Caryophyllene synthase mutant as well as preparation method and application thereof

Abstract

The invention discloses a caryophyllene synthase mutant as well as a preparation method and application thereof. The mutant is characterized in that the 67th residue of an amino acid sequence in caryophyllene synthase (AaQHS1 / AcHS3 / TPS7) from artemisia apiacea, agilawood and tobacco is replaced by (W / F / L), and the 71th residue of the amino acid sequence in the DDXXD structural domain is replaced by (D / E / N / Q / K / H). The mutant is beneficial to improving the purity of the synthesized product caryophyllene, and the product specificity of wild type caryophyllene synthase is improved to more than 98% from about 90%. A better production element is provided for biosynthesis of caryophyllene, and the production and separation cost of caryophyllene can be reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to a caryophyllene synthase mutant, a preparation method and application thereof, and belongs to the technical field of biotechnology. BACKGROUND

[0002] β Caryophyllene is an important bicyclic sesquiterpene compound, mainly used in the pharmaceutical and perfume industries, and recently used as a new fuel additive. Traditional caryophyllene acquisition methods mainly rely on plant extraction, which is limited by low abundance in plants. Synthesizing caryophyllene through microbial cell factories is a powerful supplement to existing acquisition methods. Caryophyllene belongs to sesquiterpenes, the biosynthesis of which depends on its common substrate farnesyl pyrophosphate. Caryophyllene synthase (CS) catalyzes the cyclization of farnesyl pyrophosphate to generate caryophyllene, which is one of the key enzymes in the caryophyllene synthesis pathway. The caryophyllene synthases reported so far have low activity and low catalytic product specificity, which limits the yield of caryophyllene and increases the cost of separation and extraction.

[0003]

[0004] To improve the efficiency of microbial cell factories for producing caryophyllene, researchers have done a lot of work in microbial cells such as E. coli and yeast. For example, introducing exogenous mevalonate pathway genes into E. coli to improve the titer of the precursor isopentenyl pyrophosphate, and then introducing a new tobacco-derived caryophyllene synthase TPS7 combined with fed-batch fermentation to successfully produce 5.142 g / L of caryophyllene (Cheng T, Zhang K, Guo J, et al. Highly efficient biosynthesis of β-caryophyllene with a new sesquiterpene synthase from tobacco [J]. Biotechnology for Biofuels and Bioproducts, 2022, 15(1): 39.). Compared with E. coli which needs to introduce an exogenous synthesis pathway, yeast has an advantage because it has a powerful sterol synthesis system, and the intermediates can be directly used as substrates for sesquiterpene synthesis. By flexibly connecting caryophyllene synthase AarTPS88 and ERG20, the probability of substrate binding to caryophyllene synthase is improved, and the mevalonate pathway is strengthened, finally achieving a yield of 15.6 g / L in S. cerevisiae (Li Z, Gan Y, Gou C, et al. Efficient biosynthesis of caryophyllene in Saccharomyces cerevisiae by combinatorial engineering of mevalonate pathway and sesquiterpene synthase [J]. Biotechnology for Biofuels and Bioproducts, 2022, 15(1): 39.). β-caryophyllene in Saccharomyces cerevisiae by β -caryophyllenesynthase from Artemisia argyi[J]. Synthetic and Systems Biotechnology, 2025,10(1): 158-164.). Furthermore, recent studies have reported that by precisely regulating key genes in yeast caryophyllene synthesis through a glucose sensing system, cell growth and product production are decoupled. Combined with the highly active caryophyllene synthase AaQHS1, the highest reported yield to date—21.4 g / L—was achieved in a fermenter (Zhang Y, Liu C, Li W, et al. Systematic engineering of the sterol synthesis pathway for Saccharomyces cerevisiae promotes the efficient production of β -caryophyllene[J]. MetabolicEngineering, 2025.).

