Sibertrienol synthase mutant and its application in the biosynthesis of sibertrienol

Abstract

This invention discloses a cephalothinyl synthase mutant and its application in the biosynthesis of cephalothinyl synthase, belonging to the fields of enzyme engineering and synthetic biology. Through rational design and mutation screening of tobacco-derived cephalothinyl synthase (NtCBTS), this invention obtained a mutant with significantly improved catalytic performance. Specifically, the cephalothinyl synthase yield of strain containing the mutant NtCBTS_K208R-A268C-K353R was increased by 231.18% compared to strains containing NtCBTS. Applying this mutant to a *Saccharomyces cerevisiae* expression system achieved highly efficient biosynthesis of cephalothinyl synthase, significantly improving the production level of the target product. The highly active enzyme mutant and its application strategy provided by this invention effectively improve the biosynthetic efficiency of cephalothinyl synthase, providing important technical support for constructing high-yield yeast cell factories and promoting its industrial production.

Description

Technical Field

[0001] This invention belongs to the fields of enzyme engineering and synthetic biology, specifically relating to a cephalothrix synthase mutant and its application in the biosynthesis of cephalothrix, particularly to a cephalothrix synthase mutant constructed using the amino acid sequence shown in SEQ ID No. 2 of the sequence listing as the starting sequence, and its application in the biological preparation of cephalothrix by recombinant expression cells. It also relates to the nucleic acid sequence encoding the enzyme, the recombinant expression vector containing the encoding nucleic acid sequence, and a method for preparing cephalothrix. Background Technology

[0002] Cembratrienol (CBT-ol) is a diterpenoid compound with a tobacco aroma, a colorless to pale yellow viscous liquid, and a diterpenoid compound with significant application potential. It is widely used as a flavoring agent in the food and beverage industry, possessing high economic value. Cembratrienol accounts for approximately 60% of the secretions from tobacco glandular trichomes and is a key secondary metabolite in plant defense mechanisms. Increasing its content can significantly enhance plant resistance to aphids and powdery mildew fungi, highlighting its potential as a biopesticide. Furthermore, cembrazolidol has shown potential in treating neurodegenerative diseases and has been shown to inhibit nicotine sensitization in rats, demonstrating its importance in the pharmaceutical field. Currently, the main industrial method for obtaining cembrazolidol is extraction from tobacco leaves. However, due to the low concentration of cembrazolidol in tobacco, limited sources, long plant cultivation cycles, low efficiency, and high costs, this method cannot be considered a sustainable means of acquisition. Furthermore, since cephalothrixol is a diterpenoid compound with a complex cyclic structure, its total chemical synthesis is complex, involves numerous steps, and is costly, making it unsuitable for industrial production. Therefore, biosynthesis technology, with its advantages of short cycle time, high efficiency, and relatively low equipment requirements, has become an ideal path for its large-scale production. In tobacco plants, cephalothrixol is synthesized from geranyl-geranyl pyrophosphate (GGPP) catalyzed by cephalothrixol synthase (CBTS). The biosynthetic precursors of GGPP—isopentene pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP)—are mainly synthesized via the mevalonic acid (MVA) pathway and the 1-deoxy-D-xylitol-5-phosphate (DXP) pathway (or MEP pathway).

[0003] Recently, microbial cell factories have been widely used in the production of compounds. Currently, expressing cephalothinol synthase in microorganisms enables heterologous synthesis of cephalothinol, but the yield levels are still low. The main reason is the low activity of existing cephalothinol synthases, often requiring complex metabolic engineering modifications to achieve higher yields. Therefore, obtaining cephalothinol synthases with high enzyme activity is a key factor in achieving high cephalothinol production. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a cephalothrix synthase mutant and its application in the biosynthesis of cephalothrix alcohol, thereby solving the problems of low cephalothrix synthase activity and low cephalothrix alcohol yield in the prior art.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] This invention provides a cibarbiturate synthase mutant. The previously reported cibarbiturate synthase NtCBTS is derived from tobacco (Nicotiana tabacum), and its amino acid sequence is shown in SEQ ID No. 2. Compared to NtCBTS, the cibarbiturate synthase mutant contains at least one amino acid substitution selected from Q35E, K208R, A268C, and K353R. The site is defined with reference to SEQ ID No. 2. The cibarbiturate synthase mutant exhibits higher activity than NtCBTS.

