Protein of targeted peroxisome and application thereof

Abstract

The invention discloses a protein of targeted peroxisome, which comprises a protein obtained by fusion expression of farnesyl pyrophosphate synthase and peroxisome I type targeted positioning signal peptide, and / or a protein obtained by fusion expression of patchouli alcohol synthase and peroxisome I type targeted positioning signal peptide. According to the invention, terpenoid synthesis based on peroxisome regionalization is realized in a plant host, and the method can be used for heterologous synthesis of patchouli alcohol in nicotiana benthamiana.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a protein targeting peroxisome and application thereof. Background Art

[0002] Terpenoids are the most diverse natural compounds in nature. Currently, more than 80,000 terpenoids with different structures have been identified. They are widely used in the production industry because of their rich biological activities. Terpenoids are classified according to the number of isoprene unit structures they have. Among them, sesquiterpenes are a type of terpenoid compound with three isoprene units. Many sesquiterpenes are known to have unique aromas and are used to increase the natural flavor of products. Some sesquiterpenes also have multiple pharmacological activities such as anti-inflammatory, antibacterial, anti-tumor, and antioxidant.

[0003] Patchouli alcohol is one of the most widely used sesquiterpenes. It is the main component of patchouli essential oil and has a variety of beneficial activities, including anti-influenza virus, anti-inflammatory, antioxidant, anti-ulcer, anti-colitis, anti-mucositis, and protective effects on brain and lung damage. The biosynthesis steps of patchouli alcohol mainly include: the synthesis of terpene precursor isoprene units, the generation of farnesyl diphosphate (FPP) from isoprene units under the action of farnesyl diphosphate synthase (FPPS), and the production of patchouli alcohol by FPP under the action of patchouli alcohol synthase (PAS), where FPPS and PAS are both located in the cytoplasm.

[0004] In recent years, the research on the synthesis of terpenes by metabolic engineering has shown a significant growth trend, and a large number of terpenoid bioreactors have been successfully constructed. Among them, plant hosts, especially economic crop bioreactors, have the characteristics of high biomass, easy batch planting and management, and environmental friendliness. The use of metabolic engineering to construct plant terpenoid reactors has made great progress in the heterologous synthesis of sesquiterpenes such as nerolidol, patchouli alcohol, and artemisinin.

[0005] Previous studies on plant metabolic engineering mainly used chloroplasts and mitochondria in plant cells as organelles for the synthesis of target metabolites. However, chloroplasts and mitochondria in leaves are important organelles for energy synthesis. Introducing heterologous metabolic pathways for metabolic engineering may burden plant growth. The development of new organelle metabolic engineering in plants is of great significance. Peroxisomes are a type of multifunctional single-layer membrane organelles that are widely distributed in eukaryotic organisms. They show good performance in the construction of microbial terpene reactors, but there are currently no studies reporting the use of plant peroxisomes for heterologous synthesis of terpenes. Summary of the invention

[0006] The purpose of the present invention is to provide a protein targeting peroxisome and its application, so as to promote and realize the terpene synthesis in plant peroxisome in Nicotiana benthamiana host. The above purpose is achieved through the implementation of the following technical solutions:

[0007] A peroxisome-targeted protein, comprising: a protein obtained by fusion expression of farnesyl pyrophosphate synthase and a peroxisome type I targeting signal peptide, and / or

[0008] The protein obtained by fusion expression of patchouli alcohol synthase and peroxisome type I targeting signal peptide;

[0009] The amino acid sequence of the peroxisome type I targeting signal peptide is shown in SEQ ID No.1.

[0010] Optionally, the amino acid sequence of the protein obtained by fusion expression of the farnesyl pyrophosphate synthase and the peroxisome type I targeting signal peptide is as shown in SEQ ID No. 2;

[0011] The amino acid sequence of the protein obtained by fusion expression of the patchouli alcohol synthase and the peroxisome type I targeting signal peptide is shown in SEQ ID No.3.

[0012] A nucleic acid encoding the peroxisome-targeted protein according to claim 1 or 2.

[0013] Optionally, the nucleotide sequence of the nucleic acid is shown as SEQ ID No.4 or SEQ ID No.5.

[0014] A vector comprising the above-mentioned nucleic acid.

[0015] A host cell, comprising the vector according to claim 1.

[0016] Optionally, the host cell comprises Agrobacterium.

[0017] A kit comprises the above-mentioned nucleic acid or the above-mentioned vector.

[0018] Application of the above peroxisome-targeted protein, nucleic acid, vector, host cell and kit in synthesizing the sesquiterpene patchouli alcohol by peroxisome.