[0005] Despite reports of high-yield caryophyllene production, the specificity of currently reported caryophyllene synthases for the product is only between 85-90%, and byproducts such as humulene and elemol have boiling points close to caryophyllene, hindering separation and extraction. No caryophyllene synthase with a catalytic selectivity exceeding 95% has yet been reported. Even using the most efficient enzymes currently available for caryophyllene fermentation, the purity after vacuum distillation can only reach 97.2% (Zhang Y, Liu C, Li W, et al. Systematic engineering of the sterol synthesis pathway for Saccharomyces cerevisiaeepromotes the efficient production of β -caryophyllene[J]. Metabolic Engineering, 2025.). To meet the industrial application needs of caryophyllene, further purification leads to higher production costs. To address this issue and reduce post-fermentation separation costs, this invention starts from the source of caryophyllene synthesis and mutates several representative caryophyllene synthases reported so far, aiming to obtain mutants that further enhance the catalytic specificity of caryophyllene, facilitating subsequent separation and purification, thereby reducing the production cost of caryophyllene. SUMMARY

[0006] In order to solve the problem of lacking caryophyllene synthase with high catalytic activity and high specificity, the application provides a caryophyllene synthase mutant, a preparation method and application thereof.

[0007] The first technical solution provided by the application is a caryophyllene synthase mutant, which is obtained by mutating the 67th amino acid in the DDXXD domain of caryophyllene synthase from Artemisia annua, Agathodamium englerianum and Nicotiana tabacum to (W / F / L) and mutating the 71th amino acid to (D / E / N / Q / K / H); more specifically, the caryophyllene synthase is derived from Artemisia annua, Agathodamium englerianum and Nicotiana tabacum, wherein X is any amino acid.

[0008] In some embodiments, the 372nd amino acid of the caryophyllene synthase AaQHS1 derived from Artemisia annua, the 378th amino acid of the caryophyllene synthase AcHS3 (M4c) derived from Agathodamium englerianum and the 379th amino acid of the caryophyllene synthase derived from Nicotiana tabacum are leucine, phenylalanine or tryptophan (W / F / L). In some embodiments, the 372nd amino acid of the caryophyllene synthase AaQHS1 derived from Artemisia annua, the 378th amino acid of the caryophyllene synthase AcHS3 (M4c) derived from Agathodamium englerianum and the 379th amino acid of the caryophyllene synthase derived from Nicotiana tabacum are leucine, phenylalanine or tryptophan (W / F / L).

[0009] In some embodiments, the 372nd amino acid of the caryophyllene synthase AaQHS1 derived from Artemisia annua, the 378th amino acid of the caryophyllene synthase AcHS3 (M4c) derived from Agathodamium englerianum and the 379th amino acid of the caryophyllene synthase derived from Nicotiana tabacum are leucine, phenylalanine or tryptophan (W / F / L).

[0010] The second technical solution provided by the application is a gene encoding the caryophyllene synthase mutant of the first technical solution.

[0011] The third technical solution provided by the application is a recombinant vector carrying the gene of the second technical solution.

[0012] In some embodiments, the recombinant vector uses plasmid pET28a as an expression vector.

[0013] The fourth technical solution provided by the application is a recombinant cell expressing the mutant of the first technical solution, containing the gene of the second technical solution or transformed with the recombinant vector of the third technical solution.

[0014] In some embodiments, the recombinant cell uses Escherichia coli as a host.

[0015] The application further provides a preparation method of the caryophyllene synthase mutant, which uses the recombinant cell of the fourth technical solution to express the mutant of the first technical solution.

[0016] In some embodiments, the method comprises the following steps: S1: inserting the coding gene into a vector plasmid to obtain a recombinant plasmid; S2: transforming the recombinant plasmid obtained in S1 into a competent cell; S3: culturing the competent cell in S2 in resistance to obtain a single colony, and expanding the colony; S4: fermenting the colony after expansion in S3.

[0017] S5: collecting, crushing and purifying the protein after the culture in S4.

[0018] Specifically, the vector plasmid in step S1 is pET28a, and the competent cell in step S2 is an E. coli cell.