[0007] Preferred cephalothrix synthase mutants are: (1) NtCBTS_A268C; (2) NtCBTS_K208R; (3) NtCBTS_Q35E; (4) NtCBTS_K353R; (5) NtCBTS_Q35E-K208R; (6) NtCBTS_Q35E-A268C; (7) NtCBTS_Q35E-K353R; (8) NtCBTS_Q35E-A268C; (9) NtCBTS_Q35E-K353R; (10) NtCBTS_Q35E-A268C; (11) NtCBTS_Q35E-K353R; (12) NtCBTS_Q35E-A268C; (13) NtCBTS_Q35E-K353R; (14) NtCBTS_Q35E-K208R; (15) NtCBTS_Q35E-A268C; (16) NtCBTS_Q35E-K353R; (17) NtCBTS_Q35E-A268C; (18) NtCBTS_Q35E-K353R; (19) NtCBTS_Q35E-A268C; (10) NtCBTS_Q35E-K353R; (10) NtCBTS_Q35E-A268C; (11) NtCBTS_Q35E-A268C; (12) NtCBTS_Q35E-K353R; (13) NtCBTS_Q35E-A268C; (14) NtCBTS_Q35E-K353R; (15) NtCBTS_Q35E-K208R; (19) NtCB (9) NtCBTS_K208R-K353R; (10) NtCBTS_A268C-K353R; (11) NtCBTS_Q35E-K208R-A268C; (12) NtCBTS_Q35E-K208R-A268C-K353R; where all sites are defined with reference to SEQ ID No. 2.

[0008] More preferably, the cebertrienol synthase mutant is NtCBTS_K208R-A268C-K353R, which is a mutant NtCBTS_K208R-A268C-K353R in which lysine (K) at position 208 is mutated to arginine (R), alanine (A) at position 268 is mutated to cysteine ​​(C), and lysine (K) at position 353 is mutated to arginine (R). The amino acid sequence is shown in SEQ ID No. 3; the nucleotide sequence is shown in SEQ ID No. 4.

[0009] In this invention, a gene encoding the above-mentioned cephalothrix-trienol synthase mutant is provided.

[0010] This invention provides a recombinant expression vector containing the coding gene of the above-mentioned cebertrienol synthase mutant, and provides a method for preparing cebertrienol by introducing the recombinant expression vector into an engineered strain of Saccharomyces cerevisiae.

[0011] According to the present invention, experiments have confirmed that the cephalothrix synthase mutant NtCBTS_K208R-A268C-K353R described in this invention has higher activity than the NtCBTS that has been reported and widely used in the bio-production of cephalothrix, and the yield of cephalothrix is ​​increased by 231.18%, which further improves the yield level of biosynthesized cephalothrix and lays the foundation for building a high-yield yeast cephalothrix cell factory.

[0012] As used herein, the term "mutation" refers to a mutation in the enzyme in which at least one nucleotide or amino acid in the mutant gene or amino acid sequence of the cebertrienol synthase differs from the NtCBTS starting sequence being compared, and the mutation can be achieved by site-directed mutagenesis using methods conventional in the art.

[0013] As used herein, the term “nucleic acid molecule” has the meaning commonly understood by one of ordinary skill in the art. A nucleic acid molecule may be a polynucleotide as shown in SEQ ID No. 4, or a polynucleotide that also includes additional coding and / or non-coding sequences.

[0014] As used herein, the terms "vector" and "expression vector" have the meanings commonly understood by those skilled in the art. A "vector" refers to a nucleic acid medium into which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, it is called an expression vector. In the process of expressing a foreign gene using *Saccharomyces cerevisiae*, the foreign gene to be expressed must first be ligated to a vector. The vector can be any conventional vector in the art, such as commercially available plasmids, bacteriophages, viral vectors, etc. This invention preferably uses plasmid YEp352 to express the cephalothin-351 mutant gene.