[0019] A method for synthesizing the sesquiterpene patchouli alcohol by using peroxisomes comprises the following steps:

[0020] The host cells are used to perform Agrobacterium-mediated transient transformation of Nicotiana benthamiana leaves, so that a protein obtained by fusion expression of farnesyl pyrophosphate synthase and peroxisome type I targeting signal peptide and a protein obtained by fusion expression of patchouli alcohol synthase and peroxisome type I targeting signal peptide are formed in the cells of the Nicotiana benthamiana plants.

[0021] The present invention has the following beneficial effects:

[0022] The present invention provides a method and application for synthesizing sesquiterpenoid patchouli alcohol by using peroxisomes of Nicotiana benthamiana, and belongs to the field of plant metabolic engineering. The inventors have found that the fusion expression of farnesyl pyrophosphate synthase (AtFPPS2) and patchouli alcohol synthase (PcPAS) with peroxisomal type I localization signal peptide PTS1 (RSKL) can effectively change the subcellular localization of these two proteins in heterologous expression in Nicotiana benthamiana, from the original cytoplasmic localization to peroxisomal localization. At the same time, by Agrobacterium-mediated transient transformation technology, AtFPPS-RSKL and PcPAS-RSKL proteins are co-expressed in Nicotiana benthamiana leaves, and sesquiterpenoid patchouli alcohol can be produced in the leaves. The present invention realizes the synthesis of terpenoid compounds based on peroxisome compartmentalization in plant hosts, and can be used for heterologous synthesis of patchouli alcohol in Nicotiana benthamiana. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a map of the subcellular localization of fluorescent proteins. Figure 1 A in the middle is the test result of tobacco samples transferred into Mvenus-AtFPPS2. Figure 1 B in the figure is the test result of tobacco samples transferred into Mvenus-PcPAS. Figure 1 C in the middle is the test result of tobacco sample transferred with Mvenus-AtFPPS2-RSKL. Figure 1 D in the middle is the test result of the tobacco sample transferred into Mvenus-PcPAS-RSKL.

[0025] Figure 2 It is a standard curve graph of peak area and concentration.

[0026] Figure 3 This is the patchouli alcohol detection result of Example 2. DETAILED DESCRIPTION

[0027] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0028] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] The present invention provides a peroxisome-targeted protein, comprising: a protein obtained by fusion expression of farnesyl pyrophosphate synthase and a peroxisome type I targeting signal peptide, and / or a protein obtained by fusion expression of patchouli alcohol synthase and a peroxisome type I targeting signal peptide; the amino acid sequence of the peroxisome type I targeting signal peptide is shown in SEQ ID No. 1.

[0032] Farnesyl pyrophosphate synthase is a key enzyme in the mevalonate pathway, catalyzing the sequential condensation of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) to generate geranyl pyrophosphate (GPP) and farnesyl pyrophosphate (FPP). FPP is not only an important substrate for the synthesis of cholesterol and coenzyme Q, but also participates in the farnesylation modification of many small G proteins.

[0033] Patchouli alcohol synthase is the key enzyme for the synthesis of patchouli alcohol in patchouli, which can catalyze the precursor of sesquiterpenes, farnesyl diphosphate (FDP), to produce sesquiterpenes such as patchouli alcohol.

[0034] The peroxisomal type I targeting signal peptide used in the present invention has an amino acid sequence of RSKL. Fusion expression of the peroxisomal type I targeting signal peptide with the above-mentioned farnesyl pyrophosphate synthase and patchouli alcohol synthase can effectively change the subcellular localization of farnesyl pyrophosphate synthase and patchouli alcohol synthase in heterologous expression, from the original cytoplasmic localization to peroxisomal localization. Thus, the synthesis of terpenoid compounds based on peroxisomal compartmentalization can be achieved.

[0035] Example 1

[0036] In this example, primers were designed based on the CDSs sequences of Arabidopsis thaliana farnesyl pyrophosphate synthase AtFPPS2 (At4g17190) (amino acid sequence as shown in SEQ ID No.6, and the corresponding nucleotide sequence as shown in SEQ ID No.7) and Pogostemon cablin patchouli alcohol synthase PcPAS (AY508730) (amino acid sequence as shown in SEQ ID No.10, and the corresponding nucleotide sequence as shown in SEQ ID No.11) in published articles, and the full-length CDS of AtFPPS2 and PcPAS were cloned using the mixed cDNA pool of Arabidopsis and Pogostemon cablin leaves as templates. During amplification, a known peroxisome type I targeting signal peptide PTS1 (amino acid sequence SEQ ID No.1: RSKL) was introduced into the primer region, and the corresponding nucleotide sequence was 5'-AGGAGCAAGCTC-3'. PTS1 was seamlessly fused to the carboxyl termini of AtFPPS2 and PcPAS proteins to obtain the modified peroxisome targeting proteins AtFPPS2-RSKL (as shown in SEQ ID No.2) and PcPAS-RSKL (as shown in SEQ ID No.3).

