Method for producing menthol by modifying corn terpene synthesis gene ZmDLS of corn P450 gene and application thereof

Abstract

The application provides a method for producing menthol by modifying a corn P450 gene ZmDLS of a corn terpene synthesis gene, and the method comprises the following steps: constructing a recombinant strain for co-expression of the corn P450 gene ZmCYP709E4 and the corn terpene synthesis gene ZmDLS by a genetic engineering method, and producing menthol by using the obtained recombinant strain. The application also provides an application of the above method. The application uses genome and transcriptome data to screen and clone the corn terpene synthesis gene ZmDLS and the corn P450 gene ZmCYP709E4, and experimentally verifies that the ZmCYP709E4 modifies the ZmDLS to produce monoterpene alcohol-menthol.

Description

Technical Field

[0001] This invention relates to the field of plant secondary metabolism technology, and in particular to a method and application of modifying the maize P450 gene to produce menthol from the maize terpene synthesis gene ZmDLS. Background Technology

[0002] Terpenes are hydrocarbons and their oxygenated derivatives that exist in nature and have a molecular formula that is a multiple of isoprene units. These oxygenated derivatives can be alcohols, aldehydes, ketones, carboxylic acids, esters, etc. Terpenes are widely distributed in nature and are major components of the fragrances, resins, and pigments of some plants. Terpenes have many physiological activities, such as expectorant, antitussive, carminative, diaphoretic, anthelmintic, and analgesic effects.

[0003] Terpenoids are a class of natural products produced by plant secondary metabolism, derived from the precursor isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP). There are two synthetic pathways for IPP and DMAPP: the mevalonate (MVA) pathway and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. Different biosynthetic pathways of terpenoids and their subcellular localization are related; the MVA pathway is localized in the cytoplasm, while the MEP pathway is localized in the plastids. Terpenoid synthases are key enzymes in the biosynthesis of terpenoids. The peptide chains of terpenoid synthases are typically 550–850 amino acid residues long, while the peptide chains of sesquiterpene synthases are 550–580 amino acids long. Based on the different structures and reaction mechanisms of terpene synthases, they can be divided into two categories: Class I and Class II. Class I enzymes have an α-domain as their catalytic active site and two highly conserved motifs, DDXXD and NSE / DTE. Class II enzymes have a β-domain as their active site and contain one conserved motif, DXDD.

[0004] Plant cytochrome P450s (CYP450s) are monooxygenases encoded by a superfamily, and are the first enzymes to be classified as a "superfamily." These enzymes typically bind to organelle membrane systems such as the endoplasmic reticulum, plastids, mitochondria, and Golgi apparatus, participating in numerous metabolic processes during life activities. CYP450s are named for their highest absorbance at 450 nm after binding to reduced CO2. The CYP450s family is vast, comprising over 1000 families and 2500 subfamilies. A key characteristic of this family is the presence of a conserved heme-binding region (FxxGxCxG) in their structure. During plant growth and development, CYP450s interact with various substrates to participate in the synthesis and degradation of alkaloids, terpenes, fatty acids, plant hormones, signaling molecules, flavonoids, and other secondary metabolic reactions. Therefore, the CYP450s family plays a crucial regulatory role in the plant life activity network system.

[0005] However, there are currently no studies on the CYP450s-modified terpene ZmDLS synthesis gene. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method and application for producing menthol by modifying the maize P450 gene with the maize terpene synthesis gene ZmDLS.

[0007] Among them, the maize terpene synthesis gene ZmDLS is a gene that encodes D-limonene synthase, which is a key enzyme in the formation of the terpene compound D-limonene.

[0008] Menthol, also known as peppermint camphor, is a cyclic monoterpene alcohol (a type of terpene compound). Studies have shown that menthol can promote drug penetration through the skin. Due to the presence of the skin's stratum corneum barrier and the physicochemical properties of the drugs themselves, many drugs have very low skin permeability, making it difficult to reach effective therapeutic concentrations. Therefore, menthol can be added to topical preparations such as those for the skin to exert local antipruritic, analgesic, cooling, and mild local anesthetic effects.

[0009] This invention modifies the maize terpene synthesis gene ZmDLS by screening a suitable maize P450 gene, which can convert D-limonene produced by ZmDLS into menthol, thereby realizing the synthesis and production of menthol.

[0010] The present invention solves the above-mentioned technical problems by adopting the following technical solutions:

[0011] A method for producing menthol by modifying the maize P450 gene with the maize terpene synthesis gene ZmDLS involves constructing a recombinant strain that co-expresses the maize P450 gene ZmCYP709E4 and the maize terpene synthesis gene ZmDLS using genetic engineering methods, and then using the obtained recombinant strain to produce menthol; wherein the nucleotide sequence of the maize terpene synthesis gene ZmDLS is shown in SEQ ID NO.1, and the nucleotide sequence of the maize P450 gene ZmCYP709E4 is shown in SEQ ID NO.3.