[0015] This invention provides recombinant expression cells comprising the vector of this invention. These recombinant expression cells can be prepared by transforming the recombinant expression vector of this invention into host cells. The host cells can be various conventional host cells in the art, provided that the recombinant expression vector can be stably replicated and passaged, and the gene carried by it can be effectively expressed. Preferred strains are *Saccharomyces cerevisiae*, more preferably *S. cerevisiae* CEN.PK2-1Ca strain or *S. cerevisiae* PL00.

[0016] In this invention, the aforementioned mutant-related biomaterials are any one or more combinations of the following biomaterials:

[0017] (a) An expression cassette containing the above-mentioned encoded genes;

[0018] (b) Recombinant expression vectors containing the above-mentioned coding genes;

[0019] (c) A recombinant expression vector containing the expression cassette described in (a);

[0020] (d) Recombinant expression cells containing the above-encoded genes;

[0021] (e) Recombinant expression cells containing the expression cassette described in (a);

[0022] (f) Recombinant expression cells containing the recombinant expression vector described in (b) or (c).

[0023] Furthermore, the starting vector for the recombinant expression vector described in (b) and (c) is a plasmid of the YEp series, etc.; preferably, it is the YEp352 plasmid.

[0024] Furthermore, the host bacteria of the recombinant expression cells described in (d), (e), and (f) are selected from eukaryotes, etc.; the eukaryotes include Saccharomyces, etc. More specifically, the eukaryote is Saccharomyces cerevisiae, specifically S. cerevisiae CEN.PK2-1Ca or S. cerevisiae PL00.

[0025] This invention provides a method for obtaining the above-mentioned cephalothorax synthase mutant, comprising the following steps:

[0026] By designing primers containing mutation sites to perform site-directed mutagenesis on the gene encoding cebertrienol synthase NtCBTS, whose amino acid sequence is shown in SEQ ID No. 2, a cebertrienol synthase mutant was obtained.

[0027] Furthermore, primers containing mutation sites were designed to introduce mutations into the gene encoding cephalothin-1,4-diol synthase NtCBTS, as shown in SEQ ID No. 2. After correct sequencing, the gene was transformed into Saccharomyces cerevisiae for expression, resulting in a cephalothin-1,4-diol synthase mutant.

[0028] Furthermore, using plasmid YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t as a template, primers containing mutation sites were designed to introduce mutations into the gene encoding cebertrienol synthase NtCBTS. The mutants were then transformed into Saccharomyces cerevisiae, and plasmids were extracted from the transformants for sequencing.

[0029] This invention provides an application of the aforementioned mutant, encoding gene, and mutant-related biomaterials in the synthesis of cepertrienol; and further, its application in the biosynthesis of cepertrienol.

[0030] This invention provides a method for producing cepertrienol, comprising the step of fermenting the above-mentioned recombinant expression cells.

[0031] Specifically, the steps include the following:

[0032] Ferment the above-mentioned recombinant expression cells and collect the fermentation products;

[0033] Sibertrienol was extracted from the fermentation product.

[0034] The specific method involves using *Saccharomyces cerevisiae* as a host, transforming the expression vector containing the *cibertrienol synthase* mutant gene described in this invention into the host to obtain engineered *Saccharomyces cerevisiae* strains. The engineered strains are then inoculated into a uracil-deficient culture medium for fermentation. Specific reaction conditions, such as substrate concentration, pH, culture medium composition, and amount of recombinant expression transformant, can be selected according to conventional conditions for such reactions in the art. The fermentation can be carried out under shaking or stirring conditions. The fermentation time is preferably 72 hours. After the reaction is completed, the content of *cibertrienol* in the reaction mixture can be determined using conventional gas chromatography methods in the art.

[0035] The bioreactor used is, in principle, selected from equipment capable of ensuring cell proliferation and biochemical reactions of living cells and enzymes, such as small shake flasks or fermenters. The culture medium used is primarily a commonly used medium suitable for the growth of *Saccharomyces cerevisiae*, with a preference for a uracil-deficient medium in this invention. The fermentation temperature is preferably selected to maintain good cell growth and optimal enzyme catalytic performance. In one specific embodiment, a fermentation temperature of 30°C is preferred.

