Acyltransferase AcBAHD13 participating in synthesis of aescin A as well as coding gene and application of acyltransferase AcBAHD13

Abstract

The invention discloses acyltransferase AcBAHD13 participating in synthesis of aescin A as well as a coding gene and application of the acyltransferase AcBAHD13. The amino acid sequence of acyltransferase protein is shown as SEQ ID NO: 2. The nucleotide sequence of the coding gene of the acyltransferase protein is shown as SEQ ID NO: 1. The invention relates to the technical field of biotechnology. According to the acyltransferase AcBAHD13 participating in synthesis of aescin A as well as the coding gene and the application of the acyltransferase AcBAHD13, an AcBAHD13 gene is obtained based on aescin genome and transcriptome data screening, a pET28a-AcBAHD13 prokaryotic expression vector is constructed and is subjected to induced expression, and the acylation function of the AcBAHD13 is verified in vitro by taking proaescin as a substrate. The result proves that the AcBAHD13 can be used for catalyzing the proaescin to generate the 21-O-angeloyl proaescin by utilizing an exogenous angeloyl donor, and a foundation is laid for the biological research on synthesis of aescin components with important medicinal values.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and in particular to an acyltransferase AcBAHD13 involved in the synthesis of aescin A, a coding gene thereof, and an application thereof. Background Art

[0002] Aesculus hippocastanum is a deciduous tree of the genus Aesculus in the family Aesculaceae, Sapindaceae, and the 2020 edition of the Chinese Pharmacopoeia lists the dried, mature seeds of the horse chestnut tree. The main active ingredient in the seeds is aescin, which has high medicinal value and can treat cerebral hemorrhage, cerebral edema, respiratory diseases, and other conditions. Currently, a variety of new aescin-based formulations are widely used clinically both domestically and internationally, including "Reparil" (Germany, injection), "Qescusan, Germad" (Germany, tablets or suppositories), "Venostascin" (Germany, injection, capsules, or ointment), "Tochikinon" (Japan, tablets), and domestic sodium aescinate injections and tablets. With growing demand for aescin products in both the international and domestic markets, obtaining aescin from the seeds alone is no longer sufficient to meet production needs. To promote the industrialization of aescin, it is urgent to find methods to increase aescin production.

[0003] Aescin is a triterpenoid component, including aescin A / B / C / D, of which the most common is aescin A / B, and its structural formula is as follows: Figure 1 As shown. The analysis of the biosynthetic mechanism of aescin components is a research hotspot at home and abroad, and it has not yet been fully analyzed. Acylation is an important post-modification step in the biosynthesis of aescin, and the acyltransferase transfers the activated acyl group on the donor molecule to the hydroxyl group of the acceptor molecule. The team has reported that AcBAHD6 in Aesculus hippocastanum can catalyze the conversion of protoesculin to 22-O-acetylprotoesculin using acetyl as a donor, but the catalytic enzyme for angelica acyl at position 21 has not yet been reported. Based on the genome and transcriptome data of Aesculus hippocastanum, the present invention screened and identified the key enzyme that can introduce crotonyl at C21-OH, providing a key element for the synthetic biology research of aesculin. Summary of the Invention

[0004] (1) Technical problems solved

[0005] In response to the deficiencies in the prior art, the present invention provides an acyltransferase AcBAHD13 involved in the synthesis of aescin A, its encoding gene, and applications. The present invention provides a method for screening and functional identification of the acyltransferase AcBAHD13 in Aesculus hippocastanum that catalyzes the conversion of protoescinogenin to 21-O-crotonylprotoescinogenin, verifies that AcBAHD13 has the function of catalyzing the conversion of protoescinogenin to 21-O-crotonylprotoescinogenin, provides a molecular basis for elucidating the biosynthetic pathway of aescin, and has significant application value.

[0006] (2) Technical solution

[0007] To achieve the above object, the present invention is implemented through the following technical solution: Acyltransferase AcBAHD13 involved in the synthesis of aescin A, the amino acid sequence of the acyltransferase AcBAHD13 is shown in SEQ ID NO: 2.

