about 20 mg of powder into a 2.0 mL centrifuge tube, add pre-cooled 1 mL of methanol, vortex and resuspend by blowing. Centrifuge at 12000xg for 5 minutes in a 4°C centrifuge, and take the supernatant into a new 2.0 mL centrifuge tube. Add new pre-cooled 1 mL of methanol to the precipitate and repeat the extraction once, trying to extract the pigment completely. Mix the supernatants twice and filter them into a 1.5 mL brown centrifuge tube with a 0.22 μm microporous filter membrane (organic), and mark them. Subsequently, high performance liquid chromatography (HPLC) was used to detect the content of zeaxanthin and crocetin.

[0057] The following is a further explanation of the saffron-derived CsCCD2 mutant, gene and application, and recombinant model organism for producing crocetin provided by the present invention through specific examples.

[0058] Example 1

[0059] Construction of the recombinant Escherichia coli strain producing zeaxanthin according to the present invention

[0060] 1. Construction of chassis strain

[0061] ① Take out the BL21 (DE3) competent cells from -80℃ and quickly put them in ice. After 5 minutes, wait for the bacterial block to thaw, add 20-50ng of pACCAR25ΔcrtX plasmid, and gently mix by tapping the bottom of the EP tube by hand (avoid using a gun to suck), and let it stand in ice for 25 minutes.

[0062] ② Heat shock in a 42℃ water bath for 45 seconds, quickly put back on ice and let stand for 2 minutes. Shaking will reduce the transformation efficiency.

[0063] ③ Add 700 μL of sterile LB medium without antibiotics into the centrifuge tube, mix well and resuscitate at 37°C, 200 rpm for 60 min.

[0064] ④ Pipette about 100 μL of supernatant and spread it on LB+CPL (final concentration is 34 μg / mL) plate.

[0065] ⑤ Place the plate upside down in a 37°C incubator for overnight culture.

[0066] 2. Preparation of competent E. coli strains for zeaxanthin production

[0067] ① Pick three single clones from the LB+CPL (final concentration 34 μg / mL) plate and culture them in 3 ml LB+CPL (final concentration 34 μg / mL) liquid medium at 37°C and 200 rpm overnight;

[0068] ②Pick the overnight bacterial culture with the darkest β-carotene color and name it BL21(DE3), pACCAR-25ΔCrtX

[0069] ③ Take 500 μL of seed solution and add it to 50 mL of LB+CPL, culture at 37°C, 200 rpm for 2.5 h (OD600 reaches 0.4-0.5), and then place the culture on ice for 30 min;

[0070] ④ Add 500 μL of 50% glycerol to the remaining 500 μL of bacterial solution for seed preservation;

[0071] ⑤ Centrifuge at 2700 rpm for 10 min at 4°C, discard the supernatant, add 20 mL of 0.1 M pre-cooled CaCl2 solution to resuspend the cells, and place on ice for 30 min;

[0072] ⑥ Centrifuge at 2700 rpm for 10 min at 4°C and discard the supernatant;

[0073] ⑦ Add 1 mL of pre-cooled 0.1 M CaCl2 solution to suspend cells for every 50 mL of culture, then add 1 mL of 50% glycerol, mix well, and dispense on ice (70-80 μL per tube);

[0074] ⑧ After aliquoting, the cells were quickly frozen in liquid nitrogen and stored at -80°C. They were named BL21(DE3), pACCAR-25ΔCrtX.

[0075] Example 2

[0076] Crocetin shake flask fermentation experiment at 20℃ with recombinant Escherichia coli carrying wild-type CsCCD2

[0077] 1. Acquisition of exogenous functional gene elements

[0078] The wild-type CsCCD2 gene sequence, the protein-modified CsCCD2-E1 gene sequence and the wild-type NcALD gene are all codon-optimized by Escherichia coli, and the commonly used restriction enzyme sites are appropriately avoided. The wild-type CsCCD2 gene sequence, CsCCD2-E1 gene sequence and the wild-type NcALD gene fragment used in the present invention are all obtained by artificial synthesis, specifically by GenScript using pETDuet-1 plasmid as a vector to prepare the experimental plasmid.

[0079] 2. Test strains

[0080] Chassis strain: the zeaxanthin-producing strain BL21 (DE3) in Example 1, pACCAR-25ΔCrtX;

[0081] Recombinant strain 1: constructed according to the construction method of the chassis strain in Example 1, and the difference in naming is that BL21 (DE3), pACCAR-25ΔCrtX competent Escherichia coli replaced BL21 (DE3) competent Escherichia coli; pET-Duet-CsCCD-NcALD plasmid replaced pACCAR-25ΔCrtX plasmid; CPL (final concentration of 34 μg / mL) + ampicillin (AMP) (final concentration of 100ug / mL) double antibiotic LB medium plate replaced CPL (final concentration of 34μg / mL) single antibiotic LB medium plate.

[0082] 3. Induction culture

[0083] ①Pick a single clone and place it in 4 mL of LB liquid medium and culture it at 37°C overnight;

[0084] ②Take 2 mL of 30°C overnight bacterial liquid, centrifuge at 3000 rpm for 2 min, discard the supernatant, resuspend with FM medium, add to 40 mL FM liquid medium, and add corresponding antibiotics (AMP 100 μg / mL + CPL 34 μg / mL);

[0085] ③Cultivate at 37℃ for 6h until OD600=0.6-0.8, add 200μL of 0.1M IPTG solution to a final concentration of 0.5mM, and induce at 20℃ for 72h;

[0086] ④ Centrifuge at 4000 rpm for 8 min at 4℃ to collect the cells; wash the cells once with an appropriate amount of ddH2O to remove excess culture medium; freeze-dry overnight.