[0036] The present invention has the following advantages and effects compared with the prior art:

[0037] (1) This invention utilizes synthetic biology and enzyme engineering to provide a cepertrienol synthase mutant and its application in the synthesis of cepertrienol in Saccharomyces cerevisiae. By providing a cepertrienol synthase mutant with enhanced catalytic activity, the yield of cepertrienol is increased, creating conditions for the industrial production of cepertrienol.

[0038] (2) This invention, through rational design and mutation screening of tobacco-derived cephalothinol synthase (NtCBTS), obtained mutants with significantly improved catalytic performance. Among them, the strain containing the mutant NtCBTS_K208R-A268C-K353R increased the yield of cephalothinol by 231.18% compared to strains containing NtCBTS, achieving high-yield production of cephalothinol in yeast. The cephalothinol synthase mutant of this invention lays a strong foundation for the industrial production of cephalothinol.

[0039] (3) The present invention applies the mutant to the Saccharomyces cerevisiae expression system, realizing the efficient biosynthesis of cepertrienol and significantly improving the production level of the target product. The highly active enzyme mutant and its application strategy provided by the present invention effectively improve the biosynthetic efficiency of cepertrienol, providing important technical support for the construction of high-yield yeast cell factories and the promotion of its industrial production. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the synthesis route of cebertrienol.

[0041] Figure 2 These are the fermentation results of a single-point mutant of NtCBTS.

[0042] Figure 3 These are the fermentation results of the NtCBTS multi-point mutant.

[0043] Figure 4 The image shows the recombinant plasmid YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t; where AmpR is the ampicillin resistance gene. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. It is worth noting that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art with reference to existing technology. Unless otherwise specified, the reagents, materials and instruments used can be regarded as conventional products that can be obtained from commercial channels.

[0045] The pathway diagram for the synthesis of cephalothrix trienol by cephalothrix synthase in this invention is as follows: Figure 1 As shown.

[0046] The term "enzyme activity" as used in this article refers to enzyme activity measured in units of OD under the same culture conditions. 600 The corresponding amount of product generated is used as a characterization indicator for relative enzyme activity.

[0047] Example 1 Construction of the starting strain of cephalothrix trienol synthase

[0048] The codon-optimized NtCBTS gene (its nucleotide sequence is shown in SEQ ID No. 1, and the corresponding encoded amino acid sequence is shown in SEQ ID No. 2) and gerany-gerany pyrophosphate synthase GGPPS (its nucleotide sequence is shown in SEQ ID No. 5, and it also contains a 6×His sequence, and the corresponding encoded amino acid sequence is shown in SEQ ID No. 6) were synthesized by Sangon Biotech. The promoter of the NtCBTS gene expression cassette is TDH3, and the terminator is ADH1; the promoter of the GGPPS gene expression cassette is TEF1, and the terminator is CYC1. The promoters TDH3, TEF1, ADH1, and CYC1 are all derived from Saccharomyces cerevisiae.

[0049] Using the yeast expression vector YEp352 (purchased from Invitrogen) as the backbone vector and the NtCBTS and GGPPS gene expression cassettes as insert fragments, PCR was performed using PrimeSTAR Max enzyme.

[0050] The PCR primers are as follows:

[0051] 352-backbone-F: 5'-aaggctttaatttgcggatccctgcaggcatgcaa-3';

[0052] 352-backbone-R: 5'-ccaggaataaactgtgtcgacggtaaagaattcgt-3';

[0053] TDH3-NtCBTS-F: 5'-acagtttattcctggcatcc-3';

[0054] ADH1-NtCBTS-R: 5'-tctagaggatcccatagggtaggggaatttcg-3';

[0055] TEF1-GGPPS-F: 5'-cctatggggatcctctagacttcaaaatgtttctactc-3';

[0056] CYC1-GGPPS-R: 5'-gcaaattaaagccttcgagc-3'.

[0057] The target fragment was recovered and subjected to a three-fragment homologous recombination ligation reaction using ClonExpress II to obtain the recombinant plasmid YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t.

