Application of Cytochrome P450 Monooxygenase LsDHLS

Abstract

The invention discloses the application of cytochrome P450 monooxygenase LsDHLS, the gene of which is cloned from lettuce, and pEAQ-HT is used as a tobacco transient expression vector, and Agrobacterium GV3101 is used as a host cell to realize heterologous expression of the protease in tobacco, and it is proved that the protease can perform carbonylation modification on the 2-position of kauniolide to generate dehydroleucodine. The invention provides a new idea for the site-specific carbonylation modification of kauniolide and the synthesis of the carbonylation product dehydroleucodine.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of cytochrome P450 monooxygenase LsDHLS. Background Art

[0002] Dehydroleucodine is a guaiacyl sesquiterpene lactone widely found in Asteraceae plants. It can improve phytoalexin response, disease resistance and insect resistance. It has important medicinal values ​​such as antibacterial, anti-inflammatory, treatment of gastric ulcers, inhibition of lipogenesis, inhibition of tumor growth and anti-leukemia. Some structural genes of the dehydroleucodine biosynthesis pathway have been reported. Since 2002, researchers have cloned germacrene A synthase (GAS) from lettuce, which can catalyze farnesylpyrophosphate (FPP) to produce the sesquiterpene skeleton of lettuce; germacrene A oxidase (GAO) that modifies germacrene A to produce germacrene A acid; and costunolide synthase (COS) that hydroxylates costunolide A acid at the C6 position and cyclizes it to produce costunolide. Kauniolide synthase (COS) that catalyzes costunolide to produce kauniolide was identified from Tanacetum parthenium and Cichorium intybus. KLS), but the final step of how co-oniolide is catalyzed to form dehydroleucodine has not been resolved, and the in vitro reconstruction of the dehydroleucodine synthesis pathway has not been achieved, limiting the wide application of dehydroleucodine. According to the dehydroleucodine synthesis pathway, it is speculated that cytochrome P450 is the main enzyme involved in the modification of the dehydroleucodine skeleton structure.

[0003] In recent years, with the development of science and technology, the analysis of the biosynthetic pathway of natural products has ushered in new development opportunities. The comprehensive use of multiple candidate gene screening methods such as co-expression analysis, phylogenetic analysis, activity tracking, and gene cluster analysis is conducive to the efficient and accurate acquisition of candidate genes. This study took the lettuce (Lactuca sativa), a plant of the genus Lactuca in the Asteraceae family rich in dehydroleucodine, as the research object, and functionally identified the cytochrome P450 monooxygenase CYP71A6 of a lettuce. This enzyme can add a carbonyl group to the 2-position of kauniolide to generate dehydroleucodine. According to the gene function, it was named LsDHLS (Dehydroleucodine synthase). This gene is helpful to produce dehydroleucodine using metabolic engineering technology and realize the full application of dehydroleucodine and its derivatives. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide an application of cytochrome P450 monooxygenase LsDHLS in catalyzing kauniolide to generate dehydroleucodine; the second object of the present invention is to provide a method for catalyzing kauniolide to generate dehydroleucodine.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] 1. Application of cytochrome P450 monooxygenase LsDHLS in catalyzing kauniolide to generate dehydroleucodine, wherein the amino acid sequence of the cytochrome P450 monooxygenase LsDHLS is shown in SEQ ID NO.2.

[0007] 2. A method for catalyzing kauniolide to generate dehydroleucodine, wherein cytochrome P450 monooxygenase LsDHLS is reacted with kauniolide to obtain dehydroleucodine.

[0008] Preferably, the reaction is carried out in vivo or in vitro.

[0009] Preferably in the present invention, the organism is a plant or a microorganism.

[0010] Preferably in the present invention, the plant is tobacco.

[0011] The beneficial effect of the present invention is that: the present invention cloned a cytochrome P450 monooxygenase CYP71A6 that can catalyze kauniolide to generate Dehydroleucodine from lettuce for the first time, and named it LsDHLS. The gene has a full length of 1527bp and encodes 508 amino acids. Further, pEAQ-HT was used as a tobacco transient expression vector, and Agrobacterium GV3101 was used as a host cell to achieve heterologous expression of the protease, and its catalytic product was separated and identified to show that the LsDHLS protease can add a carbonyl group to the 2-position of kauniolide. That is, the present invention provides a cytochrome P450 monooxygenase with a new catalytic function, and verifies its function of carbonylation modification of kauniolide, which provides a new idea for the biosynthesis of Dehydroleucodine, provides more resources for the application of P450 monooxygenase in synthetic biology, promotes the biosynthetic production of Dehydroleucodine as a drug, is conducive to the large-scale production of Dehydroleucodine, and will make new contributions to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0013] Figure 1 The LC-MS analysis results (chromatogram) of the formation of dehydroleucodine catalyzed by LsDHLS and the standard.