[0037] The present invention prepares the above-mentioned proteins AtFPPS2-RSKL and PcPAS-RSKL in the following manner.

[0038] The pEarlyGate 100 vector was linearized by double enzyme digestion with XhoI and XbaI, and DNA encoding fragments of Mvenus, PcPAS, PcPAS-RSKL (as shown in SEQ ID No.4), AtFPPS2, and AtFPPS-RSKL (as shown in SEQ ID No.5) were amplified respectively. According to the instructions of the purchased kit, a multi-fragment homologous recombination kit (ClonExpress MultiS One Step Cloning Kit) was used to connect the four fusion protein coding sequences containing the mVenus fluorescent tag, Mvenus-PcPAS (as shown in SEQ ID No.12), Mvenus-PcPAS-RSKL (as shown in SEQ ID No.13), Mvenus-AtFPPS2 (as shown in SEQ ID No.8), and Mvenus-AtFPPS2-RSKL (as shown in SEQ ID No.9), to the pEarlyGate 100 vector, respectively, to obtain the pEarlyGate 100-Mvenus-AtFPPS2-RSKL, PEarlyGate100-Mvenus-PcPAS-RSKL, PEarlyGate 100-Mvenus-AtFPPS2, PEarlyGate100-Mvenus-PcPAS binary expression vectors.

[0039] The above vectors were respectively transferred into Agrobacterium GV3101, and 3-week-old Nicotiana benthamiana plants were taken for Agrobacterium-mediated transient transformation of leaves. The operation method is as follows:

[0040] According to the instructions of the purchased competent medium, the above vector was transferred into the competent medium of Agrobacterium GV3101 (Weidi Biotechnology, AC1001L) by chemical transfection method. The transformed Agrobacterium was resuspended in SOC medium, then revived in a shaking incubator at 28°C for 2 hours, and spread on LB plates containing rifampicin (50 mg L-1) and kanamycin (50 mg L-1). After culturing at 28°C for 2 days, a single clone was picked and transferred to LB liquid medium containing the corresponding resistance, and cultured in a shaking incubator at 28°C overnight. The bacterial liquid was enriched by centrifugation at 8000g for 2 minutes, the supernatant was discarded, and the bacteria were resuspended in the transformation liquid (10mM MES, 10mM MgCl2, 200μM acetosyringone) and allowed to stand for 90 minutes. The transformation liquid OD 600nmAdjust to 1, discard the supernatant after centrifugal enrichment, add an equal amount of transformation solution to resuspend the bacteria, and use a 1 mL syringe to inject the bacterial solution into the back of 3-week-old pre-treated (refer to the tobacco peroxisomes marked with fluorescent protein moxcerulean3, the marking method and fluorescence detection verification method can refer to the paper Comprehensive identification of plant peroxisome targeting signal type 1 tripeptides) tobacco leaves, and routinely culture the Nicotiana benthamiana plants containing the leaves for 48-72 hours, and then use a laser confocal microscope for fluorescence detection.

[0041] like Figure 1 As shown: The green signal in the figure is a fusion protein with an mVenus fluorescent tag, the red signal indicates peroxisome, and the yellow indicates that the two signals overlap. Mvenus-AtFPPS2 and Mvenus-PcPAS did not show peroxisome localization, while Mvenus-AtFPPS2-RSKL and Mvenus-PcPAS-RSKL showed peroxisome localization. It was confirmed that AtFPPS2 and PcPAS, which were originally localized in the tobacco cytoplasm, were successfully targeted to peroxisomes ( Figure 1 ).