[0058] The ligation product was transformed into *E. coli* DH5α competent cells: 10 μL of the ligation product was added to 20 μL of competent cells, incubated on ice for 30 min, then heat-shocked at 42 ℃ for 90 s, and immediately cooled on ice for 5 min; 800 μL of antibiotic-free LB liquid medium was added, and the cells were revived and cultured at 37 ℃ and 220 rpm for 1 h. After revival, the bacterial culture was centrifuged to collect the cells, some of the supernatant was discarded, and the cells were resuspended. Approximately 300 μL of the bacterial culture was spread on LB agar plates containing 100 μg / mL ampicillin and cultured at 37 ℃ for 12–16 h.

[0059] After single colonies grew on the plates, transformants were randomly selected for colony PCR identification to verify the correct insertion of the target fragment. Positive clones with correct identification were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37℃ and 220 rpm for 12–16 h. Plasmid DNA was then extracted, and colony PCR and sequencing were performed to further verify its correctness. The correctly identified recombinant plasmid was named YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t. The plasmid map is shown below. Figure 4 .

[0060] Saccharomyces cerevisiae PL00 strain, stored at -80℃, was activated by streak plating on YPD plates. Single colonies were picked and inoculated into 5 ml of YPD liquid medium and incubated at 30℃ and 200 rpm for 12–16 h until OD was reached. 600 Once the OD value reaches between 3.0 and 5.0, take a certain amount of bacterial culture and transfer it to a new 5 mL YPD liquid medium to control the initial OD. 600 Incubate at 0.2-0.4 g / L at 30°C and 220 rpm for approximately 4-6 hours to obtain the final OD value. 600The concentration should be controlled between 0.8 and 1.0. Prepare competent yeast cells using the kit: Transfer 1 mL of bacterial culture to a 1.5 mL centrifuge tube, centrifuge at 7000 rpm for 3 min, and discard the supernatant. Resuspend the cells in 1 mL of Solution I, centrifuge at 7000 rpm for 3 min, and discard the supernatant. Resuspend the cells in 100 μL of Solution II. The competent yeast cells are now obtained and aliquoted into 25 μL tubes. Transform the constructed recombinant plasmid YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t into S. cerevisiae PL00 competent cells to obtain the recombinant strain S. cerevisiae PL00 / YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t. After the transformants have grown, pick them from the plate for colony PCR to verify the correct size of the target band and then sequence them. If the verification is correct, record them as the Siberia trienol synthase control strain.

[0061] Among them, Saccharomyces cerevisiae PL00 is disclosed in "CN118792290A, an α-farnesene synthase AoFS of a brain-boosting origin and its application".

[0062] Example 2 Construction of a Siberia trienol synthase mutant strain

[0063] In screening for highly active cephalothinol synthase mutants, AlphaFold2 was used to predict and model the NtCBTS protein structure, obtaining its three-dimensional structural model. Subsequently, AutoDock Vina was used to dock the GGPP molecule to the catalytic center region, analyzing its binding conformation and interactions with surrounding amino acid residues. CAVER 3.0 was used to analyze the substrate entry channel, identifying key residues in the channel bottleneck region. Combining residue conservation analysis and spatial location, four mutation sites—Q35E, K208R, A268C, and K353R—were screened for mutation experiments.

[0064] Homologous recombination, a method commonly used in molecular cloning, was employed to construct the cebertrienol synthase mutant. Using PCR, the recombinant plasmid YEp352-TDH3p-NtCBTS-ADH1t-TEF1p-GGPPS-CYC1t was used as a template for site-directed mutagenesis to obtain a plasmid containing the NtCBTS mutant gene. The mutation sites were: Q35E, K208R, A268C, and K353R.

[0065] The primers used for PCR are as follows (uppercase letters indicate replaced codons):

[0066] 352-Tong-F:5'-caagctgtgaccgtctccgggagc-3';

[0067] 352-Tong-R:5'-gctcccggacggtcacagcttg-3';

[0068] Q35E-F:5'-actgaaataactactGAAgaaaagaatgagc-3';

[0069] Q35E-R:5'-gctcattcttttcTTCagtagttatttcagt-3';

[0070] K208R-F:5'-agagtttggagctagaAGAtatattcatatctat-3';

[0071] K208R-R:5'-atagatatgaatataTCTtctagctccaactct-3';

[0072] A268C-F:5'-gataggttggttgaaTGTtatttttggactgtt-3';

[0073] A268C-R:5'-aacagtccaaaaataACAttcaaccaacctatc-3';

