Application of caper acyltransferase and its encoding gene in the preparation of caperane diterpenoid esters

Abstract

The present invention provides a caper acyltransferase gene ElBAHD16 and uses of its encoded protein. The gene is cloned for the first time from the caper. The present invention also provides a recombinant vector and an engineered bacterium comprising the gene. The present invention can use cinnamoyl acyl-CoA, benzoyl-CoA, or acetyl-CoA as an acyl donor and 7-hydroxycaperane diterpene alcohol as a substrate to biocatalytically generate a variety of caperane diterpene esters. The present invention can also be used to increase the content of diterpenoid ester substances in plants such as the caper through genetic engineering technology, showing good application prospects.

Description

Technical Field

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

[0002] Euphorbia lathylris L. is a biennial herbaceous plant in the Euphorbiaceae family. Its mature seeds are the traditional Chinese medicine, Qianjinzi (Fructus Aurantii). The 2020 edition of the Chinese Pharmacopoeia states that Qianjinzi has the functions of purging water, dispelling blood, and eliminating symptoms. It can also be used externally to treat ringworms and warts. [1] Modern pharmacological studies have shown that the macrocyclic diterpenoid components in Caper seeds have multiple biological activities such as anti-inflammatory, anti-tumor, anti-tumor multidrug resistance and antioxidant [2] The study found that the oleanolic acid diterpenoid lipid component has the activity of reversing tumor multidrug resistance. Due to the trace distribution of oleanolic acid diterpenoid esters in Euphorbia plants, the yield of oleanolic acid diterpenoid lipid compounds extracted and separated from oleanolic acid is low, and the cost of direct extraction is high. The chemical synthesis of this component is difficult and the yield is lower than [3] Although the production of 7β-acetoxycaperol by microbial biotransformation has been reported, the specific enzyme gene responsible for acylation is unknown. [4] . It is crucial to analyze the key enzymes in the biosynthesis of caperane diterpenoid esters in plants and to apply biosynthesis methods to increase the yield of caperane diterpenoid esters. The cloning of the caperane acyltransferase (ElBAHD16) gene provides a valuable gene resource 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, there has been no public report on the caperane acyltransferase gene and its amino acid sequence mentioned in this patent application. Summary of the Invention

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

[0004] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0005] 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.

[0006] The nucleotide sequence of the ElBAHD16 is shown in SEQ ID NO.1.

[0007] The protein encoded by this gene has an amino acid sequence shown in SEQ ID NO.2.

[0008] 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 enzyme sites of pMAL-c4x to construct the pMAL-ElBAHD16 recombinant expression plasmid.

[0009] The engineering bacteria mentioned above are Escherichia coli BL21 (DE3) containing the recombinant expression plasmid, and the engineering bacteria is named BL21-pMAL-ElBAHD16.

[0010] The method for preparing dapoxetane diterpene esters catalyzed by the engineered bacteria of the present invention is as follows: Figure 1 As shown, the BL21-pMAL-ElBAHD16 engineered strain catalyzes 7-hydroxylathyrol to produce lathyrol esters.

[0011] The acyltransferase gene E1BAHD16 provided by the present invention was cloned and prepared for the first time from the caper plant. In vitro experiments demonstrated that E1BAHD16 catalyzes the acetylation, cinnamoylation, and benzoylation of the 7-hydroxycaperane diterpenoid hydroxyl group. This invention can be used to increase the content of diterpenoid esters in plants such as the caper plant through genetic engineering techniques. BRIEF DESCRIPTION OF THE DRAWINGS Description of the drawings:

[0013] 1. Figure 1 Schematic diagram of the biocatalytic production of 7-hydroxylathyrol by ElBAHD16 and in vitro catalytic results. A, Schematic diagram of the biocatalytic production of 7-hydroxylathyrol by ElBAHD16. LC-MS analysis of the catalytic production of 7-O-acetyllathyrol (B), 7-O-cinnamoyllathyrol (C), and 7-O-benzoyllathyrol (D) by ElBAHD16 from 7-hydroxylathyrol. The acyl donors were acetyl-CoA, cinnamoyl-CoA, and benzoyl-CoA.

[0014] Example 1 Isolation and prokaryotic expression of the ElBAHD16 gene

[0015] 1. Construction of pMAL-ElBAHD16 plasmid

[0016] (I) PCR amplification was performed using Phanta DNA polymerase using primers P1 and P2, using the following PCR procedure: 95°C pre-denaturation for 1 min, 95°C denaturation for 15 s, 60°C annealing for 15 s, and 72°C extension for 90 s, for 32 cycles. A 1341 bp ElBAHD16 gene fragment was obtained. The nucleotide sequence of this fragment is shown in SEQ ID No. 1.

[0017] P1: 5'-agggaaggatttcagaattcATGGGGATTGAAATAGAGTTCATTG-3'

[0018] P2: 5'-aagcttgcctgcaggtcgacTCATAACCTTGATTGTTTGATCAAGTT'

[0019] Plasmids were constructed for homologous recombination using the Vazyme Clon Express Ultra One-Step Cloning Kit. Empty plasmid pMAL-c4 was double-digested with EcoRI and SalI before use. For single-fragment homologous recombination reactions: optimal cloning vector addition amount = (0.02 × cloning vector base number) ng; optimal insert addition amount = (0.04 × cloning vector base number) ng.