[0087] 4. Extraction of Escherichia coli pigment

[0088] ① Grind the freeze-dried bacteria into powder, weigh about 20 mg of powder sample into a 2.0 mL centrifuge tube, add 1 mL of pre-cooled methanol and vortex;

[0089] ② Centrifuge at 12000xg for 5 min at 4°C and place the supernatant in a new 2.0 mL centrifuge tube;

[0090] ③ Repeat once, add 1 mL of pre-cooled methanol to the precipitate and extract once, trying to extract the pigment completely;

[0091] ④ Filter with a 0.22μm organic filter membrane into a 1.5mL brown centrifuge tube and mark it.

[0092] 5. Crocetin HPLC detection method

[0093] The chromatographic conditions were as follows.

[0094] Chromatographic column: Phenomenex l una 5um C18(2)100A, LC column 250x4.6mm

[0095] Instrument: Shimadzu LC-2023C 3D

[0096] Method name: 20min-crocet in

[0097] Mobile phase: A: ultrapure water (0.1% formic acid); B: methanol

[0098] Flow rate: 0.6mL / min

[0099] Column temperature: 35°C

[0100] Injection volume: 10 μL

[0101] Detection wavelength: 430nm

[0102] The time program is shown in Table 3:

[0103] Table 3 HPLC liquid phase procedure

[0104]

[0105] The peak time of each standard is as follows Figure 6 As shown, each standard and its corresponding peak time are Crocet in: 7.073 min and Zeaxanthin: 11.820 min.

[0106] 6. Results

[0107] The recombinant Escherichia coli strain provided by the present invention can synthesize zeaxanthin and crocetin in FM medium at 20° C. It is proved that the recombinant strain 1 carrying wild-type CsCCD2 and NcALD can realize normal synthesis of zeaxanthin, cleavage of zeaxanthin and generation of crocetin in FM fermentation medium at 20° C., that is, the recombinant Escherichia coli can normally synthesize zeaxanthin and crocetin.

[0108] Example 3

[0109] Crocetin shake flask fermentation experiment at 20℃ with recombinant Escherichia coli carrying deletion mutation CsCDD2-E1

[0110] 1. Test strains

[0111] The chassis strain is the zeaxanthin-producing strain BL21 (DE3) in Example 1, pACCAR-25ΔCrtX;

[0112] The control strains were the crocetin-producing strain BL21 (DE3) in Example 2, pACCAR-25ΔCrtX, pET-Duet-CsCCD2-NcALD;

[0113] Recombinant strain 2: constructed by referring to the method of recombinant strain 1 in Example 2, except that pET-Duet-CsCCD2-E1-NcALD plasmid was used instead of pET-Duet-CsCCD-NcALD plasmid;

[0114] The induction culture, Escherichia coli pigment extraction and crocetin HPLC detection methods were all carried out according to the method in Example 2.

[0115] Results Figure 7 and Figure 8 , the recombinant strain 2 with CsCCD2-E1 enzyme produced 1.20 mg / g of crocetin, which was higher than the 0.97 mg / g of crocetin produced by the recombinant strain 1 with wild-type CsCCD2. It was proved that the combination of mutant CsCCD2-E1 enzyme and wild-type NcALD enzyme can achieve the normal cleavage of zeaxanthin by Escherichia coli in the fermentation culture environment of FM medium at 20°C and efficiently produce crocetin, that is, compared with the wild-type CsCCD2 enzyme, the CsCCD2-E1 enzyme can still exercise the normal ability to cleave zeaxanthin after the deletion of part of the N-terminal protein, and its cleavage efficiency is higher than that of the wild-type CsCCD2 enzyme.

[0116] It should be understood that the application of the present invention is not limited to the above examples, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the mutants, recombinant vectors, strains, and construction methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

Claims

1. A CsCCD2 mutant derived from saffron, characterized in that: The nucleotide sequence encoding the CsCCD2 mutant is shown in SEQ ID NO:

1.

2. A gene expressing the CsCCD2 mutant according to claim 1.

3. Use of the gene according to claim 2 in preparing a recombinant vector expressing the CsCCD2 mutant and / or a recombinant model organism for producing crocetin; the model organism is selected from one of Escherichia coli, yeast and tobacco.

4. A recombinant vector, characterized in that: The gene according to claim 2 is introduced into the recombinant vector.

5. The recombinant vector according to claim 4, characterized in that: The recombinant vector also contains a gene encoding NcALD, and the nucleotide sequence of the NcALD gene is shown in SEQ ID NO:

3.

6. Use of the recombinant vector according to claim 4 or 5 in constructing a recombinant Escherichia coli strain producing crocetin.

7. A recombinant Escherichia coli strain producing crocetin, characterized in that: Transformed with the recombinant vector according to claim 4 or 5.

8. Use of the recombinant Escherichia coli strain according to claim 7 in the production of crocetin.

9. A method for producing crocetin, characterized in that: The recombinant Escherichia coli strain according to claim 7 is used for fermentation production.

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