[0074] K353R-F:5'-gaagaagttttagctAGAgagggtaaagctgat-3';

[0075] K353R-R:5'-atcagctttaccctcTCTagctaaaacttcttc-3'。

[0076] The upstream primer F and universal primer 352-Tong-R corresponding to each mutation site were used to amplify the upstream fragment, and the downstream primer R and universal primer 352-Tong-F corresponding to each mutation site were used to amplify the downstream fragment. PrimeSTAR Max enzyme was used for PCR amplification of the target fragment. Homologous recombination ligation of the amplified upstream and downstream fragments was performed using the ClonExpress II recombinant cloning kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.). The recombinant products were then transformed into E. coli DH5α competent cells, and colony PCR verification and plasmid extraction were performed. Sequencing verification confirmed correct results, yielding recombinant plasmids containing the gene encoding the cephalothin-1,4-diol synthase mutant. These recombinant plasmids were then transformed into S. cerevisiae PL00 competent cells to obtain recombinant strains containing the gene encoding the cephalothin-1,4-diol synthase mutant.

[0077] Example 3: Shake-flask fermentation and product detection of the sispertrienol synthase mutant strain

[0078] The recombinant Saccharomyces cerevisiae strains constructed in Examples 1 and 2 were selected for shake-flask fermentation and product detection and analysis.

[0079] First, a single colony was inoculated into 5 mL of SD-ΔUra liquid medium (SD medium lacking uracil (Ura)) and pre-cultured for 16 h at 30 ℃ and 220 rpm. Then, the colonies were cultured according to the initial OD... 600 The culture medium was transferred at a concentration of 0.05 μL to 10 mL of SD-ΔUra liquid medium and cultured in shake flasks for 72 h under the same conditions. Three biological replicates were set up for each strain to ensure the reliability of the experimental results.

[0080] After fermentation, 1 mL of fermentation broth was added to a disruption tube, along with 1 mL of extraction solvent (ethyl acetate: n-hexane = 1:1, v / v) and a suitable amount of grinding beads. The cells were disrupted at 12000 rpm for 5 min to achieve complete cell lysis. Subsequently, the mixture was centrifuged at 12000 rpm for 5 min to separate the organic phase from the cell residue. Approximately 500 μL of the supernatant was transferred to a 1.5 mL centrifuge tube, dried under nitrogen, and then reconstituted with 100 μL of ethyl acetate. After mixing, the mixture was filtered through a 0.22 μm filter membrane and transferred to a sample vial for analysis.

[0081] Gas chromatography (GC) was used for product detection and analysis. The instrument was a Shimadzu GC-2014C, equipped with an HP-5 capillary column (30 m × 0.32 mm × 0.25 μm) and a flame ionization detector (FID). The detector temperature was set to 280 ℃, and the injection port temperature was 250 ℃. Nitrogen was used as the carrier gas, with a split ratio of 5:1, and a split injection mode was employed, with an injection volume of 1 μL per injection.

[0082] The chromatographic program was set as follows: the initial temperature of the column oven was 80 ℃, held for 1 min; the temperature was increased to 200 ℃ at 15 ℃ / min, held for 1 min; then the temperature was increased to 220 ℃ at 4 ℃ / min, held for 3 min; then the temperature was increased to 240 ℃ at 30 ℃ / min, held for 2 min, and the total running time was 26 min.

[0083] Based on the GC analysis results, the fermentation products of each mutant under the same expression conditions were quantitatively analyzed, and the results are as follows: Figure 2-3 As shown in Table 1.

[0084] NtCBTS_A268C, NtCBTS_K208R, NtCBTS_Q35E, and NtCBTS_K353R all exhibited significant positive effects, with relative enzyme activities increasing by 32.06%, 34.67%, 44.93%, and 48.80% respectively compared to the control. This preliminarily suggests that these sites may be involved in optimizing the substrate binding environment or catalytic conformation.

[0085] Based on this, a stepwise combination mutation strategy was employed to verify the synergistic effect at key sites. First, using A268C, K208R, Q35E, and K353R as candidate sites, a second mutation site was introduced into the single mutants to construct double mutants NtCBTS_Q35E-K208R, NtCBTS_Q35E-A268C, NtCBTS_Q35E-K353R, NtCBTS_K208R-K353R, and NtCBTS_A268C-K353R. The results showed that NtCBTS_A268C-K353R exhibited the most significant increase in enzyme activity, increasing by 86.51%, indicating that this combination has a good synergistic effect.