[0020] (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 of 2× Clon Express Mix, and mix thoroughly by gently pipetting. Centrifuge at 3000 rpm for 30 seconds.

[0021] (3) Place in a PCR instrument, 50°C, 5 min; take out and immediately place on ice to cool for 2 min.

[0022] The recombinant solution was introduced into Escherichia coli DH5α, and positive transformants were screened on ampicillin-resistant plates and verified by sequencing to obtain a recombinant plasmid named pMAL-ElBAHD16.

[0023] 2. Transformation of plasmid into E. coli BL21(DE3)

[0024] Thaw the competent cells in an ice bath, gently and quickly add 50 μL of competent cells and 5 μL of plasmid, flicking gently with a finger. Incubate on ice for 30 minutes, heat shock at 42°C for 45 seconds, and then incubate on ice for 2 minutes. Add 500 μL of LB medium and shake at 37°C, 220 rpm for 45 minutes. Centrifuge at 5000 rpm for 3 minutes, remove 300 μL of the supernatant, resuspend the remaining 200 μL, and spread on a plate. Incubate overnight for 30 minutes, pick 10 single colonies, and transfer to LB liquid medium containing ampicillin (pMAL-ElBAHD16). Verify the positive colonies by colony PCR, and pick a correct colony for use.

[0025] Example 2 Verification of ElBAHD16 Activity

[0026] Bacterial strain: recombinant engineered Escherichia coli BL21-pMAL-ElBAHD16;

[0027] Fermentation medium (g / L): peptone 10; yeast extract 5; NaCl 10; ampicillin 100 mg / mL.

[0028] Seed culture: Transfer the glycerol-preserved strain to fresh LB liquid medium and activate it at 37°C, 200 rpm for 16 h; streak one loop of the activated strain onto LB solid medium with the corresponding antibiotic and incubate at 37°C for 12 h; pick a single colony from the fresh plate and transfer it to a 250 mL Erlenmeyer flask containing 50 mL of seed medium and incubate it at 37°C, 200 rpm for 12 h as the primary seed; then inoculate the primary seed solution into another 50 mL of seed medium at a 1% inoculum size and incubate it at 37°C, 200 rpm for 12 h as the secondary seed.

[0029] Fermentation culture and enzyme induced expression:

[0030] The secondary seed solution was inoculated at 1% inoculum into 50 mL of fermentation expression medium and cultured in a shaker at 37°C and 220 rpm. After 3 hours, IPTG was added to a final concentration of 0.1 mM and expression was induced at 16°C and 200 rpm. Expression was induced for 16 hours, and the cells were harvested and broken to obtain crude protein.

[0031] Enzyme activity reaction

[0032] Acyltransferase activity of ElBAHD16 was determined by acylation of the substrate acetyl-2-diterpene alcohol to the corresponding ester product. The reaction was performed in a 1.5 mL centrifuge tube containing 10 mM acyl-CoA donor, 100 μM 7-Hydroxylathyrol, 50 mM Tris-HCl (pH 7.5), and crude ElBAHD16 enzyme in a total volume of 100 μL. The reaction was maintained at 30°C for 6 h. The reaction mixture was ultrasonically extracted with an equal volume of methanol and centrifuged at 12,000 rpm for 10 min. The supernatant was collected and analyzed by LC-MS for reaction product analysis.

[0033] The conversion products were analyzed by LC-MS using the following method:

[0034] Chromatographic column Agilent Poroshell 120 Ec-C 18 Column (2.1 mm × 150 mm, 2.7 μm); Mobile phase: Water (A, containing 0.1% formic acid); Methanol (B); Gradient elution (B): 30-55% (0-8.0 min); 55%, 8.0-12 min; 55-100%, 12-22 min; 100%, 22-30 min; 100-30%, 30-30.1 min; 30%, 30.1-40 min. Flow rate: 0.5 mL min -1 ; Column temperature 28℃; injection volume 5μL, detection wavelength 280nm.

[0035] References

[0036] 1. Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia. 2020, 1:36.

[0037] 2. Liao Peihai, Weng Lianjin, Liu Shulan, Geng Di. Research progress on chemical components and pharmacological activities of Caper chinensis. Modern Chinese Medicine 2020, 22(02): 296-304.

[0038] 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.

[0039] 4. Cheng Zhihong, Wu Yiqing, inventors; Conversion method of diterpene alcohol derivatives of Caperata chinensis and use thereof in the preparation of anti-tumor drugs patent CN107164422B 2021-01-22.

Claims

1. Use of a gene encoding caper acyltransferase in the preparation of caperane diterpene esters, the nucleotide sequence of the encoding gene being shown in SEQ ID No.

1.

2. Use of a caper acyltransferase in the preparation of caperane diterpene esters, wherein the amino acid sequence of the caper acyltransferase is shown in SEQ ID No.

2.

3. Use of an engineered strain expressing ElBAHD16 in the preparation of argan diterpene esters, wherein the amino acid sequence of ElBAHD16 is shown in SEQ ID No. 2.

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