[0008] The present invention also provides a gene encoding the acyltransferase AcBAHD13, and the nucleotide sequence of the gene encoding the acyltransferase AcBAHD13 is shown in SEQ ID NO: 1.

[0009] The gene was named AcBAHD13, and the protein it encodes was named AcBAHD13. The details are as follows: The AcBAHD13 gene sequence is shown in Sequence 1 in the sequence listing, which contains 1209 nucleotides and encodes the protein shown in Sequence 2 in the sequence listing, which consists of 402 amino acids.

[0010] Expression cassettes, recombinant expression vectors, transgenic cell lines, or recombinant bacteria containing the encoding gene also fall within the scope of protection of the present invention. A primer pair for amplifying the full length of the encoding gene also falls within the scope of protection of the present invention, wherein one primer sequence is shown as Sequence 3 in the sequence listing, and the other primer sequence is shown as Sequence 4 in the sequence listing.

[0011] Preferably, the use of the acyltransferase AcBAHD13 as an acyltransferase also falls within the scope of protection of the present invention.

[0012] Preferably, the acyltransferase is an acyltransferase that catalyzes the conversion of protoescinogenin to 21-O-angeloylprotoescinogenin.

[0013] Preferably, the use of the acyltransferase AcBAHD13 in catalyzing the production of 21-O-angeloylprotoescin from protoescin also falls within the scope of protection of the present invention.

[0014] Preferably, the use of the encoding gene in catalyzing protoescin to generate 22-O-acetylprotoescin also falls within the scope of protection of the present invention.

[0015] (3) Beneficial effects

[0016] The present invention provides an acyltransferase, AcBAHD13, involved in the synthesis of aescin A, its encoding gene, and its use. Compared to the prior art, the present invention has the following advantages: The acyltransferase, AcBAHD13, involved in the synthesis of aescin A, its encoding gene, and its use are screened based on the genome and transcriptome data of Aesculus hippocastanum, and the AcBAHD13 gene is obtained. The pET28a-AcBAHD13 prokaryotic expression vector is constructed and induced for expression. The acylation function of AcBAHD13 is then verified in vitro using protoescinin as a substrate. The results demonstrate that AcBAHD13 catalyzes the conversion of protoescinin to 21-O-crotonylprotoescinin, laying the foundation for synthetic biology research on aescins, a class of medicinally valuable components. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the structural formula of aescin A / B;

[0018] Figure 2 Identification and evolutionary relationship of acyltransferase (BAHDs) family members based on the Aesculus hippocastanum genome;

[0019] Figure 3 The results of differential expression of BAHDs encoding genes in different tissues of Aesculus hippocastanum;

[0020] Figure 4 This is the SDS-PAGE gel image of the AcBAHD13 recombinant protein of the present invention;

[0021] Figure 5 This is the in vitro functional verification of the AcBAHD13 catalyzing the conversion of protoescin to 21-O-β-crotonylprotoescin by AcBAHD13 of the present invention; wherein, A: the reaction formula of AcBAHD13 catalyzing the conversion of protoescin to 21-O-β-crotonylprotoescin; B: the EIC graph of AcBAHD13 catalyzing the conversion of protoescin to product P1 (m / z 633.4008). DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0023] See also Figure 1-5 The present invention provides three technical solutions: Acyltransferase AcBAHD13 involved in the synthesis of aescin A and its encoding gene and application specifically include the following embodiments:

[0024] Example 1: Screening of BAHDs genes based on Aesculus hippocastanum genome and transcriptome data and their phylogenetic analysis

[0025] 1.1 Experimental methods:

[0026] Based on the Aesculus hippocastanum genome data, Arabidopsis thaliana BAHD protein sequences were extracted from the TAIR database. Homologous genes were searched using BLASTP and HMMER, and manually corrected. Multiple sequence alignment was performed using MAFFT, and a phylogenetic tree was constructed using IQtree and the maximum likelihood (ML) method, with 1000 bootstrap replicates. RNA-Seq transcriptome data from Aesculus hippocastanum roots, stems, leaves, flowers, exocarps, seeds, and fruits were aligned to the Aesculus hippocastanum genome using HiSAT2, and differential expression analysis was performed using Cufflinks. Metabolomic studies revealed that aescin A is specifically distributed in Aesculus hippocastanum seeds. Co-expression analysis was used to identify candidate AcBAHD genes consistent with the distribution of aescin metabolites.