[0012] As one of the preferred embodiments of the present invention, in the step of constructing a recombinant strain co-expressing the maize P450 gene ZmCYP709E4 and the maize terpene synthesis gene ZmDLS by genetic engineering methods, the recombinant plasmids pMAL-c2x-ZmDLS and pMAL-c2x-ZmCYP709E4 are first constructed separately, and then the recombinant plasmids pMAL-c2x-ZmDLS and pMAL-c2x-ZmCYP709E4 are simultaneously transformed into Escherichia coli BL21.

[0013] As one of the preferred embodiments of the present invention, the amino acid sequence of the protein encoded by the corn terpene synthesis gene ZmDLS is shown in SEQ ID NO.2.

[0014] As one of the preferred embodiments of the present invention, the amino acid sequence of the protein encoded by the maize P450 gene ZmCYP709E4 is shown in SEQ ID NO.4.

[0015] As one of the preferred embodiments of the present invention, the maize terpene synthesis gene ZmDLS is modified by the maize P450 gene ZmCYP709E4 to convert D-limonene produced by ZmDLS into menthol.

[0016] An application of a method for producing menthol by modifying the above-mentioned maize P450 gene with the maize terpene synthesis gene ZmDLS involves co-expressing the maize P450 gene ZmCYP709E4 and the maize terpene synthesis gene ZmDLS in a strain to obtain a high-yielding menthol strain for use in menthol production.

[0017] The advantages of this invention compared to the prior art are:

[0018] This invention utilizes genomic and transcriptomic data to screen and clone the maize terpene synthesis gene ZmDLS and the maize P450 gene ZmCYP709E4. Experiments verified that ZmCYP709E4 modification of ZmDLS produces the monoterpene alcohol menthol. First, this invention constructed mutant and overexpression lines of the terpene synthesis gene ZmDLS. Maize borer larvae fed on maize ZmDLS, B73, and ZmDLS-OE. Then, quantitative real-time analysis was used to observe changes in the expression levels of ZmDLS and ZmCYP709E4. The changes in ZmDLS and ZmCYP709E4 were similar, suggesting a possible relationship. In vitro enzyme activity analysis of ZmDLS was performed as a control. Subsequently, co-expression of ZmCYP709E4 and ZmDLS was analyzed in vitro to observe that modification with ZmCYP709E4 could convert D-limonene produced by ZmDLS into menthol. Attached Figure Description

[0019] Figure 1 This is a chromosomal mapping of the ZmDLS and ZmCYPS genes in Example 2;

[0020] Figure 2 This is a diagram of the co-expression network of the ZmDLS gene and the ZmCYPS gene in Example 2;

[0021] Figure 3 This is an analysis of the expression levels of ZmDLS and ZmCYP709E4, ZmCYP94D27, ZmCYP79A61, and ZmCYP711A13 genes in B73 corn plants, corn leaf mutant plants, and ZmDLS-OE plants (in the figure, "*" indicates p<0.05; "**" indicates p<0.01; "ns" indicates no significance, p>0.05).

[0022] Figure 4 This is an agarose gel electrophoresis image of the genes encoding ZmDLS and ZmCYP709E4 in Example 3;

[0023] Figure 5 This is a diagram of menthol, the co-expressed product of the ZmDLS gene and the ZmCYP709E4 gene in vitro, as shown in Example 3. Detailed Implementation

[0024] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. Furthermore, unless otherwise specified, the reagents and methods used in the following embodiments are all conventional reagents and methods in the art and will not be described again.

[0025] Example 1

[0026] Information related to the maize terpene synthesis gene ZmDLS:

[0027] The ZmDLS gene encodes D-limonene synthase, which, in previous studies, produces D-limonene. D-limonene synthase is a key enzyme in the formation of the terpene compound D-limonene. The nucleotide sequence of the ZmDLS gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0028] Example 2

[0029] Screening for the ZmCYP709E4 gene:

[0030] (1) Download all screened maize P450 genes from NCBI (https: / / www.ncbi.nlm.nih.gov / ), a total of 269 ZmCYPS were screened. These 269 ZmCYPS were then used for chromosome localization (e.g., Figure 1 As shown in the figure, ZmCYPs located on chromosome 9 were screened, and a total of 19 ZmCYP genes were screened.

[0031] (2) The 19 selected ZmCYP genes were co-expressed with the aforementioned ZmDLS genes, and the data were visualized as follows: Figure 2 The co-expression network diagram is shown, and four genes, Zm00001d047418, Zm00001d045188, Zm00001d046334, and Zm00001d046207, were initially screened.

