Application of euphorbia lathyris acyltransferase and coding gene thereof in preparation of euphorbia lathyris alkane diterpenoid ester

Abstract

The invention provides a euphorbia lathyris acyltransferase gene ElBAHD16 and application of a coding protein thereof. The gene is obtained by cloning from euphorbia lathyris for the first time. The invention also provides a recombinant vector and an engineering bacterium containing the gene. Cinnamoyl coenzyme A, benzoyl coenzyme A or acetyl coenzyme A can be used as an acyl donor, and 7-hydroxy euphorbia lathyris diterpenoid alcohol is used as a substrate to generate a plurality of types of euphorbia lathyris diterpenoid esters through biological catalysis. The content of diterpenoid ester substances in plants such as euphorbia lathyris can be increased through a genetic engineering technology, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of caper acyltransferase in the biosynthesis of caperane diterpene esters, and belongs to the field of medicinal plant genetic engineering. Background Art

[0002] Euphorbia lathylris L. is a biennial herbaceous plant of the Euphorbiaceae family. Its mature seeds are the traditional Chinese medicine Qianjinzi. The 2020 edition of the Chinese Pharmacopoeia records that Qianjinzi has the function of purging water, removing blood and eliminating symptoms; it can also be used externally to treat tinea and warts. [1] Modern pharmacological studies have shown that the macrocyclic diterpenoid components in Caper chinensis have multiple biological activities such as anti-inflammatory, anti-tumor, anti-tumor multidrug resistance and antioxidant [2] . Studies have found that the terpenoid lipid component of Euphorbia serrata has the activity of reversing tumor multidrug resistance. Due to the trace distribution of terpenoid esters in Euphorbia plants, the yield of terpenoid lipid compounds extracted and separated from Euphorbia serrata is low, and the direct extraction cost is high. The chemical synthesis of this component is difficult and the yield is lower than [3] Although the production of 7β-acetoxycaprylonitrile alcohol by microbial biotransformation has been reported, the specific enzyme gene responsible for acylation is unknown. [4] . It is of great importance to analyze the key enzymes in the biosynthesis of caperane diterpenoid esters in plants and to increase the yield of caperane diterpenoid esters by using biosynthesis methods. The cloning of the caperane acyltransferase (ElBAHD16) gene provides valuable gene resources for increasing the content of active ingredients in caperane diterpenoid esters by genetic engineering, and has good application prospects in the quality improvement of the medicinal plant caperane and the biosynthesis of active ingredients. Prior to the present invention, the caperane acyltransferase gene and its amino acid sequence mentioned in this patent application have not been publicly reported. Summary of the invention

[0003] The present invention provides a caper acyltransferase gene and its coding gene, and also provides a recombinant vector and a host cell (engineered strain) containing the gene. Another object of the present invention is to utilize the constructed Escherichia coli engineered strain to biocatalyze the synthesis of caperane diterpene esters.

[0004] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In the above-mentioned engineered bacteria, the method for overexpressing the capsid acyltransferase gene ElBAHD16 is to insert the gene sequence into an expression vector and then express it.

[0005] The nucleotide sequence of ElBAHD16 is shown in SEQ ID NO.1.

[0006] The protein encoded by the gene is the amino acid sequence shown in SEQ ID NO.2.

[0007] The gene fragment is introduced through a recombinant expression vector, and the expression vector specifically inserts the ElBAHD16 gene fragment between the EcoR I / Sal I restriction sites of pMAL-c4x to construct a pMAL-ElBAHD16 recombinant expression plasmid.

[0008] The engineering bacteria described above are Escherichia coli BL21 (DE3) containing the recombinant expression plasmid, and the name of the engineering bacteria is BL21-pMAL-ElBAHD16. The method for preparing arganyl diterpene esters catalyzed by the engineered bacteria of the present invention has the following route: Figure 1 As shown, the BL21-pMAL-ElBAHD16 engineered strain was used to catalyze 7-hydroxylathyrol to produce lathyrol esters.