[0027] 1.2 Results and Analysis

[0028] Based on the genome data of Aesculus hippocastanum, a total of 81 BAHD homologous genes were annotated. Phylogenetic tree analysis showed that the BADH gene family of Aesculus hippocastanum can be divided into seven subfamilies, namely: Clade Ia, Clade Ib, Clade II, Clade IIIa, Clade IIIb, Clade Va, Clade Vb ( Figure 2 Among them, Clade IIIa is often reported to be related to the acylation of secondary metabolites, etc., and the present invention selects Clade IIIa as the main candidate gene subfamily.

[0029] Based on the transcriptome data analysis of Aesculus hippocastanum, it was found that the AcBAHD13 gene was specifically expressed in Aesculus hippocastanum seeds ( Figure 3 Moreover, AcBAHD13 belongs to the Clade IIIa subfamily and is clustered with the reported AcBAHD6 ( Figure 2 Therefore, it is speculated that it is the key enzyme gene that catalyzes protoescigenin to 21-O-β-tigloylprotoaescigenin in the biosynthesis pathway of aescin in Aesculus hippocastanum.

[0030] The AcBAHD13 gene sequence is shown in Sequence 1 in the sequence listing. Sequence 1 contains 1209 nucleotides and encodes the protein shown in Sequence 2 in the sequence listing. Sequence 2 consists of 402 amino acids. The protein is named AcBAHD13.

[0031] Example 2: Construction of an expression vector encoding the candidate AcBAHD13 gene

[0032] 2.1 Experimental methods:

[0033] The pET28a-AcBAHD13 expression vector was obtained by homologous recombination. BamHI and SalI were selected as double restriction sites, and primer sequences were designed (see Table 1). The seed cDNA was used as a template and KOD one TM PCR master mix high-fidelity enzyme cloned into the AcBAHD13 gene fragment (the total volume of the cloning system program is 50μL: 25μL KOD one TM PCR master mix, 5 μL template, 1.5 μL forward primer, 1.5 μL reverse primer and 17 μL water, see Table 2 for the procedure). The obtained gene fragment was ligated into the pET28a vector (purchased from Merck Sigma-Aldrich, product number 69864) using the Novogene Recombination Kit. The ligation system was directly transformed into the TransT1 competent cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number CD501). Positive clones were selected for sequencing (the total volume of the colony PCR system was 20 μL: 10 μL 2× Taq PCR Mix, 1 μL template, 1 μL forward primer, 1 μL reverse primer and 7 μL water, see Table 3 for the procedure).

[0034] 2.2 Experimental results:

[0035] The sequencing results of the positive clones were compared with the genome annotation sequence, and it was found that the nucleotide sequence had a similarity of 93.7% with the original data. The sequence differences may be related to the tandem duplication phenomenon of the sequence gene or the quality of genome sequencing. The actual sequencing results shall prevail.

[0036] Table 1 Primer sequences for constructing pET28a-AcBAHD13 expression vector

[0037]

[0038] Table 2 KOD high-fidelity enzyme PCR reaction program

[0039]

[0040]

[0041] Table 3 Colony PCR reaction procedure

[0042]

[0043] Example 3. Functional verification of candidate AcBAHD13 encoding genes

[0044] 3.1 Experimental methods:

[0045] The pET28a-AcBAHD13 expression vector obtained above was transformed into BL21 (DE3) Escherichia coli expression strain (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., product number CB105). 2 mL of overnight culture solution was added to 40 mL of LB liquid medium (containing 50 μg / mL kanamycin resistance medium) and activated at 37°C, 200 rpm for 2-3 hours. 600 When the cell count reached 0.6-0.8, IPTG was added to a final concentration of 0.3 mM for induction at 16°C and 110 rpm. After 24 h of induction, the bacterial pellet was centrifuged for protein purification and its concentration was determined using a BCA protein kit.