[0019] The fifth technical solution provided by the application is a method for improving the catalytic selectivity of caryophyllene synthase, wherein the 372nd amino acid of caryophyllene synthase from Artemisia annua with an amino acid sequence as shown in SEQ ID NO. 1 is mutated into leucine, and the 376th amino acid is mutated into aspartic acid, glutamic acid, glutamine or histidine; or the 372nd amino acid of caryophyllene synthase from Artemisia annua with an amino acid sequence as shown in SEQ ID NO. 1 is mutated into tryptophan, and the 376th amino acid is mutated into aspartic acid, glutamic acid, glutamine or histidine; or the 376th amino acid of caryophyllene synthase from Artemisia annua with an amino acid sequence as shown in SEQ ID NO. 1 is mutated into glutamine or histidine; or the 382nd amino acid of caryophyllene synthase from Aquilaria sinensis with an amino acid sequence as shown in SEQ ID NO. 3 is mutated into aspartic acid, glutamine or histidine; or the 378th amino acid of caryophyllene synthase from Aquilaria sinensis with an amino acid sequence as shown in SEQ ID NO. 3 is mutated into tryptophan, and the 382nd amino acid is mutated into aspartic acid, glutamine or histidine; or the 378th amino acid of caryophyllene synthase from Aquilaria sinensis with an amino acid sequence as shown in SEQ ID NO. 3 is mutated into phenylalanine, and the 382nd amino acid is mutated into aspartic acid, glutamine or histidine; or the 383rd amino acid of caryophyllene synthase from Nicotiana tabacum with an amino acid sequence as shown in SEQ ID NO. 5 is mutated into aspartic acid, glutamic acid, glutamine or histidine; or the amino acid sequence of the tobacco-derived caryophyllene synthase shown in SEQ ID NO. 5 is mutated at the 379th amino acid to tryptophan and the 383rd amino acid to glutamic acid, glutamine or histidine; or the amino acid sequence of the tobacco-derived caryophyllene synthase shown in SEQ ID NO. 5 is mutated at the 379th amino acid to phenylalanine and the 383rd amino acid to aspartic acid, glutamic acid, glutamine or histidine.

[0020] The sixth technical solution provided by the present application is the use of the caryophyllene synthase mutant of the first technical solution, or the gene of the second technical solution, or the recombinant vector of the third technical solution, or the recombinant cell of the fourth technical solution, or the method of the fifth technical solution in the preparation of caryophyllene or a product containing caryophyllene.

[0021] The present application further provides the use of the caryophyllene synthase mutant in the synthesis of food, medicine, fine chemicals, health products, bioenergy and cosmetics. Specifically, the use in improving the specificity of products in the biosynthesis of food, medicine, fine chemicals, health products, bioenergy and cosmetics containing caryophyllene.

[0022] Compared with the prior art, the present application has the following beneficial effects: Compared with the wild type, the caryophyllene synthase mutant of the present application improves the specificity of catalyzing caryophyllene. The mutant improves the catalytic specificity of synthesizing caryophyllene from 90% to more than 98% compared with the wild type, significantly reduces the proportion of by-products in the biosynthesis of caryophyllene, and has similar activity and wild type; promotes the production of caryophyllene, and is conducive to reducing the production and separation cost. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A is the optimal conformation of AaQHS1 and substrate intermediate molecular docking in Example 1 of the present application, in which F372 and Y376 have interactions; Figure 1 B is the sequence alignment of AaQHS1 and homologous enzymes in Example 1 of the present application.

[0024] Figure 2 is the gas chromatogram for comparison of the catalytic products of the most preferred mutant and its wild type in Example 2 of the present application.

[0025] Figure 3 is the fermentation curve of caryophyllene-producing bacteria CaWH in Example 3 of the present application. DETAILED DESCRIPTION

[0026] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explanation of the present application and are not used to limit the present application.

[0027] The product testing method involved in the following examples is as follows: The product was extracted with n-hexane and detected using the gas chromatography method in reference (Kumeta Y, Ito M. Characterization of δ-guaiene synthases from cultured cells of Aquilaria, responsible for the formation of the sesquiterpenes in agarwood [J]. Plant physiology, 2010, 154(4): 1998-2007.).