[0009] The acyltransferase ElBAHD16 gene provided by the present invention is cloned and prepared from Caper chinensis for the first time. In vitro experiments show that ElBAHD16 has the activity of catalyzing the acetylation, cinnamylation and benzoylation of the 7-hydroxycaperane diterpene alcohol hydroxyl group. The present invention can be used to increase the content of diterpenoid ester substances in plants such as Caper chinensis through genetic engineering technology. BRIEF DESCRIPTION OF THE DRAWINGS Description of the drawings: 1. Figure 1 Schematic diagram of biocatalysis of 7-hydroxylathyrol by ElBAHD16 and the in vitro catalytic results. A, Schematic diagram of biocatalysis of 7-hydroxylathyrol by ElBAHD16. LC-MS analysis of the results of ElBAHD16 catalyzing 7-hydroxylathyrol to generate 7-O-acetyllathyrol (7-O-acetyllathyrol, B), 7-O-cinnamoyllathyrol (7-O-cinnamoyllathyrol, C) and 7-O-benzoyllathyrol (7-O-benzoyllathyrol, D). The acyl donors are acetyl-CoA, cinnamoyl-CoA and benzoyl-CoA.

[0011] Example 1 Isolation and prokaryotic expression of the ElBAHD16 gene 1. Construction of pMAL-ElBAHD16 plasmid (I) Using the cDNA of the sucrose as a template, using the polymerase DNA polymerase Phanta, and using P1 and P2 as primers, PCR amplification was performed. The PCR program was: 95°C pre-denaturation for 1 min, 95°C denaturation for 15 seconds, 60°C annealing for 15 seconds, 72°C extension for 90 seconds, 32 cycles, and a 1341 bp ElBAHD16 gene fragment was obtained. The nucleotide sequence of the fragment is shown in SEQ ID No. 1. P1: 5'-agggaaggatttcagaattcATGGGGATTGAAATAGAGTTCATTG-3' P2: 5'-aagcttgcctgcaggtcgacTCATAACCTTGATTGTTTGATCAAGTT' Vazyme Clon Express Ultra One Step Cloning Kit was used to construct plasmids for homologous recombination. Empty plasmid pMAL-c4 was used after double restriction digestion (EcoR I and Sal I). Single fragment homologous recombination reaction: optimal cloning vector addition amount = (0.02 × cloning vector base number) ng; optimal insert fragment addition amount = (0.04 × cloning vector base number) ng. (2) Calculate the required amount of DNA according to the formula in (1), mix the vector and target fragment, add ddH2O to 5 μL, add 5 μL 2×Clon Express Mix, and mix by gently pipetting. Centrifuge at 3000 rpm for 30 seconds. (3) Place in PCR instrument, 50°C, 5 min; take out and immediately place on ice to cool for 2 min. The recombinant solution was introduced into Escherichia coli DH5α, and positive transformants were screened on ampicillin resistance plates. After sequencing verification, the recombinant plasmid was obtained and named pMAL-ElBAHD16. 2. Transformation of plasmid into E. coli BL21(DE3) The competent cells were melted in an ice bath, and 50 μL competent cells and 5 μL plasmid were added gently and quickly, flicked with fingers, ice bathed for 30 minutes, heat-shocked at 42°C for 45 seconds, ice bathed for 2 minutes, and 500 μL LB medium was added. The mixture was shaken at 37°C and 220 rpm for 45 minutes. Centrifuged at 5000 rpm for 3 minutes, 300 μL of supernatant was removed, and the remaining 200 μL was resuspended and coated on a plate. After 30 minutes of overnight culture, 10 single colonies were picked and transferred to LB liquid culture medium containing ampicillin (pMAL-ElBAHD16). The positive colonies were verified by colony PCR, and a correct colony was picked for use.