[0014] Figure 2 The results are the mass spectrometry analysis of the formation of dehydroleucodine catalyzed by LsDHLS and the standard.

[0015] Figure 3 The product and standard mass spectrum of Dehydroleucodine-glutathione. DETAILED DESCRIPTION

[0016] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0017] Example 1: Cloning of the lettuce LsDHLS gene

[0018] (1) Extraction of total RNA from lettuce genome

[0019] The total RNA of lettuce genome was extracted using the RNA kit of BIOFIT. The specific steps are as follows: 100 mg of lettuce leaves were quickly ground into powder in liquid nitrogen, 1 mL of solution A was added, vortexed and vigorous shaking was performed to mix, centrifuged briefly for 5-10 seconds, 300 μL of solution B and 200 μL of dichloromethane were added, and the mixture was shaken on a shaker for 30 seconds to emulsify it, centrifuged at 12,000 g for 5 minutes, 800 μL of the supernatant in the tube was carefully pipetted into an RNase-free centrifuge tube, an equal volume of solution C was added, mixed, the solution was transferred to the adsorption column twice, centrifuged at 12,000 g for 1 minute, and the waste liquid was discarded. 700 μL of solution D was added to the adsorption column, centrifuged at 12,000 g for 1 minute, and the waste liquid was discarded. Place the adsorption column back into the collection tube, add 500 μL of rinse solution to the adsorption column, centrifuge at 12,000 g for 1 min, discard the waste solution, place the adsorption column back into the collection tube, repeat once, centrifuge at 12,000 g for 1 min, discard the waste solution, place the adsorption column back into the collection tube, centrifuge at 12,000 g for 2 min, place the adsorption column into a new RNase-free centrifuge tube, and let stand for 10 min. Add 60 μL of RNase-free dd H 2 O, stand at room temperature for 2 minutes, centrifuge at 12000g for 2 minutes to obtain RNA solution. Take 10 μL and put it in an RNase-free tube for gel running and concentration measurement; the remaining 50 μL is placed in an RNase-free centrifuge tube and stored at -80℃.

[0020] (2) Cloning of the LsDHLS gene from lettuce

[0021] The obtained lettuce genomic total RNA was reverse transcribed by reverse transcriptase to obtain the first-strand cDNA, and the upstream primer and downstream primer of the complete coding frame were designed and synthesized according to the DNA sequence disclosed in LsDHLSGenbank. The nucleotide sequence is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2, which is as follows:

[0022] Upstream primer LsDHLS-F: 5′-ATGCCGGAAGGAACAATGTTTC-3′ (SEQ ID NO. 3);

[0023] Downstream primer LsDHLS-R: 5′-TTAGGAAGAGATCGGTTTTGGC-3′ (SEQ ID NO .4);

[0024] The first-strand cDNA obtained by inversion was used as a template, and the upstream primer shown in SEQ ID NO.3 and the downstream primer shown in SEQ ID NO.4 were used as a primer pair. After PCR amplification, it was sequenced and verified by Sangon Biotechnology (Shanghai) Co., Ltd.

[0025] Example 2: Construction of a recombinant plant expression vector and engineered bacteria containing the LsDHLS gene

[0026] The LsDHLS gene was amplified again using primer pairs pEAQ-LsDHLS-F and pEAQ-LsDHLS-R to obtain the LsDHLS gene with homologous arms of the PEAQ-HT vector. pEAQ-HT was digested with Age I and Xhol I and constructed into the plant expression vector pEAQ-HT by homologous recombination.