[0028] The culture medium involved in the following examples is as follows: 1. LB medium formula: yeast powder 5 g / L, tryptone 10 g / L, NaCl 10 g / L.

[0029] 2. TB medium formula: KH2PO4 2.31 g / L, K2HPO4 12.56 g / L, yeast powder 24 g / L, tryptone 12 g / L, glycerol 4 g / L.

[0030] 3. Formula of fermentation medium: 1-3 g / L (NH4)2SO4, 5-10 g / L KH2PO4, 0.5-4 g / L yeast powder, 2-6 g / L MgSO4, 0.5-3 g / L citric acid, 0.5-2 g / L methionine, 1% E. coli culture general trace elements.

[0031] Example 1 Design and preparation of AaQHS1 mutant of Artemisia-derived caryophyllene synthase The gene sequence of Artemisia-derived caryophyllene synthase AaQHS1 obtained from the NCBI website is SEQ ID NO. 2. AlphaFold3 (https: / / alphafoldserver.com / ?golgi=true) was used to model AaQHS1; Autodock Vina software was used to construct the docking model of the catalytic intermediate cation and AaQHS1; the most reasonable conformation was selected according to the score and experimental results; and the key amino acid residues were analyzed. For example, Figure 1As shown in A, the intermediate cation and the enzyme interact with each other to deprotonate the C15 position in the last step of caryophyllene synthesis, and the hydroxyl oxygen of the tyrosine at position 376 is 4.0 Å away from C15, which is conducive to participating in the deprotonation reaction as a basic residue. Therefore, Y376 is identified as a key residue for completing the deprotonation of C15, and analysis of the surrounding residues finds that F372 and Y376 form a π-π interaction, which can fine-tune the conformation of Y376, thereby affecting the deprotonation efficiency and ultimately affecting the specificity of caryophyllene synthesis. For this reason, mutants are designed, (D / E / N / Q / K / H) are designed at position 376 to strengthen the C15 deprotonation reaction as a basic residue, and non-polar residues of different sizes (W / F / L) are designed at position 372 to fine-tune the conformation of the basic residue at position 376, so as to ultimately achieve the mutual fit of the substrate and the enzyme. As shown in B, the homologous enzymes AcHS3 and TPS7 also adopt the same strategy to design L378, L379 positions as (W / F / L) and Y382, Y383 positions as (D / E / N / Q / K / H). Figure 1 B, the homologous enzymes AcHS3 and TPS7 also adopt the same strategy to design L378, L379 positions as (W / F / L) and Y382, Y383 positions as (D / E / N / Q / K / H).

[0032] Taking AaQHS1 as an example, the pET28a-AaQHS1 plasmid is biosynthesized by GenScript. The primers are as shown in SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9, and are mixed at a molar ratio of 2:1:3. The pET28a-AaQHS1 plasmid is amplified by a two-step method using the pET28a-AaQHS1 as a template, and the PCR product is digested with Dpn 1 and then transformed into E. coli BL21 (DE3) competent cells, which are then plated on LB plates containing Kan and incubated at 37°C overnight. Single colonies are selected for sequencing identification until all combinations in the library are covered. The M4c mutant of the linaloe-derived caryophyllene synthase AcHS3 (the amino acid sequence is shown in SEQ ID NO. 3, and the nucleotide sequence is shown in SEQ ID NO. 4) can produce 80% caryophyllene, and the tobacco-derived caryophyllene synthase TPS (the amino acid sequence is shown in SEQ ID NO. 5, and the nucleotide sequence is shown in SEQ ID NO. 6) can produce 85% caryophyllene. The same method is also used for design.