[0046] The purified protein was functionally verified in vitro using the following reaction system (200 μL): 1× phosphate buffer (PBS, pH 7.4), 50 μg of purified protein, 1 mM Tigoly-CoA (crotonyl-Coenzyme A, purchased from Shanghai Kaisen Biotechnology Co., Ltd., product number FM130), and 200 μM protoescin (purchased from Chengdu Purifa Technology Development Co., Ltd., product number BP1485). The reaction was incubated at 37°C for 2 h. The reaction was terminated with methanol and then filtered through a 0.22 μm filter. The chemical composition was analyzed using UPLC-TOF-MS / MS. Instrument model: Agilent Technologies 1290 InfinityⅡ and 6530Q-TOF (Agilent Technologies, Germany). The injection volume was 1 μL, and the chromatographic column was: Waters Acquity BEH C18 column (1.7 μm, 100 × 2.1 mm), column temperature: 35°C. Mass spectrometry conditions: ESI ion source, negative ion mode, scan range 300–1700 m / z, collision energy 20 V, VCap 2000 V, dryer temperature and flow rate 300°C and 6 L / min, sheath gas temperature 300°C, sheath gas flow rate 11.0 L / min, nebulizer pressure 30 psi. Mobile phases: (A): water (containing 0.1% formic acid), (B): acetonitrile, flow rate 0.30 mL / min, elution program: 0–4 min, 40% B; 10–12 min, 100% B; 12–15 min, 40% B.

[0047] 3.2 Results and Analysis

[0048] Protein purification results are shown in Figure 4A clear protein expression band can be seen. The in vitro catalytic results showed that AcBAHD13 could catalyze protoescin to produce product peak P1. By comparing the elution time and mass spectrometry information with the standard 21-O-β-crotonyl-protoescin, the peak time was 8.691 min and the molecular weight was 633.4008 [C35H56O7+COOH]. - The product peak P1 was determined to be 21-O-β-crotonyl-protoescinogenin ( Figure 5 ), indicating that AcBAHD13 can perform crotonyl modification on protoescin.

[0049] In summary, the present invention screened and identified the AcBAHD13 gene based on genome and transcriptome data from Aesculus hippocastanum. By constructing the pET28a-AcBAHD13 prokaryotic expression vector and inducing expression, the acylation function of AcBAHD13 was verified in vitro using protoescinin as a substrate. The results demonstrated that AcBAHD13 catalyzes the conversion of protoescinin to 21-O-crotonylprotoescinin, laying the foundation for synthetic biology research on aescins, a class of medicinally important components.

[0050] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0051] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus.

[0052] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Acyltransferase AcBAHD13 involved in the synthesis of aescin A, characterized by: The amino acid sequence of the acyltransferase AcBAHD13 is shown in SEQ ID NO:

2.

2. The gene encoding the acyltransferase AcBAHD13 involved in the synthesis of aescin A according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO:

1.

3. The gene encoding the acyltransferase AcBAHD13 involved in the synthesis of aescin A according to claim 2, characterized in that: A primer pair for amplifying the full length of the coding gene, wherein the sequence of one primer is shown in SEQ ID NO: 3, and the sequence of the other primer is shown in SEQ ID NO:

4.

4. An expression cassette, recombinant expression vector, transgenic cell line or recombinant bacterium containing the coding gene according to claim 2.

5. Use of the acyltransferase AcBAHD13 according to claim 1 as an acyltransferase.

6. The use according to claim 5, characterized in that: The acyltransferase is an acyltransferase that catalyzes protoescin to generate 21-O-crotonylprotoescin.

7. Use of the acyltransferase AcBAHD13 according to claim 1 in catalyzing the production of 21-O-crotonylprotoescinogenin from protoescinogenin.

8. Use of the encoding gene according to claim 2 in catalyzing the production of 21-O-crotonylprotoescinogenin from protoescinogenin.

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