[0027] The specific sequences of pEAQ-LsDHLS-F and pEAQ-LsDHLS-R are as follows:

[0028] pEAQ-LsDHLS-F: 5'-tattctgcccaaattcgcgaccggtATGCCGGAAGGAACAATG-3' (SEQ ID NO.5);

[0029] pEAQ-LsDHLS-R: 5'-atgaaaccagagttaaaggcctcgagTTAGGAAGAGATCGGTTTTG-3' (SEQ ID NO.6);

[0030] The E. coli DH5α was connected and transformed, positive clones were screened, and then the plasmid was extracted for PCR detection and sequencing, and the correctness of the gene was confirmed by sequencing by Sangon Biotech (Shanghai) Co., Ltd. A recombinant plant expression vector containing the LsDHLS gene was obtained and named pEAQ-HT-LsDHLS vector.

[0031] The pEAQ-HT-LsDHLS vector was transformed into Agrobacterium tumefaciens GV3101, and positive clones were screened to obtain an engineered bacterium containing the pEAQ-HT-LsDHLS vector, which was named GV3101-pEAQ-HT-LsDHLS.

[0032] Example 3: Protein expression and function verification in Nicotiana benthamiana

[0033] The GV3101-pEAQ-HT-LsDHLS engineered bacteria and the control strain GV3101-pEAQ-HT were inoculated into 50 mL of LB liquid medium containing Rif (40 mg / L) + Gent (50 mg / L) + Kan (50 mg / L) and cultured at 28°C with shaking at 200 r / min for 24-48 h until OD600 was about 1.0. The bacteria were collected by centrifugation at 5000 g for 10 min and the culture medium was discarded; 15 mL of ddH 2O resuspended, 4 ℃, 5000g centrifuged for 10min, discarded the supernatant, repeated 2 times, added resuspended solution to adjust OD600 to about 1.0, placed in dark for 2-3h, injected into 4-week-old tobacco leaves, covered with black plastic bags for 24h after injection, uncovered, and injected 50μmol / L Kauniolide as substrate into the tobacco leaves injected with bacterial solution again on the 5th day, and collected samples on the 6th day. Weigh 300mg of fresh sample and freeze it with liquid nitrogen, grind it into powder, add 1mL of acidified methanol extract (volume ratio 100:0.1, methanol:formic acid), ultrasonically disrupt for 15min, let stand for 30min, centrifuge at 12000g for 10min, and take 500μL of supernatant for detection.

[0034] Example 4: Detection of Dehydroleucodine Production in Tobacco Using HPLC-MS

[0035] (1) HPLC-MS detection conditions and preparation of standard solutions

[0036] Determination of Sesquiterpenes Using AB Sciex TripleTOF 5600+ Liquid Chromatography Tandem Mass Spectrometry

[0037] Chromatographic column: Acquity UPLC HSST3 column (100 mm2.1 mm, id, 1.7 μm; Waters);

[0038] Mobile phase: 0.1% formic acid in ultrapure water (A) and 0.1% formic acid in acetonitrile (B);

[0039] Flow rate: 0.4 mL / min;

[0040] Column temperature: 40°C;

[0041] Injection volume: 4 μL;

[0042] The gradient elution program is shown in Table 1:

[0043] Table 1. Gradient elution program

[0044] Time(min) %A %B 1 85 15 6 5 95 8 5 95 10 85 15

[0045] (2) Configuration of standard products

[0046] Weigh Kauniolide and Dehydroleucodine (Chengdu Zhibiao Huachun Biotechnology Co., Ltd.) respectively and prepare them into a concentration of 1 mg mL -1 The mother solution was diluted to 1 μg mL with methanol -1 Sample loading and testing. Test results are as follows Figures 1 to 3The results of enzyme activity detection in tobacco showed that LsDHLS could convert Kauniolide into Dehydroleucodine.

[0047] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Application of cytochrome P450 monooxygenase LsDHLS in catalyzing kauniolide to generate dehydroleucodine, characterized in that: The amino acid sequence of the cytochrome P450 monooxygenase LsDHLS is shown in SEQ ID NO.

2.

2. A method for catalyzing kauniolide to produce dehydroleucodine, characterized in that: Cytochrome P450 monooxygenase LsDHLS is reacted with Kauniolide to obtain Dehydroleucodine; the amino acid sequence of the cytochrome P450 monooxygenase LsDHLS is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that: The reaction is carried out in plants or microorganisms.

4. The method according to claim 3, characterized in that: The reaction is carried out in vitro.

5. The method according to claim 4, characterized in that: The plant is tobacco.

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