[0086] Furthermore, based on NtCBTS_A268C-K353R, a third mutation site was introduced to construct triple mutants NtCBTS_Q35E-A268C-K353R and NtCBTS_K208R-A268C-K353R. Experimental results showed that the combined mutants containing K208R, A268C, and K353R exhibited the best catalytic performance. NtCBTS_K208R-A268C-K353R maintained a high level of enzyme activity enhancement, which was 231.18% higher than that of NtCBTS.

[0087] Based on NtCBTS_K208R-A268C-K353R, Q35E was introduced to construct a quad mutant NtCBTS_Q35E-K208R-A268C-K353R. The results showed that the relative enzyme activity of this quad mutant was also significantly increased, by 127.75%.

[0088] In summary, the strategy of "rational design - dominant site identification - stepwise combination optimization" effectively enhanced the activity of NtCBTS enzyme, and verified that there is a certain degree of synergistic effect among key sites.

[0089] Table 1. Relative enzyme activities of mutants

[0090]

[0091] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A cephalothorax synthase mutant, characterized in that: The amino acid sequence of the cibertrienol synthase mutant is obtained by any one of the following mutations of SEQ ID No. 2: At least one of Q35E, K208R, A268C and K353R.

2. The cephalothorax synthase mutant according to claim 1, characterized in that: The cebertrienol synthase mutants are: NtCBTS_A268C, NtCBTS_K208R, NtCBTS_Q35E, NtCBTS_K353R, NtCBTS_Q35E-K208R, NtCBTS_Q35E-A268C, NtCBTS_Q35E-K353R, NtCBTS_K208R-K353R, NtCBTS_A268C-K353R, NtCBTS_Q35E-K208R-A268C, NtCBTS_K208R-A268C-K353R, or NtCBTS_Q35E-K208R-A268C-K353R; The amino acid sequence of the mutant NtCBTS_K208R-A268C-K353R is shown in SEQ ID No.

3.

3. The gene encoding the cebertrienol synthase mutant as described in claim 1 or 2.

4. The gene according to claim 3, characterized in that: The nucleotide sequence of the gene encoding the mutant NtCBTS_K208R-A268C-K353R is shown in SEQ ID No.

4.

5. The biomaterials related to the cephalothorenol synthase mutant according to any one of claims 1 to 2, characterized in that: It can be any one or more combinations of the following biological materials: (a) An expression cassette containing the gene of claim 3 or 4; (b) A recombinant expression vector containing the gene of claim 3 or 4; (c) A recombinant expression vector containing the expression cassette described in (a); (d) Recombinant expression cells containing the gene of claim 3 or 4; (e) Recombinant expression cells containing the expression cassette described in (a); (f) Recombinant expression cells containing the recombinant expression vector described in (b) or (c).

6. The biomaterial according to claim 5, characterized in that: The starting vector for the recombinant expression vectors described in (b) and (c) is a plasmid from the YEp series; The host bacteria of the recombinant expression cells described in (d), (e), and (f) are selected from eukaryotes.

7. The use of the gene according to any one of claims 3 to 4 or the biomaterial according to any one of claims 5 to 6 in the preparation of cepertrienol synthase mutants.

8. The use of the cebertrienol synthase mutant according to any one of claims 1 to 2, the gene according to any one of claims 3 to 4, or the biomaterial according to any one of claims 5 to 6 in the biosynthesis of cebertrienol.

9. A method for producing cepertrienol, characterized in that: It includes the step of fermenting the recombinant expression cells as described in any one of claims 5 to 6.

10. A method for obtaining the cephalothrix-trienol synthase mutant according to any one of claims 1 to 2, characterized in that, The method includes the following steps: by designing primers containing mutation sites to perform site-directed mutagenesis on the gene encoding the amino acid sequence NtCBTS as shown in SEQ ID No. 2, the mutant of cephalothryn-tanyl synthase as described in any one of claims 1 to 2 is obtained.

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