[0033] Example 2 Expression of caryophyllene synthase mutants and whole-cell catalysis To characterize the performance of all mutants in the library, 10 μl of the bacterial solution from the mutant library stock tube obtained in Example 1 was inoculated into a test tube containing LB with Kan, and incubated at 37°C, 220 rpm overnight. The previous culture was inoculated into a fresh 10 ml TB flask at a 1% inoculation amount, and incubated at 37°C, 220 rpm for 3 h to OD600=0.8-1.0. IPTG was added to the medium at a final concentration of 0.2 mM to induce protein expression, and the incubation was continued at 22°C, 180 rpm for 20 h. After centrifugation, the bacterial cells were resuspended in 50 mM Tris-HCl at pH=6-9 to an OD 600 =20-40, and the final concentration of substrate FPP and 10-100 mM MgSO4 was added, and the system was sealed. The reaction was carried out at 500-800 rpm, 20-40°C for 2-24 h. The purity (%) of caryophyllene produced by each mutant is shown in Table 1, Table 2, Table 3, and the preferred mutant and its wild type comparative chromatogram is shown in Figure 1, Figure 2, Figure 3. Figure 2

[0034] The results show that for AaQHS1, the preferred mutant combination is 372W / 376H, and for AcHS3 (M4c) and TPS7, the preferred mutants are 378W / 382Q and 379W / 383H, respectively. The further preferred mutant among all mutants is AaQHS1 (F372W / Y376H), and the catalytic specificity for the synthesis of caryophyllene is increased from 90% of the wild type to more than 98%, and the byproduct 1 is completely eliminated, and only trace amounts of byproduct 2 (<1.6%) are retained (Table 1, Figure 2

[0035] Table 1 AaQHS1 372-376 library test

[0036] Table 2 AcHS3 (M4c) 378-382 library test

[0037] Table 3 TPS7 379-383 library test

[0038] Example 3 Application of AaQHS1 (F372W / Y376H) in caryophyllene-producing bacteria The plasmid pET28a-AaQHS1 (F372W / Y376H) was synthesized from the mevalonate pathway biosynthesis plasmid pCZ154, and the DNA sequence of pCZ154 is shown in SEQ ID NO. 10. The plasmid pET28a-AaQHS1 (F372W / Y376H) and the plasmid pET28a-ispA (FPPS) from E. coli were co-expressed in pET28a.​​AaQHS1 (F372W / Y376H) - ispA The sequence of the plasmid is shown as SEQ ID NO. 11. pCZ154 and pET28a- AaQHS1 (F372W / Y376H) - ispA The car S from scratch synthesis strain CaWH was obtained by co-transforming pCZ154 and pET28a- Figure 3 into E. coli MG1655EPR (DE3). The CaWH monoclonal was picked into LB liquid medium and a proper amount of chloramphenicol and kanamycin was applied; after 8-12 h of shaking culture at 37℃, it was transferred into TB medium at a ratio of 1‰, and shaking culture was carried out at 37℃ for 12-16 h; the bacterial liquid of the previous step was transferred into fermentation medium at a ratio of 10%, the initial temperature was set to 37℃, the initial glucose concentration was 10-20 g / L, and the dissolved oxygen and stirring linkage were controlled to maintain the dissolved oxygen at 30%; when the fermentation liquid OD 600 =20-40, 0.5-1 mM IPTG was added, and the temperature was reduced to 20-25℃, and 10% n-dodecane was added to extract the product in situ; after the initial sugar consumption, the sugar supplementing speed was controlled to maintain the glucose concentration in the fermentation liquid at 1-2 g / L; product and OD 600 were detected every 8 h until the activity of the bacterial cells decreased. As shown in Table 1, CaWH can produce up to 804 mg / L of car S within 48 h, and the proportion of by-products can be controlled to be less than 1.6% throughout the process. This result shows that AaQHS1 (F372W / Y376H) plays a role in reducing by-products in the synthesis of car S in E. coli, and has a stable industrialization prospect.

[0039] In addition, the wild-type AaQHS1, AcHS3, TPS7 can be efficiently and soluble expressed in E. coli, yeast and Bacillus, so the mutants of the application are also expected to be efficiently expressed in yeast, Bacillus and other sesquiterpene model chassis, for higher titer and higher purity car S production, providing more efficient production elements for efficient biosynthesis of car S, and promoting the development of car S in the fields of food, medicine, fine chemicals, health products, biological energy and cosmetics.