[0032] For ease of representation, the four genes Zm00001d047418, Zm00001d045188, Zm00001d046334, and Zm00001d046207 are represented as ZmCYP709E4, ZmCYP94D27, ZmCYP79A61, and ZmCYP711A13, respectively. The nucleotide sequence of ZmCYP709E4 is shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4. The nucleotide sequences of ZmCYP94D27, ZmCYP79A61, and ZmCYP711A13 are shown in SEQ ID NO.5, 6, and 7, respectively.

[0033] (3) Construct ZmDLS gene mutant plants “zmdls” (ZmDLS gene expression is incomplete), B73 (wild maize plant variety) and ZmDLS-OE plant (ZmDLS overexpression plant).

[0034] The construction process of the ZmDLS gene mutant plant zmdls was as follows: ZmDLS mutants were obtained through screening on the maize mutant website (www.elabcaas.cn / memd / ). zmdls exhibits a GA mutation at the 6th base of the 2nd exon region, prematurely terminating translation at amino acid 135 and lacking the DDXXD domain. Genomic DNA of the mutant was extracted (using the B73 genome as a control) and sent to Shanghai Sangon Biotech for sequencing. Sequencing results were compared to clarify the location of the mutation site, and the homozygous mutant was finally identified.

[0035] The construction process of ZmDLS-OE plants is as follows: 1305 eukaryotic expression vector was constructed, sent to Shanghai Sangon Biotech Co., Ltd., and after correct sequencing, it was handed over to Wuhan Aidijing Biotechnology Co., Ltd. to construct ZmDLS overexpressing maize plants.

[0036] (4) Using plants with ZmDLS gene mutation (incomplete ZmDLS gene expression), B73 (wild maize plant variety), and ZmDLS-OE plants (ZmDLS overexpressing plants) that were bitten by maize borers, the changes in the expression levels of the ZmDLS gene and the ZmCYP709E4, ZmCYP94D27, ZmCYP79A61, and ZmCYP711A13 genes in the corresponding plants after being bitten by maize borers.

[0037] In this step, quantitative real-time PCR was used to determine the expression levels of genes in plants under specific conditions; primers were designed using PrimerPrimier 5.0 software and synthesized by Shanghai Sangon Biotech Co., Ltd.; and a plant RNA extraction kit (Monzol) was used. TM RNA was extracted from maize B73 leaves using ReagentPro and analyzed using MonScipt. TM cDNA was synthesized via reverse transcription using the RTIII Super Mix with dsDNAse (Two-Step) kit. Reagents were added according to Tables 1 and 2, and three biological replicates were set up. The reaction was carried out under the conditions specified in Table 1, and the relative expression levels were calculated using the 2-ΔΔCt method.

[0038] The results of the expression level measurement are shown in Figure 3 It can be seen that, compared with other ZmCYPS, the expression level of ZmCYP709E4 is highly correlated with the expression level of ZmDLS, and finally, the ZmCYP709E4 gene was selected to modify the ZmDLS gene.

[0039] Table 1 Reaction system and reaction conditions

[0040]

[0041]

[0042] Table 2 Primers in the reaction system

[0043]

[0044] Example 3

[0045] Construction of a maize strain co-expressing the ZmDLS and ZmCYP709E4 genes:

[0046] I. Amplification of the maize ZmDLS and ZmCYP709E4 genes:

[0047] Based on the previously screened ZmDLS sequence and ZmCYP709E4 gene, full-length specific primers were designed using Primer Premier 5.0. Using maize B73 (eight-leaf stage) cDNA as a template, PCR was used to amplify the full-length CDS sequences of ZmDLS and ZmCYP709E4. The specific steps are as follows:

[0048] (1) Using the extracted RNA as a template, cDNA was reverse transcribed in two steps.

[0049] Step 1: Genomic DNA contamination removal, the reaction system is shown in Table 3 below:

[0050] Table 3. Step 1 of the maize leaf reverse transcription system

[0051]

[0052] PCR reaction conditions: 37°C, warm bath for 2 minutes; 55°C, warm bath for 5 minutes; 4°C, 2 minutes.

[0053] Step 2: cDNA synthesis, the reaction system is shown in Table 4 below:

[0054] Table 4. Step 2 of the maize leaf reverse transcription system

[0055]

[0056] PCR reaction conditions: 50°C, warm bath for 15 minutes: 85°C, warm bath for 5 minutes; 4°C, 2 minutes.

[0057] After the reaction is complete, store the reverse transcription product at -20°C.

[0058] (2) Obtaining the full-length cDNA sequences of ZmDLS and ZmCYP709E4.

[0059] The primer sequences for ZmDLS and ZmCYP709E4 are shown in Table 5.