[0012] Example 2 Verification of ElBAHD16 Activity Bacterial strain: recombinant engineered Escherichia coli BL21-pMAL-ElBAHD16; Fermentation medium (g / L): peptone 10; yeast extract 5; NaCl 10; ampicillin 100 mg / mL. Seed culture: transfer the glycerol-preserved strains to fresh LB liquid medium and activate them at 37°C, 200 rpm for 16 h; streak one loop of activated strains onto LB solid medium with corresponding antibiotics and culture at 37°C for 12 h; pick a single colony from the fresh plate into a 250 mL Erlenmeyer flask containing 50 mL of seed medium and culture it at 37°C, 200 rpm for 12 h as the primary seed; then transfer the primary seed liquid to another 50 mL of seed medium at a 1% inoculum and culture it at 37°C, 200 rpm for 12 h as the secondary seed. Fermentation culture and enzyme induction expression: The secondary seed solution was inoculated into 50 mL fermentation expression medium at 1% inoculation volume, and cultured in a shaking incubator at 37°C and 220 rpm. After 3 hours, the inducer IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16°C and 200 rpm. The expression was induced for 16 hours, and the bacteria were harvested and broken to obtain crude protein. Enzyme activity reaction The activity of acyltransferase ElBAHD16 was determined by acylation of the substrate 7-hydroxylathyrol to synthesize the corresponding ester product. The reaction was carried out in a 1.5 mL centrifuge tube. The reaction system contained: 10 mM acyl CoA donor, 100 μM substrate 7-Hydroxylathyrol, 50 mM Tri-HCl (pH 7.5), ElBAHD16 crude enzyme, and the total volume of the reaction system was 100 μL. The reaction was carried out at 30 ° C for 6 h, and the reaction mixture was ultrasonically extracted with an equal volume of methanol, centrifuged at 12000 rpm for 10 min, and the supernatant was taken and analyzed by LC-MS to detect the reaction product. The transformation products were analyzed by LC-MS, and the analysis method was as follows: Column: Agilent Poroshell 120 Ec-C 18 column (2.1mm×150mm, 2.7μm); mobile phase: water (A, containing 0.1% formic acid); methanol (B); gradient elution (B): 30-55% 0-8.0min; 55%, 8.0-12min; 55-100%, 12-22min; 100%, 22-30min; 100-30%, 30-30.1min; 30%, 30.1-40min. Flow rate: 0.5mL·min -1 ; Column temperature 28℃; Injection volume 5μL, Detection wavelength 280nm. References 1. Chinese Pharmacopoeia Committee. Chinese Pharmacopoeia. 2020, 1:36. 2. Liao Peihai, Weng Lianjin, Liu Shulan, Geng Di. Research progress on chemical components and pharmacological activities of Capilla ovata. Modern Chinese Medicine 2020, 22(02): 296-304. 3. L,McKerrall SJ,Kuttruff CA,Ungeheuer F,Felding J,BaranPS.14-step synthesis of(+)-ingenol from(+)-3-carene.Science 2013,341(6148):878-882. 4. Cheng Zhihong, Wu Yiqing, inventors; Conversion method of diterpene alcohol derivatives of Caperna oleifera and use thereof in the preparation of anti-tumor drugs patent CN107164422B 2021-01-22.

Claims

1. Use of a caper acyltransferase and a gene encoding the same in the preparation of a caper alkane diterpene ester, characterized by the nucleotide sequence shown in SEQ ID No.

1.

2. The protein encoded by the gene according to claim 1, characterized by the amino acid sequence listed in SEQ ID No.

2.

3. A recombinant vector containing the ElBAHD16 encoding gene as described in claim 2 or 3.

4. The transgenic engineering strain E1BAHD16 expressing claim 1, characterized in that: The Escherichia coli is E. Coli BL21 (DE3).

5. Use of the ElBAHD16 encoding gene as described in claim 1 or 2 as a molecular marker for caper-assisted breeding.

6. Use of the engineered strain expressing E1BAHD16 as claimed in claim 3 or 4 in the preparation of argan diterpene esters.

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