[0040] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A caryophyllene synthase mutant, characterized in that, The mutant is any one of the following: (1) Mutate the 372nd amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.1 to leucine and the 376th amino acid to aspartic acid, glutamine or histidine. (2) Mutate the 372nd amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.1 to tryptophan and the 376th amino acid to aspartic acid, glutamic acid, glutamine or histidine; (3) Mutate the 376th amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.1 to glutamine or histidine; (4) Mutate the 382nd amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.3 to aspartic acid, glutamine or histidine; (5) Mutate the 378th amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.3 to tryptophan and the 382nd amino acid to aspartic acid, glutamine or histidine. (6) Mutate the 378th amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.3 to phenylalanine and the 382nd amino acid to aspartic acid, glutamine or histidine. (7) Mutate the 383rd amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.5 to aspartic acid, glutamic acid, glutamine or histidine; (8) Mutate the 379th amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.5 to tryptophan and the 383rd amino acid to glutamic acid, glutamine or histidine; (9) Mutate the 379th amino acid of the caryophyllene synthase parent with the amino acid sequence shown in SEQ ID NO.5 to phenylalanine and the 383rd amino acid to aspartic acid, glutamic acid, glutamine or histidine.

2. The gene encoding the caryophyllene synthase mutant of claim 1.

3. A recombinant vector carrying the gene of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector uses plasmid pET28a as the expression vector.

5. A recombinant cell expressing the mutant of claim 1, or containing the gene of claim 2, or transformed with the recombinant vector of claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells used Escherichia coli as the host.

7. A method for preparing a caryophyllene synthase mutant, characterized in that, The mutant of claim 1 is expressed using the recombinant cells of claim 5 or 6.

8. The preparation method according to claim 7, characterized in that, The recombinant cells used Escherichia coli as the host and pET28a as the expression vector.

9. A method for improving the catalytic selectivity of caryophyllene synthase, wherein the method involves mutating the 372nd amino acid of the caryophyllene synthase parent as shown in SEQ ID NO.1 to leucine and the 376th amino acid to aspartic acid, glutamine, or histidine; Alternatively, the amino acid sequence of the caryophyllene synthase parent, as shown in SEQ ID NO.1, can be mutated to tryptophan at position 372 and to aspartic acid, glutamic acid, glutamine, or histidine at position 376. Alternatively, the 376th amino acid of the caryophyllene synthase parent, as shown in SEQ ID NO.1, can be mutated to glutamine or histidine; Alternatively, the 382nd amino acid of the caryophyllene synthase parent, as shown in SEQ ID NO.3, can be mutated to aspartic acid, glutamine, or histidine. Alternatively, the amino acid sequence of the caryophyllene synthase parent, as shown in SEQ ID NO.3, can be mutated to tryptophan at position 378 and to aspartic acid, glutamine, or histidine at position 382. Alternatively, the amino acid sequence of the caryophyllene synthase parent, as shown in SEQ ID NO.3, can be mutated to phenylalanine at position 378 and to aspartic acid, glutamine, or histidine at position 382. Alternatively, the 383rd amino acid of the caryophyllene synthase parent, as shown in SEQ ID NO.5, can be mutated to aspartic acid, glutamic acid, glutamine, or histidine. Alternatively, the amino acid sequence of the caryophyllene synthase parent, as shown in SEQ ID NO.5, can be mutated to tryptophan at position 379 and to glutamic acid, glutamine, or histidine at position 383. Alternatively, the amino acid sequence of the caryophyllene synthase parent, as shown in SEQ ID NO.5, can be mutated to phenylalanine at position 379 and to aspartic acid, glutamic acid, glutamine, or histidine at position 383.

10. The use of the caryophyllene synthase mutant of claim 1, or the gene of claim 2, or the recombinant vector of claim 3 or 4, or the recombinant cell of claim 5 or 6, or the method of claim 9 in the preparation of caryophyllene or caryophyllene-containing products.

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