[0060] Table 5 Primer names and sequences

[0061]

[0062] Using maize cDNA as a template, the full-length fragment was amplified using specific primers. The PCR reaction system is shown in Tables 6 and 7 below.

[0063] Table 6 Cloning conditions and reaction system for full-length maize ZmDLS cDNA

[0064]

[0065] Table 7 Cloning conditions and reaction system for full-length cDNA of maize ZmCYP709E4

[0066]

[0067]

[0068] After PCR, the target gene bands were gel-returned.

[0069] II. Construction of Recombinant Plasmids

[0070] The gene gel-reversion products obtained in the above steps were ligated into the pMD18-T vector, transformed into E. coli DH5α, and colony PCR was performed on bacteria showing the target gene band for sequencing. The results are as follows: Figure 4 As shown, this indicates that the cloned band is the desired target band.

[0071] Plasmids were then extracted and homologous recombination primers with restriction sites BamHI and EcoRI were designed to amplify the plasmids by PCR and gel return. The reaction system is shown in Table 8.

[0072] Table 8. Double enzyme digestion reaction system and reaction conditions

[0073]

[0074] The double digestion products were then gel-returned, and homologous recombination ligase was used to ligate the gel-returned products. The ligation reaction system is shown in Table 9.

[0075] Table 9. Homologous recombination system and reaction conditions

[0076]

[0077]

[0078] The ligation reaction was carried out at 50°C for 15 minutes. After the reaction, the ligation product was transformed into Escherichia coli DH5α, then plated on solid LB medium containing ampicillin and cultured overnight. The next day, colonies with a diameter of about 2 mm were picked for PCR identification. Positive clones were selected and 50% glycerol was added at a 1:1 ratio and stored at -80°C.

[0079] III. Construction of Recombinant Strains

[0080] The recombinant plasmids pMAL-c2x-ZmDLS and pMAL-c2x-ZmCYP709E4 obtained in the above steps were transformed into Escherichia coli expression strain BL21, and then colony PCR was performed for identification.

[0081] The positive clones of BL21 strain were successfully introduced and cultured on a large scale. After the culture became turbid, the OD was measured and adjusted using a spectrophotometer. 600 The pH was adjusted to approximately 0.6–0.8, and then IPTG was added to induce protein expression. The mixture was placed in a shaker at 16°C and induced for 12 hours to ensure full expression of the recombinant protein of the target gene in *E. coli*. After centrifugation at 4°C, the collected target gene bacterial cells were sonicated to disrupt the cell structure. The resulting supernatant, or crude target gene protein, was obtained after centrifugation and aliquoted and stored at -80°C for later use.

[0082] The collected crude protein was subjected to in vitro enzyme activity identification. GPP was added exogenously as a substrate, and headspace solid-phase microextraction was used. The reaction was carried out at 30°C for 1 hour. After 1 hour of extraction, the catalytic products were detected and analyzed by GC-MS. The detection results were confirmed by comparison with the NIST (National Institute of Standards and Technology) 2011 standard library, and the volatile terpenes expressed by the target gene were analyzed.

[0083] The in vitro enzyme activity analysis results of co-expression of ZmDLS and ZmCYP709E4 in this embodiment are as follows: Figure 5 As shown. From Figure 5 It can be seen that the co-expressed product of ZmDLS and ZmCYP709E4 is L-Menthol.

[0084] In summary, by co-expressing the maize P450 gene ZmCYP709E4 and the maize terpene synthesis gene ZmDLS in the strain, a high-yield menthol strain can be obtained for menthol production.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for producing menthol, characterized in that, Constructing the maize P450 gene using genetic engineering methods ZmCYP709E4 With corn terpene synthesis gene ZmDLS The co-expressed recombinant strain is used to produce menthol; wherein the corn terpene synthesis gene is mentioned. ZmDLS The nucleotide sequence is shown in SEQ ID NO.1, for the maize P450 gene. ZmCYP709E4 The nucleotide sequence is shown in SEQ ID NO. 3; the maize P450 gene was constructed using genetic engineering methods. ZmCYP709E4 With corn terpene synthesis gene ZmDLS In the co-expression of recombinant strains, pMAL-c2x-ZmDLS and pMAL-c2x-ZmCYP709E4 recombinant plasmids were first constructed, and then the pMAL-c2x-ZmDLS and pMAL-c2x-ZmCYP709E4 recombinant plasmids were simultaneously transformed into Escherichia coli BL21.

2. An application of the method as claimed in claim 1, characterized in that, The maize P450 gene was co-expressed in the strain. ZmCYP709E4 With corn terpene synthesis gene ZmDLS A high-yield strain of menthol was obtained and used for menthol production.

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