[0042] Example 2

[0043] The detection and quantification system of patchouli alcohol was established. Agilent 7890A GC was used, with the chromatographic column model HP-5MS and the mass spectrometer detector 5977A. The specific steps of the column oven heating program were as follows: the initial temperature was maintained at 45 for 2 minutes, and then the temperature was increased by 10°C per minute to 250°C, and the temperature was kept for 5 minutes. The ion source temperature of the mass spectrometer and the chromatographic end interface temperature was 280°C, the tuning parameters were called E.Tune, and the mass spectrometer detector used the scanning mode for qualitative detection, recording fragments of 35-400 m / z. When establishing the quantitative detection system, n-tetradecane, which has a similar structure to sesquiterpenes, was used as the internal standard, and standards of different concentrations were prepared. The concentration gradient of patchouli alcohol was 0.83 ng / μL, 1 ng / μL, 1.25 ng / μL, 1.67 ng / μL, and 2.5 ng / μL, and the concentration gradient of n-tetradecane was 0.639 ng / μL, 0.767 ng / μL, 0.959 ng / μL, 1.278 ng / μL, and 1.917 ng / μL. The peak area and concentration standard curves were drawn ( Figure 2 ).

[0044] The PEarlyGate 100 vector was linearized by double enzyme digestion with XhoI and XbaI, and the binary expression vectors PEarlyGate 100-AtFPPS2-RSKL and PEarlyGate 100-PcPAS-RSKL were constructed by the method of Example 1 using a single fragment homologous recombination kit (Novozyme, ClonExpress IIOne Step Cloning Kit). Agrobacterium GV3101 was introduced, and 3-week-old Nicotiana benthamiana plants were taken, one part as the experimental group and the other part as the control group. The experimental group was subjected to Agrobacterium-mediated transient transformation of leaves (implemented according to Example 1), and volatiles from tobacco leaves were extracted three days later. Weigh 0.2-0.3g of tobacco leaves (remove the stem veins), cut them into pieces and put them into a 5ml glass sample bottle, and record the sample weight at the same time. 3.5ml of methyl tert-butyl ether (containing 954ng / sample of n-tetradecane as an internal standard) was added to each glass sample bottle, and the leaf fragments were incubated in the extract for 3h for full extraction. The extracted solution was filtered with a 0.22 μM organic filter membrane, and the obtained filtrate was concentrated to about 150 μL by nitrogen blowing. After centrifugation at 13000 rpm for 10 min, the upper liquid was collected for subsequent GC-MS analysis. The results showed that the target compound patchouli alcohol ( Figure 3 ), while patchouli alcohol was not detected in the control group.

[0045] The patchouli alcohol detection and quantification system was used to quantitatively measure the transformed tobacco leaf samples, and the results showed that the patchouli alcohol content in the samples reached 0.95±0.36 μg / g leaf. The experiment proved that the present invention realizes the synthesis of terpenoid compounds based on peroxisome compartmentalization in plant hosts, and can be used for heterologous synthesis of patchouli alcohol in Nicotiana benthamiana.

[0046] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A protein targeting peroxisome, characterized in that include: A protein obtained by fusion expression of farnesyl pyrophosphate synthase with a peroxisome type I targeting signal peptide, and / or The protein obtained by fusion expression of patchouli alcohol synthase and peroxisome type I targeting signal peptide; The amino acid sequence of the peroxisome type I targeting signal peptide is shown in SEQ ID No.

1.

2. The peroxisome-targeting protein according to claim 1, characterized in that The amino acid sequence of the protein obtained by fusion expression of the farnesyl pyrophosphate synthase and the peroxisome type I targeting signal peptide is shown in SEQ ID No. 2; The amino acid sequence of the protein obtained by fusion expression of the patchouli alcohol synthase and the peroxisome type I targeting signal peptide is shown in SEQ ID No.

3.

3. A nucleic acid encoding the peroxisome-targeted protein according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleotide sequence of the nucleic acid is shown in SEQ ID No.4 or SEQ ID No.

5.

5. A carrier, characterized in that The vector comprises the nucleic acid according to claim 3 or 4.

6. A host cell, characterized in that The host cell comprises the vector of claim 5.

7. The host cell according to claim 6, characterized in that The host cell includes Agrobacterium.

8. A kit, characterized in that: Comprising the nucleic acid of claim 3 or 4 or the vector of claim 5.

9. Use of the peroxisome-targeting protein according to claim 1 or 2, the nucleic acid according to claim 3 or 4, the vector according to claim 5, the host cell according to claim 6, and the kit according to claim 8 in synthesizing the sesquiterpene patchouli alcohol using peroxisomes.

10. A method for synthesizing the sesquiterpene patchouli alcohol using peroxisomes, characterized in that: The steps include: The host cell of claim 6 is used to perform Agrobacterium-mediated transient transformation of leaves of Nicotiana benthamiana plants, so that a protein obtained by fusion expression of farnesyl pyrophosphate synthase and peroxisome type I targeting signal peptide and a protein obtained by fusion expression of patchouli alcohol synthase and peroxisome type I targeting signal peptide are formed in the cells of the Nicotiana benthamiana plants.

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